Phenacetin

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2022 to 08 December 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2023

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:

Phenacetin

EC Number:

200-533-0

EC Name:

Phenacetin

Cas Number:

62-44-2

Molecular formula:

C10H13NO2

IUPAC Name:

phenacetin

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Remarks:

(SkinEthic RHE model)

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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE
- Tissue batch number(s): 22-RHE-149
- Delivery date:06/12/2022
- Date of initiation of testing: 06/12/2022

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: 25 x 1 mL of DPBS
- 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: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2.
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm.
- Linear OD range of spectrophotometer: not specified.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.3 (CV=15.6%), specification OD> 0.7. Historical negative control mean OD range =0.402-1.402 (measured after a 1:2 dilution of the extracts in isopropanol).
- Barrier function: 5.4h (Specification 4.0h≤ET50≤10.0h
- Morphology: 5 cell layers
- Contamination: no.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: no interference.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is greater than 50%.

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 of the test item on 0.50 cm2 human skin model

Duration of treatment / exposure:

42 min at room temperature

Duration of post-treatment incubation (if applicable):

41 hours and 06 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

93.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1.5%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTABILITY CRITERIA:
- SD value of the % viability ≤18%
- Negative control: OD values of the 3 replicates in the range ≥ 0.8 and ≤ 3.0.
The optical density was measured a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥ 0.4 and ≤ 1.5.
- Positive control: Mean viability <40%.

Note:
- If the viability obtained for the test is greater than 50%, the test item has to be considered as non-irritant.
- If the viability obtained for the test item is less than or equal to 50% and the result of skin corrosion test is non-corrosive, the test item has to be considered as irritant.
- If the viability obtained for the test item is less than or equal to 50% and in absence of information on skin corrosion, the test item has to be considered as corrosive or irritant.

Any other information on results incl. tables

Table 1. Table of results

 

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	0.797	0.825	0.873	94.5	100.0	4.8	No Category
		0.848
		0.832
	SPL 2	0.889	0.900		103.1
		0.902
		0.909
	SPL 3	0.891	0.895		102.5
		0.886
		0.909
Positive control	SPL 4	0.013	0.014	0.013	1.6	1.5	0.1	Category 2 "Irritant"
		0.014
		0.015
	SPL 5	0.015	0.014		1.6
		0.013
		0.014
	SPL 6	0.014	0.012		1.4
		0.014
		0.010
Test item PH-22/0352	SPL 10	0.738	0.823	0.815	94.2	93.3	5.6	No Category
		0.875
		0.857
	SPL 11	0.747	0.762		87.3
		0.787
		0.753
	SPL 12	0.860	0.859		98.4
		0.867
		0.852

# mean of 3 values (triplicate of the same extract).

Applicant's summary and conclusion

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test substance can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 93.3%

Executive summary:

An in vitro skin irritation test was conducted for the test item in a reconstructed human epidermis model (EpiDerm™) according to OECD TG 439 (GLP study). Three epidermis units, previously moistened with 10 μL of distilled water, were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated for a 41 hours and 06 minutes hours post-treatment incubation period in fresh medium at 37ºC, 5% CO2, then placed into 300 μL of fresh new assay medium during 3 hours and 45 minutes, at 37ºC, 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol.Under the test conditions, the mean percent viability of the treated tissues was 93.3%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.