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:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Version 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Version 19 May 2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
Version Aug 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary
Specific details on test material used for the study:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Analytical purity: 95%
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:NMRI mice from Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 28.1 g
- Assigned to test groups randomly: yes
- Housing: single housing in Makrolon cages, type M I
- Diet: ad libitum, standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum, drinking water from bottles
- 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:
intraperitoneal
Vehicle:
- Vehicle used: corn oil
- Justification for choice of vehicle: corn oil was used as vehicle to obtain a homogeneous suspension of the test substance, which has been demonstrated to be suitable in the mouse micronucleus test and for which historical control data are available.
- Concentration of test material in vehicle: 15 mL/kg bw for low dose (of a test substance suspension with a concentration of 25 mg/mL), 15 mL/kg bw for intermediate dose (50 mg/mL) and 15 mL/kg bw for intermediate dose (100 mg/mL).
Details on exposure:
- The animals were treated once intraperitoneally with a volume of 15 mL/kg bw of the test substance, the vehicle or the positive control substances.
- To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration and controlled analytically (HPLC).
Duration of treatment / exposure:
24 - 48 h
Frequency of treatment:
Once
Post exposure period:
The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) or 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Two positive control groups receiving each cyclophosphamide (CCP) or vincristine sulfate (VCR).
- Justification for choice of positive control(s): CPP and VCR are well-established reference clastogens or aneugens, respectively.
- Route of administration: i.p. (both 10 mL/kg bw)
- Doses / concentrations: 20 mg/kg bw for CCP or 0.15 mg/kg bw for VCR.

Examinations

Tissues and cell types examined:
Bone marrow prepared from two femora of sacrificed animals (cervical dislocation).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The acute intraperitoneal toxicity of the test substance was determined in a pretest at 2000 mg/kg bw (recommended as the highest dose according to the OECD Guideline). All animals (male and female) survived, but showed severe clinical signs: piloerection, hunched posture, reduced general condition and eyelid closure. No distinct differences in the symptoms between males and females were observed. Therefore, only male animals were used for the cytogenetic investigations.
On account of the observations of the pretest, 1500 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 750 mg/kg and 375 mg/kg bw were administered as further doses.

DETAILS OF SLIDE PREPARATION: 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 (about 2 mL/femur). The suspension was then mixed thoroughly with a pipette and centrifuged. The supernatant was removed and the precipitate was resuspended in fresh FCS. One drop of this suspension was dropped onto clean microscopic slides and smears were prepared using slides with ground edges. The preparations were then dried in the air and subsequently stained.

METHOD OF ANALYSIS: the slides were stained with eosin and methylene blue (modified May-Gruenwald solution or Wrights solution), briefly rinsed and then soaked in purified water, subsequently stained with Giemsa, then rinsed twice in purified water and clarified in xylene, and mounted in Corbit-Balsam.
2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group. In total 10000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored as well as the number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4; [d = diameter of micronucleus, D = cell diameter]).

OTHER:
Clinical examinations: after treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
(1) the quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs.
(2) The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
(3) The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
(4) The two positive control substances have 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.

A finding is considered positive if the following criteria are met:
(1) statistically significant and dose-related increase in the number of PCEs containing micronuclei.
(2) The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.

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 vehicle control value and is within the range of the historical vehicle control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Clinical signs of toxicity in test animals:
The single intraperitoneal administration of the vehicle in a volume of 15 mL/kg body weight was tolerated by all animals without any signs or symptoms. The administration of the test substance led to distinct clinical signs of toxicity. Neither the single administration of the positive control substance cyclophosphamide (20 mg/kg bw) nor that of vincristine sulfate (0.15 mg/kg bw) caused any evident signs of toxicity.

- Induction of micronuclei:
a) The administration of the vehicle led to 1.0 % polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.3 % after the 48-hour sacrifice interval, respectively.
b) After administration of the highest dose of the test substance, 1.1 % polychromatic erythrocytes containing micronuclei were found after 24 hours and 0.8 % after 48 hours.
In the two lower dose groups, rates of micronuclei of about 0.9 % (750 mg/kg) and 1.3 % (375 mg/kg) were detected after a sacrifice interval of 24 hours in each case.
c) The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (14.1 %) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected. Vincristine sulfate, a spindle poison, produced a 54.7 % increase in micronuclei in polychromatic erythrocytes. A significant portion (17.0 %) was attributable to large micronuclei.

- Ratio of PCE/NCE:
A clear inhibition of erythropoiesis (ratio of PCEs to NCEs) induced by the treatment of mice with test substance was detected after treatment with 1500 mg/kg bw at the 48-hour preparation interval.

- Statistical evaluation:
No statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.

Any other information on results incl. tables

After administration of the vehicle corn oil the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control animals was within the range of the historical vehicle control data both for PCEs and for NCEs.

In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

 

 

Table 1: Summary table - Induction of Micronuclei in bone marrow cells

Test group

Sacrifice interval (hours)

Micronuclei in PCE

Number of NCEc

total (%)a

large (%)b

Vehicle control (corn oil)

24

1.0

0.0

3187

Test substance (375 mg/kg bw)

24

1.3

0.0

5524

Test substance (750 mg/kg bw)

24

0.9

0.0

5609

Test substance (1500 mg/kg bw)

24

1.1

0.0

4809

Positive control (cyclophosphamide, 20 mg/kg bw)

24

14.1**

0.0

3490

Positive control (vincristine sulfate, 0.15 mg/kg bw)

24

54.7**

17.0**

4419

Vehicle control (corn oil)

48

1.3

0.1

4332

Test substance (1500 mg/kg bw)

48

0.8

0.1

9294

PCE = polychromatic erythrocytes; NCE = normochromatic erythrocytes;a= sum of small and large micronuclei;b= large micronuclei (indication for spindle poison effect);c= number of NCEs observed when scoring 10 000 PCEs; * = p ≤ 0.05; ** = p ≤ 0.01

 

Applicant's summary and conclusion