[No public or meaningful name is available]

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 January 10 to February 18, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

IUPAC Name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

Test material form:

solid

Method

Target gene:

Histidine operon for typhimurium strains and tryptophan operon for E. Coli

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Experiments without S9 mix
The selected dose levels were:
- 2.06, 6.17, 18.5, 55.6, 167 and 500 µg/plate for the TA 1535 and TA 100 strains in the first experiment,
- 0.686, 2.06, 6.17, 18.5, 55.6 and 167 µg/plate for the TA 1537 and TA 98 strains in the first experiment,
- 6.25, 12.5, 25, 50, 100 and 200 µg/plate for the TA 100 strain in the second experiment,
- 3.13, 6.25, 12.5, 25, 50 and 100 µg/plate for the TA 1535, TA 1537 and TA 98 strains in the second experiment,
- 312.5, 625, 1250, 2500 and 5000 µg/plate for the WP2 uvrA strain in both experiments.

Experiments with S9 mix
The selected dose levels were:
- 2.06, 6.17, 18.5, 55.6, 167 and 500 µg/plate for the TA 1535 and TA 100 strains in the first experiment,
- 0.686, 2.06, 6.17, 18.5, 55.6 and 167 µg/plate for the TA 1537 and TA 98 strains in the first experiment,
- 2.57, 7.72, 23.1, 69.4, 208 and 625 µg/plate for the TA1535, TA1537, TA 98 and TA 100 strains in the second experiment,
- 312.5, 625, 1250, 2500 and 5000 µg/plate for the WP2 uvrA strain in both experiments.

The selection of the highest dose level to be used in the main experiments was based on the level of toxicity

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for injection

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Anthramine

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation in the first experiment and preincubation in the second
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours of incubation at 37°C
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

DETERMINATION OF CYTOTOXICITY
- Method: count of the number of revertant colonies associated observation on bacterial lawn

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.
The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and WP2 uvrA strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship is noted.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the upper dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the upper dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the upper dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the upper dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Not mutagenic

Executive summary:

Method

The mutagenic effect of the test item was assessed according to the method descibed in the OECD guideline 471. A preliminary toxicity test was performed to define the dose levels of the test item, dissolved in water for injections, to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. 

Treatments were performed according to the direct plate incorporation method, except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). 

Four strains of bacteria Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and one strain of Escherichia coli (WP2 uvrA) were used. Each strain was exposed to at least five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Results 

The test item was freely dissolved in the vehicle (water for injections) at 50 mg/mL, after a minimum of 30 minutes of magnetic stirring. Since the test item was toxic in the preliminary test, the selection of the highest dose level to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose levels (i.e.including at least three non-cytotoxic dose levels) for each strain and test condition. The study was therefore considered to be valid. 

Experiments without S9 mix

The selected dose levels were:

- 2.06, 6.17, 18.5, 55.6, 167 and 500µg/plate for the TA 1535 and TA 100 strains in the first experiment,

- 0.686, 2.06, 6.17, 18.5, 55.6 and 167µg/plate for the TA 1537 and TA 98 strains in the first experiment,  

- 6.25, 12.5, 25, 50, 100 and 200 µg/plate for the TA 100 strain in the second experiment,

- 3.13, 6.25, 12.5, 25, 50 and 100µg/plate for the TA 1535, TA 1537 and TA 98 strains in the second experiment,

- 312.5, 625, 1250, 2500 and 5000 µg/plate for the WP2 uvrA strain in both experiments.          

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.

A strong toxicity was noted at dose levels >= 50 µg/plate in the TA 1537 and TA 98 strains, >= 55.6 µg/plate in the TA 1535 strain and >= 167 µg/plate in the TA 100 strain.

No noteworthy toxicity was noted in the WP2 uvrA strainat any of the tested dose levels.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results met the criteria of a negative response.

Experiments with S9 mix

The selected dose levels were:

-  2.06, 6.17, 18.5, 55.6, 167 and 500µg/plate for the TA 1535 and TA 100 strains in the first experiment,

- 0.686, 2.06, 6.17, 18.5, 55.6 and 167µg/plate for the TA 1537 and TA 98 strains in the first experiment,

-  2.57, 7.72, 23.1, 69.4, 208 and 625µg/plate for the TA1535, TA1537, TA 98 and TA 100 strains in the second experiment,

- 312.5, 625, 1250, 2500 and 5000 µg/plate for the WP2 uvrA strain in both experiments.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.

A strong toxicity was noted at dose levels >= 69.4 µg/plate in the TA 1537 strain, >= 167 µg/plate in the TA 98 strain, >= 208 µg/plate in the TA 1535 strain and >= 500 µg/plate in the TA 100 strain.

No noteworthy toxicity was noted in the WP2 uvrA strainat any of the tested dose levels.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results met the criteria of a negative response.

 

Conclusion 

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli strains, either in the presence or absence of a rat liver metabolizing system.