Biomedical Engineering Reference
In-Depth Information
chromosomal changes. The chromosomal changes in the test
exposed cells are compared to the vehicle exposed cells to deter-
mine the presence of chromosomal damage.
Under the testing recommendations of ISO 10993-3 [
3
],
in vivo genotoxicity testing is not required unless a genotoxic
response is noted in one of the in vitro assays for the device. As a
note, some countries do not recognize ISO 10993-3 as a consensus
standard and require in vivo genotoxicity assays as part of the
overall genotoxicity assessment regardless of the response in the
in vitro assays. When in vivo genotoxicity assays are required, in vivo
tests for chromosomal damage in rodent hematopoietic cells are
typically used. The two most common assays in this category are (1)
the in vivo mouse micronucleus assays and (2) the in vivo chromo-
somal aberration assay.
The in vivo mouse micronucleus assay currently is the more
popular of the two in vivo assays. The methodology is based on
OECD 474,
Mammalian Erythrocyte Micronucleus Test
[
22
]. The
assay is conducted in young rodents, either mice or rats, but mice
are used most commonly. Assays are conducted with concurrent
negative (vehicle) and positive controls. A minimum of five males
and five females per group are used. Both sexes are used unless a single
sex is justified. Animals are dosedwith the device extract or solutionby
an appropriate route. The intravenous route is typically used for saline
extracts (insoluble devices) and the intraperitoneal route for other
extracts and solutions. The oral route of administration may be appli-
cable for devices that have exposure through the gastrointestinal
system. If bone marrow is used, the animals are sacrificed at appropri-
ate times after treatment, the bone marrow extracted, and prepara-
tions made and stained. When peripheral blood is used, the blood is
collected at appropriate times after treatment and smear preparations
aremade and stained. For studies with peripheral blood, smears canbe
prepared as with bone marrow specimens or samples can be analyzed
by flow cytometry. With both methods, preparations are analyzed for
the presence of micronuclei. Numbers of micronuclei in test animals
are compared to the negative control to determine whether the
treatment caused an increase in micronuclei.
The basic methodology for the in vivo chromosomal aberration
assay is based on OECD 475,
Mammalian Bone Marrow Chromo-
some Aberration Test
[
23
]. As with the mouse micronucleus assay,
the test is conducted in young rodents with similar numbers and
groups. Animals are dosed once daily for two consecutive days
(multiple or split dosing may be justified) and the dose is based
on a maximum volume per kg body weight or mg/kg with extract
residues. Animals are sacrificed at 1.5 cell cycle hour times after the
last treatment, which is approximately 12-18 h for mice. Prior to
sacrifice (3-5 h for mice), animals are treated with a metaphase-
arresting agent (Colchicine). Bone marrow cells are harvested,
slides are prepared, and metaphase cells are scored for different
