Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

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:

Experimental Starting Date: 07 January 2014; Experimental Completion Date: 03 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

other: USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

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:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

EC Number:

289-056-7

EC Name:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Cas Number:

85959-61-1

Molecular formula:

C162H180N12O18Cl6+O3.P2O5.xWO3.yMoO3

IUPAC Name:

heptakis(N-[(3E)-6-(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-3H-xanthen-3-ylidene]ethan-1-aminium) dioxomolybdenumbis(olate) dioxotungstenbis(olate) phosphate

Test material form:

other: granular solid

Details on test material:

Identification: Pigment Red 81:4
Batch: S-3101
Purity: 97%
Expiry Date: 01 June 2014
Storage Conditions: Room temperature in the dark

Formulated concentrations were adjusted to allow for the stated water/impurity content (3%) of the test item.

Method

Target gene:

Histidine for Salmonella
Tryptophan for E. Coli

Species / strain

Species / strain / cell type:

other: S. typhimurium strains TA1535, TA1537, TA98 and TA100. E. coli strain WP2uvrA

Details on mammalian cell type (if applicable):

Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

Test concentrations with justification for top dose:

Experiment 1 (Plate incorporation Method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (Pre-Incubation Method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: In solubility checks performed in–house, the test item was insoluble in sterile distilled water and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL but was soluble in dimethyl sulphoxide and dimethyl formamide at 50 mg/mL. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

The positive controls ENNG, 9AA and 4NQO were used in the series of plates without S9-mix. 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP) were used in the series of plates with S9-mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
-Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.

Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical analysis of data as determined by UKEMS.

Results and discussion

Test results

Species / strain:

other: S. typhimurium strains TA1535, TA1537, TA98 and TA100. E. coli strain WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no toxicologically significant increases in the frequency of revertant colonies

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results for details

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In Experiment 1 (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the tester strains in both the absence and presence of S9-mix, initially from 1500 µg/plate (TA1537) and at 5000 µg/plate (remaining strains). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second experiment. In Experiment 2 (pre-incubation method) the test item induced a stronger toxic response with either a visible reduction in the growth of the bacterial background lawn and/or a substantial reduction in the frequency of revertant colonies noted to all of the tester strains, initially from 500 µg/plate (E.coli strain WP2uvrA, TA1537 with and without S9 and TA1535 in the absence of S9 mix only) and from 1500 µg/plate to the remaining strains. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. In Experiment 1 (plate incorporation method), an intense pink test item induced coloration was noted from 1500 µg/plate with an associated precipitate (powdery in appearance) observed at 5000 µg/plate. In Experiment 2, after the change in test methodology, an intense pink test item induced coloration was only noted at 1500 µg/plate and the associated precipitate was much thicker at 5000 µg/plate. None of these observations prevented the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Small, statistically significant increases in TA1537 revertant colony frequency were noted in the absence of S9 mix at 150 and 500 µg/plate. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 and 500 µg/plate were within the in house historical untreated/vehicle control range for the tester strain. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Pigment Red 81:4 was considered to be non-mutagenic under the conditions of this test.