Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): tert-amyl hydroperoxide
- Physical state: liquid
- Analytical purity: 83.2% tert-amyl hydroperoxide in water
- Lot/batch No.: 11071D1480
- Expiration date of the lot/batch: 23/11/2021
- Storage condition of test material: refrigerator (+5±3°C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 5 to 6 weeks old
- Weight at study initiation: between 165 g and 194 g (male)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: polypropylene cages
- Diet (ad libitum): A04C-10 from SAFE
- Water (ad libitum): softened by reverse osmosis and filtered on 0.22 µm membrane
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: 50, 32, 20, 10 and 5 mg/mL
- Amount of vehicle (if gavage or dermal): 10ml/kg
Details on exposure:
The stability of the test item in the solvent was demonstrated for 9 days in previous repeated dose toxicity studies. Preparations for treatment were performed just before use.
Duration of treatment / exposure:
2 days
Frequency of treatment:
daily
Post exposure period:
3 to 6 hours after the last treatment.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200, 320 and 500 mg/kg
Basis:
other: Toxicity assays
Remarks:
Doses / Concentrations:
50, 100 and 200 mg/kg
Basis:
other: Genotoxicity assay
No. of animals per sex per dose:
5-7 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Methylmethane sulfonate (MMS) at a dose of 100 mg/kg PO, 24 hours and 3 to 6 hours before sacrifice.

Examinations

Tissues and cell types examined:
Hepatocytes and glandular stomach and forestomach cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The OECD guideline recommends that the test be carried out at the maximum tolerated dose. The maximum tolerated dose is described as the highest dose which causes no mortality, but which may give rise to the appearance of signs of toxicity (OECD No. 489, 2014; Hayashi et al, 1994).
In absence of data regarding differences in systemic toxicity, metabolism, bioavailability, bone marrow toxicity, etc. including e.g. in a range-finding study, the preliminary toxicity test was performed in both sexes.
In an effort to reduce the use of laboratory animals required and to obtain more accurate information, the range- finding toxicity test was performed according to the improved experimental design recommended by Fielder et al. (1992). For the preliminary assay, groups of 2 male and 2 female rats 5 to 6 weeks old were treated. For the confirmatory assay, 10 animals were used (5 male and 5 female rats 5 to 6 weeks old).

DETAILS OF SLIDE PREPARATION:
The 5 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 was removed to reveal the abdominal cavity.
The whole liver, and the whole stomach were removed. The stomach and a portion of the liver that was previously cut were washed in the cold mincing buffer until as much blood as possible has been removed.

Isolation of hepatocytes
The portion was minced 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 haemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.

Isolation of glandular stomach and forestomach cells
A portion of the stomach (containing both glandular stomach and forestomach) was removed as provision for histopathology (see § 10). The remaining stomach was separated in glandular stomach and forestomah to proceed to the comet assay on each specialised part of the organ.
The portions were minced with to release the cells. The cell suspensions were stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.
It is noteworthy that the whole stomachs were compacted when compared to the control organs. Moreover, at the highest dose of 200 mg/kg, the alimentary bolus was semiliquid, particularly at the forestomach level.

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 µL 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) (75µL/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 (3 slides were actually read).

METHOD OF ANALYSIS:
Protocol for the Comet assay
Lysis
After the top layer of agarose has solidified, the glass coverslips were removed and the slides were immersed overnight 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 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 ca. 20 minutes to allow the DNA unwinding, and expression of single-strand breaks and alkali-labile sites. Next, electrophoresis was conducted for ca. 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 ca. 5 minutes with 0.4 M Tris (pH 7.5) and the DNA was exposed for ca. 5 minutes to absolute ethanol in order to preserve all the Comet assay slides. Subsequently, the slides were air-dried and then stored at room temperature until they were scored for DNA migration.

Image analysis
Just prior to scoring, the DNA was stained using propidium iodide (final concentration of 20 µg/mL sterile water; 25 µL/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. Five animals were retained per group, i.e. at least 15 slides per group, at least 750 analysed nuclei per group.

Tail parameters
Recent publications focused on the interpretation of the results through the analysis of the median of the percentage of DNA in tail, with the animal as statistical unit (D. Lovell and T. Omori, 2008).
In fact, this parameter appears to be the most linearly related to dose (B. Burlinson et al., 2007).

OTHER:
Cytotoxicity was evaluated through the enumeration of hedgehogs.
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 concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside 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
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
PRELIMINARY TOXICITY ASSAY
500 mg/kg/day (x1)
The preliminary assay performed on 2 male and 2 female rats at the dose of 500 mg/ kg/ day (x1) per os demonstrated 15 minutes after the treatment, in all animals a slight decrease in spontaneous motor activity, mydriasis and slight loss of muscular tonus in males, 25 minutes after the treatment.
20 to 30 minutes after the treatment, the females demonstrated titubation. Moreover, all animals presented 45 to 60 minutes after the treatment a moderate decrease in spontaneous motor activity, titubation, and a moderate loss of muscular tonus. Finally, 75 minutes after the first treatment, piloerection; was visible in all animals and the females demonstrated ptosis.
Under these conditions, for ethical reason, animals were sacrificed and the lower doses of 320 and 200 mg/kg were tested.

320 mg/kg/day (x2)
The dose of 320 mg/kg induced 15 min after the 1st treatment in all animals a slight increase in spontaneous, motor activity, moderate sniffing, and coprophagy. One hour after the 1st treatment, 1 male and 1 female demonstrated a moderate decrease in spontaneous motor activity, and the other male and female rats presented a moderate decrease in muscular tonus, slight tremors and a moderate chewing.
Moreover, 15 min after the 2nd treatment, the test item induced in all animals a moderate decrease in spontaneous motor activity and chewing, and 2 to 4 hours after the 2nd treatment all animals demonstrated a moderate decrease in spontaneous motor activity with slight decrease in muscular tonus.

200 mg/kg/day (x2)
The dose of 200 mg/kg induced 15 minutes after each treatment a slight decrease in spontaneous motor activity in all animals.

Under these conditions, the dose of 200 mg/kg was retained to be tested in the confirmatory toxicity assay.

CONFIRMATORY TOXICITY ASSAY
The dose of 200 mg/kg induced 15 minutes after each treatment a slight decrease in spontaneous motor activity in all animals.

The highest dose retained for the comet assay was set at 200 mg/kg/day (x2). Two inferior doses of 100 and 50 mg/kg/day (x2) were also tested.

As no sex-specific toxicity difference was observed between sexes in the preliminary toxicity assays, the comet test was performed using groups of 5 males only (7 males for the high dose group) treated at different dose levels. Indeed, as there was no substantial difference between sexes in toxicity, testing in a single sex is sufficient (OECD No. 489, 2014).

RESULTS OF DEFINITIVE STUDY
No statistically significant increases in the median percentage of DNA in tail of the hepatocytes at the three analysed doses of 200, 100 and 50 mg/kg/day (x2),vs.the negative control. Indeed, the means of median for the percentage of DNA in tail were of 1.20, 1.57 and 3.10 % at 200, 100 and 50 mg/kg/day (x2)vs. 1.70% for the vehicle control group

No statistically significant increases in the median percentage of DNA in tail of the glandular stomach cells at the three analysed doses of 200, 100 and 50 mg/kg/day (x2),vs.the negative control. Indeed, the means of median for the percentage of DNA in tail were of 30.50, 21.59 and 25.69 % at 200, 100 and 50 mg/kg/day (x2)vs. 22.49% for the vehicle control group.

No statistically significant increases in the median percentage of DNA in tail of the forestomach cells at the three analysed doses of 200, 100 and 50 mg/kg/day (x2),vs.the negative control. Indeed, the means of median for the percentage of DNA in tail were of 7.03, 11.47 and 13.26 % at 200, 100 and 50 mg/kg/day (x2) vs. 8.44% for the vehicle control group.

Any other information on results incl. tables

ORGAN: LIVER

 

in vivo COMET ASSAY IN ISOLATED RAT LIVER CELLS

 

 

 

GROUPS

 

 

 

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- Wallis1

 

p Mann- Whitney2

 

Relative ratio of hedgehogs3

 

p

Negative control

 

sterile water

0

1.70

 

 

 

 

N.S.

-

-

-

 

 

TREATED

 

 

tert-amyl hydroperoxide

200

1.20

N.S.

2.42

<0.01

100

1.57

N.S.

2.31

<0.01

50

3.10

N.S.

1.51

N.S.

Positive control

Methylmethane Sulfonate

 

100 mg/kg/day (x2)

53.23

-

<0.01

1.91

<0.05

ORGAN: GLANDULAR STOMACH

 

in vivo COMET ASSAY IN ISOLATED RAT GLANDULAR STOMACH CELLS

 

 

 

GROUPS

 

 

 

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- Wallis1

 

p Mann- Whitney2

 

Relative ratio of hedgehogs3

 

p

Negative control

 

sterile water

0

22.49

 

 

 

 

N.S.

-

-

-

 

 

TREATED

 

 

tert-amyl hydroperoxide

200

30.50

N.S.

0.56

<0.01

100

21.59

N.S.

0.82

N.S.

50

25.29

N.S.

0.78

N.S.

Positive control

Methylmethane Sulfonate

 

100 mg/kg/day (x2)

71.08

-

<0.01

0.99

N.S.

ORGAN: FORESTOMACH

 

in vivo COMET ASSAY IN ISOLATED RAT FORESTOMACH CELLS

 

 

 

GROUPS

 

 

 

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- Wallis1

 

p Mann- Whitney2

 

Relative ratio of hedgehogs3

 

p

Negative control

 

sterile water

0

8.68

 

 

 

 

N.S.

-

-

-

 

 

TREATED

 

 

tert-amyl hydroperoxide

200

7.03

N.S.

0.60

<0.01

100

11.47

N.S.

1.00

N.S.

50

13.26

N.S.

1.42

<0.05

Positive control

Methylmethane Sulfonate

 

100 mg/kg/day (x2)

48.83

-

<0.01

0.80

N.S.

1: Total group without positive control

2: Mean values obtained in treated group compared to Mean values obtained in solvent control group

3: Corresponds to the percentage of hedgehogs per treated group / percentage of hedgehogs in negative control group.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under these experimental conditions, tert-amyl hydroperoxide induced no statistically or biologically significant increases in DNA strand breaks at 200, 100 and 50mg/kg/day(x2) in male rat isolated Liver, Glandular Stomach and Forestomach cells after oral administration. Therefore, tert-amyl hydroperoxide is considered having no genotoxic activity in these organs.
Executive summary:

The genotoxic potential of tert-amyl hydroperoxide was investigated in the in vivo mammalian alkaline comet assay performed under alkaline conditions, i.e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) in liver, glandular stomach and forestomach of male rats in compliance with OECD Guideline 489 (2014) using 2 successive daily treatments at the maximum dose compatible with the toxicity of the test item, i.e. 200 mg/kg/day (x2), followed by one sampling time 2 to 6 hours after the last treatment. The two lower doses of 100 and 50 mg/kg/day (x2) were also analysed.

No statistically significant increases in the median percentage of DNA in tail of the hepatocytes, glandular stomach and forestomach cells were observed at the three analysed doses of 200, 100 and 50 mg/kg/day (x2), vs. the negative control.

Indeed, the means of median for the percentage of DNA in tail were 1.20, 1.57 and 3.10 % at 200, 100 and 50 mg/kg/day (x2) vs. 1.70% for the vehicle control group in hepatocytes, 30.50, 21.59 and 25.69 % vs. 22.49% in glandular stomach cells and 7.03, 11.47 and 13.26 % vs. 8.44% in forestomach cells, respectively.

Therefore, tert-amyl hydroperoxide is considered having no genotoxic activity in these organs.