Dihydrogen (ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, aluminium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

 

Chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

in vitro mammalian gene mutation:

The given test material does not induce mutation in the L5178Y (Tk+/-) mouse lymphoma cells in the absence of S9 metabolic activation system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Data available from study report.

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 476 and the ICH guidelines S2A and S2B

Principles of method if other than guideline:

The mutagenic potential of Patent Blue V was assessed in the in vitro mammalian cell gene mutation assay using L5178Y (Tk+/-) mouse lymphoma cells.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

In vitro mammalian cell gene mutation assay using L5178Y (Tk+/-) mouse lymphoma cells

Species / strain / cell type:

other: L5178Y (Tk+/-) mouse lymphoma cells

Details on mammalian cell type (if applicable):

No data available

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0.86 to 2500 μg/ml

Vehicle / solvent:

No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: In medium
- Exposure duration: 24 hrs
NUMBER OF REPLICATIONS: No detailed data available

DURATION
- Exposure duration: 24 hrs

Number of Replications: Duplicate

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

other: L5178Y (Tk+/-) mouse lymphoma cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

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 without metabolic activation

The given test material does not induce mutation in the L5178Y (Tk+/-) mouse lymphoma cells in the absence of S9 metabolic activation system.

Executive summary:

The mutagenic potential of Patent Blue V was assessed 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 cellsin the absence of S9 metabolic activation system. Hence the test material is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting 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 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic response of the test chemical

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster fibroblast cell line (CHL)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Properly maintained: yes by 4 day passages
- 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

Cytokinesis block (if used):

No data

Metabolic activation:

without

Metabolic activation system:

Metabolic activation system

Test concentrations with justification for top dose:

At three different doses with 2500 µg/plate being the maximum dose concentration

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Physiological saline
- Justification for choice of solvent/vehicle: The test chemical was soluble in physiological saline

Untreated negative controls:

yes

Remarks:

Untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

Physiological saline

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 medium

DURATION
- Preincubation period: No data available
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hr

SELECTION AGENT (mutation assays): No data available

SPINDLE INHIBITOR (cytogenetic assays): No data available

STAIN (for cytogenetic assays): 1.5% Geimsa stain at pH 6.8

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: 100 well spread metaphases were observed

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

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

- Determination of endoreplication: No data available

- Other: The incidence of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%

Statistics:

No data available

Species / strain:

other: Chinese hamster fibroblast cell line

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

No data

Conclusions:

The incidence of chromosome aberration in Chinese hamster fibroblast cell line for the test chemical was considered to be more than 10% in the absence of metabolic activation system during the 48 hrs study duration.

Executive summary:

Chromosomal aberration test was performed for the test chemical using Chinese hamster fibroblast cell line CHL. The cells were exposed to the test material at three different doses with 2.5 mg/plate being the highest dose for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The incidence of chromosome aberration in Chinese hamster fibroblast cell line for the test chemical was considered to be more than 10% in the absence of metabolic activation system during the 48 hrs study duration and was mutagenic in the absence of S9 metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 test chemical.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

other: TA 92. TA 1535, TA 100, TA 1537, TA 94, TA 98

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with

Metabolic activation system:

The liver microsome fraction (S-9) was prepared from the liver of Fischer rats

Test concentrations with justification for top dose:

6 different concentrations were used; 5.0 mg/plate was the maximum concentration

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Phosphate buffer
- Justification for choice of solvent/vehicle: The test chemical was soluble in Phosphate buffer

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Phosphate buffer

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

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

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).

Statistics:

No data

Species / strain:

S. typhimurium, other: TA 92. TA 1535, TA 100, TA 1537, TA 94, TA 98

Metabolic activation:

with

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:

The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment

Remarks on result:

other: No mutagenic potential

Conclusions:

Test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 5.0 mg/plate being the maximum concentration. The chemical was dissolved in phosphate buffer. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify 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

Ames Test:

Data available for the test chemicals were reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

 

Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 5.0 mg/plate being the maximum concentration. The chemical was dissolved in phosphate buffer. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

 

In another study, Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. The study was performed as per the qualitative plate test using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in glass distilled water or DMSO and used at dose levels of 0, 50, 250 or 1000 µg/plate. Concurrent solvent, negative, historical and positive control chemicals were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98 in the presence and absence of S9 metabolic activation system and hence it is considered to be non-mutagenic.

 

Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. The study was performed as per the qualitative plate technique using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in glass distilled water or DMSO and used at dose levels of 0, 50, 250 or 1000 µg/plate. Concurrent solvent, negative, historical and positive control chemicals were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98 in the presence and absence of S9 metabolic activation system and hence it is considered to be non mutagenic.

Based on the data available, the test chemical did not induce gene mutation in sallmonela strain in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Chromosomal Abberation:

Data available for the test chemicals were reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

 

Chromosomal aberration test was performed for the test chemical using Chinese hamster fibroblast cell line CHL. The cells were exposed to the test material at three different doses with 2.5 mg/plate being the highest dose for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The incidence of chromosome aberration in Chinese hamster fibroblast cell line for the test chemical was considered to be more than 10% in the absence of metabolic activation system during the 48 hrs study duration and was mutagenic in the absence of S9 metabolic activation system.

 

In another study, Chromosomal aberration tests were carried out to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in medium and used at dose levels of up to 5 mg/mL. In the assays for chromosomal aberrations, the top dose (TD) was based on toxicity, solubility, or the upper testing limit (5 mg/ml). In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1 µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay. In these tests the cell growth period was extended to about 20 hr. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed. All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials, etc.). These data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. The test chemical did not induce chromosome aberrations in Chinese hamster ovary cells (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

In vitro mammalian chromosome aberration test was also performed to determine the mutagenic nature of test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in serum free culture medium and used at dose level of 0, 1000, 1600, 3000 or 5000 µg/plate. Concurrent solvent and negative control chemicals were also included in the study. Approximately 24 hr prior to cell treatment, 1.2 x 10⁶cells were seeded per 75 cm2 flask. For assays without metabolic activation, the cells were treated for about 10 hr. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off. In the test protocol for assays with metabolic activation cells were harvested approximately 11 hr after removal of the S9 fraction. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. The test chemical did not induce ABS at doses up to 5000µg/ml in either the presence or the absence of S9. Based on the observations made, the test chemical did not induce chromosome aberration in Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

 

in vitro mammalian gene mutation:

The mutagenic potential of Patent Blue V was assessed 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 cellsin the absence of S9 metabolic activation system. Hence the test material is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce mammalian gene mutation in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and in vitro mammalian gene mutation assay. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.