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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
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

Constituent 1
Reference substance name:
Quaternary ammonium compounds, dicoco alkyldimethyl, chlorides
EC Number:
263-087-6
EC Name:
Quaternary ammonium compounds, dicoco alkyldimethyl, chlorides
Cas Number:
61789-77-3
Molecular formula:
Details not available
IUPAC Name:
Quaternary ammonium compounds, dicoco alkyldimethyl, chlorides
additive 1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
additive 2
Chemical structure
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
Test material form:
liquid

Method

Target gene:
Histidine: hisC3076, hisD3052 pKM101, hisG46, hisG46 pKM101
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Controlsopen allclose all
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
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
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
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).