Quaternary ammonium compounds, tri-C8-10-alkylmethyl, chlorides

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, tri-C8-10-alkylmethyl, chlorides

EC Number:

264-120-7

EC Name:

Quaternary ammonium compounds, tri-C8-10-alkylmethyl, chlorides

Cas Number:

63393-96-4

Molecular formula:

Unspecified

IUPAC Name:

Quaternary ammonium compounds, tri-C8-10-alkylmethyl, chlorides

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 38-043-11
- Purity/Content: 90.6%
- Expiry date: 02 January 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was verified analytically.
- Solubility and stability of the test substance in the solvent/vehicle: Stable in DMSO over a period of 4 hours

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO.
All formulations were prepared freshly before treatment and used within two hours of preparation.

FORM AS APPLIED IN THE TEST: diluted in DMSO

Method

Target gene:

HPRT

Species / strain

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.

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.