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Toxicological information

Genetic toxicity: in vivo

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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: OECD Guideline study, GLP
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform
Details on test material:
Z-Cote HP1
zinc oxide (and) triethoxycaprylylsilane (coating)
ZnO-content [%]: 96-99
< 200 nm
Specific surface area [m²/g]: 12 -24

- Physical state: solid, beige
- Lot/batch No.: CNFC0701
- Stability under test conditions: guaranteed until 30 Jan 2012
- Storage condition of test material: room temperature
- Other: more details see analytical report (study code 06L00180)

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: 30.0 g
- Assigned to test groups randomly: yes, under following basis: appropriate computer program
- Fasting period before study: no data
- Housing: single
- Diet: ad libitum, standardized pelleted feed (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Fetal claf serum (FCS).
- Justification for choice of solvent/vehicle: suitable for dispersion of nanoparticular substances to reach a relatively low amount of agglomerates
- other: The substance was disperged in FCS and the preparations were stirred in a closed vessel for 24 hours at about 1 000 rpm at room temperature for homogeneity.
Duration of treatment / exposure:
intraperitoneally with a volume of 10 mL/kg bw
Frequency of treatment:
once
Post exposure period:
24 h (and 48 h for control and highest dose group)
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 30 and 60 mg/kg bw
Basis:
other: i.p. with a volume of 10 mL/kg bw
No. of animals per sex per dose:
5 animals per dose and time
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive controls:
vincristine sulfate 0.15 mg/kg bw.
cyclophosphamide 20 mg/kg bw

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (= normocytes, NCE) from the bone marrow of both femora per animal.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In pretests about the acute intraperitoneal toxicity, deaths were observed down to 80 mg/kg body weight. At 60 mg/kg, all animals survived but distinct clinical signs were observed. Therefore, doses of 15, 30 and 60 mg/kg bw were selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- treatmetn: once i.p. with a volume of 10 mL/kg bw
- sacrifice: 24 h (all groups) and 48 h (highest dose, vehicle) after the treatment

DETAILS OF SLIDE PREPARATION:
Smears of one drop of bone marrow , suspended in FCS, were air dried on the slides and stained with eosin/methylene blue and subsequently with Giemsa solution. Preparations were clarified in xylene and mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
- Evaluation of 2 000 polychromatic erythrocytes (PCE) per animal for the occurrence of micronuclei. The normochromatic erythrocytes (= normocytes / NCE) were also scored.
- Recorded parameters: Number of PCE, PCE containing micronuclei, NCE and NCE containing micronuclei; ratio of PCE to NCE, number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4); [d = diameter of micronucleus, D = cell diameter]
Evaluation criteria:
- sufficient number of analyzable cells
ACCEPTANCE CRITERIA:
- ratios of PCE/NCE in the concurrent vehicle control within the normal range
- number of cells containing micronuclei in vehicle control within the historical range
- increased number of PCE containing small or large micronuclei in both positive controls within the historical range or above

ASSESSMENT CRITERIA:
- positive findings, if number of PCEs containing micronuclei are statistically significant and dose-related increased or exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
- negative, if no statistical significant increase above the concurrent vehicle control value and within the range of the historical vehicle control data
Statistics:
asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON), using the program system MUKERN.
Statistical significances: * p ≤ 0.05, ** p ≤ 0.01.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MORPHOLOGY OF TEST MATERIAL:
Representative TEM images of the test material are shown in the attached document.

Any other information on results incl. tables

Induction of micronuclei in bone marrow cells:

 Test group Sacrifice interval [hrs] Animal No.  Total micronuclei in PCE [‰] Large micronuclei in PCE [‰] Number of NCE
vehicle control FCS  24 5  0.9 0.1  5778
test substance 15 mg/kg bw  24  5  1.2  0.0  5067
test substance 30 mg/kg bw  24 5  0.9  0.0  4858
test substance 60 mg/kg bw 24  5  1.3  0.1  5468
cyclophosphamide 20 mg/kg bw (PC)  24  5  14.6**  0.5  4483
vincristine sulfate 0.15 mg/kg bw (PC)  24 5  66.2**  20.1**  6156
vehicle control FCS  48  5  1.2  0.0  3769
 Test substance 60 mg/kg bw  48  5  1.0  0.1  5824

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions chosen here, the test substance Z-Cote HP1 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.