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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:
12 December 2000 to 22 February 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

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 OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Remarks:
Study in compliance with GLP based on Art.19a of German Chemical Act and OECD principles
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione
EC Number:
213-561-3
EC Name:
5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione
Cas Number:
980-26-7
Molecular formula:
C22H16N2O2
IUPAC Name:
2,9-dimethyl-5,12-dihydroquino[2,3-b]acridine-7,14-dione
Test material form:
solid: nanoform

Method

Target gene:
his- for S. typhimurium strains
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced S9 fraction (liver homogenate fraction from male rats
Test concentrations with justification for top dose:
Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system: 50, 160, 500, 1600 and 5000 ug/plate in both studies
The test compound was suspended in DMSO and a stock suspension of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: aminoanthracene (all strains with S9)
Evaluation criteria:
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold .increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
Statistics:
not required

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Key result
Species / strain:
S. typhimurium TA 102
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
Additional information on results:
The test compound was suspended in DMSO and a stock suspension of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 ug/plate were used in all experiments.
Visible precipitation of the test compound on the plates was observed at concentrations of 160 ug/plate and above in the plate incorporation test and in the preincubation test at dose levels of 500 ug/plate and above.
Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 500 ug/plate and lower concentrations in the plate incorporation test and in the preincubation test at dose level of 1600 ug/plate down to the lowest concentration of 50 ug/plate.
The test compound proved to be not toxic to the bacterial strains in the plate incorporation test and in the preincubation experiment. In the preincubation test with the strain TA 1535 in the presence of S9-mix the number of revertant colonies was slightly decreased.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mean mutant number ratios treated/solvent control

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 50 -- 160 -- 500 -- 1600 -- 5000

TA100 -- 0.9 -- 1.0 -- 1.0 -- 0.9 -- 0.9

TA1535 -- 0.9 -- 0.8 -- 0.8 -- 0.9 -- 0.6

TA1537 -- 1.5 -- 1.3 -- 1.5 -- 1.1 -- 1.5

TA98 -- 1.1 -- 1.4 --1.1 -- 1.1 -- 0.7

TA102 -- 1.0 -- 0.9 -- 1.2 -- 1.1 -- 1.1

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 50 -- 160 -- 500 -- 1600 -- 5000

TA100 -- 1.0 -- 1.0 -- 1.1 -- 0.9 -- 1.2

TA1535 -- 1.4 -- 1.4 -- 1.3 -- 1.4 -- 1.3

TA1537 -- 0.9 -- 1.2 -- 0.8 -- 1.2 -- 0.7

TA98 -- 0.9 -- 0.6 --1.1 -- 0.9 -- 0.8

TA102 -- 1.0 -- 1.1 -- 1.3 -- 1.1 -- 1.0

Exp. II: pre-incubation method without S9 mix

Concentrations given in µg/plate

Strain -- 50 -- 160 -- 500 -- 1600 -- 5000

TA100 -- 1.2 -- 1.1 -- 1.2 -- 1.2 -- 1.1

TA1535 -- 1.1 -- 0.9 -- 1.2 -- 0.9 -- 0.9

TA1537 -- 0.8 -- 0.7 -- 0.8 -- 0.7 -- 0.6

TA98 -- 1.0 -- 0.8 --0.9 -- 0.6 -- 0.6

TA102 -- 1.0 -- 1.0 -- 0.9 -- 0.9 -- 0.9

Exp. II: pre-incubation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 50 -- 160 -- 500 -- 1600 -- 5000

TA100 -- 1.0 -- 1.0 -- 1.2 -- 1.0 -- 0.9

TA1535 -- 0.6 -- 0.4 -- 0.5 -- 0.6 -- 0.6

TA1537 -- 1.2 -- 1.7 -- 1.3 -- 1.2 -- 1.1

TA98 -- 1.2 -- 1.2 --1.4 -- 1.3 -- 1.3

TA102 -- 1.0 -- 1.0 -- 1.0 -- 0.9 -- 1.1

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item was considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 of Salmonella typhimurium.

Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate.

For both studies, the compound was suspended in DMSO, and each bacterial strain was exposed to 5 dose levels.

Concentrations for both studies were 50, 160, 500, 1600 and 5000 ug/plate.

Visible precipitation of the test compound on the plates was observed at concentrations of 160 ug/plate and above in the plate incorporation test and in the preincubation test at dose levels of 500 ug/plate and above. Because of heavy precipitation of the test compound the bacterial lawn could only be

evaluated at the dose level of 500 ug/plate and lower concentrations in the plate incorporation test and in the preincubation test at dose level of 1600 ug/plate down to the lowest concentration of 50 ug/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range.

All the positive control compounds showed the expected increase in the number of revertant colonies. The number of revertant colonies of the positive compounds with the strains TA 1535 in the absence and with the strain TA 102 in the presence of S9 -mix were slightly out of the historical control data range, but the criteria for the positive response were succeeded.

Toxicity: In the plate incorporation test and in the preincubation experiment toxicity was not observed with and without metabolic activation.

Mutagenicity: In the absence and in the presence of the metabolic activation system the test item did not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated that the test item was not mutagenic in this bacterial mutation test at any dose level either in the absence or presence of an exogenous metabolic activation.