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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-06-23 to 2021-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016-06-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentapotassium bis(peroxymonosulphate) bis(sulphate)
EC Number:
274-778-7
EC Name:
Pentapotassium bis(peroxymonosulphate) bis(sulphate)
Cas Number:
70693-62-8
Molecular formula:
H3K5O18S4
IUPAC Name:
pentapotassium bis((hydroperoxysulfonyl)oxidanide) hydrogen sulfate sulfate
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han) (Full Barrier)
Details on species / strain selection:
The Wistar rat is a commonly used strain for this type of assay. Furthermore, a wide historical control database is available for this strain at the testing laboratory.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 315-366 g
- Assigned to test groups randomly: Yes, randomization was performed by a validated software or by excel-file.
- Fasting period before study: No
- Housing: In groups of 2-3 animals / sex / group / cage in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding
- Diet: Ad libitum (Altromin 1324 maintenance diet for rats and mice)
- Water: Ad libitum (Tap water)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 1

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle: Deionised water
- Justification for choice of solvent/vehicle: Solubility properties and non-toxicity in the test system
- Concentration of test material in vehicle: 15, 30, 60 and 75 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Batch No: 2005066
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulations were kept under magnetic stirring until visual homogeneity was achieved. The test item formulations were prepared freshly on each administration day within one hour prior to administration. The prepared formulations were stored protected from light and at room temperature.
Duration of treatment / exposure:
Approx. 28 hours (animals were sacrificed 4 hours after the last treatment) for treatment and negative control groups or 4 hours for the positive control group
Frequency of treatment:
2 treatments at 24 (± 1) hour intervals for test substance treatment groups and negative control group; 1 treatment 4 hours before sacrifice for the positive control group
Post exposure period:
4 hours
Doses / concentrationsopen allclose all
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 male animals per group
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate
- Justification for choice of positive control: According to the JaCVAM validation trial EMS was selected as positive control and was administered orally by gavage 4 h before animal sacrifice.
- Route of administration: Oral gavage
- Doses: 250 mg/kg bw

Examinations

Tissues and cell types examined:
Liver, forestomach, glandular stomach and duodenum
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Doses were selected based on the results of a dose-range finding study. The highest dose administered in the dose range-finding study was 2000 mg/kg bw/day. Further doses of 75, 125, 250, 500, 750 and 1000 mg/kg bw were administered as well. The animals received the test item twice at 0 h and 24 h after the first administration. The application was done in parallel with male and female rats. Toxic effects leading to humane killing (prone position, reduced spontaneous activity, ataxia, piloerection, half eyelid closure, and lacrimation) occured after the the first administration of 2000 mg/kg bw in one male and one female rat. Reduction of the dose to 1000 mg/kg bw/day in one male and one female animal lead to similar clinical signs and necropsy showed dark and reddened stomach mucosa which was dilated by gas. Administration of 750 mg/kg bw to three male and female rats led to similar clinical signs. No difference between sexes were found. The test item caused inflammatory and degenerative lesions in the stomach from 250 mg/kg bw onwards. From 750 mg/kg bw onwards, there were subacute submucosal inflammation associated with edema, erosion and/or ulceration in the stomach and clinical signs. The findings in the stomach at a dose of 750 mg/kg bw were considered adverse in nature. Based on these local effects, doses selected for the main study were 150, 300, 600 and 750 mg/kg bw/day to demonstrate a dose-dependency in histopathological findings and to induce clinical symptoms in the highest dose of 750 mg/kg bw.

TREATMENT AND SAMPLING TIMES: After euthanasia of the animals, the abdominal aorta was cut and the blood was released. The liver, the forestomach, the glandular stomach and the duodenum were removed, rinsed with cold mincing buffer to remove residual blood and kept in ice-cold mincing buffer on ice until further processing. The times to remove the tissues until the preparations of the slides were recorded in the raw data.

DETAILS OF SLIDE PREPARATION: A portion of the respective tissue was minced, further crushed (stomach tissue) and cell suspension was kept for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 μL of the supernatant was pipetted into a tube and mixed with 270 μL low-melting agarose (LMA) solution. The slides used were pre-coated with normal-melting agarose. A volume of 75 μL of cell suspension embedded in LMA was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue). Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts. Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis solution for 20 min. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order. After electrophoresis, the slides were neutralized by rinsing with neutralization buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards. The cells were stained by applying 75 μL gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS: Comet slides are analyzed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software “Comet Assay IV” (Perceptive Instruments, Software version 2.1.2). Each slide is screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. Calculations were done automatically by the software but might be corrected manually. All cells of the visual field were scored, except of e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (heavily damaged cells). Therefore, cells will be classified into three categories: scorable, non-scorable and “hedgehog”. To avoid artefacts only scorable cells (defined round to oval nucleus) and at least 150 cells per sample on two slides (75 cells per slide) were scored. The %-tail intensity was evaluated as the main parameter for interpretation of DNA damage. It was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus.
Evaluation criteria:
Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value.

Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).

To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.
Statistics:
Statistical analysis was performed by testing for normality according to Kolmogorov-Smirnov-test. Statistical significances in comparison with the negative control were evaluated with one-way ANOVA and Dunnett’s post-hoc test at a 5 % level (p < 0.05). In addition, dose-dependency was tested with a linear trend test at the 5 % level (p < 0.05).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 75, 125, 250, 500, 750, 1000 and 2000 mg/kg bw
- Solubility: Soluble in water at all concentrations
- Clinical signs of toxicity in test animals: Toxic effects leading to humane killing (including prone position, reduced spontaneous activity, ataxia, piloerection, half eyelid closure, and lacrimation) occured after the the first administration of 2000 mg/kg bw in one male and one female rat. Reduction of the dose to 1000 and 750 mg/kg bw/day lead to similar clinical signs. No difference between sexes were found.
- Evidence of cytotoxicity in tissue analysed: The test item caused inflammatory and degenerative lesion in the stomach from 250 mg/kg bw onwards. From 750 mg/kg bw onwards, there were subacute submucosal inflammation associated with edema, erosion and/or ulceration in the stomach. The findings in the stomach at a dose of 750 mg/kg bw were considered adverse in nature. Dose ranges for the main study were based on these local effects and were supported by histopathological examination of liver, forestomach, glandular stomach and duodenum.
- Harvest times: 4 hours after the last treatment

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The dose levels were shown to be appropriate as the different doses induced graded signs of toxicity. The highest dose induced clinical signs like reduction of spontaneous activity, prone position and ataxia after the first and second application. Rats treated with the second highest dose only showed a reduction of spontaneous activity. No clinical signs were present in the two lowest dose groups. The test substance caused inflammatory and reactive lesions in the gastrointestinal tract at ≥300 mg/kg bw and in the duodenum at ≥600 mg/kg bw in a dose-dependent manner. Thus, it was shown that the test substance reached target tissues and the oral route of exposure was appropriate. The oral route is also the likely route of human exposure.
- Statistical evaluation: The statistical evaluation showed no significant differences in any of the parameters examined between vehicle control and dose groups.

Any other information on results incl. tables

Dose Formulation Analysis


Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.


 


Clinical signs


Animals treated with the highest dose (HD2) showed slight to moderate toxic effects such as reduction of spontaneous activity, prone position and ataxia after the first application. One animal additionally showed piloerection after 30 minutes. After 24 h before the second administration, no symptoms were left. After the second administration, reduction of spontaneous activity, prone position and ataxia were present after 4h. Rats treated with 600 mg/kg bw (HD1) only showed a reduction of spontaneous activity. Rats in the dose groups 300 mg/kg bw (MD) and 150 mg/kg bw (LD) showed no clinical signs.


 


Histopathology


For evaluation of possible toxicity in liver, stomach (fore and glandular stomach) and duodenum induced by repeated administration of the test item, a histopathological evaluation was performed. Under the conditions of this study, KMPS Triple Salt caused inflammatory and reactive lesions in the gastrointestinal tract at ≥300 mg/kg bw and in the duodenum at ≥600 mg/kg bw in a dose-dependent manner.

Applicant's summary and conclusion

Conclusions:
In an in vivo Mammalian Alkaline Comet Assay according to OECD guideline 489, the test item KMPS Triple Salt did not induce biologically relevant DNA-strand breaks in liver, forestomach, glandular stomach and duodenum after oral administration to rats under the experimental conditions reported. Therefore, KMPS Triple Salt is considered to be non-DNA damaging under these experimental conditions in the in vivo mammalian Alkaline Comet Assay.
Executive summary:

In an in vivo Mammalian Alkaline Comet Assay according to OECD guideline 489 and GLP, the genotoxic potential of KMPS Triple Salt was assessed by measuring its ability to induce DNA-strand breaks in the liver, the forestomach, the glandular stomach and the duodenum of rats. The organs were selected to cover three first-contact organs of chemicals upon peroral exposure and the liver as the primary organ for the metabolism of absorbed chemicals.


In addition, a formulation analysis for verification of concentration of KMPS Triple Salt in formulation samples was performed using an iodometric titration method. Nominal concentrations of KMPS Triple Salt Salt in formulation samples were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%. A histopathological evaluation was performed to evaluate the possible toxicity in liver, stomach (fore and glandular stomach) and duodenum induced by repeated administration of KMPS Triple Salt. Under the conditions of this study, KMPS Triple Salt inflammatory and reactive lesions in the gastrointestinal tract at ≥300 mg/kg bw and in the duodenum at ≥600 mg/kg bw in a dose-dependent manner. The test item was suspended in deionized water and administered to 5 rats/sex and dose by daily gavage at 10 mL/kg bw (body weight) for two consecutive days. The organs were collected 4 h after the second administration of the test item. Based on local and systemic effects observed in the dose range finding study, a dose of 750 mg/kg bw was selected as the highest dose. In the main experiment, four dose levels (LD:150 mg/kg bw, MD: 300 mg/kg bw, HD1: 600 mg/kg bw/d and HD2: 750 mg/kg bw/d) were used covering a range from the little or no toxicity to the maximum tolerated dose. The animals treated with the LD and the MD showed no signs of systemic toxicity. The animals treated with the HD1 showed reduced spontaneous activity, while animals treated with the highest dose (HD2) showed moderate signs of systemic toxicity such as reduction of spontaneous activity, prone position, piloerection and ataxia. Cells from the liver, forestomach, glandular stomach and duodenum were isolated, embedded in agarose and lysed. DNA was allowed to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated. DNA migration during electrophoresis was determined and expressed as tail intensity.


The validity criteria were met:


The tail intensities of the negative control group were within the historical control limits and therefore accepted for addition to the laboratory control data set. Ethyl methanesulfonate (250 mg/kg bw) administered orally was used as positive control and induced a statistically significant increase in DNA damage for all evaluated organs. The mean values noted for the dose groups, which were treated with the test item, were within the range of the concurrent negative control and within the historical control limits. No biologically relevant increase of tail intensity was found after treatment with the test item in any of the dose groups and organs evaluated compared to the negative controls. The test item did not induce DNA damage under the conditions tested.


 


In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item KMPS Triple Salt did not induce biologically relevant DNA-strand breaks in liver, forestomach, glandular stomach and duodenum after oral administration to rats. Therefore, KMPS Triple Salt is considered to be non-DNA damaging under these experimental conditions in the in vivo mammalian Alkaline Comet Assay.