3-Quinolinecarboxylic acid, 7-[6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2020 - 27 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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)

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-Quinolinecarboxylic acid, 7-[6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

EC Number:

610-204-7

Cas Number:

446299-90-7

Molecular formula:

C30H29N3O4

IUPAC Name:

3-Quinolinecarboxylic acid, 7-[6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Remarks:

(SkinEthic RHE® model)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

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® model
- Tissue batch number(s): 20-RHE-104
- Production date: N/A
- Shipping date: 25.08.2020
- Delivery date: 25.08.2020
- Date of initiation of testing: 25.08.2020

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 and 5 min at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.0 (Acceptance criterion: >0.7). Historical negative control mean OD range = 0.530-1.211 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5)
- Barrier function: 5.5 h (Acceptance criterion: 4.0h ≤ ET50 ≤10.0h)
- Morphology: 6 Cell layers (specification ≥ 4). Multi-layered, highly differenciated epidermis consisting of organized basal, spinous and granular layers and a multilayered stratum corneum.
- Contamination: No

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: there is no direct interaction between the test item and MTT.

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The 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 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.

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 (32 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 min at room temperature.

Duration of post-treatment incubation (if applicable):

41 hours and 29 min at 37ºC, 5% CO2.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

90.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

valid

Remarks:

1.9% viability (5% SDS)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin model, plus a reduced validation with the SkinEthic RHE model, having into account that both models are very similar. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD = 0.892 (OD measured after 1:2 dilution in isopropanol; criterion for acceptability should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, mean viability = 1.9% (criterion for acceptability should be < 40%).
- Acceptance criteria met for variability between replicate measurements: yes. SD of negative, positive and test item replicates were 8.3, 0.5 and 5.2% respectively (criterion for acceptability, SD ≤ 18%).

Any other information on results incl. tables

Table 1. Table of results

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Meanviability %	SD viability	Conclusion
		0.911
	SPL1	0.795	0.848		95.1
		0.838
Negativecontrol	SPL2	0.859 0.838 0.855	0.850	0.892	95.3	100.0	8.3
		0.996
	SPL3	0.979	0.977		109.6
		0.956
		0.028
	SPL4	0.020	0.022		2.5
		0.020
Positive control	SPL5	0.018 0.016 0.017	0.017	0.017	1.9	1.9	0.5	Irritant
		0.014
	SPL6	0.013	0.013		1.5
		0.014
		0.887
	SPL10	0.822	0.846		94.9
		0.830
Test item PH-20/0572	SPL11	0.762 0.756 0.750	0.756	0.807	84.8	90.5	5.2	Non irritant
		0.832
	SPL12	0.813	0.820		92.0
		0.817

# 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 90.5% in the in vitro RhE test.

Executive summary:

An in vitro skin irritation test was conducted for the test item in a reconstructed human epidermis model ( SkinEthic™) 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 the post-treatment incubation period for 41 hours and 29 minutes post-treatment incubation period in growth medium (Episkin SA, batch No. 20 SGM 064) 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 90.5%, versus 1.9% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.