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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:
Experimental start date: 10 October 2017 Experimental completion date: 05 January 2018
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)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bisisobutyryl peroxide
EC Number:
222-340-0
EC Name:
Bisisobutyryl peroxide
Cas Number:
3437-84-1
Molecular formula:
C8H14O4
IUPAC Name:
2-methylpropanoyl 2-methylpropaneperoxoate
Details on test material:
- Name of test material (as cited in study report): Bisisobutyryl peroxide solution
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Storage condition of test material: In freezer (≤ -25°C) in the dark


Specific details on test material used for the study:
Identification: Bisisobutyryl peroxide (CAS# 3437-84-1)
Lot/Batch Number: 1705447077
Physical state/Appearance: Clear colourless liquid
Expiry Date: 16 July 2018
Purity:
Tert-Butyl Hydroperoxide (0.566%)
Diisobutyryl peroxide (38.91%)
Isododecane (60.5%)
No adjustment for purity was made.
Storage Conditions: Stored at approximately -20 °C in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Envigo (UK). At the start of the main test the males weighed 186.0 g to 219.1 g, and were approximately eight to ten weeks old. Details of the individual animal weights, group means and standard deviations for the animals used in the main test are presented in Table 1. After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.
The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study. The diet and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The animals were provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). These enrichment items were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil.
Details on exposure:
Determination by analysis of the concentration, homogeneity and stability of the test item preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.

Positive Control Preparation
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant Batch no.3012436).
Duration of treatment / exposure:
28 hours
Frequency of treatment:
dosed twice with a 24-hour interval
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Groups each of five male rats per dose
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
N-Nitroso-N-methylurea (MNU). MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.

Examinations

Tissues and cell types examined:
Liver
Glandular Stomach
Testes
Details of tissue and slide preparation:
Comet Test
Groups each of five male rats were dosed twice with a 24 hour interval via the oral route with the test item at 2000, 1000 or 500 mg/kg. The groups of rats from each dose level were killed by humane euthanasia (carbon monoxide asphyxiation) approximately 4 hours following the second administration, 28 hours after the start of the test. In addition, two further groups of rats were included in the study; one group (five male rats) was dosed twice with a 24-hour interval via the oral route with the vehicle alone (Arachis oil) and a second group (three male rats) was dosed twice orally with 24-hour interval with N-Nitroso-N-methylurea (MNU). MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test. The vehicle and positive controls were killed approximately 4 hours following the second administration, 28 hours after the start of the test.

Tissue Sample Requirements
Humane euthanasia was performed on the animals at the end of the exposure period using a method that did not affect the integrity of the required tissues (carbon monoxide asphyxiation). Samples of liver, glandular stomach, and testes were obtained from each animal for comet processing.
Sub-samples of the liver, glandular stomach and testes were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathology investigations. Assessment of cytotoxicity by histopathology may be conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.
The tissue samples were processed under subdued lighting and over ice to provide single cell suspensions, providing sufficient cells for scoring for the comet assay as follows:
Liver - A small piece of liver (approximately 1 cm3) was washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered through gauze to provide a single cell suspension.

Glandular Stomach– The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides.

Testes – These were dissected from each animal. One was retained for possible histopathology. The second one was placed in liver buffer and chopped with scissors to provide a single cell suspension which was filtered through gauze before use for the comet slides.


Slide Preparation
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
Approximately 30 µL of the cell suspension was added to 270 µL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period, the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.
Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.


Scoring
Comet slides from the liver and glandular stomach were scored. Comet slides from the testes were prepared but were not scored at the request of the Sponsor.
The slides were coded prior to scoring to allow “blind” scoring. The slides are stained just prior to analysis for comets. To each dry slide, 75 L of propidium iodide (20 g/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, i.e. Comet IV version 4.3.1.
Two slide gels for each tissue per animal were scored with a maximum of 75 cells per slide gel giving an accumulative total of 150 cells per tissue per animal. Care was taken to guarantee that a cell is not scored twice. The slide score data from each experiment was processed using the Excel macro program provided in Comet IV. Comparisons between the vehicle control group response and that of the test item dose groups were made. The primary end-points are percentage tail DNA (%Tail intensity) and median percentage tail intensity.
Each slide was assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.
Evaluation criteria:
Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly Negative if:
• None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
• There is no evidence of a dose-related response
• The results are within the laboratory historical vehicle control range
• There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved
The test item is then considered unable to induce DNA strand breakage in the tissues studied in the test system.
Providing that all the 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 compared to the concurrent negative control.
• The response is considered to be dose related
• The results are substantially outside the laboratory historical vehicle control range.
The test item can be considered to induce DNA strand breakage in a particular tissue if all three conditions are met.
There is no requirement for verification of a clearly positive or negative response.
Although most experiments will be expected to give clear negative or positive results in rare cases the data set will preclude making a definite judgment. This may require the scoring of additional slides to increase the number of cells and, therefore, add more power to the data. If this does not resolve the issue then the result will be given as equivocal or questionable, and may require the histopathological assessment of the tissues to see if cell toxicity may be the causative agent rather than any genotoxic mechanism.
Statistics:
When a less than clear response is observed a comparison will be made between the vehicle control groups and each corresponding treatment dose group, on the percentage tail intensity data using individual slide scored values.

Results and discussion

Additional information on results:
Range-Finding Toxicity Test
In animals dosed with test item there were no premature deaths and no clinical signs observed.
Bone marrow slides were prepared for quantitative assessment from the final range-finding experiment at 2000 mg/kg.
The quantitative assessment revealed that moderate bone marrow toxicity had occurred at 2000 mg/kg in one of the animals. This was considered to give an indication of systematic absorption of the test item in the absence of clinical signs.
Based on the above data the maximum recommended dose (MRD) of the test item, 2000 mg/kg, was selected for use in the main test, with 1000 and 500 mg/kg as the lower dose levels.

Comet Assay
Mortality Data and Clinical Observations
One premature death was observed in the 2000 mg/kg (MRD) dose group and one in the 1000 mg/kg group prior to the second dose being administered. In the 2000 mg/kg dose group one animal (11) exhibited hunched posture and one animal (13) exhibited hunched posture, lethargy and increased salivation one hour after the first dose was administered. Hunched posture was observed in three animals in the 2000 mg/kg dose group prior to termination. No clinical signs were seen in the animals of the 1000 mg/kg or the 500 mg/kg dose groups.
It is considered that the premature deaths were not due to true test item toxicity and resulted in each group not meeting the minimum animal number of five per group as stated in the test guideline. However, the Comet assay results for both tissues were well within the historical control range and did not indicate any genotoxic activity and, therefore, it was considered there was no impact on the integrity or purpose of the study.

Evaluation of Comet Assay Slides
The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach, comparable with the laboratory historical control range for these tissues. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

There were no marked increases in percentage tail intensity for any of the test item dose levels in the glandular stomach or liver tissues which exceeded the current historical control range for a vehicle, confirming the test item did not induce DNA damage in the liver or glandular stomach.

There was no marked increase in hedgehog frequency for any of the test item dose levels in either of the tissues investigated. High numbers of hedgehogs observed in the glandular stomach are characteristic of gastro-intestinal tract where there is a high turnover of cells.

Any other information on results incl. tables

Range-Finding Toxicity Test

The mortality data are summarized as follows:

Dose Level
(mg/kg)

Sex

Number of Animals Treated

Route

Deaths on Day

Total Deaths

0

1

450

Male

2

oral

0

0

0/2

600

Male

2

oral

0

0

0/2

1000

Male

2

oral

0

0

0/2

2000

Male

2

oral

0

0

0/2

2000

Male

2

oral

0

0

0/2

Bone marrow slides were prepared for quantitative assessment from the final range-finding experiment at 2000 mg/kg. The slides were scored per 1000 cells and the data is presented below.  

Animal Code

Dose level (mg/kg)

Number of polychromatic erythrocytes

Number of normochromatic erythrocytes

PCE/NCE ratio

4-0

2000

536

464

1.16

4-1

404

596

0.68

 

Summary of Results for Percentage Tail Intensity, Median Percentage Tail Intensity and Percentage Hedgehogs for each Tissue

Liver

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity per Animal

Vehicle
(Arachis oil)

0

0.29

0

500 mg/kg (MRD/4)

0

0.16

0

1000 mg/kg (MRD/2)

0

0.17

0

2000 mg/kg (MRD)

0

0.16

0

Positive control
(N-Nitroso-N-Methylurea)

2.07

21.10

20.95

 

Glandular Stomach

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity per Animal

Vehicle
(Arachis oil)

7.65

5.48

3.61

500 mg/kg (MRD/4)

7.32

4.06

2.09

1000 mg/kg (MRD/2)

6.65

4.39

2.53

2000 mg/kg (MRD)

8.90

4.15

1.60

Positive control
(N-Nitroso-N-Methylurea)

10.22

30.41

28.82

Applicant's summary and conclusion

Conclusions:
The test item, Bisisobutyryl peroxide (CAS# 3437-84-1) did not induce any increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.
Executive summary:

 Introduction

The Comet Assay has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC 1999, as described by Ticeet al., 2000. The method is designed to be compatible with the procedures indicated in the OECD 489 Guideline (2016).

The primary target tissues of the comet assay were liver and glandular stomach. Comet slides were also prepared from the testes but these were not scored.

 

Methods

A range-finding test was performed to find suitable dose levels of the test item. The Comet assay main test was conducted at the maximum recommended dose (MRD), 2000 mg/kg with 1000 and 500 mg/kg as the lower dose levels. Groups of rats (five per group) were dosed a 0 hours and 24-hours and were killed at 28 hours (4 hours after the second dose administration). 

In addition, two further groups of rats were included in the study; one group (five rats) were dosed via the oral route with the vehicle alone (arachis oil) at 0 and 24 hours, and a second group (three rats) were dosed orally with N-Nitroso-N-methylurea (NMU) at 0 and 24 hours to act as the positive control.

 

The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 28 hours after the start of the test. The glandular stomach, liver, and testes were processed for comet slides. Samples of liver, testes and glandular stomach were preserved in formalin for possible histopathology in the event of a positive response. 

 

The slides prepared from the liver and glandular stomach were scored for the presence of Comets.

 

Results

The presence of clinical signs indicated that systemic absorption had occurred.

One premature death was observed in the 2000 mg/kg (MRD) dose group and one in the 1000 mg/kg group prior to the second dose being administered. The following clinical signs were observed in some animals dosed with test item at 2000 mg/kg: hunched posture, lethargy and increased salivation.It is considered that the premature deaths were not due to true test item toxicity and resulted in each group not meeting the minimum animal number of five per group as stated in the test guideline. However, the Comet assay results for both tissues were well within the historical control range and did not indicate any genotoxic activity and, therefore, it was considered there was no impact on the integrity or purpose of the study.

 

The positive control group induced a marked increase in percentage tail intensity and median percentage tail intensity in the liver and glandular stomach indicating that the test method itself was operating as expected. The vehicle control groups all had percentage tail intensity and median percentage tail intensity values within the expected range.

 

There was no statistically significant marked increase in percentage tail intensity or median percentage tail intensity for any of the test item dose groups in the liver or glandular stomach when compared to the vehicle control, confirming that the test item did not induce DNA damage in the tissue investigated under the conditions of the test.

 

Conclusion

The test item,Bisisobutyryl peroxide (CAS# 3437-84-1) did not induce any increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.