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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

Link to relevant study records

Reference

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

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

Target gene:

Human Lymphocytes

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

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).

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'.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity In vitro

Studies of Fast Green FCF were reviewed for genetic toxicity in vitro from reliable sources having Klimisch rating 2

The summary of the results are presented below:

Sr. No	Type of genotoxicity	Result	Species	Remarks
1.	Gene mutation	Positive	S. typhimurium TA 100	Experimental data for target chemical
2.	Chromosome aberration	Positive	Chinese hamster lung fibroblasts (V79)	Experimental data for target chemical
3.	Gene mutation	Negative	Embryo cells isolated from Fischer rat	Experimental data for target chemical
4.	Gene mutation	Negative	Bacillus subtilis/: H17(rec+)and M45(Rec-)	Experimental data for target chemical
5.	Gene mutation	Negative	Diploid Yeast cells/ BZ 34	Experimental data for target chemical
6.	Chromosome aberration	Positive	Human lymphocytes	Experimental data for target chemical
7.	Gene mutation	Negative	Salmonella typhimurium	Experimental data for target chemical

 

On the basis of studies summarised in the above table, it can be observed that the substance gives positive as well as negative results for genetic toxicity. Genetic toxicity In vivo

Studies of Fast Green FCF were reviewed for genetic toxicity in vivo from reliable sources having Klimisch rating 2

The summary of the results are presented below:

Sr. No	Type of genotoxicity	Result	Species	Remarks
1.	DNA damage and/or repair	Positive	Mouse	Experimental data for target chemical
2.	DNA damage and/or repair	Negative	Rat	Experimental data for target chemical
3.	Chromosome aberration	Positive	Mouse	Experimental data for target chemical
4.	DNA damage and/or repair	Negative	Rat	Experimental data for target chemical

On the basis of studies summarised in the above table, it can be observed that the substance gives positive results for genetictoxicity in mice whereas it gives negative results in rats

Justification for selection of genetic toxicity endpoint
Data is from peer reviewed journal and from a recent study (2011) done on human lymphocytes.

Justification for classification or non-classification

Based on the available data and adopting a weight of evidence approach, the chemical dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl) amino](4-hydroxy-2-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt is shown to exhibit genetic toxicity to the various Salmonella strains of bacteria as well as chromosomal abberation in Chinese hamster lung fibroblasts. Thus, the chemical is likely to be classified as Mutagen category 2 based upon the mutagenic effects observed in animals.