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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 April 2010 to 02 June 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Because the study was performed with a substance analogue and the data are read across, the Klimisch score is 2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Potassium nitrate
EC Number:
231-818-8
EC Name:
Potassium nitrate
Cas Number:
7757-79-1
IUPAC Name:
potassium nitrate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Potassium nitrate
- Substance type: White crystalline powder with lumps
- Physical state: Solid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Species strain
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 666 and 1011 µg/mL
Without S9-mix, 24 hours treatment: 33, 100, 333, 666 and 1011 µg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 1, 3, 10, 33, 100, 333, 666 and 1011 µg/mL
Experiment 2
Without and with S9-mix, 24 hours treatment: 1, 3, 10, 33, 100, 333, 666 and 1011 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium
- Justification for choice of solvent/vehicle:Test compound was soluble in RPMI 1640 medium and RPMI 1640 medium has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Positive control substance:
cyclophosphamide
Remarks:
with S9; 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 1011 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest test substance concentration of 1011 μg/ml

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest tested dose level in both experiments.

Applicant's summary and conclusion

Conclusions:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 16- and 8.6-fold for MMS in the absence of S9-mix, and by 13- and 16-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Potassium nitrate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Potassium nitrate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Potassium nitrate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.