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
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, for read-across justification refer to IUCLID section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
refer to confidential details on test material

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: Mean body weight = 29.54 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program.
- Fasting period before study: no
- Housing: Makrolon cages, type M II; single housing, Dust-free wooden bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): no data; Fully air-conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 - 18.00 hours; 12 hours darkness from 18.00 - 6.00 hours

IN-LIFE DATES: From: 07 July 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: solution of the test substance in the vehicle was prepared immediately before administration

Duration of treatment / exposure:
24 h (all test substance concentrations, vehicle, positive control) and 48 h (highest test substance concentration, vehicle)
Frequency of treatment:
once
Post exposure period:
no
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide

- Route of administration: oral, gavage
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range finding study

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 positive control substance has 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:
• A 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:
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.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: good solubility of the test substance in the vehicle corn oil
- Clinical signs of toxicity in test animals: piloerection


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): not obeserved
- Ratio of PCE/NCE (for Micronucleus assay): clearly influenced at the highest administered dose
- Appropriateness of dose levels and route: limit dose
- Statistical evaluation: yes

Any other information on results incl. tables

The single oral administration of the vehicle corn oil in a volume of 10 mL/kg body weight led to 2.3‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.0‰ after the 48-hour sacrifice interval, respectively. After the single administration of the highest dose of 2 000 mg/kg body weight, 1.3‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 0.7‰ after 48 hours. In the two lower dose groups, rates of micronuclei of 1.7‰ were detected at a sacrifice interval of 24 hours at 500 and 1 000 mg/kg group, each. The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (19.9‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals. A clear inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at the highest recommended test substance dose of 2 000 mg/kg body weight at 48 hours sacrifice interval. Thus, bioavailabilty of the test substance in the target organ after oral administration was confirmed.

The administration of the test substance led to weak clinical signs of toxicity at the top dose of 2 000 mg/kg body weight within the first day of exposure (piloerection). The single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight caused no evident signs of toxicity.

The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of 90% - 110% of the nominal concentration. Based on the recovery rates it has to be considered that the test substance was stable at room temperature in the vehicle corn oil at least over a period of 30 minutes.

According to the results of the present study, there are thus 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 (500 mg/kg, 1 000 mg/kg and 2 000 mg/kg) 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. The ratio of PCE/NCE was clearly influenced at the highest administered dose of 2 000 mg/kg body weight at delayed 48-hour preparation interval as indication for target organ toxicity. Thus, bioavailabilty of the test substance in the target organ after oral administration was confirmed. In this study, after single oral 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 for PCEs. In addition, the positive control substance, cyclophosphamide, induced a statistically significant increase in the number of PCEs containing small micronuclei within the range of the historical positive control data or above.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative