Disodium peroxodisulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Disodium persulfate did not show any mutagenic properties in two bacterial reverse mutation assays and a study covering unscheduled DNA synthesis. An in vitro chromosome aberration assay was not performed due to the fact that an adequate in vivo study is available with disodium persulfate.

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:

02-05-1990 to 13-08-1990

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: FIFRA Guideline 84-1

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

A Salmonella Mammalian-Mircosome Mutagenicity Assay (Ames test) was conducted with sodium persulfate, E608244, Lot 0021. Five tester strains of Salmonella typhimurium: TA98, TA100, TA1535, TA1537 and TA1538 were utilized. The assay was conducted in the presence and absence of metabolic activation by Aroclor 1254 induced rat liver microsomes.

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254

Test concentrations with justification for top dose:

The test concentrations were: 10000, 3333, 1000, 333, 100 µg/plate.

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, except sodium azide (sterile, deionized water)

True negative controls:

yes

Positive controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For the S. typhimurium TA98, TA1538 without metabolic activation

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For the S. typhimurium TA100 and TA1535 without metabolic activation.

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For the S. typhimurium TA1537 without metabolic activation.

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For the S. typhimurium TA98, TA100, TA1535, TA 1537, TA 1538 with metabolic activation.

Details on test system and experimental conditions:

Five doses of the test article were plated with all five tester strains (TA98, TA100, TA 1535, TA1537, TA1538) with and without metabolic activation. All solvent controls and test article doses were plated in triplicate. Without metabolic activation, 100 µL of tester strain and 50 µL of solvent or test article and 0.5 of S-9 mix were added to 2.0 mL of molten selective top agar at 45 ± 2 °C. After vortexing, the mixture was overlayed onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 - 72 hours at 37 ± 3 °C. Plates which were not counted immediately following the incubation period were stored at 4 ± 2 °C until such time that colony counting could be conducted.

Evaluation criteria:

Evaluation of mutagenicity assay data:
For a test article to be considered positive, it must cause at least a doubling in the mean number of revertants per plate of at least one strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article. In those cases where the observed dose response increase in TA1537 revertants per plate is less than three-fold, the response must be reproducible.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Two independent mutagenicity assays were performed.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the study experimental conditions, disodium persulfate did not cause a positive response in any of tester strains with or without metabolic activation. Therefore, disodium persulfate was considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The purpose of the study was to establish the potential of disodium persulfate to induce gene mutations in Salmonella typhimurium: TA98, TA100, TA1535, TA1537 and TA1538, using Salmonella/Mammalian-Microsome plate incorporation mutagenicity Assay (Ames test), performed according to FIFRA Guideline 84 -1. Disodium persulfate was tested at five dose levels ranging from 100 to 10000 µg/plate. The dose levels were based on a preliminary toxicity test. The assay was conducted in the presence and absence of metabolic activation by Aroclor 1254 induced rat liver microsomes (S9 Mix). Revertant colonies were counted. During the tests positive and negative controls were run concurrently. The reference mutagens (sodium azide, 9 -aminoacridine, 2 -nitrofluorene, 2 -anthramine) showed a distinct increase of induced relevant colonies. Results with the test substance showed that disodium persulfate did not cause a positive response in any of the tester strains with or without metabolic activation. Therefore, disodium persulfate was considered non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-16 to 2017-10-26

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:

21st July, 1997

Deviations:

yes

Remarks:

only one strain tested

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

yes

Remarks:

only one strain tested

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

yes

Remarks:

only one strain tested

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use

Version / remarks:

June 2012

Deviations:

yes

Remarks:

only one strain tested

Principles of method if other than guideline:

Only one strain was tested as requested by ECHA. An older study with Disodium peroxodisulphate (SPS) does include 5 bacterial strains; however non of these strains does detect cross-linking mutagens. Therefore an additional bacterial reverse mutation assay was performed using only Escherichia coli WP2 uvrA. This study was designed to fill the data gap for the detection of cross linking mutagens.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).
±S9 Mix: 5000; 160; 500; 160; 50 and 16 μg/plate (Experiment I - plate incorporation method)
±S9 Mix: 5000; 160; 500; 160; 50 and 16 μg/plate (Experiment II - pre-incubation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ultrapure water (ASTM Type I) was applied as vehicle of the test item and the positive control substance MMS (Methyl methanesulfonate); and DMSO was applied as vehicle for positive control substances 2AA.
- Justification for choice of solvent/vehicle: In the study two vehicle control groups were used depending on the solubility of the test item and the solubility of positive control chemicals.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2AA (2-Aminoanthracene)

Remarks:

With metabolic activation (+S9), 50 µg

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without Metabolic activation (-S9), 2 µL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 hours in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
The toxicity of the test item was determined with Escherichia coli WP2 uvrA in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each.

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response (the number of reversions is at least three times higher than the reversion rate of the vehicle control) for at least one of the dose groups occurs in the examined strain with or without metabolic activation.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- The Escherichia coli WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 109 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at the tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- clearing or diminution of the background lawn (reduced background lawn development occurs).

Species / strain:

S. typhimurium TA 100

Remarks:

Please refer to Key 002-Genetic toxicity in vitro-Ames.FMC, 1990

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

Please refer to Key 002-Genetic toxicity in vitro-Ames.FMC, 1990

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Please refer to Key 002-Genetic toxicity in vitro-Ames.FMC, 1990

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Please refer to Key 002-Genetic toxicity in vitro-Ames.FMC, 1990

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

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 was observed on the plates in the examined bacterial strain at any examined concentration level (±S9 Mix) throughout the study.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with Escherichia coli WP2 uvrA in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
The revertant colony numbers of vehicle control plates in the examined strain with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies.
In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
No precipitation of the test item was observed on the plates in the examined bacterial strain at any examined concentration levels (±S9 Mix).

HISTORICAL CONTROL DATA (Please refer to "Any other information on results incl.tables")
In the Initial Mutation Test all of the obtained higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control) remained within the corresponding historical control data ranges, were far below the biologically relevant threshold for being positive (3 fold higher mutation rate of control) and were considered to reflect the biological variability of the applied test system.
In the Confirmatory Mutation Test all of the decreased revertant colony numbers (when compared to the revertant colony numbers of the vehicle control) remained in the corresponding historical control data ranges of the ultrapure water vehicle control, and were without any biological significance. The background lawn development was not affected in any case.
In the performed experiments the revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in the different experimental phases were slightly higher or lower than the ultrapure water control plates. The higher or lower revertant counts of these controls remained in the historical control data ranges.

Table 1: Summary Table of the Results of the Concentration Range Finding Test

Concentration Range Finding Test (Informatory Toxicity Test)
Concentrations (µg/plate)	Escherichia coli WP2 uvrA
	-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR
Untreated Control	32.0	1.09	29.0	0.83
DMSO Control	-	-	34.7	1.00
Ultrapure Water Control	29.3	1.00	35.0	1.00
5000	25.7	0.88	28.0	0.80
1600	33.3	1.14	31.7	0.90
500	33.7	1.15	31.3	0.90
160	28.7	0.98	33.7	0.96
50	32.7	1.11	33.3	0.95
16	29.0	0.99	29.0	0.83
5	36.3	1.24	33.7	0.96
MMS (2 µL)	773.3	26.36	-	-
2AA (50 µg)	-	-	220.7	6.37

MR: Mutation Rate

Remarks:Ultrapure water was applied as vehicle of the test item and the positive control substance: MMS and the DMSO was applied as vehicle for positive control substance: 2AA. The mutation rate of the test item, MMS and untreated control is given referring to the ultrapure water; the mutation rate of 2AA is given referring to DMSO.

 

Table 2: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (µg/plate)	Escherichia coli WP2 uvrA
	-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR
Untreated Control	29.0	1.02	37.3	0.94
DMSO Control	-	-	38.0	1.00
Ultrapure Water Control	28.3	1.00	39.7	1.00
5000	28.3	1.00	29.0	0.73
1600	34.3	1.21	40.0	1.01
500	32.0	1.13	41.0	1.03
160	32.7	1.15	32.7	0.82
50	33.3	1.18	32.3	0.82
16	34.7	1.22	33.7	0.85
MMS (2 µL)	768.0	27.11	-	-
2AA (50 µg)	-	-	174.0	4.58

MR:Mutation Rate

Remarks:Ultrapure water was applied as vehicle of the test item and the positive control substance: MMS and the DMSO was applied as vehicle for positive control substance: 2AA. The mutation rate of the test item, MMS and untreated control is given referring to the ultrapure water; the mutation rate of 2AA is given referring to DMSO.

Table 3: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (µg/plate)	Escherichia coli WP2 uvrA
	-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR
Untreated Control	29.3	0.86	38.7	0.84
DMSO Control	-	-	50.7	1.00
Ultrapure Water Control	34.0	1.00	46.0	1.00
5000	16.7	0.49	36.7	0.80
1600	31.0	0.91	45.0	0.98
500	27.7	0.81	42.0	0.91
160	31.0	0.91	40.3	0.88
50	31.7	0.93	42.0	0.91
16	27.3	0.80	33.3	0.72
MMS (2 µL)	750.7	22.08	-	-
2AA (50 µg)	-	-	180.7	3.57

MR:Mutation Rate

Remarks:Ultrapure water was applied as vehicle of the test item and the positive control substance: MMS and the DMSO was applied as vehicle for positive control substance: 2AA. The mutation rate of the test item, MMS and untreated control is given referring to the ultrapure water; the mutation rate of 2AA is given referring to DMSO.

 

Table 4: Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

		Bacterial stain
Historical control data for untreated control	-S9		E. coli
		Average	25.4
		SD	5.2
		Minimum	11
		Maximum	45
	+S9		E. coli
		Average	33.9
		SD	5.2
		Minimum	17
		Maximum	56
		Bacterial Strain
Historical control data of DMSO control	-S9		E. coli
		Average	24.7
		SD	4.6
		Minimum	11
		Maximum	45
	+S9		E. coli
		Average	33.7
		SD	5.0
		Minimum	16
		Maximum	57
		Bacterial Strain
Historical control data of Water control	-S9		E. coli
		Average	26.1
		SD	5.5
		Minimum	12
		Maximum	48
	+S9		E. coli
		Average	34.9
		SD	4.9
		Minimum	18
		Maximum	57
Historical control data of positive controls	-S9		E. coli
		Average	724.5
		SD	65.0
		Minimum	320
		Maximum	1313
	+S9		E. coli
		Average	257.7
		SD	72.5
		Minimum	140
		Maximum	477

 Abbreviations: E. coli: Escherichia coli WP2uvrA; SD: Standard deviation

Conclusions:

The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by base-pair substitution in the genome of the Escherichia coli WP2 uvrA used. Therefore, Disodium peroxodisulphate (SPS) is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay according OECD guideline 471, EU method B.13/14 and EPA OPPTS 789.5100 was performed to investigate the mutagenic potential of Disodium peroxodisulphate (SPS)  in two independent experiments, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test).
The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: 5000, 1600, 500, 160, 50 and 16 μg/plate. Each assay was conducted with and without metabolic activation (±S9 Mix).  The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strain, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of the investigated Escherichia coli WP2 uvrA tester strain following treatment with Disodium peroxodisulphate (SPS) at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in the independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. No precipitation of the test item was observed on the plates in the examined bacterial strain at any examined concentration level (±S9 Mix) throughout the study.
The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by base-pair substitution in the genome of the Escherichia coli WP2 uvrA used. Therefore, Disodium peroxodisulphate (SPS) is considered non-mutagenic in this bacterial reverse mutation assay.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-05-1 to 1990-09-21

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Unscheduled DNA Synthesis

Species / strain / cell type:

hepatocytes: rat liver hepatocytes

Details on mammalian cell type (if applicable):

Primary rat hepatocytes derived from the livers of normal adult male Fischer 344 rats were used in this study. The procedure for obtaining rat hepatocytes was as follows: Each rat used was sacrificed by inhalation of metofane. The animal was dissected and the liver perfused first 0.5 mM EGTA solution and then with a collagenase solution. The liver was removed from the animal and the cells were dissociated, counted and seeded into 35 mm dishes (5x10E5 viable cells/dish). The cells were seeded in William's Medium E (WME) supplemented with 10 % foetal bovine serum, 2 mM L-glutamine and 50 µg/mL gentamicin (complete medium). The cultures were incubated at 37 ± 1 °C in a humidified % ± 1 % CO2 incubator for 90 to 180 minutes, washed with complete medium, referred with serum-free medium and used in the test.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

250, 150, 50, 15, 5 µg/mL (analytical concentrations)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

3.0 and 5.0 µg/mL

Details on test system and experimental conditions:

Preliminary Cytotoxicity Test:
A preliminary cytotoxicity test was performed to establish an appropriate dose range for the test article. Ten doses ranging from 0.15 to 5000 µg/mL were tested. Replicate cultures of rat hepatocytes were washed with complete medium and refed with serum-free medium 90-180 minutes after seeding. The cultures were then treated with the test article. Eighteen to twenty hours later, an aliquot of culture fluid was removed, centrifugated, and the level of lactic acid dehydrogenase (LDH) activity in the culture fluid determined. Two replicate plates were used for LDH measurement at each dose level the relative toxicities were obtained by comparing the LDH activity in the treated cultures to the LDH activity in the solvent control cultures.
Unscheduled DNA synthesis test:
The UDS assay was repeated once. In the first assay, a dose which exhibited toxicity and also had cells whose nuclear morphology allowed them to be evaluated for UDS was not obtained. Therefore, a second UDS assay was performed. The dose levels used in the second UDS assay were based on the results of the parallel cytotoxicity test from the first UDS assay. Three replicate plates seeded with 5 x 10E5 rat hepatocytes/plate were treated with eight concentrations (1.5 to 500 µg/mL) of sodium persulfate. WME, which was used to dissolve the test article, was used as the solvent control for the test article. DMBA, at 3.0 and 5.0 µg/mL, was used as the positive control. DMSO, which was used to dissolve DMBA, was used as the solvent control for the positive control. Each test article and control dish received 3H-thymidine at a final concentration of 10 µCi/mL. in parallel with the test plates, two cultures per dilution were treated with the test article and control compounds for a parallel toxicity test. For further information on test performance, see sect. "Any other information on materials and methods..."

Evaluation criteria:

The results of the study were evaluated according to the following criteria. If the mean net nuclear count was increased by at least five counts over the control, the results for a particular dose level were considered significant. A test article was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of the control. In the absence of a dose response, a test article which showed a significant increase in the mean net nuclear grain count in at least two successive doses was considered positive. If a test article showed a significant increase in the net nuclear grain count at one dose level without any dose response, the activity of the test article was considered to be equivocal. The test article was considered negative if no significant increase in the net nuclear grain counts at any dose level was observed.

Statistics:

For each treatment slide, the net nuclear counts were averaged and the standard deviation (S.D.) determined and recorded on a summary form. Also reported are the grand mean and S.D. for each dose level as well as the percent of cells in repair (cells with >= 5 net nuclear grains). Means, standard deviations and percent survivals were computed using a LOTUS 1-2-3 program on an IBM PC or compatible computer.

Key result

Species / strain:

hepatocytes: rat liver hepatocytes

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Microscopic examination of the hepatocyte cultures indicated high levels of toxicity at 500 and 400 µg/mL and moderate levels at 250 µg/mL. Normal cellular morphology was observed at lower concentrations. Microscopic examination of the fixed and stained cells indicated that cells exposed to 500 and 400 µg/mL could not be evaluated for UDS due to excessive toxicity. Cells exposed to 250 µg/mL exhibited signs of toxicity such as small and irregularly shaped nuclei. However, it was possible to find one hundred-fifty nuclei with acceptable morphology at the 250 µg/mL dose level to allow this dose level to be evaluated for UDS. The next four lower concentrations of 150, 50, 15 and 5.0 µg/mL were evaluated.

Remarks on result:

other: strain/cell type: rat liver hepatocytes from adult male Fischer 344 rats

The results of the UDS assay are summarized in the table.

Table: Summary of UDS assay with sodium persulfate

TREATMENT	RELATIVE SURVIVAL	SLIDE No.	PERCENT SCORABLE NUCLEI	NO. OF NUCLEI COUNTED	AVERAGE NET GRAINS PER NUCLEUS		S.D.	GRAND MEAN		S.D.	PERCENT CELL WIT 5 OR MORE NET NUCLEAR GRAINS
Sodium persulfate
500 ug/ml	41%	57A	0%	0	Too Toxic to be Evaluated for UDS
		57B	0%	0
		57C	0%	0
400 ug/ml	38%	55A	0%	0	Too Toxic to be Evaluated for UDS
		55B	0%	0
		55C	0%	0
250 ug/ml	84%	53A	64%	50	0.7	+/-	2.5	0.3	+/-	2.1	3%
		53B	32%	50	- 0.2	+/-	1.8
		53C	52%	50	0.3	+/-	2.0
150 ug/ml	99%	56A	92%	50	0.3	+/-	2.2	0.1	+/-	2.0	1%
		56B	88%	50	0.3	+/-	1.7
		56C	80%	50	- 0.3	+/-	2.0
50 ug/ml	100%	54A	80%	50	- 1.4	+/-	1.7	- 2.3	+/-	2.3	0%
		54B	84%	50	-2.6	+/-	1.8
		54C	88%	50	-3.1	+/-	2.9
15 ug/ml	99%	52A	96%	50	- 2.7	+/-	2.3	-2 2	+/-	2.3	0%
		52B	96%	50	- 1.6	+/-	2.1
		52C	88%	50	- 2.3	+/-	2.5
5.0 ug/ml	102%	58A	84%	50	- 1.8	+/-	2.6	- 1.1	+/-	2.1	0%
		58B	88%	50	- 0.6	+/-	2.1
		58C	84%	50	- 1.0	+/-	1.5
DMBA
5.0 ug/ml	94%	12A	60%	50	15.2	+/-	5.1	19.6*	+/-	7.5	100%
		12B	64%	50	23.9	+/-	7.9
		12C	68%	50	19.8	+/-	6.7
3.0 ug/ml	99%	16A	68%	50	19.1	+/-	5.8	20.1*	+/-	6.5	100%
		16B	76%	50	24.4	+/-	6.4
		16C	84%	50	16.7	+/-	4.9
DMSO (Solvent Control for DMBA)
10 ul/ml	100%	13A	100%	50	- 5.0	+/-	4.1	- 3.7	+/-	3.3	0%
		13B	92%	50	- 2.6	+/-	2.8
		13C	96%	50	- 3.5	+/-	2.5
WME (Solvent Control for Test Article)
	100%	11A	84%	50	- 3.7	+/-	3.7	- 3.8	+/-	3.3	3%
		11B	88%	50	- 3.8	+/-	2.6
		11C	80%	50	- 3.9	+/-	3.5

Relative survival = 100% - relative toxicity S.D. Standard Deviation * Significant (See Protocol: Section 8.0, Evaluation of Test Results) Slides treated with sodium persulfate or DMBA were compared to the appropriate solvent control. According to the criteria set for evaluating the test results, both doses of the positive control compound, DMBA, induced a significant increase in the average net nuclear count of silver grains. Although none of the test article doses caused a significant increase ( an increase of at least five grain counts over the solvent control) in the mean net nuclear counts, an apparent dose-related increase in net nuclear grain counts was observed. All criteria for a valid test were met.

Conclusions:

The results of the UDS assay indicated that under the test conditions, disodium persulfate did not cause a significant increase in the unscheduled DNA synthesis as measured by the mean number of net nuclear grain counts, at any dose level.

Executive summary:

Disodium persulfate was tested in the rat hepatocyte unscheduled DNA synthesis assay. The test substance was tested at eight dose levels ranging from 1.5 to 500 µg/mL and was fully evaluated at five dose levels of 5.0, 15, 50, 150 and 250 µg/mL. In this study the positive control, 7,12 -Dimethylbenz(a)anthracene (DMBA), induced significant increases in the mean number of net nuclear grain counts over that in the solvent control. All criteria for a valid test were met. The results of the UDS assay indicated that under the test conditions, disodium persulfate did not cause a significant increase in the unscheduled DNA synthesis as measured by the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the solvent control), at any dose level. Thus, the test substance was considered not mutagenic.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Results of in vivo tests with disodium persulfate showed no clastogenic effect in a mouse micronucleus test and no mutagenic activity in an in vivo unscheduled DNA synthesis assay in rat livers. Thus, disodium persulfate was considered not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-04-27 to 1990-09-21

Reliability:

1 (reliable without restriction)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The purpose of this study is to evaluate the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice.
In vivo cytogenetic analysis provides a valuable tool for the detection of chromosome breakage. Micronuclei usually arise from acentric chromosome fragments or whole chromosomes which are lost from the nucleus during replication of erythrocyte precursor cells and remain in the cytoplasm of young, anucleate (polychromatic) erythrocytes after expulsion of the nucleus. The increase in incidence of micronucleated cells has two primary causes, chromosome breakage and malfunction of the spindle apparatus.
The clastogenic potential of the test article is measured by its ability to significantly increase the incidence of micronucleated polychromatic erythrocytes when compared with control animals.
see also:
Heddle, J.A., et al.; Mut. Res. 123, pp 61 - 118, 1983;
Matter, B.E., Grauwiler, J.; Mut. Res. 23, pp 239 - 249, 1973.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley Inc., Frederick, MD or Charles River Breeding Laboratories, Kingston, NY or Raleigh, NC
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Toxicity studies: Males 24 - 34 grams; Females: 21 - 25 grams; Micronucleus assay: Males: 27 - 35 grams; Females. 21 - 27 g; all weights measured at dose administration
- Assigned to test groups randomly: yes
- Fasting period before study: not indicated
- Housing: five per cage in autoclavable plastic cages with hardwood chips as bedding material
- Diet: certified laboratory rodent chow ad libitum
- Water: tap water ad libitum
- Acclimation period: not less than 5 days


ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 26.7 °C
- Humidity: 50 ± 20 % R.H
- Air changes: not indicated
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

intraperitoneal

Vehicle:

Distilled water

Details on exposure:

For the initial toxicity study, animals were randomly assigned to five groups of five males and five females each. All animals were weighed immediately prior to dose administration.
For the micronucleus assay, the animals were assigned to thirteen experimental groups of five males and five females, each based on a computer-generated randomization program. An additional group of five males and five females were designated as replacement animals in the event of mortality prior to the scheduled sacrifice time and were dosed with the test article high dose level. Each animal was given a sequential number and identified by ear tag.
The test article-vehicle mixture or the vehicle alone were administered by IP injection at a constant rate of 10 mL/kg bw. The positive control, TEM, was injected IP at a dose of level of 0.25 mg/kg. All mice in the experimental and control groups were weighed immediately prior to dose administration and the dose volumes based on individual body weights . Animals were observed after dose administration for clinical signs of chemical effect.

Duration of treatment / exposure:

Scheduled sacrifice time after administration of the test article: 24; 48; 72 h

Frequency of treatment:

One treatment

Post exposure period:

24; 48; 72 h

Dose / conc.:

85 mg/kg bw/day (nominal)

Dose / conc.:

169 mg/kg bw/day (nominal)

Dose / conc.:

338 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

yes; TEM - Triethylene melamine; 0.25 mg/kg bw

Tissues and cell types examined:

Bone marrow of the femur

Details of tissue and slide preparation:

At the scheduled sacrifice time, five mice per sex were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL FBS. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded.

Evaluation criteria:

The test article is considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes is observed relative to the vehicle control (p <= 0.05, Kastenbaum-Bowman tables). The positive response must be dose-dependent or must be observed at a single dose level at adjacent sacrifice times. If a single treatment group is significantly elevated at one sacrifice time, the assay is considered a suspect or unconfirmed positive and a repeat assay will be recommended.

Statistics:

Statistical significance will be determined using the Kastenbaum-Bowman tables which are based on binomial distributions.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

For the initial toxicity study, sodium persulfate was administered by IP injection to male and female ICR mice at five treatment levels and a vehicle control. 208, 292, 408, 571 or 800 mg test article/kg body weight which was administered in a total volume of 10 mL test article-vehicle mixture/kg body weight. Mortality which occurred shortly after dose administration and up to four days later, was 0/10 at 208 and 292 mg/kg, 6/10 at 408 mg/kg and 10/10 at 571 and 800 mg/kg. The LD50 was calculated by probit analysis to be approximately 422 mg/kg. The high dose for the micronucleus test was set at 338 mg/kg which was estimated to be 80 % of the LD50.

TABLE : MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES IN BONE MARROW: SUMMARY 

TREATMENT	SEX	TIME (HR)	NUMBER OF MICE	PCE/TOTAL ERYTHROCYTES	MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES
					NUMBER PER 1000 PCE’S			NUMBER PER PCE'S SCORED
					(MEAN	±	S.D.)
Water
10 ml/kg	M	24	5	0.57	0.6	±	0.89	3 / 5000
		48	5	0.48	1.6	±	0.89	8 / 5000
		72	5	0.57	0.4	±	0.89	2 / 5000
	F	24	5	0.62	1.2	±	0.84	6 / 5000
		48	5	0.53	1.2	±	0.84	6 / 5000
		72	5	0.54	1.0	±	1.00	5 / 5000
Sodium persulfate, E608244, Lot 0021
85 mg/kg	M	24	5	0.60	0.6	±	0.55	3 / 5000
		48	5	0.48	1.6	±	0.55	8 / 5000
		72	5	0.50	1.0	±	1.00	5 / 5000
	F	24	5	0.65	1.4	±	1.14	7 / 5000
		48	5	0.59	1.8	±	0.84	9 / 5000
		72	5	0.60	0.8	±	1.10	4 / 5000
169 mg/kg	M	24	5	0.60	1.4	±	0.89	7 / 5000
		48	5	0.59	0.8	±	0.45	4 / 5000
		72	5	0.64	1.0	±	1.00	5 / 5000
	F	24	5	0.63	0.8	±	0.84	4 / 5000
		48	5	0.65	1.2	±	0.45	6 / 5000
		72	5	0.45	1.2	±	1.10	6 / 5000
338 mg/kg	M	24	5	0.51	0.2	±	0.45	1 / 5000
		48	5	0.54	1.0	±	0,71	5 / 5000
		72	4	0.47	1.0	±	0.82	4 / 4000
	F	24	5	0.56	1.0	±	0.71	5 / 5000
		48	5	0.57	2.4	±	1.52	12 / 5000
		72	3	0.37	2.7	±	2.08	8 / 3000
TEM
0.25mg/kg	M	24	5	0.55	59.8	±	22.42	299 / 5000*
	F	24	5	0.54	49.6	±	8.38	248 / 5000*

1*, p <= 0.05 (Kastenbaum-Bowman Tables)

Conclusions:

No significant increase in micronucleated polychromatic erythrocytes was observed at 24, 48 or 72 hours after dose administration in male or female ICR mice. Thus, disodium persulfate was considered to be not clastogenic.

Executive summary:

In the presented mouse micronucleus assay, male and female ICR mice were exposed to 85, 169 or 338 mg/kg of disodium persulfate which was administered at a constant rate of 10 mL/kg as a single IP injection. The high dose level was calculated to be 80 % of the LD50. Bone marrow cells, collected 24, 48 and 72 hours after treatment and were examined microscopically for micronucleated polychromatic erythrocytes. A reduction in the ratio of polychromatic erythrocytes to total erythrocytes was observed in female mice at 72 hours after administration of 169 mg/kg and in male and female mice at 72 hours after administration of 338 mg/kg, indicated that the test substance did induce bone marrow toxicity. No significant increases in micronucleated polychromatic erythrocytes was observed at 24, 48 or 72 hours after dose administration in males or females. The results of the assay indicated that under the conditions described disodium persulfate did not induce a significant increase in micronucleated polychromatic erythrocytes in male or female ICR mice. Disodium persulfate was concluded to be negative in the mouse micronucleus assay. Thus, disodium persulfate was considered to be not clastogenic.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-11-12 to 1991-05-02

Reliability:

1 (reliable without restriction)

Qualifier:

no guideline followed

Principles of method if other than guideline:

The experimental design described in this study is modified from that of Mirsalis et al. (1) and Butterworth et al. (2)
(1): John C. Mirsalis, et al., Detection of genotoxic carcinogens in the in vivo - in vitro hepatocyte DNA repair assay. Environmental Mutagenesis 4: 553-562, (1982).
(2): Byron E. Butterworth, et al., A protocol and guide for the in vivo rat hepatocyte DNA-repair assay. Mutation Res. 189, 113-121, (1987).

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Indianapolis, In
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: weight ranges as recommended in Guide for Care and Use of Laboratory Animals, DHEW, (NIH) No. 86,23
- Assigned to test groups randomly: yes
- Fasting period before study: not indicated
- Housing: in groups of five in polycarbonate cages with hardwood bedding
- Diet: Purina certified rodent chow ad libitum
- Water: tap water ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 21 °C
- Humidity: 30 - 70 % R.H.
- Air changes: not indicated
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

oral: gavage

Vehicle:

Distilled water

Details on exposure:

All rats were weighed immediately prior to dose administration. The dose volume was based on individual body weights. Animals were observed after dose administration and daily thereafter for 7 days for clinical signs of chemical effect. Body weights were recorded prior to dose administration and no less than 1 and 3 days after dose administration.

Duration of treatment / exposure:

five groups were exposed 2 - 4 hours
five groups were exposed 12 - 18 hours

Frequency of treatment:

one treatment

Post exposure period:

7 days

Dose / conc.:

41 mg/kg bw/day (nominal)

Dose / conc.:

164 mg/kg bw/day (nominal)

Dose / conc.:

820 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose

Control animals:

yes

Positive control(s):

yes;
MMS at 100 mg/kg bw was used in the 2 -4 hour exposure group
2AAF at 100 mg/kg bw was used in the 12 - 18 hour exposure group

Tissues and cell types examined:

primary cultures of hepatocytes

Details of tissue and slide preparation:

Each rat used was sacrificed by inhalation of metofane. The animals were dissected and the liver perfused first with a 0.5 mM EGTA solution and then with a collagenase solution. The liver was removed from the animal, and the cells dissociated, counted, and seeded into 35 mm dishes (5 x 10E5 viable cells/dish) containing coverslips. The cells were seeded in William's Medium E supplemented with10 % foetal bovine serum, 2 mM L-glutamine and 50 µg/mL gentamicin (complete WME). Five cultures per rat, containing coverslips were incubated at 37 ± 1 °C in a humidified 5 ± 10 % CO2 incubator.

Evaluation criteria:

The results of this study were evaluated according to the following criteria:
If the mean net nuclear count was increased by at least five counts over the control, the results for a particular dose level were considered significant. A test substance was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of the control. In the absence of the dose response, a test article which showed a significant increase in the mean net nuclear grain count at one dose level without any dose response, the test article was considered to have a marginal positive activity. The test article was considered negative if no significant increase in the net nuclear grain counts at any dose was observed.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Clinical observations/mortality:
No abnormal clinical signs were noted for the 2 -4 hour study. In the 12 - 18 hour study, the following clinical signs were observed less than 1 hour after dose administration: 1/5 animals at 100 mg/kg of 2AAF had a bloody mouth and was breathing irregularly. All other animals appeared normal. Clinical signs which were noted 12 - 14 hours after dose administration indicated diarrhoea in 1/5 animals at 820 mg/kg, and lethargy in 1/5 animals at 820 mg/kg.
Unscheduled DNA synthesis:
Table 1 (see sect. "Remarks on results incl. tables and figures") presents the UDS data for animals exposed to sodium persulfate, MMS and ddH2O for 2 to 4 hours. Hepatocytes from animals exposed to sodium persulfate showed no increase in UDS when compared to animals exposed to the negative control. The means of the net nuclear grain counts for groups receiving 820, 164 and 41 mg/kg of sodium persulfate were -3.1, -2.5 and -1.8, respectively. The mean of the net nuclear grain counts for the negative control was -2.6. The MMS treated group had a significant increase (greater than 5 net nuclear counts above the control) in UDS. The mean of the net nuclear counts for MMS was 4.8. Thus, according to the criteria set for evaluating the test, the animals receiving MMS were judged positive in the test and none of the animals in the 2 -4 hour exposure groups receiving sodium persulfate exhibited a positive response. All criteria for a valid test were met.
Table 2 (see sect. “Overall remarks") presents the UDS data for animals exposed to sodium persulfate, 2AAF and ddH2O for 12 to 18 hours. Hepatocytes from animals exposed to sodium persulfate showed no increase in UDS when compared to animals exposed to the negative control. The means of the net nuclear grain counts for groups receiving 820, 164 and 41 mg/kg of sodium persulfate were -2.2, -2.1 and -2.2, respectively. The mean of the net nuclear grain counts for the negative control was -3.0. The 2AAF treated group had a significant increase (greater than 5 net nuclear counts above the control) in UDS. The mean of the net nuclear counts for 2AAF was 7.9. Thus, according to the criteria set for evaluating the test, the animals receiving 2AAF were judged positive in the test and none of the animals in the 12 -18 hour exposure groups receiving disodium persulfate exhibited a positive response. All criteria for a valid test were met.

TABLE 1: SUMMARY OF UDS ASSAY WITH DISODIUM PERSULFATE (2-4 HOURS)

TREATMENT	ANIMAL NO.	SLIDE DESIGNATION	NO. OF NUCLIE COUNTED	AVERAGE NET GRAINS PER NUCLEUS		S.D.	MEAN PER ANIMAL		S.D.@	CELLS IN REPAIR	MEAN PER GROUP		S.D.#
Sodium Persulfate
820 mg/kg	6	29A	50	-3.4	+/-	1.8	-2.8	+/-	0.6	0%
		29B	50	-2.7	+/-	1.5		+/-
		29C	50	-2.3	+/-	1.8		+/-
	7	26A	50	-3.1	+/-	1.8	-3.0	+/-	0.1	0%	-3.1	+/-	0.3
		26B	50	-2.9	+/-	1.9		+/-
		26D	50	-3.0	+/-	1.6		+/-
	8	23A	50	-3.1	+/-	1.4	-3.4	+/-	0.4	1%
		23B	50	-3.9	+/-	1.5
		23C	50	-3.3	+/-	1.7
164 mg/kg	11	24A	50	-3.1	+/-	1.5	-3.0	+/-	0.2	0%
		24B	50	-2.8	+/-	1.6
		24C	50	-3.1	+/-	1.8
	12	24A&C	50	-2.0	+/-	2.3	-2.0	+/-	0.0	0%	-2.5	+/-	0.5
		25D, E&F	26@	-2.0	+/-	1.8
	13	21A	50	-2.7	+/-	1.7	-2.6	+/-	0.3	0%
		21B	50	-2.8	+/-	1.6
		21C	50	-2.3	+/-	1.3
41 mg/kg	16	20D	50	-1.9	+/-	-1.4	-1.9	+/-	0.6	0%
		20C	50	-2.5	+/-	1.2
		20F	50	-1.3	+/-	1.5
	17	19C	50	-1.8	+/-	1.7	-1.9	+/-	0.3	0%	-1.8	+/-	0.2
		19D	50	-1.6	+/-	1.9
		19E	50	-2.2	+/-	1.8
	18	17A	50	-1.9	+/-	1.6	-1.6	+/-	0.4	1%
		17D	50	-1.1	+/-	2.3
		17E	50	-1.8	+/-	1.5
MMS (Positive Control)
100 mg/kg	21	16B	50	4.3	+/-	2.4	3.7	+/-	0.7	37%
		16C	50	3.9	+/-	1.8
		16E	50	2.9	+/-	2.2
	22	22A	50	6.3	+/-	2.9	5.5	+/-	0.8	61%	4.8	*+/-	0.9
		22B	50	4.7	+/-	3.5
		22C	50	5.5	+/-	3.2
	23	18A	50	5.0	+/	1.8	5.1	+/-	0.1	63%
		18B	50	5.0	+/	1.5
		18C	50	5.2	+/	1.6
Water (Vehicle Control for Test Article)
10 ml/kg	1	30A	50	-3.5	+/-	2.4	-2.8	+/-	0.7	0%
		30B	50	-2.9	+/-	2.3
		30C	50	-2.1	+/-	2.0
	2	28A	50	-2.3	+/-	1.2	-2.1	+/-	0.2	0%	-2.6	+/-	0.4
		28B	50	-2.1	+/-	1.1
		28C	50	-1.9	+/-	1.2
	3	27A	50	-2.6	+/	1.5	-2.7	+/-	0.1	1%
		27B	50	-2.8	+/	1.6
		27C	50	-2.8	+/	2.0

* Significant (See Protocol: Section 8.0, Evaluation of Test Results)

S.D.@ Standard Deviation Reflecting Slide to Slide Variation

S.D.# Standard Deviation Reflecting Animal to Animal Variation

@ Only 26 Nuclei Were Acceptable for UDS Evaluation

Conclusions:

The results of the in vivo/in vitro UDS assay indicated that under the test conditions, disodium persulfate did not cause a significant increase in the main net nuclear grain counts in hepatocytes isolated from the treated animals. Therefore, disodium persulfate was considered negative in this study.

Executive summary:

Disodium persulfate was tested in the In vivo - In vitro rat hepatocyte unscheduled DNA synthesis assay. The test substance was administered to the animals via oral gavage at three dose levels of 41, 164 and 820 mg/kg bw. The hepatocytes were harvested 2 - 4 and 12 - 18 hours after administration of the test substance. The results of the in vivo/in vitro UDS assay indicated that under the test conditions, the test substance did not cause a significant increase in the mean net nuclear grain counts (i.e., an increase of at least 5 counts over the vehicle control) in hepatocytes isolated from treated animals (a negative result). Therefore, disodium persulfate was considered not mutagenic .

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Disodium persulfate was tested for genetic toxicity in two in vitro tests. A gene mutation assay with bacteria (Ames test) according to FIFRA guideline 84-1 was negative. An unscheduled DNA synthesis test with rat liver hepatocytes according to FIFRA guideline 84-2 showed no signs of mutagenic activity. An additional bacterial reverse mutation assay with Escherichia coli WP2 uvrA did not reveal any mutagenic properties.

The purpose of the first study was to establish the potential of disodium persulfate to induce gene mutations in Salmonella typhimurium: TA98, TA100, TA1535, TA1537 and TA1538, using Salmonella/Mammalian-Microsome plate incorporation mutagenicity Assay (Ames test), performed according to FIFRA Guideline 84-1. Disodium persulfate was tested at five dose levels ranging from 100 to 10000 µg/plate. The dose levels were based on a preliminary toxicity test. The assay was conducted in the presence and absence of metabolic activation by Aroclor 1254 induced rat liver microsomes (S9 Mix). Revertant colonies were counted. During the tests positive and negative controls were run concurrently. The reference mutagens (sodium azide, 9 -aminoacridine, 2 -nitrofluorene, 2 -anthramine) showed a distinct increase of induced relevant colonies. Results with the test substance showed that disodium persulfate did not cause a positive response in any of the tester strains with or without metabolic activation. Therefore, disodium persulfate was considered non-mutagenic in this bacterial reverse mutation assay.

Disodium persulfate was additionally tested in the rat hepatocyte unscheduled DNA synthesis assay. The test substance was tested at eight dose levels ranging from 1.5 to 500 µg/mL and was fully evaluated at five dose levels of 5.0, 15, 50, 150 and 250 µg/mL. In this study the positive control, 7,12 -Dimethylbenz(a)anthracene (DMBA), induced significant increases in the mean number of net nuclear grain counts over that in the solvent control. All criteria for a valid test were met. The results of the UDS assay indicated that under the test conditions, disodium persulfate did not cause a significant increase in the unscheduled DNA synthesis as measured by the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the solvent control), at any dose level. Thus, the test substance was considered not mutagenic.

A second bacterial reverse mutation assay according OECD guideline 471, EU method B.13/14 and EPA OPPTS 789.5100 was performed to investigate the mutagenic potential of disodium peroxodisulphate (SPS)  in two independent experiments with Escherichia coli WP2 uvrA, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test). This additional study was performed because the available study with Disodium peroxodisulphate (SPS) does include 5 bacterial strains; however non of these strains does detect cross-linking mutagens. Therefore an additional bacterial reverse mutation assay was performed using Escherichia coli WP2 uvrA.

The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: 5000, 1600, 500, 160, 50 and 16 μg/plate. Each assay was conducted with and without metabolic activation (±S9 Mix).  The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strain, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of the investigated Escherichia coli WP2 uvrA tester strain following treatment with Disodium peroxodisulphate (SPS) at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in the independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. No precipitation of the test item was observed on the plates in the examined bacterial strain at any examined concentration level (±S9 Mix) throughout the study.
The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by base-pair substitution in the genome of the Escherichia coli WP2 uvrA used. Therefore, Disodium peroxodisulphate (SPS) is considered non-mutagenic in this bacterial reverse mutation assay.

Genetic toxicity in vivo

Disodium persulfate was tested in two in vivo tests for mutagenic activity. In the mouse micronucleus test, no mutagenic activity could be detected, as well as in an in vivo unscheduled DNA synthesis assay in rat livers. Based on the obtained results, disodium persulfate was considered not genotoxic.

In the mouse micronucleus assay, male and female ICR mice were exposed to 85, 169 or 338 mg/kg of disodium persulfate which was administered at a constant rate of 10 mL/kg as a single IP injection. The high dose level was calculated to be 80 % of the LD50. Bone marrow cells, collected 24, 48 and 72 hours after treatment and were examined microscopically for micronucleated polychromatic erythrocytes.

A reduction in the ratio of polychromatic erythrocytes to total erythrocytes was observed in female mice at 72 hours after administration of 169 mg/kg and in male and female mice at 72 hours after administration of 338 mg/kg, indicated that the test substance did induce bone marrow toxicity.

No significant increases in micronucleated polychromatic erythrocytes was observed at 24, 48 or 72 hours after dose administration in males or females. The results of the assay indicated that under the conditions described disodium persulfate did not induce a significant increase in micronucleated polychromatic erythrocytes in male or female ICR mice. Disodium persulfate was concluded to be negative in the mouse micronucleus assay. Thus, disodium persulfate was considered to be not clastogenic.

 

Disodium persulfate was additionally tested in the in vivo rat hepatocyte unscheduled DNA synthesis assay. The test substance was administered to the animals via oral gavage at three dose levels of 41, 164 and 820 mg/kg bw. The hepatocytes were harvested 2 - 4 and 12 - 18 hours after administration of the test substance.

The results of the in vivo/in vitro UDS assay indicated that under the test conditions, the test substance did not cause a significant increase in the mean net nuclear grain counts (i.e., an increase of at least 5 counts over the vehicle control) in hepatocytes isolated from treated animals (a negative result). Therefore, disodium persulfate was considered not mutagenic.

Justification for classification or non-classification

Based on the results obtained, substances of the Persulfate Category were not classified and labelled for genetic toxicity according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.