One of the newest biocompatibility evaluation tools is extractable and leachable (E&L) testing. A correctly run E&L study, with an accompanying toxicological evaluation, can be used to replace traditional tests like systemic toxicity, genotoxicity, reproductive toxicity, and carcinogenicity. The data gained from these studies can help understand the total risk of your device to an intended population of users; but unlike the traditional animal tests, it comes with separate risks. These tests are not your typical “stamped” tests, where every lab gives a similar quality of results. Because of this, FDA has refined a strict, detailed, list of parameters that should be included in every test. This list is very dynamic and is changing rapidly; the best way to make sure you are performing the correct version of the test is to learn from the most recent FDA feedback on studies. TAKEAWAY ITEMS: • Understand recent FDA feedback and dissect what FDA is asking/looking for • Learn how to address these concerns and develop a protocol to make sure you don’t receive similar questions • Recognize how FDA is using the new ISO 10993-18 and where they deviate from that standard This session took place live at the Greenlight Guru True Quality Virtual Summit, a three-day event for medical device professionals to learn to get their devices to market faster, stay ahead of regulatory changes, and use quality as their multiplier to grow their device business.

1. FDA Feedback Regarding Chemistry for
Toxicological Risk Assessment –
How to Make Sure FDA will Accept Your Protocol
Matthew R. Jorgensen, PhD, DABT
Chemist, Materials Scientist, Toxicologist
mjorgensen@nelsonlabs.com
Thor Rollins, BS, RM(NRCM)
Director, Toxicology and E&L Consulting
trollins@nelsonlabs.com

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3. Toxicology Consulting Group
Thor Rollins
Director of Consulting
Biocompatibility Wizard
Dr. Matt Jorgensen, DABT
Chemist, Materials Scientist,
and Board Certified Toxicologist
Mtn Dew Enthusiast

4. Outline
3
What is ChemTox?
• One Slide Overview of Fundamentals of Toxicology
• Basic Principles of Chemistry for Toxicology
FDA Feedback Outlining Changes to Expectations
• Extractions (solvents, exaggerated/exhaustive conditions)
• Sample Preparation (solvent exchange and concentrations)
• Method Suitability (reference standards, spike and recovery)
How to Address FDA Concerns
• Overlap Between Expectations and 10993-18
• Development of an FDA Approvable Protocol

5. Systemic Toxicological Effects
4
Endpoints Required for Evaluation:
• Cytotoxicity
• Sensitization
• Irritation
• Material Mediated Pyrogenicity
• Acute Systemic Tox
• Subacute/Subchronic Tox
• Chronic Tox
• Hemocompatibility
• Genotoxicity
• Carcinogenicity
• Implantation

6. Systemic Toxicological Effects: ChemTox Accepted by FDA
5
Required Endpoint for Evaluation:
• Cytotoxicity
• Sensitization
• Irritation
• Material Mediated Pyrogenicity
• Acute Systemic Tox
• Subacute/Subchronic Tox
• Chronic Tox
• Hemocompatibility
• Genotoxicity
• Carcinogenicity
• Implantation

7. One Slide Intro to Toxicology
6
Seeks an answer to a fundamental question:
How much of a good thing is too much?
• Route of exposure
• Duration of exposure
• Potential negative outcomes
Oral Caffeine mg/kg to Dr Dew
(Acidic, Aqueous, Delicious Vehicle)
Irritability
New “Normal”
Arrythmia
Tremors
50% Chance
of Death
Death
Hallucinations
~150 mg/kg
11.5 g or 100 Red Bulls

8. Dose/Response Curves: Xenobiotics
7
Dose mg/kg, Oral, Rat
PercentResponse
AdverseEffect
Highest Dose
Where Nothing Bad
Happened
(NOAEL) LOAEL
Dose Where Something Bad
Happened 50% of the Time
Max
Response

9. Chemistry Strategy Framed by Problem

10. • ANY substance that could
realistically leave the device during
use and enter the body
– Metals
– Production oils and other residuals
– Plastic additives
– Byproducts
• Ignore substances that are
impossible/improbable
Chemistry Strategy Framed by Problem

11. Suite of Analytical Methods
ANY Substance
(That Could Leave The Device)
Inorganic
(not carbon based)
Metals
And
Metal Oxides
Organic
(carbon based)
Very Small Molecules -
Volatile Organics (VOCs)
Small Molecules - Semi-
Volatile Organics (SVOCs)
Big Molecules – Non-
Volatile Organics (NVOCs)
Small molecules
evaporate easily,
great for
gas chromatography
(GC)
Big molecules
do not evaporate easily,
great for
liquid chromatography
(LC)

12. Critical Criteria for Chemistry: Sufficient Breadth and Sensitivity
11
Dose mg/kg, Oral, Rat
PercentResponse
AdverseEffect
Highest Dose
Where Nothing Bad
Happened
(NOAEL) LOAEL
Dose Where Something Bad
Happened 50% of the Time
Max
Response
Where is the Point of
Departure (POD)?

13. Changed Expectations: Choice of Extraction Vehicles
12
𝛿 𝑠𝑜𝑙 = 𝑓𝑎 𝛿 𝑎 + 𝑓𝑏 𝛿 𝑏 + ⋯
𝛿10% 𝐸𝑡𝑂𝐻 = 0.9 ∙ 80 + 0.1 ∙ 24.3 = 74.4
Water
Blood
Brain
Water = 80
Blood = 70-75
Brain Tissue = 43-58
IPA = 18.3
Hexane = 2.0
IPA
Hexane

14. Changed Expectations: Choice of Extraction Vehicles
13
Recent FDA Feedback
In your repeat testing, you conducted chemical characterization for the DEVICE using
exhaustive extraction conditions in polar (ultra purified water, UPW), non-polar (hexane), and
mixed polarity (40% ethanol in ultra purified water) solvents with analysis by HS-GC-MS, GC-
MS, UHPLC-HRAM, ICP-OES, and ICP-MS.
The polarity of 40% ethanol in water is very close to the polarity of water and therefore FDA
does not consider 40% ethanol in water to be a mid-polar solvent. Please repeat exhaustive
extractions using isopropanol as your mid-polar solvent as was done in your original chemical
characterization report.
Yet, in the new 10993-18,

15. Changed Expectations: Choice of Extraction Vehicles
14
Recent FDA Feedback
We recommend that for implants with permanent contact (> 30 days), exhaustive
extraction should be conducted with polar, mid polar, and non-polar solvents to
generate worst-case extractables profiles. Therefore, we recommend that you
conduct exhaustive extraction with final finished implantable devices using
appropriate solvents (e.g., water, isopropyl alcohol, and hexane).
Lack of flexibility on this point observed across many device classes, including
limited contact devices and gas-path devices.
“Leachables may leave the device and remain in the body, therefore the
device is permanent”

16. Changed Expectations: Exhaustive Extractions
15
Source: ISO 10993-18 (2020)

17. Changed Expectations: Exhaustive Extractions
16
• NVR can still be used to demonstrate exhaustively
• TOC, THC, and ICP/MS have been successfully used
• Full work-up at 24 hour intervals by GC/MS, LC/MS, and ICP/MS have been successful
Recent FDA Feedback
You state that the total mass of Non-Volatile Residue water extracts was 40.1 mg. However,
based on the information provided, FDA calculates that there was a total water extract amount
of 3.12 mg for semi-volatile organic compounds as determined by GC-MS and 7.42 mg for non-
volatile organic compounds as determined by UHPLC-MS. Based on your data, FDA calculates
that there is 81% of unaccounted mass for the water extracts. FDA is concerned that there is a
significant amount of mass that is unaccounted for...

18. Changed Expectations: Method Suitability
17
• FDA has been requesting evidence of method suitability that is becoming more close to
what is expected in an EPA study:
• Method validation including precision and accuracy for a wide range of compounds
• Matrix spike and recovery through analytical process (including solvent exchange)
• Laboratory control samples, calibration curve verifications, etc.
• Defense or explanation of accuracy of non-validated compounds

19. The Analytical Evaluation Threshold
Final AET
ISI
All compounds above the AET:
Reported, identified and
quantified
2
1
3 4 5 6
7
8

20. Determining the Required Analytical Sensitivity
• Goal is to minimize risk related to undetected
compounds below the analytical sensitivity
• Consideration must be given to uncertainty in
accuracy of measurement (generally a factor of
2 to 4)

21. The Analytical Evaluation Threshold
Recent FDA Feedback (in regards to AET calculation)
We asked you to justify the analytical Uncertainty Factor (UF) of 4. In response to this question,
your Q-sub refers to the papers by Jordi and Jenke to justify an uncertainty factor of 4. However,
the references do not unequivocally recommend using a UF of 4. According to paper by Jordi et
al, “It is recommended that the AET be lowered by applying either a 50% reduction or a
correction factor equal to the % RSD of the relative response factor database. The approach
which provides the largest reduction should be applied.” Although you used a conservative UF
value of 4, you did not provide any data to justify the UF of 4. You should provide calculations
you made based on the %RSD value of the relative response factors to justify using a UF of 4.
𝐴𝐸𝑇
𝜇𝑔
𝑑𝑒𝑣𝑖𝑐𝑒
=
𝐷𝐵𝑇
𝜇𝑔
𝑑𝑒𝑣𝑖𝑐𝑒
𝑈𝐹

22. How Does E&L Work: Identification and Quantification
• GC/MS: Electron Ionization (EI)
– Literally shooting molecules with an
electron machine gun
– Highly standardized and reproducible
• LC/MS and LC/HRAM
– More variable from lab to lab
– Atmospheric Pressure Chemical
Ionization (APCI±)
– Electrospray Ionization (ESI±)
– MS is “low resolution” and good for
targeted lists
– High resolution accurate mass (HRAM)
good for screening but requires special
expertise

23. Extra Caution Needed with Identifications
O
O
O
O
O
O
NIST, Automatic ID
Internal DB
With
Expert Review

24. Extra Caution Needed with Identifications

25. Extra Caution Needed with Identifications
Regarding Identifications:
• We do not recommend choosing a structure associated with the highest
match factor without providing a justification for eliminating other
potential matches.
• All potential identifications should be provided so that they can be
considered in the toxicological risk assessment.
• Substances for which only partial structure data is available should be
designated as unknown in the test report and for toxicological risk
assessment.
• If a class of substances is to be listed as the identification, then all potential
members of the class should be provided.

26. Extra Caution Needed with Identifications
Regarding Identifications:
• If a class of substances is to be listed as the identification, then all potential
members of the class should be provided.
C4 Alkanes: 2 members
C5 Alkanes: 3 members
C6 Alkanes: 5 members
C12 Alkanes: 355 members
C32 Alkanes: 27.7 billion members

27. Leachables Studies: When to Conduct Them and How?
Typical Extractables Analytical Test Matrix
Analytical Method
Polar
(water)
Mid-Polar
(IPA)
Non-Polar
(hexane)
Elements
ICP/MS and ICP/OES
X N/A N/A
VOCs
Headspace GC/MS
X N/A N/A
SVOCs
Direct Injection GC/MS
X X X
NVOCs
HRAM-UPLC/MS, APCl ±
N/A X X
NVOCs
HRAM-UPLC/MS, APCI/ESI ±
X N/A N/A
• The device is extracted per ISO
10993 standards
– 50 °C for 72 hours
– Polar, Mid-Polar, and Non-Polar
Solvents
• If there are problems, then
non-standard conditions can
be used
– 37 °C for 24 hours
– 10% EtOH in water
– Other conditions which closely
mimic clinical use
Even if we know that an extractables study will “fail,” it
is still conducted, followed by a leachables (simulated
use extractable) study targeted to the problem.

28. Feedback from FDA on Reporting
Regarding Reporting, the Following Should be Included:
• Calibration data that demonstrates that suitability of the calibration
method across the range needed for quantification.
• Chromatographic data that includes labeling to discern retention time and
relative signal intensity.
• Substance information when higher than the AET:
– (1) Retention time, (2) proposed identity(ies), (3) chemical name (e.g. IUPAC name), (4)
CAS number, (5) structural descriptor (e.g. SMILEs), (6) identification confidence level, (7)
type of identification data (e.g. spectra library match), (8) quantification method (e.g.
fully quantitative using an authentic standard, semi-quantitative based on a relative
response factor, or semi-quantitative using a surrogate response factor), (9)
concentration (e.g. in μg/ml), and (10) quantity (μg/device).

29. What the FDA Wants
In General:

30. Reports Include-
• Introduction and Study Standards: “This is a medical device extractables study conducted
per ISO 10993-18:2020”
• Photos of the devices before and after extraction
29

31. Reports Include-
30

32. Reports Include-
31

33. Reports Include-
32

34. Reports Include-
33

35. Reports Include-
34

36. Conclusion on ChemTox and FDA
• It should be understood that ChemTox is often viewed as a requirement and
undergoing a period of intense scrutiny by regulators
• The process should begin with a collaboration between the toxicologist and
the chemistry lab; erring on the side of providing more detail
• A detailed testing plan is recommended; this should be given to the FDA as
part of their pre-sub program
• Goals: Save animal lives, save time, save money, and IMPROVE PATIENT
SAFETY!

37. Questions?
36