“Subchronic Inhalation Exposure of Rats to Libby Amphibole and Amosite Asbestos: Effects at 18 Months Post Exposure.” Willson GA (presenter), Dodd DA, Roberts KC, Wall HG, Jarabek AM, Gavett SH. The 33rd Society of Toxicologic Pathology Annual Meeting. Washington, DC. June 22-26, 2014. “Subchronic Inhalation Exposure of Rats to Libby Amphibole and Amosite Asbestos: Effects at 18 Months Post Exposure.” Dodd DA (presenter), Willson GA, Roberts KC, Wall HG, Jarabek AM, Gavett SH. The 53rd Annual Society of Toxicology Meeting. Phoenix, AZ. March 23-27, 2014. For full-resolution viewing, please open or save as a PDF.

1. Innovative Research for a Sustainable FutureInnovative Research for a Sustainable Futurewww.epa.gov/research
SUBCHRONIC INHALATION EXPOSURE OF RATS TO LIBBY AMPHIBOLE AND
AMOSITE ASBESTOS: EFFECTS AT 18 MONTHS POST EXPOSURE
G.A. Willson1, D.E. Dodd2, K.C. Roberts2, H.G. Wall1, S.H. Gavett3.
1Experimental Pathology Laboratories, Inc., RTP, NC, 2The Hamner Institutes for Health Sciences, RTP, NC, 3U.S. EPA, RTP, NC.
Abstract
Increased asbestosis, lung cancer, and mesothelioma rates are evident
after exposures to Libby amphibole (LA). To support dosimetry model
development and compare potency, a subchronic nose‐only inhalation
study (6 hr/d, 5 d/wk, 13 wk) was conducted in male F344 rats. Rats
were exposed to air (control), LA (LO, MED, HI; 1.01, 3.32, 10.06
mg/m3; 159, 693, 1522 fibers/cc), or amosite (AM; 3.34 mg/m3; 230
f/cc). Toxicity endpoints, pathology, and fiber burden evaluation were
determined 18 mo post‐exposure. Fiber exposure had no effect on
survival. Mononuclear cell leukemia was the main cause of death prior
to scheduled necropsy in all groups except the LA 3.3 group. BAL cell
numbers, LDH, and protein in AM and LA groups were not statistically
different from controls (n=8 rats/group), indicating resolution of
earlier inflammation. Histopathology of the left lung, trachea, sternum,
pleura, epididymis and testes, and gross tissue lesions was conducted
on 50 rats/group. Alveolus inflammation, pleural fibrosis, lung
interstitial fibrosis, and foreign bodies were noted in all fiber‐exposed
groups. A greater incidence of chronic tracheal inflammation was
noted in the LA groups. Alveolar bronchiolar adenoma occurred in 2
rats in each of the AM, MED LA, and HI LA groups, and 1 alveolar
bronchiolar carcinoma was observed in the HI LA group. No pleural
mesotheliomas were observed in any group. In conclusion, both AM
and LA induced dose‐related lung fibrotic responses; tumor incidences
were apparently increased but not beyond historical control ranges.
(This abstract does not represent US EPA policy.)
Exposure Fiber Characterization
• Overall, LA fiber is slightly longer and has a slightly greater aspect ratio compared
to UICC Amosite.
• UICC amosite had a higher proportion of particles (L/D <3) compared to LA.
• Other exposure atmosphere characteristics (T, %RH, airflow) were equivalent.
Air
Control
UICC
Amosite
LA
low
LA
medium
LA
high
Target Concentration
(mg/m3)
0.0 3.30 1.00 3.30 10.0
Actual Concentration
(mg/m3)
0.0 3.32 1.01 3.34 10.0
APS: Count Median Aero‐
dynamic Diameter (m)
1.41 0.93 1.05 1.02 1.13
APS: g 2.01 1.40 1.51 1.46 1.52
APS: Particles
Mean #/cc
1.2 404 43 171 280
SEM: All Objects
Mean #/cc
‐ 3101 1158 3728 6718
SEM: Mean Fiber
Concentration (f/cc)
(L/D> 3)
‐ 1627 663 2753 5093
SEM: Mean Fiber
Concentration (f/cc)
(L/D > 3; L > 5)
‐ 230 159 693 1522
SEM: Mean Fiber Length
(µm) (L/D > 3)
‐ 3.05 3.94 4.16 4.63
SEM: Mean Fiber Diameter
(µm) (L/D > 3)
‐ 0.33 0.39 0.41 0.42
Experimental Design
• Samples: LA obtained from the USGS collection in 2007. Amosite
(Amo, UICC) obtained from Dr. Phil Cook (EPA, Duluth, MN).
• Exposure, analysis: Samples lofted and aerosolized by rotary brush
aerosol generators were analyzed by SEM (JEOL JSM‐840A) and
Image‐Pro Plus (v. 5.0.1.11) to count, measure particle and fiber size.
• Exposure (nose‐only) of male 10‐wk old F344 rats (89‐98/group)
for 6 hr/d, 5 d/week, 13 weeks, at target concentration levels:
• Air control
• Amosite: 3.3 mg/m3
• LA Low: 1.0 mg/m3, LA Med: 3.3 mg/m3, LA High:10 mg/m3
• Major endpoints at necropsy (1‐day, 1‐mo, and 3‐mo post‐exposure,
n=7‐8/group) were bronchoalveolar lavage (BAL) fluid cytology and
biochemistry, and lung histopathology. At 18‐mo post‐exposure,
necropsy included up to 50/group for lung, trachea, sternum, pleura,
epididymis, testes, and relevant gross tissue lesions histopathology,
n=8 for BAL, and n=3‐4/tissue/groupfor fiber burden analysis.
• Data not reported here: Tissue fiber burden analyses are being
determined for individual or combined lung lobes, trachea/larynx,
lung lymph nodes, upper respiratory tract, and pleura at each time
point.
Background
• Vermiculite ore from Libby, Montana contains several types of
amphibole asbestos, including winchite, richterite, tremolite, and
magnesioriebeckite, with crystalline forms ranging from asbestiform
to acicular or prismatic (1).
• Occupational exposure to Libby amphibole (LA) is associated with
significant increases in asbestosis, lung cancer, and mesothelioma
compared to the rest of the U.S. population (2‐4).
• The overall goal of this research is to inform the scientific basis for
the risk assessment of LA‐contaminated communities by conducting
animal toxicology studies to help define key determinants of internal
dose and provide additional insight on key health or pathologic
endpoints.
• The specific aims of the study are to determine the biological potency
of inhaled LA fibers over the near‐life span of the rat compared to the
potency of inhaled UICC amosite, a known fibrogenic amphibole
asbestos fiber, and to develop fiber burden data to use in a dosimetry
model of amphibole fiber deposition, clearance and retention in the
respiratory tract.
Summary and Conclusions
• SEM results of exposure atmospheres indicate that the LA
fiber is slightly longer and has a greater aspect ratio
compared to the AM fiber.
• At 18 months post‐exposure, the mean survival percentages
were similar. The main cause of early animal
deaths/animals euthanized moribund was mononuclear cell
leukemia (MCL), but group incidences of MCL (6‐14%) were
below NTP’s historical control average of 36% for male
F344 rats.
• Results of BAL cytology and enzymology (data not shown)
at 18 months were unremarkable, indicating a reversal of
inflammatory effects observed at earlier time points.
• Mesothelioma was not detected in the thoracic cavity of any
rats, including the AM group. Primary thoracic
mesotheliomas are rare in NTP studies (0.037% of 66941
control and treated rats, Willson, Soc. Toxicol. Pathol., 2003).
• Fibers could be identified in the majority of animals from
each fiber exposure group, indicating a persistence of the
fiber throughout 18 months post‐exposure. The fibers
continued to be sequestered in macrophages and sometimes
syncytial giant cells were observed.
• Throughout the 18 months post‐exposure, the most
significant histopathological findings were observed in the
lungs of rats exposed to LA HI (10 mg/m3).
• Lung effects at 1‐day, 1‐mo, 3‐mo, and 18‐mo post‐exposure
were similar between the AM and LA groups exposed to the
same fiber concentration of 3.3 mg/m3.
• Dosimetry model predictions of various internal dose
metrics may provide further insight on factors determining
this pathogenesis.
Air Control
UICC
Amosite
LA
Low
LA
Medium
LA
High
1 D
1
Mo
3
Mo
18
Mo
1 D
1
Mo
3
Mo
18
Mo
1 D
1
Mo
3
Mo
18
Mo
1 D
1
Mo
3
Mo
18
Mo
1 D
1
Mo
3
Mo
18
Mo
Bronchiole Epithelial
Hyperplasia
0 0 0 0 0
1
(0.1)
0 0 0 0 0 0 0 0
0at
this
0
8
(1.0)
8
(1.0)
0 0
Alveolar Epithelial
Hyperplasia
0 0 0
1
(0.1)
0 0 0
9
(0.3)
0 0 0
6
(0.2)
0 0 0
6
(0.2)
0
1
(0.1)
1
(0.3)
6
(0.3)
Alveolus Inflammation 0 0 0 0
8
(1.0)
8
(1.1)
8
(1.0)
45
(1.0)
8
(1.0)
8
(1.0)
8
(1.0)
40
(0.9)
8
(1.0)
8
(1.0)
8
(1.1)
47
(1.1)
8
(2.0)
8
(2.0)
8
(1.4)
48
(1.1)
Interstitial
Fibrosis
0 0 0 0
8
(1.0)
8
(1.0)
8
(1.0)
42
(0.8)
8
(1.0)
7
(0.9)
8
(1.0)
29
(0.6)
8
(1.0)
8
(1.0)
8
(1.0)
43
(0.9)
8
(1.0)
8
(1.0)
8
(1.0)
42
(0.9)
Pleural Fibrosis ‐ ‐ ‐ 0 ‐ ‐ ‐
25
(0.5)
‐ ‐ ‐
16
(0.3)
‐ ‐ ‐
19
(0.4)
‐ ‐ ‐
22
(0.4)
Foreign Body
(Fibers; not graded)
0 0 0 0 8 8 8 47 8 8 8 40 8 8 8 48 8 8 8 49
Bronchiolization 0 0 0 0
8
(1.0)
7
(0.9)
8
(1.0)
5
(0.1)
7
(0.9)
7
(0.9)
8
(1.0)
5
(0.1)
8
(1.0)
7
(0.9)
8
(1.0)
0
8
(1.0)
8
(1.4)
8
(1.0)
0
Alveolar Bronchiolar
Adenoma
0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 2 0 0 0 2
Alveolar Bronchiolar
Carcinoma
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
This research was supported under the US EPA’s ORD Libby Action Plan and via EPA
Contract #EP‐W‐08‐051 to The Hamner Institutes for Health Sciences. The views
expressed in this poster do not necessarily represent the views or policies of the U.S.
Environmental Protection Agency. References available upon request.
Values shown are incidence, number animals with a finding, n=8 or 50 (18‐mo) animals examined per group, and mean severity score (in parentheses). Severity score: 1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe.
Survival Curves Post‐Exposure
50%
60%
70%
80%
90%
100%
0 20 40 60 80
Survival
Weeks Post-Exposure
Air
AM 3.3
LA 1.0
LA 3.3
LA10.0
mcl mcl mcl mcl mcl
oms
oms oms
oms
pa
pa
pa
pa
pm
pgc
ne
ne
un
un
un
pm
os
os
sgc
sf
ms
tfc
labc
mf
me
acc
tfc
ss
Severity Score Severity Score
18 Months
Bronchoalveolar Lavage Cells
Significant Differences (p < 0.05):
† vs. all others * vs. Air
# vs. LA 1.0 § vs. LA 3.3
‡ vs. Amo 3.3
Incidence, n = 50
LA 10.0
Alveolar Epithelial Hyperplasia,
Inflammation, Interstitial Fibrosis
Causes of Early Death/Moribund Necropsy
Interstitial Fibrosis Pleural Fibrosis
Alveolar Bronchiolar Adenoma (AM 3.3)
Tracheal Inflammation
Incidence, n = 50
Major Histopathology
Findings (18 Months)
• Alveolus inflammation, interstitial
fibrosis, foreign bodies (fibers), and
pleural fibrosis were observed in the
lungs of all fiber exposure groups.
Pleural fibrosis was not observed at
earlier post‐exposure time points.
• Alveolar epithelial hyperplasia was
diagnosed in all fiber exposure groups.
Except in the LA HI group, this lesion
had not been observed at earlier post‐
exposure time points.
• Alveolar bronchiolar adenomas were
observed in the AM, LA MED, and LA
HI groups and a single alveolar
bronchiolar carcinoma was diagnosed
in the LA HI group.
• All rats with mesotheliomas also had
tumors in the testes/epididymides;
the tumors are thought to originate in
the tunica vaginalis.
• Pulmonary neoplasms were evident
only in fiber exposure groups at this
21 month time, while NTP’s historical
control incidence range for 24 months
is 0‐8%.