N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantimonate

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:

From Jan. 20, 2004 to Feb. 2, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1998

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:

-

EC Number:

457-280-6

EC Name:

-

Cas Number:

126803-19-8

Molecular formula:

C16H20F6NOSb

IUPAC Name:

λ⁵-antimony(5+) N-[(4-methoxyphenyl)methyl]-N,N-dimethylanilinium hexafluoride

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M Corporate Toxicology

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark with desiccant

Method

Target gene:

TA 1537: his C 3076 (Frameshift mutation)
TA98: his D 3052 (Frameshift mutation)
TA 1535: his G 46 (Base-pair substitution)
TA 100: his G 46 (Frameshift and base-pair substitution)
WP2uvrA: trpE (Base-pair substitution)

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Aroclor 1254-induced rat liver S9
- method of preparation of S9 mix: The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9 or Sham mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each bulk preparation of S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TAl00.To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: The dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg per plat.
Confirmatory mutagenicity assay: The dose levels tested were 75, 200, 600, 1800 and 5000 μg per plate. The top dose was selected based on the findings of the initial toxicity-mutation assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: A solubility test was conducted to select the vehicle. The test was conducted using dimethyl sulfoxide (DMSO). The test article was tested to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration, up to 500 mg/mL. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other:

Remarks:

With S9
All salmonella strains and WP2uvrA: 2-aminoanthracene:
Without S9:
TA98: 2-nitrofluorene
TAI00, TA1535: Sodium azide
TA1537: 9-aminoacridine
WP2uvrA: Methyl methanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate in the initial toxicity-mutation assay, triplicate in the confirmatory Mutagenicity Assay.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 10^9 cells/mL
- Test substance added in: agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 to 72 hours at 37±2°C.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility Test
Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article forms a soluble and clear solution in dimethyl sulfoxide (DMSO) at 500 mg/mL, the highest concentration tested.

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

Initial toxicity-mutation assay
The maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg per plate. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose pIated in the confirmatory mutagenicity assay was 5000 μg per plate. Νo positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation (Table 1).

Confirmatory Mutagenicity Assay
The dose levels tested were 75, 200, 600, 1800 and 5000 μg per plate. Neither precipitate nor appreciable toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation (Table 2).

Any other information on results incl. tables

 

Table 1. Average Revertants Per Plate ± SD. Experiment No: B1
Liver Microsomes: None
Dose (μg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle	18 ± 0	120 ± 2	16 ± 4	6 ± 0	17 ± 4
2.5	18 ± 4	109 ± 4	17 ± 3	7 ± 1	18 ± 1
7.5	15 ± 1	129 ± 10	16 ± 2	3 ± 0	17 ± 1
25	16 ± 1	126 ± 16	20 ± 3	4 ± 1	13 ± 1
75	15 ± 3	121 ± 8	22 ± 4	7 ± 6	17 ± 1
200	16 ± 7	110 ± 7	22 ± 1	6 ± 1	12 ± 6
600	21 ± 2	142 ± 10	17 ± 1	5 ± 3	16 ± 4
1800	16 ± 1	119 ± 14	19 ± 0	9 ± 1	16 ± 6
5000	15 ± 2	124 ± 19	18 ± 1	7 ± 5	17 ± 1
Positive control	190 ± 15	566 + 43	331 + 39	659 + 28	122 + 27
Liver Microsomes: Rat liver S9
Dose (μg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle	26 ± 5	145 ± 6	16 ± 4	8 ± 1	16 ± 1
2.5	26 ± 6	147 ± 6	14 ± 2	10 ± 2	19 ± 1
7.5	32 ± 11	166 ± 22	15 ± 3	8 ± 2	15 ± 6
25	29 ± 0	168 ± 18	14 ± 1	8 ± 0	21 ± 1
75	37 ± 4	161 ± 17	17 ± 1	8 ± 1	25 ± 5
200	33 ± 1	179 ± 24	11 ± 0	9 ± 1	19 ± 3
600	35 ± 2	150 ± 16	15 ± 1	10 ± 1	26 ± 4
1800	30 ± 1	185 ± 4	13 ± 4	8 ± 1	19 ± 1
5000	33 ± 1	180 ± 6	16 ± 4	12 ± 1	20 ± 4
Positive control	740 ± 33	852 ± 42	120 ± 1	82 ± 19	525 ± 143

 

Table 2. Average Revertants Per Plate ± SD. Experiment No: B2
Liver Microsomes: None
Dose (μg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle	16 ± 2	115 ± 19	21 ± 3	6 ± 2	15 ± 5
75	16 ± 1	112 ± 15	23 ± 1	7 ± 1	15 ± 1
200	17 ± 1	128 ± 11	23 ± 10	8 ± 2	15 ± 3
600	16 ± 6	123 ± 15	18 ± 6	8 ± 3	12 ± 1
1800	15 ± 3	125 ± 4	21 ± 3	8 ± 2	13 ± 2
5000	15 ± 2	131 ± 6	18 ± 5	7 ± 1	18 ± 2
Positive control	356 ± 27	537 ± 33	314 ± 33	455 ± 28	87 ± 8
Liver Microsomes: Rat liver S9
Dose (μg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle	27 ± 1	129 ± 7	14 ± 2	9 ± 1	15 ± 6
75	24 ± 3	131 ± 18	14 ± 3	10 ± 1	17 ± 1
200	25 ± 3	154 ± 36	15 ± 2	9 ± 2	19 ± 1
600	23 ± 5	150 ± 14	15 ± 4	6 ± 4	16 ± 2
1800	29 ± 6	140 ± 10	13 ± 2	9 ± 1	13 ± 1
5000	33 ± 2	143 ± 10	14 ± 4	10 ± 2	16 ± 2
Positive control	908 ± 37	967 + 98	126 + 16	94 + 18	690 + 125

 

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantimonate did not cause a positive response in the presence and absence of metabolic activation and thus it is negative in the Bacterial Reverse Mutation Assay.

Executive summary:

The mutagenic potential of N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantimonate was assessed in the Bacterial Reverse Mutation Assay using Salmonella typhimuriu1n tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9.

The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells.

In the initial toxicity-mutation assay, the dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg per plate. No positive mutagenic response was observed in the presence or absence of metabolic activation. Neither precipitate nor appreciable toxicity was observed. In the confirmatory mutagenicity assay, the dose levels tested were 75, 200, 600, 1800 and 5000 μg per plate. Νo positive mutagenic response was observed in the presence or absence of metabolic activation. Neither precipitate nor appreciable toxicity was observed.

Under the conditions of this study, N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantimonate was concluded to be negative in the Bacterial Reverse Mutation Assay.