Reaction mass of Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[[2-(sulfooxy)Vinyl]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)], trisodium salt and Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)]-, sodium and Cuprate(4-), [4,5-dihydro-4-[[8-hydroxy-7-[[2-hydroxy-5-methoxy-4-[2-(sulfooxy)Ethanol]phenyl]azo]-6-sulfo-2-naphthalenyl]azo]-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylato(6-)], trisodium salt

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:

supporting study

Study period:

From June 13 to July 7, 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Complete read across justification is attached in section 13. Source study has reliability 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

RA Substance 02

IUPAC Name:

RA Substance 02

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 µg/plate.
Main test: 312.5; 625; 1250; 2500; and 5000.0 µg/plate. The highest dose level was 5000 µg/plate, chosen as test substance was freely-soluble and non toxic in the preliminary test. All concentrations were expressed as active ingredient, taking into account a purity of 94 %.

Vehicle / solvent:

Water for injections (batch EVBO3A) was used as vehicle, based on the solubility properties of test substance.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1µg/plate: TA1535 and TA100 without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate: TA1537 without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

0.5 µg/plate: TA98 without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 µg/plate: TA102 without S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2 µg/plate: Escerichia coli WP2 uvra without S9

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: a-anthramine

Remarks:

2 µg/plate: TA1535, TA1537, TA98 and TA100 with S9

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

10 µg/plate: TA102 and E.C. WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- direct plate incorporation: preliminary test, both experiments without S9 mix, first experiment with S9 mix. This method was applied as follows: test item solution (0.1 ml), S9 mix when required or phosphate buffer pH 7.4 (0.5 ml) and bacterial suspension (0.1 ml) were mixed with 2 ml of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45 °C). After rapid homogenisation, the mixture was overlaid onto a Petri plate containing minimum medium.
- preincubation method: second experiment with S9 mix. This method was applied as follows: test item solution (0.1 ml), S9 mix (0.5 ml) and the bacterial suspension (0.1 ml) were incubated for 60 minutes at 37 °C under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate.

After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter. Perceptive Instruments, Suffolk CB9 7 BN, UK).

NUMBER OF REPLICATIONS:
- preliminary test was performed with 6 doses with and without S9 mix, 1 plate/dose level.
- main assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

DETERMINATION OF CYTOTOXICITY: toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

Evaluation criteria:

Acceptance criteria
This study is considered valid if the following criteria are fully met: the number of revertants in the vehicle controls is consistent with the historical data of the
testing facility; the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with historical data.

Evaluation criteria
A reproducible 2-fold increase (for TA 98, TA 100, TA 102 and WP2 uvrA strains) or 3-fold increase (for TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

Preliminary toxicity test

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. A yellow coloration was noted in the plates when scoring the revertants at dose-levels > 500 µg/plate. No noteworthy toxicity was noted towards the four strains used, with and without S9 mix.

Mutagenicity experiments

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six strains.

Applicant's summary and conclusion

Conclusions:

Under experimental conditions reported, test substance did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

Executive summary:

Method

The substance was tested in water for injections. A preliminary toxicity test was performed to define the dose-levels of test substance to be used for the mutagenicity study. This was carried out in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37 °C).

Five strains of bacteria Salmonella typhimurium, i.e. TA 1535, TA 1537, TA98, TA100 and TA 102, and one strain of Escherichia coli, i.e. WP2 uvrA, were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level).

After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Results

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the intemational guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9 mix.

No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six strains.