Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In- vitro gene mutation toxicity test was performed for Green S (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) by performng microsomal rec assay in E. coli strains. The rec assay was performed using E. coli WP100 trp uvrA recA. The bacteria was exposed for 5.5 hrs to 10 mg/mL Green S and then incubated overnight before the plates were counted. Green S failed to show mutation using E. coli WP100 trp uvrA rec A and hence is not likely to classify 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:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In- vitro gene mutation toxicity test was performed for Green S by performng microsomal rec assay in E. coli strains

GLP compliance:

not specified

Type of assay:

other: Rec assay

Specific details on test material used for the study:

- Name of the test material: Green S
- EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
- Molecular formula: C27H26N2O7S2.Na
- Molecular Weight: 576.623 g/mol
- Substance type: Organic
- Smiles: c12c(\C(c3ccc(N(C)C)cc3)=C3/C=C\C(=[N+](/C)C)C=C3)c(O)c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O-])cc2.[Na+]

Target gene:

Tryptophan

Species / strain / cell type:

E. coli, other: WP100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: trp uvrA recA

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Calcium-precipitated microsomes from the livers of phenobarbitone-induced Sprague-Dawley rats

Test concentrations with justification for top dose:

0 or 10 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

other: Ethidium bromide (167 µg), furacin (1 µg)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Preincubation period: No data available
- Exposure duration: 5.5 hours
- Expression time (cells in growth medium): Overnight
- 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: No data available

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:

Coefficient of survival was measured

Statistics:

No data

Species / strain:

E. coli, other: WP100

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:

No data available

Conclusions:

Green S failed to show mutation using E. coli WP100 trp uvrA rec A and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

In- vitro gene mutation toxicity test was performed for Green S (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) by performng microsomal rec assay in E. coli strains. The rec assay was performed using E. coli WP100 trp uvrA recA. The bacteria was exposed for 5.5 hrs to 10 mg/mL Green S and then incubated overnight before the plates were counted. Green S failed to show mutation using E. coli WP100 trp uvrA rec A and hence is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo gene mutation assay was performed for the test chemical Lissamine Green B in male Canton-S Drosophila melanogaster by the injection route. Adult male flies were injected with dye in 0.7% NaCl solution at a dose level of 0.0025 or 0.025 M . Alternatively Drosophila larval stages were reared on standard semolina-treacle-agar culture medium containing dye. The positive control for these experiments was ethylmethane sulphonate (EMS), adults having been fed for 72 hr and third instar larvae for 48 hr on a yeasted suspension containing EMS. The frequency of sex-linked recessive lethal mutations in Drosophila melanogaster was estimated by the Basc method. Mutants were scored by mating 12 treated 4-7 day old Canton-S males to Basc females, three females for each male. with successive broods from fresh virgin females at 72-hr intervals. All cultures were maintained at 25 deg C and suspected lethals were checked in the F3 generation. No evidence of recessive lethal mutations was found among the first brood progeny of either adults (0/240) or larvae (0/94) treated with Lissamine Green B and hence is considered to be negative for gene mutation in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo insect germ cell study: gene mutation

Remarks:

Drosophila SLRL test

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:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In- vivo gene mutation assay was performed for the test chemical Lissamine Green B in Canton-S Drosophila by the injection route of exposure

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Specific details on test material used for the study:

- Name of the test material: Lissamine Green B
- EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
- Molecular formula: C27H26N2O7S2.Na
- Molecular Weight: 576.623 g/mol
- Substance type: Organic
- Smiles: c12c(\C(c3ccc(N(C)C)cc3)=C3/C=C\C(=[N+](/C)C)C=C3)c(O)c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O-])cc2.[Na+]
- Purity: 89%

Species:

Drosophila melanogaster

Strain:

other: Canton S

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data available
- Age at study initiation: 4-7 day old
- Weight at study initiation: No data available
- Assigned to test groups randomly: [no/yes, under following basis: ] No data available
- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To: No data available

Route of administration:

other: Injection

Vehicle:

- Vehicle(s)/solvent(s) used: 0.7% NaCl
- Justification for choice of solvent/vehicle: The chemical was soluble in 0.7% NaCl
- Concentration of test material in vehicle: 0.0025 or 0.025 M
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Lissamine B was dissolved in 0.7% NaCl to give a dose level of 0.0025 or 0.025 M.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

Duration of treatment / exposure:

72 hrs

Frequency of treatment:

Continuous for 72 hrs

Post exposure period:

No data

Remarks:

0.0025 or 0.025 M

No. of animals per sex per dose:

Total: 48
12 males and 36 females (three females for each male)

Control animals:

not specified

Positive control(s):

Ethylmethane sulphonate (EMS)
- Justification for choice of positive control(s): No data
- Route of administration: Oral
- Doses / concentrations: Positive control adults were fed 0.025 M-ethyl methane sulphonate (EMS) and third instar larvae were fed on a yeasted suspension containing 0.0025 M-EMS.

Tissues and cell types examined:

Brood progenies were observed for recessive lethal mutations

Details of tissue and slide preparation:

No data

Evaluation criteria:

An increase in the frequency of sex-linked recessive lethal mutations

Statistics:

No data

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Conclusions:

Lissamine Green B failed to induce sex linked recessive lethal mutation in Canton S Drosophila melanogaster and hence is considered to be negative for gene mutation in vivo.

Executive summary:

In vivo gene mutation assay was performed for the test chemical Lissamine Green B in male Canton-S Drosophila melanogaster by the injection route. Adult male flies were injected with dye in 0.7% NaCl solution at a dose level of 0.0025 or 0.025 M . Alternatively Drosophila larval stages were reared on standard semolina-treacle-agar culture medium containing dye. The positive control for these experiments was ethylmethane sulphonate (EMS), adults having been fed for 72 hr and third instar larvae for 48 hr on a yeasted suspension containing EMS. The frequency of sex-linked recessive lethal mutations in Drosophila melanogaster was estimated by the Basc method. Mutants were scored by mating 12 treated 4-7 day old Canton-S males to Basc females, three females for each male. with successive broods from fresh virgin females at 72-hr intervals. All cultures were maintained at 25 deg C and suspected lethals were checked in the F3 generation. No evidence of recessive lethal mutations was found among the first brood progeny of either adults (0/240) or larvae (0/94) treated with Lissamine Green B and hence it is considered to be negative for gene mutation in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation in vitro:

Various peer reviewed publications were reviewed to determine the mutagenic nature of

Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt. The studies are summarized as below:

In- vitro gene mutation toxicity test was performed by Haveland-Smith et al ( Food and cosmetics toxicology,1979) for Green S (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) by performing microsomal rec assay in E. coli strains. The rec assay was performed using E. coli WP100 trp uvrA recA. The bacteria was exposed for 5.5 hrs to 10 mg/mL Green S and then incubated overnight before the plates were counted. Green S failed to show mutation using E. coli WP100 trp uvrA rec A and hence is not likely to classify as a gene mutant in vitro.

In the same study as mentioned above, Gene mutation toxicity study was performed to determine the mutagenic nature of Green S. Liquid fluctuation assay was performed using Salmonella typhimurium strain TA1538 and E. coli WP2 uvrA. The test was performed both in the presence and absence of Calcium-precipitated microsomes from the livers of phenobarbitone-induced Sprague-Dawley rats. The test chemical was tested at dose level of 0.01 or 0.1 mg/mL with microsomal activation and 0.01 mg/mL without microsomal activation. A compound was considered positive only if it resulted in significantly more turbid tubes in a treated series when compared with the untreated set of tubes. Green S failed to induce turbid tubes in a treated series when compared with the untreated set of tubes and hence is considered to be negative for gene mutation in vitro.

Gene mutation toxicity study was also performed by Bonin et al (Mutation reasearch, 1981) to determine the mutagenic nature of Lissamine Green B (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt). The study was performed using Salmonella typhimurium strain TA98, TA100, TA1535, TA1537 and TA1538 in the presence and absence of S9 metabolic activation system at dose levels of 0, 32, 100, 320 or 1000 µg/plate. The plates were incubated for 72 hrs at 37 deg C and then were observed for a dose dependent increase n the number of revertants/plate. Lissamine Green B failed to induce mutation in Salmonella typhimurium strain TA98, TA100, TA1535, TA1537 and TA1538 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Bonin et al (Mutation research, 1981) performed gene mutation toxicity study determine the mutagenic nature of Lissamime Green B (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt). The study was performed using Salmonella typhimurium strain TA98 in the presence of fresh S9 metabolic activation system. The doses used for the study were 0, 625, 1250, 2500 or 5000 µg/plate. The plates were observed for a dose dependent increase in the number of revertants/plate. Lissamine Green B induced a dose related increase in the number of revertant colonies in Salmonella typhimurium strain TA98 in the presence of S9 metabolic activation system and hence is likely to classify as a gene mutant in vitro. However, the weak mutagenicity was attributed to the minor dye components present in Lissamine Green B and the positive effect observed was only within narrow dose ranges.

In another gene mutation toxicity study was performed by Angus et al (Mutation research, 1981) and Bonin et al (Mutation research, 1981) to determine the mutagenic nature of Lissamine Green B (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt). The study was performed in the presence and absence of S9 metabolic activation system at dose levels of 0, 1.85 X 10^-6(32), 5.78 X 10^-6(100), 1.85 X 10^-5(320) or 5.78 X 10^-5(1000) µg/plate. 100µL bacterial broth culture, 100µL aliquots of dye solution and 500 µL S9, mix (as required) were incorporated in 2 mL top agar. This was mixed and plated over 28 ml of minimal agar containing trace quantities of histidine and biotin. Lissamine Green B failed to induce mutation in Salmonella typhimurium strain TA100, TA1537 and TA1535 both in the presence ad absence of S9 metabolic activation system. It however induce mutation in strains TA98 and TA1538 only in the presence of S9 metabolic activation system. It was found that a significant doubling of revertant colonies only occurred at dye concentrations of 3.2 x 10 -4 or more (120 ± 7.4 revertants; n = 10) as compared with control plates (60 ± 3.7 revertants; n = 10). This food colouring was found to be less mutagenic/weakly mutagenc to strain TA98 and TA1538 and negative for strains TA100, TA1537 and TA1537. However, minor dye components were responsible for the weak mutagenicity of Lissamine Green B and the positive effect observed was only within narrow dose ranges.

Based on the key study and its supporting data, Lissamine Green B (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) is not likely to classify as a gene mutant in vitro. Study 4 and study 5 reflected positive gene mutation in Salmonella typhimurium strain TA98 and TA1538 but the mutagenicity observed was because of the minor dye components were responsible for the weak mutagenicity of Lissamine Green B and the positive effect observed was only within narrow dose ranges.

Gene toxicity in vivo:

Various peer reviewed publications were reviewed to determine the mutagenic nature of

Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt in vivo. The studies are summarized as below:

In vivo gene mutation assay was performed by Angus et al ( Food and cosmetics toxicology, 1981) for the test chemical Lissamine Green B (IUPAC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) in male Canton-S Drosophila melanogaster. Adult male flies were either fed for 72 hr on dye solution dissolved in 5% sucrose injected with dye in 0.7% NaCl solution at a dose level of 0.0025 or 0.025 M . Alternatively Drosophila larval stages were reared on standard semolina-treacle-agar culture medium containing dye. The positive control for these experiments was ethylmethane sulphonate (EMS), adults having been fed for 72 hr and third instar larvae for 48 hr on a yeasted suspension containing EMS. The frequency of sex-linked recessive lethal mutations in Drosophila melanogaster was estimated by the Basc method. Mutants were scored by mating 12 treated 4-7 day old Canton-S males to Basc females, three females for each male. with successive broods from fresh virgin females at 72-hr intervals. All cultures were maintained at 25 deg C and suspected lethals were checked in the F3 generation. No evidence of recessive lethal mutations was found among the first brood progeny of either adults (0/240) or larvae (0/94) treated with Lissamine Green B and hence is considered to be negative for gene mutation in vivo by the oral and injection route of exposure.

Giri et al ( Environmental and Molecular Mutagenesis, 1992) and Chetelat et al (Mutation reaserch, 1992) also performed gene mutation in vivo study to determine the mutagenic nature of Green S (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) in Swiss albino mice. 10 -12 weeks old male mice recieved three doses of 100, 200, and 400 mg/kg body weight of Green S were dissolved in distilled water and intraperitoneally (i.p.) injected in a volume of 150 µl/mouse. Five male animals were used for each dose group and for the control group. Negative control mice were injected with equal volume of distilled water while positive control animals received 25 mg/kg of cyclophosphamide. After 22 hr the animals were injected (i.p.) with colchicine (2 mgikg) and 2 hr later they were sacrificed by cervical dislocation. Bone marrow chromosomes were prepared. All slides were coded and stained with Giemsa (1:20 dilution). All slides were scored by a single observer. A total of 100 well-spread metaphase cells were scored per animal from each of 5 animals at each dose tested. Mitotic index (MI) were calculated from 1000 cells/animal and expressed as percentage. CA were scored. The aberration frequencies per cell for chromatid and chromosome types were calculated. Gaps were recorded but not included in the frequency of aberrations per cell. Except for the lowest dose (100 mgikg), the two higher doses shows significant increase in the percentage of CA when compared with negative control. The minimum tested dose which induced CA was 200 mg/kg body weight. A significant increase in the mitotic index (MI) was observed in the highest dose (400 mgikg) tested when compared with negative control. The trend tests for the evidence of dose response effects were also significant. No significant differences were observed in cell replication kinetic (RI) analysis. Green S can therefore be considered to not likely induce chromosome aberration upto a dose level of 100 mg/Kg bw.

However, an unknown parameter of the testing conditions induced an impressively uniform increase in Chromosome Aberration and on this basis Green S is not likely to be classified as a gene mutant in vivo (Chetelat et al, 1992).

In the same study by Giri et al ( Environmental and Molecular Mutagenesis, 1992), In vivo Sister Chromatid Exchange assay was performed using Swiss albino mice to determine the mutagenic potential for the test compound Green S (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt). 10 -12 week ols male and female Swiss albino mice were intaperitoneally injected a dose level of 0, 25, 50, 100 or 200 mg/kg bw. This tratement was preceeded Paraffin coated (-80% of the surface) BrdU tablets (50 mg each) being implanted subcutaneously in the flank of mice under ether anaesthesia following the methodology of McFee ct al for in vivo SCE study and cell replication kinetic analysis (RI). The test chemical was administered as a single i.p. injection 1 hr after tablet implantation. Negative control mice were injected with 100 p,l of distilled water while mitomycin C was used as a positive control at a dose of 1.5 mg/kg of body weight. For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 hr after BrdU tablet implantation. Two hours later bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCI at 37°C) for 20 min cells were fixed three times with methanol: acetic acid (3: 1). The slides were prepared and chromosomes were differentially stained with fluorescence-plus- Giemsa technique. All slides were coded and 30 second division metaphase cells (40 2 2 chromosomes) per animal scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested. Randomly selected metaphase cells ( 100/animal) were scored for replicative indices analysis by their staining pattern as first (M1), second (M2), and third (M3) division metaphases. Except the lowest dose (25 mgikg) a significant increase in the SCE frequency was observed for all the doses (SO, 100, and 200 mgikg) tested when compared with negative control. No significant differences were observed in the cell replicative kinetics (RI) when compared to the control. The minimum effective dose to induce the significant SCE was 50 mg/kg of body weight which was just ten times the previous acceptable daily uptake (ADI).

However, an unknown parameter of the testing conditions induced an impressively uniform increase in Sister Chromatid Exchange and on this basis Green S is not likely to be classified as a gene mutant in vivo (Chetelat et al, 1992).

Based on the studies summarized, Green S is not likely to classify as a gene mutant in vivo. The positive effect mentioned in in- vivo studies 3 and 4 is basically because of some unknown parameter of the testing conditions (Chetelat et al, 1992) and hence the positive effect is ignored for classification.

Justification for classification or non-classification

Based on the key study and its supporting data, Lissamine Green B (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) does not exhibit gene mutation toxicity in vitro and in vivo. Hence the chmeical is not likely to classify as a gene mutant in vitro and in vivo.