Juan Carlos Carrillo Shell Key points for discussion Mouse skin painting studies IP346 Total aromatics MOAH and carcinogenicity Conclusions MOCRINIS2 JuanCarlos Carrillo 17102017 Mouse skin painting studies ID: 1034747
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1. Toxicity of MOAH:The Impact Of Molecular StructureJuan Carlos Carrillo (Shell)
2. Key points for discussionMouse skin painting studiesIP346 Total aromatics (MOAH) and carcinogenicityConclusionsMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
3. Mouse skin painting studiesMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
4. The Mouse Skin Painting Bioassay And Manufacturinghttp://www.bio-protocol.org/attached/image/20160911/20160911234503_5045.jpgMOCRINIS-2 Juan-Carlos Carrillo 17/10/20171
5. The Mouse Skin Painting Carcinogenicity BioassayUndiluted oil, single doseShaving of dosing site before dosingRepeat 2-3 times/weekhttp://www.bio-protocol.org/attached/image/20160911/20160911234503_5045.jpghttp://www.ratbehavior.org/RatsMice.htmhttp://www.phytojournal.com/vol2Issue2/images/40.1.pnghttps://www.euromabnet.com/img/antibody/507-TEJ.RATON.SKIN.PAPILLOMA.HUGO291.copia.jpgNecropsies on all mice and all macroscopic observations recordedAll tumours (benign & malignant) and skin lesions are recordedTumor latency and regressions recordedTHE gold standard for studying carcinogenesisSystemic metastasis is common in mice with skin malignant tumoursHistopathology of skin and major organs Skin tumor classification:benign or malignantcontrolsn = 40-96undiluted oil treatmentn = 40-5078-104 weeksMOCRINIS-2 Juan-Carlos Carrillo 17/10/20172
6. Tissue Sensitive to B[a]P-induced Tumors is Reflected in Average Rodent DMEL ValuesDermal route is the worst case scenario for PAH (PAC) mediated carcinogenicity. Slide credits: D. Adenuga - ExxonMobilMOCRINIS-2 Juan-Carlos Carrillo 17/10/20173
7. Why the Mouse Skin Painting Model?Extensively used as a holistic multistage carcinogenesis model1Principles studied in this model are relevant to other epithelial tissuesCarcinogenicity and potency of naked ring and alkylated PAH and isomerism has been studied in this model2, 3Model of choice to study the impact of petroleum refining on modulating carcinogenicity of petroleum substances4Main route of exposure for petroleum products1. Rundhaug et al., 2010. Cancers; 2(2): 436–482.2. La Voie el al., 1985. Carcinogenesis; 6(10): 1483-1488.3. Luch A., 2009. Mol. Clin. Env. Tox. (1): 151-1794. Bingham et al., 1980. J. Env Path Tox; (3)483-563.MOCRINIS-2 Juan-Carlos Carrillo 17/10/20174
8. Tissues rich in CYP activity – skin, liver etc.B[a]PEpoxideo-quinonecationCYP1A1/1B1/1A2Tumor promotion:Local irritation, cytotoxicity, inflammatory responseMost sensitive tumor sites – dermal, oral (forestomach, oral cavity, GIT)B[a]P Rodent Tumor Mode of Action Dictates Tumor Location Slide credits: D. Adenuga - ExxonMobilMOCRINIS-2 Juan-Carlos Carrillo 17/10/20175
9. MOAH Molecular Structure Determines CarcinogenicitySteric HindranceCYPenzymesMOAH substrateMOAH substrateCYPenzymesMOCRINIS-2 Juan-Carlos Carrillo 17/10/20176
10. Tumour initiation and promotion model – Testing of “MOAH” fractionsWhat type of aromatics in an oil are carcinogenic?Fractionation of a carcinogenic oil demonstrates where the hazard is1:What can we learn from this experiment?Fractions of a carcinogenic oil were tested with or without a promotor. There is a type of MOAH that is NOT carcinogenic with or without promotor.It is the > 3 ring MOAH fraction which is potentially carcinogenic.The interaction of ALL fractions causes the carcinogenic effect. Therefore, especially with UVCB’s, it is imperative to test SUBSTANCE (the actual oil), and NOT the fractions thereof. There are refined aromatic oils, with high level of aromatics which are not carcinogenic2Substance or fractionLive animals after 40 weeksRe-treatment of live animals with a promotorCarcinogenic oilTumours in all animals-Fraction I+II+IIITumours in all animals-Fraction I (PAH “free”)No tumoursNo tumoursFraction II (2 and 3 rings)No tumoursNo tumoursFraction III (> 3 rings)No tumoursTumours in all animals1. Agarwal et al., 1988; 2.Doak et al.,1985MOCRINIS-2 Juan-Carlos Carrillo 17/10/20177
11. Toxicologists focus on PAC* not MOAHMouse model is the gold standard to assess PAC carcinogenicityPrinciples studied on mouse skin are relevant to other epithelial tissues and humansIndustry dermal mouse studies and protocols are fit for purpose and reliable Dermal route is the worst case scenario compared to oral routeCarcinogenicity potential depends on molecular structure and route of exposureIt is the >3 ring PAC which are the potentially carcinogenic speciesThere are MOAH structures that have no carcinogenic potential !Take Home Message for Mouse Skin Painting Studies in Mineral Oils*PAC = polycyclic aromatic compounds (PAH) + Sulphur, Nitrogen atoms in polyaromatic-ring structuresMOCRINIS-2 Juan-Carlos Carrillo 17/10/20178
12. IP346MOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
13. Mouse skin painting studies are the gold standard, but time consuming limit manufacturing flexibilityanimal and cost intensiveCarcinogenicity screening method should be fit for purpose to mineral oilsrapid, reliable, specific, simple, low costreflect variability in feedstock and manufacturing conditionsanimal free testReflect toxicological hypothesispotentially hazardous are the 3-7 PACPAC are bare or with few and short alkyl substituents highly correlated to mouse skin painting dataThe Necessity For A Carcinogenicity Screening MethodMOCRINIS-2 Juan-Carlos Carrillo 17/10/20179
14. Boiling Point < 300 ⁰C at 1bar substituted benzenes and naphthalenesMineral Oils boiling range > 300 ⁰C at vacuum The PAC found in mineral oil are found in the 340-565⁰C boiling point range (BP)BP 340-565⁰C (vacuum) 3-7 PAC Phenanthrene BP 340⁰CCoronene BP 535⁰CThe higher the alkylation, the higher the BPMethod should collective assess of all isomers and alkylation levels within given BP rangeThus method links PAC to boundaries set by manufacturing and toxicological relevanceScreening Method Is In Line With Manufacturing MOCRINIS-2 Juan-Carlos Carrillo 17/10/201710
15. Screening Method Should Be Selective To Toxicologically Relevant PACScreening method based on DMSO DMSO shows special interaction with PAH (PAC) Pi-systemCan be modulated by alkyl substitution and halogenationCan be interrupted by polar and non-polar solventsAllow selective extraction based steric hindrance of highly alkylated PACExtraction efficiency drops with decreasing number of fused aromatic rings per molecule, and length of alkyl chainsDMSO-extract compositionreflect refinement efficacy in PAC removalMechanistic explanation of biological process: bioavailability and bio-activation of the type of DMSO-extracted PACrelevant in the prediction of mouse skin painting studiesHigh affinityPoor affinityMOCRINIS-2 Juan-Carlos Carrillo 17/10/201710
16. DMSO extract composition Aromatic extracts* DMSO extract%Av. alkyl chain lengthExtract A29.32.4Extract B4.54.3DMSO extract composition Base Oil*DMSO extract%Av. alkyl chain lengthA. Low viscosity0.66.2B. High viscosity0.112.5*Note:At equivalent cut extractionDMSO extract aromatic compositionRelationship to Refinement MOCRINIS-2 Juan-Carlos Carrillo 17/10/201711
17. Screening Method By ChromatographyPLC-MS Chromatography was considered in 1970:Not selective Only PAH are determined, S and N containing PAC are not accurately reflectedNo distinction of alkylation degree – blow up effect Does not correlate with tox studies Carcinogenic Oil N2 has lower “MOAH” than non-carcinogenic Oil B MOAH chromatographic values don’t reflect manufacturing!Complex, time consuming, expensive and not simple to transfer or implementPLC-MS is the 1970’s equivalent approach to today’s “MOAH” !PAC Analysis (1970)Oil typeChromatographyDMSO extract%Av. alkyl chain lengthCancerOil N2 Low viscosity2.96.83.4YESOil B. High viscosity5.70.112.5NOMOCRINIS-2 Juan-Carlos Carrillo 17/10/201712
18. Conclusions On Screening Method Based On DMSO ExtractionDMSO based screening method is selective towards toxicologically relevant PACsThere will be aromatics in refined oils but these are not carcinogenicThe method considers manufacturing boiling rangerefinement process which determine PAC levels and alkylation degree composition of mineral oils carcinogenic potentialSteric hindrance will heavily influence DMSO extract efficiency, reflecting the enzyme-substrate behavior of highly alkylated PACToxicological data is aligned with DMSO extract carcinogenic potentialThus, it is not surprising that mineral oil refinement level and mouse skin painting studies show high correlationThis is the basis of the regulatory standard: IP346MOCRINIS-2 Juan-Carlos Carrillo 17/10/201713
19. IP346 – The Carcinogenicity Screening MethodCarcinogenicity cut-offIP346 validation:DMSO-based screening method validated with animal data1:1 relationship same oil mouse skin painting studies and its own DMSO-extractDetermine a “cut-off”: % DMSO extract that is correlated to non-carcinogenic oilCut-off solely on a hazard basis: Pass/fail in carcinogenicity assessmentPass/fail is binary. “Pass” means safe (and not safer, safest, etc…)Carcinogenicity criteria for validation:No discrimination between benign or malignant tumorsPotency (time of appearance of first skin tumour) is not considered Tumor incidence (4%) and not tumour formation stages used for IP346 validationMOCRINIS-2 Juan-Carlos Carrillo 17/10/201714
20. IP346 And Mouse Skin Painting Studies – Data BaseCompletely eliminated carcinogenicity testing on animalsAdopted in the 90’s in the EU and in other countries (e.g. Australia, Malaysia) as regulatory standard for carcinogenicity assessment IP 346 < 3% oil is not carcinogenic IP 346 > 3% is carcinogenicReferenceData points (2 year studies)CONCAWE 6/16CONCAWE 94/51133 * 104Chasey et al., 199394McKee et al., 19899Doak et al., 1983 and (1985)12 (6)Blackburn et al., 1996120Roy et al., 198839Negative predictivity = 95%Accuracy = 89% (because of false positives)*Including all studies cited, without repetitionsMOCRINIS-2 Juan-Carlos Carrillo 17/10/201715
21. MOCRINIS-2 Juan-Carlos Carrillo 17/10/201716
22. IP346 – Take Home MessageCompletely eliminated carcinogenicity animal testingCritical and rapid quality control tool to ensure in-situ refinement efficacyVital specification to ensure product safety for its release downstreamThe extracted 3-7 PAC material is related to intrinsic carcinogenicity of the undiluted oilNot just an analytical method but rather a fit for purpose tool to assess carcinogenicityThus, IP 346 is NOT to measure PAC content, but rather indicative of the relationship between the refinement history and the PAC biological activity of the oil in mouse skin painting assays (are the PAC active or not?)The IP346 (DMSO extract) encompass PAC (also low alkylated PAC), and other substances that per se may not be biologically active, but together in the oil may decrease/potentiate carcinogenic activityIP 346 is not intended for risk assessment purposes – only hazard assessmentThus, IP346 is a gatekeeper and the method for assessing refinement effectiveness: “green light” for further processing in order to meet other regulatory standards (e.g. pharmacopeia)It is the only validated analytical method with biological significanceMOCRINIS-2 Juan-Carlos Carrillo 17/10/201717
23. Total Aromatics (MOAH) and Carcinogenicity MOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
24. Why Is MOAH “High”? – The MOAH ParadoxExample Microcrystalline WaxMOAH (HPLC-GC FID) typical levels: 1-5 %.MOAH content < C35virtually absentContent of aromatic protons (NMR):~ 0,1 – 0,5 %Typical av. mol weight microwax: 700 (C50H102)3-7 rings aromatics: trace levels (specific UV test / Grimmer etc.)High alkylation of a small number of aromatic carbons leads to high MOAH values (everything is interpreted as aromatic)The higher the MW the greater the MOAH MOAH paradox: the more aliphatic, the more “aromatic”MOCRINIS-2 Juan-Carlos Carrillo 17/10/201718
25. Former Material Is Representative For Today – Decades Long Consistency In Manufacturing Recent HPLC-GC measurements on old and new production samples of several (EU) manufacturers (2015) confirm that MOAH was always present – nothing new!Historic concentrations used for fundamental toxicological studies were at least as high or even higher than those in products presently on the market<1980 Concawe 84-60 Samples1990 BIBRA Study Samples 2015 Recent production samples of several EU ManufacturersMOCRINIS-2 Juan-Carlos Carrillo 17/10/201719
26. Do We Need A Sophisticated MOAH Method?The measurement of Total Aromatics (MOAH) is nothing new DAB 8 UV-method did the sameBest correlation with Oils Oils have shorter MOAH`sLonger MOAH chains are not toxicologically relevantReplaced by UV-methods including DMSO extraction to focus on PAHnot biased by MWDAB-8MOAHData presented in collaboration with the company H&RMOCRINIS-2 Juan-Carlos Carrillo 17/10/201720
27. MOAH HPLC Does Not Correlate With DMSO-PAC MeasurementsNo correlation between MOAH content* and UV absorption according to the pharmacopoeia PAC test Amount of PAHs found in products is independent of measured MOAH content*Kirchhoff method, July 2015Data presented in collaboration with the company H&RMOCRINIS-2 Juan-Carlos Carrillo 17/10/201721
28. ConclusionsMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
29. IP 346Carcinogencity TestingRefining Methods3-7 ringPACsMouse skin paintingModified AmesBioactivationHydrogenationSolvent extractionAcid treatmentRegulatory standard methodDMSO selectivityValidation to animal dataCan't See The Forest For The TreesMOCRINIS-2 Juan-Carlos Carrillo 17/10/201722
30. The Carcinogenicity Weight of EvidenceMOCRINIS-2 Juan-Carlos Carrillo 17/10/201723
31. MOAH Take Home Message IMOAH is an integral part of the substance, can’t exist in isolationMOAH as “catch all” term is confusing for substance assessment – no toxicological contextToxicologists focus on what matters: 3-7 PACOnly manufacture determines type of MOAH in mineral oil products i.e. the MW of the intended final productBad MOAH: 3-7 PAC (eliminated through refinement) Harmless MOAH: highly alkylated aromatics (what is left after 3-7 PAC elimination)MOCRINIS-2 Juan-Carlos Carrillo 17/10/201724
32. MOAH Take Home Message IIIf refinement history is known, MOAH can be put into context, its logical but not the other way aroundcompliance focuses on regulating 3-7 PAHtherefore MOAH from unknown sources should target the bad MOAH (3-7 ring PAH)Harmless MOAH levels vary at each refinement step and increase with MWAlways been present, at same levels, nothing newCan be measured with a simple UV test (e.g. DAB 8)PAC by DMSO extraction are better descriptors of cancer hazardWhite Oil purity with a simple UV test (e.g. EuPharm 9)Refined mineral oil products are safe even if MOAH is presentThis includes all process oils e.g. printing ink oil, lubricating oilsMOCRINIS-2 Juan-Carlos Carrillo 17/10/201725
33. Thank YouMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
34. Back-upMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
35. Selectivity of a DMSO extractionSolute partitionK = Conc. in DMSO/conc. CyclohexaneNaphthalene boils at 218 °C – does not occur in mineral oilsDo notice the impact of alkylation on K and extractionMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
36. IP346 – in regulatory contextDMSO based extraction method developed in the early 80’s as a screening method to link content and activity of PACs to refinement processesKnown as Petroleum Industry Standard IP346/92 (2004)Adopted in the 90’s in the EU as standard for carcinogenicity assessment IP 346 < 3% oil is not carcinogenic IP 346 > 3% is carcinogenicApplicability domain (under CLP): LBO, Foots Oils and TDAE (OIN L)For other petroleum categories, the IP346 method is a gatekeeper and it may only be used for informational purposes about “upstream” refinement history (OIN N)Adopted in other countries (e.g. Australia, Malaysia) as the regulatory standard for mineral oilsOIN* L: The classification as a carcinogen need not apply if it can be shown that the substance contains less than 3 % DMSO extract as measured by IP 346 ‘Determination of polycyclic aromatics in unused lubricating base oils and asphalthene free petroleum fractions — DMSO extraction refractive index method’, Institute of Petroleum, London. This note applies only to certain complex oil-derived substances in Part 3 (harmonized classification). OIN* N: The classification as a carcinogen need not apply if the full refining history is known and it can be shown that the substance from which it is produced is not a carcinogen. This note applies only to certain complex oil derived substances in Part 3 (harmonized classification). IP346 – In Regulatory Context (CLP)* OIN = Oil Industry NoteMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
37. IP346 method procedureWeighed oil sample (bp>300°C)Dissolve in cyclohexaneExtract 2 x with DMSOCombine extractsDilute with aqueous NaCl solutionReextract with 2 x with cyclohexaneCombine extractsExtract in DMSO solutionPAH extract in cyclohexane solutionWash with waterDryEvaporate cyclohexaneWeigh and RI determinedPAH extract Little and unalkylated PAH’sPolar CompoundsLeft BehindExtractedRe ExtractionMOAHAliphatics, Olefins, Mono, di-aromatics, Highly alkylated PAH’sPolar compounds, Little and unalkylated PAH’sLeft BehindExtract1 st ExtractionMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
38. MOAH is not correlated to carcinogenic potential: no carcinogenicity even at 40 wt% total aromaticsEven at 40% (wt) aromatics and 55-100 ppm PAH no carcinogenicityNeither MOAH nor PAH levels is correlated to carcinogenic potentialOnly refinery treatment conditions and mouse data determine mineral oil hazard (“known refinement history”). IP346 was introduced to link manufacture and carcinogenicity McKee et al., 1989MOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
39. MOAH – as a measurement of “total aromatics” is not indicative of hazardMOAH* measurement comparable to method by STIWA The decades long observation that decreased levels of aromatics influence carcinogenicity is still true for recent MOAH analysisHowever, putative reduction of aromatics, or even direct measurements of PAC has long been shown NOT to be correlated with carcinogenicity.MOAH as a measurement of “total aromatics” is not indicative of hazardProcess stepMOAH*IP346Mod. Ames, MI indexHazard ratingDistillate>30%Not applicable> 1CarcinogenicWaxy Raffinate>10%< 3%< 1Non-carcinogenicBase Oil>15%< 3%< 1Non-carcinogenicTech. White oil0.5 - 5%< 3%< 1Non-carcinongenicPharm. White oil<0.5< 3%< 1Non-carcinogenicAromatic extract >60%> 3%> 1CarcinogenicMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017
40. Total aromatics in tox testsDoak et al., 1985For aromatic extracts IP346 gives false positivesHowever, the point here is that putative reduction of aromatics or reliance on aromatic content is NOT indicative of carcinogenicityMOCRINIS-2 Juan-Carlos Carrillo 17/10/2017