Hydrogen bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From September 29 to October 08, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01 - AY059

IUPAC Name:

Similar Substance 01 - AY059

Method

Target gene:

histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity ( uvrB); ampicillin resistance (R factor plasmid).
E. coli WP2 uvrA is checked for UV sensitivity.
Histidine and tryptophan auxotrophy is checked in each experiment via the spontaneous rate.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat/hamster liver S9 mix

Test concentrations with justification for top dose:

1st Experiment
Doses: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
2nd Experiment
Doses: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Choice of the vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With rat liver S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: 2-aminoanthracene; benzidine

Remarks:

With hamster liver S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

sovent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine;

Remarks:

Without S9 mix

Details on test system and experimental conditions:

TEST SYSTEM
For testing, deep-frozen (-70 °C to -80 °C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) are thawed at room temperature, and 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g/l Difco nutrient broth + 5 g/l NaCl) and incubated in the shaking water bath at 37 °C for about 12 - 16 hours. As a rule, a germ density of ≥ 108 bacteria/ml is reached. These cultures
grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strains mentioned is in accordance with the current scientific recommendations for the conduct of this assay.
The Salmonella strains TA 98, TA 100 and TA 1537 were obtained from KNOLL Aktiengesellschaft, Ludwigshafen, Germany, on 30 Oct 1989. The Salmonella strain TA 1535 and the Escherichia coli strain were obtained from Merck KGaA, Darmstadt, Germany (22 Apr 2010 and 09 Sep 1991, respectively).
TEST SUBSTANCE PREPARATION
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethylsulfoxide (DMSO). To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before administration
ANALYSIS OF TEST SUBSTANCE PREPARATION
The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was verified analytically. The analyses were carried out as a separate study at the test facility Competence Center Analytics, BASF SE, Ludwigshafen, Germany. The study was carried out in compliance with the Principles of Good Laboratory Practice.

EXPERIMENTAL PROCEDURE
Mutagenicity tests
Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al.
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 ml agar (0.8 % [w/v] agar + 0.6 % [w/v] NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle (negative control)
0.1 ml fresh bacterial culture
0.5 ml rat S9 mix (with metabolic activation)
or
0.5 ml phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml purified water
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Escherichia coli
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8 % [w/v] agar + 0.6 % [w/v] NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle (negative control)
0.1 ml fresh bacterial culture
0.5 ml rat S9 mix (with metabolic activation)
or
0.5 ml phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J., with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 ml solution B (agar)
100 ml solution A (saline solution)
8 ml solution C (glucose solution)
10 ml solution D (casein solution)
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

Prival Preincubation Test
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al. and has been modified further to include reductive conditions by Prival et al.
0.1 ml test solution or vehicle (negative control), 0.1 ml bacterial suspension and 0.5 ml hamster S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 30 °C for 30 minutes using a shaker. Subsequently, 2 ml soft agar which consists of 100 ml agar (0.8 % [w/v] agar + 0.6 % [w/v] NaCl in purified water) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants:
0.5 mM histidine + 0.5 mM biotin or 0.5 mM tryptophan) is added. After mixing, the samples are poured onto the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Titer determination
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments. In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. Test tubes containing 2-ml portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C,
and the remaining components are added in the following order:
0.1 ml vehicle (without and with test substance)
0.1 ml fresh bacterial culture (dilution: 10-6)
0.5 ml rat S9 mix
In the Prival preincubation test, 0.1 ml of the overnight cultures is diluted to 10-6 in each case. 0.1 ml vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL hamster S9 mix are incubated at 37 °C for 30 minutes using a shaker. Subsequently, 2 ml of soft agar containing maximal amino acid solution for titer determination (5 mM histidine + 0.5 mM biotin or 5 mM tryptophan) is added.
After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

EVALUATION
Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.

Titer
The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Toxicity
Toxicity detected by a:
• decrease in the number of revertants;
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer
is recorded for all test groups both with and without S9 mix in all experiments and indicated in the table.

Solubility
Precipitation of the test material is recorded and indicated in the tables. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 333 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 333 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prival Incubation test
- No increase in the number of his+ or trp+ revertants.
- bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants, slight reduction in the titer) was observed depending on the strain and test conditions from about 1 000 μg/plate onward.

SOLUBILITY
Test substance precipitation was found from 1 000 μg/plate onward with and without S9 mix.

Applicant's summary and conclusion

Conclusions:

Not genotoxic

Executive summary:

Method

The test substance was tested for its mutagenic potential, according to the OECD guideline 471, based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival preincubation test). The modified Bacterial Reverse Mutation Test according to Prival facilitates azo reduction and is therefore the most approriate method for the investigation of azo-dyes and diazo compounds.

Observation

The test substance did not lead to an increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (Ames standard plate test and Prival preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Conclusion

Thus, under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic substance in the bacterial reverse mutation test (Ames standard plate test and Prival preincubation assay) in the absence and the presence of metabolic activation.