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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 July 2002 to 08 August 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
19 May 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Lot/batch No.: B 02011

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: about 29 g
- Assigned to test groups randomly: yes, under following basis: randomized plan prepared with an appropriate computer program
- Housing: Makrolon cages, type MI, housed individually from start of the treatment until the end of the test
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the hydrolytical sensitivity of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 12.5 g/100 ml, 25.0 g/100 mL and 50.0 g/100 mL.
- Amount of vehicle: 4 mL/kg bw.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.
Duration of treatment / exposure:
single dose
Frequency of treatment:
once, single dose
Post exposure period:
24 or 48 hours
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 animals per dose for the 24 hour sacrifices and 5 animals per dose for the 48 hour sacrifices
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP) and vincristine sulphate (VCR)
- Justification for choice of positive controls: The stability of CPP and VCR is well-defined under the selected conditions, since both positive control articles are well-defined clastogens and aneugens respectively.
- Route of administration: The positive controls, both, dissolved in purified water were administered to male animals once orally or intraperitoneally each in a volume of 10 ml/kg body weight.
- Doses / concentrations: 20 mg CPP//kg body weight for clastogenic effects and 0.15 mg VCR/kg body weight for aneugenic effects.

Examinations

Tissues and cell types examined:
In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored .
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline were survived by all animals (male and female) without any clinical signs. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the híghest 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 groups,the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues. 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 which was at 37°C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are 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 solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (dD/4) (d = diameter of micronucleus, D= cell diameter)

The size of micronuclei may indicate the possible mode of action of the test substance, i .e . a clastogenic or a spindle poison effect. Slides were coded before microscopic analysis. Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative values can occur.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes.
- The proportion of cells with micronuclei in negative control animals was within the normal range of the historical control data.
- The two positive control chemicals induced a significant increase in the number of cells containing small and large micronuclei within the range of the historical control data or above.

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
- The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN.
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians . Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <=0.05, ** for p<=0.01) were printed with the group means in the tables. This test was performed one-sided.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control and within the range of historical control data.
With 19.7%° the positive control substance cyclophosphamide for clastogenicity, led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei.
With 61 .0%° the positive control vincristine for spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e . 14 .2%°.
- Ratio of PCE/NCE: No inhibition of erythropoesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Any other information on results incl. tables

Summary tables

Table 1:Polychromatic and normochromatic erythrocytes

 

Interval: 24 hours

Total No. of MN (‰) in

Interval: 48 hours

Total No. of MN (‰) in

PCE’s

NCE’s

PCE’s

NCE’s

PCE’s

NCE’s

PCE’s

NCE’s

Vehicle

DMSO

10000

2955

0.9

1.0

10000

2801

0.8

0.0

 

500 mg/kg

10000

2492

1.9*

1.2

 

 

 

 

 

1000 mg/kg

10000

2875

1.0

2.1

 

 

 

 

 

2000 mg/kg

10000

3540

1.3

1.1

10000

2812

1.7*

0.4

CCP

20 mg/kg

10000

3201

19.7**

0.9

 

 

 

 

VCR

0.15 mg/kg

10000

3749

61.0**

0.5

 

 

 

 

Wilcoxon Test (one-sided): * : p <= 0.05, ** : p <= 0.01

A pairwise comparison of each dose group with the vehicle control group.

 

Table 2: Polychromatic erythrocytes: differentiation between small and large micronuclei

 

Interval: 24 hours Total No. of Cells (‰) with

Interval: 48 hours Total No. of MN (‰) with

Of PCE’s

MN. D<D/4

MN.d=>D/4

Of PCE’s

MN. D<D/4

MN.d=>D/4

Vehicle

DMSO

10000

0.9

0.0

10000

0.8

0.0

 

500 mg/kg

10000

1.9*

0.0

 

 

 

 

1000 mg/kg

10000

1.0

0.0

 

 

 

 

2000 mg/kg

10000

1.3

0.0

10000

1.7*

0.0

CPP

20 mg/kg

10000

19.7**

0.0

 

 

 

VCR

0.15 mg/kg

10000

46.8**

14.2**

 

 

 

Wilcoxon Test (one-sided): * : p <= 0.05, ** : p <= 0.01

A pairwise comparison of each dose group with the vehicle control group.

 

 

Historical Negative Control Data / Mice (NMRI)

 

Table 3: Route Oral

2000 evaluated Polychromatic Erythrocytes per Animal

Means of 5 Animals per Group

Vehicle Control Water

micronuclei in polychromatic erythrocytes ‰

small

large

total

mean

1.4

0.0

1.4

min

0.6

0.0

0.6

max

2.4

0.2

2.6

SD

0.5

0.1

0.6

n

32

 

Vehicle control Olive oil

micronuclei in polychromatic erythrocytes ‰

small

large

total

mean

1.2

0.0

1.2

min

0.8

0.0

0.8

max

1.8

0.1

1.8

SD

0.4

0.0

0.4

n

17

 

Table 4: Route Oral

1000 evaluated Polychromatic Erythrocytes per Animal

Means of 5 Animals per Group

Vehicle Control DMSO

micronuclei in polychromatic erythrocytes ‰

small

large

total

mean

1.5

0.0

1.5

min

1.0

0.0

1.0

max

2.0

0.1

2.1

SD

0.3

0.0

0.4

n

12

 

Historical Positive Control Data / Mice (NMRI)

 

Table 5: Cyclophosphamide 20 mg/kg bw

2000 evaluated Polychromatic Erythrocytes per Animal

Means of 5 Animals per Group

Route oral

micronuclei in polychromatic erythrocytes‰

small

large

total

mean

12.8

0.1

12.9

min

8.7

0.0

8.9

max

17.9

0.9

17.9

SD

2.4

0.2

2.4

n

6

 

Table 6: Vincristine 0.15 mg/kg bw

1000 / 2000 evaluated Polychromatic Erythrocytes per Animal

Means of 5 Animals per Group

2000 PCEs, Route intreperitoneal

micronuclei in polychromatic erythrocytes ‰

small

large

total

mean

60.5

11.9

72.2

min

21.9

3.8

26

max

101.9

22.3

113.7

SD

17

4.8

19.8

n

51.0

 

Applicant's summary and conclusion