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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-08-2015 - 11-08-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: bone marrow chromosome aberration

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
particle size not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: N10415
- Expiration date of the lot/batch: 30 Jun 2015
- Date of production: Nov 2013

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator, under light exclusion, light sensitive
- Stability under test conditions: assumed to be stable under storage conditions.
- Solubility and stability of the test substance in the solvent/vehicle: good water solubility
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test species was selected according to the
recommendations of the OECD and EU or based on the
results published so far. Moreover, there has been up to now most experience with or most data for Wistar
rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 6-10 weeks
- Weight at study initiation: Males 217.8 +- 14.81 g, 169.4 +- 7.99 g
- Assigned to test groups randomly: yes, under following basis: computer program WinRando
- Fasting period before study: not stated
- Housing: in groups of 6
- Diet (e.g. ad libitum): standardized pelleted feed
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): not stated, but fully air-conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, deionized water was used as vehicle
- Concentration of test material in vehicle: 50 mg/L, 100 mg/L, 200 mg/L
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight
- Type and concentration of dispersant aid (if powder): none, to achieve solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in deionized water.
To achieve a solution of the test substance in the vehicle, the test substance preparation was
shaken thoroughly.
All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
The animals were dosed 48 hours, 24 hours and 3 hours before sacrifice.
Frequency of treatment:
three times
Post exposure period:
3 hours
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Justification for choice of positive control(s): This substance is a well-established positive control for genotoxicity e.g. DNA strand breaks and induction of micronuclei
- Route of administration: oral, gavage
- Doses / concentrations: 50, 100 and 200 mg/kg body weight

Examinations

Tissues and cell types examined:
lateral lobe of the liver, both femora
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
Preparing the cellulose columns
- A 13-mm filter disk with a pore size of 8 μm was placed at the bottom of a 10-mL plastic
syringe.
- Equal parts by weight of microcrystalline cellulose, Sigmacell® type 50 and α-cellulose
fibers, were mixed thoroughly and suspended with HBSS (Hanks Balanced Salt Solution
with Ca and Mg).
The cellulose suspension was added to the column of the 10-mL plastic syringe until the
3 mL calibration mark of the syringe was reached. Then, 5 mL HBSS was added.

Preparation of the bone marrow
The bone marrow was prepared according to the method described by Schmid and
Salamone et al. and Romagna et al.
- The animals were anesthetized with isoflurane. Subsequently, they were sacrificed by
cervical dislocation. Then the two femora were prepared by dissection and all soft tissues
were removed.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a
separate centrifuge tube using a cannula filled with fetal calf serum (FCS with EDTA) which was pre-heated up to 37°C (about 3 mL/femur).

Cell separation (Removal of nucleated cells)
- The bone marrow suspension was mixed gently before transferring to the cellulose column.
As soon as the suspension was fully soaked into the cellulose column 5 mL HBSS was
added.
- The eluate containing erythrocytes was centrifuged at 300 x g for 5 minutes. The
supernatant was removed gently and the precipitate was resuspended with 4 - 8 mL PBS
(Phosphate Buffered Solution with Ca and Mg) depending on the cell count.
- Labeled slides equipped with cell funnels were clamped into the rotor of the cytospin
(Cellspin I-12; whole equipment from Tharmac GmbH, Waldsolms, Germany). Then, 200 μL
cell suspension was transferred to each cell funnel and was centrifuged at 1 200 rpm
(approx. 220 x g) for 7 minutes (at least four slides per animal).
- After drying overnight the slides were stained.

Staining of the slides
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.

METHOD OF ANALYSIS:
Microscopic evaluation
Evaluation criteria:
Acceptance criteria
The rodent Micronucleus test is considered valid if the following criteria are met:
• The quality of the slides allows the evaluation ≥ 4000 PCE per animal and a clear differentiation between PCE and NCE.
• The ratio of PCE/NCE in the vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in negative control animals has to be within the range of the historical vehicle control data for PCEs (95% control limit).
• The positive control substance has to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Assessment criteria
A finding is considered positive if the following criteria are met:
• A statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent negative control value and the range of the historical negative control data (95% control limit).
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent negative control value and is within the range of the historical negative control data (95% control limit).
A scientific judgment is required in case of equivocal data.
Statistics:
The statistical evaluation of the data was carried out using an appropriate computer program.
For analysis of the rate of micronucleated polychromatic erythrocytes the Jonckheere-Terpstra test was used which is a non-parametric trend test. In addition, a pair-wise comparison of each test group with the control group was carried out using the Wilcoxon test (one-sided+). The statistical unit is the animal and therefore the rate per animal was used. The calculation was performed using R.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: yes, 2000 mg/kg bw tested
- Solubility: not tested
- Clinical signs of toxicity in test animals: none
- Evidence of cytotoxicity in tissue analyzed: not examined

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): comparable to solvent control
- Ratio of PCE/NCE (for Micronucleus assay): 500 mg/kgbw: 101%, 1000 mg/kgbw: 88%, 2000 mg/kgbw: 83%,
- Appropriateness of dose levels and route: toxicological effects (inhibition of erytrhopoiesis) indicates bioavailability
- statistical evaluation: performed, no noticable findings

Applicant's summary and conclusion

Conclusions:
not genotoxic in the Micronucleus Test in Bone Marrow Cells
Executive summary:

According to the results of the present study, the three times oral administration of Azo Initiator VA044 did not lead to any biologically relevant increase in the number of polychromatic erythrocytes containing micronuclei in bone marrow