α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

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 July 31st to October 9th, 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

EC Number:

246-042-5

EC Name:

α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

Cas Number:

24155-42-8

Molecular formula:

C11H10Cl2N2O

IUPAC Name:

1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-30ºC, in tightly closed container.

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN-SM

Source species:

human

Cell type:

other: adult human-derived epidermal keratinocytes

Cell source:

other: human adult donors, not specified.

Source strain:

not specified

Details on animal used as source of test system:

EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:13-EKIN-030, Expiry Date: 09 September 2013)

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM (Manufacturer: SkinEthic, France)
- Tissue batch number(s): 13-EKIN-030
- Expiry Date: 09 September 2013
- Date of initiation of testing: 05 September 2013

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Exposure of test material was terminated by rinsing thoroughly with PBS 1x solution. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).
- Observable damage in the tissue due to washing: no.
- Modifications to validated SOP: no.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 15 minutes exposure is less than 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure is greater than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- The test item was applied in its original form, no formulation was required.
- Amount(s) applied (volume or weight with unit): 10 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 20 µL
- Concentration: PBS 1x

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 20 µL
- Concentration: Sodium Dodecyl Sulphate (SDS) 5%

Duration of treatment / exposure:

15 min (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 h (± 1h)

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 / 1

Value:

56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 / 2

Value:

54

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 / 3

Value:

66

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

59

Vehicle controls validity:

not applicable

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: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439 (data not included).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1. Summary of results.

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1 x PBS	mean	0,631	100
	standard deviation (SD)		3,11
Positive Control: SDS (5%aq.)	mean	0,228	36
	standard deviation (SD)		1,33
Test Item: IMIDAZOLE ETHANOL	mean	0,370	59
	standard deviation (SD)		6,51

Following exposure with the test item, the mean cell viability was 59% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non irritant to skin. In conclusion, in thisin vitro EPISKIN model test with the test item, the results indicate that the test item is non irritant to skin.

The mean OD value of the three negative control tissues was 0.631, the positive control result showed 36 % viability, each standard deviation value (SD) of the % viability was below 18 (see table below in section 9.3). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria.

Conclusions:

The test item is not irritant to skin, with a cell viability of 59%.

Executive summary:

An in vitro skin irritation test of the test item was performed in an EPISKIN-SM reconstructed human epidermis model, according to OECD TG 439 (GLP study). Disks of EPISKIN (3 units / chemical) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and PBS treated epidermis were used as positive and negative controls, respectively. All validity criteria were met.

The mean cell viability was 59%, compared to the negative control. Therefore, the test item was not irritating to the skin.