Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 23, 1990 to May 10, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

EC Number:

269-924-1

EC Name:

Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Cas Number:

68391-05-9

Molecular formula:

Substance is an UVCB (representative molecular formula for 2 major constituents: C12-C12 DAQ: C26H56Cl1N1; C18-C18 DAQ: C38H80Cl1N1)

IUPAC Name:

N-C12-C18(even numbered)-alkyl-N,N-dimethyl-C12-C18(even numbered)-alkyl-1-aminium chloride

additive 1

Reference substance name:

Propan-2-ol

EC Number:

200-661-7

EC Name:

Propan-2-ol

Cas Number:

67-63-0

Molecular formula:

C3H8O

IUPAC Name:

propan-2-ol

additive 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Details on test material:

- Chemical name: Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides (C12-18 DAQ)
- EC number: 269-924-1

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant

Method

Target gene:

Histidine: hisC3076, hisD3052 pKM101, hisG46, hisG46 pKM101

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix obtained from Aroclor 1254 induced male Wistar or Sprague-Dawley rats

Test concentrations with justification for top dose:

Preliminary toxicity test:
1.0, 3.3, 10, 33.3, 100, 333, 1000, 3330, 5000 µg/plate

Experiment 1
with S9-mix
0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
without S9-mix
0.1, 0.33, 1.0, 3.3, 10.0 µg/plate

Experiment 2
with S9-mix
0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
without S9-mix
0.1, 0.33, 1.0, 3.3, 10.0 µg/plate

Vehicle / solvent:

Dimethylsulphoxide (DMSO) of spectroscopic quality

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA1535), 9-aminoacridine (TA1537), daunomycine (TA98), methylmethanesulfonate (TA100)

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all 4)

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: until 10^9 cells/mL had been obtained
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: Five different doses of the test substance have been tested in triplicate in each strain. An independentrepeat of the experiment was performed.

NUMBER OF CELLS EVALUATED: All colonies were counted.

DETERMINATION OF CYTOTOXICITY
Method: A preliminary toxicity test was performed with TA100 (with and without S9-mix), 9 concentrations were tested in duplicate. The survival of the TA100 culture was determined by comparing the number of colonies on the plate + test substance with those onthe solvent control plate.

Evaluation criteria:

An Ames test was considered acceptable if it met the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory
historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times
the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding
test with strain TA100 or should extend to 5 mg/plate (active ingredient).

A test substance was considered negative (not mutagenic) i n the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance was considered positive (mutagenic)in the Ames test if :
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent
control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two
concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.

Statistics:

Not reported

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in preliminary test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in preliminary test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in preliminary test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in preliminary test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No data

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity study with TA100 showed that survival without S9-mix was not or only slightly reduced up to test substance concentration of 10.0 µg/plate and eliminated at and above 33.3 µg/plate. In the presence of S9-mix the survival of strain TA100
was slightly reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. Based on these data, the test substance was tested up to a concentration of 10.0 µg/plate in the absence of S9-mix and up to 33.3 µg/plate in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values fell within the testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Any other information on results incl. tables

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was considered to be non mutagenic in the Ames test.

Executive summary:

A study was conducted to evaluate the mutagenic potential of the test substance, C12 -18 DAQ (76.4% active in hydroalcoholic solution) in the Ames test, according to the OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. In the study S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 strains were used and S9 liver mix obtained from Aroclor 1254 induced male Wistar or Sprague-Dawley rats was used as metabolic activating system. A preliminary toxicity study with TA100 showed that survival without S9-mix was not or only slightly reduced up to test substance concentration of 10.0 µg/plate and eliminated at and above 33.3 µg/plate. In the presence of S9-mix the survival of strain TA100 was slightly reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. Based on these data, the test substance was tested up to a concentration of 10.0 µg/plate in the absence of S9-mix and up to 33.3 µg/plate in the presence of S9-mix. Experiment 1: (a) with S9-mix - 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate (b) without S9-mix - 0.1, 0.33, 1.0, 3.3, 10.0 µg/plate. Experiment 2: (a) with S9-mix - 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate (b) without S9-mix - 0.1, 0.33, 1.0, 3.3, 10.0 µg/plate. The negative and strain-specific positive control values fell within the testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly. All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. Under the study conditions, the test substance was considered to be non mutagenic in the Ames test (Scheres, 1990).