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:
other: GLP study acc. to OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE; Experimental Toxicology and Ecology
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation:1st Experiment: 28.3 g, 2nd Experiment: 27.2 g
- Housing: Makrolon cages, type M II; single housing
- Diet: 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:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: good solubility
- Concentration of test material in vehicle: 25, 50, 100 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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:
24, 48h
Frequency of treatment:
single treatment
Post exposure period:
24, 48h
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500, 1000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide: 20 mg/kg bw.
Vincristine sulfate: 0.15 mg/kg bw.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Data from pretest.

DETAILS OF SLIDE PREPARATION:
After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis. Cell suspension was separated in cellulose columns and transferred to slides via cell funnels and centrifugation. Slides were stained with pure May-Grünwald solution and with Giemsa solution.
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of
micronuclei from each animal of every test group, so in total at least 10 000 PCEs were
scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also
scored.
Evaluation criteria:
Acceptance 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 for PCEs.
• 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.

Assessment criteria
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.
Statistics:
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.
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs at 1000 mg/kg bw (piloerection, hunched posture, reduced general condition)
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a pretest for the determination of the acute oral toxicity in males and females, deaths were
observed down to a dose of 1 500 mg/kg body weight. At 1 000 mg/kg, all animals survived
but distinct signs of toxicity were observed The clinical signs observed were piloerection,
hunched posture and reduced general condition. However, there were no distinct differences
in the clinical observations between males and females. Thus, only male animals were used
for the cytogenetic investigations as requested by the current OECD Guideline 474.
Based on the data of the pretest a dose of 1 000 mg/kg body weight was defined as MTD
(maximum tolerated dose) and was selected as the highest dose in the present cytogenetic
study.

RESULTS OF DEFINITIVE STUDY (see Appendix 1)
- Induction of micronuclei (for Micronucleus assay):
In the 1st Experiment , the single oral administration of Rose oxide 90 led to a weak, dose related
increase in the number of polychromatic erythrocytes containing micronuclei when
scoring an increased sample of 4 000 PCEs per animal in some selected test groups due to
large inter-animal variability. However, no statistical significance was calculated for any of the
dose groups. The values after administration of 500 and 1 000 mg/kg body weight (3.1‰ and
4.2‰, respectively) slightly exceeded our historical vehicle control data range (0.3 – 3.0‰
micronucleated PCEs; smear method) whereas the concurrent vehicle control at 48-hour
sacrifice interval showed a slightly higher rate of micronucleated PCEs (3.2‰). Besides, the
inter-animal variability of micronucleated PCEs per 2 000 PCEs was higher in the vehicle
control group (2.5 - 11.5) than in the intermediate and top doses (4 – 8.5 and 7 – 10.5,
respectively). Additionally, the highest value scored for a calculated sample size of
2 000 PCEs was obtained in the vehicle control group at 24-hour sacrifice interval.

Finally, due to a large inter-animal variability and failing statistical significance this
observation has to be regarded as biologically irrelevant. However, to corroborate this
conclusion a repeat experiment, designated 2nd Experiment, was performed.

In the 2nd Experiment, the single oral administration of Rose oxide 90 led again to a weak,
dose-related increase in the number of polychromatic erythrocytes containing micronuclei.
However, no statistical significance was calculated for any of the dose groups. The number
of micronucleated polychromatic erythrocytes at 1 000 mg/kg body weight (3.2‰) at 24-hour
sacrifice interval slightly exceeded either the range of the concurrent vehicle control values
(2.1 – 2.5‰) or our laboratory’s negative control data range (0.3 - 3.0‰; smear method).
However, this value was similar to the value found in the 1st Experiment in the vehicle control
group at 48-sacrifice interval. Besides, when comparing the individual animal data in the
2nd Experiment the inter-animal variability of micronucleated PCEs per 2 000 PCEs was
almost similar in the vehicle control groups (3 - 9) and in the dose groups (0 - 9). The highest
individual animal value per 2 000 PCEs was observed in the vehicle control group as well as
in the top dose group.
In conclusion, due to a similar large inter-animal variability, similar maximum PCE values per
animal and failing statistical significance the weak, dose-related increase in the frequency of
micronucleated PCEs in the 2nd Experiment has to be regarded as biologically irrelevant.

- Ratio of PCE/NCE (for Micronucleus assay):
No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected as indication of organ toxicity.

Any other information on results incl. tables

As vehicle control, male mice were administered merely the vehicle, corn oil, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes at the upper border of our historical vehicle control data range or slightly above. It has to be assumed that this deviation is based on the modification of the preparation method. Our historical negative control database presents data that were obtained after scoring slides prepared by bone marrow smear procedure. In this study we used the column separation method with cytospin centrifugation. The column separation method is commonly used in several laboratories worldwide and it is used in rats since years in our laboratory. The modified version with cytospin centrifugation was established and validated inhouse prior to use. The current negative control database shows a slight increase in mean micronuleus rates per test goups compared to the classical smear method. However, only a small number of test groups have been scored until now, so no reliable data concerning the bias of data obtained with the new method are currently available. Based on this fact the weak increase in the number of micronucleated polychromatic erythrocytes in the vehicle controls compared to our historical negative control database (bone marrow smear method) have no impact on the validity of this study.

Applicant's summary and conclusion