Registration Dossier

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:
05/12/2017 -20/02/2018
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:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
The number of animals used for the positive control group was reduced to 3 rather than 5 as stated in the study plan. This was to reduce the number of animals used in the study and it complies with the OECD 489 guideline which states 3 animals are sufficient for the positive control group.
Deviations:
yes
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / Batch :0727/16
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 99%
- Physical state/Appearance: Off white powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
No analysis was carried out to determine the 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.
- Solubility and stability of the test substance in the solvent/vehicle: test item was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item was freshly prepared as required as a suspension at the appropriate concentration in arachis oil :
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
HsdRccHan™WIST
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (UK)
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: 184.4 to 209.4 g
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding.
- Diet and Water : at libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%relative humidity
- Air changes (per hr): 15
- 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: arachis oil
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 200, 100 and 500 mg/mL
- Amount of vehicle (if gavage or dermal): 10mL
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
Identification: Arachis Oil
Supplier: W M Hodgson & Co
Label Information: ZZZ08613
Purity: Treated as 100%
Expiry Date: 12 May 2018
Storage Conditions: Room temperature
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
Duration of treatment / exposure:
24h
Frequency of treatment:
Groups of male rats were dosed twice with a 24 hour interval
Post exposure period:
4 hours following the second administration
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals for the 1000 and 500 mg/kg bw dose level
7 animals for the 2000 mg/kg bw dose level
5 animals for the vehicle control
3 animals for the positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
N-Nitroso-N-methylurea (MNU)
- Justification for choice of positive control(s):
- Route of administration: oral route, gavage. MNU is a positive control item that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.
- Doses / concentrations: 25 mg/kg bw


Identification: N-Nitroso-N-methylurea (MNU)
Supplier: AstaTech Inc
Supplier’s Lot Number: 4486-027
Physical state/ Appearance: Pale orange solid
Purity: 90%
Expiry: 05 December 2018
Storage Conditions: Approximately -20 ºC
Solvent: Distilled water

For the purpose of this study the positive control item was freshly prepared as required as a solution at a concentration of 2.5 mg/mL in distilled water (Laboratoire Aguettant batch no. 3012436).

Examinations

Tissues and cell types examined:
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 and glandular stomach were obtained from each animal for comet processing.
Sub-samples of the liver and glandular stomach 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.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 24 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

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 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.

DETAILS OF SLIDE PREPARATION:
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 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.

METHOD OF ANALYSIS:
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, i.e. Comet IV.
Two slide gels for each tissue per animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per animal. Care was taken to guarantee that a cell was not scored twice. The slide score data were 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.

OTHER:
Evaluation criteria:
The following criteria will be used to determine a valid assay:
• The concurrent negative control is comparable with the laboratory historical negative control range.
• The positive controls induce responses that are comparable with those in the laboratory historical positive control range.
• Adequate numbers of cells and doses have been analyzed.
• The highest dose level selected meets the requirements of the guideline and the study plan.
Statistics:
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 made when there was an increase over the vehicle control value.

Statistical analysis was performed on the 1000 mg/kg and the 2000 mg/kg dose groups of the liver compared to the vehicle control group where increases in mean percentage tail intensity and median % tail intensity were observed.

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:
not valid
Additional information on results:
RESULT OF THE RANGE-FINDING TOXICITY TEST /
In animals dosed with test item (2000 mg/kg bw) there were no premature deaths and no clinical signs observed.
Bone marrow slides were prepared for quantitative assessment from the range-finding experiments. The slides were scored per 1000 cells.
The quantitative assessment revealed that moderate bone marrow toxicity was observed at 2000 mg/kg in one of the animals. Although not conclusive since the other animals in the range finding experiments did not demonstrate any marked toxicity it 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. 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.

RESULTS OF THE BONE MARROW QUANTITATIVE ASSESSMENT :

The quantitative assessment revealed that moderate bone marrow toxicity was observed at 2000 mg/kg in one of the animals. Although not conclusive since the other animals in the range finding experiments did not demonstrate any marked toxicity it was considered to give an indication of systematic absorption of the test item in the absence of clinical signs.

RESULTS OF DEFINITIVE STUDY : COMET ASSAY

Mortality Data and Clinical Observations :
There were no premature deaths seen in any of the test item dose groups and no clinical signs were observed.

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 or median percentage tail intensity for any of the test item dose levels in the glandular stomach which exceeded the current laboratory historical control range for a vehicle, confirming the test item did not induce DNA damage in the glandular stomach.

The liver did demonstrate a small but statistically significant response at the maximum dose level (2000 mg/kg). The response does appear to be dose related but the values for all three exposure groups are well within the historical laboratory control range for a vehicle. The values for the lowest dose group (500 mg/kg) are less than that for the concurrent vehicle control. In the 2000 mg/kg dose group, animal 14 had higher mean percentage tail intensities than the rest of the group and this skewed the data for this group resulting in the higher response. The ‘B’ replicate slide for animal 12 also had a much higher percentage tail intensity than its partner and was not consistent with the rest of the slides in the dose group. The higher standard deviations for these slides indicates a greater spread in the response which can be attributed to a few cells with higher percentage tail intensities rather than a consistent increase in all cells scored. The response seen in the liver at 2000 mg/kg is therefore considered to be of no toxicological significance. It does not meet the requirements of the study plan for a positive in that the results are not substantially outside the laboratory historical vehicle control range. The test item was therefore considered to be unable to induce DNA strand breakage in the liver under the conditions of the test.

There was no marked increase in hedgehog frequency for any of the test item dose levels in either of the tissues investigated.

Any other information on results incl. tables

Mortality data for the Range-Finding Toxicity Study :

Dose Level (mg/kg) Sex Number of Animals Treated Route Deaths on Day
0 1
2000 male 1 oral 0 0
2000 female 1 oral 0 0
2000 male 1 oral 0 0
2000 female 1 oral 0 0

Scored bone marrow slides from the range-finding experiments :

Animal code Sex Dose level (mg/kg) Number of Polychromaatic erythrocytes (PCE) Number of normochromatic erythrocytes (NCE) PCE/NCE (ratio)
1-0 male 2000 313 687 0,46
2-0 female 2000 514 486 1,06
3-0 male 2000 486 514 0,95
3-1 female 2000 542 458 1,18

Applicant's summary and conclusion

Conclusions:
The test item did not induce any toxicologically significant 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:

A range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex.

In a Comet assay test (Morris A., 2018, ENVIGO) performed according to the OECD 489 Guideline, GLP complaince, with male animals at the Maximum Recommended Dose (MRD)

7 rats of the 2000 mg/kg dose level, 5 rats of the 1000 mg/kg dose level and 5 rats of the 500 mg/kg were dosed by oral gavage at time 0 and 24 hours after the initial dosing and were killed 4 hours after the second dose administration, at a sampling time of 28 hours. The glandular stomach and liver tissues were sampled and processed, the slides were then prepared prior to scoring for the presence of Comets.

Further groups of rats were given a double oral dose of arachis oil (5 rats) or methyl nitrosourea (3 rats), to serve as vehicle and positive controls respectively.

The quantitative assessment revealed that moderate bone marrow toxicity was observed at 2000 mg/kg in one of the animals. Although not conclusive since the other animals in the range finding experiments did not demonstrate any marked toxicity it was considered to give an indication of systematic absorption of the test item in the absence of clinical signs.

There was no evidence of an increase in the glandular stomach in the percentage tail intensity or median percentage tail intensity in the test item dose groups when compared to the concurrent vehicle control group.

The liver did demonstrate a small but statistically significant increase in both the percentage tail intensity and the median percentage tail intensity at the maximum dose level (2000 mg/kg). However, the increases were within the current laboratory historical range for a vehicle and much of the increase could be attributed to one animal. As we do not meet the three requirements in the study plan to designate a positive response we consider this to be of no toxicological significance.

The positive control item produced a marked increase in the % tail intensity value in the liver and glandular stomach, indicating that the test method was working as expected. The vehicle control group for the liver and the glandular stomach had % tail intensity values which were consistent with the current laboratory historical range for a vehicle.

The test item did not induce any toxicologically significant 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.