Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
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:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-3 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Purity: 92.1%

Test animals

Species:
mouse
Strain:
other: Crl:CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 or 7 weeks old
- Weight at study initiation: Male 26.0 - 33.0 g; Female 22.0 - 27.1 g
- Fasting period before study: No
- Housing: Mice of the same sex were housed up to five per rodent Micro-Barrier cage
- Diet: Harlan 2018C Certified Global Rodent Diet was provided ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 or 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 1.7 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: A solution of 0.1% Tween-80 in aqueous methylcellulose, 4000 cP (0.5%), was used as the vehicle for the test substance and also as the vehicle control in the definitive micronucleus study.
Details on exposure:
PREPARATION OF TEST SUBSTANCE FORMULATION: The test substance dose formulations were prepared fresh for each phase of the study prior to dose administration. The amount of test substance used in preparing each concentration was adjusted for the test substance purity using a correction factor of 1.09. Each formulation was prepared by combining an appropriate amount of the test substance with an appropriate volume, 80% of the targeted volume of the vehicle. Each formulation was vortexed (1 minute), homogenized (2 minutes) and stirred using a magnetic stir bar/plate for 10 minutes. Remaining volume of the vehicle was added to achieve the targeted volume. Each formulation was stirred for an additional 20 minutes.
Duration of treatment / exposure:
single oral administration
Frequency of treatment:
single oral administration
Post exposure period:
24 or 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
Range Finding Study
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested
Definitive Micronucleus Study
No. of animals per sex per dose:
5/sex/concentration except 10 dosed for 2000 mg/kg 24 hour sampling
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive Control: Cyclophosphamide monohydrate
- Justification for choice of positive control(s): not reported
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg)

Examinations

Tissues and cell types examined:
PCEs and NCEs from mouse bone marrow taken from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In absence of toxicity data for the test substance, a Dose Range Finding (DRF) study was conducted exposing 3 male and 3 female mice, each at 2000 mg/kg, the highest dose recommended by the OECD test guideline (OECD TG 474). In absence of mortality in the DRF study, a dose of 2000 mg/kg was tested as high dose in the definitive micronucleus study. Two lower doses, one fourth and one half of the high dose, at 500 and 1000 mg/kg were also tested.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In absence of mortality in the DRF assay, a dose of 2000 mg/kg was tested as high dose in the micronucleus assay. Two lower doses, one fourth and one half of the high dose, at 500 and 1000 mg/kg were also tested. The vehicle and positive control substances were run concurrently. Bone marrow cells [polychromatic erythrocytes (PCEs)] collected from all groups at 24 hours post –dose and from the vehicle and 2000 mg/kg treatment groups at 48 hour post-dose were examined microscopically for the presence of micronuclei (mnPCEs). In addition, the ratio of polychromatic erythrocytes to total erythrocytes (PCEs/EC ratio) was evaluated in the test substance groups relative to the respective vehicle control groups to reflect the test substance’s cytotoxicity.

DETAILS OF SLIDE PREPARATION: Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a labelled centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at about 100 x g for about five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Slides were labelled with the harvest date, experiment and animal number using a computer generated label. Two slides were prepared from each mouse. The slides were air dried and fixed in methanol. One set of slides from all treatment groups at 24 hours post dose and from the vehicle control and high dose group at 48 hours post dose were was stained with a nucleic acid-specific stain, acridine orange, and was used in microscopic evaluation. The second set of slides was packaged for storage until finalization of the report.

METHOD OF ANALYSIS: Bone marrow was evaluated by fluorescent microscopy. The staining procedure permits the differentiation by colour of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost-like, dark green NCEs, respectively). Slides initially were scanned using medium magnification to locate suitable areas where the cells are well spread and stained. Next, cells were scored using high power oil immersion.
Evaluation criteria:
The criteria for the identification of micronuclei are those of Schmid. Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring is based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus are counted as one mnPCE, not two (or more) micronuclei.
At least 2000 PCEs/animal were scored for the presence of micronuclei (mnPCEs) whenever possible. In addition, at least 1000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity.

Once the criteria for a valid assay have been met, the results are evaluated as follows:
• Test substance is considered to be positive if it induces a significant increase in mnPCE frequency (p ≤ 0.05) at any dose level or sampling time compared to the concurrent vehicle control.
• The test substance is considered to be negative if no significant increase in mnPCE frequency is observed (p > 0.05) compared to the concurrent vehicle control.
• Other criteria may be used in reaching a conclusion about the study results (e.g., magnitude of any increase,
Statistics:
The frequency of mnPCEs and the proportion of PCEs to total erythrocytes were determined for each animal and treatment group. Statistical significance (p ≤ 0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).
The MNPCE frequency of the vehicle controls must be within the historical vehicle control range, and the positive control must induce a significant increase (p ≤ 0.05, Kastenbaum-Bowman Tables) in MNPCE frequency as compared to concurrent vehicle control.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Solubility: soluble
- Clinical signs of toxicity in test animals: no
- Evidence of cytotoxicity in tissue analysed: no
- Harvest times: 48 hrs


RESULTS OF DEFINITIVE STUDY:
• No mortality was observed in any of the treatment groups. All mice in the control substance (vehicle or positive) groups and in the test substance treated groups appeared normal during the study period.
• Reductions up to 12% in the ratio of polychromatic erythrocytes to total erythrocytes (PCEs/ECs ratio) in the male or female test substance groups relative to the respective vehicle control groups were observed at 24 hours post-dose. Reductions of these magnitudes suggest that the test substance did not induce cytotoxicity. No reductions in the PCEs/EC ratio were observed in the test substance treated group compared to the vehicle control at 48 hours post dose.
• No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, binomial distribution, Kastenbaum-Bowman Tables).
• CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p≤ 0.05, binomial distribution, Kastenbaum-Bowman Tables) in both male and female mice.
• The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

Any other information on results incl. tables

No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed at any dose.

Summary of Bone Marrow Micronucleus Analysis

Treatment

Sex

Time (hr)

Number of Animals

PCE/Total Erythrocytes

Mean

S.D.

Number of mnPCE/1000 PCE

Mean

S.D.

Number of mnPCE/PCE Scored

Vehicle*

M

24

5

0.468

0.05

0.6

0.42

6/10000

 

F

24

5

0.482

0.06

0.8

0.27

8/10000

 

500 mg/kg

M

24

5

0.442

0.02

0.5

0.35

5/10000

 

F

24

5

0.463

0.04

0.1

0.22

1/10000

1000 mg/kg

M

24

5

0.457

0.02

0.4

0.42

4/10000

 

F

24

5

0.425

0.02

0.4

0.42

4/10000

200- mg/kg

M

24

5

0.459

0.03

0.6

0.22

6/10000

 

F

24

5

0.452

0.02

0.5

0.50

5/10000

 

Cyclophosphamide (50 mg/kg)

M

24

5

0.385

0.05

17.9

2.79

179/ 10000**

 

F

24

5

0.437

0.05

22.0

3.08

220/

10000**

 

Vehicle*

M

48

5

0.460

0.03

0.5

0.35

5/10000

 

F

48

5

0.450

0.02

0.6

0.22

6/10000

 

2000 mg/kg

M

48

5

0.465

0.04

0.2

0.27

2/10000

 

F

48

5

0.449

0.02

0.6

0.22

6/10000

Vehicle* = 0.1% Tween-80 in aqueous methylcellulose (4000 cP, 0.5%)

** Statistically significant increase compared to vehicle control, p≤0.05 (binomial distribution, Kastenbaum-Bowman Tables)

Applicant's summary and conclusion

Conclusions:
The test substance was concluded to be negative in the mouse micronucleus assay.
Executive summary:

An oral micronucleus study was conducted in mice to determine whether the test material induces an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow.  In this study, groups of male and female Crl:CD-1 mice were orally dosed with 500, 1000 and 2000 mg/kg bw and observed for 24 to 48 hours. Bone marrow smears were prepared approximately 24 and 48 hours after administration and 2000 polychromatic erythrocytes per animal were evaluated for the presence of micronuclei.  In addition, the ratio of polychromatic erythrocytes to total erythrocytes (PCEs/EC ratio) was evaluated in the test substance groups relative to the respective vehicle control groups to reflect the test substance’s cytotoxicity.

Under the conditions of the study conduct as described in this report, a single oral administration of the test substance at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female Crl:CD-1 (CD-1) mice. Therefore, the test substance was concluded to be negative in the mouse micronucleus assay.