1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jul - 07 Aug 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 18 Jun 2019;
corrected: 26 Jun 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

EC Number:

855-228-0

Cas Number:

4497-59-0

Molecular formula:

C14 H19 N O

IUPAC Name:

1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200)

Source strain:

other: not applicable (human skin model)

Details on animal used as source of test system:

not applicable (human skin model)

Justification for test system used:

The used test system is in line with OECD TG 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200), MatTek Corporation, Bratislava, Slovakia
- Tissue batch number(s): 30993
- Date of initiation of testing: 29 Jul 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5°C (5 ± 0.5% CO2) for 35 min and at room temperature for further 25 min
- Temperature of post-treatment incubation: 37 ± 1.5 °C; 5 ± 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material. No information on volume was given. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL MTT assay solution in DMEM (300 µL/well)
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices) using software SoftMax Pro Enterprise v.4.7.1
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: A MTT cell viability test was performed by MatTek. The results were provided in the Certificate of Analysis of the EpiDerm™ Reconstructed Human Epidermis. The determined OD (540 - 570 nm) was 1.864 ± 0.078 (acceptance criteria: 1.0 - 3.0). It was documented that the tissues treated with the negative control are stable in culture (similar viability measurements within a defined historical acceptance range) for the duration of the test exposure period.
- Barrier function: A barrier function test was performed by MatTek. The results were provided in the Certificate of Analysis of the EpiDerm™ Reconstructed Human Epidermis. The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.7 h (acceptance criteria: 4.77 - 8.72 h).
- Morphology: The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL®, 10 mm ∅).
- Contamination: A contamination test was performed by MatTek. The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses (HIV-1, Hepatitis B and C), bacteria, yeast and other fungi. No contamination was detected.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No possible interference with the MTT measurement (OD 540 nm), due to colour changes or direct interacting with the MTT assay reagent, was noted. Therefore, no additional control tissues were needed.

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

PREDICTION MODEL / DECISION CRITERIA
The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP "Category 2" or "Category 1", if the meant issue viability of three indivudual tissues after exposure and post-incubation is ≤ 50% of the mean viability of the negative controls. Since this assay covered by OECD 439 cannot resolve between UN GHS Categories 1 and 2 further information on skin corrosion will be required to decide on its final classification. In case the test item is found to be non-corrosive the test item is considered to be irritant to skin in accordance with UN GHS Category 2. The test item is identified as non-irritant to skin in accordance with UN GHS / EU CLP “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µL

NEGATIVE CONTROL
- Amount(s) applied: 30 µL

POSITIVE CONTROL SUBSTANCE:
- Amount(s) applied: 30 µL
- Concentration: 5% aqueous solution (SDS)

Duration of treatment / exposure:

total 60 min (35 min at 37 °C and 25 min at room temperature)

Duration of post-treatment incubation (if applicable):

ca. 42 h

Number of replicates:

triplicate tissues

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min exposure

Value:

93.47

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:
- Direct-MTT reduction: No direct interaction with the MTT assay reagent was observed.
- Colour interference with MTT: The test item did not change color when mixed with deionised water and isopropanol.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency data for the in vitro skin irritation assay (OECD 439) was demonstrated in March 2014 (non-GLP), and the results of the proficiency study are attached in the study report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 (value: 1.927; see 'Any other information on results incl. tables', table 1).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 h was <15% compared to the negative control (value: 3.91%; see 'Any other information on results incl. tables', table 1).
- Acceptance criteria met for standard deviation: The standard deviation calculated from individual % tissue viabilities of the 3 identically treated replicates was below the limit of acceptance of 18% (range 0.7 - 2.1, see 'Any other information on results incl. tables', table 1).

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 2

Any other information on results incl. tables

Table 1: Results in vitro Skin Irritation Test after 60 min exposure

Treatment group	Tissue no.	OD 570 nm			Mean OD of 3 Wells	Mean OD of 3 Wells  (blank corrected)	Mean OD of 3 tissues	Rel. Viability [%] Tissue 1, 2 +3	Mean relative viability [%]	Standard deviation
		Well 1	Well 2	Well 3
Blank		0.038	0.040	0.041	0.040
Negative control	1	2.035	1.967	1.972	1.991	1.952	1.927	101.273	100.0	1.7
	2	1.989	1.984	1.965	1.980	1.940		100.667
	3	1.923	1.934	1.931	1.929	1.890		98.060
Positive control	1	0.141	0.131	0.121	0.131	0.091	0.075	4.738	3.91	0.7
	2	0.106	0.106	0.106	0.106	0.066		3.442
	3	0.108	0.109	0.108	0.108	0.069		3.562
Test item	1	1.911	1.820	1.873	1.868	1.828	1.801	94.867	93.47	2.1
	2	1.866	1.864	1.850	1.860	1.820		94.462
	3	1.800	1.794	1.792	1.795	1.755		91.089

 

Table 2: Historical control data of negative and positive controls: 60 sets of controls shared between 226 studies performed from Aug 2015 until May 2020

Positive control; OD at 570 nm after exposition to 5% SDS solution in deionized water		Negative control OD at 570 nm DPBS
Tissue Viability [%]	3.93	Mean OD	1.71
Standard Deviation	0.95% points	Standard deviation	0.18
Ranges of Viabilities	2.24% – 6.19%	Ranges of ODs	1.28 - 2
Mean OD	0.07	* should be ≥ 0.8 and ≤ 2.8 (OECD 439/ MatTek)
Standard deviation	0.02
Ranges of ODs	0.03 – 0.11

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance 1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone did not possess irritating properties towards reconstructed human epidermis tissue in the EpiDerm™ model.

Executive summary:

This in vitro study was performed to assess the irritation potential of of the test item [trade name] by means of the Human Skin Model Test.

The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic color was not intensive and it did not change color when mixed with deionised water and isopropanol. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item the mean relative viability value was 93.47% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, [trade name] is non-irritant to skin.