Prop-2-yn-1-yl 1H-imidazole-1-carboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

Version 439, adopted 26. Jun. 2020

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:

adopted 06. Jul. 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-yn-1-yl 1H-imidazole-1-carboxylate

EC Number:

695-595-2

Cas Number:

83395-38-4

Molecular formula:

C7H6N2O2

IUPAC Name:

prop-2-yn-1-yl 1H-imidazole-1-carboxylate

Test material form:

solid

Specific details on test material used for the study:

Name: 2-propyn-1-yl 1H-imidazole-1-carboxylate
Batch no.: 0802-2#
CAS no.: 83395-38-4
Composition: 2-propyn-1-yl 1H-imidazole-1-carboxylate
Storage: fridge (2 - 8 °C) under inert gas
Expiry date: 11. Jul. 2021
Stability : stable under storage conditions
Appearance: white solid
Purity: 99.1 %
Homogeneity: homogeneous
Production date: 13. Oct. 2020
EC no.: 695-595-2
Molecular formula: C7H6N2O2
Molecular weight: 150.13 g/mol

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratisla-va.

Source strain:

not specified

Details on animal used as source of test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
Origin: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratisla-va.
Designation of the kit: EPI-200-SIT
Day of delivery: 16. Mar. 2021
Batch no.: 34134

Justification for test system used:

The SkinEthic RHE® model has been validated for irritation testing and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 2 (Step 1 is rinsing the test item thoroughly with DPBS from the tissues; Step 2 is rinsing the outside of the inserts at the end of the incubation time with MTT)
- Observable damage in the tissue due to washing: no
DYE BINDING METHOD
- Dye used in the dye-binding assay: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT)
- Spectrophotometer: Plate spectrophotometer
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Tissue1: 25.5 mg; Tissue2: 25.7 mg; Tissue3: 25.9 mg
The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): DPBS buffer

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS-solution

Duration of treatment / exposure:

24 hours at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity

Duration of post-treatment incubation (if applicable):

18 hours and 30 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

ca. 2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 2.4%+-0.1%

Other effects / acceptance of results:

The mean value of relative tissue viability of the test item was reduced to 2.4% after the treatment. This value is below the threshold for skin irritation (50%).
Therefore, the test item is considered as at least irritant to skin.
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: yes. The test item was soluble in H2O demin. and the resulting solution was slightly turbid. The binding capacity was not tested, as in the main test the test item is considered at least irri-tant to skin and a false negative result is excluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. The validity of the skin irritation study at LAUS GmbH was demonstrated in a non-GLP proficiency study. For this purpose, 10 proficiency chemicals (indicated by the OECD 439 guideline) were tested. All of the 10 proficiency chemicals were correctly categorized. Therefore, the proficiency of the skin irritation study was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. OD of negative control is 1.653 (acceptablility criteria: >= 0.8 and <= 2.8).
- Acceptance criteria met for positive control: yes. % tissue viability of positive control SDS is 2.8% (acceptablility criteria: < 20% of negative control).
- Acceptance criteria met for variability between replicate measurements: yes. SD of mean viability of the tissue replicates (%) is 2.7% (negative control), 0.1% (positive control) and 0.1% (test item) (acceptablility criteria: <=18% )

Any other information on results incl. tables

Table1: Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.037	0.034	0.035	0.035	0.034	0.034	0.035	0.036	0.035

Table2: Absorbance Values negative control, test item and positive control (OD 570 nm)

Designation	Measurement	Negative Control	Test Item	Positive Control
Tissue 1	1	1.739	0.075	0.084
	2	1.735	0.076	0.083
Tissue 2	1	1.641	0.073	0.080
	2	1.654	0.073	0.080
Tissue 3	1	1.682	0.074	0.081
	2	1.678	0.073	0.080

Table3: Mean Absorbance Values

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.702	0.041	0.049
Mean – blank (tissue 2)	1.613	0.038	0.045
Mean – blank (tissue 3)	1.645	0.039	0.046
Mean of the three tissues	1.653	0.039	0.047

Table4: % Tissue Viability

Designation	Test Item2-propyn-1-yl 1H-imidazole-1-carboxylate	Positive Control
% Tissue viability (tissue 1)	2.5%	3.0%
% Tissue viability (tissue 2)	2.3%	2.7%
% Tissue viability (tissue 3)	2.4%	2.8%
% Tissue viability (mean)	2.4%	2.8%
± SD of mean tissue viability (%)	0.1%	0.1%

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

According to the Reconstructed human Epidermis (RhE) Test Method. After the treatment, the mean value of relative tissue viability was reduced to 2.4%. This value is below the threshold for skin irritation (50%). This value is below the threshold for skin irritation potential (50%). The test item tert-Butylbenzene that induce values below the threshold of 50% are considered at least irritant to skin.
The OECD guideline 439 addresses the human health endpoint skin irritation. Therefore, further testing on skin corrosion potential with another suitable in vitro study (e.g. OECD 431) may be required.
Therefore, the test item 2-propyn-1-yl 1H-imidazole-1-carboxylate is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Executive summary:

Two experiments were performed. In the first experiment the standard deviation of the negative control was 24.5% and therefore too high (required: <= 18.0%). Only the results of the second experiment are used for evaluation and are reported here.

The data of the invalid experiment are not reported, but the raw data are kept in the GLP-archive of the test facility.

Three tissues of the human skin model EpiDerm^TMwere treated withthe test itemfor 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 <= mean OD <= 2.8, OD was 1.653.

The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to2.8% (required:<=20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: <= 18%).

 

After the treatment with the test item, the mean value of relative tissue viability was reduced to 2.4%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin. Therefore, the test item are considered as at least skin irritant (cat 2).