Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar - Sep 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)
Version / remarks:
26 Sep 2014
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
Type of assay:
other: Mammalian Erythrocyte Micronucles Test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation: liquid
- Density:1.138 g/mL
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 12 Feb 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the vehicle: Due to the good solubility of the test substance in deionized water, deionized water was selected as vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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.

Test animals

Species:
mouse
Strain:
other: Crl: NMRI
Details on species / strain selection:
The animals were selected according to the recommendations of the OECD and EU or on the basis of results published so far. Moreover, there has been up to now most experience with or most data for NMRI mice.
Only animals that were free of evident signs of illness were used. A health check was carried out on arrival from the breeder and before administration.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 - 8 weeks (information from the breeder)
- Weight at study initiation: 28.3 +/- 1.54 g
- Assigned to test groups randomly: yes, under following basis: The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program (WinRando, BASF SE).
- Housing: single housing in polycarbonate cages, type II
- Diet (e.g. ad libitum): standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water from bottles was available ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) useddeionized water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in deionized water, deionized water was selected as vehicle.
- Concentration of test material in vehicle: 50 mg/mL (low dose), 100 mg/mL (intermediate dose), 200 mg/mL (top dose)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
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 treated once orally (gavage).
Frequency of treatment:
once
Post exposure period:
24 h
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:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): The stability of cyclophosphamide is well-defined under the selected conditions, since it is a well-established reference clastogen.
- Route of administration: once orally (gavage)
- Doses / concentrations: 10 mL/kg bw of a 20 mg/kg bw solution in deionized water

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In the pretest to determine the acute oral toxicity in males and females, the recommended highest dose of 2000 mg/kg body weight was tolerated without any signs of toxicity. Based on the data of the pretests a dose of 2000 mg/kg body weight (limit dose) was administered as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses. Besides, no indication for a gender specific toxicity was obtained in the pretests. Thus, only male animals were used in the main experiment as requested by the current OECD Guideline 474.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle. The positive control CPP, dissolved in deionized water, was administered to male animals once orally in a volume of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance concentrations, vehicle control, positive control) and 48 hours (highest test substance concentration, vehicle control) after the treatment, respectively. The weight of each animal was measured and both femora each were excised for bone marrow preparation.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by Schmid and Salamone et al.
- The animals were anesthesized with isoflurane. Subsequently, they were sacrificed either by decapitation in the course of the blood sampling or 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 centrifuge tube using a cannula filled with fetal calf serum (FCS with EDTA) which was pre-heated up to 37 °C (about 2 mL/femur).
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 alpha-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.
Cell separation:
- 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 1 - 4 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. Then, 200 µL cell suspension was transferred to each cell funnel and was centrifuged at 1200 rpm (approx. 220 x g) for 7 minutes (at least 2 slides per animal).
- After drying overnight the slides were stained.
Staining of the slides:
- The slides were stained with eosin and methylene blue for about 5 minutes.
- After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL deionized water) for about 15 minutes.
- After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS: Microscopic evaluation
The slides for the evaluation of micronucleated erythrocytes were analyzed by use of the image analysis system Metafer4 MetaCyte. Polychromatic and nonmonochromatic erythrocytes (PCE or NCE) that fulfill the criteria for scoring were automatically detected under the light microscope and were recorded by the system. Then, a trained scorer performed manually the analysis based on these preselected images on the screen. In case of low quality of images, e.g. artefacts of staining etc., the cells were assessed directly on the slides by light microscopy.
In general, 4000 polychromatic erythrocytes (PCE) were evaluated for the occurence of micronuclei from each animal of every test group, so in total 20000 PCE were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
The microscopic analysis was run on coded slides.
Since the absolute values shown were rounded, but further calculation was based on unrounded values, there may be deviations in the relative values given.
Evaluation criteria:
Acceptance criteria
The mouse 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 polychromatic erythrocytes containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data for PCE (95 % control limit).
- The positive control substance has to induce a distinct increase in the number of PCE containing small and / or large micronuclei within the range of the historical positive control data or above.
- Exposure of the bone marrow has to be demonstrated by change in the PCE / NCE ratio, analysis of blood samples ar ADME data obtained in an independent study using the same route and species.

Assessment criteria
A finding is considered positive if the following criteria are met:
- A statistically significant and dose-related increase in the number of PCE containing micronuclei.
- The number of polychromatic erythrocytes containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data (95 % control limit).
A test substance is considered to be negative if the following criteria are met:
- The number of polychromatic erythrocytes containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data (95 % control limit).
A scientific judgement is required in case of equivocal data.
Statistics:
For analysis of the rate of micronucleated polychromatic erythrocytes the Jonckheere-Terpstra test were 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.
The results were listed in the tables. If the results of these tests were significant, symbols were used in the tables.
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
observed after administration at the top dose at the 48 h sacrifice interval
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg body weight
- Solubility: Due to the good solubility of the test substance in deionized water, deionized water was selected as vehicle.
- Clinical signs of toxicity in test animals: nothing abnormal detected
- Evidence of cytotoxicity in tissue analyzed: the dose of 2000 mg/kg body weight was tolerated without any signs of toxicity
- Rationale for exposure: determination of the acute oral toxicity in males and females

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The single oral administration of the test substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing micronuclei. The rate of micronuclei was within the range of the concurrent vehicle controls in all dose groups and at all sacrifice intervals and within the range of 95 % control limit of the historical negative control data.
- Ratio of PCE/NCE (for Micronucleus assay): A relevant stimulation of erythropoiesis determined from the ratio of PCE to NCE was detected after test substance administration at the top dose of 2000 mg/kg body weight at the delayed 48 hours sacrifice interval. Thus, bioavailability of the test substance after oral administration was confirmed by biological effects.
- Appropriateness of dose levels and route: Dose selection was based on the results of a preliminary range finding test in both sexes. No gender difference was observed and, therefore, only males were used in the main test.

Any other information on results incl. tables

Table 1: Summary table - Induction of Micronuclei in bone marrow cells after single oral administration (gavage)

Test group

Sacrifice interval [hrs]

Animal No.

Micronuclei in PCE

PCE per 4000 erythrocytesc

Meana[‰]

Rangeb[abs.]

[abs]

[rel.]

Vehicle control deionized water

24

5

4.0

10 – 20

2144

100 %

Test substance 500 mg/kg bw

24

5

3.0

10 – 15

1918

89 %

Test substance 1000 mg/kg bw

24

5

3.7

7 – 22

1997

93 %

Test substance 2000 mg/kg bw

24

5

3.8

8 – 19

2038

95 %

Positive control Cyclophosphamide 20 mg/kg bw

24

5

18.4**

45 – 110

2500

117 %

Vehicle control deionized water

48

5

2.8

6 – 22

1047

100 %

Test substance 2000 mg/kg bw

48

5

3.0

7 - 15

2413

118 %

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

Bw: body weight

Abs.: absolute value

Rel.: relative value compared to the respective vehicle control

a: mean of micronucleated PCEs per test group

b: number of micronucleated PCEs per animal (minimum versus maximum)

c: calculated number of PCEs per 4000 erythrocytes (PCE + NCE) when scoring a sample of 20000 PCE per test group

*: p ≤ 0.05

**: p ≤ 0.01

Table 2: Results of the analytical investigation

Sample ID

Analytical value

Nominal concentration (absolute)

Nominal concentration (relative)

Mean recovery rate (relative)

1

2

3

69 mg/mL

68 mg/mL

68 mg/mL

50 mg/mL

137 %

136 %

135 %

136.0 %

7

8

9

112 mg/mL

112 mg/mL

114 mg/mL

100 mg/mL

112 %

112 %

114 %

112.7 %

13

14

15

230 mg/mL

232 mg/mL

234 mg/mL

200 mg/mL

115 %

116 %

117 %

116.0 %

Table 3: Biometry - Wilcoxon Test (one-sided+)

Negative control versus test group

p-value

Significance

500 mg/kg bw test substance; 24 h

0.9444

-

1000 mg/kg bw test substance; 24 h

0.6429

-

2000 mg/kg bw test substance; 24 h

0.7103

-

Positive control CPP; 24 h

0.004

+

2000 mg/kg bw test substance; 48 h

0.222

-

- not significant

+ significant (* for p ≤ 0.05; ** for p ≤ 0.01)

Table 4: Biometry - Jonckheere-Terpstra trend test

Main experiment

p-value

Significance

24 h sacrifice interval

0.4928

-

- not significant

+ significant (* for p ≤ 0.05; ** for p ≤ 0.01)

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mic ein vivo.
Executive summary:

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in deionized water, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case.

In a preliminary experiment and in the main experiment at 24 -hour sacrifice interval blood samples were taken to analyze the bioavailability of the test substance in the target organ by oral route.

Dose selection was based on the results of a preliminary range finding test in both sexes. No gender difference was observed and, therefore, only males were used in the main test.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control group, the 24 -hour sacrifice interval was investigated only. After staining of the preparations, 4000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occuring per 4000 polychromatic erythrocytes were also recorded.

As vehicle control, male mice were administered merely the vehicle, deionized water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the 95 % control limit of the historical vehicle control data range.

The positive control substance cyclophosphamide led to the expected increase in the rate of polychromatic erythrocytes containing micronuclei.

The test substance is a highly complex plant extract. Unfortunately, it was not feasible to find a realiable analytical method for detection of the test substance in this biological matrix (plasma).

A relevant stimulation of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected after test substance administration at the top dose of 2000 mg/kg body weight at the delayed 48 hours sacrifice interval. Thus, bioavailability of the test substance after oral administration was confirmed by biological effects.

According to the results of the presend study, the single oral administration of the test substance did not lead to any relevant increase in the number of polychromatic eryhtrocytes containing micronuclei.

The rate of micronuclei was within the range of the concurrent vehicle controls in all dose groups and at all sacrifice intervals and within the range of 95 % control limit of the historical negative control data.

Thus, under the experimental conditions of this study, the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.