Endocrine activities of phthalate alternatives; Assessing the safety profile of furan dicarboxylic acid esters using a panel of human cell based reporter gene assays

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DOI: 10.1039/C9GC04348A

ARTICLE

Endocrine activities of phthalate alternatives; assessing the safety

profile of furan dicarboxylic acid esters using a panel of human

cell based reporter gene assays

Received 00th January 20xx,

Accepted 00th January 20xx

Barbara M. A. van Vugt-Lussenburg,*^aDaan S. van Es,*^bMatthijs Naderman,^aJerome le Notre,^bFrits

van der Klis,^bAbraham Brouwer,^a,cand Bart van der Burg^b

DOI: 10.1039/x0xx00000x

FDCA esters are highly relevant biobased alternatives for currently used benzene dicarboxylic acid esters. Despite all the

developments on 2,5-FDCA applications, to the best of our knowledge thus far no toxicological data were available for 2,5-

FDCA esters. In the present study we aimed to fill this gap, by using an in vitro reporter gene assay approach to compare the

activity profile of commonly used phthalates to that of their furan-based counterparts. The assay selection was aimed at the

detection of endocrine activity, since several phthalates are heavily scrutinised for their endocrine disrupting properties.

However, to avoid missing other relevant toxicological endpoints, several assays able to detect various forms of cellular

stress were also included in the panel. The results showed that the (ortho)benzene dicarboxylic acid esters were

predominantly active on several of the endocrine assays. In comparison, six of the seven furan dicarboxylic acid based

diesters tested here showed no activity in any of the 13 assays used. Only the isobutyl derivative DIBF showed moderate

estrogenic activity on one assay, compared to much more pronounced activities on four assays for the ortho-phthalate

analogue. Overall, the results presented in this paper are a strong indication that 2,5-FDCA based diesters in general are not

only technically viable alternatives to phthalates, but also offer significant toxicological benefits, which supports a non-

regrettable substitution.

ethylene glycol is already produced from renewable biomass

(on small industrial scale)⁴, the diacid component, i.e. biobased

terephthalic acid (TA), is still under development ^4-6. Given the

Introduction

Global climate change and the depletion of finite feedstocks are

strong drivers for the developing transition from a mainly fossil

fuels and feedstocks based linear economy to a more

sustainable circular one. As a consequence, the industrial

production of chemicals and materials will increasingly be based

on renewable energy and feedstocks such as wind- and solar

energy and biomass. True circular end-of life options for

materials require the possibility to depolymerise the material to

its constituent chemical building blocks, followed by

purification, and rebuilding the materials to the required

specifications. Hence polyesters, which can be efficiently

depolymerised by hydrolysis, are materials with a very high

circular potential ^1-3. Furthermore, by changing to biobased

monomers, increasing greenhouse gas emissions and the use of

finite fossil resources can be eliminated altogether.

enormous production volume of TA (> 80 Mton/a) ^{7, 8}, which is

expected to continue to grow in the coming decades, biobased

TA, or analogues, should be based on abundantly available

biobased feedstocks, which do not compete with primary food

production. Glucose, which can be obtained from

polysaccharides like starch and cellulose, is therefore the most

promising feedstock for the industrial production of biobased

TA. Despite continuing efforts, the efficient conversion of highly

functionalised sugars to TA remains a challenge ⁴. An alternative

approach is the development of a functional analogue of TA, the

biobased furan-2,5-dicarboxylic acid (or 2,5-FDCA) ⁹. This diacid,

which was already reported by Fittig in 1876 ¹⁰can be obtained

from sugars with much higher efficiency than TA. While

functionally comparable to TA, 2,5-FDCA shows different

chemical and physical behaviour in various applications. When

used in polyesters such as the PET analogue PEF (polyethylene-

2,5-furanoate) it was for instance found that various properties,

such as gas barrier properties, were significantly improved by

using 2,5-FDCA as diacid ^11-13. Many investigators have already

shown that 2,5-FDCA based polyesters are functionally

comparable to their TA based analogues, in applications such as

films, foils, bottles and fibres ^{14, 15}. Other polymer applications

of 2,5-FDCA include polyamides and unsaturated polyesters

resins ^{15, 16}. As the dimethyl ester of terephthalic acid (DMT) is

an important building block and hence a high-volume chemical

Currently, the dominant industrial polyester is polyethylene

terephthalate (PET), well known for its application in e.g.

beverage bottles, textile fibres, etc. While the diol component

^a.BioDetection Systems bv, Science Park 406, 1098XH Amsterdam.

^b.Wageningen Food and Biobased Research (WFBR), Bornse Weilanden 9, 6708WG,

Wageningen, The Netherlands.

^c.VU University, Faculty of Sciences, Department of Animal Ecology, De Boelelaan

1085, 1081HV Amsterdam, the Netherlands.

† Footnotes relating to the title and/or authors should appear here.

Electronic Supplementary Information (ESI) available: Full substance list, synthesis

methods and full bioassay results tables. See DOI: 10.1039/x0xx00000x

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substance, it is of particular interest to compare its toxicity was performed on a panel of effect-based CALUX reporter gene

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DOI: 10.1039/C9GC04348A

profile with that of its (relatively) new FDCA based counterpart. assays.

In analogy, also medium to long chain diesters of TA and

phthalic acid (PA) are industrially important high volume

17

Results and discussion

chemicals, which mainly find use as plasticisers for PVC

.

Especially phthalate esters are subject to continuous scrutiny

related to (potential) adverse health effects ^18-20. However,

complete substitution of disputed phthalates is challenging due

to both economic as well as performance issues. Hence the

development of cost effective, biobased, plasticisers that offer

environmental, health and performance benefits compared to

existing phthalates is highly desirable. 2,5-FDCA esters can

potentially offer such benefits, as was already shown for their

technical performance as plasticiser in PVC ^21-23. As a next step,

more insight is required into the potential health effects of

these substances.

Given these developments, it can be expected that industrial

2,5-FDCA production will take off in the near future, and that

the number of applications will grow, especially at first in

applications where 2,5-FDCA has a clear performance benefit.

This could include a positive ecological and toxicological profile.

The replacement of disputed phthalate plasticisers by 2,5-FDCA

esters is an interesting opportunity.

Despite all the developments on 2,5-FDCA applications, to the

best of our knowledge thus far no toxicological data were

available for 2,5-FDCA esters. An EFSA report on 2,5-FDCA,

however, is available; the document reports 2,5-FDCA as

‘negative’ in in vitro genotoxicity tests, and a 90-day oral toxicity

study in rats resulted in a NOAEL of 300 mg/kg/day.

Furthermore, no concern for accumulation in man was

reported²⁴. In order to come to a non-regrettable long-term

substitution of e.g. TA by 2,5-FDCA it is of utmost importance to

get an early insight into the toxicity of these potentially high-

volume chemicals.

Substance selection

A list of 27 relevant substances (phthalates, non-phthalate

plasticizers, plastic additives, residual monomers and

(potential) degradation products) was selected to establish an

in vitro reporter gene assay profile as ‘point of departure’.

Additionally, furans (dimethyl- to diisodecyl diesters) were

selected as test substances, as well as the related free acids. The

three benzene dicarboxylic acid isomers tested in this study are

all produced on (large) industrial scale, for application in

polyesters and polyamides (IPA, TA), as well as plasticisers (PA)

and resins (PA). Exposure to these substances can be the result

of occupational exposure during production or processing, by

leaching/migration from products, or by (bio)degradation or

metabolism of e.g. plasticisers or polyesters. Of the three FDCA

isomers tested in this study, only the 2,5-isomer is expected to

be commercialised on short to medium term. The other two

isomers are included to study the effects of positional

isomerism. All substances and their abbreviations are listed in

ESI Table 1. The synthesis methods of the substances (when

applicable) are described in the ESI.

Assay selection

The phenolic reference substances as well as the benzene

dicarboxylic acids analysed in the current study have been

previously described as endocrine active substances in vivo

33-

³⁹. In several in vitro studies they have been identified as

androgen receptor antagonists and/or estrogen receptor

agonists ^40-46. Therefore, CALUX assays were selected which

cover the nuclear hormone receptor endpoints for estrogen

agonism (ERα) and androgen antagonism (anti-AR). The OECD

guidance document on standardised test guidelines for

evaluating chemicals for endocrine disruption ⁴⁷, additionally

mentions thyroid hormone interference as a possible mode-of-

action of endocrine disrupting chemicals. Therefore, a CALUX

assay assessing thyroid receptor antagonism (anti-TRβ) was also

included. Additionally, two more assays were added that are

often targeted by endocrine active substances: progesterone

antagonism (anti-PR) and glucocorticoid antagonism (anti-GR).

These assays have been well validated, and several have been,

or are currently in the process of being included in OECD test

guidelines ^48-52. Additionally, for the benzene dicarboxylic acids,

effects on peroxisome proliferation and lipid homeostasis have

been described ^53-55. To be able to detect such effects, two

This prompted us to study a broad series of FDCA esters (short

to long chain) by means of in vitro human reporter gene assays.

These in vitro human cell-based CALUX® reporter gene assays

have been designed to detect interaction of a substance with a

specific nuclear receptor or cell signalling pathway, rather than

the overall effect of a substance on a complex biological system.

The advantage of this approach is that the results are

straightforward to interpret, since complicating factors such as

metabolism, tissue distribution or receptor crosstalk do not play

a role in these reporter gene assays. The panel generates an

activity profile that can be used to provide clues on the possible

mode-of-action of a substance, to visualise trends for structural

analogues, and as a starting point for further investigation ^25-32

.

The substances were analysed on a reporter gene assay panel

covering a broad range of endpoints, including nuclear receptor

hormone interaction, DNA damage, oxidative stress and cellular

stress pathways. This panel of assays has been shown to be

predictive for major human toxicological endpoints, including

endocrine disruption, reproductive toxicity, genotoxicity and

peroxisome proliferator assays, PPARα and PPARγ CALUX ^{56, 57}

,

involved in lipid homeostasis, were included in the assay

selection. Additionally, a cytotoxicity assay was included in the

panel ³². This assay detects cell death, but can also be used to

identify nonspecific effects of the substances, for example on

cell proliferation or luciferase stability.

acute toxicity ^25-32

.

Here we report on the in vitro toxicological effects of geometry

and substitution of a range of isomeric benzene- and furan

dicarboxylic acids and esters. In vitro analysis of the substances

In addition to these key assays directed specifically to the

detection of endocrine active substances, several more general

2 | J. Name., 2012, 00, 1-3

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assays to assess toxicity of substances were included. The aryl particular assay ⁵⁹. Finally, activation of p53 GENTOX CALUX is

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hydrocarbon receptor (AhR) CALUX ⁵⁸, for example, assesses indicative for DNA damage ^{29, 32}

.

DOI: 10.1039/C9GC04348A

toxicity of dioxin-like substances and PAHs. AP-1 CALUX detects This resulted in a panel of thirteen CALUX assays (Table 1). To

substances which interfere with cell cycle control, while ESRE be confident that this selection would enable the detection all

CALUX measures an early stage of unfolded protein response as effects elicited by the benzene dicarboxylic acids without

a result of cellular stress. Activity on the Nrf2 CALUX is indicative missing other important endpoints, these substances were also

for oxidative stress; several phenolic substances have been analysed on the twelve non-selected CALUX assays available in

known to undergo redox cycling, which would trigger this our research facility (anti-ERα, AR, GR, PR, TRβ, RAR, LXR, PAH,

Table 1 - CALUX panel results for a list of phenols and benzene dicarboxylic acids. Only the results of the selected CALUX assays are shown; for the results on the assays that were

not selected, see ESI. Results are presented as lowest observed effect concentrations (LOEC) in LogM. - = no activity observed; NA = Not Assessed. The top row shows the LOEC

value of the assay’s reference substance.

Reference substance

Bisphenol F

-6.6

-

-12.2

-6.6

-7.7

-7.2

-5.1

-7.7

-5.4

-6.8

-6.0

-5.8

-11.0

-4.8

-5.5

-6.1

-5.5

-9.5

-6.9

NA

-4.2

-

-9.7

-7.7

-12.3

-9.5

-7.5

-4.7

-4.3

-

-5.4

-9.0

-3.3

NA

-

1

2

3

4

5

620-92-8

80-05-7

-

Bisphenol A

-4.0

-5.5

-4.9

-4.5

-

-4.5

-

140-66-9

104-40-5

4-tert-Octylphenol

4-Nonylphenol

-

-4.6

NA

4376-18-5

131-70-4

2528-16-7

4376-20-9

131-11-3

1459-93-4

120-61-6

84-66-2

Monomethyl phthalate

Monobutyl phthalate

Monobenzyl phthalate

Mono(2-ethylhexyl) phtalate

Dimethyl pthtalate

-

NA

-

NA

6

-

7

-

8

-3.5

-

-5.5

-4.7

-

-3.2

9

-

-4.7

-3.1

-

-3.6

-

10

11

12

13

14

15

16

17

18

19

20

21

Dimethyl isophthalate

Dimethyl terephthalate

Diethyl phthalate

-

-3.3

-

-3.5

-

-4.0

-

-5.0

-

-4.3

-

636-53-3

636-09-9

84-74-2

Diethyl isophthalate

Diethyl terephthalate

Dibutyl phthalate

-

-4.0

-4.7

-5.0

-

-5.2

-5.3

-5.0

-5.3

-6.3

-3.9

-4.5

-5.0

-5.5

-5.4

-5.5

-

84-69-5

Diisobutyl phthalate

Di(n-hexyl) phthalate

Dicyclohexyl phthalate

Butylbenzyl phthalate

-4.5

-3.5

-4.5

-3.9

-

-4.5

-5.1

-

84-75-3

NA

-

-4.0

NA

-3.7

-

-4.2

NA

-

NA

-

84-61-7

-

-4.5

-

85-68-7

-5.6

-

117-81-7

Di(2-ethylhexyl) phthalate

(synthesized)

-

117-81-7

Di(2-ethylhexyl) phthalate

(commercial)

-

-4.0

-

22

137-89-3

Di(2-ethylhexyl) isophthalate

-

23

24

25

26

27

6422-86-2

117-84-0

Di(2-ethylhexyl) terephthalate

Dioctyl phthalate

NA

-

28553-12-0

26761-40-0

Diisononyl phthalate

Diisodecyl phthalate

-

CALUX activity profiles of phenolic substances

Hif1α, TCF, NFκB, p21). ESI Table 2 shows that none of these

assays were activated by any of the test substances, which

confirms that the current selection of assays was adequate for

the purpose of this study.

Table 1 shows the results for the 13 selected CALUX assays. The

values in the table represent lowest observed effect

concentrations (LOECs) in LogM. In the absence of

internationally established threshold values, substances were

arbitrarily considered ‘positive’ if LOEC < 1E^-5M as reported in a

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previous study on reproductive toxicity using the same CALUX anti-androgenic or anti-progestagenic activity. The long chain

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assays ²⁸. Since the aim of the current study was to compare the phthalates dioctyl, diisononyl and diisod^De^Ocy^I:l¹p⁰h^.1t⁰h³a^9/la^Ct⁹e^GC(e⁰n⁴t³r⁴i⁸e^As

activity profile of phthalate- vs furan-based substances rather 25-27) showed no activity at all on any of the assays.

than to classify substances as ‘positive’ or ‘negative’, this These results are in agreement with what is known about these

threshold was not applied in the current study, but rather a substances ^{34, 41, 42, 65-68}; high molecular weight phthalates such

comparative approach was taken.

as diisononyl- and diisodecyl phthalate are included in REACH

For the phenolic substances in the list, most of the activity as ‘not toxic for human health’ ^{69, 70}. The lower molecular weight

observed was, as expected, on the nuclear hormone receptor phthalates dibutyl-, diisobutyl-, butylbenzyl- and di(2-

assays. The bisphenols (entries 2-3) and alkyl phenols (entries 4- ethylhexyl) phthalate on the other hand, are classified in REACH

5) were mainly active as estrogen receptor agonists, and as ‘very dangerous’, in Category 1B “substances regarded as

androgen- and progesterone receptor antagonists, with a toxic to reproduction”. The two shortest phthalates in this test,

potency in the (sub-) micromolar range. This corresponds with the methyl and ethyl diesters, do show some adverse effects in

their known activity as endocrine active substances ^{44, 46}. 4-Tert- vivo, but not the reproductive tract developmental

octylphenol additionally activated the Nrf2 CALUX, a pathway abnormalities that have been described for the C4- to C6 chain

66,

indicative of oxidative stress. Indeed, oxidative stress has been diesters

reported for this substance ⁵⁹

⁶⁸. However, in concordance with our results,

.

estrogen receptor agonism and androgen receptor antagonism

has been observed in vitro for diethyl phthalate ^{40, 42, 66, 68}

.

The most prominent representative of the C2-C6 diesters is

butylbenzyl phthalate (BBP); in the current study this substance

is able to act as an agonist on the estrogen receptor and as an

antagonist on the androgen- and progesterone receptor with

higher potency than any of the other phthalates tested. This

correlates well with other studies; its endocrine disrupting

CALUX activity profiles of benzene dicarboxylic acids

A wide range of benzene dicarboxylic acid derivatives was

tested in this study. In table 1 substances were ranked by

degree of substitution (entries 6-9: mono; entries 10-27: di),

and by increasing chain length. Most of the reporter gene

activity is observed on the nuclear hormone receptor related

assays, for the di-substituted phthalates of medium chain

length (C4-C6).

activity has resulted in an industrial phase out of BBP ^{71, 72}

.

Although di(2-ethylhexyl) phthalate (DEHP) is highly scrutinised

for its endocrine disrupting properties, two preparations of this

substance (synthesized in-house (entry 21) versus commercially

available (entry 22)) showed very little activity, apart from

estrogen receptor agonism at relatively high LOEC (1 mM). Also

the corresponding monoester (entry 9) and presumed active

metabolite in vivo, did not show in vitro estrogen- or androgen

receptor activation or antagonism. Although in vivo studies in

rat, as well as epidemiological studies in men, suggest that

DEHP/MEHP exposure results in reproductive tract anomalies

and reduced sperm motility indicative for an anti-androgenic

It is known that in vivo, di-substituted phthalates are readily

60

hydrolysed to their mono-esters

. In rodent in vivo

experiments, exposure to the monoesters results in similar

adverse effects as exposure to the diesters ^{36, 61}. As a result, the

mono-substituted phthalates are thought to be the endocrine

active metabolites of the corresponding diesters, displaying

endocrine disrupting activity ^{34, 36, 41, 42}. Therefore, in the current

study, we determined the in vitro activity profile of four

monoesters of (ortho)phthalic acid (entries 6-9) as well as 17

diesters (entries 10-27). Of the four monoesters tested only

mono(2-ethylhexyl) phthalate (MEHP) was active (entry 9), yet

not on the endocrine assays but on the peroxisome proliferator-

assays PPARα and PPARγ, which is in agreement with previous

reports ^{53-55, 62}. It has been observed before that the phthalate

monoesters appear to be able to exert effects in vivo such as

reproductive tract anomalies and decreased testosterone

mode-of-action ^{38, 61}, other in vitro studies have also shown a

41, 42, 63, 68

lack of estrogen- and androgen receptor interaction

.

It has been suggested that these substances exert their effects

via other mechanisms, for example by influencing

steroidogenesis through CYP19 inhibition, rather than by direct

interaction with the androgen receptor ^{61, 68}. This is supported

by results of two in vitro assays, the H295R steroidogenesis

assay and human testis explants, where both MEHP and DEHP

synthesis, which are indicative of an anti-androgenic mode of

35, 36,

action

⁶¹, but that the phthalates fail to show anti-

were shown to significantly inhibit testosterone synthesis ^{61, 73}

.

androgenic activity in in vitro androgen receptor interaction

assays ^{38, 42, 63, 64}, suggesting an indirect, non-receptor mediated

mode of action.

For the isobutyl-, butyl- and butylbenzyl diesters, in vitro

estrogen receptor transactivation has been reported

34, 36, 41, 42, 66, 68

previously, as well as in vivo endocrine effects

,

While the short-chain diesters dimethyl- and diethyl phthalate

showed no or low estrogenic- and anti-androgenic activity, the

medium chain diesters (dibutyl (entry 16), diisobutyl (entry 17),

di(n-hexyl) (entry 18), dicyclohexyl (entry 19) and butylbenzyl

(entry 20)) showed estrogenic, anti-androgenic and anti-

progestagenic activity with LOECs in the micromolar range.

Butylbenzyl phthalate displayed the highest potency for all

three endocrine assays. Further increasing the chain length to

di(2-ethylhexyl) phthalate (entry 21/22) resulted in a marked

decrease in estrogenic potency (10-100 fold), and no detectable

which is in line with our findings. Nonetheless, in the ECHA

support document for BBP ⁷⁴, its adverse effects are considered

to be primarily related to effects on steroidogenesis, rather

than through direct interaction with steroid hormone

receptors.

Trends in positional isomerism and ester chain length

For the methyl-, ethyl- and 2-ethylhexyldiesters, all three

phthalic, isophthalic and terephthalic isomers were tested (all

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commercially available materials). For the dimethyl esters of 2,5-FDCA is closer to that of isophthalic acid (IPA). In fact, the

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moderate effects were observed for DMP and DMIP, while DMT highly unsymmetrical 2,4-FDCA is th^De^OIm^{: 10}o^.s¹⁰t³⁹li^/n^Ce^9Gar^C04fu³⁴ra⁸n^A

did not give any response (Table 1, entries 10-12). Increasing derivative. The differences between PA and 2,5-FDCA are even

the alcohol chain length to C2 results in a dramatic increase in larger than for TA and the latter. A more suitable structural

activity of the ortho-phthalate (DEP) on several endocrine comparison would hence be based on PA vs 3,4-FDCA. Another

assays, while both other isomers are virtually inactive (Table 1, difference between the analogous benzene and furan

entries 13-15). For the 2-ethylhexyl derivatives, only the ortho- dicarboxylic acid derivatives is the higher polarity of the latter

phthalate is active, while again no activity is seen in case of the due to different symmetries, the presence of the oxygen, and

other isomers (Table 1, entries 21-24). The variation in the more pronounced diene character in the furan ring. These

positional isomerism conclusively shows that ortho-phthalates differences are also related to the significantly higher acidity of

have the most pronounced endocrine activity. Whether this can the FDCA isomers, as is clear from their pKa values.

be attributed to steric or electronic effects is not clear, and Interestingly, while 2,5-FDCA more closely resembles IPA with

while highly interesting falls beyond the scope of the present respect to geometry, the aqueous solubility is closer to (or

study.

actually surpasses) that of PA.

The data in table 1 clearly show a dependency of the endocrine Hence, FDCA’s are in general more polar than their benzene

activity of the ortho-phthalates on the ester chain length, which counterparts. Based on these differences, we expect different

appears to have an optimum at C4-C6. Further increasing the effects in the interactions with human nuclear receptors and

chain length to C8 (DEHP entries 21/22 and DOP entry 25)

TA

IPA

PA

results in a complete lack of activity. This could be due to the

dramatic decrease in aqueous solubility of the longer chain

phthalates (10^-9M for n-octyl versus 10^-5M for n-butyl, see also

ROOC

COOR

ROOC

COOR

ESI table 4), resulting in a significantly reduced bioavailability ^75-

ROOC

COOR

79

⁷⁸. Furthermore, Thomsen et al.

reported an inverse

ROOC

COOR

relationship between phthalate solubility and temperature; e.g.

for DBP solubility drops from 14.6 mg/L at 25°C to 5.5 mg/L at

35°C. Since our cell assays are performed at an incubation

temperature of 37°C, it is reasonable to assume that the actual

solubilities for the phthalate esters are even lower than those

reported for the 20-25°C range. Hence, for the long chain

phthalates (≥C8) (lack of) bioavailability could simply be the

cause for observed lack of activity. A similar lack of bioactivity

for long chain phthalates (≥C8) was reported by Ejlertsson et al.

in the degradation of phthalic acid esters under methanogenic

O

COOR

2,4-FDCA

O

3,4-FDCA

2,5-FDCA

Chart 1 - structures of benzene-, and related furan dicarboxylic acid (FDCA) isomers

used in this study; TA = terephthalic acid, IPA = isophthalic acid, PA = phthalic acid.

cell signalling pathways.

conditions ⁸⁰

.

CALUX activity profiles of furan- vs benzene dicarboxylic acids

Another observation that can be made from the results in table

1 is that there is no significant effect of ester chain branching on

endocrine activity (compare DBP (entry 16) and DIBP (entry 17),

or DHP (entry 18) and DCHP (entry 19), respectively). Note that

in general chain branching leads to a slightly higher aqueous

solubility; e.g. approximately two times higher for DIBP

After having generated an activity profile for benzene

dicarboxylic acid esters in the CALUX assays, we switched our

focus to the analogous furan dicarboxylic acid esters, which are

potential biobased alternatives to the former.

Most of the diesters that are the focus of this study are

prepared from the corresponding diacids. Furthermore, under

environmental and in vivo conditions it can be expected that all

diesters will (eventually) undergo hydrolysis to the monoesters

first, and subsequently to the diacids ^{78, 81, 82}. Hence, in order to

exclude any toxicological effects of the parent diacids

themselves, they were analysed on the CALUX assay panel.

Interestingly, none of the free acids used in this study showed

any effect on the assays used (data not shown, see ESI Table 3).

This could imply that the free acids are not active in the cells.

However, lack of activity could also be due to inability to enter

the cells. Under physiological conditions, i.e. pH 7.2, all of the

diacids used will be deprotonated given their pKa values (see

Table 2), which could inhibit uptake in the cells, explaining the

compared to DBP (see also ESI table 4) ⁷⁸

.

Overall it can be concluded that solubility, and hence

bioavailability is probably the most important factor

determining the bioactivity of the phthalate esters.

Physico-chemical properties of furan- vs benzene dicarboxylic

acids

There are significant physico-chemical differences between

terephthalic and phthalic acid on the one hand and 2,5-FDCA on

the other. For instance, as can be seen from chart 1 and Table

2, due to the different geometries of the benzene vs furan

nucleus both the angles and the interatomic distances between

carboxylic acid groups differ significantly for the respective

analogues. While TA is linear (angle 180°), the angled structure

lack of observed effect ^{83, 84}

.

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 5

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DOI: 10.1039/C9GC04348A

ARTICLE

Table 2 - ^aAngle between carboxylic acid groups ^{82,85 b}Distance between carboxylic acid groups ⁸²; ^cData from ^{83 d}Solubility under ambient conditions in mg/ml (data from

; ;

https://pubchem.ncbi.nlm.nih.gov/); ^eNR: Not Reported.

Substance

TA

2,4-FDCA

IPA

2,5-FDCA

PA

3,4-FDCA

Angle (°)^a

180

150

120

129

D (Å)^b

5.73

5.08

4.40

4.83

2.67

3.37

pKa1^c

3.51

2.63

3.46

2.60

2.98

2.55

pKa2^c

4.82

3.77

4.46

3.55

5.28

7.23

Solubility^d

0.015

NR^e

0.12

1

60

83

0.7

NR^e

As discussed previously, while the dimethyl esters of phthalic In order to investigate the effect of ester chain length, next the

acid (DMP) and isophthalic acid (DMIP) showed activity, no diethyl esters of the phthalate family were compared with

effects were found for the terephthalic acid (DMT) isomer. In diethyl 2,5-FDCA (purified commercial material). As discussed in

contrast, none of the furan derivatives tested were active on the previous chapter, diethyl phthalate and diethyl

any of the assays (Table 3, entries 28-33). The 2,5-FDCA and 3,4- terephthalate showed moderate to low activity on the

FDCA dimethyl esters were commercial samples that were endocrine assays, while the isophthalic acid analogue was

subsequently purified, while the 2,4-FDCA derivative was inactive. Once again, no activity was found for the analogous

prepared and purified in our labs according to a previously 2,5-FDCA derivative (Table 3, entries 34-37).

published procedure (see ESI) ⁸⁶

.

In the current study, we observed a significant increase in both

For the furan dicarboxylic acid esters no effect of isomerism, the potency and the number of active assays for the C4

phthalate derivatives (Table 1, entries 16-17). To investigate if

2,5-FDCA diesters with similar chain length likewise show an

O

increased activity compared to their diethyl- and dimethyl

counterparts, the CALUX profile of diisobutyl phthalate was

compared to the profile of diisobutyl-2,5-FDCA (Chart 2). While

diisobutyl phthalate (DIBP) was the second most active

phthalate derivative tested, showing micromolar-range activity

as an estrogen, anti-androgen and anti-progestin, in contrast

diisobutyl-2,5-FDCA (DIBF) acted as a weak estrogen only (Table

3, entries 38-39). While DIBF is the only active furan derivative

tested thus far, the observed effects are not as pronounced as

those observed with DIBP.

O

O O

DIBP

DEHP

O

DIBF

DEHF

Chart 2 - chemical structures of isobutyl and 2-ethylhexyl diester of PA (DIBP and DEHP)

and 2,5-FDCA (DIBF and DEHF).

and hence of symmetry or dipole moment, is observed under

the conditions tested in our assays.

Further increasing the alcohol chain length from isobutyl to 2-

ethylhexyl (2EH), i.e. an extension of the isobutyl motif (Chart

2), results in a significant reduction of effect in our assays, as is

apparent from the weak estrogenic activity only in the case of

DEHP, with very similar LOEC values for the synthesized and the

commercial preparation (Table 3, entries 40 and 41). The

absence of any response of the iso- and terephthalate isomers

in our assay (as opposed to the ortho-phthalate) shows that

positional isomerism in the phthalate family has a significant

impact (Table 3, entries 40-43). The furan-based analogue of

DEHP, di(2-ethylhexyl)-2,5-FDCA, prepared for this study using

A possible reason why the FDCA methyl esters show no effect

while their benzene analogues do, could lie in a different

susceptibility to (enzymatic) hydrolysis of these esters. Oae et

al. reported significant differences between the rate of

hydrolysis of dimethyl 2,5-FDCA and 3,4-FDCA versus DMT (17

and 1.3 times higher respectively), which could result in a

relatively fast hydrolysis of the furan derivatives, successively to

the respective mono esters and diacids ⁸⁷. The latter were found

to be completely inactive (vide supra).

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two different methods, did not activate any of the CALUX assays

(entries 44 and 45).

View Article Online

DOI: 10.1039/C9GC04348A

Further increasing the chain length of the ester group to

isodecyl results in complete absence of effects, both for the

phthalate (DIDP) and the FDCA diester (DIDF) (Table 3, entries

46-47). This observation is in line with the industrial move from

DEHP as general purpose plasticiser to the longer chain

analogues DINP and DIDP ^{70, 88}

.

Table 3- CALUX assay panel results for di-substituted phthalates (bold) versus furans, arranged by order of increasing chain length. Results are presented as lowest effect

concentrations (LogM). - = no activity observed up to 1E-3M. N/A: not applicable; DIBF and DIDF have not been CAS-registered.

28

29

30

31

32

33

Dimethyl phthtalate

Dimethyl

isophthalate

DMP

-

-4.7

-3.6

-

131-11-3

1459-93-4

120-61-6

4282-33-1

4282-32-0

1710-13-0

DMIP

-3.3

-3.1

-

Dimethyl

terephthalate

Dimethyl-3,4-

furandicarboxylate

Dimethyl-2,5-

furandicarboxylate

Dimethyl-2,4-

furandicarboxylate

DMT

-

DM-3,4-FDCA

DM-2,5-FDCA

DM-2,4-FDCA

34

35

Diethyl phthalate

Diethyl isophthalate

Diethyl

DEP

DEIP

-3.5

-

-4.0

-

-5.0

-

-4.3

-

84-66-2

636-53-3

36

37

38

39

terephthalate

Diethyl-2,5-

furandicarboxylate

DET

DEF

-

-4.0

-

636-09-9

53662-83-2

84-69-5

N/A

-

-4.5

-

-5.0

-

-5.0

-

Diisobutyl phthalate

Diisobutyl-2,5-

furandicarboxylate

DIBP

DIBF

-5.3

-4.3

Di(2-ethylhexyl)

phthalate

Di(2-ethylhexyl)

phthalate

Di(2-ethylhexyl)

isophthalate

Di(2-ethylhexyl)

terephthalate

Di(2-ethylhexyl)-2,5-

furandicarboxylate

Di(2-ethylhexyl)-2,5-

furandicarboxylate

DEHP

(synthesized)

DEHP

(commercial)

40

41

42

43

44

45

46

47

-

-3.9

-

117-81-7

137-89-3

6422-86-2

158099-01-5

26761-40-0

N/A

-4.0

DEHIP

-

DEHT

DEHF (method

1)

DEHF (method

2)

Diisodecyl phthalate

Diisodecylfuran-2,5-

dicarboxylate

DIDP

DIDF

diisobutyl phthalate, dimethyl isophthalate, di(2-ethylhexyl)

terephthalate, dimethyl phthalate, dimethyl-3,4-furan

dicarboxylic acid ester, phthalic acid and terephthalic acid were

obtained from Sigma-Aldrich. Monoethylhexyl phthalate was

obtained from Wako. Butylbenzyl phthalate, di(2-ethylhexyl)

phthalate, dimethyl terephthalate and isophthalic acid were

Experimental

Origin of chemicals

4-Nonylphenol, 4-tert-octylphenol, bisphenol A, bisphenol F,

dibutyl phthalate, dicyclohexyl phthalate, diethyl phthalate,

J. Name., 2013, 00, 1-3 | 7

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Journal Name

obtained from Fluka. Di(n-hexyl) phthalate was obtained from µl/well cell suspension using a MicroFlo Sele_Vc_it_ewd_Ari_ts_icp_lee_On_ns_lie_ner

DOI: 10.1039/C9GC04348A

Dr. Ehrenstorfer. Di(2-ethylhexyl) isophthalate was obtained (BioTek). After 24 h, exposure medium was prepared by adding

from TCI. Dimethyl-2,5-furandicarboxylic acid ester was 0.2% of test substance dilution series in DMSO to a 96-wells

obtained from Bepharm (China). 2,5-Furandicarboxylic acid was plate with assay medium. Of this exposure mixture, 30 µl was

obtained from V&V Pharma Industries (India) and Bepharm added to the assay plates containing the CALUX cells, resulting

(China), and by independent synthesis from galactaric acid in final DMSO concentrations of 0.1%. The final concentrations

according to ⁸⁹

.

of the substances in the wells were 1E^-4– 3E^-10M in 0.5 log unit

increments. Additionally, DMSO blanks and a full dose response

curve of the relevant reference substance were included on

each plate. All samples were tested in triplicate. The

preparation of the substance dilution series as well as the

exposure of the cells were performed on a Hamilton Starlet

liquid handling robot coupled to a Cytomat incubator. In order

to be able to detect receptor antagonism, the assays were also

performed in antagonistic mode. The assay procedure was as

described above, with the only exception that the EC50

concentration of the reference agonist was present during the

exposure.

Cell lines

CALUX® assays have been constructed and are licensed world-

wide by BioDetection Systems BV, Amsterdam, The

Netherlands. The CALUX® battery of stable reporter gene assays

²⁹comprised assays and test conditions that were selected from

a larger panel of CALUX cells because of their possible

relevance. The selected panel consisted of: the DR CALUX,

consisting of rat H4IIE liver cells expressing the aryl hydrocarbon

receptor endogenously ⁵⁸. In addition a range of highly selective

human U2-OS cell based lines was used including doubly (i.e.

reporter gene and receptor) transfected estrogen receptor

subtype alpha (ERα)-, progesterone receptor (PR)-,

glucocorticoid receptor (GR)- and androgen receptor (AR)-

CALUX cell lines ⁹⁰, and an extension of the panel with

After 24 h exposure the exposure medium was removed and 10

µl/well triton-lysis buffer was added by the MicroFlo Select.

Subsequently, the luciferase signal was measured in

a

luminometer (Infinite Pro reader coupled to a Connect stacker,

both TECAN), essentially as described before ⁹⁰

.

peroxisome proliferator activated receptor PPARα- and PPARγ

Data analysis

56, 57

CALUX assays

and thyroid receptor subtype beta (TRβ)-

CALUX ²⁹. This screening panel was completed with a range of

U2-OS-based singly transfected lines expressing a reporter gene

only, which are designed to selectively measure the activity of

main intracellular signalling pathways. This included the assays

to assess transcriptional activation by the p53 protein (p53

CALUX), the oxidative stress responsive nrf-2 pathway (Nrf2

CALUX)³², the endoplasmic reticulum stress pathway (ESRE

CALUX), and the activator protein 1 pathway (AP1 CALUX)²⁹. The

Cytotox CALUX was used to determine cytotoxicity of the

substances; it consists of human U2-OS cells stably transfected

with an expression construct constitutively expressing the

The luminometer data was analysed as follows; the average of

the triplicate wells was determined, and the average blank

(DMSO) value was subtracted. The maximum response elicited

by the reference substance was set to 100% (full receptor

activation), and the other values were scaled accordingly. The

lowest observed effect concentrations (LOECs) were

determined; for agonist assays, the LOEC was defined as the

PC₁₀value, while for antagonist assays a PC₂₀value was used.

The PC₁₀concentration was defined as the concentration where

the response elicited by the test substance equals 10% of the

maximum response of the reference substance. For antagonist

experiments, PC₂₀values were determined instead, which was

defined as the concentration where the test substance causes

20% inhibition of the basal signal elicited by the receptor

agonist. In this case, the maximum inhibition achieved by the

reference antagonist was set to 100%.

luciferase gene ³²

.

CALUX assay procedure

The CALUX cells were cultured essentially as described before

⁹⁰. U2-OS cells were routinely subcultured every 3–4 days in

growth medium consisting of DMEM (Gibco) supplemented

with 7.5% fetal calf serum, 1×nonessential amino acids (Gibco)

and 10 U/ml penicillin and 10 µg/ml streptomycin. H4IIE-CALUX

cells were routinely subcultured every 3–4 days in growth

medium consisting of αMEM (Gibco) supplemented with 10%

fetal calf serum. All cell types were maintained at 37 °C and 5%

CO₂at all times.

All CALUX assays were performed, as described in detail in the

publicly available DB ALM protocol 197, in assay medium,

consisting of DMEM without phenol-red indicator (Gibco)

supplemented with 5% DCC-stripped fetal calf serum, 1× non-

essential amino acids (Gibco) and 10 U/ml penicillin and 10

µg/ml streptomycin. For seeding, a cell suspension in assay

medium was made of 1×10⁵cells/ml (U2-OS) or 4×10⁵cells/ml

(H4IIE), and the white 384-wells plates were seeded with 30

Conclusions

FDCA esters are highly relevant biobased alternatives for

currently used benzene dicarboxylic acid esters, and can in the

case of (ortho) phthalate based plasticisers offer a sustainable

alternative. However, in contrast to the ubiquitous phthalate

family, thus far no toxicological data were available for 2,5-

FDCA esters, although a recent paper mentions that DEHF

shows no cytotoxicity in mouse 3T3-L1 cells up to 500 µM for

72h ⁹¹

.

In the present study, an in vitro reporter gene assay approach

was used to compare the activity profile of commonly used

phthalates to that of their furan-based counterparts on a broad

series of toxicological endpoints. The assay selection was aimed

8 | J. Name., 2012, 00, 1-3

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ARTICLE

‡ Footnotes relating to the main text should appear here. These

at the detection of endocrine activity, since several phthalates

are heavily scrutinised for their endocrine disrupting properties.

However, to avoid missing other relevant toxicological

endpoints, several assays able to detect various forms of cellular

stress were also included in the panel.

The results showed that the benzene dicarboxylic acid esters

were predominantly active on the endocrine assays ERα, anti-

AR and anti-PR, while effects on other endpoints such as

peroxisome proliferation (PPARs) or genotoxicity (p53) were

seen only sporadically. The results obtained for the benzene

dicarboxylic acid esters correspond well with literature,

showing that endocrine activity (mainly estrogenic and anti-

androgenic) increases with ester chain length, reaching a

maximum at C4-C6, while longer chains result in a rapid

decrease in activity.

might include comments relevant to but n_Do_Ot c_I:e₁n₀t_.1r₀a₃l^V₉tⁱ_/o^e_C^wt₉h^A_G^re^t_C^icm₀^le₄a^O₃tⁿ₄t^l₈ⁱeⁿ_A^er

under discussion, limited experimental and spectral data, and

crystallographic data.

§

§§

etc.

1.

2.

3.

4.

5.

J. H. Clark, T. J. Farmer, L. Herrero-Davila and J.

Sherwood, Green Chem., 2016, 18, 3914-3934.

M. Hong and E. Y. X. Chen, Green Chem., 2017,

19, 3692-3706.

I. A. Ignatyev, W. Thielemans and B. Vanderꢀ

Beke, ChemSusChem, 2014, 7, 1579-1593.

J. Pang, M. Zheng, R. Sun, A. Wang, X. Wang

and T. Zhang, Green Chem., 2016, 18, 342-359.

M. Volanti, D. Cespi, F. Passarini, E. Neri, F.

Cavani, P. Mizsey and D. Fozer, Green Chem.,

2019, 21, 885-896.

Furthermore, it was shown that positional isomerism in the

phthalate family has significant effects, with ortho-phthalates

being by far the most active substances.

In comparison, six of the seven furan dicarboxylic acid based

diesters tested here showed no activity in any of the 13 assays

used. Only the isobutyl derivative DIBF showed moderate

estrogenic activity on one assay, compared to much more

pronounced activities on four assays for the ortho-phthalate

analogue.

6.

7.

D. I. Collias, A. M. Harris, V. Nagpal, I. W.

Cottrell and M. Schultheis, Ind. Biotechnol.,

2014, 10, 91-105.

P. Insight, Purified Terephthalic Acid (PTA)

Properties, Production, Price, and Market,

https://www.plasticsinsight.com/resin-

intelligence/resin-prices/purified-terephthalic-

acid-pta/).

G. V. Research, Purified Terephthalic Acid

Market Size, Share & Trends Analysis Report By

Application,

https://www.grandviewresearch.com/industry-

analysis/purified-terephthalic-acid-market).

E. de Jong, M. A. Dam, L. Sipos and G. J. M.

Gruter, in Biobased Monomers, Polymers, and

Materials, American Chemical Society, 2012,

vol. 1105, ch. 1, pp. 1-13.

As a follow-up of the current study it would be relevant to also

assess whether the 2,5-FDCA based diesters are able to

interfere with steroidogenesis, since it has been suggested for

phthalates that they exert their adverse activity not only via

direct interaction with nuclear steroid hormone receptors, but

also by affecting steroidogenesis ^{61, 68, 73}. This could be assessed

using a steroidogenesis assay (H295R, OECD TG456), or with

enzyme inhibition assays for the main enzymes involved in

steroidogenesis, CYP17 and CYP19. Additionally, since plastics

often end up in the environment, it would be advisable to

investigate the biodegradation- and ecotoxicological properties

of 2,5-FDCA based diesters and derived products. For example,

Jia et al have shown that PEDF-30+ co-polyesters are

8.

9.

enzymatically degradable ⁹²

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11.

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1199.

S. K. Burgess, O. Karvan, J. R. Johnson, R. M.

Kriegel and W. J. Koros, Polymer, 2014, 55,

4748-4756.

Overall, the results presented in this paper are a strong

indication that 2,5-FDCA based diesters in general are not only

technically viable alternatives to phthalates, but also offer

significant toxicological benefits, which supports a non-

regrettable substitution.

12.

13.

S. K. Burgess, R. M. Kriegel and W. J. Koros,

Macromolecules, 2015, 48, 2184-2193.

S. K. Burgess, J. E. Leisen, B. E. Kraftschik, C. R.

Mubarak, R. M. Kriegel and W. J. Koros,

Macromolecules, 2014, 47, 1383-1391.

R. J. I. Knoop, W. Vogelzang, J. van Haveren and

D. S. van Es, J. Polym. Sci., Part A: Polym.

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Conflicts of interest

There are no conflicts to declare.

14.

15.

Acknowledgements

Funding: This work was supported by the BE Basic consortium.

Special thanks to Dr. A. E. Frissen (WFBR) for NMR analyses, and

Dr. S. Thiyagarajan (WFBR) for substance purification.

A. F. Sousa, A. C. Fonseca, A. C. Serra, C. S. R.

Freire, A. J. D. Silvestre and J. F. J. Coelho,

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Endocrine activities of phthalate alternatives; Assessing the safety profile of furan dicarboxylic acid esters using a panel of human cell based reporter gene assays

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