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Toxicological information

Genetic toxicity: in vitro

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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:
From 27 September, 1993 to 02 February, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)-4-methylbenzenesulphonamide
EC Number:
201-369-2
EC Name:
N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)-4-methylbenzenesulphonamide
Cas Number:
81-68-5
Molecular formula:
C22H18N2O5S
IUPAC Name:
N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)-4-methylbenzenesulphonamide
Specific details on test material used for the study:
Test material: FAT 36034/E
Batch No.: 9300102
Expiry date: July, 1998
Purity: 93 %
Solubility: in water <0.1 g/L
Colour: red
Storage: at room temperature

Method

Target gene:
Histidine gene

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
The histidine auxotrophic strains of Salmonella typhimurium (TA 98, TA 102, TA 1535 and TA 1537) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Ltd., Basel, Switzerland. Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NBagar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment. The characteristics of the strains were checked monthly. Histidine auxotrophy of the strains was demonstrated by the requirement for 1-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (strains TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Strain TA 102 additionally was checked for tetracycline resistance (presence of multicopy plasmid pAQ1). The presence of the uvr+ gene was demonstrated by the resistance of strain TA 102 against UV-light. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Cytotoxicity test: 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate
Mutagenicity tests: 61.7, 185.2, 555.55, 1666.7 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on solubility
Controlsopen allclose all
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 100 and TA 1535 without metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
for TA 102 without metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for TA 98 without metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA 1537 without metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
for TA 100, TA 102, TA 98 and TA 1537 with metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide*H20
Remarks:
for TA 1535 with metabolic activation
Details on test system and experimental conditions:
Method of application: in agar (plate incorporation)

Setting up of the test plates: 0.1 mL of the overnight cultures were mixed with 2 mL of top agar, either 0.5 mL of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 mL of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. It was supplemented with 10 % of 0.5 mM 1-histidine and 0.5 mM (+)biotin dissolved in water.

Preliminary toxicity/range-finding test: A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 h at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

Mutagenicity test: The mutagenicity test was performed with strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 without and with microsomal activation. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 µg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. The plates were inverted and incubated for about 48 h at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.
Evaluation criteria:
Assay acceptance criteria: A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
Statistics:
In deviation to the OECD guideline, a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(only a weak/slight decrease in the number of back mutants were observed)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA102, TA1535 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
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:
Toxicity test/range finding test:
Six concentrations ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. The numbers of revertant colonies were not reduced. Normal back-ground growth was observed. The test substance exerted no toxic effect on the growth of the bacteria. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

Mutagenicity test, original experiment:
In the experiment performed with metabolic activation, treatment of strain TA 98 with the test substance led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 185.2 and 555.6 µg/plate, followed by a weak decrease in the number of back mutants at the concentrations of 1666.7 and 5000 µg/plate. A marginal increase in the number of revertant colonies occurred on strain TA 1537 at the concentrations of 555.6 to 5000 µg/plate. No effects were observed with the other strains. In the experiment carried out without metabolic activation, a marginal increase was observed in the number of revertant colonies at the concentrations of 1666.7 and 5000 µg/plate. No effects were seen with the other strains.

Mutagenicity test, confirmatory experiment:
In the experiment carried out with activation on strain TA 98, a slight, concentration dependent increase in the number of revertant colonies occurred at the concentrations of 555.6 and 1666.7 µg/plate, followed by a slight decrease in the number of back-mutants at the concentration of 5000 µg/plate. A marginal increase in the number of revertant colonies occurred on strain TA 1537 at the concentrations of 555.6 to 5000 µg/plate. Again, no effects occurred on the other strains. In the experiment performed without metabolic activation, a marginal increase was observed in the number of revertant colonies at the concentration of 5000 µg/plate only . Again, no effect was seen with the other strains.

Applicant's summary and conclusion

Conclusions:
The test substance exerted a marginal mutagenic action on strain TA 1537. The metabolites of the test substance were weakly mutagenic with strain TA 98 and marginally mutagenic with strain TA 1537.
Executive summary:

An in vitro study was performed to investigate the potential of the test substance (of ca. 93 % purity) to induce gene mutations according to OECD Guideline 471, EU Method B.14 and EPA OTS 798.5265 in compliance with GLP. The concentration range of the test substance to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the same concentrations. In the preliminary experiment without and with metabolic activation performed on strain TA 100, no toxic effects were observed. In the original experiment performed, a slight increase in the number of revertant colonies at the concentrations of 185.2 to 5000 µg/plate was observed with TA 98 with metabolic activation, followed by a weak decrease in the number of back mutants at the concentrations of 1666.7 and 5000 µg/plate. A marginal increase in the number of revertant colonies occurred on strain TA 1537 at the concentrations of 555.6 to 5000 µg/plate. In the experiment without metabolic activation, a marginal increase was observed in the number of revertant colonies at the concentrations of 1666.7 and 5000 µg/plate. In the confirmatory experiment, a slight, concentration dependent increase in the number of revertant colonies occurred at the concentrations of 555.6 and 1666.7 µg/plate with activation on strain TA 98, followed by a slight decrease in the number of back-mutants at the concentration of 5000 µg/plate. A marginal increase in the number of revertant colonies occurred on strain TA 1537 at the concentrations of 555.6 to 5000 µg/plate. In the experiment without metabolic activation, a marginal increase was observed in the number of revertant colonies at the concentration of 5000 µg/plate only. In both the mutagenicity tests with metabolic activation, normal background growth was observed with all strains. The number of revertant colonies was not reduced. The test substance exerted no toxic effect on the growth of the bacteria. Under the study conditions, the test substance exerted a marginal mutagenic action on strain TA 1537. The metabolites of the test substance were weakly mutagenic with strain TA 98 and marginally mutagenic with strain TA 1537.