Anthra[2,1,9-mna]naphth[2,3-h]acridine-5,10,15(16H)-trione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Start of experimental phase: 23 June 2017;End of experimental phase 24 July 2017; Study completion:11 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 1997

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:

Anthra[2,1,9-mna]naphth[2,3-h]acridine-5,10,15(16H)-trione

EC Number:

221-897-7

EC Name:

Anthra[2,1,9-mna]naphth[2,3-h]acridine-5,10,15(16H)-trione

Cas Number:

3271-76-9

Molecular formula:

C31H15NO3

IUPAC Name:

anthra[2,1,9-mna]naphth[2,3-h]acridine-5,10,15(16H)-trione

Test material form:

solid: particulate/powder

Method

Target gene:

the test item for the ability to induce gene mutations in Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these
stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures,
which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement No Growth onMinimal plates+Biotin.Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement No Growth onMinimal agar plates.Growth onMinimal plates+Tryptophan.
-uvrA, uvrB : Sensitivity to UV irradiation.
-rfa : Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

Preliminary Toxicity test: 2500, 791, 250, 79.1 and 25.0 μg/plate.

Main Assay I: 2000, 1000, 500, 250 and 125 μg/plate.

Main Assay II ( confirmatory experiment): 2000, 1000, 500,250 and 125 μg/plate with TA1537 and TA98 tester strains in the presence of S9 metabolism.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Details on test system and experimental conditions:

The preliminary toxicity test and the Main Assay I and II were perfomed using a plate-incorporation method.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate ( Chu et al. 1981); Regression line

Results and discussion

Test results

Key result

Species / strain:

other: S.typhimurium TA1535, TA1537, TA98 and TA100; E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

the test item Vat Green 3 induces reverse mutation by frameshifts in Salmonella typhimurium in the presence of S9 metabolism, under the reported experimental conditions

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item Vat Green 3 induces reverse mutation by frameshifts in Salmonella typhimurium in the presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Vat Green 3 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

The test item was used as a solution in dimethylsulfoxide (DMSO).

Toxicity test

The test item Vat Green 3 was assayed in the toxicity test at a maximum concentration of

2500 μg/plate and at four lower concentrations spaced at approximately half-log intervals:

791, 250, 79.1 and 25.0 μg/plate.

Two-fold increases in revertant numberswere observed with TA1537 and TA98 tester strains in

the presence of S9 metabolic activation at the highest or three highest dose levels, respectively.

Dose-related precipitation of the test item was observed with all tester strains at the two highest dose levels, both in the absence and presence of S9 metabolism. At 2500 μg/plate, the abundant precipitate interfered with the scoring of the background lawn and the resulting evaluation of possible toxic effects at this dose level. No toxicity was observed at lower dose levels with any tester strain, in the absence or presence of S9 metabolic activation. Based on these results, 2000 μg/plate was selected as the top dose to be used in Main Assay I.

Main Assays

In Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 2000, 1000, 500, 250 and 125 μg/plate. Dose-related precipitation of the test item was seen at the two highest dose levels both in the absence and presence of S9 metabolic activation. At the end of the incubation period, no toxicity of the test item was observed, with any tester

strain, at any dose level, in the absence or presence of S9 metabolism. Dose-related increases in revertant numbers were observed with TA1537 (2.4-fold) and TA98 (5.3-fold) tester strains in the presence of S9 metabolism. A confirmatory experiment (Main Assay II) was performed, in which TA1537 and TA98 tester strains were treated in the presence of S9 metabolism at the dose levels of 2000, 1000, 500, 250 and 125 μg/plate. Dose-related precipitation of the test item was seen at the two highest dose levels. Dose-related increases in revertant colonies were reproduced for both tester strains. The number of revertant colonies after TA1537 exposure did not reach twice the concurrent negative control values, although it fell out the historical control range for this

tester strain at higher concentrations. A clear positive effect was confirmed with TA98 tester strain, where numbers of revertant colonies over two fold (up to 4.95) the concurrent vehicle control were observed at all dose levels.

Since an obvious positive response was observed, no further experiment was undertaken.

Conclusion

It is concluded that the test item Vat Green 3 induces reverse mutation by frameshifts in Salmonella typhimurium in the presence of S9 metabolism, under the reported experimental conditions.