Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

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:

experimental study

Adequacy of study:

key study

Study period:

From August 04, 2017 to August 16, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: See remarks

Remarks:

rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galacto se metabolism; chl: mutation in nitrate reductase; bio defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced Aroclor 1254

Test concentrations with justification for top dose:

Treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. The highest concentration of the test substance used in the mutation assays was 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines).

Vehicle / solvent:

Purified water for the test substance
DMSO or purified water for positive controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or saline

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

-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.

The test substance was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments without and with S-9 for test substance, vehicle and positive controls. These plating’s were achieved by the following sequence of additions to molten agar at 45±1°C:

• 0.1 mL bacterial culture
• 0.1 mL of test substance solution/vehicle control or 0.05 mL of positive 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 and incubated at 37±1°C protected from light for 2 to 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertants colonies were counted.

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 substance, vehicle control solution 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.

Rationale for test conditions:

- Based on the most recent OECD and EC guidelines.
- First mutation experiment

Evaluation criteria:

For valid data, the test substance was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. The positive trend/effects described above were reproducible.

The test substance was considered positive in this assay if both of the above criteria were met.
The test substance was considered negative in this assay if neither of the above criteria were met.

Statistics:

No formal hypothesis testing was done.
- Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria were counted.

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- The test substance was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.
- All test substance treatments in this study were performed using formulations prepared in water for irrigation (purified water), and all concentrations are expressed in terms of pure compound.
- Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed in all strains from 50 or 160 μg/plate in the absence of S-9 and from 500 μg/plate in the presence of S-9.
- Mutation Experiment 2 treatments 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 strain specific toxicity observed in Mutation Experiment 1. Narrowed concentration intervals were employed covering the ranges 2.5-160 μg/plate or 10-500 μg/plate, in order to examine more closely those concentrations of the test substance approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was observed in all strains at concentrations of 80 and 160 μg/plate in the absence of S-9 and additionally in TA98 at 320 and 500 μg/plate and in TA102 at 40 μg/plate. In the presence of S-9, evidence of toxicity was noted in all strains at concentrations of 320 and 500 μg/plate and additionally in TA102 at 160 μg/plate.
- No precipitation was observed on the test plates following incubation. Vehicle and positive control treatments were included for all strains in both experiments.
- The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following test substance treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control.
- This study was considered therefore to have provided no evidence of any test substance mutagenic activity in this assay system it was concluded that the test substance did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Conclusions:

Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium bacterial reverse mutation assay.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity according to OECD Guideline 471, in compliance with GLP. The test substance was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9 mix), in two separate experiments. All test substance treatments were performed in purified water. In Experiment 1 treatments of all the tester strains were performed in +/- of S9 mix, using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following treatment, evidence of toxicity was observed in all strains from 50 or 160 μg/plate in the absence of S9 and from 500 μg/plate in the presence of S9. In Experiment 2 treatments of all the tester strains were performed in the presence and absence of S9. For all strains, the maximum test concentration was reduced based on strain-specific toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 2.5 -160 μg/plate or 10 -500 μg/plate, in order to examine more closely those concentrations of the test substance approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S9 were further modified by the inclusion of a pre-incubation step. Following treatment, evidence of toxicity was observed in all strains at 80 and 160 μg/plate in the absence of S9 mix and additionally in TA98 at 320 and 500 μg/plate and in TA102 at 40 μg/plate. In the presence of S9, evidence of toxicity was noted in all strains at 320 and 500 μg/plate and additionally in TA102 at 160 μg/plate. No precipitation was observed on the test plates following incubation. Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following test substance treatment in the presence and absence of S9, no increase in revertant numbers was observed that was greater than 1.5 -fold (in strain TA102), ≥2 -fold (in strains TA98 or TA100) or ≥3 -fold (in strains TA1535 or TA1537) of the concurrent vehicle control. Therefore, no evidence of test substance-induced mutagenic activity in this assay system was observed. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Dreher, 2017).

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 11, 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 concentrations: 18.14, 30.23, 50.39, 83.98, 140.0, 233.3, 388.8, 648.0, 1080, 1800, 3000 and 5000 µg/mL
Micronucleus experiment (3+21 Hour Treatments -S-9): 1.250, 2.500, 5.000, 10.00, 15.00, 20.00, 25.00, 30.00, 40.00, 50.00, 60.00, 100.0 and 200.0 µg/mL
Micronucleus experiment (3+21 Hour Treatments +S-9): 1.250, 2.500, 5.000, 10.00, 15.00, 20.00, 25.00, 30.00, 40.00, 50.00, 60.00, 100.0 and 200.0 µg/mL
Micronucleus experiment (24+24 Hour Treatments -S-9): 2.500, 5.000, 7.5, 10.00, 12.50, 15.00, 20.00, 25.00, 30.00, 35.00, 40.00, 50.00, 60.00, 100.0 µg/mL

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

Vehicle / solvent:

Vehicle: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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 purified water at concentrations up to at least 266.1 mg/mL, with the aid of vortex mixing, ultrasonication and warming at 37˚C. The maximum concentration prepared for the media solubility was 210.6 mg/mL. The solubility limit in culture medium was in the region of 21060 µg/mL as indicated by the presence of a slight precipitation upon test substance addition which cleared on agitation. A maximum concentration of 5000 µg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the maximum recommended concentration for UVCB compounds, according to current regulatory guidelines. Concentrations for the Micronucleus Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment. Test substance stock solutions were prepared by formulating test substance under subdued lighting in purified water, with the aid of vortex mixing and warming at 37˚C, 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 made using purified water. The test substance solutions were protected from light and used within approximately 3 h 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 numbers 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. Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at very toxic concentrations.

Statistics:

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

Key result

Species / strain:

lymphocytes:

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:

It is concluded that test substance did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an aroclor-induced rat liver metabolic activation system (S 9). Maximum concentrations analysed were limited by toxicity or post treatment precipitate (in line with current regulatory guidelines for the in vitro micronucleus assay).

Remarks on result:

other: Non genotoxic

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	34	157	9	200	0.88
	B	42	149	9	200	0.84
	Total	76	306	18	400	0.86	-
18.14	A	65	134	1	200	0.68	20
30.23	A	68	130	2	200	0.67	22 E
50.39	A	99	101	0	200	0.51	41 E H
83.98	A	NE	-	-	-	-	- E H
140.0	A	NE	-	-	-	-	- 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

 

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	58	135	7	200	0.75
	B	36	151	13	200	0.89
	Total	94	286	20	400	0.82	-
18.14	A	48	152	0	200	0.76	7
30.23	A	60	140	0	200	0.70	14 E H
50.39	A	78	120	2	200	0.62	24 E H
83.98	A	NE	-	-	-	-	- E H
140.0	A	NE	-	-	-	-	- 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

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	8	122	70	200	1.31
	B	11	113	76	200	1.33
	Total	19	235	146	400	1.32	-
18.14	A	73	104	23	200	0.75	43
30.23	A	111	72	17	200	0.53	60 E
50.39	A	NE	-	-	-	-	- E H
83.98	A	NE	-	-	-	-	- E H
140.0	A	NE	-	-	-	-	- 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 (5000 µg/mL) following 24+24 h treatments in the absence of S-9, as compared to the concurrent vehicle controls (measured data not reported). Osmolality and pH for the 3+21 h treatment in the absence and presence of S-9 were measured in the Micronucleus Experiment.

The results of the cytotoxicity Range-Finder Experiment were used to select suitable concentrations for the Micronucleus Experiment.

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	71	383	46	500	0.95
	B	90	377	33	500	0.89
	C	74	375	51	500	0.95
	D	62	401	37	500	0.95
	Total	297	1536	167	2000	0.94	-
1.250	A	90	365	45	500	0.91
	B	72	389	39	500	0.93
	Total	162	754	84	1000	0.92	1
2.500	A	91	384	25	500	0.87
	B	90	371	39	500	0.90
	Total	181	755	64	1000	0.88	6
5.000	A	103	380	17	500	0.83
	B	92	379	29	500	0.87
	Total	195	759	46	1000	0.85	9
10.00	A	109	381	10	500	0.80
	B	117	359	24	500	0.81
	Total	226	740	34	1000	0.81	14 #
15.00	A	134	353	13	500	0.76
	B	155	341	4	500	0.70
	Total	289	694	17	1000	0.73	22
20.00	A	131	362	7	500	0.75
	B	141	355	4	500	0.73
	Total	272	717	11	1000	0.74	21
25.00	A	187	306	7	500	0.64
	B	180	315	5	500	0.65
	Total	367	621	12	1000	0.65	31
30.00	A	148	342	10	500	0.72
	B	170	325	5	500	0.67
	Total	318	667	15	1000	0.70	25 #
40.00	A	177	320	3	500	0.65
	B	175	316	9	500	0.67
	Total	352	636	12	1000	0.66	29
50.00	A	185	314	1	500	0.63
	B	239	256	5	500	0.53
	Total	424	570	6	1000	0.58	38
60.00	A	243	255	2	500	0.52
	B	268	231	1	500	0.47
	Total	511	486	3	1000	0.49	47 E H #
100.0	A	404	96	0	500	0.19
	B	378	121	0	499	0.24
	Total	782	217	0	999	0.22	77 E H

 

(continued): 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 (%)
200.0	A	NE	-	-	-	-
	B	NE	-	-	-	-
	Total	-	-	-	-	-	- E H
MMC, 0.20	A	128	366	6	500	0.76
	B	121	373	6	500	0.77
	Total	249	739	12	1000	0.76	18 #
MMC, 0.30	A	183	314	3	500	0.64
	B	161	333	6	500	0.69
	Total	344	647	9	1000	0.67	29

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

 

# 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	68	365	67	500	1.00
	B	57	361	82	500	1.05
	C	66	367	67	500	1.00
	D	54	371	75	500	1.04
	Total	245	1464	291	2000	1.02	-
1.250	A	57	396	47	500	0.98
	B	58	389	53	500	0.99
	Total	115	785	100	1000	0.99	4
2.500	A	73	377	50	500	0.95
	B	57	386	57	500	1.00
	Total	130	763	107	1000	0.98	4
5.000	A	72	386	42	500	0.94
	B	37	414	49	500	1.02
	Total	109	800	91	1000	0.98	4
10.00	A	57	412	31	500	0.95
	B	60	425	15	500	0.91
	Total	117	837	46	1000	0.93	9
15.00	A	63	416	21	500	0.92
	B	79	398	23	500	0.89
	Total	142	814	44	1000	0.90	12
20.00	A	72	415	13	500	0.88
	B	85	397	18	500	0.87
	Total	157	812	31	1000	0.87	15 #
25.00	A	91	403	6	500	0.83
	B	107	383	10	500	0.81
	Total	198	786	16	1000	0.82	20
30.00	A	142	353	5	500	0.73
	B	163	333	4	500	0.68
	Total	305	686	9	1000	0.70	31
40.00	A	142	351	7	500	0.73
	B	176	319	5	500	0.66
	Total	318	670	12	1000	0.69	32 #
50.00	A	191	307	2	500	0.62
	B	226	273	1	500	0.55
	Total	417	580	3	1000	0.59	43
60.00	A	237	263	0	500	0.53
	B	273	226	1	500	0.46
	Total	510	489	1	1000	0.49	52 #
100.0	A	219	35	0	254	0.14
	B	NE	-	-	-	-
	Total	219	35	0	254	0.14	87 E H
200.0	A	NE	-	-	-	-
	B	NE	-	-	-	-
	Total	-	-	-	-	-	- E H

 

(continued): 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 (%)
CPA, 2.00	A	77	410	13	500	0.87
	B	111	375	14	500	0.81
	Total	188	785	27	1000	0.84	18
CPA, 3.00	A	71	399	30	500	0.92
	B	73	398	29	500	0.91
	Total	144	797	59	1000	0.92	11 #

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

 

# 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	42	378	80	500	1.08
	B	34	365	101	500	1.13
	C	47	384	69	500	1.04
	D	42	372	86	500	1.09
	Total	165	1499	336	2000	1.09	-
2.500	A	78	386	36	500	0.92
	B	98	371	31	500	0.87
	Total	176	757	67	1000	0.89	18 #
5.000	A	133	356	11	500	0.76
	B	125	364	11	500	0.77
	Total	258	720	22	1000	0.76	30 #
7.500	A	186	307	7	500	0.64
	B	179	315	6	500	0.65
	Total	365	622	13	1000	0.65	40
10.00	A	220	275	5	500	0.57
	B	237	262	1	500	0.53
	Total	457	537	6	1000	0.55	49
12.50	A	259	239	2	500	0.49
	B	271	227	2	500	0.46
	Total	530	466	4	1000	0.47	56 #
15.00	A	293	206	1	500	0.42
	B	329	168	3	500	0.35
	Total	622	374	4	1000	0.38	65
20.00	A	368	130	2	500	0.27
	B	372	121	7	500	0.27
	Total	740	251	9	1000	0.27	75
25.00	A	407	91	2	500	0.19
	B	391	105	4	500	0.23
	Total	798	196	6	1000	0.21	81
30.00	A	349	142	9	500	0.32
	B	341	153	6	500	0.33
	Total	690	295	15	1000	0.33	70
35.00	A	349	146	5	500	0.31
	B	307	187	6	500	0.40
	Total	656	333	11	1000	0.36	67
40.00	A	310	183	7	500	0.39
	B	327	166	7	500	0.36
	Total	637	349	14	1000	0.38	65 E
50.00	A	313	183	4	500	0.38
	B	291	204	5	500	0.43
	Total	604	387	9	1000	0.41	63 E
60.00	A	339	158	3	500	0.33
	B	326	172	2	500	0.35
	Total	665	330	5	1000	0.34	69 E H
100.0	A	NE	-	-	-	-
	B	NE	-	-	-	-
	Total	-	-	-	-	-	- E H

Table continued overleaf

 

 

(continued): 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 (%)
VIN, 0.04	A	197	257	46	500	0.70
	B	186	238	76	500	0.78
	Total	383	495	122	1000	0.74	32 #
VIN, 0.06	A	192	223	85	500	0.79
	B	221	216	63	500	0.68
	Total	413	439	148	1000	0.74	32

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

 

# Highlighted concentrations selected for analysis

 

No significant changes in osmolality or pH were observed at the highest concentration tested (200 µg/mL) following 3+21 h treatments in the absence and presence of S‑9, as compared to the concurrent vehicle controls (measured data not reported).

 

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

The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures.

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

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.

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.

The maximum concentration analysed under each treatment condition met the criteria.

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, using human peripheral blood lymphocytes, according to OECD Guideline 487, in compliance with GLP. The 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. Treatment covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9) from Aroclor 1254 -induced rats. The test substance was formulated in purified water and tested in the following concentrations in range finding study 18.14, 30.23, 50.39, 83.98, 140.0, 233.3, 388.8, 648.0, 1080, 1800, 3000 and 5000 µg/mL. Concentrations tested in the micronucleus experiment are as follows:1.25, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 100 and 200 µg/mL(3 +21 h treatment in the absence and presence of S-9) and 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 50, 60 and 100 µg/mL (24 +24 h treatment in the absence of S-9), were limited by toxicity and were determined following a preliminary cytotoxicity range finding experiment.Three assays (two short and one continuous exposure period) with and without metabolic activation, both with and without recovery periods were performed with test substance concentrations. In the first two experiments, no biologically or statistically significant increase in the number of MNBN cells were observed. The test substance was thus considered as non-genotoxic under this condition by the study author. In the continuous exposure experiment of 24 h at 2.50 to 100 µg/mL, without metabolic activation, no biologically or statistically significant increase in the number of MNBN cells was observed. Overall, the study author concluded that, the test substance induced no biologically or statistically significant increase in the number MNBN cells either with or without metabolic activation, either with a short or with a continuous treatment. The acceptance criteria for the assay were fulfilled. The study is thus considered as valid. Under study conditions, the test substance was considered to be non-genotoxic with or without metabolic activation in the micronucleus assay (Hargreaves, 2017).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 21, 2017 to December 12, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1°C. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
The preliminary cytotoxicity test was initially performed with a test substance dose range of 156.3 to 5000 μg/mL. However due to excessive toxicity seen in the initial experiment the test was repeated with modified ranges,0.7813 to 400 μg/mL in the absence and presence of S9.

Mutagenicity test - Experiment 1
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of test substance (µg/mL) 3 h without S9: 2.5, 5, 10, 20, 30, 40, 50, 60, 80 and 100 μg/mL
3 h with S9: 5, 10, 20, 30, 40, 50, 60, 70 80, 100 μg/mL

Mutagenicity test - Experiment 2
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of test substance (µg/mL) 3 h with S9: 10, 20, 30, 40, 45, 50, 60, 80, 100 μg/mL

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

Test System
The test system was suitably labelled to clearly identify the study number, test substance concentration (if applicable), positive and vehicle control, presence and absence of S-9.

Cytotoxicity Range-Finder Experiments
Treatment of cell cultures for the cytotoxicity Range-Finder Experiments were as described below for the Mutation Experiments. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL. Following 3 h treatment, cells were centrifuged (200 g), washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air for 7 to 8 days. Wells containing viable clones were identified by eye using background illumination and counted.

Mutation Assays
Treatment of Cell Cultures
At least 107 cells in a volume of 16 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2 mL vehicle or test substance or 0.2 mL positive control solution was added (plus 1.8 mL purified water). S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL. After 3 h’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described. Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies. Osmolality and pH measurements on post-treatment media were taken in the initial cytotoxicity Range-Finder Experiment.


Plating for Survival
Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from
these were diluted to 8 cells/mL as shown in following table:
Initial Cell Conc (A) Dilution Intermediate
Cell Conc
(B) Dilution Final Cell
Conc
Survival 2 x 105/mL* mL
A mL
RPMI 10 2 x 103/mL mL
B mL
RPMI 20 8/mL
0.1 9.9 0.2 50
* Where fewer than 2 x 105 cells/mL survived treatment, an alternative dilution to 2 x 103 cells/mL was
used
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7-9 days). Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period
Cultures were maintained in flasks for a period of 7 days during which the hprt mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance as shown in the following table:






Mutation Experiment 1 (μg/mL)
`-S9 `+S9
0 0
2.5 5
5 10
10 20
20 30
30 40
40 50
50 60
60
80
NQO 0.15 B[a]P 2
NQO 0.20 B[a]P 3

Mutation Experiment 2 (μg/mL)
`-S9
0
10
20
30
35
40
45
50
60
B[a]P 2
B[a]P 3

Plating for Viability
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL as shown in the following table:
Initial Cell Conc (A) Dilution Intermediate
Cell Conc
(B) Dilution Final Cell
Conc
Viability 1 x 105/mL mL
A mL
RPMI 10 2 x 103/mL mL
B mL
RPMI 20 8/mL
0.2 9.8 0.2 50
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (8-11 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 6TG resistance:

At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (12-13 days) and wells containing clones were identified as above and counted.

Analysis of results
All calculations were performed by computer using validated software. From the zero term of the Poisson distribution the probable number of clones/well (P) on microtitre plates in which there are EW empty wells (without clones) out of a total of TW wells is given by:
P = -ln (EW/TW).

Cloning Efficiency (CE) in any given culture is therefore:
CE = P/No of cells plated per well
and as an average of 1.6 cells/well were plated on all survival and viability plates,
CE = P/1.6.

Percentage relative survival (%RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus:
%RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 h treatment period, percentage relative survival values for each concentration of test substance were adjusted as follows:

Adjusted %RS
= %RS x Post-treatment cell concentration for test substance treatment Post-treatment cell concentration for vehicle control All %RS values were adjusted as described above.

Mutant frequency (MF) is usually expressed as "mutants per 106 viable cells". In order to calculate this, the cloning efficiencies of both mutant and viable cells in the same culture were calculated:
MF = [CE (mutant)/CE (viable)] x 106.
From the formula given and with the knowledge that 2 x 104 cells were plated/well for mutation to 6TG resistance,
CE (mutant) = P (mutant)/2 x 104
CE (viable) = P (viable)/1.6
where, in each case, P = -ln (EW/TW).
Therefore,
MF = [P (mutant)/2 x 104] x [1.6/P (viable)] x 106
= {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 80.
Statistical significance of mutant frequencies was carried out according to the
UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test substance treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Acceptance criteria
The assay was considered valid if all of the following criteria were met:

1. The MF in the vehicle control cultures was considered acceptable for addition to the laboratory historical negative control database
2. The MF in the concurrent positive controls induced responses that were comparable with those generated in the historical positive control database and gave a clear, unequivocal increase in MF over the concurrent negative control
3. The test was performed with and without metabolic activation
4. Adequate numbers of cells and concentrations were analysable.

Evaluation criteria
For valid data, the test substance was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-response relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/ 2 standard deviations.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Evaluation criteria:

Please see 'Assay Acceptance criteria', in details on test system and conditions section.

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:

not applicable

Positive controls validity:

valid

Additional information on results:

It is concluded that test substance did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9). The maximum concentrations tested were limited by toxicity in the absence of S-9 and by post treatment precipitate in the presence of S-9.

Range finder experiment 1: 3 h treatment in the absence and presence of S-9

Concentration μg/mL	3 Hour Treatment –S-9 %RS	3 Hour Treatment +S-9 %RS
0	100	100
156.3	0	0
312.5 PP	0	0
625 P, PP	NP	NP
1250 P, PP	NP	NP
2500 P, PP	NP	NP
5000 P, PP	NP	NP

%RS- Percent Relative Survival

P - Precipitation noted at time of treatment

PP - Precipitation noted at end of treatment incubation period

NP - Not plated as discarded following treatment incubation due to precipitation.

Range - finder experiment 2: 3 h treatment in the absence and presence of S-9

Concentration μg/mL	3 Hour Treatment –S-9 %RS	3 Hour Treatment +S-9 %RS
0	100	100
0.7813	81	76
1.563	98	89
3.125	86	91
6.25	91	81
12.5	72	77
25	44	57
50	13	29
100	0	0 PP
200	0 PP	NP PP
400	NP PP	NP PP

%RS- Percent Relative Survival

PP - Precipitation noted at end of treatment incubation period

NP - Not plated as discarded following treatment incubation due to precipitation.

Mutation Experiment - 3 Hour treatment in the Absence and Presence of S-9

3 Hour Treatment –S-9			3 Hour Treatment +S-9
Concentration μg/mL	%RS	MF §	Concentration μg/mL	%RS	MF §
0	100	3.82	0	100	3.15
2.5	102	6.87 NS	5	97	3.54 NS
5	99	5.99 NS	10	73	4.3 NS
10	96	3.49 NS	20	67	8.41*
20	77	3.59 NS	30	72	8.46*
30	75	6.02 NS	40	61	4.81 NS
40	65	8.33 NS	50	26	8.02*
50	63	5.07 NS	60 PP	34	6.12 NS
60	48	5.2 NS
80	16	5.31 NS
NQO 0.15	56	38.8	BP 2	67	17.96
NQO 0.2	49	60.25	BP 3	89	33.7

Linear trend –S-9: Not significant

Linear trend +S-9: **

§ 6-TG resistant mutants/106 viable cells 7 days after treatment

%RS Percent relative survival adjusted by post treatment cell counts

PP Precipitation noted at end of treatment incubation period

NS Not significant

* Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

*, **, *** Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively.

Mutation Experiment 2 - 3 Hour treatment in the Presence of S-9

3 Hour Treatment –S-9
Concentration μg/mL	%RS	MF §
0	100	3.78
10	88	0.35 NS
20	75	1.32 NS
30	82	0.63 NS
35	84	1.01 NS
40	81	3.03 NS
45	69	1.76 NS
50	73	1.84 NS
60 PP	64	1.52 NS
BP 2	38	12.85
BP 3	39	19.94

Linear trend +S-9: *

§ 6-TG resistant mutants/106 viable cells 7 days after treatment

%RS Percent relative survival adjusted by post treatment cell counts

PP Precipitation noted at end of treatment incubation period

NS Not significant

*, **, *** Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively.

Conclusions:

Under study conditions, the test substance was therefore considered to be non-mutagenic to L5178Y mouse lymphoma cells at the HPRT locus, with and without metabolic activation

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the test substance, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of L5178Y mouse lymphoma cells, according to OECD Guideline 476, in compliance with GLP. The technique used was a plate assay in tissue culture flasks and 6-thioguanine (6­TG) as the selective agent. L5178Y mouse lymphoma cells were treated with the test substance at various dose levels, in duplicate, together with vehicle (solvent) and positive controls. Three treatment conditions were used for the test. In Experiment 1, a 3 h exposure in the presence of an induced rat liver homogenate metabolising system (S9) (at 5, 10, 20, 30, 40, 50, 60, 70, 80 and 100 μg/mL) and a 3 h exposure in the absence of metabolic activation (S9) (at 2.5, 5, 10, 20, 30, 40, 50, 60, 80 and 100 μg/mL). In Experiment 2, the 3 h exposure with addition of S9 was repeated (at 10, 20, 30, 40, 45, 50, 60, 80 and 100 μg/mL). The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity tests. The vehicle (solvent) controls produced mutation frequencies within the range expected in L5178Y cells at the HPRT locus. The positive control treatment, both in the presence and absence of metabolic activation, produced significant increases in the mutation frequency indicating satisfactory performance of the test and of the metabolising system. The test substancedemonstrated statistically significant increases in MF in Experiment 1, that exceeded the historical range at 20, 30 and 50 μg/mL and a statistically significant linear trend (p≤0.01), indicating a positive result. Although all the criteria for a positive result were achieved, statistically significant increases were only observed at sporadic concentrations and therefore a confirmatory experiment was conducted under the same treatment conditions. Following 3 h treatment in the presence of S-9 in mutation Experiment 2, no statistically significant increase in MF compared to the vehicle control was observed at any concentration analysed.A significant linear trend (p≤0.05) was noted but in the absence of any statistically significant increase in MF this observation was considered not biologically relevant. Under study conditions, the test substance was therefore considered to be non-mutagenic to L5178Y mouse lymphoma cells at the HPRT locus(Hargreaves, 2018).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study:1 (Ames assay)

A study was conducted to determine the in vitro genetic toxicity according to OECD Guideline 471, in compliance with GLP. The test substance was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9 mix), in two separate experiments. All test substance treatments were performed in purified water. In Experiment 1 treatments of all the tester strains were performed in +/- of S9 mix, using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following treatment, evidence of toxicity was observed in all strains from 50 or 160 μg/plate in the absence of S9 and from 500 μg/plate in the presence of S9. In Experiment 2 treatments of all the tester strains were performed in the presence and absence of S9. For all strains, the maximum test concentration was reduced based on strain-specific toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 2.5 -160 μg/plate or 10 -500 μg/plate, in order to examine more closely those concentrations of the test substance approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S9 were further modified by the inclusion of a pre-incubation step. Following treatment, evidence of toxicity was observed in all strains at 80 and 160 μg/plate in the absence of S9 mix and additionally in TA98 at 320 and 500 μg/plate and in TA102 at 40 μg/plate. In the presence of S9, evidence of toxicity was noted in all strains at 320 and 500 μg/plate and additionally in TA102 at 160 μg/plate. No precipitation was observed on the test plates following incubation. Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following test substance treatment in the presence and absence of S9, no increase in revertant numbers was observed that was greater than 1.5 -fold (in strain TA102), ≥2 -fold (in strains TA98 or TA100) or ≥3 -fold (in strains TA1535 or TA1537) of the concurrent vehicle control. Therefore, no evidence of test substance-induced mutagenic activity in this assay system was observed. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Dreher, 2017).

Study 2: (Micronucleus assay)

A study was conducted to determine the genotoxic potential of the test substance, using human peripheral blood lymphocytes, according to OECD Guideline 487, in compliance with GLP. The 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. Treatment covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9) from Aroclor 1254 -induced rats. The test substance was formulated in purified water and tested in the following concentrations in range finding study 18.14, 30.23, 50.39, 83.98, 140.0, 233.3, 388.8, 648.0, 1080, 1800, 3000 and 5000 µg/mL. Concentrations tested in the micronucleus experiment are as follows: 1.25, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 100 and 200 µg/mL(3 +21 h treatment in the absence and presence of S-9) and 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 50, 60 and 100 µg/mL (24 +24 h treatment in the absence of S-9), were limited by toxicity and were determined following a preliminary cytotoxicity range finding experiment. Three assays (two short and one continuous exposure period) with and without metabolic activation, both with and without recovery periods were performed with test substance concentrations. In the first two experiments, no biologically or statistically significant increase in the number of MNBN cells were observed. The test substance was thus considered as non-genotoxic under this condition by the study author. In the continuous exposure experiment of 24 h at 2.50 to 100 µg/mL, without metabolic activation, no biologically or statistically significant increase in the number of MNBN cells was observed. Overall, the study author concluded that, the test substance induced no biologically or statistically significant increase in the number MNBN cells either with or without metabolic activation, either with a short or with a continuous treatment. The acceptance criteria for the assay were fulfilled. The study is thus considered as valid. Under study conditions, the test substance was considered to be non-genotoxic with or without metabolic activation in the micronucleus assay (Hargreaves, 2017).

Study 3: (HRPT assay)

An in vitro study was conducted to determine the mutagenic potential of the test substance, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of L5178Y mouse lymphoma cells, according to OECD Guideline 476, in compliance with GLP. The technique used was a plate assay in tissue culture flasks and 6-thioguanine (6­TG) as the selective agent. L5178Y mouse lymphoma cells were treated with the test substance at various dose levels, in duplicate, together with vehicle (solvent) and positive controls. Three treatment conditions were used for the test. In Experiment 1, a 3 h exposure in the presence of an induced rat liver homogenate metabolising system (S9) (at 5, 10, 20, 30, 40, 50, 60, 70, 80 and 100 μg/mL) and a 3 h exposure in the absence of metabolic activation (S9) (at 2.5, 5, 10, 20, 30, 40, 50, 60, 80 and 100 μg/mL). In Experiment 2, the 3 h exposure with addition of S9 was repeated (at 10, 20, 30, 40, 45, 50, 60, 80 and 100 μg/mL). The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity tests. The vehicle (solvent) controls produced mutation frequencies within the range expected in L5178Y cells at the HPRT locus. The positive control treatment, both in the presence and absence of metabolic activation, produced significant increases in the mutation frequency indicating satisfactory performance of the test and of the metabolising system. The test substance demonstrated statistically significant increases in MF in Experiment 1, that exceeded the historical range at 20, 30 and 50 μg/mL and a statistically significant linear trend (p≤0.01), indicating a positive result. Although all the criteria for a positive result were achieved, statistically significant increases were only observed at sporadic concentrations and therefore a confirmatory experiment was conducted under the same treatment conditions. Following 3 h treatment in the presence of S-9 in mutation Experiment 2, no statistically significant increase in MF compared to the vehicle control was observed at any concentration analysed.A significant linear trend (p≤0.05) was noted but in the absence of any statistically significant increase in MF this observation was considered not biologically relevant. Under study conditions, the test substance was therefore considered to be non-mutagenic to L5178Y mouse lymphoma cells at the HPRT locus (Hargreaves, 2018).

Justification for classification or non-classification

Based on the results of the Ames test, Micronucleus assay and HRPT gene mutation assay with the test substance, Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-tallow alkyltrimethylenedi-, bis(Me sulfates) (salts), is concluded not to warrant classification for genotoxicity according to EU CLP criteria (Regulation 1272/2008/EC).