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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 June 2021 - 29 March 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
corrected June 26, 2020
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed on 23 Oktober 2019
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium dithionite
EC Number:
231-890-0
EC Name:
Sodium dithionite
Cas Number:
7775-14-6
Molecular formula:
H2O4S2.2Na
IUPAC Name:
disodium dithionite
Test material form:
solid: particulate/powder
Details on test material:
- CAS No.: 7775-14-6
- EC No.: 231-890-0
- Molecular weight: 174.107 g/mol
- Molecular formula: Na2S2O4
- Physical State / Appearance: Solid white powder

Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: At room temperature, from moisture protected
- Expiry Date: 2031
- Stability in Solvent: Not stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: All formulations were prepared freshly before treatment and used within two hours of preparation.

Method

Target gene:
hisD (TA 98), hisG (TA 100, TA 1535), hisC (TA 1537), and trpE (WP2 uvr A pKM101)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: The S9 was prepared in-house. Phenobarbital/β-naphthoflavone induced male Wistar rat liver S9 was used as the metabolic activation system.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL S9 mix (containing 10% (v/v) S9)
- quality controls of S9: sterility and metabolic capability
Test concentrations with justification for top dose:
- Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
- Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate
The top concentration (5000 µg/plate) was selected, since it is the recommended maximum test concentration for soluble non-cytotoxic substances according to OECD guideline 471 (2020). The concentration selection was adjusted to purity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981)*.

*References:
- Maron, D.M., J. Katzenellenbogen, and B.N. Ames (1981). Compatibility of organic solvents with the Salmonella/Microsome Test. Mutation Res. 88, 343-350.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
CONCENTRATION SELECTION
In the pre-experiment the concentration range of the test item was 3 - 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II: 33, 100, 333, 1000, 2500, and 5000 μg/plate

BACTERIAL REVERSE MUTATION ASSAY
For each strain and dose level, including the controls, three plates were used.

Experiment I (Plate Incorporation):
The following materials were mixed in a test tube and poured onto the selective agar plates: 100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL bacteria suspension, 2000 μL overlay agar

Experiment II (Pre-Incubation):
The following materials were mixed in a test tube and incubated at 37°C±1.5°C for 60 minutes: 100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL Bacteria suspension. After pre-incubation 2 mL overlay agar (45°C) was added to each tube.

The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C±1.5°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2 mL overlay agar and poured on minimal agar plates.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colonies were counted using a validated computer system (Petri Viewer Sorcerer Colony Counter 3.0 (Instem, Suffolk IP33 3TA, UK) with the software program Ames Study Manager (v1.24) and Ames Archive Manager (v1.01)).
Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA (pKM101)) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
- An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

TOXICITY
- The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
- No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

GENOTOXICITY RESULTS
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sodium dithionite at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix) (please refer to "Attached background material: Results"). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

ASSAY VALIDITY
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in the number of revertant colonies, which fell in the expected range (please refer to "Attached background material: Historical control data").
- Vehicle and untreated control treatments were included for all strains in both experiments. The mean number of revertant colonies fell within acceptable ranges of the historical control database.
Thus, the controls demonstrated sensitivity of the test systems and the validity of the assay.

Applicant's summary and conclusion

Conclusions:
No toxicity (thinning of the background lawn or a reduction in the number of revertants) was found in both experiments. No precipitation was observed on the test plates. Sodium dithionite, tested up to the recommended maximum concentration, did not induce biologically relevant increases in the number of revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. All validity criteria were met. The study was fully compliant with OECD 471 (2020).

Therefore, Sodium dithionite is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.