Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 1995 to 1 September 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

EC Number:

421-660-1

EC Name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

Molecular formula:

Hill formula: C50 H96 N2 O10 CAS formula: C42 H74 O8. 2(C4 H11 N O)

IUPAC Name:

bis((2-hydroxyethyl)dimethylazanium) (4E)-3-{[2-({3-carboxylato-3-[(2E)-hexadec-2-en-2-yl]propanoyl}oxy)ethoxy]carbonyl}-4-methyloctadec-4-enoate

Constituent 2

Reference substance name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

IUPAC Name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

Method

Target gene:

Not required

Species / strain

Species / strain / cell type:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E.coli WP2 uvrA

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

other: Histidine dependant

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 0 - 5000 µg/plate
Concentration range in the main test (without metabolic activation): 0 - 5000 µg/plate

Vehicle / solvent:

- Solvent: Acetone
- Justification for choice of solvent/vehicle: Solubility of test material

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) for WP2uvrA, TA100 and TA1535; 9-Aminoacridine (9AA) for TA1537; 4-Nitroquinoline-1-oxide (4NQO) for TA98; 2-Aminoanthracene (2AA) was used in the S9 series of plates.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate incorporation method: Known aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten trace histidine/tryptohan supplemented top agar at 45 °C, 0.1 mL of the appropriately diluted test material or vehicle control and either 0.5 mL of the S9 liver microsome mix or 0.5 mL of pH 7.4 buffer. The contents of each tube were mixed and equally distributed onto the surface of the agar plates (one tube per plate).
The plates were then incubated at 37 °C for approximately 48 hours and the number of revertant colonies counted.

A range-finding study was also performed.

DURATION
- Preincubation period: Overnight sub-cultures of the appropraite coded stock cultures were prepared in nutrient broth and incubated at 37 °C for approximately 10 hours.
- Exposure duration: Approxiamtely 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: Three replicates of each bacterial strain, each concentration of the test material with and without S9 mix and each control plate were prepared.


NUMBER OF CELLS EVALUATED: Cell viability at the end of pre-culture:

Range finding study:
S. typhimurium TA 1535: 18.72x10^9 cells/mL
S. typhimurium TA 1537: 7.03x10^9 cells/mL
S. typhimurium TA 98: 12.96x10^9 cells/mL
S. typhimurium TA 100: 8.67x10^9 cells/mL
E.coli WP2 uvrA: 16.74x10^9 cells/mL

Main study:
S. typhimurium TA 1535: 7.64x10^9 cells/mL
S. typhimurium TA 1537: 0.55x10^9 cells/mL
S. typhimurium TA 98: 6.49x10^9 cells/mL
S. typhimurium TA 100: 0.82x10^9 cells/mL
E.coli WP2 uvrA: 9.07x10^9 cells/mL


DETERMINATION OF CYTOTOXICITY
- Method: N/A


OTHER EXAMINATIONS: N/A


OTHER: Prior to being used, characterisation checks were carried out on the bacterial strains to determine the amino-acid requirement, presence of rfa, R factors, urvB mutation and the spontaneous reversion rate.

A preliminary study was performed in order to select the appropriate dose levels for use in the main study using the bacterial strains TA100 and WP2uvrA. The dose range of the test material was 0, 50, 150, 500, 1500 and 5000 µg/plate.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold at each dose level employed.

Statistics:

NDA

Results and discussion

Test results

Test results 1

Species / strain:

bacteria, other: S. typhimurium TA100 and E.coli WP2 uvrA

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The test material was non-toxic.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Test results 2

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increase in the frequency of revertant colonies was recorded.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxicity to the bacterial background lawn was exhibited to any of the strains of bacteria used, although small and inconsistent decreases in revertant colony frequency were observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

OBSERVATIONS:
Small, inconsistent decreases in revertant colony frequency were noted, especially with TA 1535 (with or without activation). However, no weakening of the bacterial background lawn was noted.

In both the first and second assays (with or without activation), there were no significant increases in the frequency of revertant colonies noted for any of the bacterial strains at any dose level.

Marked increases in the frequency of revertant colonies (with or without activation) were observed in all of the positive control chemicals. Normal ranges of revertant colonies were produced by the vehicle (acetone).


TEST-SPECIFIC CONFOUNDING FACTORS: None - the results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.


RANGE-FINDING/SCREENING STUDIES: The range finding studies showed no evidence of cytotoxicity or mutagenicity.


COMPARISON WITH HISTORICAL CONTROL DATA: NDA


ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material was not cytotoxic at the dose levels tested.

Remarks on result:

other: other: preliminary test and all strains tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was found to be non-mutagenic under the conditions of this test.

Executive summary:

 In a reverse gene mutation assay in bacteria, strains S. typhimurium and E. coli were exposed to OS 114451A in acetone at concentrations of 0, 50, 150, 500, 1500 and 5000  µg/plate in the presence and absence of mammalian metabolic activation.

 

OS 114451A was tested up to a limit concentration of 5000 µg/plate.  The positive controls induced the appropriate responses in the corresponding strains.  There was no evidenceof induced mutant colonies over background.