Download The present application is directed towards an increased

Dnr 100160
Ej för publicering förrän referenserna 7 och 8 blivit accepterade
1
Slutrapport för projekt: Förhindra allergiskt kontakteksem vid arbete med epoxiharts och
andra epoxider Dnr 100160 (Beviljningsdatum för projektet: 2011-04 -05)
Projektet har resulterat i åtta originalartiklar, en doktorsavhandling, två mastersarbeten och ett
kandidatarbete under perioden 2011-2013. En patentansökan har skickats in. Projektets resultat
kommer att presenteras och diskuteras i ett möte på Arbetsmiljöverket den 19 mars 2014 samt vid
ett seminarium inom LO:s kemikaliegrupp och nätverket för hudexponering – DENNIS, den 18
mars 2014 i Stockholm.
Background
Allergic contact dermatitis caused by epoxy resin systems (ERS) is an important occupational
health problem containing extreme sensitizers with an extensive usage. ERS are used when strong,
flexible, light weight construction materials are required. The reported prevalence of contact
allergy to ERS for occupational settings is ca 12%. The epoxy resin monomers, diglycidyl ethers
of bisphenol A (DGEBA) and bisphenol F (DGEBF) containing epoxy groups (epoxides) are
considered to be the major allergens. Phenyl glycidyl ether (PGE) known to be a strong allergen is
a reactive diluent in the ERS (Fig.1). Specific research efforts in society are made in developing
methods for dermal exposure measurements to help in the occupational hygiene activities to
diminish the exposure from work with epoxy resin. In contrast, the present research project is
directed towards an increased understanding of the underlying molecular mechanism of epoxy
resin allergy with the goal to prevent sensitization to occur.
Figure 1. Important allergens in epoxy resin systems (ERS)
There are very few reports of the sensitizing effects of monomers other than DGEBA and
DGEBF. Over all, the allergenic activity of epoxides per se is very little studied. According to
clinical and experimental experience, the sensitization potential of a compound depends on its
structural and physico-chemical properties. In our research, we have noticed that the sensitizing
effect of epoxides varies considerably depending on the structure of the compound. Thus, it is
important to identify the structural features that contribute to the skin sensitization potential of
epoxides. The obtained results could also be important considering prevention of exposure to
possible mutagenic and carcinogenic glycidyl ethers used in ERS.
The research with regard to synthesis of the final polymer, i.e., reaction between a diepoxide and a
polyamine, will add new knowledge into the area of thermosetting polymers based on epoxy
resins. Although these polymers have been on the market for several decades, little is known
about the relation between the chemical structure of the starting diepoxide and the chemical and
physical characteristics of the final polymer.
Results
Alternative structures to DGEBA and DGEBF with reduced sensitizing potency and
retained polymerization properties
It was found that the contact allergenic effects of the epoxy resin monomers depend on the
reactive epoxide groups (Fig. 2) (3). However, compounds without the epoxides are not suitable
from a technical point of view since the reactivity is necessary to get polymerization.
Dnr 100160
2
Figure 2. Contact allergenic properties depend on the reactive epoxide groups (3).
Therefore, to maintain the technical properties i.e. the polymerization capacity, we must keep the
epoxides but try to reduce their reactivity to diminish the sensitizing capacity of the epoxy resin
monomers. This is an act of balance since rectivity also increases the risk of allergenic activity.
Based on a series of experiments using design and synthesis of relevant analogues of epoxy resin
monomers, testing of reactivity with peptides, predictive allergy testing and testing of curability
we developed an overall strategy for identification of features that provide a decreased sensitizing
capacity of the bisepoxides (Fig. 3).
Figure 3: Summary of the overall strategy developed. Identification of substructures important for
decreasing the sensitizing potency but maintaining the curability of the bisepoxides.
Synthesis of DGEBF and DGEBA analogues with reduced sensitizing potency that might still be
able to polymerize was performed based on our obtained experience. The impact on chemical
reactivity, sensitization potential and polymerization properties when the structures were varied
(variation of chain lengths, removal of aromatic rings, removal of oxygen atom) was investigated.
Altered reactivity to thiols (allergenicity) and amines (curability) can be important for the
usefulness of a bisepoxide. The syntheses to obtain analogues with the desired properties are
demanding and time consuming. Not only should new synthesis routes be developed but the new
compound must be synthesized in amounts enough for testing of reactivity, polymerization
properties and sensitization potential. The results obtained are presented in Figures 4-7.
Dnr 100160
3
Figure 4. Three DGEBA analogues (1, 3, 5) and three DGEBF analogues (2, 4, 6) were
identified as promising alternatives.
A.
B.
5
Figure 5A: Reduced sensitizing potency of the
analogues 1, 2, 4, 6 compared to DEGEBA and
DGEBF (red lines far to the left) according to the
local lymph node assay (LLNA) in mice. B:
Enlarged portion showing concentrations from 0
to 0.2 M. An equal sensitizing potency was
obtained between the pairs DGEBA and DGEBF
as well as between 1 and 2. Thus, the sensitizing
potency was investigated only for one of the
compounds in the pairs 3, 4 and 5, 6 due to ethical
reasons.
Figure 6. Results from the modified LLNA
experiment using single-cell suspensions of the
local lymph nodes from individual mice.
Statistical analysis using non-parametric MannWhitney U test showed that when using 0.064 M
of either epoxide, DGEBA (□) was significantly
more potent in inducing lymph node cell
proliferation compared to analogue 5 (○)
(P<0.0001).
Figure 7. Thermogravimetric thermograms
showing % weight loss at increasing temperatures
of epoxy resins based on different epoxy resin
monomers under N2. DGEBA and analogues:
dashed lines; DGEBF and analogues: solid lines.
Dnr 100160
4
The LLNA experiments according to the ordinary protocol showed that the analogues had
decreased sensitizing potency (EC3 = 2.2 – 2.6%) compared to DGEBA and DGEBF (EC3 = 1%)
(Fig. 5). The analogues are classified as moderate sensitizers (EC3>2% w/v) in accordance with
regulatory guidelines in comparison to the commercially available monomers DGEBA and
DGEBF which are classified as strong sensitizers (0.2<EC3≤2% w/v). In the LLNA with nonpooled lymph nodes which allows for statistical calculations, DGEBA was a significantly more
potent sensitizer compared to 5 (P<0.0001) (Fig.6).
Thermal properties and suitability for polymerization of the new epoxy resin monomers were
investigated. Epoxy resins were prepared using the common curing agent triethylenetetramine
(TETA). The thermal properties of the new epoxy resins were assessed using differential scanning
calorimetry (DSC) and thermogravimetric analysis (TGA) and the results were compared to epoxy
resins based on DGEBA and DGEBF. The DSC analyses demonstrated that the enthalpy involved
in the phase transition was approximately the same for all polymers. Initial decomposition
temperature indicates the apparent thermal stability of the epoxy resin, i.e., the failure temperatures
of the resin in processing and molding, and is determined from the onset of weight loss of the
sample in a thermogravimetric (TGA) thermogram. The maximum weight loss rate (Rmax) and the
temperature at maximum rate of weight loss (Tmax) were taken from the peak values of the
differential thermograms. Epoxy resins based on 1, 2, 5, and 6 showed almost the same thermal
stability profiles as the epoxy resins based on DGEBA and DGEBF. This demonstrates that the
reactivity with TETA was approximately the same for 1, 2, 5 and 6 as for DGEBA and DGEBF and
also that the degree of cross-linking obtained was almost the same. The initial decomposition
temperature of the epoxy resins based on 3 and 4 was considerably lower than that of the other
epoxy resins, indicating that these two compounds would be less suitable as replacements for
DGEBA and DGEBF. The activation energy (Ea) of the epoxy resins based on 5 and 6 was of the
same magnitude as for resins based on DGEBA and DGEBF. The TGA data indicate that from a
polymerization point of view 5 and 6 are preferred, both giving polymers with TETA with a thermal
stability close to that obtained with DGEBA and DGEBF (Fig. 7).
Patch testing in individuals with known contact allergy to DGEBA
We currently perform patch testing with synthesized DGEBA analogues in individuals with
known contact allergy to DGEBA to reveal a possible cross-reactivity (grants from FAS Dnr
2011-1432). Each individual is tested with DGEBA and analogues 1 and 5 since 3 showed a lower
activation energy at decomposition and thus is not suitable for technical reasons. The work is
performed in collaboration with the Dermatology Clinic at Sahlgrenska University Hospital. The
compounds are applied in concentrations 1% - 1 ppm where 1% is the ordinary patch test
concentration when screening for contact allergy to epoxy resin monomers among dermatitis
patients at dermatology clinics. Of nine persons tested so far, all reacted to DGEBA while nobody
reacted to compound 5. Three persons with very strong reactions to DGEBA showed weaker
reactions to the highest concentrations of compound 1. It is a very promising result showing that
individuals allergic to DGEBA can handle compound 5 without risk of elicitation of eczema if
handled with care. This could also apply to compound 1 if the individual is not strongly allergic.
Based on the similarity in sensitization in the LLNA and in polymerization studies, compounds 6
and 2 can also be regarded as suitable alternatives. The results obtained in the clinical study show
analogues 5 and 6 as slightly more interesting compared to 1 and 2.
Peptide reactivity and in vivo testing – structure-activity relationship (SAR) studies and
possibilities to perform read-over determinations for DGEBA/F analogues
Peptide reactivity. The kinetics of the peptide reactivity and the sensitizing potency according to
the LLNA correlated well. The more reactive compound caused more depletion of the unreacted
Dnr 100160
5
peptide after mixing the compounds and peptide for the same period of time. In subsequent LLNA
experiments on the same compounds the sensitizing potency was clearly correlated to the
reactivity (Fig. 8).
LLNA studies
Increased sensitizing
potency
Peptide depletion
100
Peak Area (%)
80
60
40
b
a
c, d, e
20
f
PGE
0
0
20
40
60
80
Time (min)
Figure 8: Peptide reactivity and sensitizing potency for a series of PGE analogues.
Thus, it is possible to use peptide reactivity to screen for an initial judgement of which alternatives
within a series of compounds that could be of interest to further investigate for technical
properties. Only those providing an appropriate polymer quality should then be investigated in
depth for skin sensitizing potency.
In vitro testing. Using the same series of PGE analogues as described in Figure 8, a cell based
assay “KeratinoSens™ was performed. A strong correlation was obtained between the LLNA data
and the luciferase-inducing concentrations and the cytotoxicity (Fig. 9 A) (2).
A
B
C
a
g
g
DGEBA/F
DGEBA/F
h
h
Figure 9. Correlation between LLNA and KeratinoSens™ test results (2)
The KeratinoSens™ method was used to predict the sensitizing potencies of two other monomeric
epoxides (g, h) which were analogues to those in the first series. The prediction was in good
agreement with the values obtained in the subsequent LLNA. However, the sensitizing potencies
of DGEBF and DGEBA (the bidentate analogues to the PGE analogues (Fig. 1)) were
overpredicted in the KerationSens™ assay based on the correlation data obtained for the
monomeric PGE analogues (Fig. 9 B). Also the cytotoxicity data measured in the KerationSens™
assay gave the same pattern (Fig. 9 C). This indicates that accurate potency estimation by readacross based on the in vitro data could be restricted to a relatively narrow applicability domain
and does not allow read-across correlation between the monoepoxides and the bidentate
epoxides. It would therefore be of great interest in the future to also investigate the correlation
between the DGEBA/F analogues 1-6 to see if the method can be used as a screening method to
identify low allergenic bidentate epoxides.
In this subproject we have collaborated with Dr Andreas Natsch at Givaudan Schweiz AG, to
investigate the SARs for our produced series of epoxides and epoxy resin monomers using the
Dnr 100160
6
KeratinoSens™ assay. The assay is based on a stable transgenic keratinocyte cell line with a
luciferase gene under control of an antioxidant response element. The Nrf2-Keap1-ARE regulatory
pathway is a toxicity pathway which is activated by cysteine-reactive skin sensitizers. It is one of
the most promising in vitro test methods for replacement of animal experiments in predicitive
testing of sensitization potential. It has recently been recommended by the European Union
Reference Laboratory for alternatives to animal testing (EURL ECVAM) as a suitable component
of integrated hazard and potency assessment for alternatives to animal testing.
In dept structure-activity relationship (SAR) investigations of epoxides
Methods used: Design and synthesis of relevant epoxides and compounds able to form epoxides due
to bioactivation in the skin or air oxidation outside skin have been performed. These compounds
have been investigated using metabolic activation in vitro, kinetics in experiments with model
peptides, computational chemistry, and predictive allergy testing in vivo and in vitro.
We have noticed that the allergenic activity of epoxides strongly depends on the total structure of
the molecule. As seen for the epoxy resin monomers, the terminal epoxides are very reactive and
strongly sensitizing, in which cases we have managed to reduce the allergenicity by manipulating
the overall structure of these compounds (see above). In order to further investigate how the
sensitizing potency is influenced by structural changes of compounds containing epoxides
additional deeper investigations were performed. Epoxides are commonly formed as metabolites
due to bioactivation in the skin and also through autoxidation when unstable compounds are
exposed to air. Therefore, these activation pathways were also studied to be able to predict
increased risks of sensitization from certain compounds due to formed epoxides.
One designed series (A) of epoxyaldehydes and another series (B) with their corresponding
epoxyalcohols were investigated (Fig. 10). SAR analysis with this precision has never been
undertaken on structures that are containing epoxides or are able to form epoxides within or
outside the skin.
Our results showed that even small changes in the chemical structure result in differences in
sensitizing potency. In series A, the general SARs identified conjugated aldehydes and 2,3epoxyaldehydes (compounds 7, 8, 9, 10) as sensitizers while non-conjugated aldehydes and
aliphatic epoxides (compounds 11, 12) had low reactivity inducing a low sensitizing potency.
Series B of epoxyalcohols was used to study the impact of activation. For this purpose also
cinnamic alcohol was investigated (Fig. 10 series C). It was found that both air oxidation and
metabolic activation occurred forming among others strongly sensitizing epoxides but also
epoxides that were not sensitizing. In agreement with the results from the studies on the aldehydes
in series A, the importance of conjugation to an active site was important for the sensitizing
potential of the epoxides. Thus, plausible bioactivation/autoxidation of a non-conjugated double
bond to form a non-conjugated epoxide is not sufficient to significantly increase the sensitizing
potency of a compound. Our results reinforce the importance of the double bond conjugation in
contact allergy (4-6, 8).
The results show that the strategies used to modify the DGEBA and DGEBF structures to obtain
less sensitizing epoxy resin monomers are valid in a broader perspective of skin sensitization
caused by epoxides.
Dnr 100160
7
Fig 10: SAR studies on the impact on the sensitizing potency depending on the conjugation of epoxides to
other active sites in the structure (series A). In series B and C bioactivation and/or activation outside skin by
autoxidation (air oxidation) of compounds able to form epoxides are investigated (4-6, 8).
We observed a good correlation between the chemical reactivity and the sensitizing potency seen
in the LLNA. The different series of epoxides were also tested in vitro in the KeratinoSens™
assay to further explore the possibilities and limits of in vitro predictions of the sensitization
potential of epoxides. Moreover, computational chemistry was used to explain the mechanisms
behind the results obtained in these studies.
Based on the achieved results the prediction of the sensitization potential of new epoxy resin
monomers and other epoxides could rely more on chemical reactivity, in vitro data and SAR
analyses saving animal testing for confirmation as a final step for the most interesting compounds
in the future.
Studies on cytotoxicity and investigations of a possible induction of DNA mutations by
glycidyl ethers
Cytotoxicity tests were performed with PGE, DGEBA, DGEBF and various analogues both in our
own laboratory and in collaboration with Andreas Natsch using KeratinoSens™. The results
obtained from the two assays are in accordance although the transfected HaCaT cells in the
KeratinoSens™ assay and the Chinese hamster lung fibroblast cells (V79-4) have different
sensitivity to the compounds. The results show that bidentate epoxides (DGEBF and DGEBA) in
general are more cytotoxic than the monomers (PGE analogues). Moreover, the length of the
carbon chain affects the compounds cytotoxicity, the longer the chain the less toxic is the
compound. The toxicity decreases when the oxygen in the beta-position to the epoxide is
removed. The cytotoxicity is also highly dependent on the aromaticity of the sixmembered rings.
The differing cytotoxic profiles of the biaromatic and monoaromatic compounds are potentially
due to differing mechanisms of cell death. The biaromatic compounds can target nuclear
receptors, such as the nuclear transcription factor peroxisome proliferator-activated receptorgamma (PPARγ), where complementarity between ligand and receptor is the most important
feature for activity, rather than the presence of reactive groups that can form covalent bonds. The
Dnr 100160
8
biaromatic compounds DGEBA, DGEBF and its mono-methylated analogue could also
theoretically bind to and damage DNA via their terminal epoxide group(s) (3). A protocol for in
vitro mammalian chromosome abberation test based on the cell line V79-4 was also started. The
OECD guideline description is general and could not be used directly for the investigations of our
compounds making the optimization of the method very time consuming. As we had difficulties in
identifying possible cooperative laboratories better equipped for these investigations this part was
not continued.
Insatser som skett och planeras för att resulatet ska komma till praktisk använding i
arbetslivet
1. The results obtained will be presented in a seminar arranged by “LO’s kemikaliegrupp och
nätverket för hudexponering – DENNIS”, Stockholm March 18th 2014. Hopefully, this
will lead to an engagement from LO to work for a change since the present epoxy resin
monomers are strong work- related skin sensitisers.
2. A presentation and discussion of the results obtained in the project will take place March
19th 2014 at Arbetsmiljöverket. It has been shown that it is very difficult to protect against
epoxy resin allergy and allergic contact dermatitis only by regulatory work and
occupational hygiene. Therefore, the people in charge of the regulation are very interested
to get information and discuss what we have achieved in this ground-breaking work. Is it
possible to act based on the material we present or is it nessecary to continue along the
road to get more important material and information? Another question that will be
discussed is the possibility to use starting materials free from the hormone mimetic
bisphenol A. This was a question that there was no time to focuse on in the present work
since the importance of the strong contact allergenic effect of the bisepoxides was
prioritized.
New original articles
1. Niklasson I B, Delaine T, Luthman K, Karlberg A-T. Impact of a heteroatom in a
structure-realtionship study on the analogues of phenyl glycidyl ether (PGE) from epoxy
resin systems. Chem Res Toxicol 2011: 24: 542-548.
2. Delaine T, Niklasson I B, Emter R, Luthman K, Karlberg A-T, Natsch A. Structure-activity
relationship between the in vivo skin sensitizing potency of analogues of phenyl glycidyl
ether and the induction of Nrf2-dependent luciferase activity in the KeratinoSens in vitro
assay. Chem Res Toxicol 2011: 24: 1312- 1318.
3. O'Boyle N M, Delaine T, Luthman K, Natsch A, and Karlberg A-T. Analogues of the epoxy
resin monomer diglycidyl ether of bisphenol F: Effects on contact allergenic potency and
cytotoxicity. Chem Res Toxicol 2012: 25: 2469-2478.
4. Delaine T, Hagvall L, Rudbäck J, Luthman K, Karlberg A-T. Structure-Activity
Relationships for epoxyaldehydes – Importance of conjugation for skin sensitization.
Chem Res Toxicol 2013: 26: 674−684.
5. Niklasson IB, Delaine T, Islam M N, Karlsson R, Luthman K, Karlberg A-T. Cinnamyl
alcohol oxidizes rapidly upon air exposure. Contact Dermatitis 2013: 68: 129–138.
6. Niklasson IB, Ponting D, Luthman K, Karlberg A-T. Bioactivation of Cinnamic Alcohol
Forms Several Strong Skin Sensitizers. Chem Res Toxicol dx.doi.org/10.1021/tx400428f.
7. O’Boyle NM, Niklasson IB, Tehrani-Bagha AR, Delaine T, Holmberg K, Luthman K,
Karlberg A-T. Improvements in epoxy resin polymer systems: new monomers with
reduced skin sensitizing potency. Submitted
Dnr 100160
9
8. Delaine T, Ponting D, Niklasson IB, Emter R, Hagvall L, Norrby P-O, Natsch A, Luthman
K, Karlberg A-T. Epoxyalcohols: bioactivation and conjugation required for skin
sensitization Manuscript
Doctoral, Master and Bachelor Theses
Niklasson Belogorcev I. Epoxides as Contact Allergens. Formation, Sensitising Potency and
Structure-Activity Relationships DOCTORAL THESIS Göteborgs Universitet, October 21,
2013. ISBN 978-91-628-8835-0. Available online at: http://hdl.handle.net/2077/33972
Westerberg D. A structure activity relationship study of epoxides (epoxy resins) as contact
allergens. Degree project for master science 60 hec 2011: 26 University of Gothenburg.
Bolbaran K. Evaluation of the sensitizing effect of the epoxy groups in DGEBF. Synthesis and
analysis of a DGEBF analogue. Master thesis in pharmacy 30 hec 2012 University of
Gothenburg.
Helgegren K. Peptidreaktivitet av kanelalkohol och dess metaboliter Examenskurs i Farmaci 15
hp (FAR200) VT 2013
Patent Applicaton
Resin compositions as specified in UK patent application no. AWE/P211125GB 2013
Presentations in Conferences
Niklasson IB, Delaine T, Emter R, Luthman K, Karlberg AT, Natsch A: SAR study on
analogues of phenyl glycidyl ether in the KeratinoSens assay. The 23rd Meeting of the
ERGECD (European Research Group on Experimental Contact Dermatitis) Gothenburg June
9 – 11, 2011.
Delaine T, Rudbäck J, Hagvall L, Luthman K, Karlberg A-T. (2012) A stucture activity
relationship study of geranial derivatives. In Contact Dermatitis, 11th Congress of the
European Society of Contact Dermatitis p 49, Malmö, Sweden.
O’Boyle N, Niklasson I, Delaine T, Luthman K, Natsch A, Karlberg A-T. (2012) StructureActivity Relationships of the Epoxy Resin DGEBF. In Contact Dermatitis, 11th Congress of the
European Society of Contact Dermatitis p 49, Malmö, Sweden.
Delaine, T., Rudbäck, J., Hagvall, L., Luthman, K., and Karlberg, A.-T. SAR studies on
geranial: skin sensitization and reactivity. The 24th Meeting of the ERGECD (European
Research Group on Experimental Contact Dermatitis) Oct. 10 – 12, 2012, Trier, Germany.
Delaine, T., Rudbäck, J., Hagvall, L., Luthman, K., and Karlberg, A.-T. SAR studies on
geranial: skin sensitization and reactivity. The Science and Technology Day 2012, Chalmers
Technology University and University of Gothenburg, Göteborg.
Niklasson IB, O’Boyle NM, Tehrani-Bagha AR, Delaine T, Holmberg K, Luthman K, Karlberg
A-T. Improvements in epoxy resin polymer systems: new monomers with reduced skin
sensitizing potency. The 25th Meeting of the ERGECD (European Research Group on
Experimental Contact Dermatitis) June 12 – 13, 2014, Paris, France (to be presented).
Ponting D, Delaine T, Niklasson IB, Emter R, Hagvall L, Norrby P-O, Natsch A, Luthman K,
Karlberg A-T. Epoxyalcohols: bioactivation and conjugation required for skin sensitization. The
25th Meeting of the ERGECD (European Research Group on Experimental Contact Dermatitis)
June 12 – 13, 2014, Paris, France (to be presented).
Dnr 100160
10
Ethical considerations
We perform the local lymph node assay (LLNA) in mice on a regular basis with ethical
permission from Göteborgs djurförsöksetiska nämnd (Dnr 53-2009 and Dnr 39-2012). The
method is the preferred method within the OECD Guidelines for predictive testing of skin
sensitizers. Ethical permission to patch test research persons with known contact allergy to
DGEBA was achieved from Regionala Etikprövningsnämnden i Göteborg (Dnr 607-13).
Communication
See “New articles and reports published or in press” and “Presentations in conferences”.
A press release from University of Gothenburg with regard to Ida B Niklasson’s dissertation in
October 2013.
Presentations and discussions have been held within Gothenburg Centre for Skin Research
(SkinResQU) which is an interdisciplinary centre within University of Gothenburg and Chalmers
University of Technology involving also the industry in the area of Gothenburg (e. g.
AstraZencea, SCA and AKZO).
A presentation of the results obtained in the project will take place in a seminar arranged by LO’s
kemikaliegrupp och nätverket för hudexponering – DENNIS, Stockholm March 18th 2014.
A presentation and discussion of the results obtained in the project will take place March 19th
2014 at Arbetsmiljöverket.