2-year Rat Carcinogenicity Studies Research Articles

ICH S1 prospective evaluation study and weight of evidence assessments: commentary from industry representatives

Industry representatives on the ICH S1B(R1) Expert Working Group (EWG) worked closely with colleagues from the Drug Regulatory Authorities to develop an addendum to the ICH S1B guideline on carcinogenicity studies that allows for a weight-of-evidence (WoE) carcinogenicity assessment in some cases, rather than conducting a 2-year rat carcinogenicity study. A subgroup of the EWG composed of regulators have published in this issue a detailed analysis of the Prospective Evaluation Study (PES) conducted under the auspices of the ICH S1B(R1) EWG. Based on the experience gained through the Prospective Evaluation Study (PES) process, industry members of the EWG have prepared the following commentary to aid sponsors in assessing the standard WoE factors, considering how novel investigative approaches may be used to support a WoE assessment, and preparing appropriate documentation of the WoE assessment for presentation to regulatory authorities. The commentary also reviews some of the implementation challenges sponsors must consider in developing a carcinogenicity assessment strategy. Finally, case examples drawn from previously marketed products are provided as a supplement to this commentary to provide additional examples of how WoE criteria may be applied. The information and opinions expressed in this commentary are aimed at increasing the quality of WoE assessments to ensure the successful implementation of this approach.

ICH S1 prospective evaluation study: weight of evidence approach to predict outcome and value of 2-year rat carcinogenicity studies. A report from the regulatory authorities subgroup.

Introduction: The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) initiated a process in 2012 to revise the S1B Guideline "Testing for Carcinogenicity of Pharmaceuticals". Previous retrospective analysis indicated the importance of histopathological risk factors in chronic toxicity studies, evidence of endocrine perturbation, and positive genetic toxicology results as potentially predictive indicators of carcinogenic risk. In addition, a relationship between pharmacodynamic activity and carcinogenicity outcome in long-term rodent studies has been reported. It was postulated that these factors could be evaluated in a Weight-of-Evidence (WoE) approach to predict the outcome of a 2-year rat study. Methods: The ICH S1B(R1) Expert Working Group (EWG) conducted a Prospective Evaluation Study (PES) to determine the regulatory feasibility of this WoE approach. Drug Regulatory Authorities (DRAs) evaluated 49 Carcinogenicity Assessment Documents (CADs), which describe the WoE for submitted pharmaceutical compounds. Each compound was categorized into a carcinogenic risk category including a statement of the value of the 2-year rat study. The outcome of the completed 2-year rat studies was evaluated in relation to the prospective CAD to determine the accuracy of predictions. Results: Based on the results of the PES, the EWG concluded that the evaluation process for assessing human carcinogenic risk of pharmaceuticals described in ICH S1B could be expanded to include a WoE approach. Approximately 27% of 2-year rat studies could be avoided in cases where DRAs and sponsors unanimously agreed that such a study would not add value. Discussion: Key factors supporting a WoE assessment were identified: data that inform carcinogenic potential based on drug target biology and the primary pharmacologic mechanism of the parent compound and major human metabolites; results from secondary pharmacology screens for this compound and major human metabolites that inform carcinogenic risk; histopathology data from repeated-dose toxicity studies; evidence for hormonal perturbation; genotoxicity data; and evidence of immune modulation. The outcome of the PES indicates that a WoE approach can be used in place of conducting a 2-year rat study for some pharmaceuticals. These data were used by the ICH S1B(R1) EWG to write the R1 Addendum to the S1B Guideline published in August 2022.

Developing a pragmatic consensus procedure supporting the ICH S1B(R1) weight of evidence carcinogenicity assessment.

The ICH S1B carcinogenicity global testing guideline has been recently revised with a novel addendum that describes a comprehensive integrated Weight of Evidence (WoE) approach to determine the need for a 2-year rat carcinogenicity study. In the present work, experts from different organizations have joined efforts to standardize as much as possible a procedural framework for the integration of evidence associated with the different ICH S1B(R1) WoE criteria. The framework uses a pragmatic consensus procedure for carcinogenicity hazard assessment to facilitate transparent, consistent, and documented decision-making and it discusses best-practices both for the organization of studies and presentation of data in a format suitable for regulatory review. First, it is acknowledged that the six WoE factors described in the addendum form an integrated network of evidence within a holistic assessment framework that is used synergistically to analyze and explain safety signals. Second, the proposed standardized procedure builds upon different considerations related to the primary sources of evidence, mechanistic analysis, alternative methodologies and novel investigative approaches, metabolites, and reliability of the data and other acquired information. Each of the six WoE factors is described highlighting how they can contribute evidence for the overall WoE assessment. A suggested reporting format to summarize the cross-integration of evidence from the different WoE factors is also presented. This work also notes that even if a 2-year rat study is ultimately required, creating a WoE assessment is valuable in understanding the specific factors and levels of human carcinogenic risk better than have been identified previously with the 2-year rat bioassay alone.

Regulatory Experience Assessing the Carcinogenic Potential of a Monoclonal Antibody Inhibiting PCSK9, Bococizumab, Including a 2-Year Carcinogenicity Study in Rats.

Bococizumab is an anti-PCSK9 monoclonal antibody that was intended for the treatment of hypercholesterolemia. After reviewing the 6-month rat toxicity study data, in which there was a low spontaneous tumor incidence, unrelated to bococizumab administration, the U.S. FDA granted a carcinogenicity waiver request based on a weight-of-evidence assessment of low carcinogenic risk. Subsequently, after reviewing 6-month rat toxicity study data from another anti-PCSK9 antibody, RN317, with a similar low tumor incidence (unrelated to RN317), the U.S. FDA rescinded the bococizumab carcinogenicity study waiver and requested a full 2-year rat carcinogenicity study be conducted. The resulting 2-year carcinogenicity study demonstrated no bococizumab-related increase in tumors, confirming the weight-of-evidence evaluation and alleviating concerns regarding the carcinogenic potential. Here we report the scientific and regulatory background that led to the request for a rat carcinogenicity study, the feedback on the design of the carcinogenicity study, and the results from this study which affirmed the original weight-of-evidence assessment of low carcinogenic risk.

Role and potential of histopathological specimens in the toxicological evaluation of pharmaceuticals and chemicals

With the development of molecular-targeted drugs, the demand for utilization of histopathological specimens and pathological diagnosis is increasing in the field of clinical pathology. In particular, in case of companion diagnostics, the results of immunohistochemical staining have become beyond diagnostic assistance, definitive diagnosis, and now indispensable for selection of therapeutic agents. Histopathological examination also serves an important role in non-clinical toxicological evaluation. Particularly, it is pivotal for obtaining data on organ-specific toxicity and carcinogenicity. On the other hand, a weight of evidence approach is currently being considered in International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) S1 as an option to replace the 2-year rat carcinogenicity study, in which case the evaluation of the 26-week-repeated dose toxicity study will be more important. In this article, We would like to introduce the usefulness of γ-H2AX-based rat bladder carcinogenicity evaluation using histopathological specimens obtain from 28-day repeated-dose study and the application of mass spectrometric imaging currently under consideration in our laboratory, thus discuss the role of histopathological examination in non-clinical toxicological and safety evaluation and its future perspective.

Safety assessment of central nervous system tumors in 2-year rat carcinogenicity studies

Safety assessment of central nervous system tumors in 2-year rat carcinogenicity studies

AhR Activation in Pharmaceutical Development: Applying Liver Gene Expression Biomarker Thresholds to Identify Doses Associated With Tumorigenic Risks in Rats.

Aryl hydrocarbon receptor (AhR) activation is associated with carcinogenicity of non-genotoxic AhR-activating carcinogens such as 2,3,7,8-tetrachlorodibenzodioxin (TCDD), and is often observed with drug candidate molecules in development and raises safety concerns. As downstream effectors of AhR signaling, the expression and activity of Cyp1a1 and Cyp1a2 genes are commonly monitored as evidence of AhR activation to inform carcinogenic risk of compounds in question. However, many marketed drugs and phytochemicals are reported to induce these Cyps modestly and are not associated with dioxin-like toxicity or carcinogenicity. We hypothesized that a threshold of AhR activation needs to be surpassed in a sustained manner in order for the dioxin-like toxicity to manifest, and a simple liver gene expression signature based on Cyp1a1 and Cyp1a2 from a short-term rat study could be used to assess AhR activation strength and differentiate tumorigenic dose levels from non-tumorigenic ones. To test this hypothesis, short-term studies were conducted in Wistar Han rats with 2 AhR-activating carcinogens (TCDD and PCB126) at minimally carcinogenic and noncarcinogenic dose levels, and 3 AhR-activating noncarcinogens (omeprazole, mexiletine, and canagliflozin) at the top doses used in their reported 2-year rat carcinogenicity studies. A threshold of AhR activation was identified in rat liver that separated a meaningful "tumorigenic-strength AhR signal" from a statistically significant AhR activation signal that was not associated with dioxin-like carcinogenicity. These studies also confirmed the importance of the sustainability of AhR activation for carcinogenic potential. A sustained activation of AhR above the threshold could thus be used in early pharmaceutical development to identify dose levels of drug candidates expected to exhibit dioxin-like carcinogenic potential.

Central nervous system tumors in 2-year rat carcinogenicity studies: perspectives on human risk assessment.

Rodent in vivo carcinogenicity bioassays are required for human risk assessment and have been utilized in this capacity for decades. Accordingly, there is an abundance of data that could be accessed and analyzed to better understand the translatability of xenobiotic-induced rodent tumors to human risk assessment. In the past decade, various groups have published assessments of the value garnered by these life-time rodent studies. Results and recommendations from the International Council for Harmonization Expert Working Group (ICH-S1 EWG) on the predictability of the current testing paradigm and proposal for an integrated approach to human carcinogenicity risk assessment are pending. Central nervous system (CNS) tumors in rats are rare and translatability to human remains unknown. This review focuses on microglial cell tumors (MCT) of the CNS in rats including its classification, nomenclature, incidence and translatability to human risk assessment. Based on emerging immunohistochemistry (IHC) characterization, glial tumors previously thought of astrocytic origin are more likely MCTs. These may be considered rodent specific and glucose dysregulation may be one component contributing to their formation. Based on review of the literature, MCTs are rarely diagnosed in humans, thus this tumor type may be rat-specific. We propose to include MCTs as a tumor type in revised International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) classification and all glial tumors to be classified as MCTs unless proven otherwise by IHC.

Non-neoplastic lesions found only in the two-year bioassays but not in shorter toxicity studies of rats

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has been conducting a prospective evaluation period to validate the criteria for waiving some carcinogenicity studies in rats. Before the waiving strategy is practiced in ICH, it is crucial to elucidate whether non-neoplastic lesions are found only in 2-year rat carcinogenicity studies. To confirm possible importance of 2-year bioassays for evaluating chronic toxicity but not carcinogenicity, we retrospectively surveyed 59 pharmaceuticals approved by the Ministry of Health, Labour and Welfare (MHLW) from 2007 to 2010 in Japan for non-neoplastic lesions observed in carcinogenicity studies. Non-neoplastic histopathological lesions observed only in 2-year carcinogenicity studies but not in 6-month chronic toxicity studies using rats were compared with clinical adverse drug reactions (ADRs). Thirteen non-neoplastic lesions that may correlate with clinical ADRs were classified into three categories: Category 1, lesions not predictable from other nonclinical data except those from 2-year rat carcinogenicity studies; Category 2, lesions predictable mainly from chronic toxicity studies; Category 3, lesions predictable mainly from pharmacological actions. In the present survey, non-neoplastic lesions only found in 2-year rat carcinogenicity studies were neither significant in terms of frequency and severity nor useful for clinical risk management.

Regulatory Forum.

Revision of the International Council for Harmonization (ICH) S1 guidance for rat carcinogenicity studies to be more selective of compounds requiring a 2-year rat carcinogenicity study has been proposed following extensive evaluation of rat carcinogenicity and chronic toxicity studies by industry and drug regulatory authorities. To inform the ICH S1 expert working group in their potential revision of ICH S1, a prospective evaluation study was initiated in 2013, in which sponsors would assess the pharmacologic and toxicologic findings present in the chronic toxicity studies and predict a positive or negative carcinogenicity outcome using a weight of evidence argument (a carcinogenicity assessment document [CAD]). The Scientific and Regulatory Policy Committee was asked by the Society of Toxicology Pathology (STP) executive committee to track these changes with ICH S1 and inform the STP membership of status changes. This commentary is intended to provide a brief summary of recent changes to the CAD guidance and highlight the importance of STP membership participation in the process of CAD submissions.

Effects of the Janus Kinase Inhibitor, Tofacitinib, on Testicular Leydig Cell Hyperplasia and Adenoma in Rats, and on Prolactin Signaling in Cultured Primary Rat Leydig Cells.

Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis. Tofacitinib preferentially inhibits receptor signaling through JAK3 and JAK1, relative to JAK2. In the 2-year rat carcinogenicity study, there were tofacitinib, dose-related increases in the incidences of testicular Leydig cell hyperplasia and benign adenomas in male rats, and decreased incidences of mammary tumors and duct dilatation/galactocele in female rats. Such findings in rats are typical of agents, such as dopamine agonists, which decrease prolactin (PRL) activity. Since prolactin signals through the JAK2 pathway, we hypothesized that these findings were off-target effects due to inhibition of PRL signaling via JAK2. The studies reported here were designed to investigate the interruption of PRL signaling pathways in Leydig cells. In isolated primary rat Leydig cells, PRL increased phosphorylated Signal Transducer and Activator of Transcription-5 protein, and mRNA levels for luteinizing hormone receptor. Tofacitinib, at concentrations observed in the rat carcinogenicity study, dose-dependently inhibited these effects. These observations illustrate a novel mechanism, the inhibition of prolactin signaling by which modulation of JAK activity can modulate PRL signaling pathways to induce Leydig cell tumors in rats. Since human Leydig cells lack this PRL dependence for normal function, these rodent tumors do not indicate a health risk to human patients.

Evaluation of potential activity of luseogliflozin on vascular proliferation in the mesenteric lymph node with or without vascular tumors in Sprague-Dawley rats in a carcinogenicity study.

The incidence of mesenteric lymph node vascular tumors can vary in rats, and appropriate assessment of potential risk of tumorigenicity is needed when the incidence is higher in treated groups than in a control group. In a 2-year rat carcinogenicity study of luseogliflozin, a selective sodium-dependent glucose co-transporter 2 inhibitor for the treatment of type 2 diabetes mellitus, there was a slight but statistically significant increase in the total number of hemangiomas and hemangiosarcomas in the mesenteric lymph nodes in males at a high-dose. As part of the risk assessment for luseogliflozin, its effect on the vascular proliferation potential in the mesenteric lymph nodes was examined in a rat carcinogenicity study by performing an image analysis using specimens with double immunohistochemical staining for PCNA and CD34 in control and high-dose males. In addition, immunohistochemical staining for VEGF was performed to detect enhanced angiogenesis. In the high-dose males that did not have a hemangioma/hemangiosarcoma, neither an increased number of PCNA/CD34-positive cells nor changes in the expression pattern of VEGF was observed. On the other hand, in the high-dose males that had a hemangioma/hemangiosarcoma, the number of PCNA-positive cells was increased in the tumor areas, and the number in the hemangioma/hemangiosarcoma was approximately one-half of that in the hemangiosarcoma in the control male. In conclusion, no potential change leading to vascular proliferation/tumors was detected in the mesenteric lymph nodes of high-dose males receiving luseogliflozin.

Injection-site Malignant Fibrous Histiocytomas in a Pegvisomant Carcinogenicity Study in SD Rats

In a 2-year rat carcinogenicity study, pegvisomant injected subcutaneously on a daily basis at doses of 0, 2, 8, or 20 mg/kg/day produced malignant fibrous histiocytomas (MFHs) at the injection sites of 3 male rats (5%) given 8 mg/kg/day and 5 males (8%) given 20 mg/kg/day. MFH was characterized by unencapsulated dermal and subcutaneous sheets of fusiform and spindle-shaped cells sometimes with areas of round and/or irregular, pleomorphic cells and variable numbers of large multinucleated giant cells. Some regions of MFH had a fibroblastic appearance with streaming cells forming storiform patterns, while other areas consisted primarily of round to plump irregular cells with more giant cells. Pegvisomant did not increase the incidence of MFH in female rats and did not produce any other neoplastic responses in rats. In the dermis and subcutis at the injection sites of many males and females, pegvisomant produced dose-related increased incidences and severity of histiocytic infiltrates consisting of vacuolated macrophages with variable mature or immature fibrous tissue. Neoplasms at injection sites did not result in marketing restrictions or a label warning for human cancer risk, highlighting that injection-site neoplasms in rats have low relevance for human risk assessment.

Nonclinical Assessment of Carcinogenic Risk and Tumor Growth Enhancement Potential of Prasugrel, a Platelet-Inhibiting Therapeutic Agent

Prasugrel, a thienopyridine ADP receptor antagonist, is an orally administered prodrug requiring in vivo metabolism to form the active metabolite that irreversibly inhibits platelet activation and aggregation mediated by the P2Y12[sub 12] receptor. A comprehensive nonclinical safety assessment including genotoxicity and carcinogenicity studies supported the chronic use of prasugrel in patients with atherothrombotic disease. In addition, a special assessment of the potential for prasugrel to enhance tumor growth was undertaken to address regulatory concerns relating to increases in human cancers. Prasugrel demonstrated no evidence of genotoxicity and was not oncogenic in a 2-year rat carcinogenicity study. In the 2-year mouse study, an increase in hepatocellular adenomas was considered secondary to enzyme induction and not relevant to human safety. Further, the absence of any increase in common background tumors at any other organ site in either rodent study indicated a lack of tumor promoting activity (apart from the CYP450 induction-related increase in mouse liver tumors). Cell culture studies with 3 human tumor cell lines (lung, colon, prostate) demonstrated that exposure of serum-starved cells to prasugrel's active and major circulating human metabolites does not increase cell proliferation relative to starved cells stimulated to proliferate by addition of 10% FBS. Prasugrel also did not increase tumor growth relative to vehicle controls in nude mice implanted with 3 human tumor cell lines. Thus, traditional genotoxicity and 2-year bioassays as well as specially designed tumor growth enhancement studies in human tumor cell lines and mouse xenograft models clearly demonstrated prasugrel's lack of tumorigenic potential.

An Evaluation of Chronic 6- and 12-Month Rat Toxicology Studies as Predictors of 2-Year Tumor Outcome

Chronic 6- and 12-month rat toxicology studies were evaluated for their ability to predict tumor outcome in 2-year rat carcinogenicity studies for 80 pharmaceuticals from commercial and Merck databases. The data consisted of 62 (6-month) and 54 (12-month) studies, which included 30 rat carcinogens and 50 noncarcinogens in 2-year studies. The histopathology findings considered as evidence of potential preneoplasia in the chronic studies were hyperplasia, cellular hypertrophy, and atypical cellular foci. The authors hypothesized that a whole animal-based approach should be taken, wherein (1) evidence of potential preneoplasia in any tissue may serve as a sensitive predictor of tumor outcome in any tissue in the whole animal and not necessarily the same tissue and (2) the absence of evidence for potential preneoplasia in all tissues may serve as a strong negative predictor of tumor outcome in any tissue. Based on this whole animal approach, 25 of 30 rat carcinogens showed histopathologic signals in chronic toxicology studies, yielding a test sensitivity of 83%. The negative predictivity of the absence of histopathology findings in chronic toxicology studies of 50 nontumorigenic compounds was 88%. The value of the extra 6-month treatment was not apparent. The 5 false negatives (negative chronic studies but positive 2-year studies) are for marketed drugs approved for non-life-threatening conditions and associated with rat-specific mechanisms. The absence of preneoplasia in the whole animal is a reliable predictor of negative tumor outcome in 2-year rat studies, and approximately 50% rat carcinogenicity studies could be eliminated for the 80 pharmaceuticals examined, with no risk to humans and with a substantial reduction in animal usage and drug development time.

S03: PPAR-induced sarcoma: Cell proliferation and altered gene expression in tumour precursor cells

The development of the dual PPAR α/γ agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year rat carcinogenicity study with focus on the findings of subcutaneous fibrosarcomas with a predominant spindle cell appearance that occurred at the highest dose level of 10 μmol/kg in both sexes. This dose level was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis and expression of PPARγ in subcutaneous adipose tissues was assessed in rats administered 1, 3 and 10 μmol/kg tesaglitazar for 2 weeks, 3 and 6 months. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased dose dependently after 2 weeks. The increase in DNA synthesis was still statistically significant at later time points but only at 10 μmol/kg, the dose producing fibrosarcoma. Immunohistochemical analyses showed no detectable PPARγ protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARγ in these cells. Gene expression analysis of adipose tissue revealed that by six months the cells were less responsive to PPARγ stimulation and that markers of adipose tissue differentiation were down regulated. Furthermore, increased gene expression of inflammatory cytokines was associated with increased occurrence of necrotic or fibrotic lesions in subcutaneous adipose tissues. In summary, the data suggest that high dose tesaglitazar increases cell turnover in mesenchymal interstitial cells that results in depletion of adipogenic precursor cells and preneoplastic lesions.