N,N”-hexane-1,6-diylbis(N’-alkylaryl)urea

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 - 15 Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): trade name given
- Substance type: white, crystalline powder
- Analytical purity: 99.5%
- Solubility: water: < 0.1 g/L
- Lot/batch No.: 1592ZG-076
- Expiration date of the lot/batch: Aug 2015
- Storage condition of test material: at room temperature 20 ± 5 °C

Test animals

Species:

human

Strain:

other: EpiDermTM

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEST METHOD
The EpiDermTM tissue consists of 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 test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential.

ADAPTATION TO CELL CULTURE CONDITIONS
Tissues were transferred into 6-well plates (three per plate, upper row) containing 900 µL assay medium per well and preincubated in an incubator for 1 h (37 °C, 5% CO2). After the preincubation, every tissue was transferred into a well of the lower row containing 900 µL fresh assay medium . All 6-well-plates were set into the incubator at 37 °C and 5% CO2 for 18 hours.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37
- CO2 gas concentration (%): 5

Test system

Type of coverage:

other: open, in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: Concurrent control tissues treated with DPBS buffer served as negative controls, positive controls were exposed to 5% SDS.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 24.9 mg (mean)
The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size.

POSITIVE CONTROL SUBSTANCE
- Positive control substance: SDS, 5%

NEGATIVE CONTROL SUBSTANCE
- Negative control substance: DPBS buffer

Duration of treatment / exposure:

60 min (35 min at 37°C and 25 min at RT)

Observation period:

Not applicable.
Post-treatment incubation period: 42 ± 2 h

Number of animals:

Not applicable.
The test was performed in triplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.63 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with DPBS buffer.
- Time after start of exposure: 60 min
- Post-treatment incubation period: 42 ± 2 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed 42 ± 2 h h after the incubation period. Therefore, tissues were incubated in 300 µL freshly prepared MTT-reagent for 3 h ± 5 min at 37 ± 1 °C and 5% CO2. After aspiration of the MTT reagent, tissues were washed several times in DPBS buffer followed by tissue drying. Extraction of the formazan product was carried out in 2 mL isopropanol for 2 h. The optical density was measured at 570 nm wave length in a plate spectral photometer.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls (DPBS buffer). Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

2.2

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (5% SDS). Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

101.8

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 60 min. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18%).
After the treatment with the test substance, the relative absorbance values increased compared to the negative control to 101.8 %. This value is well above the threshold for irritation potential (50%).

Any other information on results incl. tables

Table 4. MTT assay after 60 min exposure.

	Negative control			Positive control			Test substance
Tissue sample	1	2	3	1	2	3	1	2	3
OD570	2.079 2.072	2.213 2.195	1.917 1.921	0.082 0.082	0.086 0.082	0.081 0.079	2.040 2.020	2.046 2.211	2.180 2.120
OD570 (mean-blank)	2.039	2.167	1.882	0.045	0.045	0.043	1.993	2.092	2.113
Relative SD (%)	7.0			3.1			4.4
OD570(mean values of replicates)	2.029			0.045			2.066
Viability (%)	100			2.2			101.8

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified