Onium compounds, morpholinium, 4-ethyl-4-soya alkyl, Et sulfates

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:

03 May - 02 Jun 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)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Onium compounds, morpholinium, 4-ethyl-4-soya alkyl, Et sulfates

EC Number:

263-167-0

EC Name:

Onium compounds, morpholinium, 4-ethyl-4-soya alkyl, Et sulfates

Cas Number:

61791-34-2

Molecular formula:

Not available for UVCB substance

IUPAC Name:

4-ethyl-4-(hexadecanoyloxy)morpholin-4-ium 4-ethyl-4-[(9E)-octadec-9-enoyloxy]morpholin-4-ium 4-ethyl-4-[(9E,12E)-octadeca-9,12-dienoyloxy]morpholin-4-ium triethyl sulfate

Method

Target gene:

"his operon" (for S. typhimurium strains) and "trp operon" (for E. coli strains)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphtha flavone

Test concentrations with justification for top dose:

Pre-experiment for toxicity testing: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate for all strains with and without metabolic activation

Based on the results of the pre-experiment, the following concentrations were chosen for the main experiments:
First experiment: 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate without metabolic activation for TA 100, TA 1535, TA 98 and TA 1537
First experiment: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for WP2uvrA
First experiment: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA 100, TA 98, TA 1537 and WP2uvrA
First experiment: 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate with metabolic activation for TA 1535

Second experiment: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate without metabolic activation for TA 100, TA 1535, TA 98 and TA 1537
Second experiment: 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate without metabolic activation for WP2uvrA
Second experiment: 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate with metabolic activation for TA 100, TA 98 and TA 1537
Second experiment: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate with metabolic activation for TA 1535
Second experiment: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate with metabolic activation for WP2uvrA

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was insoluble in sterile distilled water at 50 mg/mL but fully soluble in DMSO at the same concentration therefore DMSO was selected as the vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene: +S9: 1, 2 or 10 µg/plate for TA100, TA1535 and TA1537, and WP2uvrA, respectively

Details on test system and experimental conditions:

METHOD OF APPLICATION: First Experiment: in agar (plate incorporation); Second Experiment: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: Approximately 48 h (plate incorporation and preincubation method)

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Reduction of the bacterial background lawn (thinning)

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 item 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 item 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

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: at 150 µg/plate with and without S9 mix; Experiment 2: at 150 and 50 µg/plate with and without S9 mix, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: at 150 µg/plate with and without S9 mix; Experiment 2: at 15 and 5 µg/plate with and without S9 mix, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: at 500 µg/plate with and without S9 mix; Experiment 2: at 500 and 50 µg/plate with and without S9 mix, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: at 500 and 150 µg/plate with and without S9 mix; Experiment 2: at 150 and 15 µg/plate with and without S9 mix, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

Experiment 1: at 150 and 50 µg/plate with and without S9 mix, respectively; Experiment 2: at 150 and 5 µg/plate with and without S9 mix, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was not observed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The respective positive control values of all strains were within the range of the historical control data and thus the values were considered as valid (please refer to Table 3 under "any other information on results incl. tables).
- Negative (solvent/vehicle) historical control data: The solvent control values of TA98, WP2uvrA, TA 98 and TA 1537 were within the range of the historical control data and thus the values were considered as valid. Two counts of the solvent control for TA 1535 were just outside the historical control data, but still considered acceptable as the other solvent control counts were within expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9-mix (please refer to Table 3 under "any other information on results incl. tables).

Any other information on results incl. tables

Table 1: Results of Experiment I (plate incorporation)

Metabolic Activation	Test Group	Dose Level (µg/plate)	Revertant Colony Counts (Mean ±SD)
			TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Without Activation	Deionised water		81 ± 3.6	36 ± 4.6	29 ± 13.8	30 ± 2.1	11 ± 4.9
	Test substance	0.05	77 ± 11.5	36 ± 4.6	nt	20 ± 7.1	10 ± 4.0
		0.15	77 ± 5.1	30 ± 2.5	nt	17 ± 2.6	12 ± 7.5
		0.5	69 ± 2.9	28 ± 2.1	nt	24 ± 3.1	11 ± 2.6
		1.5	74 ± 8.0	26 ± 5.0	24 ± 12.2	26 ± 5.2	11 ± 6.7
		5	81 ± 13.6	28 ± 1.0	26 ± 6.1	21 ± 3.5	9 ± 5.0
		15	86 ± 9.0	25 ± 3.8	24 ± 2.5	28 ± 6.2	14 ± 4.5
		50	46 ± 11.8 (S)	27 ± 4.5	27 ± 5.1	15 ± 2.0 (S)	0 V
		150	0 T	28 ± 2.3 (S)	18 ± 4.0 (S)	0 T	0 T
		500	nt	0 V	0 V	nt	nt
		1500	nt	nt	0 V	nt	nt
		5000	nt	nt	0 V	nt	nt
	ENNG	3	425 ± 46.2	nt	nt	nt	nt
		5	nt	461 ± 45.9	nt	nt	nt
		2	nt	nt	704 ± 38.1	nt	nt
	4NQO	0.2	nt	nt	nt	269 ± 38.0	nt
	9AA	80	nt	nt	nt	nt	356 ± 71.6
With Activation	Deionised water		114 ± 7.5	23 ± 7.0	38 ± 7.5	34 ± 3.6	10 ± 2.5
	Test substance	0.05	nt	22 ± 2.1	nt	nt	nt
		0.15	nt	18 ± 3.5	nt	nt	nt
		0.5	nt	19 ± 2.6	nt	nt	nt
		1.5	108 ± 5.9	23 ± 3.1	34 ± 1.0	21 ± 5.3	9 ± 2.9
		5	111 ± 6.4	20 ± 1.2	39 ± 5.5	29 ± 10.5	9 ± 2.0
		15	105 ± 13.0	16 ± 4.0	41 ± 2.0	31 ± 6.1	10 ± 3.0
		50	93 ± 6.1	20 ± 3.2 (S)	38 ± 7.4	19 ± 1.5	10 ± 1.0
		150	0 V	0 V	29 ± 2.5	12 ± 6.0 (S)	0 V
		500	0 T	nt	0 V	0 T	0 T
		1500	0 T	nt	0 T	0 T	0 T
		5000	0 T	nt	0 T	0 T	0 T
	2-AA	1	594 ± 168.6	nt	nt	nt	nt
	2-AA	2	nt	205 ± 24.6	nt	nt	567 ± 11.2
	2-AA	10	nt	nt	462 ± 44.7	nt	nt
	BP	5	nt	nt	nt	281 ± 2.9	nt
	2-AA	2	nt	nt	nt	nt	nt

nt: not tested

2 -AA: 2 -Aminoanthracene

BP: Benzo(a)pyrene

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4 -NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

T: Toxic, no bacterial background lawn

V: Very weak bacterial background lawn

S: Sparse bacterial background lawn

Table 2: Results of Experiment 2 (preincubation)

Metabolic Activation	Test Group	Dose Level (µg/plate)	Revertant Colony Counts (Mean ±SD)
			TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Without Activation	Deionised water		86 ± 7.0	8 ± 1.7	26 ± 4.0	18 ± 8.1	10 ± 4.0
	Test substance	0.015	86 ± 9.1	10 ± 1.5	nz	18 ± 11.9	10 ± 1.2
		0.05	83 ± 12.2	9 ± 1.0	23 ± 1.5	14 ± 6.7	9 ± 2.6
		0.15	72 ± 4.9	8 ± 0.6	26 ± 8.1	10 ± 3.2	12 ± 2.1
		0.5	91 ± 10.1	10 ± 3.5	22 ± 3.8	16 ± 3.5	7 ± 3.1
		1.5	66 ± 9.2	8 ± 1.2 (S)	21 ± 0.6	13 ± 3.5	6 ± 3.1
		5	45 ± 9.6 (S)	0 V	27 ± 11.4	10 ± 4.0 (S)	5 ± 1.5 (S)
		15	39 ± 6.2 (S)	0 V	21 ± 9.3 (S)	0 V	5 ± 3.5 (S)
		50	0 V	0 T	0 V	0 T	0 V
		150	nt	nt	0 T	nt	nt
	ENNG	3	623 ± 54.2	nt	nt	nt	nt
		5	nt	1165 ± 362.7	nt	nt	nt
		2	nt	nt	572 ± 41.2	nt	nt
	4NQO	0.2	nt	nt	nt	136 ± 18.7	nt
	9AA	80	nt	nt	nt	nt	106 ± 4.4
With Activation	Deionised water		80 ± 10.5	12 ± 2.6	46 ± 3.8	24 ± 5.5	11 ± 5.0
	Test substance	0.015	nt	9 ± 0.6	nt	nt	nt
		0.05	74 ± 7.8	8 ± 0.0	nt	27 ± 2.6	11 ± 4.2
		0.15	70 ± 6.0	0 ± 1.7	35 ± 4.7	22 ± 1.7	10 ± 3.5
		0.5	81 ± 8.0	14 ± 1.5	44 ± 3.1	16 ± 1.5	9 ± 4.6
		1.5	85 ± 3.6	12 ± 0.6	42 ± 3.5	14 ± 0.6	7 ± 3.5
		5	74 ± 6.7	8 ± 1.7	40 ± 7.5	19 ± 2.9	9 ± 3.5
		15	87 ± 16.9	5 ± 1.7	37 ± 0.0	15 ± 0.6	8 ± 5.1
		50	76 ± 4.4	0 V	53 ± 3.2	22 ± 1.2	9 ± 1.5
		150	0 V	nt	39 ± 2.0	12 ± 2.0 (S)	0 V
		500	nt	nt	0 V	nt	nt
	2-AA	1	1173 ± 36.1	nt	nt	nt	nt
	2-AA	2	nt	136 ± 4.9	nt	nt	180 ± 17.9
	2-AA	10	nt	nt	247 ± 14.0	nt	nt
	BP	5	nt	nt	nt	204 ± 28.0	nt
	2-AA	2	nt	nt	nt	nt	nt

nt: not tested

2 -AA: 2 -Aminoanthracene

BP: Benzo(a)pyrene

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4 -NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

T: Toxic, no bacterial background lawn

V: Very weak bacterial background lawn

S: Sparse bacterial background lawn

Table 3: Historical Control Data (2016)

Strain		without S9 mix				with S9 mix
		Min	Max	Mean	SD	Min	Max	Mean	SD
TA 1535	Solvent control	8	34	15	4.5	8	39	15	5.2
	Positive control	84	2994	854	664.9	92	879	240	62.1
TA1537	Solvent control	3	24	12	3.5	4	23	13	3.5
	Positive control	95	1413	406	227.0	101	639	290	97.7
TA 98	Solvent control	8	49	21	4.8	12	51	25	5.7
	Positive control	100	449	486	49.8	96	4357	188	230.8
TA 100	Solvent control	63	154	90	14.5	66	156	93	14.3
	Positive control	21	2222	724	320.4	284	2994	854	664.9
WP2uvrA	Solvent control	10	53	22	5.8	13	53	27	6.3
	Positive control	107	1611	718	338.6	102	637	240	98.2