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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 7-9 weeks old
- Weight at study initiation: not reported
- Housing: housed 2-3 animals / sex / group / cage in IVC cages
- Diet: Altromin 1324 maintenance diet for rats and mice provided ad libitum
- Water: tap water provided ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no: MKCD1021 (pre-experiment); MKCG 3257 (main experiment)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulations were kept under magnetic stirring for approximately 10 minutes or until visual homogeneity was achieved.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
400 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5 males per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Justification for choice of positive control(s): Selection was based on the results of the JaCVAM validation trial
- Route of administration: oral
- Doses / concentrations: 200 mg/kg bw

Examinations

Tissues and cell types examined:
Cells from the liver, glandular stomach and duodenum were isolated, embedded in agarose, lysed and DNA allowed to migrate under electrophoresis conditions.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose levels were based on the results of a dose-range finding acute toxicity study.The maximum dose applied in the dose range-finding study was 2000 mg/kg bw. The maximum volume administered was 10 mL/kg bw and was compatible with physiological space available. Since only slight toxic effects were determined after the first and second administration of 2000 mg/kg bw in one animal of each sex, the same dose was applied in two additional animals of each sex. Lower doses were not tested.

TREATMENT AND SAMPLING TIMES: Animals of the vehicle control group and the three test item dose groups were treated orally by gavage daily for a period of 2 consecutive days. The animals of the positive control were treated once by oral gavage 4 h before sacrifice. During the period of administration, the animals were observed precisely each day for signs of toxicity. 4 h after last treatment, animals were deeply anaesthetized using ketamine/xylazine. The abdominal aorta was cut and the blood was released. Organs assigned to comet assay (liver, glandular stomach and duodenum) were removed, rinsed with cold mincing buffer to remove residual blood and stored in ice-cold mincing buffer on ice until further processing. The times to remove the tissues until the preparation of the slides were recorded in the raw data. A part of each analysed organ was preserved in 10% neutral-buffered formalin for histopathological evaluation.

DETAILS OF SLIDE PREPARATION: During the comet assay procedure individual cells were embedded in a thin agarose gel on a microscope slide. All cellular proteins were then removed from the cells by removing the cover slips and then incubating the slides overnight in chilled lysing solution. The DNA was allowed to unwind by incubating the slides in an alkaline (pH > 13) electrophoresis solution. Following the unwinding, the liberated DNA underwent electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus. This was accomplished by placing the slides in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order. After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times and incubated again in ice-cold ethanol and air-dried afterwards. After dehydration, cells were stained by applying a gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS: 150 cells/animal/tissue were analysed, if available. Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated. Each slide was screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet were adjusted manually if they are not set correctly automatically. All cells of the visual field were scored, except of e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail, were considered to be heavily damaged cells). Therefore, cells were classified into three potential categories scorable, non-scorable and “hedgehog” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail are considered to be heavily damaged cells). To avoid artefacts only scorable cells (defined round to oval nucleus) and at least 150 cells per sample on two slides (75 cells per slide) were scored. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus.
Evaluation criteria:
The alkaline comet assay is considered acceptable if it meets the following criteria according to OECD guideline 489:
- the concurrent negative control data are considered acceptable for addition to the laboratory historical control database;
- the concurrent positive controls should induce responses that are compatible to those previously generated and included in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control; and
- three doses and if available 150 cells per organ of each animal have been analysed

Increases in DNA damage in the presence of clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value. The positive control should produce a positive response, and if not, the study data will not be acceptable.

The test material is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- 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.

The test material is considered to be clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).
Statistics:
All slides, including those of positive and vehicle controls were independently coded before microscopic analysis and subsequently scored blinded. The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.

For each tissue type, the mean of the individual animal means was then determined to give a group mean. Statistical analysis was done using a variety of approaches.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
High-dose animals showed moderate toxic effects such as reduction of spontaneous activity, prone position, half eyelid closure and ataxia directly after the first administration. The same symptoms were present in the mid-dose group were less pronounced.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the registration substance did not induce biologically relevant DNA-strand breaks in any of the tissues evaluated. The result indicates no adverse effect of the test item on the DNA of liver, glandular stomach and duodenum cells after oral administration in rats.