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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:
11 Junuary 2021 - 22 September 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium chlorite
EC Number:
231-836-6
EC Name:
Sodium chlorite
Cas Number:
7758-19-2
Molecular formula:
ClHO2.Na
IUPAC Name:
sodium chlorite
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
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 old
- Assigned to test groups randomly: yes
- Housing: the animals were kept in groups of 2-3 animals/sex/group/cage
- Diet: maintenance diet ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water (aqua ad iniectabilia)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no.: 911506
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A correction factor of 3.15 was applied to consider the purity of the active compound of the test item. 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 for approximately 10 minutes or until visual homogeneity was achieved. The test item formulations were prepared freshly on each administration day within one hour prior to administration. The prepared formulation was stored protected from light and at room temperature.
Duration of treatment / exposure:
Daily over a period of 2 days (0h, 24h ± 1h).
Frequency of treatment:
24 h
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
5 animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethylmethanesulphonate (EMS)
- Justification for choice of positive control(s): JaCVAM validation trial
- Route of administration: Oral gavage 4h before animal sacrifice.
- Doses / concentrations: 230 mg/kg bw (in 0.9% NaCl).

Examinations

Tissues and cell types examined:
Liver (primary site of xenobiotic metabolism), glandular stomach and duodenum (sites of contact).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the dose range-finding study performed with male and female rats, 100 mg/kg bw/day was chosen as the maximum tolerated dose used in the main experiment. As no relevant differences between males and females were noted, only male rats were tested in the main study

TREATMENT AND SAMPLING TIMES:
Animals were treated daily over a period of 2 days (administration time points were 0h and 24h). The sampling time was 4 h after the last treatment for all dose groups evaluated.

DETAILS OF SLIDE PREPARATION:
A total of 25 samples of liver, 25 samples of glandular stomach and 25 samples of duodenum were analysed for DNA strand breaks. The samples were kept in ice-cold mincing buffer. The fresh samples were directly prepared according to the following procedure.
1) Isolation of primary hepatocytes: A portion of the liver was minced with a pair of scissors to isolate the cells. The cell suspension was kept for not more than 15 seconds until bigger fragments of the liver 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.
2) Isolation of duodenum cells: The duodenum was flushed with a syringe filled with cold mincing buffer to wash out the food. Afterwards a portion of the duodenum was minced with a pair of scissors. The 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 LMA solution.
3) Isolation of glandular stomach cells: The stomach was cut open and washed free of food using cold water. A portion of the glandular stomach was minced with a pair of scissors. The pieces were further crushed with a pistel to release single cells. The suspension was kept for less than 15 seconds to allow large clumps to settle. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution. For all tissues, the cell pellets were used for preparing comet slides.

Slide preparation:
The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 µL of cell suspension embedded in low-melting temperature agarose was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).

Lysis:
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.

Unwinding of DNA and electrophoresis:
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.

Neutralization and dehydration of slides:
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.

DNA staining:
Following dehydration, 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:
Analysis of DNA-strand breaks:
Comet slides were analyzed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated.
Cells were classified into three potential categories scorable, non-scorable and “hedgehog” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail are considered to be heavily damaged cells). To avoid artefacts only scorable cells and at least 150 cells per sample were scored, if available. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and 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:
Evaluation criteria:
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
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.

Acceptability criteria:
1) The concurrent negative control data are considered acceptable for addition to the laboratory historical control database.
2) The concurrent positive controls should induce responses that are compatible to those previously generated and included in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
3) Three doses and if available 150 cells per organ of each animal have been analyzed.
Statistics:
For each tissue type, the mean of the individual animal means was then determined to give a group mean. Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p-value was used as a limit in judging for significance levels in comparison with the corresponding Negative Control.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
ambiguous
Remarks:
Glandular stomach
Toxicity:
yes
Remarks:
Toxicity: Only at 100 mg/kg bw Cytotoxicity: At 100 and 50 mg/kg bw
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Liver
Toxicity:
yes
Remarks:
Only at 100 mg/kg bw
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Duodenum
Toxicity:
yes
Remarks:
Only at 100 mg/kg bw
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 1000, 500, 250, 150, 120 and 100 mg/kg bw
- Animales: Male and female rats (1 to 3 per dose).
- Solubility: Soluble in water
- Clinical signs of toxicity in test animals: Different clinical signs observed depending on the dose received (see the report for details). At 100 mg/kg bw (2 rats per sex and dose), reduced spontaneous activity, prone position, moving the bedding, half eyelid to eyes closed and piloerection were observed after administration of the second dose. After 4h the only symptom left was reduced spontaneous activity or piloerection, which was gone on the next day.
- Evidence of cytotoxicity in tissue analysed: Not assessed in the range-finding study. No differences were observed between male and female rats.
- Route of administation: The route was chosen in consideration of the anticipated route of human exposure and to ensure adequate exposure of the target tissue (please refer to toxicokinetics).

RESULTS OF DEFINITIVE STUDY
- Effects observed: DNA-strand breaks.
- Appropriateness of dose levels and route: The dose levels (25, 50 and 100 mg/kg bw) and route of exposure (oral gavage) were determined to be appropriate.
- Statistical evaluation: The values of the concentrations 50 and 100 mg/kg bw in glandular stomach cells were statistically significant increased compared to the negative control. The significance determined at 50 and 100 mg/kg bw in glandular stomach cells of male rats was regarded as biologically relevant. No dose-related increase was noted in liver and duodenum. Despite a slight tendency, no concentration-related increase was noted in glandular stomach cells.

Any other information on results incl. tables

Toxicity:


All animals treated with 100 mg/kg bw showed slight to moderate toxic effects such as reduction of spontaneous activity, piloerection and half eyelid closure (one rat) after the first application. Before the second administration after 24h, all of the symptoms were gone. After the second administration, the same symptoms were present, but more pronounced. Additionally, moving bedding (3 rats), prone position (4 rats), hunched posture (4 rats) and wasp waist (1 rat) were observed. Rats treated with 50 mg/kg bw and 25 mg/kg bw showed no signs of toxicity.


Body weight:


The body weight variation in the main experiment (±6.3 %) was below 20%. Four out of five male rats in the negative control group gained weight during the observation period. A reduction of body weight was noted in four out of seven male animals in the HD group. In the lower concentrations tested, one out of five male rats lost weight in the mid and low dose group.


Analysis of DNA-strand breaks:


Liver: The tail intensity of the LD (1.96 %), MD (1.47 %) and HD (1.42 %) was within the historic control limits of the test facility (0.07 – 3.82 %). No statistical significant increase compared to the negative control was indicated for the values of all doses evaluated in liver cells. No concentration-dependency was noted.


Tail intensity of liver cells:





































































Nº animalPC (EMS)NC (Water)LD (25 mg/kg bw)MD (50 mg/kg bw)HD (100 mg/kg bw)
17.570.991.471.230.83
27.391.500.771.630.78
39.820.900.621.452.19
47.441.595.301.801.08
56.630.431.671.252.25
Mean7.771.081.961.471.42
±SD1.200.471.920.250.73

Glandular stomach: The group mean tail intensity of the LD (4.12 %) was within the range of the historic negative control limits of the test facility. However, the tail intensities of male rats of the MD (6.01 %) and HD (7.44 %) were above the range of the historic negative control limits. Additionally, both tail intensities showed a statistical significant increase compared to the corresponding negative control. Despite a tendency, no statistical significant concentration-related increase was noted. 


Sodium Chlorite caused degenerative inflammatory and reactive changes in the stomach of several dosed animals. The overall cause of the gastric changes was the irritative action of the test item on the glandular stomach mucosa. Comparing the individual Comet Assay results with the individual histopathology data, there is a correlation between the severity of the observed gastric changes and the results of the Comet assay for High Group animal.


Tail intensity of glandular stomach cells:





















































































Nº animalPC (EMS)NC (Water)LD (25 mg/kg bw)MD (50 mg/kg bw)HD (100 mg/kg bw)
114.693.503.573.042.99
211.981.782.983.384.80
311.471.894.7012.2010.28
412.121.224.474.797.30
511.531.134.876.637.88
6----8.53
7----10.33
Mean12.361.904.126.017.44
±SD1.330.950.813.742.72

Duodenum: The tail intensity of the LD (2.85 %), MD (2.78 %) and HD (3.81 %) was within the historic control limits. No statistical significance compared to the negative control. No concentration-dependency was noted for the tail intensities.


Negative control: All tail intensities were within the laboratory historical control data: Liver (1.08%), glandular stomach (1.90%), duodenum (1.55%).


Positive control: The tail intensity increases of EMS (230 mg/kg bw) demonstrated the validity of the assay: Liver (7.77 %), glandular stomach (12.36%), duodenum (8.02%).


Historical control data (tail intensity):


Negative control: Liver cells: 0.07 – 3.82 %; Glandular stomach cells: 1.30 – 5.46 %; Duodenum cells: 0.89 – 4.06 %.


Dose formulation analysis:


The mean recoveries observed in the LD, MD and HD groups were 100.8%, 100.5%, and 100.8% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

Applicant's summary and conclusion

Conclusions:
The test item resulted to be negative in liver and duodenum. In the contrary, the substance induced DNA-strand breaks in glandular stomach. Sodium Chlorite caused degenerative inflammatory and reactive changes in the stomach of several dosed animals. The overall cause of the gastric changes was the irritative action of the test item on the glandular stomach mucosa. Comparing the individual Comet Assay results with the individual histopathology data, there is a correlation between the severity of the observed gastric changes and the results of the Comet assay for High Group animals. Further testing is deemed necessary in order to conclude whether the observed effects are due to cytotoxicity and/or genotoxicity.
Executive summary:

The in vivo mammalian alkaline comet assay was conducted according to OECD 489 (GLP study). Wistar rats (5 per group) were exposed to the substance at 25 (LD), 50 (MD) and 100 (HD) mg/kg bw for two consecutive days (administration time points: 0h and 24h). The liver (as the metabolising organ) and glandular stomach and duodenum (as the first-contact organs upon peroral exposure) were collected 4h after the second administration of the test item. The tail intensity of 150 cells per animal and tissue were evaluated. The results obtained with the negative control (vehicle) and the positive control (Ethyl methanesulfonate) were within the historical control limits and therefore accepted. No biologically relevant increase of tail intensity was found after treatment with the test item in the liver and the duodenum in any of the dose groups evaluated compared to the negative controls. Moreover, the mean values of the dose groups, were within the range of the concurrent negative control group. In the glandular stomach, a biologically relevant increase of the tail intensity was found after treatment with the test item in the MD and HD group. The tail intensities of MD and HD were above the range of the historic negative control limits. Additionally, both tail intensities showed a statistical significant increase compared to the corresponding negative control. Despite a tendency, no statistical significant concentration-related increase was noted. Due to the fact that a positive result was observed in the glandular stomach (first site of contact) in the in vivo comet assay, a histopathological analysis was performed to determine whether cell death due to increased cytotoxicity of the test material could be associated with increased levels of DNA strand breaks. Here, sodium chlorite caused degenerative inflammatory and reactive changes in the stomach of several dosed animals. The overall cause of the gastric changes was the irritative action of the test item on the glandular stomach mucosa. Comparing the individual Comet Assay results with the individual histopathology data, there is a correlation between the severity of the observed gastric changes and the results of the Comet assay for High Group animals. Since genotoxic effects of sodium chlorite cannot be excluded under these experimental conditions in the in vivo mammalian Alkaline Comet Assay, further testing would be necessary and is recommended.