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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The reported data of the OECD471 mutagenicity assay show that under the experimental conditions applied, the substance Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in the study OECD471 (Ames test negative).

Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) tested up to the maximum cytotoxic cconcentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system, but caused perturbation in the cell cycle (OECD473 negative).

Study OECD 476 in progress (05/2018).

Based on available information the substance is considered not a genotoxic substance and the test item is not classified with genetic toxicity according to the CLP regulation 1272/2008/EC.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

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

Specific details on test material used for the study:

Expiring date: 10 January 2018

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 / 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.

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.

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August - 14 September, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29th July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Expiring date: 10 January 2018

Target gene:

Chromatid and chromosome type aberrations in metaphase cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79:Chinese hamster lung male
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).

Metabolic activation:

with and without

Metabolic activation system:

liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 300 (150-150) well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:

Experiment A with 3/20 h treatment/sampling time
without: 125, 250 and 500 *g/mL test item
with S9 mix: 250, 500 and 1000 *g/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 250, 500 and 1000 *g/mL test item

Vehicle / solvent:

DME (Dulbecco’s Modified Eagle’s)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

The V79 cell line is well established in toxicology studies.
This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in liquid nitrogen. Checking for mycoplasma infections was carried out. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 ¿C in an incubator with a humidified atmosphere, set at 5 % CO2. The V79 cells for this study was grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with
L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 ¿g/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.
Mammalian Microsomal Fraction S9 Mix

An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential when they are metabolised by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were, 33.7 and 33.8 mg/mL.

Rat Liver S9 Fraction

The S9 fraction of phenobarbital (PB) and ß-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.

The S9 Mix (with Rat Liver S9)

The complete S9 Mix was freshly prepared containing components with the following ratios:
S9 fraction 3 mL
HEPES* 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP** 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
*= N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**= ß-Nicotinamide Adenine Dinucleotide Phosphate
Before adding to the culture medium the S9 mix was kept in an ice bath.

Rationale for test conditions:

Acceptability of the Assay

The Chromosome Aberration Assay is considered acceptable because it meets the following criteria:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data, .
– concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control,
– cell proliferation in the solvent control is adequate,
– adequate number of cells and concentrations are analyzable,
– all requested experimental conditions were tested unless one resulted in a positive result
– the criteria for the selection of top concentration is adequate.

Evaluation criteria:

Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.

Interpretation of Results

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative because:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data

Statistics:

For statistical analysis, CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

Solubility and Dose Selection

Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) was dissolved in DME (Dulbecco’s Modified Eagle’s). A clear solution was obtained up to a concentration of 100 mg/mL. There was no precipitation in the medium at any concentration tested. Concentration selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays (Experiment A and B), both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by cell counting and results noted. Based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Detailed results of the cytotoxicity assay with Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) are presented in Table 2A - 2C. These results were used to select concentrations of test item for the Chromosome Aberration Assays.
Based on the results of the cytotoxicity assay the following concentrations were selected for the Chromosome Aberration Assay.
All concentrations were run in duplicates (incl. negative and positive controls) and at least 300 (150-150) well-spread metaphases were assessed:

Experiment A with 3/20 h treatment/sampling time
without: 125, 250 and 500 *g/mL test item
with S9 mix: 250, 500 and 1000 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 250, 500 and 1000 *g/mL test item

Chromosome Aberration Assay

In Experiment A, Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) did not induce an increase in the number of cells with aberrations without gaps at any examined concentration, neither in the absence nor in the presence of metabolic activation. There were no statistically significant differences between test item treatment and control groups and no dose-response relationship was noted. .
In this part of Experiment the test item caused perturbation in the cell cycle, because endoreduplication were observed at concentration of 500 *g/mL without S9 mix and at concentrations of 250, 500 and 1000 *g/mL with S9 mix. In the presence of S9 mix the observed perturbation was dose associated.

In Experiment B, Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) was examined without S9 mix, over a long treatment period and the sampling was made at approximately 1.5 cell cycles (20 hours after treatment start). The cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent solvent and historical controls.

There was no increase in the number of cells with aberrations without S9 mix following exposure over a long treatment period of 20 hours and sampling at approximately 2 cell cycles (28 hours after treatment start).

A 3-hour treatment in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations without gaps compared to the concurrent solvent and historical controls.

In Experiment A and in Experiment B no statistically significant differences between test item treatment and controls (solvent and historical) groups and no dose-response relationships were noted.

Tables 10-11 show the occurrence of polyploidy and endoreduplicated metaphases.
In the Experimental A endoreduplication were observed at concentration of 500 *g/mL without S9 mix and at concentrations of 250, 500 and 1000 *g/mL with S9 mix. In the presence of S9 mix the number of endoreduplicated metaphases showed dose associated.

pH and osmolality values of control and test item treatment solutions were measured. In Experiments A and B no significant differences between test item treatment and control groups were observed.

In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was less than 5 %, confirming the suitability of the cell line used.

The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 *L/mL) and Cyclophosphamide (5 *g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Remarks on result:

other: Test item tested up to the maximum cytotoxic cconcentrations, did not induce structural chromosome aberrations (with/without metabolic act.).Thus, the test item is considered as not clastogenic in this system, but caused perturbation in the cell cycle.

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

3-hour treatment without and with S9 mix / 20-hour sampling time

 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2000000	1950000	1993750	-	-	-
	-	B	–	2050000	1900000
	-	C	–	2000000	2000000
	-	D	–	2000000	2050000
Solvent control (DME)	-	A	–	7350000	7100000	7287500	5293750	100,00	0,00
	-	B	–	7500000	7200000
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)	125	A	–	7200000	7200000	7200000	5206250	98,35	1,65
	250	A	–	6450000	6800000	6625000	4631250	87,49	12,51
	500	A	–	4700000	4600000	4650000	2656250	50,18	49,82
	1000	A	–	150000	0	75000	-1918750*	-36,25**	136,25***
	2000	A	–	0	0	0	-1993750*	-37,66**	137,66***
	5000	A	–	0	0	0	-1993750*	-37,66**	137,66***
	EMS 1 µL/mL	A	–	4700000	4600000	4650000	2656250	50,18	49,82
Solvent control (DME) medium	-	A	+	7200000	7050000	7175000	5181250	100,00	0,00
	-	B	+	7350000	7100000
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)	125	A	+	7100000	7100000	7100000	5106250	98,55	1,45
	250	A	+	6700000	7000000	6850000	4856250	93,73	6,27
	500	A	+	6000000	6000000	6000000	4006250	77,32	22,68
	1000	A	+	4000000	4200000	4100000	2106250	40,65	59,35
	2000	A	+	150000	50000	100000	-1893750	-36,55	136,55
	5000	A		0	0	0	-1993750	-38,48	138,48
	Cycl. 5µg/mL	A	+	4500000	4550000	4525000	2531250	48,85	51,15

RICC=Relative Increase in Cell Counts                                 *: cell number decrease

Cytotoxicity= 100-RICC                                                         **: zero RICC value,

DME: (Dulbecco’s Modified Eagle’s)medium                       ***: 100% cytotoxicity

EMS: Ethyl methanesulfonate (EMS)

Cycl: Cyclophosphamide monohydrate

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

 

20-hour treatment without S9 mix / 20-hour sampling time

 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2000000	1950000	1993750	-	-	-
	-	B	–	2050000	1900000
	-	C	–	2000000	2000000
	-	D	–	2000000	2050000
Solvent control (DME)	-	A	–	7250000	7600000	7387500	5393750	100,00	0,00
	-	B	–	7200000	7500000
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)	15.7	A	–	7400000	7300000	7350000	5356250	99,30	0,70
	31.3	A	–	7100000	7100000	7100000	5106250	94,67	5,33
	62.5	A	–	6250000	5950000	6100000	4106250	76,13	23,87
	125	A	–	5900000	5600000	5750000	3756250	69,64	30,36
	250	A	–	4200000	4300000	4250000	2256250	41,83	58,17
	500	A	–	2100000	1850000	1975000	-18750*	-0,35**	100,35***
	EMS 1 µL/mL	A	–	4700000	4600000	4650000	2656250	49,25	50,75

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

DME: (Dulbecco’s Modified Eagle’s)medium

EMS: Ethyl methanesulfonate (EMS)

*:cell number decrease,

**:zero RICC value,

***:100% cytotoxicity

  

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

 

20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time

 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2000000	1950000	1993750	-	-	-
	-	B	–	2050000	1900000
	-	C	–	2000000	2000000
	-	D	–	2000000	2050000
Solvent control (DME)	-	A	–	8550000	8700000	8700000	6706250	100,00	0,00
	-	B	–	8900000	8650000
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)	15.7	A	–	8400000	8700000	8550000	6556250	97,76	2,24
	31.3	A	–	8400000	8250000	8325000	6331250	94,41	5,59
	62.5	A	–	6800000	7150000	6975000	4981250	74,28	25,72
	125	A	–	6750000	6600000	6675000	4681250	69,80	30,20
	250	A	–	5000000	4800000	4900000	2906250	43,34	56,66
	500	A	–	2250000	2100000	2175000	181250	2,70	97,30
	EMS 1 µL/mL	A	–	5500000	5400000	5450000	3456250	51,54	48,46
Solvent control (DME)	-	A	+	9400000	9500000	9412500	7418750	100,00	0,00
	-	B	+	9300000	9450000
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)	125	A	+	9450000	9250000	9350000	7356250	99,16	0,84
	250	A	+	9000000	9000000	9000000	7006250	94,44	5,56
	500	A	+	7000000	7100000	7050000	5056250	68,16	31,84
	1000	A	+	5000000	4800000	4900000	2906250	39,17	60,83
	2000	A	+	0	0	0	-1993750*	-26,87**	126,87***
	5000	A	+	0	0	0	-1993750*	-26,87**	126,87****
	Cycl. 5µg/mL	A	+	5650000	5800000	5725000	3731250	50,29	49,71

RICC=Relative Increase in Cell Counts                                 *:cell number decrease

Cytotoxicity= 100-RICC                                                         **:zero RICC value,

DME: (Dulbecco’s Modified Eagle’s)medium                       ***:100% cytotoxicity

EMS: Ethyl methanesulfonate (EMS)

Cycl: Cyclophosphamide monohydrate

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT A

 

 

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
				incl. gaps	excl. gaps
Negative (Solvent) control	-	3 h	20 h	7	4
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
125 µg/mL	-	3 h	20 h	6	3
250 µg/mL	-	3 h	20 h	7	3
500 µg/mL	-	3 h	20 h	7	3
Pos. Control (Ethyl methanesulphonate)	-	3 h	20 h	41**	36**
Negative (Solvent) control	+	3 h	20 h	6	3
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
250 µg/mL	+	3 h	20 h	8	4
500 µg/mL	+	3 h	20 h	7	4
1000 µg/mL	+	3 h	20 h	9	3
Pos. Control (Cyclophosphamide)	+	3 h	20 h	47**	41**

Positive control (-S9): Ethyl methanesulphonate (1.0L/mL)

Positive control (+S9): Cyclophosphamide (5.0g/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

 

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B

 

 

Concentration (µg/mL)		S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
					incl. gaps	excl. gaps
	Negative (Solvent) control	-	20 h	20 h	8	3
	Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
	31.3 µg/mL	-	20 h	20 h	7	4
	62.5 µg/mL	-	20 h	20 h	6	3
	125 µg/mL	-	20 h	20 h	8	3
	250 µg/mL	-	20 h	20 h	8	4
	Pos. Control (Ethyl methanesulphonate)	-	20 h	20 h	46**	37**
	Negative (Solvent) control	-	20 h	28 h	6	3
	Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
	31.3 µg/mL	-	20 h	28 h	7	3
	62.5 µg/mL	-	20 h	28 h	6	3
	125 µg/mL	-	20 h	28 h	6	3
	250 µg/mL	-	20 h	28 h	8	3
	Pos. Control (Ethyl methanesulphonate)	-	20 h	28 h	49**	39**

Positive control (-S9): Ethyl methanesulphonate (0.4L/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

 

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B

 

 

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
				incl. gaps	excl. gaps
Negative (Solvent) control	+	3 h	28 h	7	4
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
250 µg/mL	+	3 h	28 h	7	3
500 µg/mL	+	3 h	28 h	7	3
1000 µg/mL	+	3 h	28 h	9	5
Pos. Control (Cyclophosphamide)	+	3 h	28 h	49**	38**

Cyclophosphamide: 5.0g/mL

** = p < 0.01 to the concurrent negative control and to the historical control

 

 

NUMBER OF POLYPLOID AND ENDOREDUPLICATED CELLS

 

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS 

EXPERIMENT A

 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	3/20 h	0.0	0.0
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
125 µg/mL	-	3/20 h	0.0	0.0
250 µg/mL	-	3/20 h	0.0	0.0
500 µg/mL	-	3/20 h	0.0	1.5
Pos. Control (Ethyl methanesulphonate)	-	3/20 h	0.0	0.0
Negative (Solvent) control	+	3/20 h	0.0	0.0
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
250 µg/mL	+	3/20 h	0.0	0.5
500 µg/mL	+	3/20 h	0.0	1.0
1000 µg/mL	+	3/20 h	0.0	6.0
Pos. Control (Cyclophosphamide)	+	3/20 h	0.0	0.0

Ethyl methanesulphonate: 1.0mL/mL

Cyclophosphamide: 5.0g/mL

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS

 

EXPERIMENT B

 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	20/20 h	0.0	0.0
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
31.3 µg/mL	-	20/20 h	0.0	0.0
62.5 µg/mL	-	20/20 h	0.0	0.0
125 µg/mL	-	20/20 h	0.0	0.0
250 µg/mL	-	20/20 h	0.0	0.0
Pos. Control	-	20/20 h	0.0	0.0
Negative (Solvent) control	-	20/28 h	0.0	0.0
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
31.3 µg/mL	-	20/28 h	0.0	0.0
62.5 µg/mL	-	20/28 h	0.0	0.0
125 µg/mL	-	20/28 h	0.0	0.0
250 µg/mL	-	20/28 h	0.0	0.0
Pos. Control	-	20/28 h	0.0	0.0

Positive control (-S9):Ethyl methanesulphonate(0.4L/mL)

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

 

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS

 

EXPERIMENT B

 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	+	3/28 h	0.0	0.0
Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %)
250 µg/mL	+	3/28 h	0.0	0.0
500 µg/mL	+	3/28 h	0.0	0.0
1000 µg/mL	+	3/28 h	0.0	0.0
Pos. Control	+	3/28 h	0.0	0.0

Cyclophosphamide: 5.0g/mL

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

Conclusions:

Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) tested up to the maximum cytotoxic cconcentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.

Thus, the test item is considered as not clastogenic in this system, but caused perturbation in the cell cycle.

Executive summary:

The test item, Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) was tested in a Chromosome Aberration Assay in V79 cells.The test item was dissolved inDME (Dulbecco’s Modified Eagle’s) mediumand the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using rodent S9 mix).In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 300 (150-150) well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:

 

Experiment Awith 3/20 h treatment/sampling time

without:                           125, 250and 500g/mLtest item

with S9 mix:                   250, 500 and 1000g/mLtest item

Experiment Bwith 20/20 h treatment/sampling time

without S9 mix:              31.3, 62.5, 125 and 250g/mLtest item

Experiment Bwith 20/28 h treatment/sampling time

without S9 mix:              31.3, 62.5, 125 and 250g/mLtest item

Experiment Bwith 3/28 h treatment/sampling time

with S9 mix:                   250, 500 and 1000g/mLtest item

 

In the performed Chromosome Aberration Assay the concentration levels were chosen mainly based on the cytotoxicity.

 

Following treatment (exposure) and sampling (expression) time cells were exposed to selection agent Colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Following harvesting cells were treated with fixative for ca. 10 min. before being placed on slides and stained. Chromosome aberration frequencies were then scored for at least 300 well-spread metaphase cells.

 

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted. In this part of Experiment the test item caused perturbation in the cell cycle, because endoreduplication were observed at concentration of 500g/mL without S9 mix and at concentrations of 250, 500 and 1000g/mLwith S9 mix.

In the presence of S9 mix the observed perturbation was dose associated.

 

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to the maximum cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum cytotoxic concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted.The observed chromosome aberration rates were within the ranges of historical control data.

 

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

 

The number of aberrations found in the solvent controls was in the range of historical laboratory control data.The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and Cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

 

Benzene, 1,1’oxybis(methyl-, sulfonated, ammonium salt (ca 50 %) tested up to the maximum cytotoxic cconcentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.

 

Thus, the test item is considered as not clastogenic in this system, but caused perturbation in the cell cycle.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018 - study in progress

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Remarks on result:

other: preliminary results, study in progress

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available