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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:
2002-05-08 - 2007-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Remarks:
2005-12-16
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Synonyms: ITC 908, vinylidenephosphonic acid tetrasodium salt (VDPA tetrasodium salt)
- Lot/batch No.of test material: 562CH/646
- Purity: 87.33-88.33%
- Storage condition of test material: Room temperature, in the dark

Method

Target gene:
Histidine producing gene for S. typhimurium
Tryptophan producing gene for E. coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Molecular Toxicology, INC, Boone, NC 28607, USA
- method of preparation of S9 mix: from liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route
- concentration or volume of S9 mix and S9 in the final culture medium: S9 fraction in S9 mix = 10% (v/v)
- quality controls of S9: tested and validated by Molecular Toxicology for its ability to activate benzo(a)pyrene and 2-anthramine to mutagenic intermediates
Test concentrations with justification for top dose:
Mutagenicity experiments
312.5, 625, 1250, 2500 and 5000 µg/plate with and without S9 mix

Preliminary toxicity test
To assess the toxicity of the test item to the bacteria, six dose-levels were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and without S9 mix.
Concentrations: 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No noteworthy toxicity was noted toward the four strains used, with and without S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Anthramine (2-Aminoanthracene)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
The direct plate incorporation method was performed as follows: test item solution (0.1 ml), S9 mix when required (0.5 ml) and bacterial suspension (0.1 ml) were mixed with 2 ml of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45 °C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test item solution (0.1 ml), S9 mix (0.5 ml) and the bacterial suspension (0.1 ml) were incubated for 60 minutes at 37 °C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the nubmer of revertant colonies and/or a thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100, TA 102 and WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: All strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Moderate to strong thinning of the bacterial lawn was sometimes noted at dose-levels >= 2500 µg/plate (without S9 mix) or at 5000 µg/plate (with S9 mix in the preincubation method)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the six strains.
Executive summary:

Under the experimental conditions, the test item VDPA Tetra Sodium Salt did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.