2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October to 06 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 05 July 2016/ signed on 28 October 2016)

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

EC Number:

815-961-9

Cas Number:

1374760-95-8

Molecular formula:

C17H17N3O2S

IUPAC Name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Test material form:

solid: particulate/powder

Details on test material:

Appearance: white to beige / white powder (solid)
Stability under test : Not specified, assumed to be stable
Storage conditions: Room temperature, protected from light, in the tightly closed original container.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- EpiSkin™ Tissues (0.38cm2) lot number: 17-EKIN-044
- Maintenance Medium lot number: 17-MAIN3-046
- Assay Medium lot number: 17-ESSC-043
- Production date: not reported
- Shipping date: not reported
- Delivery date: 31 October 2017
- Expiry date: 06 November 2017
- Date of initiation of testing: 31 October 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: IC50 = 2.0 mg/ml (1.5 mg/ml ≤ IC50 ≤ 3.0 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: All values for the control groups were within the ranges obtained by the testing laboratory in the preceding six months. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: Not a MTT reducer

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

PREDICTION MODEL / DECISION CRITERIA
- Classification of irritation potential is based upon relative mean tissue viability following the 15 - minute exposure period followed by the 42 - hour post-exposure incubation period
Relative mean tissue viability is ≤50%: Irritant
Relative mean tissue viability is >50%: Non-Irritant

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10 mg (26.3 mg/cm2) of the test item was applied to the epidermal surface. 5 µL of sterile distilled water was topically applied previously to the epidermal surface in order to improve contact between the test item and the epidermis.
- Concentration (if solution): The test item was used as supplied.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

Triplicate tissues for test item, negative and positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

105.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

7.2

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; the mean OD570 for the negative control treated tissues was 0.844 and the standard deviation value of the viability was 7.8%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: Yes; the relative mean tissue viability for the positive control treated tissues was 7.2% relative to the negative control treated tissues and the standard deviation value of the viability was 2.5%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.9%. The test item acceptance criterion was therefore satisfied.
- Historical Control Data Comparison: All values for the control groups were within the ranges obtained by the testing laboratory in the preceding six months.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.914	0.844	0.066	108.3	100*	7.8
	0.784			92.9
	0.834			98.8
Positive Control Item	0.072	0.061	0.021	8.5	7.2	2.5
	0.074			8.8
	0.037			4.4
Test Item	0.831	0.892	0.058	98.5	105.7	6.9
	0.947			112.2
	0.899			106.5

 

OD=Optical Density

SD= Standard deviation

*= The mean viability of the negative control tissues is set at 100%

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 105.7% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The mean OD₅₇₀ for the negative control treated tissues was 0.844 and the standard deviation value of the viability was 7.8%. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 7.2% relative to the negative control treated tissues and the standard deviation value of the viability was 2.5%. The positive control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.9%. The test item acceptance criterion was therefore satisfied.

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.