1-(2,2,4-trimethyl-1-quinolyl)ethanone

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 - 27 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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Test No. 431: In Vitro Skin Corrosion: Reconstructed Human Epidermis (Rhe) Test Method
adopted 14 Jun 2019

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-1,2-dihydroquinolin-1-yl)ethan-1-one

EC Number:

665-911-3

Cas Number:

5855-23-2

Molecular formula:

C14 H17 N O

IUPAC Name:

1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one

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 431.

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): 30887
- Date of initiation of testing: 20 Aug 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure); 37 ± 1.5 °C (60 ± 5 min exposure)
- Temperature of post-treatment incubation: incubation with MTT assay solution at 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 exposure period the tissues were removed from the 6-well plate and gently rinsed with PBS (phosphate buffered saline) to remove any residual test material for several times. No information on the volume was given. Excess PBS was then removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues have been rinsed.
- 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 optical density (OD; 540 - 570 nm) was 1.872 ± 0.1333 (acceptance criteria: 1.0 - 3.0). The negative control tissue has been shown to be stable in culture (provided similar viability measurements) for the duration of the test exposure period. The tissue employed has been shown to demonstrate reproducibility over time and between laboratories. Moreover, it has been shown to be capable of predicting the corrosive potential of the reference items when used in the testing protocol selected.
- 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 4.82 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 multi-layered 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 direct MTT reduction was noted, but colour interference when the test substance was mixed with water or isopropanol, took place. Thus, it is presumed to have the potential to stain the tissue and therefore, additional controls (colored controls = CC) were necessary.
- Fresh tissues / killed tissues: viable tissues (DPBS-treated color control tissues (NC_CC) and test item-treated color control tissues (TI_CC). These tissues were incubated in medium without MTT solution in the MTT assay.
- N. of replicates : 2
- Method of calculation used: Data Correction Procedure I (for details, see "any other information on materials and methods")

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered to be corrosive to skin and classified as sub-category 1A, if the viability after 3 min exposure is less than 25%. According to the data generated in view of assessing the usefulness of the RhE test methods for supporting sub-categorisation, it was shown that around 29%, 31% and 33% of the Subcategory 1A results of the EpiDERM™ test method may actually constitute Subcategory 1B or Subcategory 1C substances/mixtures (i.e. over-classifications).
- The test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 min exposure is greater than or equal to 25%.
- The test item is considered to be non-corrosive to skin, if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL SUBSTANCE:
- Amount(s) applied: 50 µL
- Concentration: 8.0 N KOH

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

2 tissues were used per treatment and exposition time;, negative and positive control included (12 tissues in total).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

99.51

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

99.68

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:
- Visible damage on test system: not reported
- Direct-MTT reduction: A direct interaction with the MTT assay reagent was not observed.
- Colour interference with MTT: The test item changed colour when mixed with isopropanol. Consequently, additional tests with viable tissues to determine the correction factor for calculating the true viability in the main experiment were necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY: A certificate of proficiency (11 Dec 2019) certifying that the testing institution has demonstrated proficiency in the EpiDerm Skin Corrosion Test (OECD TG 431) by correctly predicting the corrosive potential of proficiency chemicals, as well as correctly assigning chemicals to sub-classes of corrosiveness as defined by the OECD TG 431 has been issued.

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 for every exposure time (values 2.102 for 3 min exposure, and 1.977 for 60 min exposure; see 'any other information on results incl. tables', table 1 and 2, respectively).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was <15% compared to the negative control (11.84% for 3 min and 2.69% for 60 min exposure; see 'any other information on results incl. tables', table 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates is ≤30%.

HISTORICAL CONTROL DATA
See 'any other information on results incl. tables', table 3

Any other information on results incl. tables

Table 1: Results in vitro Skin Corrosion Test after 3 min exposure

	Blank	Negative control (NC)		Positive control (PC)		Test item (TI)		Blank	Negative control viable tissue		Test item viable tissue (TI_CC)
Tissue sample		1	2	1	2	1	2		1	2	1	2
OD570	0.039 0.040 0.040	2.194 2.120 2.143	2.136 2.131 2.125	0.311 0.287 0.272	0.289 0.287 0.285	2.078 2.103 2.099	2.178 2.183 2.203	0.039 0.040 0.040	0.046 0.045 0.046	0.048 0.049 0.048	0.050 0.050 0.049	0.049 0.047 0.048
OD570 (mean of 3 wells, blank corrected)		2.113	2.091	0.251	0.247	2.054	2.149		0.006	0.009	0.010	0.009
SD (3 wells)		0.038	0.005	0.020	0.002	0.013	0.013		0.000	0.000	0.000	0.001
OD570 (mean values of tissue replicates, blank corrected)		2.102		0.249		2.101			0.007		0.009
Mean rel. viability (%)*		100.00		11.84		99.96			0.35		0.45
CV (%)		0.7		0.9		3.2			24.8		13.9
Corrected viability (%)**						99.51

* mean viability (%) = 100 x mean_OD(TI/PC/NC) / mean_OD(NC)

**Corrected viability (%) = TI viability – TI_CC viability

 

Table 2: Results in Vitro Skin Corrosion test after 60 min exposure

 

	Blank	Negative control (NC)		Positive control (PC)		Test item (TI)		Blank	Negative control viable tissue		Test item viable tissue (TI_CC)
Tissue sample		1	2	1	2	1	2		1	2	1	2
OD570	0.039 0.040 0.040	2.086 2.048 2.040	1.989 1.980 1.955	0.112 0.102 0.091	0.085 0.083 0.083	2.083 2.117 2.138	1.943 1.938 1.939	0.039 0.040 0.040	0.050 0.052 0.053	0.049 0.048 0.046	0.047 0.047 0.045	0.067 0.063 0.065
OD570 (mean of 3 wells, blank corrected)		2.018	1.936	0.062	0.044	2.073	1.900		0.012	0.008	0.007	0.026
SD (3 wells)		0.025	0.018	0.011	0.001	0.028	0.003		0.001	0.002	0.001	0.002
OD570 (mean values of tissue replicates, blank corrected)		1.977		0.053		1.987			0.010		0.016
Mean rel. viability (%)*		100.00		2.69		100.50			0.51		0.82
CV (%)		3.0		24.3		6.1			29.3		81.5
Corrected viability (%)**						99.68

* mean viability (%) = 100 x mean_OD(TI/PC/NC) / mean_OD(NC)

**Corrected viability (%) = TI viability – TI_CC viability

 

Table 3: Historical data of negative and positive controls (Sep 2015 - May 2020)

 

3 min treatment
Positive control		Negative control
Mean Viability	20.50%	Mean OD	1.68
Standard Deviation	8.06 p.p.	Standard deviation	0.20
Ranges of Viabilities	4.60% - 39.83%	Ranges of ODs	1.25 – 2.07
60 min treatment
Positive control		Negative control
Mean Viability	5.00%	Mean OD	1.64
Standard deviation	2.98 p.p.	Standard Deviation	0.23
Ranges of Viabilities	1.07% - 14.77%	Ranges of ODs	1.23 – 2.16

 

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

Under the present test conditions, 1-(2,2,4-trimethylquinolin-l (2H)-yl)ethanone tested at two exposure periods of 3 min and 1 h was non-corrosive to reconstructed human epidermis tissue in the EpiDerm™ model.
There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.

Executive summary:

 

This in vitro study was performed to assess the corrosive potential of test item [trade name] by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not reduce MTT (pre-test for direct MTT reduction), but it changed colour when mixed with isopropanol (pre-test for colour interference). Consequently, additional tests with viable tissues to determine the correction factor for calculating the true viability in the main experiment were necessary.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (Dulbecco's Phosphate-Buffered Saline (DPBS)) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value was 99.51% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 99.68%. Both values did not reach the threshold for corrosivity which is defined to be < 50% after the 3 minutes exposure or ≥ 50% after 3 minutes exposure and < 15% after the 1 hour exposure.

Therefore, the test item is considered to be not corrosive.