N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000!32/EC, L 1362000. Anllex 4D", dated May 19, 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: "Kanpoan No. 287 -- Envlrorlment Protection Agency" Eisei No. 127 -- Ministry 01 Health & Welfare" "Heisei 09/10131 Kikyoku No. 2 -- Ministry of In!erna!ional Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

EC Number:

274-040-4

EC Name:

N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Cas Number:

69563-51-5

Molecular formula:

C21H16N2O5S

IUPAC Name:

N-(4-amino-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)benzenesulfonamide

Test material form:

other: solid

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 09979EB5
- Expiration date of the lot/batch: March 14, 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Method

Target gene:

Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mixType and composition of metabolic activation system:
- source of S9 : rat liver
- method of preparation of S9 mix:
The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx, 220 - 320 g induced by applications of 80 mglkg b.w. Phenobarbital Lp (Desitin; D-22335 Hamburg) and ß-Naphthoflavone p,o. (Aldrich, D-89555 Steinheim) each on three consecutive days, The livers are prepared 24 hours after the last treatment The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supenatant are frozen and stored in ampoules at -80 °C. Small numbers of Ihe ampoules can be kept at -20 °C for up to one week. Each balch of S9 mix is routinely tested wilh 2-aminoanthracene as well as benzo(a}pyrene.

Test concentrations with justification for top dose:

3; 10; 33; 100: 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100; without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 1537,TA98; without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA; without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535, TA 1537. TA 98, TA 100, WP2 uvrA ; With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold al only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent Increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential If reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolite activation. A moderate but dose dependent increase in revertant colony numbers was observed following treatment with FAT 360341G in experiment I in strain TA 1537 in the absence of metabolite activation (S9 mix) and in strain TA 98 in the presence of metabolite activation. In strain TA 1537 the threshold of three times the number of the corresponding solvent control was exceeded at 5000 µg/plate. In strain TA 98 the required threshold of twice the number of the corresponding control was reached at 2500 µg/plate and exceeded at 5000 µg/plate. No comparable increase of the mutation frequency occurred in the other strains. A confirmatory experiment was performed under identically conditions 10 reproduce this minor increase. The results of the confirmatory confirmed the minor increase in the number of the revertants in strain TA 1537 without 89 mix and in strain TA 98 with S9 mix. In this experiment in strain TA 1537 the threshold of three limes the number of the corresponding solvent control was exceeded at 5000 µg/plate. In strain TA 98 the required threshold of twice the number of the corresponding control was reached at 5000 µg/plate.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

FAT 36034/G is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 36034/G to induce gene mutations in the plate incorporation test (experiment I) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA as per OECD 471 guideline. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment l and l a: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. A moderate but dose dependent increase in revertant colony numbers was observed following treatment with FAT 360341G in experiment in strain TA 1537 in the absence of metabolic activation (S9 mix) and in strain TA 98 in the presence of metabolic activation. In strain TA 1537 the threshold of three times the number of the corresponding solvent control was exceeded at 5000 µg/plate. In strain TA 98 the required threshold of twice the number of the corresponding control was reached at 2500 ug/plate and exceeded at 5000 µg/plate, No comparable increase of the mutation frequency occurred in the other strains. A confirmatory experiment was performed under identical conditions to reproduce this minor increase. The results of the confirmatory confirmed the minor increase in the number of the revertants In strain TA 1537 without S9 mix and in strain TA 98 with S9 mix. In this experiment in strain TA 1537 the threshold of three times the number of the corresponding solvent control was exceeded at 5000 µg/plate. In strain TA 98 the required threshold of mice the number of the corresponding control was reached at 5000 µg/plate. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frame shifts in the genome of the strains TA 1537 (without S9 mix) and TA 98 (with S9 mix). Therefore, FAT 36034/G is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.