Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
In vivo Mammalian Alkaline Comet Assay - incorporated into the study design of OECD 422 Combined 28-day Repeated Dose Toxicity Study and Reproductive/Developmental Screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2018 to TBC
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29th July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test and genotoxicity screening"
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Storage Conditions:Stable at ambient temperature (10-30°C). Store in darkness; may be used/formulated in light.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST
Details on species / strain selection:
Rat - Wistar Han™
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Ltd., Oxon, UK
- Age at study initiation: Male 8 - 11 weeks Female: 13-14 weeks
- Weight at study initiation: Not reported.
- Assigned to test groups randomly: assigned dose groups using a randomization procedure based on body weight at approximately five days prior to the start of treatment
- Fasting period before study: No
- Housing: Individually in wooden chew blocks and cardboard fun tunnels
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Pelleted Diet
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least fourteen days to allow exclusion of females not showing appropriate estrous cycling.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C)
- Humidity (%): 30-70%
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light)

IN-LIFE DATES: tbc

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
PEG 400 - more details to follow

- Vehicle(s)/solvent(s) used: PEG 400
- Justification for choice of solvent/vehicle: 

- Concentration of test material in vehicle: 

- Amount of vehicle (if gavage or dermal): 

- Type and concentration of dispersant aid (if powder):

 - Lot/batch no. (if required):

 - Purity:
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Oral gavage
Five males from each dose group (Groups 1 to 4) were dosed once daily with the test item for 45 consecutive days and killed approximately 3 hours after the last dose. Another set of three males were dosed with N-nitroso-N-methylurea (NMU) on Days 44 and 45 only (approximately 27 and 3 hours before euthanasia) and used as positive controls.
Duration of treatment / exposure:
45 days
Frequency of treatment:
Daily
Post exposure period:
N/A

Examinations

Tissues and cell types examined:
Liver, glandular stomach and jejunum were assessed
Details of tissue and slide preparation:
-CRITERIA FOR DOSE SELECTION:
Dose selection was based on the dose range finder study where the animals were exposed to dose range of 500, 750 and 1000 mg/kg bw/day at a treatment volume of 4 mL/kg. Based on the observations results of the study, it was considered that dose levels of 0 (control), 100, 350 and 750 mg/kg bw/day, utilizing a dose volume of 6 mL/kg, would be appropriate for further investigation of effects of the Test Item on reproduction or toxicity.

- TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
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.

A small section of the glandular stomach was immersed in stomach buffer 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

Approximately a 2 cm piece of Jejunum was processed and immersed briefly in stomach buffer and then scraped gently to remove any contents, incubated in stomach 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.

A small piece of liver (approximately 1 cm3) was washed in liver buffer, before being minced and filtered to provide a single cell suspension.

- DETAILS OF SLIDE PREPARATION:
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. 
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 in order 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. Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath filled with chilled electrophoresis buffer (pH>13) at low temperature (2-10 °C). The slides were then left for 20 minutes after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The slides were 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, after which they were stored prior to staining and scoring.
For each tissue, two of the four processed slide gels were scored (A and B replicates) and the remaining two slides were stored as backup slides (C and D replicates).

METHOD OF ANALYSIS:
The processed Comet slides were coded to allow “blind” scoring using a computer generated code and stained just prior to analysis for comets. To each dry slide gel, 75 µL of propidium iodide (20 µg/mL) was placed on top of the slide and 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 version 4.3.1). Two slide gels for each tissue for each animal were scored with a maximum of 75 cells per slide gel giving an accumulative total of 150 cells per tissue per animal. The slide score data for each tissue was processed using the Excel macro program provided in Comet IV version 4.3.1

 OTHER: N/A
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
Statistics:
Where considered appropriate, statistical analysis was performed on the mean % Tail Intensity and median % Tail Intensity data using a Students t-test on transformed data using a √(x+1) transformation.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 2. Comet Assay - Summary of Group Data on Glandular stomach

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Group Mean Standard Deviation of % Tail Intensity

Vehicle

0

1.44

0.47

2.25

Low

0

1.30

0.40

2.09

Intermediate

0.09

2.81

1.86

2.97

High

0

1.45

0.55

2.00

Positive (MNU)

20.54

38.26***

35.93***

14.39

Table 2b. Comet Assay - Summary of Group Data on Jejunum

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Group Mean Standard Deviation of % Tail Intensity

Vehicle

0

2.30

1.31

2.64

Low

0.10

2.17

1.21

5.69

Intermediate

0

2.19

1.24

2.56

High

0

1.52

0.62

2.15

Positive (MNU)

13.78

34.71***

32.42***

14.99

Table 2c. Comet Assay - Summary of Group Data on Liver

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Group Mean Standard Deviation of % Tail Intensity

Vehicle

0

0.24

0.01

0.72

Low

0

0.29

0.01

0.88

Intermediate

0

0.40

0.02

1.04

High

0

0.34

0.02

0.97

Positive (MNU)

2.80

45.30***

46.07***

10.09

MNU = N-nitroso-N-methylurea

 *** = P<0.001

Applicant's summary and conclusion

Conclusions:
Under the condition of the study, the test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver and was considered to not induce DNA damage in these tissues. The test item does not meet the criteria for classfication for mutagenicity in accordance with the Regulation (EC) 1272/2008.
Executive summary:

In comet assay assessment OECD 489 - 2019: Five males from each dose group (Groups 1 to 4) were dosed once daily with the test item at concentration of 0, 100, 350 & 750 mg/kg bw/day for 45 consecutive days and killed approximately 3 hours after the last dose.  Another set of three males were dosed with N-nitroso-N-methylurea (NMU) on Days 44 and 45 only (approximately 27 and 3 hours before euthanasia) and used as positive controls.

Samples of liver, glandular stomach and jejunum were taken at necropsy from these males and transferred to the Cell and Molecular Sciences Department and processed to provide slides for the comet assay.  

The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control data ranges.  The positive control item (MNU) produced a statistically significant increase in the percentage tail intensity and median percentage tail intensity in the liver, glandular stomach and jejunum indicating that the test method itself was operating as expected and was considered to be valid under the conditions of the test.

The glandular stomach did not demonstrate any marked increases in the percentage tail intensity or the median percentage tail intensity over the vehicle control. One individual slide, the ‘B’ replicate slide for animal 56 in the intermediate dose group had a  percentage tail intensity which was much greater than the remaining slides in the group and it exceeded the upper limit of the laboratory historical control range for a vehicle.  The reason for this is unclear since the ‘A’ replicate of the same animal and tissue had percentage tail intensities which were consistent with the rest of the group.  However, since the group as a whole had no statistically significant increases in percentage tail intensity, the increase observed in one slide is considered to be artefactual and of no significance in the overall result.  Therefore, it is considered that the test item did not induce DNA damage in the glandular stomach tissue investigated under the conditions of the test.  There were no dose-related increases in the percentage hedgehog frequencies in the dose groups of the glandular stomach.  There were no significant increases in the percentage mean tail intensity or median tail intensity in the jejunum or liver for any of the test item dose groups when compared to the vehicle control.  The incidence of hedgehog cells in both tissues, however, in the jejunum was lower than minimum of the historical control range for a vehicle but this was seen in both the vehicle and the test item dose groups and was therefore considered to be of no consequence.

Under the condition of the study, the test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver and was considered to not induce DNA damage in these tissues. The item does not meet the criteria for classification for mutagenicity in accordance with the Regulation (EC) 1272/2008.