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Toxicological information

Genetic toxicity: in vitro

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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:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
E. coli WP2 strain missing; Second experiment performed as a plate incorporation assay, but not as a preincubation assay.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1955

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides
EC Number:
271-756-9
EC Name:
Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides
Cas Number:
68607-24-9
Molecular formula:
The substance is a quarternary ammonium chloride derived from fatty alcohol. The general chemical structure is R-N(Me)3+ Cl-, whereas the alkylrest R is variable for the main components C20 and C22 but also shorter and longer C-Chains (in smaller amounts)
IUPAC Name:
N,N,N-trimethyl-C20-22-(even numbered)-alkyl-1-aminium chloride

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Pre-Experiment and Experiment I: 0 (control), 4, 20, 100, 500, 2500, 5000 µg/plate
Experiment II: 0 (control), 4, 20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below for additional information
Details on test system and experimental conditions:
The assay was performed in two independent experiments:

experiment I: plate incorporation assay with and without induced rat liver S9 mix
experiment II: plate incorporation assay with and without induced rat liver S9 mix

Rat liver S9 mix from Aroclor 1254 induced rats was used.

Top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. After mixing, the liquid was poured into a petridish with minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (wlv) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (hi? revertants) were counted.

DURATION
- Exposure duration: after solidification the plates were incubated upside down for approx. 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: The first experiment was performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculating an appropriate dose range. A reduced rate of spontaneously occuring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
In combination with the second experiment, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 108 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

POSITIVE CONTROL SUBSTANCES:
without metabolic activation: sodium azide (TA 1535, TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98);
with metabolic activation: 2-aminoanthracene (all strains)
Evaluation criteria:
A test article is classified as mutagenic if it has either of the following effects:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.

Results and discussion

Test results
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:
see below for additional information
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test compound was tested at doses of 4 to 5000 microgram/plate and proved to be toxic to most of the bacterial strains at doses of 2500 microgram/plate and above. Thinning of the bacterial lawn and a reduction in the number of colonies were observed at this dose.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mean mutant number

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 176.7 -- 168.0 -- 201.0 -- 217.0 -- 25.0 -- 1.3 -- 1.0

TA1535 -- 10.7 -- 6.3 -- 8.3 -- 8.0 -- 1.3 -- 1.0 -- 0.3

TA1537 -- 7.0 -- 10.7 -- 9.7 -- 11.3 -- 5.0 -- 0.0 -- 0.0

TA98 -- 27.7 -- 24.0 -- 31.3 -- 23.0 -- 7.3 -- 1.0 -- 0.0

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 137.7 -- 207.0 -- 182.7 -- 185.0 -- 165.7 -- 43.0 -- 6.0

TA1535 -- 9.3 -- 11.7 -- 14.7 -- 12.3 -- 10.0 -- 5.0 -- 1.0

TA1537 -- 11.7 -- 8.7 -- 10.0 -- 8.3 -- 6.3 -- 2.3 -- 0.7

TA98 -- 36.3 -- 34.7 -- 36.7 -- 41.7 -- 37.0 -- 15.0 -- 2.0

Exp. II: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 141.7 -- 184.3 -- 146.3 -- 145.7 -- 2.0 -- 1.0 -- 1.0

TA1535 -- 16.0 -- 12.7 -- 7.7 -- 8.7 -- 3.3 -- 0.0 -- 0.0

TA1537 -- 7.7 -- 7.7 -- 8.7 -- 8.3 -- 4.3 -- 0.0 -- 0.0

TA98 -- 26.3 -- 24.3 -- 22.0 -- 31.0 -- 9.3 -- 1.3 -- 0.0

Exp. II:

plate incorporation method with rat liver S9 mix

Concentrations given in µg/plate

Strain -- 0 -- 4 -- 20 -- 100 -- 500 -- 2500 -- 5000

TA100 -- 176.3 -- 172.7 -- 170.3 -- 170.3 -- 123.3 -- 98.7 -- 24.0

TA1535 -- 19.3 -- 13.0 -- 16.7 -- 13.3 -- 15.0 -- 1.3 -- 1.0

TA1537 -- 8.0 -- 7.0 -- 9.7 -- 10.3 -- 6.0 -- 9.3 -- 0.3

TA98 -- 36.7 -- 33.3 -- 36.3 -- 29.3 -- 30.7 -- 8.3 -- 1.3

Applicant's summary and conclusion

Conclusions:
The genotoxicity of the registration substance was investigated according to the guideline OECD 471. Negative result was obtained.
Executive summary:

The registration substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium.

The registration substance did not increase the number of revertants in any of the bacterial strains with and without metabolic activation. No significant mutagenicity was found.