Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)amino](4-hydroxy-2-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity of Synthetic Food Colorants

Author:

V. R. Swaroop, D. Dinesh Roy and T. Vijayakumar

Year:

2011

Bibliographic source:

Journal of Food Science and Engineering 1 (2011) 128-134

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Cytokinesis Block Micronucleus (CBMN) Assay) was performed for Fast green FCF using lymphocytes isolated from human volunteers. 

Principles of method if other than guideline:

Cytokinesis Block Micronucleus (CBMN) Assay) was performed for Fast green FCF using lymphocytes isolated from human volunteers. 

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)amino](4-hydroxy-2-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

EC Number:

219-091-5

EC Name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)amino](4-hydroxy-2-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Cas Number:

2353-45-9

Molecular formula:

C37H36N2O10S3.2Na

IUPAC Name:

disodium 2-({4-[ethyl(3-sulfonatobenzyl)amino]phenyl}{4-[ethyl(3-sulfonatobenzyl)iminio]cyclohexa-2,5-dien-1-ylidene}methyl)-5-hydroxybenzenesulfonate

Details on test material:

- Name of test material (as cited in study report): Fast Green FCF (2353-45-9)- Molecular formula (if other than submission substance): C37-H36-N2-O10-S3.2Na- Molecular weight (if other than submission substance): 808.8576 g/mol- Substance type: Organic- Physical state: Solid- Purity: No data available- Impurities (identity and concentrations): No data available

Method

Target gene:

Human Lymphocytes

Species / strain

Species / strain / cell type:

other: Human

Details on mammalian cell type (if applicable):

- Type and identity of media: No data available- Properly maintained: No data available- Periodically checked for Mycoplasma contamination: No data available- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

100, 200 and 500 μg mL-1

Vehicle / solvent:

water

Controls

Untreated negative controls:

not specified

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 mediumBlood samples from healthy young adult volunteer without any history of chronic illness, drug intake and or any life style associated bad habits were collected with informed consent. Lymphocytes were separated from these samples using Lymphoprep. Four parallel Lymphocyte micro cultures in duplicate were set up for each sample to evaluate the extent of genotoxicity. Culture A was kept as control and coloring agents were added to Culture B, C and D with varying concentrations of 100, 200 and 500 μg mL-1, Cytochalasin B (Sigma) at a concentration of 4.5 μg mL-1 was added to all the 4 cultures at 44th hour to block the cell division at Cytokinesis. The cultures were harvested at 72nd hour; slides were prepared and stained with 10% Giemsa stain. Observed under a plan achromatic microscope for enumerating the frequency of micronuclei among 1000 binucleated cells and recorded.DURATION- Preincubation period: No data available- Exposure duration: 72 hrs- Expression time (cells in growth medium): No data available- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells):SELECTION AGENT (mutation assays): Giemsa stainSPINDLE INHIBITOR : Cytochalasin BSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: No data availableNUMBER OF CELLS EVALUATED: 1000 binucleated cells examinedDETERMINATION 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 availableOTHER: No data available

Evaluation criteria:

The criteria for enumeration were: (1) cells should have two nuclei of approximately equal size, (2)the 2 nuclei may be attached by a fine nucleoplasmic bridge, (3) the two nuclei may overlap slightly or toucheach other at the edges and (4) Cells should not contain more than 6 micronuclei.

Statistics:

Statistical Package for Social Sciences (SPSS).

Results and discussion

Test results

Species / strain:

other: Human

Metabolic activation:

not specified

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: other: Human lymphocytes

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):positiveFast green FCF is found to induce gene mutation in the lymphocytes isolated from human volunteers in the Cytokinesis Block Micronucleus (CBMN) Assay performed.

Executive summary:

Cytokinesis Block Micronucleus (CBMN) Assay was performed for the test chemicalFast green FCF using lymphocytes isolated from human volunteers.

Blood samples from healthy young adult volunteer without any history of chronic illness, drug intake or any life style associated bad habits were collected. Lymphocytes were separated from the samples.Four parallel Lymphocyte micro cultures in duplicate were set up for each sample to evaluate the extent of genotoxicity. Culture A was kept as control and coloring agents were added to Culture B, C and D with varying concentrations of 100, 200 and 500 μg mL^-1,Cytochalasin B was added to all the 4 cultures at 44th hour to block the cell division at Cytokinesis.The cultures were harvested at 72nd hour; slides were prepared and stained with 10% Giemsa stain. Observed under a plan achromatic microscope for enumerating the frequency of micronuclei among 1000 binucleated cells and recorded.

The criteria for enumeration were:

(1) cells should have two nuclei of approximately equal size,

(2)the 2 nuclei may be attached by a fine nucleoplasmic bridge,

(3) the two nuclei may overlap slightly or touch

each other at the edges and

(4) Cells should not contain more than 6 micronuclei.

Fast green FCF is found to induce gene mutation in the lymphocytes isolated from human volunteers in theCytokinesis Block Micronucleus (CBMN) Assay performed.

According to the publication, the test material Fast Green FCF is a positive mutagen.