Reaction products of 6-hydroxy-5-nitrosonaphthalene-2-sulfonic acid, chelated with iron (3+), sodium salts.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

December 1992

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Green 001

IUPAC Name:

Acid Green 001

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Salmonella strains were obtained from KNOLL Aktiengesellschaft on October 30, 1989. Eschericha coli strains were obtained from Merck on September 9, 1991 A germ density of 10^9 bacteria/mI is reached.

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver metabolic fraction S9

Test concentrations with justification for top dose:

0, 26, 130, 650, 3250 and 6500 μg/plate.
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.

Vehicle / solvent:

- Vehicle used: water.
- Justification for choice of solvent/vehicle: good solubility of test substance in water.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2.5 μg/plate in DMSO for TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate in DMSO for Escherichia coli WP2 uvrA

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 4-nitro-o-phenylendiamine 10 μg/plate in DMSO for TA98; N-methyl-N'-nitro-N-nitrosoguanidine 5 μg/plate in DMSO for TA1535, TA100

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1st experiment: in agar (plate incorporation)
2nd experiment: preincubation

TISSUE PREPARATION
S-9 fraction: at least 5 male Sprague-Dawley rats (200 - 300 g) receive a single intraperitoneal injection of 500 mg Aroclor 1254 (as a solution in corn oil with a concentration of 20 g/100 ml) per kg b.w, 5 days before sacrifice. During this time, the animals are housed in Makrolon cages in air-conditioned rooms in which centrol air conditioning guaranteed a range of temperature of 20 - 24 °C and a relative humidity of 30-70 %. The daylnight rhythm is 12 hours (light period from 6.00-18.00 hrs and dark period from 18.00-6.00 hours). Standardized pelleted feed and tap water from bottles are availlable ad libitum. Five days after administration, the rats are sacrificed, and the Iivers are prepared (all preparation steps for obtaining the liver miorosome enzymes arecarried out using sterile solvents and glassware at a temperature of +4 °C). The livers are weighed and washed in an equivalent volume of a 150 mM KCI solution (1 ml for 1 g wet liver), then cut into small pieces and homogenized in three volumes of KCI solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4 °C, 5 ml portions of the supernatant (so-called S-9 fraction) are quickly deep-frozen in dry ice and stored at -70 °C to -80 °C.

S-9 mix: the S-9 mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S-9 fraction is thawed at room temperature and 1 volume of S-9 fraction is mixed with 9 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, is kept on ice until used. The concentrations of the cofactors in the S-9 mix are: MgCI2 8 mM, KCI 33 mM, Glucose-6-phosphatase 5 mM, NADP 4mM, Phosphate buffer (pH 7.4) 15 mM. The phosphate buffer is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4:1. To demonstrate the efficacy of the S-9 mix in this assay, the S-9 batch was characterized with benzo(a)pyrene.

MUTAGENICITY TESTS
METHOD OF APPLICATION: in agar (plate incorporation)
-Salmonella typhimurjum
Test tubes containing 2-mI portions of soft agar (overlay agar), which consists of 100 ml agar (0.6 % agar + 0.6 % NaCI) 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 45 °C, and the remaining components are added in the following order: 0.1 ml test solution or vehicle, 0.1 ml fresh bacterial culture, 0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests 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 aqua dest., 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 (histidine revertants) are counted.
- Escherichia coli
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.6 % agar + 0.6 % NaCI) 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 45 °C, and the remaining components are added in the following order: 0.1 ml test solution or vehicle, 0.1 ml fresh bacterial culture, 0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. The composition of the minimal agar (SAI 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 (tryptophane revertants) are counted.

METHOD OF APPLICATION: preincubation.
0.1 ml test solution or vehicle, 0.1 ml bacterial Suspension and 0.5 ml S-9 mix are incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, 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.

- Titer determination: 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 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 S-9 mix.
In the 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 S-9 mix are incubated at 37 °C for about 20 minutes using a shaker. Subsequently, 2 ml of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) 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.

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETECTION OF TOXICITY
Toxicity is detected by a:
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced histidine or tryptophan background growth)
- reduction in the titer

OTHER: 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 firist experiment (plate incorporation).

* Green, M.H.L.; Muriel, W.J. Mutagen testing using trp+ reversions in Escherichia coli. Mut. Res., 38 3 - 32 (1976)

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met: a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if: the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

Standard deviation for all dose groups, positive and negative controls.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in the number of his+ or trp+ revertants.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance precipitation was found.

COMPARISON WITH HISTORICAL CONTROL DATA: the comparison with historical control data showed that the number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain. The comparison also showed that the positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.

RESULTS OF STERILITY CONTROLS: the sterility controls revealed no indication of bacterial contamination.

INFORMATION ON TOXICITY: nto bacteriotoxic effect was observed.

Remarks on result:

other: plate incorporation and preincubation tests

Applicant's summary and conclusion

Conclusions:

The substance is non mutagenic both with and without metabolic activation.

Executive summary:

Method

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum and in tryptophan-requiring strains of Escherichia coli. The following strains were used: TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA.

The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) in a Standard Plate Test (SPT) and Preincubation Test (PIT). The compound was dissolved in water and tested at five concentrations in the range of 26 to 6500 μg/plate both in the presence and the absence of a metabolic activation system. 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. Parallel with each experiment, negative controls were carried out in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control)

Observations

In both experiments, performed with and without metabolic activation, none of the tested concentrations led to an increase in the incidence of histidine- or tryptophan- revertants. Furthermore, the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Conclusion

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium and Escherichia coli used.