EXTERNAL LETTER PEER REVIEW
OF A REPORT BY BIO-RESEARCH LABORATORIES, LTD.:
"SUPPORT: FINAL REPORT FOR A 90-DAY
INHALATION NEUROTOXICITY AND TOXICITY STUDY
BY EXPOSURE TO A DRY POWDER
AEROSOL OF CERIC OXIDE IN THE ALBINO RAT
WITH COVER LETTER DATED 013095."
FINAL REPORT
Prepared for
Integrated Risk Information System (IRIS) Program
Office of Research and Development
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Prepared by
ORISE IRIS Technical Assistance Team
Oak Ridge Institute for Science and Education
Oak Ridge Associated Universities
August 2006
This document was prepared for the EPA by ORISE under Interagency Agreement
No. DW-89939822-01-0 between EPA and the U.S. Department of Energy.
ORISE is managed by Oak Ridge Associated Universities under a contract with DOE.

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EXTERNAL PEER REVIEWERS
Rodney R. Dietert, Ph.D.
College of Veterinary Medicine
Cornell University
Ithaca, NY
Jie Liu, Ph.D.
National Cancer Institute at NIEHS
Research Triangle Park, NC
Mark David Noble, Ph.D.
University of Rochester
School of Medicine and Dentistry
Rochester, New York
David Warheit, Ph.D.
Haskell Laboratory
Newark, DE
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PEER REVIEW PROJECT
Martin W. Gehlhaus, III
U. S. Environmental Protection Agency
Leslie Shapard, Peer Review Manager
Oak Ridge Institute for Science and Education
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Ceric Oxide Exposure Study Review
TABLE OF CONTENTS
REVIEWER COMMENTS BY QUESTION
Question 1	1
Question II	6
Question III	7
Question IV 	8
Question V	10
Other Comments	12
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External Letter Peer Review of a report by Bio-Research Laboratories, Ltd.:
"Support: Final Report for a 90-day Inhalation Neurotoxicity
and Toxicity Study by Exposure to a Dry Powder Aerosol of Ceric Oxide
in the Albino Rat with cover letter dated 013095."
The U.S. Environmental Protection Agency's (EPA) National Center for Environmental
Assessment (NCEA) is developing a "Toxicological Review of Cerium and Cerium Compounds."
The Bio-Research Laboratories, Ltd (BRL, 1994) Final Report is a proposed principal study, but
has not undergone a formal peer review. It is important that selected outside experts evaluate the
accuracy of the experimental procedures, results, and interpretation and discussion of the findings
presented in this investigational report.
The BRL (1994) study exposed four groups of Sprague-Dawley rats, 15 rats/ sex/ group, via
nose-only inhalation to a dry powder aerosol of ceric oxide for 6 hours a day, 5 days a week, for
13 weeks at dose levels of 0, 5, 50, and 500 mg/m3. Behavorial effects of exposure were not
observed, nor were effects on the liver or kidney function or reproductive organs. Increased lung
weights were evident and histological examination of the lung revealed pigment accumulation at
all dose levels and alveolar hyperplasia at the two highest doses. Pigment accumulation was also
observed in the nasal cavity, bronchi and trachea, and liver and spleen. The bronchial lymph
nodes were macroscopically enlarged and microscopic findings included pigment accumulation
and hyperplasia. Segmented neutrophil counts were elevated in females at all dose levels and the
two highest dose levels for males.
1. Based on your knowledge of toxicological protocols, please comment on the experimental
design of this investigation. Do you see any significant issues with the test system or article,
inhalation exposure equipment and monitoring of atmosphere, endpoints recorded, terminal
procedures, statistical analyses, and quality assurance?
R. R. Dietert. Ph.D.
The study design incorporated 15 Sprague-Dawley rats of each sex for each of four groups: a
control and three cerix oxide dose groups (exposure via inhalation). Sexes were started into the
protocol in a slightly staggered time course to permit complete animal assessment to be
accomplished within labor/facility constraints. The exposure protocol as well as the atmosphere
monitoring were appropriate and with the stated exception of a single animal death due to an
equipment sizing problem during exposure, all seems to have gone as designed. Atmospheric
levels of the ceric oxide powder were in the ranges anticipated and the fluctuations across the
exposure period were not problematic. The data collection, quality assurance oversight, and
statistical analyses were conducted appropriately. Some neurotoxicologic endpoints had to be
evaluated as qualitative or semi-quantitative assessments, but this was to be expected given
protocols at the time the study was conducted. Overall, the study was well-conceived and
appears to have been executed with considerable care.
Jie Liu. Ph.D.
The experimental design fits well with the title (goal) of this report "A 13-week inhalation
toxicity and neurotoxicity study by nose-only exposure of a dry powder aerosol of ceric oxide in
the albino rat." Inhalation to ceric oxide aerosols occurs at workplace where pollucite is mined or
cerium compounds are manufactured. Inhalation exposure to airborne ceric oxide is also a
possible route for the general population. Thus, to study the health effects from inhalation of ceric
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oxide is of toxicological significance. Adult SD rats were selected because rat is a commonly
used rodent species for inhalation toxicity and for neurotoxicity studies. A total of 120 qualified
adult rats (15 male and 15 female rats/group x 4 groups) are sufficient and typical in such a 90-
day subchronic study. The dose levels (0, 5, 50, and 500 mg/m3) were selected according to the
potential human exposure (TLV of 5 mg/m3). Thus, the three dose level selections were
appropriate and the highest dose is 100 times the TLV value for eerie oxide. All rats were
examined before, during, and after exposure for any overt signs of reaction to treatment,
including clinical observation, food consumption, body weight, Functional Observation Battery,
laboratory investigations, and ophthalmologic examinations. At the end of the experiments, a
complete necropsy and histopathology were performed. This was a well-designed and well-
conducted study.
Overall, no specific issues are raised with the entire test system.
a.	Animals. The 60 qualified animals/sex were selected from 85 rats and randomly assigned
to different test groups and acclimated for two weeks prior to treatment as detailed on Page
13.	This is a very good approach. Additional 10 rats were used to obtain the normal values
for laboratory investigations and gross pathology. All animals were clearly labeled to trace
their reactions to treatment as reported in the entire experiment (a minor comment: air
control males were actually numbered 1001-1017, not from 1001 to 1015, similar to other
groups). The animal housing, diet and water system were certified (page 1293-1310), and
met the criteria of NIH Guidelines for Humane Use and Care of Animals. No significant
issues in animals were found.
b.	Test chemical. The identity and chemical analysis of test chemical were detailed on page
14,	with certification on page 1311-1312. The stability of the test compounds during the
experiment was also analyzed (page 1344 to 1352). No questions about the purity of eerie
oxide used in the study.
c.	Inhalation exposure equipment. The "nose-only" exposure equipment as depicted in Fig. 2
(page 48) was described in detail on page 14-16. No questions on the equipment system.
d.	Monitoring of the experimental atmosphere. The exposure atmospheres were adequately
described (page 16-17). For the three eerie oxide groups, daily camber concentrations
(page 51-66), daily nominal concentrations (page 67-68), weekly particulate size (page 69-
76), all were documented in detail. No questions.
e.	Observations. The observations were adequately described in general on page 17 to 22,
and documented in details in later sections:
i. Clinical examinations and mortality were summarized on page 26-27. A
accidental death was described in detail and the conclusion that it is unrelated to
treatment was valid. Table 6 (page 77-90) summarized the reddish staining
among groups (Minor: please check page 79, on Day 37, group 3, pre-staining).
The frequencies were low and also seen in controls, and the conclusion that these
reactions were associated with exposure, not treatment, is valid. Other clinical
signs were recorded in detail on page 278-282, and these occasionally occurred
clinical symptoms were not consistent and are unlikely related to cerix oxide
treatment.
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ii. The animal body weight, body weight gain, food consumption were summarized
in table 7-9 (page 91-96), with detailed individual animals on page 283-307. The
individual numbers were correctly summarized.
Hi. Ophthalmology summary reports (page 27, page 33) and the individual
documentations (page 308-310) supported the conclusion that eerie oxide
inhalation did not produce any ocular changes.
iv.	Functional Observation Battery for neurotoxicty was a major goal of this study,
and the comprehensive observations included home cage observation, removal
observation, arena observation, handling, grip strength, and body temperature
from pre-study, on Day 1, week 4, 8, and 13, were documented in detail. These
observations were complete and summarized in table 10 (page 97-187), with
individual animal report from page 311-670. These huge individual data were
appropriately summarized, and supported the conclusion that no clear behavioral
changes are associated with eerie oxide treatment (page 27). Minor: The page
438 (female week 8, group 4) should be placed after page 462 (female, week8,
group 3).
v.	Motor Activities are a major endpoint for neurotoxicity. They were depicted in
Fig. 1 (page 40-47), summarized in Table 11 (page 188-195), with individual
records from page 671-801. All the data supported the conclusion that no
toxicologically significant differences in total motor activity between control and
eerie oxide treatments were evident. (Minor: page 716, animal 3011 could be
"T." as the number was too low and the same animal was normal on the 13 week,
page 732).
vi.	Laboratory Investigations. Detailed descriptions of Methods, Equipments,
Symbols, and Criteria are included as Appendix 7 (page 802-812). Complete
batteries of laboratory investigations were clearly documented. These included
hematology summary in Table 12 (page 196-219) with individuals on appendix 8
(page 813-903), clinical biochemistry in Table 13 (page 220-237) with
individuals on appendix 9 (page 904-958), and urinalysis with individuals in
appendix 10 (page 959-995). These investigations were complete and supported
the conclusion that eerie oxide treatment did not cause alterations on these 65
parameters. Minor: Clotted blood occurred on week 8 sampling (about 40% on
page 835 and page 854), but clotted blood was avoided at week 13 sampling,
which is the most important time point. Animal 4503 at 13 weeks had slightly
higher ALT (page 956), but histology did not reveal significant change (page
1252), and other parameters were in normal range. Thus, this slight increase in
ALT does not matter and does not affect the conclusion.
b. Terminal
i. Organ weights. At animal termination, the weights of 11 major organs were
recorded in detail (appendix 11, page 998-1021) and summarized in Table 14.
Ceric oxide exposure increased lung organ weight in both male and females in a
clear dose-dependent fashion. The same conclusions were reached when the
absolute organ weights were normalized to body weight (appendix 12, page
1022-1045, and Table 15, page 244-249), or to brain weight (appendix 13, page
1046 to 1069, Table 16, page 250-255). All these data clearly indicate the
increases in lung weight are related to ceric oxide exposure. However, slightly,
but significantly increases in spleen weights (only in male high dose group 4)
may be related to ceric oxide exposure. The spleen weights (may be related)
would be better separated from lung weights (clearly related to ceric oxide
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exposure) on page 29 description. Increases in thymus weights in group 3 of
male and female rats are also significant when normalized with the body weight,
and thus they may also be related to eerie oxide exposure, rather than "treatment-
unrelated" (page 29), which could be grouped together with spleen, as both
organs are likely involved in clearance of transferred eerie oxide, as pigment
accumulation was evident in spleen, and lymphonodes (page 29-32, although
thymus was not examined, a few hemorrhage was seen in males at the high dose
(page 267).
ii.	Histopathology. Gross histopathology was summarized in Table 17 (page 256-
260). In addition to the lung, the gross alterations in lymphonodes were evident
(page 29-32, and Table 18, page 261-275).
iii.	Histology procedures were described in detail on page 23-25. A detailed
individual histopathology was reported on appendix 14, page 1070-1291, and
partially summarized on page 29-32. In general, Histopathology was well
conducted and the pathology findings supported laboratory analysis and gross
pathological observations. Minor comments: some pages were misplaced: page
1210 and 1211 should be exchanged; page 1254-1262 should be followed by
page 1216; page 1218 should be placed before page 1168. These pathological
reports were appropriately summarized as notable pathological findings on page
34-36, and the conclusions reached are valid.
c.	Statistics. The procedures of statistics were described adequately on page 25. Data were
expressed as mean and SD, and analyzed ANOVA, followed by Dunnett's, Kruskal-Wallis,
or Dunn's test. The frequency data and pathology were analyzed by Fisher's exact test. All
these analysis are correct.
d.	Quality Assurance. Quality assurance statement was described on page 25, and the
statements were presented on Page 38-39. No questions on quality assurance issues.
Mark Noble. Ph.D.
The behavioral tests employed are not subtle and would be expected to only reveal dramatic
alterations in function. Observational tests (locomotor activity level, arousal, grooming,
defecation, urination, olfactory response, handling, salivation, etc.) all also represent crude tests,
and there are few toxic insults in an environmentally relevant range that would affect any of these
parameters. For example, it is not even clear whether any of these tests would detect toxicity of
lead (Pb) when applied at environmentally relevant levels that now are believed to be associated
with clear evidence of adverse effects on development of the CNS.
It is also noted that the standard deviation on some of these tests may be so large as to make it
impossible to observe differences that are less than catastrophic. For example, consider on
document pg. 188 the motor activity scores. Mean scores in the range of 255-290 are associated
with standard deviations ranging from 89-135. From the small number of observations made, it
would be statistically impossible to discern differences between groups.
David B. Warheit, Ph.D.
This is, in general, a well conducted study with a couple of limitations. There are, however, a
few weaknesses which, while they do not compromise the integrity of the study, would have
strengthened the quality of the study. These include the following:
1) There appears to be a significant lack of physicochemical characterization of the test
article/test sample. This Reviewer makes a distinction between what would have been
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required today (2006) vs. what should have been required in 1993, when the study was
undertaken. In 2006, more extensive physicochemical characterization would have
included the following: mean particle size and size distribution of the bulk material,
surface area determinations, aggregation potential, surface coatings (if any), crystal
structure, AS WELL AS the characterization that should have been included in 1993 -
i.e., data regarding the composition, purity, solubility, and contaminants of the test
article. This is rather important because the inhaled eerie oxide particles appeared to
translocate from alveolar regions to extrapulmonary organs such as the liver, spleen and
other organs - therefore it would be important to know whether the eerie oxide particles
were soluble or insoluble.
2)	The absence of an additional postexposure (sacrifice) period of evaluation is unfortunate.
The experimental design described a 13 week inhalation exposure period, followed by an
immediate postexposure sacrifice period. In order to gauge the reversibility of the effects
observed or measured, it would have been necessary to add an additional 1 or 3-month
postexposure period of evaluation.
3)	It is very interesting that the authors have concluded that inhalation of eerie oxide
particles produced antigenic stimulation. This would seem to be an unusual response
following inhalation of dust particles. It seems likely to this Reviewer that inhaled
particles could translocate from sites of particle deposition (bronchoalveolar junctions) to
the corresponding interstitial compartment and connecting lymph nodes, wherein the
particles may lie freely or be phagocytized by macrophages residing in the lymph nodes.
However, the finding of lymphoid hyperplasia seems to be an unusual response for an
inhaled dust. Therefore, it would have been very helpful to have information on the
solubility of the eerie oxide dust and to have scheduled an additional postexposure time
period to gauge the possible reversibility of the response.
4)	It seems very interesting, given that eerie oxide particles are a white powder, that the
pathologist reported that the pigment present both intra- and extracellularly within the
respiratory system presented histologically as distinctly, round, blackish green material.
Again, it would have been interesting to have more information on the physicochemical
characteristics of the eerie oxide test article - as well as an additional postexposure time
period.
Do you see any significant issues with the test system or article, inhalation exposure equipment
and monitoring of atmosphere, endpoints recorded, terminal procedures, statistical analyses, and
quality assurance?
1) As stated above, this appears to be a well conducted study. This Reviewer has already
discussed some significant issues related to the lack of physicochemical characterization
of the test article and the experimental design of the study. The inhalation exposure
equipment, monitoring of atmospheres, endpoints recorded, terminal procedures,
statistical analyses, and quality assurance appear to be acceptable. It is unclear whether
the authors considered the pigmentation/discoloration to be an adverse effect.
Some of the authors' conclusions were a bit confusing. Moreover, some aspects of the
study results were interesting. These are detailed below:
1) In the summary and conclusion sections (page 32) it is written "An overall no-effect
level cannot be established based on marginally reduced body weight gains recorded
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in Group 4 animals, as well as other changes recorded in treated groups including
increased segmented neutrophil counts, higher lung and spleen weights	"
The reference to the body weight gains recorded in Group 4 animals is confusing.
According to the assessment of this Reviewer - the reason that a no-effect level could
not be established was because - there were clearly significant biological effects vs.
controls in Groups 3 and 4 animals; and more importantly, in Group 2 females -
there was a significant increase in segmented neutrophil counts (hematology),
concomitant with bronchial lymph node hyperplastic effects in both male and female
group 2 animals as well as mediastinal lymph node hyperplasia in group 2 female
animals.
2)	The authors concluded that systemic dissemination of the test article was evident in
Group 4 animals (without defining the term "systemic dissemination"). Was there
not translocation (i.e., systemic dissemination) of eerie oxide particles from sites of
particle deposition to systemic circulation (i.e., spleen, liver, etc.) at all exposure
levels?
3)	This Reviewer was intrigued by the relative lack of dose response effects when
comparing the low (5 mg/m3) and higher exposure concentrations (50 and 500
mg/m3). The differences in exposure concentrations and corresponding doses (lung
burdens were not measured) between 5 and 500 mg/m3 are substantial (100-fold!)
Yet, although there was a (minor) dose response trend, with respect to the measured
endpoints, it is remarkable that there were not greater histopathological differences
between the responses to the low and high exposure concentrations.
4)	This Reviewer was also surprised that exposures to 500 mg/m3 eerie oxide did not
produce more substantial pulmonary effects such as (lung fibrosis). The major
pulmonary effects were alveolar hyperplasia, lung weight increases and pigment
accumulation. However, metaplasia in the larynx was noted for all treatment groups;
and the lymph node effects have also been noted.
5)	One must conclude, however, that since an overall no-effect level could not be
established from this 13-week inhalation study, the currently proposed "nuisance
dust" exposure level of 5 mg/m3 for eerie oxide particulates should be reduced.
2. Are there endpoints that should have been monitored that were not part of the
investigation ?
R. R. Dietert. Ph.D.
Given the time of the study (1993-1994), the endpoints monitored were appropriate for general
toxicity and neurological toxicity evaluation. Most of the neurotoxicologic endpoints pertained to
motor function rather than behavioral assessment. If the study were redone today, additional
behavioral measures such as learning and memory-related endpoints might have been added.
Some of the endpoints evaluated might have been quantitative instead of semi-quantitative as
well. The same is true for lung, blood and lymphoid organ assessment. The measures obtained in
1993 were appropriate with the toxicological assessment norm for that time. However, if the
study were repeated today, additional parameters would have been assessed to adequately analyze
the hematopoietic, inflammatory and immune considerations. Lung cytokines could be evaluated
and immune cell populations examined in lymph nodes, spleen and thymus. Again, these
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evaluations were not all routine in rats in 1993 and, hence, the study parameters were appropriate
given the study is now over a decade old.
Jie Liu, Ph.D.
For the goal and scope of this 90-day inhalation study, all the endpoints used are sufficient and no
additional endpoints are necessary.
Mark Noble, Ph.D.
The information provided in this report clearly indicates a number of endpoints that should have
been part of this investigation. These are considered in greater detail under heading 5, but are
equally applicable to this question.
a.	How long does the inflammation caused by eerie oxide particle inhalation last? It is clear
that industrial and environmental exposure will last for far longer than the two week end
point utilized in this study. The truncated time point used provides no information on
delayed effects, or changes over time.
b.	Are the hyperplastic and neoplastic changes seen indications of carcinogenic potential of
exposure? When metaplastic changes are seen in the context of an examination in the
clinic, one's immediate concern is to determine whether such changes are associated with
pre-neoplastic states. Experiments that could be used to provide such information are
considered under point 5.
c.	How do effects change with repeated exposure? It is suggested by the submitters of this
trial that that the inflammatory changes seen are associated with stimulation of an
immune response. Yet, there were no attempts to define whether this is so, nor what the
consequences would be in the context of repeated challenge (let alone on an asthmatic
background, for example).
d.	How do results differ in different strains? The results of analysis within a single strain are
impossible to interpret in a meaningful way. Recommendations on other strains to use
are provided under point 5.
e.	Combined insults: Single insult paradigms are highly artificial, and provide little
resemblance to the effects of exposure in the real word. Co-exposures that would be
likely to occur with eerie oxide exposure (e.g, other industrial contaminants likely to be
found in the same workplace, exposure via diesel emissions.
David B. Warheit, Ph.D.
The endpoints monitored for this study were adequate.
3. Please comment on the strength, credibility, and relevance of the toxicological results.
Were the results of the individual rodents correctly summarized and interpreted?
R. R. Dietert, Ph.D.
The toxicological results of the study do reflect the profiles from the individual rodents as far as
the parameters evaluated. The observations appear to have been recorded with care and with
appropriate oversight. As previously-mentioned some parameters were only semi-quantitative
and if performed today, might have been done using quantitative imaging programs that would
permit more extensive comparisons. Having said that, the histological results are clear-cut and
the results did not suffer from the methodologies employed. In fact, there are very few gray areas
in the study such as might have occurred where serious individual problems were noted but were
not reflected as statistical analyses were performed. Such examples were minimal to non-
existent. In summary, the results do reflect underlying individual rodent observations and the
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profile of results can be viewed as credible.
Jie Liu. Ph.D.
This is a well-designed and well-performed study. The results obtained are solid, the data
interpretations are appropriate. The conclusions reached are sound. All the individual data are
correctly presented and summarized. These observations should be of toxicological significance.
Mark Noble, Ph.D.
Within the tests that do show changes, there also some peculiar observations that raise concern
about the outcomes reported. For example in gross pathological observations of the lung, 15/15
animals in the moderate exposure group showed pale areas in the lung, but 0/15 animals in the
low or high dose exposure groups (page 256). How is it that such effects are seen in all animals
in an intermediate exposure group, but in none of the animals receiving either a lower exposure or
a higher exposure? The most likely interpretation is of a flaw in the investigation, an issue that is
un-addressed.
Bronchial and mediastinal lymph nodes in moderate and high exposure groups had a high
frequency of enlargement and discoloration, and the potential negative implications of such
finding were un-explored.
David B. Warheit, Ph.D.
As discussed above, the strength, credibility, and relevance of the toxicological results are
adequate - under the conditions of this study. The study would have been strengthened if the
characterization of the test material had been more robust and an additional postexposure period
of evaluation (i.e., sacrifice) had been added to the experimental design of the study.
The results of the individual rodent data appear to be correctly summarized in this report.
4. Were the conclusions supported by the data? Are there any observations that were excluded
from the conclusion that should have been included? More specifically, were there any
observations that were excluded from the conclusions that are contradictory?
R. R. Dietert, Ph.D.
The most significant questions with the present study concern the interpretations of the findings,
the fact that additional tests would have been conducted had the study been performed in this
decade, and the projected ramifications of the reported findings.
In the overall conclusions the report states there were no behavioral effects observed associated
with the test chemical treatment (page 1). Yet, the decrease in forelimb grip in the high dose
females was significant and rather marked (page 27, data on page 177). It was, however,
restricted to the one sex. It remains an open question in terms of the significance of this one dose
group one sex effect.
Other issues surround the immune-inflammatory responses. The report states that the
inflammation in the lungs (and also reflected by swelling in the spleen and lymph nodes)
reflected little more than normal clearance of the particles. This led to a dramatic increase in
circulating neutrophils as well as lung infiltration. Alveolar macrophages were largely
responsible for particle uptake and this was particularly seen at lower exposure doses (page 35).
The report speculates on the likelihood that the observed inflammatory findings would have been
reversed following an appropriate recovery period (page 2). That is certainly one possibility and
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might be the case. However, it is only one of several possibilities given the full range of
observations in this report. The report indicates that lymphoid hyperplasia occurred at even low
doses in both sexes in several lymph nodes and the lung (page 35). The reason for this is
speculated to be immune stimulation by the test chemical. If indeed the report's authors are
correct, then the test compound is antigenic and has induced a significant lymphoid immune
response, the profile of it is uncharacterized. In effect, it is not known if this response would
prove to be clinically uneventful and subside as such or if it might become problematic elevating
the risk of hypersensitivity/autoimmune reactions. None of the parameters measured address the
nature of the host immune stimulation beyond the clear lymphoid hyperplasia response.
Therefore, it remains unanswered whether the host response is not problematic or potentially
similar to what might be seen with exposure to certain nickel compounds. Clearly, the longer-
term disposition of the lung inflammatory response is highly dependent upon the underlying
nature of the lymphoid response via antigenic stimulation. For this reason, long term immune
effects resulting from these exposures are simply unknown and were not addressed within this
specific protocol.
Jie Liu. Ph.D.
All the data are carefully analyzed and appropriated interpreted. The major conclusions are
supported by the data obtained, and no contradictory is evident. Thus, inhalation exposure of rats
to eerie oxide at doses up to 100-fold the TLV value (500 mg/m3) for 6 hours/day, 5 days a week
for 13 weeks did not produce neurotoxicity, did not affect liver and kidney function or
reproductive organs. The notable changes were the increased lung weights with pigment
accumulation in a dose-dependent fashion following subchronic eerie oxide inhalation exposure.
Pigment accumulation was also evident throughout the respiratory tract, and the immune organs
(spleen, liver, lymphonode, and likely in thymus) with slight organ enlargement at the higher
doses. All these changes could be envisioned as the adaptive mechanism to eerie oxide exposure,
and the "threshold" to produce overt pathology changes is perhaps not reached. In addition,
segmented neutropil counts were elevated in females at all doses, and in males at the higher
doses. The significance of such an increase is not clear, but may also be related to immune
reaction to eerie oxide exposure.
Mark Noble, Ph.D.
There is clear evidence of inhalation toxicity of eerie oxide. While the authors contend this is not
evidence of direct toxicity, that is solely because experimental design was not such as to
distinguish between direct and indirect effects. Moreover, it is of concern that changes of both
hyperplasia and metaplasia were observed. While it is stated that "it is probable that the observed
findings would be reversible following an appopriate recovery period," no evidence is provided
to support such a claim.
While it is stated (page 39, document page 31) that "the presence of lymphoid hyperplasia in the
lymph nodes and lungs is consistent with antigenic stimulation by the compound," it is also
consistent with more adverse effects. Of particular concern in this regard was the metaplasia seen
in the larynx.
While it was suggested that the lymphoid hyperplasia could be interpreted as antigenic
stimulation by the compound, no tests on whether a T- or B-cell response is initiated were
offered. This is of particular importance in the context of repeated exposure. If this is a
substance that is allergenic, this would be of great concern in raising the possibility that reactions
would become more serious over time. This critical question was not addressed.
While it was reported that "the pulmonary change was remarkable for the somewhat contained
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inflammatory response despite the copious amount of pigmented material present" but was a
finding "compatible with the minimal clinical signs noted" it is striking how discussion of
worrisome data is consistently skewed as being of no significance when a more negative
interpretation of the data is in fact more reasonable.
In he larynx, at the low exposure levels already 3/15 animals showed evidence of metaplasia,
increasing to 9/15 and 13/15 in moderate and high dose respectively (pg 263). Hyperplasia was
seen in bronchial and mediastinal lymph nodes in almost all animals, even in the lwo exposure
group (pg. 267, 275). The laryngeal metaplasia was interpreted as being adaptive to the insult
and "can be reversible." Once again, however, no observations are supplied to support this view
and the data is automatically interpreted in favor of the there being no cause for concern.
David B. Warheit, Ph.D.
In general, the conclusions were supported by the data. Comments on the experimental design
and characterization of the test material are detailed above.
5. In your opinion, was this investigation properly planned, conducted, and reported? Are
there any procedures, observations or analyses that would have added to the quality of this
investigation ?
R. R. Dietert. Ph.D.
The study was well-conceived and conducted with care. The overall group summaries do reflect
the underlying individual observations. In general, the conclusions are fully supported by the
data with the possible exception of some speculation regarding outcomes that would extend
beyond the timeframe of the direct observations. The study employed appropriate assessment
tools available at the time of the investigation. This reviewer would consider the study protocol
and subsequent results to be highly reliable to the extent parameters were examined.
If the study were conducted today, assessment measures would certainly be different. For
example, with compounds such as cerix oxide, neurobehavioral assessment might well include
parameters associated with learning and memory. Even some histological assessment would
probably employ more quantitative measures using imaging equipment and software programs vs.
qualitative subjective scoring. Additionally, it is likely bronchial-alveolar lavage would be
collected for animals and analyzed for inflammatory cell profiles as well as cytokine profiles.
This would provide a much better indication of the nature and potential changes in lung
inflammation during the course of exposure. More information concerning the extensive
lymphoid hyperplasia would be important as well. At present, little can be concluded relative to
potential immunotoxicity based on the parameters examined.
In summary, the study appears to have been well conceived and the conclusions should be
reliable. The utility of the findings is somewhat limited because of the historical nature of the
study. It was performed more than a decade ago and, in some cases, used less sensitive
assessment tools for neurobehavioral toxicity and immunotoxicity than are available today.
Jie Liu. Ph.D.
In summary, this 90-day subchronic study was well planed, professionally performed, detailed
documented, and appropriately reported.
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Mark Noble, Ph.D.
These studies raise serious concerns about potential toxicity of eerie oxide that need to be
addressed with more precisely targeted investigations. Information that would seem of particular
interest in establishing a better foundation for appropriate regulation of eerie oxide is as follows:
a.	How long does the inflammation last? Considering the known data (discussed in Section
B) on clearance of eerie oxide, it is possible that the changes in induced are not readily
resolved. Proper evaluation of eerie oxide toxicity requires information on longer
exposure times (as individuals in the industrial environment, in particular, are likely to be
exposed for periods far longer for 15 days) and also on effects that mayh occur at delayed
time points after exposure ceases.
b.	Are the hyperplastic and neoplastic changes seen indications of carcinogenic potential of
exposure? When one observes such morphological changes in tissue structure, the issue
of transition to a neoplastic phenotype becomes of primary concern. The question of
carcinogenic potential could be addressed in several ways. For example, inhalation
exposure in one of the multiple mice strains that harbor a cancer-predisposing gene
(generically referred to as "Onco-mice") could be used to provide a test of carcinogenic
potential. In the present era, one could also conduct microrarray studies if a sufficient
proportion of cells in the tissue are affected (or one could use laser-capture to cut out
affected regions and conduct microarray analysis on mRNA amplified from these
regions) to look for changes that would be indicative of neoplastic initiation and/or
progression.
c.	How do effects change with repeated exposure? It is suggested that the inflammatory
changes seen are associated with stimulation of an immune response. If this is the case,
then exposing animals for 15 days, resting them for a month, and then re-exposing them
would be associated with a more dramatic response in the second exposure trial. Due to
the large number of individuals in the population with asthma, the question of whether
eerie oxide can stimulate an immune response that might trigger asthmatic attack is
considered of great concern. (In this respect, one is also concerned with the question of
what effects eerie oxide exposure, in general, might have when combined with another
inflammatory insult.)
d.	How do results differ in different strains? It has become absolutely clear that different
strains of experimental animals provide very different outcomes in situations of toxicant
exposure. One example of such differences is in the effects of methylmercury and
thimerosal on different mouse strains. In the SJL strain it is very easy to detect adverse
neurological effects of exposure to these substances at environmentally relevant levels,
while in other strains of mice effects may be more limited. It is therefore recommended
that studies need to consider this possibility, as choosing a particularly healthy strain of
laboratory animals may provide a false sense of security (particularly in the context of the
controlled diet of such animals, as discussed below).
As breeding of SJL mice is problematic, a compromise strain that appears to be
readily susceptible to chemical insults would be C57B1/6. As most rat strains are not truly
syngeneic (as determined by the need for immunosuppression when carrying out
transplantation experimentation), this introduces another problem in interpretation of
toxicological studies. The one strain of rats that is sufficiently inbred to allow cell
transplantation without use of immunosuppressive drugs (the most rigorous test of
whether animals are truly syngeneic) is the Fisher 344 strain.
e.	Combined insults: All toxicological insults in the real world represent combined insults.
Some combinations that are relevant are combinations of environmental toxicants, which
are well-established to cause different outcomes when applied in combination than when
they are applied alone. For example, recent studies have shown that the combination of
paraquat and maneb exposure in the rat produces CNS damage like that seen in
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Parkinson's disease, while exposure to either agent alone does not have such effects.
Other co-exposures need to be thought of in the context of the exposure itself. For
example, exposure via diesel emissions might have different interacting factors than
exposure in the context of glass polishing.
f.	Dietary effects: In the context of industrial or ambient exposure to eerie oxide, dietary
contributions may be of particular concern. The diets used for care of animals are often
far superior to diets in human populations, in containing large amounts of fibre and
vitamins, no refined sugars, and little or no trans-saturated fatty acids of the sort available
in "junk food." As vitamin deficiencies, hyperglycemia and trans-saturated fatty acids all
are known to cause oxidative stress to cells, any or all of these dietary problems could
exacerbate the effects of exposure to a physiological stressor. For example, it has been
shown that exposure to omega-3 fatty acids improve the healing response to spinal cord
injury, while omega-6 fatty acid exposure actually worsens outcome (King et al., (2006)
J. Neurosci., 26:4672-4680). For a more general review of the role of diet in
neuroprotection, see, e.g., Mattson et al. (2002), Physiol. Rev. 82:637-642).
g.	Particle size: It has become very clear that nano-particles can exhibit physicochemical
properties very different from larger particles made out of the same substance, and may
in fact be more reactive. As fine particulates will contain a contribution from nano-
particles, and the use of nano-scale eerie oxide is already increasing (and can be predicted
to increase still further), it is felt to be important to implement studies that allow the
comparative properties of particulates of eerie oxide and nano-scale particulates of eerie
oxide to be directly compared as the nano-scale particulates may be more toxic than those
examined in the submitted studies. In these studies, concerns regarding strain
differences and dietary contributions also pertain.
David B. Warheit. Ph.D.
In general, the conclusions were supported by the data. Comments on the experimental design
and characterization of the test material are detailed above.
Other comments
Jie Liu. Ph.D.
This 90-day subchronic inhalation toxicity study of eerie oxide aerosol was well-designed, nicely
performed, and all the procedures and results were very detailed reported. The data obtained
were sound and appropriately analyzed. The conclusions reached are solid. There is no similar
cerium toxicity and neurotoxicity study following inhalation exposure available in the literature,
and this work would add to our understanding of toxicological profile of cesium.
Mark Noble. Ph.D.
A. Summary
This is a toxicity study on a hydrolytically stable high molecular weight cationic polymer of eerie
oxide. Testing was via inhalation for 13 weeks in groups of 15 male and female rats. Animals
were examined monthly for signs of toxicity, neurological testing (functional observational
battery and motor activity testing), standard laboratory tests (hematology, clinical biochemistry,
urinalysis), ophthalmoscopy and gross pathology.
This study was completed in 1994.
This study provides sufficient evidence of eerie oxide toxicity in the lungs as to provide cause for
concern. There are also multiple examples of flaws in the study design. These include the use of
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tests that would not readily detect subtle effects and/or have standard deviations so large that
recognition of anything other than catastrophic results might not be possible. Moreover,
evidence of adverse effects is consistently interpreted with a bias towards concern not being
necessary, despite the fact that no data is presented to obviate the concerns raised.
Even with the flaws in this study, the evidence indicating a need for concern is readily apparent,
and needs to be addressed by further experimentation. Specific recommendations for
experimentation to resolve critical issues are offered, along with discussion of the flaws in study
design.
B. Background
There is an increasing recognition that substances believed to inert, or otherwise toxicologically
benign, actually may have significant adverse effects on the organism. One of the most recent
examples of such a change in our understanding of the toxicological potential of a widely used
compound is the 4-carbon molecule diacetyl, which is used to provide the aroma of butter in
microwave popcorn and other baking uses. For many years of usage, no toxicological effects
were reported. More recently, however, it has become clear that inhalation exposure to diacetyl,
at least in industrial settings, causes extensive and very severe lung damage.
B.l. Ceric oxide:
The following information is summarized from the Chemical Information Profile for Ceric Oxide
[CAS No. 1306-38-3] prepared in February 2006 for the National Toxicology Program by
Integrated Laboratory Systems, Inc. (Research Triangle Park, NC) under Contract No. N01-ES-
35515. Information provided in this previous report is considered highly pertinent to analysis of
the present report. Detailed references for all of the following information is provided in this
report, which is available at http://ntp.niehs.nih.gov/files/Ceric_oxide2.pdf.
Ceric oxide is used in an increasing variety of industrial applications, including petroleum
refining (catalytic cracking catalysts), glass products, polishing powder, ceramics, crystals (e.g.,
for lasers and garnets), phosphors, automotive catalytic converters, as an additive to promote
combustion of diesel fuels, as a pigment in dermatological preparations, and in nanoparticulate
form as a carrier for otic and ophthalmic compositions. Projected uses include usages in
cosmetics and lipsticks, as a matrix for anti-bacterial or bacteriostatic compositions. There has
also been developing interest in nanoscale ceric oxide, which is being used as a fuel additive for
diesel powered vehicles to increase fuel efficiency. Nanoparticles of ceric oxide are also used in
the semi-conductor industry in chemical-mechanical polishing/planarization. Proposed
nanoparticle usage includes as catalysts for chemical scrubers, nanoparticle coatings, as
components of fuel cells, in electro-optical devices and even as an additive to cigarettes that emit
low sidestream smoke.
Current exposure is not limited, by any regulations other than as might relate to exposure to
particulates that are not otherwise regulated. These values are 10 mg/m3 time weighted average
(TWA) inhalable particulate containing no asbestos and <1% crystalline silica; 3 mg/m3 TWA
(respirable particulate); OSHA PEL: 15 mg/m3 TWA (total dust) and 5 mg/m3 TWA (respirable
fraction)
B.2. Occupational Exposure to ceric oxide
NIOSH considered ceric oxide exposure in a report from 1981-1983.. At this time it was
suggested that 25,130 workers (13,436 females) were potentially exposed to Ce02 in three
industrial categories and 11 job categories including electrical and electronic technicians (17,417
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[8,599 females]); grinding, abrading, buffing, and polishing machine operators (2,946 [2,448
females]); and optical goods workers (2,188 [2,448 females]). Exposure was confirmed by
autopsy of workers with and without pneumoconiosis and workers who used carbon arc lamps or
lens grinders.
B.3. Exposure to ambient particles
Data from the Health Effects Institute (HEI, 2001) suggests that there may be an increase in
ambient eerie oxide particles in air (currently approximately 1 ng/m3) in areas of high traffic to
levels of >l(ig/m3. Diesel engine emissions are a particular source of interest due to the use of
eerie oxide as a fuel additive, and emission factors for fuel containing 100 ppm Ce02 have been
calculated to be 0.3 to 3.3 mg/km, depending on vehicle type. In tests with a Pt/Ce catalyst (0.5
ppm Pt and 7.5 ppm Ce in the diesel fuel), filtered emissions had 4.7 |ig Ce/bhp-hr and 1.1 fig
Pt/bhp-hr (brake-horsepower hour). It is estimated that by 2010 cerium emissions from use of
diesel fuel in the European Union could total 1.3 million pounds annually (worst-case scenario:
22 million pounds).
Another source of eerie oxide emissions is municipal and hospital waste incineration. The mean
cerium concentration found in bottom ash from incineration of various wastes was: food scrap -
8.57 ppm, animal waste - 23.5 ppm, horticultural wastes - 27.3 ppm, sewage sludge - 35.4 ppm,
and municipal waste - 24.6 ppm.
In a subchronic inhalation toxicity study of microscale eerie oxide, there were significant
increases in lung weights, concentration-related metaplasia of the larynx, and alveolar epithelial
hyperplasia for mid- and high-dose male and female rats. The human equivalent Lowest
Observable Effect Level (LOEL) derived from this study was 1 mg Ce/m3. Using data from this
study and an uncertainty factor of 3000, a human equivalent Reference Concentration (RfC) of
0.3 (ig Ce/m3 was developed (TERA, 1999). Intratracheal instillation of fine particles of Ce02
(size not given) induce primary lung lesions (i.e., pulmonary fibrosis and alveolar proteinosis and
granulomas) but coarse particles did not.
No toxicological studies of nanoscale eerie oxide are available, and there are no adequate studies
in mice for evaluating the potential inhalation hazard of either nanoscale or microscale eerie
oxide. Given the concern that nanoscale metal oxides may be more toxic per unit mass than
microscale metal oxides (due to the larger surface area per unit mass), the toxicological potency
of nanoscale eerie oxide may be greater per unit mass. Consequently a human equivalent RfC for
nanoscale eerie oxide may be considerably lower than 0.3 |ig Ce/m3 and far lower than that
predicted to occur from the use of nanoscale eerie oxide as a diesel additive.
B.4. Animal Studies:
Several interesting pieces of information pertinent to analysis of eerie oxide are available from
previous animals studies, including the ones that are the subject of the present analysis. This
information is contained in the February 2006 Chemical Information Profile for Ceric Oxide
(Supporting Nomination for Toxicological Evaluation by the National Toxicology Program). The
Chemical Information Profile included the statement that, for both sexes, "statistically significant
lung weight increases, concentration-related metaplasia of the larynx, and alveolar epithelial
hyperplasia for mid- and high- dose; pigment accumulated in lungs at all doses; and significant
increase in lymphoid hyperplasia in bronchial lymph nodes that correlated with pigment volume."
Based on previous studies, a No Observed Adverse Effect Level (NOAEL) of 0.41-0.43 mg
Ce/m3 was determined for alveolar epithelial hyperplasia and of 0.55 mg Ce/m3 for increased
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lung weight. Critically, the LOAEL (i.e., the lowest amount or concentration of an agent, found
by experiment or observation, that causes an adverse alteration of morphology, functional
capacity, growth, development or life span in an organism, system or (sub)population) was only
marginally higher, and was suggested as 0.85 mg/m3 in males and 0.82 mg Ce/m3 in females
(equivalent to -1.0 mg Ce02/m3).
What is important about the above information is that it indicates defining 5 mg/m3 as low
exposure and 50 mg/m3 as moderate exposure, as in the studies under review, appears to be
excessive in respect to suggested calculated LOAEL levels.
Other potentially pertinent information that has emerged from animal studies is that:
-Repeated doses increased retention half-times.
-	Intratracheal administration of coarse particles did not induce lesions in rat lung, but finer
particles did (size specifics not provided). Typical lesions were pulmonary fibrosis, alveolar
proteinosis and granulomas.
B.5. Human Toxicity:
Studies on human toxicity are relatively rare. RE pneumoconiosis has been reported in several
case studies of workers exposed to eerie and other RE oxides via inhalation, but these workers
were also exposed to other particulates. Other rare observations have also been made, of which
the most pertinent seems to be a report from Russia on children near a phosphate fertilizer plant
in Russia, who were exposed to cerium concentrations decreasing from 10.2 ng/m3 at 200 m to
3.6 ng/m3 at 2500 m were 1.5 times more likely to have respiratory diseases, chronic
inflammation of the tonsils, etc. (Volokh et al., 1990; PMID:2169646).
What has emerged as of particular interest from previous studies is that the less-soluble forms of
inhaled cerium (e.g., eerie oxide) may remain in the lung and lymph nodes for years. Cerium
deposits were found in the alveoli and interstitial tissue of an optical lens grinder 20 years after
exposure to Ce02 powder particulates (<1 - 10 |im): some cerium deposits were also found inside
the cells.
B.6. Other information of potential interest:
-	When inhaled as Ce02, cerium precipitates in the lysosomes of alveolar macrophages as
insoluble phosphates in fine needles or granules.
-	Lung clearance rate is measured in years.
-	Approximately 10% of absorbed cerium is excreted in the feces and urine with retention of 45%
in the liver, 35% in the skeleton, and 10% in other organs (primarily, the spleen and kidneys).
-	Ln3+ ions (which have the same valency shell as cerium) are well known inhibitors of Ca2+-
dependent physiological processes such as those involved in blood clotting (e.g., prothrombin
activation) and neuronal and muscular functions. It has been shown that trivalent cerium
compounds inhibit active transport of Ca2+ through mitochondrial membranes, calcium and
potassium channels, calcium-dependent hemolysis in burn patients, calcium- dependent enzymes,
and contractility in cardiac, skeletal, and smooth muscle (e.g., intestinal) (Jakupec et al., 2005;
PMID: 15674649).
-	Possible connection between cerium toxicity/magnesium deficiency and endomyocardial
fibrosis has been reported (Brown et al., 2004; PMID: 15275858; Eapen et al., 1996;
PMID:8720088). The combination has promoted fibrogenesis in rat heart (Kumar et al., 1996;
PMID:8694866). Cerium had an inhibitory effect on protein synthesis in cultured cardiac
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myocytes and lung fibroblasts exposed to normal- and low-levels of Mg2+ (Shivakumar and
Nair, 1991; PMID: 2051999).
C. Evaluation of the current submission:
C.l. Summary of effects noted in the submission:
No effects were seen at a gross level, but higher lung weights were seen in the two higher dose
levels of 50 and 500 mg/m(3). Lungs in all dose groups had pale areas macroscopically. There
was pigment accumulation at all doses levels with alveolar hyperplasia. All treatment groups
showed pigment accumulation in the nasal cavity, and the two higher doses levels showed
pigment accumulation and hyperplasia. The highest dose showed pigment accumulation in liver
and spleen.
Notably, bronchial lymph nodes were enlarged macroscopically at all dose levels in males and the
two higher doses in females, and bronchial and other lymph nodes showed pigmenta
accumulation and hyperplasia at all dose levels. The larynx of animals in all treatment groups
showed pigment accumulation and metaplasia.
In addition, there were higher neutrophil counts in all dose levels in females and the two higher
dose levels in males.
Pigment was thought to be evidence of failed clearance of the eerie oxide. That pigment was also
found in the spleen and liver, it is apparent that respiratory exposure was associated with
distribution in non-respiratory sites.
David B. Warheit, Ph.D.
Reviewer's notes-
A 13-week inhalation toxicity and neurotoxicity study by nose-only exposures of a dry
powder aerosol of eerie oxide in the albino rat.
Fours groups each - 15 males and 15 females
0, 5, 50 and 500 mg/m3 6 hr/d 5 d/wk for 13 weeks nose-only
Groups 1 (0) ; 2 (5 mg/m3); 3 (50 mg/m3) and 4 (500 mg/m3)
MMAD = 1.8-2.2 jim
Body weights
Food consumption - weekly
Behavioral testing
Functional observation battery (FOB)
Activity levels?
hematology
clinical biochemistry
urinalysis
ophthalmological exams
gross pathology
histopathology
Results
• No clinical signs
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•	Overall body weight gain and food consumption of Group 4 was marginally inferior to
controls - treatment related
•	Treatment related effects in hematology were observed as higher segmented neutrophils
counts in Group 2 females and Groups 3 and 4 of both sexes at 6 and 13 weeks
•	No behavioral changes
•	A trend for higher lung weights of males and females from Groups 2,3, and 4, and higher
spleen weights of males from Group 4, were considered to be related to eerie oxide
treatment
•	Macroscopically, pertinent changes in the lungs - such as discoloration or pale areas (30
each in Groups 3 and 4), pale foci (4 animals in Group 2), and uncollapsed parenchyma
(30 in Group 4 and 2 in Group 3). In addition, enlargement and/or pale discoloration of
the bronchial lymph nodes (30 in Groups 3 and 4, and 28 in Group 2) and mediastinal (20
in Group 4, 18 in Group 3, 12 in Group 2 and 1 in Group 1) and pancreatic lymph nodes
(3 in Group 1, 1 each in Groups 3 and 4) were primarily evident in animals treated with
eerie oxide and considered to be induced by the test material.
•	Histopathologically, pigment accumulation and/or alveolar epithelial/lymphoid
hyperplasia in the lungs (30 each in Groups 2,3, and 4), lymphoid hyperplasia of the
bronchial (30 each in Groups 3 and 4, 24 in Group 2), mediastinal (18 each in Groups 3
and 4, 12 in Group 2) and pancreatic (1 in Group 3) lymph nodes, metaplasia and/or
pigment accumulation ion the larynx (22 in Group 4, 16 in Group 3, and 9 in Group 2),
and pigment accumulation in the bronchial lymph node (30 each in Groups 3 and 4, and
27 in Group 2), nasal cavity (30 in Group 4, 26 in Group 3 and 15 in Group 2), bronchi
(30 in Group 4, 9 in Group 3 and 1 in Group 2), trachea (28 in Group 4, 2 in Group 3),
mediastinal lymph node (18 in Group 4, 17 in Group 3 and 12 in Group 2), liver (11 in
Group 4), mandibular lymph node (12 in Group 4); spleen (9 in Group 4) and pancreatic
lymph node (1 each in Groups 3 and 4) were considered to be induced by the test
material.
•	Conclusions —
•	An overall no-effect level cannot be established based on marginally reduced body
weight gains recorded in Group 4 animals?, as well as other changes recorded in treated
groups and including increased segmented neutrophil counts, higher lung and spleen
weights, discoloration of the lungs and discoloration/enlargement of lymph nodes.
•	Histopathologically, pigmented material accumulation in the lungs, bronchial,
mandibular and mediastinal or pancreatic lymph nodes, trachea, bronchi, larynx, nasal
cavity, liver and spleen, as well as alveolar epithelial hyperplasia (lungs), metaplasia
(larynx) and lymphoid hyperplasia (bronchial and mediastinal or pancreatic lymph nodes,
lungs) were seen in all treated groups. Systemic dissemination of the test article was
evident in Group 4 animals.
Results
Clinical signs - not significant
Body weights
• Statistically significant lower mean body weight gains were recorded in high
dose males vs. controls in weeks 2 and 8.
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•	The overall body weight gain of Group 4 was marginally inferior vs. controls -
not statistically significant
•	Statistically significant lower mean body weight gains were recorded in Group 2
females at week 7 and Group 2,3 and 4 females at week 10 vs. controls
Food consumption - NS
Ophthamalogy - NS
FOBs - No clear behavior changes were observed between the control and treated groups
for qualitative assessment at any of the time points measured. However, a significantly
reduced forelimb grip strength was recorded in Group 4 females at week 13.
Motor Activity - NS
Hematology
•	Treatment-related effects were observed in segmented neutrophil counts in
Group 2 females and Groups 3 and 4 of both sexes.
•	Statistically significant elevated segmented neutrophils counts in Group 2 and 3
females and Group 4 animals - recorded at weeks 6 and/or 13 vs. controls.
Clinical Biochemistry - NS
Urinalysis - NS
Terminal Studies
Organ weights
•	Treatment-related effects were seen in lung and spleen weights when expressed
as absolute or relative to organ and brain weights.
•	Higher lung weights were statistically significant in Groups 3 and 4 vs. controls
•	Higher spleen weights (relative to body wt) were stat significant in Group 4
males vs. controls
•	Spleen and lung weight changes correlated with gross and/or microscopic
findings and were considered to be treatment-related.
•	Significantly higher thymus wt recorded in Group 3 males vs. controls - not
considered to be treatment-related.
Histopathology
• Histopathologically, pigment accumulation and/or alveolar epithelial/lymphoid
hyperplasia in the lungs (30 each in Groups 2,3, and 4), lymphoid hyperplasia of the
bronchial (30 each in Groups 3 and 4, 24 in Group 2), mediastinal (18 each in Groups 3
and 4, 12 in Group 2) and pancreatic (1 in Group 3) lymph nodes, metaplasia and/or
pigment accumulation ion the larynx (22 in Group 4, 16 in Group 3, and 9 in Group 2),
and pigment accumulation in the bronchial lymph node (30 each in Groups 3 and 4, and
27 in Group 2), nasal cavity (30 in Group 4, 26 in Group 3 and 15 in Group 2), bronchi
(30 in Group 4, 9 in Group 3 and 1 in Group 2), trachea (28 in Group 4, 2 in Group 3),
mediastinal lymph node (18 in Group 4, 17 in Group 3 and 12 in Group 2), liver (11 in
Group 4), mandibular lymph node (12 in Group 4); spleen (9 in Group 4) and pancreatic
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lymph node (1 each in Groups 3 and 4) were considered to be induced by the test
material.
• A no-effect level was not present in Groups 2, 3 and 4, and systemic dissemination of the
test article was evident in Group 4 animals. A dose-related effect of the compound was
clearly evident.
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