Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Fluctuation test was performed for the test chemical Patent blue V used at a concentration of 500 mg/kg in liquid medium for72-96 hrs. The tester strains used were Salmonella typhimurium TA 1538 and Escherichia coli WP2uvrA. The assay was performed for each bacterium in three separate experiments.

The given test material failed to inducegenotoxicity in the bacteriaSalmonella typhimuriumTA 1538 andEscherichia coliWP2uvrA.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: Data is from Journal with permission

Principles of method if other than guideline:

The induction of mutations was studied in modified liquid fluctuation tests using a tryptophan-requiring E. coli strain (sensitive tobase substitutions) and a histidine auxotroph of Salmonella typhimurium (specific for frameshifts)for the test compound Patent Blue V.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Modified Liquid Fluctuation test:Salmonella typhimurium TA 1538 and Escherichia coli WP2 uvrA

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

500 mg/L

Vehicle / solvent:

Deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumDURATION- Preincubation period: No data available - Exposure duration: 72-96 hrs- Expression time (cells in growth medium): 72-96 hrs- 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 availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: TriplicateNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data availableOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data available OTHER: The food colour was made up in deionized water and membrane-sterilized prior to use. The test material was tested at its maximum sublethal concentration.

Evaluation criteria:

The test material was considered positive only if it resulted in significant more turbid tubes in a treated series when compared with an untreated set of tubes

Statistics:

Chi square test

Species / strain:

E. coli WP2 uvr A

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:

not specified

Species / strain:

S. typhimurium TA 1538

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:

not specified

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):negative with and withoutPatent blue V failed to induce gene mutation in the bacterial tester strains Salmonella typhimurium TA 1538 and Escherichia coli WP2 uvrA

Executive summary:

Fluctuation test was performed for the test chemical Patent blue V used at a concentration of 500 mg/kg in liquid medium for 72-96 hrs. The tester strains used were Salmonella typhimurium TA 1538 and Escherichia coli WP2 uvrA. The assay was performed for each bacterium in three separate experiments.

The given test material failed to induce genotoxicity in the bacteria Salmonella typhimurium TA 1538 and Escherichia coli WP2 uvrA and hence does not classify for gene mutation in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Gene mutation in vitro:

Various peer reviewed publications were reviewed to determine the mutagenic nature of the test compound Patent blue V in vitro. The study is as summarized below:

 

Salmonella/ mammalian microsome mutagenicity assay was performed by Bonin et al (1981) to study the mutagenic potential of Patent Blue V (CAS no 129-17-9) both in the presence and absence of metabolic activator S9 mix. To each 2 ml of top agar at 42°C was added 100 µL bacterial broth culture, 100 µL test compound dissolved in DMSO various concentrations, and 500 µL S9 mix as required. Plates were incubated at 37°C for 72 hrs before counting his⁺revertant colonies and each dose point was determined from at least two plates, unless indicated otherwise. Criteria for mutagenicity were (a) a dose-response and, (b) reproducibility of the result. Dose-responses were not always evident at concentratrations selected for initial testing. Since no mutagenicity was detected, Patent Blue V is negative for the induction of gene toxicity in vitro. Hence the test chemical is not likely to classify for gene mutation in vitro.

Fluctuation test was performed (Haveland- Smith, 1979) for the test chemical Patent blue V (CAS no 129-17-9) used at a concentration of 500 mg/L in liquid medium for 72-96 hrs. The tester strains used wereSalmonella typhimuriumTA 1538 andEscherichia coliWP2uvrA. The assay was performed for each bacterium in three separate experiments. The given test material failed to induce genotoxicity in the bacteriaSalmonella typhimuriumTA 1538 andEscherichia coliWP2uvrA and hence does not classify for gene mutation in vitro.

 

Patent Blue V (CAS no 129-17-9) was used at a concentration of 5000 mg/L and its genotoxic effect was studied (Haveland- Smith, 1979) in the bacteriaE. coli strains WP2 trp uvrA and WP100 trp uvrA recAbyrecplate assay in the agar medium. The given test material is negative for genotoxicity in the bacteriaE. coli strains WP2 trp uvrA and WP100 trp uvrA recAand hence is not likely ti classify for gene mutation in vitro.

An Ames test with Patent Blue V (CAS no 129-17-9) was performed (EFSA Journal, 2013) according to the OECD guideline No. 471. Five histidine-dependent strains of S. typhimurium (TA98, TA100, TA1535, TA1537 and TA102) were used to evaluate the mutagenic potential of Patent Blue V, both in the absence and presence of rat liver S9 metabolism. The study was carried out using both the plate incorporation and the pre-incubation methods. Patent Blue V was tested as a dark blue solution in water for injection. The experiment performed using the plate incorporation method was carried out using the dose range of 52-5000 µg/plate, both in the absence and presence of rat S9 metabolism. Following treatment, no precipitation and cytotoxicity were noted in the absence or in the presence of metabolic activation. Statistically and biologically significant increases in the number of revertants were observed only with the TA98 tester strain in the presence of metabolic activation. Patent Blue V is positive to induce mutation only in theSalmonella typhimuriumTA98 strain in the presence of S9 metabolic activation system. The positive outcome obtained in the presence of metabolic activation and at higher dose levels only, strongly indicates that mutagenicity could have been caused by the presence of impurities given the low purity of the test item (86 %). It does not induce mutation in the strainsTA100, TA1535, TA1537 and TA102 in the presence or absence of S9 metabolic activation system. Hence the test chemical is not likely to classify as a gene mutant in vitro.

 

An Ames test with Patent Blue V (CAS no 129-17-9) was performed (EFSA Journal, 2013) according to the OECD guideline No. 471. Five histidine-dependent strains of S. typhimurium (TA98, TA100, TA1535, TA1537 and TA102) were used to evaluate the mutagenic potential of Patent Blue V, both in the absence and presence of rat liver S9 metabolism. The study was carried out using both the plate incorporation and the pre-incubation methods. Patent Blue V was tested as a dark blue solution in water for injection. The pre-incubation method was carried out in the dose range of 492-5000 µg/plate, both in the absence and presence of rat liver S9 metabolism. Following treatment, no precipitation or cytotoxicity were noted in the absence or in the presence of metabolic activation. Statistically and biologically significant increases in the number of revertants were confirmed with the TA98 tester strain in the presence of metabolic activation. The positive outcome obtained in the presence of metabolic activation and at higher dose levels only, strongly indicates that mutagenicity could have been caused by the presence of impurities given the low purity of the test item (86 %). Patent Blue V is positive to induce mutation only in the Salmonella typhimurium TA98 strain in the presence of S9 metabolic activation system. It does not induce mutation in the strains TA100, TA1535, TA1537 and TA102 in the presence or absence of S9 metabolic activation system. Hence, the test material is not likely to classify a gene mutant in vitro.

 

The mutagenic potential of Patent Blue V (CAS no 129-17-9) was assessed (EFSA Journal, 2013) in the in vitro mammalian cell gene mutation assay using L5178Y (Tk+/-) mouse lymphoma cells. The study was conducted in compliance with the OECD guideline No. 476 and the ICH guidelines S2A and S2B. Long treatment (approximately 24 hours) without metabolic activation was performed. Replicate cultures were set up at each experimental point. No statistically or biologically significant increases in the mutant frequency were noted up to the maximum tested dose level of 2500 μg/ml at any treatment time in the absence of metabolic activation. Patent Blue V does not induce mutation in the L5178Y (Tk+/-) mouse lymphoma cells in the absence of S9 metabolic activation system. Hence the test material is not likely to classify as a gene mutant in vitro.

 

The mutagenic potential of Patent Blue V (CAS no 129-17-9) was assessed (EFSA Journal, 2013) in the in vitro mammalian cell gene mutation assay using L5178Y (Tk+/-) mouse lymphoma cells. The study was conducted in compliance with the OECD guideline No. 476 and the ICH guidelines S2A and S2B. Short treatment (approximately 4 hours) with and without metabolic activation was performed. Replicate cultures were set up at each experimental point. No statistically or biologically significant increases in the mutant frequency were noted up to the maximum tested dose level of 2500 μg/ml at any treatment time in the presence or absence of metabolic activation. Patent Blue V does not induce mutation in the L5178Y (Tk+/-) mouse lymphoma cells in the presence or absence of S9 metabolic activation system. Hence the test compound is not likely to classify as a gene mutant in vitro

 

Gene mutation in vivo:

 

Various references for the test compound and the prediction data are used to summarize the mutageinc nature of the test compound in vivo:

The DNA damaging capabilities of Patent Blue V (E 131) (CAS no 129-17-9)were assessed (EFSA Journal, 2013) in the in vivo single cell gel/Comet assay in rats. Patent Blue V (E 131) was examined for genotoxic properties by evaluating the induction of DNA damage in cell suspensions isolated from liver, jejunum/ileum and peripheral blood of rats after in vivo treatment using the alkaline (pH>13) version of the Comet Assay. In the main experiment, only male animals were treated since no substantial inter-sex differences in toxicity were observed. Groups of 5 Sprague-Dawley male rats were treated twice at 24 hour intervals with the vehicle only (sterile distilled water of injectable grade), or Patent Blue V (E 131) at the dose levels of 500, 1000 and 2000 mg/kg/day. Ethyl methanesulphonate (EMS), at 200 mg/kg/day served as positive control. Animals were sacrificed approximately 3-4 hours after the second dosing. Peripheral blood and cell suspensions isolated from liver and jejunum/ileum were embedded in agarose gel on microscope slides. No statistically significant increases in tail moment and tail intensity values compared with the vehicle control values were observed at any dose-level in the treated groups. Patent Blue V (E 131) does not induce any effect on DNA migration in rat liver, jejunum/ileum and peripheral blood. The given test material gives negative gene toxicity in vivo response on the Sprague Dawley male rats and hence is not likely to classify for gene mutation in vivo.

 

The mutagenic potential of Patent Blue V (CAS no 129-17-9)was assessed (EFSA Journal, 2013) in the in vivo bone marrow micronucleus assay in Sprague-Dawley rats. On the basis of a preliminary experiment in which mortality was observed at 600 mg/kg bw, the study was carried out using groups of 5 male and 5 female rats administered dose levels of 75, 150 and 300 mg/kg, by a single intravenous injection. Saline (0.9 % NaCl) was used as vehicle. Animals were sacrificed 24 and 48 hours after administration. Clinical signs were noted only at the highest dose level of 300 mg/kg bw and consisted of irregular breathing and/or lowered activity in some animals. When compared to the negative control group (0.9 % NaCl), no biologically significant decreases in the polychromatic/normochromatic erythrocytes ratio (PCEs/NCEs) were noted in any animal treatment groups. For either sex or both sexes combined, when compared to the negative control group (0.9 % NaCl), no biologically significant increase in the mean frequencies of micronucleated polychromatic erythrocytes (MNPCEs) was noted in the animal groups treated with Patent Blue V up to the highest dose level of 300 mg/kg. Patent Blue V does not prove to induce micronuclei in rat bone marrow erythrocytes and hence is negative for inducing mutation in vivo.

 

In vivo bone marrow chromosome aberration assay was performed (CAS no 129-17-9) in C57Bl6 mice to assess for the potential clastogenicity activity of Patent Blue V (EFSA Journal, 2013). The test item was administered by oral gavage at daily dose levels of 0.08 and 0.8 mg/kg bw for 5 days. Animals were sacrificed 6 hours after the administration of the last dose level. Mice also received colchicines in the last 2 hours at 2.5 mg/kg bw to accumulate cells in metaphases. A minimum number of 100 well spread metaphases were scored for each animal in the control and treated groups. Results obtained indicated that Patent Blue V did not induce statistically significant increases of chromosomal aberrations in the mouse bone marrow cells erythrocytes. The given test material gives negative gene toxicity in vivo response in mice and hence is not likely toclassify for gene mutation in vivo.

 

Based on the available data summarized, the test chemical patent Blue V is not likely to classify as a gene mutant in vitro and in vivo. Though some mutagenic activity is reported for the test chemical in the data summarized for in vitro toxicity but it has been strongly indicated that mutagenicity could have been caused by the presence of impurities given the low purity of the test item. Thus the test chemical is likely to be not classififed for gene mutation.

Justification for selection of genetic toxicity endpoint

Data from K2 publication

Justification for classification or non-classification

Based on the available data summarized, the test chemical patent Blue V is not likely to classify as a gene mutant in vitro and in vivo.