N-tert-butylacrylamide

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:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted: July 21 1997, Corrected: June 26 2020

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-tert-butylacrylamide

EC Number:

203-505-6

EC Name:

N-tert-butylacrylamide

Cas Number:

107-58-4

Molecular formula:

C7H13NO

IUPAC Name:

N-tert-butylacrylamide

Test material form:

solid

Details on test material:

- Name of test material: N-tert-butylacrylamide
- Molecular formula : C7H13NO
- Molecular weight: 127.19 g/mol
- Substance type: Organic
- Physical state: Solid

Method

Target gene:

Histidine

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:

S9 Homogenate
Aroclor 1254-induced rat liver microsomal enzymes (S9 homogenate) procured commercially was used for the assay. Certificate of analysis received from the supplier was included in the report. Efficiency check of S9 was performed during the main assay. The protein concentration in the S9 fraction was 34.6 mg/ml (Annexure: 1).

S9 Mix
S9 mix cofactors solution (Cofactor ingredients: D-glucose-6-phosphate, β-NADP, magnesium chloride, potassium chloride, sodium phosphate and liver homogenate] was prepared before use. The post-mitochondrial fraction (liver homogenate) was used at the concentration of 10 % (v/v) in the S9 mix.

Test concentrations with justification for top dose:

Concentration tested were 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. These concentrations were selected based on solubility, precipitation test and preliminary cytotoxicity test.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was selected as a vehicle for the test item in this study.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

The mutagenic potential of N-tert-butylacrylamide [TBAA] [CAS No. 107-58-4] was tested in two independent experiments (Trial I and Trial II) and both in the presence (10% v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102.

Trial I and II: 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate
Trial I was performed according to the plate incorporation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item together with the vehicle, negative and positive controls, was tested in triplicates. The Test item doses were selected using concentration spacing factor of 2. No increase in the number of revertant colonies or inhibition in background lawn was observed up to the highest concentration of 5 mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data.

Trial II was performed to confirm the negative results observed in Trial I.
Trial II was conducted according to the preincubation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item together with the vehicle, negative and positive controls, was tested in all tester strains in triplicates. There was no increase in the number of revertant colonies or inhibition of the background lawn up to the highest concentration of 5 mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data.
The numbers of revertant colonies of the vehicle, negative (spontaneous revertant colonies), and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test.

Evaluation criteria:

A Test item is considered as a mutagen if a biologically relevant increase in the mean number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
A dose-dependent increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if it is reproduced in an independent experiment.

Statistics:

The colonies were counted manually. The mean number of revertant colonies and the standard deviation within the plates for each concentration were compared to the spontaneous reversion rates of the control. Microsoft Office Excel-based calculations were used for descriptive statistical analysis.

Results and discussion

Test results

Test results 1

Key result

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Test results 2

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

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No Mutagenic pottential

Any other information on results incl. tables

Table1          Mean Revertant Colony Count – Preliminary Cytotoxicity Assay

 

Test Item Concentration (mg/plate)	TA 98				TA 100
	- S9		+ S9		- S9		+ S9
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC	20.33	3.21	22.33	2.89	103.33	9.50	103.67	7.02
VC	20.33	2.52	21.00	1.73	100.67	4.73	102.00	8.00
T1 0.0390625	20.67	2.52	21.67	1.15	93.00	3.46	96.00	6.24
T2 0.078125	19.00	1.73	22.00	1.73	104.33	8.08	107.67	8.50
T3 0.15625	21.33	1.53	22.00	2.65	95.33	7.09	96.00	6.24
T4 0.3125	21.00	1.73	21.00	3.61	96.33	6.51	108.33	5.69
T5 0.625	20.33	2.52	21.67	2.31	96.00	6.24	100.33	8.50
T6 1.25	21.00	3.46	20.33	2.52	93.67	5.03	98.00	8.00
T7 2.5	19.33	1.15	21.67	1.15	109.67	6.66	91.33	2.52
T8 5.0	20.67	2.08	20.33	2.31	96.00	6.24	95.00	1.73
PC	401.33	18.01	389.00	26.06	703.00	10.54	703.33	15.53

Key:NC = Negative control,VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, NI = No Inhibition.

Note: Since there was no reduction in the revertant count and noinhibition of thebackground lawn observedat 5 mg/plate, Trial I (plate incorporation method) was performed with 5 mg/plate as thehighest concentration.

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 (absence of metabolic activation)
Benzo[a]pyrene	:	TA98 and TA100 (presence of metabolic activation)

Mean Revertant Colony Count -Trial I

 

Plate Incorporation Method [Absence of metabolic activation (-S9)]
Test Item Concentration (mg/plate)	TA 1535		TA 1537		TA 102
	Mean	SD	Mean	SD	Mean	SD
NC	14.00	1.73	6.67	1.15	235.67	7.77
VC	12.67	1.53	7.33	0.58	238.00	5.00
T1(0.3125)	11.33	1.53	5.33	1.15	228.00	22.61
T2(0.625)	12.33	2.31	5.00	0.00	227.00	9.85
T3(1.25)	12.33	3.21	5.00	1.73	219.33	3.51
T4(2.5)	15.00	1.73	5.67	1.15	219.33	13.32
T5(5.0)	13.33	2.52	5.33	1.15	215.33	10.21
PC	390.00	18.33	226.67	9.45	1562.67	42.67

 

Plate Incorporation Method [Presence of metabolic activation (+S9 10 % v/v S9 Mix)]
Test Item Concentration (mg/plate)	TA 1535		TA 1537		TA 102
	Mean	SD	Mean	SD	Mean	SD
NC	13.67	1.15	7.33	1.15	229.33	10.02
VC	14.67	1.15	7.00	1.00	214.00	14.00
T1(0.3125)	15.67	2.52	7.33	2.31	216.67	11.02
T2(0.625)	13.67	0.58	9.33	1.53	222.33	17.04
T3(1.25)	14.00	1.00	8.67	2.31	224.33	9.07
T4(2.5)	13.00	2.00	6.33	1.53	225.33	7.77
T5(5.0)	14.67	1.15	6.33	0.58	233.33	6.03
PC	385.33	27.47	230.67	12.01	1499.67	20.60

 

Key:NC = Negative control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.

 

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102(absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table3          Mean Revertant Colony Count -Trial II

 

Preincubation Method [Absence of metabolic activation]
Test Item Concentration (mg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC	21.67	2.89	98.33	8.39	14.00	1.73	9.00	1.00	231.67	5.69
VC	21.67	2.65	100.67	8.50	12.67	1.53	6.33	0.70	225.33	5.69
T1(0.3125)	19.67	1.00	105.67	6.81	14.33	0.58	9.00	1.42	215.67	4.63
T2(0.625)	20.67	3.79	98.00	4.58	11.67	1.15	7.67	1.21	234.33	5.28
T3(1.25)	19.67	1.00	104.67	7.64	13.00	2.65	5.33	0.84	207.00	7.57
T4(2.5)	22.00	2.00	102.67	10.02	12.00	1.73	6.67	1.05	217.67	13.01
T5(5.0)	22.00	2.65	92.33	4.16	12.00	1.73	7.33	1.16	215.00	14.00
PC	263.00	9.54	810.00	27.18	383.67	8.74	220.00	34.74	1726.00	6.11

 

Preincubation Method [Presence of metabolic activation (+S9 10% v/v S9 Mix)]
Test Item Concentration (mg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC	22.33	2.89	102.00	5.57	15.33	0.58	7.33	1.00	216.67	17.62
VC	21.00	2.65	103.67	11.37	13.67	0.58	6.67	0.91	224.00	13.00
T1(0.3125)	20.00	1.00	107.33	9.07	12.67	0.58	7.67	1.15	235.33	6.66
T2(0.625)	21.33	3.79	107.00	7.94	12.00	1.73	9.00	1.35	219.00	8.00
T3(1.25)	20.00	1.00	98.67	8.02	12.00	1.73	7.33	1.10	237.00	9.54
T4(2.5)	21.00	2.00	101.33	11.15	13.00	2.00	7.33	1.10	216.67	11.68
T5(5.0)	21.00	2.65	104.33	7.57	11.67	1.15	7.00	1.05	207.00	4.00
PC	368.00	9.54	822.33	31.97	375.00	11.53	212.67	31.90	1699.00	44.98

Key:NC = Negative control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.

 

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102(absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

 

Fold Increase

 

Trial I - Plate Incorporation Method
Test Item Concentration (mg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
NC	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
VC	1.00	1.02	0.90	1.10	1.01	1.00	1.02	0.90	1.10	1.01
T1(0.3125)	1.03	0.93	0.89	0.73	0.96	1.03	0.93	0.89	0.73	0.96
T2(0.625)	1.00	0.93	0.97	0.68	0.95	1.00	0.93	0.97	0.68	0.95
T3(1.25)	1.03	0.91	0.97	0.68	0.92	1.03	0.91	0.97	0.68	0.92
T4(2.5)	0.95	1.06	1.18	0.77	0.92	0.95	1.06	1.18	0.77	0.92
T5(5.0)	1.02	0.93	1.05	0.73	0.90	1.02	0.93	1.05	0.73	0.90
PC	19.74	6.80	30.79	30.91	6.57	19.74	6.80	30.79	30.91	6.57

 

Trial II – Preincubation Method
Test Item Concentration (mg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
NC	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
VC	0.94	0.98	1.07	0.95	0.93	0.94	0.98	1.07	0.95	0.93
T1(0.3125)	1.00	1.06	1.07	1.05	1.01	1.00	1.06	1.07	1.05	1.01
T2(0.625)	1.03	0.98	0.93	1.33	1.04	1.03	0.98	0.93	1.33	1.04
T3(1.25)	0.97	0.96	0.95	1.24	1.05	0.97	0.96	0.95	1.24	1.05
T4(2.5)	1.03	0.90	0.89	0.90	1.05	1.03	0.90	0.89	0.90	1.05
T5(5.0)	0.97	0.93	1.00	0.90	1.09	0.97	0.93	1.00	0.90	1.09
PC	18.52	6.90	26.27	32.95	7.01	18.52	6.90	26.27	32.95	7.01

Key:NC = Negative control,VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation.

S9 Efficiency Check- Summary

 

Summary of S9 efficiency check
	TA100		TA1535
	Mean	SD	Mean	SD
VC Distilled water (-S9)	100.67	4.73	12.67	1.53
VC Distilled water (+S9)	102.00	8.00	14.67	1.15
PC Benzo[a]pyrene (-S9)	103.67	7.02	12.67	1.53
PC Benzo[a]pyrene (+S9)	703.33	15.53	385.33	27.47

Key:VC = Vehicle control, PC = Positive control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.

Individual Revertant Colony Count-Trial I

 

Trial I- Plate Incorporation Method- Absence of Metabolic Activation
Test ItemConcentration (mg/plate)	R	TA1535	TA1537	TA102
NC	1	13	6	238
	2	16	8	242
	3	13	6	227
VC	1	11	7	233
	2	13	7	243
	3	14	8	238
T1  (0.3125)	1	13	6	203
	2	11	6	247
	3	10	4	234
T2  (0.625)	1	11	5	219
	2	11	5	238
	3	15	5	224
T3  (1.25)	1	11	4	216
	2	10	7	223
	3	16	4	219
T4 (2.5)	1	14	5	208
	2	14	5	216
	3	17	7	234
T5  (5)	1	11	6	211
	2	13	4	208
	3	16	6	227
PC	1	410	216	1518
	2	386	234	1603
	3	374	230	1567

Key:NC = Negative control, VC = Vehicle control, PC = Positive Control, R = Replicate, TI-T5 = Test Item concentration from lower to higher concentration.

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102(absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Individual Revertant Colony Count-Trial II

 

Trial II- Preincubation Method- Presence of Metabolic Activation
Test Item Concentration (mg/plate)	R	TA98	TA100	TA1535	TA1537	TA102
NC	1	18	94	16	8	238
	2	23	108	13	10	227
	3	24	93	13	9	230
VC	1	20	91	11	6	227
	2	21	107	13	6	219
	3	24	104	14	7	230
T1 (0.3125)	1	18	111	14	9	221
	2	18	108	15	10	219
	3	23	98	14	8	207
T2 (0.625)	1	21	103	11	8	233
	2	22	97	11	8	229
	3	19	94	13	7	241
T3 (1.25)	1	17	103	10	6	197
	2	23	113	14	4	208
	3	24	98	15	6	216
T4 (2.5)	1	21	93	11	6	207
	2	21	113	11	8	219
	3	17	102	14	6	227
T5 (5.0)	1	24	91	13	7	216
	2	25	89	13	7	209
	3	20	97	10	8	220
PC	1	378	780	386	219	1673
	2	391	833	374	234	1811
	3	364	817	391	207	1694

 

Key:NC = Negative control, VC = Vehicle control, PC = Positive Control, R = Replicate, TI-T5 = Test Item concentration from lower to higher concentration.

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102(absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

 

Applicant's summary and conclusion

Conclusions:

The registered substance, N-tert-butylacrylamide (CAS No. 107-58-4) is non-mutagenic (negative) as it does not induce (point) gene mutations by base-pair changes or frameshift in the histidine operon of Salmonella typhimurium tester strains (TA1537, TA1535, TA98, TA100 or TA102 neither in the present nor in the absence of S9 metabolic activation system.

Executive summary:

Ames test of-tert-butylacrylamide [TBAA] [CAS No.107-58-4] was conducted according to the plate incorporation and preincubation methods. This study was performed as per the OECD guideline No. 471 (Adopted: July 21 1997, Corrected: June 26 2020). Based on the solubility test, dimethyl sulfoxide (DMSO) was selected as a vehicle for the test item in the study. The mutagenic potential of N-tert-butylacrylamide [TBAA] [CAS No. 107-58-4] was tested in two independent experiments (Trial I and Trial II) and both in the presence (10% v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. The Test item was tested along with solvent-vehicle control (DMSO), negative (distilled water), and concurrent positive control substances in triplicates. A preliminary cytotoxicity assay was performed using the strains TA98 and TA100 with the following concentrations along with the vehicle, negative and positive controls both in the presence (10% v/v S9 mix) and absence of metabolic activation in triplicates: 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. In the tester strains, TA98 and TA100, no reduction in the revertant count and no inhibition in background lawn were observed at any of the concentrations, either in the presence (10% v/v S9 mix) or the absence of metabolic activation, when compared to the vehicle control data. Based on the preliminary cytotoxicity test results, the main study was performed with the following concentrations: Trial I and II: 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate

Trial I was performed according to the plate incorporation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item and the vehicle, negative and positive controls, were tested in triplicates. The Test item doses were selected using a concentration spacing factor of 2. No increase in the number of revertant colonies or inhibition in the background lawn was observed up to the highest concentration of 5mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data.

Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item and the vehicle, negative and positive controls, were tested in all tester strains in triplicates. There was no increase in the number of revertant colonies or inhibition of the background lawn up to the highest concentration of 5 mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. The numbers of revertant colonies of the vehicle, negative (spontaneous revertant colonies), and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test.

Based on the results of this study, it is concluded that N-tert-butylacrylamide [TBAA] [CAS No. 107-58-4] is non-mutagenic as it does not induce (point) gene mutations in the histidine operon by base-pair changes or frameshifts, in the presence and the absence of metabolic activation system, in the tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100, and TA102).