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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The genotoxicity of Trisodium Hydrogendicarbonate is expected to be driven by its constituents – namely sodium ions, carbonate ions and hydrogencarbonate ions. A study conducted on trisodium hydrogendicarbonate in 1982 using methods equivalent to OECD 471 found the substance to be negative for gene mutation in bacteria both with and without metabolic activation. The results of QSAR predictions on the in vitro and in vivo genotoxicity of the substance and its constituents were negative. Further, the negative result for chromosome aberration in mammalian cells are supported by limited, but well documented and reliable, results from the scientific literature.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted before the implementation of GLP according to a method similar to OECD Testing Guideline 471. Sufficient information is provided in the study report on the methods and materials used and the results and conclusions.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
plate incorporation method
Deviations:
yes
Remarks:
S. typhimurium TA1538 was used instead of E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. Highest concentration tested during the study was above the highest concentration recommended by OECD 471
Principles of method if other than guideline:
The strains used in this study varied from those suggested in the OECD 471 test guideline. S. typhimurium histTA1538 was used in place of E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. This deviation is not expected to affect the results of the study as E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 are recommended for detecting mutation in hydrazine or oxidising substances and cross-linking agent which may not be detected in the other recommended S. typhimurium strains. Sodium sesquicarbinate is not expected to be oxidising or to function as a cross-linking agent. Further, the highest concentration tested during the study was above the highest concentration recommended by OECD 471.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
The target genes were not named in the study. The strains used has three mutations; histidine mutation to make the strain auxotrophic to histidine, a mutation which effects losses of the excision repair mechanism and a mutation which effects losses to the lippopolysacharide portion of cell walls.
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: LT2
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: LT2
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 (Arcoclor 1254 induced)
Test concentrations with justification for top dose:
Mutagenicity spot testing: 50 μg/plate with and without metabolic activation in all strains
Plate incorporation testing: 50 μg/plate with and without metabolic activation in all strains plus addition of a 0.1 ml solution containing 50 μg of a known mutagen added over the surface of the agar
Vehicle / solvent:
Sterile, double distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-amino-anthracene and 4-nitro-o-phenylene diamine
Remarks:
Made with both the spot test and plate incorporation test
Details on test system and experimental conditions:
The agar plates used in the study were Vogel Bonner Medium E minmial agar plates.

Mutagenicity spot testing was conducted as an initial screening test. Post-mitochondrial preparations (S9) from the livers of male Sprague-Dawley rats (150 - 200 g) from Charles River Laboratories were made. The rat livers were induced with 500 mg/kg Aroclor 1254 in corn oil, administered in one dose on day one. The liver extracts were prepared after the rats were killed on day 6. The liver homogenate supernatent was stored in 1 ml aliquots at -70°C and was checked for bacterial contamination. The S9 activation system was checked by assessing the reversion of all strains by 5 μg/plate of 2-aminoanthracene, a known mutagen.

The spot test was conducted using 50 μg of sodium sesquicarbonate in all strains with and without S9 mixture, with the S9 mixture present at 50 μl/plate. Plates were incubated for 2 days at 37°C in the dark.

A plate incorporation test was conducted under similar conditions as the spot test other than the sodium sesquicarbonate being added to the top agar before the bacteria and S9 mix. The agar and S9 mix were prepared in the same way as in the spot test. 0.05 mg/plate, 0.5 mg/plate and 5 mg/plate of sodium sesquicarbinare was tested in duplicate against each strain in at least six independent tests. Plates were incubated at 37°C for 2 days and counted using a Quebec colony counter.

Each plate was examined for 'background lawn' and positive and negative controls, along with sterility controls, were run for each test.

Negative controls included plates containing only bacteria and plaes containing bacteria and the solvent to be used in the test, in each case both with and without S9 mix.

Positive controls with known mutagens were used. The plates were prepared using the same procedure as for the spot test with the addition of 0.1 ml of a solution containing 50 μg of the teh mutagen. The solution was added over the surface of the agar. The substances used as positive controls were: 2-aminoanthracene in DMSO, 4-nitro-o-phenylene diamine in DMSO, sodium azide in water and 9-aminoacridine in ethanol.

Sterility control plates were also examined for the sodium sesquicarbonate, the solvent (water), top agar and the S9 mix.

All experimental plates and control plates were prepared at least in duplicate.
Evaluation criteria:
The mutagenic toxicity of the sodium sesquicarbonate was assessed based on either a clearing of the bacterial lawn or the appearance of pinpoint colonies. Plate counts were performed before and after exposure to sodium sesquicarbonate to determine the percentage survival of colony-forming units per complete growth medium plate. If there was a significant increase i.e. a reproducible dose related increase in the number of revertent colonies in the number of colonies on the plate when compared with the negative control plate and the sodium sesquicarbonate plate then the test substance was considered to be mutagenic.
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The results provided above relate to the plate incorporation test using sodium sesquicarbonate. The substance had previously been shown to be positive for genotoxicity in the spot test on TA100 without metabolic activation, however, in the plate incorporation test sodium sesquicarbonate was found to be negative for genotoxicity for all strains tested both with and without S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In the plate incorporation test sodium sesquicarbonate was found to be negative for genotoxicity for all strains tested both with and without S9 mix. A negative result in the plate incorporation test was defined as the absence of a reproducible increase in revertants where at least 5 mg per plate or the maximum non-inhibitory level has been tested.

Sodium sesquibarbonate gave a tow- to three-fold increase over the solvent control count in strain TA1538 with metabolic activation, and a two-fold increase over the solvent control count in TA1538. However, these increases were not dose-related and therefore were not considered to demonstrate genotoxicity of the substance.

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Sodium sesquicarbonate was found to be negative for genotoxicity in five mutated strains of Salmonella typhimurium up to a concentration of 5 mg/plate both with and without metabolic activation.
Executive summary:

A study was carried out using a method equivalent to the OECD Testing Guideline 471 (Bacterial Reverse Mutation Test) to determine the mutagenicity of 25 cosmetic product ingredients including sodium sesquicarbonate. Five mutated strains of Salmonella typhimurium (S. typhimurium LT2 hisTA98, hisTA100, hisTA1535, hisTA1537 and hisTA1538) were exposed to sodium sesquicarbonate first in a spot test for screening and subsequently in a plate incorporation test. The test substance was found to be positive for genotoxicity in the spot test on TA100 without metabolic activation, however, in the plate incorporation test sodium sesquicarbonate was found to be negative for genotoxicity for all strains tested both with and without S9 mix. Positive and negative controls, along with sterility controls, were conducted and all were found to be valid.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A study was carried out using a method equivalent to the OECD Testing Guideline 471 (Bacterial Reverse Mutation Test) to determine the mutagenicity of 25 cosmetic product ingredients including sodium sesquicarbonate. Five mutated strains of Salmonella typhimurium (S. typhimurium LT2 hisTA98, hisTA100, hisTA1535, hisTA1537 and hisTA1538) were exposed to sodium sesquicarbonate first in a spot test for screening and subsequently in a plate incorporation test. The test substance was found to be positive for genotoxicity in the spot test on TA100 without metabolic activation, however, in the plate incorporation test sodium sesquicarbonate was found to be negative for genotoxicity for all strains tested both with and without S9 mix. Positive and negative controls, along with sterility controls, were conducted and all were found to be valid. This negative result was supported by the results of limited but well documented and reliable studies found in the scientific literature.

No study was found in the literature on the potential of trisodium hydrogendicarbonate to induce either chromosome aberration in mammalian cells or gene mutation in mammalian cells. The substance is expected to dissociate into its constituent parts - sodium hydrogencarbonate and sodium carbonate - in the typical pH range of mammalian cells; based on this, QSAR predictions were carried out for the in vitro and in vivo genotoxicity of the substance and its constituents. All predictions were found to be negative; this was further supported by a study identified in the literature which found sodium hydrogencarbonate to be negative for chromosomal aberration in mammalian cells.

No literature information on the potential of the substance or its constituents to induce gene mutation in mammalian cells was identified, however, considering the weight of evidence available for the geneotoxic potential of trisodium hydrogendicarbonate, it is considered the case that an experimental study on the potential of the substance to induce gene mutation in mammalian cells is not necessary since it is unlikely to provide additional relevant data about the in vivo mutagenicity potential of the substance. This is in accordance with ECHA’s Endpoint Specific Guidance on Genotoxicity (ECHA, 2015), which states that the potential of a substance to induce gene mutation in mammalian cells does not need to be evaluated, if it can be demonstrated that it will not provide any further useful information about the potential in vivo mutagenicity of a substance


Justification for selection of genetic toxicity endpoint
This study was performed on the registered substance Trisodium Hydrogendicarbonate. The study was well documented and considered equivalent to OECD 471, therefore, the negative result is considered to be representative of the genotoxicity of Trisodium Hydrogendicarbonate.

Justification for classification or non-classification

Based on the available information, the substance is not classified according to EU CLP Regulation (EC) No 1272/2008.