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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
OO-tert-butyl O-(2-ethylhexyl) peroxycarbonate
EC Number:
252-029-5
EC Name:
OO-tert-butyl O-(2-ethylhexyl) peroxycarbonate
Cas Number:
34443-12-4
Molecular formula:
C13H26O4
IUPAC Name:
3-({[(tert-butylperoxy)carbonyl]oxy}methyl)heptane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 5-10 weeks old
- Weight at study initiation: 171 g and 223 g in males and between 174 g and 210 g in females
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: three or two animals per cage.
- Diet (e.g. ad libitum): A04C-10 from SAFE (batch 16216).
- Water: drinking water, softened by reverse osmosis and filtered on 0.22 ìm membrane, was provided ad libitum.
- Acclimation period: 7 or 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: 200, 100, 50 or 25 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The stability of the test item in the vehicle was 21 days at ambient temperature.
Preparations for treatments in the dose-range finding were performed just before use.
The concentration of LUPEROX® TBEC in dosing formulations were controlled analytically.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily
Post exposure period:
Tissue samples were taken 2 to 6 hours after the last treatment.
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
group 4
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
group 5
No. of animals per sex per dose:
4 groups of 5/sex, and one group of 7/sex (highest dose)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Methylmethane sulfonate
- Justification for choice of positive control(s): historical data
- Route of administration: oral, 24 hours and 2 to 6 hours before sacrifice.
- Doses / concentrations: 100 mg/kg/day

Examinations

Tissues and cell types examined:
Isolated glandular stomach, liver, duodenum and kidney cells.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A Dose Range Finding assay was performed with 4 groups of 3 male and 3 female rats receiving 2 daily treatments at 24-hour intervals at the doses of 2000, 1000, 250 and 0 mg/kg/day per os for the determination of the MTD (Maximal Tolerated Dose) and the assessment of the cytotoxicity of the test item on the glandular stomach of all animals by both a histological analysis and the halo test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treatment took the form of 2 successive administrations at 24-hour intervals by oral route (gavage). Samples were taken 2 to 6 hours after the last treatment.
The animals were treated according to the experimental program below:

GROUP NUMBER – TREATMENT PER DAY (x2) NUMBER OF ANIMALS
Males Females
1. Vehicle alone (negative control), PO 5 5
2. Methylmethane sulfonate 100 mg/kg/day PO (x2) 5 5
3. Test item: 2000 mg/kg/day (x2)a, PO 5 (+2)b 5 (+2)b
4. Test item: 1000 mg/kg/day (x2) , PO 5 5
5. Test item: 500 mg/kg/day (x2) , PO 5 5
PO: per os (oral)
a As the maximum tolerated dose was equal or higher than 2000 mg/kg, this dose was administered in line with OECD (2016) and HAYASHI et al. (2007).
b Two animals per gender studied in the high-dose group were treated, in parallel. They were sacrificed but were not examined as no mortality was observed in the five first treated animals.

DETAILS OF SLIDE PREPARATION:
- Tissue sampling and cell isolation
The 5 males and the 5 females1 of each group were assigned for cell isolation and assessed for DNA fragmentation.
Individual animals were anaesthetized with isoflurane and exsanguined.
A 'V' shaped incision will be made from the center of the lower abdomen to the rib cage. The skin and muscles will be removed to reveal the abdominal cavity.
A portion of the Glandular Stomach, Liver, Duodenum and one of the 2 Kidneys were removed and washed in the cold mincing buffer until as much blood as possible has been removed (see also § 9.2 for histological assessment). The portion was minced with a pair of fine scissors to release the cells. The cell suspension were stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Moreover, a part of the stomach cell suspension from each animal (control and treated) was smeared and fixed on 2 labelled slides for eventual subsequent cytological evaluation (see § 9.3).
Cells were enumerated on a hemocytometer, and an adequate number of cells was harvested from each cell suspension for proceeding to slides preparation with 20 x 103 cells per slide whatever the organ assessed.
Single cell preparations were done within one hour after animal sacrifice.
- Protocol for the Comet assay
Dried slides preparation (pre-layering)
Conventional slides were dipped in hot 1.5 % normal melting point agarose in PBS. After gentle removal, the underside of the slides were wiped in order to remove excess agarose. The slides were then laid in a tray on a flat surface to dry
- Slide preparation
Before use, a volume of 85 ìL of 0.8% of Normal Agarose (NA) was added on the microscope slide pre-layered with 1.5% of NA and then covered with a glass coverslip. Slides were placed at +2-8°C until the agarose layer hardens (3 to 5 minutes). The cells of the different doses tested were mixed with 0.5% of Low Melting Point Agarose (LMPA) (75 ìL/slide) kept at ca. 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at +2-8°C.
Four slides per animal were prepared for the Comet assay.
Lysis
After the top layer of agarose has solidified, the glass coverslips were removed and the slides were immersed for one night at ca. + 4 °C in the dark in a lysing solution.
Unwinding, electrophoresis and staining
After this incubation period, the slides were then removed and placed on a horizontal gel electrophoresis unit and the unit filled with freshly prepared alkaline buffer (pH > 13) to around 0.25 cm above the slides. In order to avoid excessive variation across the groups during each electrophoretic run, only one of the replicate slides were processed in each run for each animal (DNA – unwinding and electrophoresis). The cells were exposed to the alkali for 20 minutes to allow the DNA unwinding, and expression of single-strand breaks and alkali-labile sites. Next, electrophoresis was conducted for 20 minutes at <10°C by applying an electric current of 0.7 V / cm (25 V / 300 mA). All these steps were conducted protected from daylight to prevent the occurrence of additional DNA damage. After electrophoresis at pH >13, the slides were neutralized twice for 5 minutes with 0.4 M Tris (pH 7.5) and the DNA was exposed for 5 minutes to absolute ethanol in order to preserve all the Comet assay slides. Subsequently, the slides were airdried and then stored at room temperature until they were scored for DNA migration.
- DNA staining and image analysis
Slides were coded by a person not involved in the reading (coding sheets are presented in Appendix No. 7)
Just prior to scoring, the DNA was stained using propidium iodide (final concentration of 20 ìg/mL sterile water; 25 ìL/slide).
Slides were examined with a 200 x magnification, using a fluorescent microscope (Leica Microsystems SAS - DM 2000, Heerbrugg, Switzerland), equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated monochrome CCD IEEE1394 FireWire video camera (Allied Vision Technologies), to the Comet Assay IV Image Analysis System, version 4.11 with Windows XP Pro Software (Perceptive Instruments Ltd, Suffolk, UK).
For all groups three slides were analysed with 50 nuclei per slide randomly scored. The five animals were used, i.e. 15 slides per group, 750 analysed nuclei per group.
- Scoring and parameters
According to the OECD guideline (489, 2016), the percentage of DNA in tail (percent tail intensity) are considered as the most relevant DNA damage parameter. This parameter appears to be the most linearly related to dose (B. Burlinson et al., 2007). Then, the median of the percentage of DNA in tail measured in the different nuclei for each slide and for each animal were calculated, considering the animal as statistical unit (D. Lovell and T. Omori, 2008). Finally the mean of medians obtained for the different animals was calculated for each group.
In addition, each slide was also examined for presence of hedgehogs. The hedgehogs, also known as clouds or ghost cells, are morphological indicative of highly damaged cells often associated with severe genotoxicity, necrosis and apoptosis (therefore possible indicator of toxicity). 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. Hedgehogs were excluded from image analysis data collection and calculation of percent tail intensity. Their frequency might be useful for data interpretation. Statistical evaluation was done with the X2 test to determine significance of differences in group values between each group versus the vehicle control.
In the 2000, 1000 and 500 mg/kg/day (x2) treated groups the frequencies of hedgehogs were inferior to 50% whatever the organ assessed when compared to the relative vehicle control, in both male and female rats.
Otherwise, only a slight statistically significant increase in the percent hedgehogs was noted in the stomach of female rat at the intermediary dose of 1000 mg/kg/day(x2) with 2.51% of hedgehogs vs. 0.78% in the relative control group. It was without any incidence for the assessment of data for genotoxicity.
- Acceptance criteria for results of the Comet assay
A study is accepted if the following criteria are fulfilled:
- The mean of medians of percentage of DNA in tail of the concurrent negative controls should be within the control limits of the distribution of the laboratory’s historical negative control database (extreme deviations).
- The concurrent positive controls should induce means of medians of percentage of DNA in tail that are comparable to the historical positive control data and produce a statistically significant increase compared with the concurrent negative control.
- The appropriate number of doses and cells must be analyzed.
Moreover:
- In the vehicle group, an eventual increase in the frequency of hedgehogs must not be >50%.
The validity criteria for the test were considered as fulfilled with 2 exceptions regarding the results for positive control in male and female duodenum. Test was validated.
Evaluation criteria:
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 negative 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 negative control data.
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 doses exhibits a statistically significant increase compared with the concurrent negative control,
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are within the distribution of the historical negative control data for a given species, vehicle, route, tissue, and number of administrations
- 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.
Statistics:
In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5.
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
Sex:
male/female
Genotoxicity:
negative
Remarks:
glandular stomach, liver, duodenum and kidney cells
Toxicity:
no effects
Remarks:
but tested up to the limit dose
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals:
A slight stereotypy (slight head movements and chewing) was observed in all male and female rats 15 minutes after the first and the second treatment with 2000 mg/kg. Difficulty to breath for 1 male 15 minutes after the first treatment at this dose level was also noticed. No other clinical signs were noted whatever the period of observation or at both 1000 and 250 mg/kg/day (x2). Otherwise, during the sampling of stomachs for histological assessment and the halo assay (see below), it was noted that the stomachs from the highest dose group (2000 mg/kg) were larger than the ones from the other treatment groups.
- Evidence of cytotoxicity in tissue analyzed:
The histological assessment of a portion of the stomach (glandular part and forestomach) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of toxicity (i.e. assessment of necrosis).
As well, the halo assay performed on a portion of the stomach (glandular part only) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of either necrotic or apoptotic cells when compared to the concurrent vehicle control.
- Rationale for exposure:
From the overall results obtained in the dose range finding assay (i.e. clinical assessment, histological assessment of stomach cells and halo test), the highest dose of 2000 mg/ kg/ day (x2) was retained for the comet assay. Two inferior doses of 1000 and 500 mg/kg/day (x2) per os were also tested.

RESULTS OF DEFINITIVE STUDY
The mean of medians of percentage of DNA in tail of the concurrent negative controls were within the control limits of the distribution of the laboratory’s historical negative control database (extreme deviations).
In all organs, the concurrent positive control induced statistically significant increases of percentage of DNA in tail compared with the negative control. These values are comparable to the historical positive control data with 2 exceptions in male and female duodenum with values of 20.08 and 20.49%, respectively vs. 28.45 – 73.93%.
No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in Isolated glandular stomach, liver, duodenum and kidney cells of OFA Sprague Dawley male and female rats.

Applicant's summary and conclusion

Conclusions:
LUPEROX® TBEC is not genotoxic in glandular stomach, liver, duodenum and kidney from male and female rats treated once a day for 2 days with up to 2000 mg/kg.
Executive summary:

The evaluation of the genotoxic potential of LUPEROX® TBEC (98.2 %w/w of OO-tert-butyl O-(2-ethylhexyl) peroxycarbonate) was done through the in vivo comet assay performed under alkaline conditions,i.e.pH > 13 (Alkaline Single Cell Gel Electrophoresis, OECD TG no. 485) in both male and female OFA Sprague Dawley rats, in isolated glandular stomach, liver, duodenum and kidney cells.

A Dose Range Finding assaywas performed with 4 groups of 3 male and 3 female rats receiving 2 daily treatments at 24-hour intervals at the doses of 2000, 1000, 250 and 0 mg/kg/day per os for the determination of the MTD (Maximal Tolerated Dose) and the assessment of the cytotoxicity of the test item on the glandular stomach of all animals by both a histological analysis and the halo test.

A slight stereotypy (slight head movements and chewing) was observed in all male and female rats 15 minutes after the first and the second treatment with 2000 mg/kg. Difficulty to breath for 1 male 15 minutes after the first treatment at this dose level was also noticed. No other clinical signs were noted whatever the period of observation or at both 1000 and 250 mg/kg/day (x2). Otherwise, during the sampling of stomachs for histological assessment and the halo assay (see below), it was noted that the stomachs from the highest dose group (2000 mg/kg) were larger than the ones from the other treatment groups.

The histological assessment of a portion of the stomach (glandular part and forestomach) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of toxicity (i.e.assessment of necrosis). As well, the halo assay performed on a portion of the stomach (glandular part only) did not demonstrate any difference between treated groups and vehicle control groups regarding the appearance of either necrotic or apoptotic cells when compared to the concurrent vehicle control.

From the overall results obtained in the dose range finding assay (i.e.clinical assessment, histological assessment of stomach cells and halo test), the highest dose of 2000 mg/ kg/ day (x2) was retained for the comet assay. Two inferior doses of 1000 and 500 mg/kg/day (x2)per oswere also tested.

For the comet assay, groups of male and females rats (5/sex for the controls and low and mid dose levels, and 7/sex for the top dose level) were treated twice at 24-hour interval by oral route (gavage) with the test item at dose levels of 2000, 1000 and 500 mg/kg/day in corn oil, or with methylmethane sulfonate at 100 mg/kg/day in sterile water as positive control, or the vehicle corn oil under 10 ml/kg as negative control. All animals were sacrificed following one expression time of 3 to 6 hours after the last treatment. One hundred and fifty cells (50 cells/slide, 3 slides /animal) observed per animal.

The determination of LUPEROX® TBEC in treatment formulations was performed by a GLP- compliant laboratory using a validated analyticin treatment formulations used in the main assays were satisfactory with less than 10% deviation from target concentrations.

The mean of medians of percentage of DNA in tail of the concurrent negative controls were within the control limits of the distribution of the laboratory’s historical negative control database (extreme deviations).

In all organs, the concurrent positive control induced statistically significant increases of percentage of DNA in tail compared with the negative control. These values are comparable to the historical positive control data with 2 exceptions in male and female duodenum with values of 20.08 and 20.49%, respectively vs. 28.45 – 73.93%.

 

Glandular stomach cells

 

in vivo COMET ASSAY IN ISOLATED RAT MALE GLANDULAR STOMACH CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

4.48

  

 

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

2.50

<0.05

0.50

N.S.

1000

2.50

<0.05

1.35

N.S.

500

2.73

<0.05

1.64

N.S.

Positive control

Methylmethane Sulfonate

 

100

39.39

-

<0.01

2.24

<0.01

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley male rats.

Statistically significant decreases in the mean of medians of percentage of DNA in tail were noted at all doses. This effect was considered to be without biologically significance and any incidence in terms of genotoxic hazard.

The test item was thus not genotoxic toward the glandular stomach cells in male rat.

 

in vivo COMET ASSAY IN ISOLATED RAT FEMALE GLANDULAR STOMACH CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

1.93

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

2.10

N.S.

1.15

N.S.

1000

2.01

N.S.

3.22

<0.01

500

2.23

N.S.

1.82

N.S.

Positive control

Methylmethane Sulfonate

 

100

43.17

-

<0.01

3.75

<0.01

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley female rats.

The test item was thus not genotoxic toward the glandular stomach cells in female rat.

 

Liver cells

 

in vivo COMET ASSAY IN ISOLATED RAT MALE LIVER CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

0.29

  

 

<0.05

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

0.13

N.S.

0.38

N.S.

1000

0.16

N.S.

0.38

N.S.

500

0.45

N.S.

0.75

N.S.

Positive control

Methylmethane Sulfonate

 

100

25.52

-

<0.01

0.61

N.S.

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley male rats. A statistically dose-dependent effect was noted but was considered without biologically significance.

The test item was thus considered as not genotoxic toward the liver cells in male rat.

 

in vivo COMET ASSAY IN ISOLATED RAT FEMALE LIVER CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

0.51

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

0.17

N.S.

2.01

N.S.

1000

0.23

N.S.

1.99

N.S.

500

0.31

N.S.

2.66

N.S.

Positive control

Methylmethane Sulfonate

 

100

24.34

-

<0.01

2.65

N.S.

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley female rats.

The test item was thus not genotoxic toward the liver cells in female rat.

 

Duodenum cells

 

in vivo COMET ASSAY IN ISOLATED RAT MALE DUODENUM CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

2.54

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

3.33

N.S.

0.65

N.S.

1000

2.41

N.S.

0.73

N.S.

500

4.54

N.S.

1.06

N.S.

Positive control

Methylmethane Sulfonate

 

100

20.08

-

<0.01

1.32

N.S.

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley male rats.

The test item was thus not genotoxic toward the duodenum cells in male rat.

 

in vivo COMET ASSAY IN ISOLATED RAT FEMALE DUODENUM CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

2.83

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

2.24

N.S.

0.88

N.S.

1000

2.72

N.S.

0.83

N.S.

500

1.99

N.S.

0.44

N.S.

Positive control

Methylmethane Sulfonate

 

100

20.49

-

<0.01

0.89

N.S.

N.S.: not statistically significant at the threshold p=0.05

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley female rats.

The test item was thus not genotoxic toward the duodenum cells in female rat.

 

Kidney

 

in vivo COMET ASSAY IN ISOLATED RAT MALE KIDNEY CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

1.05

 

  

<0.05

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

0.31

<0.01

0.67

N.S.

1000

0.41

N.S.

0.89

N.S.

500

0.77

N.S.

0.82

N.S.

Positive control

Methylmethane Sulfonate

 

100

24.14

-

<0.01

0.18

<0.001

N.S.: not statistically significant at the threshold p=0.05

 

A statistically and dose-related significant decrease in the mean of medians of percentage of DNA in tail was observed at the highest tested doses of 2000 mg/kg/day (x2) in OFA Sprague Dawley male rats. This effect was considered to be without biological significance.

The test item was thus considered as not genotoxic toward the kidney cells in male rat.

 

in vivo COMET ASSAY IN ISOLATED RAT FEMALE KIDNEY CELLS

 

 

 

GROUP

 

 

 

TEST ITEM

 

 

DOSES in

mg/kg/day (x2)

 

 

% of DNA in tail Mean of medians per animal (/5 animals)

NON PARAMETRIC

statistical assessment

 

Hedgehogs

 

p Kruskall- Wallis

 

p Mann- Whitney

 

Relative ratio of hedgehogs

 

p

Vehicle control

Corn oil

0

0.43

 

  

N.S.

-

-

-

 

 

TREATED

 

 

LUPEROX® TBEC

2000

0.33

N.S.

1.44

N.S.

1000

1.36

N.S.

2.27

N.S.

500

3.20

N.S.

1.98

N.S.

Positive control

Methylmethane Sulfonate

 

100

24.04

-

<0.01

1.71

N.S.

N.S.: not statistically significant at the threshold p=0.05

 

No statistically significant decrease or increase in the mean of medians of percentage of DNA in tail was observed at the 3 tested doses of 2000, 1000 and 500 mg/kg/day (x2) in OFA Sprague Dawley female rats.

The test item was thus considered as not genotoxic toward the kidney cells in female rat.

 

In conclusion, LUPEROX® TBEC (98.2 %w/w of OO-tert-butyl O-(2-ethylhexyl) peroxycarbonate) induced no biologically significant increase in DNA strand breaks. Therefore, LUPEROX® TBEC has no genotoxic activity in glandular stomach, liver, duodenum and kidney cells of male and female Sprague Dawley rats.

 

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