Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-04-02 - 2003-11-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP, guideline study; four bacterial strains used instead of five. Triplicate plating used only for vehicle controls

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Homogenous Liquid
Specific details on test material used for the study:
Trade name: CYPHOS IL 101 Phosphonium Salt
Physical state: liquid
Composition of test material, percentage of components: Trihexyl(tetradecyl)phosphonium chloride (258864-54-9), > 95%
Lot/batch No.: WE01-001
Expiration date of the lot/batch: >1 year when stored at room temperature and protected from direct contact with water (hydrophobic)

Method

Target gene:
Histidine locus
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 from Aroclor-induced rat liver (from Molecular Toxicology, Inc)
Test concentrations with justification for top dose:
1.00, 3.33, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test article was observed to form a transparent, colorless, non-viscous solution in ethanol at a concentration of approximately 100 mg per mL.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene, ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period:
- Exposure duration: 72 hours at 37°C

NUMBER OF REPLICATIONS: All doses of test article and positive controls were plated at one plate per dose.Vehicle controls were plated in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the background bacterial lawn is evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity.
Evaluation criteria:
A test material is considered mutagenic if there is a reproducibly increasing dose-response curve of induced revertant colonies for at least 3 test concentrations. The minimal criteria for a positive response are a 2- to 3-fold increase in the number of revertants (at least 15 colonies) over the spontaneous number for the TA1535, TA1537, TA1538 and TA98 strains. In addition, a positive response must not be observed only at concentrations near toxic dose levels.

Results and discussion

Test results
Species / strain:
other: TA1537, TA1535, TA100, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Mutagenicity assay Results - Individual Plate Counts

Concentration (µg test solution) TA98 TA100 TA1535 TA1537
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 (vehicle control) 11 11 9 27 26 22 59 66 63 90 93 93 14 7 8 15 14 20 10 7 5 6 5 6
10±1 25±3 63±4 92±2 10±4 16±3 7±3 6±1
1.00 14 20 74 86 6 9 7 7
3.33 12 17 74 106 7 10 10 1
10.0 4 20 54 79 10 17 9 6
33.3 3 20 21 105 3 23 1 5
100 0 14 0 66 0 6 0 4
333 0 0 0 0 0 0 0 0
1000 0 0 0 0 0 0 0 0
3330 0 0 0 0 0 0 0 0
5000 0 0 0 0 0 0 0 0
Positive control
2-Nitrofluorene 243 - - - - - - -
benzo[a]pyrene - 219 - - - - - -
Sodium azide - - 1160 - 893 - - -
2-Aminoanthracene - - - 477   58 - 80
ICR-191 - - - - - - 942 -

Applicant's summary and conclusion

Conclusions:
negative with and without metabolic activation

Trihexyltetradecylphosphonium chloride was not mutagenic in a bacterial reverse mutation assay up to cytotoxic concentrations.
Executive summary:

Trihexyltetradecylphosphonium chloride was examined for mutagenicity activity in the Salmonella/Mammalian-Microsome Reverse Mutation Screening Assay (Ames Test). This assay evaluated the test article for its ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains TA-98, TA-100, TA-1535 and TA-1537 both in the presence and absence of an exogenous metabolic activation system.

Positive controls (sodium azide, 2-Nitrofluorene, benzo[a]pyrene, ICR-191 and 2-aminoanthracene) were tested under the same experimental conditions.

No significant increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at levels from 1.00 to 5000 µg per plate, either in the presence or absence of the S-9 mix.

Under the conditions of this study, the test material Trihexyltetradecylphosphonium chloride was devoid of mutagenic activity.