Reaction mass of Tetrasodium 3-{[5-({4-[alkyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-6-fluoro- heteromonocycle-2-yl}amino)-2-sulfonatophenyl]diazenyl}-4-hydroxy-5-(alkanoylamino)naphthalene-2,7-disulfonate and Trisodium 3-[{5-[(4-{[3-(ethenylsulfonyl)phenyl](alkyl)amino}-6-fluoro-heteromonocycle-2-yl)amino]-2-sulfonatophenyl}diazenyl]-4-hydroxy-5-(alkanoylamino)naphthalene-2,7-disulfonate

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:

Experimental starting date: 10 April 2015 and Experimental Completion date: 05 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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:

Please see "Principles of method if other than guideline" field.

Principles of method if other than guideline:

The following deviation from the protocol occurred during the conduct of this study:
Event No. 209365: In the initial mutagenicity assay, the top dose formulation was to be prepared at a total volume of 41.0 mL. After adding an initial volume of vehicle to the top dose, the technician measured 31.0 mL of formulation. According to the test substance dilution scheme, only an additional 9.0 mL was added to the top formulation for a total of 40.0 mL. The technician indicated that a three-step dilution process was used, and an additional 1.0 mL was added, but the third step was not documented. The technician would not have had enough volume to dose this study if the volumes were used as documented on the test substance dilution scheme in the workbook. Because there was ample volume to dose the assay, this was determined to be a documentation error, and 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.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

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 uninduced hamster liver S9 activation (reductive)

Test concentrations with justification for top dose:

- Preliminary toxicity assay: 6.7/10.0/33.0/67.00/100/333/667/1000/333 and 5000 μg per plate.
- Initial mutagenicity assay: 15/50/150/500/1500 and 5000 μg per plate.
- Retest of the initial mutagenicity assay: 15/50/150/500/1500 and 5000 μg per plate.
- Confirmatory mutagenicity assay: 15/50/150/500/1500 and μg per plate.

Vehicle / solvent:

Water was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells.
The test substance formed a clear solution in sterile water at approximately 10 mg/mL and workable suspensions from approximately 25 to 50 mg/mL in the solubility test conducted at BioReliance.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

Diluted in DMSO

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9 activation (rat). 2-AA at 1.0 μg/plate for TA98 and TA1535. 2-AA at 2.0 μg/plate for TA100 and TA1537 and at 15.0 μg/plate for WP2 uvrA.

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

Diluted in DMSO

Positive control substance:

congo red

Remarks:

With S9 activation (hamster). Red Congo at 100 μg/plate for TA98.

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

Diluted in DMSO

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation (2- nitroflurene at 1.0 μg/plate for TA98).

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

Diluted in sterile water

Positive control substance:

sodium azide

Remarks:

Without metabolic activation (sodium azide at 1.0 μg/plate for TA100 and TA1535).

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

Diluted in DMSO

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation (9-aminoacridine at 75 μg/plate for TA1537).

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

Diluted in DMSO

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (methyl methansulfonate at 1000 μg/plate for WP2 uvrA).

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: 37 +/- 2°c for approxymately 12 hours
- Exposure duration: 48 to 72 hours at 37 +/- 2 °C


NUMBER OF REPLICATIONS:
- Preliminary toxicity assay: one plate per dose.
- Initial and confirmatory mutagenicity assay: In triplicate.

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

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:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Solubility Test

Water was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water at approximately 10 mg/mL and workable suspensions from approximately 25 to 50 mg/mL 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.

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 10.0 mg/mL and a 500 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the initial mutagenicity assay was 5000 μg per plate.Preliminary Toxicity Assay

Initial and Confirmatory Mutagenicity Assays

In Experiment B1 (Initial Mutagenicity Assay), the dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the absence of S9 activation or the presence of oxidative S9 activation, or with tester strains TA98, TA1535, TA1537 and WP2 uvrA in the presence of reductive S9 activation. Neither precipitate nor background lawn toxicity was observed. Due to an unacceptable vehicle control value, tester strain TA100 in the presence of reductive S9 activation was not evaluated for mutagenicity but was retested under identical conditions in Experiment B2. Since there is no positive control for TA100 in the presence of reductive S9 of S9 activation, as well as TA98 in the presence of reductive S9 activation.

In Experiment B2 (Retest of the Initial Mutagenicity Assay), no positive mutagenic response was observed with tester strain TA100 in the presence of reductive S9 activation. Neither precipitate nor toxicity was observed.

In Experiment B3 (Confirmatory Mutagenicity Assay), the dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the absence of S9 activation or in the presence of oxidative or reductive S9 activation. Neither precipitate nor toxicity was observed.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, FAT 40870/A TE did not cause a positive mutagenic response with any of the tester strains in either the absence of S9 activation or in the presence of oxidative or reductive S9 activation. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

The test substance, FAT 40870/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 three 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 and third phases, the initial and confirmatory mutagenicity assays, were used to evaluate and confirm the mutagenic potential of the test substance.

Water was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water at approximately 10 mg/mL and workable suspensions from approximately 25 to 50 mg/mL 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 10.0 mg/mL and a 500 μL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the initial mutagenicity assay was 5000 μg per plate.

In the initial mutagenicity assay, the dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the absence of S9 activation or the presence of oxidative S9 activation, or with tester strains TA98, TA1535, TA1537 and WP2 uvrA in the presence of reductive S9 activation. Neither precipitate nor background lawn toxicity was observed. Due to an unacceptable vehicle control value, tester strain TA100 in the presence of reductive S9 activation was not evaluated for mutagenicity but was retested under identical conditions. Since there is no positive control for TA100 in the presence of reductive S9 activation, vehicle and positive controls were dosed in the retest assay for TA100 in the absence of S9 activation, as well as TA98 in the presence of reductive S9 activation.

In the retest of the initial mutagenicity assay, no positive mutagenic response was observed with tester strain TA100 in the presence of reductive S9 activation.Neither precipitate nor toxicity was observed.

In the confirmatory mutagenicity assay, the dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the absence of S9 activation or in the presence of oxidative or reductive S9 activation. Neither precipitate nor toxicity was observed.

Under the conditions of this study, FAT 40870/A TE was concluded to be negative in the Bacterial Reverse Mutation Assay.