Acetylcysteine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 21, 2018 - June 22, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

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)

Version / remarks:

July 28, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ECVAM 2009, Performance Standards for In-Vitro Skin Irritation test Methods boased on Reconstructed Human Epidermis (RHE)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: SkinEthic Skin Irritation Test Standard Operating Procedure (SOP): Using the Reconstructed Human Epidermis (RHE) model, INVITTOX

Version / remarks:

Version 2.1, 2009

GLP compliance:

yes (incl. QA statement)

Remarks:

Merck KGaA, Non-Clinical Safety, Frankfurter Strasse 250, 64293 Darmstadt, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Acetylcysteine

EC Number:

210-498-3

EC Name:

Acetylcysteine

Cas Number:

616-91-1

Molecular formula:

C5H9NO3S

IUPAC Name:

N-acetylcysteine

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: SkinEthic RHE-model RHE/S/17 obtained from Episkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 18-RHE-041 (1st run) , 18-RHE-068 (2nd run)

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: gently rinsing with minimum volume of 25 mL DPBS using pipette
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.3
- Barrier function: 4.6h
- Morphology: well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL
- The test substance is considered as non-irritant to skin if the viability after exposure and post-treatment incubation is greater than 50 %. A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is equal or greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 16 mg per tissue

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration: 5% aqueous solution

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st run

Value:

82.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

3.6%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2nd run

Value:

98.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6.7%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

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: Results of the 1st Run

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	2.034	104.1%	1.912	97.9%	1.913	98.0%	1.953	100.00	3.6%
Positive Control	0.031	1.6%	0.028	1.4%	0026	1.3%	0.028	1.4%	14.3%
Test item	1.661	85.0%	1.646	84.3%	1.537	787%	1.615	82.7%	4.2%

 

Table 2: Results of the 2nd Run

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.794	107.7%	1.607	96.5%	1.594	95.7%	1.665	100.00	6.7%
Positive Control	0.025	1.5%	0.021	1.3%	0.021	1.3%	0.022	1.4%	7.1%
Test item	1.562	93.8%	1.581	95.0%	1.798	108.0%	1 647	98.9%	8.0%

Table 3: Acceptability of the Test (Run 1)

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
	Acceptance Criterion	Result
Negative control OD	≥ 0.8 and ≤ 3.0	1.912 to 2.034
Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
	Acceptance Criterion	Result
Mean OD negative control	≥1.2	1.953
Mean viability positive control	< 40%	1.4%
SD of group-mean value	≤18%	14.3% (positive control) 3.6% (negative control)
Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
	Acceptance Criterion	Result
Mean OD negative control	≥1.453	1.953
Mean viability positive control	≤ 2.89%	1.4%
Test Item Data Acceptance Criteria:
	Acceptance Criterion	Result
SD of group-mean value	≤ 18%	4.2%

Table 4: Acceptability of the Test (Run 2)

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
	Acceptance Criterion	Result
Negative control OD	≥ 0.8 and ≤ 3.0	1.594 to 1.794
Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
	Acceptance Criterion	Result
Mean OD negative control	≥1.2	1.665
Mean viability positive control	< 40%	1.4%
SD of group-mean value	≤18%	7.1% (positive control) 6.7% (negative control)
Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
	Acceptance Criterion	Result
Mean OD negative control	≥1.450	1.665
Mean viability positive control	≤ 2.87%	1.4%
Test Item Data Acceptance Criteria:
	Acceptance Criterion	Result
SD of group-mean value	≤ 18%	8.0%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the substance is not considered to possess an irritant potential to skin (UN GHS: No Category).

Executive summary:

A study according OECD TG 439 was conducted to investigate the potential of the test item to induce skin irritation in an in vitro human skin model (SkinEthic RHE-model).

The substance was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Because of non-concordant replicate results after treatment with the substance between the first (non-valid) run and the second run, a third run was performed. Only the two valid runs performed are reported as run 1 and run 2. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 82.7% in run 1 and 98.9% in run 2 and thus, higher than 50% in both runs, i.e. according to OECD TG 439 the test item is considered as non-irritant to skin (UN GHS: No Category).