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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20-09-2022 to 21-10-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

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

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method (e.g. ICE, EIT, RhCE) and considerations regarding applicability: The EpiOcular™ model and the corresponding test method have been validated for irritation testing in an international va
lidation study and its use is recommended by the relevant OECD guideline (OECD No. 492), and the method is applicable to mixtures, solids, liquids, semi-solids and waxes. Therefore, it was considered to be suitable for this study.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The 0.60 cm² Reconstructed human Cornea-like Epithelium (EpiOcularTM OCL-212, supplied by MatTek Corporation, batch No. 34989) were received on 19 October 2022.The same day, the tissues in their well shipping container were equilibrated to room temperature for 40 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed culture medium (MatTek Corporation, batch No. 101722ISA) and incubated for 22 hours and 01 minutes at standard culture conditions.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

17 hours and 45 minutes

Number of animals or in vitro replicates:

2 tissues per test substance and for each control were used.

Details on study design:

- Details of the test procedure used: Two tissues (0.6 cm2) were pre-wetted with 20 μL DPBS buffer, incubated for 30 min, then dosed topically with 50 μL test item and exposed for 30 min at 37ºC, 5%
CO2, ≥ 95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 12 min. Then, they were transferred to fresh medium again and incubated for 125 min at 37ºC, 5% CO2. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution between 36.4ºC and 36.7ºC, 5% CO2. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically.
- Doses of test chemical and control substances used: test item applied at the dose of 50 mg and controls (negative and positive) 50 μL.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): exposure 6 hours (±0.25 h) in culture medium at 37±2ºC, 5±1% CO2, ≥95% RH; post-exposure immersion 25 min (±2 min) at RT in culture medium; post-exposure incubation 18 h (±0.25 h) in culture medium at 37±2ºC, 5±1% CO2, ≥95% RH
- Number of tissue replicates used per test chemical and controls: 2 tissues per test substance and for each control were used.
- For hCE cells: number of runs and of hCE models used within each run: 2 runs
- Description of the method used to quantify MTT formazan, if applicable: Optical density by microtiter plate photometer.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to OECD 492, the percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Therefore, the test item is identified as not requiring classification if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. Otherwise, the test item is identified as potentially requiring classification and labelling.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The validity of the RhCE EpiOcular™ test at laboratory facility was demonstra
ted in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized.
- Positive and negative control means and acceptance ranges based on historical data: The OD values of the two replicates with negative control are practically 0.4.
- Acceptable variability between tissue replicates for positive and negative controls: The difference of viability between the 2 tissue replicates treated with the positive and negative controls is higher than 20%.
The test has correctly classified the positive control (Category 2 and 1). The test is therefore able to classify a product. Moreover, the OD mean value of the test item is higher than the OD mean values of negative and positive controls. Therefore, it can be concluded on the classification of the test item.
- Acceptable variability between tissue replicates for the test chemical: yes, 7.8% below 20% (OECD Guideline 492).

Results and discussion

In vitro

Results

Irritation parameter:

other: Cell viability (5)

Run / experiment:

2

Value:

111.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD 570 of the negative control tissues must be > 0.8 and < 2.8. The mean OD values for negative control was 0.398. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.25 for the negative control. Difference for tissues treated with negative control was higher than 20%. The test has correctly classified the positive control (Category 2 and 1). The test is therefore able to classify a product. Moreover, the OD mean value of the test item is higher than the OD mean values of negative and positive controls. Therefore, it can be concluded on the classification of the test item
- Acceptance criteria met for positive control: The mean relative viability of the positive control (methyl acetate) should be below 50% The mean viability of positive control tissues was 30.9 %. Difference for tissues treated with positive control was higher than 20%. The test has correctly classified the positive control (Category 2 and 1). The test is therefore able to classify a product. Moreover, the OD mean value of the test item is higher than the OD mean values of negative and positive controls. Therefore, it can be concluded on the classification of the test item

Any other information on results incl. tables

Table 1. Second run 

	Well ID	OD	Mean OD / disc	Mean OD / product	Viability %	Mean viability %	SD viability	Viability difference between replicates %	Conclusion
Negative control	SPL1	0.315 0.301 0.341	0.319	0.398	80.2	100.0	28.1	39.7	No Category
	SPL 2	0.456 0.489 0.485	0.477		119.8
Positive control	SPL 3	0.202 0.193 0.185	0.193	0.123	48.5	30.9	24.9	35.2	UN GHS Category 2 or 1
	SPL 4	0.049 0.055 0.056	0.053		13.3
Test item PH-22/0484	SPL 5	0.432 0.433 0.425	0.430	0.446	108.0	111.9	5.5	7.8	No Category
	SPL 6	0.471 0.453 0.460	0.461		115.8

Applicant's summary and conclusion

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item T-A10 does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.

Executive summary:

An in vitro EIT study according to OECD 492 was conducted under GLP conditions to assess the irritation potential of the test item.EpiOcular™ tissues were used as test system. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.Two tissues were pre-wetted with 20 μL DPBS buffer, incubated for 30 min, then dosed topically with 50 μg test item and exposed for 6 hours in culture medium at 37±2ºC, 5±1% CO2, ≥95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 18 h at 37ºC, 5% CO2, ≥ 95% RH. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 2 hours and 45 minutes with MTT solution. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically. The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 111.9 % (considered as 100 %), versus 24.9 % in the positive control (Methyl acetate).Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.