Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 487, Draft Version 1

Deviations:

no

Principles of method if other than guideline:

OECD Guideline Draft Proposal for a new Guideline No. 487, Version 1

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

EC Number:

271-756-9

EC Name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Cas Number:

68607-24-9

Molecular formula:

The substance is a quarternary ammonium chloride derived from fatty alcohol. The general chemical structure is R-N(Me)3+ Cl-, whereas the alkylrest R is variable for the main components C20 and C22 but also shorter and longer C-Chains (in smaller amounts)

IUPAC Name:

N,N,N-trimethyl-C20-22-(even numbered)-alkyl-1-aminium chloride

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Seromed, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5% air).

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Exp. I: with and without S9 mix: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0, and 400.0 µg/mL
Exp. IIA: with and without S9 mix: 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, and 25.0 µg/mL
Exp. IIB: without S9 mix: 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.5, and 15.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: solubility

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

and colcemid and cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 20 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours (48 hours for Exp. II with S9 mix) after start of treatment with the test item.

METHOD OF APPLICATION: in minimal essential medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (48 hours for Exp. II with S9 mix)

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa

NUMBER OF REPLICATIONS: 1.5 - 2

NUMBER OF CELLS EVALUATED: 2000

EVALUATION: Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index

OTHER EXAMINATIONS:

Evaluation criteria:

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical control data (0.0-2.0% micronucleated cells), and
- either a concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the historical control data (0.0-2.0% micronucleated cells), and/or
- no concentration-related increase in the number of micronucleated cells is observed.
Statistical significance can be confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria above mentioned for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

Statistical significance can be confirmed by means of the Chi square test.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see textbelow

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item Behentrimonium chloride, suspended (Experiment I) or dissolved (Experiments IIA and llB) in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vifro in the absence and the presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without metabolic activation. In Experiment IIA and IIB, the exposure period was 20 hrs without S9 mix and in Experiment 11, the exposure period was 4 hours with metabolic activation. The cells were prepared 24 hours (Exp. I, IIA and llB) and 48 hrs (Exp. IIA) after start of treatment with the test item.
In each experimental group two parallel cultures were set up. Per culture 1000 cells were scored for micronuclei.
Evaluated experimental points after treatment with Behentrimonium chloride:
Exp I: 3.1, 6.3 and 12.5 µg/ml, without S9 mix, 4 hrs exposure, 24 hrs preparation interval
Exp IIA: 3.1, 6.3 and 12.5 µg/ml, without S9 mix, 20 hrs exposure, 24 hrs preparation interval
Exp IIB: 5.0, 6.0, and 7.0 µg/ml, without S9 mix, 20 hrs exposure, 24 hrs preparation interval
Exp I: 6.3, 12.5 and 25.0 µg/ml, with S9 mix, 4 hrs exposure, 24 hrs preparation interval
Exp IIA: 3.1, 6.3 and 12.5 µg/ml, with S9 mix, 4 hrs exposure, 48 hrs preparation interval

In Experiment I, no relevant increase of the pH value or osmolarity was observed (solvent control 271 mOsm, pH 7.3 versus 288 mOsm and pH 7.4 at 400.0 µg/mL). Test item precipitation was observed at a concentration of 50.0 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 12.5 µg/mL (11.3 % of control) and above in the absence of S9 mix. In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.
In the absence and presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the test item in the absence of S9 mix (0.95 - 1.55 %) and in the presence of S9 mix (0.85 - 1.55 %) were below the corresponding solvent control values (2.00 and 1.70 %, respectively) and within the range the laboratory's historical control data: 0.0 - 2.0 % micronucleated cells.
In Experiment IIA, test item precipitation was observed at a concentration of 6.3 µg/mL and above in the absence of S9 mix and at 25.0 µg/mL and above in the presence of S9 mix. Clear cytototoxicity (indicated by reduced cell numbers when compared to control values) of about 60% or above were observed after treatment with 12.5 µg/mL (70 % cytotoxicity) and above in the absence of S9 mix, and with 12.5 µg/mL (58 % cytotoxicity) and above in the presence of S9 mix.
In the presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluated concentration of 12.5 µg/mL. The rates of micronucleated cells after treatment with the test item (0.60 - 1.00%) were close to the corresponding solvent control value (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. In the absence of S9 mix, dose-related increases in the percentage of micronucleated cells (1 -65 %, 1.75 %, and 2.40 %) were observed at the three evaluated concentrations (3.1, 6.3, and 12.5 µg/mL, respectively). Although all three values were statistically significantly increased compared to the corresponding solvent control value (0.85%), the percentages of micronucleated cells at 3.1 and 6.3 µg/mL (1.65 % and 1.75 %, respectively) were within the laboratorie's historical control data range (0.0 - 2.0 % micronucleated cells), and these increases have to be regarded as biologically irrelevant. At the highest concentration evaluated (12.5µg/mL), given the high cytotoxicity observed (70%), the biological relevance of the increased incidence of micronucleated cells remained ambiguous.
Accordingly, two repeat experiments (Experiment IIB) within a narrow concentration range were performed, in order to verify the observation in Experiment IIA.
The first repeat experiment could not be evaluated, since only two concentrations were available for cytogenetic evaluation, which is not in compliance with the guideline recommendations. In both experiments, concentrations of 7.5 µg/mL and 8.0 µg/mL and above were not scorable for cytogenetic damage due to strong test item induced cytotoxicity.
In the second repeat experiment, test item precipitation was observed at a concentration of 6.0 µglmL and above in the absence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 7.0 µg/mL (62 % cytotoxicity). No biologically relevant increase in the percentage of micronucleated cells was observed up to the highest scorable concentration. The rates of micronucleated cells after treatment with the test item (0.05 - 1.05 %) were close to the corresponding solvent control values (0.55 %) and within the range of our historical control data: 0.0 - 2.0 % micronucleated cells. Accordingly, the increased incidence of micronucleated cells observed at a concentration associated with high cytotoxicity
(Experiment IIA in the absence of S9 mix) could not be reproduced, and this finding was considered to bear no biological significance.
In Experiment IIB, test item precipitation was observed at a concentration of 6.0 µg/mL and above in the absence of S9 mix. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 7.0 µg/mL.
Colcemid (25 ng/mL to 10 µg/mL), Mitomycin C (0.03 or 0.1 µg/mL) or CPA (2.5 to 25 µg/mL) were evaluated as positive controls and showed a distinct increase in the percentage of micronucleated cells.
In conclusion, it can be stated that under the experimental conditions reported, the test item Behentrimonium chloride did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation.
Therefore, Behentrimonium chloride has to be considered as non-mutagenic in this in vitro test system, when tested up to cytotoxic test item concentrations (Experiment I, with metabolic activation, Experiment IIA with and without metabolic activation, and Experiment IlB, without metabolic activation) or the highest scorable concentration (Experiment I, with metabolic activation).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: V79

Any other information on results incl. tables

Dose selection

Dose selection was performed following the current OECD Guideline for in vitro chromosome aberration studies (OECD Guideline no. 473). The highest concentration chosen for the evaluation of genotoxicity should produce clear toxicity with reduced cell growth, determined by the mean of the cell count prior to cell seeding on slides, by > 60 % and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible.

With respect to the molecular weight of the test item, 5000.0 µg/mL of Behentrimonium chloride (approx. 9.7 mM) were applied as top concentration in Experiment I. Test item concentrations between 9.8 and 5000.0 µg/mL (with and without S9 mix) were chosen for the treatment of the cultures. Due to strong toxic effects indicated by reduced cell numbers at test item concentrations of 9.8 µg/mL and above in the absence and presence of metabolic activation, Experiment I was repeated with test item concentrations between 0.8 and 400.0 µg/mL (with and without S9 mix). With regard to the purity of the test item, the corresponding amount of the active substance in the respective test item concentrations was 78.8 %.

In Experiment I, precipitation of the test item in culture medium was observed with 50.0 µg/mL and above in the absence of S9 mix, and with 25.0 µg/mL and above in the presence of S9 mix. Using reduced cell numbers of about 40 % of control or below as an indicator for toxicity in Experiment I, clear toxic effects were observed after 4 hrs treatment with 12.5 µg/mL and above in the absence of S9 mix, and with 50.0 µg/mL and above in the presence of S9 mix. With respect to the results obtained in Experiment I, 25.0 µg/mL was chosen as top concentration for Experiment IIA in the absence and presence of S9 mix. In a confirmatory experiment, designated Experiment llB, concentrations between 2.5 and 20.0 µg/mL, in the absence of S9 mix, were chosen in order to verify the results obtained in Experiment IIA. Due to strong test item induced cytotoxicity at concentrations of 7.5 µg/mL and above, only two concentrations were scorable for cytogenetic damage. Therefore, this experimental part was repeated with test item concentrations between 0.5 and 15.0 µg/mL.

Summary of results of the micronucleus test with Behentrimonium chloride

Exp.	Preparation	Test item	Cell number	Micronucleated
	interval	concentration	In %	cells
		in µg/mL	of control	in %
Exposure period 4 hrs without S9 mix
I	24 hrs	Negative control	103	0.55
		Solvent control^1	100	2.00
		Positive control^2	78.9	5.1S
		Positive control^3	80.2	3.25S
		Positive control^4	78.1	4.70S
		Positive control^5	45.8	17.95S
		3.1	88.9	0.95
		6.3	62.7	1.55
		12.5	11.3	1.15
Exposure period 20 hrs without S9 mix
IIA	24 hrs	Negative control	99.5	1.05
		Solvent control^1	100	0.85
		Positive control^2	51.8	28.3S
		Positive control^3	44.4	28.80S
		Positive control^4	72.3	9.45S
		Positive control^5	61.4	13.85S
		3.1	97.4	1.65S
		6.3 P	57.5	1.75S
		12.5 P	29.9	2.40S
Exposure period 20 hrs without S9 mix
IIB	24 hrs	Negative control	90.0	0.30
		Solvent control^1	100	0.55
		Positive control^2	40.6	66.35S
		Positive control^3	33.8	50.35S
		Positive control^4	64.0	6.85S
		Positive control^5	69.8	6.00S
		5.0	66.4	1.05
		6.0 P	47.1	0.70
		7.0 P	37.5	0.05
Exposure period 4 hrs with S9 mix
I	24 hrs	Negative control	158.3	0.90
		Solvent control^1	100	1.70
		Positive control^6	49.0	8.40S
		Positive control^7	41.0	5.15S
		6.3	159.4	1.55
		12.5	113.2	1.55
		25.0 P	57.4	0.85
Exposure period 4 hrs with S9 mix
IIA	48 hrs	Negative control	100.8	0.95
		Solvent control^1	100	0.55
		Positive control^8	67.7	15.55S
		Positive control^9	19.9	44.70S
		3.1	82.2	0.70
		6.3	80.9	1.00
		12.5	42.4	0.60

 

P         precipitation occurred

S        number of micronucleated cells statistically significant higher than corresponding control values

1        deioinised water 10%(v/v)

2         colcemid 7.5µg/mL

3         colcemid 10.0 µg/mL

4         mitomycin C 0.03 µg/mL

5         mitomycin C 0.1 µg/mL

6         cyclophosphamide 10 µg/mL

7         cyclophosphamide 25 µg/mL

8         cyclophosphamide 2.5 µg/mL

9         cyclophosphamide 5.0 µg/mL

Applicant's summary and conclusion

Conclusions:

The genotoxicity of the registration substance was investigated according to the guideline OECD 487. Negative result was obtained.

Executive summary:

The registration substance was assessed for its clastogenicity according to the guideline OECD 487.

The registration substance did not induce micronuclei in V79 cells (Chinese hamster cell line) in the absence and the presence of metabolic activation at up to the concentration associated with clear cytoxicity. No significant clastogenicity was found.