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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- negative: Ames test with S. typhimurium TA98, TA100, TA102, TA1535 and TA1537 (met. act.: with and without) (OECD TG 471; GLP); cytotoxicity: yes

- negative: Mammalian cell gene mutation assay with V79 cells of the Chinese hamster

(HPRT) (met. act.: with and without) (OECD TG 476; GLP); cytotoxicity: yes; read across from Quarternary Ammonium compounds, tri-C8-C10-alkylmethyl, chlorides

- negative: In vitro mammalian chromosome aberration test with human lymphocytes

(met. act.: with and without) (OECD TG 487; GLP); cytotoxicity: yes; read across from Quarternary Ammonium compounds, tri-C8-C10-alkylmethyl, chlorides

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:

06.11.2019 - 19.02.2020

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:

30 May 2008

Deviations:

no

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

Specific details on test material used for the study:

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

Target gene:

histidine operon

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

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

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

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

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

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

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

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

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)

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.

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

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

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

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

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”.

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

   

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood samples from healthy non-smoking donors not receiving medication
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Sex, age and number of blood donors if applicable: pre-test: one female donor (32 years old); Experiment I: one male donor (33 years old); Experiment II: one female donor (31 years old)
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Phenobarbital/b-Naphthoflavone induced rat liver S9)

Test concentrations with justification for top dose:

Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test.
With regard to the purity (90.6%) of the test item, 2208 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 2.8 to 2208 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 26.2 μg/mL and above in the absence of S9 mix and at 45.8 μg/mL and above in the presence of S9 mix. Due to strong cytotoxic effects at all applied concentrations, this preliminary test was repeated with a top dose of 15.0 μg/mL and designated Experiment I, since the new cultures fulfilled the requirements for cytogenetic evaluation. Clear toxic effects were observed after 4 hours treatment with 2.8 μg/mL and above in the pre-test and with 0.53 μg/mL and above in Experiment I in the absence of S9 mix. In the presence of S9 mix clear toxic effects were observed after 4 hours treatment with 2.8 μg/mL and above in the pre-test and with 1.63 μg/mL and above in Experiment I. Considering the toxicity data of the pre-test and Experiment I, 2.5 μg/mL (without S9 mix) were chosen as top concentration in Experiment II.

Concentrations applied:
Experiment I: With and without S9 mix; 4 h exposure: 0.06; 0.10; 0.17; 0.30; 0.53; 0.93; 1.63; 2.86; 5.0; 15.0* µg/mL
Experiment II: Without S9 mix; 20 h exposure: 0.01; 0.02; 0.03; 0.05; 0.09; 0.15; 0.27; 0.47; 0.82; 1.43; 2.5 µg/mL
* Phase separation was observed by the unaided eye at the end of treatment.

Vehicle / solvent:

- DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine (100 ng/mL; without metabolic activation; continuous treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h incubation with PHA (mitogen) to stimulate lymphocyte proliferation
- Exposure duration: 4 h (pulse exposure); 20 h (continuous exposure; without S9 mix)
- Expression time (cells in growth medium): 16 h (recovery period; pulse exposure only) + another 20 h after addition of Cytochalasin B
- Fixation time (start of exposure up to fixation of cells): 40 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 μg/mL)

STAIN (for cytogentic assay): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cultures were harvested by centrifugation (5 min)
- cells were resuspended in approximately 5 mL "saline G" and spun down once again (5 min)
- cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes
- 1mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added and the cells were resuspended
- after removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold
- slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide
- cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).
- The micronuclei have to be stained in the same way as the main nucleus.
- The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: determination of CBPI (Cytokinesis-block proliferation index) in 500 cells per culture; A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
CBPI = ((Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)) / Total number of cells

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).

An increase in the number of micronucleated mononucleate cells may indicate that the test item has aneugenic potential.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no

HISTORICAL CONTROL DATA
- Positive historical control data: see table 1
- Negative (solvent/vehicle) historical control data: see table 1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In Experiment I in the absence and presence of S9 mix, clear cytotoxicity was observed to the highest evaluated concentration. In Experiment II in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

Table 1: Historical control data (2015 - 2016)

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
No. of experiments	78	79
Mean	0.60	0.57
95 % Ctrl limit	0.08 – 1.12	0.12 – 1.03
1x SD	0.26	0.23
Min – Max	0.15 – 1.65	0.05 – 1.35

 

Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
No. of experiments	96
Mean	0.62
95 % Ctrl limit	0.16 – 1.08
1x SD	0.23
Min – Max	0.15 – 1.30

 

Positive Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
	MMC	Demecolcine
No. of experiments	78	81
Mean	12.48	3.72
95 % Ctrl limit	1.44 – 23.52	1.43 – 6.01
1x SD	5.52	1.15
Min – Max	4.15 – 30.30	2.10 – 7.25

 

Positive Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
	CPA
No. of experiments	165
Mean	5.16
95 % Ctrl limit	0.84 – 9.49
1x SD	2.16
Min – Max	2.10 – 11.90

Table 2: Summary of results

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells	95% Ctrl limit
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	40 hrs	Solvent control^1	1.86		0.65	0.08 – 1.12
		Positive control^2	1.65	24.6	19.15^S	1.44 – 23.52
		0.17	1.82	4.5	0.65
		0.30	1.65	25.0	0.45
		0.53	1.44	48.8	0.35
Exposure period 20 hrs without S9 mix
II	40 hrs	Solvent control^1	1.82		0.80	0.12 – 1.03
		Positive control^3	1.47	42.2	3.50^S	1.43 – 6.01
		0.09	1.78	4.4	0.50
		0.15	1.66	18.9	0.40
		0.27	1.54	34.4	0.55
Exposure period 4 hrs with S9 mix
I	40 hrs	Solvent control^1	1.99		1.05	0.16 – 1.08
		Positive control^4	1.66	33.0	8.70^S	0.84 – 9.49
		0.53	1.93	6.2	0.95
		0.93	1.66	32.8	1.15
		1.63	1.50	48.9	0.70

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S The number of micronucleated cells is statistically significantly higher than corresponding control values

1 DMSO 0.5 % (v/v)

2 MMC 0.8 µg/mL

3 Demecolcine 100 ng/mL

4 CPA 17.5 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or to the highest evaluable concentration.

Executive summary:

The test item was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I
Stimulation period	48 hrs	48 hrs	48 hrs
Exposure period	4 hrs	20 hrs	4 hrs
Recovery	16 hrs	-	16 hrs
Cytochalasin B exposure	20 hrs	20 hrs	20 hrs
Total culture period	88 hrs	88 hrs	88 hrs

In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2208 μg/mL of the test item) was chosen with regard to the purity (90.6%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix, clear cytotoxicity was observed to the highest evaluated concentration. In Experiment II in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In the presence of S9 mix, however, one value (1.15 % micronucleated cells) after treatment with 0.93 μg/mL slightly exceeded the 95% control limit (0.16 – 1.08 %), but was in the min-max range (0.15 – 1.30%) of the historical control data. Neither is this value statistically significant increased nor was a dose-dependency observed. Therefore, this observation is regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University; D-64287 Darmstadt)
- Suitability of cells: Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.
- Doubling time: 12 - 16 hours
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37° C in 75 cm^2 plastic flasks. About 2-3 x 10^6 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium) containing Hank’s salts supplemented with 10 % fetal calf serum (FCS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were subcultured twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration: MEM (Minimal Essential Medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During 4 hours treatment no FBS was added to the medium. During 24 hours treatment the medium was supplemented with 10% FBS. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 (98.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, before freezing
- Periodically checked for karyotype stability: yes, before freezing
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor-supplemented rat liver S9 (Phenobarbital/f3-Naphthoflavone induced)

Test concentrations with justification for top dose:

Experiment 1:
without S9 mix; 4h exposure: 0.08; 0.16; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0*; 20.0* µg/ml
with S9 mix, 4h exposure: 0.4; 0.8; 1.6; 3.13; 6.25; 12.5; 18.75*; 25.0*; 30.0* µg/mI
Experiment 2:
without S9 mix; 24h exposure: 0.04; 0.08; 0.16; 0.31; 0.63; 1.25; 2.5; 3.75; 5.0; 10.0* µg/mI
with S9 mix; 4h exposure: 0.3; 0.63; 1.25; 2.5; 5.0; 10.0; 15.0; 20.0*; 25.0* µg/mI
* phase separation visible at the end of treatment

The concentration range of the first main experiment was chosen according to the data generated in the first pre-experiment. The concentration range of the second experiment was chosen based on the results of the second pre-experiment (24 h without metabolic activation) and of the first main experiment (with metabolic activation).

In experiment I the cultures at the two highest concentrations with and without metabolic activation were not continued due to exceedingly severe cytotoxic effects. The cultures at the lowest concentration in experiment I with and without metabolic activation were not continued as a minimum of only four analyzable concentrations is required by the guidelines. In experiment II the cultures at the lowest concentration with metabolic activation were not continued for the same reason. The cultures at the four highest concentrations in experiment II without metabolic activation, and the two highest concentrations with metabolic activation were not continued due to exceedingly severe cytotoxicity.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: Approximately 0.7 to 1.2×10^7 cells/flask

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 hours; 24 hours (without S9 mix)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 8 days
- Fixation time (start of exposure up to fixation): approx. 15 days

SELECTION AGENT (mutation assays): thioguanine (6-TG); 11 μg/mL

STAIN (for cytogenetic assays): 10 % methylene blue in 0.01% KOH solution

STAINING TECHNIQUE USED:
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment.
Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of approx. 7 days five 80 cm^2 cell culture flasks were seeded with about 4 - 5 x 10^5 cells each in medium containing 6-TG. Two additional 25 cm^2 flasks are seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures are incubated at 37°C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methyhene blue in 0.01 % KOH solution.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- cloning efficiency

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).

Statistics:

A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05.
A t-Test was performed using a validated test script of “R” to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. A t-test was judged as significant if the p-value (probability value) was below 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no

HISTORICAL CONTROL DATA:
- Positive historical control data: see table 1
- Negative (solvent/vehicle) historical control data: see table 1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures was observed in experiment I at 5.0 μg/mL and above without metabolic activation and at 18.75 μg/mL with metabolic activation. In experiment II cytotoxic effects as described above was observed at 0.16 μg/mL and above without metabolic activation and at 15.0 μg/mL with metabolic activation.

Table 2: Mutagenicity data (Mutation rates), Experiment 1, Culture 1

	conc.		S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 10^6cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with DMSO			-	4	7	1	4	3	3.8	2.2	10.9
Positive control with EMS	300.0		-	80	84	93	79	89	85.0	6.0	231.6
Test item	0.08		-	culture was not continued^##
Test item	0.16		-	6	2	5	9	7	5.8	2.6	18.9
Test item	0.31		-	6	7	8	5	8	6.8	1.3	24.4
Test item	0.63		-	8	6	5	5	3	5.4	1.8	16.3
Test item	1.25		-	6	6	5	6	6	5.8	0.4	16.2
Test item	2.5		-	4	9	10	4	12	7.8	3.6	19.7
Test item	5.0		-	6	5	6	2	1	4.0	2.3	11.0
Test item	10.0	PS	-	culture was not continued^#
Test item	20.0	PS	-	culture was not continued^#
Solvent control with DMSO			+	4	11	4	10	7	7.2	3.3	26.1
Positive control with DMBA	2.3		+	34	41	29	36	25	33.0	6.2	129.5
Test item	0.40		+	culture was not continued^##
Test item	0.80		+	10	11	7	6	6	8.0	2.3	34.8
Test item	1.60		+	4	4	4	5	5	4.4	0.5	18.9
Test item	3.13		+	7	9	9	10	8	8.6	1.1	32.2
Test item	6.25		+	3	3	5	6	4	4.2	1.3	18.7
Test Item	12.5		+	11	5	6	5	11	7.6	3.1	34.3
Test item	18.75	PS	+	13	20	13	21	19	17.2	3.9	62.6
Test item	25.0	PS	+	culture was not continued^#
Test item	30.0	PS	+	culture was not continued^#

PS = phase separation visible at the end of treatment

# culture was not continued based on exceedingly serve cytotoxicity

## culture was not continued as a minimum of only four analyzable concentrations are required

Table 3: Mutagenicity data (Mutation rates), Experiment 1, Culture 2

	conc.		S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 10^6cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with DMSO			-	2	6	7	6	6	5.4	1.9	22.3
Positive control with EMS	300.0		-	71	59	77	68	75	70.0	7.1	329.0
Test item	0.08		-	culture was not continued^##
Test item	0.16		-	4	6	7	5	6	5.6	1.1	21.4
Test item	0.31		-	9	3	6	6	4	5.6	2.3	24.6
Test item	0.63		-	7	4	5	6	6	5.6	1.1	27.0
Test item	1.25		-	3	6	9	8	8	6.8	2.4	29.4
Test item	2.5		-	3	3	4	3	5	3.6	0.9	13.6
Test item	5.0		-	6	7	3	9	4	5.8	2.4	20.8
Test item	10.0	PS	-	culture was not continued^#
Test item	20.0	PS	-	culture was not continued^#
Solvent control with DMSO			+	7	8	10	2	6	6.6	3.0	19.9
Positive control with DMBA	2.3		+	71	65	63	58	49	61.2	8.3	299.4
Test item	0.40		+	culture was not continued^##
Test item	0.80		+	5	7	6	9	5	6.4	1.7	18.6
Test item	1.60		+	4	6	4	5	7	5.2	1.3	16.7
Test item	3.13		+	5	2	6	5	3	4.2	1.6	13.9
Test item	6.25		+	9	4	10	6	7	7.2	2.4	22.5
Test Item	12.5		+	8	8	7	5	8	7.2	1.3	21.9
Test item	18.75	PS	+	15	12	9	16	17	13.8	3.3	48.0
Test item	25.0	PS	+	culture was not continued^#
Test item	30.0	PS	+	culture was not continued^#

PS = phase separation visible at the end of treatment

# culture was not continued based on exceedingly serve cytotoxicity

## culture was not continued as a minimum of only four analyzable concentrations are required

Table 4:   Mutagenicity data (Mutationrates), Experiment 2, Culture 1

	conc.		S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 10^6cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with DMSO			-	5	5	7	10	12	7.8	3.1	23.4
Positive control with EMS	300.00		-	257	240	261	272	259	257.8	11.5	876.0
Test item	0.04		-	9	9	6	7	11	8.4	1.9	28.7
Test item	0.08		-	6	7	4	11	6	6.8	2.6	21.1
Test item	0.16		-	8	6	5	7	8	6.8	1.3	18.8
Test item	0.31		-	10	5	9	15	17	11.2	4.8	33.2
Test item	0.63		-	10	7	6	10	10	8.6	1.9	26.3
Test item	1.25		-	5	5	4	4	5	4.6	0.5	13.0
Test item	2.50		-	culture was not continued^#
Test item	3.75		-	culture was not continued^#
Test item	5.00		-	culture was not continued^#
Solvent control with DMSO			+	4	5	7	5	5	5.2	1.1	16.4
Positive control with DMBA	2.30		+	29	36	41	28	37	34.2	5.5	126.3
Test item	0.30		+	culture was not continued^##
Test item	0.63		+	3	4	3	4	3	3.4	0.5	10.5
Test item	1.25		+	6	2	4	4	2	3.6	1.7	10.7
Test item	2.50		+	7	2	5	7	2	4.6	2.5	14.5
Test item	5.00		+	2	4	4	1	7	3.6	2.3	11.6
Test Item	10.00		+	3	2	5	7	5	4.4	1.9	13.5
Test item	15.00		+	7	3	2	4	4	4.0	1.9	15.1
Test item	20.00	PS	+	culture was not continued^#
Test item	25.00	PS	+	culture was not continued^#

PS = phase separation visible at the end of treatment

# culture was not continued based on exceedingly serve cytotoxicity

## culture was not continued as a minimum of only four analyzable concentrations are required

Table 5:   Mutagenicity data (Mutationrates), Experiment 2, Culture 2

	conc.		S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 10^6cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with DMSO			-	2	5	4	5	7	4.6	1.8	20.5
Positive control with EMS	300.00		-	120	128	114	118	141	124.2	10.7	498.3
Test item	0.04		-	8	8	8	10	7	8.2	1.1	28.9
Test item	0.08		-	4	3	6	5	6	4.8	1.3	16.4
Test item	0.16		-	3	2	5	3	4	3.4	1.1	10.5
Test item	0.31		-	7	6	6	3	2	4.8	2.2	19.0
Test item	0.63		-	6	6	5	4	5	5.2	0.8	16.1
Test item	1.25		-	1	1	4	4	4	2.8	1.6	14.5
Test item	2.50		-	culture was not continued^#
Test item	3.75		-	culture was not continued^#
Test item	5.00		-	culture was not continued^#
Solvent control with DMSO			+	5	2	2	1	2	2.4	1.5	10.2
Positive control with DMBA	2.30		+	32	30	44	46	28	36.0	8.4	158.0
Test item	0.30		+	culture was not continued^##
Test item	0.63		+	5	6	5	2	3	4.2	1.6	15.9
Test item	1.25		+	5	4	7	6	6	5.6	1.1	20.9
Test item	2.50		+	8	6	6	5	5	6.0	1.2	20.8
Test item	5.00		+	3	3	3	4	5	3.6	0.9	12.6
Test Item	10.00		+	1	3	4	3	2	2.6	1.1	10.2
Test item	15.00		+	7	5	3	4	3	4.4	1.7	16.9
Test item	20.00	PS	+	culture was not continued^#
Test item	25.00	PS	+	culture was not continued^#

PS = phase separation visible at the end of treatment

# culture was not continued based on exceedingly serve cytotoxicity

## culture was not continued as a minimum of only four analyzable concentrations are required

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each.

The first main experiment was performed with a treatment period of 4 hours with and without microsomal activation. The second main experiment was performed with a 24 hours treatment period without microsomal activation and a 4 hours treatment period with microsomal activation.

The maximum test item concentration of the pre-experiment (2208.0 μg/mL) was chosen with respect to the current OECD guideline 476 regarding the content of the test item. The test item was dissolved in DMSO.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A reverse gene mutation assay in bacteria according to OECD guideline 471 is available for the target substance Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates. A mammalian cell gene mutation assay, and an in vitro mammalian chromosome aberration test are available for the structurally closely related source substances Quarternary Ammonium compounds, tri-C8-C10-alkylmethyl, chlorides. A justification for read-across is attached to IUCLID section 13.2.

 

In vitro gene mutation study in bacteria:

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. 

In vitro gene mutation study in mammalian cells:

The study was performed to investigate the potential of the read-across source substance Quarternary Ammonium compounds, tri-C8-C10-alkylmethyl, chlorides to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each.

The first main experiment was performed with a treatment period of 4 hours with and without microsomal activation. The second main experiment was performed with a 24 hours treatment period without microsomal activation and a 4 hours treatment period with microsomal activation.

The maximum test item concentration of the pre-experiment (2208.0 μg/mL) was chosen with respect to the current OECD guideline 476 regarding the content of the test item. The test item was dissolved in DMSO.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

 

In vitro chromosome aberration study in mammalian cells:

The read-across source substance was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments.

In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2208 μg/mL of the test item) was chosen with regard to the purity (90.6%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix, clear cytotoxicity was observed to the highest evaluated concentration. In Experiment II in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In the presence of S9 mix, however, one value (1.15 % micronucleated cells) after treatment with 0.93 μg/mL slightly exceeded the 95% control limit (0.16 – 1.08 %), but was in the min-max range (0.15 – 1.30%) of the historical control data. Neither is this value statistically significant increased nor was a dose-dependency observed. Therefore, this observation is regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or to the highest evaluable concentration.

Justification for classification or non-classification

Based on the available data, the target substance Quarternary ammonium compounds, tri-C8-C10-alkylmethyl, Me sulfates does not need to be classified and labelled according to the CLP Regulation (EC) No 1272/2008 with respect to mutagenicity.