Mechanism of Action of TiO2: Recommendations to Reduce Uncertainties Related to Carcinogenic Potential

The Risk Assessment Committee of the European Chemicals Agency issued an opinion on classifying titanium dioxide (TiO2) as a suspected human carcinogen upon inhalation. Recent animal studies indicate that TiO2 may be carcinogenic through the oral route. There is considerable uncertainty on the carcinogenicity of TiO2, which may be decreased if its mechanism of action becomes clearer. Here we consider adverse outcome pathways and present the available information on each of the key events (KEs). Inhalation exposure to TiO2 can induce lung tumors in rats via a mechanism that is also applicable to other poorly soluble, low-toxicity particles. To reduce uncertainties regarding human relevance, we recommend gathering information on earlier KEs such as oxidative stress in humans. For oral exposure, insufficient information is available to conclude whether TiO2 can induce intestinal tumors. An oral carcinogenicity study with well-characterized (food-grade) TiO2 is needed, including an assessment of toxicokinetics and early KEs.

Comparative carcinogenicity study of a thick, straight-type and a thin, tangled-type multi-walled carbon nanotube administered by intra-tracheal instillation in the rat

Background Multi-walled carbon nanotubes can be divided into two general subtypes: tangled and straight. MWCNT-N (60 nm in diameter) and MWCNT-7 (80–90 nm in diameter) are straight-type MWCNTs, and similarly to asbestos, both are carcinogenic to the lung and pleura when administered to rats via the airway. Injection of straight-type MWCNTs into the peritoneal cavity also induces the development of mesothelioma, however, injection of tangled-type MWCNTs into the peritoneal cavity does not induce carcinogenesis. To investigate these effects in the lung we conducted a 2-year comparative study of the potential carcinogenicities of a straight-type MWCNT, MWCNT-A (approximately 150 nm in diameter), and a tangled-type MWCNT, MWCNT-B (7.4 nm in diameter) after administration into the rat lung. Crocidolite asbestos was used as the reference material, and rats administered vehicle were used as the controls. Test materials were administered by intra-Tracheal Intra-Pulmonary Spraying (TIPS) once a week over a 7 week period (8 administrations from day 1 to day 50), followed by a 2-year observation period without further treatment. Rats were administered total doses of 0.5 or 1.0 mg MWCNT-A and MWCNT-B or 1.0 mg asbestos. Results There was no difference in survival between any of the groups. The rats administered MWCNT-A or asbestos did not have a significant increase in bronchiolo-alveolar hyperplasia or tumors in the lung. However, the rats administered MWCNT-B did have significantly elevated incidences of bronchiolo-alveolar hyperplasia and tumors in the lung: the incidence of bronchiolo-alveolar hyperplasia was 0/20, 6/20, and 9/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively, and the incidence of adenoma and adenocarcinoma combined was 1/19, 5/20, and 7/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively. Malignant pleural mesothelioma was not induced in any of the groups. Conclusions The results of this initial study indicate that tangled-type MWCNT-B is carcinogenic to the rat lung when administered via the airway, and that straight-type MWCNT-A did not have higher carcinogenic potential in the rat lung than tangled-type MWCNT-B.

Long-Term Nonclinical Pulmonary Safety Assessment of Afrezza, a Novel Insulin Inhalation Powder

Afrezza delivers inhaled insulin using the Gen2 inhaler for the treatment of patients with type 1 and type 2 Diabetes. Afrezza was evaluated in long-term nonclinical pulmonary safety studies in 2 toxicology species. Chronic inhalation toxicology studies in rat (26 weeks) and dog (39 weeks) and an inhalation carcinogenicity study in rats were conducted with Technosphere insulin (Afrezza) and with Technosphere alone as a vehicle control. Respiratory tract tissues were evaluated by histopathology and cells expressing proliferating cell nuclear antigen (PCNA) were quantified in lungs of rats. Microscopic findings in rats exposed to Afrezza were attributed to the Technosphere particle component, were confined to nasal epithelia, and consisted of eosinophilic globules and nasal epithelial degeneration. There were no Afrezza-related changes in pulmonary PCNA labeling indices in alveoli, large bronchioles, or terminal bronchioles. Microscopic findings in rats exposed to Technosphere particles included eosinophilic globules, mucus cell hyperplasia, and epithelial degeneration in the nasal cavities. PCNA labeling indices were increased in large bronchioles and terminal bronchioles but not in alveoli. There were no Technosphere particle-related findings in the dog study. Afrezza did not exhibit carcinogenic potential in the 2-year study in rats. These nonclinical inhalation studies support the use of Afrezza in humans over extended periods.

Incidental Ultrastructural Findings in the Sural Nerve and Dorsal Root Ganglion of Aged Control Sprague Dawley Rats in a Nonclinical Carcinogenicity Study

Xenobiotic-induced peripheral nerve damage is a growing concern. Identifying relative risks that a new drug may cause peripheral nerve injury over long periods of administration is gathering importance in the evaluation of animal models. Separating out age-related changes in peripheral nerves of rats caused by compression injury from drug-induced effects has been difficult. Biopsy of the sural nerve is utilized in humans for investigations of peripheral neuropathy, because it is largely removed from the effects of nerve compression. This study used transmission electron microscopy to identify incidental findings in the sural nerves and dorsal root ganglia of aged control rats over time. The goal was to establish a baseline understanding of the range of possible changes that could be noted in controls compared to rats treated with any new investigative drug. In this evaluation, most sural nerve fibers from aged control rats had few ultrastructural abnormalities of pathologic significance. However, glycogenosomes, polyglucosan bodies, swollen mitochondria, autolysosomes, split myelin, Schwann cell processes, and endoneural macrophages with phagocytosed debris (considered an indication of ongoing degenerative changes) were occasionally noted.

Risk Assessment of the Plant Protection Product PROMAN – with the Active Ingredient Metobromuron

Proman is a new product containing the active substance metobromuron. The intended use is as a broad spectrum selective herbicide for potatoes grown outdoors. VKM’s Panel on Plant Protection Products has discussed the questions raised by The Norwegian Food Safety Authority in the Terms of reference and has stated the following opinion: On the relevance of the carcinogenic effects observed in the rat carcinogenicity study; fibrosarcomas in females and pheochromocytomas and Leydig cell tumours in males: It is the opinion of VKM’s Panel for Plant Protection Products that the relevance of the observed incidence in mammary gland tumours and Leydig cell tumours in the rat carcinogenicity study is strengthened by the fact that the tumours are observed in hormone responsive tissues. The panel concludes that the carcinogenic effects observed in the rat carcinogenicity study are likely to be relevant for tumours that are influenced by the endocrine system, also in humans. On the higher incidences of still dumbbell-shaped centres of thoracic vertebrae and nonossification of the 13th rib observed in the rat developmental toxicity study and whether these are considered to be malformations: VKM’s Panel on Plant Protection Products has discussed the classification of the different types of incomplete ossifications and concluded that incomplete ossification of sternebrae and non-ossification of the 13th rib in rats should by itself be considered to be variations, and not adverse developmental effects. On the other hand, the Panel agrees with ECHA that the “thoracic vertebral centres still dumbbell-shaped” should be considered as malformations, due to limited data and understanding of the mechanism underlying the observed slow reversal of these anomalies. Furthermore, it is the view of the Panel that the different types of retarded ossification induced by the exposure of metobromuron should be considered as a whole when assessing for developmental effects. On the establishment of the NOAEL for the developmental toxicity study in rats and the reference value (ARfD): VKM’s Panel on Plant Protection Products supports the proposal of an ADI value of 0.008 mg/kg bw/day based on a NOAEL of 0.8 mg/kg bw/day from the 2-year study in mouse, and VKM Report 2015: 03 5. AOEL of 0.016 mg/kg bw/day based on the NOAEL of 1.6 mg/kg bw/day from the 1-year feeding study in dog. An UF of 100 is applied. The panel suggests on the other hand an ARfD of 0.03 mg/kg bw based on a LOAEL of 10 mg/kg bw /day with the observations of incomplete ossification in the rat developmental study. An UF of 300 is applied. On the possible anti-androgenic potential of metobromuron: It is the view of the Panel that the rat carcinogenicity study suggests that metobromuron may possess endocrine disrupting potency. The data from the Hershberger in vivo rat study and the in vitro studies is also suggestive of a weak anti-androgenic effect. Thus, it is the opinion of the VKM Panel on Plant Protection Products that an anti-androgenic effect of metobromuron cannot be excluded.

Generation of TD50 values for carcinogenicity study data

Carcinogenic potency is a key factor in the understanding of chemical risk assessment. Measures of carcinogenic potency, for example TD50, are instrumental in the determination of metrics such as the threshold of toxicological concern (TTC), acceptable intake (AI) and permitted daily exposure (PDE), which in turn impact on human exposure. The Carcinogenic Potency Data Base (CPDB) has provided a source of study information, complete with calculated TD50 values. However, this is no longer actively updated. An understanding of carcinogenic potency, which can be derived from dose–response data, can be used as part of human risk assessments to generate safety thresholds under which cancer risk is judged to be minimal. The aim of this paper is to produce a transparent methodology for calculating TD50 values from experimental data in a manner consistent with the CPDB. This was then applied across the same data as used in the CPDB and analysis done on the correlation with the CPDB TD50 values. While the two sets of values showed a high level of correlation overall, there were some significant discrepancies. These were predominantly due to a lack of clarity in the CPDB methodology and inappropriate use of a linear model in TD50 calculation where the data was not suitable for such an approach.