Report of a Workshop to Assess the Modes of Action of Lung Tumors in Mice from Exposures to Styrene Ethylbenzene, and Naphthalene

Home , Ethylbenzene

SEPTEMBER 17, 2013

WORKSHOP PANEL: Michael L. Dourson, William Farland, David R. Mattie, M.E. (Bette) Meek

WORKSHOP ORGANIZED BY: Styrene Information & Research Center

REPORT PREPARED FOR THE PANEL BY: Toxicology Excellence for Risk Assessment

1 | November 14, 2013 Appendix A: Workshop Agenda

WORKSHOP TO ASSESS THE MODES OF ACTION OF LUNG TUMORS IN MICE From Exposure to Styrene, Ethylbenzene, & Naphthalene

AGENDA 9:00 Welcome (J. Snyder) A. Introductions B. Welcome and review purpose and goal of workshop

9:15 Introduction (G. Cruzan): Potential Modes of Action for Chemicals that cause lung tumors in mice, but not rats, that are metabolized by CYP2F2 A. Cytotoxicity and/or mitogenesis from CYP2F2 metabolism B. Genotoxicity from these compounds or their metabolites

9:30 Presentation 1 (G. Cruzan): Cytotoxicity from CYP2F2 Metabolism of styrene and ethylbenzene Description of toxicity, necessity of CYP2F2 metabolism – pharmacologic inhibitors, knockout mice (2E1 and 2F2), metabolites produced by CYP2F2 and relationship to major pathways, non-tumorigenic

10:00 Presentation 2 (L. Van Winkle): Pathology and MOA data for Naphthalene Lung toxicity and proliferation patterns, evidence for CYP2F2 metabolism (inhibitors, Km, knockout mice). Summary of evidence for metabolites in relation to toxicity and genotoxicity.

10:30 Break

10:45 Presentation 3 (B. Gollipudi): Alternative Hypothesis: Genotoxicity

11:15 Presentation 4 (J. Bus): Human Relevance Analysis of the MOA data using the Human Relevance Framework

12:00 Lunch

26 | November 14, 2013 1:00 Panel Discussion (Moderator: M. Dourson)

1. Is the CYP2F2-‐mediated mode of action proposed for mouse lung toxicity and tumors adequately defined in animals? If not, what data are necessary, or useful, to enhance confidence in the MOA?

2. Are the data on the three chemicals mutually consistent with the hypothesized MOA, or are there discordances that diminish the plausibility of the MOA?

3. Do the data indicate whether the hypothesized MOA, if established in animals, is likely to be qualitatively irrelevant to humans?

4. If the hypothesized MOA is qualitatively relevant for humans, is it characterized as quantitatively irrelevant (or relevant) to human?

5. If necessary, what new data might further clarify the question of human relevance?

6. Is there a set of criteria, based on short-‐term experimental protocols (≤ 90 days) that can be applied to other chemicals to determine if they operate by the same mode of action as that presented?

3:45 Wrap up and Next Steps

27 | November 14, 2013 Appendix B: Presentation Slides

28 | November 14, 2013 Styrene Tumor Styrene Tumor Mode Mode Action of George Cruzan, PhD, DABT George Cruzan, PhD, DABT ToxWorks This research was sponsored by the Styrene DC Washington, Information and Research Center,

29 | November 14, 2013 Naphthalene Styrene Ethylbenzene The chemicals   

30 | November 14, 2013 Ethylbenzene – no studies in increase Naphthalene – limited data; no Styrene – 13 cohort studies; no styrene- lung tumors reported related increases in lung tumors. One study et al., 1994; Collins 1990; Wong (Wong, al, 2013) increased lung tumors, inverse dose-response, attributed to smoking Human Cancer Data Human Cancer   

31 | November 14, 2013 1988 NCI, 1979a, b, Ponomarkov and Tomatis, 1978, Conti et al., al., et Conti 1978, Tomatis, and Ponomarkov b, 1979a, NCI, 1985 al., et Beliles – no increases in tumors in tumors 4 Gavage studies – no increases in 1 Drinking water study – no increases tumors tumors   Rat Studies  

32 | November 14, 2013 ppm 1 year exposure exposure year 1 ppm ppm – 2 years years – 2 1000 ppm 25-300 mammary tumors tumors mammary decreased years; 2 ppm 200 and 50 at tumors malignant mammary tumors at 500 and 1000 ppm mammary tumors, but within historical control control historical within but tumors, mammary not at at 600 ppm, range Conti et al., 1988 - Increased malignant Conti et al., 1988 - Increased Cruzan et al., 1998 – No increase in mammary Jersey et al., 1978 – Increased malignant 3 Inhalation studies    Rat Studies 

33 | November 14, 2013 Ponomarkov and Tomatis, and Tomatis, Ponomarkov in tumors - in tumors in lung tumors at 40, 80 and 160 ppm in lung tumors at 40, 80 and 160 ppm Suggestive increase in lung tumors – increase No in males, 20, 40, 160 females - Cruzan et al., 20, 40, 160 females in males, 2001 Ponomarkov and Tomatis, 1984; NCI, 1979a Tomatis, and Ponomarkov 1984; NCI, 1979b 2- 2- Increase 1 Inhalation Study 4 Gavage studies    Mouse Studies Mouse Studies  

34 | November 14, 2013 Are Humans Like Mice or Rats? Why Mice and Not Rats? Mode Mode Action of  

35 | November 14, 2013 Single exposure – inc. LDH, Cells and protein in Up to 2 weeks – inc. BrdU labeling, dec. CCSP 3 months – decreased staining of Clara cells; bronchioles in terminal – hyperplasia 12 months bronchioles in terminal – hyperplasia 18 months 24 months – lung tumors, mostly benign BALF cellular crowding in terminal bronchioles extending into alveolar ducts Progression of Toxicity in Mice Toxicity of Progression      

36 | November 14, 2013 metabolism of styrene by CYP2F2 styrene by CYP2F2 of Lung metabolism produces ring-oxidized metabolites that are toxic to Clara cells. Continued exposure results in continual cell replication, leading to cells Clara formed Newly hyperplasia. lack production of CCSP(CC10), which reduces resistance to lung tumors. Proposed MOA MOA Proposed 

37 | November 14, 2013 positive cells positive Normal levels of BrdU labeled cells and CCSP Normal levels of BrdU labeled cells and CCSP Inhibit 2F2 (5P1P), reduce toxicity - (BALF, Inhibit 2F2 (5P1P), reduce toxicity - (BALF, Eliminate 2F2 (knockout mouse), eliminate Eliminate 2F2, eliminate toxicity from SO BrdU) BrdU) toxicity from styrene – (BALF,  Evidence for CYP2F2 MOA Evidence for CYP2F2 MOA   

38 | November 14, 2013 Cruzan et al., 2012 Cruzan Labeling index (% of cells labeled with BrdU) in type and CYP2F2(-/-) wild of bronchioles terminal mice exposed to styrene, or styrene oxide, for 5 days. 

39 | November 14, 2013 6-10% of styrene urinary metabolites in metabolites urinary styrene 6-10% of No lung tumors in mice from 3-methyl- 4-Hydroxystyrene (4HS, 4-vinylphenol) more mice are ring-oxidized; 0.5% in rats styrene or 4-methylstyrene (no ring oxidation possible) 2 mg/kg/ toxic than styrene or SO (NOAEL day over 2 weeks compared to 100 mg/kg/ styrene) day for Role of Ring Oxidation Role   

40 | November 14, 2013 WT mice produce 4HS, 3,4-dihydroxystyrene WT Limited ring oxidation of styrene in 2F2-KO Limited ring oxidation of 4HS in 2F2-KO Some toxicity from 4HS in 2F2-KO mice and 4-hydroxystyrene oxide from styrene in lung mice mice Role of Ring Oxidation Role    

41 | November 14, 2013 BrdU Labeling index in terminal bronchioles and KO mice exposed to 4- of WT, hydroxystyrene for 5 days

42 | November 14, 2013 Default MOA Hypothesis Hypothesis MOA Default Styrene is genotoxic carcinogen via metabolism to SO. will be discussed later.  Alternative MOA of genotoxicity via SO Alternative MOA

43 | November 14, 2013 metabolite metabolite Hypothesis that SO is toxic Styrene metabolized to styrene-7,8-oxide (SO) Level of SO from liver through blood to lung much Level of SO in mouse lung much greater than rat Administration of SO causes lung toxicity in mice primarily by CYP2E1 primarily by CYP2E1 greater in mice than rats at same exposure concentration lung at same exposure concentration    

44 | November 14, 2013 Mice exposed to SO did not get lung tumors Blood level of SO in rats at 1000 ppm (no Level of SO in perfusate from isolated lungs No reduction in lung toxicity of styrene tumors) is 100 fold higher than in mice at 40 ppm (lung tumors) in rats exposed to 1000 ppm is 8 fold higher than in mice at 40 ppm. from inhibition of CYP2E1 or in CYP2E1- mice. knockout Data Do Not Support Hypothesis    

45 | November 14, 2013 in forestomach in forestomach 6 2 rat studies – forestomach necrosis and tumors and tumors necrosis – forestomach studies 2 rat and tumors necrosis – forestomach study 1 mouse <1/10 adducts DNA Increased cell replication parallels tumors in tumors parallels cell replication Increased forestomach Styrene Oxide   males dose low tumors liver  

46 | November 14, 2013 Styrene oxide at end of 6-hour exposure Styrene oxide at end of 6-hour exposure during chronic inhalation studies

47 | November 14, 2013 2.05 2005, whole lung explants 1.83 3.16 et al., SO level in rat lung: SO level in rat SO level in mouse lung From Hofmann  SO in perfusate of isolated lungs exposed to styrene through trachea  Styrene ppm: 50 160 475 985 SO (ng/ml) .06 .50 1.05 Styrene ppm: 42 164 407 SO (ng/ml) .25 

48 | November 14, 2013 Hypothesis Hypothesis Alternative Toxicity is seen primarily in tissues is seen primarily in tissues Toxicity Therefore, R-SO is toxic that produce high levels of R-SO nasal olfactory and mouse (rat epithelium and mouse lung bronchioles; terminal metabolite.  

49 | November 14, 2013 the toxic metabolite must be R- position SO contains a chiral carbon at α position In rats R:S-SO ratio is slightly < than 1 In mouse liver R:S ratio is slightly > than 1 3 In mouse lung R:S ratio is about R-SO in mouse lung is estimated to be about Therefore, 15-fold higher than in rat lung SO or derived from R-SO Hypothesis 2: R-SO is toxic metabolite      

50 | November 14, 2013 in toxicity between R and S in toxicity between R and S No difference difference No difference No following single exposure (BALF – Carlson) following 5 days of exposure (BrdU – Cruzan 2012) et al., Data Do Not Support This MOA This MOA Data Do Not Support  

51 | November 14, 2013 SO – No lung tumors related to to related Mouse lung tumors not No lung tumors in absence of ring oxidation – 3-,4-MeS SO 4HS toxic at much lower dose than styrene or CYP2F2 of in absence Some 4HS toxicity even CYP2F2 creates ring-oxidized metabolites that are toxic from SO metabolism metabolism to Clara cells cells Clara to Summary of Mouse Lung Tumor MOA MOA Tumor Summary of Mouse Lung     

52 | November 14, 2013 Human isoform is CYP2F1 CYP2F1 is Human isoform 3- metabolize to shown and 2F1 2F2 Developed transgenic mouse to study 2F1 methylindole; 2F1 metabolizes naphthalene 2F1 metabolizes methylindole; indication preliminary rate; 2F2 1% of about styrene not metabolized by 2F1 ability to produce toxic metabolites Human Relevance of CYP2F2 MOA MOA CYP2F2 of Human Relevance   

53 | November 14, 2013 level of of level th Inserted transgene containing DNA for CYP2F1 (lung Inserted transgene containing DNA 2F1 and 2A13 protein expressed in lung nasal TG mouse lung at 1/40 CYP2F1 present in Lung microsomes from TG mice metabolize Lung microsomes from and nasal), 2A13 (lung 2B6 (liver) mice CYP2F2-KO into mice these of tissue 2F2 in WT mice (greater than amount in human 2F2 in WT Clara cells) naphthalene (Ding) and 3-methylindole (Yost) CYP2F1 Transgenic Mouse CYP2F1 Transgenic    

54 | November 14, 2013 No lung toxicity from styrene or SO in TG No lung toxicity from styrene or SO in TG mice Some toxicity from 4HS in mice CYP2F1 Transgenic Mouse CYP2F1 Transgenic  

55 | November 14, 2013 Females 7.5 (3.0) 3.5 (0.9) 6.0 (2.9) 6.8 (2.0) 22.9 (8.3) TG Mice Males Males 5.2 (2.1) 2.7 (1.1) 6.3 (2.2) 4.3 (3.0) 12.3 (6.6) Females 7.2 (2.7) 50.0 (8.9) 42.0 (8.5) 42.3 (1.1) 51.7 (1.4) WT Mice WT Males Males 4.7 (2.0) 35.4 (9.6) 35.6 (2.5) 27.5 (17.0) 48.0 (1.7) Control Control Styrene - 200 R-SO - 200 S-SO - 200 4HS - 105 Labeling index (% of cells labeled with BrdU) in terminal bronchioles of wild type and CYP2F2(-/-), 2F1-tg mice exposed to styrene for 5 days.

56 | November 14, 2013 Mouse lung tumors from styrene exposure the toxic metabolite Styrene oxide is NOT sufficient produce not does Human CYP2F1 Mouse lung tumors from styrene do not caused by ring-oxidized metabolites metabolism by CYP2F2 produced metabolites to cause toxicity or tumors styrene from cancer of indicate human risk Conclusions Conclusions    

57 | November 14, 2013 58 | November 14, 2013 Ethylbenzene

59 | November 14, 2013 EB Cancer Data NTP developed series of genotoxicity tests NTP 2-yr bioassays on some + Performed 2-yr bioassays on – chemicals to Performed EB negative in gene tox battery; expected to be surrogate for 2-yr bioassay accuracy show chemicals to show specificity - NOT bioassay be – in 2-yr to    

60 | November 14, 2013 Increase in lung tumors male in male in kidney tumors Increase at 250 ppm increases No mice and liver tumors in female in female mice and liver tumors mice at 750 ppm and testicular rats and female at 750 ppm rats in male tumors   EB Cancer Data 

61 | November 14, 2013 EB Lung MOA Data Data EB Lung MOA Decreased staining of Clara cells; in males cell proliferation Increased hyperplasia of terminal bronchioles bronchioles terminal of hyperplasia extending into alveolar ducts (referred of metaplasia bronchiolar as to report) in NTP ducts alveolar exposed to 750 ppm  

62 | November 14, 2013 Active EB Metabolites in Lung Active EB Metabolites in Lung 1-PE not tumorigenic in 2-yr bioassay labeling BrdU increased cause not does 1-PE Primary metabolism of EB is by CYP2E1 metabolism of EB is by CYP2E1 Primary in mouse lung to 1-phenylethanol (1-PE) (1-PE) 1-phenylethanol to   

63 | November 14, 2013 Lung metabolism produces 2- and 4- 4- from cell proliferation Increased EB lung cell proliferation inhibited by 5P1P ethylphenol (2- and 4-hydroxyethylbenzene) ethylphenol (2- and 4-hydroxyethylbenzene) ethylphenol (unpublished BASF report) Active EB Metabolites in Lung    (evidence of CYP2F2) CYP2F2) of (evidence

64 | November 14, 2013 Mouse lung tumors not related to main Mouse lung tumors not related to main EB less potent than styrene (750 ppm but parallels data limited, MOA metabolism (CYP2E1) (CYP2E1) metabolism vs 40 ppm) styrene EB Conclusion   

65 | November 14, 2013 Sept 2013 Mouse lung Tumor workshop Tumor Mouse lung [email protected] [email protected] Center for Health and the Environment the for Health and Center Adjunct Professor/Research Cell Biologist Cell Professor/Research Adjunct University of California at Davis California of University Laura S. Van Winkle, Ph.D., DABT Van Winkle, S. Laura

School of Veterinary Medicine: Anatomy, Physiology and Cell Biology Anatomy, Veterinary Medicine: School of Pathology and Mode of Action for Naphthalene Pathology and Mode of

66 | November 14, 2013 naphthalene exposure exposure naphthalene Mouse lung tumors and Mouse lung

67 | November 14, 2013 of – alveolar/bronchiolar of carcinogenic activity. of carcinogenic activity. clear evidence adenomas of the respiratory epithelium yr inhalation bioassay NTP 2 NTP Male and Female rats – adenomas of the lung increased in high dose group. Male mice - no evidence Female mice – some evidence carcinogenesis: and neuroblastomas of the olfactory epithelium in nose. No lung tumors ------A/J Adkins found increased tumors in Additional study by mice exposed to 10 or 30 ppm for 6 mos.

68 | November 14, 2013 Toxicopathology

69 | November 14, 2013 depends on depends chronic) chronic) sex and age of the animal and sex route of exposure exposure of route dose vs (acute duraOon

of NA of injury? toxicopathology toxicity: What do we know about the do What toxicity: NA injures Clara cells regardless of route of exposure of regardless cells Clara injures NA airway in the DistribuOon Toxicity In general, tumors are found at sites of found are tumors general, In

70 | November 14, 2013 DistribuOon of target/ of DistribuOon -‐ cells sensiOve to repair capability -‐ injury following cells and Mouse Clara all intra and cells in ciliated airways. extrapulmonary Clara cells. to 70-‐85% Up have a cells Clara Primates: distribuOon restricted more LocaOon, LocaOon, LocaOon LocaOon, LocaOon,

71 | November 14, 2013 Naphthalene is toxic to mouse Clara cells Images from Van Winkle et al 1999 Winkle Images from Van

72 | November 14, 2013 From West et al 2001 West From 200 mg/kg 100 mg/kg 30 PPM 10 PPM and terminal bronchiolar Clara cells bronchiolar terminal and

73 | November 14, 2013

Naphthalene Species and Site Selective Toxicity of Toxicity Species and Site Selective Plopper et al., 1992; 1993; West et al, 2001; Lee al., 2005 Plopper et al., 1992; 1993; West

74 | November 14, 2013 Oliver et al 2009 Oliver Female Mice Cell ProliferaOon Male vs ProliferaOon Cell

75 | November 14, 2013 + NA + . 151 (443-‐459) 1997 (443-‐459) . 151 from Van Winkle et al . Am J Pathol Van Winkle from Naphthalene and the Cycle of Injury and Repair Cycle of and the Naphthalene

76 | November 14, 2013 -NA i.p . tolerance Lakritz et al 1996; O’Brien 1989 -NA -NA inhalation tolerance West et al 2003 inhalation tolerance West -NA

Naphthalene causes “Tolerance” Naphthalene causes “Tolerance” Repeated Inhalation or Injection of Tolerance is resistance to a high challenge Tolerance dose following a week or more of exposure to repeated exposures

77 | November 14, 2013 . Sci. et al. Toxicol West J A A West J A 2003;75:161-168 Nodules in tolerant mice Tolerant + challenge Tolerant Tolerance to inhaled NA to inhaled NA Tolerance © 2003 Society of Toxicology Toxicology © 2003 Society of

78 | November 14, 2013 et al 1996 Lakritz CCSP Expression in Tolerant Mice in CCSP Expression Tolerant Tolerant Control Control

79 | November 14, 2013 i.p . exposures. Repeated Exposure Sutherland et al 2012 et al is facilitated by induction of gamma GCS. West Tolerance 2002. Repeated exposure results in cells that are unlike the normal markers such as CCSP, steady state in terms of differentiation the presence of focal regions cellular hyperplasia and resistance to additional injury. It is unclear what role the tolerant conducting airway epithelium (or alveolar epithelium) plays in formation of bronchiolar and chronic bioassay. alveolar adenomas as described in the NTP In female mice, tolerance is incomplete following

80 | November 14, 2013 How does NA cause toxicity? NA does How Glutathione depleOon Glutathione metabolism P450 mediated metabolites reacOve of binding Protein in sensiOve species more robust is NA of Metabolism target regions tract respiratory and in speciﬁc are metabolites downstream and epoxide Naphthalene Clara cells to toxic cell toxicity-‐ Clara lung to mouse contributes CYP2F2 knockout mouse from the lessons tell us about species and metabolism do kineOcs What to the mouse in relaOon toxicity speciﬁc • • • • • • •

81 | November 14, 2013 LUNG in vivo-dose threshold-mouse Warren et al., 1982 Warren Formation of reactive naphthalene metabolites

82 | November 14, 2013 - - Phimister et al 2004 Whole Lung Covalent Binding

- - toxicity Airway epithelial Injury Pretreatment None Piperonyl Butoxide Diethyl maleate reactive, glutathione depleting metabolites in Warren et al., 1982; DEM finding confirmed for inhalation of 15ppm NA in et al., 1982; DEM finding confirmed for inhalation of 15ppm NA Warren Evidence for the involvement of P450 generated

83 | November 14, 2013 DEM No toxicity Toxicity Toxicity Glutathione depletion is a necessary but not sufficient event for toxicity GSH cytotoxicity Reactive Metabolites GSH Depletion NA Possible sequence of events leading to Cell is left without normal protective mechanisms protein thiol oxidation ensues leading to protein unfolding. The cell attempts to correct this but critical proteins involved in protein folding are adducted.

84 | November 14, 2013 Mechanism of Toxicity Mechanism of Toxicity Metabolite Protein Binding in the Importance of Understanding Reactive Reactive metabolite formation precedes earliest signs of toxicity and is dose dependent Reactive metabolite binding much higher in airway cell proteins than whole lung Reactive metabolite binding generally coincides with changes in toxicity • • •

85 | November 14, 2013

Airways 500 µM and 50 µM).

50 µ M

500 µ M

Total reactive metabolite formation in Total dissected monkey airways incubated ( with NA Trachea Proximal Medial Respiratory Parenchyma Trachea

MG PROTEIN PROTEIN MG NMOLE / 1 : ~ 0.3 ). Cho et al., 500 µM airways: mice vs. rhesus macaques A. Total reactive metabolite A. Total formation in dissected mouse airways incubated ( with NA 1994 Total reactive metabolite formation in dissected Total

86 | November 14, 2013 Metabolism Naphthalene

87 | November 14, 2013 Naphthalene Metabolism Species Comparisons of Rates

in Whole Lung Microsomal Incubations- Nmoles/min/mg protein protein Nmoles/min/mg

88 | November 14, 2013 Plopper et al 1991; Van Winkle et al 1996 1991; Van et al Plopper Airway microdissecOon microdissecOon Airway

89 | November 14, 2013 Total Metabolites Metabolites Total

90 | November 14, 2013 Which Metabolites are Toxic? are Metabolites Which

91 | November 14, 2013

naphthoquinone naphthol Naphthalene oxide Evidence that the Epoxide causes Toxicity Data from Chichester et al., 1994 May not reflect local Quinones may not have Toxicity of naphthalene and Toxicity Metabolites in isolated mouse Clara Cells • concentrations • crossed membrane naphthalene

92 | November 14, 2013 naphthoquinone Buonarati et al., 1989 naphthol naphthalene oxide H-‐Naphthalene Oxide is rapid Oxide H-‐Naphthalene 3

Covalent Binding of RadioacQvity from Binding of RadioacQvity Covalent naphthalene

93 | November 14, 2013

metabolites perfused mouse lung in response to naphthalene or response in perfused mouse lung Morphometric alteraQons in the airway epithelium of the the in alteraQons Morphometric Kanekal et al., 1991. Concentrations were infused over 60 min followed by 4 hrs with Waymouth’s medium Kanekal et al., 1991. Concentrations were infused over 60 min followed by 4 hrs with Waymouth’s

94 | November 14, 2013 Summary H-‐naphthalene oxide suggesOng that the epoxide that oxide suggesOng H-‐naphthalene 3 may be the key step in this process key step the be may In a variety of model systems (perfused lung, variety of In a naphthalene naphthalene, cells) Clara hepatocytes, cytotoxic are and naphthoquinones epoxide toxicant in Clara potent more a oxide is Naphthalene than are the perfused lung isolated cells and . quinones of radioacOvity from binding is no lag in the There • • •

95 | November 14, 2013 may be related to: Differences in the catalytic activity of P450 Differences 2F in the amounts of protein Differences expressed in the airways in the amounts of P450/cell Differences (hot spots in the lung) Contribution of other P450 Mouse/rat/human/primate differences Mouse/rat/human/primate differences • • • •

96 | November 14, 2013 naphthalene epoxide Without conversion to the intermediate epoxide, naphthalene is toxicologically inert Overall P450 levels in primate lung are low-therefore high is essential if enzyme to be important catalytic efficiency Amounts of catalytically active protein present per cell are important Importance of Kinetics Metabolism • • •

97 | November 14, 2013

-1 -1 max max V 107 min 104 min M K 3 µ M 3 µ M No detectable P450 No detectable P450 8 1 9 7 8 0 human human Sequence similarity to similarity to Mice, Rats and Monkeys Comparison of Recombinant P450s from Shultz et al., 1999; 2001; Baldwin al, 2005 Rat CYP2F4 Monkey CYP2F5 Species/ CYP Mouse CYP2F2

98 | November 14, 2013 recombinant P450 proteins P450 recombinant Comparison of catalyOc acOviOes of human Comparison of Cho, Rose, Hodgson: Drug Metab. Dispo, 2006

99 | November 14, 2013

µ M 3 7 3 m 111 111 58.6 58.6 10.1 60.7 K Cho, Rose and Hodgson, DMD, 2006 pmoles/ 9.1 9.1 8.4 8.1 107 107 35.8 35.8 20.2 max pmoles/min pmoles/min V Comparison of kinetics naphthalene P450 isoform 1A1 2E1 3A4 2F2 (mouse) 1A2 2B6 metabolism in recombinant human P450s

100 | November 14, 2013 CYP2F2 Knockout mice Knockout CYP2F2

101 | November 14, 2013 Li et al 2011 Li

102 | November 14, 2013 0.2 Km 100 Km Vmax from 0.5 Km 1.5 uM Km Vmax plots for in vitro NA-‐GSH in for plots 1.0 WT and Cyp2F2-‐null Km 34.3 Km Vmax 160-‐fold) lower catalyQc eﬃciency toward NA. eﬃciency catalyQc lower 160-‐fold) -‐null mice, compared with WT mice, have much (up to mice, WT with compared mice, Cyp2f2 -‐null ∼ formaOon by lung microsomes formaOon Eadie-‐Hofstee

103 | November 14, 2013 H-‐naphthalene oxide suggesOng that the epoxide is that oxide suggesOng H-‐naphthalene Summary of Metabolite Toxicity Metabolite Summary of 3 a key step in this process step in key a In a variety of model systems (perfused lung, variety of In a naphthalene naphthalene, cells) Clara hepatocytes, cytotoxic are and naphthoquinones epoxide toxicant in Clara potent more a oxide is Naphthalene than are the perfused lung isolated cells and quinones of radioacOvity from binding is no lag in the There • • •

104 | November 14, 2013 ) -1 min

) than any of the recombinant m Summary and lower K max Mouse – rat differences in rates of airway metabolism Mouse – rat differences of the amounts are due to substantial differences CYP2F protein present CYP2F1 (human) metabolizes naphthalene poorly. Mouse CYP2F has much higher catalytic activities (higher V CYP2F2 (mouse) and CYP 2F4 (rat) show high catalytic CYP2F2 (mouse) and CYP turnover of naphthalene (>100 human proteins tested. Cyp2F2 null mice have a 160 fold decrease in NA metabolism • • • • •

105 | November 14, 2013 ? et al 2012) assays that have either posiOve or have either that assays Genotoxicity genotoxicity negaOve results for lung. results negaOve are quite robust, capabiliOes detoxiﬁcaOon cell levels and lung GSH However, lung tumors is unknown to ﬁndings relevance of these so the Short term mutagenesis assays for naphthalene are mostly naphthalene for assays mutagenesis term Short negaOve vivo in No -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐ -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐ tesOng reverse mutaOons in S typhimurium causes NQ 2 1, chromaOd exchanges in vitro. sister cause NQ 4 1, and NQ 2 1, causes DNA strand scission. and adducts forms DNA NQ 2 1, NA increases micronucleus that shown have 2013) et al studies ( Recio Recent an added S9 fracOon. Further in vitro with in human lymphocytes formaOon been correlated with have children in lymphocytes changes chromosomal naphthalene ( Orjuela to exposure

106 | November 14, 2013 Conclusion re: mouse tumors re: mouse Conclusion P450 acOvaOon is required for toxicity required is P450 acOvaOon correlate to sites of lung the toxicity in of Sites formaOon tumor NA extremely metabolizes mouse Cyp2F2 in the well toxicity of CYP2F2 diminishes Knockout of NA Cyp2F2 metabolism that likely It is the mouse formaOon in to tumor contributes

107 | November 14, 2013 Xinxin Ding Lei Li

Acknowledgements Dexter Morin Winkle Lab Van Winkle receives grant support from: Van Dr. NIEHS -National Institute of Env Health Sciences Air Resources Board CARB - Calif. American Petroleum Institute API - Alan Buckpitt

108 | November 14, 2013 Midland, MI 17 September 2013 Senior Managing Scientist MoA for Lung Tumors B. Bhaskar Gollapudi, Ph.D. Induced by Styrene, Ethyl Benzene, and Naphthalene Consideration of Mutagenic Workshop On On Workshop environmental • failure analysis & prevention • health • technology development environmental • failure analysis & prevention health technology development & Naphthalene Ethylbenzene & Naphthalene to Styrene, Exposure in Mice From Tumors Action of Lung Modes of

109 | November 14, 2013 2 ., 2011 et al ., 2011 Swenberg Foreword mutation in one of a number assays is not sufficient to conclude that it causes specific tumors by a mutagenic MOA or that mutation is the only key event in pathway to tumor induction” (EPA Draft MMOA Framework, 2007 ). DNA Adduct(s) ≠ Mutations. They are markers of exposure. “The determination that a chemical carcinogen can induce

. .

110 | November 14, 2013 3

111 | November 14, 2013 4 Styrene

112 | November 14, 2013 5 neoplasia . Styrene is activated to reactive metabolites by mouse lung Clara cells. Some of these metabolites induce cytotoxicity while other(s) produce a very low frequency of DNA adducts. In the presence regenerative cell proliferation, some of the DNA adducts will be fixed into mutations. Sustained cell proliferation leads to amplification of both spontaneous and induced mutations in critical genes, hyperplasia However, this is a species-specific phenomenon that not applicable to humans due to quantitative differences in the activity of cytochrome P450 orthologs responsible for the production of cytotoxic metabolite(s). Hypothesized Mutagenic Mode of Action for Styrene

113 | November 14, 2013 6

MoA adduct formation. compensatory cell proliferation. spontaneous mutations and hyperplasia. Metabolism of styrene to reactive metabolite(s) leading DNA Ring oxidized metabolites of styrene induce cytotoxicity and Cell proliferation leads to fixation of DNA adducts into mutations. Sustained cell proliferation leading to amplification of induced/ Progression of hyperplasia into adenomas and carcinomas. Key Events in the Mutagenic 1. 2. 3. 4. 5.

114 | November 14, 2013 7 WoE : SO from S efficiently detoxified in lung tissue Styrene (S) metabolized to styrene oxide by Clara cells S induces low levels of DNA adducts in mouse lung. Extremely weak covalent binding index for lung: < 1 N7 and O6 guanine adducts are most frequent.

. . . . KE 1: Metabolism of styrene to reactive metabolites leading to DNA adduct formation

115 | November 14, 2013 8 8 Cruzan et al., 2012 & 2013 KE 2: Cytotoxicity from ring oxidized metabolites

116 | November 14, 2013 9 in SCE

) ( increase in CA & questionable  increase in CA tumor target tissue : weak mutagenicity in vivo; no  WoE KE 3: Fixation of DNA Adducts into Mutations - Summary of Literature (modified from NTP)

117 | November 14, 2013 1 0 SO itself is not a lung tumorigen Efficient detoxification of SO

. . vivo mutagen, including in tumor target tissue. enhanced by evaluating dose response and temporality for mutations induced in a neutral gene (e.g., cII ) and an oncogene K- ras in mouse lung. Mutagenicity not likely responsible for lung tumors Weight of evidence suggests that S is not likely an in Confidence in this assessment can be further

. Conclusions for Styrene . .

118 | November 14, 2013

1 1 Styrene Ethylbenzene

119 | November 14, 2013 1 2 + ( ) In Vitro Damage In Vitro Oxidative DNA Mutagenicity of Ethylbenzene (modified from Henderson et al ., 2007)

120 | November 14, 2013 1 3

MoA : Mutagenic not considered to be critical given the WoE based on available mutagenicity data.

. potential data gap, the etiology of mouse lung tumors. The confidence in this assessment is very high. Lack of mutagenicity data in tumor target tissue is a WoE suggests that ethylbenzene is not a mutagen. It is unlikely that mutagenicity as an early key event in

Ethylbenzene . . . .

121 | November 14, 2013 1 4

Naphthalene Ethylbenzene Styrene

122 | November 14, 2013 1 5 evidence of genotoxicity for naphthalene and that some the studies which reported positive finding were technically unsuited to study this class of chemicals and, therefore, generated unreliable data..” in most genotoxicity and mutagenicity tests; however, the role of genotoxicity in the MOA has not been characterized target tissues ” indicates that naphthalene is not genotoxic.” suggest that naphthalene is not likely to be genotoxic in vivo.” Brusick (2008): “……. 80% of the studies reported found no Flowers et al. (2006): “ Naphthalene has produced negative results EU Risk Assessment (2003): “ Overall, the balance of evidence Schreiner (2003): “ Results of standard genetic toxicity assays

Reviews on Genotoxicity Naphthalene . . . .

123 | November 14, 2013 1 6 in vitro (naked DNA), ex vivo (nasal epithelium), and (skin, non-tumor target), no in vivo data following systemic exposure

. . No activity in gene mutation assays with bacterial and mammalian cell cultures. Negative results from in vitro and vivo UDS. Non-genotoxic in rodent bone marrow micronucleus tests. Low or no activity in SCE tests Some evidence for DNA reactivity of metabolites, Clastogenic to cells in culture.

Naphthalene Genotoxicity Highlights ......

124 | November 14, 2013 1 7 et al ., 2012) Naphthalene (+S9): Dose Response for MN in TK6 Cells MN in Naphthalene (+S9): Dose Response for ( Recio

125 | November 14, 2013 1 8 et al ., 2012) Naphthalene (+S9): Dose Response for MN in TK6 Cells MN in Naphthalene (+S9): Dose Response for ( Recio

126 | November 14, 2013 1 9 et al ., 2012) Naphthalene (+S9): Dose Response for MN in without (A) and MN in without (A) and Naphthalene (+S9): Dose Response for with (B) 5 mM GSH Supplementation ( Recio

127 | November 14, 2013 2 0 can be enhanced by evaluating dose response and temporality for DNA adducts and mutations in a neutral gene (e.g., cII ) and an oncogene K- ras in mouse lung.

. of naphthalene-induced lung tumors. an in vivo mutagen. Confidence in this assessment is moderate. Mutagenicity is not an initial key event in the etiology Weight of evidence suggests that naphthalene is not

. . Conclusions for Naphthalene .

128 | November 14, 2013 1 Exponent, Inc. Cincinnati, OH, September 17, 2013 James S. Bus PhD, DABT, ATS James S. Bus PhD, DABT, “Workshop to Assess the Modes of Action Lung Tumors in Mice” Human Relevance: Analysis of the MoA Data using the Human Relevance Framework

129 | November 14, 2013 2 . Toxicol From: Cohen et al., From: Crit. Rev. 33: 581-589, 2003 Human Relevance Framework: Key Questions

130 | November 14, 2013 3 – level of confidence

Weight of evidence linking key event, precursor of evidence linking key event, precursor Weight response lesions, and tumor Postulated MoA Key events and temporal associations Dose-response and specificity of association consistency, Strength, with key events response tumor • Biological plausibility and coherence Alternative MoA (s) Uncertainties, inconsistencies, and data gaps Assessment of MoA • • • • • • • • Answering Framework Questions: A Structured Approach

131 | November 14, 2013 4 reviewed in: reviewed analyses are chemical specific analyses are . Pharmacol 55: 205-218, 2009. Toxicol Mouse specific lung tumors from Cruzan et al., Mouse specific lung tumors from An CYP2F2-mediated cytotoxic metabolism: multiple data from where endpoint/toxic response chemicals converge to support a mode of action. Regul . Most MoA Accumulating body of evidence from multiple chemicals mutually supportive of CYP2F2-mediated mouse lung specific toxicity and tumorigenicity Hypothesized MoA • Mouse Lung Tumors Mediated through CYP2F2 Mouse Lung Specific Metabolism • • •

132 | November 14, 2013 5 From: Cruzan et al., 2009 Chemicals Potentially in MoA Class

133 | November 14, 2013 6 Cruzan et al. , 2009 Mouse vs Rat Lung Tumor Development

134 | November 14, 2013 7 “Metabolism of several chemicals by CYP2F2 in terminal bronchiolar Clara “Metabolism of several chemicals by CYP2F2 in terminal bronchiolar in the localized generation of cytotoxic metabolites and cells in mice results These metabolites generally are cell proliferation. subsequent reparative -benzene-ring derivatives. hydroxylated leads to cellular cell replication the increased …On continued exposure, As the and then to hyperplasia in the terminal bronchioles. crowding ducts. Some of alveolar hyperplasia continues, it expands into the surrounding to form adenomas in the mouse lung. Depending on these hyperplasias proceed to carcinomas. a few of the adenomas may progress the severity of stress, …The analogous CYP2F4 in rats may be as capable of forming these cytotoxic levels of rats have much lower metabolites as mouse CYP2F2; however, sufficient levels of these and do not produce CYP2F4 in terminal bronchioles lung tumors” hyperplasia, or metabolites to cause cytotoxicity, Hypothesized MoA (Cruzan et al., 2009)

135 | November 14, 2013 8 Delivery of chemical to the respiratory system Delivery of chemical to the respiratory Metabolism in lung by CYP2F2 metabolites Cytotoxicity mediated by reactive CYP2F2 metabolism formed from hyperplasia Sustained reparative to late-developing tumors Progression • • • • • Key Events of MoA

136 | November 14, 2013 9 MoA as Illustrated by Styrene (Cruzan et al., 2009)

137 | November 14, 2013 1 0 Cruzan et al., 2009 Key Events: Dose & Temporal Relationships

138 | November 14, 2013 1 1 pass metabolism attenuates amount of chemical st delivered to systemic circulation to systemic circulation delivered , ethylbenzene , cumene α - methylstyrene Styrene, napthalene , divinylbenzene ethylbenzene , coumarin napthalene Styrene, toxic to lung following oral benzofuran : all are dosing 1 Strong likelihood of delivery to lung resulting from from likelihood of delivery to lung resulting Strong inhalation exposures • (oral Delivery to lung via systemic circulation absorption) • • • • Key Event Weight of Evidence: Delivery to Respiratory System

139 | November 14, 2013 1 2 Cruzan et al., 2009 Lung toxicity of styrene, SO, and napthalene blocked in CYP2F2 KO mice Lung toxicity of styrene, and SO blocked in CYP2F1 humanized mice Lung toxicity of styrene Key Event Weight of Evidence: Metabolism Drives Toxicity • •

140 | November 14, 2013 1 3 Cruzan et al., 2009 Qual. Human Relevance Quant. Rel. Key Event Weight of Evidence: Cytotoxicity Leads to Hyperplasia Event Specificity Biological Plausibility

141 | November 14, 2013 1 4 Cruzan et al., 2009 Key Event Weight of Evidence: Sustained Cell Proliferation Leads to Lung Tumors y Qual. Human Relevance Quant. Human Rel. Event Specificity Biological Plausibilit y Qual. Human Relevance Quant. Rel.

142 | November 14, 2013 1 5 1-Phenylethanol, primary ethylbenzene metabolite, is tumorigenic not lung toxic or metabolite, is not cytotoxic in SO, primary styrene CYP2F2 KO mice and is not a mouse lung carcinogen is not a lung carcinogen 3,4-Dihydroxycoumarin • • • Non-ring oxidized metabolites are non-cytotoxic and/or non-cytotoxic and/or Non-ring oxidized metabolites are non-tumorigenic Structural analogs that cannot form ring oxidized vinyl not tumorigenic: 4-methylstyrene; metabolites are toluene • • Key Events Weight of Evidence: Ring-Oxidized Metabolites Drive Cytotoxicity and Lung Tumors

143 | November 14, 2013 1 6 not lung toxic in CYP2F2 KO or CYP2F1-humanized mice. not lung toxic in CYP2F2 KO or and cytotoxic agent, however, SO is oxide (SO) hypothesized as putative genotoxic and cytotoxic agent, however, Styrene cannot form alkyl-epoxide metabolite, yet still produces lung tumors. Ethylbenzene cannot form alkyl-epoxide metabolite, yet still produces Alternative MoA : Genotoxicity • •

144 | November 14, 2013 1 7 MoAs All chemicals in proposed class are non- or weakly genotoxic non- or class are All chemicals in proposed Human lung metabolism preparations obtained from whole obtained from Human lung metabolism preparations lung; low metabolism due to dilution of unidentified target cell metabolism? Humanized mice compensate? human epidemiology not supportive of lung toxicity, However, animal tumorigenicity dose overlaps styrene, even though for human occupational exposures sites, e.g., CAR- Plausible alternative MoAs operating at other tumors; accentuation of rat kidney mouse liver receptor α -2u-globulin male rat tumorigenicity or nephropathy • • Most of chemicals in class cause tumors at multiple sites • and napthalene studied in CYP2F2 KO and/or Only styrene CYP2F1 mice; possible alternative metabolic activation Human lung metabolism: difficult to obtain and study • • • • Uncertainties, Inconsistencies, Data Gaps

145 | November 14, 2013 1 8 . Toxicol From: Cohen et al., From: Crit. Rev. 33: 581-589, 2003 X x ? Human Relevance Framework: CYP2F2-Mediated Mouse Lung Tumors

146 | November 14, 2013 Appendix C: Post Workshop Materials

147 | November 14, 2013

Response

– Data for Data

Relationships Relationships Implications of Implications R/Temporal R/Temporal Dose Postulated MOAs MOAs Postulated D - Specificity Consistency, Biological Plausibility From: Meek & Klaunig, 2010 Kinetic & Dynamic & Dynamic Kinetic

Confidence? Confidence? Confidence?

in animals?

differences differences establish the in key events? MoA Q1. Is the weight of evidence sufficient evidenceto sufficient differences in key differences events? Q2. Fundamental qualitative Q3. Fundamental quantitative

IPCS/ILSI MOA/HR MOA/HR IPCS/ILSI (WOE) Framework Criteria” of “Key biology humans relevant relevant between between Events” & Events” Key Events” Events” Key established animals & animals “ Comparison based on “Hill on “Hill based

148 | November 14, 2013

effect Adverse Key events Mode of action assessment assessment Implications for risk

and and on events) confidence of confidence based Weight ofWeight (key Bradford Hill concordance Evidence for Hypothesized considerations mode of action Qualitative Qualitative quantitative human Level Level Level ofLevel

gaps identified gaps

data data Critical

data data Critical - - Modified MOA Framework Modified MOA generation generation specific data Assessment specific data Assessment

149 | November 14, 2013 3

Initial

Conclusion Likely or not? Likely or not? Likely or not?

From: Meek From: Meek & Klaunig, 2010

Genotoxicity Compounds)

Nature Nature of Evidence First Step First (Chemical Specific or Related Specific or (Chemical Pattern inconsistentPattern Pattern of Pattern Results Multiple studies on key studiesMultiple events Possible MOAs Possible -

prolifera /

MOA Possible regeneration

Oxidative Oxidative metabolism/sustained cytotoxicity Direct Acting Direct Acting Mutagen? Metabolic Reductive Pathway? tive

150 | November 14, 2013 4

From: Meek et al. submitted ‒ Are the key key events Are the ‒

‒ Are the key key events Are the ‒

Dose response response Dose toxic effect of the incidence the Is key for the that than less consistent events? reversible if sequence of events the Is key event or a is stopped dosing prevented? Is the pattern across of effects the with consistent species/strains mode of action? hypothesized observed at doses below to or similar at doses below observed associated those with the apical effect? Temporality order? in hypothesized observed of action mode hypothesized the Does sense make based on broader established (e.g., biology, knowledge action)? of mode

Modified BHModified Considerations Response & Temporal Response & Temporal

Dose

Concordance specificity Consistency, Biological plausibility

151 | November 14, 2013

+ ++ + = severity 52 weeks 52 107 weeks 107 Key event Key 3 From: Meek & Klaunig, 2010 & From: Meek Klaunig,

+ ++ +++ weeks 52 weeks 13 weeks 52

Key event 2 event Key

Temporal + ++ +++ 4 weeks 4 weeks 4 weeks Key event 1 event Key )

Response and Temporality

–

1 4 0.2

Dose Dose (2 ppm) (10 ppm) (10 ppm) (40 mg/kg bw/day mg/kg (

Response Dose Dose -

152 | November 14, 2013 500 500 500 Rat Mouse Rat Mouse Rat Mouse 400 400 400

300 300 300

- 200 200 200 100 100 100 DNA Adducts DNA Adducts DNA Adducts DNA 0 0 0 0 0 0 50 50 50

Quant. Dose Resp. 450 400 350 300 250 200 150 100 450 400 350 300 250 200 150 100 450 400 350 300 250 200 150 100

HPG (fmol/ug DNA) (fmol/ug HPG - N7 DNA) (fmol/ug HPG - N7 DNA) (fmol/ug HPG - 7 N

in in vitro

data

From: Meek et al. submitted - Response PBPK model incorporating metabolic rates, affinitiesenzyme and distribution on based data human by supported vivo Quantitative Concordance No data No data

highly highly humans humans limited but relevant torelevant possible in in animals, In animals; plausible in in animals,; limited data humans but but humans Considerable Considerable Considerable Considerable Considerable Strength

humans Relevant Relevant Possible enzyme in in enzyme kidney and liver kidney target organs target in Liver and kidney and kidney Liver Human

induce tumours doses that Correlation Correlation Mice & rats metabolites In all cases at with with binding of Animal

;

cytotoxicity proliferation) Key Event Key repair ( Liver tumours & Liver kidney Metabolism by CYP2E1 Concordance Table Table with Concordance Dose Sustained cell cell and damage Sustained

153 | November 14, 2013

From: Meek et al. submitted Mutagenic Mode of Action Mode Mutagenic

Comparative Comparative Weight of Evidence Cytotoxic Mode of Mode Cytotoxic Action

154 | November 14, 2013 References Cited Meek, M.E., Klaunig, J., 2010. Interpreting available data and identifying critical data gaps for benzene risk assessment. Chem-Biol Interact. 184, 279-285. Meek, M.E., Boobis, A.R., Cote, I., Dellarco, V., Fotakis, G., Munn, S., Seed, J., Vickers, C., in press. New developments in the evolution and application of the WHO/IPCS framework on mode of action/species concordance analysis. Appl. Toxicol.

155 | November 14, 2013