Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Solid, yellow
- Analytical purity: > 99%
- Lot/batch No.: 081012P040
- Storage condition of test material: Room temperature
- Other: Date of production: 29 Mar 2008

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories GmbH, Germany
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: mean: 30.0 g
- Assigned to test groups randomly: yes, under following basis: randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type M I; single housing
- Diet (ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: at least 5 days


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


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 500 mg/kg bw (25 mg/mL), 1000 mg/kg bw (50 mg/mL), 2000 mg/kg bw (100 mg/mL)
- Amount of vehicle (if gavage or dermal): The usual application volume of 10 mL/kg body weight led to a non-applicable mass and therefore the volume was increased to 20 mL/kg body weight.

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To achieve homogeneity of the test substance in the vehicle, the test substance preparation was stirred with an ultraturrax.
All test substance formulations were prepared immediately before administration.
The stability of the test substance at room temperature in the vehicle corn oil was determined analytically.
To keep the test substance homogeneously in the vehicle, the test substance preparation was constantly stirred.
Duration of treatment / exposure:
24 h (all test substance concentrations, vehicle control and both positive controls)
48 h (highest test substance concentration and vehicle control)
Frequency of treatment:
single oral administration
Post exposure period:
The animals were sacrificed 24 hours and 48 hours after the treatment, respectively.
The animals were examined for any clinically evident signs of toxicity several times.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP) and Vincristine sulfate (VCR)
- Justification for choice of positive control(s): Both substances are well-established reference clastogens and aneugens, respectively.
- Route of administration: orally (CPP), intraperitoneal (VCR)
- Doses / concentrations: 20 mg/kg bw (CPP), 0.15 mg/kg bw (VCR)
The positive control substances were given with a volume of 10 mL/kg bw.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2 000 mg/kg body weight, recommended as the highest dose according to the OECD Guideline, was tolerated by all animals (male and female) without any clinical sign. Thus, only male animals were used for the cytogenetic investigations.
Therefore, a dose of 2 000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1 000 mg/kg and 500 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
Bone marrow/FCS (fetal calf serum) suspension (about 2 mL/femur), preheated up to 37°C and 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 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 with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
After briefly rinsing in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL purified water) for about 15 minutes.
After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs 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.)
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/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 effect (d < D/4) or a spindle poison effect (d ≥ D/4)).
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• 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.
• 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.
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. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

A finding is considered positive if the following criteria are met:
• 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 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.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: The administration of the test substance did not lead to any clinical signs of toxicity

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): summary table see below
- Ratio of PCE/NCE (for Micronucleus assay): test compound: 0.7, CPP: 0.97, VCS: 0.47

Any other information on results incl. tables

Induction of Micronuclei in bone marrow cells

Substance

Dose (mg/kg)

 

sex

post exposure period (h)

Micronuclei in PCE

totala []

largeb [‰]

Number of NCEc

vehicle

corn oil

male

24

0.80

0.0

3442

test substance

500

male

24

1.10

0.0

2753

test substance

1000

male

24

0.90

0.0

3094

test substance

2000

male

24

2.2*#

0.1

2495

vehicle

corn oil

male

48

0.90

0.0

2542

test substance

2000

male

48

1.30

0.0

3216

positive control cyclophosphamid

80

male

24

15.0**

0.0

2057

positive control vincristine sulfate

20

male

24

61.1**

22.6**

4253

PCE = polychromatic erythrocyts (2000 were scored for micronuclei)

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

(# = historical control range 0.7 - 3.0)

Under the experimental conditions chosen here, the test substance Paliotol Gelb K 2142 does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative