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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The purpose of this study was to assess toxic and genotoxic effects of Sodium salicylate on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.
GLP compliance:
not specified
Type of assay:
other: mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Sodium salicylate
- Molecular Formula: C7H5NaO3
- Molecular Weight: 160.10 g/mol
- Substance type: Organic
- Physical state: Solid
- Analytical purity: ≥ 99.5%
- Consistency: White powder/flakes
- Activity (Clinical Indication): Industrial Chemical
- Stability under test conditions: Stable
- Storage condition of test material: As per requirements mentioned in guidance for safe use
Specific details on test material used for the study:
- Name of test material (as cited in study report): Sodium salicylate
- Molecular Formula: C7H5NaO3
- Molecular Weight: 160.10 g/mol
- Substance type: Organic
- Physical state: Solid
- Analytical purity: ≥ 99.5%
- Consistency: White powder/flakes
- Activity (Clinical Indication): Industrial Chemical
- Stability under test conditions: Stable
- Storage condition of test material: As per requirements mentioned in guidance for safe use

Method

Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
no data
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.0625, 0.125, 0.25 or 0.5 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Phosphate-buffered saline (PBS)
Justification for choice of solvent/ vehicle: Sodium salicylate was easily dissolved in PBS.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Phosphate-buffered saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT (mutation assays):
6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays):
Not applicable

STAIN (for cytogenetic assays):
Crystal violet

NUMBER OF REPLICATIONS:
A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED:
5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
Rationale for test conditions:
no data available.
Evaluation criteria:
No data available.
Statistics:
No data available.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No mutagenic effect were observed.

Any other information on results incl. tables

Table 1A.Effect of sodium salicylate exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO2. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO2. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

 

With S9

Without S9

 

with TG

without TG

with TG

without TG

Neg. control

0/4

184/4

0/4

208/4

Pos. control

0/4

221/4

12/4

143/4

0.0625 mM

2/4a

218/4

0/4

198/4

0.125 mM

0/4

226/4

0/4

185/4

0.25 mM

0/4

177/4

0/4

181/4

0.5 mM

0/4

185/4

0/4

198/4

 

a)2 very diffuse colonies were found in one single well.

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to sodium salicylate in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

 

With S9

Without S9

Neg. control

N/A

N/A

Pos. control

N/A

3.08x10-4

0.0625 mM

N/Aa

N/A

0.125 mM

N/A

N/A

0.25 mM

N/A

N/A

0.5 mM

N/A

N/A

a)Since only diffuse colonies were found in one single well (see Table 1A), these diffuse colonies were not regarded as reliable and true colonies since the cells seemed to be apoptotic.


 

Applicant's summary and conclusion

Conclusions:
Sodium salicylate in the concentration of 0, 0.0625, 0.125, 0.25 or 0.5 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical, in the presence or abscence of metabolic activation.
Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of Sodium salicylate (CAS No. 54-21-7) when administered to Chinese Hamster Ovary (CHO) cells.

In the genotoxicity test, sodium salicylate was administered to CHO cells for 3 hrs at the dose levels of 0.0625, 0.125, 0.25 or 0.5 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.

Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at the concentration 0.0625 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the two spots were only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of sodium salicylate, i.e. these concentrations did not show any evidence of diffuse or clear colonies present.

When the mutation frequency was determined, a frequency of 3.08 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of sodium salicylate in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that sodium salicylate does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.0625, 0.125, 0.25 or 0.5 mM for 3 hrs.

Based on the results of the current study, we conclude that sodium salicylate does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.0625, 0.125, 0.25 or 0.5 mM for 3 hrs, in the presence or abscence of metabolic activation. Therefore Sodium salicylate was considered to be non mutagenic and hence cannot be classified as gene mutant in vitro.