[[4-[[4-(anilino)phenyl][4-(phenylimino)-2,5-cyclohexadien-1-ylidene]methyl]phenyl]amino]benzenesulphonic acid

Genetic toxicity: in vitro

Administrative data

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:

from November 6, 2003 to December 12, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Alkali Blue Dry 6100

IUPAC Name:

Alkali Blue Dry 6100

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

name: Alkali Blue Dry 6100/Lot 2236308
Test substance no. 03/0437-3
Batch no.: Lot 2236308
CAS no. 1324-76-1
date of filling: September 18, 2003
Purity/composition: 88% main component
appearance, consisitency: blue powder
storage: room temperature

Method

Target gene:

salmonella typhimurium and Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

from rat livers induced by Aroclor 1254

Test concentrations with justification for top dose:

1st experiment: salmonella strains and E. coli WP2 uvrA 0; 23; 115; 575; 2 875 and 5 750 µg/plate
2nd experiment: salmonella strains 0, 0.8, 4, 20, 100 and 500 µg/plate
3rd experiment: salmonella strains and E. coli WP2 uvrA : 0; 4; 20; 100; 500 and 1 500 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with S-9 mix: 2.5 µg/plate

Positive control substance:

other: 2-aminoanthracene(2-AA)

Remarks:

strains: TA 1535, TA 100, TA 1537, TA 98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with s-9 mix: 60 µg/plate

Positive control substance:

other: 2-aminoanthracene(2-AA)

Remarks:

strain: Escherichia coli WP2 uvrA

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

5 µg/plate without S-9 mix

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

strains: TA 1535, TA 100

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

10 µg/plate without S-9 mix

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

strain: TA 98

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

100 µg/plate without S-9 mix

Positive control substance:

9-aminoacridine

Remarks:

strain: TA 1537

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

5 µg/plate without S-9 mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

strain: E. coli WP2 uvrA

Details on test system and experimental conditions:

EXOGENOUS METABOLIC ACTIVATION
S-9 fraction
The S-9 fraction is prepared according to Ames.
At least 5 male Sprague-Dawley rats {200 - 300 g, Charles River Deutschland GmbH} 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 body weight 5 days before sacrifice.
During this time, the animals are housed in Makrolon cages in air-conditioned rooms in which central air conditioning guaranted a range of temperature of 20 - 24°C and a relative humidity of 30 - 70%. The day/night rhythm is 12 hours (light period from 6.00 -18.00 hours and dark period from 18.00 - 6.00 hours).
Standardized pelleted feed and tap water from bottles are available ad libitum.
5 days after administration, the rats are sacrificed, and the livers are prepared (alt preparation steps for obtaining the liver microsome enzymes are carried 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, then cut into small pieces and homogenized in three volumes of KCI solution. After centrifligation of the homogenate at 9 000 xg for 10 minutes at +4°C, 5 ml portions of the supernatant (so-called S-9 fraction) are 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:
MgCb 8mM
KCI 33 mM
Glucose-6-phosphatase 5 mM
NADP 4mM
Phosphate buffer (pH 7.4) 15 mM
The phosphate buffer (6) is prepared by mixing an Na2HP04 solution with an NaH2P04 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.
BACTERIA
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 Difco nutrient broth + 5 g NaCi/liter) and incubated in the shaking water bath at 37°C for about 10 -12 hours. As a rule, a germ density of > 10s 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 were obtained from KNOLL Aktiengesellschaft on October 30, 1989. The Escherichia coli strain was obtained from Merck on September 9, 1991.
Salmonella typhimurium
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his*) to histidine prototrophy (his+) is determined (2, 3, 4), The tester strains TA 1535,TA 1537,TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GO base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.
Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions . The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.
Checking out the tester strains
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid).
E. coli WP2 uvrA is checked for UV sensitivity.
Histidine and tryptophan auxotrophy is automatically checked in each experiment via the spontaneous rate.

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 丨east 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:

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 S-9 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 S-9 mix in all experiments and indicated in the tables.
Solubiiity
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 and TA 100

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

Test results 2

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

The substance was tested for mutagenicity in the Salmonella typhimurium I Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing system (S-9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA.
A bacteriotoxic effect (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed in the standard plate test using the Salmonella strains at doses > 575µg/plate. With E. coli WP2 uvrA only a slight decrease in the number of trp+ revertants was found at doses > 2 875 µg/plate.
In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants) was occasionally observed at about 1 500 µg/plate.
Test substance precipitation was found from about 100µg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the experimental conditions in this study, it can be concluded that test article is not a mutagenic agent in a bacterial reverse mutation test.

Executive summary:

This study was conducted to assess the mutagenic potetial of test article 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. Dose range was:0.8µg - 5 750µg/plate (SPT; Salmonella strains), 23.0µg -5 750µg/piate (SPT;E.coli WP2 uvrA) and 4.0µg -1 500µg/plate (PIT; all tester strains) A bacteriotoxic effect was observed under all test conditions. An increase in the number of his⁺or trp⁺revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.