Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

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:

Start of experimental phase: 14 Oct 2016; End of experimental phase: 14 Oct 2016; Study completion: the signature date of Final Report

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

EC Number:

268-655-7

EC Name:

Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Cas Number:

68132-91-2

Molecular formula:

C38H20Cl2CuN14O18S5. H. 5Na C38H21Cl2CuN14Na5O18S5

IUPAC Name:

Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Test material form:

solid: particulate/powder

Method

Target gene:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.

Species / strain

Species / strain / cell type:

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

Remarks:

The E.coli used for this study was the strain WP2 uvrA.

Details on mammalian cell type (if applicable):

Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement: No Growth onMinimal plates+Biotin; Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement: No Growth onMinimal agar plates; Growth onMinimal plates+Tryptophan.
- uvrA, uvrB: Sensitivity to UV irradiation.
- rfa: Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from rats pre-treated with Phenobarbital and 5,6-Benzoflavone for plate-incorporation and S9 mix-Prival modification for pre-incubation.

Test concentrations with justification for top dose:

Preliminary toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate (based on solubility test).

Main Assay I:
- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate
- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate
-TA100 :+ S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

Main Assay II:
- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate
- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate
-TA100 :+ S9: 5000, 2500, 1250, 625, 313,156 and 78.1 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water for injection
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

methylmethanesulfonate

other: 2-aminoanthracene and Trypan Blue

Remarks:

Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Details on test system and experimental conditions:

The preliminary toxicity test and the first experiment were performed using a plate-incorporation method. The second experiment was performed using a pre-incubation method.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate (Chu et al. 1981); Regression line.

Results and discussion

Test results

Key result

Species / strain:

other: S.typhyimurium TA1535, TA1537, TA98 and TA100; E.coli WP2 uvrA

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 test item did not induce two-fold increases in the number of revertant colonies, in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Reactive Blue 160 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone (standard metabolic activation) in Main Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification), in Main Assay II. The test item was used as solution in sterile water for injection.

Toxicity test: The test item Reactive Blue 160 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. A slight reduction in revertant colonies was observed with TA100 tester strain at higher dose levels, both in the absence and presence of S9 metabolism. No relevant increases in revertant colonies were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

Main Assays: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate

-TA100 :+ S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

Slight toxicity was observed with TA100 tester strain at the highest dose level in the absence of S9 metabolism. No relevant increase in revertant numbers, was observed with any tester strain at any dose levels in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed. Based on the chemical structure of the test item (azo-dyes), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactor). The dose-range was slightly modified to take into account the results ofMain Assay I.

The test item was assayed at the following dose levels:

- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate

-TA100 :+ S9: 5000, 2500, 1250, 625, 313,156 and 78.1 µg/plate

Neither relevant toxicity nor precipitation of the test item was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism, in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.

Conclusion: It is concluded that the test item Reactive Blue 160 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.