Bisisopropyl peroxydicarbonate

Genetic toxicity: in vivo

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:

23 May 2022 - 19 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Bisisopropyl peroxydicarbonate

EC Number:

203-317-4

EC Name:

Bisisopropyl peroxydicarbonate

Cas Number:

105-64-6

Molecular formula:

C8H14O6

IUPAC Name:

2-[({[(propan-2-yloxy)carbonyl]peroxy}carbonyl)oxy]propane

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is the species recommended by OECD guidelline for this test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 8 weeks old
- Weight at study initiation: 201 g to 230 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in polypropylene cages
- Diet : A04C-10 from SAFE (batch 19330), ad libitum
- Water : ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %,
- Air changes (per hr): not precised, but ventilated system
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Dose volume : 10 mL/kg b.w.
emulsion

In the main genotoxicity assay, three preparations of the test item at the concentrations of 100 - 50 and 25 mg/mL were prepared, giving final doses of 1000 - 500 and 250 mg/kg b.w./day (x2), respectively when administered at 10 mL/kg b.w..
Preparations for treatment were performed on the day of the 1st treatment and the formulations were used within 24 hours for the 2nd treatment.

Duration of treatment / exposure:

2 daily treatments at 24-hour intervals

Frequency of treatment:

2 daily treatments at 24-hour intervals

Post exposure period:

2-6 hours after the second treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5 males/group, except for the high dose group : 7

Control animals:

yes, concurrent vehicle

Positive control(s):

A total of 5 animals were treated orally twice with Methylmethane sulfonate (Aldrich, batch MKCL6261) in sterile water (Fresenius, Batch 13RPD081), 100 mg/kg b.w./day, gavage, 2 daily treatments at 24-hour intervals and sacrified 2 to 6 hours after the last treatment.

Interpretation of the results
For a test item to be considered positive in the comet assay, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the concurrent vehicle control,
- This increase is dose-related when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical vehicle control data and/or the intervals of historical data for individual values.
When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage in the tissues studied in this test system.

A test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control,
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical vehicle control data and/or the intervals of historical data for individual values
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.

Examinations

Tissues and cell types examined:

Two to six hours after the second treatment, the rats are killed and cells from the selected target organs (i.e. liver, glandular stomach and duodenum) are isolated for the comet assay.
Five males of each group were assigned for cell isolation and assessed for DNA fragmentation.
Individual animals were anaesthetized with isoflurane and exsanguined.

Single cell preparations were done within one hour after animal sacrifice.
A 'V' shaped incision was made from the centre of the lower abdomen to the rib cage. The skin and muscles were removed to reveal the abdominal cavity.
A portion of the liver, the glandular stomach and the duodenum was removed and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced or scrapped with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a hemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.

Number of cells observed per animal: 150
Number of cells observed per dose: 750

Details of tissue and slide preparation:

After isolation, single cells are embedded in agarose on microscope slides and the obtained microgels are successively submitted to lysis, unwinding and electrophoresis in alkaline conditions and under dimmed light to prevent any additional DNA damage. After neutralization, slides are dried and could then be stained with a fluorescent dye (e.g. propidium iodide) before analysis and scoring. The method used for quantifying DNA migration involves a computerized image analysis system in order to collect comet data; then, the dedicated software allows the calculation of metrics for DNA migration.

Determination of the cytotoxicity of the test item
Statistically significant increases in the percentage of hedgehogs were observed in the 1000 and 250 mg/kg b.w./day (x2) treated groups when compared to the vehicle group (See Table 12) with values of 22.68 and 16.21%, respectively, vs. 12.79% in the relative vehicle control. The values were however clearly below 50%.

Evaluation criteria:

Expression of the results
- the median per slide of the percentage of DNA in tail for at least 50 cells
- the mean of medians of the percentage of DNA in tail per animal (i.e. 3 slides, 150 cells)
- the mean of medians per concentration (i.e. 5 animals, 750 cells)
In addition, each slide was also examined for presence of hedgehogs (possible indicator of toxicity and or apoptosis). Hedgehogs were excluded from image analysis data collection. However, determining their frequency might be useful for data interpretation. Therefore, the percentage of hedgehogs was recorded for each slide per animal and per organ. The hedgehogs, also known as clouds or ghost cells, are morphological indicative of highly damaged cells often associated with severe genotoxicity, necrosis and apoptosis. A hedgehog results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum.
A study is accepted if the following criteria are fulfilled:
- Concurrent vehicle controls should be within the control limits of the distribution of the laboratory’s historical vehicle control database and in the intervals of historical data for individual values.
- The concurrent positive controls should induce responses that are comparable to the historical positive control data and produce a statistically significant increase compared with the concurrent vehicle control.
- The appropriate number of doses and cells must be analysed.

Moreover, in the vehicle group, an eventual increase in the percentage of hedgehogs, must not be >50%.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software GraphPad Version 3.10.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Remarks:

on liver cells

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Sex:

male

Genotoxicity:

negative

Remarks:

on duodenum cells

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A slight decrease in spontaneous motor activity was noted at 1000 mg/kg b.w. 15 minutes and between 2 to 4 hours after the 2nd treatment.

Liver cells
Determination of the cytotoxicity of the test item
A statistically increase in the percentage of hedgehogs was observed in the 1000 mg/kg b.w./day (x2) treated groups when compared to the vehicle group with 4.12% of ghost cells vs. 2.31% in the relative control group.

Determination of the genotoxicity of the test item
No statistically significant increase in the mean of medians for the percentage of DNA in tail was induced at the three analysed doses of 1000, 500 and 250 mg/kg b.w./day (x2), vs. the vehicle control. Indeed, the means of medians of the percentage of tail DNA ranged from 0.13 to 0.68% vs. 0.09% in the respective vehicle control.
No assessment of systemic exposure was done, however clinical signs such as decrease in spontaneous motor activity were observed at the top dose tested and a slight increase in the percentage of ghost cells was noted.
The test item Bisisopropyl peroxydicarbonate was thus not genotoxic in rat liver. Indeed, no primary DNA damage was detected.

Duodenum cells
Determination of the cytotoxicity of the test item
No statistically significant increase in the percentage of hedgehogs was noted at the doses tested of 1000, 500 or 250 mg/kg b.w./day (x2) treated groups when compared to the vehicle group.

Determination of the genotoxicity of the test item
No statistically significant increases in the mean of medians for the percentage of DNA in tail was induced at the three tested doses of 1000, 500 and 250mg/kg b.w./day (x2), vs. the vehicle control. Indeed, the means of medians of the percentage of tail DNA ranged from 1.23 to 2.29% vs. 2.79% in the respective vehicle control.
The test item Bisisopropyl peroxydicarbonate was thus not genotoxic in rat duodenum. Indeed, no primary DNA damage was detected.

Glandular stomach cells
In progress.

Analytical results:
The control of concentrations of Bisisopropyl peroxydicarbonate in treatment preparations was performed in a GLP compliant laboratory following a validated method. The resuslts are reliable; concentrations were determined to be within the range 85-115% of the treatment with percent recovery between the theoretical and actual concentrations of -1 or -0.7% at the concentrations ranging from 25 to 100 mg/mL (report in progress).

Applicant's summary and conclusion

Conclusions:

Under our experimental conditions, the test item does not present DNA strand breaks and/or alkali-labile sites inducer activities toward the liver and the duodenum from CD Sprague-Dawley male rats.
Investigations on the glandular stomach are in progress.

Executive summary:

The search for potential genotoxic activity of Bisisopropyl peroxydicarbonate (CAS 105-64-6) was assessed using the in vivo comet assay in the liver, glandular stomach and duodenum in the rat.
The treatment was carried out by oral route (gavage), using 1 daily treatment for 2 consecutive days with the OCDE guideline 489 (2016) at the dose-levels of 1000 – 500 – 250 mg/kg b.w./day base on the results of preliminary assay up to 2000 mg/kg b.w./day.

No increase in the percentage of DNA in tail was observed in liver and duodenum.

Investigations on the glandular stomach are in progress.