INTERNATIONAL SCIENTIFIC SYMPOSIUM FOR TITANIUM DIOXIDE. Paris November 15-16, 2016

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1 INTERNATIONAL SCIENTIFIC SYMPOSIUM FOR TITANIUM DIOXIDE Paris November 15-16, 2016 Evaluation of TiO 2 based on the MAK documentation (2012) on granular biopersistent particles (GBP) Helmut Greim Technical University Munich, Germany

2 Content 1. Background: ANSES 2016 proposal for C&L TiO 2 as Canc. Cat. 1B Carcinogen 2. ECHA CLP Criteria for Carcinogens 3. MAK and SCOEL classification of threshold carcinogens 4. MAK and SCOEL classification of TiO 2 as a threshold carcinogen 5. Conclusion

3 1. Background: ANSES proposal for C&L TiO 2 as Canc. Cat. 1B Carcinogen Cat 1B is justified considering the increase of both malignant (females only) and benign lung tumours in one species, reported in two studies by inhalation (in rats in an overload context) and two studies by instillation. Although the full mode of action is still unclear, an inflammatory process and indirect genotoxic effect through ROS seems to be the major mechanism to explain the effects induced by TiO 2. However, a genotoxic effect by direct interaction with DNA cannot be excluded since TiO 2 was found in the cell nucleus in various in vitro and in vivo studies. However: intra-nuclear location does not mean DNA-interaction!

4 Life time rat inhalation studies: Thyssen 1978: SD 16 mg/m 3 : inflammation, no malignant tumours Muhle 1989: F-344, 5 mg/m 3 : inflammation, no malignant tumours Lee 1985: CD, 0, 10, 50, 250 mg/m 3 : adenomas in f and m, squamous lesions in f at 250, impairment of clearance at 50 mg/m 3. No malignant tumours. Heinrich 1995: f Wistar: 7.2, 14.8 and 9.4 mg/m 3 for 4, 4 and 16 mo, resp.: benign and malignant squamous cell tumours, adenomas and adenocarcinomas. Mice negative. Conclusion: Malignant tumours in females of one species.

5 2. ECHA CLP Hazard categories for carcinogens Classification criteria for substances Category 1A: human evidence Category 1B: animal experiments for which there is sufficient evidence to demonstrate animal carcinogenicity e.g. from conventional long-term bioassays increased incidence of malignant neoplasms or an appropriate combination of benign and malignant neoplasms (presumed human carcinogen). Category 2: Suspected human carcinogens: Limited evidence from human or animal studies.

6 Limited evidence: the data suggest a carcinogenic effect but are limited for making a definitive evaluation because, e.g. (a) (b) (c) (d) the evidence of carcinogenicity is restricted to a single experiment; there are unresolved questions regarding the adequacy of the design, conduct or interpretation of the studies; the agent increases the incidence only of benign lesions of uncertain neoplastic potential; or the evidence of carcinogenicity is restricted to studies that demonstrate only promoting activity in a narrow range of tissues or organs. (e) If tumours are seen only in one sex of an animal species, the mode of action should be carefully evaluated to see if the response is consistent with the postulated mode of action. Effects seen only in one sex in a test species may be less convincing than effects seen in both sexes unless there is a clear patho-physiologiacl difference consistent with the mode of action to explain the single sex response.

7 i. Consideration of differences in toxicokinetics: Annex I: Certain tumour types in animals may be associated with toxicokinetics or toxicodynamics that are unique to the animal species tested and may not be predictive of carcinogenicity in humans. j. Confounding effect of excessive toxicity at test doses that do not alter animal's normal longevity from effects other than carcinogenicity. k. Mode of action and its relevance to humans, such as mutagenicity, cytotoxicity with growth stimulation, mitogenesis, immunosuppression. The existence of a secondary mechanism of action with the implication of a practical threshold above a certain dose level (e.g. hormonal effects on target organs or on mechanisms of physiological regulation, chronic stimulation of cell proliferation) may lead to a downgrading of a Category 1 to Category 2 classification.

8 Conclusion Malignant tumours in females in one of two rat studies. There is no indication of different toxic mechanisms in males and females. Two negative inhalation studies in rats. Not carcinogenic in the 2. species mice. Direct interaction with DNA more than questionable. Inert particles and organelles float in the viscous intracellular environment and organelles will avoid contact. DNA damage may occur but will be dose dependent. Supporting evidence: threshold mechanism of TiO 2. This is limited evidence rather than evidence for carcinogenicity: canc. Cat. 2 rather than 1B.

9 3. MAK and SCOEL classification of threshold carcinogens MAK Category 4: Substances that cause cancer in humans or animals or that are considered to be carcinogenic for humans and for which a MAK value can be derived. A non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the MAK and BAT values are observed. Under these conditions no contribution to human cancer risk is expected. The classification is supported especially by evidence that, for example, increases in cellular proliferation, inhibition of apoptosis or disturbances in cellular differentiation are important in the mode of action. The classification and the MAK and BAT values take into consideration the manifold mechanisms contributing to carcinogenesis and their characteristic dose-time-response relationships. SCOEL Group C: Genotoxic carcinogens for which a practical threshold is supported.

10 Carcinogenicity of Particles (19 dusts study) MAK Cat. 4 carcinogens? A publication (the 19 dusts study*) and previous studies indicate that almost all granular biopersistent particles (GBP) are potential carcinogens. Questionable attempts to extrapolate to lower human exposure to demonstrate linearity. Mechanistic data will allow for deciding whether an NOAEC can be identified to derive a health based exposure limit. *Intratracheal application of carbon black and TiO2 of low and high surface area, Diesel emission particles of low surface area, amorphous silica of high surface area.

11 GBP-criteria, mechanism and MAK derivation 1. Definition of granular biopersistent particles (GBP): Particle effect without other substance specific effects 2. Carcinogenic mechanism: primarily inflammation 3. Determination of NOAEC 4. Extrapolation to humans

12 Pathogenic sequence of effects of granular biopersistent particles or fibres Particle exposure * Relevant for NOAEC Macrophage activation Inflammation*, impaired clearance Particle accumulation Oxidative stress DNA-adducts, mutations Metaplasia Fibrosis Cell proliferation (hyperplasia) Chronic inflammation Modified from P. Borm Tumours

13 Schematic illustration of particle-induced pulmonary effects in rats. Adapted from Oberdörster. Exposure- Duration, Dose increase Inflammatory cells (AM, PMN) Biochemical lavage parameters Alveolar epithelial damage Lung weight INFLAMMATION AM clearance function Particle retention AM aggregation Interstitialisation of particles (LN) PARTICLE KINETICS Cell proliferation Type II cells, Clara cells Fibroblasts Bronchiolization Collagen deposition and degradation Fibrotic loci Emphysema Benign and malign tumors MORPHOLOGY FUNCTIONAL CHANGES CHRONIC EFFECTS

14 Conclusion 1. Inflammation is seen as the underlying mechanism of GBP tumor induction. 2. Inflammation leads to secondary genotoxicity. Avoidance of inflammation will prevent genotoxicity and tumor induction. 3. Sensitive parameters to detect inflammation are cellular changes and enzyme activities in BAL. 4. BAL effects correlate with lung histopathology. 5. Effects are seen at particle concentrations below 1 mg/m Classification of TiO 2 as a threshold carcinogen: SCOEL Category C, (MAK Category 4 so far no OEL derived: Category 3A).

15 NOAECs for inflammation (short term exposure) Salvi et al (2000): Volunteers, 1 hr diesel exhaust, BAL and Endobronchial biopsies: 0.3 mg/m 3 IL-8 in biopsies in BAL cells increased. Onkogen-alpha expression in bronchial epithelial cells indicate increased cell proliferation; leukocyte infiltration. Nightingale et al (2000):, 2 hrs DEP, sputum: 0.2 mg/m 3 Neutrophils, myeloperoxidase (slightly) increased, Macrophages decreased. 15

16 NOAECs of TiO 2 Hext et al 2002, Bermudez et al 2004 Rats, 13 w exposure to 0.5, 2 and 10 mg/m 3 Parameters used to derive an NOAEC: Increased proliferation of terminal bronchiolar cells and of alveolar cells at 2 mg/m 3, not at 0.5 mg/m 3. NOAEC: 0.5 mg/m 3 Muhle et al 1991: rats 2 y inhalation 5 mg/m 3 at 24 mo no indication of inflammatory reactions (leucozytes, LDH, protein content) no fibrosis NOAEC: 5 mg/m 3 (used for HEC calculation)

17 Two models to calculate HEC (Human Equivalent Concentration) A MPPD (Multiple-Path-Particle-Dosimetry)-Model of US EPA (2004) based on lung surfaces of rats and humans. HEC for TiO 2 considering density 4: 1 mg/m 3 B MPPD-Model based on macrophage volumes of rats and humans. HEC for TiO 2 considering density 4: 2 mg/m 3

18 Lung-(alveolar) Surface Critical for model A. According to US-EPA (2004), Brown et al (2005): rat: 0,3 m 2, humans: 57 m 2 (functional residual capacity) Other values: Gehr et al (1978): 143 m 2 (true values m 2 ) Hasleton (1972): m 2 Ochs et al (2004): 60 m 2

19 Volumes of makrophages Critical for model B According to Pauluhn (2011) rat: 1166 x 27 µm³ man: 4990 x 7000 µm³ Ration man/rat: 1110

20 Normalisierungsmaß Ratte Mensch Verhältnis Mensch/Ratte Lungenoberfläche (Hartwig, 2011)[m²/Lunge] (SD-Ratte) Fläche der Atemwege (conducting airways, bronchi, bronchioli) (Mercer et al., 1994)[total, cm²] (SD-Ratte) Oberfläche Respirationstrakt (TB+AV) [cm²], (Ratte, ohne Stammangabe) (Oberdörster, 2010) Anzahl Makrophagen (Geiser, 2010), x 10 6, (SD-Ratte) Anzahl Makrophagen; (Jarabek et al., 2005) x 10 6 (Ratte, Stamm?) Anzahl Makrophagen, (Pauluhn, 2011) x 10 6, (Wistar-Ratte) Gesamtmakrophagenvolumen (Geiser, 2010) x 10 6 (F344-Ratte) Gesamtmakrophagenvolumen (Geiser, 2010) x 10 6 (SD-Ratte) Gesamtalveolarmakrophagenvolumen Pauluhn (2011), x10 6 ; (CD-/Wistar) 0,295 56, ,5 27, , ,8 26, , , , x 29,1 µm³ 1058 x 26,9 µm³ 1166 * 27 µm³ 1474 x 5990 µm³ 1474 x 5990 µm³ 4990 * 7000 µm³ 474,82 310, ,52 20 Gebel (BAuA) 2013

21 Final Conclusion on TiO 2 According the ECHA CLP guidelines canc. classification of TiO2 allows Cat. 2 rather than 1B. The major effect in the respiratory tract is inflammation, which under overload conditions may lead to tumour formation. Derivation of an OEL by MAK is pending. So far canc. Group 3A (Group 4 if a MAK value can be derived) is recommended. SCOEL: 8 hour TWA: 0.3 mg/m 3 (respirable fraction of biopersistent granular dusts at a material density of 1 g/cm 3 ).

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