Keywords

REACH, read-across, RDT, systemic toxicity, NAM, uncertainty, mode of action

Objective

In-vivo evidence indicates that short branched carboxylic acids –such as the drug valproic acid (VPA)– induce microvesicular liver steatosis in animals and humans. Would an analogue compound induce the same adverse outcome? To investigate this, we investigated a category of branched carboxylic acids by characterizing their toxicology profile with an in-vitro test battery. This study aimed to explore how far biological data from NAMs can be used in a read-across scenario to prove a shared mode of action and similar toxicokinetics.

Testing Strategy

Based on available in-vivo data of three structurally related analogues, the target compound 2-ethylbutyric acid (2-EBA) was assumed to be a liver toxicant with special concern for hepatic steatosis.
Toxicokinetics and toxicodynamics properties of 2-EBA were analysed and compared to a category of structurally highly similar carboxylic acids that vary only with regard to their aliphatic side chain length.
To characterize the toxicodynamics, published signalling pathways/AOPs leading to steatosis were compiled from literature and described in an AOP network. Two high-throughput reporter gene assays (CALUX and stress pathway responses in HepG2) were used to measure some of the described molecular initiating events (MIEs). Furthermore, three liver models were used to measure lipid accumulation as a direct in-vitro surrogate for in-vivo steatosis. It was shown that the number of activated MIEs and the induction of lipid accumulation increased with the side chain length of the tested carboxylic acids, whereas short-chain analogues like 2-EBA remained inactive up to the highest tested dose in vitro. The low activity of short-chain analogues was in good agreement with known in-vivo animal data.
To characterize the toxicokinetics, a PBPK model was developed and parameterized with in-vitro ADME data. Bioavailable concentrations were predicted and QIVIVE extrapolation was performed using PBPK-based reverse dosimetry.
Overall, the NAM data proved the initial read-across hypothesis to be valid by showing a consistent trend concerning toxicokinetics and toxicodynamics within grouped compounds.

Publications

van Vugt-Lussenburg et al. 2018 [link]; Leist et al. 2017 [link]; Tolosa et al. 2017 [link]; Fisher et al. 2018 [link]; Gadaleta et al. 2018 [link]; Wink et al. 2018 [link]; Niemeijer et al. 2018 [link]; Escher et al. 2022 [link]; Escher et al. 2019 [link]; Rovida et al. 2021 [link]

OECD IATA report

Report [link]; Annex II [link]; Annex III [link]

Contributors

ITEM, UL, UM, RISE, INERIS, BDS, TNO, DC, IRFMN, SYMCYP, VUA, UOF, UNIVIE, HULAFE, UCPH, CRX, IS, UHEI, L’OREAL, LHASA.

Keywords

REACH, read-across, DART

Objective

2-Methylhexanoic acid (MHA) is mostly used as a food additive. Developmental and reproductive toxicity (DART) data for MHA were taken to be lacking. The aim was to find structural analogues for which DART data is available and to attempt to show that a NAM-based read-across would rightly predict the DART profile of MHA.

Testing Strategy

MHA and its selected chemical analogues were tested in a battery of in-vitro tests relevant to developmental toxicity, including the zebrafish embryotoxicity test (ZET), the mouse embryonic stem cell test (mEST), an iPSC-based neurodevelopmental model (UKN1), and a series of CALUX reporter assays. Also, toxicokinetic models were developed and applied to calculate effective cellular concentrations and associated in-vivo exposure doses.
The applied NAM-based read-across was used to predict the in-vivo developmental toxicity of MHA from the developmental toxicity data of selected source chemicals. This data would also allow us to further explore the relationship between structure and developmental toxicity within this series of aliphatic carboxylic acids. We also investigated the potential to inhibit histone deacetylase in these test models, as this enzyme is postulated to be the molecular target initiating neural tube defects, an observed developmental toxicity for some of these analogues. NAM results showed that VPA, PHA, EHA, and 4-ene-VPA were correctly predicted as in-vivo developmental toxicants, and EBA and DMPA as non-developmental toxicants. The results indicate that MHA, which is a non-developmental toxicant, cannot be classified as fully negative based on this NAM-based read-across.

Publications

Shinde et al. 2016 [link]; Waldman et al. 2016 [link]; van Vugt-Lussenburg et al. 2018 [link]; Fisher et al. 2017 [link]; Simeon et al. 2020 [link]; Escher et al. 2019 [link]; Brotzmann et al. 2021 [link]; Koch et al. 2021 [link]; Escher et al. 2019 [link]; Rovida et al. 2021 [link]

OECD IATA report

Report [link]; Annex II [link]; Annex III [link]

Contributors

TNO, UKN, UM, INERIS, BDS, IFADO, SIMCYP, LUMC, ROCHE, UHEI, NC3Rs, UNIVIE, RISE.

Keywords

REACH, read-across, RDT, systemic toxicity, NAM, metabolomics

Objective

The phenoxy acetic/propionic acid herbicides form a group of structurally similar herbicides are known to induce similar systemic toxicity in rat studies. The main toxicological effects observed are liver toxicity due to peroxisome proliferation as well as kidney toxicity associated with oxidative stress. Since the liver effects are mediated through activation of peroxisome proliferation, other non-herbicidal peroxisome proliferators – such as phthalates or pharmaceutical peroxisome proliferators– were tested to evaluate the NAMs in terms of general detection of peroxisome proliferation.

Testing Strategy

Data from CALUX assays, HepG2 metabolomics, and stress responses showed that the biological effects observed can be linked to the toxicological mode of action in the liver. The metabolite profile indicated changes in lipid metabolism as had been seen for peroxisome proliferators in vivo. The data for the herbicides were well in line with published in vivo findings (van Ravenzwaay et al., 2016) and demonstrated that the in vitro data might be used to substantiate a read-across based on in vitro methodologies. PBPK modelling for the compounds and test substances will be performed.

Publications

Van Ravenzwaay et al. 2016 [link]; Escher et al. [link]

Contributors

BASF, RISE, KI, BDS, ITEM, DC, MUI, VU.

Keywords

REACH, read-across, RDT, DART, NAM, AOP

Objective

A large group of pesticides used in the agrochemical sector targets mitochondria functionality. The synthetic strobilurin fungicides, derived from the naturally occurring strobilurin A and B, belong to this group. Strobilurin fungicidal mode of action is known. The chemical binds to the quinol oxidation site of cytochrome b of complex III (CIII) of the mitochondria. The degree of in vivo inhibition of the mitochondrial respiratory system depends on the respiratory activity and thereby tissues like the brain can be more susceptible if exposed. Evidence of potential neurotoxicity was also shown in in vitro studies. The objective of this case is to characterise potential CIII-mediated neurotoxicity of target compound azoxystrobin by NAM-enhanced read-across. The objective is to answer the question: ‘Can the absence of a neurotoxic potential of azoxystrobin-mediated inhibition of complex III of mitochondria be predicted by toxicodynamic and toxicokinetic NAM data?’

Testing Strategy

Source compounds selected in this case study are other strobilurin fungicides. Existing regulatory in vivo data was collected for the source and target compounds with a focus on ADME, neurotoxicity, as well as target organ toxicity data. Source compounds showed no signs of neurotoxicity, neither in neurotoxicity studies nor in other RDT studies. Overall, based on the generated data on kinetics and effect data, there is no evidence for a stronger neurotoxic potential of azoxystrobin mediated by a CIII inhibitory mode of action as compared to the source compounds. Since source compounds did not elicit neurotoxicity in vivo, we concluded that also the target compound azoxystrobin is not a neurotoxicant.

Publications

Wink et al. 2018 [link]; Hiemstra et al. 2019 [link]; Delp et al. 2018 [link]; Terron et al. 2019 [link];  van der Stel W et al. 2020 [link]; Fisher et al. 2017 [link];  Delp et al. 2019 [link]; Hemmerich et al. 2020 [link]; Troger et al. 2020 [link]; Delp et al. 2019 [link]; Delp et al. 2018 [link]; Delp et al. 2021 [link]; Hemmerich et al. 2020 [link]; van der Stel et al. 2021 [link]; van der Stel et al. 2020 [link]; Troger et al.  et al. 2020 [link]; Vrijenhoek et al. 2022 [link]; Escher et al. 2019 [link]; Rovida et al. 2021 [link]

OECD IATA report

Report [link], Annex I [link]

Contributors

UL, UKN, BASF, Unilever, RISE, KI, BDS, ITEM, VU, UPF, UNIVIE, HULAFE, CRX, DTU, L’Oreal

Keywords

REACH, read-across, RDT, DART, NAM, AOP

Objective

There is an anticipated hazard for agrochemicals that inhibit complex I of the mitochondrial respiratory chain to cause toxicity to the nigrostriatal neurons leading to symptoms that reflect Parkinson's disease. This effect has been comprehensively described in an OECD-validated AOP (Terron et al. 2019). This case study aims to assess the application of an AOP approach in a read-across safety assessment of structurally related mitochondrial complex I inhibitors, deguelin, and rotenone.
Epidemiological studies indicate that exposure of workers to rotenone is statistically associated with an increased incidence of Parkinson's disease; moreover, rotenone is known to induce parkinsonian phenotypes in experimental animals. Therefore, rotenone was used as the source substance. Deguelin can induce parkinsonian-like phenotypes in rats. Whether deguelin has such a parkinsonian hazard liability in humans is currently unclear and therefore deguelin was the target substance for this case study.

Testing Strategy

Based on the AOP, structural modelling approaches were applied to define the binding of rotenone and deguelin to mitochondrial complex I, the molecular initiating event of the AOP. Then, previously established and routinely applied assays that reflect the various key events in this AOP were defined. Multiple human-based in vitro test systems to monitor mitochondrial effects of rotenone and deguelin were additionally integrated. To assess the effect on neuron toxicity, high-content imaging approaches to measure degeneration of neuronal neurites were applied. Finally, both biokinetic evaluation of cellular exposure to rotenone and deguelin, as well as PBPK modelling, were used to evaluate the relevance of observed effects in vitro towards a likely in vivo exposure situation. Both substances inhibit complex I activity and cause mitochondrial dysfunction. Similarly, rotenone is also more potent than deguelin in disrupting neurites.
The target compound deguelin displayed a similar mode-of-action as rotenone, but with minor potency. The case study showed how practical application of an AOP approach through the integration of specific technologies and test systems might find broader application in a read-across safety assessment of structurally related substances.

Publications

Wink et al. 2018 [link]; Hiemstra et al. 2019 [link]; Delp et al. 2018 [link]; Terron et al. 2019 [link];  van der Stel W et al. 2020 [link]; Fisher et al. 2017 [link];  Delp et al. 2019 [link]; Hemmerich et al. 2020 [link]; Troger et al. 2020 [link]; Delp et al. 2019 [link]; Delp et al. 2018 [link]; Delp et al. 2021 [link]; Hemmerich et al. 2020 [link]; van der Stel et al. 2021 [link]; van der Stel et al. 2020 [link]; Troger et al.  et al. 2020 [link]; Vrijenhoek et al. 2022 [link]; Escher et al. 2019 [link]; Rovida et al. 2021 [link]

OECD IATA report

Report [link]; Annex I [link]

Contributors

UL, UKN, BASF, Unilever, RISE, KI, BDS, ITEM, VU, UPF, UNIVIE, HULAFE, CRX, DTU, L’Oreal

Keywords

RDT, liver toxicity, NAM

Objective

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. Also, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI concerning oral doses and blood concentrations.
Despite intensive research, it is currently not possible to reliably predict whether repeated exposure to a certain dose of a chemical leads to an increased risk of hepatotoxicity or can be considered harmless.

The main objectives of this case study are:

1. To predict blood concentrations of chemicals that cause an increased risk of hepatotoxicity and to identify concentration ranges that can be considered harmless.
2. To predict oral doses of chemicals that cause an increased risk of hepatotoxicity (reverse modelling).

Testing Strategy

The developed novel in vitro/in silico method can be used to estimate DILI risk if the maximal blood concentration  of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations.
To systematically optimize the in vitro system, two novel test performance metrics were introduced: the toxicity separation index (TSI), which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI), which measures how well hepatotoxic blood concentrations in vivo can be estimated.
In vitro test performance was optimized for a training set of compounds, based on TSI and TEI, to identify the most performing concentrations and incubation time. Additionally, metrics were moderately improved by adding gene expression to the test battery and evaluation of pharmacokinetic parameters were evaluated. With a support vector machine-based classifier, the cross-validated sensitivity, specificity, and accuracy for hepatotoxicity prediction were highly relevant, respectively 100, 88, and 93%. Currently, the novel in vitro/in silico method is validated in a set of >200 test compounds. A constantly ongoing activity in this project is the identification of key events of DILI that can be tested in vitro in order to integrate them into our in vitro test battery.

Publications

Albrecht et al. 2019 [link]; Gu et al. 2018 [link]; Leist et al. 2017 [link]; Sachinidis et al. 2019 [link]; Ghallab et al. 2018 [link]; Jansen et al. 2016 [link]; Kappenberg et al. 2021 [link]; Gupta et al. 2020 [link]; Kappenberg et al. [link]; Krebs et al. 2020 [link]; Hengstler et al. 2020 [link]; Fasbender et al. 2020 [link]; Ruoß et al. 2020 [link]; Campos et al. 2020 [link]; Ghallab et al. 2019 [link]

Contributors

IFADO, UL, UKN, UM, UNILEVER, RISE, KI, CE, TNO, ITEM, DC, SimCyp, EBI, UNIVIE, UCPH, TissUse, JHSPH, IS, L’Oreal.

Keywords

Read-across, RDT, REACH, respiratory disease, NAM

Objective

The alpha-diketone diacetyl (2,3- butadiene) is known to induce the so-called “popcorn lung” condition. This disease was frequently observed among microwave popcorn manufacturing employees who inhaled the butter flavour vapour of diacetyl. This condition represents an obstructive pulmonary disease in which the airway epithelium is the initial target of injury. The disease is called bronchiolitis obliterans and is characterized by fibroproliferative airway lesions.
In this case study, the effects of alpha-diketone have been investigated together with two structurally similar groups (β and γ diketone) which seem to have a different mode of action based on available information.
This approach can show how far selected NAMs are also able to differentiate the α, β, and γ diketone specific toxicity in the selected in vitro test systems. The three groups of structurally related compounds are sufficiently data-rich to allow the exploration of NAMs to adequately perform chemical hazard identification and herewith replacing the regulatorily required animal study. EU-ToxRisk tools were applied to predict toxicokinetics properties of the target compounds (e.g. PBPK and metabolism). Furthermore, we explored to which extent chemical similarity can be enriched by biological data, e.g. derived from omics investigations or cellular readouts.

Testing Strategy

All case study compounds were evaluated in fluorescent cell stress reporter assay high-throughput screens. Alpha diketones are known to have a high electron affinity and can transfer electrons and, thus, can induce ROS and oxidative stress. Αlpha-diketones as well as the beta-diketone 2,4-pentanedione were shown to induce activation of oxidative stress and DNA damage response pathways. The air-liquid interface (ALI) exposure setup for diacetyl and analogues was preferred in further experiments since dose control is ensured by online measurements of volatile test atmosphere employing FT-IR spectroscopy. Cultures of primary airway epithelial cells (PBECs) as well as human precision cut lung slices (PCLuS) were set up for ALI exposures. Additionally, transcriptomics as well as cytokine data are currently being analysed. Kinetic studies with diacetyl aiming at the determination of diacetyl uptake and intracellular concentrations are ongoing.

Contributors

ITEM,  UL,  UKN, DC, SimCyp, LUMC, Mario Negri IRCCS

Keywords

Cosmetics regulation, industry-sponsored case study, read-across, ab-initio

Objective

This case study aims at establishing a proof-of-concept approach for the use of NAM-enhanced read-across in a next-generation risk assessment context. Parabens are esters of para-hydroxybenzoic acid (pHBA) that are widely used as preservatives in diverse product sectors including agrochemical, pharmaceutical, food, and cosmetics. In this case study, the use of in silico information, in vitro toxicodynamic (toxicogenomics, endocrine activity) and toxicokinetic data were combined to support biological similarity among analogues and establish potency trends to inform the selection of the best source chemical from within a category. This approach, along with consideration of aggregate exposure, was here used in an example safety assessment of low-toxicity chemicals. The chemical category under consideration is short linear chain parabens.

Testing Strategy

The external dermal cosmetic exposure to four parabens of primary interest (methyl, ethyl, propyl, and butyl) was analysed. Deterministic external exposure values were used to predict human plasma systemic exposure to the compounds.
A comprehensive human in vitro dataset was obtained, which enabled PBPK modelling of systemic plasma concentrations of the parent compounds following dermal exposure in humans. This data comprised human skin penetration data; biotransformation data in skin, liver, enterocytes (Caco-2 cells), and plasma, which characterized major metabolites and intrinsic clearance rates; plus plasma protein binding. The parabens are readily hydrolysed to p-hydroxy benzoic acid in presence of metabolic activation. The human ADME parameters were used to develop PBPK-based multi-compartment models of human systemic plasma parabens concentrations following dermal external exposure to compounds.
Parabens were shown to lack specific target organ toxicity at very high doses in repeated dose toxicity studies. However, they are active in some uterotrophic assays and were assigned a conservative point of departure (PoD) by the Scientific Committee for Consumer Safety. Also, data have emerged in the literature that show parabens exhibit low activity in in vitro assays relevant for endocrine activity. These in vitro assays suggest that parabens (the parent compounds but not the main metabolite) possess very weak activity on some nuclear receptors involved in endocrine homeostasis, many orders of magnitude lower than natural estrogens/androgens. In this case study, NAMs have been applied to explore this oestrogenic activity as an approach to inform on biological similarity and relative potency of the category members. This relative potency is then used to adjust the conservative PoD derived from butylparaben to carry out a theoretical risk assessment for propylparaben based on read-across.
For the read-across risk assessment, a margin of safety was derived using internal concentrations: margin of internal exposure –MoiE- which is protective of human health.
In conclusion this case study demonstrated the value added by NAMs in identifying, characterizing analogues, in informing similarities/differences on toxicokinetic and toxicodynamic properties and in the calculation of the internal margin of safety.

Publications

Berggren et al. (2017) [link]

OECD IATA report

Report [link]

Contributors

L’OREAL, UL, P&G, BASF, Unilever, Clariant, INERIS, CE, BDS, TNO,  ITEM, LHASA, University of Liverpool.

Keywords

REACH, RDT, LOEL

Objective

The majority of toxic chemicals cause their most sensitive toxicological effects in liver, kidney or the respiratory tract in studies with repeated exposure. Nevertheless, there are compounds that show other types of toxicity. However, even if unaffected at the lowest observed effect level (LOEL), there is a 95% probability that effects on liver or kidney are observed in vivo at the next higher dose. The objective of this case study is to gain knowledge on how toxicological data derived from neuro, lung, liver, or kidney in vitro models can be used to predict the LOEL of test compounds and whether an additional safety factor will be needed.

Testing Strategy

In this case study, compounds that show specific toxicity effects in target organs different from the EU-ToxRisk test method panel have been selected for testing. In this way, knowledge will be gained in how far we can use the results of neuro/lung/liver or kidney models to predict the LOEL of such compounds. This case study applies cross-system testing and a 3-tiered approach: hypothesis generation, test cellular assays, PBPK modeling. Finally, data will be analyzed and the next tests will be refined based on the information gained in the first phase.

Contributors

ITEM, UL, BDS, VU, LUMC, HULAFE, IRFM, CRX, SIMCYP.

Keywords

RDT/DART ab initio

Objective

Currently, for an enormous proportion of REACH registrations, no safety information is available. This particularly applies to 'lower tonnage' chemicals. Here there is a need to prioritize chemicals based on the likelihood of safety issues. The main objective of the case study is to demonstrate the overall feasibility of high-throughput NAM hazard-based information to identify substances of high-toxicity concern.

Testing Strategy

The driving hypothesis is that only toxic substances will strongly impact cell biology. This would be reflected by the activation of specific transcriptional networks that will provide mechanistic mode-of-action information. We anticipate that high-throughput NAM approaches could provide this mechanistic insight and thereby allow to rank substances based on their mode-of-action.
To demonstrate the feasibility to prioritize chemicals for further safety assessment, a training set of high tonnage compounds, for which in vivo safety data is available, will be used. These compounds will all be tested in dose-response scenarios in high-throughput test systems to assess the impact on the cell biology, and include: in silico approaches (metabolism, QSAR, target prediction); high-throughput reporter assays; targeted transcriptomics in selected cell types. All compounds will be ranked and we will relate our findings to the available in vivo adversity and potency information.

Contributors

UL, UKN, BASF, Unilever, BDS, TNO, ITEM, EWC, IRFMN, LUMC, UPF, EMBL-EBI, UNIVIE, HULAFE, CRX, L’Oreal, LHASA, VU, RISE.

Keywords

DART, OECD DNT TG426, IATA

Objective

In 2013, the European Food Safety Authority (EFSA) published a scientific opinion raising concerns regarding the DNT potential based on newer mechanistic in vitro data and insufficient hazard characterization based on available in vivo DNT tests of two neonicotinoid pesticides, acetamiprid and imidacloprid. This action has resulted in a proposal for revision of the human reference values. The objective of the case study is the development of an IATA to support hazard identification/characterization of selected neonicotinoids and to validate the biological plausibility and sufficiency of data from available datasets.

Testing Strategy

Data on neonicotinoid compounds in a combination of NAMs have been contextualized in an IATA. The selected NAMs span from molecular receptor docking, gene expression, assays covering endpoints involved in key neurodevelopmental processes as well as zebrafish embryos, and PBPK modelling.
Many endpoints of the in vitro test battery were not affected by high concentrations (up to 100 µM) of neonicotinoids. However, the functional properties of neurons were affected by a subset of test compounds in similar directions as observed with the well-known DNT toxicant nicotine. The case study now focuses on a more detailed molecular characterization of the observed hazard and potency estimates. Results across the testing battery expand the knowledge of available in vivo data and can, therefore, contribute to a better risk assessment. This approach aligns with the OECD program on „OECD DNT guidance on the interpretation of in vitro DNT data that can be used in an IATA", a global effort to test up to 120 DNT compounds in an in vitro test battery, generation of a database, and generation of IATA cases supporting endorsed guidance.

Publications

Loser et al. 2021 [link]; Loser et al. 2021 [link]; Loser et al. 2021 [link]

Contributors

DTU, UKN INERIS, BIOT, SIMCYP, UHEI, VU, SU.

Keywords

REACH, RTD, hepatotoxicity, nephrotoxicity, metabolism, kinetics

Objective

Halogenated alkenes are a family of chemicals that have been widely used in various industrial applications, like pesticides, solvents, and dry cleaning, and pose a significant hazard to human health. Occupational investigations and animal studies have indeed demonstrated that halogenated alkenes – especially trichloroethylene (TCE), perchloroethylene (PER), and hexachlorobutadiene (HCBD) – can cause various types of toxicity, including hepatotoxicity and nephrotoxicity. Mechanistic in vivo and in vitro animal studies have demonstrated that the renal toxicity of these chemicals is not caused by the parent compounds but is a result of several enzymatic reactions (GST, GGT, dipeptidase, and β-lyase) at hepatic and renal levels. The resulting reactive metabolites will lead to oxidative stress and mitochondrial dysfunction, which are considered key events in renal proximal tubule toxicity.
The case study aims to develop a quantitative model for halogenated alkene metabolism, distribution and target organ toxicity for halogenated alkenes, focusing on TCE.

Testing Strategy

An integrative workflow that includes in vitro hepatic studies for biotransformation of parent compounds, monitorisation of renal metabolism of hepatic conjugates and toxicity testing in hepatic, renal, and neuro in vitro systems has been developed. NAMs have been integrated into the testing approach. An in vitro model was developed utilizing human liver preparations, human recombinant enzymes, synthetic chemistry, and the human renal cell proximal tubule cell line RPTEC/TERT1. Metabolism stages and eventual mitochondrial toxicity have been evaluated in this system. LC-MS based analytical methods were applied to monitor the kinetics of parent compounds and tested metabolites which will also provide data for the support of an improved physiologically based pharmacokinetic (PBPK) model for human in vivo TCE, PER, and HCBD exposures.

Publications

Zgheib et al. 2018 [link]; Limonciel et al. 2018 [link]; van der Stel et al. 2020 [link]; Aschauer et al. 2015 [link]; Capinha et al. 2021 [link]; Capinha et al. 2021 [link]

Contributors

VU, UL, UKN, BIOT, EwC, IRFMN, SIMCYP.