N-2-hydroxyethylacetamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 14, 2017 to July 11, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction mass of N-2-hydroxyethylacetamide and N,O-diacetyl-2-aminoethanol

Molecular formula:

C4H9NO2 (amide) & C6H11NO3 (amido ester)

IUPAC Name:

Reaction mass of N-2-hydroxyethylacetamide and N,O-diacetyl-2-aminoethanol

Test material form:

liquid

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:

Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate
The maximum concentration was 5000 µg/plate (the maximum recommended dose level)

Vehicle / solvent:

The test substance was fully miscible in sterile distilled water at 50 mg/mL in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

Identity: Sterile distilled water, Supplier: Aguettant, Batch number (purity): 3012436 (N/A), Expiry: 10/2018

Negative solvent / vehicle controls:

yes

Remarks:

Identity: Sterile distilled water, Supplier: Aguettant, Batch number (purity): 3012436 (N/A), Expiry: 10/2018

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: Identity: 2-Aminoanthracene (2AA), CAS No.: 613-13-8

Details on test system and experimental conditions:

Test System and Supporting Information
Bacteria: The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
Strains: Genotype: Type of mutations indicated
TA1537: his C 3076; rfa-; uvrB-: frame shift
TA98: his D 3052; rfa-; uvrB-;R-factor: frame shift
TA1535: his G 46; rfa-; uvrB-: base-pair substitution
TA100: his G 46; rfa-; uvrB-;R-factor: base-pair substitution

Escherichia coli
Strain: Genotype: Type of mutations indicated
WP2uvrA: trp-; uvrA-: base-pair substitution
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000). The bacteria used in the test were obtained from: University of California, Berkeley, on culture discs, on 04 August 1995 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 6147883 03/21) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 44673 07/17 and 44674 07/17).

Study controls
The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate. The positive control substances used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix;
and The maximum dosing solution of the test substance in the absence of S9-mix only (test in singular only).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA

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:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test substance formulation was also shown to be sterile. These data are not given in the report. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data were for concurrent untreated control plates performed on the same day as the Mutation Test. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method).

Table1: Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Plate Incorporation)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
100		24		25		18		8
93	(91)	20	(20)	18	(24)	18	(18)	15	(12)
80		16		28		18		14

Experiment 2 (Pre-Incubation)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
91		21		34		26		15
96	(90)	12	(16)	29	(29)	19	(21)	10	(12)
83		16		25		19		12

Table 2 Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period		From: 26 June 2017					To: 29 June 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	114 93 118	(108) 13.4#	21 21 23	(22) 1.2	22 28 20		(23) 4.2	17 20 20	(19) 1.7	15 13 12	(13) 1.5
	1.5 µg	104 94 103	(100) 5.5	16 22 15	(18) 3.8	24 30 20		(25) 5.0	22 22 18	(21) 2.3	12 7 12	(10) 2.9
	5 µg	110 131 119	(120) 10.5	28 25 28	(27) 1.7	18 24 26		(23) 4.2	24 18 20	(21) 3.1	18 24 12	(18) 6.0
	15 µg	104 99 124	(109) 13.2	22 24 14	(20) 5.3	21 19 29		(23) 5.3	20 17 26	(21) 4.6	9 15 9	(11) 3.5
	50 µg	76 97 98	(90) 12.4	19 21 21	(20) 1.2	24 14 31		(23) 8.5	24 23 27	(25) 2.1	9 13 10	(11) 2.1
	150 µg	99 97 99	(98) 1.2	19 24 16	(20) 4.0	26 18 24		(23) 4.2	18 14 20	(17) 3.1	9 15 9	(11) 3.5
	500 µg	102 107 99	(103) 4.0	24 25 14	(21) 6.1	27 22 26		(25) 2.6	15 25 19	(20) 5.0	10 14 13	(12) 2.1
	1500 µg	78 82 100	(87) 11.7	27 18 25	(23) 4.7	25 31 28		(28) 3.0	23 20 25	(23) 2.5	17 11 13	(14) 3.1
	5000 µg	102 104 102	(103) 1.2	25 23 16	(21) 4.7	26 19 24		(23) 3.6	18 25 18	(20) 4.0	10 16 13	(13) 3.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		501 541 622	(555) 61.6	502 478 470	(483) 16.7	281 269 308		(286) 20.0	177 196 188	(187) 9.5	177 329 245	(250) 76.1

Table 3 Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 26 June 2017					To: 29 June 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	113 103 106	(107) 5.1#	22 20 22	(21) 1.2	18 26 24		(23) 4.2	27 20 25	(24) 3.6	11 10 12	(11) 1.0
	1.5 µg	92 103 113	(103) 10.5	21 11 19	(17) 5.3	17 20 22		(20) 2.5	27 21 26	(25) 3.2	8 8 10	(9) 1.2
	5 µg	93 92 95	(93) 1.5	31 13 22	(22) 9.0	16 22 20		(19) 3.1	32 26 31	(30) 3.2	8 10 11	(10) 1.5
	15 µg	110 93 92	(98) 10.1	20 19 17	(19) 1.5	23 24 16		(21) 4.4	22 22 22	(22) 0.0	9 7 8	(8) 1.0
	50 µg	93 91 95	(93) 2.0	22 20 17	(20) 2.5	23 14 22		(20) 4.9	16 24 23	(21) 4.4	6 8 9	(8) 1.5
	150 µg	91 111 87	(96) 12.9	16 23 23	(21) 4.0	19 20 20		(20) 0.6	20 22 20	(21) 1.2	14 4 10	(9) 5.0
	500 µg	109 89 100	(99) 10.0	20 21 21	(21) 0.6	17 27 16		(20) 6.1	32 32 13	(26) 11.0	11 10 14	(12) 2.1
	1500 µg	90 102 92	(95) 6.4	21 22 22	(22) 0.6	19 20 24		(21) 2.6	29 23 30	(27) 3.8	9 11 10	(10) 1.0
	5000 µg	85 100 82	(89) 9.6	24 15 21	(20) 4.6	21 18 18		(19) 1.7	24 25 30	(26) 3.2	11 11 11	(11) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		850 818 865	(844) 24.0	189 224 245	(219) 28.3	393 496 412		(434) 54.8	198 213 231	(214) 16.5	400 412 423	(412) 11.5

Table 4 Test Results: Experiment 2 – Without Metabolic Activation(Pre-Incubation)

Test Period		From: 07 July 2017					To: 10 July 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	103 94 115	(104) 10.5#	17 10 17	(15) 4.0	27 25 29		(27) 2.0	24 21 32	(26) 5.7	17 11 10	(13) 3.8
	15 µg	76 103 82	(87) 14.2	11 13 11	(12) 1.2	28 25 22		(25) 3.0	32 36 19	(29) 8.9	4 13 16	(11) 6.2
	50 µg	89 76 122	(96) 23.7	12 11 17	(13) 3.2	19 20 28		(22) 4.9	32 17 25	(25) 7.5	7 13 13	(11) 3.5
	150 µg	109 114 94	(106) 10.4	12 12 16	(13) 2.3	35 28 28		(30) 4.0	24 32 24	(27) 4.6	8 11 15	(11) 3.5
	500 µg	112 102 108	(107) 5.0	17 16 17	(17) 0.6	20 22 37		(26) 9.3	25 21 21	(22) 2.3	15 17 18	(17) 1.5
	1500 µg	119 103 118	(113) 9.0	18 14 14	(15) 2.3	34 22 31		(29) 6.2	17 26 25	(23) 4.9	12 10 11	(11) 1.0
	5000 µg	90 98 85	(91) 6.6	8 8 10	(9) 1.2	31 26 24		(27) 3.6	22 22 22	(22) 0.0	11 14 12	(12) 1.5
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		824 885 772	(827) 56.6	594 576 778	(649) 111.8	767 721 759		(749) 24.6	394 439 469	(434) 37.7	461 365 598	(475) 117.1

Table 5 Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 07 July 2017					To: 10 July 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	90 107 92	(96) 9.3#	11 14 13	(13) 1.5	40 36 27		(34) 6.7	22 35 22	(26) 7.5	17 12 13	(14) 2.6
	15 µg	88 110 106	(101) 11.7	11 11 9	(10) 1.2	24 29 30		(28) 3.2	23 24 22	(23) 1.0	17 10 18	(15) 4.4
	50 µg	100 104 120	(108) 10.6	11 11 11	(11) 0.0	21 39 37		(32) 9.9	30 15 19	(21) 7.8	10 8 10	(9) 1.2
	150 µg	106 131 92	(110) 19.8	12 7 11	(10) 2.6	26 22 33		(27) 5.6	30 26 27	(28) 2.1	9 18 9	(12) 5.2
	500 µg	102 100 100	(101) 1.2	8 11 11	(10) 1.7	32 31 31		(31) 0.6	24 23 27	(25) 2.1	10 11 12	(11) 1.0
	1500 µg	97 94 122	(104) 15.4	8 15 7	(10) 4.4	31 36 27		(31) 4.5	27 28 31	(29) 2.1	9 12 15	(12) 3.0
	5000 µg	86 84 86	(85) 1.2	11 6 11	(9) 2.9	28 29 32		(30) 2.1	33 27 35	(32) 4.2	15 14 13	(14) 1.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1182 1403 1213	(1266) 119.7	301 258 232	(264) 34.8	193 157 187		(179) 19.3	191 182 169	(181) 11.1	368 361 391	(373) 15.7

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames test, the substance was determined to be non-mutagenic with or without metabolic activation.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance (purity: assumed to be >96%) in Salmonella typhimurium and Escherichia coli according to OECD Guideline 471, EU Method B13/14 and the USA, EPA OCSPP Harmonized Guideline, Bacterial Reverse Mutation Test (Ames Test), in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the substance using both the Ames plate incorporation and the pre-incubation methods at up to eight dose levels (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 ranged from 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended to 15 to 5000 µg/plate, following the results of Experiment 1. Six test substance concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method. Under the study conditions, the test substance was determined to be non-mutagenic in the Ames test, with or without metabolic activation.