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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
25 March 2008 to 24 February 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study on a structural analogue (Klimisch score 2 is the maximal score in case of read across)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Anydrous Sodium Perchlorate
- Substance type: Technical material
- Physical state: White powder
- Analytical purity: 98.21%
- Impurities (identity and concentrations): Not reported
- Purity test date: 10 January 2008
- Lot/batch No.: Lot moyen du 10/01/08 test
- Expiration date of the lot/batch: February 2010
- Stability under test conditions: Not reported
- Storage condition of test material: at room temperature

Method

Target gene:
TK (thymidine kinase) locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level selected for the main test was 5000 μg/mL.
Using a treatment volume of 100 μL/20 mL, the selected dose-levels were 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL for both mutagenicity
experiments with and without S9 mix.
Vehicle / solvent:
- Vehicle used: Water for injection; batch number IVE14A, supplied by Fresenius-Kabi (92316 Sèvres, France).
- Justification for choice of vehicle: Solubility (the substance is freely soluble in water)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
vehicle only
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethane sulfonate (without S9), Cyclophosphamide (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation; in suspension;

DURATION
- Preincubation period: one week
- Exposure duration: 3 hours and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 days


SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 2 per dose concentration including negative and positive controls


NUMBER OF CELLS EVALUATED:
Viability plates:
An average of 1.6 cells/well (two 96-well plates/culture = four plates/dose-level) to define the number of viable cells (CE2). After at least 7 days of
incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted.
Mutant plates:
2000 cells/well (four 96-well plates/culture = eight plates/dose-level) to select the TFTR (trifluorothymidine resistant) mutant cells (for the
determination of CEmutant). After 11-12 days of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air in the presence of 4 μg TFT/mL of culture medium, the clones were counted, differentiating small and large colonies:
⋅ size of small colonies: < 25% of the diameter of the well,
⋅ size of large colonies: > 25% of the diameter of the well.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth (adjusted)
For scoring of colonies in mutant plates, the following parameters were considered:
⋅ Well containing mutant colony (small or large),
⋅ Well not containing mutant colony,
⋅ When both small and large colonies are present in the same well both mutant colonies were counted (one small and one large).


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:
Evaluation criteria:
at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor
(126 x 10-6 for the microtiter method),
⋅ and a dose-related trend is demonstrated by a statistically significant trend test.
Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (RTG lower than 10%), but with no evidence of
mutagenicity at dose-levels with RTG between 10 and 20%, is considered as positive result. A test item may be determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if (e):
⋅ there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG,
⋅ there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and
1% Adj. RTG.
Statistics:
No statistical analyses were performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: slight at 5000 µg/mL in the second experiment shown by a a 39% decrease in adjusted relative total growth
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects; the pH values were equivivalent to those of the vehicle control culture
- Effects of osmolality: No effects; the osmalality values were equivalent to those of the vehicle control culture
- Evaporation from medium: Not reported
- Water solubility: The test substance is freely soluble in water
- Precipitation: None
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
Using a treatment volume of 100 μL/20mL of culture medium, the final dose-level of 5000 μg/mL showed no precipitate. At this dose-level, the pH was approximately 7.4 (as for the vehicle control) and the osmolality equal to 344 mOsm/kg H2O (296 for the vehicle control).
The dose-levels used for treatment were 10, 100, 500, 1000, 2500, 5000 μg/mL.
No noteworthy toxicity was noted, either following the 3- and 24-hour treatments without S9 mix or following the 3-hour treatment with S9 mix.



COMPARISON WITH HISTORICAL CONTROL DATA:
Control data were included as an appendix in the Report, which covered experiments during the the period April 2005 to September 2006. It includedresults from 12 experiments without metabolic activation (S9 mix) and 24 experiments with metabolic activation using negative controls and using
the positive controls methylmethanesulfonate (MMS) and cyclophosphamide (CPA). The results are in line with those of the current study.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Not reported
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiments without S9 mix:

Cytotoxicity:

Following the 3-hour treatment and the 24-hour treatment, no noteworthy toxicity was noted.

Mutagenicity: No significant increase in the mutation frequency was noted, either following the 3-hour treatment or following the 24-hour treatment.

Experiments with S9 mix:

Cytotoxicity:

In the first experiment, no noteworthy toxicity was noted. In the second experiment, a slight toxicity was noted at the dose-level of 5000 μg/mL, as shown by 39% decrease in Adj. RTG.

Mutagenicity:

No significant increase in the mutation frequency was noted following the 3-hour treatment in either experiment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions, the test item ANHYDROUS SODIUM PERCHLORATE (batch No. "Lot moyen du 10/01/08 test"; purity: 98.21%) did
not show any mutagenic activity in the mouse lymphoma assay.
Executive summary:

A study was performed at CIT Laboratories France to investigate the potential of Anhydrous Sodium Perchlorate to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. The study was performed according to the international guidelines (OECD 476 and Commission Directive No. B17) and in compliance with the Principles of Good Laboratory Practice Regulations. After a preliminary toxicity test, Anhydrous Sodium Perchlorate was tested in two independent experiments, in the absence and presence of a rat liver metabolising system (S9 mix). Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Using a treatment volume of 100 μL/20 mL, the selected dose-levels were 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL for both mutagenicity experiments with and without the S9 mix. Following the 3-hour treatment and the 24-hour treatment without S9 mix, no noteworthy cytotoxicity was found. Similarly there was no evidence of mutagenicity, either following either the

3-hour or the 24-hour treatment. Experiments with the S9 mix revealed no noteworthy cytotoxicity in the first experiment but the second experiment showed evidence of slight toxicity at the highest dose level (5000 µg/mL), shown by a 39% decrease in Adj. RTG. However, there was no significant mutation frequency noted following the 3-hour treatment in either experiment. Under the experimental conditions, the test item did not show any mutagenic activity in the mouse lymphoma assay.