Registration Dossier

Administrative data

genetic toxicity in vivo, other
In vivo alkaline comet (single cell get electrophoresis) assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2018 - 18 May 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
Adopted 29 July 2016
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Test material form:
solid: particulate/powder
Details on test material:
Physical state / appearance: Light yellow powder
Storage conditions: Room temperature in the dark

Test animals

Details on test animals and environmental conditions:
- Age at study initiation: Eight to ten weeks
- Weight at study initiation: 188.0 to 218.1 g
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Up to five animals per sex in solid-floor polypropylene cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Minimum of five days

- Temperature: 19 to 25°C
- Humidity: 30 to 70%
- Air changes: Approximately 15 air changes per hour
- Photoperiod: 12 hours light / 12 hours dark

IN-LIFE DATES: From: 04 April 2018 To: 18 May 2018

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used:arachis oil
- Concentration of test material in vehicle: 50 - 200 mg/mL
- Amount of vehicle: 10 mL/kg
Details on exposure:
For the purpose of this study the test item was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within 2 hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.
Duration of treatment / exposure:
Approximately 28 hours (two doses approximately 24 hours apart; animals were killed 4 hours after the second dose)
Frequency of treatment:
Two doses over a 24 hour period
Post exposure period:
4 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Vehicle control
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Maximum recommended dose
No. of animals per sex per dose:
2 males and 2 females - range finding group
5 males only - 500 and 1000 mg/kg
7 males - 2000 mg/kg
Control animals:
yes, concurrent vehicle
Positive control(s):
- Justification for choice of positive control: Substance has been found in-house to induce strand breaks and damage to DNA under the conditions of the test
- Route of administration: Oral gavage
- Doses / concentrations: Three male rats received two administrations of 25 mg/kg (10 mL/kg of a 2.5 mg/mL solution in water) approximately 24 hours apart


Tissues and cell types examined:
Liver, glandular stomach, duodenum
Details of tissue and slide preparation:
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 dioxide asphyxiation).
Samples of liver, glandular stomach, and duodenum were obtained from each animal for comet processing.

Sub-samples of the liver, glandular stomach and duodenum 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 have been conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may have been 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 to provide a single cell suspension.

Glandular Stomach – A small section of the glandular 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 glandular stomach was removed by gentle scraping and then a single cell suspension was obtained by scraping the remaining
tissue into a small volume of stomach buffer.

Duodenum - Approximately a 2 cm piece of Duodenum was processed. This was immersed briefly in liver buffer and then scraped gently to remove any contents, incubated in liver
buffer (approximately 10 mL), on ice for 5 to 10 minutes. A single cell suspension was obtained by gentle scraping into approximately 1 mL of fresh liver buffer.

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 molten 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 cover slip, 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 cover slips 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.
Evaluation criteria:
The slides were stained just prior to analysis for comets. To each dry slide, 75 μL of propidium iodide (20 μg/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, Comet IV.

Two slides for each tissue per animal were scored with a maximum of 75 cells per slide giving an accumulative total of 150 cells per tissue per animal. Care was taken to guarantee that a cell was not scored twice. The slide score data for each tissue was processed using the Excel macro program provided in Comet IV version 4.3.1. Comparisons between the vehicle control group response and that of the test item dose groups was made. The primary endpoint was percentage DNA in the tail (percentage Tail intensity and median percentage Tail intensity).

Each slide was also assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity.
A comparison was made between the vehicle control groups and the positive control groups. The individual slide score data for the percentage tail intensity and median percentage tail intensity was compared using a Students t-test with a √1+x transformation. Comparisons between the vehicle control groups and the test item dose groups were also made when it was considered that there was a marked increase over the vehicle control value.

Results and discussion

Test results
Key result
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
- Dose range: 2000 mg/kg, 2 males and 2 females
In animals dosed with test item there were no premature deaths and no clinical signs were observed.
Bone marrow slides were prepared from the range-finding experiments for quantitative assessment.
The quantitative assessment revealed that there was very modest bone marrow toxicity observed in one male and one female at 2000mg/kg which was considered to give an indication that systematic absorption of the test item had occurred.
Based on the above data the maximum tolerated dose (MTD) 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. There was no noticeable difference in clinical signs between the male and female animals and therefore only male animals were used for the main test.

One animal from the 1000 mg/kg dose group was killed in extremis after the initial dose but this death was considered to be due to a technical error and not test item related. No clinical signs were observed in animals dosed with the test item, other than the animal which was killed prematurely. The loss of one animal from the 1000 mg/kg dose group is considered to have no impact on the study outcome since there were sufficient animals in the MTD group (2000 mg/kg) to meet the requirements of the guideline.

There were no statistically significant increases in percentage tail intensity for any of the test item dose levels in the liver, glandular stomach or duodenum tissues which exceeded the current historical control range for a vehicle, confirming the test item did not induce DNA damage in the liver, glandular stomach or duodenum. One animal in the 2000 mg/kg dose group had very high tail intensities in the glandular stomach compared with the rest of the group but this was considered to be an exception for one animal and although it increased the overall tail intensity for the group the result was not statistically significant.

There were no marked increases 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 and duodenum are characteristic of the gastro-intestinal tract where there is a high turnover of cells.

Applicant's summary and conclusion

The test item did not induce any statistically significant increases in the percentage tail intensity or median percentage tail intensity in the liver, glandular stomach or duodenum and therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo under the conditions of the test.
Executive summary:

As the available in-vitro data indicated the possibility of a mutagenic effect, an in-vivo Comet assay was conducted (Envigo, 2018) according to OECD TG 489 and in accordance with GLP principles. Three groups of male rats received two doses of BMI (500, 1000 and 2000 mg/kg) approximately 24 hours apart. Following dosing the rats were humanely euthanised and tissue samples were collected from the liver, glandular stomach and duodenum. The tissue samples were processed and scored for the presence of Comets which are indicative of DNA strand breakage in the assessed tissues.

The test item did not induce any statistically significant increases in the percentage tail intensity or median percentage tail intensity values in any of the tissues investigated when compared to the concurrent vehicle control group of the liver, glandular stomach or duodenum. The test item was considered to be unable to induce DNA strand breakage to the liver, glandular stomach or duodenum in vivo, under the conditions of the test.