Reaction products of 1-(substitutedphenyl)urea coupled with diazotated potassium sodium substituted-5-{[2-(substituted)ethyl]sulfonyl}benzenesulfonate, further condensed with 2,4,6-trichloro-1,3,5-triazine, further converted with disubstituted benzene-1,4-disulfonic acid in aq. sodium hydroxide

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:

Experimental Starting Date: 29 September 2015 and Experimental Completion Date: 25 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP 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)

Deviations:

no

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

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 and hamster liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

- Initial toxicity-mutation assay (Experiment B1), the dose levels tested were: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate.
- In the confirmatory mutagenicity assay (experiment B2), the dose levels tested were: 50.0, 150, 500, 1500, 5000 μg per plate.

To achieve a solution, the most concentrated dilution was vortexed for 3 minutes and sonicated at 35.1 to 35.9ºC for 10 to 13 minutes in each assay. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Vehicle / solvent:

Water was used as the vehicle.

- Vehicle: Water
- CAS Number: 7732-18-5
- Supplier: Sigma-Aldrich
- Lot Number: RNBD5189/RNBD7780
- Purity: Sterile-filtered
- Expiration Date: November 2016/April 2017

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2-AA: Positive control used for test performed with metabolic activation S9 from rat liver (oxidative). Used at 1.0 μg/plate for TA98 and TA1535. Used at 2.0 μg/plate for starin TA100 and 1537. Used at 15 μg/plate with starin WP2 uvrA.

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

Positive control used for test performed with metabolic activation S9 from hamster liver (reductive).

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Positive control used at 1.0 ug/plate, without matebolic activation for strain TA98 .

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control used at 1.0 umg/plate without metabolic activation for strain TA100 and TA1535

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Posotive control used at 75 ug/plate without metabolic activation for strain TA1537.

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control used at 1000 ug/plate without metabolic activation for starin WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-incubation method.

CONDITIONS OF PRE-CULTURE:
Time of pre-culture: Approxymately 12 hours
Culture vessel (shape and volume): cylinder, 150 ml
Volume of culture medium: 30 to 50 mL
Amount of starin inoculated: 1 colony or around 100 uL


PRE-INCUBATION:
Temperature: 37°C (for without S9 and with oxidative S9)
30°C (for reductive S9)

Time: 60 minutes (for without S9 and with oxidative S9)
30 minutes (for reductive S9)

INCUBATION:
Temperature: 37°C
Time: 48 to 72 hours

NUMBER OF REPLICATIONS:
- Initial toxicity-mutation assay (experiment B1): 2 replicates
- In the confirmatory mutagenicity assay (experiment B2): 3 replicates

COLONY COUNTING METHOD: Automatic counting

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:

Not avaialble

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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 3

Key result

Species / strain:

S. typhimurium TA 98

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 4

Key result

Species / strain:

S. typhimurium 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 5

Key result

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

Additional information on results:

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:
Tester Strain
TA98 TA100 TA1535 TA1537 WP2 uvrA
Experiment Titer Value (x 109 cells per mL)
B1 2.1 1.4 1.4 1.8 1.7
B2 2.4 2.0 1.4 1.9 2.8


Initial Toxicity-Mutation Assay:
The results of the preliminary toxicity assay conducted at dose levels of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate in water.
The maximum dose of 5000 μg per plate was achieved using a concentration of 50.0 mg/mL and a 100 μL plating aliquot. Neither precipitate nor toxicity was observed.

Confirmatory Mutagenicity Assay
Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 50.0, 150, 500, 1500, 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.


CONCLUSION
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 40871/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.

Remarks on result:

other: Neither precipitate nor toxicity was observed

Any other information on results incl. tables

Historical Negative and Positive Control Values 2014 Revertants per plate

		Activation
		None					Rat Liver
Strain	Control	Mean	SD	Min	Max	95% CL	Mean	SD	Min	Max	95% CL
TA98	Neg	16	5	5	42	6 -26	24	7	5	53	10 -38
TA98	Pos	232	258	57	2691		400	165	109	1382
TA100	Neg	94	14	66	152	66 -122	102	18	63	164	66 -138
TA100	Pos	681	176	213	1767		681	259	186	2793
TA1535	Neg	11	4	2	31	3 -19	13	5	2	36	3 -23
TA1535	Pos	586	226	16	2509		117	99	23	1060
TA1537	Neg	7	3	1	19	1 -13	9	4	1	23	1 -17
TA1537	Pos	411	355	32	2921		72	52	10	562
WP2 uvrA	Neg	25	7 7	62		11 -39	28	8	10	55	12 -44
WP2 uvrA	Pos	376	123	99	1026		302	102	91	687
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Historical Negative and Positive Control Values Hamster Liver S9 (2011-2014) revertants per plate

ACTIVATION/HAMSTER LIVER

Strain	Control	Mean	SD	Min	Max	95% CL
TA98	Neg	32	8	13	47	16 -48
TA98	Pos	304	389	93	1711
TA100	Neg	94	14	72	127	66 -122
TA100	Pos	1498	868	597	2727
TA1535	Neg	14	5	6	27	4 -24
TA1535	Pos	398	107	269	589
TA1537	Neg	14	4	5	19	6 -22
TA1537	Pos	487	123	328	743
WP2 uvrA	Neg	25	9	10	42	7 -43
WP2 uvrA	Pos	301	34	267	345
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Applicant's summary and conclusion

Conclusions:

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 40871/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.

Executive summary:

The test substance, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli 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). Water was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 50.0, 150, 500, 1500, 5000 μg per plate. Neither precipitate nor toxicity was observed. 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.

These results indicate wthat the test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in either the absence of S9 activation or in the presence of oxidative or reductive S9 activation.