Reaction mass of Ethanaminium, 2-hydroxy-N,N-dimethyl-N-[2-[(1-oxooctadecyl)oxy]ethyl]-, chloride and Ethanaminium, N,N-dimethyl-2-[(1-oxohexadecyl)oxy]-N-[2-[(1-oxooctadecyl)oxy]ethyl]-, chloride

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:

2008-06-06 to 2008-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

Cas Number:

1079184-43-2

Molecular formula:

n.a. (UVCB)

IUPAC Name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

Details on test material:

- Name of test material (as cited in study report): Reaction products of C16-18 (C18 unsatturated) fatty acid wiht mehtyl diethanolamine, MeCl quaternized
- Diethylester dimethyl ammonium chloride
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): Average-692 g/mol
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: Active
- Physical state: White powder

Method

Target gene:

Histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

other: Tester strains TA98 and TA1537 are reverted by frameshift mutagens. Tester strain TA1535 is reverted by basepair substitution mutagens. Tester strain TA100 is reverted by frameshift and basepair substitution mutagens.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

other: WP2 uvrA pKM 101 is sensitive to basepair substitution mutagens. See below for additional information.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Initial Assay/range-finding (used to establish the dose-range for the confirmatory assay)- 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Confirmatory Assay (used to evaluate and confirm the mutagenic potential of the test substance)- 15, 50, 150, 500, 1500 and 5000 µg/plate

Analysis of Dose Levels: Duplicate aliquots of the dosing formulations {1.0 mL form the top, middle and bottom regions of the 50 mg/mL concentration, 2.0 mL for the top, middle and bottom regions of the 15 mg/mL concentrations (Salmonella strains only), 10 mL from the middle region of the 5.0 mg/mL concentration (lowest analyzable dose) and vehicle} were collected from the confirmatory mutagenicity assay and its retest.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol (Lot No. B0514580, Exp. Date- 2011-03)
- Justification for choice of solvent/vehicle: A solubility test was conducted using water, dimethyl sufoxide, ethanol, acetone and isopropyl alcohol to determine the highest soluble or workable stock concentration, up to 50 mg/mL for water and up to 500 mg/ml for the organic vehicles. In the solubility test, the test substance formed a workable suspension in ethanol at a maximum concentration of 75 mg/mL. Ethanol was selected as the solvent of choice based on the Sponsor's request, solubility of the test substance and compatibility with the target cells.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (With metabolic activation)

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: (Without metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Initial assay and Confirmatory assay- in agar (plate incorporation)

DURATION
- Preincubation period: N/A
- Exposure duration: 48-72 hours (initial and confirmatory assays)
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): 48-72 hours (initial and confirmatory assays); simultaneous with exposure
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): L-histidine (Salmonella tester strains), L-tryptophan (E. coli tester strain)
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 0.4-11.3 x 10^8 cells were seeded


DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the bacterial background lawn


OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Other: N/A


OTHER: To confirm the sterility of the test substance, the highest test substance dose level used in the initial and confirmatory mutagenicity assay was plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay. In addition, to confirm the sterility of the S9 and Sham mixes, a 0.5 ml aliquot of each was plated on selective agar.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.
For the test substance to have induced a positive response in the assay, it must have caused a dose-related increase in the mean number of revertants per plate in at least one tester strain. The mean number of revertants per plate at the peak of the dose response should have been equal to or greater than 2.0 times the mean vehicle control values for tester strains TA98, TA100 and WP2 uvrA (pKM101) and equal to or greater than 3.0 times the mean vehicle control values for tester strains TA1535 and TA1537.

Statistics:

The use of statistics was limited to the calculation of means and standard deviations where appropriate.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Initial and Confirmatory assays

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Initial (TA98, TA100 and TA1537 in the absence of S9 activation) and Confirmatory (TA98, TA100, and TA1537 in the absence of S9 activation and TA100 and TA1535 in the presence of S9 actaivation) assays

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Initial and Confirmatory Assays

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Confirmatory only (absence of S9 activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: N/A
- Effects of osmolality: N/A
- Evaporation from medium: N/A
- Water solubility: N/A
- Precipitation: Initial and Confirmatory Assay-Precipitate was observed beginning at 1500 or at 5000 ug/plate.
- Other confounding effects: Sterility results- Several contaminant colonies (3-7) were observed on the pre-dosing sterility plates for the sham mix and the 50 mg/ml dilution in the confirmatory experiment and the post-dosing sterility plate for the 1.5 mg/ml dilution in the confirmatory experiment. In addition, no contaminants were observed on the assay plates for the confirmatory assay so the presence of contaminant colonies on the sterility plates did not invalidate the results of the study. No other contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions and the S9 and sham mixes.


RANGE-FINDING/SCREENING STUDIES: An initial assay (Ames) was performed as described in the materials and methods section to determine concentrations to be used in the confirmatory assay. Results are reported in applicable sections.


COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle and positive control values in the presence and absence of metabolic activation were within historical control ranges.


ADDITIONAL INFORMATION ON CYTOTOXICITY: Initial Assay- Toxicity was observed in the absence of S9 activation beginning at 1500 or at 5000 ug/plate with tester strains TA98, TA100 and TA1537. Confirmatory Assay- Toxicity was observed beginning at 1500 or at 5000 ug/plate with tester strains TA98, TA100, TA1537 and WP2 uvrA (pKM101) in the absence of S9 and with tester strains TA100 and TA1535 in the presence of S9 activation.

OTHER: Initial assay- no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Confirmatory assay- no positive mutagenic responses were observed with any of the Salmonella strains in either the presence or absence of S9 activation. Due to unacceptable vehicle control values, tester strain WP2 uvrA (pKM101) in the presence and absence of S9 activation was not evaluated but was retested. In the retest experiment, no positive mutagenic responses were observed in the presence or absence of S9 activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Initial and Confirmatory assays

Any other information on results incl. tables

Analysis of Dose Levels: The concentration analysis indicated that the actual concentrations of the analyzed dose levels were 96.9 to 112 % of their respective targets and the most concentration formulation met the acceptance criteria for homogeneity. No test substance was detected in the vehicle samples. The lowest analyzed dose level in the confirmatory experiment was above the acceptance criterion, which indicated that the intended dose level at this concentration was exceeded. Since the most concentration formulations were within 80 to 120 % of target in each trial, the formulations were considered stable for the purpose of the study. This indicated that the regulatory-required top dose level was achieved in each case and the results support the validity of the study conclusion.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative With and without metabolic activation.

MDEA-Esterquat C16-18 and C18 unsatd. was evaluated in the bacterial reverse mutation assay (Ames) using Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2 uvrA (pKM101) in the presence and absence of Aroclor-induced rat liver S9. Under the conditions of the study, the test substance was negative for mutagenic potential.

Executive summary:

The purpose of the study was to evaluate the mutagenic potential of MDEA-Esterquat C16-18 and C18 unsatd. by the Bacterial Mutation Test (GLP Ames). The test substance was tested in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2 uvrA (pKM101) in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial mutagenicity assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.

Ethanol was selected as the solvent of choice based on the Sponsor's request, solubility of the test substance and compatibility with the target cells. In the solubility test, the test substance formed a workable suspension in ethanol at a maximum concentration of 75 mg/ml.

In the initial mutagenicity assay, the maximum dose tested was 5000 ug/plate; this dose was achieved using a concentration of 50 mg/ml and a 100 ul plating aliquot. In the initial mutagenicity assay, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500, and 5000 ug/plate. No positive mutagenic responses were observed. Precipitate was observed beginning at 1500 or at 5000 ug/plate. Toxicity was observed in the absence of S9 activation beginning at 1500 or at 5000 ug/plate with tester strains TA98, TA100 and TA1537.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 ug/plate. Precipitate was observed beginning at 1500 or at 5000 ug/plate. Toxicity was observed beginning at 1500 or at 5000 ug/plate with tester strains TA98, TA100, TA1537 and WP2 uvrA (pKM101) in the absence of S9 and with tester stains TA100 and TA1535 in the presence of S9 activation.

Under the conditions of this study, the test substance was concluded to be negative with Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA (pKM101) in the presence and absence of Aroclor-induced rat liver S9.