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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation toxicity study was performed to determine the mutagenic nature of C.I. Pigment Red 81. The study was performed using Salmonella typhimurium strains  TA100, TA1535, TA1537, TA 97 and TA98 with and without rat liver S9 and hamster liver S9 as the metabolic activation system. Preicubation assay was performed at dose levels of 0, 100, 333, 1000, 3333 or 10000 µg/plate. The plates were preincubated for 20 mins following an expression duration of 48 hrs. The plates were observed for a dose dependent increase in the number of revertants/plate. Pigment Red 81 is negative in the preincubation assay on the Salmonella typhimurium strains  TA100, TA1535, TA1537, TA 97 and TA98 with and without rat liver S9 and hamster liver S9 as the metabolic activation system and hence is not likely to classsify as a gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to determine the mutagenic nature of C.I. Pigment Red 81

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of the test material: Pigment Red 81
- EC name: Xanthylium, 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethyl-, molybdatetungstatephosphate
- Molecular formula: C28H31N2O3.xUnspecified
- Molecular weight: 443.5639 g/mol
- Substance type: Organic
- Physical state: No data available
- Purity: ≈ 50%
- Impurities (identity and concentrations): No data available

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA1537, TA1538, TA97 and TA98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

10% and 30% hamster liver S9 and 10% and 30% rat liver S9

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333 or 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Pigment Red 81 was soluble in water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98 and TA1538; Without S9) and 2-aminoanthracene (With S9; all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

The plates werwe observed for a dose dependent increase in the number of revertants/plate.

Evaluations were made at both the individual trial and chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?),
or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in hisf revertants in repeat trials.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537, TA 97 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Pigment Red 81 was run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100 or the system developed by Waleh et al. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Conclusions:

Pigment Red 81 is negative in the preincubation assay on the Salmonella typhimurium strains TA100, TA1535, TA1537, TA 97 and TA98 with and without rat liver S9 and hamster liver S9 as the metabolic activation system and hence is not likely to classsify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of C.I. Pigment Red 81. The study was performed using Salmonella typhimurium strains  TA100, TA1535, TA1537, TA 97 and TA98 with and without rat liver S9 and hamster liver S9 as the metabolic activation system. Preicubation assay was performed at dose levels of 0, 100, 333, 1000, 3333 or 10000 µg/plate. The plates were preincubated for 20 mins following an exporession duration of 48 hrs. The plates werwe observed for a dose dependent increase in the number of revertants/plate. Pigment Red 81 is negative in the preincubation assay on the Salmonella typhimurium strains  TA100, TA1535, TA1537, TA 97 and TA98 with and without rat liver S9 and hamster liver S9 as the metabolic activation system and hence is not likely to classsify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene toxicity in vitro:

Peer reviewed publications for the target chemical and its read across chemicals were reviewed to determine the mutagenic nature of C.I. Pigment Red 81. The studies are as mentioned below:

Gene mutation toxicity study was performed by Zeiger et al ( Environmental and Molecular Mutagenesis, 1992) to determine the mutagenic nature of C.I. Pigment Red 81 (EC name: Xanthylium, 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethyl-, molybdate tungstate phosphate). The study was performed using Salmonella typhimurium strains  TA100, TA1535, TA1537, TA 97 and TA98 with and without rat liver S9 and hamster liver S9 as the metabolic activation system. Preicubation assay was performed at dose levels of 0, 100, 333, 1000, 3333 or 10000 µg/plate. The plates were preincubated for 20 mins following an expression duration of 48 hrs. The plates were observed for a dose dependent increase in the number of revertants/plate. Pigment Red 81 is negative in the preincubation assay on the Salmonella typhimurium strains  TA100, TA1535, TA1537, TA 97 and TA98 with and without rat liver S9 and hamster liver S9 as the metabolic activation system and hence is not likely to classsify as a gene mutant in vitro.

The data for the target chemical is further supported by the data from structurally and/or functionally similar read across chemicals as mentioned below:

Zeiger et al ( Environmental Mutagenesis, 1987) performed Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters to evaluate the mutagenic nature of the test compound Rhodamine 6G (RA CAS no 989 -38 -8; IUPAC name: 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride) using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins. The test compound was used at a dosage level of 0.0, 0.3, 1.0, 3.3, 6.7, 10.00, 16.7, 33.00, 67.0, 100.00 or 333.0 µg/plate in the preincubation assay of 48 hrs. Rhodamine 6G did not induce a reproducible, dose-related increase in his⁺revertants over the corresponding solventin the S. typhimurium tester strains TA1535, TA1537, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is negative for mutation in vitro.

In a study for another read across chemical by Weubbles et al (Environmental Mutagenesis, 1985), gene mutation toxicity study was performed for functionally similar read across chemical Rhodamine 590 tetrafluoroborate (RA CAS no 54854 -14 -7; IUPAC name: 9-[2-(Ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium tetrafluoroborate). Bacteria were grown overnight in Oxoid nutrient broth, then refrigerated at 4-5^OC for a few hours before use. 0.1 ml of bacterial culture was added to 2 ml of 45°C molten top agar containing 0.01 mg histidine HCI and 0.012 mg biotin/ml, followed by the test sample at dose levels of 0, 0.1 or 1 mg/plate in ≤0.2 ml DMSO. Finally, 0.5 ml of sodium phosphate buffer, pH 7.4 (no activation), or 0.5 ml of Aroclor-induced rat S9 mixture was added, and the mixture was poured on minimal glucose agar plates. Histidine revertant colonies were counted on a Biotran II automated colony counter after a 2-day incubation at 37°C. Duplicate plates were run on all tests. All results were verified in repeat experiments. A sample was judged mutagenic if it produced greater than twice the spontaneous background colonies at more than one dose or at the highest dose tested. Rhodamine 590 tetrafluoroboratedid not induce gene toxicity in the Salmonella typhimurium strains TA1538 and TA100 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant.

Based on the information observed for the test chemical, it is summarized that C.I. Pigment Red 81 is not likely to exhibit genetic toxicity. Thus, the chemical is not classified as a genetic toxicant

Justification for classification or non-classification

Based on the information observed for the test chemical, it is summarized that C.I. Pigment Red 81 (CAS no 12224 -87 -5) is not likely to exhibit genetic toxicity. Thus, the chemical is not classified as a genetic toxicant as per the criteria mentioned in CLP regulation.