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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6.-20.11.2019

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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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 chemico test system

Details on the study design:

PRINCIPLE OF TEST METHOD
The DPRA is a chemistry-based assay. Nucleophile-containing synthetic peptides (cysteine peptide­Ac-RFAACAA-COOH; lysine peptide- Ac-RFAAKAA-COOH) are used to screen for skin sensitisation potential by measuring peptide depletion following incubation with allergens and non-allergens.
Synthetic heptapeptides containing either cysteine or lysine are incubated with the test item for 24 hours. Depletion of the peptide in the reaction mixture is measured by high pressure liquid chromatography (HPLC) using UV detection. Average peptide depletion data for cysteine and lysine are then calculated.

SOLVENT SELECTION
Solvent selection was performed according to DB-ALM (INVITTOX) Protocol No.154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing, 2012. According to this protocol, acetonitrile is the preferred solvent for test chemicals. An approximately 100 mM solution of ATIBA-A in acetonitrile was prepared. Test item was not dissolved completely. Therefore, been prepared 100 mM solution of ATIBA-A in ultra pure water, then in isopropanol and acetone, but test item was not dissolved completely.
Further test item was attempted to dissolve in 300 microliters of dimethyl sulfoxide and diluted the resulting solution with 2700 microliters of acetonitrile. Vial was placed in sonicating bath for 1 minute, but item was not dissolved completely.
Further test item was attempted to dissolve in 1500 microliters of dimethyl sulfoxide and diluted the resulting solution with 1500 microliters of acetonitrile. Vial was placed in sonicating bath for 1 minute, but item was not dissolved completely.
So test item was attempted to dissolve in 3000 microliters of dimethyl sulfoxide. Vial was placed in sonicating bath for 5 minutes, item was dissolved completely. However, it is important to note that DMSO may lead to peptide dimerisation and as a result, it may be more difficult to meet the acceptance criteria.
As a last resort, was tested lower soluble concentration of the test item. Was prepared 50mM solution of ATIBA-A dissolved in 1500 microliters of dimethyl sulfoxide and diluted the resulting solution with 1500 microliters of acetonitrile. Vial was placed in sonicating bath for 1 minute, item was dissolved completely. In the case of negative prediction (lack of reactivity), no firm conclusion should be drawn.

PREPARATION OF TEST ITEM SOLUTION
The weighed amount of ATIBA-A (approximately 213.6 mg) was dissolved in 3 ml of dimethyl sulfoxide.
The weighed amount of ATIBA-A (approximately 106.8 mg) was dissolved in 1500 µl of dimethyl sulfoxide and diluted with 1500 µl acetonitrile.

REFERENCE CONTROLS AND CO-ELUTION CONTROL
Three types of "Reference Controls" were included in this study. Reference Control is a peptide solution where the test chemical is replaced by the solvent used to dissolve it.
Reference Control A was made with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification.
Reference Control B was made with acetonitrile and its replicates are injected in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
Reference Controls C was made with acetonitrile and DMSO/ACN (1:1) (solvent used to solubilise the test item). They are used to verify that the solvent does not impact the Percent PeptideDepletion. The appropriate Reference Controls C for each chemical are used to calculate Percent Peptide Depletion.
Co-elution control was constituted by the test item alone without the addition of peptides. Instead of the addition of peptides, the appropriate amount of buffer was added. The co-elution test serves to verify that the test item does not have the same retention time and does not absorb at 220 nm as the Cysteine and Lysine peptides.

PREPARATION OF PEPTIDE STOCK SOLUTION
The necessary amount of peptide was estimated. For each analysis with addition of peptide, 800 µl of stock solution is needed. 25 ml of the stock solution of Cysteine or Lysine peptide (0.667 mM) was prepared.

ANALYSES
Liquid chromatograph: Shimadzu Nexera LC-30AC with DAD
Column: Agilent Zorbax SB-C18, 100x2.1 mm, 3.5µm
Precolumn: Phenomex security guard C18, 4.0 x 2.0 mm
Mobile phase A: 0.1% Trifluoracetic acid in water
Mobile phase B: 0.085% Trifluoracetic acid in acetonitrile
Time programmer:
0 min 10 % B
10 min 25 % B
11 min 90 % B
13 min 90 % B
13.5-20 min 10 % B
Column temperature: 30 °C
Sample temperature: 25°C
Flow rate: 0.35 mL/min
Injection volume: 10 μL
Detection: 220 nm, 258 nm

Results and discussion

Positive control results:

Cinnamaldehyde was used as positive control at a concentration of 100 mM in acetonitrile.

The mean percent peptide depletion value at 220 nm for the positive control Cinnamaldehyde is 77.2 % for the Lysine peptide. This was due to the ambiguous integration of the lysine peptide peak after incubation with Cinnamaldehyde. Therefore, the peak area of lysine peptide was also evaluated at 258 nm. The mean percent peptide depletion value at 258 nm for the positive control Cinnamaldehyde is 66.2 % for the Lysine peptide. This does not affect the outcome of the study, as the test item appears to be negative.

In vitro / in chemico

Results

Key result

Run / experiment:

other: 1

Parameter:

other: mean of percent Cysteine and percent Lysine depletion values

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The following criteria should be met for a run to be considered valid:
a) the standard calibration curve should have an r2˃0.99
b) the mean percent peptide depletion value of the three replicates for the positive control Cinnamaldehyde should be between 60.8% and 100% for the Cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be <14.9% for the percent Cysteine depletion and <11.6% for the percent lysine depletion
c) the mean peptide concentration of reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%. If one or more of these criteria is not met the run should be repeated.

The following criteria should be met for a test item results to be considered valid:
a) the maximum standard deviation for the test item replicates should be <14.9% for the percent Cysteine depletion and <11.6% for the percent lysine depletion
b) the mean peptide concentration of the free Reference controls C in the appropriate solvent should be 0.50±0.05 mM.

Any other information on results incl. tables

RESULTS EVALUATION

Since the use of DMSO as the sample solvent led to its significant dimerization, the results of determination mean of cysteine and Lysine percent depletion were not included in the final assay.

Since the test chemical was not dissolved in a suitable solvent at a concentration of 100 mM, the concentration of the test chemical was reduced to half (50 mM) a test item was prepared by adding 1500 µl DMSO to the sample and diluted the resulting solution with 1500 µl ACN.

In this case too, the effect of dimerization was apparent. To obtain approximate results, but was neglected.

Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used.

 

Determining the reactivity class and the DPRA prediction forATIBA-A

Test item	CAS No.:	Mean of Percent Cysteine depletion %	Mean of Percent Lysine depletion %	Mean of Cysteine and Lysine Percent depletion %	Reactivity Class	DPRA prediction
ATIBA-A	76801-93-9	-6.7	-2.9	0.0	No reactivity	Negative

 

The mean of Cysteine and Lysine percent depletion values was0.0. Thetest item ATIBA-A was included in the reactivity class – no reactivity and was determination as negative in the DPRA prediction. Due to the fact that the test substance does not dissolve in any solvent at the recommended concentration of 100 mM and in the case of negative prediction (lack of reactivity), no firm conclusion can be drawn.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

All study acceptance criteria have been successfully met.
The test item, ATIBA-A, has been found negative in the DPRA prediction; it was assigned to reactivity class “no or minimal reactivity” for both peptides.
Due to the fact that the test item does not dissolve in any solvent at the recommended concentration of 100 mM and due the fact that the test item was negative in the DPRA prediction, no firm conclusion about covalent binding of test item to proteins can be drawn.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

The purpose of the test is to contribute to the evaluation of the skin sensitisation potential of ATIBA-A. The study is a part of test item hazard evaluation for registration of industrial chemicals.

The test was performed according to OECD TG 442C, In Chemico Skin Sensitisation: Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins (Adopted: 18 June 2019).

The test item, ATIBA-A, has been found negative in the DPRA prediction; it was assigned to reactivity class “no or minimal reactivity” for both peptides.

Due to the fact that the test item does not dissolve in any solvent at the recommended concentration of 100 mM and due the fact that the test item was negative in the DPRA prediction, no firm conclusion about covalent binding of test item to proteins can be drawn.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.