Reaction mass of 5-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium methyl sulphate and 3-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium methyl sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Basic Red 046 Methylsulfate was considered not to be a genotoxicant.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

none

Target gene:

The experiments were performed to detect any properties of the test material or its metabolites to induce gene mutations in histidine-requiring strains of Salmonella typhimurium.

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction

Test concentrations with justification for top dose:

25, 75, 225, 675 and 2025 µg/0.1 ml

Vehicle / solvent:

Dimethylsulfoxyde

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunorubicin-HCl for TA 98; 2-nitrofluorene for TA 1538

Remarks:

Without microsomal activation

Details on test system and experimental conditions:

Bacterial cultures were prepared from frozen stocks or from colonies on plates, and on the following days the Standard Plate Test was carried out with and without the addition of activation mixture
(rat liver microsomes and co-factors) .
The test was performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. The substance was dissolved in DMSO. DMSO alone was used for the negative controls. Each Petri dish contained:
1) approx. 20 ml of minimum agar (Difco agar noble, Difco Laboratories, Detroit, Michigan, U.S.A., Art.No.0142-01, plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth: Bacto Nutrient Broth dehydrated, Difco Laboratories, Detroit, Michigan, U.S.A., Art.No.0003 0.8 % plus 0.5 % NaCl) in 2.0 ml of soft agar. The soft agar was composed of:
100 ml of 0.6 % agar solution (Difco agar noble) with 0.6 % NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland, Art.No.14400) and +biotin 0.5 mM (Fluka, Buchs, Switzerland,
Art .No .53320). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 125 4 (Analabs, Inc., North Haven, Connecticut, U.S.A.,, No.RCS-088), 8 ymoles MgCl , 33 µmoles KCl, 5 µmoles glucose-6-phosphate, 4 µmoles NADP and 100 µmoles phosphate buffer, pH 7.4.
Positive control experiments were carried out simultaneously with the following substances: 1) for Strain TA 98: daunorubicin-HCl (DAÜNOBLASTIN , Farmitalia, Montedison Farmaceutica GmbH, Freiburg
i.Br., Germany), 5 and 10 µg/0.1 ml phosphate buffer; 2) for Strain TA 1538: 2-nitrofluorene (Fluka, Buchs, Switzerland, Art. No.73330), 5 and 10 µg/0.1 ml DMSO. The activation mixture was tested with Strain TA 15 35 and cyclophosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 250 µg/0.1 ml phosphate buffer.
In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In the positive control experiments two Petri dishes were used per strain and per group.

The plates were incubated for about 48 hours at 37 degree C in darkness.

Evaluation criteria:

The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration .

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

other: TA 98 and TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: Cytotoxicity was observed at the highest tested concentration of 2000 µg/0.1 ml

In the experiments performed without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 31 016/C revealed no marked differences.

In the experiments with microsomal activation, on the other hand, treatment with FAT 31 016/C led to an increase in the number of back-mutant colonies of Strains TA 98 and TA 1538. This effect was observed at the concentrations of 75 µg/0.1 ml and above. At the highest concentration there was again a reduction in the number of back-mutant colonies, due to a growth-inhibiting effect of the substance on the bacteria.

Conclusions:

FAT 31016/C was found to exert a mutagenic effect on strains TA 98 and TA 1538 in the presence of metabolic activation.

Executive summary:

FAT 31016/C was tested for mutagenic effects on histidine auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. In the experiments performed without microsomal activation, comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 31016/C revealed no marked deviations. In the experiments in which activation mixture was added to the cultures, the number of back-mutant colonies of Strains TA 98 and TA 1538 was distinctly greater after treatment with FAT 31016/C than in the controls. Hence, it can be concluded that FAT 31016/C was found to exert a mutagenic effect on strains TA 98 and TA 1538 in the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

None

Qualifier:

no guideline available

Principles of method if other than guideline:

None

GLP compliance:

no

Type of assay:

other: Microbial Mutagenecity

Specific details on test material used for the study:

None

Target gene:

None

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Induced rat liver enzymes with Aroclor 1254

Test concentrations with justification for top dose:

0., 2.0, 20, 200, and 2000 µg (or nl) per petri dish.

Vehicle / solvent:

FAT 31016/C was diluted in sterile water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: N-methyl-N'-nitro_N_nitrosoguanidine at 1.6 microgr. for strains TA 1535 and TA 100 ; 9-aminoacridine at 50 microgr. for TA 1537 and daunomycine at 5 microgr for TA98

Remarks:

Without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine at 25 microgr. (TA 1535, TA 1537, TA 98 and TA 100)

Remarks:

With S9 mix

Details on test system and experimental conditions:

None

Evaluation criteria:

CRITERIA OF MUTAGENICITY:
The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Statistics:

No data

Key result

Species / strain:

other: S. Typhimurium TA 1535 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Mutagenic effect was observed at a concentration of 200 µg of FAT 31016/C per Petri dish tested in the presence of a liver microsomal enzyme preparation (S9-mix) from male rats pretreated with Aroclor 1254. In the absence of such an activation no mutagenic effect existed. A toxic effect was observed at 2000 µg with each strain in the absence of the liver microsomal enzyme preparation and in the presence of such an activation only a decrease of the number of revertants was noted.

Remarks on result:

other: mutagenic effect observed at 200 µg only

None

Conclusions:

FAT 31016/C was found to be mutagenic for S. typhimurium strain TA 98 without metabolic activation at the concentration of 200 µg only.

Executive summary:

FAT 31016/C was tested for mutagenicity in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of in vitro activation by microsomal enzymes from rat liver (Ames test). The doses used were 0.2, 2, 20, 200 and 2000 ml per petri dish. The test substance was diluted in sterile water and every concentration was tested in triplicate.

 

Positive controls were also used to check the efficacy of the test system. MNG, 9 -aminoacridine and daunomycine were used as positive control without S9. 2 -anthramine was used as positive control with S9.

 

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, product FAT 31016/C was found to be mutagenic for S. typhimurium strain TA 98 without metabolic activation at the concentration of 200 µg only. In the absence of such an activation no mutagenic effect existed. A toxic effect was observed at 2000 µg with each strain in the absence of the liver microsomal enzyme preparation and in the presence of such an activation only a decrease of the number of revertants was noted.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 26, 1985 to May 9, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

None

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

other: Mammalian cell point mutation test

Specific details on test material used for the study:

- Test substance: FAT 31 016/I
- Batch No: RZ 3068/37
- Purity: 94.3 %
- Validity: October 2012
- Stability: October 2012

Target gene:

hypoxanthine-guanine phosphoribosyl transferase gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal fraction S9

Test concentrations with justification for top dose:

Without microsomal activation: 0.075, 0.15, 0.30, 0.60, 1.20, 1.80, 2.40 and 3.00 µg/ml
with microsomal activation: 0.50, 1.00, 2.00, 4.00, 8.00, 12.00, 16.00 and 20.00 µg/ml.

Vehicle / solvent:

Distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Evaluation criteria:

A test substance is considered mutagenic in this test system, if the analysis of the data demonstrates either :
- a dose-dependent increase in the mutant frequency and an increase in the highest mutant frequency with respect to the negative control by a factor of at least 2.5; or
- an increase in any mutant frequency by a factor of 3.0 or more with respect to the negative control at any concentration tested and an absolute difference of at least 20 clones per 10 cells plated between the negative control and substance treated dishes.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Preliminary toxicity tests:

In the preliminary toxicity tests, a 90% reduction in the viability of the cells treated with FAT 31 016/I was obtained at a concentration between 1.2 µg/ml and 2.4 µg/ml in the experiment without microsomal activation and at a concentration between 9.8 µg/ml and 19.5 µg/ml in the experiment with microsomal activation. Due to the narrow concentration range of the slightly toxic and highly toxic concentrations, it was decided to perform the mutagenicity test without microsomal activation with concentrations of FAT 31 016/I ranging from 0.075 µg/ml to 3.00 µg/ml and the mutagenicity test with microsomal activation at concentrations of FAT 31 016/I ranging from 0.5 µg/ml to 20.0 µg/ml.

In the mutagenicity test without activation the mutant frequency values in the negative (medium) controls were lower than 4x10^-6 (the lower limit of the sensitivity of the test) either in those cultures screened with 8-azaguanine or in those screened with 6-thioguanine. The mutant frequencies of the treated cultures selected with 8 -AG were smaller than 4x10^-6 at all concentrations tested. The corresponding mutant factors were therefore 1.0. The positive control treated with 300 nl ethylmethanesulfonate / ml medium gave a mutant frequency of 1.15x10^-3 and a corresponding mutant factor of 287.9 . The cultures treated with FAT 31 016/I and screened with 6-TG gave mutant frequencies <4x10^-6 at 0.08 µg/ml, 0.30 µg/ml and 1.80 µg/ml (factor 1.0), 4.4x10^-6 at 0.60 µg/ml (factor 1.1), 5.0x10^-6 at 2.40 µg/ml (factor 1.3), 7.9x10^-6 at 1.20 µg/ml (factor 2.0) and 8.6x10^-6 at 3.00 µg/ml (factor 2.2) and 8.8x10^-6 at 0.15 µg/ml (factor 2.2). All these mutant frequencies are in the range of the historical negative control.

The mutant frequency in the microsomal-activated cultures was smaller than 4.0x10^-6 in the negative (medium) control when selected with 8-AG and was 5.5x10^-6 when selected with 6-TG. The metabolically activated cultures treated with FAT 31 016/I and selected with 8-AG showed mutant frequencies <4.0x10^-6at 0.5 µg/ml, 1.0 µg/ml, 2.0 µg/ml and 4.0 µg/ml resulting in mutant factors of 1.0 and showed a mutant frequency of 4.8x10^-6 at 8.0 ug/ml (factor 1.2). At 12.0 µg/ml, the mutant frequency was 6.1x10^-6 and the mutant factor was 1.5 . The two highest concentrations were too toxic and there were not enough cells to be subjected to the mutant selection procedure. The positive control treated with 1 µl dimethylnitrosamine /ml medium revealed a mutant frequency of 1.35x10^-4 , giving a mutant factor of 33.8 . The microsomal activated cultures selected with 6-TG gave mutant frequencies <4x10^-6 at 1.0 µg/ml, 2.0 µg/ml, 4.0 µg/ml and 16.0 µg/ml and a mutant frequency of 5.6x10^-6 at 0.5 µg/ml. In all these cultures the mutant factor was 1.0 . In the culture treated with 12.0 µg/ml FAT 31 016/I the mutant frequency was 14.3x10^-6 giving a mutant factor of 2.6 . The two highest concentrations were too toxic and there were not enough cells to be subjected to the mutant selection procedure. The mutant frequency of the concurrent positive control (1 µl DMN /ml medium) was increased to 2.47x10^-4 and the mutant factor was consequently enhanced to 44.8.

Conclusions:

FAT 31016/I was found to be not mutagenic in this point mutation assay.

Executive summary:

FAT 31 016/I was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The experiments were performed without microsomal activation at concentrations of 0.075, 0.15, 0.30, 0.60, 1.20, 1.80, 2.40 and 3.00 µg/ml and with microsomal activation at concentrations of 0.50, 1.00, 2.00, 4.00, 8.00, 12.00, 16.00 and 20.00 µg/ml.

In the experiment performed with and without microsomal activation, comparison of the number of mutant colonies in the controls and in the cultures treated with various concentratons of the

test substance revealed no significant deviation. Hence, it was concluded, that under the given experimental conditions FAT 31 016/I and its metabolites induced no mutagenic effects in this forward mutation system.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 14, 1998 to January 11,1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998, adopted July 21, 1997,

Deviations:

yes

Remarks:

this study was performed as a screening test in one experiment with a single preparation interval (18 h) and evaluation of the highest possible test article concentration.

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

EEC Directive 92/69, L 383 A, Annexe V, B 10, dated December 29, 1992.

Deviations:

yes

Remarks:

this study was performed as a screening test in one experiment with a single preparation interval (18 h) and evaluation of the highest possible test article concentration.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro chromosomal aberration assay

Specific details on test material used for the study:

- Test article name: FAT31016/Q
- Batch-No.: EN-Nr.: 0021469.D7
- Aggregate state at room temperature: solid
- Colour: red
- Content of active ingredient: about 95 %
- pH-value: pH 7 (1 g/l)
- Solubility in solvent: 40 g/l (at 20°C)
- Storage: at room temperature
- Expiration date: August 2002

Target gene:

V79 cell line of the Chinese hamster

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from the livers of 8 - 12 weeks old male rats,

Test concentrations with justification for top dose:

Pre-test: 26.56, 53.13, 106.25, 212.5, 425.0, 850.0, 1700.0 and 3400.0 µg/ml
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests.

Main test: Without S9 mix: 1.25, 2.5, 5.0, 10.0 and 20.0 µg/ml; With S9 mix: 1.875, 3.75, 7.5, 15.0 and 30.0 µg/ml
The top concentrations were chosen taking into account the results of the pre-test, where, in the absence of S9 mix and the presence of S9 mix reduced cell numbers below 50 % of the corresponding control were observed after treatment with the lowest applied concentration of 26.56 µg/ml.

Untreated negative controls:

yes

Remarks:

Concurrent solvent controls (MEM) were performed

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Evaluation criteria:

A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.

Statistical significance was confirmed by means of the Fischer's exact test (10) (p < 0.05). However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (10) (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Neither in the absence nor in the presence of S9 mix substantial reductions of the mitotic indices were observed (111.9 % and 96.1 % of control). Reduced cell numbers were observed in the absence of S9 mix after treatment with 5 ug/ml (55 % of control). However, evaluation of cultures after treatment with the next higher concentrations was not feasible, since no or less mitotic cells were found.

In the absence and presence of S9 mix, neither a significant nor a statistically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test article (0.5 %) were within the range of the solvent control values (0.5 % - 1.0 %) and within the range of our historical control data: 0.0 % - 4.0 %.

No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test article (4.7 % - 5.1 %) as compared to the rates of the solvent controls (2.5 % - 6.1 %). EMS (600 µg/ml) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

FAT 31016/Q did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

Executive summary:

The test article FAT 31016/Q, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. The following test concentrations were used in the main test:

Without metabolic activation: 1.25, 2.5, 5.0, 10.0 and 20.0 µg/ml; With metabolic activation: 1.875, 3.75, 7.5, 15.0 and 30.0 µg/ml

The exposure period was 4 h with and 18 h without metabolic activation. The chromosomes were prepared 18 h after start of treatment with the test article. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

Neither in the absence nor in the presence of S9 mix substantial reductions of the mitotic indices were observed (111.9 % and 96.1 % of control). Reduced cell numbers were observed in the absence of S9 mix after treatment with 5 µg/ml (55 % of control). However, evaluation of cultures after treatment with the next higher concentrations was not feasible, since no or less mitotic cells were found.

In the absence and presence of S9 mix, neither a significant nor a statistically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test article (0.5 %) were within the range of the solvent control values (0.5 % - 1.0 %) and within the range of our historical control data: 0.0 % - 4.0 %.

No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test article (4.7 % - 5.1 %) as compared to the rates of the solvent controls (2.5 % - 6.1 %).

EMS (600 µg/ml) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article FAT 31016/Q did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Studies evaluating genetic toxicity potential of Basic Red 046 Methylsulfate are not available. However, Basic Red 046 Bromide was evaluated for genetic toxicity potential in bacterial reverse mutation assays, a mammalian cell gene mutation assay and a chromosomal aberration assay, which is being used to fulfil the data requirements and complete the genetic toxicity assessment of Basic Red 046 Methylsulfate.

Bacterial reverse mutation assays

Arni, P. (1979)

FAT 31016/C (Basic Red 046 Bromide, purity 85 %) was tested for mutagenic effects on histidine auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. In the experiments performed without microsomal activation, comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 31016/C revealed no marked deviations. In the experiments in which activation mixture was added to the cultures, the number of back-mutant colonies of Strains TA 98 and TA 1538 was distinctly greater after treatment with FAT 31016/C than in the controls. Hence, it can be concluded that FAT 31016/C was found to exert a mutagenic effect on strains TA 98 and TA 1538 in the presence of metabolic activation.

Fouillet, X. (1979)

FAT 31016/C (BAasic Red 046 Bromide, purity: 85%) was tested for mutagenicity in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of in vitro activation by microsomal enzymes from rat liver (Ames test). The doses used were 0.2, 2, 20, 200 and 2000 ml per petri dish. The test substance was diluted in sterile water and every concentration was tested in triplicate. Positive controls were also used to check the efficacy of the test system. MNG, 9 -aminoacridine and daunomycine were used as positive control without S9. 2 -anthramine was used as positive control with S9. Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, product FAT 31016/C was found to be mutagenic for S. typhimurium strain TA 98 without metabolic activation at the concentration of 200 µg only. In the absence of such an activation no mutagenic effect existed. A toxic effect was observed at 2000 µg with each strain in the absence of the liver microsomal enzyme preparation and in the presence of such an activation only a decrease of the number of revertants was noted.

Mammalian cell gene mutation in vitro

FAT 31016/I (Basic Red 046 Bromide 0.075, 0.15, 0.30, 0.60, 1.20, 1.80, 2.40 and 3.00 µg/ml and with microsomal activation at concentrations of 0.50, 1.00, 2.00, 4.00, 8.00, 12.00, 16.00 and 20.00 µg/ml. In the experiment performed with and without microsomal activation, comparison of the number of mutant colonies in the controls and in the cultures treated with various concentratons of the test substance revealed no significant deviation. Hence, it was concluded, that under the given experimental conditions FAT 31 016/I and its metabolites induced no mutagenic effects in this forward mutation system.

Chromosomal aberration assay in vitro

The test article FAT 31016/Q (Basic Red 046 Bromide, purity: approx. 52%), dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. The following test concentrations were used in the main test: Without metabolic activation: 1.25, 2.5, 5.0, 10.0 and 20.0 µg/ml; With metabolic activation: 1.875, 3.75, 7.5, 15.0 and 30.0 µg/ml

The exposure period was 4 h with and 18 h without metabolic activation. The chromosomes were prepared 18 h after start of treatment with the test article. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. Neither in the absence nor in the presence of S9 mix substantial reductions of the mitotic indices were observed (111.9 % and 96.1 % of control). Reduced cell numbers were observed in the absence of S9 mix after treatment with 5 µg/ml (55 % of control). However, evaluation of cultures after treatment with the next higher concentrations was not feasible, since no or less mitotic cells were found. In the absence and presence of S9 mix, neither a significant nor a statistically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test article (0.5 %) were within the range of the solvent control values (0.5 % - 1.0 %) and within the range of our historical control data: 0.0 % - 4.0 %. No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test article (4.7 % - 5.1 %) as compared to the rates of the solvent controls (2.5 % - 6.1 %). EMS (600 µg/ml) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article FAT 31016/Q did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

The outcome of bacterial reverse mutation assays indicated that Basic Red 046 Bromide may possess potential to induce reverse mutations in bacterial cells. However, no mutagenic effect was observed in the mammalian cell gene mutation assay. Since, mutatgenic effect was absent in the mammalian cells, the substance was considered to be not mutagenic. Further, the substance was found to have no clastogenic potential in an in vitro chromosomal aberration assay. Hence, based on the outcome of the genetic toxicity studies in mammalian cells in vitro, Basic Red 046 Bromide as well as Basic Red 046 Methylsulfate were considered not to be genotoxic.

Justification for classification or non-classification

Based on the available information, Basic Red 046 Methylsulfate was considered not to be a genotoxicant, hence it does not warrant the classification for mutagenicity as per the Regulation EC No. 1272/2008 (CLP) criteria.