N-alkylester N-aryl substituted aryl diazo aryl amine

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:

26 February to 06 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to OECD 471 and GLP guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

Due to the positive test result in the Initial Mutation Test (standard plate incorporation method), the same test method was used in the Confirmatory Mutation Test. The Prival modification was therefore skipped.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

Name: DYNS 2246

Method

Target gene:

Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan dependent

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Initial Mutation Test and Confirmatory Mutation Test : 500; 158.1; 50; 15.81; 5; 1.581; 0.5 µg/plate

Vehicle / solvent:

aqua bidest.

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:

w/o S9

Migrated to IUCLID6: TA100, TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

w/o S9

Migrated to IUCLID6: TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine

Remarks:

TA98: w/o S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

w/o S9

Migrated to IUCLID6: WP2uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9: TA98, TA100, TA1535, TA1537, WP2uvrA

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: plate incorporation test

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Spontaneous Reversion of Tester Strain Laboratory's historical control values for spontaneous revertants (revertants/plate) in the period of 1999 to 2007 (as guide) as follows: (-S9) Salmonella typhimuriumTA98: 10-54, TA100: 72-211, TA1535: 4-31, TA1537: 1-24, Escherichia coli WP2 uvrA: 9-66.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive Salmonella strains

In conclusion, the test item DYNS 2246 had strong mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item DYNS 2246 was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post-mitochondrial supernatant (S9 fraction) prepared from livers of phenobarbital/b-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Plate Incorporation Method). This last test replaced the Prival Modification test because the strong positive response in the first main test caused a change in study design, in agreement with the Sponsor.

The test item concentrations in the two independently performed main experiments (Initial Mutation Test and Confirmatory Mutation Test) were: 500; 158.1; 50; 15.81; 5; 1.581 and 0.5 µg/plate.

Biologically relevant increases in the number of revertant colonies compared to the solvent control plates were observed in the Initial Mutation Test in all Salmonella strains with and without metabolic activation and in Escherichia coli strain without metabolic activation. Also, biologically relevant increases in the number of revertants compared to the solvent control were observed in the Confirmatory Mutation Test in all Salmonella strains with and without metabolic activation. The effect of the test item on the number of revertant colonies in these cases was dose-dependent.

The observed substantial increases were higher in tester strains sensitive to frameshifts (Salmonella typhimurium TA98 and TA1537) than in tester strains sensitive to base pair substitutions (Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA). Higher numbers of revertants were observed in experiments carried out without metabolic activation than in experiments with metabolic activation in all of the five bacterial strains used in this study.

The revertant colony numbers of vehicle control plates without S9 mix were within the historical control data range. The reference mutagens showed the expected increase in induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item DYNS 2246 hasstrong mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.