Quaternary ammonium compounds, benzyl-C16-18-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 23 January, 2012 to 02 Feburary, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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:

Quaternary ammonium compounds, benzyl-C16-C18 (even numbered)-alkyldimethyl, chlorides

EC Number:

939-290-7

Cas Number:

68607-20-5

Molecular formula:

Representative molecular formula of the major constituents, as the substance is an UVCB: C16 ADBAC: C27 H50 CL1 N1 C18 ADBAC: C29H54CL1N1

IUPAC Name:

Quaternary ammonium compounds, benzyl-C16-C18 (even numbered)-alkyldimethyl, chlorides

Test material form:

solid

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 0.1, 0.3, 1, 3, 10 and 33 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
Experiment 2:
TA1535, TA1537 and TA100
Without S9-mix: 0,1, 0,3, 1, 3, 10 and 33 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
TA98
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
WP2uvrA
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 1, 3, 10, 33, 100 and 333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Controls 2

Positive control substance:

2-nitrofluorene

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Controls 4

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Controls 5

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

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:

without S9: 10 µg/plate and above and with S9: 33 µg/plate and above

Vehicle controls validity:

valid

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:

without S9: 3 µg/plate and above and with S9: 10 µg/plate and above

Vehicle controls validity:

valid

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:

without S9: 100 µg/plate and above and with S9: 10 µg/plate and above

Vehicle controls validity:

valid

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:

without S9: 10 µg/plate and above and with S9: 33 µg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 66 µg/plate and above and with S9: 333 µg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 10 μg/plate and above in the absence of S9-mix and at 33 µg/plate in the presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 100 μg/plate and above in the absence of S9-mix and at 333 µg/plate in the presence of S9-mix.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 10 µg/plate and above and with S9: 33 µg/plate and above
TA1537: without S9: 3 µg/plate and above and with S9: 10 µg/plate and above
TA98: without S9: 100 µg/plate and above and with S9: 10 µg/plate and above
TA100: without S9: 10 µg/plate and above and with S9: 33 µg/plate and above
WP2uvrA: without S9: 66 µg/plate and above and with S9: 333 µg/plate and above

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was not mutagenic in S. typhimurium strains TA1535, TA1537, TA98, TA100 and in the Escherichia coli strain WP2 uvr A,with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the test substance, C16-18 ADBAC (98.2% active), in S. typhimurium strains TA1535, TA1537, TA98, TA100 and in the Escherichia coli strain WP2 uvr A, according to OECD 471 Guideline and EU Method B.13/14, in compliance with GLP. Six concentration levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system (S9). The concentration-range was determined in a preliminary toxicity assay and was 0.1 to 66 µg/plate in the first experiment. The experiment was repeated on a separate day using the same concentration-range, fresh cultures of the bacterial strains and fresh test substance formulations. The test substance did not induce a significant concentration-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9. These results were confirmed in an independent repeat. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was not mutagenic in S. typhimurium strains TA1535, TA1537, TA98, TA100 and in the Escherichia coli strain WP2 uvr A,with and without metabolic activation (Verspeek-Rip, 2012).