Quaternary ammonium compounds, C12-14-alkyltrimethyl, Me sulfates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 05 December, 2002 to 13 January, 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80°C, until use)

Test concentrations with justification for top dose:

Preliminary test concentrations:10, 100, 500, 1000, 2500 and 5000 μg/plate for both with and without S9-mix

Main test concentrations: Experiments without S9 mix:
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for all tester strains in the first experiment,
• 0.41, 1.23, 3.7, 11.1 and 33.3 μg/plate, for all tester strains in the second experiment.

Experiments with S9 mix:
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for all tester strains in the first experiment,
• 3.7, 11.1, 33.3, 100 and 200 μg/plate, for all tester strains in the second experiment, except for the TA 1537 strain,
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for the TA 1537 strain in the second experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO), batch No. K30379650 214 (Merck Eurolab, Fontenay-Sous-Bois, France).

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1 μg/plate: for TA 1535 and TA 100 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 μg/plate: for TA 1537 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

0.5 μg/plate: for TA 98 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 μg/plate: for TA 102 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-anthramine

Remarks:

2 μg/plate: for TA 1535, TA1537, TA 98 and TA 100 in the presence of S9 mix and 10 μg/plate: TA 102 in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation method) as well as preincubation method

DURATION
- Incubation period: 48 to 72 h at approximately 37°C

NUMBER OF REPLICATIONS: Three/dose

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies were counted, with an automatic counter (Artek counter, model 880, OSi, 75015 Paris, France or cardinal counter, Perceptive instruments, Suffolk CB9 7 BN, UK)

Evaluation criteria:

- Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.

- Acceptance criteria
This study is considered valid if the following criteria are fully met:
• the number of revertants in the vehicle controls is consistent with the historical data of the testing facility (appendix 1),
• the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

- Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 33.3 μg/plate (experiments without S9). A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 100 μg/plate (with S9).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test: Yes

Refer to the attached report (pages 15 to 19) for full details

Conclusions:

Under the study conditions, the test substance is not considered to be mutagenic in the presence and absence of exogenous metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the read across substance, C12-14 ADMAES, according to OECD 471 Guideline and EU Method B.13/14, in compliance with GLP. The test substance was examined for mutagenic activity in five strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, using the plate incorporation and pre-incubation methods. The studies were performed in the absence and presence of metabolic activation (S9-mix). The concentrations ranged from 1.23 to 100 µg/plate in first experiment and 0.41 to 200 µg/plate in second experiment. No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test substance at ≥100 µg/plate. Under the study conditions, the test substance was not considered to be mutagenic in the presence and absence of exogenous metabolic activation (Haddouck, 2003).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 11, 2017 to October 24, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation (S-9) from Aroclor 1254-induced rats

Test concentrations with justification for top dose:

Range finding test (1) concentrations: 18.14, 30.23, 50.39, 83.98, 140.0, 233.3, 388.8, 648.0, 1080, 1800, 3000 and 5000 µg/mL
Range finding test (2) concentrations: 0.3628, 0.6047, 1.008, 1.680, 2.799, 4.666, 7.776, 12.96, 21.60, 36.00, 60.00 and 100.0 µg/mL
Micronucleus experiment (3+21 Hour Treatments -S-9): 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7.5 and 10 µg/mL
Micronucleus experiment (3+21 Hour Treatments +S-9): 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7.5, 10 and 15 µg/mL
Micronucleus experiment (24+24 Hour Treatments -S-9): 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 4 and 5 µg/mL

Dose range for micronucleus tests were selected based on the cytotoxicity results of the reange finding study 1 and 2.

Vehicle / solvent:

Vehicle: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: See remarks

Details on test system and experimental conditions:

Test system: The test system was suitably labelled (using a colour-coded procedure) to clearly identify the study number, assay type, experiment number, treatment time, donor sex, test substance concentration (if applicable), positive and vehicle controls, in the absence and presence of S-9 mix.

Test substance solution preparation: Preliminary solubility data indicated that test substance was soluble in water for irrigation (purified water) at concentrations up to at least 202.84 mg/mL. The solubility limit in culture medium was in excess of 20284 μg/mL, as indicated by the absence of precipitation 24 hours after test article addition. A maximum concentration of 5000 μg/mL was selected for the initial cytotoxicity range finding experiment, in order that treatments were performed up to the maximum recommended concentration for UVCB compounds, according to current regulatory guidelines. Test article stock solutions were prepared by formulating under subdued lighting in purified water, with the aid of vortex mixing and ultrasonication, to give the maximum required treatment concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 mm filter, 0.2 μm pore size) and subsequent dilutions were made using purified water. The test article solutions were protected from light and used within approximately 4 hours of initial formulation.

Metabolic activation system: The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from molecular toxicology incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).

Blood cultures: Blood from two healthy, non-smoking male volunteers from a panel of donors: No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 h. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes on the day prior to culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 7.6 mL pre-warmed (in an incubator set to 37±1°C) HEPES-buffered RPMI (Roswell Park Memorial Institute) medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix/KCl and the test substance in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1°C for approximately 48 h and rocked continuously.

Evaluation criteria:

For valid data, the test substance was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of binucleate cells with micronuclei (MNBN) cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).

The test substance was considered positive in this assay if all of the above criteria were met.

The test substance was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result.

Statistics:

Fisher's exact test used for the evaluation of statistical significance

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

Treatment of cells with test substance in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly higher (at the p≤0.05 level) than those observed in concurrent vehicle controls for all concentrations analysed (3+21 hour treatments in the absence and presence of S-9 and 24+24 hour treatments in the absence of S-9). The MNBN cell frequencies of all test substance treated cultures fell within the normal ranges. No test substance related increases in cells with NPBs were observed.

Results

Selection of Concentrations for Micronucleus Analysis

The results of the RI determinations from the cytotoxicity Range-Finder Experiment were as follows:

Data for 3+21 H Treatments -S-9, Range-Finder - Male Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	32	155	13	200	0.91
	B	38	145	17	200	0.90
	Total	70	300	30	400	0.90	-
18.14	A	177	23	0	200	0.12	20
30.23	A	NE	-	-	-	-	-
50.39	A	NE	-	-	-	-	-
83.98	A	T	-	-	-	-	- E
140.0	A	T	-	-	-	-	- E
233.3	A	NM	-	-	-	-	- E H
388.8	A	NM	-	-	-	-	- E H
648.0	A	NM	-	-	-	-	- E H
1080	A	NM	-	-	-	-	- E H
1800	A	NM	-	-	-	-	- E H
3000	A	NM	-	-	-	-	- E H
5000	A	NM	-	-	-	-	- E H

 

Data for 3+21 H Treatments +S-9, Range-Finder - Male Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	25	146	29	200	1.02
	B	37	147	16	200	0.90
	Total	62	293	45	400	0.96	-
18.14	A	153	48	0	201	0.24	75
30.23	A	NE	-	-	-	-	-
50.39	A	NE	-	-	-	-	-
83.98	A	T	-	-	-	-	- E
140.0	A	T	-	-	-	-	- E
233.3	A	NM	-	-	-	-	- E H
388.8	A	NM	-	-	-	-	- E H
648.0	A	NM	-	-	-	-	- E H
1080	A	NM	-	-	-	-	- E H
1800	A	NM	-	-	-	-	- E H
3000	A	NM	-	-	-	-	- E H
5000	A	NM	-	-	-	-	- E H

NE = Not evaluated - no scoreable cells

NM = Not made

E = Precipitation observed at the end of treatment incubation

H = Precipitation observed at harvest

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

Data for 24+24 H Treatments -S-9, Range-Finder – Male Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	15	150	35	200	1.10
	B	14	130	56	200	1.21
	Total	29	280	91	400	1.16	-
18.14	A	NE	-	-	-	-	-
30.23	A	T	-	-	-	-	-
50.39	A	T	-	-	-	-	- E H
83.98	A	T	-	-	-	-	- E H
140.0	A	T	-	-	-	-	- E H
233.3	A	NM	-	-	-	-	- E H
388.8	A	NM	-	-	-	-	- E H
648.0	A	NM	-	-	-	-	- E H
1080	A	NM	-	-	-	-	- E H
1800	A	NM	-	-	-	-	- E H
3000	A	NM	-	-	-	-	- E H
5000	A	NM	-	-	-	-	- E H

NE = Not evaluated - no scoreable cells

NM = Not made

E = Precipitation observed at the end of treatment incubation

H = Precipitation observed at harvest

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

No marked changes in osmolality or pH were observed at the highest concentration tested in the initial range finder (5000 μg/mL) as compared to the concurrent vehicle controls (individual data not reported).

As complete/extreme toxicity was observed at all timepoints, appropriate concentrations could not be selected for the micronucleus experiment based on these data. In order to select concentrations for the micronucleus experiment, an additional range finder treatment was performed using a lower concentration range (Range-Finder Experiment 2).

Range finder experiment 2 were as follows:

Data for 3+21 H treatments -S-9, range finder 2 male donors

Treatment (μg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	29	159	12	200	0.92
	B	34	156	10	200	0.88
	Total	63	315	22	400	0.9
0.3628	A	34	154	12	200	0.89	1
0.6047	A	34	156	10	200	0.88	2
1.008	A	51	144	5	200	0.77	14
1.68	A	50	143	7	200	0.79	13
2.799	A	80	117	3	200	0.62	31
4.666	A	114	86	0	200	0.43	52
7.776	A	147	53	0	200	0.27	70
12.96	A	186	14	0	200	0.07	92
21.6	A	186	14	0	200	0.07	92
36	A	192	8	0	200	0.04	96
60	A	179	21	0	200	0.11	88
100	A	`-	`-	`-	`-	`-	E H

Data for 3 +21 H treatments +S-9, range finder 2 – male donors

Treatment (μg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	27	158	15	200	0.94
	B	43	133	24	200	0.91
	Total	70	291	39	400	0.92
0.3628	A	21	171	8	200	0.94	0
0.6047	A	34	140	26	200	0.96	0
1.008	A	21	161	18	200	0.99	0
1.68	A	34	154	12	200	0.89	4
2.799	A	49	145	6	200	0.79	15
4.666	A	87	113	0	200	0.57	39
7.776	A	114	86	0	200	0.43	53
12.96	A	165	35	0	200	0.18	81
21.6	A	189	11	0	200	0.06	94
36	A	191	9	0	200	0.05	95
60	A	190	10	0	200	0.05	95 E
100	A	NE	-	-	-	-	`-E H

Data for 24+24 H treatments -S-9, range finder 2 male donors

Treatment (μg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	19	150	31	200	1.06
	B	23	143	34	200	1.06
	Total	42	293	65	400	1.06	`-
0.3628	A	27	156	17	200	0.95	10
0.6047	A	38	150	12	200	0.87	18
1.008	A	35	154	11	200	0.88	17
1.68	A	73	124	3	200	0.65	39
2.799	A	115	85	0	200	0.43	60
4.666	A	161	39	0	200	0.2	82
7.776	A	180	20	0	200	0.1	91
12.96	A	179	21	0	200	0.11	90
21.6	A	163	37	0	200	0.19	83
36	A	174	26	0	200	0.13	88
60	A	NE	-	-	-	-	`-E H
100	A	NE	-	-	-	-	`-E H

NE = Not evaluated - no scoreable cells

E = Precipitation observed at the end of treatment incubation

H = Precipitation observed at harvest

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

The results of the RI determinations from the micronucleus experiment were as follows:

Data for 3+21 H Treatments -S-9, Micronucleus Experiment - Male Donors

Treatment (μg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	95	359	46	500	0.9
	B	87	365	48	500	0.92
	C	88	373	39	500	0.9
	D	79	384	37	500	0.92
	Total	349	1481	170	2000	0.91	-
0.5	A	138	344	18	500	0.76
	B	93	379	28	500	0.87
	Total	231	723	46	1000	0.82	10#
1	A	137	348	15	500	0.76
	B	139	344	17	500	0.76
	Total	276	692	32	1000	0.76	17
2	A	142	343	15	500	0.75
	B	147	342	11	500	0.73
	Total	289	685	26	1000	0.74	19
2.5	A	186	306	8	500	0.64
	B	188	306	6	500	0.64
	Total	374	612	14	1000	0.64	30#
3	A	190	303	7	500	0.63
	B	212	281	7	500	0.59
	Total	402	584	14	1000	0.61	33
3.5	A	205	291	4	500	0.6
	B	199	296	5	500	0.61
	Total	404	587	9	1000	0.61	34
4	A	256	243	1	500	0.49
	B	297	202	1	500	0.41
	Total	553	445	2	1000	0.45	51#
4.5	A	293	204	3	500	0.42
	B	258	239	3	500	0.49
	Total	551	443	6	1000	0.46	50
5	A	297	201	2	500	0.41
	B	287	210	3	500	0.43
	Total	584	411	5	1000	0.42	54
6	A	317	183	0	500	0.37
	B	358	141	1	500	0.29
	Total	675	324	1	1000	0.33	64
7.5	A	374	123	3	500	0.26
	B	362	138	0	500	0.28
	Total	736	261	3	1000	0.27	71
10	A	401	99	0	500	0.2
	B	393	107	0	500	0.21
	Total	794	206	0	1000	0.21	77
MMC, 0.2	A	176	304	20	500	0.69
	B	119	369	12	500	0.79
	Total	295	673	32	1000	0.74	19#
MMC, 0.3	A	166	326	8	500	0.68
	B	168	317	15	500	0.69
	Total	334	643	23	1000	0.69	24

 

Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index # Highlighted concentrations selected for analysis

 

Data for 3+21 H Treatments +S-9, Micronucleus Experiment - Male Donors

Treatment (μg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	71	372	57	500	0.97
	B	52	384	64	500	1.02
	C	64	370	66	500	1
	D	92	379	29	500	0.87
	Total	279	1505	216	2000	0.97	-
1	A	71	373	56	500	0.97
	B	82	384	34	500	0.9
	Total	153	757	90	1000	0.94	3#
2	A	111	349	40	500	0.86
	B	87	373	40	500	0.91
	Total	198	722	80	1000	0.88	9
4	A	151	330	19	500	0.74
	B	184	312	4	500	0.64
	Total	335	642	23	1000	0.69	29#
5	A	199	295	6	500	0.61
	B	230	265	5	500	0.55
	Total	429	560	11	1000	0.58	40
6	A	211	282	7	500	0.59
	B	238	256	6	500	0.54
	Total	449	538	13	1000	0.56	42
6.5	A	239	255	6	500	0.53
	B	241	252	7	500	0.53
	Total	480	507	13	1000	0.53	45
7	A	262	236	2	500	0.48
	B	273	224	3	500	0.46
	Total	535	460	5	1000	0.47	51#
7.5	A	311	185	4	500	0.39
	B	256	239	5	500	0.5
	Total	567	424	9	1000	0.44	54
8	A	309	189	2	500	0.39
	B	295	202	3	500	0.42
	Total	604	391	5	1000	0.4	59
9	A	299	199	2	500	0.41
	B	324	172	4	500	0.36
	Total	623	371	6	1000	0.38	60
10	A	342	157	1	500	0.32
	B	328	170	2	500	0.35
	Total	670	327	3	1000	0.33	66
15	A	424	76	0	500	0.15
	B	436	63	1	500	0.13
	Total	860	139	1	1000	0.14	85
CPA, 2	A	124	366	10	500	0.77
	B	124	366	10	500	0.77
	Total	248	732	20	1000	0.77	20
CPA, 3	A	162	330	8	500	0.69
	B	173	323	4	500	0.66
	Total	335	653	12	1000	0.68	30#

Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index # Highlighted concentrations selected for analysis

Data for 24+24 H Treatments -S-9, Micronucleus Experiment - Male Donors

Treatment (μg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	56	391	53	500	0.99
	B	62	389	49	500	0.97
	C	81	365	54	500	0.95
	D	68	379	66	513	1
	Total	267	1524	222	2013	0.98	-
0.5	A	83	370	47	500	0.93
	B	93	374	33	500	0.88
	Total	176	744	80	1000	0.9	8#
1	A	136	348	16	500	0.76
	B	121	361	18	500	0.79
	Total	257	709	34	1000	0.78	21
1.25	A	140	344	16	500	0.75
	B	188	301	11	500	0.65
	Total	328	645	27	1000	0.7	29#
1.5	A	210	277	13	500	0.61
	B	200	297	3	500	0.61
	Total	410	574	16	1000	0.61	38
1.75	A	253	246	1	500	0.5
	B	232	266	2	500	0.54
	Total	485	512	3	1000	0.52	47
2	A	272	223	5	500	0.47
	B	268	231	1	500	0.47
	Total	540	454	6	1000	0.47	52#
2.25	A	311	188	1	500	0.38
	B	259	240	1	500	0.48
	Total	570	428	2	1000	0.43	56
2.5	A	282	215	3	500	0.44
	B	326	173	1	500	0.35
	Total	608	388	4	1000	0.4	59
2.75	A	353	146	1	500	0.3
	B	322	179	6	507	0.38
	Total	675	325	7	1007	0.34	66
3	A	365	132	3	500	0.28
	B	347	151	2	500	0.31
	Total	712	283	5	1000	0.29	70
4	A	416	82	2	500	0.17
	B	417	83	0	500	0.17
	Total	833	165	2	1000	0.17	83
5	A	463	35	2	500	0.08
	B	452	48	0	500	0.1
	Total	915	83	2	1000	0.09	91
VIN, 0.04	A	238	171	91	500	0.71
	B	218	177	105	500	0.77
	Total	456	348	196	1000	0.74	24#
VIN, 0.06	A	221	155	134	510	0.83
	B	220	155	125	500	0.81
	Total	441	310	259	1010	0.82	16

Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index # Highlighted concentrations selected for analysis

 

Micronucleus analysis

Raw data

The raw data for the observations on the test substance plus positive and vehicle controls are retained by Covance Laboratories Ltd.

Validity of study

1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of

MNBN cell frequency) between replicate cultures.

2. The frequency of MNBN cells in vehicle controls fell within the normal ranges.

3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range.

4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.

5. The maximum concentration analysed under each treatment condition met the criteria specified in selection of concentrations for micronucleus analysis (Micronucleus Experiment Only) (see above).

Conclusions:

Under study conditions, the test substance was determined to be non-genotoxic with or without metabolic activation in the micronucleus assay.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, using human peripheral blood lymphocytes, according to the OECD Guideline 487, in compliance with GLP. Test substance was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test substance was formulated in purified water and tested in the following concentrations in range finding study: 0, 18.14, 30.23, 50.39, 83.98, 140.0, 233.3, 388.8, 648.0, 1080, 1800, 3000 and 5000 µg/mL. As complete/extreme toxicity was observed at all timepoints, appropriate concentrations could not be selected for the micronucleus experiment based on the study. In order to select concentrations for the micronucleus experiment, an additional range finder experiment was performed using a lower concentration range (range finder experiment 2): 0, 0.3628, 0.6047, 1.008, 1.680, 2.799, 4.666, 7.776, 12.96, 21.60, 36.00, 60.00 and 100.0 µg/mL. The results of this experiment were used to select suitable maximum concentrations for the micronucleus experiment. Concentrations tested for the micronucleus experiments were as follows: 0, 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7.5 and 10 µg/mL (3 +21 h treatment in absence of S-9) and 0, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7.5, 10 and 15 µg/mL (3 +21 h treatment in presence of S-9) and 0, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 4 and 5 µg/mL (24 +24 h treatment in the absence of S-9). These three assayswith the test substance resulted in frequencies of MNBN cells which were similar to and not significantly higher (at the p≤0.05 level) than those observed in the concurrent vehicle controls and fell within the normal ranges.The test substance was thus considered as non-genotoxic under this condition by the study author. The acceptance criteria for the assay were fulfilled. The study is thus considered as valid. Under study conditions, the test substance was determined to be non-genotoxic with or without metabolic activation in the micronucleus assay (Hargreaves, 2018).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Mouse lymphoma assay

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 15 October, 2001 to 05 March, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

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:

other: Commission directive 2002/32/EC and U.K Environmental Mutagen Society

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

For Experiment 1: 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL for both with and without S9-mix
For Experiment 2: 0.313, 0.625, 0.938, 1.25, 2.5 and 5 µg/mL (without S9 mix) and 2.5, 5, 10, 15, 20 and 30 µg/mL (with S9-mix)

Vehicle / solvent:

RPMI 1640 without serum

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

in the absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in the presence of S9-mix

Details on test system and experimental conditions:

After a preliminary toxicity test, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L was selected for the first experiment. The entire experiment was repeated to confirm the results of the first experiment. 3 h exposure were used both with and without S9 mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the dose range was 2.5 to 30 µg/L with S9 mix and 0.313 to 5 µg/L without S9 mix.

Evaluation criteria:

For a test substance to give a significant result then two or more of the following criteria should be met:
a) A greater than three-fold increase in the mutant frequency/survivor over the negative control value.
b) A dose-related increase in the mutant frequency per survivor.
c) An increase in the absolute number of mutants.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Yes

There was evidence of toxicity with the test substance in both the presence and absence of S9-mix as indicated by relative suspension growth (RSG), this was confirmed by the decrease in relative total growth (RTG). There was no evidence of a reduction in Day 2 viability, therefore indicating that no residual toxicity occurred, in either the presence or absence of S9-mix. Optimum level of toxicity was achieved, in both the presence and absence of S9-mix. The toxicity observed at 30 µg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%; therefore it was excluded from the statistical analysis.

Conclusions:

Based on the results of the read across study, the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, can also be considered to be not mutagenic with and without metabolic activation in the mouse lymphoma assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the read across substance, Coco TMAC in L5178Y mouse lymphoma cells accoding to OECD Guideline 476 and EU Method B.17, in compliance with GLP. After a preliminary toxicity study, doses of 0, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L were selected as the test concentrations for the first experiment. The experiment was repeated to confirm the results of the first experiment. 3 h exposure was used both with and without S9 -mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the concentration range was 2.5 to 30 µg/L with S9 -mix and 0.313 to 5 µg/L without S9 -mix. The test substance did not induce a statistically significant or concentration-related increase in the mutant frequency at any concentration level, either with or without metabolic activation. Adequate levels of toxicity were achieved in all exposure groups.Under the study conditions, the test substance was not found to be mutagenic in mouse lymphoma assay (Nolan, 2002).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1:

A study was conducted to determine the mutagenic potential of the read across substance, C12-14 ADMAES, according to OECD 471 Guideline and EU Method B.13/14, in compliance with GLP. The test substance was examined for mutagenic activity in five strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, using the plate incorporation and pre-incubation methods. The studies were performed in the absence and presence of metabolic activation (S9-mix). The concentrations ranged from 1.23 to 100 µg/plate in first experiment and 0.41 to 200 µg/plate in second experiment. No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test substance at ≥100 µg/plate. Under the study conditions, the test substance was not considered to be mutagenic in the presence and absence of exogenous metabolic activation (Haddouck, 2003).

Study 2:

A study was conducted to determine the genotoxic potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, using human peripheral blood lymphocytes, according to the OECD Guideline 487, in compliance with GLP. Test substance was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test substance was formulated in purified water and tested in the following concentrations in range finding study: 0, 18.14, 30.23, 50.39, 83.98, 140.0, 233.3, 388.8, 648.0, 1080, 1800, 3000 and 5000 µg/mL. As complete/extreme toxicity was observed at all timepoints, appropriate concentrations could not be selected for the micronucleus experiment based on the study. In order to select concentrations for the micronucleus experiment, an additional range finder experiment was performed using a lower concentration range (range finder experiment 2): 0, 0.3628, 0.6047, 1.008, 1.680, 2.799, 4.666, 7.776, 12.96, 21.60, 36.00, 60.00 and 100.0 µg/mL. The results of this experiment were used to select suitable maximum concentrations for the micronucleus experiment. Concentrations tested for the micronucleus experiments were as follows: 0, 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7.5 and 10 µg/mL (3 +21 h treatment in absence of S-9) and 0, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7.5, 10 and 15 µg/mL (3 +21 h treatment in presence of S-9) and 0, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 4 and 5 µg/mL (24 +24 h treatment in the absence of S-9). These three assays with the test substance resulted in frequencies of MNBN cells which were similar to and not significantly higher (at the p≤0.05 level) than those observed in the concurrent vehicle controls and fell within the normal ranges. The test substance was thus considered as non-genotoxic under this condition by the study author. The acceptance criteria for the assay were fulfilled. The study is thus considered as valid. Under study conditions, the test substance was determined to be non-genotoxic with or without metabolic activation in the micronucleus assay (Hargreaves, 2018).

Study 3:

A study was conducted to determine the mutagenic potential of the read across substance, Coco TMAC (active ingredient 35%) in L5178Y mouse lymphoma cells accoding to OECD Guideline 476 and EU Method B.17, in compliance with GLP. After a preliminary toxicity study, doses of 0, 0.625, 1.25, 2.5, 5, 10 and 20 µg/L were selected as the test concentrations for the first experiment. The experiment was repeated to confirm the results of the first experiment. 3 h exposure was used both with and without S9 -mix while for the second experiment, the exposure time was increased to 24 h. For the second experiment, the concentration range was 2.5 to 30 µg/L with S9 -mix and 0.313 to 5 µg/L without S9 -mix. The test substance did not induce a statistically significant or concentration-related increase in the mutant frequency at any concentration level, either with or without metabolic activation. Adequate levels of toxicity were achieved in all exposure groups. Under the study conditions, the test substance was not found to be mutagenic in mouse lymphoma assay (Nolan, 2002).

Justification for classification or non-classification

Based on the results of the Micronucleus test with the test substance as well as two in vitro assays with the read across substance, the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, does not warrant classification for genotoxicity according to EU CLP (1272/2008/EC) criteria.