5-Amino-N,N'-Bis(2,3-Dihydroxypropyl)-2,4,6-Triiodoisophtalamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.8.-28.8.2019

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:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Adopted: June 18th, 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

474-080-4

EC Name:

-

Cas Number:

76801-93-9

Molecular formula:

C14H18I3N3O6

IUPAC Name:

5-amino-N1,N3-bis(2,3-dihydroxypropyl)-2,4,6-triiodobenzene-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: 00104016
- Expiration date of the lot/batch: 12/2020

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

Remarks:

a reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek)

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SK)
- Tissue batch number(s): Lot No. 30820, kit A

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Tissues were thoroughly rinsed and blotted to remove the test item/control

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)
- Incubation time: 3 hr
- Spectrophotometer: Libra S22. Isopropyl alcohol serves as a blank.
- Wavelength: 570±30 nm

NUMBER OF REPLICATE TISSUES: 3

EXPERIMENTAL PROCEDURES
1. Complementary experimets - Direct MTT reduction (test item did not reduce MTT directly) and colour interference (colour of test item did not interfere with the endpoint)
2. MTT Test
3. OD50 measuring

DECISION CRITERIA
According to the OECD TG 431 as well as to the EU Method B.40, the test substance is considered to be corrosive to skin:
i) if the viability after 3 minutes exposure is less than 50 %, or
ii) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
The test substance is considered to be non-corrosive to skin:
i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test item: 25 mg, no vehicle
NC: water for injection (50 μL)
PC: 8N KOH (50 μL)

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 3 min treatment

Value:

99

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 60 min treatment

Value:

101.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS: All assay acceptance criteria have been met.
Negative control: The assay meets the acceptance criterion - OD570 of the NC tissues was 1.741(3 min) and 1.716 (60 min) which is ≥ 0.8 and ≤ 2.8.
Positive control: Viability of tissues treated with 8N KOH after 60 minutes treatment was 5.5% which is ＜15%.

Any other information on results incl. tables

MTT test results

time	treatment		OD570			mean	SD	CV	% NC
			tissues
			1	2	3
3min	NC	water	1.729	1.751	1.742	1.741	0.009	0.005
	C1	227/19	1.752	1.646	1.771	1.723	0.055	0.032	99.0
	PC	8N KOH	0.172	0.118	0.120	0.137	0.025	0.183	7.9
60 min.	NC	water	1.765	1.643	1.740	1.716	0.053	0.031
	C1	227/19	1.890	1.764	1.579	1.744	0.128	0.073	101.7
	PC	8N KOH	0.096	0.082	0.106	0.095	0.010	0.104	5.5

NC         negative control

PC         positive control

C2         test item

mean    arithmetic mean

% NC     viability of single tissues compared with negative control

SD         standard deviation

CV        coefficient of variance

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, the test item ATIBA-A was non-corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.

Executive summary:

The test item ATIBA-A was assayed for in vitro skin corrosion in the human epidermal model EpiDermTM. The test was performed according to OECD Test Guideline 431, In Vitro Skin Corrosion: Human Skin Model Test, Adopted: June 18th, 2019.

A test for assessing the interference of colour excluded the influence of colour on the endpoint.

A test for direct reduction demonstrated that the test item did not reduce MTT directly. Thus, no steps for correction of results were performed.

In the skin corrosion test, the test item (25 mg) was placed atop previously moistened tissue. The length of exposure was 3 and 60 minutes. Nine tissues were used for each timepoint: three per test item (C1), three for positive control (PC) and three for negative control (NC).

After rinsing, the tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol overnight at room temperature with shaking. The OD570 of the isopropyl alcohol extracts was were measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

All the assay acceptance criteria were met.

Under the above-described experimental design, the average viability of tissues treated with the test item ATIBA-A was 99.0 % of the negative control average value after 3 minutes treatment and 101.7 % after 60 minutes of treatment.

The test item is considered to be non-corrosive to skin:

i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.

In the experiment arrangement described above, the test item ATIBA-A was non-corrosive in the EpiDermTM model.