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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:
February - April 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: Cetylpyridinium Chloride (CPC) monohydrate
- Molecular formula: C21H3Na.Cl(.H20)
- Molecular weight: 358.1
- Physical state: White Powder
- Analytical purity: 100%
- Lot/batch No.: 00227993
- Expiration date of the lot/batch: 30th June, 2011
- Stability under test conditions:Stable at higher temps (Melts at 80 deg C). Stable in water for at least 48 hours and soluble in water at 32 deg C
- Storage condition of test material: At room temperature in the dark

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes/no
Metabolic activation:
with and without
Metabolic activation system:
10% Rat Liver S9
Test concentrations with justification for top dose:
Without S9: 0.3, 1.0, 3.0, 10.0 and 20.0 µg/plate (Some strains used concentrations up to 33.0 µg/plate or from 0.1, 0.3, 1.0, 3.0 and 10.0 µg/plate).
With S9: 0.3, 1.0, 3.0, 10.0, 20.0 and 33.0 µg/plate (Some strains used concentrations up to 100.0 µg/plate).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water.
- Justification for choice of solvent/vehicle: Test substance completely soluble and stable in the vehicle of choice (water).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Remarks:
With and without metbolic activation
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
Solvents for reference substances: Saline, DMSO, Milli-Q water
Details on test system and experimental conditions:
Test system Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale Recommended test system in international guidelines (e.g. OECD, EEC etc.)
Source Salmonella typhimurium strains:
Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames): TA100 received on 14-06-2006, used batches: TA100.210807 and TA100.130208
TA98 received on 14-06-2006, used batch: TA98.290108 TA1537 received on 14-06-2006, used batch: TA1537.210807 TA1535 received on 14-06-2006, used batch: TA1535.210807 Escherichia coli strain:
Trinova Biochem GmbH, Germany (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) WP2uvrA received on 06-02-2008, used batch: EC.270208

The characteristics of the different Salmonella typhimurium strains were as follows: Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA 1535 hisG46 Base-pair substitutions
TA 100 hisG46/R-factor* Base-pair substitutions
Each tester strain contained the following additional mutations: rfa : deep rough (defective lipopolysaccharide cellcoat)
gm : mutation in the galactose metabolism
chi : mutation in nitrate reductase bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

Evaluation criteria:
Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 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 (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA 1535, TA 1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
Statistics:
none

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction in bacterial lawn was extreme at high doses of 33.0, 66.0 or 100.0 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Precipitation: No precipitation of CPC (monohydrate) on the plates was observed at the start or at the end of the incubation period.

COMPARISON WITH HISTORICAL CONTROL DATA: No reduction in the number of revertant colonies, no reduction of bacterial background lawn.
No reduction in the number of revertant colonies less than the minimal value of the historical control data range. Fluctuations in the number of revertant colonies below the laboratory historical control data range were observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Since in tester strain TA1537, TA98, TA100 and WP2 uvrA in the second experiment no toxicity or precipitate on the plates was observed in the presence of S9-mix, a third mutation experiment was performed with these strains in the presence of S9-mix. The following dose range was selected for the third mutation experiment: 10, 33, 66 and 100 µg/plate.

In the third mutation experiment, slight cytotoxicity was observed at 33 µg/plate and extreme cytotoxicity was observed at 66 µg/plate, the dose ranges were considered appropriate. There was no increase in the number of revertants was observed upon treatment with CPC (monohydrate) under all conditions tested.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

CPC (monohydrate) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.