Tetrabutylammonium bromide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study contains experimental data of the registered substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted: 21 July, 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrabutylammonium bromide

EC Number:

216-699-2

EC Name:

Tetrabutylammonium bromide

Cas Number:

1643-19-2

Molecular formula:

C16H36N.Br

IUPAC Name:

tetrabutylazanium bromide

Details on test material:

- Name of test material (as cited in study report): Tetrabutylammonium bromide
- IUPAC name: 1-Butanaminium, N,N,N-tributyl-, bromide
- Molecular formula: C16H36BNr
- Molecular weight: 322.37 g/mol
- Substance type: Organic
- Physical state: White crystalline powder
- Purity: No data
- Impurities (identity and concentrations): No data

Specific details on test material used for the study:

Purity: 100%
Appearance: White crystalline powder

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented S9 microsomal fraction was used. The S9 fraction was obtained from Aroclor 1254-injected rats.
Composition of S9 mix (in 500 ml of RO water): D-glucose-6-phosphate (0.8 g), β-NADP (1.75 g), MgCl2 (1.0 g), KCl (1.0 g), NAD2HPO4 (6.4 g), NADH2 PO4 H2O (1.4 g)

Test concentrations with justification for top dose:

Test concentrations: 0.0, 0.050, 0.158, 0.50, 1.582 and 5 mg/plate

Justification: Test concentrations were selected based on a preliminary cytotoxicity experiment. The pre-experiment was performed according to the plate incorporation method using TA 98 and TA 100. Eight concentrations, i.e., 0.0 (VC), 0.002, 0.005, 0.016, 0.05, 0.158, 0.501, 1.582 and 5 mg/plate and positive controls were tested for toxicity in the presence and absence of S9 mix using triplicates. No colony reduction or background lawn inhibition was observed in the concentration range of 0.002-5 mg/plate in both strains in the presence and absence of S9 metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test chemical was solulble in distilles water at 50 mg/ml.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (10 ug/plate, TA 1537, TA 98, without S9); 2-Aminoanthracene (2.5 ug/plate, TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)
DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls, was tested in triplicate in two independent experiments performed.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was determined by the reduction in the number of mutant colonies and/or inhibition of the background lawn.

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

The colonies were counted manually. The mean values of the plates for each concentration together with standard deviation were compared to the spontaneous reversion rate. Microsoft office excel based calculations were used for descriptive statistical analysis.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

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

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and precipitation check:
The test substance was found soluble in distilled water at 50 mg/ml. Precipitation was checked as insolubility to assess precipitation in the final mixture under the actual test conditions and evident to the unaided eye. No precipitation formation was observed in distilled water at 50 mg/ml.
Cytotoxicity:
A preliminary cytotoxicity experiment was performed according to the plate incorporation method using TA 98 and TA 100. Eight concentrations, i.e., 0.0 (VC), 0.002, 0.005, 0.016, 0.05, 0.158, 0.501, 1.582 and 5 mg/plate and positive controls were tested for toxicity in the presence and absence of S9 mix using triplicates. No colony reduction or background lawn inhibition was observed in the concentration range of 0.002-5 mg/plate in both strains in the presence and absence of S9 metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0 (NC), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9). (for tabular data, refer to Any other information on results incl. tables).

Remarks on result:

other: Non-mutagenic

Any other information on results incl. tables

Table 1: REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	104	22	116	28
	R2	106	20	120	25
	R3	108	24	118	21
T1 (0.002)	R1	82	12	90	20
	R2	84	14	86	18
	R3	90	12	88	16
T2 (0.005)	R1	85	14	84	19
	R2	80	12	82	21
	R3	86	14	80	15
T3 (0.016)	R1	88	18	96	18
	R2	94	15	92	21
	R3	82	14	88	18
T4 (0.050)	R1	92	18	86	20
	R2	88	16	90	18
	R3	90	16	82	20
T5 (0.158)	R1	86	14	84	18
	R2	92	18	90	22
	R3	86	16	82	20
T6 (0.501)	R1	90	18	92	20
	R2	92	19	88	18
	R3	96	15	84	18
T7 (1.582)	R1	94	20	88	20
	R2	90	16	86	22
	R3	92	18	86	22
T8 (5)	R1	98	16	94	24
	R2	96	20	104	22
	R3	92	20	102	24
PC	R1	1240	976	1456	1008
	R2	1288	1008	1480	1128
	R3	1256	1032	1448	1104

NC           =     Negative control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)	R	In the presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	8	14	28	116	282
	R2	6	12	25	120	288
	R3	8	14	21	118	282
T1 (0.050)	R1	5	10	20	86	226
	R2	5	10	18	90	240
	R3	5	10	20	82	234
T2 (0.158)	R1	7	10	18	84	236
	R2	5	11	22	90	252
	R3	6	11	20	82	246
T3 (0.501)	R1	5	10	20	92	240
	R2	7	10	18	88	248
	R3	5	11	18	84	232
T4 (1.582)	R1	6	12	20	88	260
	R2	7	10	22	86	248
	R3	6	12	22	86	232
T5 (5)	R1	7	12	24	94	282
	R2	7	10	22	104	268
	R3	6	14	24	102	272
PC	R1	142	448	1008	1456	1266
	R2	164	472	1128	1480	1544
	R3	176	456	1104	1448	1368

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	12	22	104	274
	R2	6	15	20	106	268
	R3	7	15	24	108	288
T1 (0.050)	R1	5	10	18	92	232
	R2	4	10	16	88	244
	R3	4	8	16	90	238
T2 (0.158)	R1	5	12	14	86	228
	R2	5	10	18	92	240
	R3	4	10	16	86	234
T3 (0.501)	R1	5	12	18	90	242
	R2	6	15	19	92	238
	R3	5	10	15	96	244
T4 (1.582)	R1	5	12	20	94	240
	R2	5	10	16	90	236
	R3	5	14	18	92	260
T5 (5)	R1	6	12	16	98	264
	R2	6	14	20	96	252
	R3	7	12	20	92	240
PC	R1	168	1208	976	1240	1584
	R2	156	1232	1008	1288	1648
	R3	184	1264	1032	1256	1632

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100                                             

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]

Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	12	27	114	256
	R2	5	15	24	122	264
	R3	7	14	24	120	257
T1 (0.050)	R1	4	10	20	102	244
	R2	3	12	22	110	232
	R3	4	12	24	104	238
T2 (0.158)	R1	5	12	22	102	242
	R2	4	10	20	106	238
	R3	5	14	22	110	244
T3 (0.501)	R1	5	13	24	100	240
	R2	4	10	20	108	236
	R3	4	12	24	105	242
T4 (1.582)	R1	6	13	22	110	246
	R2	4	12	24	106	238
	R3	5	12	24	108	242
T5 (5)	R1	4	12	22	112	246
	R2	6	12	24	110	254
	R3	6	14	26	112	250
PC	R1	178	448	1482	1444	1712
	R2	189	496	1504	1460	1796
	R3	202	502	1548	1528	1808

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	14	25	122	246
	R2	6	12	24	118	252
	R3	7	15	26	112	264
T1 (0.050)	R1	3	10	18	100	236
	R2	4	8	20	110	242
	R3	4	12	18	106	240
T2 (0.158)	R1	5	10	22	112	240
	R2	3	8	18	106	244
	R3	4	14	20	108	246
T3 (0.501)	R1	5	12	20	106	246
	R2	4	13	18	106	240
	R3	5	10	20	106	236
T4 (1.582)	R1	4	12	22	108	248
	R2	4	9	20	114	232
	R3	5	12	22	110	256
T5 (5)	R1	6	10	24	112	252
	R2	4	12	22	110	248
	R3	6	14	22	118	244
PC	R1	184	1076	808	1208	1504
	R2	196	1136	916	1364	1620
	R3	210	1208	1024	1344	1748

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.33	1.15	13.33	1.15	24.67	3.51	118.00	2.00	284.00	3.46
T1 (0.050)	5.00	0.00	10.00	0.00	19.33	1.15	86.00	4.00	233.33	7.02
T2 (0.158)	6.00	1.00	10.67	0.58	20.00	2.00	85.33	4.16	244.67	8.08
T3 (0.501)	5.67	1.15	10.33	0.58	18.67	1.15	88.00	4.00	240.00	8.00
T4 (1.582)	6.33	0.58	11.33	1.15	21.33	1.15	86.67	1.15	246.67	14.05
T5 (5)	6.67	0.58	12.00	2.00	23.33	1.15	100.00	5.29	274.00	7.21
PC	160.67	17.24	458.67	12.22	1080.00	63.50	1461.33	16.65	1392.67	140.63

 

 

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.67	0.58	14.00	1.73	22.00	2.00	106.00	2.00	276.67	10.26
T1 (0.050)	4.33	0.58	9.33	1.15	16.67	1.15	90.00	2.00	238.00	6.00
T2 (0.158)	4.67	0.58	10.67	1.15	16.00	2.00	88.00	3.46	234.00	6.00
T3 (0.501)	5.33	0.58	12.33	2.52	17.33	2.08	92.67	3.06	241.33	3.06
T4 (1.582)	5.00	0.00	12.00	2.00	18.00	2.00	92.00	2.00	245.33	12.86
T5 (5)	6.33	0.58	12.67	1.15	18.67	2.31	95.33	3.06	252.00	12.00
PC	169.33	14.05	1234.67	28.10	1005.33	28.10	1261.33	24.44	1621.33	33.31

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD (TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.00	1.00	13.67	1.53	25.00	1.73	118.67	4.16	259.00	4.36
T1 (0.050)	3.67	0.58	11.33	1.15	22.00	2.00	10.33	4.16	238.00	6.00
T2 (0.158)	4.67	0.58	12.00	2.00	21.33	1.15	106.00	4.00	241.33	3.06
T3 (0.501)	4.33	0.58	11.67	1.53	22.67	2.31	104.33	4.04	239.33	3.06
T4 (1.582)	5.00	1.00	12.33	0.58	23.33	1.15	108.00	2.00	242.00	4.00
T5 (5)	5.33	1.15	12.67	1.15	24.00	2.00	111.33	1.15	250.00	4.00
PC	189.67	12.01	482.00	29.60	1511.33	33.61	1477.33	44.60	1772.00	52.31

 

 

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	5.67	1.53	13.67	1.53	25.00	1.00	117.33	5.03	254.00	9.17
T1 (0.050)	3.67	0.58	10.00	2.00	18.67	1.15	105.33	5.03	239.33	3.06
T2 (0.158)	4.00	1.00	10.67	3.06	20.00	2.00	108.67	3.06	243.33	3.06
T3 (0.501)	4.67	0.58	11.67	1.53	19.33	1.15	106.00	0.00	240.67	5.03
T4 (1.582)	4.33	0.58	11.00	1.73	21.33	1.15	110.67	3.06	245.33	12.22
T5 (5)	5.33	1.15	12.00	2.00	22.67	1.15	113.33	4.16	248.00	4.00
PC	196.67	13.01	1140.00	66.09	916.00	108.00	1305.33	84.88	1624.00	122.05

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100,  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

Applicant's summary and conclusion

Conclusions:

The registered substance, 1-Butanaminium, N,N,N-tributyl-, bromide (CAS: 1643-19-2) was tested non-mutagenic (negative) in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537and TA 102 when tested up to 5 mg/plate, in the presence and absence of S9 metabolic activation system.The test was performed according to OECD TG 471 and GLP.

Executive summary:

The potential of the registered substance, 1-Butanaminium, N,N,N-tributyl-, bromide (CAS: 1643-19-2) to induce gene mutation and/or frameshift in Salmonella typhimurium TA 98, TA 100, TA1535, TA1537 and TA 102 was tested according to OECD TG 471. The test was performed in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented S9 microsomal fraction was used as a metabolic activation system. The S9 fraction was obtained from Aroclor 1254-injected rats. Test concentrations were selected based on solubility and precipitation check and a preliminary cytotoxicity test. Distilled water was selected as a vehicle for the test substance. The preliminary cytotoxicity test was performed according to the plate incorporation method using TA 98 and TA 100. Eight concentrations, i.e., 0.0 (VC), 0.002, 0.005, 0.016, 0.05, 0.158, 0.501, 1.582 and 5 mg/plate and positive controls were tested for toxicity in the presence and absence of S9 mix using triplicates. No colony reduction or background lawn inhibition was observed in the concentration range of 0.002-5 mg/plate in both strains in the presence and absence of S9 metabolic activation. Hence, the following test item concentrations were chosen for the main study: 0.0 (VC), 0.050, 0.158, 0.50, 1.582 and 5 mg/plate with and without S9 metabolic activation. The main test consisted of two trials. Trial I was performed with five test substance concentrations along with negative and positive control substances according to the plate incorporation method with and without metabolic activation. Trial II was carried out with five test substance concentrations along with negative and positive control substances according to the preincubation method with and without metabolic activation. Results: No substantial increase in the number of revertant colonies compared to the vehicle control was observed at concentrations tested in any tester strains in both trials in the absence and presence of S9 metabolic activation. There was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Positive control substances induced unequivocal increases in revertant counts in all tester strains. The spontaneous reversion rates in the negative control were within the range of the historical laboratory data. Conclusion: The registered substance, 1-Butanaminium, N,N,N-tributyl-, bromide (CAS: 1643-19-2) did not induce point mutation and/or frameshift in the histidine operon of Salmonella typhimurium tester strains when tested concentrations up to 5 mg/plate both in the presence and absence of S9 metabolic activation.