Acetylcysteine

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:

February 5, 2018 - March 15, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetylcysteine

EC Number:

210-498-3

EC Name:

Acetylcysteine

Cas Number:

616-91-1

Molecular formula:

C5H9NO3S

IUPAC Name:

N-acetylcysteine

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from beta-Naphthoflavone and Phenobarbital pretreated Wistar rats

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system. 5000 µg/plate was chosen as the appropriate maximum concentration.
+/- S9-Mix: 5.00;15.8; 50.0; 158; 500; 1580; 5000 µg/plate (1st Experiment)
+/- S9-Mix: 50.0; 158; 500; 1580; 5000 µg/plate (2nd Experiment)

Vehicle / solvent:

- Solvent used: ultrapure water
- Justification for choice of solvent: The selection of the solvent for this assay was based on the available information from a preliminary solubility test. Ultrapure water showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without S9 Mix, TA 98

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Mix, E.coli WP2 uvrA

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 Mix, TA 100, TA 1535

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 Mix, TA 1537

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 Mix, all tester strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h at 37°C

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test material was to be defined as positive or mutagenic in the assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA98,TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above-mentioned criteria are met

Statistics:

None

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:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summery of Results (1st Run)                                                                                

Metabolic Activation	Test material	Concentration  [µg/plate]	Revertants per plate (mean ± SD)
			TA 98	TA100	TA1535	TA1537	WP2 uvrA
Without Activation	H20		36 ±14	115 ± 9	18 ± 4	5 ± 3	28 ± 4
	Test item	5.00	40 ± 2	107 ±6	18 ± 6	5 ± 4	31 ± 7
		15.8	37 ± 3	104 ± 16	18 ± 6	7 ± 3	25 ± 10
		50.0	37 ± 2	115 ± 10	19 ± 4	5 ± 3	30 ± 6
		158	39 ± 7	121 ± 10	23 ± 2	4 ± 3	26 ± 14
		500	36 ± 9	122 ± 17	17 ± 3	8 ± 4	27 ± 4
		1580	361± 10	121 ± 9	20 ± 12	3 ± 2	31 ± 9
		5000	32 ± 8	112 ± 9	14 ± 3	5 ± 4	28 ± 7
	DAUN	2.00	1130 ±115
	NaN3	2.00		1386 ± 47	891 ± 21
	9-AA	50.0				1331 ± 341
	NQO	2.00					2038 ± 69
With Activation	H20		48 ± 14	127 ±5	21 ± 2	7 ± 3	34 ± 6
	Test item	5.00	38 ±7	125 ± 11	17 ± 4	7 ± 2	35 ± 5
		15.8	33 ± 2	138 ± 11	18 ± 9	8 ± 7	39 ± 5
		50.0	42 ± 8	130 ± 22	26 ± 10	6 ± 4	35 ± 8
		158	43 ± 10	129 ± 22	19 ± 2	10 ± 5	33 ± 9
		500	42 ± 2	110 ± 14	13 ± 4	6 ± 2	28 ± 9
		1580	46 ± 5	132 ± 34	15 ± 2	6 ± 3	30 ± 6
		5000	32 ± 3	130 ± 15	17 ± 3	7 ± 4	29 ± 6
	2-AA	2.00	1154 ± 122	2219 ± 392
	2-AA	5.00			100 ± 28	180 ±25
	2-AA	10.0					396 ±52

NaN3 = Sodium azide 2-AA = 2-Aminoanthracene 9-AA = 9-Aminoacridine DAUN = Daunomycin NQO = 4-Nitroquinoline-N-oxide     

 

Table 2: Summery of Results (2nd Run)    

Metabolic Activation	Test material	Concentration  [µg/plate]	Revertants per plate (mean ± SD)
			TA 98	TA100	TA1535	TA1537	WP2 uvrA
Without Activation	H20		39 ±5	127 ± 9	16 ± 3	6 ± 4	37 ± 8
	Test item	50.00	39 ± 9	120 ± 3	19 ± 4	5 ± 2	31 ± 10
		158	30 ± 2	116 ± 10	19 ± 4	6 ± 3	36 ± 5
		500	34 ± 4	128 ± 9	15 ±3	7 ± 3	40 ± 10
		1580	35 ± 7	106 ± 10	21 ± 7	6 ± 2	32 ± 10
		5000	34 ± 3	118 ± 0	18 ± 2	7 ± 2	31 ± 6
	DAUN	2.00	670 ± 26
	NaN3	2.00		1590 ± 37	922 ± 117
	9-AA	50.0				2681 ± 576
	NQO	2.00					1814 ± 141
With Activation	H20		42 ± 6	124 ±15	16 ± 7	10 ± 4	39 ± 9
	Test item	50.00	37 ± 6	111 ± 16	15 ± 8	13 ± 5	44 ± 4
		158	46 ± 4	111± 10	16 ± 6	6 ±1	36 ± 12
		500	42 ± 10	104 ± 16	10 ± 4	11± 5	30 ± 6
		1580	45 ± 11	108 ± 6	14 ± 5	8 ± 2	41 ± 6
		5000	43 ± 2	136 ± 21	10 ± 3	9 ± 1	25 ± 3
	2-AA	2.00	430 ± 20	698 ± 79
	2-AA	5.00			228 ± 96	200 ± 22
	2-AA	10.0					252 ± 22

NaN3 = Sodium azide2-AA = 2-Aminoanthracene9-AA = 9-AminoacridineDAUN = DaunomycinNQO = 4-Nitroquinoline-N-oxide     

The historical data have been obtained in experiments between 01/2017 and 12/2017.

Table 3: Historical data - Negative Controls

Strain	TA 98		TA 100
S9 Mix	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent
Total Plates	486	486	482	488
Number of values	90	90	89	90
Minimum	19	20	94	90
Maximum	52	58	159	172
Mean	36	42	118	123
Standard Deviation	7.1	7.3	12.1	13.4
Strain	TA1537		WP2 uvrA		TA 1538
S9Mix	Without	With	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent
Total Plates	394	394	434	433	44	44
Number of Values	68	68	77	77	11	11
Minimum	4	6	19	28	6	11
Maximum	11	16	44	47	15	21
Mean	8	10	30	37	12	15
Standard Deviation	15	23	49	43	2.9	2.7

Table 4: Historical data - Positive Controls

Strain	TA 98			TA100
S9 Mix	Without	With		Without	With
Compound	DAUN	2-AA		NaN3	2-AA
Total Plates	243	243		242	244
Number of Values	90	90		89	90
Minimum	68	112		821	437
Maximum	779	3015		2376	3429
Mean	243	738		1550	1386
Standard Deviation	134 2	508.4		213.6	724.3
Strain	TA 1537			WP2 uvrA		TA 1538
S9 Mix	Without		With	Without	With	Without	With
Compound	9-AA		2-AA	NQO	2-AA	2-NF	2-AA
Total Plates	203		203	217	217	22	22
Number of Values	70		70	77	77	11	11
Minimum	247		72	317	154	1087	461
Maximum	1485		705	2275	696	2511	1323
Mean	736		293	1677	353	1909	1038
Standard Deviation	284.9		161.7	381.9	129.2	470 3	285.3

Applicant's summary and conclusion

Conclusions:

The substance was not mutagenic under the described experimental conditions.

Executive summary:

A study according to OECD TG 471 was conducted to examine the mutagenic potential in an in vitro bacterial reverse mutation test employing Salmonella typhimurium TA 98, TA 100. TA 1535 and TA 1537 and Escherichia coli WP2 uvrA as indicator organisms.

The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/p-Naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used.Treatments of all tester strains were performed using test item formulations prepared in ultrapure water in the absence and in the presence of S9 mix using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls. The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values. The strain-specific positive controls, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide and 9-aminoacridine in the absence of S9 mix yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Following treatment with the substance, neither precipitation of the test material, nor toxicity to the bacteria was observed. Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix. According to the criteria for negative and positive results predetermined in the study, the substance was not mutagenic under the described experimental conditions.