N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06.11.2019 - 19.02.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

EC Number:

850-698-3

Cas Number:

2387913-24-6

Molecular formula:

not applicable

IUPAC Name:

N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

Test material form:

other: solified melt

Details on test material:

- Name of test material ((as cited in study report):
Product name: V10069,
Systemic name: Quaternary ammonium compounds, tri-C8-10-alkylmethyl-methylsulfate

Specific details on test material used for the study:

- Characteristics: solid
- Storage conditions: stored at (+10 °C to +25 °C)

Method

Target gene:

histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB-, rfa-, pKM 101, resistance to Ampicillin

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB-, rfa-, pKM 101, resistance to Ampicillin

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: rfa-, pKM 101/pAQ1, resistance to Ampicillin

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: uvrB-, rfa-, non-resistance to Ampicillin

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: uvrB-, rfa-, non-resistance to Ampicillin

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, Aroclor 1254-induced, from male Sprague-Dawley rat liver (Trinova Biochem GmbH, Gießen, Germany)

Test concentrations with justification for top dose:

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at concentrations of 10 and 31.6 µg test item/plate in
the experiment without and with metabolic activation, respectively.
Hence, 10 and 31.6 µg test item per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Batch no. STBG9092; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 0.1 mL of approximately 1.00E+08 viable cells in the late exponential or early stationary phase
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 37°C for 48 -72 h
NUMBER OF REPLICATIONS:
- Plates: 3 per concentration and experiment
- Experiments: 2 independent experiments, each with and without metabolic activation
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Insolubility could have been assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.

Evaluation criteria:

Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by the testing facility. The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20-60
TA100: 100-200
TA102: 240-320
TA1535: 10-35
TA1537: 3-20
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment. Cytotoxicity is defined as a reduction in the number of colonies by more than 50 % compared with the vehicle control and/or a scarce background lawn.
Historical background data of revertant colony numbers of the negative and positive controls without and with metabolic activation for the experiments of the years 2016 to 2018 (most recent background data, not audited by the QAU-department) are given in Appendix 4 in section “any other details on results incl. tables”.

Statistics:

Interpretation of results:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium. A test item for which the results do not meet the above-mentioned criteria is considered as non-mutagenic in the AMES test.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolarity: not reported
- Evaporation from medium: not reported
- Water solubility: the test item is insoluble in water
- Precipitation: see section “any other information on results incl. tables”.
RANGE-FINDING/SCREENING STUDIES: see section “any other information on results incl. tables”.
HISTORICAL CONTROL DATA see tables 1 and 2 in section “any other information on results incl. tables”.

Any other information on results incl. tables

Preliminary test:

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at concentrations of 10 and 31.6 µg test item/plate in the experiment without and with metabolic activation, respectively.

Hence, 10 and 31.6 µg test item per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Main study:

Six concentrations ranging from 0.0316 to 10 or 0.1 to 31.6 µg PC-2019-853 /plate were employed in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Cytotoxicity: 

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentrations of 10 and 31.6 µg PC-2019-853/plate in the plate incorporation test and preincubation test for the experiments without and with metabolic activation, respectively, in all test strains.

Mutagenicity:

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a concentration of 10 and 31.6 µg /plate, in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are withinthe historical control rangegenerated by LPT. Hence, all acceptance criteria are met.

In conclusion,under the present test conditions, test item tested up to a cytotoxic concentration of 10 and 31.6 µg /plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

History profile of negative and positive control values of the years 2016 to 2018 (n= 90 studies). Data obtained from plate incorporation and preincubation tests.

Negative reference item
Strain	TA 98		TA 100		TA 102		TA 1535		TA 1537
S9-mix	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
Mean	30.5	31.7	144.2	143.4	278.9	280.4	19.3	19.3	6.9	6.6
SD	5.9	6.2	21.0	20.8	17.2	18.7	4.2	4.4	1.8	1.9
Min	18	20	95	100	245	203	10	10	2	2
Max	48	53	200	209	333	324	32	30	10	10
Positive reference item
Strain	TA 98		TA 100		TA 102		TA 1535		TA 1537
S9-mix	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
	2-nitro-fluorene	Benzo[a]-pyrene	Sodium azide	2-amino-anthracene	Mitomycin C	Benzo[a]-pyrene	Sodium azide	2-amino-anthracene	9-amino-acridine	Benzo[a]-pyrene
Mean	164.8	163.9	956.4	957.6	1017.5	1012.9	151.0	152.7	67.7	68.0
SD	26.1	27.5	107.4	107.7	96.1	98.7	44.7	47.6	28.8	25.6
Min	102	104	701	703	709	759	62	67	25	24
Max	301	304	1576	1412	1437	1433	406	404	187	184

 Table 1: Preliminary cytotoxicity test without metabolic activation. Plate incorporation test with TA 100.

Test item (µg/plate)	Background lawn	Revertants plate 1	Revertants plate 2
0.316	normal	135	106
1	normal	101	99
3.16	normal	103	121
10	scarce background lawn	25	30
31.6	scarce background lawn	36	44
100	scarce background lawn	0	0
316	scarce background lawn	1	0
1000	scarce background lawn	0	0
3160	scarce background lawn	0	1
5000	scarce background lawn	3	6
Solvent control 100 µL/plate	normal	103	116

#  not evaluable due to the intense test item precipitation

Table 2: Preliminary cytotoxicity test with metabolic activation. Plate incorporation test with TA 100.

Test item (µg/plate)	Background lawn	Revertants plate 1	Revertants plate 2
0.316	normal	131	104
1	normal	115	128
3.16	normal	99	130
10	normal	87	110
31.6	scarce background lawn	27	44
100	scarce background lawn	41	29
316	scarce background lawn	1	0
1000	scarce background lawn	0	0
3160	scarce background lawn	0	0
5000	scarce background lawn	9	7
Solvent control 100 µL/plate	normal	137	98

# not evaluable due to the intense test item precipitation

Table 3: Plate incorporation test without metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
0.0316	25.3	103.7	305.7	15.3	5.7
0.1	21.7	142.7	290.3	14.7	5.3
0.316	25.7	146.3	274.3	12.7	5.3
1	25.3	168.7	306.7	15.0	3.7
3.16	21.0	153.0	311.3	11.7	5.7
10	16.7 #	18.3 #	212.3 #	3.0 #
Vehicle control 100 µL/plate	23.0	117.0	289.7	13.7	5.0
Positive reference item	2-nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-amino-acridine
Concentration µg/plate	10	10	10	10	100
	140.7	763.3	1183.7	201.0	69.0

# scarce background lawn

Table 4: Plate incorporation test with metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
0.1	23.3	143.7	273.7	19.3	3.7
0.316	21.7	126.3	271.0	12.0	3.3
1	24.3	110.7	285.0	14.7	3.3
3.16	25.3	139.7	307.0	15.3	3.0
10	26.3	117.7	294.3	15.7	4.3
31.6	8.0 #	12.0 #	277.3 #	4.0 #	1.0 #
Vehicle control 100 µL/plate	22.7	124.0	311.3	11.0	4.3
Positive reference item	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene
Concentration µg/plate	10	2	10	2	10
	142.7	1037.7	1048.7	229.3	69.0

 # scarce background lawn

Table 5: Preincubation test without metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
0.0316	42.0	138.3	288.0	37.0	6.7
0.1	20.3	150.0	282.0	26.0	7.3
0.316	29.0	136.0	281.0	25.3	5.0
1	23.3	152.3	258.7	25.0	6.0
3.16	13.7	91.7	243.7	24.0	6.7
10	11.3 #	36.7 #	168.7 #	7.0 #	1.0 #
Vehicle control 100 µL/plate	27.3	128.7	283.3	19.7	6.7
Positive reference item	2-nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-amino-acridine
Concentration µg/plate	10	10	10	10	100
	161.0	1122.3	871.0	217.3	76.0

 # scarce background lawn

Table 6: Preincubation test with metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
0.1	29.3	101.7	282.0	13.7	8.3
0.316	26.3	114.7	266.3	17.7	6.7
1	25.3	105.0	269.3	15.3	4.3
3.16	28.7	117.3	286.0	17.3	4.3
10	22.0	108.0	277.3	12.3	5.3
31.6	8.3 #	16.0 #	231.3 #	6.3 #	2.0 #
Vehicle control 100 µL/plate	29.3	132.7	275.3	13.7	8.0
Positive reference item	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene
Concentration µg/plate	10	2	10	2	10
	157.3	1153.7	1139.3	235.3	79.3

 # scarce background lawn

   

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 10 and 31.6 µg /plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

Executive summary:

The potential of Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates to induce gene mutations was examined in 5 Salmonella typhimurium strains in two independent experiments without and with metabolic activation (plate incorporation test and preincubation test).

The test item was was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle DMSO was employed as the negative control.

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at concentrations of 10 and 31.6 µg test item/plate in the experiment without and with metabolic activation, respectively.

Hence, 10 and 31.6 µg Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates

per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Six concentrations ranging from 0.0316 to 10 or 0.1 to 31.6 µg test item /plate were employed in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentrations of 10 and 31.6 µg test item/plate in the plate incorporation test and preincubation test for the experiments without and with metabolic activation, respectively, in all test strains.

No increase in revertant colony numbers as compared with control counts was observed for

Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates

, tested up to a concentration of 10 and 31.6 µg /plate, in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the historical control range generated by LPT. Hence, all acceptance criteria are met.

In conclusion, under the present test conditions, Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates

tested up to a concentration of 31.6 µg/plate, that led to test item precipitation, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.