N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24--27.9.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

EC Number:

616-392-7

Cas Number:

76820-34-3

Molecular formula:

C14H19N3O8

IUPAC Name:

N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Not available

IUPAC Name:

Unknown impurities

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00601016
- Expiration date of the lot/batch: 03/2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.
- Stability under test conditions: Stable

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek ver. 2.0, Bratislava, SK)
- Tissue batch number(s): Lot No. 30827

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min room temperature and 35 min at culture conditions

REMOVAL OF TEST MATERIAL AND CONTROLS
- Tissues were thoroughly rinsed with PBS (Phosphate Buffered Saline)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: Tissues were transferred to 24-well plates containing MTT medium (1 mg·mL-1)
- Spectrophotometer: Libra S22. Isopropyl alcohol serves as a blank.
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

EXPERIMENTAL PROCEDURES
1. Complementary experimets - part of the study in vitro skin corrosion (228/19/4AC)
2. MTT Test
3. OD50 measuring

MTT test
a) Application:
25 mg of the test item was dosed directly on the tissue previously moistened with 25 μL of DPBS. The item was spread over the entire tissue surface. A single experiment, composed of three replicate tissues, was run.
b) Tissues preparation and treatment:
On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After 60 minutes of pre-incubation, medium was replaced and pre-incubation continued for other 19 hours and 11 minutes.
After pre-incubations, tissues were topically exposed to the test item for 60 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Tissues were then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium.
After 23 hours and 57 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 17 hours and 52 minutes.
c) MTT assay:
Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg·mL-1). After 180 minutes of MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol (for 164 minutes, room temperature, shaking) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.
d) OD570 measuring:
OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was used.

DECISION CRITERIA
The cut-off values for the prediction of irritation are given below; these values are stated in OECD Test Guideline No. 439, par. 36:
a. In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS * Category 2.
b. The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

Amount/concentration applied:

Test item: 25 mg
NC: DPBS (Dulbecco’s phosphate buffered saline) (30 μL)
PC: 5% SDS (sodium dodecyl sulphate) water solution (30 μL)

Number of replicates:

3

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

93.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE CRITERIA
All study acceptance criteria were fulfilled.
The mean OD570 of the NC tissue was 1.651 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.
The mean viability of the PC tissues expressed as % of the negative control tissues was 3.0 % which meets the acceptance criterion of ≤ 20 %.
The SD calculated from individual % tissue viabilities of the identically treated replicates was 0.32 % for the positive control, 1.57 % for negative control and 7.12 % for the test item what is < 18 % in all cases.

Any other information on results incl. tables

MTT Test

Treat-ment	OD570	Tissue			Avg	SD	Average viability
		1	2	3			(% NC)
NC DPBS	OD	1.615	1.674	1.665	1.651	0.026	100.0
	%	97.80	101.37	100.83	100.00	1.572
C3  228/19	OD	1.444	1.664	1.429	1.547	0.118	93.7
	%	87.44	100.77	86.54	93.65	7.115
PC 5% SDS	OD	0.050	0.056	0.043	0.050	0.005	3.0

DPBS    Dulbecco’s Phosphate Buffered Saline – delivered from MatTek

NC        negative control

PC         positive control

SDS       sodium dodecyl sulphate

C3, 228/19         test item 

avg       arithmetic mean

SD         standard deviation calculated from individual % tissue viabilities

viability (%)        viability of single tissues compared with negative control

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test item NBA-A was 93.7 % of the negative control average value i.e. viability was > 50 %. The effect of the test item was negative in the EpiDermTM model.

Executive summary:

The test item, NBA-A, was assayed for in vitro skin irritation in the human epidermal model EpiDermTM. The main methodical documents were OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2019) and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

In the main MTT experiment, after pre-incubation of tissues, 25 mg of the test item was placed directly on the moistened tissue and spread over the entire surface for 60 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used for the test item and for positive and negative controls.

After removal of the test item, tissues were post-incubated in culture medium for about 42 hours. After three hours incubation with MTT, samples were extracted with isopropyl alcohol for 2 hours and 44 minutes Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

In complementary experiments, performed as a part of study 228/19/4AC: NBA-A - In vitro Skin Corrosion Test (EpiDermTM Model); VUOS-CETA, Report No. 19-353, 2019, direct MTT reduction and colour interference were not found. Therefore, the results of the MTT test did not require correction.

Under the above-described experimental design, the average viability of test item-treated tissues was 93.7±7.1%, i.e. viability was > 50%.

The effect of test item was negative in the EpiDermTM model (i.e. not irritating).