Benzamide, N-(5-bromo-3-methyl-2-pyridinyl)-N-methyl-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02-08-2022 to 25-08-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Standard deviation between tissue repplicates was higher tham 18% for negative control. Positive control is compliant which means that the experiment is able to classify a test item.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(5-bromo-3-methylpyridin-2-yl)-N-methylbenzamide

EC Number:

610-202-6

Cas Number:

446299-80-5

Molecular formula:

C14H13BrN2O

IUPAC Name:

N-(5-bromo-3-methylpyridin-2-yl)-N-methylbenzamide

impurity 1

Reference substance name:

N-(3-bromo-5-methyl pyridine-2-yl)-N-methylbenzamide

Molecular formula:

C14H13BrN2O

IUPAC Name:

N-(3-bromo-5-methyl pyridine-2-yl)-N-methylbenzamide

impurity 2

Reference substance name:

Benzoic acid

EC Number:

200-618-2

EC Name:

Benzoic acid

Cas Number:

65-85-0

Molecular formula:

C7H6O2

IUPAC Name:

Benzoic acid

impurity 3

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

impurity 4

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Remarks:

(SkinEthic RHE model)

Source species:

human

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
- Tissue batch number(s): 22-RHE-112
- Production date: N/A
- Shipping date: -
- Delivery date: 23.08.2022
- Date of initiation of testing: 23.08.2022

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 41 minutes at 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 at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.4 (Acceptance criterion: 1.0-3.0). Historical negative control mean OD range= 0.402-1.402 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.4)
- Barrier function: 5.4 h (Acceptance criterion: 4.77h < ET50< 8.72h)
- Morphology: Tissue viability and barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functinal stratum corneum, a viable basal cell layer and intermediate spinous and granular layers.
- 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 posttreatment 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

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:

41 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41 hours and 01 minutes at 37ºC, 5% CO2

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

98.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

valid

Remarks:

3.6% 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 EpiDerm 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 = 0.900 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.4).
- Acceptance criteria met for positive control: yes, mean viability = 0.014% (acceptability criteria should be < 20%).
- Acceptance criteria met for variability between replicate measurements: no. SD of negative, positive and test item replicates were 30.0, 0.1 and 5.4% respectively (acceptablility criteria, SD ≤ 18%).

Any other information on results incl. tables

Table 1. Table of results 

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	0.648 0.536 0.585	0.589	0.900	65.4	100.0	30.0	No Category
	SPL 2	1.177 1.007 0.922	1.035		115.0
	SPL 3	1.067 1.103 1.060	1.076		119.6
Positive control	SPL 4	0.012 0.013 0.014	0.013	0.014	1.4	1.5	0.1	Category 2 "Irritant"
	SPL 5	0.015 0.015 0.013	0.014		1.6
	SPL 6	0.015 0.014 0.014	0.014		1.6
Test item PH-22/0484	SPL 16	0.801 0.811 0.890	0.834	0.890	92.7	98.9	5.4	No Category
	SPL 17	0.866 0.933 0.965	0.921		102.3
	SPL 18	0.873 0.926 0.949	0.916		101.8

#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 98.9% 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 RHE model) according to OECD guideline 439 (GLP study). Three epidermis units, previously moistened with 10 μL of distilled water, were treated with 16 mg test item for 41 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 a 41 hours and 01 minutes post-treatment incubation period in culture plate filled with 300 μL of fresh medium 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 1 hour 55 minutes 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 98.9%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). Standard deviation between tissue repplicates was higher tham 18% for negative control. Positive control is compliant which means that the experiment is able to classify a test item.Therefore, the test item is considered as not irritant to the skin.