(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 8, 1990 to August 20, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Tested on Salmonella typhimurium strains: TA 1535, TA 1537, TA 98 and TA 100

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

EC Number:

407-240-9

EC Name:

(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

Cas Number:

159604-94-1

Molecular formula:

C66H88Cu2N20O10S2.C3H5O3.C2H3O2

IUPAC Name:

7,7'-bis[4-(3-diethylaminopropylamino)-6-(3-diethylammoniopropylamino)-1,3,5-triazin-2-ylamino]-{μ-4,4'-dihydroxy-1:2k2O4:O4'-3,3'-[3,3'-dihydroxy-1:2k2O3:O3'-biphenyl-bisazo-1:2(N3,N4-η:N3',N4'-η)]dinaphthalene-2-sulphonato(6-)}dicuprate(2-), mixed (1:1) acetic/lactic acid salts

Test material form:

solid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: CCR
- method of preparation of S9 mix: The S9 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male Wistar rats, strain WU (SAVO—Ivanovas, med. Versuchstierzuchten GmbH, D-7964 Kisslegg, F . R. G. ; weight ca. 150 - 200 g) which received a single i . p. injection of 500 mg/kg b. w. Aroclor 1254 (Antechnika, D—7500 Karlsruhe, F. R. G. ) in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCI and homogenised . The homogenate, diluted 1 : 3 in KCI was centrifuged cold at 9.000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at —70°C. Small numbers of the ampoules are kept at —20°C for only several weeks before use. The standardization of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-8000 München: Bio—Rad protein assay, Catalogue 500 000 6 ( 6 ).
The protein concentration in the S9 preparation was 24.7 mg/ml (lot 171089) and 30.3 mg/ml (lot 191289).

- concentration or volume of S9 mix and S9 in the final culture medium:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4

Test concentrations with justification for top dose:

10.0, 100.0, 333.3, 1000.0, 5000.0 µg/plate experiment 1
10.0, 100.0, 333.3, 1000.0, 2500.0, 5000.0 µg/plate experiment 2
The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 ans TA 100, respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua bidest

- Justification for choice of solvent/vehicle: because of its solubility properties.

Controls

Controls 1

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation).

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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:

S. typhimurium TA 1537

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 3

Species / strain:

S. typhimurium TA 98

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 4

Species / strain:

S. typhimurium 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 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Any other information on results incl. tables

Toxic effects, evidenced by a reduction in the number of revertants, occurred in nearly all strains with and without metabolic activation in both experiments .

The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant nurnber exists. The presence of liver microsomal activation did not influence these findings .

A slight increase in the revertant colonie numbers was observed in strain TA 1535 in experiment II with S9 mix at 100.0 µg/plate. This effect is considered being irrelevant as it could not be reproduced in the independent experiment. In strain TA 100 in experiment I in the presence of S9 mix a concentration-dependent enhancement was observed from 10.0 µg/plate to 1000.0 µg/plate.

At 5000.0 µg/plate the revertant number of revertant colonies decreased due to toxic effects of the test article. According to these results an additional concentration (2500.0 µg/plate) was tested in all strains in experiment Il. According to the results in experiment II the observed effects in strain TA 100 in experiment I are considered not to be relevant. They could not be reproduced.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:

The test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

The 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. The test article was tested at the following concentrations:

Exp. I:

10.0; 100.0; 333.3; 1000.0; and 5000.0; µg/plate.

Exp. II:

10.0; 100.0; 333.3; 1000.0; 2500.0; and 5000.0; µg/plate,

Toxic effects, evidenced by a reduction in the number of revertants, occurred in nearly all strains with and without metabolic activation in both experiments.

The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.