Imidazolium compounds, 2-C17-unsatd.-alkyl-1-(2-C18-unsatd. amidoethyl)-4,5-dihydro-N-methyl, Me sulfates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007-10-11 to 2007-11-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L 1362000, Annex 4A: ”Mutagenicity – In vitro Mammalian Chromosome Aberration Test“, May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

partially unsaturated IQAC, DMS quaternised

IUPAC Name:

partially unsaturated IQAC, DMS quaternised

Constituent 2

Reference substance name:

Imidazolium compounds, 2-(C15-17(odd numbered), C17-unsatd. alkyl)-1-[2-(C16-18(even numbered), C18-unsatd. amido)ethyl]-4,5-dihydro-N-methyl, Me sulfates)

IUPAC Name:

Imidazolium compounds, 2-(C15-17(odd numbered), C17-unsatd. alkyl)-1-[2-(C16-18(even numbered), C18-unsatd. amido)ethyl]-4,5-dihydro-N-methyl, Me sulfates)

Constituent 3

Reference substance name:

Fatty acids, C16-18 (even numbered) and C18 unsatd., reaction products with diethylene triamine, di-Me sulfate quaternized

EC Number:

937-237-2

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Fatty acids, C16-18 (even numbered) and C18 unsatd., reaction products with diethylene triamine, di-Me sulfate quaternized

Method

Target gene:

Chromosome aberration assays detect the induction of chromosome breakage (clastogenesis). Although mutagenic substances produce structural
chromosome aberrations by a variety of mechanisms, the endpoint is a discontinuity in the chromosomal DNA which is left unrejoined, or rejoined
inaccurately, thus producing a mutated chromosome. Chromosome aberrations are generally evaluated in first post treatment mitoses. The majority of chemical mutagens induce aberration of the chromatid type, but chromosome type aberrations also occur.

Species / strain

Species / strain / cell type:

other: human lymphocytes

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication.
Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks.
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1; Life Technologies GmbH, 76339 Eggenstein, Germany) containing 10 % FCS (fetal calf serum) provided by PAA Laboratories GmbH (35091 Cölbe, Germany), the antibiotic solution containing 10,000 U/mL penicillin and 10,000 µg/mL streptomycin (SEROMED, D-12247 Berlin). Additionally, the medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), the anticoagulant heparin (25,000 U.S.P.-U/mL, NATTERMANN, 50829 Köln, Germany), and
HEPES (final concentration 10 mM, Serva, 69115 Heidelberg, Germany).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar HanIbm rats.

Test concentrations with justification for top dose:

Experiment I
with metabolic activation: 11.9, 20.8, 36.4, 63.7, 111.4, 195.0, 341.2, 597.1, 1044.9, 1828.6 and 3200 µg/mL
without metabolic activation: 20.8, 36.4, 63.7, 111.4, 195.0, 341.2, 597.1, 1044.9, 1828.6 and 3200 µg/mL

Experiment II
with metabolic activation: 10.0, 20.0, 50.0, 125.0, 250.0, 500.0, 750.0, 1000.0, 1250.0 and 1500.0 µg/mL
without metabolic activation: 8.1, 14.2, 25.9, 43.5, 76.2, 133.3, 233.2, 408.2, 714.3 and 1250 µg/mL
Results from the rangefinding assay were used to determine the dose range to be used in the chromosomal aberrations assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol 0.5%
- Justification for choice of solvent/vehicle: solubility

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION:

Range-finder
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by
the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay.
The pre-test phase was performed with 10 concentrations without S9 mix and 11 concentrations with S9 mix of the test item and a solvent and
positive control. All cell cultures were set up in duplicate. Exposure times were 4 hrs (with and without S9 mix). The preparation interval was 22 hrs
after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 µg/mL) to reassure the replication time of the cultured lymphocytes.

Dose Selection
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests
requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, 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 ability to formulate a homogeneous suspension of the test item, 3200 µg/mL of test substance
(approx. 4.8 mM) were applied as top concentration for treatment of the cultures in the pre-test. Doses over 3200 µg/mL led to an inhomogeneous
suspension in ethanol that was not applicable. Test item concentrations between 11.9 and 3200 µg/mL, and between 20.8 and 3200 µg/mL (with and without S9 mix, respectively) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed
before start of treatment at 36.4 µg/mL and above in the absence of S9 mix, and at 20.8 µg/mL and above in the presence of S9 mix. Since the
cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, toxic effects of about 50 % of control were observed after 4 hrs treatment
with 597.1 µg/mL and above in the absence and presence of S9 mix. Considering these toxicity data, 1250 µg/mL (without S9 mix) and 1500 µg/mL
(with S9 mix) were chosen as top concentrations in Experiment II.
The cytogenetic evaluation of higher concentrations in the respective preparation interval (with and without S9 mix) was impossible due to strong test item-induced toxic effects (low metaphase numbers, partially paralleled by poor metaphase quality and precipitation on the slides).

Exposure time 4 hours
The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test item.
For the treatment with metabolic activation 50 µL S9 mix per mL medium were used.
Concurrent solvent and positive controls were performed. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The
supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G" solution (NaCl -8000 mg, KCl -400 mg, glucose x H2O -1100 mg, Na2HPO47H2O - 290 mg, KH2PO4 --150 mg). The washing procedure was repeated once as described.
After washing the cells were re-suspended in complete culture medium and cultured until preparation.

Exposure time 22 hours (without S9 mix)
The culture medium was replaced with complete medium (with 10 % FCS) containing the test item without S9 mix. The culture medium at continuous
treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
All cultures were incubated at 37° C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

Preparation of the Cultures
Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by
dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa (MERCK, 64293 Darmstadt, Germany) or according to the Fluorescent plus Giemsa technique, respectively.

Analysis of Metaphase Cells
The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" [4]) using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. At least 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphase plates were scored. Only metaphases with 46 +/-1 centromer regions were included in the analysis. To describe a
cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 250 metaphase cells (% polyploid
metaphases) was scored.

Evaluation criteria:

A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 %
aberrant cells, excluding gaps).
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding
gaps) and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be
considered together. If the criteria 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.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human lymphocytes

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:

With respect to the ability to formulate a homogeneous suspension of the test item, 3200 µg/mL of the test substance (approx. 4.8 mM) were applied as top concentration for treatment of the cultures in the pre-test. Doses over 3200 µg/mL led to an inhomogeneous suspension in ethanol that was not applicable.
Test item concentrations between 11.9 and 3200 µg/mL, and between 20.8 and 3200 µg/mL (with and without S9 mix, respectively) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed before start of treatment at 36.4 µg/mL and above in the absence of S9 mix, and at 20.8 µg/mL and above in the presence of S9 mix.

Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.
 

Polyploid metaphases:
Polyploid metaphases occured, but in both experiments, no biologically relevant increase in the rate of polyploid metaphases was found
after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.8 %).


POSITIVE CONTROLS
In both experiments, EMS (880 and 770 µg/mL, respectively) and CPA (37.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Any other information on results incl. tables

 

Summary of results of the chromosomal aberration study with PC-2007-140 (W 575 no solvent)

Exp.	Preparation interval	Test item concentration in µg/mL	Polyploid cells in %	Mitotic indices in % of control	Incl. gaps	Aberrant cells in % excl. gaps	With exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control	0.8	100.0	2.0	2.0	1.0
		Positive control	0.2	64.2	10.0	9.0^S	0.0
		20.8	0.2	73.4	3.0	2.0	0.0
		36.4 P	0.4	82.6	2.5	1.5	0.5
		341.2 P	0.2	61.3	2.0	1.5	0.0
		597.1 P	0.4	50.5	0.5	0.5	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control	0.4	100.0	2.0	2.0	0.0
		Positive# control	0.0	25.3	50.0	49.0^S	11.0
		43.5##	0.2	72.3	5.0	4.5	0.0
		76.2 P	0.2	67.9	0.0	0.0	0.0
		133.3 P	0.0	53.6	3.0	3.0	0.0
		233.2 P	0.0	29.2	2.5	2.0	0.0
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control	0.2	100.0	2.0	1.5	0.0
		Positive control	0.4	49.7	8.5	8.5^S	2.0
		11.9	0.0	84.7	1.0	1.0	0.0
		20.8 P	0.4	88.9	1.5	1.0	0.5
		341.2 P	0.6	65.3	0.0	0.0	0.0
		597.1 P	0.4	73.4	2.5	2.0	0.0
II	22 hrs	Solvent control	0.0	100.0	2.0	1.0	0.0
		Positive control	0.0	34.6	22.0	20.0^S	1.0
		20.0	0.0	120.1	1.5	1.5	0.0
		50.0 P##	0.0	111.0	3.5	3.3^S	0.0
		125.0 P	0.0	110.2	1.5	1.5	0.0

 

P -Test item precipitation was observed

S - Aberration frequency statistically significant higher than corresponding control values

# - Evaluation of 50 metaphases per culture

## - Evaluation of 200 metaphases per culture

Applicant's summary and conclusion

Conclusions:

It can be stated that under the experimental conditions reported, the test itempartially unsaturated IQAC, DMS quaternised (no solvent) did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.

Executive summary:

In a mammalian chromosome aberration test performed according to OECD Guideline 473 (1997), human lymphocyte cultures were exposed to partially unsaturated IQAC, DMS quaternised (no solvent), (100% a.i.), suspended in ethanol at concentrations between 11.9 - 3200 µg/mL with metabolic activation and 20.8 - 3200 µg/mL without metabolic activation.

Partially unsaturated IQAC, DMS quaternised (no solvent) was tested up to cytotoxic or precipitating concentrations. 

The following experimental points were microscopically evaluated:11.9, 20.0, 20.8, 50.0, 125.0, 341.2, 597.1 µg/mL with metabolic activation and 20.8, 36.4, 43.5, 76.2, 133.3, 233.2, 341.2, 597.1 µg/mL without metabolic activation.

In the absence of S9 mix, reduced mitotic indices of about or below 50 % of control were observed at the highest evaluated concentrations. In the presence of S9 mix, concentrations showing clear cytotoxic effects were excluded from scoring for the endpoint cytogenicity.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.

 Positive controls induced the appropriate response. There was no evidence of Chromosome Aberration induced over background.

This study is classified as acceptable. It satisfies the requirement for Test Guideline In vitro mammalian Chromosome Aberration test OECD 473 for in vitro cytogenetic mutagenicity data.