5-amino-2,4,6-triiodo-1,3-benzenedicarbonyldichloride

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:

From January 3rd, 1995 to January 12th, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: We need more information to make the conlusions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

according to EEC Directive 92/69 B14

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26. 1983

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-2,4,6-triiodo-1,3-benzenedicarbonyldichloride

EC Number:

417-220-1

EC Name:

5-amino-2,4,6-triiodo-1,3-benzenedicarbonyldichloride

Cas Number:

37441-29-5

Molecular formula:

C8H2Cl2I3NO2

IUPAC Name:

5-amino-2,4,6-triiodobenzene-1,3-dicarbonyl dichloride

Test material form:

solid: particulate/powder

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 100 - 5000 µg/plate
Concentration range in the main test (without metabolic activation): 100 - 5000 µg/plate

The test substance was tested in strain TAl00 at concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
At the start and the end of the incubation period precipitation of the test substance on the plates was not observed. The test substance did not reduce the bacterial background lawn and the number of revertants. Based on these data, the test substance was tested up to and including a concentration of 5000 µg/plate in the absence and presence of S9-mix.

Vehicle / solvent:

solvent DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

The vehicle of the test article, which was DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycine for TA98

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Remarks:

The vehicle of the test article which was DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene for TA1535, TA1537, TA98, TA100

Remarks:

with S9

Details on test system and experimental conditions:

CELL CULTURE
Preparation of bacterial cultures:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking bath (37°C, 150 spm), until the cultures reached an O.D. of 0.4 at 700 nm (10*9 cells/ml). Freshlygrown cultures of each strain were used for a test.

Agar plates:
Agar plates ( Ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid) in Vogel-Bonner Medium E, 10 g glucose, 0.5 mg biotin and 0.6 mg histidine.

Top agar:
Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121°C.

Environmental conditions:
All incubations were carried out in the dark at 37°C. The temperature was monitored during the experiment.


METABOLIC ACTIVATION SYSTEM
Preparation of S9-homogenate:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from BRL, Switzerland.

The animals were housed at the testing facility laboratory in a special room under standard laboratory conditions. The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil.

Five days later , they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C).

The S9-batch used for the preliminary test was no. 94-7 (preparation date: 10-10-1994; final concentration of S9-fraction in the S9-mix was 2.36 nmol/ml).

The S9-batch used for experiment1 was no. 94-8 (preparation date: 10-10-1994; final concentration of S9-fraction in the S9-mix was 2.45 nmo/lml).

The S9-batch used for experiment 2 was no. 94-6 (preparation date: 10-10-1994 final concentration of S9-fraction in the S9-mix was 2.23 nmo/lml).

S9-batches were produced by the testing facility.

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice during the test. S9-mix contained per 10 ml : 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml aqua bidest; 2 m l 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgC12 solution; 1 ml 0.33 M KC1 solution, and 0.5 m l S9. The above solutions were mixed and filter (0.22 µm)-sterilized (apart from the S9- fraction, which was added after filter - sterilization of the S9-mix components).


EXPERIMENTAL PROCEDURE
Selection of dose levels
Selection of an adequate range of doses was based on a preliminary test with strain TA100, both with and without S9-mix. Eight concentrations (3, 10, 33, 100, 333,1000, 3330 and 5000 µg/plate) were tested in triplicate (this preliminary test was reported as a part of the experiment of the mutagenesis assay).

The highest concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate or the level at which the test substance inhibited bacterial growth unless the test substance exhibited limited solubility or was not uniformly dispersable in the solvent of choice.

Direct plate incorporation assay:
Five different doses (with approximately half - log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both with and without S9-mix in each strain, in two independent experiments.

Top agar in top agar tubes was melted and heated to 45°C. The following solutions were successively added to 3 ml top agar: 0.1 ml of a fresh bacterial culture ( 10*9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48 h. After this period revertant (histidine independent) colonies were counted.

Colony counting
The revertant colonies (histidine independent) were counted automatically with an Artek model 880 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

DATA EVALUATION AND STATISTICAL PROCEDURES

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the Ames test if :
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic)in the Ames test if :
a) It induces at least a 2 - fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester
strains, either with or without metabolic activation .
However, any mean plate count of less than 20 is considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>5000 µg/plate

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>5000 µg/plate

Positive controls validity:

valid

Any other information on results incl. tables

The bacterial strains showed the following dose-related increases in the number of revertants:

Strain	Experiment 1 Without S9-mix    With S9-mix	Experiment 2 Without S9-mix    With S9-mix
TA1535	-                              -	-                            -
TA1537	-                              -	-                            -
TA98	5-fold                     4-fold	7-fold                    4-fold
TA100	5-fold                     5-fold	10-fold                    9-fold

-No increase on the number of revertants.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
positive with metabolic activation
positive without metabolic activation
The test substance is mutagenic in the Ames Salmonella/microsome assay.

Executive summary:

The mutagenic effect of the test item was assessed according the method described in the EU Method B14 guideline.

The test substance was tested in the Ames Salmonella/microsome plate test with four histidine-requi ring strains of Salmonella typhimurium (TA1535, TA1537, TAlOO and TA98).

The test substance was tested up to and including 5000 µg/plate in the absence and presence of S9-mix in the Salmonella strains. The test substance induced a dose-related 5-to 7-fold increase in the number of revertant (His+) colonies in strain TA98 in the absence of S9-mix and a dose-related 4-fold increase in the number of revertant (His+) colonies in the presence of S9-mix. In strain TA100 the test substance induced a dose-related 5-to 10-fold increase in the number of revertant (His+) colonies in the absence of S9-mix and a dose-related 5- to 9-fold increase in the number of revertant (His+) colonies in the presence of S9-mix.

No dose-related increases in the tester strains TA1535 and TA1537 were observed.

Based on the results of this study it is concluded that the test substance is mutagenic in the Ames Salmonella/microsome assay.