Discussion of definitions of adversity followed by defining NOAELs using practical case examples of adverse, non-adverse and adaptive responses along with how to deal with lesion reversibility and exacerbation of background lesions in preclinical toxicology studies. Includes comments on justifying results to regulatory authorities.

View as PDF

Adverse, Non-adverse and Adaptive Responses in Toxicologic Pathology

View as PDF
1. Adverse, Non-adverse and Adaptive Responses in Toxicologic Pathology International Academy of Toxicology Pathology (IATP) Lecture Robert R. Maronpot Website: focusontoxpath.com32nd JSTP Annual Meeting, Takamatsu, Japan
2. Acknowledgements • Photomicrographs – National Toxicology Program Archives – NTP Non-neoplastic Lesion Atlas – Dr. Rick Hailey – Dr. Peter Mann – Dr. David Malarkey • Japanese translations – Dr. Katsuhiko Yoshizawa
3. Outline of Presentation • Definitions – Adverse response – Adaptive response – Reversible response – Exacerbation of background lesions • For the Toxicology Report & Regulatory Submission • Paradigm Shift & Determining Adversity • Practical Examples of Adverse and Adaptive Responses in Preclinical Studies
4. Adverse Response There is no perfect definition of an adverse response in a preclinical study. 前臨床試験におけるadverse(有害性) 反応の完璧な定義はない
5. Adverse Response • A biochemical, morphological or physiological change (in response to a stimulus) that either singly or in combination adversely affects the performance of the whole organism or reduces the organism’s ability to respond to an additional environmental challenge Lewis et al., Toxicol Pathol 30:66-74 (2002)
6. Adverse Response • In very broad terms, an adverse finding may be considered to be a change (biochemical, functional, or structural) that may impair performance and generally has a detrimental effect on growth, development, or life span of a non-clinical toxicology model. • More specifically, an adverse effect in a non- clinical toxicology study should be an effect that would be unacceptable if it occurred in a human clinical trial (FDA Guidance, 2002). Dorato & Englehardt, Reg Tox & Pharmacol 42:265-274 (2005)
7. Adverse Effect • The judgment on the adverse nature of an observation in a non-clinical toxicology study is subject to discussion, challenge, and reinterpretation. • 前臨床試験で観察されたadverse(有害性)の判断 は、ディスカッション、チャレンジおよび解釈を必要と する。 Dorato & Englehardt, Reg Tox & Pharmacol 2005
8. Adverse Response • A change in morphology, physiology, growth, development, reproduction, or life span of a cell or organism, system, or (sub)population that results in: – impairment of functional capacity – 機能的なキャパシティ‐を損なう – impairment of the capacity to compensate for additional stress or – 付加的なストレスを補うキャパシティーを損なう – increase in susceptibility to other influences – 他の影響に対する感受性が増加する Keller et al., Toxicol Sci 2012
9. No Observed Adverse Effect Level (NOAEL) • Highest dose or exposure that does not cause a toxicologically relevant increase in frequency or severity of effects between exposed and control groups based on careful biological and statistical analysis. • Minimum toxic or pharmacodynamic responses may occur at the NOAEL and may not endanger human health or be precursors to serious events with continued duration of exposure. Dorato & Englehardt 2005
10. NOAEL (Keller et al., 2012) • Highest exposure level with no significant increases in the frequency or severity of adverse effects between exposed population and its appropriate control. • 投与群と適切な対照群との間で、adverse effect(有害作用) の頻度・程度に関して有意な増加がみられない最も高い暴 露量 • Significance is considered with regard to biological significance in the test species and may also incorporate statistical significance. • 有意性とは、試験動物において生物学的に意味があるとい うことであり、統計学的な有意差を含んでいることもある。
11. Two Recent Publications • Scientific and Regulatory Policy Committee: Recommended (“Best”) Practices for Determining, Communicating, and Using Adverse Effect Data from Nonclinical Studies – Kerlin et al., 2015 Toxicologic Pathology (DOI:10.1177.0192623315623265) • Characterizing “Adversity” of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop – Palazzi et al., 2016 Toxicologic Pathology (In Press)
12. STP Recommendations • Judgment if a test article effect in a nonclinical study is adverse or nonadverse must be clearly stated in the study report • 前臨床試験において、被験物質の影響がadverse(有害性)である かの判断は、試験報告書に明確に言及しなければならない • Adversity in a nonclinical study should be applied only to the test species used in that study • 前臨床試験におけるadversity(有害性)は、その試験で使用した 動物種のみに適応すべきである • Setting a NOAEL for a test article should be stated in an overview document based on data from multiple studies. • 被験物質に対するNOAELの設定は、多くの試験データに基づいた 概要書に言及されるべきである
13. STP Recommendations • All available data from nonclinical studies must be evaluated together to define potential toxicities and predict human risk. • 潜在的な毒性の明確化とヒトへのリスク予測のために、前臨床 試験の全ての利用できるデータは評価されなければならない • Communication of adverse findings and NOAEL should include direct participation of contributing scientific disciplines (toxicologists, pathologists, risk assessore, etc.) in assessing and communicating human risk. • Adverse(有害)所見とNOAELに関するコミュニケーションは、ヒト のリスク評価・コミュニケーションにおいて、関係した科学者(毒 性学者、病理学者、リスク評価者など)の直接的参加が含まれ るべきだ
14. Factors to Consider in an Adversity Call • Are there related pathological findings? • 関連する病理所見はあるか? • Is there a known or biologically plausible underlying mechanism? • 既知あるいは生物学的に妥当なメカニズムがあるか? • What are the severity criteria? • 重篤度のクライテリアは何か? • What is the background incidence (historical control)? • 背景データでの頻度はどうか(ヒストリカルコントロー ル)?
15. Calling a Finding ADVERSE Should NOT be Based On: • Extrapolation across species (including humans) • 種をまたがる外挿性(ヒトを含む) • Whether the effect is an exacerbation of a background lesion • その変化は背景病変を増悪化させたものかどうか • Whether the effect is a presumed supra-pharmacological effect • その変化は薬理効果を増強させたものと予想できるかどうか • Whether the effect is primary, secondary, tertiary, etc,. • その変化は、直接的、二次的、三次的なもの……かどうか • Whether the lesion is transient or reversible • その病変は一時的なものか回復可能なものか • Solely on statistics • 単に統計学的なものか
16. Are some findings non-adverse?
17. Are some findings non-adverse? • Bile duct hyperplasia • Lymphoid hyperplasia • Microsomal enzyme induction • Decreased serum ALT and AST • Extramedullary hematopoiesis in liver
18. Are some findings non-adverse? • Bile duct hyperplasia • Lymphoid hyperplasia • Microsomal enzyme induction • Decreased serum ALT and AST • Extramedullary hematopoiesis in liver When findings such as these do not compromise normal tissue physiology, do not impair functional capacity, and do not increase susceptibility to other influences, then they may be considered non-adverse.
19. Some Examples of Non-Adverse Findings Thymic involution in a chronic rat study Congenital cyst in a rodent study
20. Some Examples of Non-Adverse Findings Bile duct hyperplasia in a chronic rat study
21. Some Examples of Non-Adverse Findings Bile duct hyperplasia in a chronic rat study However, this degree of bile duct hyperplasia in a 90-day rat study would probably be an adverse response.
22. Some Examples of Non-Adverse Findings Extramedullary hematopoiesis in the liver – a secondary response to bone marrow suppression Pigment in a lymph node – a normal function in a draining lymph node
23. Some Examples of Non-Adverse Findings Extramedullary hematopoiesis in the liver – a secondary response to bone marrow suppression Pigment in a lymph node – a normal function in a draining lymph node These secondary responses could be considered adaptive responses.
24. Adaptive Response • In the context of a toxicology study, the process whereby a cell or organism responds to a xenobiotic so that the cell or organism will survive in the new environment that contains the xenobiotic without impairment of function. • Adaptive responses to toxicant exposure may be characterized by reversibility.
25. Adaptation • Evolutionary strategy to insure survival in a new environment where the xenobiotic is present • 生体異物の存在する新しい環境において、生き残るた めの進化的戦略である • Adaptation can be adverse • Adaptation(適応)がadverse(有害性)になりうる • Example: Tracheal squamous metaplasia • Adaptive changes are not always reversible • 適応性変化は必ずしも回復性があるとは限らない • Example: Hepatic fibrosis
26. Adaptive Responses • Adaptive change allows organism to respond to environmental change & is usually beneficial but not necessarily desirable • 生物体の環境変化への反応として適応性変化がお こるが、通常は有益であるが、必ずしも望ましいもの ではない • Can result in a new functional steady state in a tissue or organ • 組織や器官において、新たな機能的安定状態にな ることができる
27. Adaptive Response (cont) • Adaptive changes are often early homeostatic adjustments (metabolism or gene expression/transcriptomic changes) • 適応性変化はしばしば初期の恒常性調節機構である(代謝 あるいは遺伝子発現/トランスクリプトームな変化) • May temporarily result in a new homeostatic steady-state • 一次的に新たな恒常性の定常状態になるかもしれない • Adaptive changes may ultimately result in return to a normal homeostatic condition • 適応性変化は最終的に正常の恒常状態に戻るかもしれない
28. Adaptive Responses • An adaptive change can sometimes be adverse • 適応性変化は時にadverse(有害性)となりうる
29. Adaptive Responses • An adaptive change can sometimes be adverse • 適応性変化は時にadverse(有害性)となりうる Squamous metaplasia of the trachea occurred following inhalation exposure to smoke.
30. Adaptive Responses • An adaptive change can sometimes be adverse • 適応性変化は時にadverse(有害性)となりうる Squamous metaplasia affects normal cilliary function in the trachea and is, therefore, an adaptive response that is adverse.
31. Adaptive Responses • An adaptive change can sometimes be adverse • 適応性変化は時にadverse(有害性)となりうる Squamous metaplasia affects normal ciliary function in the trachea and is, therefore, an adaptive response that is adverse. Even thought it is potentially reversible after the cause is removed, it is still an adverse response.
32. Reversibility • Disappears after treatment is stopped • Typically determined using recovery groups in toxicity studies
33. What Determines if a Lesion is Reversible? • Depends upon the regenerative capacity of the tissue or organ • Depends of the type of lesion – Proliferative/non-proliferative • Depends on the severity of the lesion • Depends upon length of time without further treatment – Partial/complete reversibility
34. Reversibility • An adverse lesion may or may not be reversible • Adverse(有害性)な変化は回復性がある場合やない 場合がある • If an adverse lesion is reversible, then it can be: • もしadverse(有害性)な変化に回復性があるならば、 A key component in weight-of-evidence in study interpretation – 試験の解釈でのweight-of-evidenceにおけるキーポイント である。 – May indicate a lower level of concern – より低用量に懸念があるかもしれない Perry et al., 2013. Toxicologic Pathology 41: 1159-1169 Sewell et al., 2014. Regulatory Toxicology & Pharmacology 70: 414-429
35. What About Exacerbation?
36. Exacerbation Exacerbation = an increase in the incidence and/or severity of an age-related and/or strain-specific common background lesion seen in control animals 増悪化とは :対照群の動物で観察される加齢性変化や系統に特異的 に観察される背景データに関して、その発現頻度や程度 が増加することである。
37. Exacerbation Can exacerbated background lesions be adverse? Exacerbation = an increase in the incidence and/or severity of an age-related and/or strain-specific common background lesion seen in control animals 増悪化とは :対照群の動物で観察される加齢性変化や系統に特異的 に観察される背景データに関して、その発現頻度や程度 が増加することである。
38. Exacerbation Can exacerbated background lesions be adverse? Yes, if – • the exacerbation is a biologically plausible primary effect of the test agent • the exacerbation shows a clear dose-response • the exacerbation exceeds historical control Exacerbation = an increase in the incidence and/or severity of an age-related and/or strain-specific common background lesion seen in control animals 増悪化とは :対照群の動物で観察される加齢性変化や系統に特異的 に観察される背景データに関して、その発現頻度や程度 が増加することである。
39. • Definitions – Adverse response – Adaptive response – Reversible response – Exacerbation of background lesions • For the Toxicology Report & Regulatory Submission • Paradigm Shift & Determining Adversity • Practical Examples of Adverse and Adaptive Responses in Preclinical Studies Outline of Presentation
40. What is needed in the toxicology report for submission to regulatory authorities? 規制当局に提出する毒性レポートで何 が必要とされるか?
41. For the Toxicology Report and Regulatory Submission • Need detailed description of what is adverse for each health-related endpoint • どの健康に関連するエンドポイントに対しても、何が adverse(有害性)であるかについて詳細な記載が必要で ある • Provide details on pathogenesis & mechanism • 病因とメカニズムを詳細に提供すること • Explain morphological criteria for diagnoses and severity scoring • 診断の形態学的クライテリアと病変程度のスコアーにつ いて説明すること
42. For the Toxicology Report and Regulatory Submission • Clearly characterize relevant changes & explain judgments regarding why a change is adverse or non-adverse • 関連のある変化を明確に特徴付けることと、変化が adverse(有害性)かどうかの判断した根拠について 説明すること • Consider use of outside peer review & expert reports • 外部のピアレビューや専門家の報告を利用すること を考慮すること
43. For the Toxicology Report and Regulatory Submission • Cite relevant literature • For example : using alveolar histiocytosis Control Treated
44. For the Toxicology Report and Regulatory Submission • Cite relevant literature • For example : using alveolar histiocytosis Treated • Inhalation study • Serum enzymes are normal • Minimal to mild increase in alveolar histiocytes • No evidence of inflammation • No epithelial hyperplasia Sponsor considered the alveolar histiocytosis to be adaptive and non-adverse because…
45. For the Toxicology Report and Regulatory Submission • Cite relevant literature • For example : using alveolar histiocytosis Treated • Inhalation study • Serum enzymes are normal • Minimal to mild increase in alveolar histiocytes • No evidence of inflammation • No epithelial hyperplasia Sponsor considered the alveolar histiocytosis to be adaptive and non-adverse because… In submitting their report to a regulatory authority, the sponsor cited some relevant literature
46. For the Toxicology Report and Regulatory Submission • Citing relevant literature for histiocytosis example Nikula et al., 2014. Toxicologic Pathology 42:472-486. “An STP working group focused on distinguishing adaptive versus adverse responses in rodents and concluded that increases in alveolar macrophage number and/or size are not adverse if there are no other lung changes such as inflammation and/or hyperplastic epithelial responses.”
47. What about the paradigm shift? • What is the paradigm shift? – In accordance with Tox21 and related global opinion, there is the recommendation to move away from animal testing and to rely on new molecular measurements and molecular pathways • High throughput screening • Toxicity pathways • Adversity outcome pathways • In vitro testing using human cells • Computational systems biology Ankley et al., (2010) Environ Toxicol Chem 29:730-41
48. What about the paradigm shift? Now there is an interest in defining adversity at the molecular level using new molecular biology and related methods.
49. What about the paradigm shift? Now there is an interest in defining adversity at the molecular level using new molecular biology and related methods. The question is: Can adverse effects be defined at the molecular level?
50. What about the paradigm shift • Development of new molecular methods to replace animal testing • 動物実験に置き換わる新たな分子学的手法の開発 • Tox21, high throughput screening, toxicogenomics, toxicity pathways, altered gene expression, adverse outcome pathways • Use of these new molecular methods to identify adversity will require establishing scientific validation • Adversity(有害性)を見つけ出すこれらの新たな分子学的手法の 利用には、科学的なバリデーションの確立が必要となる • Until new methods are validated, we continue to rely on classical toxicity testing to identify adverse data for risk assessment • リスク評価のためのadverse(有害性)のデータを明らかにするため に、新たな手法がバリデートされるまで、我々はクラシカルな毒性 試験に頼り続けざるを得ない
51. What about the paradigm shift? Can adverse effects be defined at the molecular level? Answer: Not yet.
52. A Final Definition of Adverse • ‘…change in the morphology, physiology, growth, development, reproduction, or life span of an organism, system, or (sub) population that results in an impairment of functional capacity, an impairment of the capacity to compensate for additional stress, or an increase in susceptibility to other influences’ (IPCS, 2004). • Based on “apical responses” – Clinical signs, lesions, traditional biomarkers – And always within the context of the specific study
53. • Definitions – Adverse response – Adaptive response – Reversible response – Exacerbation of background lesions • For the Toxicology Report & Regulatory Submission • Paradigm Shift & Determining Adversity • Practical Examples of Adverse and Adaptive Responses in Preclinical Studies Outline of Presentation
54. Practical Examples • Restricted to one sex for purposes of demonstrating potential adverse responses • However, an effect seen in both genders would be of more concern • Examples are based on reasonably expected study outcomes
55. Dr. John Seely’s car
56. Sprague-Dawley 12-Month Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 20 n= 20 n= 20 n= 20 Chronic Progressive Nephropathy % Incidence 10 15 25 30 Minimal 2 2 1 0 Mild 0 0 2 2 Moderate 0 0 2 4 Severity Average 1.0 1.0 2.2 2.7 Treatment associated exacerbation of chronic progressive nephropathy
57. Sprague-Dawley 12-Month Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 20 n= 20 n= 20 n= 20 Chronic Progressive Nephropathy % Incidence 10 15 25 30 Minimal 2 2 1 0 Mild 0 0 2 2 Moderate 0 0 2 4 Severity Average 1.0 1.0 2.2 2.7 Treatment associated exacerbation of chronic progressive nephropathy Is this an adverse response?
58. Sprague-Dawley 12-Month Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 20 n= 20 n= 20 n= 20 Chronic Progressive Nephropathy % Incidence 10 15 25 30 Minimal 2 2 1 0 Mild 0 0 2 2 Moderate 0 0 2 4 Severity Average 1.0 1.0 2.2 2.7 Treatment associated exacerbation of chronic progressive nephropathy Is this an adverse response? Yes, it is adverse
59. Sprague-Dawley 12-Month Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 20 n= 20 n= 20 n= 20 Chronic Progressive Nephropathy % Incidence 10 15 25 30 Minimal 2 2 1 0 Mild 0 0 2 2 Moderate 0 0 2 4 Severity Average 1.0 1.0 2.2 2.7 Treatment associated exacerbation of chronic progressive nephropathy Is this an adverse response? Yes, it is adverse Dose-related increase incidence and increased severity
60. Sprague-Dawley 12-Month Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 20 n= 20 n= 20 n= 20 Chronic Progressive Nephropathy % Incidence 10 15 25 30 Minimal 2 2 1 0 Mild 0 0 2 2 Moderate 0 0 2 4 Severity Average 1.0 1.0 2.2 2.7 Treatment associated exacerbation of chronic progressive nephropathy Is this an adverse response? NOAEL
61. 9-Month Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure KIDNEY n= 15 n= 15 n= 15 n= 15 Karyomegaly 0 0 15 15 Tubular hyperplasia 0 0 1 0 Tubular adenoma 0 0 0 0 Renal Tubular Karyomegaly
62. 9-Month Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure KIDNEY n= 15 n= 15 n= 15 n= 15 Karyomegaly 0 0 15 15 Tubular hyperplasia 0 0 1 0 Tubular adenoma 0 0 0 0 Renal Tubular Karyomegaly Boorman et al., 1992. Toxicologic Pathology 20:236-245 Frazier et al., 2012. Toxicologic Pathology 40: 7S-86S • The toxicological significance of this change is unknown • There is no evidence that it progresses to neoplasia • Not considered adverse by subject matter experts
63. 9-Month Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure KIDNEY n= 15 n= 15 n= 15 n= 15 Karyomegaly 0 0 15 15 Tubular hyperplasia 0 0 1 0 Tubular adenoma 0 0 0 0 Renal Tubular Karyomegaly Boorman et al., 1992. Toxicologic Pathology 20:236-245 Frazier et al., 2012. Toxicologic Pathology 40: 7S-86S • The toxicological significance of this change is unknown • There is no evidence that it progresses to neoplasia • Not considered adverse by subject matter experts Non-adverse
64. Renal Tubular Karyomegaly Boorman et al., 1992. Toxicologic Pathology 20:236-245 Frazier et al., 2012. Toxicologic Pathology 40: 7S-86S • The toxicological significance of this change is unknown • There is no evidence that it progresses to neoplasia • Not considered adverse by subject matter experts
65. Renal Tubular Karyomegaly Boorman et al., 1992. Toxicologic Pathology 20:236-245 Frazier et al., 2012. Toxicologic Pathology 40: 7S-86S • The toxicological significance of this change is unknown • There is no evidence that it progresses to neoplasia • Not considered adverse but subject matter experts 9-Month Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure KIDNEY n= 15 n= 15 n= 15 n= 15 Karyomegaly 0 0 15 15 Tubular hyperplasia 0 0 3 6 Tubular adenoma 0 0 0 1 Everything is the same except
66. Renal Tubular Karyomegaly Boorman et al., 1992. Toxicologic Pathology 20:236-245 Frazier et al., 2012. Toxicologic Pathology 40: 7S-86S • The toxicological significance of this change is unknown • There is no evidence that it progresses to neoplasia • Not considered adverse by subject matter experts Probably adverse because of tubular hyperplasia and adenoma
67. Renal Papillary Mineralization
68. 90-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 Kidney – Mineralization Minimal 1 2 1 3 Mild 0 0 1 1 Moderate 0 0 1 1 Total 1 2 3 5 Kidney – Mineralization • Treatment may exacerbate this background lesion • May not be a direct effect of the test agent • Mechanism is usually unknown
69. 90-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 Kidney – Mineralization Minimal 1 2 1 3 Mild 0 0 1 1 Moderate 0 0 1 1 Total 1 2 3 5 Kidney – Mineralization • Treatment may exacerbate this background lesion • May not be a direct effect of the test agent • Mechanism is usually unknown Presumption – a perturbation of calcium homeostasis
70. 90-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 Kidney – Mineralization Minimal 1 2 1 3 Mild 0 0 1 1 Moderate 0 0 1 1 Total 1 2 3 5 Kidney – Mineralization • Treatment may exacerbate this background lesion • May not be a direct effect of the test agent • Mechanism is usually unknown Presumption – a perturbation of calcium homeostasis Conservative NOAEL is the low dose
71. Adrenal Cortical Necrosis
72. Adrenal 90-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 Adrenal Necrosis, diffuse, severe 0 0 2 8 Hemorrhage, cortex, focal 0 0 1 6
73. Adrenal • Adrenal cortical necrosis is adverse • The 2/10 incidence at the medium exposure is a severe response • The NOAEL is the low exposure
74. Alveolar Histiocytosis Control Exposed
75. Lung – Alveolar histiocytosis • Inhalation study • Serum enzymes are normal • Minimal to mild increase in alveolar histiocytes
76. Lung – Alveolar histiocytosis • Inhalation study • Serum enzymes are normal • Minimal to mild increase in alveolar histiocytes Is this adverse?
77. Lung – Alveolar histiocytosis • Inhalation study • Serum enzymes are normal • Minimal to mild increase in alveolar histiocytes Is this adverse? This degree of histiocytosis is not considered adverse by lung experts.
78. Lung – Alveolar histiocytosis 14-Day Recovery
79. Lung – Alveolar histiocytosis 14-Day Recovery Not considered adverse, even without recovery
80. Minimal to mild alveolar histiocytosis reflects a macrophage response to changes in the local environment & represents a tissue adaptation to maintain normal lung function
81. Minimal to mild alveolar histiocytosis reflects a macrophage response to changes in the local environment & represents a tissue adaptation to maintain normal lung function But severe alveolar histiocytosis as in this example represents an adverse response, even if it is reversible.
82. Lesion severity is important in defining an adverse effect
83. Lymph Node Histiocytic Infiltrate
84. Lymph Node 90-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 Infiltrates, histiocytic Minimal 2 3 5 4 Mild 1 1 3 3 Moderate 0 0 1 3 Total 3 4 9 10 Necrotic foci Minimal 0 0 1 0
85. 90-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 Infiltrates, histiocytic Minimal 2 3 5 4 Mild 1 1 3 3 Moderate 0 0 1 3 Total 3 4 9 10 Necrotic foci Minimal 0 0 1 0 Lymph Node • Clearance via lymph node drainage is a part of normal lymph node physiology • Not adverse unless severe and associated with necrosis in the affected lymph node
86. Lymph Node Although there is a dose response and increased severity, this change may be non-adverse. A recovery study would help clarify this issue. 90-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 Infiltrates, histiocytic Minimal 2 3 5 4 Mild 1 1 3 3 Moderate 0 0 1 3 Total 3 4 9 10 Necrotic foci Minimal 0 0 1 0 • Clearance via lymph node drainage is a part of normal lymph node physiology • Not adverse unless severe and associated with necrosis in the affected lymph node
87. Lymph Node 90-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 Infiltrates, histiocytic Minimal 2 3 5 4 Mild 1 1 3 3 Moderate 0 0 1 3 Total 3 4 9 10 Necrotic foci Minimal 0 0 1 0 • Clearance via lymph node drainage is a part of normal lymph node physiology
88. Progressive Cardiomyopathy in Rats
89. Progressive Cardiomyopathy – Rat Slide courtesy of Dr. Rick Hailey
90. Progressive Cardiomyopathy – Rat • Background lesion in rats in prechronic studies • Especially common in Sprague Dawley rats • Difficult to find at low magnification • Typically very mild in severity unless exacerbated by treatment • Randomly distributed in the myocardium Slide courtesy of Dr. Rick Hailey
91. Progressive Cardiomyopathy – Rat • Background lesion in rats in prechronic studies • Especially common in Sprague Dawley rats • Difficult to find at low magnification • Typically very mild in severity unless exacerbated by treatment • Randomly distributed in the myocardium Pathogenesis: myocardial fiber degenerationmononuclear inflammatory responsephagocytosis of degenerating myofibersfocal fibrosis Slide courtesy of Dr. Rick Hailey
92. Examples of Progressive Cardiomyopathy Slide courtesy of Dr. Rick Hailey
93. Slide 1 of 3 Cardiomyopathy in a 13-Week Study Slide courtesy of Dr. Rick Hailey
94. Slide 2 of 3 Medium Magnification Slide courtesy of Dr. Rick Hailey
95. Slide 3 of 3 High Magnification Slide courtesy of Dr. Rick Hailey
96. Progressive Cardiomyopathy Adverse or Non-adverse?
97. Progressive Cardiomyopathy Non-Adverse No dose-response All lesions are of minimal severity
98. Progressive Cardiomyopathy Adverse or Non-adverse?
99. Progressive Cardiomyopathy Non-Adverse No convincing dose-response All lesions are of minimal severity
100. Progressive Cardiomyopathy 13-Week Rat Study Progressive Cardiomyopathy Control Low-Dose Medium-Dose High-Dose 1/10 3/10 3/10 4/10 (1) (1) (1.5) (2) ( ) = Severity score: 1= minimal, 2= mild, 3 = moderate, 4 = marked Same lesion but different incidence !!
101. Progressive Cardiomyopathy 13-Week Rat Study Progressive Cardiomyopathy Control Low-Dose Medium-Dose High-Dose 1/10 3/10 3/10 4/10 (1) (1) (1.5) (2) ( ) = Severity score: 1= minimal, 2= mild, 3 = moderate, 4 = marked Adverse or Non-adverse? Same lesion but different incidence !!
102. Progressive Cardiomyopathy 13-Week Rat Study Progressive Cardiomyopathy Control Low-Dose Medium-Dose High-Dose 1/10 3/10 3/10 4/10 (1) (1) (1.5) (2) ( ) = Severity score: 1= minimal, 2= mild, 3 = moderate, 4 = marked Adverse Dose-response – yes Increased severity – yes NOAEL
103. Acute Centrilobular Necrosis (Prechronic Mouse Study) Liver enzymes increased > 5-fold
104. Acute Centrilobular Necrosis (Prechronic Mouse Study) This change is adverse Liver enzymes increased > 5-fold
105. Acute Periportal Necrosis Liver enzymes increased > 5-fold
106. Acute Periportal Necrosis This change is adverse Liver enzymes increased > 5-fold
107. Acute Liver Response Return to normal in 3 to 7 days This represents an example of reversibility
108. Acute Liver Response Return to normal in 3 to 7 days Liver enzyme levels may or may not still be elevated when the liver structure has returned to normal depending upon their half-life in the circulation. This represents an example of reversibility
109. Acute Liver Response Return to normal in 3 to 7 days Liver enzyme levels may or may not still be elevated when the liver structure has returned to normal depending upon their half-life in the circulation.What is adverse or what is non-adverse may depend on when you take the sample. This represents an example of reversibility
110. Acute Liver Response Return to normal in 3 to 7 days What is adverse or what is non-adverse may depend on when you take the sample. This represents an example of reversibility The fact that a lesion is reversible is an important piece of information that should be considered in a weight-of-evidence approach.
111. Cholangitis Control Treated Treatment-associated exacerbation of cholangitis Common background change in older rats Adverse?
112. Cholangitis Control Treated Adverse? In general, minimal treatment-associated exacerbations of background lesions are non-adverse but mild, moderate, and marked changes are adverse.
113. Cholangitis Control Treated Increase in inflammatory cells; some are neutrophils; there is necrosis of adjacent hepatocytes (arrows) Common background change in older rats Slide courtesy of R. Hailey Adverse?
114. Cholangitis Control Treated Increase in inflammatory cells; some are neutrophils; there is necrosis of adjacent hepatocytes (arrows) Common background change in older rats Slide courtesy of R. Hailey Adverse-YES
115. Biliary epithelial hyperplasia: Adverse? Are These Examples of Bile Duct Hyperplasia Adverse? Slide courtesy of R. Hailey
116. Biliary epithelial hyperplasia: Adverse? Mitotic figures Proliferation outside the portal triad Are These Examples of Bile Duct Hyperplasia Adverse? Slide courtesy of R. Hailey
117. Biliary epithelial hyperplasia: Adverse? Mitotic figures Hepatocellular necrosis Proliferation outside the portal triad Are These Examples of Bile Duct Hyperplasia Adverse? Slide courtesy of R. Hailey
118. Biliary epithelial hyperplasia: Adverse? Mitotic figures Hepatocellular necrosis Proliferation outside the portal triad Yes, They Are Adverse! Slide courtesy of R. Hailey
119. Is this adverse? Proliferation outside the portal triad and cellular atypia Increases in ALT and AST (up to 20X) and ALP (1.4X) and bilirubin (13X) Another Example of An Adverse Response Affecting Bile Ducts
120. Can Occur Spontaneously Control- no change Control- spontaneous biliary epithelial hyperplasia Bile ducts In Contrast This Degree of Bile Duct Hyperplasia is Non-Adverse
121. Hepatic Foci of Cellular Alteration
122. Hepatic Foci of Cellular Alteration • Occur spontaneous in older rodents • May be induced by treatment in younger rodents • Can be present with or without hepatotoxicity • 1 Hepatocellular tumor per 10,000 foci Kaufmann et al., Am J Pathol 119:171-174 (1985)
123. Are Foci Predictors of Liver Tumor Response? Are foci adverse?
124. Hepatic Foci of Cellular Alteration * p< 0.05 ** p< 0.01
125. Hepatic Foci of Cellular Alteration * p< 0.05 ** p< 0.01 • A treatment-related increase in foci at the medium & high exposures per se may not be considered adverse, but this is controversial • 中間並びに高用量群で投与に関連した変異 細胞巣の増加が認められ、本質的にadverse (有害性)とは考えられないかもしれないが、 これは議論の余地があるだろう • A single hepatocellular adenoma can occur by chance, even in a 90-day study • 90日試験でさえも、肝細胞腺腫が1例に偶発 所見としてみられている
126. Hepatic Foci of Cellular Alteration * p< 0.05 ** p< 0.01 • This incidence of foci in a 12-month study is of some concern since it may predict a potential hepatocarcinogenic response in a long-term study. • The concern may be sufficient to stop development of a new drug or pesticide • However, aside from its predictive potential, a liver focus response by itself does not compromise liver function sufficiently to be classified as adverse.
127. Hepatic Foci of Cellular Alteration * p< 0.05 ** p< 0.01 • This incidence of foci in a 12-month study is of some concern since it may predict a potential hepatocarcinogenic response in a long-term study. • The concern may be sufficient to stop development of a new drug or pesticide • However, aside from its predictive potential, a liver focus response by itself does not compromise liver function sufficiently to be classified as adverse. It is OK to disagree with my opinion on this.
128. Hepatic Foci of Cellular Alteration * p< 0.05 ** p< 0.01 • This incidence of foci in a 12-month study is of some concern since it may predict a potential hepatocarcinogenic response in a long-term study. • The concern may be sufficient to stop development of a new drug or pesticide • However, aside from its predictive potential, a liver focus response by itself does not compromise liver function sufficiently to be classified as adverse. It is OK to disagree with my opinion on this. You may feel that development of foci is part of a continuum known to progress to adversity.
129. Hepatomegaly Control Treated
130. Liver Weight Increases Without Other Changes • < 10% liver weight – non-adverse • >40-50% increase liver at 12-months – leads to cancer (it is predictive but not necessarily adverse) Carmichael et al., Environ Hlth Perspectives 105: 1196-1203 (1997) Haseman et al., Toxicol Pathol 25:256-263(1997) Maronpot et al., Toxicol Pathol 38: 7786-795 (2010
131. Liver Weight Increases Without Other Changes • < 10% liver weight – non-adverse • >20% liver weight increase = adverse – According to some regulatory authorities • >40-50% increase liver at 12- months – leads to cancer (predictive) • This is controversial!! • 20% liver weight increases are common responses to xenobiotic exposures • In the absence of any other changes, a 20% increase should not be considered adverse Carmichael et al., Environ Hlth Perspectives 105: 1196-1203 (1997) Haseman et al., Toxicol Pathol 25:256-263(1997) Maronpot et al., Toxicol Pathol 38: 7786-795 (2010
132. Chemicals That Cause Liver Enlargement • Chemicals that cause liver enlargement (hypertrophy/hyperplasia) at low doses often will cause degeneration at higher doses • 低用量で肝腫大(肥大/過形成)を引き起こす化学物質は、より 高用量では変性をしばしば誘発する • Saturation of normal clearance mechanisms – Alternate metabolic routes can damage cellular macromolecules • Presence of degeneration is adverse • 変性の存在はadverse(有害性)である • Resulting functional loss may not be completely reversed (e.g., heptic fibrosis) • 結果としてみられる機能消失は完全に回復しないかもしれない (例えば肝線維化)
133. Hepatomegaly in Prechronic Studies is a Frequent Reflection of Microsomal Enzyme Induction Control Treated
134. Hepatic Enzyme Induction • Centrilobular hepatocyte hypertrophy
135. Microsomal Enzyme Induction
136. N N Smooth Endoplasmic Reticulum (SER) Proliferation Control Treated Arrows = smooth endoplasmic reticulum
137. Microsomal Enzyme Induction Evidence of enzyme induction without any other changes is typically an adaptive change that is not adverse.
138. Hepatic Enzyme Induction • Centrilobular hepatocyte hypertrophy Adaptive and non-adverse when there is only hepatocellular hypertrophy Typically reversible when exposure is stopped
139. Enzyme Induction with other changes should be considered an adverse response Cytoplasmic vacuolation & bile duct hyperplasia Single cell necrosis
140. 1. extreme hypertrophy leading to reduced sinusoidal blood circulation, hypoxia, and necrosis and/or 2. metabolic activation forming more toxic active metabolites Proposed Mechanisms for Hepatocyte Necrosis Adverse
141. Extreme Hepatocyte Hypertrophy • Enlarged hepatocytes • Increased cytoplasm • Multiple nuclei • Karyomegaly Adverse
142. Liver Case #1 90-Day Rat Study Microscopic Findings Control Low Exposure Low Medium Exposure High Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 n=10 Liver –Centrilobular hypertrophy 0 4 (1) 7 (1.5) 9 (1.5) 9 (2) Hepatocyte fatty change 6 (1.5) 9 (2) 10 (3) 9 (3) 8 (3) ( ) = Severity: 1 – minimal 2 – mild 3 = marked
143. Liver Case #1 This is very similar to an actual case that was considered by a regulatory agency. 90-Day Rat Study Microscopic Findings Control Low Exposure Low Medium Exposure High Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 n=10 Liver –Centrilobular hypertrophy 0 4 (1) 7 (1.5) 9 (1.5) 9 (2) Hepatocyte fatty change 6 (1.5) 9 (2) 10 (3) 9 (3) 8 (3) ( ) = Severity: 1 – minimal 2 – mild 3 = marked
144. Liver Case #1 • Generally low severity centrilobular hepatocyte hypertrophy • No cytotoxicity or hepatocellular degeneration • Liver weight increased up to 35% • ALT & AST increased 2.6 to 4.5 at 2 highest exposures • Plasma cholesterol increased 25 to 40% at 3 highest doses 90-Day Rat Study Microscopic Findings Control Low Exposure Low Medium Exposure High Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 n=10 Liver –Centrilobular hypertrophy 0 4 (1) 7 (1.5) 9 (1.5) 9 (2) Hepatocyte fatty change 6 (1.5) 9 (2) 10 (3) 9 (3) 8 (3) ( ) = Severity: 1 – minimal 2 – mild 3 = marked
145. Liver Case #1 HYPERTROPHY CONSIDERED ADAPTIVE • Typical low severity centrilobular hepatocyte hypertrophy & no cytotoxicity or hepatocellular degeneration • Liver weight increased up to 35% FATTY CHANGE CONSIDERED ADVERSE (Alteration in lipid metabolism) • ALT & AST increased 2.6 to 4.5 at 2 highest exposures • Plasma cholesterol increased 25 to 40% at 3 highest doses ( ) = Severity: 1 – minimal 2 – mild 3 = marked 90-Day Rat Study Microscopic Findings Control Low Exposure Low Medium Exposure High Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 n=10 Liver –Centrilobular hypertrophy 0 4 (1) 7 (1.5) 9 (1.5) 9 (2) Hepatocyte fatty change 6 (1.5) 9 (2) 10 (3) 9 (3) 8 (3)
146. Liver Case #1 NOAEL HYPERTROPHY CONSIDERED ADAPTIVE • Typical low severity centrilobular hepatocyte hypertrophy & no cytotoxicity or hepatocellular degeneration • Liver weight increased up to 35% FATTY CHANGE CONSIDERED ADVERSE (Alteration in lipid metabolism) • ALT & AST increased 2.6 to 4.5 at 2 highest exposures • Plasma cholesterol increased 25 to 40% at 3 highest doses 90-Day Rat Study Microscopic Findings Control Low Exposure Low Medium Exposure High Medium Exposure High Exposure n= 10 n= 10 n= 10 n= 10 n=10 Liver –Centrilobular hypertrophy 0 4 (1) 7 (1.5) 9 (1.5) 9 (2) Hepatocyte fatty change 6 (1.5) 9 (2) 10 (3) 9 (3) 8 (3)
147. Liver Case #2 • Increased liver weight up to 30% • Increased liver enzymes in 3 of the high exposure rats 28-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure LIVER n= 10 n= 10 n= 10 n= 10 Centrilobular hypertrophy 0 0 5 (1) 7(1) Hepatocyte vacuolation 2(1) 1(1) 1(1) 2(1) Degeneration/necrosis 0 0 0 3(1.5) Increased mitoses 0 0 0 4 (1) ( ) = Severity: 1 – minimal 2 – mild 3 = marked SIMILAR TO DATA SUBMITTED FOR REGULATORY REVIEW
148. Liver Case #2 • Hypertrophy and vacuolation considered adaptive • Degeneration/necrosis and increased mitoses considered adverse 28-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure LIVER n= 10 n= 10 n= 10 n= 10 Centrilobular hypertrophy 0 0 5 (1) 7(1) Hepatocyte vacuolation 2(1) 1(1) 1(1) 2(1) Degeneration/necrosis 0 0 0 3(1.5) Increased mitoses 0 0 0 4 (1) ( ) = Severity: 1 – minimal 2 – mild 3 = marked • Increased liver weight up to 30% • Increased liver enzymes in 3 of the high exposure rats
149. Liver Case #2 • Hypertrophy and vacuolation considered adaptive • Degeneration/necrosis and increased mitoses considered adverse 28-Day Rat Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure LIVER n= 10 n= 10 n= 10 n= 10 Centrilobular hypertrophy 0 0 5 (1) 7(1) Hepatocyte vacuolation 2(1) 1(1) 1(1) 2(1) Degeneration/necrosis 0 0 0 3(1.5) Increased mitoses 0 0 0 4 (1) ( ) = Severity: 1 – minimal 2 – mild 3 = marked NOAEL identified as medium exposure • Increased liver weight up to 30% • Increased liver enzymes in 3 of the high exposure rats
150. Three Other Practical Examples
151. Epididymis – Tunica vaginalis mesothelioma Male 2-Year F344 Study Microscopic Findings Control Low Exposure Low Medium Exposure High Medium Exposure High Exposure Tunica vaginalis mesothelioma 2/48 4% 2/48 4% 1/48 2% 3/48 6% 8/48 17% Historical control range = 3.3% to 6.4%
152. Epididymis – Tunica vaginalis mesothelioma Male 2-Year F344 Study Microscopic Findings Control Low Exposure Low Medium Exposure High Medium Exposure High Exposure Tunica vaginalis mesothelioma 2/48 4% 2/48 4% 1/48 2% 3/48 6% 8/48 17% Historical control range = 3.3% to 6.4% Adverse
153. 14-Day Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 3 n= 3 n= 3 n= 3 BRAIN Inflammation, acute 1 0 0 1 Inflammation, subacute 0 0 0 1 Dog Study No clinical signs or gross lesions Slide courtesy of Dr. Peter Mann
154. 14-Day Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure n= 3 n= 3 n= 3 n= 3 BRAIN Inflammation, acute 1 0 0 1 Inflammation, subacute 0 0 0 1 Dog Study No clinical signs or gross lesions Slide courtesy of Dr. Peter Mann A difficult call because of the small number of study animals but probably not adverse.
155. Female 2-Year F344 Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure Mononuclear cell Leukemia 8/50 16% 7/50 14% 19/51* 37% 16/50 32% * p < 0.05 Fischer’s Exact Test; p < 0.05 Life Table test Mononuclear cell leukemia • No tumors in males • No other tumors in females in this study Historical Control 14% to 36%
156. Female 2-Year F344 Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure Mononuclear Cell Leukemia 8/50 16% 7/50 14% 19/51* 37% 16/50 32% * p < 0.05 Fischer’s Exact Test; p < 0.05 Life Table test Mononuclear cell leukemia • No tumors in males • No other tumors in females in this study Historical Control 14% to 36% NTP considered this response was equivocal evidence of carcinogenicity
157. Female 2-Year F344 Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure Mononuclear Cell Leukemia 8/50 16% 7/50 14% 19/51* 37% 16/50 32% * p < 0.05 Fischer’s Exact Test; p < 0.05 Life Table test Historical Control Range = 14% to 36% Is this an exacerbation of leukemia and is it an adverse response?
158. Female 2-Year F344 Study Microscopic Findings Control Low Exposure Medium Exposure High Exposure Mononuclear Cell Leukemia 8/50 16% 7/50 14% 19/51* 37% 16/50 32% * p < 0.05 Fischer’s Exact Test; p < 0.05 Life Table test Historical Control Range = 14% to 36% Is this an exacerbation of leukemia and is it an adverse response? Because of its high & variable background incidence, a positive mononuclear cell leukemia response should take the historical control range into account in determining if there is a real treatment- related increased incidence.
159. • Definitions – Adverse response – Adaptive response – Reversible response – Exacerbation of background lesions • For the Toxicology Report & Regulatory Submission • Paradigm Shift & Determining Adversity • Practical Examples of Adverse and Adaptive Responses in Preclinical Studies Outline of Presentation
160. Adverse Response There is no perfect definition of an adverse response in a preclinical study. 前臨床試験におけるadverse反応の完 璧な定義はない
161. Criteria for Defining an Adverse Response • ‘…change in the morphology, physiology, growth, development, reproduction, or life span of an organism, system, or (sub) population that results in an impairment of functional capacity, an impairment of the capacity to compensate for additional stress, or an increase in susceptibility to other influences’ (IPCS, 2004). • At the present time based on “apical responses” – Clinical signs, lesions, traditional biomarkers – And always within the context of the specific study
162. If a finding fulfills the criteria for an adverse effect, then it is considered adverse even if: • it is transient (disappears during treatment) • 一過性の変化(投与期間中に消失) • it is reversible (disappears after treatment is stopped) • 回復性がある(投与中止後に消失) • it is caused by exaggerated pharmacology • 薬理作用が増強されたことによって発現 • It is secondary to some other change or secondary to stress • 他の所見に対する二次的な変化、あるいはストレスに対す る二次的な変化 もし、ある所見がadverse effect(有害性作用)のクライテリアを満た すならば、たとえ下記の件が該当するとしても、adverseであると考 えられる
163. Thanks to Dr. Katsuhiko Yoshizawa for Japanese translations And Thanks to you for your kind attention
164. You can view this presentation at focusontoxpath.com/adverse-responses