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
Study period:
2015-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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 (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 99.0%
Solid, yellow
Storage conditions: Room temperature
Expiry date: 26 Jun 2024

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
- Mean weight at study initiation: 28.7 g
- Assigned to test groups randomly: yes, under following basis: appropriate computer program
- Housing: Makrolon cages, type M II; single housing
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water (e.g. ad libitum): Drinking water from bottles was available 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(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Using the vehicle corn oil a homogeneous suspension of the test substance was obtained. Therefore, corn oil was used as vehicle, which has been demonstrated to be suitable in the in vivo Micronucleus test and for which historical control data are available.
- Concentration of test material in vehicle: 50, 100 or 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was stirred with a spatula and shaken thoroughly. All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
24 or 48 hrs
Frequency of treatment:
once
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
5 males per treatment
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive controls, both dissolved in deionised water, were administered to male animals once orally (cyclophosphamide, CPP) or intraperitoneally (vincristine sulphate, VCR) each in a volume of 10 mL/kg body weight. The animals were sacrificed after 24 hours.

Examinations

Tissues and cell types examined:
The bone marrow was prepared according to the method described by Schmid (1976, 1977) and Salamone et al. (1980).
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then 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 foetal calf serum (FCS) which was preheated up to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette 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 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.
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 clinical signs of toxicity. Additionally, discoloured faeces were observed from 4 hours after test substance administration until sacrifice of the animals.
On account of the results of the pretest, 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.

TREATMENT AND SAMPLING TIMES:
sacrifice interval 24 h
vehicle control, 500 mg/kg bw TS, 1000 mg/kg bw TS, 2000 mg/kg bw TS, 20 mg/kg bw CPP, 0.15 mg/kg bw VCR

sacrifice interval 48 h
vehicle control, 2000 mg/kg bw TS

DETAILS OF SLIDE PREPARATION:
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in deionised water, the preparations were soaked in deionised water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL deionised water) for about 15 minutes.
- After rinsing twice in deionised water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.
Evaluation 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:
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:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The single oral administration of the vehicle corn oil in a volume of 10 mL/kg body weight led to 1.5‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.6‰ after the 48-hour sacrifice interval, respectively. The single oral administration of the vehicle corn oil in a volume of 20 mL/kg body weight led to 2.5‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 2.6‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 2000 mg/kg body weight, 2.8‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 2.2‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of 1.4‰ (1000 mg/kg group) and 3.2‰ (500 mg/kg group) were detected at a sacrifice interval of 24 hours in each case. The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (29.5‰) 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.
No inhibition of erythropoiesis induced by the treatment of mice was detected. The ratio of polychromatic to normochromatic erythrocytes was close to the range of the vehicle control values in all dose groups.

The single oral administration of the vehicle in a volume of 20 mL/kg body weight was tolerated by all animals without any clinical observations.
The administration of the test substance did not lead to distinct clinical signs of toxicit. However, at both top dose groups discolored feces was
observed 4 and 24 hours after test substance administration. Additionally, all animals treated with 2000 mg/kg body weight showed excretion of discolored urine 24 hours after oral administration. The single administration of the positive control substance cyclophosphamide in a dose of
20 mg/kg body weight did not cause any evident signs of toxicity.

Any other information on results incl. tables

Induction of Micronuclei in bone marrow cells

Test group Sacrifice interval [hrs] Animal No.  Micronuclei in PCE - total[‰]a  Micronuclei in PCE - large MN[‰]b          PCE per 2000 erythrocytes [‰]c
Vehicle control corn oil 24 5 2.5 0.1 1041.0
Test substance 500 mg/kg bw. 24 5 3.2 0.0 999.0
Test substance 1 000 mg/kg bw. 24 5 1.4 0.0 1055.0
Test substance 2 000 mg/kg bw. 24 5 2.8 0.0 1105.0
Positive control cyclophosphamide 20 mg/kg bw. 24 5 29.5** 0.0 1275.0
Vehicle control corn oil 48 5 2.6 0.0 1092.0
Test substance 2 000 mg/kg bw. 48 5 2.2 0.0 1101.0

PCE = polychromatic erythrocytes
NCE = normochromatic erythrocytes
bw. = body weight
a = sum of small and large micronuclei
b = large micronuclei (indication for spindle poison effect)
c = calculated number of PCEs per 2 000 erythrocytes (PCE + NCE) when scoring a sample of 20 000 PCE per test group (4 000 PCE per animal)
* = p ≤ 0.05
** = p ≤ 0.01

Depending on the dose, about 82% - 100% of the theoretical values were determined analytically. Regarding the use of test substance suspension in the vehicle corn oil the concentration control analyses of all concentrations revealed that the values were within the expected range of the target concentrations, i.e. a range of 80% - 120% 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 45 minutes.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

Under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.