2-heptadecyl-1H-imidazole

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May - 27 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit, Schwabach, Deutschland

Test material

Test material information

1

Reference substance name:

2-heptadecyl-1H-imidazole

EC Number:

245-589-7

EC Name:

2-heptadecyl-1H-imidazole

Cas Number:

23328-87-2

Molecular formula:

C20H38N2

IUPAC Name:

2-heptadecyl-1H-imidazole

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200, MatTek)
- Tissue batch number: 25834
- Delivery date: 2 Aug 2017
- Date of initiation of testing: 3 Aug 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min exposure), 37 ± 1 °C (60 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed using a wash bottle containing phosphate buffered saline (PBS) to remove any residual test material (20 times).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.866 ± 0.143 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.02 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was observed.

NUMBER OF REPLICATE TISSUES: 2 replicates for each treatment condition (3 min and 60 min experiment)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT and showed no colouring as compared to the solvent, an additional test with freeze-killed or viable tissues was not performed.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg test subsatnce + 25 µL distilled water

NEGATIVE CONTROL
- Amount(s) applied (volume): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume): 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

The test was performed on a total of 4 tissues per dose group, two replicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

100.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

106.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction:
Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT:
The test substance did not change colour, when mixed with deionised water and isopropanol and thus passed the colour interference pre-test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control OD, both for the 3 and 60 min exposure period, was in the range of ≥ 0.8 and ≤ 2.8 for every exposure time thereby confirming the acceptable quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 min exposure period (13.3%) and for the 60 min exposure period (6.3%) thus confirming the validity of the test system and the specific batch of tissue models (acceptance criteria: mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%)
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation in the range 20 - 100% viability between tissue replicates was ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

Any other information on results incl. tables

Results of the Pre-experiment

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple.Therefore, non-specific reduction of MTT (NSMTT) equalled 0%.

The mixture of 25 mg test item per 300 µL aqua dest.and per 90 µL isopropanolshowed no colouring as compared to the solvent.Therefore non-specifc colour (NSC) equalled 0%.

Table 3: Results of 3 min experiment

Name	Negative Control		Test Substance		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.732	1.762	1.713	1.834	0.214	0.322
	1.741	1.845	1.732	1.840	0.212	0.334
	1.794	1.785	1.742	1.888	0.223	0.340
OD570 - Blank Corrected	1.688	1.718	1.669	1.790	0.170	0.278
	1.697	1.801	1.688	1.796	0.168	0.290
	1.750	1.741	1.698	1.844	0.179	0.296
Mean OD570 of 3 Aliquots (Blank Corrected)	1.712	1.753	1.685	1.810	0.172	0.288
SD OD570 of 3 Aliquots	0.034	0.045	0.027	0.036	0.025	0.025
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.733*		1.748		0.230
SD OD570 of 2 Replicate Tissues	0.030		0.088		0.082
Mean Relative Tissue Viability [%]	100.0		100.9		13.3
Coefficient Of Variation [%]^***	1.7		5.0		35.5

*         corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

***      coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

SD = Standard deviation

OD = Optical density

Table 4: Result of the 60 min experiment

Name	Negative Control		Test Substance		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.777	1.697	1.900	1.968	0.164	0.134
	1.884	1.842	1.834	1.966	0.176	0.144
	1.853	1.748	1.799	2.002	0.173	0.137
OD570 - Blank Corrected	1.733	1.653	1.856	1.924	0.120	0.090
	1.840	1.798	1.790	1.922	0.132	0.100
	1.809	1.704	1.755	1.958	0.129	0.093
Mean OD570 of 3 Aliquots (Blank Corrected)	1.794	1.718	1.800	1.935	0.127	0.094
SD OD570 of 3 Aliquots	0.055	0.070	0.052	0.030	0.025	0.024
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.756*		1.867		0.110
SD OD570 of 2 Replicate Tissues	0.054		0.095		0.023
Mean Relative Tissue Viability [%]	100.0		106.3		6.3**
Coefficient Of Variation [%]^***	3.1		5.1		21.1

*         corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

**        mean relative tissue viability of the 60 min positive control < 15%,

***       coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

SD = Standard deviation

OD = Optical density

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiDerm™ model.
CLP: non-corrosive