Tetrabutylammonium bromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation test:

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.

In vitro mammalian chromosome aberration assay:

The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2) was tested non-clastogenic (negative) up to the concentration of 2 mg/ml in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and GLP.

In vitro mammalian cell gene mutation assay:

An in vitro gene mutation test in mammalian cells according to OECD TG 476 with the registered substance has been initiated. The results will be reported as soon as possible.

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

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.

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

Specific details on test material used for the study:

Purity: 100%
Appearance: White crystalline powder

Target gene:

Histidine operon

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.

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.

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

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]

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.

  

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Justification for type of information:

The study contains experimental data of the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

Accepted: 29th July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Appearance :White crystalline powder
Batch Number: L217961707
Purity: 100%

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was obtained from the liver of phenobarbitone and β-naphthoflavone-induced rats.

Test concentrations with justification for top dose:

Test concentrations:
0.0 (VC), 0.5, 1.0 and 2.0 mg/ml

Justification:
Test concentrations were selected based on a preliminary cytotoxicity test. In this pre-test, CHO cells were exposed to 0.0 (VC), 0.125, 0.25, 0.5, 1.0 and 2.0 mg/ml. No cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item at ≤2 mg/ml, either in the presence or absence of metabolic activation. RICC values were 21.43 (-S9) and 24.14 (+S9).

Vehicle / solvent:

Distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Single
- Number of independent experiments: Phase I-III
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 × 106 cells/flask
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: In medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: Phase I-II: 4hrs, Phase IiI: 24 hrs,
- Harvest time after the end of treatment (sampling/recovery times): Recovery time for Phase I-II: 20 hrs, Harvesting time for Phase I-III: 24 hrs
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Colchicine, final conc: 1 µg/ml, for 2 hrs
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: NA
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): At harvest time, CHO cells were washed with plain RPMI-1640 medium. Cells were trypsinized with Trypsin EDTA, and the cell suspensions were centrifuged. Cells were resuspended in approximately 5 ml of freshly prepared hypotonic solution (Potassium chloride 0.075 M). The cell suspensions were allowed to stand at room temperature for 10 minutes. After hypotonic treatment, the cultures were centrifuged to discard the supernatant. Carnoy’s fixative (3:1 methanol: acetic acid solution) was added to each tube and incubated at room temperature for 10 minutes. Cells were centrifuged at 1000 rpm for 10 minutes, and the cell suspension was prepared by discarding the supernatant.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):NA
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). As slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, cells which that contain the number of centromeres equal to the number 2n ± 2 were scored. In CHO cells, the number of centromeres equal to the modal number is 22 ± 2.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was determined by the Relative Increase in Cell Count (RICC).

Evaluation criteria:

Acceptance of a test is based on the following criteria:
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
- The positive controls induce responses that are compatible with the laboratory historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
- Cell proliferation criteria in the solvent control should be fulfilled: Cytotoxicity evaluation of solvent control by Relative Increase in Cell Count both in the presence and absence of metabolic activation. Assessment of Relative Increase in Cell Count of solvent control to ensure the sufficient number of cells reached mitosis during the treatment period. Cytotoxicity evaluation of the solvent control both in the presence and absence of metabolic activation
- All three experimental conditions were tested unless one resulted in positive results.
- An adequate number of cells and concentrations are analysable.

Statistics:

Statistical analysis was performed to assess a possible dose-dependent increase of aberrant cell frequencies using Fisher’s Exact Test (NCSS statistics software). The percentage of aberrant cells from the Test Item treated group was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The cytotoxicity (%) was 78.05, 76.74 and 76.11at 2 mg/ml in the absence and presence of S9 mix (short term incubation) and without S9 mix (continuous incubation), respectively.

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, precipitation and pH checks: The test substance was found to be soluble in distilled water. No precipitation was observed at the concentration of 2 mg/ml. The pH of the test concentrations in the media was unchanged after 0 and 4 hours incubation.
Cytotoxicity: In the preliminary cytotoxicity assay, no cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed at ≤2 mg/ml, either in the presence or absence of metabolic activation. The cytotoxicity (%) was 78.05, 76.74 and 76.11at 2 mg/ml in the absence and presence of S9 mix (short term incubation) and without S9 mix (continuous incubation), respectively.

Remarks on result:

other: non-clastogenic

Appendix 1: Relative Increase in Cell Counts – Preliminary Cytotoxicity Assay

Dose Level	Conc. (mg/ml)	Absence of Metabolic activation				Presence of Metabolic activation
		Cell count		RICC	% Cytotoxicity	Cell count		RICC	% Cytotoxicity
		Starting	Final			Starting	Final
VC	-	1000000	3240000	100.00	0.00	1000000	3220000	100.00	0.00
T1	0.125	1000000	3160000	96.43	3.57	1000000	3084000	93.87	6.13
T2	0.25	1000000	2980000	88.39	11.61	1000000	2986000	89.46	10.54
T3	0.5	1000000	2968000	87.86	12.14	1000000	2846000	83.15	16.85
T4	1	1000000	2846000	82.41	17.59	1000000	2824000	82.16	17.84
T5	2	1000000	2760000	78.57	21.43	1000000	2684000	75.86	24.14

Appendix 2: Relative Increase in Cell Counts- Main Study

Dose Level	Conc.	Phase I -Absence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
VC	-	1000000	3342000	100.00	0.00
T1	0.5 mg/ml	1000000	2985000	84.76	15.24
T2	1 mg/ml	1000000	2940000	82.84	17.16
T3	2 mg/ml	1000000	2828000	78.05	21.95
PC	20 µg/ml	1000000	2568000	66.95	33.05

Dose Level	Conc.	Phase II -Presence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
VC	-	1000000	3236000	100.00	0.00
T1	0.5 mg/ml	1000000	2880000	84.08	15.92
T2	1 mg/ml	1000000	2824000	81.57	18.43
T3	2 mg/ml	1000000	2716000	76.74	23.26
PC	30 µg/ml	1000000	2545000	69.10	30.90

Dose Level	Conc.	Phase III -Absence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
VC	-	1000000	3294000	100.00	0.00
T1	0.5 mg/ml	1000000	2962000	85.53	14.47
T2	1 mg/ml	1000000	2884000	82.13	17.87
T3	2 mg/ml	1000000	2746000	76.11	23.89
PC	20 µg/ml	1000000	2532000	66.78	33.22

Appendix 3: Individual Data on Chromosome Aberrations- Phase I: Absence of metabolic activation (short term)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
VC 0 mg/ml	300	1 Ctg	1	0
T1 0.5 mg/ml	300	1 Ctb, 2 Ctg	3	1
T2 1 mg/ml	300	1 dic	1	1
T3 2 mg/ml	300	2 dic	2	2
PC 20 µg/ml	300	1 Ctg, 11 Ctb, 2 fragments, 1 ring, 6 dic, 2 minute	21	20

Appendix 4: Individual Data on Chromosome Aberrations- Phase II:Presence of metabolic activation (short term)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
VC 0 mg/ml	300	1 dic	1	1
T1 0.5 mg/ml	300	1 Ctg, 1 dic	2	1
T2 1 mg/ml	300	1 Ctb, 1Csb, 1 ring	2	2
T3 2 mg/ml	300	1 Csg, 1Ctb, 1fragment	2	2
PC 30 µg/ml	300	1 Ctg,3 Csg, 5 Ctb,3 Csb, 3 fragment, 1 ring, 1 exchange, 10 dic	21	20

Appendix 5: Individual Data on Chromosome Aberrations- Phase III: Absence of metabolic activation (Continuous)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
VC 0 mg/ml	300	1 Ctb	1	1
T1 0.5 mg/ml	300	1 fragment, 1 Csb	2	2
T2 1 mg/ml	300	1 dic	1	1
T3 2 mg/ml	300	1 Csg, 1 exchange	2	2
PC 20 µg/ml	300	11 Ctb, 3 fragements, 2 rings, 9 dic, 3 minute	25	25

Key: VC = Vehicle Control (distilled water), PC = Positive Control (methyl methanesulfonate), mg = milligram, µg = microgram, ml = milliliter, T3-T1 = Test Item concentration from higher to lower, Ctg = Chromatid gap, Csg = Chromosome gap, Ctb = Chromatid break, Csb = Chromosome break, dic = dicentric.

Appendix 6: Summary Dataon Chromosome Aberrations - Phase I

Dose Level	Concentration	Absence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
VC	-	0	0.00
T1	0.5 mg/ml	1	0.33
T2	1 mg/ml	1	0.33
T3	2 mg/ml	2	0.67
PC	20 µg/ml*	20	6.67

Appendix 7: Summary Data on Chromosome Aberrations - Phase II

Dose Level	Concentration	Presence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
VC	-	1	0.33
T1	0.5 mg/ml	1	0.33
T2	1 mg/ml	2	0.67
T3	2 mg/ml	2	0.67
PC	30 µg/ml*	20	6.67

Appendix 8: SummaryData on Chromosome Aberrations - Phase III  

Dose Level	Concentration	Absence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
VC	-	1	0.33
T1	0.5 mg/ml	2	0.67
T2	1 mg/ml	1	0.33
T3	2 mg/ml	2	0.67
PC	20 µg/ml*	25	8.33

Conclusions:

The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2) was tested non-clastogenic (negative) up to the concentration of 2 mg/ml in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and GLP.

Executive summary:

The ability of the registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), to induce chromosome aberration in cultured Chinese Hamster Ovary (CHO) cells was tested according to OECD TG 473 both in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was obtained from the liver of phenobarbitone and β-naphthoflavone-induced rats. Distilled water was selected as a vehicle for the test substance. Test concentrations were selected based on the solubility, precipitation and pH checks, as well as a preliminary cytotoxicity test. In the cytotoxicity test, CHO cells were exposed to the test substance at concentrations of 0.0 (vehicle control/distilled water), 0.125, 0.25, 0.5, 1.0 and 2.0 mg/ml, both in the absence and presence of S9 metabolic activation. No cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item at ≤2 mg/ml, either in the presence or absence of metabolic activation. Hence, the following concentrations were employed in the chromosome aberration test: 0.0 (VC), 0.5, 1 and 2 mg/ml. Positive control substances were also included in the test, i.e., Methyl methanesulfonate (20 µg/ml, without S9) and Benzo (a) pyrene (30 µg/ml, with S9). The chromosome aberration test consisted of three phases. In Phase,s I-II, CHO cells (1 × 106 cells/flask ) were treated with test substance formulation and vehicle/ positive controls for 4 hours (short-term treatment) in the presence and absence of S9 metabolic activation which was followed by a 20 hours recovery period. In Phase III, CHO cells (1 × 106 cells/flask) were treated with the test substance and vehicle and positive controls for 24 hours (continuous treatment). The cultures were harvested 24 hours (short term treatment and continuous treatment) after the beginning of treatment. After the treatment period, CHO cells were fixed with Carnoy’s fixative (3:1 methanol: acetic acid solution) and stained with 5 % Giemsa stain. At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations. Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). The cytotoxicity was assessed by the Relative Increase in Cell Counts (RICC) in all phases. Results: In Phase I, no significant increase in the mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992) was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). Average RICC values were 100 % (vehicle control), 84.76 % (at 0.5 mg/ml), 82.84 % (at 1 mg/ml) and 78.05 % (at 2 mg/ml). In Phase II experiment, no significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed  compared to the vehicle control (the mean % aberrant cells 0.33 %). Average RICC values were 100 % (vehicle control), 84.08 % (at 0.5 mg/ml), 81.57 % (at 1 mg/ml) and 76.74 % (at 2 mg/ml). In Phase III, the average RICC values were 100 % (vehicle control), 85.53 % (at 0.5 mg/ml), 82.13 % (at 1 mg/ml) and 76.11 % (at 2 mg/ml).  No significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed when compared to the vehicle control (the mean % aberrant cells 0.33 %). Conclusion: The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), did not induce chromosome aberration in cultured CHO cells up to 2 mg/ml neither in the present nor in the absence of S9 metabolic activation under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

The study is ongoing and this information will be submitted later based on ECHA communication/decision number CCH-D-2114461478-39-01/F.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation test:

Study 1

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.

Study 2

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 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA/pKM101 was tested according to the preincubation method in a guideline study (according to OECD TG 471). The test was performed in the presence and absence of S9 metabolic activation. Distiled water was chosen as a vehicle for the test substance.Tes concnetrations were selected based on a preliminary dose-range finding study. In this pre-test, bacterial cells were exposed to eight doses, i.e., 0.0 (VC), 2.29, 6.86, 20.6, 61.7, 185, 556, 1667, 5000 μg/plate (all strains). The test substance did not increase in number of revertant colonies more than twice that of the negative control (solvent control) in any tester strain with or without metabolic activation. Growth inhibition of the bacterial lawn with the chemical was observed at ≥1667 µg/ml in TA100, TA1535, TA98 and TA1537 with or without metabolic activation.Hence, the main study was perfomed with the following concentrations: 0, 39.1, 78.1, 156, 313, 625, 1250, 2500 μg/plate (S. typhimurium strains, with and without S9 mix) and 0, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/plate (E. coli, with and without S9). Concurrent solvent and positive control plates were also included in the study. Results: The test substance did not increase the number of revertant colonies more than twice that of the negative control in any strain with or without metabolic activation. Bacterial cell growth inhibition was observed at ≥1250 ug/plate with and without S9 in Salmonella typhimurium tester strains. The positive control substances assayed increased the numbers of revertant colonies more than twice that of the solvent control. The numbers of revertant colonies for the negative and positive controls obtained in the present test were within the range of standard values derived from historical control data in our laboratory, which suggested that the tests were performed adequately. Conclusion: Tetrabutylammonium bromide (CAS: 1643-19-2) was tested non-mutagenic (negative) in the reverse mutation test in Salmonella typhimurium, and E. coli WP2 uvrA/pKM101 tester strains in the presence and absence of S9 metabolic activation.

In vitro mammalian chromosome aberration assay:

The ability of the registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), to induce chromosome aberration in cultured Chinese Hamster Ovary (CHO) cells was tested according to OECD TG 473 both in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was obtained from the liver of phenobarbitone and β-naphthoflavone-induced rats. Distilled water was selected as a vehicle for the test substance. Test concentrations were selected based on the solubility, precipitation and pH checks, as well as a preliminary cytotoxicity test. In the cytotoxicity test, CHO cells were exposed to the test substance at concentrations of 0.0 (vehicle control/distilled water), 0.125, 0.25, 0.5, 1.0 and 2.0 mg/ml, both in the absence and presence of S9 metabolic activation. No cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item at ≤2 mg/ml, either in the presence or absence of metabolic activation. Hence, the following concentrations were employed in the chromosome aberration test: 0.0 (VC), 0.5, 1 and 2 mg/ml. Positive control substances were also included in the test, i.e., Methyl methanesulfonate (20 µg/ml, without S9) and Benzo (a) pyrene (30 µg/ml, with S9). The chromosome aberration test consisted of three phases. In Phase,s I-II, CHO cells (1 × 106 cells/flask ) were treated with test substance formulation and vehicle/ positive controls for 4 hours (short-term treatment) in the presence and absence of S9 metabolic activation which was followed by a 20 hours recovery period. In Phase III, CHO cells (1 × 106 cells/flask) were treated with the test substance and vehicle and positive controls for 24 hours (continuous treatment). The cultures were harvested 24 hours (short term treatment and continuous treatment) after the beginning of treatment. After the treatment period, CHO cells were fixed with Carnoy’s fixative (3:1 methanol: acetic acid solution) and stained with 5 % Giemsa stain. At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations. Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). The cytotoxicity was assessed by the Relative Increase in Cell Counts (RICC) in all phases. Results: In Phase I, no significant increase in the mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992) was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). Average RICC values were 100 % (vehicle control), 84.76 % (at 0.5 mg/ml), 82.84 % (at 1 mg/ml) and 78.05 % (at 2 mg/ml). In Phase II experiment, no significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed  compared to the vehicle control (the mean % aberrant cells 0.33 %). Average RICC values were 100 % (vehicle control), 84.08 % (at 0.5 mg/ml), 81.57 % (at 1 mg/ml) and 76.74 % (at 2 mg/ml). In Phase III, the average RICC values were 100 % (vehicle control), 85.53 % (at 0.5 mg/ml), 82.13 % (at 1 mg/ml) and 76.11 % (at 2 mg/ml).  No significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed when compared to the vehicle control (the mean % aberrant cells 0.33 %). Conclusion: The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), did not induce chromosome aberration in cultured CHO cells up to 2 mg/ml neither in the present nor in the absence of S9 metabolic activation under the experimental conditions described.

In vitro gene mutation test in mammalian cells

Justification for classification or non-classification

The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), was tested non-mutagenic in bacterial cells and induced no structural chromosomal aberration in mammalian cells. Therefore, an in vitro gene mutation study in mammalian cells has been initiated with the Substance in order to fulfil the standard information requirement of Annex VIII of REACH.