**Preclinical Review/additional information**

P030004

**DATE:** 7/25/03

**Review**

Additional information regarding DMSO/coil compatibility, the effect of
radiation on the polymer, endotoxin quantities of the catheter and
syringe, the amount of silicone oil placed into the syringe and polymer
solidification time was requested. As mentioned in the first mail-out,
the repeat injection issue related to DMSO potential toxicity is
discussed. Finally, additional review information is provided in this
email as excerpted from the manufacturing reviewer/chemist's and
preclinical reviewer's review of the chemistry and toxicology of the
device.

[1. DMSO/coil compatibility]{.underline}

In the experiment the sponsor used HPLC to detect any leachable chemical
entities from platinum, GDC or Cook fiber coils due to contact with
DMSO. The concomitant use of coils and the polymeric embolization agent
is likely to occur in the presurgical embolization of AVMs. A minor peak
had been identified that was different from control in 6 of 12 coils
evaluated. I reviewed the control and suspect chromatograms. The control
chromatogram shows a broad peak in the area where the additional peak
appears in 6 of 12 samples. In those 6 samples the peak appears to have
been split into two. FDA agrees with the sponsor's chemist that the peak
identifies a minor chemical entity and that the splitting of the broad
peak identified in the control into 2 minor peaks is of minor
importance, chemically, and does not necessarily indicate a new
degradation by-product or leached chemical. The sponsor has adequately
addressed the deficiency.

[2. Effect of radiation on polymer]{.underline}

The sponsor had conducted evaluations to determine if radiation could
cause degradation of the polymer in vivo. Although the device is
intended for use as a presurgical embolication agent, in some cases a
patient's AVM may not be resected due to various reasons. In those
situations, an alternative means for "resecting" the AVM is to irradiate
it. Radiation causes the tissue to undergo fibrosis and thereby,
stabilization. The sponsor had not included the IR spectra or GPC
chromatograms to support their contention that the material had not
degraded after having been irradiated.

The IR spectra provided of material (n = 2) irradiated with 30 Gy were
identical, or nearly identical. The control and irradiated material
spectra are qualitatively the same. The GPC chromatograms (n = 2) of
irradiated material and control EVOH were nearly superimposable.

In addition, the sponsor has provided information that adequately
addresses the concerns raised regarding the endotoxin amounts, the
silicone oil used in the syringe and the polymer solidification time.

[3. Repeat injection issue]{.underline}

In the first mail-out FDA provided the following draft preclinical
question for consideration:

*Preclinical animal evaluations have shown that the rate and amount of
DMSO can cause vasospasm and vascular wall damage. Patients undergoing
staged embolization procedures for Cerebral Arteriovenous Malformations
will be exposed repeatedly to the potential for DMSO-mediated vessel
damage. Do you believe additional animal evaluation should be conducted
to more completely assess for repeat-DMSO vessel wall exposure and
potential adverse effects? Do you have any recommendations regarding the
amount of DMSO a patient should be exposed to over a 24 hour period or
the length of time between embolization procedures?*

FDA indicated that this issue would be discussed in more detail in the
second mail-out. The following information is a summary of relevant data
that you may find helpful in determining how you think this issue is
best resolved.

Onyx requires a two-step procedure employing anhydrous DMSO. MTI data
shows that the initial catheter-priming step used 0.2 -- 0.26 mL DMSO
injected intra-arterially at ≤ 0.4 mL/min. The second step involves slow
injection (mean rates = 0.115 mL/min) of a mixture of EVOH polymer and
tantalum powder dissolved in pure DMSO. Animal studies have shown that
if concentrated DMSO is given too quickly, severe vasospasm and
angiotoxicity will occur. In early evaluations of the embolic agent,
Chaloupka et al studied the device in the swine rete mirabile. The
investigators encountered visualization, catheter-compatibility and
vascular toxicity complications. The DMSO infusion caused moderate to
severe vasospasm immediately; subarachnoid hemorrhage or stroke occurred
frequently. Histopathology showed variable endothelial denuding,
thrombosis, and internal elastic lamina disruption acutely; an intense
mixed inflammatory response with organized thrombus formation and
transmural necrosis with extravasation was noted in subacute and chronic
specimens. The investigators concluded that undiluted DMSO was
angiotoxic.

Additional studies have shown that if a low dose of DMSO is administered
using a very slow injection rate, vasospasm and angiotoxicity is not
observed. Murayama et al., evaluated the embolic agent for acute and
chronic effects after intra-arterial delivery. The study looked at the
importance of slow infusion in reducing arterial damage caused by
concentrated DMSO. Injections of 0.5 mL DMSO were given over 5, 15, 30,
60, and 120 seconds; the EVOH mixture used a priming dose of 0.3 mL DMSO
administered over 40 seconds followed by 0.3-0.5 mL of the EVOH/DMSO
mixture given over 20-40 seconds. Special attention was directed to
findings of focal or diffuse angionecrosis, arterial revascularization,
and perivascular inflammation. When 0.5 mL DMSO alone was given over
5-15 seconds, vasospasm and endothelial necrosis developed. The same
dose infused over 15 seconds yielded focal vasospasm, but no laminal
disruption or angionecrosis. No toxicity of any kind was noted if 0.5 mL
anhydrous DMSO was given slowly over 30, 60 or 120 seconds. The authors
concluded that the two most important elements in controlling vascular
toxicity precipitated by intravenous injection of concentrated DMSO
were:

-   Contact time with the arterial wall

-   DMSO volume

Murayama et al. concluded that "slow, controlled intra-arterial delivery
of DMSO shows minimal endothelial inflammatory response and no
histological evidence of necrotizing arteritis". The preclinical
information provided in the PMA and found in the scientific research
literature clearly indicates that DMSO can cause vascular toxicities if
the rate of its infusion is not carefully controlled. In addition, the
preclinical information in the PMA also shows that if the rate of
injection is controlled, vasospasm and vascular toxicities are avoided
in single infusion experience. Training physicians with regard to the
use of the product and how to avoid causing DMSO-mediated vascular
toxicity is important to the safe use of the product. The sponsor has an
established training program for physicians learning how to use the
product that includes the following elements:

-   Theoretical presentation: includes discussion of Onyx formulations
    (Onyx 18 (6%) and Onyx 34 (8%) and rationale of when to use each
    formulation; overview of preclinical testing; use of DMSO (research
    papers, animal studies, clinical experience to date), and complete
    review of Onyx LES (liquid embolic system) tips and techniques,
    i.e., material preparation, rate of injection of DMSO, compatible
    micro-catheters, injection technique.

-   In vitro bench workshop: bench model that replicates AVM flow
    characteristics used to provided physicians experience with
    injecting Onyx 18 and Onyx 34 at various flow rates

-   In vivo animal injections or clinical observation: physician is
    offered opportunity to perform embolizations in the swine rete
    mirabile, renal arteries or external carotid arteries, or to observe
    a clinical case performed by the Onyx proctor.

-   Case review: training physician shares case films to provided
    reference regarding clinical use of Onyx -- overall clinical
    experience from Europe, selected case videos, and films are reviewed

-   Clinical representative attends the physician's first case

And the product label contains the following information regarding the
use of DMSO:

-   A DMSO compatible delivery micro catheter that is indicated for use
    in the neuro vasculature (e.g. Rebar™ or UltraFlow™ HPC catheters)
    is used to access the embolization site.

-   Direction and Warning: Based on clinical practice, it is recommended
    that Onyx be injected at a slow, steady rate of 0.16 mL/min (0.25
    mL/90 sec). Do not exceed 0.3 mL/min. Do not exceed 0.3 mL/min
    injection rate. Animal studies have shown that rapid injection of
    DMSO into the vasculature may lead to vasospasm and /or
    angionecrosis.

The training program and the label instructions/warnings appear to
adequately inform the user about the dangers of rapid DMSO vascular
infusion. However, little research information or clinically meaningful
information is available regarding the safety of repeat infusions of
DMSO as might occur during staged embolizations of the product. In an
Onyx-unrelated animal assessment of the repeat intravenous
administration of DMSO, Willson et al found that undiluted DMSO given to
dogs at 0.3, 0.6, 1.2 and 2.4 g/kg/day six times per week for 4 weeks
caused injection-site vein occlusion.

Limited information to address repeat DMSO injection as related to the
Onyx device and its potential resultant toxicity was provided by the
sponsor. In tissue surgically excised from 7 patients of the Mexico City
Embolyx Pilot Study and the International Brain Arteriovenous
Malformation Clinical study the investigators assessed Onyx for its
potential chronic, histotoxic effects. Seven BAVMs embolized with Onyx
were surgically excised and submitted for evaluation to a board
certified histopathologist. Prior to surgery, 1 of 7 patients received a
single embolization treatment, 2 of 7 received 2 treatments, 1 of 7
received 3 treatments and 3 of 7 received 4 treatments. The timing
between embolization and surgery ranged from 1 week to 19 months while
all patients had pre-surgical embolization periods of at least 3 months,
i.e., no one had one embolization procedure and then within 1 week went
to surgery whereas some patients may have had multiple embolization
procedures with the last one being 1 week prior to surgery. There were
no indications of vascular necrosis, rupture or extravasation of the
Onyx material. Numerous vessels were observed with disruption of the
internal elastic lamina, but there did not appear to be any serious
adverse effect on the vessel wall. The information is obviously very
limited.

A review of CT, MRI and flat film skull x-rays obtained from patients
whose BAVMs were treated with Onyx or n-BCA was performed for MTI by a
central reader to determine if any direct neurotoxicity due to Onyx can
be detected in the brain post-embolization. A total of 54 patients were
studied in the Onyx group and 19 in the n-BCA group (total = 73). The
central reader was blinded as to treatment. All MRI and CT studies were
evaluated for the presence or absence of gliosis, encephalomalacia,
edema, leptomeningeal or parenchymal enhancement and hemorrhage. These
parameters were pre-defined based on specific imaging characteristics.
The average time post-embolization for all imaging studies was 23
months, with a range of 9 to 50 months. Forty-one patients of the 73 had
imaging findings that required an assessment as to whether the finding
was due to the device. Twelve of the 19 n-BCA patients had imaging
changes that were due to: concurrent neurosurgical resection of the AVM,
changes in the brain related to neurosurgery, or due to the natural
history of the AVM. Twenty-nine of 54 patients in the Onyx groups
demonstrated imaging findings post-embolization that were not present
pre-embolization. The reader (Director, Clinical Image Processing
Service for UCLA Department of Radiological Sciences) asserts that "in
all cases the etiology of the post-embolization findings was found to be
due to events unrelated to the presence of Onyx." The findings were
believed to be due to radiosurgery, surgical resection of the AVM and
the natural history of the AVM. An FDA radiologist reviewed the images
and found no reasons to disagree with the central reader's
interpretation that the image post-embolization observations were due to
events unrelated to the presence of Onyx.

The following parameters of the investigational study should be taken
into account:

Number of n-BCA (n = 54) Onyx (n = 46)

[embolization procedures \# % \# %]{.underline}

1 34 63 26 56.5

2 9 16.7 11 23.9

3 7 13 6 13

4 2 3.7 1 2.2

5 2 3.7 1 2.2

6 0 0 0 0

7 0 0 1 2.2

From the sponsor's clinical experience outside of the United States:

Number of Onyx (n = 161)

[embolization procedures \# %]{.underline}

1 113 70.2

2 32 19.9

3 12 7.4

\>3 4 2.5

So, the information gathered in the sponsor's U.S. clinical study and
their experience outside the U.S. clearly indicates that although the
majority of AVM patients will undergo one embolization procedure, there
is a subpopulation of individuals that will undergo two or more
infusions of the embolic agent. The mean volume of Onyx injected in this
\[U.S.\] investigational study was 0.5 mL and the mean volume of DMSO
injected was 0.27 mL, whereas clinical experience outside the U.S. found
that the mean volume of DMSO per treatment was 1.57 mL and the maximum
dose of DMSO ever delivered was 8.36 mL.

It could be argued that since the embolic agent is targeted to a
vascular abnormality that the physician intends to remove from the
patient, long term evaluation regarding repeat DMSO injections is of
little interest. However, not all patients in the study went on to have
their AVM surgically excised. Of the 100 patients in the ITT population,
86 had total resection and 89 had total or partial resection. The
patients enrolled in the study were identified as surgical candidates
but because surgical resection was not in the patients' best interest,
surgical resolution did not occur in every case. With respect to
vasculature that has been embolized with DMSO more than once we have
very limited information in terms of numbers of patients and in length
of time of follow-up.

Please consider this information in preparation for discussion of the
repeat-injection DMSO toxicity panel question.

[4. Chemistry of Device]{.underline}

Ethylene vinyl alcohol copolymer (EVOH) is synthesized by polymerizing a
mixture of ethylene gas and vinyl acetate. The resulting ethyl vinyl
acetate is treated in a basic pH environment with sodium hydroxide and
methanol to hydrolyze the acetate from the polymeric chain resulting in
ethyl vinyl alcohol. The EVOH polymer is washed with methanol to remove
the acetate and other low molecular weight oligomers.

The EVOH co-polymer requires the use of anhydrous DMSO (Onyx = EVOH in
DMSO plus tantalum for radiopacification) as a solvent for delivery
through the micro-catheter to the AVM site. If Onyx comes into contact
with saline, it will immediately precipitate and block the catheter. A
small amount of anhydrous DMSO (0.2-0.26 mL) is used to prime the
micro-catheter. After Onyx reaches the aqueous environment of the
embolization site, DMSO from the EVOH/DMSO mixture will be diluted by
water in the blood and surrounding tissues. Water contact will cause the
EVOH polymer to precipitate and produce an embolus that will conform to
the tissues of the embolization site. Formation of the embolic plug
begins at its outer surfaces and proceeds inward. Complete embolus
formation requires a prolonged period of time, from 3-20 minutes,
depending on blood flow and the amount of material injected. Micronized
tantalum is added to the EVOH/DMSO solution to provide for fluoroscopic
visualization. It is important to note that the catheter priming amount
of DMSO represents the free, non-solvent DMSO device component in that
it is used simply to prime the catheter. The priming volume of DMSO will
be readily transported from the embolization site intravascularly and/or
into the interstitial space of the site. DMSO solvating the EVOH will
diffuse more slowly from the site, than the free DMSO priming amount, as
it is gradually released from the precipitating embolus.

Master file and release specifications for each device component have
been reviewed. From the manufacturing/polymer chemist's perspective, the
"three major components of the subject device, Ethylene Vinyl Alcohol
Copolymer (EVOH), Dimethyl Sulfoxide (DMSO) and Tantalum do not contain
significant amounts of impurities and are spectrascopically pure
materials."

There are 2 formulations used in the Onyx LES for AVM treatment: Onyx 18
and Onyx 34. The maximum concentration of DMSO is approximately 90% by
volume. In MTI's studies to date the average amount of DMSO used per
treatment was 1.57 mL and the maximum volume of DMSO used in any one
treatment was 8.36 mL (data collected from 222 procedures). For the
calculation of the potential maximum DMSO dose observed in AVM
treatments, 8.36 mL x 1.10 (specific gravity of DMSO) equals 9.2 g DMSO,
thus yielding (9.2 g/70 kg) a maximal DMSO dose of **[131
mg/kg]{.underline}**. If this maximum dose is calculated to be used as
the first of a series of staged embolizations, it is reasonable to
assume that subsequent embolization treatments would require smaller,
more average, quantities of Onyx. Using the average amount of DMSO (1.57
mL) used for 3 additional treatments (1.57 mL x 3 treatments x 1.10
\[spec. gravity of DMSO\]), plus 9.2 g DMSO per the first treatment, the
total maximal dose of DMSO a patient would be likely to be exposed to is
14.37 g DMSO/70 kg, or **[205 mg/kg]{.underline}**. \[Please note that
these values are numerically larger than what was used in the U.S.
clinical study, i.e., 0.27 mL free DMSO plus 0.5 mL Onyx.\] To calculate
a lower range value, using the mean, average dose for the first
treatment instead of the maximal dose ever observed, the lower potential
DMSO exposure would be, 1.57 mL x 1.10 = 1.73/70 kg = 0.025 g/kg x 4
treatments = **[100 mg/kg]{.underline}**. Therefore the range of DMSO
concentration that a patient could be exposed to is **[100-205
mg/kg]{.underline}**. It is important to note that the embolization
procedures would be done over a period of time and the patient would not
be exposed to the 100-205 mg/kg total all at once.

Metabolic studies in man and lower animals indicate that the primary
metabolites of DMSO are dimethyl sulfone (DMSO~2~) and dimethyl sulfide
(DMS).

[5. DMSO Biocompatibility: Toxicities of DMSO and DMSO
metabolites]{.underline}

MTI provided a white paper on DMSO toxicity which identified research
information regarding the absorption, distribution, metabolism, and
excretion of DMSO. The following information is a very brief summary of
the most pertinent information of that white paper. DMSO, when used in
the treatment of interstitial cystitis in humans is considered a drug.
For a point of comparison, Rimso-50^®^ consists of a 50% solution of
DMSO, and as instilled in the bladder as a 50 mL dose, equals a 393
mg/kg dose for a 70 kg person.

DMSO has chemical properties which facilitate its absorption into and
distribution throughout biological systems by all routes of
administration. DMSO can also carry other substances along with it due
to its solvating power. DMSO is a polar nucleophile which has free
electron pairs at its sulfur and oxygen terminals. It is considered an
aprotic solvent since it does not normally donate hydrogen atoms in
chemical reactions. Hydrogen bonding of DMSO with water is 1⅓ times
stronger than the hydrogen boding between water molecules themselves,
thus yielding DMSO's hygroscopic character. Intravenous administration
of DMSO appears to be well-tolerated at concentrations lower than 50%.
Higher concentrations, given repeatedly, injure the injected vessels
causing fibrosis proportional to the concentration and number of
injections. Persistent damage to the blood vessel causes a narrowing of
the lumen. As noted above, the Willson study observed injection site
vein occlusion in dogs given daily injections of DMSO for 4 weeks. The
injection rate of DMSO has been observed to determine toxicity. As noted
in a 1971 text, *Toxicology of DMSO in Animals* (Mason), a 5 mL rapid
intravenous infusion of DMSO caused death in a dog whereas a dose of 100
mL infused over 4 hours did not cause death.

Distribution studies regarding DMSO reveal that the molecule is rapidly
distributed in a wide-spread manner. Denko et al. found that DMSO
accumulated more in soft tissues and it was found in tissues with low
and high lipid content. Tissue vascularity or permeability appeared to
offer no preferred mode of action. In a study by Nishimura et al.
looking specifically at distribution of DMSO in brain and vascular
tissue in the rat, calculated tissue (muscle, liver and gray matter) to
plasma ratios were observed to be 1:1 two hours after infusion (of a
1g/kg/hour) was initiated. White matter approached this ratio after one
hour and had declined some by 2 hours. In the mouse, peak plasma
concentration was reached one minute after a bolus injection into the
tail vein and it diminished in a biexponential fashion; its rapid
distributive phase showed a t½ of 1.5 minutes, while the longer terminal
half-life was 90 minutes. In summary, MTI believes that the DMSO in the
Onyx solution will be distributed rapidly by the vascular system, a
portion likely bound to serum or plasma proteins, or dispersed into
interstitial spaces of surrounding tissues. The priming dose (of the
catheter) will likely be distributed via the vascular system very
quickly whereas the DMSO that slowly elutes from the embolus will likely
gain entrance to the endothelial lining cells of the blood vessel and to
the interstitial space.

Many metabolic studies in man and animals show that the primary
metabolites of DMSO are dimethyl sulfone and dimethyl sulfide. The
extent to which DMSO is converted to dimethyl sulfone by non-primates
seems to vary somewhat by route and species. All studies reviewed in the
white paper indicated that DMSO is not excreted unchanged to some
extent, most of it will be either oxidized to dimethyl sulfone and
excreted in the urine or reduced to dimethyl sulfide and exhaled. The
garlic-breath of individuals treated with DMSO is thought to result from
the conversion to dimethyl sulfide. Although unsupported by data, MTI
believes, based on animal studies including human and non-human
primates, that approximately half of the DMSO in Onyx will not be
metabolized with 20-25% being converted to dimethyl sulfone and a small
fraction being converted to dimethyl sulfide. Dimethyl sulfoxide is a
constituent of plant materials. Dimethyl sulfone is found in milk and
dimethyl sulfide has been found in prepared foods. The manufacturer of
the DMSO provided limited LD~50~ information regarding the acute
toxicity of dimethyl sulfide in the rat. The oral, inhalation and dermal
values were 3.7 g/kg, 40,250 ppm, and 10.2 g/kg, respectively. The small
amounts of dimethyl sulfone and dimethyl sulfide should not, based on
available toxicology information, cause risk to the patient.

LD~50~s after intravenous dosing reveals that anhydrous DMSO caused
acute toxicity in cats, dogs, monkeys, rabbits and rats in the range of
**[2.5 g/kg-11g/kg]{.underline}**. Some investigators found that
repeated intravenous injections of undiluted DMSO were damaging to the
veins of dogs and rats. However other investigators found no negative
effects of injecting 40% DMSO into dogs for 33 days. The sponsor cites a
1963 reference that determined the LD~0.1~, or that dose likely to kill
1/1000 animals dosed, for anhydrous DMSO after intravenous
administration to be 400 mg/kg in the mouse. As the sponsor notes, this
dose is approximately 3 times that of the largest single DMSO dosage
used in Onyx clinical experience to date (i.e., 131 mg/kg) and 14-16
times the average dosages for Onyx clinical treatments.

There is sufficient evidence provided in the research literature that
demonstrates DMSO to be hemolytic. A number of animal studies noted
hematuria and hemoglobinuria -- specifically after intravenous
administration. Emmerling et al in a 1991 study report to NCI found that
male rats given 70% DMSO via a rate of 2 mL/kg/hr/120 hrs showed marked
decreases in hematocrit and hemoglobin by the second day. In a study by
Bennet et al concentrations of DMSO of 10, 20 and 40% infused at 1g/kg
caused decreases in hematocrit of 1.5, 4.9 and 5% in humans. In the
study by Willson, anemia, hemoglobinuria, bilirubinuria, increased SGOT
levels and slight liver pathology was observed at a dose of 0.3
g/kg/day/6 days/week/4 weeks.

In conclusion, Onyx administration is likely to produce some hemolysis,
primarily after the priming dose is given. In addition, endothelial cell
damage is possible but should be minimized by adherence to the slow rate
of administration determined in the sponsor's animal model
investigations evaluating for angiotoxicity. The severity of vasospasm
and the occurrence of angionecrosis in swine were reduced when the
volume of DMSO was reduced from 0.8 to 0.5 mL. As a result of the
recommendation by Murayama et al, that 0.3 mL delivered over 40 seconds
was a safe dose, the recommended priming doses now used for Onyx is 0.26
over 40 seconds. The amount of DMSO a patient has been maximally exposed
to falls 3-4 times below levels noted in animal toxicology studies to
cause adverse effects. As evidenced by the sponsor's U.S. clinical
study, the amounts of DMSO most commonly used in the presurgical
embolization of AVMs is much lower than the maximal amount reported. The
extensive biocompatibility assessments and animal performance
evaluations indicate that the product was biocompatible and did not
cause adverse tissue responses different than, or greater than what is
seen with approved embolic agents.