1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with C18-unsatd. fatty acids, Me sulfates (salts)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-06-03 to 1996-07-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18-unsatd. fatty acids, chlorides

Cas Number:

1079184-43-2

Molecular formula:

n.a. (UVCB)

IUPAC Name:

Ethanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18-unsatd. fatty acids, chlorides

Details on test material:

- Name of test material (as cited in study report): N,N-dimethyl-2-(stearoyloxy)-N-[2-(stearoyloxy)ethyl]ethanaminium chloride
- Molecular formula (if other than submission substance): Not available
- Molecular weight (if other than submission substance): 689
- Smiles notation (if other than submission substance): Not available
- InChl (if other than submission substance): Not available
- Structural formula attached as image file (if other than submission substance): Not available
- Substance type: active
- Physical state: white flakes

Method

Target gene:

Thymidine kinase (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: P0P Medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes. Each lot of cryopreserved cells was tested using the agar culture and Hoechst staining procedures-found to be free of contamination.
- Periodically checked for karyotype stability:no
- Periodically "cleansed" against high spontaneous background: yes. Prior to use in the assay, L5178Y cells were cleansed of spontaneous TK +/- cells by culturing in a restrictive medium.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Assay: 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 36 µg/mL. (presence and absence of S9-activation)
Mutagenesis Assay: 22.5, 25, 27.5, 30, 32.5, 35, 40, 50, 60, and 75 µg/mL (non-activated system) and 25, 27.5, 30, 32.5, 35, 40, 50, 60, and 75 µg/mL (S9-activated system).
Confirmatory Mutagenesis Assay: 5, 10, 20, 35, 60, 75, 90, 100, 110, 120 and 130 µg/mL (non-activated system) and 50, 60, 75, 90, 100, 110, 120, 130, 140, and 150 µg/mL (S9-activated system).
3rd Mutagenesis Assay: 35, 50, 60, 75, 90, 100, 110, 120, 130, 140 and 150 µg/mL (non-activated system) and 110, 120, 130, 140, 150, 200, 300, 400, 500, and 550 µg/mL (S9-activated).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone (CAS 67-64-1). The dosing solutions were adjusted to the acitve content of the test substance. Aliquots of dosing solution preparations were returned to the Sponsor for chemical analysis.
- Justification for choice of solvent/vehicle: acetone was chosen based on the Sponsor's request and compatibility with the target cells. The test substance was workable in acetone at 1.0 mg/mL. Concentrations of greater than or equal to 1.0 mg/mL were delivered to the test system as suspensions.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: (with metabolic activation)

Controls 2

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: (without metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium, combining 6 x 10 E6 L 5178Y/TK+/- cells and 100 µL dosing solution of test or control article in solvent or solvent alone in a total volume of 10 mL F0P medium or S9 activation mixture.

DURATION
- Preincubation period: Not applicable
- Exposure duration: 4 hours with mechaniclal mixing
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): TFT Medium (Trifluorthymidine)

NUMBER OF REPLICATIONS: 3 petri dishes for each sample/ 3 indipendent experiments

NUMBER OF CELLS EVALUATED: 1x10E6/plate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: Not available.
- Determination of endoreplication: Not available.

Evaluation criteria:

- Cytotoxic effects: of each treatment condition were expressed relative to the solvent-treated control for suspension growth over 2 days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection).
- Mutant frequency: (number of mutants per 10E6 surviving cells) was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding V.C. plates and multiplying by the dilution factor (2x10E-4).
- Induced mutant frequency: calculated by subtracting the average mutant frequency of the solvent controls from the mutant frequency of the test substance treated cultures. Due to the use of non-rounded numbers by the computer system in calculations, the induced mutant frequencies presented in the data tables may indicate a slight discrepancy (typically by a value of 1) from the difference between the average solvent control mutant frequency (a rounded number) and the mutant frequency of the test substance treated cultures (rounded numbers).

-Positive: a result was considered positive if there was a positive dose response and one or more of the three highest doses in the 10% or greater total growth range exhibited a mutant frequency which was greater than or equal to 100 mutants per 10E6 clonable cells over the background level.
-Equivocal: a result was considered equivocal if the mutant frequency in treated cultures was between 55 and 100 mutants per 10E6 clonable cells over background level.
-Negative: a result was considered to be negative if the test articles which produced fewer than 55 induced mutants per 10E6 clonable cells at dose levels with greater than or equal to 10 % total growth.

Statistics:

Not applicable

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not available.
- Effects of osmolality: Preliminary Toxicity Assay-osmolality of the solvent control was 202 mmol/kg and the osmolality of the top dose, 36 µg/ml, was 323 mmol/kg. Osmolality of higher concentrations not measured; pH not measured.
- Evaporation from medium: not applicable.
- Water solubility: not available.
- Precipitation: Preliminary Toxicity Assay--treatment medium was cloudy but with no visible precipitate at a concentration of 36 µg/ml. Initial Mutagenesis Assay--treatment medium was cloudy but with no visible precipitate at concentrations of greater than or equal to 15 µg/ml. Confirmatory Mutagenesis Assay--treatment medium was cloudy but with no visible precipitate at concentrations of greater than or equal to 60 µg/ml. Third mutagenesis Assay-treatment medium was cloudy but with no visible precipitate at concentrations of greater than or equal to 60 µg/ml. The highest achievable dose was 550 µg/ml. No justification given. Might be precipitation.


RANGE-FINDING/SCREENING STUDIES: not available.


COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data for solvent and positive controls were given. Solvent controls of second test with metabolic activation were considered to be unacceptable due to high mutant frequencies of 136 and 167 induced mutants, corresponding historical data range of mean mutation frequency is 35,3 – 50,3 (1993 - 1995).


ADDITIONAL INFORMATION ON CYTOTOXICITY: not available.

Any other information on results incl. tables

PRELIMINARY TOXICITY ASSAY: The maximum dose tested in the preliminary toxicity assay was 36 µg/mL. Concentrations of less than or equal to 10 µg/mL were soluble in treatment medium. Suspension growth relative to the solvent controls was 78 % at 36 µg/ml without activation and 92 % at 36 µg/ml with S9 activation. Based on the results of the toxicity test and the Sponsor's request, the doses chosen for the initital mutagenesis assay ranged from 1.0 to 75 µg/mL for both the non-activated and S9 -activated cultures.

INITIAL MUTAGENESIS ASSAY: In the non-activated system, cultures treated with test substance concentrations were cloned and produced a range in suspension growth of 28-81 %. In the S9 -activated system cultures treated with test substance concentrations were cloned and produced a range in suspension growth of 87-95 %. Concentrations of less than or equal to 10 µg/mL were soluble in treatment medium.

No treated cultures exhibited greater than or equal to 100 induced mutants per 10E6 clonable cells over the background level; three S9-activated cultures exhibited between 55 and 99 induced mutants per 10E6 clonable cells over the background level (40,60, and 75 µg/mL).

The total growths ranged from 23-76 % for the non-activated cultures at concentrations of 22.5-75 µg/mL and 70 -92 % for the S9 -activated cultures at concentrations of 25 -75 µg/mL. At the sponsor's request, the dose levels chosen for the confirmatory assay ranged from 1.0-150 µg/mL.

CONFIRMATORY MUTAGENESIS ASSAY: in the non-activated system, cultures treated with test substance concentrations of 5 to 120 µg/mL were cloned and produced a range in suspension growth of 17 -105 %. In the S9 -activated system cultures treated with test substance concentrations of 50 - 150 µg/mL were cloned and produced a range in suspension growth of 81 -95 %. Treatment medium was cloudy but with no visible precipitate at concentrations of  ≥ 60 µg/mL. Concentrations of less than or equal to 50 µg/mL were soluble in treatment medium.

No treated cultures exhibited greater than or equal to 100 induced mutants per 10E6 clonable cells over the background level. A dose-response trend was not observed in the non-activated or S9 -activated systems. The total growths ranged from 13 -90 % for the non-activated cultures at concentrations of 5 -120 µg/mL and 72 -103 % for the S9 -activated cultures. The solvent controls for the S9 -activated portion were unacceptable due to high mutant frequencies; however, the data are included in the report for completeness, along with the data from a repeat of the assay with S9 activation.

THIRD MUTAGENESIS ASSAY: performed since no cultures exhibited < 20 % total relative growth in the initial mutagenesis assay (despite the use of test substance concentrations that were cloudy in treatment medium) and the solvent controls for the S9 -activated portion were unacceptable in the confirmatory mutagenesis assay. At the Sponsor's request, an attempt was made to achieve a high dose of 1000 µg/mL in the S9 portion. The highest achievable dose was 550 µg/mL. The doses used in the third mutagenesis assay ranged from 35 -140 µg/mL without activation and from 110 -550 µg/mL with S9 activation. In the non-activated system, cultures treated with test substance concentrations were cloned and produced a range in suspension growth of 10 -66 %. In the S9 -activated system cultures treated with test substance concentrations were cloned and produced in a range suspension growth of 44 -78 %. Treatment medium was cloudy but with no visible precipitate at concentrations of ≥ 60 µg/mL. Concentrations of less than or equal to 50 µg/mL were soluble in treatment medium.

No treated cultures with greater than or equal to 10% total growth exhibited greater than or equal to 100 induced mutants per 10E6 clonable cells over the background level, one S9 -activated culture (300 µg/ml) exhibited 55 induced mutants per 10E6 clonable cells over the background level. A dose-response trend was not observed in the non-activated or S9 activated system. The total growths ranged from 5 – 40 % for the non –activated cultures at concentrations of 35 – 140 µg/mL and 20 – 68 % for the S9 –activated cultures at concentrations of 110 – 550 µg/mL.

For the 3 mutagenesis assays the TFT colonies for the positive and solvent control cultures were sized according to diameter over a range from approx. 0.2 to 1.1 mm.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

The test substance Reaction products of C16-18/C18 unsaturated fatty acid with methyl diethanolamine, MeCl quaternized was concluded to be negative with and without activation in the L5178Y/TK Mouse Lymphoma Mutagenesis Assay.

Executive summary:

In a mammalian cell gene mutation assay thymidine kinase locus comparable to OECD guideline 476, L5178Y mouse lymphoma cells cultured in vitro were exposed to MDEA-Esterquat C16-18 and C18 unsatd. at the following concentrations in the presence and absence of mammalian metabolic activation (S9- mix of Arochlor 1254 induced rat liver).

Preliminary Toxicity Assay: 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 36 µg/mL (presence and absence of S9-activation)

Mutagenesis Assay: 22.5, 25, 27.5, 30, 32.5, 35, 40, 50, 60, and 75 µg/mL (non-activated) and

25, 27.5, 30, 32.5, 35, 40, 50, 60, and 75 µg/mL (S9 -activated).

Confirmatory Mutagenesis Assay: 5, 10, 20, 35, 60, 75, 90, 100, 110, 120 and 130 µg/mL (non-activated) and 50, 60, 75, 90, 100, 110, 120, 130, 140, and 150 µg/mL (S9-activated).

3rd Mutagenesis Assay: 35, 50, 60, 75, 90, 100, 110, 120, 130, 140 and 150 µg/mL (non-activated system) and 110, 120, 130, 140, 150, 200, 300, 400, 500, and 550 µg/mL (S9-activated).

Acetone was selected as solvent and was workable at concentrations of 1.0 mg/mL. Higher concentrations were delivered to the test system as suspensions.

MDEA-Esterquat C16-18 and C18 unsatd. was tested up to cytotoxic concentrations of ≥ 110 µg/mL without metabolic activation in the second and third assay. With metabolic activation in the third test a relative total growth of 20 % was observed at a concentration of 500 µg/ml (at 550 µg/mL the relative total growth was 31 %). According to the authors 550 µg/mL was the highest achievable concentration.

No treated cultures without metabolic activation with ≥ 10 % total growth exhibited ≥  55 induced mutants per 10E6 clonable cells over background level. No treated cultures with metabolic activation with ≥ 10 % total growth exhibited ≥ 100 induced mutants per 10E6 clonable cells over background level. Whereas 4 cultures with metabolic activation with ≥ 10 % total growth show induced mutant frequency of ≥ 55 induced mutants per 10E6 clonable cells over background at a concentration of 300 µg/mL in the third. assay and  40, 60 and 75 µg/mL in the first assay, respectively. The positive controls did induce the appropriate response. 

The results of the L 5178Y/ TK Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of this study MDEA-Esterquat C16-18 and C18 unsatd. did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative under the conditions of this study.