3-((C12-18)-acylamino)-N-(2-((2-hydroxyethyl)amino)-2-oxoethyl)-N,N-dimethyl-1-propanaminium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

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:

-

EC Number:

427-370-1

EC Name:

-

Cas Number:

164288-56-6

Molecular formula:

C21H44N3O3Cl

IUPAC Name:

{[(2-hydroxyethyl)carbamoyl]methyl}dimethyl(3-octadecanamidopropyl)azanium chloride

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Specific details on test material used for the study:

Purity: 94%
Lot number: 1514MP
Description: orange solid gel
Storage conditions: room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The ceIl-cycle time for the lymphocytes from the donor used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 14 hours under optimal growth conditions.

Metabolic activation:

with and without

Metabolic activation system:

Lot No. Aro. S9/22/08/98 was prepared, in-house, from the livers of male Sprague-Dawley rats weighing 250g. These had received a single injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation.

Test concentrations with justification for top dose:

Experiment 1
Final concentration of the test item (ug/mL)
(4) 16h -S9 0, 39.07, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000, EMS 750
(4) 16h +S9 0, 39.07, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000, CP 25

EMS: Ethyl methanesulphonate
CP: Cyclophosphamide

Experiment 2
Final concentration of the test item (pg/ml)
20h -S9 0, 2.44, 4.88, 9.77, 19.54, 39.07, 78.13, 156.25, 312.5, EMS 500
(4) 16h +S9 0, 4.88, 9.77, 19.54, 39.07, 78.13, 156.25, 312.5, 625, CP 25

Controls

Controls 1

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Controls 2

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

CelI Culture
CeIls were grown in Eagle’s minimal essential media, (supplemented with sodium bicarbonate, HEPES buffer, L-glutamine, pen iciIin/streptomycin, amphotericin B and 15% foetal calf serum) at 37 °C with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 ug/mL final concentration.

The test material was accurately weighed, dissolved in Minimal Essential Media (MEM) and serial two-fold dilutions prepared. The maximum dose level tested was 5000 ug/mL which was the maximum recommended dose level. There was no observable change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOSM. Chemical analysis of the test material formulations was not performed because it is not a requirement of the test method.

Culture Conditions - Experiment 1
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05 to 9.05 ml MEM, 15% (FCS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

Treatment With-activation:
After approximately 48 hours incubation at 37°C, 5% C02 in humidified air, the with-S9 cultures were centrifuged after transfer into tubes and approximately 9 ml of the culture medium removed, reserved and replaced with MEM (including serum) and 0.1 ml of the appropriate solution of positive control or 1 ml of appropriate solution of vehicle or test material was added to each culture. The final concentrations of the test material were 39.07, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 ug/ml. 1 ml of 10% S9 (je 1 % final concentration of S9) in standard co-factors was added and the cultures returned to the incubator.
After 4 hours at 37 °C the with-S9 cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 16 hours.

Treatment Without-activation:
After approximately 48 hours incubation at 37°C, 5% C02 in humidified air the cultures were decanted into centrifuge tubes and centrifuged, as for the treatment with-activation. Most of the culture media was drawn off and stored. The cells were then resuspended with fresh MEM (including serum).
The total volume for each culture was a nominal 9.9 ml for non-aqueous vehicles and 9.0 mIs for aqueous vehicles. The final concentrations were the same as for the with-activation treatments.

Culture Conditions - Experiment 2
These were as in previous section, except that there was continuous exposure in the absence of metabolic activation. The exposure in the presence of activation was for 4 hours, followed by a 16-hour expression period as performed in Experiment 1. The final concentration of S9 was increased, 1 ml of 20% S9 (ie 2% final concentration) in standard co-factors was added and the cultures returned to the incubator. The final concentrations of the test material were 2.44, 4.88, 9.77, 19.54, 39.07, 78.13, 156.25, 312.5 and 625 ug/ml.

Evaluation criteria:

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. CeIls with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) in either the presence or absence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16-hour expression period and a 4-hour exposure in the absence of activation with a 16-hour expression period, this was Experiment 1. In Experiment 2 the 4-hour exposure with addition of S9 was repeated (using 2% final S9 concentration), whilst in the absence of activation the exposure time was increased to 20 hours.

The method used followed that described in the OECD Guidelines for the Testing of Chemicals (1997) No. 473 “Genetic Toxicology: Chromosome Aberration Test” and Method B10 of Commission Directive 92/69/EEC.

 

Cytotoxicity was observed with the tested concentrations.

 

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

 

AIl the positive control treatments gave statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either of two separate experiments. The test material was shown to be non-clastogenic to human lymphocytes in vitro.