101
4
Gadol inium Containing
Contrast Agents for
Magnetic Resonance
Imaging (MRI)
Investigations on
the Environmental
Fate and Effects
Claudia Neubert, Reinhard Länge,
and Thomas Steger-Hartmann
Contents
4.1 Introduction 102
4.2 Methods 104
4.2.1 BiodegradabilityofDimeglumine Gadopentetate, Gadobutrol,
Gadoxetic Acid, Disodium, and Gadofosveset Trisodium 104
4.2.2 Acute Toxicity Test of Dimeglumine Gadopentetate,
Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset
Triso dium with Fish 106
4.2.3 Acute Immobilization Test of Dimeglumine Gadopentetate,
Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset
Trisodium with Daphnia magna 107
4.2.4 GrowthInhibitionTestofDimeglumineGadopentetate,
Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset
Trisodium on Green Algae 107
4.2.5 GrowthInhibitionTestofDimeglumineGadopentetateon
Different Microorganisms 108
4.3 Results 109
4.3.1 Biodegradability of Dimeglumine Gadopentetate, Gadobutrol,
Gadoxetic Acid Disodium, and Gadofosveset Trisodium 109
4.3.2 Acute Toxicity of Dimeglumine Gadopentetate, Gadobutrol,

3
proposesatieredsysteminwhichexposureestima-
tion and risk screening are included, as well as the determination of physicochemical
properties of new human pharmaceuticals and diagnostic agents.
To assess the potential effects of contaminants on the aquatic environment, a
battery of selected organisms, each representing a specic level of the aquatic eco
-
sy
stem (see Figure 4.1),isi
nvestigated. Furthermore, in order to assess persistence
and thus temporal development of exposure, tests on biodegradation are conducted.
Screeningtestsforbiodegradationallowarstqualitativeassessmentofthepoten
-
ti
alofsewagetreatmentplantsornaturalsurfacewaterstodegradethecompound
of interest.
Among the rst pharmaceutical compounds that were analytically detected in
the aquatic environment
5
and subsequently assessed for their ecotoxicological risk
were iodinated X-ray contrast agents.
6
Fewerdataarecurrentlyavailableforthesec-
ond class of contrast agents used in MRI, even though those compounds have been
detected in ground water as early as in 1996.
7
4.3.3 Acute Immobilization Test of Dimeglumine Gadopentetate,
Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset
Trisodium with Daphnia magna 110
4.3.4 GrowthInhibitionTestofGadobutrol,Dimeglumine

11–13
clinicaluseisonlypossibleinacomplexed
form. Commonly used chelating agents are polyamino-polycarboxylic ligands such
as diethylenetriaminepentaacetic (DTPA). The complexes formed by the different
chelatescanbegrouped,accordingtotheirsizeandstructure,into:
macrocyclicchelatessuchasgadobutrol(Gadovist
®
)and
linear chelates such as dimeglumine gadopentetate (Gd–DTPA) (Magnev-
ist
®
) or Gadodiamide, Gd–diethylenetriamine pentaacetate bismethylamide
(Gd–DTPA–BMA) (Omniscan
®
)
Duetotheexceptionalstabilityofthesehighlyhydrophilicchelatesandthelack
of human metabolism, the contrast media are quantitatively excreted unchanged
afteradministrationwithinhours,andaresubsequentlyemittedintotheaquatic
•
•
Producers
(photosyn.
organisms)
Consumers
(e.g.,
zooplankton)
Consumers
(fish)
Consumers
(predator fish)

in
s
creening tests.
4.2 METHODS
All described tests were performed according to internationally standardized guide-
lines and in accordance with the good laboratory practice (GLP) principles. Dime-
gl
umine gadopentetate, gadobutrol, and gadoxetic acid disodium were manufactured
by Bayer Schering Pharma AG, Germany, gadofosveset trisodium by Mallinckrodt
Inc., United States. Table 4.1 shows the structures and selected physicochemical
properties of the tested compounds.
4.2.1 BIODEGRADABILITYOF DIMEGLUMINE GADOPENTETATE, GADOBUTROL,
G
ADOXETIC ACID, DISODIUM, AND GADOFOSVESET TRISODIUM
Test systems for ready biodegradability were originally established for household
detergents and are required by the European Reserach Area (EU ERA) guideline
to assess the degradation of a human pharmaceutical. The test compounds dime
-
gl
umine gadopentetate, gadoxetic acid disodium, and gadofosveset trisodium were
investigatedaccordingtothetestguidelineoftheOrganizationforEconomicCoop
-
er
ation and Development (OECD), 301E.
17
Briey, the compounds were incubated
in aqueous solutions including nutrients with microorganisms from a municipal
sewagetreatmentplantfor62days(testcompound:dimegluminegadopentetate,in
duplicate)and28to29days(testcompound:gadoxeticaciddisodium,gadofosveset
trisodium, in triplicate).

Complex, dimeglumine salt
Water solubility: c469 g/L
N
N
O
N
O
O
–
O
–
O
O
–
O
O
–
O
O
–
Gd
3+
H
3
C
NH
2
+
HO
HO

O
O
–
O
O
–
Gd
3+
Compound:
Primovist®
Active agent: Gadoxetic
acid disodium
Molecular weight: 725.7
IUPAC name:
(4S) –4–(4–Ethoxybenzyl)
– 3, 6, 9 – tris (carboxy-
latomethyl) – 3, 6, 9 –
triazaundecanedioic acid,
Gadolinium – Complex,
Disodium salt)
Water solubility:1057 g/L
N
N
O
O
–
O
O
–
N

yloxy)phosphinato – kappa
O]oxy} propyl]glycinato(6 - )
– kappa N, kappa O}
gadolinate(3 -)
Water solubility: c247 g/L
O
P
O
O
O
–
N
N
O
O
–
O
N
O
O
–
O
O
–
O
O
–
Chiral
Gd
3+

2
.CO
2
production was determined by titration of the
Ba(OH)
2
solution as described in the guideline.
4.2.2 ACUTE TOXICITY TEST OF DIMEGLUMINE GADOPENTETATE,
GADOBUTROL, GADOXETIC ACID DISODIUM,
AND GADOFOSVESET TRISODIUM WITH FISH
Fish represent the nonmammalian consumer of an aquatic ecosystem (Figure 4.1).
In order to assess the toxicity of the test compound to representative species of this
trophiclevel,theacutetoxicityofgadobutrolandgadoxeticaciddisodiumwas
determined with rainbow trout (
Oncorhynchus mykiss)o
nthebasisoftheguideline
Freshwater Fish Acute Toxicity, Environmental Assessment Technical Assistance
Handbook, Technical Assistance Document 4.11
19
withatestdurationof96hours.
The acute toxicity of dimeglumine gadopentetate and gadofosveset trisodium to the
zebrash (
Danio rerio)
w
as conducted in accordance with the test guideline OECD
203
20
and the EC Guideline Part 2—Testing Methods, Part C. 1.
21
Tenshwereusedforeachconcentrationofthetestcompoundandforthecon-

22
whereas
the test compounds dimeglumine gadopentetate, gadoxetic acid disodium, and gado-
fo
svesettrisodiumwereinvestigatedaccordingtotheguidelineoftheOECD202
and the EC guideline part C.2.
23,24
Different guidelines were used for these tests
becausetheywereperformedfortheuseindifferentregulatoryregions.
Thetestwasperformedwithvejuveniledaphniaineachvesselandfourrepli-
cate
sforeachconcentration.Thecrustaceanswereexposedforaperiodof48hours
understaticconditions.Immobilizationwasrecordedat24and48hours.ThepH
value, oxygen concentration, and temperature were measured at 0 and 48 hours.
Thetestsolutionshadnominalconcentrationsof0.1,1.0,10.0,100.0,and
1000.0 mg/L (test compound: gadobutrol); 100 mg/L (test compounds: dimeglumine
gadopentetate and gadoxetic acid disodium); and 90 mg/L (test compound: gadofos
-
ve
set trisodium).
Samplesfortheconcentrationanalysisofgadobutrolandgadoxeticaciddiso-
di
umbyICP/MSweretakendaily.ThemethodincludedadetectionofGd,andthe
nal concentrations for gadobutrol and gadoxetic acid disodium were calculated
accordingly.
Fo
r dimeglumine gadopentetate and gadofosveset trisodium only nominal val-
ue
s were available. Since these compounds are very well soluble in water (≤469 g/L
for dimeglumine gadopentetate and ≤247 g/L for gadofosveset trisodium, respec-

Thealgaewereexposedtoeachconcentrationintriplicate.Sixvesselswere
prepared for the control. The algae were incubated in an incubator shaker under
continuous light. As a parameter for the growth of the algae population, the cell con
-
c
e
ntrations of the test and control solutions were counted with an electronic particle
counter(“CoulterCounter”)atapproximately24,48,and72hours.ThepHvalue
wasmeasuredatthebeginningandattheendofthetest.
For the study with dimeglumine gadopentetate and gadofosveset trisodium, an
incubatingapparatus(AbimedAlgenTestXT)wasused.Inthiscasethecellnumber
was determined via measurement of chlorophyll uorescence. The increase of biomass
and the growth rate was calculated on the basis of the cell counts. The calculated bio-
mas
s and growth rate of each concentration were compared to those of the controls,
and the inhibition was calculated. Concentration analysis was not performed.
4.2.5 GROWTH INHIBITION TEST OF DIMEGLUMINE
G
ADOPENTETATE ON DIFFERENT MICROORGANISMS
Microorganismsplayaroleasdegradersintheaquaticenvironment,thuslowering
the exposure with introduced contaminants. Furthermore, some of the microorga-
nisms (bluegreen algae) also represent the trophic level of producers.
Thegrowthinhibitiontestofdimegluminegadopentetatewasconductedin
agreement with the standard DIN 38 412 L8.
27
It was incubated in an aqueous solu-
tion including nutrients, with a bacterial population containing Pseudomonas putida
forthetestdurationofapproximately16hours.
The test concentrations were 0.1, 1.0, 10.0, 100.0, and 1000.0 mg/L and a con-
trol.Alltestconcentrationswereincubatedinduplicate.Asaparameterforthetest

gadopentetate,whichwaslikelyduetothedegradationofmeglumine(seeSection
4.4).
The
individualdegradationcurvesofdimegluminegadopentetateandsodium
acetate are depicted in
Figure 4.3. Degradation o
f the test compound started between
day15andday21,anddegradationvaluesofapproximately40%werereachedafter
43 days.
Figure 4.4 shows the concentrations of dimeglumine gadopentetate [mg/L] mea-
suredbyHPLC/UV.Theyvariedbetween53.6and62.1mg/L.Theanalysisforfree
Gd was negative, indicating that no Gd was released from the chelate. The results of
the degradation of gadoxetic acid disodium, gadobutrol, and gadofosveset trisodium
showed that none of these compounds was readily biodegradable and none of the
compoundswastoxictothedegradingbacteria.
4.3.2 ACUTE TOXICITY OF DIMEGLUMINE GADOPENTETATE, GADOBUTROL,
G
ADOXETIC ACID DISODIUM, AND GADOFOSVESET TRISODIUM TO FISH
The measured substance concentrations were approximately 90 to 120% of the
nominalvalues.Thetimecourseoftheresultsdemonstratesthatthesubstance
solutionswerestableduringthewholeexposureperiod.Theresultsofthemeasured
Biological Degradation (%)
0
10
20
30
40
50
60
70

Table 4.2 summa-
rizestheresultsofthemeasuredconcentrationsofthetestcompoundsofthestudies.
4.3.4 GROWTH INHIBITION TEST OF GADOBUTROL, DIMEGLUMINE
G
ADOPENTETATE, GADOXETIC ACID DISODIUM, AND
G
ADOFOSVESET TRISODIUM ON GREEN ALGAE
Figure 4.5givestheinhibition[%]ofthegrowthofChlorella vulgaris after72hours
exposure to gadobutrol on the basis of the biomass (integral) and the growth rate.
Inordertoillustratethedataonwhichtheinhibition[%]iscalculated,cellnumbers
Day of Sampling (d)
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Biological Degradation (%)
0
10
20
30
40
50
60
70
80
90
100
Reference (sodium acetate)
Dimeglumine Gadopentetate
FIGURE 4.3 Biologicaldegradationofdimegluminegadopentetateandthereferencecom-
poundsodiumacetate[%]inthemodiedOECDscreeningtest.
© 2008 by Taylor & Francis Group, LLC
Gadolinium Containing Contrast Agents for MRI 111

Concentrations]
Measured Concentrations in
the Acute Toxicity Tests on
Waterflea [% of the Nominal
Concentrations]
Dimeglumine gadopentetate 106.01 (mean) —
Gadobutrol 90–120* 91–100
+
Gadoxetic acid disodium 90–100 90
Gadofosveset trisodium 97.71 —
*
An exceptionally low concentration at the nominal value of 1.0 mg/L (72 hours) was excluded from fur-
ther calculations.
+
The analysis of the control solution yielded a detectable concentration of gadobutrol after 24 hours
(mean value (MV) = 0.671 mg/L, standard deviation (SD) = 0.0009).
© 2008 by Taylor & Francis Group, LLC
112 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
Figure 4.6 shows the percentage inhibition of the growth rate and the biomass of
gadoxeticaciddisodiumafter72hoursexposuretime,includingcellnumbers.
Noadverseeffectswereobservedinthegrowthinhibitiontestofdimeglu
-
mi
negadopentetateuptoaconcentrationof100mg/Landinthegrowthinhibition
testofgadofosvesettrisodiumuptoaconcentrationof80mg/L.TheNOECand
EC
50
-values are summarized in Table 4.3.
4.3.5 GROWTH INHIBITION TEST OF DIMEGLUMINE GADOPENTETATE
AND

5
Inhibition (%)
–40
–20
0
20
40
60
80
100
Cell Numbers (cells/mL × 10
3
) at 72 h
0
500
1000
1500
2000
2500
3000
Biomass
Growth Rate
Cell Number
FIGURE 4.5 InhibitionofthegrowthrateandthebiomassofChlorella vulgaris [%] and cell
numbers(cells/mLx10
3
±SD)ofChlorella vulgaris after 72-hour exposure to gadobutrol.
© 2008 by Taylor & Francis Group, LLC
Gadolinium Containing Contrast Agents for MRI 113
of scientic progress and experiences in the laboratory. For these reasons the studies

Nominal Concentration of Gadoxetic Acid Disodium (mg/L)
10
1
10
2
10
3
10
4
10
5
Inhibition (%)
0
10
20
30
40
50
60
70
80
90
100
Cell Numbers (cells/mL × 10
3
) at 72 h
0
500
1000
1500

modynamic stability constants of the tested compounds indicate equilibrium far on
thesideofthecomplex:
Dimegluminegadopentetate logK=22.52
30
Gadobutrol logK=21.75
30
Gadoxeticacid logK=23.46
31
and
Gadofosveset logK=22.06
32
Evenifsmallamountsofthechelateswoulddecomplex,theresultingfreeligands
wouldnotnecessarilybereadilydegradable.Pitteretal.(2001)foundintheZahn-
Wellenstestforinherentbiodegradabilitythatthebiodegradabilityofethylene(pro
pylene)di(tri)amine-based complexing agents depends on the character and number
of substituents and nitrogen atoms in the molecule. Tetra(penta)substituted deriva-
ti
veswithtwoormoretertiarynitrogenatomsandcarboxymethylor2-hydroxy-
ethylgroupsinthemolecule(ethylenediaminetetraaceticacid[EDTA],DTPA,
propylenediaminetetraacetic acid [PDTA], hydroxyethylethylenediaminetriacetic
acid[HEDTA])showahighstabilityunderenvironmentalconditions.Ontheother
hand, disubstituted derivatives with two secondary nitrogen atoms in the molecule
(e.g.,ethylenediaminediaceticacid[EDDA])arepotentiallybiodegradable.Readily
degradable are analogous compounds with substituents, which can be hydrolyzed
(e.g.,
ac
etylderivativeswith–COCH
3
groups)asN,N’-diacetylethylenediamine
(DAED) and N,N,N’,N’-tetracetylethylenediamine (TAED).

bis(carboxymethyl)-10-([N-carboxymethyl)-N-(4-cyclohexylphenyl)carbamoyl]me
thyl)cyclododecyl)acetic acid, monosodium gadolinium salt (Gd[CPA-DO3A])
–
at
various concentrations. Gd–DTPA
2–
and Gd(HP-DO3A) produced no lethality up to
200 g/L, and Gd(CPA-DO3A)
–
producedlethalityof17%and31%at24-hourexpo-
suresof100g/Land200g/L.
34
Thetoxicityofmetalsandtheirchelatesisinuencedbytheuptakeintothe
organisms. The bioconcentration of rare earth elements (REEs) in algae was stud-
ie
dbySunetal.
35
The authors were able to show that bioconcentration was largely
dependent on chemical speciation. Adding organic ligands (EDTA, Nitrilotriacetic
acid [NTA], Citrate [Cit]), which can form RE-organic complex species, led to major
reductionoftheREEsbioconcentrationinalgae.Theorderfromhightolowwas
REE
3+
>REE–Cit>REE–NTA>REE–EDTA complex, which is in the reverse order
of the thermodynamic stability constants. The authors found that the relationship
of REEs concentration in algae and their concentration in culture medium can be
described by the Freundlich adsorption isotherm equation. They concluded that an
adsorption process which is rate-limiting controls the rate of the uptake. The pres
-
en

proposes the following equation for the calculation:
PEC
surfacewater
=
Dose
water pen
inhab
xF
Wastew x Dilution
where:
PEC
surfacewater
Local surface water concentration
Wastew
inhab
Amount of wastewater per inhabitant per day
(200 L inh
–1
d
–1
)
Dilution Dilution factor (10)
DOSE
ai
Maximum daily dose of active ingredient (42,000 mg)
F
pen
Percentage of market penetration (0.0008 % for Germany)
&""&"
"%(#&

withpotentialtocauseadverseeffectsareidentiedintheeffectsassessments.
4
IntheacutetoxicityteststhelowestNOECwasobservedfordimegluminegado-
pentetateinalgaewith100mg/L.Ifanassessmentfactorof1000isapplied,aPNEC
in water of 0.10 mg/L (equivalent to 100 µg/L) is obtained. Thus, the ratio of PEC
(0.17µg/L)toPNECfordimegluminegadopentetateintheaquaticcompartment
is 1.7 × 10
–3
,farbelowthecriticalPEC/PNECthresholdof1.ThelowPEC/PNEC
ratio for dimeglumine gadopentetate clearly indicates that the introduction of this
diagnosticproductintosurfacewaterisoflittleenvironmentalrisk.
Gadobutrol,gadoxeticaciddisodium,andgadofosvesttrisodiumhavealower
market volume and are therefore assumed to occur in lower concentrations in the
aquatic environment.
Considering the estimated environmental concentrations and the results of the
ecotoxicologicalinvestigationsofthetestedcompounds,theyarenotassumedto
represent a risk for the aquatic environment. The calculated PECs can
be viewed
in relation to the environmental concentrations of Gd reported in literature. The
geogenic background concentrations of Gd in surface waters are generally low. Bau
and Dulski
7
reported Gd concentrations in Swedish and Japanese rivers, which drain
thinlypopulated,nonindustrializedareastovarybetween0.001and0.012µg/L.
Signicantly higher Gd concentrations are found at sites close to sewage efuent dis-
ch
arges, especially if these efuents receive contributions from hospital wastewater,
which in turn contains excreted MRI contrast media. For instance, at the wastewater
dischargeofthelargetreatmentplantBerlin/Ruhleben(Germany),Gdconcentra-
ti

. Furthermore, long-term tests with the MRI-contrast agents to assess the
chronictoxicityarebeingperformed.
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