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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
genetic toxicity in vivo, other
Remarks:
in vivo combined mammalian somatic cell study: cytogenicity / erythrocyte micronucleus and comet assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-12-03 to 2014-03-05
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Qualifier:
according to
Guideline:
other: In vivo Mammalian Alkaline Comet Assay - Draft OECD Guideline for the Testing of Chemicals
Qualifier:
according to
Guideline:
other: ICH S2 (R1)
Version / remarks:
2011
GLP compliance:
yes
Type of assay:
other: in vivo combined mammalian somatic cell study: cytogenicity / erythrocyte micronucleus and comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Source of the test substance was not reported. Batch No: 236/2013
- Expiration date of the lot/batch:
28 Feb 2015
- Purity test date: No details reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Determined by the Sponsor. No further details reported.
- Stability under test conditions:
Determined by the Sponsor. No further details reported.
- Solubility and stability of the test substance in the solvent/vehicle:
Suspensions of the test item were freshly prepared before testing.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
No details reported.



TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Suspensions of the test item were freshly prepared before testing.
- Preliminary purification step (if any):
No details reported.
- Final dilution of a dissolved solid, stock liquid or gel:
No details reported.
- Final preparation of a solid:
No details reported.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 8-10 weeks
- Weight at study initiation: approximately 300 grams
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: up to 5 animals/cage, in polisulphone H-temp solid bottomed cages with nesting material provided into suitable bedding bags
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +- 2°C
- Humidity (%): 55% +- 15%
- Photoperiod (hrs dark / hrs light): 12 hour light/ 12 hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% methylcellulose
- Justification for choice of solvent/vehicle: based on results obtained from solubility trials and the preliminary tests
Details on exposure:
Test item:
Treatment: three times
Dose volume: 20 mL/kg body weight.

Positive control (comet assay):
Treatment: twice
Dose volume: 10 mL/kg body weight.

Positive control (micronucleus assay):
Treatment: once
Dose volume: 10 mL/kg body weight.

A reserve group of 3 males was dosed at the high dose level to substitute animals dying during the treatment period. Moreover, a satellite group of 3 males was included for blood sampling.



Duration of treatment / exposure:
- Animals were sacrifced 3-4 hours after last dosing (test item group and positive control group of comet assay)
- Animals were sacrifced 24 hours after dosing (positive control group micronucleus assay)
Frequency of treatment:
test item: at 0 (Day 1), 24 (Day 2) and 45 (Day 3) hours
positive control (comet assay): 21 hour interval
positive control (micronucleus assay):once
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:
5 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Comet assay: EMS
Micronucleus assay: Mitomycin C:
- Route of administration: oral gavage (test item, positive control comet assay), intraperitoneal injection (positive control micronucleus assay)

Examinations

Tissues and cell types examined:
Comet assay: liver tissue
Micronucleus assay: bone marrow
Details of tissue and slide preparation:
Collection of blood samples for exposure determination:
Before the first dosing, blood samples were collected pre-dose. Additional blood samples were collected 1 and 3 hours after dosing from 3 animals treated at the high dose level (satellite group). Approximately 1 mL of blood was collected from the tail of each animal and placed into a tube containing lithium heparin as anticoagulant. Blood samples were centrifuged at room temperature. The plasma was separated immediately after centrifugation and transferred into two separate pre-labelled tubes. Approximately 250 mL of plasma was transferred in the first set tube (aliquot A) and the remaining plasma in the second set tube (aliquot B). The plasma aliquots were stored at -80°C until analysis. Plasma analysis was performed by the Analytical Chemistry Department at RTC according to a validated method.

Comet assay:

Preparation of single cells from liver:
The animals were sacrifced at the appropriate time after last treatment by asphyxiation with carbon dioxide. From each animal, a section of liver was removed, cut into 3 or more pieces and gently agitated in a Petri dish full of ice-cold phosphate buffered saline (DPBS) containing 20 mM EDTA and 10% DMSO (mincing solution) to remove as much blood as possible. The sample was placed into ice-cold mincing solution and minced into finer pieces. The cell suspension was filtered and centrifuged at 500 rpm for 10 minutes at approximately +4°C. The pellet was resuspended in ice-cold mincing solution and the cell concentration was adjusted to a final concentration of approximately 1×106 cells/mL using mincing solution. The cell suspension was used for slide preparation. Prior to preparation of single cell suspensions, a sample of liver from each vehicle control and test item treated animal was taken. These samples were fixed and preserved in 10% neutral buffered formalin. No samples were retained from rats treated with the positive control solution.

Slide preparation:
For each cell suspension, 60 µL of cell suspension was added to 390 µL of 0.7% (w/v) low melting point agarose (LMA), pre-warmed at 37°C. An aliquot of 75 µL of each suspension was dispensed onto glass microscope slides pre-coated with a layer of 1% (w/v) normal melting point agarose (NMA). The slides were covered with a clean coverslip and the gels were allowed to solidify. The coverslips were removed and further 75 µL of LMA added over the cell layer and covered with a clean coverslip. The gels were allowed to solidify and when applied the coverslips were carefully removed. At least four slides were prepared. Theslideswereimmersedinfreshlypreparedpre-cooledlysissolutionovernight at approximately 4°C in the dark. After the lysis step, one slide from each animal was rinsed three times for 5 minutes in 0.4 M Tris, pH 7.5 and then dried and stored at room temperature. These ‘diffusion’ slides were used to estimate the degree of highly damaged cells in the cell suspensions. All slides, with the exception of the “diffusion” slides, were incubated for 25 minutes in alkaline (pH>13) electrophoresis buffer to produce single-stranded DNA and to express ALS and SSB. After alkali unwinding, electrophoresis was carried out at 1-10°C for 25 minutes, at 30 V (1 V/cm) and 300 mA, using a Bio-Rad power supply. Since not all slides can be processed at the same time, a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run. After electrophoresis, the slides were immersed in 0.3 M sodium acetate in ethanol for 30 minutes. Microgels were dehydrated in absolute ethanol for 2 hours and immersed for 5 min in 70% ethanol. Slides were air-dried and stored at room temperature. Immediately before scoring, slides were stained with 12 µg/mL ethidium bromide.

Micronucleus assay:

Slide preparation:
One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end and irrigated with 2 mL foetal calf serum using a syringe. The suspension of cells was aspirated and this procedure was repeated several times. The cells were centrifuged and a concentrated suspension prepared. The suspension thus obtained was centrifuged at 1000 rpm for 5 minutes and the supernatant completely removed. The cells of the sediment were then resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried overnight and then fixed with methanol for 10 minutes. Subsequently slides were stained with haematoxylin and eosin solutions. Finally slides were rinsed in distilled water and allowed to dry.
Evaluation criteria:
Comet Assay:

Acceptance criteria:
The assay is considered to be valid if the following criteria are met:
– The positive control treatment shows a statistically signifcant increase in comet parameters over the concurrent vehicle control.
– At least 4 animals per group are available for slide analysis.

Criterion for outcome of the assays:
For the test item to be considered positive, a significant increase (indicative of strand breaks) or decrease (indicative of cross-links) in DNA migration must be observed in any tissue evaluated, in at least one dose group. In addition there must be evidence of a dose-related increase or decrease in DNA migration measurements. It is important to consider any cytotoxic effect in conjunction with a positive response in DNA migration.

Micronucleus Assay:

Acceptance criteria:
The assay is considered to be valid if the following criteria are met:
- The incidence of micronucleated PCEs of the vehicle control group falls within the historical negative control range.
- The positive control item induces a significant increase in the frequency of micronucleated PCEs.
- At least 5 animals per group are available for slide analysis.

Criterion for outcome of the assays:
The test item is considered to induce micronuclei if a statistically significant and biologically meaningful increase in micronucleus incidence (at p<0.05) is observed in any treatment group. Where increases in the incidence of micronucleated PCEs are observed which are statistically signifcant, but fall within the range of negative control values of this laboratory, then these data are used to demonstrate that these increases do not have biological significance. Historical controls are available.
Statistics:
Comet assay:
DNA damage was evaluated as extent of DNA migration and the metric end points used were tail moment and tail intensity. The statistical significance of the differences amongst groups was assessed by analysis of variance followed by Dunnett’s or Cochran’s test.

Micronucleus assay:
Two thousand polychromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei. The results obtained were subjected to statistical analysis using a modifed Chi-squared test.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
other: Negative
Remarks:
Micronucleus Assay
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Sex:
male
Genotoxicity:
other: Comet assay: All treatments demonstrated low levels of cytotoxicity. Micronucleus assay: No inhibitory e¿ect on erythropoietic cell division was induced by the test item in any treatment group.
Vehicle controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Comet assay:

Comet parameters of the vehicle control treatment are within historical control values with the exception of one animal which presented high values of tail intensity and tail moment. These values were markedly higher than those observed in animals of the same group or in animals from historical negative controls and were probably attributable to an e¿ect induced by mechanical reasons (e.g cell suspension preparation). No increases in tail migration were observed in any treatment group. Good responses were observed after treatment with the positive control item ethyl methanesulfonate, indicating the correct response of the comet test system.

At least four animals per group were available for slide analysis. The positive control ethyl methanesulphonate induced clear and statistically significant increases in DNA migration parameters over the concurrent controls. The study was accepted as valid. No evidence of a dose-related increase or decrease in DNA migration measurements was observed, nor statistically significant difference between vehicle and test item treated groups. Based on the stated criteria, the test was considered valid and the test substance was not considered positive in the comet assay.

Micronucleus assay:

An increase in the incidence of micronucleated PCEs over the control value was observed at the low dose level. The incidence was slightly above the range of historical values observed in our laboratory . No increases in the numbers of micronucleated PCEs were observed in other treatment groups. For one animal, possibly due to misdosing during Mitomycin-C administration, no increase of micronucleated PCEs was observed. The animal was excluded from the evaluation of data. Marked increases in the incidence of micronucleated PCEs were observed in other positive control group animals, indicating the correct functioning of the test system.

The incidence and distribution of micronucleated PCEs in vehicle control groups were consistent with the laboratory’s historical control data. A statistically significant increase in the incidence of micronucleated PCEs over the control values was seen in the positive control group, indicating the correct functioning of the test system. Five animals were available for slide analysis in all treatment groups. The study was accepted as valid. Following treatment with the test item, a statistically significant increase (p<0.05) in the incidence of micronucleated cells over the control was observed. The incidence slightly exceeded the range of historical values for negative controls.

Since the increase was only observed in the low dose group and was attributable to the incidence noted in one animal, it was considered to be due to a chance event and therefore not biologically relevant.

Applicant's summary and conclusion

Conclusions:
It is concluded that the test substance did not induce DNA damage in the liver of male rats following oral gavage administration of doses of 500, 1000 and 2000 mg/kg/day. At these same dose levels, the test item did not induce significant increases in micronucleated polychromatic erythrocytes in the bone marrow.