In vitro cytotoxic effects of DEHP-alternative plasticizers and their primary metabolites on a L929 cell line

Article abstract of DOI:10.1016/j.chemosphere.2017.01.026

Phthalic acid esters have been widely used to improve the plasticity of PVC medical devices. They carry a high exposure risk for both humans and the environment in clinical situations. Our study focuses on the cytotoxicity of alternative plasticizers. Postulated primary metabolites were synthesized, not being commercially available. Cytotoxicity assays were performed on L929 murine cells according to the ISO-EN 10993-5 standard design for the biocompatibility of medical devices. The tested concentrations of plasticizers (0.01, 0.05 and 0.1 mg/ml) covered the range likely to be found in biological fluids coming into direct contact with the medical devices. DEHP, DINP and DINCH were cytotoxic at the highest concentration (0.1 mg/ml) for 7 days of exposure. Their corresponding metabolites were found to be more cytotoxic, for the same concentration. By contrast, TOTM and its corresponding metabolite MOTM were not found to be cytotoxic. DEHA showed no cytotoxicity, but its corresponding monoester (MEHA) produced a cytotoxic effect at 0.05 mg/ml. In clinical situations, medical devices can release plasticizers, which can come into contact with patients. In vivo, the plasticizers are quickly transformed into primary metabolites. It is therefore important to measure the effects of both the plasticizers and their corresponding metabolites. Standard first-line cytotoxicity assays should be performed to ensure biocompatibility.

Full text of DOI:10.1016/j.chemosphere.2017.01.026

Accepted Manuscript
In vitro cytotoxic effects of DEHP-alternative plasticizers and their primary metabolites
on a L929 cell line
Teuta Eljezi, Pierre Pinta, Damien Richard, Jérémy Pinguet, Jean-Michel Chezal,
Marie-Christine Chagnon, Valérie Sautou, Gaël Grimandi, Emmanuel Moreau
PII:
S0045-6535(17)30029-2
10.1016/j.chemosphere.2017.01.026
CHEM 18638
DOI:
Reference:
To appear in: ECSN
Received Date: 4 July 2016
Revised Date: 3 December 2016
Accepted Date: 4 January 2017
Please cite this article as: Eljezi, T., Pinta, P., Richard, D., Pinguet, J., Chezal, J.-M., Chagnon, M.-C.,
Sautou, V., Grimandi, G., Moreau, E., In vitro cytotoxic effects of DEHP-alternative plasticizers and their
primary metabolites on a L929 cell line, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.01.026.
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ACCEPTED MANUSCRIPT
Graphical abstract
Significant decrease of
L929 cells proliferation
Non Significant decrease of
L929 cells proliferation
°DEHT
°DEHA
°TOTM
DINCH
DEHP
DINP
Legend
°DEHP ‰MEHP
°DEHT ‰MEHT
°DEHA ‰MEHA
0.01
0.01
0.05
0.05
0.1
Plasticizers
Tested C°
(mg/mL)
L929 cells
°DINP ‰MINP,
MMeOP
Primaries
metabolites
0.1
°DINCH ‰MINCH,
MMeOCH
MEHT
MEHT
°TOTM ‰MOTM
MEHP
MINP
MEHA
MEHA
MINCH, MMeOCH
MOTM,
MMeOP

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In vitro cytotoxic effects of DEHP-alternative plasticizers and their primary metabolites
on a L929 cell line
b,
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Teuta Eljezi,a,
Pierre Pinta,c, Damien Richard,d Jérémy Pinguet,d Jean-Michel
f
Chezal,a Marie-Christine Chagnon,e Valérie Sautou,b, Gaël Grimandi,c Emmanuel
Moreau,a, *
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a Clermont Université, Université d'Auvergne, Imagerie Moléculaire et Thérapie Vectorisée, BP 184, F-
63005 Clermont-Ferrand, France. Inserm, U 990, F-63000 Clermont-Ferrand, France. Centre Jean
Perrin, F-63011 Clermont-Ferrand, France.
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b CHU Clermont-Ferrand, Pôle Pharmacie, rue Montalembert, 63003 Clermont-Ferrand, France
c INSERM, U791, LIOAD, Nantes, F-44042, France Université de Nantes
d Service de Pharmacologie, Hôpital Gabriel-Montpied, CHU de Clermont-Ferrand, Clermont-Ferrand,
France
eNutrition Physiology and Toxicology Team (NUTox), UMR U866 INSERM, Université de Bourgogne
Franche-Comté, AgroSup Dijon, 1 esplanade Erasme, 21000 Dijon, France.
f
Clermont Université, Université d'Auvergne, EA 4676C-BIOSENSS, 63000 Clermont-Ferrand,
France.
* corresponding author: emmanuel.moreau@udamail.fr
1 both authors contributed equally
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Abstract
Phthalic acid esters have been widely used to improve the plasticity of PVC medical
devices. They carry a high exposure risk for both humans and the environment in
clinical situations. Our study focuses on the cytotoxicity of alternative plasticizers.
Postulated primary metabolites were synthesized, not being commercially available.
Cytotoxicity assays were performed on L929 murine cells according to the ISO-EN
10993-5 standard design for the biocompatibility of medical devices. The tested
concentrations of plasticizers (0.01, 0.05 and 0.1 mg/ml) covered the range likely to
be found in biological fluids coming into direct contact with the medical devices.
DEHP, DINP and DINCH were cytotoxic at the highest concentration (0.1 mg/ml) for 7
days of exposure. Their corresponding metabolites were found to be more cytotoxic,
for the same concentration. By contrast, TOTM and its corresponding metabolite
MOTM were not found to be cytotoxic. DEHA showed no cytotoxicity, but its
corresponding monoester (MEHA) produced a cytotoxic effect at 0.05 mg/ml. In
clinical situations, medical devices can release plasticizers, which can come into
contact with patients. In vivo, the plasticizers are quickly transformed into primary
metabolites. It is therefore important to measure the effects of both the plasticizers
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and their corresponding metabolites. Standard first-line cytotoxicity assays should be
performed to ensure biocompatibility.
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Keywords
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Plasticizers, metabolite synthesis; cytotoxicity, DEHP-alternative plasticizers, phthalates
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Abbreviations
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ARMED®, Assessment and Risk Management of Medical Devices in Plasticized
Polyvinylchloride; DEHA, diethylhexyladipate; DEHP, diethylhexyl phthalate; DEHT, di-
ethylhexylterephthalate; DINCH, diisononyl cyclohexane-1,2-dicarboxylate; DINP, di-
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isononylphthalate;
MEHP,
monoethylhexyl
phthalate;
MMeOP,
mono(4-
methyloctyl)phthalate; MMeOCH, mono(4-methyloctyl) cyclohexane-1,2-dicarboxylate;
MEHT, monoethylhexylterephthalate; MINCH, monoisononyl cyclohexane-1,2-dicarboxylate;
MINP, monoisononylphthalate; MOTM, monooctyltrimellitate; SCENIHR, Scientific
Committee on Emerging and Newly-Identified Health Risks; TOTM, trioctyltrimellitate.
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1. Introduction
PVC (polyvinyl chloride) is widely used in medical devices, in particular to produce flexible
tubing such as in infusion sets or extracorporeal circulation lines. To confer and maintain
flexibility, plasticizers are added to the PVC matrix. However, it is now generally agreed that
plasticizers can migrate from the PVC matrix into infused drug solutions or biological fluids,
and thereby come into contact with the patient. Some of these chemicals are likely to be
hazardous for patients, as demonstrated for diethylhexyl phthalate (DEHP), now classified as
CMR 1B (carcinogenic, mutagenic or toxic to reproduction) under the CLP Regulation
(European Union, R 2008). It is also well known that the monoethylphthalate MEHP,
produced in vivo by enzymatic hydrolysis of the plasticizer, is even more toxic than the
plasticizer itself (ECB, 2004; CRHER, 2005; SCENIHR, 2015). The use of DEHP in PVC
medical devices was therefore challenged by the European authorities (European Union, R
2007), and in 2012 a French law banned the use of DEHP in plasticized PVC medical tubing
in neonatology and maternity services as from July 1, 2015 (French law, 2012). Supported by
physical and chemical properties, and some animal toxicology data, manufacturers turned to
trioctyltrimellitate (TOTM), di-ethylhexylterephthalate (DEHT), diisononyl cyclohexane-1,2-
dicarboxylate (DINCH), di-isononylphthalate (DINP) and diethylhexyladipate (DEHA), to
replace DEHP in PVC medical devices (Figure 1). However, data on the migration of these
additives from medical devices, and on their potential human toxicity are still insufficient.
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To be marketed, medical devices have to meet certain essential requirements. These mainly
concern their performance, safety, toxicity and biocompatibility. The last three properties
concern interactions between tissues in contact with the materials, but other tissues may also
be affected. The evaluation of a device has to take into account not only the substances
released from it, but also those derived from their degradation. Lastly, the duration of contact
with the body must be considered. To study toxicity and biocompatibility, in vivo and in vitro
tests are proposed in the standard EN ISO 1993. Devices have to undergo tests prescribed
according to the level of risk to which patients are exposed. The cytotoxicity test described in
EN ISO 10993-5 is mandatory, regardless of the medical device: the authors of the standard
consider that the toxicity assessment of a medical device, material or additive on a cell model
can be predictive of in vivo toxicity. An apparent toxicity in this model does not necessarily
demand rejection of the product concerned, but calls for further investigation. Recommended
tests depend on the final use of the medical device. For example, an infusion set will not
need to meet the same requirements as a central catheter coming directly into contact with
blood. Manufacturers mostly test the medical device itself and not the separate materials
used in its composition. Secondary materials can include bonding substances and
accessories such as fittings. Most often no data is available on the additives used for either
the main material or secondary ones. Many additives serve to facilitate the manufacture or
use of the medical device: for example, manufacturers often use plasticizers, lubricants or
dyes. The biocompatibility of these additives, which affect the ergonomic properties of
medical devices, is poorly evaluated. We studied the cytotoxicity of plasticizers added to
PVC medical devices using the MTT test, and the cell line (L929 cell line) described in EN 10
993-5. The concentrations studied were those found in biological fluids during direct contact
with the medical devices (Takahashi et al., 2008; Kambia et al., 2011; Scenirh 2015; Eckert
et al., 2016)
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Information is lacking on the toxicity of the primary metabolites of DEHP-alternative
plasticizers. The aim of our study was to assess the toxicity of several such alternative
plasticizers and their primary metabolites, with cytotoxicity assays performed according to
the EN 10993-5 standard using the MTT assay. The primary metabolites of TOTM, DEHT,
DEHA, DINCH, DINP and DEHA are not commercially available, so had to be chemically
synthesized. The production of pure metabolites enabled us to study their respective
cytoxicities, and to compare them with that of MEHP in the same conditions.
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Figure 1. Plasticizers most often used in medical devices and their corresponding primary
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metabolites
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2. Materials and methods
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2.1.
Chemicals, biochemical and reagents
Unless otherwise stated, all manipulations were performed under argon; all reagents were
purchased from the following commercial suppliers: Sigma-Aldrich, Acros Organics, Carlo
Erba, TCI Europa, and Alpha Aesar. Anhydrous DMF and anhydrous triethylamine were
purchased from Acros Organics. THF was distilled over benzophenone and sodium.
Dichloromethane was distilled over calcium hydride. Nuclear magnetic resonance (NMR)
spectra were acquired on a Bruker AC-200 instrument operating at 200 MHz and 50 MHz for
1H NMR and 13C NMR, respectively. All 1H NMR spectral peaks are reported in δ units, parts
per million (ppm), and the coupling constants are indicated in hertz (Hz). The following
abbreviations are used for spin multiplicity: s = singlet, d = doublet, t = triplet, q = quadruplet,
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m = multiplet, and br = broad. The chromatographic system consisted of an HTC PAL
autosampler, and a Transcend TLX-1 HTLC System (Thermo Fisher Scientific, San Jose,
United States), The HRMS analysis was performed using a Thermo Exactive benchtop
Orbitrap® instrument. TLC was performed on pre-coated silica gel sheets (POLYGRAM®
60F254 plates) and visualized under UV light (254 nm). Column chromatography was
performed using silica gel normal phase (35–70 µm). Uncorrected melting points (Mp) were
recorded on an Electrothermal IA9300 apparatus. Infrared spectra (IR) were recorded on a
Bruker FT Vector 22 instrument.
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DEHP (Ref: D201154, CAS: 117-81-7), TOTM (ref: 538140, CAS: 3319-31-1), DEHA (ref:
524197 CAS: 103-23-1), DINP (ref: 376663, CAS: 28553-12-0), and DEHT (ref: 525189,
CAS: 6422-56-2) were purchased from Sigma Aldrich, France. DINCH (CAS: 166-412-78-8)
was supplied by BASF, France. The primary metabolites MEHP, MEHT, MINP, MINCH,
MEHA, MMeOP, MMeOCH and MOTM were synthesized and characterized by the UMR 990
team, Clermont-Ferrand, France.
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Dimethylsulfoxide DMSO (ref: D8418 CAS: 67-68-5), Mouse fibroblasts L929 (ref: 85011425
lot: 10L019), MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, ref:
M2128) were also purchased from Sigma Aldrich. Dulbecco’s modified Eagle Medium
(DMEM) (fetal calf serum (SVF), phosphate saline buffer (PBS), penicillin (P), streptomycin
(S), and actinomycin D were purchased from Life technologies SAS, France.
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2.2.
Methods for the synthesis of primary metabolites
Metabolites MEHP (3), MOTM (5), MEHA (8), MINCH (19), MINP (21), MeMINCH (30) and
MeMINP (31) were synthesized from the appropriate anhydrides (1a, b), (7), (18) or (19) and
the appropriate alcohol derivatives 2-ethylhexanol (2), 7-methyloctanol (17) or 4-
methyloctanol (29) as previously described (F. Nüti et al., 2005). Scheme 1S, 2S, 3S, 5S and
6S depict the synthesis of the primary metabolites listed above (See Supporting Information).
Briefly, to a solution of the appropriate anhydride (1a, b), (7), (18) or (19) (1 eq.) dissolved in
dry pyridine, was added 2-ethylhexanol (2), 7-methyloctanol (17) or 4-methyloctanol (29)
(1 eq.). The resulting mixture was stirred at 125 °C (external temperature) until the anhydride
derivative had disappeared (TLC monitoring). The mixture was quenched with cold water
(50 mL) and extracted with diethyl ether (8 × 40 mL). The combined organic layer was
washed with a 10% solution of hydrochloric acid (100 mL). Finally, the mixture was extracted
with 0.4 M K2CO3 (100 mL). The aqueous basic layer was acidified to pH 1 with a solution of
hydrochloric acid (1M) and then extracted with Et2O (4 × 100 mL). The combined organic
layer was washed with brine (150 mL), dried over MgSO4 and evaporated under reduced
pressure.
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The metabolite MEHT (12) was obtained by esterification of the terephthalate derivative (11)
and 2-ethylhexanol (2) (See Supporting Information, scheme 4S).
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2.3.
Biocompatibility assays
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2.3.1. Solubilization of the plasticizers
All the plasticizers and their metabolites were solubilized in DMSO (stock solutions) at 1%
concentration (10 mg/mL). The final concentrations of the respective stock solutions were
checked by GC-MS (Supporting Information, Table S1). Work solutions of each plasticizer
were obtained by dilution of the stock solutions to the concentrations 0.01 mg/ml (0.1%);
0.05 mg/ml (0.5%) and 0.1 mg/ml (1%) in the culture medium (DMEM).
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2.3.2. Cells cultures
L929 cells were cultured in 96-well plates (without plasticizers) with DMEM supplemented
with 10% of SVF, 100 IU/mL of penicillin and 100 µg/mL of streptomycin (complete DMEM)
at 37°C with 5% of CO2.
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2.3.3. MTT assay procedure
The MTT reagent was used at a concentration of 5 mg/mL in PBS. At the end of the cell
cultures, the media were removed, and 30 µL of the MTT reagent was added to each well.
Cells were then incubated for 3 h at 37 °C with 5% of CO2. The MTT reagent was removed,
and 200 µL of dimethyl sulfoxide was added to each well (multiwell plates without
plasticizers). After 1 h of formazan dissolution, the optical density (OD) of each well was
measured at 570 nm (spectrophotometer VICTORTM Multilabel HTS Counter PerkinElmer).
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2.3.4. Specificity of the MTT assay
L929 cells were seeded in triplicate at 104 cells/well. After 24 h, the medium was replaced by
200 µL of the complete DMEM (control) or 200 µL of the complete DMEM supplemented with
0.1% of actinomycin D. Cells were then cultured for 5 days with a medium change every day.
MTT assays were performed every day for each condition. Cell viability percentage was
calculated using the formula: ((OD test − OD control) ⁄ OD control).
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2.3.5. Linearity of the MTT assay
L929 cells were seeded in triplicate at increasing numbers of cells per well (5×103 - 10×103 -
50×103 - 100×103 - 150×103 - 200×103 - 250×103 - 300×103 - 350×103) in 96-well plates.
After 8 h of adherence, the MTT assay was performed as described above.
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2.3.6. Viability assay for the DMSO solvent
According to the work plasticizer solutions, L929 cells were cultured in complete DMEM
supplemented with 0.1%, 0.5%, 1%, 2.5% and 5% of DMSO. Cells were seeded in triplicate
at 104 cells/well. After 24 h, the medium was replaced every day by 200 µL of the complete
DMEM for the control and 200 µL of the complete DMEM supplemented with DMSO at the
selected concentrations. Cells were cultured for 7 days. An MTT assay was performed every
day according to the MTT assay procedure.
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2.3.7. Viability assays for the plasticizers
For the viability assay of the plasticizers, cells were seeded in triplicate at 104 cells/well. After
24 h, the media were replaced by 200 µL of the followings solutions: (i) control conditions
with complete medium, (ii) control DMSO conditions with the complete medium
supplemented with the same concentration of DMSO as the plasticizer work solutions, and
(iii) work solutions (0.1%, 0.5 % and 1%) of the plasticizers. Cells were cultured for 7 days
with a medium change every day. MTT assay and quantification of the viability percentage
were carried out every day.
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2.3.8. Statistical analysis
The experiments performed in triplicate were repeated at least twice, and the outcomes of
one representative data set of each plasticizer are reported. Differences in viability
percentages between the control conditions and the test conditions were analyzed using the
Mann-Whitney non-parametric test. A p value less than 0.05 was considered significant.
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3. Results
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3.1.
Synthesis of metabolites
Primary metabolites MEHP (3) (F. Nüti et al., 2005), MOTM (5), MEHA (8), MINCH (19),
MINP (21), MMeOCH (30) and MMeOP (31) were synthesized by esterification of the
appropriate anhydride ((1a, b), (7), (18) and (20)) by the appropriate alcohol: 2-ethylhexanol
(2), 7-methyloctanol (17) or 4-methyloctanol (29) (Figure 2) as previously described (Nüti et
al., 2005). For MOTM, a condensation of anhydride (1b) and (2) yielded a mixture of two
isomers, which was not separated. MEHA (8) was straightforwardly obtained from anhydride
(7) (Cisneros, 2012) and alcohol (2) with good yield. We also synthesized the MEHT
metabolite. Starting material terephthalic acid (9), after protection with benzyl bromide, then
treatment with thionyl chloride followed by an esterification with (2), and a final catalytic
hydrogenation, afforded MEHT (12).
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Figure 2. Access to MEHT, MEHA, MOTM, MINCH, MINP, MMeOCH and MMeOP from 2-
ethylhexanol (2), 7-methyloctanol (17) or 4-methyloctanol (29)
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MINCH and MINP were obtained by condensation of 7-methyloctan-1-ol (17) and dihydro-
2H-pyran-2,6-(3H)-dione (18) or hexahydroisobenzofuran-1,3-dione (20), respectively.
Compound 7-methyloctan-1-ol (17) was commercialized, but was very costly. We therefore
synthesized it in four steps as described in Scheme 5S. MMeOCH and MMeOP were
obtained by a similar procedure with 4-methyloctan-1-ol (29) (Scheme 6S). All intermediates
and final compounds were analyzed by IR, 1H and 13C NMR, and HRMS. For all details, see
Supporting Information. The purity of all our synthesized metabolites and their corresponding
intermediates exceeded 95%.
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Biocompatibility assay
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3.2.1. Specificity of MTT assay
Figure 3 shows the optical density obtained after MTT assay on L929 cells cultured in
complete medium with and without actinomycin D for 5 days. The optical density of control
wells gradually increased from D1 to D5; L929 cells proliferated normally. The optical density
in the wells cultured in the presence of actinomycin D fell significantly from D1 compared with
the control, and tended to zero at D3. The apoptosis-inducting effect of actinomycin D (Kleeff
et al., 2000) time-dependently caused cell death, which was close to 100% at D3 in the MTT
assay.
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Figure 3. Specificity of the MTT assay. Optical density of the control wells vs. the optical
density of the actinomycin D wells. L929 cells were seeded at 104 cells/well, and cultured in
Complete DMEM with and without actinomycin D (0.1%) for 5 days. MTT assays were
carried out every day for each condition. *p < 0.05 control vs. test (DMEM + %DMSO)
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3.2.2. Linearity of MTT assay
Figure 4 shows the linearity of the MTT assay. A linear correlation (r2 = 0.9812) was found
between the number of seeded cells and the optical density obtained at the end of the MTT
assay after 8 h of adhesion.
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Figure 4. Linearity of MTT assay. L929 cells were seeded at increasing numbers of cells per
well (10×103 - 50×103 - 100×103 - 150×103 - 200×103 - 250×103 - 300×103 - 350×103) in
complete medium. After 8 h of adherence, the MTT assay was performed for each well.
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3.2.3. DMSO solvent effect on cell viability
Figure 5 shows the proliferation of L929 cells cultured in complete medium with and without
DMSO (0.1%, 0.5%, 1%, 2.5% and 5%) for 7 days. There was no significant difference in cell
proliferation between control (DMEM) and the cells cultured in DMEM supplemented with
0.1% and 0.5% of DMSO. Cells cultured in DMEM supplemented with 1% of DMSO
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proliferated for the 7 days of culture, but their proliferation was significantly lower than that of
the control. Beyond 1% of DMSO, L929 cells did not proliferate.
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Figure 5. Viability assays of the solvent DMSO. L929 cells were seeded at 104 cells/well, and
cultured in complete medium with and without DMSO (0.1%, 0.5%, 1%, 2.5% and 5%) for 7
days. MTT assay was carried out every day. *p < 0.05 control vs. test (DMEM + %DMSO)
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3.2.4. Viability of cells when exposed to the plasticizers and their primary
metabolites
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Figure 6 shows the results of viability tests, at day 7 (D7), performed on L929 cells cultured
in the presence of three concentrations of plasticizers after 7 days of contact. Plasticizers
were not toxic to the cells at the concentration of 0.01 mg/ml compared with the control
containing DMSO at the same concentration. A decrease in cell viability was observed for
DEHP and DINCH from the concentration of 0.05 mg/mL. For these two plasticizers, this
decrease in viability was higher than 50%. DINP reduced cell proliferation from a
concentration of 0.1 mg/mL (40%). For the other plasticizers (TOTM, DEHA and DEHT),
there were no significant differences compared with DMSO at any of the three concentrations
tested.
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Figure 6. Cytotoxicity test of plasticizers at 0.01 mg/mL (a), 0.05 mg/mL and 0.1 mg/mL, day
7. Outcomes of the cell viability analysis using MTT assay. L929 cells were seeded at 104
cells/well and cultured for 7 days in three conditions with medium change every day: control
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(complete medium), control DMSO (complete medium supplemented with DMSO 0.1%, 0.5%
and 1%) and test (work solutions of plasticizer in complete medium at 0.1% (0.01 mg/mL),
0.5% (0.05 mg/mL) and 1% (0.1 mg/mL). MTT assay and quantification of the viability
percentage were carried out every day. *p < 0.05 DMSO control vs. Test.
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Figure 7 depicts the results of the viability tests on primary metabolites of the plasticizers.
From 0.01 mg/mL, only MEHP had an impact on cell growth. MEHP expressed a cytotoxicity
of 50% and 70% respectively at 0.05 mg/mL and 0.1 mg/ml. Compared with MEHP, the
effect on the L929 proliferation was different for all the other primary metabolites studied
except for MINCH and MEHT: at 0.05 mg/ml, these latter were as cytotoxic as MEHP (about
50% cell death). From 0.1 mg/ml, they were much more cytotoxic than MEHP (98% and 99%
of cell death observed for MEHT and MINCH, respectively versus 70% for MEHP). For
MMeOCH, a structural isomer of MINCH, results were close to those obtained for MINCH
from 0.05 to 0.1 mg/mL. By contrast, the MMeOP, a structural isomer of MINP, never
significantly decreased cell viability at any of the concentrations tested. For MOTM, no
significant decrease in proliferation was observed compared with the DMSO control at any of
the concentrations tested. MEHA was weakly toxic, its cytotoxicity lying in the range 20–30%
at 0.05 and 0.1 mg/mL, respectively.
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Figure 7. Cytotoxicity test of primary metabolites at 0.01 mg/mL (a), at 0.05 mg/mL and at
0.1 mg/mL, day 7. Outcomes of the cell viability analysis using MTT assay. L929 cells were
seeded at 104 cells/well and cultured for 7 days in three conditions with medium change
every day: control (complete medium), control DMSO (complete medium supplemented with
DMSO 0.1%, 0.5% and 1%) and test (work solutions of plasticizer in complete medium at
0.1% (0.01 mg/mL), 0.5% (0.05mg/mL) and 1% (0.1 mg/mL). MTT assay and quantification
of the viability percentage were carried out every day. *p < 0.05 DMSO control vs. Test
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In summary, we observed that MOTM and MMeoP were not cytotoxic at any of the
concentrations tested. MEHA was weakly cytotoxic. MEHP, MEHT, MINP, MINCH and
MMeOCH were cytotoxic with a concentration effect.
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4. Discussion
Since the plasticizer DEHP was classified as CMR 1B, manufacturers have used various
alternative plasticizers with high molecular weights or different solubility properties, such as
other phthalates, trimellitates, citrates, alkyl sulfonic phenyl esters (ASEs), C10-21-alkanes,
acetylated monoglycerides of hydrogenated castor oil (COMGHAs), or acetates. In PVC
medical devices, the plasticizers most commonly added are TOTM and DINCH, but DINP
and DEHA are also frequently found. DEHT is seldom used in MDs as the main plasticizer,
but it is also a contaminant of TOTM (Gimeno et al., 2014, Bourdeaux et al., 2016). Here we
focus on plasticizers commonly used in medical devices. Special attention was of course
paid to all phthalate substitutes because of their endocrine-disrupting and reprotoxic
properties. Except for TOTM, there are many studies on alternative PVC plasticizers used by
manufacturers. For DEHA, carcinogenic properties were observed toward female B6C3F1,
and a disturbance of the estrous cycle and increased ovarian follicle atresia were detected in
rats (Miyata et al., 2006). For DEHT, no effects were reported on reproductive tissues,
organs, kidneys, liver hepatocytes or peroxisomes, which are known targets of DEHP-toxicity
(Wirnitzer et al., 2011; Deyo, 2008). DINP and DINCH have been studied more thoroughly.
Levels of DINP exposure are far below those that have no observed adverse effects in
animals, and also below health-based exposure guidance values set by regulatory authorities
and other authoritative bodies as acceptable (Borch et al., 2004; Patynaa et al., 2006;
Kransler et al., 2012). DINCH has been studied by Schütze and co-workers. DINCH is
neither a reproductive toxicant nor an endocrine disruptor in rodents. Thyroid hyperplasia
and signs of renal toxicity were only observed at relatively high dose levels in animals of
either sex (Schütze et al., 2015; Schütze et al., 2014).
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Until now, all these numerous studies have estimated the kinetics of metabolism of DEHP
from the urinary excretion of its main primary and secondary metabolites, or the parent
substance, after administration of the PVC plasticizer (Fromme et al., 2016; Schütze et al.,
2012, 2014; Anderson et al., 2011; Koch et al., 2011, 2012, 2013). For other studies,
analyses were conducted using deuterated plasticizers (D2 or D4-DEHP, D2 or D4-DINCH,
D2 or D4-DINP): after administration to mice, urine samples were analyzed by LC-MS to
measure metabolite concentrations (Anderson et al., 2001, 2011; Koch et al., 2005, 2007).
Besides these results, the literature reports that plasticizers are metabolized in vivo. In a first
metabolism step, they are cleaved into monoesters (or diester for TOTM), which are further
oxidized in various ways into alcohol, ketone or acid derivatives. These secondary
metabolites are recovered in urine (Martis et al., 1987). These metabolites are not all
currently available on the market, and several were produced after oral or intravenous
administration of the plasticizers to animals and collected as glucuronated derivatives.
Primary and secondary metabolites were obtained by hydrolysis of these derivatives. The
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low quantities obtained by these methods do not always allow full cytotoxicity studies. To
overcome this limitation, we undertook here for the first time the total synthesis of the
postulated primary metabolites of these plasticizers, namely MOTM, MEHA, MEHT, MINP,
MINCH, MMeOP (isomer of MINP) and MMeOCH (isomer of MINCH) as they are described
in the literature.
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All the primary metabolites were successively synthesized (for details, see Figure 2 and
supplementary material). The synthesized metabolites were chemically conformant (purity
95%), and so the results of the cytotoxicity assays can be considered as reflecting the
synthesized substance and not impurities. The objective of our preliminary work was to
compare the effect of these plasticizers and their metabolites on the in vitro cell culture
models used to evaluate the biocompatibility according to the EN 10993. However, authors
report difficulties applying the EN 10993-5 standard for plasticizers: these substances are
hydrophobic, and cannot be used as they are for direct contact in a culture medium. It is
therefore necessary to use a solvent for these materials, which is itself non-cytotoxic toward
the cells, to perform the tests. DMSO was used for this purpose (Wang et al., 2012).
Solubility tests showed that the plasticizers were soluble up to a concentration of 1% in
DMSO (data not shown). We validated the DMSO maximum concentrations that can be used
in the presence of L929 according to our experimental protocol (7 days of cell culture at an
initial cell density of 104 cells/well). DMSO could thus be used up to a maximum
concentration of 1%. To avoid bias, all tests were compared with a control DMSO (medium
supplemented with DMSO at the same concentration).
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All the plasticizers and their corresponding primary metabolites were cultured directly with
murine L929 fibroblasts at three different concentrations (0.01, 0.05 and 0.1 mg/mL) for 7
days. Concerning cytotoxicity of plasticizers, no effect was observed at 0.01 mg/mL. DEHP
and DINCH caused a decrease in L929 cell proliferation from the concentration of
0.05 mg/mL, and their effect was massive at the highest concentration, with total cell death.
DINCH was as toxic as DEHP for the L929 fibroblast cells. These results were unexpected
because the chemical structures of the two substances are widely different. DEHP is a
phthalate with two ethylhexanyl chains, whereas DINCH has no aromatic scaffold, and two
more lipophilic chains grafted on the ester function. Except for DINP, which caused cell
proliferation inhibition (20–30%) from 0.1 mg/mL, all the other plasticizers (e.g. TOTM, DEHA
and DEHT) showed no significant differences compared with DMSO at any of the three
concentrations tested. By contrast, TOTM, one of the most widely used alternative
plasticizers, though banned in food applications, presented a favorable toxicity profile, If we
consider the cytotoxicity threshold described in the standard EN 10993-5, DEHP and DINCH
were equivalent in terms of cytotoxicity (viability threshold < 70%).
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These results show a clear difference in the cytotoxicities of the plasticizers evaluated.
Köksal et al. studied the cytotoxicity of cyclohexyl butyl phthalate (BHP) with the MTT assay
on L929 cell lines. They found an ICC at 0.29 µg/ml for BHP. (Köksal et al., 2016).
Alternative plasticizers evaluated in our study showed significantly lower cytotoxicities. The
most cytotoxic (except for DEHP) did not lower cell viability at a concentration of 0.01 mg/ml,
30 times higher than the IC50 of BHP.
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Concerning the primary metabolites of the PVC plasticizers, their effects on L929 cell viability
were very different when they were tested at the same three concentrations. From
0.05 mg/mL, all the metabolites were cytotoxic except for MOTM. MEHT and MINCH were as
toxic as MEHP. Their corresponding parent plasticizers (DEHT and DINCH) had no effect at
this concentration. At 0.1 mg/mL, MEHT and MINCH were as toxic as DEHP and more toxic
than MEHP. MINP was weakly toxic at 0.05 mg/mL, but cell viability fell sharply at 0.1 mg/mL
to reach 80% cell death, i.e. more toxic than MEHP and with an effect similar to that
observed with DEHP. The isomer of MINCH (MMeOCH) had a similar cytotoxic activity at
0.05 mg/mL (15% cell death), but was also more toxic than MEHP and behaved like DEHP
at 0.1 mg/mL.
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The metabolization of plasticizers increases their cytotoxicity. For example, in the literature,
MEHP, the DEHP bioactive metabolite (Frederiksen et al., 2007), was shown to be 10 times
more potent than DEHP (Huber et al., 1996). MEHP is a well-known activator of the PPAR
family of nuclear receptors (Hurst and Waxman, 2003; Maloney and Waxman, 1999). The
toxicities of the other metabolites are not well known. A recent study reports that MINCH (50,
100 µM) like MEHP (50µM), is also a potential PPAR-α agonist and a metabolic disruptor,
able to induce SVF preadipocyte differentiation, which may interfere with the endocrine
system in mammals (Campioli et al., 2015). The implications of primary metabolite toxicities
higher than that of plasticizers themselves depend on the in vivo metabolism and species.
This information is not available, and so there is a need to perform in vivo animal toxicity
studies on primary and secondary metabolites identified for each plasticizer. Koch et al.
(2013) have measured the amount of MINCH excreted in urine after oral absorption of
DINCH. However, they do not report the absorption percentage of MINCH, its metabolism
rate, or the rate of metabolization of MINCH to its secondary metabolites (Cx-MINCH, OH-
MINCH and OXO-MINCH). The value of 1% for MINCH measured in urine is therefore not
predictive of the amount of MINCH formed and excreted from the vascular system. The
literature shows that absorption of plasticizers after oral exposure is very low in rodents (50%
for DEHP (Anderson et al., 2011))
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5. Conclusion
The main finding of our study is that TOTM showed lower toxicity on L929 cells than the
other plasticizers. Its primary metabolite, MOTM, did not display any toxicity compared with
the other metabolites. This finding is important because we recently demonstrated a
predominance of TOTM in 32 PVC medical devices, accompanied by some DEHP (<0.1%
w/w), DEHT, and sometimes DEHA (Bourdeaux et al., 2016). Under conditions appropriate
for obtaining biosimilar model results, TOTM thus presents a favorable profile as a plasticizer
for medical devices. Our results still have to be interpreted and correlated with plasticizer
extraction tests performed in clinical conditions of use. An infusion set used once in the life of
a patient for 24 hours presents a lower risk than a hemodialysis circuit used for 4 hours three
times a week. Hence to assess patient exposure risk, it will be necessary to determine the
amount of plasticizer extracted per unit mass of PVC in clinical conditions. Such work is
particularly necessary for medical devices used for vascular access. One objective of the
ARMED project is to propose a migration simulation model (Bernard et al., 2015): the
SCENIHR 2015 reported different migration profiles among plasticizers. Given the primary
metabolite toxicities of several DEHP alternatives (MEHT, MINP, MINCH and MMeOCH), it
will also be necessary to look for the presence of their secondary metabolites, and study their
toxicities.
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Declaration of interest
This study is a part of the ARMED® project, and received financial support from the French
National Agency for the Safety of Medicines and Health Products (ANSM).
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Highlights
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All plasticizers induce an action on cell proliferation at 0.1 mg/ml
DEHP, ATBC and DINCH are considered as potentially toxic in the standard EN 10993-5
All plasticizers primaries metabolites cause a decrease in cell viability except MOTM
MEHT, MINP, MINCH reduce significantly the cell proliferation at 0.1 mg/ml
MINCH causes a very high inhibition of cell proliferation