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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.8. - 23.9.2019

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.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Method

Target gene:

gene for histidine or tryptophan synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

supernatant of rat liver and a mixture of cofactors

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 μg
The maximum test item concentration used has been determined with respect to results of previous cytotoxicity test.

Vehicle / solvent:

DMSO, Penta lot. 2307200718, rec.retest date 07/2023
- Justification for choice of solvent/vehicle: solubility of the substance

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene

Details on test system and experimental conditions:

Salmonella typhimurium come from Czech Collection of Microorganisms (CCM), Brno (CZ). Lots of strains used for this study are: TA 1535 CCM Cat. No. 3814, lot. No. 2101200916917, TA 98, Cat. No. CCM 3811, lot No. 0102201220053, TA 100 Cat. No.CCM 3812, lot No. 0102201220054 and TA 1537 Cat. No. CCM 3815, lot No. 2101200916918.
Escherichia coli WP2 uvrA was obtained from Xenometrix (lot No. U20).

NUMBER OF REPLICATIONS: two series

DETERMINATION OF CYTOTOXICITY: The cytotoxicity experiment was performed in Salmonella typhimurium TA 100 as plate incorporation test without metabolic activation and at concentrations 10, 100, 500, 1000, 2500 and 5000 μg per plate, which were applied to plates in volume of 0.1 mL.

Evaluation criteria:

The main criterion used for the evaluation of reversion results was a modified two-fold increase rule, which is compatible with the application of statistical methods. Per this rule, the result is positive if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached (Rt – number of revertants at tested dose, Rc – number of revertants of the solvent control).
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD TG 471, the biological relevance is the criterion for the interpretation of results, and a statistical evaluation of the results is not necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA: Spontaneous reversions, solvent controls and positive controls were compared with historical controls in the laboratory (see table below).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item was dissolved in DMSO up to the highest concentration recommended in the OECD TG 471 – 5 mg per 0.1 millilitre (plate). For cytotoxicity experiment the highest concentration was diluted to the other 5 concentrations within the range of 3 orders of magnitude. The concentration series was tested for toxicity in strain TA 100 without metabolic activation. Neither cytotoxicity nor precipitation was observed in any concentration.

Any other information on results incl. tables

TableA:Current historical ranges ofrevertant numbers in bacterial strains used in the studyand live bacteria count used in experiments

Control                    Strain	Spont.rev.	DMSO	PC - S9	PC + S9	Number of CFU/mL
S.t. TA 100	69-177 (859)	66-170 (546)	341-597 (44)	875-3015 (40)	1.46*10^9
S.t. TA 1535	9-29 (669)	11-27 (422)	434-730 (40)	64-420 (38)	1.82*10^9
S.t. TA 98	13-49 (915)	13-45 (567)	1310-3346 (42)	1904-5024 (42)	1.76*10^9
S.t. TA 1537	5-21 (710)	4-20 (441)	1419-4139 (38)	5-405 (31)	4.00*10^8
E.c. WP2 uvrA	11-51 (691)	8-44 (397)	479-1919 (39)	216-1056 (12)	5.50*10^9

Applicant's summary and conclusion

Conclusions:

Under the above-described experimental design, the test item, ATIBA-A, was non mutagenic for all the used indicator strains in experiments with and without metabolic activation. Change of experimental conditions performed in the second experiments had no influence on study results.

Executive summary:

The test item, ATIBA-A, was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test item was diluted in dimethyl sulfoxide (DMSO) and assayed in concentrations 50, 150, 500, 1500 and 5000 μg per plate. The maximum test item concentration used has been determined with respect to results of previous cytotoxicity test.

The first mutagenicity experiments were performed as plate incorporation test without and with the metabolic activation using a supernatant of rat liver (volume of S9 was 30 μL per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 50-5000 μg per plate, which were applied to plates in volume of 0.1 mL.

In the of first series of mutagenicity experiments no cytotoxicity, precipitation or signs of mutagenicity were observed.

In the second experiments the same concentrations, which were applied to plates in volume of 0.05 mL were used but experiments were performed with 30 minutes of pre-incubation at 37±1°C and the metabolic activation was slightly modified (volume of S9 was 50 μL per plate). Experimental conditions were changed due to improve the contact of bacteria with the test item and metabolic activation, according to OECD requirements.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

In the arrangement given above, the test item, ATIBA-A, was non-mutagenic for all the used indicator strains in experiments with and without metabolic activation.

Change of experimental conditions performed in the second experiments had no influence on study results.