4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 October 2021 to April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to the OECD TG 439 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

14 June 2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

EC Number:

262-911-1

EC Name:

4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

Cas Number:

61698-32-6

Molecular formula:

C11H12N2O2

IUPAC Name:

[4-(hydroxymethyl)-2-phenyl-1H-imidazol-5-yl]methanol

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: white to light-pink powder
- Storage: Keep away from heat and sources of ignition. Store in tightly closed container. Store in a well-ventilated place. Store in a cool, dark place.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in a dry place
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable under storage conditions
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: NA
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: NA
- Reactivity of the test material with the incubation material used (e.g. plastic ware): NA

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): NA
- Preliminary purification step (if any): NA
- Final concentration of a dissolved solid, stock liquid or gel: NA
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): NA

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: NA

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE/ Human Epidermis (RHE/S/17)
- Tissue batch number: 21-RHE-147
- Production date: 21 September 2021 (expiry date: 27 September 2021)
- Shipping date: 21 September 2021
- Delivery date: 21 September 2021
- Date of initiation of testing: 21 September 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1mL washes with Phosphate Buffer Saline (DPBS)
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm
- Filter: n.a.
- Filter bandwidth: n.a.
- Linear OD range of spectrophotometer: Not stated

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Specification: O.D. > 0.7, Result O.D = 1.1 (CV = 7.1%)
- Barrier function: Specification: 4.0h < ET50 < 10.0h
- Morphology: Multi-layered, highly differentiated epidermis consisting of organised basal, spinous and granular layers, and a multilayered stratum corneum.
- Contamination: Cell have been verified the absence of bacteria, fungus and mycoplasma
- Reproducibility: Not stated

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours (± 1 hour) of post-treatment incubation is > 50%.
- The test substance is considered to be non-corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours (± 1 hour) of post-treatment incubation is ≤ 50
- Justification for the selection of the cut-off point(s) if different than recommended in TG 439: n/a (same as OECD TG 439)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg
- Concentration (if solution): NA

VEHICLE
- Amount(s) applied (volume or weight with unit): NA
- Concentration (if solution): NA
- Lot/batch no. (if required): NA
- Purity:

NEGATIVE CONTROL
- Amount applied (volume) : 16 µL
- Concentration (if solution): NA

POSITIVE CONTROL
- Amount(s) applied (volume): 16 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours ± 1 hour

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

123.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

98.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

87.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none observed
- Direct-MTT reduction: The direct interaction of MTT with the test item was checked by adding 16 mg of the test item to 300 µL of the solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow solution with white test item was observed after 3 hours of incubation between 37.2°C and 37.4°C, 5% CO2.
Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT: The spectral properties at 570 nm of test item in isopropanol were checked by adding 16 mg of the test item to 1.5 mL of isopropanol (same conditions as in the main test). A white solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking.
The solution was centrifuged for 1 minute at 16000 g before dosing the supernatant.
The mean of the corrected OD after centrifugation was 0.319 which is higher than 0.08 (value corresponding to approximately twice the OD of the extracting solvent).
Therefore, the test item was identified as causing colour interference with the viability assay (mean of the corrected OD > 0.08) and two viable control tissues were added to the study which underwent the entire testing procedure but were incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSC living) control.


DEMONSTRATION OF TECHNICAL PROFICIENCY: yes (cf. 'Attachment')

ACCEPTANCE OF RESULTS: ('cf. Attachment')
- Acceptance criteria met for negative control: yes, OD value of the 3 replicates in the range ≥ 0.8 and ≤ 3.0 and the SD value of the % viability ≤ 18%.
- Acceptance criteria met for positive control: yes, Mean Viability < 40%, and SD value of the % viability ≤ 18%.
- Acceptance criteria met for variability between replicate measurements: OD values of the 3 replicates in the range ≥ 0.8 and ≤ 3.0, and SD value of the % viability is 18.4%.
- Range of historical values if different from the ones specified in the test guideline: cf. attachment.

Any other information on results incl. tables

Table 1: Test item - assessment of the skin irritation individual and average values of OD after 42 minutes exposure

	Well ID	OD	Mean OD/ disc	Mean OD/product	Viability %	Mean viability %	SD	Conclusion
Negative control	SPL_1	0.945 0.921 0.923	0.930	0.868	107.1	100.0	7.9
	SPL_2	0.762 0.778 0.841	0.794		91.4
	SPL_3	0.852 0.896 0.895	0.881		101.5
Positive control	SPL_4	0.011 0.011 0.011	0.011	0.011	1.3	1.3	0.1	Irritant
	SPL_5	0.012 0.012 0.011	0.012		1.4
	SPL_6	0.010 0.013 0.011	0.011		1.3
Test Item PH-21/0755	SPL_7	1.073 1.043 1.096	1.071	0.895	123.3	103.1	18.4
	SPL_8	0.857 0.852 0.861	0.857		98.7
	SPL_9	0.790 0.725 0.759	0.758		87.3
Test Item PH-21/0755 NSC living	SPL_10	0.001 0.001 0.001	0.001	0.001	0.1	0.1	0.1
	SPL_11	0.000 -0.001 -0.001	0.000		0.0
Test item PH-21/-755 Corrected						103.0		Non Irritant

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corrected percent viability of the treated tissues was 103.0%. Not classified according to CLP and GHS criteria.

Executive summary:

In an in vitro skin irritation study (Reconstructed Human Epidermis Test Methods - SkinEthic RHE^® model) performed according to the OECD TG No. 439 and in compliance with GLP, 16 mg of undiluted test item was applied to three living reconstructed epidermis for 42 minutes. The application was followed by a rinse with 25 mL of PBS and a 41 hours incubation period at 37°C, 5% CO₂. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, 2 living Human skin model surfaces were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living color controls.

 

The mean corrected percent viability of the treated tissues was 103.0% for the test item and 1.3% for the positive control (5% SDS).

The quality criteria required for acceptance of results in the tests were satified:

• SD ≤ 18%


• Negative control: OD value of the 3 replicates in the range ≥ 0.8 and ≤ 3.0 and the SD value of the % viability ≤ 18%.


• Positive control: Mean Viability < 40%, and SD value of the % viability ≤ 18%.

 

In accordance with the Regulation EC No. 1272/2008, the test item CUREZOL 2PHZ-PW is not considered to be irritating to the skin and therefore classified as a Non-irritant UN GHS No Category.