Bis(hydroxyethyl)methyloleylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro

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.

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 mutationin four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).

In addition to the above two supporting studies on a read-across substance are presented :-

1. The genetic toxicity of the test substance, Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9, was investigated in a study conducted according to OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

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).

Under the conditions of the study the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes and therefore is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Micronucleus Assay.

2. The genetic toxicity of the test substance, Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9, was investigated in a study conducted according to OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) / EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test) / EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test / International Workshop on Genetic Testing (IWGT) Recommendations.

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

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).

In the described mutagenicity test under the experimental conditions reported, the test item Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Link to relevant study records

Reference

Reference 1

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

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

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.

Target gene:

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

Species / strain / cell type:

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

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).

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.

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

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

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

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

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

 

 

 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

 

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).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011 - 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Justification for type of information:

Read-across data is presented from quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808-53-2, EC Number 276-038-9. This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

CAS No.: 71808-53-2 (old CAS No. 70750-47-9)
Batch No.: S001700
Physical state at RT: solid
Storage: at room temperature
Expiry Date: 28-04-2019

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

The following concentrations were used in the main experiments:
Experiment I:
without metabolic activation: 10, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450 and 500 µg/mL
with metabolic activation: 5, 10, 15, 25, 50, 75, 100 and 250 µg/mL
Experiment II:
without metabolic activation: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 and 70 µg/mL
The following concentrations were selected in the main experiment for the microscopic analyses:
Experiment I:
without metabolic activation: 10, 25 and 50 µg/mL
with metabolic activation: 25, 50, 75 and 100 µg/mL
Experiment II:
without metabolic activation: 35, 40, 45 and 50 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium (RPMI 1640)

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

cell culture medium

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

clastogenic control without metabolic activation

Positive controls:

other: colcemide

Remarks:

aneugenic control without metabolic activation

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

clastogenic control with metabolic activation

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation)
44 hours (Experiment II without metabolic activation)

FIXATION INTERVAL:
44 hours (Experiment I and II)

STAIN (for cytogenetic assays): acridine orange

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 2000 cells per concentration (1000 cells per culture)

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI)

Evaluation criteria:

There are several criteria for determining a positive result:
- a concentration-related increase or reproducible increase in the number of cells containing micronuclei
- a biologically relevant increase in the number of cells containing micronuclei for at least one of the dose groups, which is higher than the laboratory negative control range.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of cells with micronuclei above concurrent control levels, at any dose group.

Statistics:

Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determination of a positive response.
In the study the nonparametric chi test was performed to verify the results in the experiments.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Additional information on results:

The test item was dissolved in cell culture medium (RPMI medium). No precipitate of the test item was noted in any of the dose groups evaluated in experiment I and II.

Toxicity

In experiment I without metabolic activation no decrease of the relative CBPI below 70% was noted up to a test item concentration of 25 µg/mL. At a concentration of 50 µg/mL a relative CBPI of 42% was noted.In experiment I with metabolic activation no decrease of the relative CBPI below 70% was noted up to a test item concentration of 50 µg/mL. At a concentration of 75 µg/mL a relative CBPI of 66% and at a concentration of 100 µg/mL a relative CBPI of 49% was noted. In experiment II without metabolic activation no decrease of the relative CBPI below 70 % was noted up to a test item concentration of 40 µg/mL. At a concentration of 45 µg/mL a relative CBPI of 65% and at a concentration of 50 µg/mL a relative CBPI of 58% was noted.

Clastogenicity / Aneugenicity

In experiment I without metabolic activation the micronucleated cell frequency of the negative control (2.42%) was slightly above the range of the historical negative control data (0.4% – 2.3%). Since the upper limit was only slightly exceeded the data were considered as acceptable. The mean values of micronucleated cell frequencies found after treatment with the test item were 2.10% (10 µg/mL), 1.53% (25 µg/mL) and 1.40% (50 µg/mL). The micronucleated cell frequencies found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control and were within the range of the historical negative control data.

In experiment I with metabolic activation the micronucleated cell frequency of the negative control (1.40%) was within the historical control data of the negative control (0.6% – 2.1%). The mean values of micronucleated cell frequencies found after treatment with the test item were 0.85% (25 µg/mL), 0.90% (50 µg/mL), 1.35% (75 µg/mL) and 1.28% (100 µg/mL). The micronucleated cell frequencies found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control and were within the range of the historical negative control data.

In experiment II without metabolic activation the micronucleated cell frequency of the negative control (2.25%) was within the range of the historical negative control data (0.4% – 2.3%). The mean values of micronucleated cell frequencies found after treatment with the test item were 2.40% (35 µg/mL), 1.38% (40 µg/mL), 1.65% (45 µg/mL) and 2.08% (50 µg/mL). The micronucleated cell frequencies found in the groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. Moreover, all values were within the range of the historical negative control data except the value observed for a test item concentration of 35 µg/mL that slightly exceeded the upper range of the historical negative control data.

The nonparametric chi Test was performed to verify the results in the experiments. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in any of the dose groups of the test item evaluated in experiment I and II.

Ethylmethanesulfonate (EMS, 600 µg/mL) and Cyclophosphamide (CPA, 7.5 µg/mL) were used as clastogenic controls. Colcemide (0.08-0.8 µg/mL) was used as aneugenic control. All induced distinct and biologically relevant increases of the micronucleus frequency. This demonstrates the validity of the assay.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item C12-18-alkylbis(hydroxyethyl)methyl, chloride did not induce structural and/or numerical chromosomal damage in human lymphocytes.
Therefore, C12-18-alkylbis(hydroxyethyl)methyl, chloride is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Micronucleus Assay.

Executive summary:

The genetic toxicity of the test substance, Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9, was investigated in a study conducted according to OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

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).

Under the conditions of the study the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes and therefore is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Micronucleus Assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Justification for type of information:

Read-across data is presented from quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808-53-2, EC Number 276-038-9. This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Workshop on Genetic Testing (IWGT) Recommendations

Deviations:

no

GLP compliance:

yes

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Name: C12-18-alkylbis(hydroxyethyl)methyl, chloride
Product (common name): Ethoquad C/12
CAS No.: 71808-53-2 (old CAS No. 70750-47-9)
Batch No.: S001700
Physical State: solid
Storage Conditions: at room temperature
Expiry Date: 28-04-2019

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment I with and without metabolic activation: 0.1, 1, 10 and 50 µg/mL
Pre-experiment II without metabolic activation (24 h long-term exposure): 0.02, 0.2, 1, 5, 10 and 20 µg/mL

Experiment I
with metabolic activation: 1, 2.5, 5, 7.5, 10, 15, 20 and 25 µg/mL
without metabolic activation: 0.1, 0.5, 1, 2.5, 5, 7.5, 10 and 15 µg/mL

Experiment II
with metabolic activation: 0.3, 0.8, 2, 4, 8, 12, 16 and 22 µg/mL
without metabolic activation: 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2 and 3 µg/mL

Vehicle / solvent:

RPMI cell culture medium was used as solvent (RPMI + 5% HS).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 2.5 µg/mL

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 200 µg/mL and 300 µg/mL

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)

Evaluation criteria:

The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 106 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:

The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Executive summary:

The genetic toxicity of the test substance, Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9, was investigated in a study conducted according to OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) / EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test) / EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test / International Workshop on Genetic Testing (IWGT) Recommendations.

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

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).

In the described mutagenicity test under the experimental conditions reported, the test item Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)