Bis(hydroxyethyl)methyloleylammonium chloride

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:

28th November 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(hydroxyethyl)methyloleylammonium chloride

EC Number:

242-332-0

EC Name:

Bis(hydroxyethyl)methyloleylammonium chloride

Cas Number:

18448-65-2

Molecular formula:

C23H48NO2.Cl

IUPAC Name:

bis(2-hydroxyethyl)(methyl)[(9Z)-octadec-9-en-1-yl]azanium chloride

Test material form:

solid

Specific details on test material used for the study:

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride (CAS number 18448-65-2; EC number 242-332-0), batch number SC171117, was a yellow/amber/brown waxy solid. It was received on 06 December 2017 and stored at 15-25°C, protected from light and dessicated. Purity was stated as ca. 96%, mono-constituent substance and the expiry date was given as November 2018, see Certificate of Analysis.

Method

Target gene:

Histidine (in S. Tryhimurium); Tryptophan (in E-coli)

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:

Rat liver metabolising system (S-9).

Test concentrations with justification for top dose:

Experiment 1 : Ttreatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Bis (2-hydroxyethyl) oleyl methyl ammonium chloride at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity ranging from a slight thinning of the background bacterial lawn, with or without a marked reduction in revertant numbers, to a complete killing of the background bacteria was observed at 16 or 50 µg/plate and above (depending on the strain) in all strains in the absence of S-9, and at 50 µg/plate or above (depending on the strain) in all strains in the presence of S-9 except strain WP2 uvrA where it was observed at 500 µg/plate and above.

Experiment 2 : Ttreatments of all the tester strains were performed in the absence and in the presence of S-9. For all strains the maximum test concentration was reduced based on toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 0.05-50 µg/plate (TA98, TA100, TA1535 and TA1537 in the absence of S-9), 0.16-160 µg/plate (WP2 uvrA in the absence of S-9 and TA98, TA100, TA1535 and TA1537 in the presence of S-9) or 0.5–500 µg/plate (WP2 uvrA in the presence of S-9), in order to examine more closely those concentrations of Bis (2-hydroxyethyl) oleyl methyl ammonium chloride approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity

Vehicle / solvent:

Anhydrous analytical grade dimethyl sulphoxide (DMSO).

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

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 1-aminoanthracene

Details on test system and experimental conditions:

Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, absence or presence of S-9 mix.

Mutation Experiments
Bis (2-hydroxyethyl) oleyl methyl ammonium chloride was tested for mutation (and toxicity) in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) in two separate experiments at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution

followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted, incubated at 37±1°C and protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration Section 4.4).
As the results of Mutation Experiment 1 were negative, treatments in the presence of S-9 in Mutation Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Volume additions for the Mutation Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMSO) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or split in agar or a very thin bacterial lawn affected the accuracy of the automated counter.

Evaluation criteria:

Evaluation Criteria

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3 fold (in strains TA1535 and TA1537) the concurrent vehicle control values
2. The positive trends/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if either of the above criteria were met.

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:

valid

Untreated negative controls validity:

not examined

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:

valid

Untreated negative controls validity:

not examined

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:

valid

Untreated negative controls validity:

not examined

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:

valid

Untreated negative controls validity:

not examined

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:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

 

 

 Raw Plate Counts and calculated Mutagenicity Data, Experiment 1, without S-9

Strain

Compound

Conc Level (µg/plate)

Mean

Standard Deviation

Fold Increase

Revertant Number Per Plate

TA98	DMSO	-	26.3	9.5	-	26, 36, 17
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	22.3	2.5	0.8	20, 25, 22
		16	18.3	6.7	0.7	14 S, 26 S, 15 S
		50	-	-	-	- T, - T, - T
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	2NF	5	908.7	22.5	34.5	932, 907, 887

 

TA100	DMSO	-	83.7	5.0	-	79, 83, 89
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	87.3	9.0	1.0	92, 77, 93
		16	70.0	5.3	0.8	74 S, 72 S, 64 S
		50	-	-	-	- T, - T, - T
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	NaN3	2	627.3	58.8	7.5	673, 648, 561

 

TA1535	DMSO	-	8.0	2.6	-	5, 9, 10
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	12.7	3.8	1.6	10, 17, 11
		16	8.3	1.2	1.0	9 S, 9 S, 7 S
		50	-	-	-	- T, - T, - T
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	NaN3	2	454.7	7.0	56.8	462, 448, 454

 

TA1537	DMSO	-	18.0	1.7	-	19, 16, 19
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	16.0	1.0	0.9	17, 16, 15
		16	14.0	6.2	0.8	21 S, 12 S, 9 S
		50	-	-	-	- T, - T, - T
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	AAC	50	493.0	69.1	27.4	569, 476, 434

 

WP2uvrA	DMSO	-	21.7	4.9	-	16, 25, 24
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	20.0	3.5	0.9	22, 22, 16
		16	15.0	4.0	0.7	19, 15, 11
		50	7.3	4.2	0.3	6 S, 12 S, 4 S
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	NQO	2	363.3	88.7	16.8	418, 411, 261

 

 

 Raw Plate Counts and Calculated Mutagenicity Data, Experiment 1, with S-9

Strain

Compound

Conc Level (µg/plate)

Mean

Standard Deviation

Fold Increase

Revertant Number Per Plate

TA98	DMSO	-	37.3	8.4	-	47, 33, 32
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	38.7	8.3	1.0	36, 48, 32
		16	27.7	2.5	0.7	25, 30, 28
		50	42.0	4.6	1.1	47, 38, 41
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	B[a]P	10	323.7	14.6	8.7	340, 312, 319

 

TA100	DMSO	-	128.3	12.3	-	125, 118, 142
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	108.7	16.0	0.8	92, 110, 124
		16	96.3	24.5	0.8	111, 110, 68
		50	84.7	14.4	0.7	79 S, 101 S, 74 S
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	AAN	5	1690.0	141.4	13.2	1763, 1780, 1527

 

TA1535	DMSO	-	11.7	4.5	-	12, 16, 7
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	12.7	3.1	1.1	12, 10, 16
		16	17.3	2.9	1.5	19, 19, 14
		50	9.3	2.5	0.8	7 S, 12 S, 9 S
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	AAN	5	182.0	1.0	15.6	181, 182, 183

 

TA1537	DMSO	-	12.0	2.0	-	14, 10, 12
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	16.7	3.8	1.4	15, 21, 14
		16	11.7	4.9	1.0	15, 14, 6
		50	9.7	4.6	0.8	7 S, 7 S, 15 S
		160	-	-	-	- T, - T, - T
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	AAN	5	359.7	21.7	30.0	376, 368, 335

 

WP2uvrA	DMSO	-	25.3	3.1	-	26, 28, 22
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	5	23.0	9.5	0.9	22, 14, 33
		16	22.7	1.2	0.9	24, 22, 22
		50	25.3	2.3	1.0	24, 24, 28
		160	26.3	2.1	1.0	24, 27, 28
		500	-	-	-	- T, - T, - T
		1600	-	-	-	- T, - T, - T
		5000	-	-	-	- T, - T, - T
	AAN	15	227.3	7.6	9.0	224, 222, 236

 

   

 Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, without S-9

Strain

Compound

Conc Level (µg/plate)

Mean

Standard Deviation

Fold Increase

Revertant Number Per Plate

TA98	DMSO	-	19.3	6.8	-	27, 17, 14
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.05	20.0	10.0	1.0	10, 30, 20
		0.16	19.7	4.0	1.0	16, 19, 24
		0.5	21.7	9.5	1.1	31, 12, 22
		1.6	17.3	3.5	0.9	17, 21, 14
		5	21.7	3.8	1.1	20, 26, 19
		16	24.0	6.6	1.2	30, 17, 25
		50	-	-	-	- T, - T, - T
	2NF	5	1000.3	66.8	51.7	955, 969, 1077

 

TA100	DMSO	-	94.0	11.4	-	89, 86, 107
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.05	107.0	4.6	1.1	111, 102, 108
		0.16	97.0	15.1	1.0	81, 111, 99
		0.5	108.7	5.0	1.2	114, 104, 108
		1.6	106.3	12.5	1.1	106, 119, 94
		5	89.3	17.6	1.0	108, 87, 73
		16	84.0	13.9	0.9	93 S, 68 S, 91 S
		50	-	-	-	- T, - T, - T
	NaN3	2	645.7	44.7	6.9	594, 672, 671

 

TA1535	DMSO	-	10.7	2.9	-	9, 9, 14
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.05	15.7	3.5	1.5	16, 19, 12
		0.16	13.7	3.5	1.3	10, 14, 17
		0.5	10.3	1.2	1.0	9, 11, 11
		1.6	10.0	0.0	0.9	10, 10, 10
		5	10.0	5.6	0.9	15, 4, 11
		16	12.3	4.6	1.2	15 S, 7 S, 15 S
		50	-	-	-	- T, - T, - T
	NaN3	2	518.7	57.8	48.6	455, 533, 568

 

TA1537	DMSO	-	9.3	3.8	-	11, 5, 12
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.05	7.7	1.2	0.8	7, 9, 7
		0.16	12.7	2.3	1.4	14, 14, 10
		0.5	16.3	3.2	1.8	20, 15, 14
		1.6	11.3	0.6	1.2	12, 11, 11
		5	6.3	2.5	0.7	4, 9, 6
		16	12.0	4.4	1.3	10 S, 17 S, 9 S
		50	-	-	-	- T, - T, - T
	AAC	50	353.3	51.3	37.9	310, 410, 340

 

WP2uvrA	DMSO	-	18.0	4.4	-	15, 16, 23 M B
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.16	17.3	1.5	1.0	17, 19, 16
		0.5	20.7	6.0	1.1	27, 20, 15
		1.6	20.0	7.9	1.1	29, 17, 14
		5	19.0	5.3	1.1	15, 17, 25
		16	25.0	9.5	1.4	35, 16, 24
		50	9.7	2.5	0.5	12, 7, 10
		160	-	-	-	- T, - T, - T
	NQO	2	437.7	58.3	24.3	505, 404, 404

 

  Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, with S-9

Strain

Compound

Conc Level (µg/plate)

Mean

Standard Deviation

Fold Increase

Revertant Number Per Plate

TA98	DMSO	-	22.3	11.8	-	36, 16, 15
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.16	31.7	8.0	1.4	24, 40, 31
		0.5	27.3	11.9	1.2	41, 22, 19
		1.6	20.3	5.8	0.9	17, 27, 17
		5	31.3	9.0	1.4	40, 32, 22
		16	30.0	4.6	1.3	29, 35, 26
		50	28.7	4.7	1.3	25, 27, 34
		160	27.3	2.3	1.2	30 S, 26 S, 26 S
	B[a]P	10	433.3	60.9	19.4	412, 502, 386

 

TA100	DMSO	-	99.0	8.9	-	92, 96, 109
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.16	100.3	5.8	1.0	107, 97, 97
		0.5	114.0	12.1	1.2	112, 127, 103
		1.6	98.3	4.0	1.0	94, 99, 102
		5	118.3	16.2	1.2	137, 109, 109
		16	106.3	20.5	1.1	89, 101, 129
		50	101.0	10.4	1.0	96, 113, 94
		160	-	-	-	- T, - T, - T
	AAN	5	1739.7	42.6	17.6	1758, 1691, 1770

 

TA1535	DMSO	-	11.7	3.8	-	9, 10, 16
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.16	12.7	2.1	1.1	12, 11, 15
		0.5	9.7	2.5	0.8	7, 10, 12
		1.6	13.7	1.5	1.2	12, 14, 15
		5	16.3	4.5	1.4	21, 12, 16
		16	15.0	4.6	1.3	11, 14, 20
		50	10.3	0.6	0.9	11, 10, 10
		160	7.3	2.1	0.6	9 M V, 8 M V, 5 M V
	AAN	5	165.0	13.5	14.1	179, 164, 152

 

TA1537	DMSO	-	13.0	1.7	-	14, 14, 11
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.16	15.0	4.0	1.2	11, 15, 19
		0.5	15.3	1.5	1.2	17, 14, 15
		1.6	11.3	2.5	0.9	14, 9, 11
		5	11.3	5.0	0.9	6, 16, 12
		16	10.0	1.7	0.8	9, 9, 12
		50	12.0	6.1	0.9	5, 16, 15
		160	5.0	1.7	0.4	3 M V, 6 M V, 6 M V
	AAN	5	157.7	15.6	12.1	143, 174, 156

 

WP2uvrA	DMSO	-	28.0	3.5	-	30, 24, 30
	Bis (2-hydroxyethyl) oleyl methyl ammonium chloride	0.5	29.3	4.5	1.0	29, 34, 25
		1.6	25.0	9.8	0.9	22, 36, 17
		5	45.3	18.4	1.6	50, 25, 61
		16	32.3	2.3	1.2	31, 35, 31
		50	43.3	6.4	1.5	48, 36, 46
		160	32.7	5.1	1.2	37, 27, 34
		500	-	-	-	- T, - T, - T
	AAN	15	124.3	13.5	4.4	111, 124, 138

 

Applicant's summary and conclusion

Conclusions:

Bis(2-hydroxyethyl)methyloleylammonium chloride, CAS No. 18448-65-2, EC No. 242-332-0, did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a rat liver metabolic activation system (S-9) in the OECD 471, Bacterial Reverse Phase Mutation study : Negative.

Executive summary:

A study was performed to evaluate the mutagenic potential of the test material (Bis(2-hydroxyethyl)methyloleylammonium chloride, CAS No. 18448-65-2, EC No. 242-332-0).

The study was conducted according to OECD guideline 471 and in accordance with GLP.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

These test conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) and a toxic concentration, in the absence and in the presence of a rat liver metabolic activation system (S-9) in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).

Under the conditions of the study it was concluded that Bis(2-hydroxyethyl)methyloleylammonium chloride did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).