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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The available data on genetic toxicity allow a conclusive statement on the genetic toxicity for thiosulfates and contain read-across information from different thiosulfates, i.e. sodium thiosulfate and ammonium thiosulfate (details see below).

Sodium 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.

Ammonium thiosulfate:The in vitro genotoxicity of ammonium thiosulfate has been evaluated in a bacterial gene mutation assay (acc. to OECD TG 471, 1997), in a mammalian cell gene mutation assay (acc. to OECD 476, 1997), and in a chromosomal aberration test (acc. to OECD TG 473, 1997). These studies were performed according to the current OECD guidelines in force at the time of conduct and in compliance with GLP. These studies are considered to be reliable without restrictions.

Link to relevant study records

Referenceopen allclose all

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 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
- 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.
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:
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.

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

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.

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).
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 - 27 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 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.
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:
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:
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:
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:
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 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:
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:
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

Endpoint conclusion
Endpoint conclusion:
no study available

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++SO32-   2HSO3-<->H2O +S2O52-

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 S2O42-+ H2O→2HSO3-+ S2O32-

 [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 (S2O32-); 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+ H2ONa2S2O3+ 2 NaHSO3(anaerobic conditions) (1), as described above

 

Na2S2O4+ O2+ H2ONaHSO4+ 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 S2O42-+ 6 OH-5 SO32-+ S2-+ 3H2O

weakly alkaline to weakly acidic medium: 2 S2O42-+ H2O2 HSO3-+ S2O32-

acidic medium: 2 H2S2O43 SO2+ S + 2 H2O

strongly acidic medium: 3 H2S2O45 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

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

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

Genetic toxicity in vitro

Gene mutation in bacteria

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].

 

Gene mutation in mammalian cells

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 S9, 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].

 

In vitro clastogenicity and aneugenicity

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].

 

Conclusion on in vitro genotoxicity

There was no evidence whatsoever for any mutagenic activity of thiosulfates in the bacterial reverse mutation assay, HPRT assay, or cytogenicity studies (micronucleus test and chromosome aberration assay) up to the recommended maximum concentration. Consequently, thiosulfates are 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 thiosulfates does not require classification as a germ cell mutagen.

Justification for classification or non-classification

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

Genotoxicity studies with thiosulfates did not show any effects in bacterial reverse mutation, mammalian cell gene mutation (HPRT assay), and cytogenicity (chromosomal aberration test and micronucleus test) tests. Thus, there is no evidence for a potential to induce gene, chromosome, or genome mutations in the test systems used.

 

The classification criteria acc. to regulation (EC) 1272/2008 as germ cell mutagen are not met, thus no classification is required.