title: Part 1. Assessment of carcinogenicity and biologic responses in rats after lifetime inhalation of new-technology diesel exhaust in the ACES bioassay.
abstract: The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to 2007-compliant new-technology diesel exhaust (NTDE*). The a priori hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions "... will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used ... although some biological effects may occur." This hypothesis was tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay. Indicators of pulmonary toxicity in rats were measured after 1, 3, 12, 24, and 28-30 months of exposure. Similar indicators of pulmonary toxicity were measured in mice, as an interspecies comparison of the effects of subchronic exposure, after 1 and 3 months of exposure. A previous HEI report (Mauderly and McDonald 2012) described the operation of the engine and exposure systems and the characteristics of the exposure atmospheres during system commissioning. Another HEI report described the biologic responses in mice and rats after subchronic exposure to NTDE (McDonald et al. 2012). The primary motivation for the present chronic study was to evaluate the effects of NTDE in rats in the context of previous studies that had shown neoplastic lung lesions in rats exposed chronically to traditional technology diesel exhaust (TDE) (i.e., exhaust from diesel engines built before the 2007 U.S. requirements went into effect). The hypothesis was largely based on the marked reduction of diesel particulate matter (DPM) in NTDE compared with emissions from older diesel engine and fuel technologies, although other emissions were also reduced. The DPM component of TDE was considered the primary driver of lung tumorigenesis in rats exposed chronically to historical diesel emissions. Emissions from a 2007-compliant, 500-horsepower-class engine and after treatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors. components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hours/day (overnight, during the rats' most active period), 5 days/week. Responses to exposure were evaluated via hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, histopathology, and pulmonary function. The exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components from exhaust were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of DPM and volatile and semi-volatile organic compounds (VOCs and SVOCs). Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to NTDE. The major outcome of this study was the absence of pre-neoplastic lung lesions, primary lung neoplasia, or neoplasia of any type attributable to NTDE exposure. The lung lesions that did occur were minimal to mild, occurred only at the highest exposure level, and were characterized by an increased number and prominence of basophilic epithelial cells (considered reactive or regenerative) lining distal terminal bronchioles, alveolar ducts, and adjacent alveoli (termed in this report "Hyperplasia; Epithelial; Periacinar"), which often had a minimal increase in subjacent fibrous stroma (termed "Fibrosis; Interstitial; Periacinar"). Slight epithelial metaplastic change to a cuboidal morphology, often demonstrating cilia, was also noted in some animals (termed "Bronchiolization"). In addition to the epithelial proliferation, there was occasionally a subtle accumulation of pulmonary alveolar macrophages (termed "Accumulation; Macrophage") in affected areas. The findings in the lung progressed slightly from 3 to 12 months, without further progression between 12 months and the final sacrifice at 28 or 30 months. In addition to the histologic findings, there were biochemical changes in the lung tissue and lavage fluid that indicated mild inflammation and oxidative stress. Generally, these findings were observed only at the highest exposure level. There was also a mild progressive decrease in pulmonary function, which was more consistent in females than males. Limited nasal epithelial changes resulted from NTDE exposure, including increases in minor olfactory epithelial degeneration, hyperplasia, and/or metaplasia. Increases in these findings were present primarily at the highest exposure level, and their minor and variable nature renders their biologic significance uncertain. Overall, the findings of this study demonstrated markedly less severe biologic responses to NTDE than observed previously in rats exposed similarly to TDE. Further, the effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors.
authors.forename: Jacob D
authors.initials: JD
authors.lastname: McDonald
authors.forename: Melanie
authors.initials: M
authors.lastname: Doyle-Eisele
authors.forename: JeanClare
authors.initials: J
authors.lastname: Seagrave
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Gigliotti
authors.forename: Judith
authors.initials: J
authors.lastname: Chow
authors.forename: Barbara
authors.initials: B
authors.lastname: Zielinska
authors.forename: Joe L
authors.initials: JL
authors.lastname: Mauderly
authors.forename: Steven K
authors.initials: SK
authors.lastname: Seilkop
authors.forename: Rodney A
authors.initials: RA
authors.lastname: Miller
mesh_terms: D000280:Administration, Inhalation; D000393:Air Pollutants / Q000494:pharmacology / Q000633:toxicity*; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002248:Carbon Monoxide / Q000633:toxicity*; D015197:Carcinogenicity Tests; D016207:Cytokines / Q000378:metabolism; D005260:Female; D008297:Male; D051379:Mice; D009569:Nitric Oxide / Q000633:toxicity*; D009585:Nitrogen Dioxide / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects; D052638:Particulate Matter / Q000633:toxicity*; D051381:Rats; D011919:Rats, Inbred Strains; D012737:Sex Factors; D013997:Time Factors; D001335:Vehicle Emissions / Q000633:toxicity*; D055549:Volatile Organic Compounds / Q000633:toxicity
chemical_list: D000393:Air Pollutants; D016207:Cytokines; D052638:Particulate Matter; D001335:Vehicle Emissions; D055549:Volatile Organic Compounds; D009569:Nitric Oxide; D002248:Carbon Monoxide; D009585:Nitrogen Dioxide
references.citation: Lancet. 2003 Jun 7;361(9373):1939-44
references.pmid: 12801737
references.citation: Environ Res. 2007 Jul;104(3):420-32
references.pmid: 17445792
references.citation: J Epidemiol Community Health. 2010 Mar;64(3):223-8
references.pmid: 19679705
references.citation: Environ Sci Technol. 2009 Oct 15;43(20):7778-83
references.pmid: 19921893
references.citation: Environ Health Perspect. 2009 Dec;117(12):1919-24
references.pmid: 20049212
references.citation: Sci Total Environ. 2013 Mar 15;448:38-47
references.pmid: 23149275
references.citation: Environ Sci Technol. 2013 Jan 15;47(2):889-98
references.pmid: 23190276
references.citation: Sci Total Environ. 2013 Jan 15;443:375-86
references.pmid: 23202383
references.citation: Environ Health Perspect. 2013 Mar;121(3):324-31
references.pmid: 23308401
references.citation: Sci Total Environ. 2013 Jun 1;454-455:348-57
references.pmid: 23562687
references.citation: Am J Respir Crit Care Med. 2013 Sep 1;188(5):593-9
references.pmid: 23805824
references.citation: Environ Sci Technol. 2014 Aug 19;48(16):9609-15
references.pmid: 25019919
references.citation: Environ Sci Technol. 2015 Apr 21;49(8):5236-44
references.pmid: 25826745
references.citation: Environ Sci Technol. 2017 Jun 20;51(12):6981-6989
references.pmid: 28166626
references.citation: Air Qual Atmos Health. 2017 Jun;10(5):611-625
references.pmid: 30245748
references.citation: J Air Waste Manag Assoc. 1998 May;48(5):418-26
references.pmid: 9602552