Sodium dithionite

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sodium dithionite: The in vitro genetic toxicity of sodium dithionite has been evaluated in a bacterial gene mutation assay (acc. to OECD TG 471), in a mammalian cell gene mutation assay (acc. to OECD TG 476), and in a mammalian cell cytogenicity test (acc. to OECD TG 487). All these studies were performed according to the current guidelines and in compliance with GLP and are considered to be reliable without restrictions.

The available data on genetic toxicity allow a conclusive statement on the genetic toxicity for sodium dithionite, which is supported by read-across information from the degradation products sulfites and thiosulfates (details see below).

Sulfites: The in vitro genetic toxicity of sodium sulfite and disodium disulfite has been evaluated in bacterial gene mutation assays (acc. to OECD TG 471), in mammalian cell gene mutation assays (acc. to OECD TG 476), and in mammalian cell cytogenicity tests (acc. to OECD TG 487). These studies were performed according to the current guidelines and in compliance with GLP and are considered to be reliable without restrictions. The existing in vivo data base was evaluated in a weight-of-evidence approach. A high-quality in vivo study with sodium sulfite via subcutaneous injection in mice did not show an increase of micronuclei formation up to the maximum tolerated dose. This finding is supported by a negative dominant lethal test in rats after single and repeated oral administration (feed) in rats. A number of in vivo clastogenicity studies were assessed as being of limited reliability, since these exhibit reporting and/or other experimental deficiencies and lack biological plausibility.

Thiosulfates: The in vitro genotoxicity of the thiosulfates has been evaluated using sodium thiosulfate and ammonium thiosulfate. The in vitro genotoxicity of sodium thiosulfate has been evaluated in a bacterial gene mutation assay (acc. to OECD TG 471) and in a mammalian cell cytogenicity study (acc. to OECD TG 487). These studies were performed according to the current guidelines and in compliance with GLP and are considered to be reliable without restrictions. Moreover, ammonium thiosulfate has been evaluated in a bacterial gene mutation assay (acc. to OECD TG 471, 1997) and in a mammalian cell gene mutation assay (acc. to OECD 476, 1997). These studies were performed according to the current OECD guideline in force at the time of conduct and in compliance with GLP. These studies are considered to be reliable without restrictions.

Overall, the comprehensive set of in vitro genotoxicity assays for sodium dithionite, sulfites, and thiosulfates in combination with reliable in vivo studies for sodium sulfite confirm a lack of genotoxicity for sodium dithionite (details see below). 

Link to relevant study records

Reference

Reference 1

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

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

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.

Target gene:

hisD (TA 98), hisG (TA 100, TA 1535), hisC (TA 1537), and trpE (WP2 uvr A pKM101)

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2021 - 18 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 23 October 2019

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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.

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster lung fibroblasts (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: Each master cell stock is screened for mycoplasma contamination.
- Number of passages if applicable: Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH allowing the repeated use of the same cell culture batch in experiments.
- Cell doubling time: 12-16 hours in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Thawed stock cultures were propagated at 37°C in 75 cm² plastic flasks. About 2-3x10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% FBS, neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

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: 14.9, 29.8, 59.5, 119.1, 238.1, 476.3, 952.5, and 1905 µg/mL
- Main experiment: 59.5, 119.1, 238.1, 476.3, 952.5, and 1905 µg/mL
The maximum test item concentration of the pre-experiment and main experiment was 1905 μg/mL corresponding to 10 mM, regarding the purity of the test item with respect to the OECD guideline 476 (2016). The test item concentrations were corrected for purity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (MEM)

- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative non-toxicity to the cell cultures (Easterbrook et al., 2001)*.

*References:
- Easterbrook, J., Lu, C., Sakai, Y. and Li, A.P. (2001) Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes Drug Metabolism and Disposition, 29, 141-144.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

PRE-EXPERIMENT ON TOXICITY
A pre-test was performed in order to determine the toxicity of the test item. The osmolarity and the pH value were determined in culture medium of the solvent control and of the highest concentration. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (with and without metabolic activation) (duplicate cultures per concentration level).
Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinised and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to the corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

CONCENTRATION SELECTION
Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 59.5, 119.1, 238.1, 476.3, 952.5, and 1905 µg/mL.

HPRT MUTAGENICITY EXPERIMENT
- Seeding: Two to four days after sub-cultivation stock cultures were trypsinised at 37°C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7-1.2x10^7 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2x10^6 cells per experimental point (concentration series plus controls) were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37±1.5°C in a humidified atmosphere with 1.5%±0.5 CO2.
The colonies used to determine the relative survival (RS) were fixed and stained approximately 8±2 days after treatment as described below.
Three or four days after the first sub-cultivation, at least 2x10^6 cells per experimental point were again, sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days, five 75 cm² cell culture flasks were seeded with 4-5x10^5 cells each in medium containing 6-TG (11 μg/mL). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37°C±1.5°C in a humidified atmosphere with 1.5%±0.5 CO2.
After 8 days (evaluation for viability) and 9±2 days (mutation analysis) the colonies were stained with 10% methylene blue in 0.01% KOH solution.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS USED TO DETERMINE pH, OSMOLALITY AND PRECIPITATION
- The osmolarity and pH of the test item formulated in culture medium was determined by using an osmometer (Gonotec, Model Osmomat 030) or a pH meter (TW, Model Vario pH), respectively, in the pre-experiment without metabolic activation.
- Precipitation or phase separation was be evaluated at the beginning and at the end of treatment by the unaided eye.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits) (please refer to “Attached background material: Historical control data”).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits) (please refer to “Attached background material: Historical control data”).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiment.
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible concentration dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance will be considered together.
A t-test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory’s historical negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT ON TOXICITY
- Test item concentrations between 14.9 μg/mL and 1905 μg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The maximum concentration in the pre-experiment was chosen corresponding to 10 mM, regarding the purity of the test item with respect to the OECD guideline 476 (2016).
- No precipitation occurred up to the highest applied concentration with and without metabolic activation.
- No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed.

TEST-SPECIFIC CONFOUNDING FACTORS
- The pH was 7.40 and 7.19 in untreated culture medium and culture medium with the test item, respectively. The osmolarity was 333 and 361 mOsm in solvent control culture medium and in culture medium mixed with sodium dithionite at the highest concentration (1905 µg/mL). Thus, there was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment.
- In the main experiment, no precipitation, as determined by the unaided eye, or phase separation occurred up to the highest concentration investigated after four hours treatment.

HPRT MUTAGENICITY ASSAY
Main experiment in absence of metabolic activation:
- Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 59.5, 119.1, 238.1, 476.3, 952.5, and 1905 μg/mL
- Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted at 952.5 μg/mL and above. The concentration of 952.5 μg/mL was the maximum evaluated concentration, achieving a relative adjusted cloning efficiency I between 10 and 20% (14.8%). Cultures of higher concentrations could not be continued due to strong cytotoxic effects (relative survival <10%).
- Consequently, the concentrations of 59.5 to 952.5 μg/mL were evaluated for mutagenicity in the absence of metabolic activation.
- The mutation frequency (MF) of the solvent control was 21.5 mutants per 10^6 cells and the mutation frequency range of the treated groups was 13.4 up to 21.0 mutants per 10^6 cells. No increase in mutant colony numbers per 10^6 cells was observed in the experiment up to the maximum concentration. The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95% control limits of the solvent historical control data (2.9 - 22.4 mutants per 10^6 cells; please refer to attached background material: “Historical control data”).
- The linear regression analysis showed a statistically significant trend in the absence of metabolic activation, but this effect is caused by an inverse trend of the mutation frequency (MF) and is therefore without any biological relevance.

Main experiment in presence of metabolic activation:
- Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 59.5, 119.1, 238.1, 476.3, 952.5, and 1905 μg/mL
- Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted at 952.5 μg/mL and above. The concentration of 952.5 μg/mL was the maximum evaluated concentration, achieving a relative adjusted cloning efficiency I between 10 and 20% (14.3%). Cultures of higher concentrations could not be continued due to strong cytotoxic effects.
- Consequently, the concentrations of 59.5 to 952.5 μg/mL were evaluated for mutagenicity in the presence of metabolic activation.
- The mutation frequency (MF) of the solvent control was 18.1 mutants per 10^6 cells and the mutation frequency range of the treated groups was 12.1 up to 19.0 mutants per 10^6 cells. No statistically significant increase in mutant colony numbers per 10^6 cells was observed in the experiment up to the maximum concentration. The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95% control limits of the solvent historical control data (2.9 - 23.7 mutants per 10^6 cells; please refer to attached background material: “Historical control data”).
- Linear regression analysis showed no statistically significant trend.

In summary, the outcome of the experiment was clearly negative in the presence and absence of metabolic activation.

ASSAY VALIDITY
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in the mutation frequency, which fell well within the historical control data range (please refer to attached background material: “Historical control data”). The concurrent solvent control cultures showed mutation frequencies, which were well within the acceptable ranges of the historical control data base. Thus, the controls demonstrated sensitivity of the test system and the validity of the assay.

Conclusions:

Increased cytotoxicity was found in the main experiment both with and without metabolic activation at concentrations of 952.5 µg/mL and above. Thus, the highest concentration evaluated was based on cytotoxicity corresponding to the criteria set out in the OECD guideline 467 (2016). Cultures treated at 952.5 µg/mL with and without metabolic activation showed a relative survival of 14.8 and 14.3%, respectively. No precipitation of the test material occurred up to the highest concentration investigated after four hours treatment both in absence and presence of metabolic activation. Sodium dithionite, tested up to cytotoxic concentrations did not induce biologically relevant increases in the mutant frequency both in presence and absence of metabolic activation. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the Hprt locus in V79 cells. All validity criteria were met. The study was fully compliant with OECD 476 (2016).
 
Therefore, sodium dithionite is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August 2021 - 10 March 2022 (Experimental completion; Only draft report available. Data will be updated upon availability of the final report.)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Only draft report available. Data will be updated upon availability of the final report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Principles of method if other than guideline:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: At room temperature, from moisture protected
- Expiry Date: 2031

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.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 23 October 2019

Type of assay:

in vitro mammalian cell micronucleus test

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.

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Lymphocytes from human peripheral blood
- Suitability of cells: Human peripheral blood lymphocytes are the most common cells used in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level (Countryman and Heddle, 1976; Evans and O’Riordan, 1975)* and are recommended in the current OECD Guideline 487 (2016).

For lymphocytes:
- Sex, age and number of blood donors: Blood samples were drawn from healthy non-smoking donors with no known illness or recent exposures to genotoxic agents (e.g. chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cells. For this study, blood was collected from a male donor (25 years old) for Experiment I and from a male donor (24 years old) for Experiment III. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohaemagglutinin (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The cell cycle time for lymphocytes from each donor has been determined with proliferation index by BrdU (bromodeoxyuridine) incorporation using the sister chromatid exchange test to assess the average generation time (AGT) for the donor pool. Additionally, the cytokinesis-block proliferation index provides data on suitability in the test system.
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: blood from different was not pooled
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX (L-glutamine source). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA 1.5% (v/v) as extract, 10% FBS (foetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37°C with 5.5% CO2 in humidified air.

*References:
- COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

- EVANS H.J. and O`RIORDAN M.L. (1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Research, 31, 135-148.

Cytokinesis block (if used):

Cytochalasin B (4 µg/mL)

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 protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL/mL culture medium. The final concentration of S9 mix in the treatment medium was 5% (v/v).
- quality controls of S9: sterility and metabolic capability

Test concentrations with justification for top dose:

- Experiment I (3 hours): 12.4, 21.7, 37.9, 66.3, 116, 203, 355, 622, 1089, and 1905 µg/mL
- Experiment II (28 hours): 73.6, 129, 226, 395, 513, 667, 867, 1127, 1465, and 1905 µg/mL (Result of the genotoxicity parameter is not reported, due to invalidity (solvent control exceeded the historical control data))
- Experiment III (28 hours): 92.2, 161, 282, 423, 466, 512, 563, 620, 682, and 750 µg/mL
The highest treatment concentration in this study, 1905 µg/mL (equivalent to 10 mM) was chosen with regard to the molecular weight (174.1071 g/mol) and the purity of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. The concentration of Experiment III was adjusted due to cytotoxicity observed in Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (DMEM/F12)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

PRE-EXPERIMENT ON TOXICITY
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. With regard to the molecular weight (174.1071 g/mol) and the purity of the test item, 1905 µg/mL (equivalent to 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 12.4 to 1905 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of 2 to 3 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 3 hours (with and without S9 mix). The preparation interval was 28 hours after start of the exposure.
In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment both in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

CONCENTRATION SELECTION
No cytotoxic effects were observed in Experiment I after the 3 3-hours treatment both in the absence and presence of S9 mix. Therefore, the test item concentration of 1905 µg/mL was chosen as top treatment concentration for Experiment II (28 hours continuous exposure). Since Experiment II was invalid (solvent control exceeded the historical control data), the experiment was repeated. Clear toxic effects were observed in Experiment II with 513 µg/mL and above in the absence of S9 mix. Considering the toxicity data of Experiment II, 750 µg/mL was chosen as top concentration in Experiment III.
MN EXPERIMENTS
- Experiment I (Pulse exposure with and without S9 mix):
About 48 hours after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test substance concentration. The culture medium was replaced with serum-free medium containing the test substance or control. For the treatment with metabolic activation S9 mix (50 µL/mL culture medium) was added. After 3 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose x H2O, 192 mg/L Na2HPO4 x 2H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10% FBS (v/v) in the presence of Cytochalasin B (4 µg/mL) and cultured for 25 hours until preparation (Clare et al., 2006, Lorge et al., 2006)*.

- Experiment II (Continuous exposure without S9 mix):
About 48 hours after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10% FBS) containing the test item and in the presence of Cytochalasin B (4 µg/mL). The cells were exposed for 28 hours until preparation (Whitwell et al., 2019)*.

- Preparation of cells:
The cultures were harvested by centrifugation 28 hours after beginning of treatment (3+25 and 28+0 hours). The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37°C for 20 minutes. A volume of 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with 10% Giemsa solution in Weise buffer for approximately 15 to 20 minutes, mounted after drying and covered with a coverslip.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Evaluation of the slides was performed using microscopes with 40 x objectives.
- Scoring criteria
The criteria for the evaluation of micronuclei were applied according to Countryman and Heddle (1976)*. At least 1000 binucleate cells were scored manually per culture for micronuclei on independently coded slides. It is advisable to use duplicate cultures; single cultures are also acceptable provided that a total of 2000 cells are scored. Only cells containing a clearly visible cytoplasm were included in the analysis.
The criteria for the evaluation of micronuclei are as follows:
- The micronucleus has to be stained in the same way as the main nucleus and the area of the micronucleus should not extend the third part of the area of the main nucleus.
- Cells containing more than two main nuclei should not be analysed for micronuclei, as the baseline micronucleus frequency may be higher in these cells.
The micronucleus frequency was reported as % micronucleated cells.
In addition, micronuclei in mononucleate cells were recorded when these events are seen, since aneuploid acting substances are known to increase the number of micronucleated mononucleate cells.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Evaluation of the slides was performed using microscopes with 40 x objectives.
- 500 cells per culture were scored on independently coded slides for the determination of the Cytokinesis-Block Proliferation Index (CBPI). Cytotoxicity will be expressed as % cytostasis.
METHODS USED TO DETERMINE pH, OSMOLALITY AND PRECIPITATION
- The osmolarity and pH of the test item formulated in culture medium was determined by using an osmometer (Gonotec, Model Osmomat 030) or a pH meter (TW, Model Vario pH), respectively, in the pre-experiment without metabolic activation.
- Precipitation or phase separation was be evaluated at the beginning and at the end of treatment by the unaided eye.

*References:
- CLARE M.G., LORENZON G., AKHURST L.C., MARZIN D., VAN DELFT J., MONTERO R., BOTTA A., BERTENS A., CINELLI S., THYBAUD V. AND LORGE E., (2006) SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes. Mutation Res., 607, 37-60.
- LORGE E., THYBAUD V., AARDEMA M.J., OLIVER J., WAKATA A., LORENZON G. and MARZIN D. (2006) SFTG international collaborative study on in vitro micronucleus test I. General conditions and overall conclusions of the study. Mutation Res., 607, 13-36.
- WHITWELL J., SMITH R., CHIROM T., WATTERS G., HARGREAVES V., LLOYD M., PHILLIPS S. and CLEMENTS J. (2019) Inclusion of an extended treatment improves the results for the human peripheral blood lymphocyte micronucleus assay. Mutagenesis 34, 217-237.
- COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions could be useful.

However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.

Statistics:

Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

A linear regression was performed using a validated test script of “R”, to assess a possible concentration-response dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field: "Additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT ON TOXICITY
- In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment both in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
- No cytotoxic effects were observed in Experiment I after the 3-hour treatment both in the absence and presence of S9 mix. Therefore, the test item concentration of 1905 µg/mL was chosen as top treatment concentration for Experiment II (28 hours continuous exposure). Since Experiment II was invalid (solvent control exceeded the historical control data), the experiment was repeated. Clear toxic effects were observed in Experiment II with 513 µg/mL and above in the absence of S9 mix. Considering the toxicity data of Experiment II, 750 µg/mL was chosen as top concentration in Experiment III.

TEST-SPECIFIC CONFOUNDING FACTORS
- No relevant influence on osmolarity was observed. The pH was measured at the beginning and at the end of treatment. No relevant influence on pH was observed.
- In Experiment I in the absence and presence of S9 mix and in Experiment III, no precipitation of the test item in the culture medium was observed.
- In Experiment I (3-hour pulse treatment) in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest concentration applied. In Experiment III in the absence of S9 mix after continuous treatment (28 hours), moderate cytotoxicity was observed at the concentrations 161 and 282 µg/mL. Clear cytotoxicity (58.8% cytostasis) was observed at the highest concentration evaluated (466 µg/mL) based on the criteria set out in the current OECD Guideline 487 (2016).

MN EXPERIMENTS

Evaluation of cytogenetic damage (Experiment I: 3-hour pulse treatment):
- Based on the absence of precipitates and cytotoxicity, the following test item concentration levels were evaluated in Experiment I: With and without S9 mix: 622, 1089, and 1905 µg/mL
- In Experiment I (3-hour pulse treatment) in the absence and presence of S9 mix, In the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item (please refer to attached background material: “Results”). In the absence of metabolic activation, the proportion of micronucleated cells was 0.35, 0.20, and 0.50% in cell cultures exposed to Sodium dithionite at concentration levels of 622, 1089, and 1905 µg/mL, respectively. In the solvent control cultures, a mean micronucleus frequency of 0.45% was observed. In presence of metabolic activation, the proportion of micronucleated cells was 0.60, 0.40, and 0.45% in cell cultures exposed to Sodium dithionite at concentration levels of 622, 1089, and 1905 µg/mL, respectively. The solvent control group showed 0.80% micronucleated cells. The mean percentage of the micronuclei in all treated conditions was within the 95% historical control limit (please refer to attached background material: “Historical control data”) and none of the values was statistically significantly increased, when compared with the solvent control. There was also no concentration related increase in micronucleus formation frequency, as judged by an appropriate trend test.

Evaluation of cytogenetic damage (Experiment II: 28-hour continuous treatment)
- Based on the presence of limiting cytotoxicity (55 ± 5%), the following test item concentration levels were evaluated in Experiment III: Without S9 mix: 161, 282, 466 µg/mL
- In Experiment III (28-hour continuous treatment), no relevant increases in the number of micronucleated cells were observed after treatment with the test item (please refer to attached background material: “Results”). The proportion of micronucleated cells was 0.55, 0.50, and 0.40% in cell cultures exposed to Sodium dithionite at concentration levels of 161, 282, and 466 µg/mL, respectively. The solvent control cultures showed 0.30% micronucleated cells. The mean percentage of the micronuclei in all treated conditions was within the 95% historical control limit (please refer to attached background material: “Historical control data”) and none of the values was statistically significantly increased, when compared with the solvent control. There was also no concentration related increase in micronucleus formation frequency, as judged by an appropriate trend test.

In summary, the outcome of the study is clearly negative.

ASSAY VALIDITY
Vinblastine (aneugen), mitomycin C (clastogen, active without metabolic activation), and cyclophosphamide (clastogen requiring metabolic activation) were used as reference mutagens. They induced distinct and statistically significant increases in the proportion of cells with micronuclei. Thus, the activity of the metabolic activation system and the sensitivity of the test system was demonstrated. Solvent control cultures were included in all experiments. The micronucleus frequencies observed in the solvent control cultures were well within the 95% historical control limit (please refer to attached background materials: “Historical control data” and “Results”).

All acceptance criteria were considered met and the study was accepted as valid.

Conclusions:

In the 3-hour pulse treatment experiment, in the absence and presence of S9 mix, no cytotoxicity was observed up to the recommended maximum concentration (1905 µg/mL; equivalent to 10 mM). In the 28 hours continuous treatment experiment III in the absence of S9 mix, limiting cytotoxicity was observed at concentrations of 466 µg/mL and above. Clear cytotoxicity (58.8% cytostasis) was observed at the highest concentration evaluated (466 µg/mL). No precipitation of the test item in the culture medium was observed after the pulse and continuous treatment experiments. Sodium dithionite, tested up to the recommended maximum concentration (3-hour pulse treatment) or cytotoxic concentrations (28-hour continuous treatment), did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2016).

Therefore, sodium dithionite is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the maximum concentration recommended or to cytotoxic concentrations.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

26 July 2021 - 11 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, from moisture protected
- Expiry Date: July 2023

Target gene:

hisD (TA 98), hisG (TA 100, TA 1535), hisC (TA 1537), and trpE (WP2 uvr A pKM101)

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.

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.

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 sulfite 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.

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

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

26 July 2021 - 09 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 23 October 2019

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, from moisture protected
- Expiry Date: July 2023

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster lung fibroblasts (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: Each master cell stock is screened for mycoplasma contamination.
- Number of passages if applicable: Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH allowing the repeated use of the same cell culture batch in experiments.
- Cell doubling time: 12-16 hours in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Thawed stock cultures were propagated at 37°C in 75 cm² plastic flasks. About 2-3x10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% FBS, neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

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 and Main experiment: 10, 20, 40, 80, 160, 320, 640, and 1280 μg/mL
- The top test item concentration of the pre-experiment and main experiment was 1280 µg/mL corresponding to 10 mM, regarding the purity of the test item with respect to the OECD guideline 476 (2016).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water

- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative non-toxicity to the cell cultures (Easterbrook et al., 2001)*.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium was 10% (v/v) corresponding to the recommendations set out in OECD guideline 476 (2016).

*References:
- Easterbrook, J., Lu, C., Sakai, Y. and Li, A.P. (2001) Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes Drug Metabolism and Disposition, 29, 141-144.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

PRE-EXPERIMENT ON TOXICITY
A pre-test was performed in order to determine the toxicity of the test item. The osmolarity and the pH value were determined in culture medium of the solvent control and of the highest concentration. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (with and without metabolic activation) (duplicate cultures per concentration level).
Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinised and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to the corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

CONCENTRATION SELECTION
Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 10, 20, 40, 80, 160, 320, 640, and 1280 μg/mL.

HPRT MUTAGENICITY EXPERIMENT
- Seeding: Two to four days after sub-cultivation stock cultures were trypsinised at 37°C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7-1.2x10^7 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2x10^6 cells per experimental point (concentration series plus controls) were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37±1.5°C in a humidified atmosphere with 1.5%±0.5 CO2.
The colonies used to determine the relative survival (RS) were fixed and stained approximately 8±2 days after treatment as described below.
Three or four days after the first sub-cultivation, at least 2x10^6 cells per experimental point were again, sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days, five 75 cm² cell culture flasks were seeded with 4-5x10^5 cells each in medium containing 6-TG (11 μg/mL). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37°C±1.5°C in a humidified atmosphere with 1.5%±0.5 CO2.
After 8 days (evaluation for viability) and 9±2 days (mutation analysis) the colonies were stained with 10% methylene blue in 0.01% KOH solution.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS USED TO DETERMINE pH, OSMOLALITY AND PRECIPITATION
- The osmolarity and pH of the test item formulated in culture medium was determined by using an osmometer (Gonotec, Model Osmomat 030) or a pH meter (TW, Model Vario pH), respectively, in the pre-experiment without metabolic activation.
- Precipitation or phase separation was be evaluated at the beginning and at the end of treatment by the unaided eye.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits) (please refer to “Attached background material: Historical control data”).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits) (please refer to “Attached background material: Historical control data”).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiment.

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible concentration dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

A t-test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. Again, a t-test is judged as significant if the p-value (probability value) is below 0.05.

However, both, biological and statistical significance will be considered together.

A t-test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory’s historical negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Please refer to the field 'Additional information on results'

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT ON TOXICITY
- Test item concentrations between 10 μg/mL and 1280 μg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The maximum concentration in the pre-experiment was chosen corresponding to 10 mM, regarding the purity of the test item with respect to the OECD guideline 476 (2016).
- The test medium was checked for phase separation and precipitation at the end of the treatment period (4 hours) before the test item was removed. No precipitation occurred up to the highest applied concentration with and without metabolic activation.
- No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed.

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: Before treatment, the pH value was 7.33 and 7.49 of the solvent control and 1280 µg/mL sodium sulfite in the culture medium, respectively. After the 4-hour treatment, the pH value was 7.38 and 7.51 of the solvent control and 1280 µg/mL sodium sulfite in the culture medium, respectively. The osmolarity was 290 and 318 mOsm for the solvent control and sodium sulfite in culture medium, respectively, before treatment. After the 4-hour treatment, the solvent control and sodium sulfite treated cultures showed an osmolarity of 294 and 320 mOsm, respectively. Thus, there was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment.
- In the main experiment, no precipitation occurred up to the highest applied concentration with and without metabolic activation.

HPRT MUTAGENICITY ASSAY
Main experiment in absence of metabolic activation:
- Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 10, 20, 40, 80, 160, 320, 640, and 1280 μg/mL
- Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted at 640 μg/mL and above. The concentration of 640 μg/mL was the maximum concentration evaluated, achieving a relative adjusted cloning efficiency I between 10 and 20% (12.1%). Cultures of higher concentrations could not be continued due to strong cytotoxic effects (please refer to attached background material: 'Results').
- Consequently, the concentrations of 40 to 640 μg/mL were evaluated for mutagenicity in the absence of metabolic activation.
- The mutation frequency (MF) of the solvent control was 18.4 mutants per 10^6 cells and the mutation frequency range of the treated groups was 7.8 up to 18.3 mutants per 10^6 cells. No increase in mutant colony numbers per 10^6 cells was observed in the experiment up to the maximum concentration. The observed mean mutant frequency of the solvent control and all evaluated concentrations was within the 95% control limits of the solvent historical control data (2.9 - 22.4 mutants per 10^6 cells) (please refer to attached background material: 'Results').
- Linear regression analysis showed no statistically significant trend.

Main experiment in presence of metabolic activation:
- Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 10, 20, 40, 80, 160, 320, 640, and 1280 μg/mL
- No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted.
- Consequently, the concentrations of 80 to 1280 μg/mL were evaluated for mutagenicity in the presence of metabolic activation.
- The mutation frequency (MF) of the solvent control was 18.4 mutants per 10^6 cells and the mutation frequency range of the treated groups was 15.8 up to 21.6 mutants per 10^6 cells. No statistically significant increase in mutant colony numbers per 106 cells was observed in the experiment up to the maximum concentration. The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95% control limits of the solvent historical control data (2.9 - 23.7 mutants per 10^6 cells) (please refer to attached background material: 'Results').
- Linear regression analysis showed no statistically significant trend.

In summary, the outcome of the experiment was clearly negative in the presence and absence of metabolic activation.

ASSAY VALIDITY
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in the mutation frequency, which fell well within the historical control data range. The concurrent solvent control cultures showed mutation frequencies, which were well within the acceptable ranges of the historical control data base. Thus, the controls demonstrated sensitivity of the test system and the validity of the assay.

Conclusions:

No relevant toxicity (10 to 20% relative survival) was found in the main experiment with metabolic activation. In the experiment without metabolic activation, limiting cytotoxicity was observed at concentrations of 640 µg/mL and above. No precipitation of the test material occurred up to the highest concentration investigated after four hours treatment both in absence and presence of metabolic activation. Sodium sulfite, tested up to the recommended maximum concentration (with metabolic activation) or cytotoxic concentrations (without metabolic activation), did not induce biologically relevant increases in the mutant frequency. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the Hprt locus in V79 cells. All validity criteria were met. The study was fully compliant with OECD 476 (2016).

Therefore, sodium sulfite is considered to be non-mutagenic in this HPRT assay.

Reference 6

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

29 July 2021 - 30 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 23 October 2019

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, from moisture protected
- Expiry Date: July 2023

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Lymphocytes from human peripheral blood
- Suitability of cells: Human peripheral blood lymphocytes are the most common cells used in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level (Countryman and Heddle, 1976; Evans and O’Riordan, 1975)* and are recommended in the current OECD Guideline 487 (2016).

For lymphocytes:
- Sex, age and number of blood donors: Blood samples were drawn from healthy non-smoking donors with no known illness or recent exposures to genotoxic agents (e.g. chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cells. For this study, blood was collected from a female donor (20 years old) for Experiment I and from a male donor (25 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohaemagglutinin (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The cell cycle time for lymphocytes from each donor has been determined with proliferation index by BrdU (bromodeoxyuridine) incorporation using the sister chromatid exchange test to assess the average generation time (AGT) for the donor pool. Additionally, the cytokinesis-block proliferation index provides data on suitability in the test system.
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: blood from different was not pooled
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX (L-glutamine source). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA 1.5% (v/v) as extract, 10% FBS (foetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37°C with 5.5% CO2 in humidified air.

*References:
- COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

- EVANS H.J. and O`RIORDAN M.L. (1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Research, 31, 135-148.

Cytokinesis block (if used):

Cytochalasin B (4 µg/mL)

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 protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL/mL culture medium. The final concentration of S9 mix in the treatment medium was 5% (v/v).
- quality controls of S9: sterility and metabolic capability

Test concentrations with justification for top dose:

- Experiment I (3 hours): 8.3, 14.6, 25.5, 44.6, 78.0, 136, 239, 418, 731, and 1280 µg/mL
- Experiment II (28 hours): 62.1, 109, 190, 333, 583, 757, 985, and 1280 µg/mL
The highest concentration applied in this study (1280 µg/mL of the test item; equivalent to10 mM) was chosen with regard to the molecular weight and the purity of the test item and with respect to the current OECD Guideline 487 (2016).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water

- Justification for choice of solvent/vehicle: The use of aqueous solvents is recommended by the current OECD Guideline 487.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium was 10% (v/v) corresponding to the recommendations set out in OECD guideline 487 (2016).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

PRE-EXPERIMENT ON TOXICITY
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. With regard to the molecular weight (126.05 g/mol) and the purity of the test item, 1280 µg/mL (equivalent to 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 8.3 to 1280 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of 2 to 3 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 3 hours (with and without S9 mix). The preparation interval was 28 hours after start of the exposure.
In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment both in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

CONCENTRATION SELECTION
No cytotoxic effects were observed in Experiment I after the 3-hour treatment both in the absence and presence of S9 mix. Therefore, the test item concentration of 1280 µg/mL was chosen as top treatment concentration for Experiment II (28 hours continuous exposure).

MN EXPERIMENTS
- Experiment I (Pulse exposure with and without S9 mix):
About 48 hours after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test substance concentration. The culture medium was replaced with serum-free medium containing the test substance or control. For the treatment with metabolic activation S9 mix (50 µL/mL culture medium) was added. After 3 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose x H2O, 192 mg/L Na2HPO4 x 2H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10% FBS (v/v) in the presence of Cytochalasin B (4 µg/mL) and cultured for 25 hours until preparation (Clare et al., 2006, Lorge et al., 2006)*.

- Experiment II (Continuous exposure without S9 mix):
About 48 hours after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10% FBS) containing the test item and in the presence of Cytochalasin B (4 µg/mL). The cells were exposed for 28 hours until preparation (Whitwell et al., 2019)*.

- Preparation of cells:
The cultures were harvested by centrifugation 28 hours after beginning of treatment (3+25 and 28+0 hours). The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37°C for 20 minutes. A volume of 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with 10% Giemsa solution in Weise buffer for approximately 15 to 20 minutes, mounted after drying and covered with a coverslip.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Evaluation of the slides was performed using microscopes with 40 x objectives.
- Scoring criteria
The criteria for the evaluation of micronuclei were applied according to Countryman and Heddle (1976)*. At least 1000 binucleate cells were scored manually per culture for micronuclei on independently coded slides. It is advisable to use duplicate cultures; single cultures are also acceptable provided that a total of 2000 cells are scored. Only cells containing a clearly visible cytoplasm were included in the analysis.
The criteria for the evaluation of micronuclei are as follows:
- The micronucleus has to be stained in the same way as the main nucleus and the area of the micronucleus should not extend the third part of the area of the main nucleus.
- Cells containing more than two main nuclei should not be analysed for micronuclei, as the baseline micronucleus frequency may be higher in these cells.
The micronucleus frequency was reported as % micronucleated cells.
In addition, micronuclei in mononucleate cells were recorded when these events are seen, since aneuploid acting substances are known to increase the number of micronucleated mononucleate cells.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Evaluation of the slides was performed using microscopes with 40 x objectives.
- 500 cells per culture were scored on independently coded slides for the determination of the Cytokinesis-Block Proliferation Index (CBPI). Cytotoxicity will be expressed as % cytostasis.
METHODS USED TO DETERMINE pH, OSMOLALITY AND PRECIPITATION
- The osmolarity and pH of the test item formulated in culture medium was determined by using an osmometer (Gonotec, Model Osmomat 030) or a pH meter (TW, Model Vario pH), respectively, in the pre-experiment without metabolic activation.
- Precipitation or phase separation was be evaluated at the beginning and at the end of treatment by the unaided eye.

*References:
- CLARE M.G., LORENZON G., AKHURST L.C., MARZIN D., VAN DELFT J., MONTERO R., BOTTA A., BERTENS A., CINELLI S., THYBAUD V. AND LORGE E., (2006) SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes. Mutation Res., 607, 37-60.
- LORGE E., THYBAUD V., AARDEMA M.J., OLIVER J., WAKATA A., LORENZON G. and MARZIN D. (2006) SFTG international collaborative study on in vitro micronucleus test I. General conditions and overall conclusions of the study. Mutation Res., 607, 13-36.
- WHITWELL J., SMITH R., CHIROM T., WATTERS G., HARGREAVES V., LLOYD M., PHILLIPS S. and CLEMENTS J. (2019) Inclusion of an extended treatment improves the results for the human peripheral blood lymphocyte micronucleus assay. Mutagenesis 34, 217-237.
- COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions could be useful.

However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.

Statistics:

Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

A linear regression was performed using a validated test script of “R”, to assess a possible concentration-response dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field: "Additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT ON TOXICITY
- In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment both in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
- No cytotoxic effects were observed in Experiment I after the 3-hour treatment both in the absence and presence of S9 mix. Therefore, the test item concentration of 1280 µg/mL (recommended maximum concentration) was chosen as top treatment concentration for Experiment II.

TEST-SPECIFIC CONFOUNDING FACTORS
- No relevant influence on osmolarity was observed. The pH was measured at the beginning and at the end of treatment. No relevant change on pH was observed.
- In Experiment I and II in the absence and presence of S9 mix, no precipitation of the test item in the culture medium was observed.
- In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest concentration applied. In Experiment II in the absence of S9 mix after continuous treatment (28 hours), limiting cytotoxicity was observed at concentrations ranging from 583-1280 µg/mL. Clear cytotoxicity (54.9% cytostasis) was observed at the highest concentration evaluated (333 µg/mL) based on the criteria set out in the current OECD Guideline 487 (2016).

MN EXPERIMENTS
Evaluation of cytogenetic damage (Experiment I: 3-hour pulse treatment):
- Based on the absence of precipitates and cytotoxicity, the following test item concentration levels were evaluated in Experiment I: 418, 731, and 1280 µg/mL with and without S9 mix
- In Experiment I in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. The data of both experimental parts were confirmed by increasing the sample size (i.e. 2000 binucleated cells per culture instead of 1000 binucleated cells per culture). In the absence of S9 mix, however, the micronucleus frequency of the solvent control (0.78%) and the cultures treated at sodium sulfite concentrations of 418 (0.75%) and 1280 µg/mL (0.73%) slightly exceeded the 95% control limit of the historical control data range (0.13 – 0.70% micronucleated cells) (please refer to attached background material: “Historical control data” and “Results”). Similarly, in the presence of S9 mix, the micronucleus frequency in cultures treated at 418 µg/mL (0.88%) was slightly above the historical data range (0.00 – 0.84% micronucleated cells). None of the micronucleus frequencies observed after treatment with sodium sulfite was statistically significantly different from the corresponding solvent control values and no concentration dependency was observed. Therefore, these outliers can be considered as biologically irrelevant.
- The historical control data of the current protocol (please refer to attached background material: “Historical control data”) are based on a rather small number of experiments (in line with the minimum requirement of OECD Guideline 487), therefore, deviations from historical control data are not unexpected. The data of the former test design (including a recovery period and a longer exposure period; please refer to attached background material: “Historical control data”) show that the expected variability is actually higher than the variability observed so far. The min-max range of the historical control data from the former test design indicated values up to 1.25% micronucleated cells for the solvent controls of all three treatment schedules (≥ 125 studies per treatment schedule collected in 2019 and 2020). Therefore, the micronucleus frequencies observed are clearly within the expected range of the biological variation of this test system. The current experiments can be considered as valid with non-mutagenic outcome.

Evaluation of cytogenetic damage (Experiment II: 28-hour continuous treatment)
- Based on the presence of limiting cytotoxicity (55 ± 5%), the following test item concentration levels were evaluated in Experiment II: 109, 190, 333 µg/mL without S9 mix
- In Experiment II in the absence of S9 mix after continuous treatment, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. The mean percentage of the micronuclei of all values was within the 95% historical control limits and none of the values were statistically significantly increased, when compared to the solvent control. A concentration related trend was not observed.

Taken together, the outcome of the current study is clearly negative.

ASSAY VALIDITY
Vinblastine (7.5 ng/mL), mitomycin C (0.6 µg/mL) and cyclophosphamide (17.5 µg/mL) were used as positive controls and showed distinct and statistically significant increases in the proportion of cells with micronuclei. Thus, the activity of metabolic activation system and the system and the sensitivity of the test system was demonstrated.

All acceptance criteria were considered met and the study was accepted as valid.

Conclusions:

In Experiment I (3-hour pulse treatment) in the absence and presence of S9 mix, no cytotoxicity was observed up to the recommended maximum concentration (1280 µg/mL; equivalent to 10 mM). In Experiment II in the absence of S9 mix after continuous treatment (28 hours), limiting cytotoxicity was observed at concentrations ranging from 583-1280 µg/mL. Clear cytotoxicity (54.9% cytostasis) was observed at the highest concentration evaluated (333 µg/mL). In Experiment I and II in the absence and presence of S9 mix, no precipitation of the test item in the culture medium was observed. Sodium sulfite, tested up to the recommended maximum concentration (Experiment I: 3-hour treatment) or cytotoxic concentrations (Experiment II: 28-hour treatment), did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2016).

Therefore, sodium sulfite is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the maximum concentration recommended or to cytotoxic concentrations.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

15 July 2021 - 11 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: At room temperature, light protected, moisture protected (only valid for storage conditions, not for test performance)
- Expiry Date: 08 July 2023

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test item was dissolved in deionised water. The pH values in the stock solution and S9 mix substitution buffer were 3.75 and 7.3, respectively. The pH value of the stock solution was adjusted with NaOH (2 M) to 6.91 in experiment I and to 6.94 in experiment II. The pH value of 100 μL stock solution in 500 μL S9 mix substitution buffer was 7.2 in both experiments. All formulations were prepared freshly before treatment and used within two hours of preparation.

Target gene:

hisD (TA 98), hisG (TA 100, TA 1535), hisC (TA 1537), and trpE (WP2 uvr A pKM101)

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).

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.

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.

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:

not 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 Disodium disulfite 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.

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. Disodium disulfite, 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, Disodium disulfite is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2010-07-21 to 2010-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15 to 25°C in the dark

Target gene:

hprt

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media containing 2.5 µg/mL Amphotericin B, 100 µg/mL Streptomycin, 100 units/mL Penicillin, 0.5 µg/mL Pluronic (ecxept for RPMI 20) and 0%, 10% or 20% (v/v) heat inactivatet horse serum for RPMI A, RPMI 10 and RPMI 20, respectively.

The master stock of L5178Y tk+/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of frozen cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: yes; each batch of frozen cells was purged of mutants.
- Periodically "cleansed" against high spontaneous background: yes
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Range-Finder:
- with and without S9-mix: 59.44, 118.9, 237.8, 475.5, 951 and 1902 µg/mL.
Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment.

Experiment I:
- with and without S9-mix: 200, 300, 400, 600, 800, 1200, 1600 and 1902 µg/mL,
Experiment II:
- with and without S9-mix: 100, 300, 600, 900, 1200, 1500 and 1902 µg/mL,
Experiment III:
- with S9-mix: 200, 400, 800, 1000, 1200, 1400, 1600, 1700, 1800 and 1902 µg/mL.

Cultures selected for mutation assessment:
Experiment I:
- with and without S9-mix: 200, 300, 400, 600, 800, 1200, 1600 and 1902 µg/mL,
Experiment II:
- with and without S9-mix: 300, 600, 900, 1200, 1500 and 1902 µg/mL,
Experiment III:
- with S9-mix: 200, 800, 1000, 1400, 1600, 1700, 1800 and 1902 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that sodium metabisulfite was soluble in water for irrigation (purified water) at concentrations up to at least 20.25 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

purified water diluted 10 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 4-nitroquinoline-1-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37°C
- Expression time (cells in growth medium): 7 days during which the hprt mutation would be expressed: After the incubation period, cultures were centrifuged (200 g), washed and resuspended in RPMI 10. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival (scoreable after 7 days).
- Selection time (if incubation with a selection agent): 12 to 14 days: At the end of the expression period, aliquots of cell suspension were placed into each well of 4 x 96 well microtitre plates (384 wells at 2 x 10^4 cells/well). Plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air and wells containing clones were identified and counted.

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

NUMBER OF REPLICATIONS: 2

EVALUATION: wells containing clones were identified and counted

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
Treatment and post treatment dilution of cell cultures for the cytotoxicity Range Finder Experiment was as described for the Mutation Experiments. However, single cultures only were used and positive controls were not included. Following treatment, cells were centrifuged (200 g) washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

OTHER:
- Probable number of clones/well (P) = -ln (empty wells (without clones) /total of TW),
- Plating efficiency (PE) = P/No of cells plated per well,
- Percentage relative survival (% RS) = % RS = [PE (test)/PE (control)] x 100,
- Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6.

Evaluation criteria:

Evaluation criteria
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Experiment I

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

Statistically significant increases in mutant frequency were observed at the highest two concentrations (1600 and 1902 µg/mL). However, there was no significant linear trend.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Experiment I and II

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No significant increases in mutant frequency were observed following treatment with sodium metabisulfite at any concentration tested and there were no significant linear trends.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Experiment II

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No significant increases in mutant frequency were observed following treatment with sodium metabisulfite at any concentration tested and there were no significant linear trends.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Experiment III

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No statistically significant increases in mutant frequency were observed following treatment with sodium metabisulfite at any concentration tested and there were no significant linear trends.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed as compared to the concurrent vehicle controls (individual data not reported).

RANGE-FINDING/SCREENING STUDIES: 6 concentrations were tested in the absence and presence of S9 ranging from 59.44 to 1902 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed gave 37% and 50% RS in the absence and presence of S9, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; comparison of controls with historical means of the positive control substances.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I, concentrations, ranging from 200 to 1902 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed was 1902 µg/mL in the absence and presence of S9, which gave 43% and 65% RS, respectively.
In Experiment II, concentrations, ranging from 100 to 1902 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed was 1902 µg/mL in the absence and presence of S9, which gave 30% and 75% RS, respectively.
In Experiment III, concentrations, ranging from 200 to 1902 µg/mL, were tested in the presence of S9. 7 days after treatment, the highest concentration analysed was 1902 µg/mL, which gave 43% RS.

Conclusions:

It is concluded that sodium metabisulfite did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study in the absence and presence of a rat liver metabolic activation system (S9).

Executive summary:

Sodium metabisulfite was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells. The study consisted of a cytotoxicity Range-Finder experiment followed by three independent experiments, each conducted in the absence and/or presence of metabolic activation (S9).

The test article was formulated inpurified water. A 3 -hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, concentrations were tested in the absence and presence of S9, ranging from 59.44 to 1902 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed gave 37% and 50% relative survival (RS) in the absence and presence of S9, respectively.

In Experiment I, concentrations, ranging from 200 to 1902 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed in the absence and presence of S9 gave 43% and 65% RS, respectively.

In Experiment II, concentrations, ranging from 100 to 1902 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed in the absence and presence of S9 gave 30% and 75% RS.

In Experiment III, concentrations, ranging from 200 to 1902 µg/mL, were tested in the presence of S9. 7 days after treatment, the highest concentration analysed gave 43% RS.

Vehicle and positive control treatments were included in each Mutation Experiment.

In Experiment I in the presence of S9, statistically significant increases in mutant frequency were observed at the highest 2 concentrations (1600 and 1902 µg/mL). However, there was no significant linear trend. These data do not fulfil all the criteria for a positive result and are therefore considered equivocal.

In Experiment I in the absence of S9 and Experiment II in the absence and presence of S9 no significant increases in mutant frequency were observed following treatment with sodium metabisulfite at any concentration tested and there were no significant linear trends, indicating a negative result. In order to confirm this negative result a confirmatory experiment in the presence of S9 was performed.

In Experiment III no statistically significant increases in mutant frequency were observed following treatment with sodium metabisulfite at any concentration tested and there were no significant linear trends, indicating a negative result.

Reference 9

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

29 July 2021 - 30 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 23 October 2019

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: At room temperature, light protected (only valid for storage conditions, not for test performance), moisture protected
- Expiry Date: 08 July 2023

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- In the stock solutions the pH was determined and adjusted to physiological value (pH 7.4 ± 1 unit). For the test item, disodium disulfite, the pH was monitored before and after treatment. The pH in the stock solution was neutralised with 2 M sodium hydroxide (Mitchell et al., 1997)*.

*References:
- MITCHELL, A.D., AULETTA, A.E., CLIVE, D., KIRBY, P.E., MOORE, M.M., MYHR, B.C. (1997). The L5178Y/tk+/- mouse lymphoma specific gene and chromosomal mutation assay a phase III report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research 394, 177-303

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Lymphocytes from human peripheral blood
- Suitability of cells: Human peripheral blood lymphocytes are the most common cells used in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level (Countryman and Heddle, 1976; Evans and O’Riordan, 1975)* and are recommended in the current OECD Guideline 487 (2016).

For lymphocytes:
- Sex, age and number of blood donors: Blood samples were drawn from healthy non-smoking donors with no known illness or recent exposures to genotoxic agents (e.g. chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cells. For this study, blood was collected from a female donor (20 years old) for Experiment I and from a male donor (25 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohaemagglutinin (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The cell cycle time for lymphocytes from each donor has been determined with proliferation index by BrdU (bromodeoxyuridine) incorporation using the sister chromatid exchange test to assess the average generation time (AGT) for the donor pool. Additionally, the cytokinesis-block proliferation index provides data on suitability in the test system.
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: blood from different was not pooled
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX (L-glutamine source). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA 1.5% (v/v) as extract, 10% FBS (foetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37°C with 5.5% CO2 in humidified air.

*References:
- COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

- EVANS H.J. and O`RIORDAN M.L. (1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Research, 31, 135-148.

Cytokinesis block (if used):

Cytochalasin B (4 µg/mL)

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 protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL/mL culture medium. The final concentration of S9 mix in the treatment medium was 5% (v/v).
- quality controls of S9: sterility and metabolic capability

Test concentrations with justification for top dose:

- Experiment I (3 hours): 12.3, 21.6, 37.8, 66.2, 116, 203, 355, 621, 1086, and 1901 µg/mL
- Experiment II (28 hours): 95.5, 167, 293, 512, 666, 865, 1125, 1462, and 1901 µg/mL
The highest treatment concentration in this study, 1901 µg/mL (equivalent to 10 mM) was chosen with regard to the molecular weight (190.1 g/mol) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water

- Justification for choice of solvent/vehicle: The use of aqueous solvents is recommended by the current OECD Guideline 487.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium was 10% (v/v) corresponding to the recommendations set out in OECD guideline 487 (2016).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

PRE-EXPERIMENT ON TOXICITY
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. With regard to the molecular weight (190.1 g/mol) of the test item, 1901 µg/mL (equivalent to 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 12.3 to 1901 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of 2 to 3 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 3 hours (with and without S9 mix). The preparation interval was 28 hours after start of the exposure.
In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment both in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

CONCENTRATION SELECTION
No cytotoxic effects were observed in Experiment I after the 3-hour treatment both in the absence and presence of S9 mix. Therefore, the test item concentration of 1901 µg/mL was chosen as top treatment concentration for Experiment II (28 hours continuous exposure).

MN EXPERIMENTS
- Experiment I (Pulse exposure with and without S9 mix):
About 48 hours after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test substance concentration. The culture medium was replaced with serum-free medium containing the test substance or control. For the treatment with metabolic activation S9 mix (50 µL/mL culture medium) was added. After 3 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose x H2O, 192 mg/L Na2HPO4 x 2H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10% FBS (v/v) in the presence of Cytochalasin B (4 µg/mL) and cultured for 25 hours until preparation (Clare et al., 2006, Lorge et al., 2006)*.

- Experiment II (Continuous exposure without S9 mix):
About 48 hours after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10% FBS) containing the test item and in the presence of Cytochalasin B (4 µg/mL). The cells were exposed for 28 hours until preparation (Whitwell et al., 2019)*.

- Preparation of cells:
The cultures were harvested by centrifugation 28 hours after beginning of treatment (3+25 and 28+0 hours). The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37°C for 20 minutes. A volume of 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with 10% Giemsa solution in Weise buffer for approximately 15 to 20 minutes, mounted after drying and covered with a coverslip.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Evaluation of the slides was performed using microscopes with 40 x objectives.
- Scoring criteria
The criteria for the evaluation of micronuclei were applied according to Countryman and Heddle (1976)*. At least 1000 binucleate cells were scored manually per culture for micronuclei on independently coded slides. It is advisable to use duplicate cultures; single cultures are also acceptable provided that a total of 2000 cells are scored. Only cells containing a clearly visible cytoplasm were included in the analysis.
The criteria for the evaluation of micronuclei are as follows:
- The micronucleus has to be stained in the same way as the main nucleus and the area of the micronucleus should not extend the third part of the area of the main nucleus.
- Cells containing more than two main nuclei should not be analysed for micronuclei, as the baseline micronucleus frequency may be higher in these cells.
The micronucleus frequency was reported as % micronucleated cells.
In addition, micronuclei in mononucleate cells were recorded when these events are seen, since aneuploid acting substances are known to increase the number of micronucleated mononucleate cells.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Evaluation of the slides was performed using microscopes with 40 x objectives.
- 500 cells per culture were scored on independently coded slides for the determination of the Cytokinesis-Block Proliferation Index (CBPI). Cytotoxicity will be expressed as % cytostasis.

METHODS USED TO DETERMINE pH, OSMOLALITY AND PRECIPITATION
- The osmolarity and pH of the test item formulated in culture medium was determined by using an osmometer (Gonotec, Model Osmomat 030) or a pH meter (TW, Model Vario pH), respectively, in the pre-experiment without metabolic activation.
- Precipitation or phase separation was be evaluated at the beginning and at the end of treatment by the unaided eye.

*References:
- CLARE M.G., LORENZON G., AKHURST L.C., MARZIN D., VAN DELFT J., MONTERO R., BOTTA A., BERTENS A., CINELLI S., THYBAUD V. AND LORGE E., (2006) SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes. Mutation Res., 607, 37-60.
- LORGE E., THYBAUD V., AARDEMA M.J., OLIVER J., WAKATA A., LORENZON G. and MARZIN D. (2006) SFTG international collaborative study on in vitro micronucleus test I. General conditions and overall conclusions of the study. Mutation Res., 607, 13-36.
- WHITWELL J., SMITH R., CHIROM T., WATTERS G., HARGREAVES V., LLOYD M., PHILLIPS S. and CLEMENTS J. (2019) Inclusion of an extended treatment improves the results for the human peripheral blood lymphocyte micronucleus assay. Mutagenesis 34, 217-237.
- COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions could be useful.

However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.

Statistics:

Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

A linear regression was performed using a validated test script of “R”, to assess a possible concentration-response dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field: "Additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT ON TOXICITY
- In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment both in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
- No cytotoxic effects were observed in Experiment I after the 3-hour treatment both in the absence and presence of S9 mix. Therefore, the test item concentration of 1901 µg/mL was chosen as top treatment concentration for Experiment II (28 hours continuous exposure).

TEST-SPECIFIC CONFOUNDING FACTORS
- No relevant influence on osmolarity was observed. The pH was measured at the beginning and at the end of treatment. No relevant change on pH was observed.
- In Experiment I and II in the absence and presence of S9 mix, no precipitation of the test item in the culture medium was observed.
- In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest concentration applied. In Experiment II in the absence of S9 mix after continuous treatment, limiting cytotoxicity was observed at concentrations ranging from 512-1901 µg/mL. Clear cytotoxicity (62.9% cytostasis) was observed at the highest evaluated concentration (293 µg/mL).

MN EXPERIMENTS
Evaluation of cytogenetic damage (Experiment I: 3-hour pulse treatment):
Based on the absence of precipitates and cytotoxicity, the following test item concentration levels were evaluated in Experiment I: With and without S9 mix: 621, 1086, and 1901 µg/mL
- In Experiment I in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. The data of both experimental parts were confirmed by increasing the sample size (i.e. 2000 binucleated cells per culture instead of 1000 binucleated cells per culture). In the absence of S9 mix, however, the micronucleus frequency of the solvent control (1.15%) and of the cultures treated at a disodium disulfite concentration level of 1086 µg/mL (0.85%) exceeded the upper limit of the 95% control limit of the historical control data range (0.13 – 0.70% micronucleated cells; please refer to attached background material: “Historical control data” and “Results”). Furthermore, in the presence of S9 mix, the micronucleus frequency of the solvent control (1.15%) and of the cultures treated at a disodium disulfite concentration level of 1901 µg/mL (1.05%) exceeded the upper limit of the 95% control limit of the historical control data range (0.00 – 0.84%. In the absence and presence of S9 mix, the micronucleus frequencies observed were not statistically different from the corresponding solvent control values and no concentration-response dependency was observed.
- The historical control data of the current protocol are based on a rather small number of experiments (in line with the minimum requirement of OECD Guideline 487), therefore, deviations from historical control data are not unexpected. The data of the former test design (including a recovery period and longer exposure periods; please refer to attached background material: “Historical control data”) show, that the expected variability of the micronucleus frequency is actually higher than the variability observed so far. The min-max range of the historical control data from the former test design indicated values of up to 1.25% micronucleated cells for the solvent controls of all three treatment schedules (≥ 125 studies per treatment schedule collected in 2019 and 2020; please refer to attached background material: “Historical control data”). Therefore, the micronucleus frequencies of the solvent controls in the absence and presence of S9 mix are clearly within the expected range of the biological variation of this test system. Even more, all values of micronucleated cells after treatment with the test item undercut the values of the respective solvent controls.

Evaluation of cytogenetic damage (Experiment II: 28-hour continuous treatment)
- Based on the presence of limiting cytotoxicity (55 ± 5%), the following test item concentration levels were evaluated in Experiment II: Without S9 mix: 95.5, 167, and 293 µg/mL
- In Experiment II in the absence of S9 mix after continuous treatment, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. The value of 0.75% micronucleated cells after treatment with 95.5 µg/mL of the test item was statistically significantly increased compared to the respective solvent control value (0.25% micronucleated cells; please refer to attached background material: “Results”). In this experimental part, the solvent control value was at a low level means at the lower end of the 95% control limit of the historical control data (0.00 – 1.22% micronucleated cells; please refer to attached background material: “Historical control data”). The statistically significant difference in the micronucleus frequency between the treatment at 95.5 µg/mL and the corresponding solvent control was due to this low level reference value. However, the statistically significant value observed at a disodium disulfite concentration of 95.5 µg/mL is clearly within the 95% control limit of the historical control data range (0.00 – 1.22% micronucleated cells) and no concentration-response dependency was observed. Thus, this finding has to be considered as biologically irrelevant.

Taken together, the outcome of the current study is clearly negative.

ASSAY VALIDITY
- Vinblastine (aneugen), mitomycin C (clastogen active without metabolic activation), and cyclophosphamide (clastogen requiring metabolic activation) were used as reference mutagens. They induced distinct and statistically significant increases in the proportion of cells with micronuclei. Thus, the activity of the metabolic activation system and the sensitivity of the test were demonstrated.

All acceptance criteria were considered met and the study was accepted as valid.

Conclusions:

In Experiment I (3-hour pulse treatment) in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest concentration applied. In Experiment II in the absence of S9 mix after continuous treatment (28 hours), limiting cytotoxicity was observed at concentrations ranging from 512-1901 µg/mL. Clear cytotoxicity (62.9% cytostasis) was observed at the highest concentration evaluated (293 µg/mL). In Experiment I and II in the absence and presence of S9 mix, no precipitation of the test item in the culture medium was observed. Disodium disulfite, tested up to the recommended maximum concentration (Experiment I: 3-hour treatment) or cytotoxic concentrations (Experiment II: 28-hour treatment), did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2016).

Therefore, disodium disulfite is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the maximum concentration recommended or to cytotoxic concentrations.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

15 July 2021 - 09 August 2021 (Experimental completion; Only draft report available. Data will be updated upon availability of the final report.)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Only draft report available. Data will be updated upon availability of the final report.

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Expiry Date: 05.07.2022
- Storage Conditions: At room temperature, moisture protected

Target gene:

hisD (TA 98), hisG (TA 100, TA 1535), hisC (TA 1537), and trpE (WP2 uvr A pKM101)

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 and IIa: 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).

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.

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.
Since the positive control in strain WP2 uvrA (pKM101) in the presence of S9 mix was invalid, this part of experiment II was repeated as a pre-incubation assay with the same concentrations as experiment II (reported as experiment IIa).
The following concentrations were tested in experiment II and IIa: 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.

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
- Since the positive control in strain WP2 uvrA (pKM101) in the presence of S9 mix was invalid, this part of experiment II was repeated as a pre-incubation assay with the same concentrations as experiment II. The repeated part is reported as experiment IIa.
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sodium thiosulfate 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.

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

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2001-04-27 until 2001-05-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

Target gene:

TA98: hisD3052
TA1535 & TA100: hisG46
TA1537: hisC3076
E. coli WP2 uvrA: trp-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

75, 200, 600, 1800 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: A solubility test was conducted to select the vehicle.

Untreated negative controls:

yes

Remarks:

Sterility control: the highest dose level was plated on selective agar. The plates were incubated under the same conditions as the test plates.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene;

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: tested in triplicate

EVALUATION: Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter er entirely by hand unless the plate exhibited toxicity.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn):
The initial toxicity-mutation assay was used to establish the dose range over which the test article would be assayed and to provide a preliminary mutagenicity evaluation. Vehicle controls, positive controls and 8 dose levels (2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 µg/plate) of the test item were plated, two plates per dose, with overnight cultures of TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA on selective minimal agar in the presence and absence of metabolic activation system.

Evaluation criteria:

For the test item to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA 1535 and TA 1537 were judged positive if the increase in the mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA 98, TA 100 and WP2 uvrA were judged positive if the increase in the mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.

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:

not examined

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:

not examined

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:

not examined

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No positive responses were observed with any of the tester strains at any tested dose level.
Neither precipitation nor appreciable toxicity was observed.

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Water was selected as the solvent of choice based on compatibility with the target cells and solubility of the test article. The test article was soluble and clear in water at approximately 50 mg/mL, the maximum concentration tested.
- Precipitation: Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

RANGE-FINDING/SCREENING STUDIES: In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg/plate. Neither precipitation nor appreciable toxocity was observed. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical negative and positive control values (1998-2000) are available.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No further details.

Conclusions:

Under the conditions of this study, the test item Ammonium thiosulfate was concluded to be negative in the Bacterial Reverse Mutation Assay.

Executive summary:

The test item, Ammonium thiosulfate, was tested in the Bacterial Reverse Mutation Assay using S. Typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli strain WP2 uvrA in the presence and absence of metabolic activation system. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay was used to establish the dose-range for the mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg/plate. Dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 µg/plate. In the initial toxicity-mutation assay, no positive response was observed. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity-mutation assay, the maximum dose plated in the mutagenicity assay was 5000 µg/plate. In the confirmatory mutagenicity assay, no positive response was observed.

Reference 12

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2010-07-21 to 2010-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15-25°C in the dark

Target gene:

hprt

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media containing 2.5 µg/mL Amphotericin B, 100 µg/mL Streptomycin, 100 units/mL Penicillin, 0.5 µg/mL Pluronic (ecxept for RPMI 20) and 0%, 10% or 20% (v/v) heat inactivatet horse serum for RPMI A, RPMI 10 and RPMI 20, respectively.

The master stock of L5178Y tk+/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of frozen cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: yes; each batch of frozen cells was purged of mutants.
- Periodically "cleansed" against high spontaneous background: yes
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Range-finder experiment:
- with and without S9-mix: 46.31, 92.63, 185.3, 370.5, 741 and 1482 µg/mL
Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment.

Experiment I:
- with and without S9-mix: 200, 400, 600, 800, 1000, 1200, 1350 and 1482 µg/mL
Experiment II:
- with and without S9-mix: 100, 300, 600, 900, 1100, 1300 and 1482 µg/mL

Cultures selected for mutation assessment:
Experiment I:
- with and without S9-mix: 200, 400, 600, 800, 1000, 1200, 1350 and 1482 µg/mL
Experiment II:
- with and without S9-mix: 300, 600, 900, 1100, 1300 and 1482 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that ammonium thiosulfate was soluble in water for irrigation (purified water) at concentrations up to at least 34.99 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

treatment with the vehicle (purified water) diluted 10 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 4-nitroquinoline-1-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37°C
- Expression time (cells in growth medium): 7 days during which the hprt mutation would be expressed: After the incubation period, cultures were centrifuged (200 g), washed and resuspended in RPMI 10. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival (scoreable after 7 days).
- Selection time (if incubation with a selection agent): 12 to 14 days: At the end of the expression period, aliquots of cell suspension were placed into each well of 4 x 96 well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air and wells containing clones were identified and counted.

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

NUMBER OF REPLICATIONS: 2

EVALUATION: wells containing clones were identified and counted

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
Treatment and post treatment dilution of cell cultures for the cytotoxicity Range Finder Experiment was as described for the Mutation Experiments. However, single cultures only were used and positive controls were not included.
Following treatment, cells were centrifuged (200 g) washed with tissue culture medium and resuspended. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

OTHER:
- Probable number of clones/well (P) = -ln (empty wells (without clones) /total of TW),
- Plating efficiency (PE) = P/No of cells plated per well,
- Percentage relative survival (% RS) = % RS = [PE (test)/PE (control)] x 100,
- Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in mutant frequency were observed following treatment with ammonium thiosulfate at any concentration tested and there were no significant linear trends.

TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed (1482 µg/mL) as compared to the concurrent vehicle controls (individual data not reported).

RANGE-FINDING/SCREENING STUDIES: 6 concentrations were tested in the absence and presence of S9 ranging from 46.31 to 1482 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed was 1482 µg/mL, which gave 103% and 65% RS in the absence and presence of S9, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; comparison of controls with historical means of the positive control substances.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I, 8 concentrations, ranging from 200 to 1482 g/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed gave 107% and 95% RS in the absence and presence of S9 respectively.
In Experiment II, 7 concentrations, ranging from 100 to 1482 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed gave 68% and 107% RS, respectively.

Conclusions:

It is concluded that ammonium thiosulfate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study in the absence and presence of a rat liver metabolic activation system (S9).

Executive summary:

Ammonium thiosulfate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells. The study consisted of a cytotoxicity Range-Finder experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9).

The test article was formulated in water for irrigation (purified water). A 3 -hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9, ranging from 46.31 to 1482 mg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed was 1482 mg/mL, which gave 103% and 65% relative survival (RS) in the absence and presence of S‑9, respectively.

In Experiment I, concentrations, ranging from 200 to 1482 mg/mL, were tested in the absence and presence of S9. 7 days after treatment all concentrations in the absence and presence of S9 were selected to determine viability and 6TG resistance. The highest concentration analysed was 1482 mg/mL, which gave 107% and 95% RS in the absence and presence of S9 respectively.

In Experiment II, concentrations, ranging from 100 to 1482 mg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 1482 mg/mL, which gave 68% and 107% RS in the absence and presence of S9, respectively.

Vehicle and positive control treatments were included in each Mutation Experiment.

In Experiments I and II no statistically significant increases in mutant frequency were observed following treatment with ammonium thiosulfate at any concentration tested in the absence and presence of S9 and there were no significant linear trends.

Reference 13

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

30 August 2021 - 23 February 2022 (Experimental completion; Only draft report available. Data will be updated upon availability of the final report.)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Only draft report available. Data will be updated upon availability of the final report.

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 23 October 2019

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Expiry Date: July 2022
- Storage Conditions: At room temperature, moisture protected

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.

Target gene:

not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Lymphocytes from human peripheral blood
- Suitability of cells: Human peripheral blood lymphocytes are the most common cells used in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level (Countryman and Heddle, 1976; Evans and O’Riordan, 1975)* and are recommended in the current OECD Guideline 487 (2016).

For lymphocytes:
- Sex, age and number of blood donors: Blood samples were drawn from healthy non-smoking donors with no known illness or recent exposures to genotoxic agents (e.g. chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cells. For this study, blood was collected from a female donor (33 years old) for Experiment I and from a male donor (25 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohaemagglutinin (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The cell cycle time for lymphocytes from each donor has been determined by BrdU (bromodeoxyuridine) incorporation to assess the average generation time (AGT) for the donor pool. Additionally, the cytokinesis-block proliferation index provides data on suitability in the test system.
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: blood from different was not pooled
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX (L-glutamine source). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA 1.5% (v/v) as extract, 10% FBS (foetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37°C with 5.5% CO2 in humidified air.

*References:
- COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

- EVANS H.J. and O`RIORDAN M.L. (1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Research, 31, 135-148.

Cytokinesis block (if used):

Cytochalasin B (4 µg/mL)

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 protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL/mL culture medium. The final concentration of S9 mix in the treatment medium was 5% (v/v).
- quality controls of S9: sterility and metabolic capability

Test concentrations with justification for top dose:

- Experiment I (3 hours): 10.3, 18.0, 31.5, 55.0, 96.3, 169, 295, 516, 903, and 1581 µg/mL
- Experiment II (28 hours): 47.0, 82.3, 144, 252, 328, 426, 554, 720, 936, 1216, and 1581 µg/mL
The highest concentration applied in this study (1581 µg/mL of the test item, equivalent to 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487 (2016).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water

- Justification for choice of solvent/vehicle: The use of aqueous solvents is recommended by the current OECD Guideline 487.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium was 10% (v/v) corresponding to the recommendations set out in OECD guideline 487 (2016).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

PRE-EXPERIMENT ON TOXICITY
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. With regard to the molecular weight (158.11 g/mol) of the test item, 1581 µg/mL (equivalent to 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 10.3 to 1581 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of 2 to 3 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 3 hours (with and without S9 mix). The preparation interval was 28 hours after start of the exposure.
In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment both in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

CONCENTRATION SELECTION
No cytotoxic effects were observed in Experiment I after the 3-hour treatment both in the absence and presence of S9 mix. Therefore, the test item concentration of 1581 µg/mL was chosen as top treatment concentration for Experiment II (28 hours continuous exposure).

MN EXPERIMENTS
- Experiment I (Pulse exposure with and without S9 mix):
About 48 hours after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test substance concentration. The culture medium was replaced with serum-free medium containing the test substance or control. For the treatment with metabolic activation S9 mix (50 µL/mL culture medium) was added. After 3 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose x H2O, 192 mg/L Na2HPO4 x 2H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10% FBS (v/v) in the presence of Cytochalasin B (4 µg/mL) and cultured for 25 hours until preparation (Clare et al., 2006, Lorge et al., 2006)*.

- Experiment II (Continuous exposure without S9 mix):
About 48 hours after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10% FBS) containing the test item and in the presence of Cytochalasin B (4 µg/mL). The cells were exposed for 28 hours until preparation (Whitwell et al., 2019)*.

- Preparation of cells:
The cultures were harvested by centrifugation 28 hours after beginning of treatment (3+25 and 28+0 hours). The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37°C for 20 minutes. A volume of 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with 10% Giemsa solution in Weise buffer for approximately 15 to 20 minutes, mounted after drying and covered with a coverslip.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Evaluation of the slides was performed using microscopes with 40 x objectives.
- Scoring criteria
The criteria for the evaluation of micronuclei were applied according to Countryman and Heddle (1976)*. At least 1000 binucleate cells were scored manually per culture for micronuclei on independently coded slides. It is advisable to use duplicate cultures; single cultures are also acceptable provided that a total of 2000 cells are scored. Only cells containing a clearly visible cytoplasm were included in the analysis.
The criteria for the evaluation of micronuclei are as follows:
- The micronucleus has to be stained in the same way as the main nucleus and the area of the micronucleus should not extend the third part of the area of the main nucleus.
- Cells containing more than two main nuclei should not be analysed for micronuclei, as the baseline micronucleus frequency may be higher in these cells.
The micronucleus frequency was reported as % micronucleated cells.
In addition, micronuclei in mononucleate cells were recorded when these events are seen, since aneuploid acting substances are known to increase the number of micronucleated mononucleate cells.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Evaluation of the slides was performed using microscopes with 40 x objectives.
- 500 cells per culture were scored on independently coded slides for the determination of the Cytokinesis-Block Proliferation Index (CBPI). Cytotoxicity will be expressed as % cytostasis.
METHODS USED TO DETERMINE pH, OSMOLALITY AND PRECIPITATION
- The osmolarity and pH of the test item formulated in culture medium was determined by using an osmometer (Gonotec, Model Osmomat 030) or a pH meter (TW, Model Vario pH), respectively, in the pre-experiment without metabolic activation.
- Precipitation or phase separation was be evaluated at the beginning and at the end of treatment by the unaided eye.

*References:
- CLARE M.G., LORENZON G., AKHURST L.C., MARZIN D., VAN DELFT J., MONTERO R., BOTTA A., BERTENS A., CINELLI S., THYBAUD V. AND LORGE E., (2006) SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes. Mutation Res., 607, 37-60.
- LORGE E., THYBAUD V., AARDEMA M.J., OLIVER J., WAKATA A., LORENZON G. and MARZIN D. (2006) SFTG international collaborative study on in vitro micronucleus test I. General conditions and overall conclusions of the study. Mutation Res., 607, 13-36.
- WHITWELL J., SMITH R., CHIROM T., WATTERS G., HARGREAVES V., LLOYD M., PHILLIPS S. and CLEMENTS J. (2019) Inclusion of an extended treatment improves the results for the human peripheral blood lymphocyte micronucleus assay. Mutagenesis 34, 217-237.
- COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions could be useful.

However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.

Statistics:

Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

A linear regression was performed using a validated test script of “R”, to assess a possible concentration-response dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes: human peripheral blood

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT ON TOXICITY
- In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment both in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
- No cytotoxic effects were observed in Experiment I after the 3-hour treatment in the absence and presence of S9 mix. Therefore, the test item concentration of 1581 µg/mL was chosen as top treatment concentration for Experiment II.

TEST-SPECIFIC CONFOUNDING FACTORS
- No relevant influence on osmolarity was observed. The pH was measured at the beginning and at the end of treatment. No relevant change on pH was observed.
- In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, no precipitation of the test item in the culture medium was observed.
- In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

MN EXPERIMENTS
Evaluation of cytogenetic damage (Experiment I: 3-hour pulse treatment):
- Based on the absence of precipitates and cytotoxicity, the following test item concentration levels were evaluated in Experiment I (cf. section 9, Table 2): With and without S9 mix: 516, 903, and 1581 µg/mL
- In Experiment I in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item.
- There was also no concentration related increase in micronucleus formation frequency, as judged by an appropriate trend test.

Evaluation of cytogenetic damage (Experiment II: 28-hour continuous treatment)
- Based on the absence of precipitates and cytotoxicity, the following test item concentration levels were evaluated in Experiment II (cf. section 9, Table 2): Without S9 mix: 720, 936, 1216, and 1581 µg/mL
- In Experiment II in the absence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item.
- A concentration related trend, however, was observed in Experiment II in the absence of S9 mix after continuous treatment. Since all values are clearly within the 95% control limit of the historical control data and none of the values were statistically significantly increased, when compared to the solvent control, this finding can be considered as biologically irrelevant.

In summary, the outcome of the study is clearly negative.

ASSAY VALIDITY
- Vinblastine (aneugen), mitomycin C (clastogen, active without metabolic activation), and cyclophosphamide (clastogen requiring metabolic activation) were used as reference mutagens. They induced distinct statistically significant increases in the proportion of cells with micronuclei. Thus, the activity of the metabolic activation system and the sensitivity of the test system was demonstrated. Solvent control cultures were included in all experiments.
- The micronucleus frequencies observed in the solvent control cultures were well within the 95% historical control limit.

All acceptance criteria were considered met and the study was accepted as valid.

Conclusions:

No relevant toxicity (55±5% cytostasis) or precipitation of the test item was found in the main experiments (Experiment I: 3-hour pulse treatment; Experiment II: 28-hour continuous treatment) both with and without metabolic activation. Sodium thiosulfate, tested up to the recommended maximum concentration did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2016).

Therefore, sodium thiosulfate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the maximum concentration recommended or to cytotoxic concentrations.

Reference 14

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2001-04-26 until 2001-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from exposure to light

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. This cell line has an average call cycle time of 10-14 hours with a modal chromosome number of 20.
- Type and identity of media: complete medium (McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS), 100 units penicillin and 100 µg streptomycin/mL, and 2 mM L-glutamine)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; The freeze lot of cells was tested using the Höchst staining procedure and found to be free of mycoplasma contamination.
- Periodically checked for karyotype stability: yes; In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20.
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

185, 370, 740 and 1480 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: A solubility test was perfomed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16-24 hours at 37°C in a humidified atmosphere of 5% CO2.
- Exposure duration: a) 4 hours and 20 hours (continuously treatment) without metabolic activation at 37°C in a humidified atmosphere of 5% CO2 and b) 4 hours at 37°C in a humidified atmosphere of 5% CO2 with metabolic activation.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours after study initiation: Two hours after addition of Colcemid, metaphase cells were harvested by trypsinisation. To prepare slides, fixed cells (fiexed in Carnoy's fixative) were centrifuged, the supernatant was aspirated and 1 mL fresh fixative was added. After removal of the fixative by centrifugation, a sufficient amount of cell suspension was dropped onto a glass slide and was air dried.

SPINDLE INHIBITOR (cytogenetic assays): Two hours pior to the scheduled cell harvest, Colcemid was added at a final concentration of 0.1 µg/mL and the flasks returned to the incubator until cell collection.
STAIN (for cytogenetic assays): Dried slides were stained with 5% Giemsa, air dried and permanently mounted.

NUMBER OF REPLICATIONS: duplicate flasks

NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth:
The preliminary toxicity assay was performed for the purpose of selecting dose levels for the chromosome aberration (CA) assay. CHO cells were seeded for each treatment condition at approximately 5 x 10^5 cells/25 cm² flask and were incubated at 37°C in a humidified atmosphere of 5% Co2 in air for 16-24 hours. Treatment was carried out for the non-activated study and for the activated study. The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9. At completion of the 4 hours exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline, refed with 5 mL complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. At 20 hours after initiation of treatment the cells were harvested by trypsinisation and counted. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.

OTHER: mitotic index:
To ensure, that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group.

Evaluation criteria:

All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test item was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p<0.05). However, values that are statistically significant but do not exceed the range of historic solvent controls may be judged as not biologically significant. Test item not demonstrating a statistically significant increase in aberrations will be concluded to be negative. Negative results with metabolic activation may need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not necessary, justification will be provided.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test item dose level, the Cochran-Armitage test was used to measure dose-responsivness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

4 hours treatment

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

The percentage of cells with structural and numeric aberrations in the test item treated groups was not significantly increased above that of the solvent control.

Cytotoxicity / choice of top concentrations:

other: absence of substantial toxicity: Toxicity in CHO cells was 5% at 1480 µg/mL. The mitotic index at the highest dose level evaluated was 4% reduced relative to the solvent control.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

20 hours treatment

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

The percentage of cells with aberrations was significantly increased at dose level 1480 µg/mL. However, the percentage was within the historical control range; therefore it is not considered to be biologically significant.

Cytotoxicity / choice of top concentrations:

other: absence of substantial toxicity: Toxicity in CHO cells was 7% at 1480 µg/mL. The mitotic index at the highest dose level evaluated was 33% reduced relative to the solvent control.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

4 hours treatment

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

The percentage of cells with structural and numeric aberrations in the test item treated groups was not significantly increased above that of the solvent control.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxicity was observed when treated for 4 hours

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test item in treatment medium was approximately 7.5.
- Effects of osmolality: The osmolarity in treatment medium of the highest concentration tested, 1480 µg/mL, was 280 mmol/kg. The osmolarity of the solvent in treatment medium was 270 mmol/kg.
- Water solubility: Water was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in water at a concentration of 50 mg/mL, the maximum concentration tested.

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity assay, the maximum dose tested was 1480 µg/mL (10mM). The test item was soluble in treatment medium at all dose levels tested. Substantial toxicity, i.e., at least 50% cell growth inhibition (relative to the solvent control), was not observed at any dose level in the 4 and 20 hour non-activated exposure groups or in the S9 activated 4 hour exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 185 to 1480 µg/mL for both the non-activated exposure groups and the S9 activated exposure group.
In the absence of both test item precipitation in the treatment medium and at least 50% toxicity, the highest dose level evaluated was 10 mM. The next two lower dose levels were included in the evaluation.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control values for structural and numerical aberrations (1997-1999) in non-activated test system and activated systems with solvent and positive controls are available.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No further details.

Conclusions:

Based on the findings of this study, Ammonium thiosulfate was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

Executive summary:

The test item, Ammonium thiosulfate, was tested in the chromosome aberration (CA) assay using Chinese hamster ovary (CHO) cells in both the absence and presence of metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The CA assay was used to evaluate the clastogenic potential of the test article. Based on the findings, the doses chosen for the chromosome aberration assay ranged from 185 to 1480 µg/mL for both the non-activated exposure groups and the S9 activated exposure group.

In the CA assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. In the absence of both substantial toxicity and test item precipitation in the treatment medium, 1480 µg/mL was chosen as the high dose for microscopic analysis for CA in all hervests. The next two lower doses were also included in the analysis.

Based on the findings of this study, Ammonium thiosulfate was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

2008-06-24 to 2008-11-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2000-05-19

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2005-10-21

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (under N2; protected against humidity)

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The animals were selected according to the recommendations of the OECD and EU or on the basis of results published so far (Boller and Schmid (1970) and Heddle (1973))*. Moreover, there has been up to now most experience with or most data for NMRI mice.

*References:
- BOLLER, K.; SCHMID, W. Chemische Mutagenese beim Säuger. Das Knochenmark des chinesischen Hamsters als in-vivo-Testsystem. Hämatologische Befunde nach Behandlung mit Trenimon. Humangenetik, 11, 35 - 54 (1970)
- HEDDLE, J. A. A rapid in vivo test of chromosomal damage. Mut. Res., 18, 187 - 190 (1973)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks (information from the breeder)
- Assigned to test groups randomly: yes, under following basis: The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program.
- Weight at study initiation: mean 29.9 g
- Housing: individually in Makrolon cages, type M I, with Type Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): Drinking water from bottles was available ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

subcutaneous

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, purified water was used as vehicle.
- Concentration of test material in vehicle: 25, 50, 100 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.
- The stability of the test substance at room temperature in the vehicle purified water was determined analytically. For the determination of the test substance concentrations in the vehicle, 6 samples of each dose were taken from the test substance preparations. These were kept at room temperature until the treatment of the last animal (approximately 1 hour) and then were kept deep-frozen. The determination of the concentrations in the vehicle was carried out by means of iodometric titration.

Duration of treatment / exposure:

single administration

Frequency of treatment:

once

Post exposure period:

24 and 48 hours

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 mice

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (20 mg/kg bw) and vincristine sulfate (0.15 mg/kg bw)

Tissues and cell types examined:

polychromatic erythrocytes of bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute subcutan toxicity, deaths were observed down to 1500 mg/kg body weight. At 1000 mg/kg body weight all animals (male and female) survived showing clinical signs. The clinical sign only observed was making rough of the administration area. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 1000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
The two femora of the animals sacrificed by cervical dislocation was prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was preheated up to 37°C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 uL fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10,000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value.
A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
[d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4). Slides were coded before microscopic analysis. Since the absolute values shown were rounded, but further calculation was based on
unrounded values, there may be deviations in the relative values given.

Evaluation criteria:

A finding is considered positive if the following criteria are met:
- Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows: * p < 0.05, ** p < 0.01. However, both biological relevance and statistical significance were considered together.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500 mg/kg bw
- Clinical signs of toxicity in test animals: deaths were observed down to 1500 mg/kg body weight. At 1000 mg/kg body weight all animals (male and female) survived showing clinical signs.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.

Results:

Test group	Interval: 24 hours				Interval: 48 hours
	Polychromatic erythrocytes	Normocytes / total amount polychromatic erythrocytes	Cells with micronuclei (%)		Polychromatic erythrocytes	Normocytes / total amount polychromatic erythrocytes	Cells with micronuclei (%)
Dose (mg/kg bw)			polychromatic	normochromatic			polychromatic	normochromatic
vehicle control	10000	4767	0.9	1.5	10000	4834	0.9	0.4
250	10000	5469	0.8	0.5
500	10000	5309	1.5	0.9
1000	10000	4211	1.4	1.4	10000	7289	1.2	0.5
20, cyclophosphamide	5000	3833	10.3	1.8
0.15, vincristine	5000	6097	48.4	1.3

Conclusions:

The substance Natriumsulfit wasserfrei food grade (E 221) / (PRD 30042389) was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in purified water, was administered once subcutan to male animals at dose levels of 250 mg/kg, 500 mg/kg and 1 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case.
Purified water was used as the vehicle control, administered by the same route. Cyclophosphamide and vincristine sulfate were used as positive control substances.
The animals were sacrificed and the bone marrow of the 2 femora was prepared 24 and 48 hours after administration in the highest dose group of 1 000 mg/kg body weight and in the vehicle controls. In the test groups of 500 mg/kg and 250 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.
According to the results of the present study, the single subcutaneous administration of Natriumsulfit wasserfrei food grade (E 221) / (PRD 30042389) did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.
Under the experimental conditions chosen here, the test substance Natriumsulfit wasserfrei food grade (E 221) / (PRD 30042389) does not have any chromosome damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Read-across concept for sulfites, hydrogensulfites, metabisulfites, dithionites and thiosulfates:

A comprehensive read-across concept has been developed for sulfites, hydrogensulfites and metabisulfites, based on the pH-dependant equilibrium in aqueous solutions which is summarised in the following equations:^{[1], [2]}

SO₂+ H₂O <->`H₂SO₃´        H₂SO₃<-> H⁺+ HSO₃^-<-> 2H⁺+SO₃^2-   2HSO₃^-<->H₂O +S₂O₅^2-

In consequence, under most physiological circumstances, sulfite and hydrogensulfite anions will be present in almost equimolar quantities, irrespective of their origin either as sulfites, hydrogensulfites and metabisulfites. Unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites is therefore considered justified. Since the nature of the cations such as sodium, potassium and ammonium is not assumed to contribute substantially to differences in toxicity and solubility (all compounds are very soluble in water), only the chemical and biological properties of the anionic sulfite moiety are considered as relevant determinants.

Further, it is well established that sodium dithionite is unstable in water, thereby disproportionating to form sodium hydrogen sulfite and sodium thiosulfate (equation II)^[1], so that this substance is also considered to be covered by the read-across concept described above. Since the substance is not stable enough under physiological conditions to fulfil the requirements of most HH test guidelines, instead the products of decomposition have to be considered:

      2 S₂O₄^2-+ H₂O→2HSO₃^-+ S₂O₃^2-

 ^[1]Hollemann Wiberg, Lehrbuch der Anorganischen Chemie, 101.Auflage

^[2]Handbook of Chemistry and Physics, Ed. Lide, DR, 88thedition, CRC Press

Like all “sulfite substances”, the dithionite anion has strongly reducing properties and decomposes/disproportionates rapidly in aqueous media (especially under acidic anaerobic conditions) to form sulfite and thiosulfate (S₂O₃^2-); under aerobic conditions, it will additionally be oxidised to finally form sulfate as the final oxiodation/decomposition product. According to Hofmann and Rüdorff (1969) and Holleman and Wiberg (1995), this process can roughly be described by the following equations (under aerobic conditions and at low concentrations, reaction (2) is favoured:

 

2 Na₂S₂O₄+ H₂O→Na₂S₂O₃+ 2 NaHSO₃(anaerobic conditions) (1), as described above

 

Na₂S₂O₄+ O₂+ H₂O→NaHSO₄+ NaHSO₃(aerobic conditions) (2)

 

According to the literature overview of Münchow (1992) the following principal decomposition patterns can be described for dithionite in relation to pH ranges at temperatures between 0°C and 32°C for 0.0025 molar solutions:

 

•strongly alkaline medium: 3 S₂O₄^2-+ 6 OH^-→5 SO₃^2-+ S^2-+ 3H₂O

•weakly alkaline to weakly acidic medium: 2 S₂O₄^2-+ H₂O→2 HSO₃^-+ S₂O₃^2-

•acidic medium: 2 H₂S₂O₄→3 SO₂+ S + 2 H₂O

•strongly acidic medium: 3 H₂S₂O₄→5 SO₂+ H₂S + 2 H₂O  

With limitations, this read-across concept also extends to the substance class of thiosulfates: although the thiosulfates are also well known to disproportionate in aqueous solution to form polythionic acids and SO₂(HSO₃^-), this requires somewhat more acidic conditions. Therefore, read-across to sulfites is primarily restricted to corresponding physiological conditions such as oral administration where the gastric passage with the strongly acidic conditions in the stomach will facilitate the chemical disproportionation described below:

      HS₂O₃^-+ H₂S₂O₃→HS₃O₃^-+ SO₂+ H₂O

 

Introductory remarks on genetic toxicity

According to the ECHA Guidance “Guidance on information requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance” (Version 2.4, February 2014), existing data shall be qualified by its (i) adequacy, (ii) reliability and (iii) relevance. Especially the reliability of data shall be considered when assessing its usefulness for hazard/risk assessment purposes. The guidance suggested the rating according to Klimisch. According to this rating scheme, existing references are rated differently in case of (i) the use of different test guidelines (compared with today's standards) (ii) the inability to characterise the test substance properly (in terms of purity, physical characteristics, etc.) (iii) the use of crude techniques/procedures which have since become refined.

A number of OECD Guidelines on genetic toxicology testing have recently been withdrawn from the portfolio of OECD test guidelines, such as:

477: Sex-Linked Recessive Lethal test in Drosophila melanogaster

478: Genetic Toxicology: Rodent dominant Lethal Test

479: in vitro Sister Chromatid Exchange in Mammalian Cells

480: Saccharomyces cerevisiae, Gene mutation assay

482: DNA Damage and Repair, Unscheduled DNA synthesis in Mammalian Cells.

According to the qualification criteria for existing data stated above, tests conducted according to these guidelines have a minor contribution to the overall assessment, since newer and more up-to-date test guidelines exist. Existing tests conducted according to the more up-to-date guidelines were therefore rated with a higher reliability and were subsequently considered with a higher contribution to the overall assessment of genetic toxicity of the category substance. Further, tests which do not directly address the endpoint genic toxicity were considered of minor relevance, such as DNA damage in bacteria tested according to the rec assay. This assay only measures differential killing and is not a mutation assay.

References on effects following inhalation of sulfur dioxide were not considered in this assessment (e.g. Meng Z. et al. 2005; Meng Z, Zhang L. 1990a and b[1]). Due to substantial local effects in the respiratory tract in animals and humans upon exposure with this severe lung irritant, subsequent physiological responses such as altered haematopoiesis may be triggered with a potential influence on levels of micronuclei formation.

For some references, only short abstracts are available. Due to the limited information content, these references were not considered for hazard assessment purposes and rated with reliability 4 “not assignable” (Valencia, 1973; Gregory, 1981; Popescu, 1984; Tsutsui, 1984).

 

In vitro genetic toxicity tests

Gene mutation in bacteria

Sodium dithionite

Chang, S. (2022) investigated the gene mutation potential of sodium dithionite in a bacterial reverse mutation assay according to OECD 471 (2020) under GLP. The Salmonella typhimurium tester strains TA 98, TA 100, TA 1535, and TA 1537 as well as Escherichia coli WP2 uvrA (pKM101) were exposed to sodium dithionite concentrations of 3 (only plate incorporation), 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate following both the plate incorporation and the pre-incubation protocol. Both experiments were performed without and with metabolic activation (rat liver S9). 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. All validity criteria were met. The study was fully compliant with OECD 471 (2020). The study is considered to be reliable without restrictions [RL-1].

The substance Hydrosulfite konz. BASF (disodium dithionite) was tested for mutagenicity in a bacterial reverse mutation assay (Engelhardt, 1989) with following Salmonella typhimurium strains: TA 1535, TA 100, TA 1537 and TA 98. The cytotoxicity was determined be investigation if various doses have an effect of the background growth. The strains were tested in the standard plate and preincubation test, respectively, both with and without metabolic activation at concentrations ranging from 20 -5000 µg/plate. No bacteriotoxic effect was observed. An increase in the number of his+ revertants was not observed both in the standard plate and in the preincubation test either without or after the addition of metabolic activation. According to the results of the present study, the test substance Hydrosulfite konz. BASF is not mutagenic in the Ames test under the experimental conditions chosen here. The study was performed according to OECD Guideline 471 (1983), however with the following restriction; neither E. coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents. The study is considered to be reliable with restrictions [RL-2].

 

Sulfites

The gene mutation potential of sodium sulfite was evaluated in a bacterial reverse mutation assay according to OECD 471 (2020) and under GLP (Chang, 2022). Salmonella typhimurium tester strains TA 98, TA 100, TA 1535, and TA 1537 as well as Escherichia coli WP2 uvrA (pKM101) were exposed to at least six different concentration levels of sodium sulfite up to the recommended maximum concentration of 5000 µg/plate. The bacteria cultures were tested following the plate incorporation and pre-incubation protocol both in presence and absence of metabolic activation. 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 sulfite, 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). The study is considered to be reliable without restrictions [RL-1].

Chang (2022) investigated the mutagenic potential of disodium disulfite in a bacterial reverse mutation assay according to OECD TG 471 (2020) and under GLP. The test was performed in Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537 as well as E. coli WP2 uvrA (pKM101) in two independent experiments using triplicate cultures. The cell cultures were exposed to the test material, both in absence and presence of metabolic activation, up to the recommended maximum concentration of 5000 µg/plate. The two independent experiments were performed according to the plate incorporation and pre-incubation procedure. 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. Disodium disulfite, 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). The study is considered to be reliable without restrictions [RL-1].

 

Sodium metabisulfite (aka disodium disulfite) was tested unequivocally negative independently by two authors in a bacterial reverse mutation assay (Simmon, 1978). S. typhimurium tester strains TA 98, TA 100, TA 1535, TA 1537, TA1538 and E. coli WP2 uvrA were exposed to doses up to 10000 µg/plate (up to 10 doses) in the presence and absence of a metabolic activation system. Tests were comparable to OECD 471 (1983) using the plate incorporation method. Cytotoxicity characterised by reduced background lawn was seen in strains TA 100, TA 1535 and E. coli WP2 at the two highest and the highest dose, respectively. In none of the assays performed was any significant increase of revertant colonies observed in any of the tester strains with or without metabolic activation. These references fulfil the requirements for chemicals risk assessment. Experiments and results are presented in adequate detail, thus both references are considered reliable without restrictions (a guideline was not available and GLP not compulsory at the time of study conduct). The study is considered to be reliable with restrictions [RL-2].

 

In a further testing programme, four sulfites were assessed for their potential to induce gene mutations in the bacterial reverse mutation assay (Engelhardt 1989a, b, c, d, e). The tests were conducted in accordance with OECD TG 471 (1983), as amended at the date of study conduct. S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 were exposed using the preincubation and plate incorporation method to sodium sulfite, sodium dithionite (as already reported above), potassium sulfite, disodium disulfite, and potassium metabisulfite (aka dipotassium disulfite) at doses of 0, 20, 100, 500, 2500, and 5000 µg/plate. Cells were incubated for 48 hours with and without metabolic activation system. Slight cytotoxic effects were observed with potassium sulfite for TA 100 at 5000 µg/plate and with disodium disulfite for TA 100 at 2500 and 5000 µg/plate. No increase in the number of revertants was observed for any of the test substances using the plate incorporation or preincubation protocol up to the maximum concentrations with or without metabolic activation. The studies were performed according to OECD Guideline 471 (1983), however with the following restriction; neither E. coli strain WP2 uvrA (pKM101) nor S. typhimurium strain TA102 were employed in this studies for the detection of oxidising mutagens and cross-linking agents. The study is considered to be reliable with restrictions [RL-2].

 

Ishidate (1984) also reported test results for (i) potassium metabisulfite (93.0% purity, aka dipotassium disulfite) (ii) sodium bisulfite, anhydrous (95.0% purity, aka disodium disulfite) and (iii) sodium sulfite, anhydrous (95.0% purity). S. typhimurium tester strains TA 92, TA 94, TA 98, TA 100, TA 1535, TA 1537 were exposed at doses of (i) 3 mg/plate (ii) 5 mg/plate (iii) 50 mg/plate in the presence and absence of a metabolic activation system. The tests were similar to OECD 471 (1983) using the preincubation method. Cytotoxicity characterised by reduced background lawn was assessed in a separate experiment and the maximum dose for the main experiments selected accordingly. In none of the assays performed, a significant increase of revertant colonies was observed in any of the tester strains with or without metabolic activation. The publication is a summary paper, reporting testing of a total of 242 substances. The reference fulfils the basic requirements for scientific publications used in chemicals risk assessment. Minor reporting or experimental deficiencies: maximum dose for sodium sulfite 10-fold above the recommended maximum dose, cytotoxicity not measured/reported for the main experiments, outcome on mutagenic effects only in binary format- no individual data. The studies are considered to be not reliable [RL-3].

 

Münzer, R. (1980) investigated the gene mutation potential of sodium hydrogensulfite (NaHSO3) in a bacterial reverse mutation assay without metabolic activation. Prior to the experiments, the test material was adjusted to pH 5.9 using NaHPO4. Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1538 were exposed to sodium hydrogensulfite at concentrations of 0.1, 0.5, and 1.0 M following both the plate incorporation and preincubation method. According to the authors, sodium hydrogensulfite induced no mutagenic response in the S. typhimurium tester strains. However, cytotoxicity was observed at the highest concentration test. Moreover, cytotoxicity precluded the analysis of the plate-incorporation experiment with hisG46 tester strain. An increase in the number of revertant colonies was observed only for hisG46 in a follow-up experiment. The short communication presented herein showed significant methodological and reporting deficiencies. The test material is poorly characterised, since information on the purity, CAS no., source, and physical appearance are missing. The preparation of the test material is not sufficiently described, since details on e.g. the solvent are missing. The description of the methodology lacks details. Only three concentrations were tested. Results are reported only for one strain. Individual culture data is completely missing. Positive and untreated control cultures are not included. Historical control data is missing. No information on the method used to determine cytotoxicity. The concentration selection was not in line with criteria for concentration selection, since cytotoxicity impeded evaluation of the mutagenic responses in at least some experiment. Based on these findings the reference is considered to be not reliable [RL-3].

The mutagenic potential of disodium disulfite was assessed in S. typhimurium TA 97 (Pagano, 1990). Bacterial cultures were exposed using the preincubation protocol to 80 mM (equivalent to 8.32 g/L) disodium disulfite at different pH and temperatures. In addition, various buffer additives were added to assess whether any mutation frequency is increased or reduced. No increase of the mutation frequency was observed at standard culture conditions (37°C, pH 7, without buffer additives). In the results section of the publications, the mutagenic potential of disodium disulfite is reported, whereas in the materials section, disodium disulfite is stated as test substance. The strain TA 97 is known for its genetic instability and its limited used for bacterial reverse mutation testing, being the reason for its replacement by the strain TA 97a. Based on the unclear test item used in this experiment, the experimental design and unsuitable strain, this reference is considered to be not reliable [RL-3] and is of no relevance for the chemicals hazard assessment.

 

Pagano, D.A. and Zeiger, E. (1987) assessed the mutagenic potential of sodium metabisulfite (aka disodium disulfite) in a range of seventeen S. typhimurium strains. Cells were exposed at concentrations of 0-0.64 M at varying pH values of 5.0 to 8.0, results not reported for all strains. The strains were preincubated for 30 minutes at 37°C, plated and incubated for further 48 hours at 37°C. Moreover, single experiments with TA 97 were performed with prolonged pre-incubation durations. According to the authors, sodium metabisulfite is a weak mutagen at pH 5 and 6 in S. typhimurium strains carrying the hisG46 and hisD6610 mutations, but is not mutagenic in strains with the hisC3076 or hisD3052 mutations. Authors report a weak increase of revertants at 0.1 M sodium metabisulfite in one strain in an initial experiment (results only shown in graph, thus no absolute fold-increase can be given). The study showed significant methodological and reporting deficiencies. The test material is insufficiently characterised, since information on purity and CAS No. are missing. The overall test design is difficult to follow, since it remains unclear at which culture conditions the results were obtained. Individual plate counts are not presented. Results are only given for a sub-set of strains without indications on toxicity. Historical control data is missing. Positive controls were not included. Scoring, acceptability, and evaluation criteria are not specified. Due to the confusing reporting and unsuitable test design with regard to culture conditions and drastic exposure concentrations, this publication is considered to be not reliable [RL-3].

 

Mallon, R.G. and Rossmann, T.G. (1981) evaluated the gene mutation potential of sodium hydrogensulfite in a bacterial reverse mutation assay using different E. coli strains. Escherichia coli tester strain WP2, WP2 (uvrA), WP5 (lexA), WP6 (polA), and WP10 (recA) were exposed to 0.1 M sodium hydrogensulfite for three days at 37°C. Afterwards, the number of trp+ revertants was scored. Negative control cultures were run concurrently. The revertant colony number after sodium hydrogensulfite treatment was comparable to the control values. The reference exhibits deficiencies in study design and reporting. The test material characterisation and description of the test material preparation are insufficient. Only one concentration level was tested, and thus concentration-response relationship evaluations are precluded. The assay was performed only in E. coli strain derived from E. coli WP2, whereas none of the required Salmonella strains was tested. The methodology is only poorly described. A confirmatory experiment was not performed. The type of negative control used is unclear. Due to the afore mentioned shortcomings, the reference is considered to be not reliable [RL-3].

 

Brusick (1975) examined performed a bacterial reverse mutation assay similar to OECD 471 (1997) with Compound FDA 73-43 (sodium sulfite). Salmonella typhimurium tester strains TA 1535, TA 1537, and TA 1538 were exposed to a concentration of 0.028% in the plate incorporation assay and concentrations of 2.5 and 5% in the pre-incubation assay. The experiments were performed both without and with metabolic activation with S9 mixes and tissue homogenates from different species (monkey, rat, and mice) and different tissues (liver, lung, and brain). Compound FDA 73-43, sodium sulfite, did not exhibit genetic activity. The study showed significant methodological and reporting deficiencies. Physical nature and purity of the test substance were not specified. Only three strains of bacteria were used. TA 1538 is not a standard strain included in the OECD test guideline. Historical control data is not provided. Only one concentration was tested in the plate incorporation test and two concentrations were tested in the suspension test. Only duplicate testing (plate test). The duration of incubation was four days during the plate test. Evaluation criteria are not specified. Based on the above-mentioned shortcomings, the study is considered to be not reliable [RL-3].

 

Thiosulfates

Chang (2022) performed a bacterial reverse mutation assay according to OECD TG 471 (2020) under GLP in order to investigate the gene mutation potential of sodium thiosulfate. Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537 as well as Escherichia coli WP2 uvrA (pKM101) were tested in two independent experiments using the plate incorporation and pre-incubation method. The tester strains were exposed to at least six different concentration levels with a top concentration of 5000 µg/plate in line with the criteria set out in the OECD guideline. Both experiments were with and without S9-mix. 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 thiosulfate, 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). The study is considered reliable without restrictions [RL-1].

 

Ammonium thiosulfate was tested in a bacterial reverse mutation assay (Wagner, 2001) according to OECD 471 (1997) using S. typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli strain WP2 uvrA in the presence and absence of metabolic activation system. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay was used to establish the dose-range for the mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article. In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg/plate. Dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 µg/plate. In the initial toxicity-mutation assay, no positive response was observed. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity-mutation assay, the maximum dose plated in the mutagenicity assay was 5000 µg/plate. In the confirmatory mutagenicity assay, no positive response was observed. The study is considered to be reliable without restrictions [RL-1].

 

SRI International examined an FDA compound identified as F76-020 (Mortelmans, 1979). Compound F76 -020 was tested as a coded chemical. After completion of a draft of this final report and acceptance of the draft by the FDA Project Officer, the identity of the chemical was made known to SRI International: Sodium thiosulfate pentahydrate. Sodium thiosulfate pentahydrate was tested in the Ames Salmonella/microsome assay for induction of reverse mutation in Salmonella typhimurium strains TA1535, TA1538, TA98, and TA 100 and in Escherichia coli WP2 (uvrA). Each assay was performed in the presence and in the absence of a rat liver homogenate metabolic activation system. Sodium thiosulfate pentahydrate was not toxic or mutagenic in these assays. Purity of the test substance is not stated. Not clear if enough indicator cells were used. No GLP, because at the time of the study conduct, GLP was not compulsory. The study is considered to be reliable with restrictions [RL-2].

 

Lawlor (1989) performed a bacterial reverse mutation assay according to EPA OPP 84-2 under GLP with ammonium thiosulfate. Salmonella typhimurium test strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 were exposed to 333, 667, 1000, 3330, 6670 and 10000 µg/plate with and without metabolic activation flowing the plate incorporation protocol. All data were acceptable and no positive increase in the number of histidine revertants per plate was observed with any of the tester strains. The results of the bacterial reverse mutation assay indicate that under the conditions of this study ammonium thiosulfate solution did not cause a positive increase in the number of histidine revertants per plate of any of the tester strains either in the presence or absence of metabolic activation. The study showed significant methodological and reporting deficiencies. The purity of the test item is not specified. Five different Salmonella strains were included, however neither E. coli strain WP2 uvrA pKM101 nor S. typhimurium strain TA 102 were employed in this study for the detection of oxidising mutagens and cross-linking agents. Salmonella tester strain TA 1538 is not recommended by the OECD guideline. A confirmatory experiment was not performed. Based on the above-mentioned shortcomings, the study is considered to be not reliable [RL-3].

 

Summary - Gene mutation in bacteria

Key studies are available for sodium dithionite, sulfites, and thiosulfates and all studies are considered to be reliable without restrictions [RL-1]. All studies returned exclusively negative results in bacterial reverse mutation assays. This finding is supported by further studies which are reliable with restrictions [RL-2]. Other studies on gene mutation in bacteria summarised above are considered to be not reliable and are provided for information purposes only.

Overall, key studies performed with sodium dithionite and other category group substances, i.e. sulfites and thiosulfates were all negative under the conditions tested. Thus, sodium dithionite is considered to be non-mutagenic in bacteria.Further details on the read-across is provided above.

 

 

Gene mutation in mammalian cells

Sodium dithionite

Sokolowski (2022) investigated on the gene mutation potential of sodium dithionite in an in vitro mammalian cell gene mutation test at the Hprt locus according to OECD TG 476 (2016) and under GLP. Chinese hamster lung fibroblasts (V79) were exposed for 4 hours to sodium dithionite up to a cytotoxic concentration of 952.5 µg/mL both with and without metabolic activation (S9 fraction from phenobarbital/β-naphthoflavone induced rat livers). The outcome of this study was judged as clearly negative both in the absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system. The study is considered to be reliable without restrictions [RL-1].

 

Sulfites

Sokolowski (2022) performed an in vitro mammalian cell gene mutation at the Hprt locus according to OECD TG 476 and under GLP in order to evaluate the mutagenicity of sodium sulfite. Cultures of Chinese hamster lung fibroblasts were exposed for four hours to five different concentrations up to concentrations of 1280 and 640 µg/mL in absence and presence of metabolic activation, respectively. No relevant toxicity (10 to 20% relative survival) was found in the main experiment with metabolic activation. In the experiment without metabolic activation, limiting cytotoxicity was observed at concentrations of 640 µg/mL and above. No precipitation of the test material occurred up to the highest concentration investigated after four hours treatment both in absence and presence of metabolic activation. Sodium sulfite, tested up to the recommended maximum concentration (with metabolic activation) or cytotoxic concentrations (without metabolic activation), did not induce biologically relevant increases in the mutant frequency. The outcome of this study was judged as clearly negative both in the absence and presence of S9 metabolic activation. All validity criteria were met. The study was fully compliant with OECD 476 (2016). The study is considered to be reliable without restriction [RL-1].

 

Disodium disulfite was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells (Stone, 2010). The test was conducted according to OECD 476 (1997) and under GLP. In a cytotoxicity dose-range finding experiment, 6 concentrations were tested in the absence and presence of S9 ranging from 59.44 to 1902 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed gave 37% and 50% RS in the absence and presence of S9, respectively. In the first main experiment, in the presence of S9, statistically significant increases in mutant frequency were observed at the highest 2 concentrations (1600 and 1902 µg/mL) but not showing a linear trend. This positive finding was not reproduced in the first main experiment without metabolic activation as well in the second main experiment with or without metabolic activation. In order to verify the isolated positive finding, a confirmatory experiment in the presence of S9 was performed. In Experiment III no statistically significant increases in mutant frequency were observed following treatment with disodium disulfite at any concentration tested and there were no significant linear trends, indicating a negative result. It is concluded that disodium disulfite does not induce mutations at the HPRT locus of L5178Y mouse lymphoma cells when tested with and without metabolic activation up to the limit dose of 1902 µg/mL (10 mM) as foreseen by the test guideline. The study is considered to be reliable without restrictions [RL-1].

 

Mallon and Rossmann (1981) investigated sodium bisulfite (aka sodium hydrogensulfite) in V79 cells for inductions of gene mutations at the Hprt locus. Cells were treated with 10 and 20 mM in a short-term exposure (15 minutes) or with 1 and 5 mM in a long-term exposure (48 hours). The toxicity of bisulfite was measured by assaying the clonal survival after exposure to the test substance for 15 minutes in PBS. UV-light (GE germicidal lamp G1578) at a fluence of 1.8 J/m² was used as positive control. Cytotoxicity was observed at concentrations above 20 mM. No increase in mutation frequency was observed after short or long-term exposure. The reference exhibits deficiencies in study design, such as non-guideline complaint exposure duration, culture conditions and selection of positive control substance, the genetic stability and sensitivity of the test system was not demonstrated. Due to the aforementioned shortcomings, reference is considered to be not reliable [RL-3].

 

In an in vitro gene mutation test in AS52 (CHO derivative), the induction of point mutations in the XPRT gene was assessed (Meng and Zhang, 1999, reference is identical with Meng & Zhang (1997) Molecular analysis of spontaneous and bisulfite-induced gpt mutants in Chinese hamster ovary cells. China Environ. Sci. 17, 171-175). Cells were exposed with 0, 5 and 10mM sodium bisulfite, ethyl methanesulfonate was used as positive control substance. Cells were exposed for 4 hours in triplicate and sub-cultured for the expression for 7 days. Cytotoxicity was expressed as percent survival relative (RS) to those from similarly plated untreated control. Cells treated with sodium bisulfite showed an increase in mutation frequency with a relative survival of 34.5% compared with the untreated control. The increase of MF was within (at 5mM) or slightly above (at 10mM) the range of historical control values by other labs. The absolute relative survival ranged from 70-90%. The high variability in RS in untreated cultures is unusual and indicates difficulties in standard cell-line culture conditions in this lab. Authors state that solid sodium bisulfite was used in the mutation experiments (purity not stated, not checked for pH effects). However, sodium bisulfite is only stable in aqueous solution and cannot be isolated in a solid form. Consequently, the test item used in these experiments cannot be verified and the reference is rated as not reliable [RL-3].

 

Thiosulfates

Ammonium thiosulfate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells (Stone, 2010). The study was performed according to OECD TG 476 (1997) under GLP. The study consisted of a cytotoxicity Range-Finder experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9). The test article was formulated in water for irrigation (purified water). A 3-hour treatment incubation period was used for all experiments. In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9, ranging from 46.31 to 1482 mg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed was 1482 mg/mL, which gave 103% and 65% relative survival (RS) in the absence and presence of S‑9, respectively. In Experiment I, concentrations, ranging from 200 to 1482 mg/mL, were tested in the absence and presence of S9. 7 days after treatment all concentrations in the absence and presence of S9 were selected to determine viability and 6TG resistance. The highest concentration analysed was 1482 mg/mL, which gave 107% and 95% RS in the absence and presence of S9 respectively. In Experiment II, concentrations, ranging from 100 to 1482 mg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 1482 mg/mL, which gave 68% and 107% RS in the absence and presence of S9, respectively. Vehicle and positive control treatments were included in each Mutation Experiment. In Experiments I and II no statistically significant increases in mutant frequency were observed following treatment with ammonium thiosulfate at any concentration tested in the absence and presence of S9 and there were no significant linear trends. The study is considered to be reliable without restrictions [RL-1].

 

Summary - Gene mutation in mammalian cells

Key studies are available for sodium dithionite, sulfites, and thiosulfates and all studies are considered to be reliable without restrictions [RL-1]. All studies returned exclusively negative results in mammalian gene mutation assays (Hprt or Xprt). Other studies on in vitro mammalian gene mutation summarised above are considered to be not reliable and are provided for information purposes only.

Overall, key studies performed with sodium dithionite and other category group substances, i.e. sulfites and thiosulfates were all negative under the conditions tested. Thus, sodium dithionite is considered to be non-mutagenic in mammalian cells.Further details on the read-across is provided above.

 

In vitro clastogenicity and aneugenicity

Sodium dithionite

Naumann (2022) examined the clastogenic and aneugenic potential of sodium dithionite in an in vitro micronucleus test with and without metabolic activation. Human peripheral blood lymphocytes were exposed to up to the recommended maximum concentration of 1905 µg/mL (equivalent to 10 mM). The cell cultures were either treated for 3 hours and incubated for a further 25 hours (pulse treatment) or for 28 hours without recovery (continuous treatment). In the 3-hour pulse treatment experiment, in the absence and presence of S9 mix, no cytotoxicity was observed up to the recommended maximum concentration (1905 µg/mL; equivalent to 10 mM). In the 28 hours continuous treatment experiment III in the absence of S9 mix, limiting cytotoxicity was observed at concentrations of 466 µg/mL and above. Clear cytotoxicity (58.8% cytostasis) was observed at the highest concentration evaluated (466 µg/mL). No precipitation of the test item in the culture medium was observed after the pulse and continuous treatment experiments. Sodium dithionite, tested up to the recommended maximum concentration (3-hour pulse treatment) or cytotoxic concentrations (28-hour continuous treatment), did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2016). The study is considered to be reliable without restrictions [RL-1].

 

Sulfites

The clastogenic and aneugenic potential of sodium sulfite was evaluated in an in vitro micronucleus test (Naumann, 2022) according to OECD TG 487 (2016) and under GLP. Human peripheral blood lymphocytes were obtained from two different donors. Sodium sulfite was tested using at least three different concentration levels up to top concentrations selected in line with the OECD guideline. The first experiment (Experiment I) was conducted as a 3-hour pulse treatment with a 25-hour recovery time both with and without metabolic activation. The second experiment was a continuous 28-hour treatment in absence of metabolic activation. In Experiment I (3-hour pulse treatment) in the absence and presence of S9 mix, no cytotoxicity was observed up to the recommended maximum concentration (1280 µg/mL; equivalent to 10 mM). In Experiment II in the absence of S9 mix after continuous treatment (28 hours), limiting cytotoxicity was observed at concentrations ranging from 583-1280 µg/mL. Clear cytotoxicity (54.9% cytostasis) was observed at the highest concentration evaluated (333 µg/mL). In Experiment I and II in the absence and presence of S9 mix, no precipitation of the test item in the culture medium was observed. Sodium sulfite, tested up to the recommended maximum concentration (Experiment I: 3-hour treatment) or cytotoxic concentrations (Experiment II: 28-hour treatment), did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2016). The study is considered to be reliable without restrictions [RL-1].

 

Naumann (2022) performed an in vitro micronucleus test according to OECD TG 487 under GLP in order to examine potential clastogenicity and aneugenicity of disodium disulfite. Human peripheral blood lymphocytes were exposed for 3 and 28 hours to disodium disulfite at three different concentration levels. The highest concentration applied in this study (1901 μg/mL; equivalent to 10 mM) was chosen with regard to the molecular weight of disodium disulfite and with respect to the current OECD Guideline 487 (2016). In Experiment I (3-hour pulse treatment) in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest concentration applied. In Experiment II in the absence of S9 mix after continuous treatment (28 hours), limiting cytotoxicity was observed at concentrations ranging from 512-1901 µg/mL. Clear cytotoxicity (62.9% cytostasis) was observed at the highest concentration evaluated (293 µg/mL). In Experiment I and II in the absence and presence of S9 mix, no precipitation of the test item in the culture medium was observed. Disodium disulfite, tested up to the recommended maximum concentration (Experiment I: 3-hour treatment) or cytotoxic concentrations (Experiment II: 28-hour treatment), did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2016). The study was fully compliant with OECD 487 (2016). The study is considered to be reliable without restrictions [RL-1].

 

In a combined study, Meng & Zhang (1992, reference is identical with Meng, Z.; Zhang, L (1994). Chromosomal aberrations, sister chromatid exchanges and micronuclei induced in human lymphocytes by sodium bisulfite (sulfur dioxide) Acta Genetica Sinica 21, 1-6) investigated clastogenic, aneugenic and DNA damaging effects in human lymphocytes exposed to sodium bisulfite (aka sodium hydrogensulfite). Results on DNA damage are discussed below. Peripheral blood lymphocytes were taken from young male and female donors and incubated under addition of a mitogen (PHA). The pre-culture duration, however, was not stated. A sodium bisulfite stock solution with pH 7 was used for all experiments. Mitotic index was calculated on the basis of 2000 lymphocytes. For the analysis of chromosome aberrations, cells from 4 different donors (sex not stated) were incubated for 48 hours at concentrations of 0, 0.05, 0.1, 0.5, 1, 2 mM. Colcemid was added 4 hours prior harvest. Cells were Giemsa stained after hypotonic treatment and drying. 200 cells per group were scored blindly for the presence of isochromatid and chromatid breaks. The dose of 2 mM could not be scored due to excessive toxicity. A dose dependent increase of chromatid breaks was observed, being significant at 0.1 mM and above. None of the reported concentrations caused excessive toxicity, i.e. mitotic index > 50% relative to untreated control. Only the maximum dose of 2 mM caused complete toxicity. No explanation was given why further structural aberrations such as fragments, deletions or exchanges were not scored. The results indicate a clastogenic effect of sodium bisulfite at concentrations with moderate cytotoxicity. In the micronucleus experiments, cells were treated under identical conditions compared with the chromosomal aberration experiments, except for the extended incubation period of 72 hours. CytoB was added to the cultures 24 hours prior harvest. Binucleated cells were scored for the presence of micronuclei. Total number of binucleated cells scored per culture was not given. A dose dependent increase in the micronucleus frequency was observed, with a statistical significance at 0.1 mM and above for 2 donors and at 1 mM for the further 2 donors. The mean values for all donors show a statistical significance at 0.5 mM and above. The total frequency of binucleated cells in all cultures was still within the range of untreated control MN frequency in other labs (i.e. 0.7-2.0%), positive controls were not used in the experiments, which would allow a statement on the specificity and sensitivity of the test system. The experimental procedures used in the above experiments are not in accordance with accepted guidelines and the findings raise questions whether a true clastogenic effect was observed. Authors only investigated chromatid breaks in the CA experiments. This type of aberration is the easiest to be analysed, whereas fragments, deletions and exchanges require more experienced evaluators. This raises questions about the overall experience of the lab with the evaluation of chromosome aberration experiments. The exposure duration of 48 hours for the chromosome aberration and 72 hours for the micronuclei experiments is too long even under conditions of continuous treatment. Usually, times representing 1.5 cell cycle lengths (i.e. approx. 24 hours for human lymphocytes) are used in those experiments. No exposure durations of 3-6 hours pulse treatment were used. Further, the mitotic index was assessed in a separate experiment, for which the incubation duration was not given. Consequently, the mitotic indices cannot directly be correlated with the conditions during mutation experiments; hence it remains unclear whether clastogenic effects were caused via unspecific cytotoxicity or a substance specific effect. Authors state that solid sodium bisulfite was used in the mutation experiments (purity not stated, not checked for pH effects). However, sodium bisulfite is only stable in aqueous solution and cannot be isolated in a solid form. Based on the above given shortcomings, the positive findings should be considered with great caution. The publication is considered to be not reliable [RL-3].

 

Chinese hamster fibroblast cells (CHL) were tested for the induction of structural aberrations (Ishidate, 1984). Cells were exposed towards (i) potassium metabisulfite (93.0% purity, aka potassium disulfite) (ii) sodium bisulfite, anhydrous (95.0% purity aka disodium disulfite) and (iii) sodium sulfite, anhydrous (95.0% purity) at max. 0.06, 0.125, and 0.5 mg/mL, respectively (3 doses, concentrations not reported) for a period of 24 and 48 hours without metabolic activation. Colcemid was added 2 hours before cell harvest and 100 well-spread Giemsa-stained metaphases were checked for incidence of polyploid cells as well as for cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations. No data on cytotoxic effects were reported. No increase of structural chromosome aberrations was reported. The publication is a summary paper, reporting clastogenic effects of 242 substances. The resulting reporting detail is very limited, stating the basic experimental parameters and results in a binary format only; only results for 48 hours exposure duration reported. Authors state that solid sodium bisulfite, anhydrous was used in the mutation experiments (not checked for pH effects). However. sodium bisulfite is only stable in aqueous solution and cannot be isolated in a solid form. Based on the above limited reporting detail and the questionable test item, the reference is considered as not reliable [RL-3].

 

Popescu and DiPaolo (1988) tested the clastogenic effect of sodium bisulfite (aka sodium hydrogensulfite) in Syrian hamster foetal cells (HFC) at concentrations of 10, 20, and 40 mM (equivalent to 1.04, 2.08, 4.16 g/L) for an exposure duration of 15 minutes. Chromosomes were prepared 6 and 24 hours after exposure by adding Colcemid 4 hours before cells harvest and Giemsa staining. 200 metaphases were analysed for chromatid or chromosome aberrations (gaps, breaks and exchanges). Chromosome aberration analysis at first (6 hours) and second (24 hours) mitosis showed no significant increases in aberrations over the control. There are deficiencies in performance and reporting of the method: No positive control was used. No data if duplicate cultures were used. Exposure period was too short for the detectable manifestation of chromosomal damage. Such damages require the completion of at least one whole cell cycle – which is not the case after 15 minutes exposure. Chromosome preparation, staining and analysis of the slides were not described in detail. Authors do not provide details on the test item, such as origin, purity, physical state, impurities, pH effects. Based on the above limited reporting detail and the questionable test item, the reference is considered as not reliable [RL-3].

 

The induction of chromosomal aberrations (CA) and micronuclei formation (MN) by potassium disulfite (99.9% purity, aka dipotassium disulfite) was tested in human peripheral blood lymphocytes (Yavuz-Kocaman, 2008). Cells from 4 healthy donors (2 females, 2 males) were used and incubated for 72 hours prior to exposure. The exposure concentrations were 25, 50, 100 and 200 µg/mL (0.13, 0.26, 0.56, 1.05 mM) for a duration of 24 and 48 hours. In the CA experiments, colchicine was added 2 hours before harvesting; ethyl methanesulfonate was added as positive control. Cells were harvested, fixed and prepared slides were stained (5% Giemsa); 100 metaphases per donor were scored for structural and/or numerical aberrations (excluding gaps). In the MN experiments Cytochalasin B was added at 44 hours of incubation to block cytokinesis. After additional 24 hours incubation at 37°C, cells were harvested, fixed and stained for MN analysis. 2000 binucleated lymphocytes were scored from each donor (8000 binucleated cells were scored per concentration). Potassium disulfite increased the percentage of MN over all concentrations and time points (by 2-3fold) but not in a dose-dependent manner. The mitotic index was increased in a dose dependent manner, cytotoxicity was <50%. Positive findings in the MN assay do not show a clear dose-dependency at both time points. Thus, a clear positive outcome was not demonstrated and in comparison with the findings of the CA assay described below is implausible. Potassium disulfite induced structural CA in a dose and time dependant manner, when compared with the negative control (4-6 fold after 24 and 4-10 fold after 48 hours). The control frequencies of CA were high (2.5%) compared to normal levels (around 0.5-1.5%). The mitotic index (MI) was reduced by 55 and 59% at 100 and 200 µg/mL after 24 hours and by 69% at 200 µg/mL after 48 hours. The assay shows a dose- and time-dependent increase of structural CA. This is an unusual finding, which was also demonstrated by the authors in a parallel in vivo clastogenicity experiment which is discussed further below. Although the CA findings appear robust, there are irregularities in study conduct and reporting. Further the credibility of the testing facility based on unusual or biologically impossible results, as discussed in detail further below is compromised. Based on the questionable results, the results reported in this reference are considered not reliable [RL-3].

 

For the conduct of an in vitro chromosome aberration test Beckman & Nordenson (1986) used peripheral blood lymphocytes taken from two different non-smoking, healthy individuals (sex not stated). The cells were cultured for 72 hours and exposed to sodium hydrogensulfite for 48 hours at a concentration of 0.4 mM. Chromosome aberrations were scored in 400 cells. Statistical significance was determined by a pairwise comparison with background cultures. Authors state that a large number of the initial assays failed due to cytotoxic effects. The number of chromosome aberrations was increased, compared with the background frequency. There are a number of relevant deficiencies in performance and reporting of the method, which renders the publication not reliable: Authors state that the test substances induce significant cytotoxicity, however no data was given to prove this; consequently, a correlation of the aberration frequency with cytotoxicity is not possible. The control (background) number of chromosome aberrations of 3% is unusually high (12 per 400 cells), which raises questions about the suitability of the test system. The culture conditions are insufficiently described, hence it is unclear how peripheral blood lymphocytes were cultivated without the use of a mitogen to induce cell proliferation. Secondly, it is unclear how the authors counted at least 400 cells per culture without the use of a spindle poison (e.g. colchicine) to arrest the cells in the M-phase with visible chromosomes. Authors state that solid sodium bisulfite was used in the mutation experiments (purity not stated, not checked for pH effects). However, sodium bisulfite is only stable in aqueous solution and cannot be isolated in a solid form. Based on the above limited reporting detail and the questionable test item, the reference is considered as not reliable [RL-3].

 

Abe (1977) tested potassium disulfite (aka dipotassium disulfite) in a Chinese hamster cell line for the induction of chromosome aberrations at doses of 0.1, 0.5 and 1 mM. For a given dose at least one culture was made. HBSS was used as vehicle and solvent control. Cells were incubated for 26 hours at 37°C in the dark (two cell cycles). 0.25 µg colchicine/ml was added 2 hours prior harvest. The cells were fixed and stained by the fluorescence or Giemsa staining technique and chromosome aberrations were examined on 100 metaphases for each dose, and frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. The Mitotic Index (MI) decreased by more than 50% compared with the solvent control value at 0.5 and 1 mM. Neither a significant increase of aberrations nor a dose dependency was observed. The publication is a summary paper, reporting clastogenic effects of 33 substances. The resulting reporting detail is very limited, stating the basic experimental parameters and results in a binary format only. The decrease of MI below 50%, indicate that the maximum tolerated dose was exceeded in the two highest doses, which invalidates the results obtained from these doses. Based on the above limited reporting detail and questionable test results, the reference is considered as not reliable [RL-3].

 

Human peripheral blood lymphocytes were tested for the induction of chromosome aberrations after sodium hydrogen sulfite exposure (Nordenson & Beckman, 1984). The stimulated lymphocytes were cultured for 70-72 hours at 37°C, during the last 48 hours incubation the cells were exposed to 0.375 mM sodium hydrogen sulfite. Cells were arrested by addition of Colcemid, fixed and stained. 200 cells per culture from coded slides were analysed for the presence of chromosomal aberrations. The classes of aberrations scored are unclear – it is possible that some of these abnormalities were gaps, which are normally not considered in the assessment of a positive response. No significant increase was observed in the number of aberrations. The reference is not suitable for hazard assessment purposes, since relevant details were not reported/measured, such as only single dose treatment conducted which is not suitable for an assessment of dose-response relationship, cytotoxicity was not determined, only one time point was investigated. The reference is considered to be not reliable [RL-3].

 

Thiosulfates

In an in vitro micronucleus test with human peripheral blood lymphocytes (acc. to OECD TG 487, GLP), Naumann (2022) examined the cytogenic potential of sodium thiosulfate. Sodium thiosulfate was tested in a 3-hour pulse treatment (Experiment I) both in absence and presence of metabolic activation and in a 28-hour continuous treatment (Experiment II) without metabolic activation. The experiment was conduct using three different sodium thiosulfate concentration levels up to top concentrations selected in line with the criteria set out in the OECD guideline. No relevant toxicity (55±5% cytostasis) was found in the main experiments (Experiment I: 3-hour pulse treatment; Experiment II: 28-hour continuous treatment) both with and without metabolic activation. In Experiment I and II in the absence and presence of S9 mix, no precipitation of the test item in the culture medium was observed. Sodium thiosulfate, tested up to the recommended maximum concentration did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2016). The study is considered to be reliable without restrictions [RL-1].

 

Ammonium thiosulfate was tested in a chromosome aberration assay (Gudi, 2001) according to OECD TG 473 (1997) and under GLP using Chinese hamster ovary (CHO) cells in both the absence and presence of metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. Based on the findings, the doses chosen for the chromosome aberration assay ranged from 185 to 1480 µg/mL for both the non-activated exposure groups and the S9 activated exposure group. The cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. In the absence of both substantial toxicity and test item precipitation in the treatment medium, 1480 µg/mL was chosen as the high dose for microscopic analysis for chromosome aberrations in all harvests. The next two lower doses were also included in the analysis. Based on the findings of this study, Ammonium thiosulfate was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells. The study is considered to be reliable without restrictions [RL-1].

 

Murli (1989) investigated on the clastogenic potential of ammonium thiosulfate in chromosomal aberration assay (EPA OPP 84-2; GLP) using Chinese hamster ovary cells. In absence of S9 mix, the cells were exposed to ammonium thiosulfate concentrations of 1270, 2550, 3820, and 5100 µg/mL. In presence of S9 mix, ammonium thiosulfate concentrations of 1260, 2510, 3770, and 5020 µg/mL were tested. With metabolic activation, the cells were exposed for 2 hours, washed and resuspended in medium. After 7.5 hours Colcemid was added. Without metabolic activation cells were exposed for 17.25 hours. The cultures were then washed with buffered saline and complete McCoy's 5a medium containing Colcemid. The cells were fixed after 20 hours without metabolic activation and after 10 hours with metabolic activation. Slight reduction in visible mitotic cells was observed in the chromosome aberration assay without metabolic activation at a concentration of 5100 µg/mL. No toxicity was evident in the other test cultures. In the chromosome aberration assay with metabolic activation no toxicity was evident in any of the test cultures. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed. The GLP study showed some deviation from the OECD TG 473: The highest test concentration is slightly over the maximum concentration required by the OECD guideline 473 (100 µg/mL over the limit). According to OECD guideline 473 testing has to be done first with and without metabolic activation for 3-6 hours. And if these tests give negative results, an additional experiment -S9 should be done with continuous treatment. In this study the procedure is not conform with this method. The study is considered to be not reliable [RL-3].

 

Summary - In vitro clastogenicity and aneugenicity

Key studies are available for sodium dithionite, sulfites, and thiosulfates and all studies are considered to be reliable without restrictions [RL-1]. All studies returned exclusively negative results in in vitro cytogenicity assays (micronucleus test or chromosome aberration assays). Other studies on in vitro cytogenicity summarised above are considered to be not reliable and are provided for information purposes only.

Overall, key studies performed with sodium dithionite and other category group substances, i.e. sulfites and thiosulfates were all negative under the conditions tested. Thus, sodium dithionite is considered non-clastogenic and non-aneugenic in mammalian cells.Further details on the read-across is provided above.

 

Summary entry – investigations on DNA damage in vitro

Several studies on in vitro DNA damage studies in mammalian cells (involving comet assays) were identified which do not fulfil the relevance, reliability and adequacy criteria as foreseen by the ECHA guidance on information requirements, chapter R.7.7. Most of the references are on mechanistic investigations to examine specific mechanisms of sulfites and are not suitable to address the endpoint of in vitro genetic toxicity. The studies are discussed below in brief for information purposes only and were included in the IUCLID as summary entry.

 

Chen (1994): Investigated the DNA base substitution in transfected cells followed by isolation and sequencing. Cells were incubated for a maximum of 54 days at sodium bisulfite concentrations up to 50mM. The test design is not suitable for the detection of potentially heritable DNA mutations, since e.g. sensitivity and specificity was not determined, the concentrations and test duration were not compliant with accepted guidelines.

Peden (1982): Published a mechanistic study on in vitro DNA base-substitutions in deletion loops. Sodium bisulfite was used as deamination reagent of the DNA bases at 2 mole/L. The test design is not suitable for the detection of potentially heritable DNA mutations, since e.g. sensitivity and specificity was not determined, the concentrations and test duration were not compliant with accepted guidelines.

 

MacRae (1979): Investigated the induction of sister chromatid exchanges in CHO cells after sodium bisulfite exposure at concentrations between 0.03 to 7.3 mM after 2 and 24 hrs exposure duration. No information on cytotoxicity available. Sister chromatid exchange detects unspecific DNA damage in mammalian cell in it therefore not a method to assess the mutagenic potential of a substance. Without information on the cytotoxic effects caused by the test item, an interpretation of the SCE frequencies is not possible.

Meng (1992): Investigated the induction of sister chromatid exchanges in human peripheral blood lymphocytes after sodium bisulfite exposure at concentrations between 0.05 to 2 mM after 72 hrs exposure duration. No information on cytotoxicity available. Sister chromatid exchange detects unspecific DNA damage in mammalian cell in it therefore not a method to assess the mutagenic potential of a substance. Without information on the cytotoxic effects caused by the test item, an interpretation of the SCE frequencies is not possible. The exposure duration is not further justified – cells are usually harvested after the completion of one cell cycle.

Doniger (1982): Study investigates substance induced DNA lesions and DNA replication rate in mammalian cells. No increase in DNA lesions was observed via gradient sedimentation. The reference exhibits several reporting deficiencies: a correlation between DNA damage and cytotoxicity was not performed; hence it remains unclear whether any positive findings were secondary to toxicity or due to direct substance interaction. Further, the amount of cells counted for DNA repair was not stated, only one concentration used which does not allow a dose-response analysis.

Popescu 1988: Investigated the induction of sister chromatid exchanges in Syrian hamster foetal cells (HFC) after sodium bisulfite exposure at concentrations of 10, 20 mM after 15 minute exposure duration. No information on cytotoxicity available. Sister chromatid exchange detects unspecific DNA damage in mammalian cell in it therefore not a method to assess the mutagenic potential of a substance per se. Without information on the cytotoxic effects caused by the test item, an interpretation of the SCE frequencies is not possible. It remains unexplained how any sister chromatid exchange could be seen after 15 minutes exposure. The manifestation of such damage requires the completion of a whole cell cycle, which is biologically impossible after 15 minutes exposure.

Yavuz-Kocaman (2008): Investigated the induction of sister chromatid exchanges in human peripheral blood lymphocytes after sodium bisulfite exposure at concentrations 25, 50, 100 and 200 µg/ml after 24 and 48 hrs exposure duration. Sister chromatid exchange detects unspecific DNA damage in mammalian cell in it therefore not a method to assess the mutagenic potential of a substance. The publication is discussed in the sections on in vitro mammalian chromosome aberration and in vivo chromosome aberration, clearly showing that the results should be treated with great caution (see discussion further below).

Beckman (1986): Investigated the induction of sister chromatid exchanges in human peripheral blood lymphocytes after sodium bisulfite exposure at a concentration of 0.4mM after at least 48 hrs exposure duration. No information on cytotoxicity available. Sister chromatid exchange detects unspecific DNA damage in mammalian cell in it therefore not a method to assess the mutagenic potential of a substance. Without information on the cytotoxic effects caused by the test item, an interpretation of the SCE frequencies is not possible. The exposure duration is not further justified – cells are usually harvested after the completion of one cell cycle. A single dose experiment does not allow the determination of a dose-response relationship.

 

Summary entry - unsuitable test systems in vitro

Several studies were identified which do not fulfil the relevance, reliability and adequacy criteria as foreseen by the ECHA guidance on information requirements, chapter R.7.7. DNA damage in bacteria, induction of SCE in mammalian cells, or tests in yeasts or drosophila are no longer recommended as part of regulatory testing by many agencies worldwide and there are no up-to-date OECD guidelines for their conduct. The majority of the references in the following summary entry are more than 30 years of age and do not comply with today’s standards in genetic toxicity testing. Interpretation of the relevance of both positive and negative results from such tests is therefore unclear and was not used for the current assessment. The studies are discussed below in brief for information purposes only and were included in the IUCLID as summary entry.

 

A range of references assessed the mutagenic potential of sulfites in bacteria (Kunz & Glickmann, 1983; Mukai, 1970; De Giovanni-Donnelly, 1985). The cells were exposed to excessively high concentrations of sulfite up to 1 M at acidic culture conditions (pH 5.7 – 5.2). The incubation duration was 0 to 30 minutes. All references reported significant cytotoxic effects, depending on the exposure duration. The aim of these studies was to assess the bactericidal effects of sulfites but not the mutagenic effects of this substance class. Due to the unsuitable test design with regard to culture conditions, drastic exposure concentrations and duration these references are considered of no relevance for chemicals hazard assessment.

 

Kunz & Glickman (1983): Detection of amber and ochre mutations at 65 individual sites within the E. coli lacI system (G:C to A:T transition).

Mukai et al. (1970): Reversion studies with Escherichia coli mutants investigated the bactericidal effects of sodium bisulfite.

De Giovanni-Donnelly, R. (1985): The bactericidal effects of sodium bisulfite on Salmonella typhimurium LT2 strains carrying the hisG46 allele were investigated.

Valencia (1973): Tested the genotoxic effects of sodium sulfite in Drosophila.

Jagiello (1975): Investigated the induction of chromosomal damage in oocytes freshly isolated from mice, ewe and cows. Cells were incubated for 5 and 15 hours for mice and 28 hours for ewe and cow. The number of cells per dose ranged from 6 to 96 – the confidence level at such low cell numbers is significantly impaired and does not allow a reliable evaluation of the clastogenic potential. It remains unclear why such an unusual test system was chosen for these experiments. There is currently no known mutagen which exclusively induces gene mutations in germ cells but not in somatic cells.

Clark (1953): Sodium sulfite and sodium bisulfite were tested in a bacterial forward mutation test in micrococcus pyogenes var, M. aureus using a preincubation method at a single concentration. A forward mutation assay is not indicative for a specific substance-induced mutagenic response. Use of a single strain, single concentration test system is not suitable to determine a reproducible dose-response relationship.

 

Conclusion on in vitro genotoxicity

There was no evidence whatsoever for any mutagenic activity of sodium dithionite, sulfites, and thiosulfates in the bacterial reverse mutation assay, HPRT/XPRT assay, or cytogenicity studies (micronucleus test and chromosome aberration assay) up to the recommended maximum concentration or top concentrations limited by cytotoxicity. Consequently, sodium dithionite is considered non-mutagenic in suitable in vitro test systems.

Therefore, the classification criteria as laid down in regulation (EC) 1272/2008 are not met and sodium dithionite does not require classification as a germ cell mutagen.

 

In vivo genetic toxicity tests

In vivo clastogenicity and aneugenicity

Sulfites

The substance sodium sulfite (purity 98.1%) was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice according to OECD 474 (1997) and under GLP (Schulz, 2008). The substance was administered once subcutaneously to male animals at dose levels of 250, 500 and 1000 mg/kg bw. The vehicle, water, was used as negative control and cyclophosphamide and vincristine as positive control substances. 24 and 48 hours after administration, animals of all dose groups and the highest dose group were sacrifices, respectively. A minimum of 2000 fixed and stained polychromatic erythrocytes were analysed for the presence of micronuclei. Bone marrow toxicity was determined by analysing the PCE/NCE ratio. The administration of the test substance led to clinical signs of toxicity at the highest administered dose of 1000 mg/kg body weight. No statistically significant increases in the micronucleated erythrocyte frequency were observed in any evaluated test substance-treated group of animals at either time point. There were no statistically significant decreases in the PCE/NCE ratio in any test-substance treated group. Under the conditions of this study, sodium sulfite did not induce formation of micronuclei in rat bone marrow up to the dose of 1000 mg/kg bw via subcutaneous route of administration up to doses showing signs of systemic toxicity. The study is considered to be reliable without restrictions [RL-1].

 

Carvalho et al. (2011) tested sodium disulfite (aka disodium disulfite) in a combined in vivo comet and micronucleus assay in CF1 mice after single oral administration. The maximum tolerated dose was determined in a previous experiment in 6 mice at doses of 0.5, 1 and 2 g/kg bw via gavage. In the main study 10 mice per group (five females and five males) were treated for 24 h with a single dose by gavage (0.1 ml/10 g body weight): (a) sodium disulfite (0.5, 1, or 2 g/kg b.w.); (b) negative control group: water; (c) positive control group: cyclophosphamide, 25 mg/kg. Bone marrow and peripheral blood smears were prepared, stained and 2000 polychromatic reticulocytes from the bone marrow or 2000 reticulocytes from the peripheral blood were scored for the presence of micronuclei. The polychromatic erythrocytes: normochromatic erythrocytes (PCE/NCE) ratio was scored in 1000 cells for the determination of bone marrow toxicity. Blood and bone marrow cells of mice treated with the higher dose of SMB (2 g/kg) showed significant increase in MN, when compared with negative control, as well as significant reduction in PCE/NCE ratio. No difference in results was observed between sexes. Again, these results appear plausible, there are however serious doubts that these results could be obtained by the described procedure. Results show a dose-dependent increase of MN frequency both in peripheral blood and bone marrow already 24 hours after administration. This finding is most unusual, since micronuclei are formed during mitosis of an erythroblast which are translocated to the immature erythrocytes, which is ultimately released form the bone marrow into the blood stream as reticulocytes. Being a sequential order of events, clastogenic effects must first be observed in the bone marrow before becoming visible in the peripheral blood. It is therefore surprising that in this publication, micronuclei formation was observed in both the bone marrow and peripheral blood at the same time to an equal degree. According to OECD Guideline 474, samples should be drawn (i) not earlier than 24hours after exposure when examining the bone marrow and (ii) not earlier than 36 hours after exposure when examining the peripheral blood. Also, the guideline foresees at least two sampling times to assess the time course of clastogenic events. Although the study design appears plausible and the positive findings appear robust, there are serious concerns as discussed above that render this publication not reliable [RL-3].

Yavuz-Kocaman (2008) investigated the induction of chromosomal aberrations in the bone marrow of albino rats. Four animals (2M/2F) per group were given a single intraperitoneal injection of 150, 300, 600 mg/kg bw potassium disulfite (aka dipotassium disulfite, purity not stated) 12 and 24 hours before sacrifice. Colchicine was given 2 hours prior sacrifice via i.p. injection. Urethane was used as positive control substance. Bone marrow of femurs was fixed and stained for microscopic analysis. 100 metaphases per animal (400 per dose group) were scored. Mitotic index was determined by scoring 3000 cells from each animal. The mitotic index showed a dose-dependent cytotoxic effect, dropping form 3.6 in the control to 1.85 at 600 mg/kg bw (51%) after 12 hours – demonstrating no excessive toxicity. A dose and time dependant increase for structural chromosome aberrations was observed (not further specified which type of aberrations were analysed). There were statistically significant increases in structural CA frequency at the top dose (2.5-fold increase) after 12 hours, and at all 3 doses (2 to 4-fold increases) at 24 hours. Although this appears to be a clear positive finding, there are reasons to question the reliability and relevance of the results, namely:

 

- The intraperitoneal route is not considered a physiologically relevant route of exposure, since this route avoids first-pass metabolism by the liver which is known to effectively eliminate sulfite by oxidation to sulfate via molybdenum cofactor by normal physiological routes (oral, inhalation, dermal). Since redox damage is considered to exhibit a threshold for genotoxic effects, by-passing of anti-redox defence mechanisms would lower the threshold and allow genotoxic effects to be manifest which would not be seen if a physiological route of administration had been used. Clearly negative results in a recent GLP and guideline study with much higher doses of sodium sulfite but also bypassing normal physiological routes of administration (subcutaneous route; Schulz, 2008) are in contradiction to the positive findings of Yavuz-Kocaman (2008).

 

- the number of animals per dose group is far too low to ensure statistical robustness; the guideline foresees at least 5 animals per dose and sex.

- Negative control CA frequencies were very high (around 5.5%). CA frequencies in the bone marrow PCE of control animals (rats and mice) are usually much lower, i.e. at 0-2%. The fact that such high control frequencies were seen in this study brings into question the health of the animals and the competency of the people who scored the slides.

 

As a major criticism, the dose- and time-related dose responses are most unusual: different sampling times were included in this assay to study the effects on different stages of the cell cycle. There are few publications where both time- and dose-related CA responses have been studied with known genotoxins. Some clearly show dose-related increases in cells with CA at one or more sampling times, but this is not always the case as the following papers indicate:

 

- McFee and Tice (1990) demonstrated time- and dose-related increases in bone marrow CA for the potent genotoxins mitomycin C and cyclophosphamide, but although DMBA gave dose-related increases in CA at 18 and 26 hr it did not induce CA at 10 hrs.

 

- Tates & Natarajan (1976) observed a clear dose response for CNU-ethanol-induced CA in bone marrow 1 day after ip dosing, but after 2 days there was no clear dose-response.

 

- Aydemir & Bilaloğlu (2003) showed that the anti-cancer drug topotecan induced dose-related CA at both 6 and 24 hr after a single ip dose. However, although gemcitabine induced dose-related increases in bone marrow CA at 24 hr, it did not induce any CA at 6 hr.

 

- There are also numerous examples of genotoxins inducing micronuclei (MN) at some sampling times but not at others. For example, Sutou et al (1990), reporting on a collaborative study by the MMS subgroup of JEMS, showed that ethylnitrosurea, ethyl methanesulphonate, ARA-C and benzene all induced dose-related increases in bone marrow MN at 6 and 24 hours but not at 48 or 72 hours sampling times after 2 or 4 doses. Thus, potent genotoxins may show dose-related increases in CA at several different sampling times, but in many cases, they only show dose-related effects at limited sampling times due to cell-cycle specific effects, death of damaged cells etc. Given the overall genotoxic profile of sulfites (i.e. they do not behave like many potent mutagens that are positive in multiple test systems) it is unlikely that it would be expected to produce dose-related increases in CA at multiple sampling times. It is strange that dose-related increases in numbers of polyploid cells were also seen. For a substance to induce polyploidy, cells need to undergo at least one mitotic division, which in a fast-dividing mammalian cell takes at least 20-24 hours. It is therefore biologically impossible to observe polyploidy as early as 12 hours after dosing with potassium disulfite. Thus, although there appears to be a significant induction of CA by potassium disulfite in this study, there are concerns, as discussed above, which render this publication not reliable [RL-3].

 

In a short communication published by Generoso (1978), the clastogenic effects of sodium bisulfite (purity not stated; aka disodium disulfite) was investigated via dominant lethal assay in male and female mice. Male (101 X C3H)F1 mice were administered intraperitoneally 20 times doses of 300 mg/kg bw/day during a 26-day period and 38 times during a 54-day period. Males were paired with two (SEC X C57BL)F1 females after the last injection. Female (C3H X 101)F1 mice were intraperitoneally administered a single dose of 550 mg/kg bw/day and mated with untreated male (101 X C3H)F1 mice. No mortalities were seen in the 550 mg/kg dose group, whereas 8 out of 46 and 5 out of 46 died in the 400 and 300 mg/kg bw/day dose group, respectively. Sodium bisulfite did not induce a detectable increase in dominant-lethal mutations in either male or female germ cells of mice. It is concluded that sodium bisulfite does not induce chromosome aberrations in the mouse germ-cell stages tested. The publication is however only a brief short communication. The resulting reporting detail is very limited, stating only the basic experimental parameters and results. Mortalities observed in the repeat-dose experiments clearly indicate that the maximum tolerated dose was exceeded, consequently results from such experiments are considered not reliable. Authors state that solid sodium bisulfite was used in the mutation experiments (purity not stated, no information on toxicity). However, sodium bisulfite is only stable in aqueous solution and cannot be isolated in a solid form. Based on the above limited reporting detail, the questionable test item and the inappropriate dose selection, the reference is considered as not reliable [RL-3].

 

In the study published by Kayraldiz and Topaktas (2007) a similar study design as used by Yavuz-Kocaman was used. Albino rats (3M/3F per group) received a single oral administration and intraperitoneal injection of 250, 500, 750, 1000 mg/kg bw potassium disulfite (aka dipotassium disulfite, purity not stated) 6, 12 and 24 hours before sacrifice. Colchicine was given 2 hours prior sacrifice via i.p. injection. Ethyl carbamate was used as positive control substance. Bone marrow of femurs was fixed and stained for microscopic analysis. 100 metaphases per animal were scored. Mitotic index was determined by scoring 3000 cells from each animal. The mitotic index showed a dose-dependent cytotoxic effect, data for control animals not presented, thus a comparison is not possible. The drop of the MI between the low and high dose group i.p. administered animals to 39% shows that the MTD was exceeded. A dose and time dependant increase for structural chromosome aberrations was observed. According to the authors frequency of chromosomal aberrations were increased in all concentrations and treatment periods, however results for the negative and positive control group was not included. The language of this reference is difficult to read and lacks precision, so that the experimental procedure and the presentation of the results is confusing. Although this appears to be a clear positive finding, there are reasons to question the reliability and relevance of the results, namely:

 

- The number of animals per dose group is too low in order to demonstrate statistical robustness; the guideline foresees at least 5 animals per dose and sex.

 

- It appears implausible that animals tolerate the identical dose via two different routes of application. Although the oral absorption is quantitative, a delayed bioavailability is expected. Via i.p. administration, almost the complete dose becomes immediately systemically available and is not metabolised via first pass effect in the liver. Consequently it is expected that the i.p. route is less well tolerated. The same group assessed the MTD via i.p. route in a similar experiment using the same rat strain at 600 mg/kg bw (Yavuz-Kocaman, 2008). A similar MTD of 550 mg/kg bw was determined by another group (Generoso, 1978). It appears implausible that doses exceeding 600 mg/kg bw are tolerated by animals without serious adverse effects.

 

- Results for the negative and positive control animals are not reported. A comparison with the exposed animals is therefore not possible. The same group published results of a similar study in which the CA frequencies were excessively high (around 5.5%), which brings into questions whether the test animals were sufficiently healthy at study initiation. Since such data is lacking in the current publication, the reliability is questionable.

 

- Dose- and time-related dose responses are most unusual. Different sampling times were included in this assay to study the effects on different stages of the cell cycle. Effects seen as early as 6 hours would mean cells were exposed (and sensitive to genotoxic effects) in the G2 phase of the cell cycle. There are very few known genotoxins which are active in G2. Most genotoxins act in G1 or S-phase which would be represented by effects at 12 hours. By 24 hours it is likely that many cells would have divided and be in the next cell cycle, so this would mean either that new damage was being induced or that damage was persistent (without being lethal) for more than 1 cell cycle. One would usually expect that cells exhibiting damage early in the cell cycle would not be able to survive through mitosis to the next cell cycle.

 

- There are few publications where both time- and dose-related CA responses have been studied with known genotoxins. Some clearly show dose-related increases in cells with CA at one or more sampling times, but this is not always the case as the following papers indicate:

- McFee and Tice (1990) demonstrated time- and dose-related increases in bone marrow CA for the potent genotoxins mitomycin C and cyclophosphamide, but although DMBA gave dose-related increases in CA at 18 and 26 hr it did not induce CA at 10 hrs.

 

- Tates & Natarajan (1976) observed a clear dose response for CNU-ethanol-induced CA in bone marrow 1 day after ip dosing, but after 2 days there was no clear dose-response.

 

- Aydemir & Bilaloğlu (2003) showed that the anti-cancer drug topotecan induced dose-related CA at both 6 and 24 hr after a single ip dose. However, although gemcitabine induced dose-related increases in bone marrow CA at 24 hr, it did not induce any CA at 6 hr.

 

- There are also numerous examples of genotoxins inducing micronuclei (MN) at some sampling times but not at others. For example, Sutou et al. (1990), reporting on a collaborative study by the MMS subgroup of JEMS, showed that ethylnitrosurea, ethyl methanesulphonate, ARA-C and benzene all induced dose-related increases in bone marrow MN at 6 and 24 hr but not at 48 or 72 hr sampling times after 2 or 4 doses. Thus, potent genotoxins may show dose-related increases in CA at several different sampling times, but in many cases, they only show dose-related effects at limited sampling times due to cell-cycle specific effects, death of damaged cells etc. Given the overall toxicokinetic and genotoxic profile of sulfites (i.e. they are rapidly metabolised by sulfite oxidase and do not show positive findings in multiple test systems) it is unlikely that it would be expected to produce dose-related increases in CA at multiple sampling times. Thus, although there appears to be a significant induction of CA by potassium disulfite in this study, there are concerns, as discussed above, which render this publication not reliable [RL-3] for hazard assessment purposes due to lack of credibility.

 

In a dominant lethal assay conducted by SRI International (Author unknown, 1979), the induction of dominant lethal mutations in rat after sodium bisulfite (aka sodium hydrogensulfite) administration was investigated. Male Sprague-Dawley rats (53 to 62 days old, bodyweight 247-339 g) were given sodium bisulfite (purity not stated) in diet, ad libitum at doses of 45 (maximum tolerated dose), 15 and 4.5 mg/kg/day over a period of 10 weeks. Animals in the positive control group received triethylenemelamine (TEM). After the 10-week treatment period, 40 male rats from the vehicle control group and 20 male rats from each treatment group were selected and mated with two adult virgin females for seven days. These females were replaced with two new females for an additional 7-day mating period. Each female was sacrificed 15-19 days after the first day of cohabitation. At the end of the 10-week treatment period, body weight gains did not show a dose-response effect. The weight gains ranged from 3% below to 8% above control values. The dominant lethal test produced no consistent responses to suggest that sodium bisulfite is mutagenic to rats. Although the overall reporting quality fulfils the criteria for its use in the chemicals safety assessment, the method is of limited relevance (as detailed in the introductory remarks above). The reference is considered reliable with restriction [RL-2] but only as supporting information in a weight of evidence assessment.

 

In a range of genetic toxicity tests, sodium metabisulfite (purity not stated) was tested in an in vivo cytogenicity and dominant lethal assay in rats (Author unknown, 1972). According to the authors, Sodium metabisulfite induced no adverse effect on metaphase chromosomes from rat bone marrow at any of the dose levels or time periods tested. Moreover, no consistent, statistically significant (at p<0.01, p<0.05, and p<0.1) responses occurred to suggest that sodium metabisulfite is mutagenic to the rat. The reference only contains data on the results (including individual raw data). There is a complete lack of information on (i) the test animals such as strain, source, age, bodyweight at study initiation (ii) housing and feeding conditions (iii) test item characterisation such as purity, impurity, vehicle, stability (iv) evaluation criteria. Although the results of the cytogenicity tests were available in detail, this reference cannot be rated [RL-4] due to the complete lack of administrative information and material and methods descriptions. Therefore, the reference is disregarded for the hazard assessment.

Pal, B.B. and Bhunya, S.P. (1992) investigated the clastogenic effect of sodium disulfite (aka disodium disulfite) in a bone marrow chromosome aberration and micronucleus test in mouse. Albino Swiss mice were given doses of 200, 300 and 400 mg/kg bw via subcutaneous, intraperitoneal injection or oral administration. The maximum dose was determined according to the authors via “trial and error method” (not further qualified). A total of 4-6 animals of unknown sex were used per dose group, control groups contained 6 or 10 animals. The animals were given the test item via different routes and different doses as follows:

 

chromosome aberration test:

1.      i.p. injection of 400 mg/kg bw, animals were sacrificed after 6, 24 and 48 hours

2.      i.p. injection of 200, 300 mg/kg bw, animals were sacrificed after 24 hours

3.      s.c. and p.o. administration of 400 mg/kg bw, animals were sacrificed after 24 hours

4.      i.p. injection of 400 mg/kg bw divided into 5 equal parts administered in 24-hour intervals, animals were sacrificed 24 hours after last injection

micronucleus test:

5.      i.p. injection of 400 mg/kg bw divided into 2 equal parts administered in 24hr intervals, animals were sacrificed 24 hours after last injection

 

Chromosomal slides were prepared by “colchicine-sodium citrate-acetic acid-alcohol-flame drying-Giemsa schedule”. A total of 75 metaphases were scored per animal. For the micronucleus experiments, the number of MN PCE and NCE was determined by scoring 1000 PCE and NCE per animal. Authors conclude a positive outcome for the chromosomal aberration and micronucleus experiments. However, this interpretation is questioned, namely:

 

- the aberration frequency is given including gaps, which are normally not considered in the assessment of a positive response. When excluding gaps, all doses, time points and applications routes do not show any significant increase in aberration frequencies whatsoever, thus in fact leading to a “negative” conclusion. No distinction was made differentiating the type of chromosomal aberrations.

 

- the elevated levels of MN PCE at 300 mg/kg bw (0.60 ±0.11) do not show a dose dependency and the values for the 200 and 400 mg/kg bw dose group (0.25% ±0.03, 0.43% ±0.17) are within or very close to the historical control values for a number of other labs (0.02%-0.38%). Due to the low number of animals used in the experiments, the lack of dose-dependency and the lack of a correlate with the chromosome aberration experiments, the elevated MN PCE levels in the 300 mg/kg bw dose group is considered incidental and lacking biological relevance.

 

- cytotoxicity was not measured for the chromosomal aberration and micronucleus experiments, likewise the report is lacking records of systemic toxicity in the test animals. It is therefore not possible to correlate clastogenic effects with cytotoxicity.

 

- the experimental procedures are very briefly described or completely lacking. According to the authors the maximum tolerated dose was determined using “trial and error method” without specifying further under which exposure conditions, duration, group size this was determined. A description of the experimental preparation for slide preparation and scoring of chromosomes is missing.

 

- basic information on animal source, health status, housing and treatment conditions were not given

Based on the comments made above the reference is considered not reliable [RL-3], since it is showing a poor reporting and experimental quality. In addition, the authors’ conclusion of a clear clastogenic finding in the chromosomal aberration experiments must be disputed.

 

DNA damage

Sulfites

Carvalho et al. (2011) tested sodium disulfite (aka disodium disulfite) in a combined in vivo comet and micronucleus assay in CF1 mice after single oral administration. The maximum tolerated dose was determined in a previous experiment in 6 mice at doses of 0.5, 1 and 2 g/kg bw via gavage. In the main study 10 mice per group (five females and five males) were treated for 24 h with a single dose by gavage (0.1 ml/10 g body weight): (a) sodium disulfite (0.5, 1, or 2 g/kg b.w.); (b) negative control group: water; (c) positive control group: cyclophosphamide, 25 mg/kg. The alkaline comet assay was performed in peripheral blood, bone marrow and liver samples obtained 24 hours post exposure. Comets were stained via silver staining. Damage index (DI) was assessed by visually examining the tail size of each cell, ranging from 0 (no tail) to 4 (maximum-length tails). The damage index was calculated by multiplying the DI with the number of cells scored, i.e. a maximum of 400 could theoretically be reached (100 cells scored x 4 (maximum damage index)). A significant increase was observed on both damage index and damage frequency values, when comparing 1 and 2 g/kg doses to negative control, for all tissues. The positive findings appear plausible. However there are reasons to question these findings: (i) silver staining is not specific for DNA, since silver cations also interact with all negatively charged functional groups in biomolecules, such as proteins, DNA, RNA. It is therefore unclear whether the measured tail was specific to DNA damage. A DNA-specific fluorophore was not used. (ii) DNA damage is usually expressed either in tail length, DNA content in tail or tail moment. Crude visual inspection is prone to subjective interpretation by the experimenter which may result in a significant intra-day and day to day variation. The metric for DNA damage should be measured using (i) micrometer in eyepieces (ii) ruler on photographs or (iii) a computerised automated system, so ensure consistent and reproducible interpretation of DNA tails. Due to the mentioned ambiguities, it is questionable whether a true substance induced DNA damage was observed in the comet assay. The study is considered to be not reliable.

Summary entry - DNA damage in vivo

The comet assay is a powerful tool to detect even low levels of DNA damage. However, this assay is also prone to positive findings not caused by the test item but by e.g. inappropriate test design. Minimum quality criteria were used to rate the comet study for its relevance in chemical safety assessment. The criteria as published by Tice et al. (2000) were used for this screening. In case a reference does not fulfil the criteria stated therein, it was rated as not rateable [RL-3] and not further considered for chemical safety assessment purposes.

 

Meng, Z. (2004, identical with Meng, Z. et al. (2005) Damage effects of sulfite sodium on DNA in cells from mice various organs. Food Sci. China 26, 203-205) investigated the DNA damage of a 3:1 sodium sulfite and sodium bisulfite (aka sodium hydrogensulfite) mixture in male mice via comet assay. Three groups of six male mice each received an i.p. dose of a mixture of sodium sulfite and sodium bisulfite (3:1 M/M) (125, 250 or 500 mg/kg bw) in 200 mL of 0.9% sodium chloride daily for 7 days, maximum dose equals the half LD50. No positive control substance was applied. Animals were killed 24 hours after final administration, assumed target organs (brain, lung, heart, liver, stomach, spleen, thymus and kidney) were removed and single cell suspensions were prepared. Cytotoxicity was determined via Trypan blue exclusion. Cells were plated in low-melting agarose, lysed and a DNA unwinding step in alkaline buffer was performed prior electrophoresis. After electrophoresis, DNA was stained with ethidium bromide. Images of 25 randomly selected cells were analysed from each slide investigated (two slides per mouse). For each group (six male mice), 300 cells were scored. Tail moment was used for the evaluation of the DNA damage. There were no signs of toxicity in either treatment group and viability of target organ cells was > 95% after isolation. The sulfite mixture significantly increased the tail moment of DNA in cells from all organs tested at all doses tested.

 

Conclusion on in vivo genetic toxicity

The in vivo data for sulfites present an inconsistent pattern. In studies administering sulfites via subcutaneous injection or the oral route, neither rats nor mice showed an increase of micronuclei in the bone marrow or an increase of dominant lethal mutations. Doses were limited by toxicity. In contrast, some studies involving intraperitoneal and oral administration appear to show a positive response. However, upon careful evaluation, the findings in these studies appear to lack biological plausibility, and also suffer from several methodological deficiencies. By weight-of-evidence, it is therefore concluded that sulfites do not cause clastogenic or aneugenic events in animals.

Due to the rapid degradation of sodium dithionate under physiological conditions (disproportionation, oxidation) to inter alia sulfites/hydrogensulfites and thiosulfates, it can scientifically be justified that the results also apply to the substance itself. Thus, it is therefore concluded that sodium dithionite will not cause clastogenic or aneugenic events in animals.

 

Conclusion on genetic toxicity

The available data on the genetic toxicity of sodium dithionite supported by the available on the read-across “sulfite substances” allow a conclusive statement on the genetic toxicity for sodium dithionite.

In the key information on in vitro genotoxicity on sodium dithionite there was no evidence for a potential to induce gene, chromosome, or genome mutations in the test systems used. This finding is further substantiated by the exclusively negative results reported for other category group substances, i.e. sulfites and thiosulfates in key information on gene, chromosome, and genome mutations. Consequently, based on the consistently negative results in genotoxicity assays for sodium dithionite and other category groups substances, sodium dithionite is considered non-mutagenic in suitable in vitro test systems.

For the in vivo genotoxicity, information is available only for the sulfites as part of the category groups of the read-across approach. Irrespective of the reporting quality of the publications, both positive and negative findings are reported in in vivo test systems, thus requiring a weight-of-evidence approach. Following rigorous relevance and reliability screening, a high-quality in vivo study with sodium sulfite was identified. In this study, sodium sulfite administered via subcutaneous injection in mice did not show an increase of micronuclei formation up to the maximum tolerated dose. This finding is supported by a negative dominant lethal test in rats after single and repeated oral administration (feed) in rats. A number of in vivo clastogenicity studies were assessed as being of limited reliability, since these exhibit reporting and/or other experimental deficiencies and lack biological plausibility.

Moreover, the genotoxicity of sulfites, as part of the category groups of the read-across concept, were most recently reviewed by other reputable scientific organisations:

This absence of a genotoxic concern is also confirmed by the EFSA panel after review of more than 60 studies with sulfur dioxide, sodium sulfite, sodium bisulfite, sodium metabisulfite and potassium metabisulfite (EFSA, 2016), with an overall conclusion as follows: “Overall, based on these data the Panel concluded that the use of sulfur dioxide and sulfites (sodium sulfite, sodium bisulfite, sodium metabisulfite, potassium metabisulfite, potassium bisulfite, calcium sulfite and calcium bisulfite) as food additives does not raise a concern with respect to genotoxicity.”

Disodium disulfite (synonym sodium metabisulfite) was subject to a recent Substance Evaluation as required by REACH Article 48 for disodium disulfite (EC No 231-673-0, CAS No 7681-57-4) by the Evaluating Member State Hungary. In their concluding report dated 30. October 2015, the following conclusion concerning the endpoint genetic toxicity was drawn by the eMS: “the evaluating Member State is of the opinion that there is very vague and inconsistent evidence of induction of genetic toxicity with relevance to humans for sulphites, and considers, based upon the available information, that the concern for mutagenicity is no longer substantiated. Thus, also classification for mutagenicity seems not warranted”.

Overall, based on the extensive in vitro data base and the supporting information from a high-quality in vivo study, there is no evidence for the induction of genetic toxicity with relevance to humans for sodium dithionite, thiosulfates, and sulfites. This conclusion is also substantiated by the conclusion of other reputable scientific organisations.

In addition to the information directly addressing the endpoint genetic toxicity, the following information should be taken into account when discussing the genetic toxicity of sulfites:

 

1. the environmental and physiological half-life is highly dependent on the solvent/vehicle, oxidation status, light and temperature; for example, under environmentally relevant conditions, sulfites are already readily oxidised to sulfate showing a half-life of 16 hours in seawater.

 

2. due to its endogenous occurrence in all mammalian species, almost all tissues show sulfite oxidase activity, with very high activities in liver, kidney and heart, whereas spleen, testes and brain show lower sulfite oxidase activity. A total background serum level in humans of 4.9 µmol sulfite/L was measured for both sexes. The overall sulfite oxidase capacity is very high in mammalian species, with a theoretical maximum oxidation rate of 750 mmol/kg/day (equivalent to 56 g of SO32-/kg/day). The total urinary excretion of sulfate resulting from sulfite metabolism was estimated to be 25mmol out of which the majority was generated from endogenous sulfite (24mmol). This amount is approx. 1000-fold the total amount of sulfite being enzymatically processed per day (assuming a total bool volume of 5L per individual).

 

3. due to their instability in an aqueous environment and their very rapid oxidation and elimination in animals and humans, it appears highly unlikely that sulfites show adverse systemic effects, when administered via physiological routes. This hypothesis is substantiated by a complete lack of adverse effects in long-term animal studies as well as in direct observations in humans:

- in a 3-generation reproductive toxicity study, groups of 20 male and 20 female Wistar rats were given doses of up to 955 mg/kg bw/day via diet over a period of 2 years. There were no signs of systemic toxicity, resulting in a NOAEL above the maximum dose of 955 mg/kg bw/day.

- taking together the results from the three animal studies on sodium and potassium metabisulfite (Tanaka et al., 1979; Til et al., 1972; Feron and Wensfoort, 1972) there was no indication that metabisulfite had any carcinogenic effect

 

- 4 reliable studies on pulp and paper mill workers were available (Milham and Demers, 1984; Robinson, et al. 1986; Anderson, et al. 1998; Rix, et al. 1997), see section 7.10.2., based upon which no carcinogenic activity must be expected for the sulfites

 

The use of sulfites as nutritional supplement and preservative in cosmetic formulations has been repeatedly reviewed by national and international authorities, concluding:

 

“Sodium metabisulfite and other sulfites are listed as GRAS (Generally Recognized as Safe) by the FDA (Food and Drug Administration) as preservatives in certain foods. Sodium metabisulfite is also used up to a concentration of 1% as an antioxidant in hair care products and as a reducing agent in cosmetic formulations (CIR 2003). [...] Sulfur dioxide and sodium metabisulfite are currently not classifiable (Group 3) as to their carcinogenicity to humans (IARC 1992). [...] Conclusions of the OECD SIDS report indicated 2% sodium metabisulfite via feed (20,000 ppm or 1,000 mg/kg/day) for 104 weeks was not carcinogenic in Wistar rats.” (EPA, 2009)

 

“The CIR expert panel concluded that sodium sulfite, potassium sulfite, ammonium sulfite, sodium bisulfite, ammonium bisulfite, sodium metabisulfite and potassium metabisulfite are safe as used in cosmetic formulations.” (CIR, 2003)

 

“Existing mutagenicity studies support information on different categories of biological endpoints. Gene mutations were analysed in a couple of in vitro tests (Ames-test with Salmonella typhimurium and E. coli, tests with yeasts). Chromosomal aberration and sister chromatid exchange (SCE) were tested in vitro (in human blood lymphocytes) as well as in vivo (micronucleus-test in mice and rats). The induction of unscheduled DNA synthesis (UDS) was analysed in rat hepatocytes after in vivo application. Dominant lethal assays in mice and rats completed the spectrum of different mutagenicity tests. Neither sodium sulfite and sodium metabisulfite nor potassium metabisulfite were found to be genotoxic in the test battery. Some of the in vitro assays performed with sodium bisulfite showed positive results, especially in the Ames test and in the chromosome aberration test. However, none of the in vivo tests have shown a genotoxic potential. To further investigate the above mentioned positive in vitro results found with sodium bisulfite, an UDS in rats and a micronucleus test in mice were added. To be sure that the bisulfite and not the disulfite was tested, an appropriate pH-value was maintained by a buffer system. Both additional tests were negative. Summarising results of the available mutagenicity tests, genotoxic potential of the specified inorganic sulfites, bisulfites and metabisulfites seems to be very unlikely.” (SCCNFP, 2003)

 

References

- Aydemir, N., Bilaloğlu, R., 2003. Genotoxicity of two anticancer drugs, gemcitabine and topotecan, in mouse bone marrow in vivo. Mutat Res. 9;537(1):43-51.

- McFee, A.F., Tice, R.R., 1990. Influence of treatment to sacrifice time and the presence of BrdUrd on chemically-induced aberration rates in mouse marrow cells. Mutat Res. 241(1): 95-108.

- Natarajan, A.T., Tates, A.D., Van Buul, P.P., Meijers, M., De Vogel, N., 1976. Cytogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes. Mutat Res. 37(1):83-9.

- Sutou, S., Mitui, Y., Toda, S., Sekijima, M., Kawasaki, K.,Kawata, N., Abe, S.,Iwai, M., Arimura, H., 1990. Effect of multiple dosing of phenacetin on micronucleus induction: A supplement to the International and Japanese cooperative studies. Mutation Research Letters 245(1): 11-14.

- Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F., 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 35(3):206-21.

- EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources Added to Food), 2016. Scientific Opinion on the re-evaluation sulfur dioxide (E 220), sodium sulfite (E 221), sodium bisulfite (E 222), sodium metabisulfite (E 223), potassium metabisulfite (E 224), calcium sulfite (E 226), calcium bisulfite (E 227) and potassium bisulfite (E 228) as food additives. EFSA Journal 2016;14(4):4438 151 pp.

- US EPA 2007. Registration Eligibility Decision- Inorganic Sulfites. Special Review and Reregistration Division Office of Pesticide Programs.

- Nair, B.; Elmore, A.R., 2003. Final report and the safety assessment sodium sulfite, potassium sulfite, ammonium sulfite, sodium bisulfite, ammonium bisulfite, sodium metabisulfite and potassium metabisulfite (Cosmetic Ingredient Review). Int. J. Toxicol. 22, 63-88

- The Scientific Committee on Cosmetic Products and non-Food Products (SCCNFP) intended for consumers opinion concerning Inorganic Sulfites and Bisulfites, 2003. Adopted at its 23rd plenary meeting of 18 March 2003.

Justification for classification or non-classification

According to regulation (EC) 1272/2008 (CLP), as amended, substances shall be classified for the endpoint germ cell mutagenicity, in case they may cause mutations in the germ cells of humans that can be transmitted to the progeny. The classification shall be based on the total weight of evidence available, using expert judgment and the relevance of the route of exposure used in the study of the substance compared to the most likely route of human exposure shall also be taken into account.

The available data on genetic toxicity allow a conclusive statement on the genetic toxicity for sodium dithionite based on substance-specific data and data for sulfites and thiosulfates as part of the read-across approach.

In the key information on the in vitro genotoxicity of sodium dithionite, there was no evidence for a potential to induce gene, chromosome, or genome mutations in the test systems used. This finding is further substantiated by the exclusively negative results reported for other category group substances, i.e. sulfites and thiosulfates in key information on gene, chromosome, and genome mutations. Consequently, based on the consistently negative results in genotoxicity assays for sodium dithionite and other category groups substances, sodium dithionite is considered non-mutagenic in suitable in vitro test systems.

For the in vivo genotoxicity, information is available only for the sulfites as part of the category groups of the read-across approach. Irrespective of the reporting quality of the publications, both positive and negative findings are reported in in vivo test systems, thus requiring a weight-of-evidence approach. Following rigorous relevance and reliability screening, a high-quality in vivo study with sodium sulfite was identified. In this study, sodium sulfite administered via subcutaneous injection in mice did not show an increase of micronuclei formation up to the maximum tolerated dose. This finding is supported by a negative dominant lethal test in rats after single and repeated oral administration (feed) in rats. A number of in vivo clastogenicity studies were assessed as being of limited reliability, since these exhibit reporting and/or other experimental deficiencies and lack biological plausibility.

Furthermore, the genotoxicity of inorganic sulfite substances (as part of the category groups of the read-across concept) was already recently reviewed by other reputable scientific organisations (incl. EFSA (2016) and Substance Evaluation by the Evaluating Member State Hungary (2015)), all concluding on an absence of concern for genotoxicity.

Overall, based on the extensive in vitro data base and the supporting weight-of-evidence information from in vivo studies, there is no evidence for the induction of genetic toxicity with relevance to humans for sodium dithionite, sulfites, and thiosulfates. Therefore, the classification criteria as laid down in regulation (EC) 1272/2008 are not met and sodium dithionite does not require classification as a germ cell mutagen.