Technical Report 122

(AOP) mouse and hamster

There are three informative sub-chronic inhalation studies from which to compare the Adverse Outcome Pathways of the rat, mouse and hamster. In all three studies, females from each of these species were exposed to the same dose-related concentrations of identical particulates for 13 weeks duration, and evaluated at the same post exposure time points by the same criteria.

In the first study with pigment-grade TiO2 particles, groups of female mice, rats and hamsters were exposed to 10, 50 or 250 mg/m3 TiO2 particles for 13 weeks and evaluated at several post-exposure time points up to 52 weeks (Bermudez et al, 2002). A variety of pulmonary endpoints were investigated, including inflammatory parameters, cytotoxicity indices, lung cell proliferation labelling kinetics and histopathological alterations. The authors noted that retained lung burdens following exposures were greatest in mice. Using particle retention data, it was determined that lung particle overload was achieved in both rats and mice at the exposure levels of 50 and 250 mg/m3. Lung inflammation and cytotoxic effects were noted initially in all three species at 50 and 250 mg/m3. The ranking order of severity of effects among the species was the following: rats > mice > hamsters.

In mice and rats, the BALF inflammatory responses remained elevated compared to controls throughout the entire post exposure recovery period in animals exposed to 250 mg/m3. In comparison, inflammation in hamsters was short-lived, likely due to the more rapid clearance of particles from the lung concomitant with the more protective, anti-inflammatory cytokine responses in this species. Pulmonary lesions were most severe in rats, where progressive epithelial- and fibroproliferative changes were observed in the particle overload 250 mg/m3 group. These epithelial proliferative changes were characterised in rats as increased alveolar epithelial cell proliferation. Associated with these foci of epithelial proliferation were interstitial particle accumulation and alveolar septal fibrosis. It was interesting to note that although exposed female mice demonstrated evidence of particle overload and pulmonary inflammation, unlike the exposed rats, high-dose TiO2-exposed mice were devoid of the fibro-proliferative and fibrotic tissue responses measured and observed in exposed rats. Moreover, when compared to identically exposed rats and mice, female hamsters had the weakest pulmonary inflammatory responses to the titanium dioxide dust burden concomitant with the fastest clearance times.

The second study of note consisted of an interspecies 13-week subchronic inhalation study; with female rats, mice and hamsters exposed to 3 concentrations of ultrafine TiO2 particles (Bermudez et al, 2004). Female rats, mice and hamsters were exposed to aerosol concentrations of 0.5, 2.0 or 10 mg/m3 of uf-TiO2 particles for 13 weeks and assessed over several post-exposure time points up to 1 year. Similar to the study effects measured with pigment-grade TiO2 particles, mice and rats had similar retained lung burdens at the end of the exposures, whereas hamsters had retained lung burdens that were significantly reduced. Lung burdens in all three species decreased with time after cessation of exposures. Moreover, at the end of the recovery period (i.e., ~ 1 year post-exposure), the percentages of particle burden remaining in the lungs of the 10 mg/m3 group were 57, 45 and 3% for rat, mouse, hamster, respectively. The retardation of particle clearance from the lungs in mice and rats of the 10 mg/m3 groups were indicators that particle overload had been achieved in these animals, but not in hamsters. BAL fluid biomarkers demonstrated that lung inflammation and cytotoxicity were apparent in rats and mice exposed to 10 mg/m3 uf-TiO2. The neutrophilic inflammatory responses in rats, but not mice, declined in a time-dependent manner correlating with a reduction in lung burdens; however, the fraction of recovered PMNs at 52 weeks post-exposure was equivalent in the two species. There were no significant changes in cellular responses, or with markers indicating toxicity in hamsters; reflecting the capacity of these animals to rapidly clear particles from the lung. Similar to the results reported in the interspecies, pigment-grade TiO2 study, progressive epithelial and fibro proliferative changes were observed in rats exposed to 10 mg/m3 TiO2 particles. Lung lesions were characterised by foci of alveolar epithelial proliferation of metaplastic epithelial cells which were located adjacent to heavily particle-laden macrophages; along with augmented alveolar epithelial cell proliferation indices. Associated with these foci of epithelial proliferation were interstitial particle accumulation and progressive alveolar septal fibrotic responses. It is noteworthy that epithelial, metaplastic and fibroproliferative changes were not noted in the lungs of either mice or hamsters.

To summarise the results of the study findings, there were significant species differences in the pulmonary responses to inhaled uf-TiO2 particles. Rats developed a more severe inflammatory and fibro proliferative response compared to mice. Clearance of particles from the lung was markedly impaired in mice and rats exposed to 10 mg/m3 uf-TiO2, whereas clearance in hamsters did not appear to be affected at any of the administered doses.

From a qualitative standpoint, these data are consistent and virtually identical with the results of the subchronic inhalation study with inhaled pigmentary TiO2 and demonstrate that the pulmonary responses of rats exposed to ultrafine particulate concentration likely to induce pulmonary overload are different from similarly exposed mice and hamsters. These differences can be explained both by pulmonary responses and by particle dosimetry differences among these rodent species.

The third study was again an interspecies, 13-week sub chronic inhalation study; with female rats, mice and hamsters exposed to three concentrations of carbon black (CB) particles (Elder et al, 2005). In this study, the authors postulated that the lung inflammation and injury induced by subchronic inhalation of CB are more pronounced in rats than in mice and hamsters. Particle retention kinetics, inflammation, and histopathology were assessed in female rats, mice, and hamsters exposed for 13 weeks to high surface area CB (HSCb) at doses chosen to span a no observable adverse effects level (NOAEL) to particle overload (0, 1, 7, 50 mg/m3). Pulmonary retention and lung effect measurements were conducted immediately after exposure as well as 3 and 11 months post-exposure; retention was also evaluated after 5 weeks of exposure. Significant decreases in body weight during exposure occurred only in hamsters exposed to high-dose HSCb. Lung weights were increased in high-dose CB-exposed animals, but this persisted only in rats and mice up to the end of the study period. Prolonged retention was measured in rats exposed to mid- and high-dose HSCb. Retention was also prolonged in mice exposed to mid- and high-dose HSCb, and in hamsters exposed to high-dose HSCb. Lung inflammation and histopathological effects were more severe and prolonged in rats when compared to mice and hamsters, and both indices were similar in rats exposed to mid-dose HSCb. The results show that hamsters have the most efficient clearance mechanisms and least severe responses of the three species.

An additional component of the CB study was published one year later, wherein the investigators assessed several pro and anti-inflammatory mediators to detect underlying mechanistic differences, which could account for the disparity in lung inflammatory responses to inhaled particulates among the rodent species (Carter et al, 2006). Here, the investigators looked at comparative dose-related responses of several key pro- and anti-inflammatory mediators in the lungs of rats, mice, and hamsters after subchronic inhalation of CB. As discussed above, rats, mice, and hamsters were exposed to air, 1, 7, or 50 mg/m3 of CB for 13 weeks and subjected to bronchoalveolar lavage 1 day, 3 months, and 11 months post- exposure. Some of the endpoints included recovered cell number and differential type, reactive oxygen and nitrogen species, and cytokine levels. Ex vivo mutational analysis of inflammatory cells was performed by co-incubating with lung epithelial cells. In addition, lung tissues were processed and evaluated for gene expression of selected anti-inflammatory mediators. The results demonstrated that a dose- and time-related effect existed with all the parameters. Rats demonstrated greater capacity for generating pro-inflammatory responses. In contrast, mice and hamsters demonstrated an increased proclivity for generating anti-inflammatory responses. The authors concluded that the cytokine differences in generating pro- and anti-inflammatory responses correlated with in vivo lung tissue responses and could play a significant role contributing to the apparent species differences in inflammation and tumourigenesis.

In summary, the results of these three above similarly designed sub chronic studies with particulates are remarkably similar in their findings. In all three studies, the rat model appeared to be the most sensitive to the development of lung inflammation, cytotoxicity, fibro-proliferative effects, and septal fibrotic response to high concentrations of particles that reached overload status. In contrast, under identical exposure circumstances, mice developed particle overload status with reduced or absent lung clearance effects, concomitant with strong evidence of robust lung inflammatory responses. However, despite reaching particle overload and lung inflammatory effects, the pulmonary tissue responses in the mouse were fundamentally different from the rat. Indeed there was no evidence of enhanced cell proliferative changes, fibro-proliferative effects or septal fibrosis – all of which are likely to lead to secondary genotoxic effects and the development of lung tumours following high-dose chronic inhalation of low solubility particulates. These extensive investigative findings separate the mouse and hamster responses from the pulmonary overload responses observed and measured in time course studies with rats. In addition, it should be noted that the hamster tends to have an accelerated particle clearance mechanisms, concomitant with reduced lung inflammatory responses to high dose particle exposures. Moreover, the reported findings by Carter et al, 2006, suggest that one potential mechanism for the differences in adverse lung responses in mouse and hamster vs. rats could be related to the enhanced generation of anti-inflammatory cytokines in mice and hamsters and the augmented generation of pro-inflammatory cytokines in the rats. This would likely lead to an imbalance in the rat response to overload particle exposures and could foster the development of the mechanistic pathological sequelae/events which have been postulated to occur in the rat lung response to high-dose particulates.