Review of Proposed ISO 10993 Changes: Blood (10993-4) and Genotoxicity (10993-3)

1. The Future of 10993: Review of
Proposed ISO 10993 Changes: Blood
(10993-4) and Genotoxicity (10993-3)
Thor Rollins B.S. RM(NRM)
Section Leader
Nelson Laboratories, Inc.
801-290-7832
trollins@nelsonlabs.com

3. Genotoxicity (10993-3)

4. Genotoxicity
Need to test for three distinct
levels; DNA, Gene, and
Chromosomal
The four tests used are: Ames, Mouse
Lymphoma, Chromosomal Aberration,
and Mouse Micronucleus (in‐vivo)

5. Ames
• Salmonella typhimurium
• Looks at the DNA level
• TAT
• 5 weeks
• Sample requirements
• 120 cm2 or 4 grams
• Uses a plate count to score
• potential mutagens

6. Ames
Employs several histidine dependent strains of S.
typhimurium
If a mutagen is present these strains revert to histidine
independent and are capable of growing on a plate.
Considered to be a potential mutagen if receives a
greater then two‐fold increase compared to the controls.

7. Mouse Lymphoma
Looks at gene level
• Mouse lymphoma cells are used to
determine whether a test material has the
capacity to induce mutations in a cultured
mammalian cell line.
TAT
• 6 weeks
Sample requirements
• 480 cm2 or 8 grams

8. Mouse lymphoma
Employs thymidine
dependent strains
mouse lymphoma
cells
Cells contain a gene,
thymidine kinase (TK),
when in the presence
of a mutagen this
gene looses activity
After exposure to the
test material. Normal
cells with intact TK
incorporate the analog
and die; mutant cells
(which lack TK activity)
survive, form colonies
and are quantified.

9. Chromosomal Aberration
Chromosomal Aberration
• Chinese Hamster Ovary cells
(CHO)
• Looks at chromosome level
• TAT
• 10‐12 weeks
• Sample requirements
• 480 cm2 or 16 grams
• Microscopic evaluation of cell’s
chromosomes

10. Chromosomal Aberration
There is evidence to
support chromosome
mutations cause
alterations in oncogenes
and tumor suppressor
genes
Possible types of
mutations
Break, gap,
interstitial deletions,
quadradial

11. Chromosomal Aberration

12. Mouse Micronucleus
Mice receive an Intraperitoneal injection at a 20 mL/Kg dose (45
male and 45 female)
At 24 hours (5 animals from each sex) and 48 hours bone marrow
is harvested and examined for micronucleated polychromatic
erythrocytes (PCEs)
The test article will be considered positive if it produces a
statistically significant increase in the total number of PCEs as
compared to the negative control

13. FDA on Genotoxicity
ISO 10993‐3 recommends 2
testing options
• Option 1: Ames and Mouse
Lymphoma (with sizing)
• Option 2: Ames, Mouse Lymphoma,
and Chromosomal Aberration
FDA recommendation
• Website references Mouse
Lymphoma or Chromosomal
Aberration, Ames, and Mouse
Micronucleus.

14. FDA on Genotoxicity
Correlation
between testing
• Ames and Mouse
Lymphoma similar
• Chromosomal Aberration
and Mouse Micronucleus
similar
FDA website:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfstan
dards/detail.cfm?id=28791

15. Hemocompatibility
(10993-4)

16. • Variety of tests that check compatibility in both the blood and immune
systems.
List of
Hemocompatibility
tests
• Thrombosis
• Coagulation
• Platelets
• Hemolysis
• Complement
Hemocompatibility

18. Hemocompatibility

19. Hemolysis

20. Hemolysis
The sample or the extraction fluid is placed in
contact with blood
Use the optical density reading to obtain the
hemolytic index and results are compared to
the negative
Hemolytic index Above the control Hemolytic Grade
0%‐2% Non‐hemolytic
2%‐5% Slightly‐hemolytic
>5% Hemolytic

21. Hemocompatibility
•Complement Activation
•Requires a predicate device
•Uses human blood
•TAT
•4 weeks
•Sample requirements
•60 cm2 or 2 grams or sample and predicate

22. Complement Activation
The complement
system is found in
plasma and consists
of two pathways,
the classical and
alternate. (C3a and
SC5b‐9)
Activation of these
pathways will show
in an absorption
measurement
This concentration
of complement
activation is
compared to
controls and a
predicate device

23. Complement Pathways
Copyright Nelson Laboratories 2010 23

24. Changes in ISO 10993‐4
•New chart will look at surface area
•More detail procedure in standard
•Round robins to look at ASTM,
Japan, and NIH methods.
New Direction
Copyright Nelson Laboratories 2010 24

25. EpiDermTM & EPISKINTM (with MTT reduction and IL‐1α release)
• These are three‐dimensional human epidermal models
• The extract or neat solution is applied to the surface and examined for cell viability
or other signs of inflammation (e.g. Interleukin 1α)
In vitro Alternatives
Copyright Nelson Laboratories 2010 25

26. Questions ??

27. Thank you!