2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: 14 October 2016; End of experimental phase: 28 October 2016; Study completion: 29 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

EC Number:

258-094-6

EC Name:

2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

Cas Number:

52673-15-1

Molecular formula:

C25H19N3O4

IUPAC Name:

17-(3-methoxypropyl)-6(or7)-methyl-3,10,17-triazahexacyclo[13.6.2.0²,¹.0,.0¹²,²².0¹,²³]tricosa-1(21),2,4(9),5,7,12(22),13,15(23),19-nonaene-11,16,18-trione

Test material form:

solid: particulate/powder

Method

Target gene:

The test item 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.

Species / strain

Species / strain / cell type:

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

Remarks:

The E.coli used for this study was the strain WP2 uvrA.

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:

S9 liver homogenate from rats pre-treated with Phenobarbital and 5,6-Benzoflavone.

Test concentrations with justification for top dose:

Preliminary toxicity test: 500, 158, 50.0, 15.8 and 5.00 µg/plate (based on solubility test).

Main Assay I:
- TA1535, TA1537, WP2 uvrA,TA98: ± S9: 500, 250, 125, 62.5 and 31.3 µg/plate
- TA100: - S9: 500, 250, 125, 62.5, 31.3 and 15.6 µg/plate
- TA100: + S9: 500, 250, 125, 62.5 and 31.3 µg/plate

Main Assay II:

TA1535, TA1537: ± S9: 500, 250, 125, 62.5, 31.3 µg/plate
WP2 uvrA, TA100: + S9: 500, 250, 125, 62.5, 31.3, 15.6 µg/plate
WP2 uvrA: − S9: 500, 250, 125, 62.5, 31.3 µg/plate
TA98 :− S9: 500, 250, 125, 62.5, 31.3, 15.6 µg/plate
TA98 : + S9: 500, 250, 125, 62.5, 31.3 µg/plate
TA100: −S9: 250, 125, 62.5, 31.3, 15.6, 7.81 µg/plate

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 first experiment were performed using a plate-incorporation method. The second experiment was performed using a pre-incubation 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.typhyimurium TA1535, TA1537, TA98 and TA100; E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item did not induce two-fold increases in the number of revertant colonies, in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Disperse Orange 32 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 Disperse Orange 32 was assayed in the toxicity test at a maximum concentration of 500 µg/plate as limited by solubility and at four lower concentrations spaced at approximately half-log intervals: 158, 50.0, 15.8 and 5.00 µg/plate. Precipitation of the test item was observed with all tester strains at the highest dose level, both in the absence and presence of S9 metabolism. Toxicity was observed with WP2 uvrA and TA98 tester strains at the highest dose level in the absence of S9 metabolism and with TA100 tester strain at the highest or two highest dose levels in the presence and absence of S9 metabolism, respectively.

Main Assay I and II: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

TA1535, TA1537, WP2 uvrA, TA98: ± S9: 500, 250, 125, 62.5, 31.3 µg/plate

TA100: −S9: 500, 250, 125, 62.5, 31.3, 15.6 µg/plate

TA100: +S9: 500, 250, 125, 62.5, 31.3 µg/plate

Slight toxicity was observed with TA98 and TA100 tester strains at the highest or two highest dose levels, respectively, in the absence of S9 metabolism and with WP2 uvrA and TA100 tester strains at the highest dose level in the presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The dose-range was slightly modified to take into account the toxicity results of Main Assay I.

The test item was assayed at the following dose levels:

TA1535, TA1537: ± S9: 500, 250, 125, 62.5, 31.3 µg/plate

WP2 uvrA, TA100: +S9: 500, 250, 125, 62.5, 31.3, 15.6 µg/plate

WP2 uvrA: −S9: 500, 250, 125, 62.5, 31.3 µg/plate

TA98: −S9: 500, 250, 125, 62.5, 31.3, 15.6 µg/plate

TA98: + S9: 500, 250, 125, 62.5, 31.3 µg/plate

TA100: −S9: 250, 125, 62.5, 31.3, 15.6, 7.81 µg/plate

Slight toxicity was observed with WP2 uvrA tester strain at the highest dose level in the presence of S9 metabolism and with TA98 tester strain at the highest dose level in the absence of S9 metabolism. Slight precipitation of the test item was observed with all tester strains at the highest dose level, both in the absence and presence of S9 metabolism, in both experiments.

Conclusion: The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.