Disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Blue 317 was assessed in three genetic toxicity studies for mutagenic potential.

Bacterial reverse mutation assay:

In the bacterial reverse mutation assay conducted according to OECD Guideline 471 and EU Method B.14 in compliance with GLP, Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were exposed to FAT 20011/D both with and without metabolic activation upto the concentration of 5000 µg/plate, which led to a very strong increase in the number of back-mutants with strains TA 98, TA 100 and TA 1537. The metabolites of FAT 20011/D led to weak increased revenant counts with strain TA 1535.

In vitro mammalian cell gene mutation assay:

In the mammalian cell gene mutation assay (HPRT locus) conducted according to OECD Guideline 476, in compliance with GLP, CHO cells cultured in vitro were exposed to the substance dissolved in culture medium at various concentrations. In the original and confirmatory experiments, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant increase of the mutant frequencies as determined by the screening with 6-TG.

In vitro mammalian chromosomal aberration assay:

In the mammalian chromosomal aberration assay conducted according to OECD guideline no. 473 and in accordance with GLP, Acid Blue 317 was considered to not induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro.

In summary, Acid Blue 317 was tested positive in the Ames test, but was negative in the mutation assay in mammalian cells. This positive effect in the bacterial mutation assay may have been a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells. It is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays. The nitro-reductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that can metabolize nitroaromatic and nitro-heterocyclic derivatives (nitro-substituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitro-reductase proteins play a central role in the activation of nitro-compounds [de Oliveira et al. 2010]. That the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect has demonstrated in the past for two compounds pharmaceutical compounds AMP397 and fexinidazole as well as for several dye chemicals harvesting one or more nitro-groups within their chemical structure.

 

In conclusion, based on the above stated mechanism between the reduction of these nitro-compounds, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effect observed in the Ames test with Acid Blue 317 is a bacteria specific effect and not relevant to mammalians. This fact is further supported with the negative outcome in the in vitro mammalian cell gene mutation assay and the in vitro chromosomal aberration assay, hence Acid Blue 317 can be considered to be not genotoxic in the mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: FAT 20011/E TE
Purity: 65 %
Molecular Weight: 791.55 g/mol
Lot No.: 1309023
Expiration Date: 30 September 2018
Description: Dark violet powder (BioReliance) Black powder (Sponsor)
Storage Conditions: Room Temperature, protected from light
Receipt Date: 02 March 2015

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10 % fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air) for 16-24 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Cytotoxicity (≥50% reduction in cell growth index relative to the vehicle control) was observed at dose levels 20, 200, 500, and 2000 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥60 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥600 μg/mL in the non-activated 20-hour exposure group.

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

For non-activated studies

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

For S9-activated studies

Details on test system and experimental conditions:

Chromosome Aberration Assays:
Seven to nine dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was based on visible precipitation of the test substance in treatment medium at the conclusion.

Treatment of Target Cells (Preliminary Toxicity Test and Chromosome Aberration Assays):
The pH of the highest dose level of dosing solution in the treatment medium was measured using pH meter. Treatment was carried out by re-feeding the cultures with 4.5 mL complete medium for the non-activated exposure or 4.5 mL S9 mix (3.5 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 500 μL of test substance dosing solution or vehicle alone. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the
S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.

After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium were aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium, and returned to the incubator under standard conditions. For the non-activated 20 hour treatment group, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts.
For the definitive assay only, two hours prior to cell harvest, Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL.

Collection of Metaphase Cells (Preliminary Toxicity Test and Chromosome Aberration Assayd)
For the preliminary toxicity test and chromosome aberration assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.
The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data was presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008). The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control (% cytotoxicity).

RICC (%) = 100 - {[(mean viable cells treated - mean viable cells baseline)/(mean viable cells solvent - mean viable cells baseline)]X100}

For the definitive assay only, cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2 to 8 °C. To prepare slides, the cells were collected by centrifugation and the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted, and identified by the BioReliance study number, dose level, treatment condition, harvest date, activation system, test phase, and replicate tube design.

Evaluation criteria:

The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤0.05), and
• the increase was concentration-related (p ≤0.05), and
• results were outside the 95 % control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Solubility Test:

Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a workable suspension in water at a maximum concentration of approximately 10 mg/mL.

Preliminary Toxicity Assay;

CHO cells were first exposed to nine dose levels of FAT 20011/E TE, ranging from 0.2 to 2000 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 metabolic activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The test substance formed workable suspensions in water at concentrations ≥ 0.06 mg/mL, while concentrations ≤ 0.02 mg/mL were soluble in water. Visible precipitate (the culture was opaque dark blue) was observed in treatment medium at the following dose levels:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 hr	≥200 µg/mL	≥200 µg/mL
	20 hr	≥200 µg/mL	≥200 µg/mL
S9-activated	4 hr	≥200 µg/mL	≥200 µg/mL

The osmolality in treatment medium was measured as follows:

Dose tested	Dose levels (µg/mL)	Osmolality (mmol/kg)
Vehicle	0	268
Highest soluble	60	270
Lowest precipitating	200	274
Highest	2000	292

The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%. The pH of the highest dose level of test substance in treatment medium was 7.46.

Cytotoxicity (50% reduction in cell growth index relative to the vehicle control) was observed at dose levels 20, 200, 500, and 2000 µg/mL in the non-activated 4-hour exposure group, at dose levels ≥60 µg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥600 µg/mL in the non-activated 20-hour exposure group. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:

Treatment Condition	Treatment Time	Recovery Time	Dose level (µg/mL)
Non-activated	4 hr	16 hr	2.5, 5, 10, 25, 60, 75, 100, 200, 300
	20 hr	0 hr	5, 10, 25, 60, 75, 100, 200, 300
S9-activated	4 hr	16 hr	5, 10, 25, 60, 75, 100, 200

Initial chromosome abberation assay:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 hr	≥ 75 µg/mL	≥ 75 µg/mL
	20 hr	≥ 75 µg/mL	≥ 75 µg/mL
S9-activated	4 hr	≥ 75 µg/mL	≥ 75 µg/mL

Repeat chromosome abberation assay:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
S9-activated	4 hr	≥ 55 µg/mL	≥ 55 µg/mL

Conclusions:

FAT 20011/E TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

FAT 20011/E TE was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473.

A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dose formulations were adjusted for the purity of the test substance (65 %), using a correction factor of 1.54.

Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a workable suspension in water at a maximum concentration of approximately 10 mg/mL. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL. Cytotoxicity (≥50 % reduction in cell growth index relative to the vehicle control) was observed at dose levels 20, 200, 500, and 2000 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 60 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥600 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 2.5 to 300 μg/mL for the non-activated 4-hour exposure group, from 5 to 200 μg/mL for the S9-activated 4-hour exposure group, and from 5 to 300 μg/mL for the non-activated 20-hour exposure group.

In the initial chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Cytotoxicity was observed at 200 μg/mL in the S9-activated 4-hour exposure group and at dose levels ≥ 200 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥75 μg/mL in all three treatment groups. The highest dose analyzed under each treatment condition exceeded the limit of solubility in treatment medium at the conclusion of the treatment period, which met the dose limit as recommended by testing guidelines for this assay.

The percentage of cells with structural or numerical aberrations in the non-activated 4 and 20-hour exposure groups was not significantly increased relative to vehicle control at any dose level (p >0.05, Fisher's Exact test).

The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (3.0 %) relative to vehicle control at 75 μg/mL (p ≤0.01, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was not significantly increased relative to vehicle control at any dose level (p >0.05, Fisher's Exact test).

In order to confirm the positive response observed, the chromosome aberration assay was repeated in the S9-activated 4-hour exposure group at dose levels ranging from 25 to 100 μg/mL. In the repeat assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥55 μg/mL. The highest dose analyzed exceeded the limit of solubility in treatment medium at the conclusion of the treatment period, which met the dose limit as recommended by testing guidelines for this assay.

In the repeat assay, the percentage of cells with structural aberrations was not significantly increased relative to vehicle control at any dose level (p >0.05, Fisher's Exact test). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (6.0 % and 5.7 %) relative to vehicle control at dose levels 25 and 50 μg/mL, respectively (p ≤0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the percentage of cells with numerical aberrations was within the historical control range of 0.0 % to 9.5 % and also within the 95 % control limit of historical data. Therefore, the statistically significant induction was considered to have no biological relevance.

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤0.01). Thus, all criteria for a valid study were met.

Conclusion

Based on the above findings, FAT 20011/E TE was considered to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA (May 20, 1987) Detection of gene mutations in somatic cells in culture. Environmental Protection Agency Health Effects Testing Guidelines 52 FR 19072 (Corr. 52 FR 26150, July 13, 1987); 798.5300

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, Annex (November 18, 1987) Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test. Official Journal of the European Communities, No L 133. Vol. 31, 61-63, May 30, 1988

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Test substance: FAT 20011/D (Lanacron Marine S-G roh feucht)
Batch No: 61
Purity: ca 52.5%
Storage conditions: Room temperature
Expiry date: 31 July 1999
Material submitted by: CIBA-GEIGY Limited, Dyestuffs Division Basle, Switzerland.

Target gene:

Hypoxanthine-guaninephosphoribosyl-transferase (HPRT).

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 Chinese hamster cells were originally derived from embryonic lung tissue. The cells were cultured in Ham's F10 medium supplemented with 10 % pre-tested fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin in tissue culture (plastic) flasks. The humidity in the incubator was adjusted to >85 % rH, the air was enriched to 5 ± 2.0 Vol% C02 and the temperature was 37±1 °C. Twice per week the growth medium was replaced by fresh one.
The frozen cell suspension contains 10 % dimethyl sulfoxide (DMSO). All stock cells were cultured in cleansing medium for three days to purge the cultures of existing hprt" mutants. Cleansing medium was growth medium supplemented with 3 uM aminopterin. The cells have a stable karyotype with a modal chromosome number of 22 ± 1. All stock cells were checked for mycoplasma contamination, using the Hoechst- Dye staining method or the 6-MPDR method, before being frozen. Thawed stock culture cells are kept not longer than for twelve passages (three months) in culture.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondral supernatant (S9) fraction from male RAI rats.

Test concentrations with justification for top dose:

Cytotoxicity test:
With metabolic activation: 0.24, 0.49, 0.98, 1.95, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL
Without metabolic activation: 0.24, 0.49, 0.98, 1.95, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 µg/mL

Mutagenicity test (Original experiment):
With metabolic activation: 18.52, 55.56, 166.67 and 500 µg/mL
Without metabolic activation: 11.11, 33.33, 100 and 300 µg/mL

Confirmatory experiment:
With metabolic activation: 62.5, 125, 250 and 500 µg/mL
Without metabolic activation: 37.5, 75, 150 and 300 µg/mL

Vehicle / solvent:

FAT 20011/D was dissolved in DMSO at room temperature.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: N-Nitrosodimethylamine (DMN)

Details on test system and experimental conditions:

- Method of application: Dissolved in medium.
- Duration: 5 hours (short term exposure) or 21 hours (long-term exposure).
- Expression time: seven to eight days.
- Selection agent: 6-thioguanine was added to a final concentration of 8 µg/mL.
- Number of replications: 2 separate experiment (I+II) with 2 replicated cultures.
- Determination of cytotoxicity: Method relative growth.

Evaluation criteria:

Criteria for a positive response
The test substance will be considered to be mutagenic if:
• The assay is valid (see assay acceptance criteria)
• The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.
• There is a significant dose-relationship as indicated by the linear trend analysis.
• The effects described above are reproducible

Exceptions
By extreme cases or if the results only partially satisfy the above criteria the Study Director (if necessary after consulting the person who will approve the report) will interpret the results from his own experience. Positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

w/o S9:cytotoxicity revealed a mean value of 19.5% in the exp II, with and without S9 cytotoxicity was determined.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity
In the preliminary toxicity test with and without metabolic activation 12 concentrations of FAT 20011/D were tested. The concentrations selected ranged from 0.24 to 500 µg/mL and separated by 2-fold intervals. In the part with metabolic activation the highest concentration produced an acute growth inhibition of 40.2 %. In the part without metabolic activation FAT 20011/D exerted a growth inhibitory effect of 99.98 % at the concentration of 500 µg/mL. The next lower concentration revealed an acute growth inhibitory effect of 70.1 %.

Accordingly, four concentrations were selected for the original experiment ranging from 18.52 to 500.0 µg/mL and from 11.11 to 300 µg/ml in the presence and absence of metabolic activation, respectively.
In the part with metabolic activation, no toxicity was observed at any concentration. In the absence of metabolic activation, the mean growth inhibitory effect determined after treatment was 82% at the highest concentration. After the expression period the determined cytotoxicity revealed a mean value of 19.5 %.
In the confirmatory experiment the part with metabolic activation was run with concentrations ranging from 62.5 to 500 µg/mL the part without activation with concentrations ranging from 37.5 to 300 µg/ml. In the presence of metabolic activation, the mean acute cytotoxicity at the highest concentration was 33.7 %. In the part without activation, the mean growth inhibition at the highest concentration of 300 µg/ml was 69.3 %.

Mutagenicity:
In the presence and absence of metabolic activation, no significant increase in mutant frequency was observed at any concentration level of FAT 20011/D tested in the original or the confirmatory experiment in comparison with the negative control. The positive controls induced a clear increase in mutant frequency.

None

Conclusions:

FAT 20011/D and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

FAT 20011/D, was tested for mutagenic effects on V79 Chinese hamster cells in vitro. This test was evaluated based on OECD test guideline 476, EPA 52FR 19072 and EEC Directive 87/302, Annex (Nov 18 1987) Part B. The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

A preliminary range finding test was run for assessing cytotoxicity. FAT 20011/D was tested at concentrations up to 500 µg/ml. Higher concentrations could not be applied due to solubility limitations in the culture vehicle. In the part with metabolic activation, at the highest concentration of 500 µg/ml an acute growth inhibiting effect of 40.2 % could be seen. Without metabolic activation treatment with FAT 20011/D proved growth inhibiting by 99.98 % at the concentration of 500 µg/ml. The next lower concentration of 250 µg/ml revealed an acute inhibition of growth of 70.1%. Accordingly, 500 µg/ml with and 300 µg/ml without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500 µg/ml. No toxicity was found at any concentration. In the confirmatory experiment, the  concentrations applied were 62.5, 125, 250 and 500 µg/ml. The highest concentration revealed a mean acute growth inhibition of 33.7 %*. N-Nitrosodimethylamine (DMN, 1.0 µl/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 11.11, 33.33, 100 and 300 µg/mL with metabolic activation. The mean growth inhibition values found at the highest concentration after treatment and expression were 82 % and 19.5 % respectively. In the confirmatory experiment the concentrations applied were 37.5, 75, 150 and 300 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 69.3 %. The mean growth inhibition after the expression period was 24 %. Ethylmethansulfonate (EMS, 0.3 µl/ml) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant increase of the mutant frequencies as determined by the screening with 6-TG.

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 20011/D and its metabolites did not show any mutagenic activity in this forward mutation system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OFFICIAL JOURNAL OF THE EUROPEAN COMMUNITIES, No. L 383 A, Volume 35, 160-162, Annex to Commission Directive 92/69/EEC of July 31, 1992: Salmonella typhimurium - Reverse Mutation Assay, December 29, 1992.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA HEALTH EFFECTS TESTING, guideline 40 CFR 798, corrected at 52 FR 26150, § 798.5265, Salmonella typhimurium Reverse Mutation Assay, July 13, 1987.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test material: FAT 20011/D (Lanacron Marine S-G roh feucht)
Batch No.: 61
Purity: Approx. 52.5 %
Appearance: Black mass
Expiry date: July 1999
Storage: Room temperature
Material submitted by: CffiA-GEIGY Limited, Dyestuffs Division Basle, Switzerland.

Target gene:

Histidine auxotrophs

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:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

- Range finding test: 20.58, 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate.
- Original experiment: 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate.
- Confirmatory experiment: 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate.

Vehicle / solvent:

Dimethylsulfoxide (DMSO).
FAT 20011/D was dissolved in DMSO at room temperature. The test substance FAT 20011/D was soluble up to the concentration of 50 mg/ml. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with DMSO. No precipitates or aggregates were noted.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

other: 2-Aminoanthracene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Details on test system and experimental conditions:

Method of application: In agar

DURATION:
- Preincubation period for bacterial strains: overnight
- Exposure duration: 48 hours
- Expression time: Not applicable
- Selection time: Not applicable

NUMBER OF REPLICATIONS: Triplicate plating

DETERMINATION OF CYTOTOXICITY: Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance criteria:
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the study director.

Criteria for a positive response:
The substance will be considered to be positive in the test system if the following condition is met.
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the strains tested. Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Range finding test

Six concentrations of FAT 20011/D (Lanacron Marine S-G roh feucht) ranging from 20.6 to 5000 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed. The numbers of revertant colonies were increased at the upper concentrations. The highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.

 

Mutagenicity test, original experiment

In the experiments performed with and without metabolic activation, treatment of strains TA 98 and TA 1537 with FAT 20011/D led to a very strong, concentration dependent increase in the number of back-mutants at all concentrations. A similar effect occurred in the experiments with and without activation on strain TA 100 at the concentrations of 185.2 to 5000 µg/plate. In the experiment carried out with activation on strain TA 1535, marginal increased revertant counts were observed at the concentration of 5000 µg/plate. No effects occurred in the experiment without activation on this strain.

 

Mutagenicity test, confirmatory experiment

In the experiments performed with and without metabolic activation, treatment of strains TA 98 and TA 1537, led to a very strong, concentration-dependent increase in the number of back-mutants at all concentrations. This effect occurred on strain TA 100 in the experiment with activation at the concentrations of 555.6 to 5000 µg/plate and in the experiment without activation at the concentrations of 185.2 to 5000 µg/plate. In the experiment carried out with activation on strain TA 1535, marginal increased revertant counts were observed at the concentration of 5000 µg/plate. No effects occurred in the experiment without activation on this strain.

 

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Conclusions:

FAT 20011/D and its metabolites exerted a very strong mutagenic action on Salmonella typhimurium strains TA 98, TA 100 and TA 1537. The metabolites of the test material also were weakly mutagenic with strain TA 1535.

Executive summary:

FAT 20011/D, identified as black mass, purity approx. 52.5 %, was tested for mutagenic effects in-vitro in histidine-requiring strains of Salmonella typhimurium. This test was conducted in accordance to OECD TG 471 (GLP). The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in the range of 61.7 to 5000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

Mutagenicity test, original experiment

In the experiments performed with and without metabolic activation, treatment of strains TA 98 and TA 1537 with FAT 20011/D led to a very strong, concentration dependent increase in the number of back-mutants at all concentrations. A similar effect occurred in the experiments with and without activation on strain TA 100 at the concentrations of 185.2 to 5000 µg/plate. In the experiment carried out with activation on strain TA 1535, marginal increased revertant counts were observed at the concentration of 5000 µg/plate. No effects occurred in the experiment without activation on this strain.

 

Mutagenicity test, confirmatory experiment

In the experiments performed with and without metabolic activation, treatment of strains TA 98 and TA 1537 with FAT 20011/D again, led to a very strong, concentration-dependent increase in the number of back-mutants at all concentrations. This effect occurred on strain TA 100 in the experiment with activation at the concentrations of 555.6 to 5000 µg/plate and in the experiment without activation at the concentrations of 185.2 to 5000 µg/plate. In the experiment carried out with activation on strain TA 1535, again, marginal increased revertant counts were observed at the concentration of 5000 µg/plate. No effects occurred in the experiment without activation on this strain.

Based on the above information, in both experiments, performed with and without metabolic activation, FAT 20011/D and its metabolites led to a very strong increase in the number of back-mutants with strains TA 98, TA 100 and TA 1537. The metabolites of FAT 20011/D led to weak increased revenant counts with strain TA 1535.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

no data available

Justification for classification or non-classification

Acid Blue 317 is considered to be not genotoxic based on the available data from genetic toxicity studies, hence it does not need to be classified according to CLP regulation (Regulation EC No.1272/2008).