Potassium sodium amino-hydroxy-[(3-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]-[(2-sulfonato-4-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-disulfonate

Genetic toxicity: in vitro

Some information in this page has been claimed confidential.

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:

Study initiation date: 17 November 2014; Experimental starting date: 18 November 2014; Experimental completion date: 25 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

See section "Deviation" in the field "Any other information on results incl. tables"

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Details on test material:

None

Specific details on test material used for the study:

None

Method

Target gene:

Induction of reverse mutations at selected loci of several strains of Salmonella typhimurium (histidine) and at the tryptophan locus of Escherichia coli strain WP2 uvrA without S9 activation, with Aroclor 1254-induced rat liver S9 activation (oxidative) and with uninduced hamster liver S9 activation (reductive).

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 activation (oxidative) and with uninduced hamster liver S9 activation (reductive).

Test concentrations with justification for top dose:

- Preliminary toxicity assay: 6,7 / 10 / 33 / 67 / 100 / 333 / 667 / 1000 / 3333 / 5000 microgr. per plate.
- Mutagenicity assay: 300 / 600 / 1000 / 3000 / 5000 microgr. per plate.
- Retest of the mutagenicity assay: 300 / 600 / 1000 / 3000 / 5000 microgr. per plate.

Vehicle / solvent:

- Vehicle: Sterile water
- CAS number: 7732-18-5
- Supplier: Mediatech, Inc.
- Lot: 25055624 and 25055615
- Purity grade: Water for injection quality
Expiration date: June 2017 and May 2017

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

steril water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA1535, TA100, TA1537 and WP2uvrA (with rat S9 metabolic activation).

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

steril water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

methylmethanesulfonate

other: Sodium azide

Remarks:

without metabolic activation.

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

steril water

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

TA 98 with Hamster S9 metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: The test system was exposed to the test substance via the preincubation methodology described by Yahagi et al. (1977), and further modified for reductive activation conditions by Prival and Mitchell (1982).

DURATION
- Preincubation period: 60±2 minutes at 37±2°C (without S9 and with oxidative S9) or for 30±2 minutes at 30±2°C (reductive S9).
- Exposure duration: 48 to 72 hours at 37°C +/-2°C

NUMBER OF REPLICATIONS: All dose levels of test substance, vehicle control and positive controls were plated in triplicate.

DETERMINATION OF CYTOTOXICITY
- The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in "Any other information on materials and methods inlc. tables" field. As appropriate, colonies were enumerated either by hand or by machine.

Evaluation criteria:

Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
- Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

The primary computer or electronic systems used for the collection of data or analysis included but were not limited to the following:

- LIMS Labware System: Test Substance Tracking
- Excel 2007 (Microsoft Corporation): Calculations
- Sorcerer Colony Counter and Ames Study Manager (Perceptive Instruments): Data Collection/Table Creation
- Kaye Lab Watch Monitoring system (Kaye GE): Environmental Monitoring
- BRIQS: Deviation and audit reporting

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Solubility Test

Water was selected as the solvent of choice based on information provided by the Sponsor, the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water for injection-quality, cell culture grade water at a concentration of approximately 50 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Tester strain Titer results:

Experiment	Tester strain
	TA98	TA100	TA1535	TA1537	WP2 uvrA
	Titer value (X10E9 cells per ml)
B1	4.2	3.8	4.1	7.7	8.0
B2	5.3	1.8	3.1	3.7	8.2

Preliminary Toxicity Assay

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Increases in revertant counts (3.0- and 3.1-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. Neither precipitate nor background lawn toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

Mutagenicity Assay

In Experiment B1 (Mutagenicity Assay), positive mutagenic responses (2.0- and 2.6-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. No positive mutagenic responses were observed with the remaining test conditions. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed; however, reductions in revertant counts were observed beginning at 3000 or at 5000 μg per plate with a few test date provided by the supplier, it was more than six months beyond its preparation date as specified in the study protocol. Therefore, the reductive metabolic activation condition was retested in Experiment B2.

In Experiment B2 (Retest of the Mutagenicity Assay), a positive mutagenic response (2.5-fold maximum increase) was observed with tester strain WP2 uvrA in the presence of reductive S9 activation. No positive mutagenic responses were observed with the remaining test conditions in the presence of reductive S9 activation. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

Deviation:

The lot of uninduced hamster liver (reductive) S9 homogenate used in the mutagenicity assay was not within six months of its preparation date at the time of use. The reductive metabolic activation condition was retested using uninduced hamster liver S9 homogenate that was within six months of its preparation date. Therefore, the Study Director has concluded that this deviation had no adverse impact on the integrity of the data or the validity of the study conclusion.

Applicant's summary and conclusion

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, FAT 40868/A TE did cause positive mutagenic responses with tester strain WP2 uvrA in the presence of both oxidative and reductive S9 activation.

Executive summary:

The test substance, FAT 40868/A TE, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the absence of S9 activation and in the presence of both Aroclor-induced rat liver S9 activation (oxidative) and uninduced hamster liver S9 activation (reductive). The assay was performed in two phases, using the preincubation method. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test substance. Water was selected as the solvent of choice based on information provided by the Sponsor, the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water for injection-quality, cell culture grade water at a concentration of approximately 50 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Increases in revertant counts (3.0- and 3.1-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. Neither precipitate nor background lawn toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, positive mutagenic responses (2.0- and 2.6-fold maximum increases) were observed with tester strain WP2 uvrA in the presence of oxidative and reductive S9 activation, respectively. No positive mutagenic responses were observed with the remaining test conditions. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed; however, reductions in revertant counts were observed beginning at 3000 or at 5000 μg per plate with a few test conditions. Although the reductive S9 used in the mutagenicity assay was within the expiration date provided by the supplier, it was more than six months beyond its preparation date as required by the study protocol. Therefore, the reductive metabolic activation condition was retested.

In the retest of the mutagenicity assay, a positive mutagenic response (2.5-fold maximum increase) was observed with tester strain WP2 uvrA in the presence of reductive S9 activation. No positive mutagenic responses were observed with the remaining test conditions in the presence of reductive S9 activation. The dose levels tested were 300, 600, 1000, 3000 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

Under the conditions of this study, FAT 40868/A TE was concluded to be positive with tester strain WP2 uvrA in the presence of both oxidative and reductive S9 activation in the Bacterial Reverse Mutation Assay.