Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

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:

26 April - 20 June, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: – ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, 2012

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

EC Number:

278-188-0

EC Name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

Cas Number:

75314-26-0

Molecular formula:

C14H13O4S . NH4 - C70H60O19S6 . 2NH4

IUPAC Name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

Constituent 2

Reference substance name:

Ammonium sulphate

EC Number:

231-984-1

EC Name:

Ammonium sulphate

Cas Number:

7783-20-2

Molecular formula:

H8N2O4S

IUPAC Name:

Ammonium tetraoxosulfate (VI)

Constituent 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

liquid: viscous

Details on test material:

ca 50% aquaous viscous solution
Batch No: INB3209089
Storage: At room temperature, protected from light
Expiring date: 2018-04-30

Specific details on test material used for the study:

Expiring date: 10 January 2018

Method

Target gene:

In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation S9 Mix; which is a cofactor-supplemented post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

The Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) concentrations investigated in the Initial and Confirmatory Mutation Tests:
5000, 1600, 500, 160, 50 and 16 µg/plate.

Vehicle / solvent:

In the preliminary Range Finding Test ultrapure water (ASTM Type 1) was found as appropriate vehicle for preparing the test item solutions. This vehicle is compatible with the survival of the bacteria and the S9 activity.

At the preparation of the test item stock solution a correction (multiplier) factor of 1.96 (1/0.511=1.96) based on the active component content was taken into consideration.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene, Dimethyl sulfoxide

Details on test system and experimental conditions:

The tester strains arrived to the test facility in a form of disc cultures. The origin of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;

Storage of Tester Strains

The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.

Storage of Tester Strains

The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

spontaneous Reversion of Tester Strains

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Procedure for Bacterial Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 11-13 hours in a 37oC Benchtop Incubator Shaker.

Viability and the Cell Count of the Testing Bacterial Cultures

The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Media

The Minimal Glucose Agar (MGA) Plates

Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates:
Supplier: VWR International;
Manufacturer: Merck Life Science GmbH, Germany.
Certificates of Analysis* were obtained from the supplier.
Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
* Batch No.: 142584; Expiry date: 22 May 2017; (used in the Informatory Toxicity Test)
143858; Expiry date: 16 August 2017; (used in the Initial and the Confirmatory Mutation Tests)

Nutrient Broth No. 2

Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Nutrient Agar

Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Top Agar for Salmonella typhimurium Strains

Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL

Top Agar for Escherichia coli Strain

Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth by (Section: 5.4.2) 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution by (Section: 5.4.4) 947.5 mL

Metabolic Activation System

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat Liver S9 Fraction

The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier1). The original Quality Control & Production Certificates of rat liver S9 are stored in the Laboratory of TOXI-COOP ZRT. The copies of the quality control certificates of the used S9 lots are given in Appendix VI. The following lots of the S9 were applied:
1) Lot Number: 3634; Expiry date: May 12, 2018; Protein content: 40.3 mg/mL
(used in the Informatory Toxicity and Confirmatory Mutation Test);
Lot Number: 3662; Expiry date: July 07, 2018; Protein content: 40.5 mg/mL
(used in the Informatory Toxicity Test);
Lot Number: 3712; Expiry date: November 03, 2018; Protein content: 34.3 mg/mL
(used in the Confirmatory Mutation Test);
Lot Number: 3727; Expiry date: December 01, 2018; Protein content: 33.7 mg/mL
(used in the Initial and Confirmatory Mutation Tests).

The S9 Mix (with Rat Liver S9)

Salt solution for S9 Mix Final concentration in S9 Mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix was kept in an ice bath before it was added to the culture medium.

Sodium Phosphate Buffer (0.2 M, pH 7.4)

Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL

Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.

Rationale for test conditions:

Based on the results of the Solubility and the Concentration Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type I). The vehicle was compatible with the survival of the bacteria and the S9 activity and appropriate historical control database is available in the testing laboratory.
Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests: 5000; 1600; 500; 160; 50 and 16 µg/plate.

At the preparation of the test item stock solution a correction (multiplier) factor of 1.96 (1/0.511=1.96) based on the active component content (of 51.1 %) was taken into consideration.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The revertant colony numbers of vehicle control (ultrapure water (ASTM Type I)) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and nearly all the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

Evaluation criteria:

The colony numbers on the untreated, vehicle control, positive control and the test item treated plates were determined visually by manual counting, and the mean values, standard deviations and the mutation rates were calculated.

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.

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

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:

Validity of the Performed Experiments

The tester strains used in this study demonstrated the specific phenotype characteristics, were in line with the corresponding historical control data ranges , and showed the adequate strain culture titer .
Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system .
Each of the investigated reference mutagens showed the expected increase (at least a
3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in both main experimental phases and the number of revertants in most cases fell in the corresponding historical control ranges , thereby meeting the criteria for the positive control in the main experimental phases, in all tester strains .
The spontaneous revertant colony numbers of the ultrapure water (ASTM Type 1) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases.
Seven concentration levels were investigated in the Informatory Toxicity Test and six in the main mutation experiments (Initial and Confirmatory Mutation Tests.
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have therefore been met.

Controls

In the performed Initial and Confirmatory Mutation Test multiple test items were tested with reference values from the common parallel controls.
In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the ultrapure water (ASTM Type 1) vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains (see in Tables of Appendices II-IV). In the Initial Mutation Test, in the case of S. typhimurium TA100 the revertant colony numbers of Sodium azide (SAZ) were above the corresponding historical control data range; however the higher counts were considered as acceptable without any effect on the final conclusion of the study .

The revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in different experimental phases were slightly higher or lower than the ultrapure water control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.

Initial and Confirmatory Mutation Tests (Plate Incorporation and Pre-Incubation Tests)

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in S. typhimurium TA1537 strain, at 50 µg/plate, in the absence of metabolic activation ( S9). This value however remained in the range of the corresponding vehicle historical control data and additional concentration related increase in revertant colony counts was not noticed. The mutation rate was 1.95, which was far below the genotoxicological threshold for being positive.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.
In the Initial Mutation Test in S. typhimurium TA100, at the concentration of 5000 µg/plate (±S9 Mix) and in the Confirmatory Mutation Test in S. typhimurium TA98, at the concentration 5000 µg/plate (+S9 Mix) the obtained revertant colony numbers were below the corresponding historical control data ranges, without any biological significance.

No precipitation of the test item was observed in the Initial and Confirmatory Mutation Tests on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).

Remarks on result:

other: Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) has no mutagenic activity on the applied bacterium tester strain under the test conditions used in this study.

Any other information on results incl. tables

Summary Table of the Results of the Concentration Range Finding Test

Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	21.7	0.82	25.7	0.95	103.7	1.10	121.0	1.24
DMSO Control	23.3	1.00	25.3	1.00	–	–	93.3	1.00
Ultrapure Water Control	26.3	1.00	27.0	1.00	94.0	1.00	97.7	1.00
5000	19.7	0.75	20.7	0.77	88.0	0.94	87.3	0.89
1600	23.0	0.87	21.3	0.79	97.3	1.04	103.7	1.06
500	29.3	1.11	24.7	0.91	84.3	0.90	102.3	1.05
160	25.7	0.97	26.3	0.98	93.0	0.99	113.3	1.16
50	21.0	0.80	29.7	1.10	100.7	1.07	110.3	1.13
16	18.7	0.71	25.0	0.93	103.7	1.10	106.7	1.09
5	18.7	0.71	25.7	0.95	105.0	1.12	115.3	1.18
NPD (4mg)	218.7	9.37	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1944.0	20.68	–	–
2AA (2mg)	–	–	1746.7	68.95	–	–	2360.0	25.29

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azide

2AA:2-aminoanthracene

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	25.3	1.10	36.3	1.06	97.0	1.17	101.3	0.84	10.7	0.82	13.7	1.24	7.7	1.64	11.3	1.36	27.0	0.80	37.3	1.09
DMSO Control	18.7	1.00	25.3	1.00	–	–	105.7	1.00	–	–	13.7	1.00	7.0	1.00	9.7	1.00	–	–	33.0	1.00
Ultrapure Water Control	23.0	1.00	34.3	1.00	82.7	1.00	120.3	1.00	13.0	1.00	11.0	1.00	4.7	1.00	8.3	1.00	33.7	1.00	34.3	1.00
5000	18.3	0.80	20.0	0.58	66.0	0.80	80.0	0.66	12.7	0.97	10.0	0.91	4.7	1.00	7.3	0.88	29.0	0.86	34.7	1.01
1600	18.3	0.80	24.3	0.71	78.7	0.95	100.3	0.83	11.0	0.85	10.7	0.97	5.0	1.07	7.0	0.84	20.7	0.61	38.7	1.13
500	20.3	0.88	21.0	0.61	83.3	1.01	96.3	0.80	11.0	0.85	10.0	0.91	6.7	1.43	10.7	1.28	30.3	0.90	52.3	1.52
160	20.7	0.90	24.7	0.72	89.7	1.08	98.3	0.82	13.3	1.03	13.7	1.24	3.7	0.79	7.0	0.84	29.3	0.87	45.3	1.32
50	18.7	0.81	26.3	0.77	88.7	1.07	96.7	0.80	14.3	1.10	12.0	1.09	6.3	1.36	8.0	0.96	23.7	0.70	44.0	1.28
16	21.7	0.94	23.7	0.69	96.7	1.17	120.0	1.00	13.0	1.00	11.0	1.00	6.3	1.36	8.7	1.04	24.7	0.73	46.0	1.34
NPD (4mg)	271.3	14.54	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	2189.3	26.48	–	–	862.7	66.36	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	914.0	130.57	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	800.0	23.76	–	–
2AA (2mg)	–	–	2397.3	94.63	–	–	2205.3	20.87	–	–	274.7	20.10	–	–	152.0	15.72	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	189.0	5.73

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	15.0	0.88	22.7	1.08	113.3	1.04	113.3	0.95	13.0	1.05	14.0	1.00	8.7	1.30	8.7	1.18	30.7	0.91	34.0	0.86
DMSO Control	16.0	1.00	19.0	1.00	–	–	113.3	1.00	–	–	11.7	1.00	6.3	1.00	8.0	1.00	–	–	31.0	1.00
Ultrapure Water Control	17.0	1.00	21.0	1.00	109.3	1.00	119.3	1.00	12.3	1.00	14.0	1.00	6.7	1.00	7.3	1.00	33.7	1.00	39.3	1.00
5000	17.0	1.00	14.3	0.68	67.7	0.62	108.7	0.91	11.3	0.92	13.7	0.98	8.7	1.30	7.0	0.95	34.3	1.02	37.3	0.95
1600	16.0	0.94	19.3	0.92	69.0	0.63	107.0	0.90	11.3	0.92	14.0	1.00	6.3	0.95	5.7	0.77	30.0	0.89	41.7	1.06
500	15.3	0.90	21.3	1.02	84.7	0.77	110.3	0.92	14.3	1.16	10.7	0.76	6.0	0.90	8.3	1.14	31.3	0.93	43.0	1.09
160	15.7	0.92	18.7	0.89	78.0	0.71	121.0	1.01	16.3	1.32	13.0	0.93	6.0	0.90	5.7	0.77	39.3	1.17	46.0	1.17
50	20.0	1.18	21.0	1.00	95.0	0.87	122.0	1.02	13.0	1.05	14.3	1.02	13.0	1.95	7.3	1.00	34.3	1.02	39.7	1.01
16	16.3	0.96	25.3	1.21	90.3	0.83	100.0	0.84	11.3	0.92	11.7	0.83	5.7	0.85	13.7	1.86	32.3	0.96	35.0	0.89
NPD (4mg)	256.0	16.00	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1584.0	14.49	–	–	1176.0	95.35	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	679.3	107.26	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1226.7	36.44	–	–
2AA (2mg)	–	–	992.0	52.21	–	–	1464.0	12.92	–	–	211.0	18.09	–	–	115.0	14.38	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	150.7	4.86

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

			Bacterial strains
Historical control data of untreated control	-S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.0	105.0	10.5	8.1	25.4
		SD	3.7	25.7	1.4	2.3	5.2
		Minimum	9	66	3	2	11
		Maximum	39	155	23	19	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.5	117.1	11.8	9.0	33.9
		SD	4.3	18.1	1.4	1.9	5.2
		Minimum	12	75	4	2	17
		Maximum	46	166	23	20	56
			Bacterial strains
Historical control data of DMSO control	-S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	20.4	100.1	10.3	7.9	24.7
		SD	3.6	24.8	1.3	2.4	4.6
		Minimum	10	64	3	2	11
		Maximum	38	147	23	20	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	26.5	113.8	11.8	8.8	33.7
		SD	4.1	18.3	1.5	1.9	5.0
		Minimum	15	71	3	3	16
		Maximum	47	162	25	20	57
			Bacterial strains
Historical control data of Water control	-S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.9	104.7	10.5	7.6	26.1
		SD	3.7	25.9	1.5	2.2	5.5
		Minimum	12	68	3	2	12
		Maximum	35	154	24	16	48
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.4	117.3	11.4	8.7	34.9
		SD	4.0	18.5	1.3	2.2	4.9
		Minimum	15	83	4	3	18
		Maximum	43	167	22	16	57
			Bacterial strains
Historical control data of positive controls	-S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	260.1	977.2	847.3	478.6	724.5
		SD	31.8	150.6	126.3	104.5	65.0
		Minimum	123	521	359	110	320
		Maximum	664	1970	1855	1601	1313
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1222.7	1436.4	164.1	147.0	257.7
		SD	274.9	318.3	33.1	20.1	72.5
		Minimum	386	583	85	69	140
		Maximum	2676	2988	498	399	477

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                               SD: Standard deviation; DMSO: Dimethyl sulfoxide

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test itemBenzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the Solubility and the Concentration Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type I). The vehicle was compatible with the survival of the bacteria and the S9 activity and appropriate historical control database is available in the testing laboratory.

Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:5000; 1600; 500; 160; 50 and 16 µg/plate.

The selection of the concentration range was based on the recommendations in OECD 471 guideline [6] for non-toxic, soluble test compounds; accordingly the test item was investigated up to and including the concentration level of 5000 µg/plate.

At the preparation of the test item stock solution a correction (multiplier) factor of 1.96 (1/0.511=1.96) based on the active component content (of 51.1 %) was taken into consideration.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

The revertant colony numbers of vehicle control (ultrapure water (ASTM Type I)) plates with and without S9 Mix demonstratedthe characteristic mean number of spontaneous revertantsthat was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant coloniesand nearly all the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment withBenzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show (see Appendix I to IV) that under the experimental conditions applied, the test itemdid not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test itemBenzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)has no mutagenic activity on the applied bacterium tester strainsunder the test conditions used in this study.