[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Aug 2019 to 10 Mar 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-2,4-dimethyl-5-(4-methylphenyl)pent-4-enal

Cas Number:

1226911-73-4

Molecular formula:

C14H18O

IUPAC Name:

(4E)-2,4-dimethyl-5-(4-methylphenyl)pent-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: approximately 4ºC, in the dark and under nitrogen
- Other: colourless liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified: origin: adult donors

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model / Human Epidermis SM/13
- Tissue batch number(s): Lot no.: 19-EKIN-033
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 13-08-2019 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: 14-08-2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37°C, 5% CO2; for 42 hours. For further information see below.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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: not applicable

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3hrs at 37°C, 5% CO2
- Spectrophotometer: Labtech LT-4500
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: 10 nm
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD values (mean of triplicate tissues=0.705) for negative control are within the TG acceptability ranges of 0.6 - 1.5.
- Barrier function: IC50 = 2.1 mg/mL (within the acceptability range of 1.5-3.0 mg/mL
- Morphology: quality control performed by the supplier showed a Multi-layered, highly differentiated epidermis consisting of organised basal, spinous and granular layers, and a multilayered stratum corneum, 8.5 cell layers
- Reproducibility: historical data are provided in the report and demonstrated reproducibility over time (SD positive historical control = 0.071; SD positive historical control = 0.092)


NUMBER OF REPLICATE TISSUES: 3 (triplicate)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): water killed then the tissues were frozen for 6 months
- N. of replicates : 3
- Method of calculation used: This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues. As the results of the assay were positive without using the results of direct MTT reduction to perform quantitative correction, it was considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating or corrosive to skin if the viability after 15-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if the viability after 15-minutes exposure / 42-hours 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): 10 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL: Dulbecco's Phosphate buffered saline with Ca2+ and Mg2+
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): used as supplied

POSITIVE CONTROL: Sodium dodecyl sulphate (SDS)
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% w/v aqueous solution (prepared within 2hrs of being applied to the test system)
- other: To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item).

Duration of treatment / exposure:

15 minutes at room temperature, after which each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++ to remove residual test item.

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C, 5% CO2

Number of replicates:

Triplicate; treatment and concurrent negative control and positive control groups

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (n=3)

Value:

32.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: It was considered unnecessary to perform IL-1α analysis. On the basis that the IL-1α analysis would not generate additional relevant information and there was an expectation of comparable results to the MTT relative mean viability endpoint results

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: The MTT solution containing the test item turned blue. An assessment found the test item was able to directly reduce MTT and an additional procedure using non-viable, water-killed, tissues was performed. However, as the results of the assay were positive without using the results of direct MTT reduction to perform quantitative correction, it was considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes..
- Colour interference with MTT: The solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD570 for the negative control treated tissues was 0.705 and the standard deviation value of the viability was 10.0%.
- Acceptance criteria met for positive control: Yes. The relative mean tissue viability for the positive control treated tissues was 3.8% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 9.4%.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data. Historic Control Data (n=30): the mean OD of the positive control was 0.085 ; range 0.030 to 0.264. In this same period the mean OD of the negative control was 0.831 ; range 0.648 – 1.067.

Any other information on results incl. tables

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

 

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.680	0.705	0.071	100*	10.0
	0.785
	0.650
Positive Control Item	0.030	0.027	0.005	3.8	0.7
	0.021
	0.029
Test Item	0.159	0.232	0.066	32.9	9.4
	0.289
	0.248

OD = Optical Density

SD = Standard deviation

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

Negative control: Phosphate buffered saline (PBS)

Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)

Applicant's summary and conclusion

Interpretation of results:

other: Under the conditions of the study, the test item demonstrated the ability to cause a positive skin irritation response. However this study alone does not allow the conclusion on whether the test item is EU CLP/UN GHS Category 1 or Category 2.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item demonstrated the ability to cause a positive skin irritation response. The following classification criteria apply:
EU CLP Category 1 - Code H314; Statement “Causes severe skin burns and eye damage” or EU CLP Category 2 - Code H315; Statement “Causes Skin Irritation”
UN GHS Category 1 or 2
This study alone does not allow the conclusion on whether the test item is EU CLP/UN GHS Category 1 or Category 2.

Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 10 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μL PBS (negative control) and 3 tissues with 10 μL 5% SDS (positive control), respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 3.8% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 32.9%. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 18%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was below 50% after 15 minutes treatment the test results are considered as positive. Under the conditions of this study the test item demonstrated the ability to cause a skin irritation response. However this study alone does not allow the conclusion on whether the test item is EU CLP/UN GHS category 1 or 2.