Accepted Manuscript Title: Chemical fingerprinting of silicone-based breast implants Author: Peter H.J. Keizers Marjo J. Vredenbregt Frank Bakker Dries de Kaste Bastiaan J. Venhuis PII: DOI: Reference:
S0731-7085(14)00437-3 http://dx.doi.org/doi:10.1016/j.jpba.2014.09.008 PBA 9719
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
20-6-2014 5-9-2014 8-9-2014
Please cite this article as: P.H.J. Keizers, M.J. Vredenbregt, F. Bakker, D. de Kaste, B.J. Venhuis, Chemical fingerprinting of silicone-based breast implants, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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*Highlights (for review)
Highlights of “Chemical fingerprinting of silicone-based breast implants”, by Keizers et al.:
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Qualitative discriminants of silicone breast implants are described Breast implants are distinguished on brand and type Barrier layers in breast implant envelopes are visualized A set of complementary spectroscopic techniques is described for market surveillance of breast implants
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Chemical fingerprinting of silicone-based breast implants
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Peter H. J. Keizers, Marjo J. Vredenbregt, Frank Bakker, Dries de Kaste, Bastiaan J. Venhuis*
4 National Institute for Public Health and the Environment, P. O. Box 1, 3721 MA, Bilthoven, the
6
Netherlands
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*Corresponding author. Tel.: +33 30274 4228. E-mail address: [email protected] (B. J.
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Venhuis)
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10 11 Abstract
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With millions of women worldwide carrying them, silicone-based breast implants represent a large
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market. Even though silicone breast implants already have a history of use of more than fifty
15
years, the discussion on their safety has not yet come to an end. To improve safety assessment,
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regulatory authorities should have the availability of a set of tests to be able to determine
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parameters of implant identity and quality. Therefore, the gels and envelopes of various brands
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and types of silicone-based breast implants have been subjected to infrared, Raman and NMR
19
spectroscopy. We show that by using a combination of complementary spectroscopic techniques
20
breast implants of various origins can be distinguished on typical chemical hallmarks. It was found
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that typical silicone-based implants display a surplus of vinyl signals in the gel, cyclosiloxane
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impurities are tolerable at low levels only and a barrier layer is present in the implant envelope.
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The techniques presented here and the results obtained offer a good starting point for market
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surveillance studies.
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Keywords: silicone implants, PIP, spectroscopy
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1. Introduction
29 Breast implants, or prostheses, find widespread application for cosmetic reasons or reconstruction
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after tissue removal by surgery. Several types are available, of which the ones with a silicone-
32
based gel and envelope are the most commonly used. They represent a large market; it was
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estimated in 1999 that 25.000 to 30.000 Dutch women carry silicone-based implants [1], and this
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number is believed to have significantly increased ever since.
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Silicone breast implants have been associated with adverse health effects, ranging from
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inflammatory reactions to cancer and autoimmune syndrome induced by adjuvants [2,3,4,5]. One
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explanation of these effects is thought to result from poor quality implants that have been
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marketed over the years.
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A notorious example in that sense is the PIP case [6,7]. The company Poly Implant Prothèse has
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used industrial grade silicones in some of their implants produced from 2001 to 2010. In 2010, the
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Dutch Health Care Inspectorate (IGZ) removed PIP implants from the Dutch market. PIP implants
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have had a higher chance on complications resulting from rupture, leaking and sweating. Three
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types of PIP implants have been produced over the years that are associated with the
44
complications; indicated as PIP-1, PIP-2 and PIP-Nusil. However, it remains unclear whether only a
45
few poor batches have been produced or if all produced PIP implants have been of questionable
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quality [8].
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At the National Institute for Public Health and the Environment (RIVM) we were requested by IGZ
48
to develop knowledge on the quality of breast implants used in the Netherlands. Specifically,
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chemical hallmarks of breast implants should be identified to be able to distinguish implants,
50
preferentially on parameters related to quality. Eventually these results are to be used in market
51
surveillance activities.
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Literature on the chemical analysis of silicon-based implants has become available in recent years
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[9,10,11,12,13]. NMR spectroscopy, light microscopy infrared spectroscopy and GC-MS have
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successfully been applied to study silicone-based implants. Specifically, the distinction between
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PIP-1 and PIP-2 type implants compared to high-end products can readily be made by high field
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NMR spectroscopy [9]. Nevertheless, PIP-Nusil implants are not easy to distinguish from other
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Nusil silicone based products. Furthermore, silicone gel contents of the implants may not fully
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explain the higher rupture rate. It is likely that the silicone-based envelope has an underlying
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cause in this.Therefore, we set out to determine chemical fingerprints of a series of implants,
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focusing on both gel and envelope using infrared, Raman and NMR spectroscopy as analytical tools.
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The results obtained were not compared to product dossiers or to essential requirements, therefore
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this research cannot be seen as a full inspection inquiry and non-conformities are not investigated.
63 2. Experimental
64 65
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2.1 Materials
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Mentor Medical Systems B.V. (Leiden, the Netherlands) and Allergan N.V. (Hoeilaart, Belgium),
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kindly provided prototypical breast implants of respectively the brands Mentor and Natrelle. A
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range of PIP implants and gel material of PIP-1 and Nusil Med 3-6300 were kindly provided by
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Läkemedelsverket (Medical Products Agency, Uppsala, Sweden). One M-implant of HansBiomed
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origin from Rofil Medical was provided by the Dutch Health Care Inspectorate (IGZ, the Hague, the
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Netherlands). Only unused implants were used in this study. D4 and D5 cyclosiloxanes were
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obtained from Sigma Aldrich (Zwijndrecht, the Netherlands). Technical grade dimethyl-, diphenyl-
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and fluoro-silicones were obtained from Applied Silicone (Santa Paula, CA, USA) and were
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subsequently prepared according to the manufacturers protocols.
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NIR measurements were performed using an Antaris II FT-NIR spectrometer and Result
software
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vs 3.0 (Thermo Scientific, Madison USA). An auxiliary transflection piece with 1.2 mm spacer was
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used to create films of equal size of the gels. Spectra were collected in the transflection mode,
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resolution 8 cm-1, spectral range 12000 – 3000 cm-1. Principle Component Analysis (PCA) was
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carried out on the first derivative of the spectra in the range of 8000 - 4000 cm-1 without additional
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spectral pre-treatments using TQ-Analyst software vs 8.4 (Thermo Scientific, Madison USA).
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2.3 Raman spectroscopy
85 86
A DXR Raman microscope (Thermo Scientific, Madison USA) was employed to record Raman
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spectra in area maps of a cross section of the envelope of an implant. Measurements were carried
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out using a 10x objective, a 780 nm laser with a laser power of 14 mW, a collection time of 10
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seconds and a slit width of 50 µm. Spectral range: 3400-50 cm-1, estimated resolution 4.7-8.7 cm-
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2.4 Nuclear Magnetic Resonance spectroscopy Silicone gels were extracted with d6DMSO (0.3-0.5 g in 1.0 mL) and CDCl3 (0.03-0.06 g in 1.0 mL)
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in glass tubes for 5 minutes, shaking at room temperature. The DMSO extracts were transferred to
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NMR tubes, CDCL3 dissolved the gels yielding slurries that were transferred as a whole into NMR
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tubes. 1H spectra were acquired at 14.1 T on a Bruker DMX 600 MHz spectrometer (Bruker,
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Wormer, the Netherlands) equipped with a TCI-Z-GRAD cryoprobe operating at 298 K, with 64
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scans, a spectral width of 12335 Hz and an acquisition time of 5.31 s. All samples were
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automatically tuned, matched and shimmed. Spectra were calibrated to the solvent peaks of CHCL3
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(7.2600 ppm) and DMSO (2.5000 ppm). Spectra were processed and analysed using Topspin 3.0
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software (Bruker, Wormer, the Netherlands).
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3. Results and discussion
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3.1 NIR spectroscopy
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To make a direct comparison between the various implant contents, these were subjected to NIR
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analysis. A PCA based on 91 spectra of a total of 8 samples (spectra of poor quality were omitted)
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of different gels revealed two main clusters, see Figure 1. Cluster 1 comprises spectra of reference
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Nusil gel and gel samples of PIP, Natrelle and Mentor implants. Cluster 2 comprises spectra of
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reference Applied Silicone gel, PIP-1 gel, and gel samples of M-implant and PIP implants. Thus, the
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PIP breast implants are found with at least two types of silicone gel. The gel of the PIP implant type
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IMGHC-TX is similar to that of PIP-1, whereas the gel of PIP implant type IMGHC-MX is more
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similar to Natrelle and Mentor. Furthermore, the M-implant is readily distinguished from the
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implants of Natrelle and Mentor. Spectroscopic bands that have a large contribution to the
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differences underlying the first principal component are those at 6064 cm-1 and 4652 cm-1 (data
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not shown). The band at 6064 cm-1 can be assigned to the first overtone of the unsaturated CH
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stretch vibration and the spectral band at 4652 cm-1 can be assigned to a combination band of C=C
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stretch and unsaturated CH stretch vibrations. The differences observed in the PCA are therefore
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possibly related to the vinylic components in the gels. With NIR spectroscopy, a direct comparison
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of types of implants is made.
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3.2 Raman spectroscopy
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For the examination of the implant envelopes, mapping of cross sections of the various envelope
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brands was performed using Raman spectroscopy. The chemigram of the Raman shift at 3050 cm-1
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(νaromatic CH) indicates that the envelope of the Natrelle and Mentor implants contain a barrier layer
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of phenyl-containing silicones (Figure 2A and 2B). A direct comparison of the Raman spectrum of
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reference diphenylsilicone with a spectrum of the Natrelle barrier layer confirms the layer to be a
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phenylsilicone (Figure 3A). The M-implant seems to contain a barrier layer of fluoro-containing
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silicone according to the chemigram of the Raman shift at 834 cm-1 (νSiF) (Figure 2C). The Raman
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spectrum of the barrier layer is subsequently compared with that of fluorosilicone (Figure 3B), and
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found to be highly similar. The cross section of the envelope of the PIP implant types IMGHC-TX
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and IMGHC-MX show no sign of a fluorosilicone or a phenylsilicone barrier layer (Figure 2D-G).
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Implant envelopes are made of silicone rubber, which is an elastomer with a filler. The exact
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composition and characteristics of the elastomer varies between the implants but mostly
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amorphous silica is used as the filler [14]. In the envelope, a barrier layer is introduced to lessen
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the diffusion of silicone fluid compounds into the surrounding tissues. Either one or two layers of
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diphenyl or other modified siloxanes, or a layer of fluorosilicone is interposed between the envelope
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and the gel contents. However, the presence of a barrier layer may have an effect on the strength
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of the elastomer. It has been reported that fluorosilicone barriers lose their effectiveness after two
139
or three years, presumably due to fracture of this weaker elastomer [15,16]. With Raman
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spectroscopy any barrier layers in the envelope are identified. Apparently, a barrier layer should be
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present in the silicone envelope of a good quality breast implant. The PIP implants investigated in
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this study are not manufactured with a barrier layer and the M-implant contains a fluorosilicone
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barrier layer. A diphenyl-based barrier layer was determined in the Mentor and Natrelle implant
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envelopes. Microscopically, several layers can be distinguished in the envelope of which only one
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contains the typical Raman signals of the phenyl group.
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3.3 NMR spectroscopy
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To study small molecule identifiers present in the implant gels, extracts were examined using NMR
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spectroscopy. Signals of vinylic protons were observed between 5.7 and 6.2 ppm in the 1H-
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spectrum of CDCl3 extracts of Natrelle and Mentor implant gels (Figure 4A). After curing of the
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silicone gel, unreacted vinyl containing silicones remain present, which are apparently small
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enough to be extracted. These vinyl proton signals were absent in PIP-1 gel material (not shown)
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and in the PIP implants of the type IMGHC-TX (Figure 4A). In the gel of PIP implant type IMGHC-
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MX however, vinylic protons were detected (Figure 4A). These findings are similar to the findings
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described in the MPA report [9], where vinylic protons were observed in PIP-Nusil gels but not in
5 Page 7 of 16
PIP-2 gels. On this basis, PIP type IMGHC-TX is tentatively assigned as PIP-2 and PIP type IMGHC-
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MX as PIP-Nusil. This assignment matches with the nomenclature of various gel formulations as
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obtained during inspection of the PIP company plant in 2010 [8]. As PIP1 and PIP2 implants are
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indicated to be inferior breast implants, made without a written production procedure [8], it seems
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that presence of the vinyl signals are a useful discriminant for good implant quality.
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Signals of vinyl groups were observed in a CDCl3 extract of the gel of an M-implant as well (Figure
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4A). The chemical shifts of the signals of the vinyl groups in the M-implant gel extract however
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differ from those observed in the other spectra. Apparently, their chemical environment is different
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which could be caused by a different source of the silicone used. The signals of the vinyl groups in
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the Applied Silicone technical grade bench-cured gels exactly matched those found in the M-
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implant sample (data not shown). This type of vinyl signals are reported to be of vinyl terminated
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silicones rather than mid-chain vinyl groups [11].
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Signals of cyclosiloxanes, specifically of D4, D5 and D6 are readily observed in DMSO extracts of
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silicone gels at 600 MHz, see Figure 4B. The cyclosiloxanes D4, D5 and D6 give single peaks at
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0.08, 0.07 and 0.06 ppm respectively. No or very small signals of D4 and D5 cyclosiloxanes were
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observed in the PIP-1 gel (not shown), and in the gels of the Natrelle implant and of the PIP
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implant type IMGHC-MX. Larger amounts of cyclosiloxanes were observed in PIP implants of the
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type IMGHC-TX and in the M-implant. Specifically the D4 content in the M-implant seems to be
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very high (Figure 4B). To small extent, cyclosiloxane D4 was present in the gel of a Mentor
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implant. For the PIP-1 type gel there was no implant available, only a sample of gel so it may be
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that cyclosiloxanes contents have decreased over time due to evaporation. Also in a bench-cured
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technical grade Applied Silicone gel, no cyclosiloxanes were observed (data not shown).
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With NMR spectroscopy the type of silicone and presence of contaminants are determined.
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Apparently, a good quality breast implant should display at least signals of residual vinyl groups
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present in the gel and contaminants in the form of cyclosiloxanes are tolerable at low levels only.
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In general, silicone based implants are made of medical grade silicones according to a protocol
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similar to as described in ISO 14949 [14,17]. A polymer consisting of vinyl containing silicones is
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added to a cross linker, consisting of silicon hydride containing silicones and cured in presence of
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an organometallic catalyst at elevated temperature. The catalyst typically contains platinum and as
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such, ethylene bridged elastomers are made. Unreacted vinyl groups containing silicone polymers
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remain present after curing, which can be extracted and detected by NMR spectroscopy. The
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absence of vinyl signals in the gel extracts of PIP implants indicates a deviation from the ISO
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14949 protocol. It seems that the vinyl containing silicone polymer was added in too little amount
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or not at all. Too short curing times, as suggested before [9], are ruled out by performing a lab
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bench curing experiment with shorter curing times, which still gave a vinyl signal in the NMR
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spectrum (data not shown). Beretta et al. also discussed PIP silicone gels to consist of a high
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percentage of free silicone oils, rather than a crosslinked cured matrix [12,13].
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Cyclosiloxanes D4, D5 and D6 (cyclic volatile siloxanes) are used as building blocks for silicone
194
polymers (ISO 14949). As these compounds are known to show some toxicity, they are washed out
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of medical grade silicones and this is the primary reason why medical grade silicones are required
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for use in implants. According to [8], a maximum level of 50 ppm D4, D5 or D6 is tolerable in
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silicone based implants. In this study we have not quantified the amounts of cyclosiloxanes present
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in the implants. By normalising on the signal at 0 ppm a qualitative comparison between the
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implants could nevertheless be made. An assay based on GC-MS has been developed by the former
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AFSSAPS, now ANSM, to quantify cyclosiloxanes. Alternatively, an estimation of cyclosiloxanes
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contents could be made using NMR spectroscopy with an internal standard.
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202 4. Conclusions
Using a variety of spectroscopic techniques we were able to distinguish breast implants of different
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origins, taking into account both silicone gel and envelope. Chemical hallmarks of a lower quality
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implant appear to be the absence of vinyl signals and the presence of high amounts of
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cyclosiloxanes in the gel and the absence of a barrier layer in the implant envelope. With the
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presented techniques, it is possible to screen series of breast implants in for instance a market
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surveillance study.
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Acknowledgement
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We thank Dr. Ian McEwen and Dr. Vendela Schnittger from the Läkemedelsverket, MPA, Uppsala,
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Sweden, for their generous gift of implant materials, the Leiden Institute of Chemistry for the use
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of the NMR spectrometer and Fons Lefeber and Dr. Karthick Babu Sai Sankar Gupta from the LIC
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for technical assistance.
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References
219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
[1] Committee on Silicone Implants, Health Risks of Silicone Breast Implants, The Hague, 1999. [2] T.F. Henriksen, L.R. Holmich, J.P. Fryzek, S. Friis, J.K. McLaughlin, A.P. Hoyer, K. Kjoller, J.H. Olsen, Incidence and severity of short-term complications after breast augmentation: results from a nationwide breast implant registry, Ann. Plast. Surg. 51 (2003) 531-539. [3] J.K. McLaughlin, L. Lipworth, D.K. Murphy, P.S. Walker, The safety of silicone gel-filled breast implants: a review of the epidemiologic evidence, Ann. Plast. Surg. 59 (2007) 569-580. [4] S.L. Brown, J.F. Todd, J.U. Cope, H.C. Sachs, Breast implant surveillance reports to the U.S. Food and Drug Administration: maternal-child health problems, J. Long Term Eff. Med. Implants 16 (2006) 281-290. [5] M.C. Maijers, C.J. de Blok, F.B. Niessen, A.A. van der Veldt, M.J. Ritt, H.A. Winters, M.H. Kramer, P.W. Nanayakkara, Women with silicone breast implants and unexplained systemic symptoms: a descriptive cohort study, Neth. J. Med. 71 (2013) 534-540. [6] ANSM, PIP Breast Implants - Situation update, ANSM, 2013, pp. 32. [7] M.G. Berry, J.J. Stanek, The PIP mammary prosthesis: a product recall study, J. Plast. Reconstr. Aesthet. Surg. 65 (2012) 697-704. [8] J.Y. Grall, D. Maraninchi, Etat des lieux de l'ensemble des controles effectues sur la societe Poly implants prothese (PIP), AFSSAPS, Paris, 2012, pp. 170. [9] L. MPA, PIP breast implants - Chemical analyses performed at the MPA (Medical Products Agency) NMR, GC and MALDI, Läkemedelsverket MPA, Uppsala, 2013, pp. 48. [10] G. Beretta, M. Malacco, Chemical and physicochemical properties of the high cohesive silicone gel from Poly Implant Prothese (PIP) breast prostheses after explantation: a preliminary, comparative analytical investigation, J. Pharm. Biomed. Anal. 78-79 (2013) 75-82. [11] A. Formes, B. Diehl, Investigation of the silicone structure in breast implants using H NMR, J. Pharm. Biomed. Anal. 93 (2014) 95-101. [12] G. Beretta, A. Richards, M. Malacco, Chemical and biochemical composition of late periprosthetic fluids from women after explantation of ruptured Poly Implant Prothese (PIP) breast prostheses, J. Pharm. Biomed. Anal. 84 (2013) 159-167. [13] G. Beretta, S. Panseri, A. Manzo, R. Hamid, A. Richards, M. Malacco, Analytical investigations on elastomeric shells of new Poly Implant Prothèse (PIP) breast and from sixteen cases of surgical explantation., J. Pharm. Biomed. Anal. 98 (2014) 144-152. [14] S. Bondurant, V. Ernster, R. Herdman, Safety of Silicone Breast Implants, National Academies Press, 1999. [15] W. Peters, D. Smith, S. Lugowski, Silicon capsule assays with low-bleed silicone gel implants, Plast. Reconstr. Surg. 97 (1996) 1311-1312. [16] L.T. Yu, G. Latorre, J. Marotta, C. Batich, N.S. Hardt, In vitro measurement of silicone bleed from breast implants, Plast. Reconstr. Surg. 97 (1996) 756-764. [17] N. Normalisatie-instituut, Chirurgische implantaten - Siliconenelastomeren - Siliconen elastomeren via een twee-componenten additiereactie (ISO 14949:2001,IDT), 2001.
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Tables and Figures
260 261
Table 1: Implants used and presence of qualitative markers
262 Cyclosiloxanes
Barrier layer type
-
+
None
-
+
None
+
-
+
++
PIP IMGHC-TX-H-230, Lot no. 36309 PIP IMGHC-TX-H-470, Lot no.
PIP IMGHC-MX-UH-535, Lot no. 27408 M-implant IMGHC-TX-H-225,
Natrelle N-27-FL100-140,
Phenyl
-
Phenyl
+
-
Phenyl
+
+/-
Phenyl
+
263 264 265
Ac
6743653
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Mentor Ref no. 334-1102, Lot no.
ed
Lot no. 2210920
6712360
Fluoro
-
M
+
2239790
Mentor Ref no. 354-3001, Lot no.
an
Lot no. 607L9 Natrelle N-TSF180, Lot no.
None
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29809
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Vinylic signals
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Implant
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266
Figure 1: Plot of the first three principal components of NIR spectra from silicone gels of five breast
267
implants and three silicone reference gels. The samples cluster in two groups. Cluster 1: Nusil Med
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3-6300 reference gel (red), PIP IMGHC-MX implant (green), Natrelle implant (light blue) and
269
Mentor implant (grey). Cluster 2: PIP-1 reference gel (dark blue), PIP IMGHC-TX implant
270
(magenta), M-implant (brown), Applied Silicone reference gel (dark green).
271 Figure 2: Pictures and chemigrams of envelope cross sections of the implants of Natrelle (A),
273
Mentor (B), M-implant (C), PIP IMGHC-MX (D and E), and PIP IMGHC-TX (F and G), determined
274
using a Raman microscope as described in the Methods section. The chemigrams of A, B, D and F
275
are recorded at 3050 cm-1, those of C, E and G at 834 cm-1.
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276
Figure 3: Raman spectra obtained from the mapping of the envelope of the Natrelle implant (A,
278
red) and a reference spectrum of diphenylsilicone (blue), and the envelope of the M-implant (B,
279
red) and a reference spectrum of fluorosilicone (cyan).
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M
280
Figure 4: 1H NMR spectra of the silicone gel extracts, focusing on the regions were vinyl and
282
cyclosiloxanes signals are expected. A: Spectra of CDCl3 extracts of Mentor (a), Natrelle (b), PIP
283
IMGHC-TX-H-470 (c), PIP IMGHC-MX-UH-535 (d) and M-implant (e) at the range 5.6 to 6.2 ppm.
284
B: Spectra of d6DMSO extracts of Mentor (a), Natrelle (b), PIP IMGHC-MX-UH-535 (c), PIP IMGHC-
285
TX-H-230 (d), M-implant (e), D4 standard (f) and D5 standard (g) at the range -0.1 to 0.2 ppm.
286
The spectra in B were normalized to the signal at 0.0 ppm. The box indicate the position of the
287
cyclosiloxanes shifts.
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Figure 2
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S0731-7085(14)00437-3 http://dx.doi.org/doi:10.1016/j.jpba.2014.09.008 PBA 9719
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
20-6-2014 5-9-2014 8-9-2014
Please cite this article as: P.H.J. Keizers, M.J. Vredenbregt, F. Bakker, D. de Kaste, B.J. Venhuis, Chemical fingerprinting of silicone-based breast implants, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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*Graphical Abstract
cr us an M
%Reflectance
20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3
6100
6050
6000 5950 5900 Wavenumbers (cm-1)
5850
5800
5750
6.15
6.10
6.05
6.00
5.95
Ac c
ep te
d
6150
5.90
5.85
0.18
5.80
0.16
5.75
0.14
5.70
0.12
5.65 ppm
0.10
0.08
0.06
0.04
0.02
0.00 −0.02 −0.04 −0.06 −0.08 ppm
Page 1 of 16
*Highlights (for review)
Highlights of “Chemical fingerprinting of silicone-based breast implants”, by Keizers et al.:
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Qualitative discriminants of silicone breast implants are described Breast implants are distinguished on brand and type Barrier layers in breast implant envelopes are visualized A set of complementary spectroscopic techniques is described for market surveillance of breast implants
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Chemical fingerprinting of silicone-based breast implants
2 3
Peter H. J. Keizers, Marjo J. Vredenbregt, Frank Bakker, Dries de Kaste, Bastiaan J. Venhuis*
4 National Institute for Public Health and the Environment, P. O. Box 1, 3721 MA, Bilthoven, the
6
Netherlands
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*Corresponding author. Tel.: +33 30274 4228. E-mail address: [email protected] (B. J.
9
Venhuis)
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10 11 Abstract
13
With millions of women worldwide carrying them, silicone-based breast implants represent a large
14
market. Even though silicone breast implants already have a history of use of more than fifty
15
years, the discussion on their safety has not yet come to an end. To improve safety assessment,
16
regulatory authorities should have the availability of a set of tests to be able to determine
17
parameters of implant identity and quality. Therefore, the gels and envelopes of various brands
18
and types of silicone-based breast implants have been subjected to infrared, Raman and NMR
19
spectroscopy. We show that by using a combination of complementary spectroscopic techniques
20
breast implants of various origins can be distinguished on typical chemical hallmarks. It was found
21
that typical silicone-based implants display a surplus of vinyl signals in the gel, cyclosiloxane
22
impurities are tolerable at low levels only and a barrier layer is present in the implant envelope.
23
The techniques presented here and the results obtained offer a good starting point for market
24
surveillance studies.
26
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Keywords: silicone implants, PIP, spectroscopy
27
1 Page 3 of 16
28
1. Introduction
29 Breast implants, or prostheses, find widespread application for cosmetic reasons or reconstruction
31
after tissue removal by surgery. Several types are available, of which the ones with a silicone-
32
based gel and envelope are the most commonly used. They represent a large market; it was
33
estimated in 1999 that 25.000 to 30.000 Dutch women carry silicone-based implants [1], and this
34
number is believed to have significantly increased ever since.
35
Silicone breast implants have been associated with adverse health effects, ranging from
36
inflammatory reactions to cancer and autoimmune syndrome induced by adjuvants [2,3,4,5]. One
37
explanation of these effects is thought to result from poor quality implants that have been
38
marketed over the years.
39
A notorious example in that sense is the PIP case [6,7]. The company Poly Implant Prothèse has
40
used industrial grade silicones in some of their implants produced from 2001 to 2010. In 2010, the
41
Dutch Health Care Inspectorate (IGZ) removed PIP implants from the Dutch market. PIP implants
42
have had a higher chance on complications resulting from rupture, leaking and sweating. Three
43
types of PIP implants have been produced over the years that are associated with the
44
complications; indicated as PIP-1, PIP-2 and PIP-Nusil. However, it remains unclear whether only a
45
few poor batches have been produced or if all produced PIP implants have been of questionable
46
quality [8].
47
At the National Institute for Public Health and the Environment (RIVM) we were requested by IGZ
48
to develop knowledge on the quality of breast implants used in the Netherlands. Specifically,
49
chemical hallmarks of breast implants should be identified to be able to distinguish implants,
50
preferentially on parameters related to quality. Eventually these results are to be used in market
51
surveillance activities.
52
Literature on the chemical analysis of silicon-based implants has become available in recent years
53
[9,10,11,12,13]. NMR spectroscopy, light microscopy infrared spectroscopy and GC-MS have
54
successfully been applied to study silicone-based implants. Specifically, the distinction between
55
PIP-1 and PIP-2 type implants compared to high-end products can readily be made by high field
56
NMR spectroscopy [9]. Nevertheless, PIP-Nusil implants are not easy to distinguish from other
57
Nusil silicone based products. Furthermore, silicone gel contents of the implants may not fully
58
explain the higher rupture rate. It is likely that the silicone-based envelope has an underlying
59
cause in this.Therefore, we set out to determine chemical fingerprints of a series of implants,
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60
focusing on both gel and envelope using infrared, Raman and NMR spectroscopy as analytical tools.
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The results obtained were not compared to product dossiers or to essential requirements, therefore
62
this research cannot be seen as a full inspection inquiry and non-conformities are not investigated.
63 2. Experimental
64 65
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2.1 Materials
66
Mentor Medical Systems B.V. (Leiden, the Netherlands) and Allergan N.V. (Hoeilaart, Belgium),
68
kindly provided prototypical breast implants of respectively the brands Mentor and Natrelle. A
69
range of PIP implants and gel material of PIP-1 and Nusil Med 3-6300 were kindly provided by
70
Läkemedelsverket (Medical Products Agency, Uppsala, Sweden). One M-implant of HansBiomed
71
origin from Rofil Medical was provided by the Dutch Health Care Inspectorate (IGZ, the Hague, the
72
Netherlands). Only unused implants were used in this study. D4 and D5 cyclosiloxanes were
73
obtained from Sigma Aldrich (Zwijndrecht, the Netherlands). Technical grade dimethyl-, diphenyl-
74
and fluoro-silicones were obtained from Applied Silicone (Santa Paula, CA, USA) and were
75
subsequently prepared according to the manufacturers protocols.
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NIR measurements were performed using an Antaris II FT-NIR spectrometer and Result
software
79
vs 3.0 (Thermo Scientific, Madison USA). An auxiliary transflection piece with 1.2 mm spacer was
80
used to create films of equal size of the gels. Spectra were collected in the transflection mode,
81
resolution 8 cm-1, spectral range 12000 – 3000 cm-1. Principle Component Analysis (PCA) was
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carried out on the first derivative of the spectra in the range of 8000 - 4000 cm-1 without additional
83
spectral pre-treatments using TQ-Analyst software vs 8.4 (Thermo Scientific, Madison USA).
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2.3 Raman spectroscopy
85 86
A DXR Raman microscope (Thermo Scientific, Madison USA) was employed to record Raman
87
spectra in area maps of a cross section of the envelope of an implant. Measurements were carried
88
out using a 10x objective, a 780 nm laser with a laser power of 14 mW, a collection time of 10
89
seconds and a slit width of 50 µm. Spectral range: 3400-50 cm-1, estimated resolution 4.7-8.7 cm-
90
1
.
91 3 Page 5 of 16
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2.4 Nuclear Magnetic Resonance spectroscopy Silicone gels were extracted with d6DMSO (0.3-0.5 g in 1.0 mL) and CDCl3 (0.03-0.06 g in 1.0 mL)
94
in glass tubes for 5 minutes, shaking at room temperature. The DMSO extracts were transferred to
95
NMR tubes, CDCL3 dissolved the gels yielding slurries that were transferred as a whole into NMR
96
tubes. 1H spectra were acquired at 14.1 T on a Bruker DMX 600 MHz spectrometer (Bruker,
97
Wormer, the Netherlands) equipped with a TCI-Z-GRAD cryoprobe operating at 298 K, with 64
98
scans, a spectral width of 12335 Hz and an acquisition time of 5.31 s. All samples were
99
automatically tuned, matched and shimmed. Spectra were calibrated to the solvent peaks of CHCL3
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(7.2600 ppm) and DMSO (2.5000 ppm). Spectra were processed and analysed using Topspin 3.0
101
software (Bruker, Wormer, the Netherlands).
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3. Results and discussion
104
3.1 NIR spectroscopy
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To make a direct comparison between the various implant contents, these were subjected to NIR
106
analysis. A PCA based on 91 spectra of a total of 8 samples (spectra of poor quality were omitted)
107
of different gels revealed two main clusters, see Figure 1. Cluster 1 comprises spectra of reference
108
Nusil gel and gel samples of PIP, Natrelle and Mentor implants. Cluster 2 comprises spectra of
109
reference Applied Silicone gel, PIP-1 gel, and gel samples of M-implant and PIP implants. Thus, the
110
PIP breast implants are found with at least two types of silicone gel. The gel of the PIP implant type
111
IMGHC-TX is similar to that of PIP-1, whereas the gel of PIP implant type IMGHC-MX is more
112
similar to Natrelle and Mentor. Furthermore, the M-implant is readily distinguished from the
113
implants of Natrelle and Mentor. Spectroscopic bands that have a large contribution to the
114
differences underlying the first principal component are those at 6064 cm-1 and 4652 cm-1 (data
115
not shown). The band at 6064 cm-1 can be assigned to the first overtone of the unsaturated CH
116
stretch vibration and the spectral band at 4652 cm-1 can be assigned to a combination band of C=C
117
stretch and unsaturated CH stretch vibrations. The differences observed in the PCA are therefore
118
possibly related to the vinylic components in the gels. With NIR spectroscopy, a direct comparison
119
of types of implants is made.
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120 121
3.2 Raman spectroscopy
122
For the examination of the implant envelopes, mapping of cross sections of the various envelope
123
brands was performed using Raman spectroscopy. The chemigram of the Raman shift at 3050 cm-1
4 Page 6 of 16
(νaromatic CH) indicates that the envelope of the Natrelle and Mentor implants contain a barrier layer
125
of phenyl-containing silicones (Figure 2A and 2B). A direct comparison of the Raman spectrum of
126
reference diphenylsilicone with a spectrum of the Natrelle barrier layer confirms the layer to be a
127
phenylsilicone (Figure 3A). The M-implant seems to contain a barrier layer of fluoro-containing
128
silicone according to the chemigram of the Raman shift at 834 cm-1 (νSiF) (Figure 2C). The Raman
129
spectrum of the barrier layer is subsequently compared with that of fluorosilicone (Figure 3B), and
130
found to be highly similar. The cross section of the envelope of the PIP implant types IMGHC-TX
131
and IMGHC-MX show no sign of a fluorosilicone or a phenylsilicone barrier layer (Figure 2D-G).
132
Implant envelopes are made of silicone rubber, which is an elastomer with a filler. The exact
133
composition and characteristics of the elastomer varies between the implants but mostly
134
amorphous silica is used as the filler [14]. In the envelope, a barrier layer is introduced to lessen
135
the diffusion of silicone fluid compounds into the surrounding tissues. Either one or two layers of
136
diphenyl or other modified siloxanes, or a layer of fluorosilicone is interposed between the envelope
137
and the gel contents. However, the presence of a barrier layer may have an effect on the strength
138
of the elastomer. It has been reported that fluorosilicone barriers lose their effectiveness after two
139
or three years, presumably due to fracture of this weaker elastomer [15,16]. With Raman
140
spectroscopy any barrier layers in the envelope are identified. Apparently, a barrier layer should be
141
present in the silicone envelope of a good quality breast implant. The PIP implants investigated in
142
this study are not manufactured with a barrier layer and the M-implant contains a fluorosilicone
143
barrier layer. A diphenyl-based barrier layer was determined in the Mentor and Natrelle implant
144
envelopes. Microscopically, several layers can be distinguished in the envelope of which only one
145
contains the typical Raman signals of the phenyl group.
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3.3 NMR spectroscopy
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To study small molecule identifiers present in the implant gels, extracts were examined using NMR
149
spectroscopy. Signals of vinylic protons were observed between 5.7 and 6.2 ppm in the 1H-
150
spectrum of CDCl3 extracts of Natrelle and Mentor implant gels (Figure 4A). After curing of the
151
silicone gel, unreacted vinyl containing silicones remain present, which are apparently small
152
enough to be extracted. These vinyl proton signals were absent in PIP-1 gel material (not shown)
153
and in the PIP implants of the type IMGHC-TX (Figure 4A). In the gel of PIP implant type IMGHC-
154
MX however, vinylic protons were detected (Figure 4A). These findings are similar to the findings
155
described in the MPA report [9], where vinylic protons were observed in PIP-Nusil gels but not in
5 Page 7 of 16
PIP-2 gels. On this basis, PIP type IMGHC-TX is tentatively assigned as PIP-2 and PIP type IMGHC-
157
MX as PIP-Nusil. This assignment matches with the nomenclature of various gel formulations as
158
obtained during inspection of the PIP company plant in 2010 [8]. As PIP1 and PIP2 implants are
159
indicated to be inferior breast implants, made without a written production procedure [8], it seems
160
that presence of the vinyl signals are a useful discriminant for good implant quality.
161
Signals of vinyl groups were observed in a CDCl3 extract of the gel of an M-implant as well (Figure
162
4A). The chemical shifts of the signals of the vinyl groups in the M-implant gel extract however
163
differ from those observed in the other spectra. Apparently, their chemical environment is different
164
which could be caused by a different source of the silicone used. The signals of the vinyl groups in
165
the Applied Silicone technical grade bench-cured gels exactly matched those found in the M-
166
implant sample (data not shown). This type of vinyl signals are reported to be of vinyl terminated
167
silicones rather than mid-chain vinyl groups [11].
168
Signals of cyclosiloxanes, specifically of D4, D5 and D6 are readily observed in DMSO extracts of
169
silicone gels at 600 MHz, see Figure 4B. The cyclosiloxanes D4, D5 and D6 give single peaks at
170
0.08, 0.07 and 0.06 ppm respectively. No or very small signals of D4 and D5 cyclosiloxanes were
171
observed in the PIP-1 gel (not shown), and in the gels of the Natrelle implant and of the PIP
172
implant type IMGHC-MX. Larger amounts of cyclosiloxanes were observed in PIP implants of the
173
type IMGHC-TX and in the M-implant. Specifically the D4 content in the M-implant seems to be
174
very high (Figure 4B). To small extent, cyclosiloxane D4 was present in the gel of a Mentor
175
implant. For the PIP-1 type gel there was no implant available, only a sample of gel so it may be
176
that cyclosiloxanes contents have decreased over time due to evaporation. Also in a bench-cured
177
technical grade Applied Silicone gel, no cyclosiloxanes were observed (data not shown).
178
With NMR spectroscopy the type of silicone and presence of contaminants are determined.
179
Apparently, a good quality breast implant should display at least signals of residual vinyl groups
180
present in the gel and contaminants in the form of cyclosiloxanes are tolerable at low levels only.
181
In general, silicone based implants are made of medical grade silicones according to a protocol
182
similar to as described in ISO 14949 [14,17]. A polymer consisting of vinyl containing silicones is
183
added to a cross linker, consisting of silicon hydride containing silicones and cured in presence of
184
an organometallic catalyst at elevated temperature. The catalyst typically contains platinum and as
185
such, ethylene bridged elastomers are made. Unreacted vinyl groups containing silicone polymers
186
remain present after curing, which can be extracted and detected by NMR spectroscopy. The
187
absence of vinyl signals in the gel extracts of PIP implants indicates a deviation from the ISO
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6 Page 8 of 16
14949 protocol. It seems that the vinyl containing silicone polymer was added in too little amount
189
or not at all. Too short curing times, as suggested before [9], are ruled out by performing a lab
190
bench curing experiment with shorter curing times, which still gave a vinyl signal in the NMR
191
spectrum (data not shown). Beretta et al. also discussed PIP silicone gels to consist of a high
192
percentage of free silicone oils, rather than a crosslinked cured matrix [12,13].
193
Cyclosiloxanes D4, D5 and D6 (cyclic volatile siloxanes) are used as building blocks for silicone
194
polymers (ISO 14949). As these compounds are known to show some toxicity, they are washed out
195
of medical grade silicones and this is the primary reason why medical grade silicones are required
196
for use in implants. According to [8], a maximum level of 50 ppm D4, D5 or D6 is tolerable in
197
silicone based implants. In this study we have not quantified the amounts of cyclosiloxanes present
198
in the implants. By normalising on the signal at 0 ppm a qualitative comparison between the
199
implants could nevertheless be made. An assay based on GC-MS has been developed by the former
200
AFSSAPS, now ANSM, to quantify cyclosiloxanes. Alternatively, an estimation of cyclosiloxanes
201
contents could be made using NMR spectroscopy with an internal standard.
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203
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202 4. Conclusions
Using a variety of spectroscopic techniques we were able to distinguish breast implants of different
205
origins, taking into account both silicone gel and envelope. Chemical hallmarks of a lower quality
206
implant appear to be the absence of vinyl signals and the presence of high amounts of
207
cyclosiloxanes in the gel and the absence of a barrier layer in the implant envelope. With the
208
presented techniques, it is possible to screen series of breast implants in for instance a market
209
surveillance study.
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Acknowledgement
212
We thank Dr. Ian McEwen and Dr. Vendela Schnittger from the Läkemedelsverket, MPA, Uppsala,
213
Sweden, for their generous gift of implant materials, the Leiden Institute of Chemistry for the use
214
of the NMR spectrometer and Fons Lefeber and Dr. Karthick Babu Sai Sankar Gupta from the LIC
215
for technical assistance.
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References
219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
[1] Committee on Silicone Implants, Health Risks of Silicone Breast Implants, The Hague, 1999. [2] T.F. Henriksen, L.R. Holmich, J.P. Fryzek, S. Friis, J.K. McLaughlin, A.P. Hoyer, K. Kjoller, J.H. Olsen, Incidence and severity of short-term complications after breast augmentation: results from a nationwide breast implant registry, Ann. Plast. Surg. 51 (2003) 531-539. [3] J.K. McLaughlin, L. Lipworth, D.K. Murphy, P.S. Walker, The safety of silicone gel-filled breast implants: a review of the epidemiologic evidence, Ann. Plast. Surg. 59 (2007) 569-580. [4] S.L. Brown, J.F. Todd, J.U. Cope, H.C. Sachs, Breast implant surveillance reports to the U.S. Food and Drug Administration: maternal-child health problems, J. Long Term Eff. Med. Implants 16 (2006) 281-290. [5] M.C. Maijers, C.J. de Blok, F.B. Niessen, A.A. van der Veldt, M.J. Ritt, H.A. Winters, M.H. Kramer, P.W. Nanayakkara, Women with silicone breast implants and unexplained systemic symptoms: a descriptive cohort study, Neth. J. Med. 71 (2013) 534-540. [6] ANSM, PIP Breast Implants - Situation update, ANSM, 2013, pp. 32. [7] M.G. Berry, J.J. Stanek, The PIP mammary prosthesis: a product recall study, J. Plast. Reconstr. Aesthet. Surg. 65 (2012) 697-704. [8] J.Y. Grall, D. Maraninchi, Etat des lieux de l'ensemble des controles effectues sur la societe Poly implants prothese (PIP), AFSSAPS, Paris, 2012, pp. 170. [9] L. MPA, PIP breast implants - Chemical analyses performed at the MPA (Medical Products Agency) NMR, GC and MALDI, Läkemedelsverket MPA, Uppsala, 2013, pp. 48. [10] G. Beretta, M. Malacco, Chemical and physicochemical properties of the high cohesive silicone gel from Poly Implant Prothese (PIP) breast prostheses after explantation: a preliminary, comparative analytical investigation, J. Pharm. Biomed. Anal. 78-79 (2013) 75-82. [11] A. Formes, B. Diehl, Investigation of the silicone structure in breast implants using H NMR, J. Pharm. Biomed. Anal. 93 (2014) 95-101. [12] G. Beretta, A. Richards, M. Malacco, Chemical and biochemical composition of late periprosthetic fluids from women after explantation of ruptured Poly Implant Prothese (PIP) breast prostheses, J. Pharm. Biomed. Anal. 84 (2013) 159-167. [13] G. Beretta, S. Panseri, A. Manzo, R. Hamid, A. Richards, M. Malacco, Analytical investigations on elastomeric shells of new Poly Implant Prothèse (PIP) breast and from sixteen cases of surgical explantation., J. Pharm. Biomed. Anal. 98 (2014) 144-152. [14] S. Bondurant, V. Ernster, R. Herdman, Safety of Silicone Breast Implants, National Academies Press, 1999. [15] W. Peters, D. Smith, S. Lugowski, Silicon capsule assays with low-bleed silicone gel implants, Plast. Reconstr. Surg. 97 (1996) 1311-1312. [16] L.T. Yu, G. Latorre, J. Marotta, C. Batich, N.S. Hardt, In vitro measurement of silicone bleed from breast implants, Plast. Reconstr. Surg. 97 (1996) 756-764. [17] N. Normalisatie-instituut, Chirurgische implantaten - Siliconenelastomeren - Siliconen elastomeren via een twee-componenten additiereactie (ISO 14949:2001,IDT), 2001.
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259
Tables and Figures
260 261
Table 1: Implants used and presence of qualitative markers
262 Cyclosiloxanes
Barrier layer type
-
+
None
-
+
None
+
-
+
++
PIP IMGHC-TX-H-230, Lot no. 36309 PIP IMGHC-TX-H-470, Lot no.
PIP IMGHC-MX-UH-535, Lot no. 27408 M-implant IMGHC-TX-H-225,
Natrelle N-27-FL100-140,
Phenyl
-
Phenyl
+
-
Phenyl
+
+/-
Phenyl
+
263 264 265
Ac
6743653
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Mentor Ref no. 334-1102, Lot no.
ed
Lot no. 2210920
6712360
Fluoro
-
M
+
2239790
Mentor Ref no. 354-3001, Lot no.
an
Lot no. 607L9 Natrelle N-TSF180, Lot no.
None
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Vinylic signals
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Implant
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266
Figure 1: Plot of the first three principal components of NIR spectra from silicone gels of five breast
267
implants and three silicone reference gels. The samples cluster in two groups. Cluster 1: Nusil Med
268
3-6300 reference gel (red), PIP IMGHC-MX implant (green), Natrelle implant (light blue) and
269
Mentor implant (grey). Cluster 2: PIP-1 reference gel (dark blue), PIP IMGHC-TX implant
270
(magenta), M-implant (brown), Applied Silicone reference gel (dark green).
271 Figure 2: Pictures and chemigrams of envelope cross sections of the implants of Natrelle (A),
273
Mentor (B), M-implant (C), PIP IMGHC-MX (D and E), and PIP IMGHC-TX (F and G), determined
274
using a Raman microscope as described in the Methods section. The chemigrams of A, B, D and F
275
are recorded at 3050 cm-1, those of C, E and G at 834 cm-1.
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276
Figure 3: Raman spectra obtained from the mapping of the envelope of the Natrelle implant (A,
278
red) and a reference spectrum of diphenylsilicone (blue), and the envelope of the M-implant (B,
279
red) and a reference spectrum of fluorosilicone (cyan).
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277
M
280
Figure 4: 1H NMR spectra of the silicone gel extracts, focusing on the regions were vinyl and
282
cyclosiloxanes signals are expected. A: Spectra of CDCl3 extracts of Mentor (a), Natrelle (b), PIP
283
IMGHC-TX-H-470 (c), PIP IMGHC-MX-UH-535 (d) and M-implant (e) at the range 5.6 to 6.2 ppm.
284
B: Spectra of d6DMSO extracts of Mentor (a), Natrelle (b), PIP IMGHC-MX-UH-535 (c), PIP IMGHC-
285
TX-H-230 (d), M-implant (e), D4 standard (f) and D5 standard (g) at the range -0.1 to 0.2 ppm.
286
The spectra in B were normalized to the signal at 0.0 ppm. The box indicate the position of the
287
cyclosiloxanes shifts.
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289
Figure 1
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293 294 295
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296
Figure 2
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Figure 3
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300 301 302 303 13 Page 15 of 16
Figure 4
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