1,3-bis[3-(dimethylamino)propyl]urea

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 June 2020

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis[3-(dimethylamino)propyl]urea

EC Number:

257-861-2

EC Name:

1,3-bis[3-(dimethylamino)propyl]urea

Cas Number:

52338-87-1

Molecular formula:

C11H26N4O

IUPAC Name:

1,3-bis[3-(dimethylamino)propyl]urea

Constituent 2

Reference substance name:

3-(dimethylamino)propylurea

EC Number:

401-950-2

EC Name:

3-(dimethylamino)propylurea

Cas Number:

31506-43-1

Molecular formula:

C6H15N3O

IUPAC Name:

3-dimethylaminopropyl urea

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: Sponsor; Batch No.: UA21111132
- Purity, including information on contaminants, isomers, etc.: not reported

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™-Standard Model (EPI-200™): normal human epidermal keratinocytes (NHEK) which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstituted three-dimensional human epidermis model

Justification for test system used:

The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, [7]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) whichmay be considered as non-irritant.

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of
normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDermTM (MatTek)
- Tissue batch number(s): 34191
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution: 1. MTT stock solution: 5 mg/mL MTT (VWR; Lot 20B2456894) in PBS (Gibco; Lot No.: 2306402); 2. MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Incubation time: 60 min
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability, Barrier function and Morphology: Tissue viability and the barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: The cells used to produce EpiDermTM tissue are screened for potential biological contaminants. Tests for each potential biological contaminant listed below were performed according to the test method give. Results of “Non detected” indicated that testing for the potential biological contaminant was not observed as determined by the stated test method.
NUMBER OF REPLICATE TISSUES: three
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates: three
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Mean Tissue Viability (% negative control) is <= 50 %-> Irritant: UN GHS -> Category 2
- Mean Tissue Viability (% negative control) is > 50% -> Non- Irritant -> UN GHS “No Category”

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 μL

Number of replicates:

Three tissues per dose group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean Relative Tissue Viability [%]

Value:

3.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

Mean Relative Tissue Viability [%]: 100

Positive controls validity:

valid

Remarks:

Mean Relative Tissue Viability [%]: 3.9 %

Other effects / acceptance of results:

Pre-Experiments: The mixture of 30 μL test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.
The mixture of 30 μL of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equaled 0%.
The test item showed non-specific reduction of MTT but no relevant colouring potential after mixturewith aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false negative results were necessary

Any other information on results incl. tables

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm^TM (MatTek) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study PU-2021-958 was applied topically to the EpiDerm^TM tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.
Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.
The test item showed non-specific MTT-reducing potential. The test item did not show water-colouring potential. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.
Since neither killed tissue controls nor controls with viable tissues were performed, no additional controls for correction of possible false-negative results were necessary.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was ≤ 50%. The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.