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

Endpoint summary

Description of key information

Skin irritation (in vitro): Key study. Test method according to OECD 439, GLP study. The mean percent viability of the treated tissues was 98.9%, versus 1.5% in the positive control (5% SDS). Therefore, the test item is not irritant to the skin. 

Serious eye damage/eye irritation: Key study. Method according to OECD guideline 492, GLP study. Under the conditions of the test, test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 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

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%).

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)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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).

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available