Rats and Humans: The Adverse Outcome Pathway Molecular, Anatomical, and Functional Aspects

Transcription

1 Rats and Humans: The Adverse Outcome Pathway Molecular, Anatomical, and Functional Aspects Relevance to OEL setting and classification for Granular Biopersistent Substances (GBS=PSP) Len Levy Emeritus Professor, Cranfield University, UK

2 Background It has been known for some time that some poorly soluble particles (PSP) can cause alveogenic lung cancer in rats. This is related to chronic inflammation as a result of lung overload in the alveolar region of the lung. [The lung cancer findings in particular appear to be unique to the rat.] These findings will affect classification and the setting of occupational exposure limits for many PSPs.

3 Adverse Outcome Pathway and Mode of Action (AOP and MOA) AOP describes the sequential progression of events evolving in an organism from the first contact of a toxicant at the molecular level, via a subset of following key effects or biological responses to a final adverse outcome at the individual or population level. The adverse outcome from the same molecular initiating event can vary significantly, and similarly, different molecular initiating events can cause the same adverse outcome. Different MOAs can share common key molecular initiating events and each step in the AOP may be influenced by other pathways ongoing and/or dominating within the biological system of interest.

4 AOP and MOA in OEL setting and Classification of PSP Translational toxicology refers to the general approach of applying toxicological findings to human settings. Discussed here to describe the approach of an interspecies comparison of the response to high exposures to PSP in experimental rodent species (in particular the rat) and humans. Main purpose conduct risk assessment and hazard classifications and to derive OELs for PSP.

5 OELs a general definition Occupational Exposure Limits (OELs) have been a feature of the industrialised world for the last hundred years or so ( the first coming from Germany). The objective of OELs is to set limits for exposure via the airborne route such that exposure, even when repeated on a regular basis throughout a working life, will not lead to adverse effects on the health of exposed persons and/or their progeny at any time (as far as can be predicted from the contemporary state of knowledge). This definition broadly applies to all OEL setting committees (MAK, SCOEL, ACGIH, DECOS, the late UK WATCH, etc.)

6 Health vs Risk OELs Health based OELs established where the available scientific data base leads to the conclusion that it is possible to identify a clear threshold dose/exposure level below which exposure is not expected to lead to adverse effects. These OELs do not take into account socioeconomic or achievability factors. Risk based OELs where it is not possible on present knowledge to define a threshold of activity (e.g. genotoxicity, carcinogenicity and respiratory sensitisation) it must be assumed that any level of exposure, however small, might carry some finite risk. OR a health based limit could be set but socioeconomic and/or achievability are taken into account

7 General procedure for setting OELs: Health based Hazard and Risk Assessment Good quality human data preferred but often unavailable. Good quality animal studies are often used. Experimental and physicochemical data. Nowadays, AOP/MOA from experimental animals to humans considered Assessment of adequacy of database.

8 Some considerations for the setting of OELs for particulates (PSPs) It is a long time since we had them all described as nuisance dusts with generic limit of 10 mg/m 3 inhalable and 5 mg/m 3 respirable. MAK Commission led the way in reducing generic limit based on human evidence UK HSE started, then did nothing. Effects in humans seen can be anywhere in the upper or lower respiratory tract (primary irritation and inflammatory changes of upper tract; possible functional changes e.g. FEV1 and inflammatory changes in lung the parenchyma, COPD). Some PSP are data rich such as carbon black and TiO2 (good human epidemiology and animal data), other are data poor (perhaps only limited animal data)

9 Some considerations for the setting of OELs for particulates (PSPs) There has been/is some confusion between hazard and risk which is complicated by the choice of the critical effect for OEL setting. Do we need different toxicological considerations for those PSPs in the nanoparticle range? Probably not just use the appropriate metric (e.g. particle surface area rather than just mass). Not good concordance (AOP/MOA) between the commonly used rat model and other rodent species (mouse, hamster), and non human simians and humans for all effects (e.g. lung tumours).

10 Some considerations for the setting of OELs for particulates (PSPs) cont Logic for good OEL setting practice data is as we do for other substances and to use all the available reliable data (a weight of evidence approach) but particularly, good human data. In fact, if a robust epidemiological data set is available, then these results are typically given more weight in hazard classification and OEL development than animal toxicology studies (which usually provide supportive evidence ).

11 Using all the relevant data The use of modelling and translational toxicology from rat data to assist prediction to human hazard and risk assessment is important. However, it can be argued that more consideration should be given to using a weight of evidence approach which takes into account the totality of the data. Important considerations include species differences in response to PSPs and findings from working populations exposed to PSPs that have been extensively investigated.

12 AOP/MOA approach for PSP Apply the AOP/MOA approach by examining toxicological studies that have investigated species specific differences with regard to the species specific responses to GBS on the cellular and molecular level. Central hypothesis in the MAK Commissions GBS document Basic assumptions are that secondary genotoxic mechanisms underpin particle genotoxicity and tumourigenicity in rats. In vitro and in vivo toxicological studies have consistently demonstrated that the tumour induction in lungs of rats by particles is closely linked to inflammation and ROS released by excessively particle loaded alveolar macrophages and by secondary elicited PMN.

13 PSP lung disposition: intra species differences (as an example) Studies comparing lung responses to inhaled particles in rats vs. monkeys/humans demonstrate divergent particle distribution patterns following chronic inhalation exposures to PSPs. The disposition and dosimetry differences between rats and monkeys/humans are very different. These are probably a key driver in the AOP/MOA

14 PSP lung disposition: intra species differences Following particle deposition: in rats inhaled PSPs are retained within alveolar ducts in rats. in monkeys and humans, inhaled particles preferentially translocate across epithelial cells to be retained/sequestered at interstitial sites Rats produce significantly augmented and sustained pulmonary inflammogenic, epithelial and fibroproliferative responses when compared to either monkeys or humans. The available data also suggest that rats are significantly more sensitive in the development of adverse lung responses to inhaled PSP exposures when compared to other rodent species

15 Comparison of initial cellular and molecular events after lung particle exposure in different experimental animals leading to pre tumour conditions: DNA damage, p53 activation and proliferation Morfeld et al. Particle and Fibre Toxicology (2015) 12:3

16 Interspecies lung responses following longterm or chronic inhalation exposure to PSPs Rats Mouse Hamster Primate/Humans Likelihood for overload High High Low questionable Neutrophilic inflammation High High Low low Epithelial and interstitial proliferation High low questionable questionable Septal fibrosis High low questionable questionable Location of particles alveolar alveolar Rapid clearance Interstitial (abstracted from Morfeld et al, Particle Fibre and Toxicology 2015; Apr 23; 12(1): 3.)

17 Comparisons of Rat Lung Responses vs. Human/Primates to Inhaled PSPs Rat Human/Primate Likelihood for Developing Particle Overload High Not determined Alveolar Macrophage Responses (long term) High (accumulation alveolar ducts) Not extensive due to greater particle translocation to interstitium Pulmonary inflammatory Responses High Low Fibroproliferative Effects High and sustained Low Location of Retained Particles in the Lung Alveolar ducts primarily Interstitium primarily Development of Particle Overload related Lung Tumors YES NO

18 Sites of Particle Retention and Lung Tissue Responses to Chronically Inhaled Diesel Exhaust and Coal Dust in Rats and Cynomolgus Monkeys [Nikula et al. (1997) EHP 105: ] Rats retained a significantly greater portion of the particulate material in the lumens of alveolar ducts and alveoli than monkeys. Conversely, monkeys retained a significantly greater portion of the particulate material in the interstitium than rats.

19 Sites of Particle Retention and Lung Tissue Responses to Chronically Diesel Exhaust and Coal Dust Inhaled in Rats and Cynomolgus Monkeys [Nikula et al. (1997) EHP 105: ] Rats, but not monkeys, had significant alveolar epithelial hyperplastic, inflammatory, and septal fibrotic responses to the retained particles. These results suggest that anatomic patterns of particle retention and lung tissue reactions in rats may not be predictive of retention patterns and tissue responses in primates that inhale PSPs at concentrations representing high occupational exposures.

20 Conceptual AOP Model of lung overload sequelae in rats following chronic PSP exposures (ECETOC 2013)

21 Human evidence from Morbidity and Mortality Studies with PSPs Extensive epidemiological evidence from carbon black (CB) and TiO2 exposed production workers. Also, extensive evidence from coal miners. Effects seem to be some non clinical reduction in FEV1 in CB workers (US and European workers), with some equivocal symptoms of chronic bronchitis. Also seen with other PSPs. CMP for coalminers No excess in lung cancer in any of above.

22 Coalmine Studies Coalmine dusts characterized as granular biopersistent particles without known specific toxicity (GBS=PSP). Over 1 million coalmine workers exposed to coal dust (exposure generally greater than that other PSPs.) Studies of coal miners provide the best evidence for using lung overload models or epidemiological findings concerning lung cancer risk. ECHA used the coalmine studies to test the hypothesis whether the lung response to high burdens of GBS can be qualitatively similar in rats and humans.

23 Coalmine dosimetry studies human evidence for overload

24 Key Findings from Coalmine Studies Dosimetry studies in humans: No lung overload Studies of coalmine workers, who have been exposed to relevant levels of dust, have not indicated an increase in lung cancer risk.

25 Summary of findings Species differences can be explored using the MoA, the AOP for all events at the biological level. This helps to better refine the way in which translational toxicology is used to exchange and interpret experimental findings between rodent species, primates and human responses to PSP. The totality of the available information at the anatomical, physiological, cellular and molecular level must be taken into account in a reliable translational exercise. Rats have been consistently shown to have a more sensitive response to the chronic inhalation of PSP compared to other species, and a unique response in relation to lung cancer.

26 Conclusions Extrapolating overload results of rats to humans need to be carefully addressed to ensure that OELs are based on appropriate scientific assumptions and metrics including other biological findings (e.g. fate of particles location in the lungs of rats and humans) AOP/MOA methodology needs to account for both concordance and discordance between rats and humans when exposed to PSP

27 Thank you you for your attention

28 Supplementary slides

29 A Cautionary Tale for Translational Rodent Models A multi centred study (Seok et al, 2013) highlighted that although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions.

30 Purpose and Results of the Investigation Carried out by 20 research institutions in the US. Prompted by the consistent finding that drugs developed to treat inflammatory conditions in humans, based on research using mouse inflammatory models were not working in humans. Among those genes seen to change significantly in humans, the murine orthologs were close to random in matching to their human counterparts (e.g., Pearson correlations (R 2 ) between 0.0 and 0.1). There were also large temporal differences in recovery from inflammation which may be a few days in mice to many months in the human counterpart.

31 Conclusions In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases. Seok et al. (2013) Genomic responses in mouse models poorly mimic human inflammatory diseases. PNAS 110:

32 Approaches to predicting toxicity from occupational exposure to dusts An appraisal of low toxicity dusts MRC Institute of Environment and Health 1999 REPORT lution/r11.pdf

33 Purpose of Workshop The UK Health and Safety Executive requested the Institute for Environment and Health to evaluate the current state of knowledge on materials described as low toxicity dusts and to identify specific gaps in knowledge that precluded the setting of robust scientifically based occupational standards, at that time.

34 Main Conclusions (1) A benchmarking approach is proposed, whereby experimental toxicity data (including results from short term bridging studies ) on dusts for which the available toxicological data, including human data, are scarce, are compared with similar data from a small number of selected benchmark dusts for which human toxicity is well characterised. The benchmarking approach can be used to assess relative toxicities of dusts and, with varying degrees of confidence depending on the available data, to predict likely human health impacts.

35 Main Conclusions (2) The relative importance, for predicting toxicological outcome, of particle surface area versus volumetric or gravimetric measures, and possible relationships between different measures should be further investigated. Further studies, including mechanistic and monitoring studies, should be conducted. In particular, studies should be conducted on ultrafine particles to investigate whether they are more toxic than other low toxicity dusts in larger mammals and human.

36 Main Conclusions (3) The mechanistic reasons for interspecies differences in susceptibility to lowtoxicity dusts and the phenomena associated with lung overload merit further investigation in both in vitro and in vivo studies. In particular, whether the rat is uniquely sensitive and whether a different animal model might be more appropriate should be investigated.

37 Main Conclusions (4) Furthermore, in order to provide a more precise indicator of what health outcomes should be investigated in epidemiological studies, species differences in the site of deposition of particulate material in the lung and the influence of the site of deposition on toxic effect (including the importance of the interstitial compartment) should be investigated further.

38 Main Conclusions (5) dust. The proposal to use a benchmarking approach to evaluate the toxicity of dusts is considered to be a positive move forward. Such an approach could be used to establish relative toxicities leading to individual standards for each substance. Even if low toxicity dusts continue to be regulated as a generic group, there is a general consensus that ultrafine particles should be considered as a separate class from otherwise unspecified low toxicity dusts. Although low toxicity dusts should be considered where possible as individual substances, a generic approach could provide a default position when lack of data precludes the setting of a specific standard.

39 Main Conclusions (6) The benchmarking approach, incorporating a comparison of experimental and human data, should be used to investigate the relevance of experimental data to the prediction of human toxicity. In particular this process can be used to establish whether quantitative, rather than merely qualitative, predictions can be made about likely human responses. Interlaboratory comparisons of short term bridging studies to be used in the benchmarking approach should be encouraged. Studies should be designed in such a way that comparisons of potency and internal dose, not simply exposure concentrations, can be made.