ORD Carpet Study
Toxicology Report:
Evaluation of Off-Gassed Carpet Sample Atmospheres
Submitted: August 6, 1993
Documents Included Pages
1. Executive Summary i-vi
2. EPA Toxicology Study EPA-1 - EPA-115
3. Anderson Labs Toxicology Study AL-1 - AL-84
4. Peer Review Comments on EPA Study and Anderson Study PR-1 - PR-27
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EXECUTIVE SUMMARY
Toxicology Report:
Evaluation of Off-Gassed Carpet Sample Atmospheres
August 6, 1993
Background
During the summer of 1992, through news releases, and in October, 1992 at a Senate hearing,
Anderson Laboratories, Inc. released information describing neurotoxicity, pulmonary irritation
and death in mice exposed to emissions from certain carpets. The information released
described tests which had four primary features. First, an exposure feature, which involved
placing carpet into an aquarium, heating the aquarium, and withdrawing air from the aquarium
into a chamber from which four mice received their breathing air. The second feature involved
modification of a standard test of sensory irritation (ASTM E 981). The standard test measures
the rate at which the mice breathe during exposures. Research has shown mat chemicals which
humans find irritating (sensory irritation), when present in sufficient concentration, decrease the
rate at which mice breathe. Further, certain chemicals, when present in sufficient concentration,
change the pattern of breathing in a way that can be interpreted to indicate pulmonary irritation.
Pulmonary irritation is considered to be more serious than sensory irritation. The standard
ASTM method was modified to include multiple exposure periods rather than a single exposure.
The third feature of the Anderson tests involves observation of the behavior of the animals after
completion of the ASTM test This behavior is scored by an observer and interpreted as an
indicator of potential neurotoxicity. The fourth feature of the Anderson tests is the observation
that death occurred in some mice. These descriptions of neurotoxic effects and death were
particularly striking because effects of this magnitude are not consistent with our current
knowledge of the emissions from carpets, and have not been described in the peer reviewed
scientific literature.
The potential public health significance of these findings prompted a vigorous effort by EPA,
with assistance from the Consumer Product Safety Commission, to evaluate their scientific
reliability, by determining whether the findings could be replicated independently. Aside from
being one of the cornerstones of science, independent replication in EPA laboratories is necessary
before subsequent studies can be done to determine the causative agent(s) and the relevance of
the observations for public health. This report describes the EPA effort to replicate independently
the Anderson Laboratories' findings. The report also includes Anderson Labs' report of their
data from the concurrently collaborative effort Comments on these two efforts provided by four
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independent peer reviewers are included with this document as well.
STUDIES bv EPA's Office of Research and Development (ORD)
EPA is aware that carpet, along with other indoor products and some indoor environments in
general has been associated with a variety of complaints, symptoms and signs. Based upon our
knowledge to date, we have no reason to believe that the nature of complaints associated with
carpet is different from the nature of complaints associated with other indoor sources. In order
to establish a firm relationship between indoor sources and complaints, EPA must have available
methods which are objective, sensitive, reliable and valid for this purpose. Methodologies which
fit these criteria are not yet available. For this reason, the EPA's indoor air research program
is identifying, developing, improving, and validating methodologies which can be used for this
purpose. It is in the context of this research program that EPA began evaluating the ASTM E
981 method some time ago. Although we concluded, in a peer-reviewed paper published in the
journal Indoor Environment last summer, that the value of ASTM E 981 for purposes of
evaluating indoor air quality is currently limited, we nevertheless had planned to further evaluate
the methodology as used by Anderson Laboratories prior to the reports of toxic effects of carpet
We were also in the process of working with other tests when the Anderson Laboratories'
information came to our attention. Given the potential significance of Anderson's reports, we
dedicated ourselves to investigating the four features of the Anderson Laboratories' testing
described above.
ORD's goal was to understand the toxic effects reported by Anderson Laboratories. Before
beginning to understand those effects, it was necessary to be able to reproduce them in our
laboratory. The process by which this is done is called independent replication. Independent
replication is also necessary to establish that a scientific finding is reliable. A scientific finding
is reliable when it can be reproduced repeatedly by scientists who are given the protocol and the
necessary equipment
From the beginning, ORD took these studies very seriously. In addition, we remained highly
confident throughout, and had every reason to believe that we would be successful in our effort
to independently replicate the Anderson Laboratories' findings. Anderson Laboratories had
indicated that their findings were reliable in their own laboratory, in that their testing of many
different carpets revealed toxic effects. ORD assembled a large team of highly skilled scientists
working on mis project, and we maintained a collegial relationship with Dr. Anderson throughout
Even more to the point, part of the study team visited Anderson Laboratories in January, 1993,
and not only witnessed a toxic exposure, but were able, using part of our apparatus and part of
theirs, to produce lethality as well There neither was, nor is, any doubt in the minds of the ORD
team of scientists that these studies in Anderson Laboratories resulted in death to some mice.
The bottom line from our studies, however, is that despite our best efforts, which were
considerable and which will be described below, we have not been able to independently replicate
the severe toxicitv described bv Anderson Laboratories. In fact we were not able to produce
any convincing signs of even mild toxicitv attributable to carpet in our tests. Our present
• •
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conclusion is that there must be an essential difference between the conditions of our experiments
and those of Anderson Laboratories, which, despite our efforts, we have not been able to identify.
Following is a more detailed description of our efforts. First, the steps we took in our attempt
to perform an independent replication of the Anderson Laboratories' observations will be
outlined. Second, the scientific practices ORD followed during the course of these studies will
be identified. Third, the results of our studies will be summarized and contrasted with the
Anderson Laboratories' results. Finally, the results of an independent peer review of the ORD
replication study will be presented.
The steps ORD took in an attempt to perform an independent replication of the Anderson
Laboratories' observations included assembling a highly skilled scientific team, assembling the
test apparatus, performing exploratory/pilot/shakedown studies, drafting a formal protocol,
reciprocal visits with Anderson Laboratories' personnel, peer review of our study protocol,
performing the formal replication study, analysis and peer review of the study results, performing
additional exploratory studies, and initiation of a dialogue with industry scientists performing
similar work.
The scientific practices ORD kept in focus during these studies were those of replication, blind
testing when subjective measurements are involved, peer review, quality assurance, and inclusion
of measurements which should help develop testable hypotheses to account for the observed
findings. The importance of replication has already been mentioned. Scientists only accept
cause-effect relationships when they can be demonstrated reliably by independent scientists.
High caliber science maintains high quality several different ways. Many scientific observations
require subjective judgment or subjective scoring, particularly in the realm of biology. Because
scientists recognize that whenever subjective evaluations are made, there is an unconscious
tendency for expectations to color observations, good scientific practice requires that critical
subjective observations be made by an individual who is not aware of the specific conditions of
the test This practice is called blinding, and hi the case of these studies refers to the fact that
the individual performing scoring should not know whether the data collected were from carpet-
exposed mice or control mice. In ORD's formal replication, effort was made to ensure blinding
of the testing laboratories by asking the Consumer Product Safety Commission to collect carpet
and randomize shipment of carpet and no carpet to the test laboratories. At EPA we established
elaborate procedures to ensure maintenance of this blinding.
Peer review refers to the process by which outside experts provide independent comment on the
quality of a research design, the data collected and the interpretation placed upon the data. Most
scientific journals send articles to peer reviewers before they will consider publication.
Scientific progress is usually measured in terms of peer-reviewed publications, and EPA judges
the performance of its scientific staff in large measure by the peer-reviewed publications they
write. The process of publishing peer-reviewed papers is a lengthy one, and when EPA must act
before important data can be published in the peer-reviewed literature, independent peer reviews
are held. In the case of our carpet study, we believed that public concern was sufficient that we
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could not wait for our studies to appear in the peer reviewed literature before they became
known. Therefore we held independent peer reviews of both our research protocol, to ensure that
the scientific design was of high quality, and of our research findings, to ensure that our data
were appropriately collected and interpreted. In addition to planning for a peer review at the end
of our study, we also arranged to have independent quality assurance audits of our procedures
and our apparatus during the study. The purpose of these audits was to ensure that all systems
were properly calibrated and operating as expected.
Finally, since ORD's expectation was that efforts would be successful in replicating the Anderson
Laboratories' findings, some measurements were included which were designed to help develop
testable hypotheses that would account for the findings. Since it was presumed that emissions
from the tested carpets would account for the findings, evaluation of the emissions from the
tested carpets was included In addition, pieces from the carpets were analyzed directly, to
determine the presence of pesticides and microbiological contaminants.
A major feature of the ORD study was collaboration with Anderson Laboratories in its design
and execution. The protocol for performing the study was agreed to by both ORD and Anderson
Laboratories. Each Lab received pieces from the same carpet to test, and the toxicology testing
done routinely by Anderson Laboratories was included. Scoring procedures were discussed and
modified to accommodate the desires of both sets of investigators. In addition, ORD performed
a large number of other lexicological and analytical measurements. The analytical measurements
characterizing carpet emissions and contaminants will be detailed in a separate document
In this collaborative study, there were three treatments. Two treatments were different carpets,
and one treatment was a control. Each of these three treatments was tested twice, so there was
a total of 6 experiments performed. CPSC collected the carpets from sources which had
previously supplied carpets to Anderson Laboratories, and which had been associated with severe
toxicity and death when tested at Anderson Laboratories. CPSC randomized the 6 different
sample sets, and sent a set simultaneously to EPA and to Anderson Labs for blind testing. After
each laboratory had completed all 6 experiments, the code was broken for data analysis purposes.
On May 26, 1993, a peer review was held of the two data sets (EPA's and Anderson
Laboratories*). The peer reviewers had received draft reports from EPA and from Anderson
Laboratories several days previously. At the peer review, each laboratory described their study
and results, and the peer reviewers asked questions and discussed findings with study participants.
The two reports, one from EPA and one from Anderson Laboratories, make clear that mere was
virtually nothing in common between the two sets of findings. EPA found no deaths in 24 tested
animals, no severe or moderate sensory irritation, no severe or moderate pulmonary irritation, and
no clear evidence of neurotoxicity. By contrast, Anderson Laboratories' findings included a total
of 8 deaths in 24 tested animals, severe pulmonary irritation, and neurotoxicity.
During the course of preparing for and trying to understand these studies, EPA performed many
exploratory studies (over 30) in which ORD examined a large number of variables and tested
carpet samples supplied to us by Anderson Laboratories. Some of these studies were performed
with as much as 10 square feet of carpet in a specially designed source chamber. Some studies
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were done with very dry air, some with normal laboratory air, and some with partially humidified
air. Some of the studies allowed for observation of animals for many days after exposure, and
some of studies involved extensive heating of the carpet samples. In all, over 140 mice have
been tested by ORD. With all of these studies, the only evidence of carpet-related neurotoxicity
which we were able to produce occurred during our visit to Anderson Labs.
The science performed by EPA was characterized independently by each peer reviewers as of
very high quality, yet we were not able to replicate the Anderson Laboratories' findings. The
conclusion with which we are left is that very subtle but very important differences exist between
the studies done bv Anderson Laboratories and those done bv us.
NEXT STEPS
We do not believe that the failure to replicate the Anderson Laboratories' findings proves that
carpet emissions do not pose adverse health effects. At the same time, however, we do not have
a sound basis for concluding that exposure to carpet emissions presents a health risk. Rather,
we see two important issues. One is the meaning of the Anderson Laboratories' findings, and
the other is the potential health effects of exposure to carpet emissions. These may be
independent issues.
EPA will continue to follow up on the Anderson Laboratories' findings. The next step we will
take is to hold a workshop at which data and hypotheses from all of the laboratories working on
this problem will be presented and discussed. Based upon the outcome of this workshop, EPA
will determine its next steps.
In addition to following up the Anderson Laboratories findings, EPA/ORD intends to continue
its efforts to develop better methodologies for detecting and studying potential health effects of
indoor sources. While many toxicology tests are available, most of them were designed to
detect health effects which are very different from die type associated with most indoor air
complaints. Our general strategy is to develop methods which can be used in humans, to be
sure that they correlate with the symptoms and signs reported following exposure to some indoor
environments. Once we have good methods for use in humans, we will develop animal models
of those methods. Such models might be useful for screening purposes. Methodologies we are
currently working on include tear film breakup for eye irritation, and trigcminal evoked potentials
for sensory irritation. As we develop appropriate animal models, we can apply mem to answer
questions about the neurobehavioral effects that have been suggested by various scientists as
related to indoor air complaints.
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Table 1.
Summary of lexicological Findings
Endpoint
EPA
Deaths
Severe Neurotoxicity
Neurotoxicity (extreme
change from control)
Pulmonary
Irritation
Sensory Irritation
General Appearance
none
none
none
none
carpet- 3/16 slight;
control: 1/8 slight
carpet: facial swelling,
lacnmation, hemorrhaging of
pinna vessels, red tears;
control: similar effects
Anderson
8 of 24 (5 carpet, 3
accidental)
none
carpet: many variables
affected;
control: only vocalization
carpef 4/16 severe;
control: none
carpet 13/16 slight;
control: none
carpet: facial swelling,
lacnmation and bleeding, ear
petechiae;
control: ear petechiae
VOCs
4-PC
Pesticides
MicrobioL
Table 2.
Summary of Analytical Chemistry/Microbiology
- Unremarkable; most compounds in ppb range
- Detected, but too little there to quantitate
• One carpet had high levels of chlorpyrifos, but not high enough to
produce acute toxicity
- Unremarkable
VI
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lexicological Evaluation of Off-Gassed Carpet Sample Atmospheres
Submitted: Augusts, 1993
Disclaimer
The research described in this article has been funded wholly by the U.S. Environmental
Protection Agency (U.S. EPA) under contract 68-D2-0056 to ManTech Environmental
Technology, Inc. It has been reviewed by the Health Effects Research Laboratory, U.S. EPA
and approved for publication. Approval does not signify that the contents necessarily reflect
the views and policies of the Agency nor does mention of trade names or commercial
products constitute endorsement or recommendation for use.
Acknowledgements
The authors wish to thank the following participants in the study for their effort and dedication
to completing this task: Mette Jackson for exposure and pulmonary function testing; Pam
Phillips for behavioral testing; Sean Dowd for computer/bioengineering support; Mary Daniels
for heart puncture and blood preparation; Denise Sailstad for performing the orbital bleeds and
blood preparation; Judy Richards and Rick Jaskot for clinical chemistries on serum and
lavage; James Lehmann for peripheral smear, lavage, lung fixation, and cell differentials;
Terisita Gabriel for hemoglobin and carboxy/methemoglobin measurements; Joel Norwood for
nasal lavage and fixation; Elias Gaillard (EPL) for necropsy and histopathology evaluations;
Lynne Gates (EPL) for performing the necropsy dissections; and Donald Doerfler for
biostatistical analysis.
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Contributors listed in alphabetical order.
Daniel L. Costa
Pulmonary Toxicology Branch, Environmental Toxicology Division, Hearth Effects Research
Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
27711
Robert S. Dyer
Office of the Associate Laboratory Director, Health Effects Research Laboratory, U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Mark A. Mason
Indoor Air Branch, Pollution Control Division, Air and Energy Engineering Laboratory, U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Virginia C. Moser
ManTech Environmental Technology, Inc., P.O. Box 12313, Research Triangle Park, North
Carolina 27709
Jeffrey S. Tepper
ManTech Environmental Technology, Inc., P.O. Box 12313, Research Triangle Park, North
Carolina 27709
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Table of Contents
Tables Titles 4
Figure Titles 5
Abstract 6
Introduction 8
Background 8
Goals 12
Caveats 12
Methods 14
Definitions 14
Study Design 16
Experimental Test Procedure 17
Exposure System 19
Animals 24
Functional Observational Battery 25
Frequency of Breathing Measurements 27
Postmortem Evaluation 30
General Data Analysis Strategy 32
Quality Assurance 38
Results 42
Exposure System 42
Body Weight 46
Irritancy Measurements 46
Functional Observational Battery 51
Postmortem Evaluation 56
Discussion 65
Interpretation of the Results 65
Potential Confounds 74
Conclusion 77
References 80
Figure Legends 84
Figures 87
Appendix A: Summary of FOB Data for EPA Formal Replication Study 104
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Tables Titles
Table 1 Test Matrix Using Aquarium Source Chamber
Table 2 Mouse Pulmonary Function Checklist
Table 3 Temperature (°C) - Bottom of Aquarium (S2)
Table 4 Temperature (°C) - Inside of Aquarium (A,)
Table 5 Temperature (°C) - Inside of Mouse Chamber (Ag)
Table 6 Percent Relative Humidify of Air Entering the Source Chamber
Table 7 Percent Decrease in Frequency of Breathing for the Mean of Four Mice
Table 8 Summary of Statistical Outcomes for Functional Observational Battery Data
Table 9 Organ Weight Data by Treatment Group
Table 10. Peripheral Blood White Cell Differential Counts
Table 11. Serum Clinical Chemistry Values
Table 12 Lung Lavage Chemistry
Table 13 Lung Lavage White Blood Cell Count Differentials (xlO4) /ml of Bronchoarvedar
Lavage
Table 14. Incidence of Selected Histopathological Lesions
Table 15. Potential Causes of Clinical Chemistry and Differential Values
Table 16. Summary of Toxicological Findings
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Figure Titles
Figure 1 Design of the Exposure System for Evaluating the Emissions
Figure 2 Change in Body Weight with Four Sequential Exposures
Figure 3 Control Period Frequency of Breathing
Figure 4 Exposure Period (a) Individual Animal Data, (b) Exposure effect
Figure 5. Recovery Period (a) Treatment Effect, (b) Exposure Effect
Figure 6. Sensory Irritation, Exposure Period
Figure 7. Pulmonary Irritation, Exposure Period
Figure 8. Disruption Index, Periods Collapsed
Figure 9. Lacrimation
Figure 10. Dilated Pinna
Figure 11. Jar Task
Figure 12. Tail Pinch
Figure 13. Click Response
Figure 14. Alertness
Figure 15. Hemoglobin
Figure 16. Clinical Chemistries: (a) protein, (b) albumin, (c) cholesterol
Figure 17. Glutathione from Nasal Lavage and Lavage Fluid Protein
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Abstract
Anderson Laboratories, Inc. of Dedham, Mass, reported that the
off-gassing of certain "complaint" carpets caused sensory and pulmonary irritation, changes in
a checklist of neurobehavioral signs and, in the most extreme cases, death in exposed mice.
This study was performed in an attempt to replicate and further investigate these findings
primarily using two in vivo biological tests. One of these tests examines the reflex change in
the breathing pattern of mice exposed to irritant airborne chemicals (American Standard Test
Method for estimating sensory irritancy of airborne chemical [ASTM E-981-84]), and the other
is an adaptation of a standard test method for evaluating the neurotoxic potential of chemicals
(functional observational battery, Subdivision F Pesticide Assessment Guidelines-FIFRA).
These two primary measures were coupled with several ancillary measures and a postmortem
assessment in an attempt to ascertain the mechanism of toxicity, if observed. The
postmortem evaluation included measurements of hemoglobin, serum clinical chemistries
(liver, kidney and cardiac enzymes), blood and lung lavage white cell counts and differential,
organ weights, and a gross necropsy with a microscopic evaluation of all major organs. The
study evaluated three treatment groups comprised of two carpet exposures and one zero-
grade air exposure. The investigators were blinded to the treatment group. Matched carpet
samples were sent to the EPA laboratory and to Anderson Laboratories by the Consumer
Product Safety Commission (CPSC). Eight mice were tested with each of the three treatment
groups. There appeared to be no severe toxic effects associated with exposure to the off-
gassing of the two tested carpets. Incidental findings of statistical significance, but unlikely to
be of biological significance, were observed. Of all the effects that were observed, only the
number of falls in the functional observational battery would be suggestive of an "adverse"
effect Several "possibly adverse" effects in each of the three categories of measurements
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(irritancy, postexposure neurobehavior, and postmortem general toxictty screen) seemed to be
randomly distributed across the three treatment groups. Most likely, these effects are false
positives because of the numerous uncorrected comparisons performed. In conclusion,
based on this assessment of irritation, neurobehavioral effects, and measures from a general
toxitity screen, there is no indication that exposure to off-gassing from these two carpets
poses a serious lexicological threat.
EPA-7
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Introduction
Background Recently, Anderson Laboratories of Dedham, Mass, distributed data
indicating irritancy and toxicrty to mice exposed to emissions from carpets collected from sites
with a history of carpet-related complaints. An in vivo test (ASTM E-981-84) was used to
estimate irritancy in mice during exposure to air passed over warmed carpet samples in a test
chamber. Briefly, carpet pieces were sealed in an aquarium that was then heated until the air
temperature reached 37 °C. This air temperature was maintained for about an hour, after
which room air was pulled through the aquarium and then through the mouse exposure
chamber for one hour.
Measurements of changes in breathing rates and patterns were used to determine
irritancy of emissions. Postexposure observations of the behavior and general condition of the
mice using a list of clinical signs were used to assess correlated neurotoxicological effects.
Death was observed in some exposed mice after multiple (up to four) one-hour exposures.
No chemical or microbiological characterizations of the emissions from the exposure system
were conducted during the tests.
These observations raise concern that some carpet emissions may adversely affect the
health of exposed humans. Therefore, the EPA considered it important to (1) systematically
replicate Anderson Laboratories' experiments to provide independent corroboration of test
results; (2) apply the resources of the Health Effects Research Laboratory to perform a
comprehensive toxitity screen to leam, if possible, what factors were responsible for the
irritancy, neurobehavioral changes, and mortality observed in the test animals; and (3)
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conduct a thorough chemical and microbial characterization of emissions from carpets.
This report deals only with the toxicology portion of the EPA evaluation. The EPA and
Anderson Laboratories have completed a study in which the protocol required each laboratory
to test three treatment conditions: subsamples of the same two carpets and an air control
exposure. For these experiments, both the EPA and Anderson Laboratories were blind to the
contents of the source chamber (carpet or air control). Upon completion of the study the EPA
and Anderson Laboratories will exchange reports and compare results (the comparison will be
the subject of a separate report). The two carpets were independently collected from their
source by a third party (Consumer Product Safety Commission [CPSC]) for distribution to the
test laboratories. Both carpets have previously been tested by Anderson Laboratories and
were shown to produce severe toxicity in some test animals and death in others. The EPA
tested subsamples of these same carpets and a zero-grade air control using methods
specified by Anderson Laboratories. Additionally, postmortem evidence of toxicity in mice
was evaluated. The EPA also attempted to characterize the carpet chemical and
microbiological contaminants and emissions under test conditions (see companion
chemistry/analytical report).
Test Method (ASTM E 981-84) Initial reports from Anderson Laboratories indicated that
exposure to carpets could produce either sensory or pulmonary irritation, or a combination of
both. This determination was made using an adaptation of the American Standard Test
Method for Estimating the Sensory Irritancy of Airborne Chemical (ASTM E-981-84). This test
method provides a quantitative estimate of the sensory irritant potential of an inhaled
chemical and has been recently reviewed (Boz et al., 1992; Tepper and Costa, 1992). Irritancy
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is detected by a characteristic change in the breathing pattern of mice, which results in a
reduction in the breathing rate, during exposure to a test atmosphere. This characteristic
response in mice has been demonstrated to qualitatively predict nose, throat, and eye irritation
in humans for 51 chemicals (Alarie, 1973). A quantitative relationship has been established
between published Threshold Limit Values and Time Weighted Averages (TLVs-TWAs) for 26
irritant chemicals and the concentrations at which the same chemicals reduce respiratory rate
by 50% (RDeo) in mice. This statistical relationship indicates that 3% of the RDM can be
used to establish interim TLVs-TWAs, if toxicity is primarily based on sensory irritation (Alarie,
1981).
Although several types of irritant responses have been identified using ASTM E 981-
84, they are broadly classified as two types, sensory and pulmonary (Alarie, 1973).
"Sensory* (upper airway) irritation is usually produced by highly water-soluble chemicals such
as ammonia, acrolein, and formaldehyde. Sensory irritation is caused by stimulation of the
trigeminal nerve, which innervates the areas of the nose, throat, and eyes. In humans,
exposures to sensory irritants readily produce a burning sensation to the eyes, often
precipitating lacrimation. If the exposure is sufficient, the larynx also can be stimulated by
these chemicals, producing cough. Analogous sensory stimulation can be readily identified in
small laboratory mammals by a decrease in breathing rate (Alarie, 1973).
Pulmonary irritation, a second type of irritant response, is caused by stimulation of
vagal nerve endings and usually occurs with less water-soluble chemicals. Prototypic
examples are ozone (Oj), phosgene (COCy, and nitrogen dioxide (NO2), chemicals that have
greater peripheral lung deposition and thus, cause more damage in the deep lung.
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Subjectively in humans, these chemicals may produce cough and substemal soreness without
the burning sensation of the upper airways and eyes produced by the sensory irritants. In
mice exposed to these irritants, breathing rate again decreases, but the breathing pattern is
qualitatively different from the pattern of response seen with sensory irritation (Alarie, 1973).
Functional Observational Battery The initial results from Anderson Laboratories also
included many signs of toxicity that implicated involvement of the nervous system (e.g.,
tremors, hindlimb weakness or paralysis, or changes in activity level). It was therefore
appropriate to include neurological testing in this study. The test chosen was a functional
observational battery that has been validated and is used by many organizations to screen
chemicals for their neurotoxic potential. This first-tier evaluation has been recommended by
several expert panels and is now required by the U.S. EPA for testing many chemicals (U.S.
EPA, 1991; for review, see Tilson and Moser, 1992). The purpose of this test battery is to
identify potential neurotoxicants, which can then be studied in more depth to characterize any
neurotoxic effects detected. The functional observational battery includes many functional
indicators of the autonomic, sensory, motor, and behavioral status of the test subject. Much
research has been conducted on the sensitivity, validity, and predictability of the functional
observational battery using rats (for example see, Moser, 1989,1990a, 1990b, 1991; Tilson
and Moser, 1992). To a lesser extent, these specific tests have also been used in mice;
however, the functional observational battery, used in rats, is based on the original screen for
neurological signs in mice described by Irwin (1968). More recently, others have used the
functional observational battery for mice with little or no modification (Tegeris, 1991; Beaton et
al., 1990). Thus, this test procedure is well-suited to assess the possible neurotoxic effects of
carpet vapors.
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Postmortem Measurements There is no information currently available suggesting a
mechanism for the toxicity observed by Anderson Laboratories. In an effort to obtain
preliminary information pertaining to mechanism of toxic effect, a comprehensive toxicity
screen was performed on each mouse in the study. The screen consisted of measurements
on the blood, lung and nasal lavage, organ weights, gross necropsy and microscopic
evaluation of several tissues. Analysis of the blood included determination of normal and
abnormal hemoglobins, evaluation of the relative percent of different types of white blood
cells (differential) and clinical chemistries to examine liver, kidney, heart, muscle and stress
responses. Lung lavage was examined for evidence of inflammation (lavage differential), cell
death and changes in lung permeability.
Goals The were two primary goals for this initial study: (1) Can we obtain comparable
results from testing the same carpets using essentially the same equipment and procedures
as those used at Anderson Laboratories? (2) If toxicity is observed in test animals, can the
EPA gather information about the biological responses of the test animals and source
emissions to determine the cause or develop plausible hypotheses to account for the findings?
Caveats This study must be considered exploratory. Due to time constraints, key
variables associated with the test apparatus, preexposure sample conditioning, environmental
conditions during testing and variability among samples have not been systematically
examined. The effects observed on test animals at Anderson Labs reportedly have ranged
from no observable effect to death within a single test that consists of four sequential one-
hour exposures of four mice over a two-day period. Abnormal behavior, defined using
Anderson's list of clinical observations, has ranged from transient to persistent and has not
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occurred at predictable times after an exposure or exposures. Variability between emissions
from subsamples of the same carpet is unknown. Quantitative and qualitative variability of
emissions over the sequence of repeated heating/exposure cycles that constitute a single test
is not known. Air temperature in the test chamber has been monitored during exposures at
Anderson Labs; however, chamber surface and carpet temperatures have not been routinely
monitored. These temperatures may or may not be critical to producing the reported effects.
Finally many biological end points, with the exception of death, used in these test
procedures rely on subjective judgments by a trained observer. The functional observational
battery, generally regarded as a screening tool that can be used to identify exposures that
warrant further study, has been modified and adapted for these experiments. The battery
requires a trained observer to make subjective evaluations of specific pre- and postexposure
behaviors and appearance of the test animals. These judgments form the basis from which a
determination is made as to severity of the effect of exposure. Furthermore, interpretation of
pulmonary irritancy requires subjective evaluations of pulmonary waveform. Criteria were
established, based on the ASTM E-981-84 for a numerical rating system to evaluate
pulmonary waveforms. However, despite attempts at objectivity, these measurements are
based on some subjective assumptions, and thus, interpretation between investigators may
vary.
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Methods
Definitions To aid the reader in understanding this document, we have attempted to
use certain words in a specific and consistent manner. The following definitions will be used
throughout this text
Study The collection of six experiments using three treatment groups as defined below.
Treatment The study evaluated three treatment groups (A-C). Two of these treatment groups
were exposed to carpet samples, whereas the third treatment group was exposed to
humidified, zero-grade air.
Experiment The study included six experiments; each experiment used four mice. A single
treatment group consisted of two replicate experiments.
Exposure Within each experiment, four one-hour exposures were performed over two days.
Period Within each exposure there were three periods: (1) a 15-minute period during which
the mice were exposed in the plethysmographs to zero-grade, humidified air; (2) a 60-minute
period during which mice were exposed to the airborne contents of the source chamber; and
(3) and a 15-minute recovery period in zero-grade, humidified air.
Control Group Two types of controls were used in this study; each group having a unique
designation. One control is the treatment group of mice exposed to only zero-grade
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humidified air from the source chamber. This group, however, is always referred to as
Treatment B. The second control used in the study refers to the non-exposed, unrestrained
cage-control group that is used for comparison purposes in all of the postmortem evaluations.
This group is referred to as the 'control" group or variant of this term (e.g., cage control,
unrestrained control, non-exposed control)
Source Chamber The source chamber was constructed from an aquarium and contained the
carpet samples. In the zero-grade air treatment group, the source chamber was empty.
Animal Exposure Chamber The heads of the mice protruded into a glass animal exposure
chamber that was connected to the source chamber during the 60-minute exposure period.
Plethysmograph Attached to the animal exposure chamber were four plethysmographs. A
plethysmograph is a device for the measurement of changes in volume. Expansion of the
chest (proportional to the volume inhaled) was sensed as a pressure change in the sealed
plethysmograph tube because the mouse inspired from air external to the plethysmograph
(i.e., from the animal exposure chamber). Frequency of breathing was instantaneously
computed by calculating the time between pressure swings.
Sensory and Pulmonary Irritation A characteristic change in the pattern and frequency of
breathing that occurs in mice exposed to irritant chemicals that stimulate the trigeminal nerve
endings of the upper respiratory tract (Sensory Irritation) or stimulate the vagal nerve endings
in the lower respiratory tract (Pulmonary Irritation).
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Functional Observational Battery The functional observational battery is a test battery that has
been primarily used to identify potential neurotoxicants that may require further, more in-depth
evaluation. The battery includes many functional indicators of the autonomic, sensory, motor,
and behavioral status of the test subject.
Study Design A total of six separate experiments were conducted, consisting of two
experiments with no carpet in the source chamber (Treatment B), and four experiments using
two different subsamples of carpets previously tested at Anderson Labs (Treatments A and C).
Because some lexicological indicators used in this experiment rely on subjective judgments or
interpretations of test animal behavior and appearance, and subjective interpretation of
pulmonary waveform data, a blinding procedure was used to minimize the potential impact of
negative or positive expectations of the experimenters on subjective observations. To ensure
experimental blind, CPSC decided the order of testing by randomizing shipments one through
six. Randomly selected subsamples of the same carpet were tested simultaneously at each
laboratory. The actual test order is listed in Table 1.
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Table 1. Test Matrix Using Aquarium Source Chamber
Treatment
A-1
B-1
B-2
C-1
C-2
A-2
Experiment #
1
2
3
4
5
6
Test Dates
3/8-3/10.
3/10-3/12
3/22-3/24
3/24-3/26
3/29-3/31
3/31 -4/2
Shipment
Carpet 1
Empty
Empty
Carpet 2
Carpet 2
Carpet 1
The CPSC shipped the appropriate subsamples (sealed in Tedlar bags) to the sample
custodian at each laboratory, who received and logged the samples, loaded the source
chamber, covered the sides and top with duct tape and placed the chamber in position for
testing. At the completion of the four, one-hour exposures, the custodian removed the source
chamber from the exposure system, removed the subsamples, and packaged and returned
them to CPSC. For control tests, CPSC shipped a package containing an empty Tedlar bag.
It was the responsibility of the experimenters to maintain the integrity of the blinding
procedure.
Experimental Test Procedure The following summary briefly describes how the series
of four, one-hour exposure tests were conducted. Details for the functional observational
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battery, the method of assessing irritation (ASTM E-981-84), and the postmortem evaluations
are described below. On the day before testing, three 1-ff subsamples of a carpet were
rolled and stapled together and then placed fiber-side down on the bottom of the source
chamber. The source chamber was sealed and moved to the biology testing laboratory and
the vinyl insulating blanket was placed on top of the source chamber. Meanwhile, six mice
meeting the selection criteria were moved to the biology laboratory and the preexperiment
functional observational battery was performed.
On the first exposure test day, the heating blankets were turned on high to achieve the
inside air target temperature of 37±3 °C and outside bottom temperature 70±5 °C. Once these
target temperatures were achieved, the carpet was allowed to bake under these static air flow
conditions for one hour. During this time, all nylon bulkhead fittings were checked: the O2and
CO2 monitors were adjusted to 20.9 and 0.03%, respectively; the flow controllers, humidify
sensor, and bottled air were turned on; and the vacuum rotameter was checked (Gilibrator
Digital Flowmeter, NIST traceable) to ensure that the flow rate was 7 ± 0.2 LPM. The vacuum
was then connected to the exhaust end of the animal exposure chamber.
Four test animals were placed in the animal exposure chamber per ASTM E-981-84
and provided with humidified clean air at 7 LPM for 15 minutes to establish baseline frequency
of breathing rates and control waveform morphology (period 1). The source chamber was
then connected to the exposure chamber and clean, zero-grade, humidified air was pulled
through the source chamber across the carpets and into the animal exposure chamber for one
hour (period 2). After one hour of exposure to effluent from the source chamber, the source
chamber was again sealed and test animals were again exposed to humidified, zero-grade air
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for a 15-minute period to evaluate if the animals recovered from the exposure (period 3).
During these three evaluation periods (control, exposure, and recovery), magnehelic pressure,
temperature from four thermocouples, humidity, and O2 and CO2 were monitored.
After the recovery period, the mice were immediately removed from their
plethysmographs and were returned to their home cage, where their behavior and appearance
was observed for 15 minutes. Two exposures were completed in one day with two hours
separating the exposure periods. During this two-hour period, the heating blankets were left on
and the temperature was monitored and adjusted to maintain, if possible, the temperature
inside the source chamber. Following the second and fourth exposures (days 1 and 2), the
mice were observed for 15 minutes, after which, each mouse was removed from the home
cage and was examined and scored using the functional observational battery. This pattern
was repeated on the following day such that one experiment included four, one-hour
exposures conducted over a two-day period. Between the second and third exposures, carpet
samples remain sealed in the source chamber overnight without additional heating.
Upon completion of the last (fourth) exposure and functional observational battery, all
mice were sacrificed for the postmortem evaluation. Additionally, the flow through the entire
exposure system was checked. This procedure was initiated to evaluate leaks into the
exposure system from sources other than the supply cylinder air (e.g., loose fittings, holes in
the duct tape front seal, etc.). The system leak check could only be performed after the fourth
exposure, otherwise carpet emissions would be potentially lost
Exposure System The design of the exposure system for evaluating the emissions of
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carpets is shown in Figure 1. Basically, the system consists of a humidified clean air system,
a modified aquarium as the source exposure chamber, carpet samples, a heating and
insulation system for the source chamber (not shown), an animal exposure chamber, and
several physical and biological monitoring systems.
Air and humidity system During the carpet exposure period (see protocol), the emissions
from the source chamber were pulled through the animal exposure chamber at 7 LPM under
approximately -0.06" of water pressure. Certified zero-grade air (National Welders) from a
compressed gas cylinder was used to supply excess air flow (approximately 14 LPM) to the
source chamber. The airflow from the cylinder was divided into two streams, with the flow
rate of each stream controlled by a mass flow controller (one 10 SLPM and one 20 SLPM
mass flow controller electronically controlled by a Tylan RO-28 Flow controller). The mass
flow controllers were calibrated just before the first experiment (Gilibrator Digital Flowmeter,
NIST traceable). One air stream passed across room-temperature distilled water held in a
one liter glass impinger, the other air stream remained dry. The two flows were recombined
and monitored for relative humidify (Omega RH411, Digital Thermo-Hygrometer) just before
entering the source chamber. Alteration of the ratio of the wet and dry flows was used to
adjust the humidity of the inlet stream. A relative humidity of 50 ± 10% was set as the target
concentration. Excess airflow (approximately 7 LPM = 14 LPM cylinder • 7 LPM vacuum)
was allowed to escape into the laboratory before entering the source chamber.
Source Chamber A commercially purchased 10 gallon (38 L) fish tank (Fish Pros, Raleigh
NC) was used as the carpet source chamber. All aquariums were first prepared by removing
the plastic rim using a hot air gun and a knife. Excess silicone adhesive was then removed
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with razor blades and precision knives (X-acto). The aquarium was turned on its side so that
the opening was facing outward (toward the animal exposure chamber) and the long side
panels (10"x 20") became the top and the bottom. The top and sides were covered in duct
tape to conceal the contents. A front cover was constructed from a 10" x 20" double thick
(0.1875") plate glass. In diagonal comers of the front cover, 3" from each side, two 0.5"ID
holes were fitted with nylon bulkhead fittings. This front cover was attached to the aquarium
with duct tape and covered with duct tape to conceal the contents of the source chamber. A
28/12 ground glass female socket was placed on the upper 0.5"! D nylon fitting, and a 28/12
ground glass male ball joint was connected to the lower fitting. Supply air entered through
this upper hole and the source chamber atmosphere exited through the lower hole into the
animal exposure chamber. An additional hole was cut in the front plate to accommodate a
teflon coated 6" thermocouple probe placed in a 0.25"ID nylon fitting located 2" below the inlet
fitting. A heating pad (Sunbeam model E12107) was used to heat the bottom of the
chamber, while the top of the chamber was heated with a wool heating throw blanket
(Sunbeam model HT-1). The top, bottom, and sides of the chamber were insulated with a
vinyl coated fiberglass blanket that was adjusted (opened or sealed) as necessary to achieve
the desired target temperatures.
Prior to use, the aquariums were baked overnight at test temperature conditions using
laboratory air at 7 LPM to flush the excess adhesive vapors. The aquariums were then
washed with an Liqui-noxR detergent solution and rinsed with deionized water. The aquariums
were air dried or dried with a clean tissue and considered suitable for use. Just prior to the
toxicology experiments, the dean dry tank was heated for one hour under test conditions and
duplicate 3L ST032 sorbents were collected from the heated aquarium to establish a system
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background before the carpet was installed. The carpet was installed in three 1-ft2 sections,
rolled fiber-side out and stacked in pyramid fashion diagonally on the aquarium bottom
surface. During control experiments, the aquarium was sealed empty (i.e., Treatment B).
Carpet source and sample collection Two carpets were selected for evaluation from
sites that have been tested at Anderson Labs and were shown to produce severe tbxicity or
death in mice under test conditions. Carpets were collected by the CPSC, according to
protocols described briefly below. The collection, transportation, and storage processes were
standardized to minimize potential differences among subsamples. Carpet samples were
collected by CPSC personnel as soon as was practical after source sites were selected and
access had been obtained.
Sample Collection Protocol Site data were collected using CPSC's Investigation
Guideline (appendix G, Carpet Test Plan). Approximately 50-100 ft2 of carpet was collected
by CPSC from the site and stored. Each sample or portion collected was tagged with an
identification number and wrapped in Tedlar. Labels with the official sample collection number
were attached to the bag. A collection report was included with the sample describing the
collection procedures, location, date of collection, and signature of the collecting agent.
Packaged samples were placed in an appropriate shipping container and shipped to CPSC
within 48 hours of collection. Samples were logged on receipt by the CPSC, inspected for
shipping damage, subdivided into 1-ft2 pieces that were labeled, randomized, and then
packaged in groups of three in sealed Tedlar bags. Bags were stored at room temperature in
a sample storage room. The CPSC maintained a bound log of all samples and subsamples.
Subsamples were shipped via air express to EPA and Anderson labs approximately 48 hours
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before testing.
Animal exposure chamber A 2.3-L glass chamber with four attached plethysmographs was
purchased (Crown Glass, Somerville, NJ) to conform to ASTM E-981-84 standards. Prior to
use of the chamber and between experiments, it was washed with analytical grade glass
deaner (Uqui-nox") and thoroughly dried in a drying oven at 180° F. During exposure, mice
were restrained in head-out plethysmograph tubes, according to ASTM E 981-84. Mice were
loaded into the plethysmographs using a rubber stopper with a plunger that sealed the back
end of the plethysmograph and pushed the hind quarter of the animal forward forcing the head
through a latex collar. The neck was sealed around a 5/16" hole in a latex dam (thin gauge
green #02146, Hygienic, Inc.). The latex dam was attached to the animal exposure chamber
using duct tape. A 1/2" hole in the duct tape aligned with the 5/16' hole in the latex to
accommodate the animal's neck. A cotton-tipped applicator was placed in front of the collar
during loading to prevent the animal from biting the collars. Between exposures, collars were
checked for potential leaks and changed as necessary. Plethysmographs were wiped dean
with tissue and distilled water after each exposure. Latex collars were always replaced
between experiments.
Exposure measurement system Three additional systems were used to monitor
conditions before and during carpet emissions. Four thermocouple probes and an electronic
thermometer (Omega HH21 Thermocouple Thermometer) were used to monitor and regulate
the source and animal exposure chambers. The probes were located in the following
positions (Figure 1): (1) 82 (chamber bottom, outside surface, target temperature =
70 ± 5 °C); (2) S, (chamber top, outside surface, expected temperature = 40 ± 3 °C); (3) A,
EPA-23
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(source chamber air, target temperature = 37 ± 3 °C); and (4) A, (animal exposure chamber,
expected temperature = 24 ± 2 °C). All thermocouples were calibrated before the initiation of
the experiment against a NIST thermometer.
Percent oxygen (O2) (Beckman Labman Oxygen analyzer) and carbon dioxide (CO2)
(SensorMedics Medical Gas analyzer LB-2) were monitored and recorded every 15 minutes
during the experiment. A single point calibration was performed daily and multipoint
calibrations using certified gas sources were performed just prior to and midway through the
study. Additionally, static pressure was measured inside the animal exposure chamber using
both positive and negative magnehelics (Dwyer Instrument Co.) that were capable of reading
between 0 and 0.5" water pressure.
Animals Weekly, 20 male Swiss-Webster mice (viral antibody free), weighing 18 to 22
grams were delivered from laconic Farms, NY. From each shipment of mice, sentinel
animals (four to five mice per experiment, total number of sentinel animals = 27) were sent for
serology, microbiology, and parasitology. The sentinel mice were found free of all common
infections, including murine virus antibody, respiratory tract pathogens, endoparasites,
ectoparasites, and fecal Pseudomonas. Mice were group housed (10 per cage) on com cob
bedding in a climate- and light- (12 hours light/12 hours dark) controlled AAALAC-accredited
vivarium for at least seven days before testing. Mouse chow (Tech Lab Agway) and tap water
were available ad libitum. Mice were considered acceptable for use after this waiting period if
they appeared healthy and their weight was between 25.5 and 28.0 grams. Prior to weighing
mice, the mouse scale accuracy was assessed using a 100 gram transfer standard weight
Particulate filter tops were used when transporting the animals between the vivarium and the
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laboratory for exposure.
Initially, six animals that met the weight criteria were tail-marked and evaluated using
the functional observational battery (see description below). Those that were acceptable
(general appearance normal and could perform the tests in the neurobehavioral screen) were
candidates for use in the exposure on the following day. On the day of exposure, four mice
were loaded into the plethysmographs. The two remaining mice were not exposed and served
as nonrestrained cage controls for postmortem evaluations. During the entire study, three
mice that were initially loaded into the plethysmograph on the first exposure day were
eliminated, according to ASTM E-981-84 guidelines, because of abnormally low baseline rates
or misshaped respiratory waveform patterns. When this occurred, the mouse was swapped
with one of the cage controls. Between exposures, the four mice in an experiment were
returned to an acrylic cage with com cob bedding located in the laboratory. This cage was
covered with a filter top through which charcoal-purified, HEPA-filtered air was forced so as to
maintain a chemical-free environment during the two days in which exposures were
performed. While the mice lived in the laboratory, they received food and water ad libitum,
except during exposure. The two non-exposed (unrestrained controls) mice from the original
six were housed similarly, but separately from the exposed group. Besides the screening
body weight measurements, body weights were also measured before each of the four
exposures. For the first 15 minutes after each exposure, general appearance and activity in
their home cage were also noted.
Functional Observational Battery
Test procedure The functional observational battery was conducted according to the
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protocol (Appendix B, U.S. EPA Carpet Test Plan), which describes the progression of tests
and standardized scoring criteria for each measure. This protocol was originally based on that
used in this laboratory for rats; modifications and additions were made to stress the possible
neuromuscular component of this toxicity syndrome, and to include measures which Dr.
Anderson, in her experience, felt were particularly sensitive.
The tests began with the trained observer hanging the mouse by one hind leg onto the
lip of a 1-gallon glass jar. The mouse had to pull up and stand upright on the jar rim, and this
was repeated three times. Next, the mouse was placed horizontally across a screen, held at
a 45* angle, and was given 20 seconds to either rotate and walk up the screen, walk straight
across, or rotate and move down the screen; this was also repeated three times. While on
the screen, the observer scored how often the mouse slipped a paw down between the wire
mesh. The screen was then placed on a table edge, and the mouse, held by the tail, was
lowered towards it. A positive forelimb placing response was noted when the mouse reached
toward the screen. The mouse was then allowed to grab the screen and was raised up until
the screen was perpendicular. The number of times the mouse dropped the screen was
counted. The mouse was then held in the observer's hand, during which time its reactivity to
being handled, lacrimation, palpebral closure, and salivation were ranked. In addition, the
presence of piloerection; exophthalmus; cyanosis; gasping; facial swelling; or blood around the
eyes, ears, or nose were noted. Body tone was scored by assessing the resistance of the
abdominal muscle to light finger pressure. Holding the mouse between the palms of the
hands, the observer flipped it over and scored the ease with which the mouse regained
normal posture.
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Open-field observations took place on the top of a laboratory cart (60 x 90 cm) with a
3" rim around the perimeter and covered with plastic-backed paper (which was changed
before each test). The mouse was observed for exactly two minutes. During this time, the
number of rears were counted, and gait characteristics, ataxia, degree of body tilt, alertness,
and overall level of activity were scored. Descriptions were recorded for body posture and
any donic or tonic motor movements. The observer also recorded any diarrhea, excessive
vocalizations, stereotypic movements, or any other atypical behaviors. The mouse's reactions
to a puff of air delivered to the face, the sound of a metal clicker, or a tail pinch using forceps
were then scored. Finally, the mouse was placed on the screen, which was then inverted.
The time required for the mouse to climb over the edge, back to the top, was recorded, with a
60-second cut-off. During the preexposure test only, mice were given up to three tries, along
with some prodding in some cases, to leam this task.
Observational testing required 5 to 10 minutes per mouse. All data were recorded on
preprinted sheets and were later entered into a computer for further analysis. The observers
(one performed the manipulations, the other recorded the data) were unaware of the treatment
conditions. Testing was conducted in the same laboratory where exposures took place. Entry
to the room was restricted during testing, and extraneous noises were held to a minimum.
Frequency of breathing measurements
Data acquisition system Four pressure transducers (Validyne DP-45) were connected
to each plethysmograph via a 7" segment of thick-walled Tygon tubing. The transducers were
connected to a chart recorder (Astro-Med, Dash-4) via preamplifiers (Hewlett-Packard, Model
8805B). A signal from the preamplifiers was also fed to a custom built frequency-to-voltage
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converter that reported the instantaneous frequency between two peaks meeting its threshold
requirements. The frequency data were then converted to a voltage and digitized by a
personal computer every five seconds. A computer program converted the voltage back to
frequency and stored and displayed the median value for the 12 samples as the
representative frequency for each mouse during the one-minute sample. Data files for each
experiment were named by date (e.g., MT921101.EXT) and stored on the hard disk.
Before each experiment, the data acquisition system was calibrated using several
independent methods. The chart recorder and frequency-to-voltage converter were first tested
using internal calibration circuits residing within the instruments. The preamplifiers were
balanced without, and then with, the electrical load contributed by the transducer. A closed
vial was loaded into the plethysmograph to simulate the displacement volume of a 25-gram
mouse. A fixed volume syringe pump (approximately 0.25 ml) was oscillated at a fixed
frequency (approximately 250 CDS) into the constant volume plethysmograph and the
amplitude of each of the four chambers was adjusted to 6 volts of the full 10-volt scale. The
frequency was then noted on the frequency-to-voltage converter and hand checked on the
chart recorder. Previous experiments had verified that the transducer/plethysmograph system
was linear between 100 and 300 breaths per minute, and that the frequency-to-voltage
converter was accurately digitized by the computer between rates of 0 and 600 cycles per
second.
Once the mice were loaded into the plethysmographs, the chart recorder was run at 10
mm/second and tidal breathing of the animals was examined to see that the signal was at
least 50%, but not more than 90%, of the width of that channel's chart and that the rates were
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between 200 and 275 breaths per minute. The chart recorder was then set to run in dual
speed mode (10 seconds at 10 mm/second, 50 seconds at 5 mm/second) and marked with
the experiment and exposure information. Similar information (investigator name, animal
species, strain, sex, date of birth, date of arrival, experiment number, exposure number and
exposure information [up to 1 line of text], animal numbers, animal weights, and exposure
protocol information [control, exposure, and recovery times]) was then entered in the
computer. The chart recorder and computer program were started simultaneously so that
samples would be time stamped and matched. At the conclusion of the exposure, the data
files were backed up to floppy disk and stored.
Subjective evaluation of waveforms During exposure, the chart recorder was run at
two speeds so that waveform morphology could be subjectively scored according to criteria
established in the ASTM E-981-84 and subsequently refined in discussions between our
laboratory and Anderson Laboratories. For each minute, for each mouse, a subjective score
between 0 and 3 (0=none, 1=slight, 2=moderate, and 3=severe) was entered onto a
preprinted data sheet in three different categories. Subjective scores were entered for
sensory irritation, pulmonary irritation, and a disruption index based on the extent and severity
of irregular waveforms not fitting into the above categories (e.g., movement artifacts). The
disruption index primarily identified sighs (large tidal excursions) and movement artifacts. Two
to three sighs or disruptions were scored as slight (1), while more frequent and prolonged
disruptions were scored moderate (approximately 2 to 10 disruptions) or severe (>10
disruptions). For sensory and pulmonary irritation, if several breaths were of the characteristic
shape, and those breaths did not precede or come immediately after a disruption, the entire
minute period was scored by those breaths. This technique would tend to overestimate the
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amount and severity of irritation compared to the decrease in frequency of breathing. All
respiratory waveforms were scored by the same person, usually during each exposure.
The minute-by-minute data (90 minutes) for each animal (N=24) for each exposure (4)
for the three different measures (sensory irritation, pulmonary irritation, and the disruption
index) (total =25,920 hand measurements) were reduced to a single score for each period
(control, exposure, and recovery) for each animal for each exposure for each measurement
type. Previous analyses indicated that the highest (1 to 3) severity score that appeared four
or more times would be significantly different from all lower scores. Thus, the highest severity
score that occurred four times was used as the score for the entire period for that animal.
The criteria for scoring a period as not zero were purposely set low (4 observations during the
60-minute exposure) to increase the sensitivity of detecting positive effects.
Postmortem Evaluation Immediately after the functional observational battery testing,
mice were deeply anesthetized with halothane and the orbital plexus was tapped to obtain
blood for analysis of %methemoglobin, %carboxyhemoglobin, and hemoglobin content
(Operator's Manual IL282 CO-Oximeter, Instrumentation; Brown, 1980). The mouse was then
exsanguinated via heart puncture, a peripheral smear was obtained, the remaining blood was
centrifuged, and serum was collected and immediately frozen at -70 °C for analysis within the
week. Peripheral blood cell differential counts were enumerated after applying Wright Giemsa
stain.
Clinical Chemistries Clinical chemistries were performed on all available serum. For most
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mice, 350 uL was obtained so that the full battery could be examined, which included
bilirubin, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate
aminotransferase (AST), creatinine, blood urea nitrogen (BUN), 5' nucleotidase (5'-ND),
glucose, alkaline phosphatase (ALP), total protein, isocitrate dehydrogenase (ICD), albumin,
triglycerides, cholesterol, sorbitol dehydrogenase (SDH), and bile acids. However, for several
mice with insufficient serum, clinical chemistries were not performed on SDH and bile acids.
All clinical chemistries were performed using a centrifugal analyzer (Cobas Fara II clinical
chemistry analyzer) using kits (Sigma) adapted for use with this analyzer.
Nasal and Lung Lavage After the mice were bled, a nasal lavage apparatus was inserted
into the posterior pharynx and 0.5 mL of warmed 0.85% saline was injected through the nose
and collected at the end of the nares. The mouse was then tracheostomized (20 gauge Luer
adaptor) and lavaged (BAL) three times using the same aliquot of saline (0.8 mL). The nasal
lavage and BAL were held on ice until centrifugation and the cell pellet and supernatant were
prepared for further biochemical analyses or for evaluation of cell count and differential (Ghio
et al., 1991). Cell count was obtained using a Coulter counter (Coulter Electronics, Inc.) and
cell differential was enumerated after Wright Giemsa staining. The supernatant of the lavage
was immediately analyzed for indicators of lung injury, protein, and LDH (Ghio et al., 1991;
Smialowicz et al., 1991) using a centrifugal analyzer. Protein was measured using the Bio-
Rad method for measurement of total protein using bovine serum albumin as a standard.
Lactate dehydrogenase was measured using a purchased kit (Sigma). Perchloric acid (17.5
uL) was added to the supernatant from the nasal lavage prior to high speed centrifugation.
The high speed supernatant was stored at -70 °C until it was analyzed for ascorbic and uric
acids, and glutathione. The high speed pellet was analyzed for total protein using the
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centrifugal analyzer as described above.
Necropsy and Histopathology Major organs (kidney, liver, brain, lung, nose, heart,
thymus, spleen, adrenal, diaphragm, thigh muscle, stomach, duodenum, and colon ) were
grossly observed and removed under the supervision of a certified veterinary pathologist
(Experimental Pathology Laboratories, Inc.)- Organ weights for brain, heart, thymus, spleen,
liver, and kidney were obtained prior to fixation. Lungs were infused with a syringe containing
a volume of 10% neutral buffered formalin equivalent to the volume removed after BAL The
removed organs and remaining carcass were appropriately fixed in 10% neutral buffered
formalin for histopathology and stored (Feldman and Seely, 1988). The following tissues were
paraffin embedded, stained with hematoxylin and eosin, and microscopically examined for
histopathological damage: kidney, liver, brain, lung, nose, heart, thymus and spleen, adrenal,
diaphragm, thigh muscle, stomach, duodenum, colon, and any other tissue with gross
abnormalities.
The relative degree of severity of inflammatory, degenerative, and proliferative changes
were graded using the following scale: minimal (1), slight/mild (2), moderate (3), moderately
severe (4), and severe/high (5). Congenital lesions were not graded, but instead were
designated as present when observed.
General Data Analysis Strategy Due to the small number of carpet samples and
test animals used in these exploratory experiments, detailed statistical analyses correlating
variables of the test system with measures of toxicity were not feasible. However, the general
data analysis strategy was designed to answer if the EPA could replicate Anderson's results
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with carpets shown to produce toxicity to mice in her system. Positive answers to any of the
four questions posed below would constitute a successful replication of toxicity observed at
Anderson Laboratories.
1. Are deaths observed at both laboratories in test animals exposed to emissions from either
carpet, but not observed from exposure to air?
2. In the absence of death, is unquestionable evidence of toxicity observed at both
laboratories using the functional observational battery criteria (e.g., paralysis or seizure-like
behaviors) from either carpet, but not air?
3. Do the detailed clinical chemistry, lung lavage analyses, and histopathologic evaluations
suggest abnormalities in mice exposed to carpet, but not air?
4. Are severe alterations in breathing pattern and rate as defined in ASTM E-981-84
observed for carpets, but not air exposures at both laboratories?
For all biological end points, descriptive statistics (means, standard deviations, and
plots) were obtained. No attempt was made to evaluate the equivalence of the replicate
exposure groups because the number of animals was too small (N=4). Treatment group A
was a carpet exposure, B was an exposure to zero-grade air, and C was an exposure to a
different carpet sample than Treatment A. For most end points, one-way analysis of variance
(ANOVA) was used as a first step to examine overall treatment effects on any single
parameter. In the event of a significant treatment effect, statistical comparisons to Treatment
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B (zero-grade air group) as well as the nonrestrained control group were made using
appropriate parametric or nonparametric tests. The significance level for accepting false
positives (Type I errors) was set at 5%. Because this study was exploratory in nature,
corrections for multiple pairwise comparisons were not made, thus increasing the likelihood of
calling a finding significant when it was not. This strategy was developed so that the results
could be used to help define potentially important parameters for testing in later studies.
Unless otherwise stated, data are presented as mean ± standard deviation for all tables and
figures.
For some end points, the analysis examined the three treatment groups, A, B, and C,
during or after four exposures. This type of data required a two-way ANOVA using treatment
and exposure as the two factors. Treatment refers to the contents of the source chamber
only, whereas exposure refers to the four consecutive exposures in any experiment regardless
of the contents of the source chamber. Thus, a significant treatment group effect would
indicate that a significant difference between treatment groups (A, B, or C) occurred, but it
was unrelated to the number of exposures or exposure procedure (i.e., the same effect was
seen after each exposure). A significant exposure effect would indicate that there were
significant difference between exposures, but that it was unrelated to the treatment group (i.e.,
all treatment groups behaved the same way). If a significant treatment-by-exposure
interaction occurred, then the effect at different exposures was different for different treatment
groups. For example, if only on the last exposure day, Treatments A and C had significant
decreases in the frequency of breathing, then a significant treatment-by-exposure interaction
would occur. If Treatments A and C showed decreases in frequency compared to Treatment
B after every exposure, there would be a significant treatment effect If all treatment groups
EPA-34
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increased the frequency of breathing with each exposure, then a significant exposure effect
would be found.
Physical exposure data Mean values from serial measurements (temperature and
humidity) and maximal deviation (magnehelic pressure, %O2, and %CO2) were reported on the
data summary sheet No formal analyses of these data were undertaken, except to note
when excursions beyond the target conditions existed.
Subjective Appearance The incidence of occurrence (yes/no) for puffy faces,
hemorrhaged ears, dilated ear vessels, and lacrimation were analyzed using a categorical
modeling procedure for the linear analysis for nonparametric data (CATMOD; SAS 1990).
Two factors, treatment and exposure, were evaluated. If no significant treatment-by-exposure
effects were observed, the data were collapsed across exposure and reanalyzed to determine
if any treatment effects could be detected.
Frequency of breathing One-minute measurements of breathing frequency data were
collected for each mouse for the three periods during each exposure. The one-minute
measurement consisted of the median of 12 instantaneous samples from the frequency-to-
voltage converter that were sampled by the computer every five seconds. The median was
used rather than the mean to filter outliers in the measurement sample. A spreadsheet
program (Excel, Microsoft Inc.) transformed the data into percent difference by dividing the
mean of the last five minutes in the control period by each minute during the exposure and
recovery period, subtracting one from the ratio and multiplying by 100. The program then
highlighted the highest percent increase and decrease from control during the exposure
EPA-35
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period. Thus, for each animal at each exposure, four values were reported, the mean of the
last five minutes of the control period, the greatest percent increase and decrease during the
exposure period, and the mean of the recovery period. All statistical analyses were done on
these data. The mean frequency of breathing for the group of four mice and the mean
percent difference from the control period were also calculated for three-minute periods as per
ASTM E-981-84.
Because of the repeated nature of these data (i.e., the same animal was measured
four times), two-way repeated measures ANOVAs were used for the analyses. The analyses
modeled treatment with three levels (A, B, and C) as one factor, and exposure was the
repeated measure with four levels (1 to 4). Four analyses were performed. The first analysis
examined the mean control period value for each animal for each exposure, to evaluate if the
animals from different treatment groups were initially the same. Additionally, the analysis
examined if the control period from subsequent exposures was the same as the initial data or
whether there was a carry-over effect Carry-over effects could result either from the
treatment or the exposure procedure, and could result in differences in subsequent control
periods. The second and third analyses evaluated effects during the exposure period. These
analyses examined whether the greatest increase or decrease percent difference from control
was affected by either the treatment or the exposure procedure. The fourth analysis evaluated
whether the recovery period was different from the control period and whether carry-over
effects occurred due to treatment or the exposure procedure.
Subjective scores of pulmonary waveforms The incidence of severity score data were
analyzed using a categorical modeling procedure for the linear analysis for nonparametric data
EPA-36
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(CATMOD; SAS 1990). Two factors, treatment and exposure, were evaluated. If no
significant treatment-by-exposure effects were observed, the data were collapsed across
exposure and reanalyzed to determine if any treatment effects could be detected. If the
treatment-by-exposure interaction was significant, one-way ANOVAs were conducted on the
treatment effects after each individual exposure.
Functional Observational Battery The two test groups for each treatment (sample)
were combined for all analyses, thus n=8 per group. Statistical analysis procedures for the
data derived from these tests have been described (Creason, 1989). Descriptive and rank
data were analyzed using a categorical modeling procedure for the linear analysis for
nonparametric data (CATMOD; SAS 1990). Initially, preexposure data were examined using a
one-way ANOVA to determine if significant differences existed between treatment groups. For
those measures that showed a significant or equivocal difference in the preexposure test, the
analyses were applied to the change from baseline (delta) values rather than actual values.
Overall, ANOVAs were conducted using treatment as the grouping factor, and repeated-
measures across time (days 1 and 2, corresponding to the tests after the second and fourth
exposures). If there was a significant treatment-by-time interaction factor, one-way ANOVAs
for each day of testing were conducted. If only the treatment effect was significant, treatment
groups were collapsed across the days and subjected to ANOVA. Treatment groups were
compared using mean contrasts (SAS 1990).
The number of rears, being the only continuous variable, was similarly analyzed except
that the GLM procedure was used (SAS, 1990). Square-root transformation was included to
more closely approach a normal distribution of the data.
EPA - 37
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In all cases, probability values <0.05 were considered significant However, p-values
that approached significance (p<0.10) were taken into account, along with time-course or
severity of effect, when evaluating the data from each measure.
Postmortem variables The analysis of most of the postmortem variables was
simplified because only one value for each measured parameter existed for each animal.
One-way ANOVA was used to evaluate the pooled (N=8) three treatment groups (Treatments
A, B, and C). If a significant treatment effect was observed (psO.05), two post-hoc Students t-
tests were performed comparing the mean of Treatment B with that of Treatment A and C. If
non-exposed control animal measurements were available for the parameter, a separate
ANOVA was performed, using the non-exposed control as a fourth treatment group in the
ANOVA. In the event of a significant effect using this analysis, the nonrestrained control group
was compared to the restrained control group (Treatment B) to see if there was a significant
effect of the exposure regimen.
Quality Assurance Because of the high visibility of this study and the importance of
this study to the EPA, an internal and external quality assurance program was instituted that
was specifically tailored to this study. For the biology studies conducted at the Health Effects
Research Laboratory, an interim Quality Assurance Project Plan, written protocols, and
operating procedures for all techniques and end points were developed. Additionally, several
internal quality control checklists were developed to ensure that all steps in all procedures
were carefully defined and followed. Table 2 is an example of the exposure checklist form.
EPA-38
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Experiment Name:
Date:
Table 2. Mouse Pulmonary Function Checklist
Exposure Number
Name:
Glass chamber is clean and all ports are connected or closed.
Dams are not jagged or punctured and completely sealed against chamber.
Create page in logbook with animal number/weights and comments as needed.
Mark beginning of chart with experiment name, exposure and animal numbers.
Check O2 setting at 20.9% and CO2 analyzer at 0.03%
Calibrate Frequency to Voltage converter (1st exposure only)
Tidal signals should be triangular and of similar bandwidth (-2/3).
Make sure corks are tight, but stoppers are pulled back behind pressure port.
Before exposure, turn Tylan and humidity sensor on
Turn air cylinder on
Humidity should read between 40 and 50%
Water vessel is appropriately filled
Set Vacuum rotameter and check flow with Gilabrator
Connect supply air and vacuum, tighten fittings
Mark beginning of exposure period.
Record pressure on report sheet and temperature and humidity every 15 minutes
Mark end of exposure period.
At end of experiment, copy data onto backup floppy.
Write O2 and CO2 peak values on report sheet
Each day analyze data and put analysis and this form in a notebook
Tm
15
—
—
—
—
90
Humidity
Inside Temp
CC)
Top Temp
fC)
BottomTemp
fC)
Mouse Temp
fC)
EPA-39
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Besides internal quality control procedures, the EPA requested an external audit by the
Research Triangle Institute, to review all procedures, ensure that all critical instruments were
operating correctly, verify that procedures were being followed, and recommend improvements
in existing procedures. Two types of quality assurance audits were performed. A technical
systems audit and a performance evaluation audit of relative humidity and temperature
sensors, positive and negative magnehelics, O2 and CO2 monitors, and indicated flow-rate
measurement instruments were performed.
The objectives of the audits were (1) to assess this study for compliance with
requirements documented in the study protocol, Quality Assurance Project Plan, and operating
procedures for the EPA carpet emissions project; and (2) to evaluate the study record-keeping
procedures for data completeness, accuracy, traceability, and defensibility.
Due to concern that the audit personnel and activities could influence the test animals,
and thereby compromise the experiment, the technical system audit was not conducted while
exposures and postexposure functional observational batteries were being performed.
Consequently, the audit was based on interviews with project personnel and review of project
records, and not on direct observation of experimental procedures. Interviews were
conducted using a checklist, and review of project records included a data tracking exercise
conducted for two animals
The objective of the performance audit was to assess the performance of instruments
to provide an independent evaluation of the quality of the data generated by them.
Performance evaluations of the carbon dioxide and oxygen analyzer, five temperature
sensors, one relative humidity sensor, two pressure sensors, and two flowrate sensors were
EPA - 40
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conducted. The oxygen analyzer was evaluated at one concentration using direct sampling of
a National Institute of Standards and Technology-Standard Reference Material (NIST-SRM).
Because this was a primary standard, no verification was necessary. The flow rates were
measured using a soap film flow meter, and the magnehelic gauges were audited using an
inclined manometer. Both of these devices are primary standards and verifications were not
necessary. Temperature and relative humidity sensors were conducted according to SOP
comparing NIST traceable sensors with test system probes.
EPA - 41
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Results
Exposure system
Temperature Two temperatures were considered critical to replicate the temperature
conditions used at Anderson Laboratories. The first critical measurement was the air
temperature in the source chamber. Although it was dear that there was a nonuniform
distribution of temperatures in the source chamber, the A, probe site was used to estimate
average source chamber air temperature. Attempts to keep this temperature stable (37±2 °C),
despite the crude method of heating, were successful for all 24 exposures (Table 3).
Similarly, attempts to keep the outside bottom temperature (S2) at 70±5 °C were successful;
however, there were specific days when we could never achieve the target temperature of 70
°C (Table 4). Despite our best attempts, the temperature in the plethysmograph hovered at
the outer limits of acceptability (24±2 °C) for 16 of the 24 exposures (Table 5). This was
primarily due to changes in the laboratory ambient temperature. With the discovery and
subsequent repair of a leaky room air conditioning unit, the final experiment more closely
approached the target temperature.
EPA-42
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Table 3. Temperature (°C) - inside of Aquarium (A,)
.TREATMENT
EXPERIMENT #
EXP1
EXP2
EXP3
EXP4
A
9317
37.7
37.0
37.3
37.7
9322
36.0
36.1
37.6
36.3
B
9318
37.4
36.9
39.3
36.9
9319
37.3
37.6
37.6
37.7
C
9320
36.9
37.3
37.6
36.9
9321
38.3
36.3
36.4
36.1
Table 4. Temperature (°C) • Bottom of Aquarium (S2)
TREATMENT
EXPERIMENT #
EXP1
EXP2
EXP3
EXP4
A
9317
69.9
71.4
71.3
71.6
9322
71.9
72.7
73.7
72.6
B
9318
64.0
64.7
64.4
64.4
9319
65.4
66.1
65.9
66.6
C
9320
71.7
67.6
70.7
70.3
9321
72.6
72.9
73.6
73.0
EPA-43
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Table 5. Temperature (°C) - Inside of Mouse Exposure Chamber
TREATMENT
EXPERIMENT #
EXP1
EXP2
EXP3
EXP4
A
9317
25.6
25.7
25.3
25.4
9322
22.1
23.1
23.1
22.7
B
9318
25.4
25.4
25.4
25.0
9319
25.6
26.4
25.9
26.0
C
9320
26.1
30.0
26.1
26.3
9321
24.7
23.6
23.4
22.7
sold numoer exceeaea study limit
Humidity The target relative humidity of 50±10% was not achieved. Although the humidity
probe was audited just prior to the study, and typically, such probes maintain their calibration
for months, unbeknownst to us, at some time just before the study the sensor stopped
working correctly. This error was discovered during the quality assurance audit after the
study was complete. Using a recently calibrated sensor, we were able to reconstruct the ratio
of dry and wet flows and calculate the actual relative humidity of air entering the source
chamber. These values are listed in Table 6. The actual humidity range was between 18.7%
and 29.2%. If experiments are examined in date order, the progressive failure of the relative
humidity sensor can be observed.
EPA-44
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Table 6. Percent Relative Humidity of Air Entering the Source Chamber
TREATMENT
EXPERIMENT #
EXPOSURE DATE
EXP1
EXP2
EXP3
EXP4
A
9317
3/9-10
29.2%
29.2%
27.6%
27.6%
9322
4/1-2
18.7%
19.9%
19.4%
18.7%
B
9318
3/11-12
27.6%
27.6%
27.2%
27.2%
9319
3/23-24
21.2%
24.2%
20.3%
20.3%
C
9320
3/25-26
19.9%
19.9%
20.8%
20.8%
9321
3/30-31
19.4%
21.0%
19.4%
20.3%
Bold numoers are outside study target range
System Flow, Static Pressure, Oxygen and Carbon Dioxide These four measures were
made to support the critical measurements described above. System flow, when measured at
the vacuum source, was always within 3% of the target flow (7 LPM) and within the limitations
of the normal house vacuum fluctuations. At the end of each experiment, flow through the
entire system was checked to evaluate possible leaks in the system. Flow through the entire
system indicated that no more than 4% leakage occurred, which is well within measurement
error, indicating that there were no leaks. Static pressure was monitored to make sure that
the animals were not exposed for prolonged periods to excessive negative or positive
pressure (±0.3" water pressure). During all conditions, static pressure was negative (vacuum
driven system) and ranged between -0.05 and -0.075" water pressure, except for very brief
excursions (<10 seconds) when the source chamber was connected to the animal exposure
EPA-45
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chamber. Larger excursions in pressure were tolerated at this time period to ensure that none
of the contents of the source chamber escaped into the laboratory. Oxygen and carbon
dioxide were monitored with no unusual findings, given the limits of accuracy and reliability of
these instalments. The poststudy audit of the flow meter, the magnehelics and the oxygen
and carbon dioxide monitors indicated that all instruments were functioning properly and were
accurate within the limitations of the instrument.
Body Weight All mice used in the exposure studies met the weight requirement for
inclusion in the study one day prior to testing (25.0 to 28.0 grams). Mice gained weight
between the pretest day and the first exposure day. Body weight on the first day of exposure
for all 24 animals ranged from 25.8 to 28.9 grams. Treatment (A, B, or C) had no significant
effect on body weight. On the other hand, the experimental procedure caused a reduction in
body weight (p<0.001), with each succeeding exposure, causing a further decrement in body
weight (Figure 2). On average, animals lost 82% of their body weight between the beginning
of the first and the end of the last exposure.
Irritancy Measurements
Rate changes Frequency of breathing was one of the primary measurements evaluated in
this study. As described in the methods section, the data were analyzed in two ways. First,
the data were analyzed according to the ASTM E-981-84 procedure using the group mean
(N=4). To increase sensitivity, a second analysis was performed using individual animal data.
ASTM E-981-84 Analysis The ASTM method specifies that the greatest percent reduction in
frequency for the mean of all four animals is to be used as "the" response for the test Thus,
statistics could not be performed because the raw data (Table 7) consisted of only six data
EPA-46
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points per exposure (i.e., two per treatment group per exposure).
Table 7. Percent Decrease in Frequency of Breathing for the Mean of Four Mice
Treatment
A-1
A-2
B-1
B-2
C-1
C-2
Sample #
1
6
2
3
4
5
Exposure 1
6.8
4.3
12.3
6.6
5.3
4.1
Exposure 2
6.0
3.2
6.3
42
2.0
5.7
Exposure 3
10.2
9.1
6.7
8.5
6.7
11.6
Exposure 4
13.3
13.3
5.8
9.1
6.4
14.2
Bold numbers indicate those groups in which the percent decrease in frequency or breathing
would be classified as slight (12-20% decrease) sensory irritation by ASTM E-981-84.
The data from Table 7 suggest that with an increased number of exposures, a
decrease in frequency of breathing is observed. When the two replicate experiments were
combined, there is some suggestion of a greater decrease in frequency of breathing during
the fourth exposure for Treatments A (13.3%) and C (10.3%), compared to Treatment group B
(7.5%).
•
Individual Animal Analyses Because there was some suggestion of an effect using
the ASTM E-981-84 criteria, a more in-depth analysis of the individual animal's response to
EPA - 47
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treatment was examined. The analysis attempted to answer three questions related to the
treatment response of individual animals with four successive exposures during the three
measurement periods.
1. Was there a treatment-related difference in frequency of breathing during the clean zero-
grade air control period? The ANOVA indicated that there was a treatment-related difference
(p=0.0129) in the control periods, with the Treatment B group having a slightly lower
frequency of breathing than Treatment A or C (Figure 3a). Additionally, there was a significant
exposure-related effect (p<0.001) without an exposure-by-treatment related interaction
(p=0.759). The lack of a significant interaction term would indicate that the treatment-related
differences that occurred were maintained across the four exposures. Thus, when the data
were collapsed across all treatment groups, frequency of breathing elevated with increasing
numbers of exposures (Figure 3b). This result would suggest that there was a carry-over
effect from the previous exposures, but it was unrelated to source chamber contents.
r
2. Were differences in treatment groups observed during the exposure period? Because
Treatment group B had a significantly lower frequency of breathing initially, and because that
difference was maintained across the four exposures, all subsequent analyses were
performed on data adjusted for this difference. Percent difference from each animal's control
response was used as the adjustment Analysis of each animal's greatest one-minute percent
decline in frequency (Figure 4a) indicated that there were significant exposure-related effects
(p<0.001), but no significant treatment (p=0.972) effects or treatment-by-exposure interactions
(p=0.639). The significant exposure effect occurred because the decrease in frequency was
less after the second exposure and greater after the fourth exposure than the decrease
observed during the first and third exposure (Figure 4b). This effect was significant only when
EPA-48
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the data were collapsed across all treatment groups. A similar analysis of each animal's
greatest one-minute percent increase in frequency indicated that there were no significant
treatment or exposure-related effects. With all treatments and exposures combined, the mean
greatest one-minute increase in frequency was 15.1%. Thus, overall, no treatment group
related increases or decreases in frequency of breathing could be detected.
3. Were differences in treatment groups observed during the recovery period? Although no
treatment-related differences were observed during the exposure period, it is possible that
treatment-related differences may become manifest during the recovery period. Another two-
way ANOVA was performed evaluating the percent difference from the control period. The
analysis indicated that both significant exposure (p=0.013) and significant treatment (p=0.044)
effects occurred; however, a significant treatment-by-exposure interaction was not observed
(p=0.529). The significant treatment-related effect occurred because, when collapsed across
all exposures, the Treatment A or B groups did not fully recover, whereas the Treatment C
group did (Figure 5a). The exposure effect occurred because the second exposure recovery
period was significantly (p=0.029) different than the percent recovery for exposures 1, 3, and 4
(Figure 5b).
Subjective Evaluations of Pulmonary Waveforms Alterations of the normal sinusoidal
respiratory waveforms were classified as either looking like sensory or pulmonary irritation, or
abnormal, but not either of the above (disruption index). The existence and severity of altered
respiratory waveforms were evaluated for each of the three exposure periods. In general,
between 70 and 90% of all periods in an exposure had less than four one-minute scores
greater than zero (i.e., no evidence of sensory-like or pulmonary irritation). Analysis indicated
that significantly more respiratory waveforms that looked like slight sensory irritation occurred
EPA - 49
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during the control period (p= 0.012) and during the second and fourth exposure periods
(p=0.005) for the Treatment B group. Figure 6 shows the incidence of sensory irritation during
the exposure period when the data are collapsed across the four exposures.
Although a significant treatment-by-exposure interaction was observed for pulmonary
irritation during the exposure period, the interaction occurred because of differences between
Treatment A and C during the fourth exposure. When collapsed across exposure periods, no
significant differences between Treatment B (zero-grade air group) versus A or C were noted
(Figure 7).
There was also a significant (p=0.001) treatment-by-exposure interaction for the control
period when the disruption index was analyzed. This interaction occurred because Treatment
A showed more disruptions than Treatment B during the third exposure. Although no
differences in the disruption index were observed during the exposure period, a marginally
significant (p=0.051) treatment effect was revealed during the recovery period because
Treatment A had more disruptions than Treatment B (p=0.039) when the data were collapsed
across all four exposures. The disruption index for all periods combined (control, exposure
and recovery) and collapsed across all exposures (1 through 4) is shown in Figure 8.
Postexposure appearance Although not part of the formal functional observational battery,
mice were observed for at least 15 minutes after each exposure. During this time, any
unusual behavior or abnormal appearance was noted. During most of these postexposure
observation periods, mice would initially be still and then after 2-3 minutes they would begin to
groom and drink water. No abnormal behavioral observations were noted for any of the
animals. However, their appearance was abnormal. These effects, which included
EPA-50
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lacrimation (tearing), dilated or hemontiaged blood vessels of the pinna (outer ear), and facial
swelling, were observed in all experimental groups after most exposures. Analysis of the
incidence of occurrence indicated that there were no differences in incidence among treatment
groups for the facial swelling and hemonrhaged pinna. For lacrimation (Figure 9) and dilated
pinna vessels (Figure 10), significant treatment differences were observed. Treatment C was
found to have significantly (p<0.001) less lacrimation, but more dilated pinna vessels
(p<0.001) than Treatment groups A or B. No treatment-by-exposure interactions were
significant on these observations.
Functional Observational Battery A summary of the data for all of the measures of
the functional observational battery is included in Appendix A. All tests were conducted at the
prescheduled times. For ease of interpretation, the functional observational battery tests have
been sorted into groups representing the neurological functions they most closely represent
(1) neuromuscular function, (2) sensorimotor function, (3) general activity and excitability, and
(4) general appearance and other measures.
Some tests were not affected at any time; they showed no variability across groups
and therefore were not subjected to statistical analysis. These were: Body Tilt, Forelimb
Placing, Tonic Movements, Excessive Vocalizations, Diarrhea, Gasping, Cyanosis,
Exophthalmus, and Salivation. The results of statistical analysis for the remaining behavioral
measures are shown in Table 8. Significant or equivocal differences between groups were
detected in 8 of 18 analyses.
EPA - 51
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Table 8. Summary of Statistical Outcomes for Functional Observational Battery Data*
FOB Measure Treatment
II (TRTMT)
Neuromuscular
Jar Task
Grip Strength
Body Tone
Righting Reflex
Body Posture
Ataxia Score
Gait Score
Inverted Screen
Test
Missteps
Sig.
NS
NS
NS
NS
Equiv.
NS
NS
NS
TRTMT-by-Time Day 1
Sig.
NS
NS
NS
NS
NS
NS
NS
NS
Sig.
A*B
Day 2
Sig.
A*B
C*B
TRTMT
NS
Sensorimotor
Air Putt
Response*
Click Response
Tail Pinch
Response*
NS
Sig.
Equiv.
NS
NS
NS
Sig.
A*B
Sig.
A*B
OB
Activity and Excitability
Activity
Alertness
Handling
Reactivity
Rearing
Clone
Movements
NS
NS
NS
NS
NS
Sig.
Sig.
NS
Equiv.
NS
NS
NS
NS
Other
Tilted Screen | Sig.
NS |
Equiv.
C*B
NS
Sig.
A*B
•DDaDUIiy Ol BiyniiiwciiH UIIIOIWIMO WBIWV
Sig.«p<0.05
NS « p>.10
Equiv. = 0.05
-------
Neuromuscular function Performance on the jar task was affected by treatment (Figure
11), showing a significant treatment-by-time interaction (p=0.042). Following the second
exposure (day 1), mice in the treatment A group had more falls than did Treatment B (air-
exposed group, p=0.002). This difference was apparent again after the fourth exposure, but
the contrast was not quite significant (p<0.052). Group C was significantly different from
Treatment B, but this was because the Treatment C mice had less falls than the air-exposed
group (p=0.017). Thus, more falls occurred after exposure to Treatment A than with
Treatment B, and better performance was observed with Treatment C after the last exposure.
The overall analysis of ataxia score produced a marginal treatment effect (p=0.084).
However, when the treatment data were collapsed across days, the significant effect (p=0.051)
was between Treatment A and C. Thus, exposure to carpets did not produce effects on this
measure that could be differentiated from the air-exposed mice.
One mouse in the Treatment B group dropped the screen twice, after both the second
and fourth exposures. There were some mice with slightly decreased body tone in all
treatment groups. In addition, there was one instance of slightly slow righting in each of
Treatments B and C, and one hunched posture in Treatment B. Four, five, and six mice
displayed somewhat abnormal gaits (score of 3) in the Treatment A, B, and C groups,
respectively; one mouse in Treatment B and one in Treatment C also showed marked gait
abnormalities (score=4). For all these measures, however, there were no statistically
significant differences in the distributions of these scores across treatment groups. Thus,
although there were some measures that showed deviations from preexposure values in these
mice, these differences were equally distributed across all treatment groups. When there
were only one or very few mice affected, most commonly it occurred in the Treatment B
EPA - 53
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group.
Sensorimotor measures There was a dear preexposure difference between treatment
groups (p=0.01) in the response to the tail pinch, and responses to the air-puff stimulus
showed a marginal (p=0.061) differences on the day before testing. Therefore the data for
these measures were analyzed using the change from preexposure values. A marginally
significant overall treatment effect (p=0.066), but no treatment-by-time interaction, was
detected in the tail pinch response; there were no changes in the air-puff response. Mice
exposed to either carpet showed less increases in response to the tail-pinch than controls;
that is, mice in Treatment B showed more increased responses than did mice in either
Treatment A or C (see Figure 12). However, the data for mice in Treatments A and C were
most like their pre-exposure values, and therefore the significance of this difference is
questionable.
There was a significant overall treatment effect with the dick response (p=0.004), but
no treatment-by-time interaction. Further analysis revealed that mice in Treatment A showed
less reactivity to the dick stimulus than did mice in Treatment B. Figure 13 shows that
following Treatment A, there were more responses rated as "slight" (rank=2), whereas with
Treatment B, more mice showed "dear" (rank=3) responses. However, as with the tail pinch
response, the scores in group A mice were more like their own preexposure data than were
the Treatment B group's data.
Activity and Excitability Changes in the ranking of handling reactivity and the number of
rears were the same in all treatment groups. One mouse in Treatment A and one in
Treatment B showed slight quivers after the fourth exposure. None of these measures were
EPA-54
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statistically significant
The overall treatment-by-time interaction for activity level was significant, but there
were no significant differences in rank distribution on any one day. A trend was detected after
the fourth exposure (rx.0772), at which time the mice in Treatment A showed lower activity
levels than did those in Treatment B.
The treatment-by-time interaction was also significant for the scoring of alertness
(p=0.024). Univariate ANOVAs showed that on day 2, the mice in both Treatments A
(p=0.007) and C (p=0.029) showed lower scores than did those in Treatment B. Review of
the data (Figure 14) showed that this difference was due to more Treatment B mice appearing
hyper-alert, which was only observed in one mouse before exposures began. Treatment C
mice, in fact, showed no changes in level of arousal across days, whereas two Treatment A
mice showed slightly lower values on day 2.
Other Measures There was a significant overall difference between treatment groups on
the direction taken on the tilted screen (p=0.03), but no treatment-by-time interaction.
Collapsing the data across days, it appeared that more mice went down the screen (2 or 3
times out of 3 trials) following exposure to Treatment A than did after exposure to Treatment
B.
General Appearance After exposures, almost all mice showed facial swelling,
lacrimation, dilation and hemorrhaging of pinna vessels, and some chromodacryorrhea
(reddish tears). Although these signs were recorded during the behavioral tests as well as
upon removal from the animal exposure chamber, the analysis of the data was conducted on
EPA - 55
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the observations made immediately after removal from all four exposures (see above).
Postmortem Evaluation Two separate one-way ANOVAs were used to analyze the
postmortem data. The first analysis examined differences between the three treatment groups
(A, B, and C), whereas the second analysis examined the three treatment groups and also
included the non-exposed cage-control mice.
Gravimetric data Gravimetric data were obtained during the necropsy for six tissues: brain,
heart, liver, kidneys, thymus, and spleen (Table 9).
Table 9. Organ Weight Data by Treatment Group
Treatment
Control
A
B
C
Brain
0.43±0.02
0.42±0.03
0.440.02
0.4310.02
Heart
0.14±0.01
0.13±0.01
0.13±0.01
0.1310.01
Liver
1.4610.13
1.2510.08
1.29±0.ir
1.18*0.07
Kidney
0.4310.04
0.3810.03
0.4010.05
0.4110.04
Thymus
0.0810.02
0.0610.02
0.0610.02*
0.0610.01
Spleen
0.1010.02
0.0810.02
0.0910.03
0.0910.01
(old number indicates significant difference from I reatment B.
* Indicates significant difference from non-exposed unrestrained cage control group.
Significant treatment differences in organ weight were found only for liver weight
(p=0.033). This difference occurred because Treatment C livers weighed less (p=0.021) than
Treatment B (air-control group). When liver weights were corrected for individual differences
in body weight, the difference was only marginally significant (p=0.055); however, there were
no significant differences in body weight between treatment groups.
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Analysis of treatment effects compared to the non-exposed control indicated that there
were significant treatment effects for liver (p< 0.001) and thymus (p=0.027). Both liver and
thymus weights of the non-exposed controls were larger than any of the treatment groups and
were significantly different from Treatment B.
Hemoglobin Measurements Three parameters were analyzed to evaluate potential
differences in the oxygen-carrying ability of blood from carpet-exposed mice. Comparison of
the three treatment groups indicated a marginally significant (p=0.059) effect of treatment on
hemoglobin concentration. When the non-exposed control group was included into the
analysis as an additional treatment group, significant differences in hemoglobin concentration
were observed (p=0.026). The analysis indicated that the non-exposed and Treatment B
groups had significantly more hemoglobin than the Treatment A or C groups (Figure 15), but
Treatment B was not different from the cage control. The other two parameters that were
evaluated, %methemoglobin and %carboxyhemoglobin, were found not to be significantly
different by treatment with or without the non-exposed controls added into the analysis.
White cell differential Analysis of the differential white cell percentages in peripheral
blood indicated that there was an overall treatment effect for neutrophils (p<0.001) when
examining the three treatment groups and the non-exposed controls (Table 10). Significantly
more neutrophils were in the peripheral blood of the Treatment B mice (p<0.001) than for the
non-exposed controls. There were no significant differences between Treatment B and
Treatments A or C.
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Table 10. Peripheral Blood White Cell Differential
%Monocyte
%Neutrophil
%Lymphocyte
%Eosinophil
%Unknown
Control
9.0 ± 12.0
15.0 ±5.1
55.9 ±15.1
1.7 ±2.3
18.5 ±8.6
Treatment A
10.7 ±7.8
49.7 ± 22.1
31.0 ±16.4
0±0
8.3 ±1.5
Treatment B
5.9 ± 3.0
40.2 ± 9.0"
42.7 ± 12.2
0.4 ± 0.7
10.7 ±4.7
Treatment C
4.3 ± 2.4
46.7 ± 8.9
41 .7 ± 7.2
0.1 ± 0.4
7.1 ± 4.9
maicates significant amerence tram non-exposea unres ramea cage control group,
Serum Clinical Chemistry Sixteen measurements indicative of liver, kidney, heart, and
stress-related responses were evaluated in the serum of treatment mice and non-exposed
controls (Table 11). Of these 16 measurements, three were found to be significantly different
in the three group treatment comparison. Total serum proteins (p=0.003), albumin (p=0.002)
and cholesterol (p=0.023) were found to be lower in Treatment C compared to Treatment B
(Figure 16). Treatment A was not different from Treatment B for any of these measurements.
When the non-exposed control group was included into the analysis as an additional treatment
group, eight of the sixteen measurements showed significant treatment differences. Of the
three measurements that were significant in the three treatment group comparison, Treatment
C was most similar to the non-exposed control group for all three measurements.
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Table 11. Serum Clinical Chemistry Values
Parameter
N
TBIL
LOH
ALT
AST
CREA
BUN
5-ND
GLUC
ALP
PRO
ICD
ALB
TRIG
CHOL
SDH
BILE
Control
12
0.429 ± 0.130
541 .7 ±187.8
37.1 ± 19.9
75.9 ± 24.6
0.583 ± 0.083
19.7 ±6.2
22.3 ±5.4
236.8 ±235
138.6 ± 27.4
4.90 ±0.38
20.7 ±11.1
2.93 ±0.21
89.1 ± 26.6
1115 ±16.9
15.0 ±3.1
45.4 ± 9.6*
Treatment A
8
0.526 ± 0.359
6335 ± 127.3
60.5 ± 24.3
123.0 ±32.7
0.614 ±0.1 77
29.0 ± 7.0
24.0 ± 4.5
207.9 ±18.0
138.6 ±29.2
5.44 ± 0.46
33.0 ± 6.9
3.35 ± 0.40
60.4 ± 12.3
136.3 ±24.0
17.1 ±6.7
103.0 ±103.8"
Treatment B
8
0.446 ± 0283
773.3 ± 356.0
475 ±21 .3
108.6 ±37.4*
0.603 ± 0.074
30.8 ± 72*
31 .4 ±17.3
21 4.8 ±22.9*
157.8 ±11. 6
5.48 ±0.36*
38.0 ± 9.0*
3.45 ± o.ir
56.7 ± 10.9*
146.1 ± 13.6*
18.3 ± 2.01
222.0 ±222.0"
Treatment C
8
0.436 ±0.1 07
806.1 ± 332.6
49.0 ± 152
157.8 ±61 5
0.546 ± 0.153
33.7 ± 12.3
26.4 ± 9.0
198.0 ±18.8
143.8 ± 29.3
4.83 ± 027
37.6 ± 13.7
2.93 ± 020
45.7 ± 132
115.7 * 22.7
16.9 ± 3.1'
2282 ± 295.8"
numbers indicate a signiricant difference irom i reaimern a.
* Indicates significant difference from non-exposed unrestrained cage control group.
**** Insufficent serum was available, N= 7,5,2, and 6, respectively
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Nasal and Bronchoatveolar Lavage Chemistry Four parameters were measured from the nasal lavage
- ascorbic acid, uric acid, glutathione, and total protein. Of these measurements, uric acid was not
detectable in the nasal lavage and no significant treatment effects occurred for ascorbic acid or total protein.
Glutathione obtained from the nasal lavage was significantly (p=0.021) greater in Treatment B than in
Treatment A, but not different than Treatment C or the non-exposed control group (Figure 17). Only two
parameters were examined in the bronchoalveolar lavage of mice - total lavageable protein and lactate
dehydrogenase. A significant (p<0.001) treatment effect for total protein was observed, indicating that
Treatment B had less protein in the lavage than Treatments A or C. However, the lavageable protein in
Treatment B was also significantly (p=0.027) less than the non-exposed controls (Table 12). No differences
in lactate dehydrogenase were noted.
Table 12. Lung Lavage Chemistry
Controls
Treatment A
Treatment B
Treatment C
Total Protein
136 ±36
144*17
108 ±15*
157 ±26
Lactate Dehydrogenase
33.5 ±8
46 ±15
41 ±15
43 ±10
Bold numoers indicate a significant oirrerence rrom i reatment b.
Indicates significant difference from non-exposed unrestrained cage control group.
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Lung Lavage White Cell Differential Between 60,00 and 100,000 cells were removed from the lungs
of both non-exposed and exposed mice, with the alveolar macrophages being the most prevalent
(approximately 80% of cells, Table 13). Analysis of variance indicated a significant treatment effect for the
number of lymphocytes when either the three treatment groups were analyzed (p=0.012) or when the non-
exposed control was an additional group in the analysis (p=0.003). No significant difference was detected
in a comparison of the number of lymphocytes in Treatment B versus the non-exposed control group.
Treatment A, but not C, had significantly (p=0.027) more lymphocytes than Treatment B.
Table 13. Lung Lavage White Cell Count and Differential (xlO4) / ml of Bronchoalveolar Lavage
White Count
Macrophage
Neutrophil
Lymphocyte
Epithelial
Unknown
Control
69.6 ± 33.8
49.1 ± 18.8
0.51 ± 0.41
0.7 ±0.1
3.1 ± 2.4
8.7 ±7.3
Treatment A
102.8 ±67.7
81 .9 ±562
0.42 ± 0.51
2.8*1.8
22 ±1.9
14.6 ±17.7
Treatment B
61 .6 ±20.4
52.7 ±18.6
021 ± 0.26
0.7 ±0.5
1.3 ±1.6
7.1 ±3.7
Treatment C
100.0 ±57.9
78.2156.5
0.15 ± 0.36
1.0 ±1.0
1.9 ±2.6
14.7 ± 155
Bora number indicates significant airrerence rrom i reatment B.
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Histopathology Several microscopic lesions were observed in treatment groups that either were not
diagnosed or occurred with a lower incidence or severity in the non-exposed, unrestrained control mice.
The incidences of these histopathological lesions are reported in Table 14. Minimal to mild
necrosis/inflammation of the heart and its arteries, focal liver necrosis, pituitary hemorrhage, thymic cortical
necrosis, and hemorrhage/inflammation of the pinnae were common findings in all treatment groups.
Table 14. Incidence of Selected Histopathological Lesions in Treated and Non-Exposed Mice
Treatment
Total Number of Tissues Examined
Heart, Necrosis
Heart Artery Inflammation (minimal)
Heart Artery Medial Necrosis/Hemorrhage
(minimal)
Heart Artery Medial Necrosis/Hemorrhage
(slight/mild)
Heart Artery Medial Necrosis/Hemorrhage
(moderate)
Liver, Focal Necrosis
Pituitary, Pars Distalis, Hemorrhage
Thymus, Cortical Necrosis (slight)
A
8
1
3
2
0
0
3
2
6
B
8
4
5
2
2
1
2
4
7
C
8
2
5
0
1
2
1
4
4
Non-
Exposed
12
0
3
1
0
0
0
0
0
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Minimal myocardial necrosis was observed in mice from all of the treatment groups, but was not
observed in the non-exposed, unrestrained controls. The incidence of necrosis was greatest with Treatment
B. This lesion was characterized by relatively small focal accumulations of cellular debris with an
associated minimal infiltration of mononudear cells and neutrophils replacing myocardial fibers.
Inflammation and medial necrosis/hemorrhage in the arteries of the heart were also observed in
some mice from all groups. The incidence of inflammation, and to a lesser degree, the medial
necrosis/hemorrhage was greatest in Treatments B and C. The severity of the medial necrosis/hemorrhage,
which ranged from minimal to moderate, was also greatest in the affected mice in Treatments B and C.
The inflammation and medial necrosis/hemorrhage involved the coronary arteries and their branches and
rarely the large elastic arteries (aorta). The inflammatory changes were characterized by a minimal
infiltration of mononudear cells and lesser numbers of neutrophils in the perivascular area and occasionally,
the walls of the arteries. Medial necrosis/hemorrhage consisted of the replacement of smooth musde fibers
in the tunica media with an eosinophilic fibrinoid material and occasionally erythrocytes. In most instances,
the changes were accompanied by a small amount of a pale basophilic, somewhat granular substance in
the perivascular area.
Focal hepatic necrosis was diagnosed in one to three mice of each treatment group, but in none of
the non-exposed control mice. The incidence of this lesion was greatest in Treatment A. The focal
necrosis was typically just beneath the hepatic capsule and was occasionally accompanied by a minimal
infiltration of inflammatory cells.
Hemorrhage of the pars distalis of the pituitary gland was also diagnosed in some mice in each of
the treatment groups, but in none of the non-exposed control mice. The incidence of pars distalis
hemorrhage was greatest in Treatments B and C. This lesion was frequently bilaterally symmetrical in
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distribution and was characterized by the focal accumulation of extravasated erythrocytes with secondary
coagulative necrosis.
Thymic cortical necrosis was diagnosed in all mice. Most of the mice in the treatment groups (A, B,
and C) had mild cortical necrosis compared to the minimal necrosis observed in the non-exposed mice.
Minimal cortical necrosis was characterized by occasional pyknotic or karyorrhectic nuclei and occasional
small foci of nuclear debris. This degree of cortical necrosis was considered to be due to normal cell
turnover (necrobiosis). In contrast, mild cortical necrosis, as seen in the treatment groups, was
characterized by more frequent small foci of nuclear debris.
The pinna of several mice in each of the treatment groups had either hemorrhage and/or subacute
inflammation. Both lesions frequently occurred together in the pinna and consisted of extravasated
erythrocytes, with or without an infiltration of neutrophils and mononuclear cells. The incidences of these
lesions were greatest in Treatment groups B and C. Subacute inflammation was not diagnosed in any of
the non-exposed control mice; however, minimal hemorrhage was present in the pinna of one non-exposed
control mouse.
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Discussion
Interpretation of the Results Overall, more than 70 different measurements were evaluated for each
mouse and statistically significant differences were detected among the three treatment groups. However,
many of these differences, although statistically significant, were not consistently observed in any carpet-
exposed treatment group and those differences from Treatment B may be of little or no biological
significance. For some parameters, it is not dear that the significant statistical or biological difference
represents an adverse effect. Furthermore, significant differences were also observed between Treatment B
(zero-air exposed group) and the non-exposed, unrestrained cage controls, indicating that the exposure
procedure itself had significant consequences associated with it. A discussion of the two primary
evaluations (measurements of irritancy and the neurobehavioraJ screen) and the postmortem assessment
follows.
Sensory and Pulmonary Irritation Irritation was evaluated (1) by an adaptation of the ASTM
method, (2) by examining the reflex changes in breathing frequency for individual animals, and (3) by
subjectively inspecting and scoring the respiratory waveform morphology. The ASTM method of combining
the four animal responses seemed to indicate that there was a greater decrease in frequency of breathing
(a sign of irritation) with increased numbers of exposure that was somewhat more prominent in Treatments
A and C than for Treatment B (the air-exposed group). This finding would provide minimal support that
irritation was associated with carpet exposure. However, the rate decreases observed would only be scored
slight, at best, according to the ASTM criteria (Table 7). To gain a further, more in-depth understanding of
the rate decrease, individual animal data were used as input to a statistical model that allowed us to
examine and separate treatment-related effects from exposure procedure effects. Visual inspection of
Figure 4a emphasizes what the conclusions from the statistical analysis indicated, which was that there
were no differences in response among the three groups to any of the four exposures. The most
conspicuous difference between groups (Figure 4a) was observed during the first exposure when more mice
EPA-65
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from Treatment B (air-exposed group) had greater decreases in frequency of breathing than carpet-exposed
mice (Treatments A or C); however, this difference was not significant. Statistical differences in breathing
frequency during the recovery period, which could be interpreted as a manifestation of toxicity, were also
observed. However, the percent difference in frequency from the control period ranged from 0 to 3% for the
three treatment groups (Figure 5a), a difference that would not be considered biologically significant
Although the rate determinations discussed above were the primary determinants of irritancy,
subjective analyses of respiratory waveforms were also evaluated to distinguish the type of irritant effect
(i.e., sensory, pulmonary, or mixed). Using a very aggressive method to detect altered waveforms,
evidence of sensory and pulmonary effects was observed for all treatment groups. During the exposure
period, a greater incidence of sensory irritation was found for Treatment B (air-exposed) as compared to
Treatments A or C (carpet-exposed), during exposures two and four. The rate analysis described above,
however, indicated that the largest rate decrease for Treatment B occurred during the first exposure period.
Thus, in contrast to the evidence of sensory irritation based on waveform morphology, the altered
waveforms did not translate into a decrement in breathing frequency. This would indicate that biologically
significant sensory irritation did not occur according to the ASTM procedure for which rate is the ultimate
determinant of effect. Although this may seem discordant it can be explained by the waveform analysis
technique. For example, during the scoring of the waveforms, if a pattern indicative of sensory irritation
appeared for several breaths (3 to 5), the entire minute rating period was scored as showing evidence of
sensory irritation. However, if only five breaths had prolonged early expiratory pauses characteristic of
sensory irritation, there would be minimal or no impact on the median rate calculated for the approximately
220 breaths. Additionally, it was observed that for many of the one-minute segments in which altered
waveform morphology was observed, these periods were also accompanied by periods of rapid breathing.
Juxtaposition of breaths with decreased rates and increased rates during the same minute would further
dilute the impact of the expiratory pause to cause a decline in frequency of breathing.
EPA-66
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Evidence of possible "pulmonary* irritation was also detected in all three treatment groups, ranging
from a slight rounding at the end of expiration (score=1, slight) to a pause between breaths (score=2,
moderate). Although there was one significant effect noted during the four exposure periods, it was
attributed to a difference between Treatments A and C and not Treatment B. Visual inspection of the data
collapsed across all exposure periods (Figure 7) would suggest that Treatment A had more pulmonary
irritation than Treatment B, and that Treatment C had the least among the three groups; however, these
differences were not significant. Again, evidence of pulmonary irritation based on waveform morphology did
not correspond with observed decrements in frequency of breathing. However, in contrast to sensory
irritation, pulmonary irritation is not solely diagnosed by a decrease in breathing frequency. Therefore, it is
possible that Treatment A showed a slight pulmonary irritant effect
Finally, in an attempt to quantify other abnormal breath shapes that would not be defined as sensory
or pulmonary irritant-like, a disruption index was formulated. Most of these abnormal waveforms were
movement artifacts related to the animals struggling in the plethysmograph, but it was of interest if such
struggling would increase or decrease in association with an inhaled toxic exposure. The statistical
analyses indicated mice in Treatment A were somewhat more disrupted than Treatment B during the control
and recovery periods, but not during the exposure periods, whereas no differences in the index were
observed between Treatment C compared to B. The interpretation of the relative adversity of this finding is
uncertain.
Subjective Appearance and Functional Observational Battery After each exposure, both the
behavior and the general appearance of the animals were observed. Formal testing using the functional
observational battery was applied only after the second and fourth exposure. In general, many of the
differences from the preexposure baseline performance were either similar across treatment groups or else
were more prominent in the air-exposed group (Treatment B). There were also several significant
differences between Treatments A and C, with Treatment C appearing more benign. Indeed, as these data
EPA-67
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were being analyzed and before the code was broken, It appeared that Treatment C was the control.
Four prominent findings related to the animal's appearance were observed after each exposure.
Virtually all of the animals had some lacrimation and dilated vessels of the pinna (outer ear), whereas many
had facial swelling and evidence of hemorrhaged vessels within the pinna. There were statistical
differences between treatment groups indicating that more mice In Treatment C (carpet-exposed) had
dilated vessels of the pinna and more mice in Treatment B (air-exposed) had lacrimation. The occurrence
of these four effects and the treatment group differences may, in part, be attributed to the high temperatures
and low humidity in the animal exposure chamber (Tables 5 and 6). Higher temperatures occurred during
both of the experiments involving Treatment B (range, 25.0 to 26.4 °C) and were the worst for the first
experiment using Treatment C (range, 26.1 to 30.0 °C). Vessel dilation is one mechanism that rodents use
to dissipate heat.
The significant treatment effects detected using the functional observational battery were not
clustered In any one neurological domain. The only significant neuromuscular effect was the altered jar task
performance observed in Treatments A and C. The increased number of falls observed in Treatment A
could be interpreted as a clear, though not severe, effect On the other hand, the Improvement of
performance in Treatment C, while also significant, would be difficult to Interpret as an adverse effect. The
other neuromuscular measures, which should also detect vestibular changes, weakness, or IncoordlnatJon
that might compromise the ability to climb on the jar, were not altered by carpet exposure. This lack of
correlative findings reduce the certainty of a significant neuromuscular/vestlbular effect of Treatment A.
The sensorimotor changes observed in Treatments A and C, although statistically significant, could
not be considered clear evidence of carpet toxldty. Although It appeared that mice exposed to carpet
(particularly Treatment A) had lower reactions to both the click and tall-pinch responses than those exposed
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to zero-grade air, in reality the Treatment B mice responded more vigorously than they did before
exposure. Moreover, In all groups, most of the responses were scored as "slight" or "clear", which are the
responses expected for "normal" mice. Therefore, It is difficult to conclude that the differences in response
between treatment groups was an adverse consequence of carpet exposure.
On the open field test, the only clearly significant effect was an apparent decrease In level of
alertness In mice exposed to carpets. The same situation applies, however, as with the sensorimotor
responses in that the effect was due not to lower scores in carpet-exposed mice, but an increase In the
score of Treatment B mice, when compared to their preexposure data as well as what has historically been
expected as "normal" behavior.
Postmortem Evaluation Although a crude indicator of toxlclty, a treatment-related change In
body weight Is often a sensitive Indicator of effect In this study, body weights declined with exposure, but
there were no treatment-related differences (Figure 2). Uver and thymus weights were found to be less
than those of the non-exposed cage control group with a somewhat larger reduction in liver weight in the
Treatment C group. Typically, liver damage produces an increase in liver weight, whereas the factors
responsible for loss of body weight (physical, humidity, and temperature stress; food and water deprivation)
may account for the reduction between treatment groups and the non-exposed animals. However, even
after corrections for the differences in body weight, Treatment C mice were still significantly affected (10%
decrease), suggesting a small, treatment-related effect.
Because the lung Is the portal of entry for the carpet vapor exposure, It might be a primary target for
toxidty. Thus, several screening measurements were conducted to evaluate the Impact of exposure.
Lactate dehydrogenase (LOH) In the BAL was unaffected, Indicating that there was no direct cytotoxlctty to
pulmonary cells. Lavage fluid protein was increased in Treatments A and C compared to Treatment B.
'EPA-69
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However, the BAL protein from the non-exposed cage control group was higher than that observed with
Treatment B. Thus, although an increase in BAL protein might be considered evidence of increased lung
permeability (leakage of plasma protein from the blood to the lung surface), the BAL protein values of the
cage control group would suggest that the effect was not biologically significant. The only other significant
effect observed in the BAL was a small, but significant, increase in the number of lymphocytes for
Treatment A, which might suggest an increased immune system activation from an antigenic protein or viral
infection. However, no other physical (particle concentration), microbiological, or histopathological evidence
would support these findings.
Histopathological examination revealed several differences between the cage control group and the
three treatment groups (Table 14). Myocardial necrosis and necrosis of the small heart vessels were more
frequently observed in Treatments B and C. Liver necrosis was seen in 3 of 8 mice exposed to Treatment
A, with Treatments B and C showing less of an effect, respectively. Similarly, thymic cortical necrosis was
observed in all mice, including the cage controls; however, the severity was much greater in the treated
mice, especially in Treatments A and B. Hemorrhage of the pars distalis of the pituitary gland was
observed predominately in Treatments B and C (50% incidence), but was also observed in Treatment A
(25% incidence). The origin of these effects is uncertain, but is possibly related to the food and water
deprivation during exposure, and physical restraint stress associated with the exposure procedure employed
in this study. Overall, the histopathological effects were minimal such that the pathologist could not dearly
distinguish which group was the air-exposed treatment group (i.e., Treatment A was incorrectly identified as
the air-exposed control group).
Several measurements were obtained from the blood that focused on alterations of hemoglobin,
white blood cell population profiles (differential), and serum chemistries. A marginally significant decrease in
hemoglobin was observed for Treatments A and C compared to both Treatment B and the non-exposed
EPA - 70
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caged controls. A decrease in hemoglobin content could signify a decrease in oxygen-carrying capacity;
however, only a 6% decrease was observed, a change that would not be considered clinically significant.
No significant differences were seen in the peripheral blood white cell differential among treatment groups.
When compared to the cage controls, all of the treatment groups had significantly elevated percentages of
neutrophils (approximately threefold increases). Although this may be a stress-induced demargination of
neutrophils, it could also signal an acute infection, chemical intoxication, acute hemorrhage or hemolysis, or
acute tissue necrosis. However, correlative pathology (described above) and serum enzyme chemistries
(described below) do not support these alternate interpretations. The fact that this condition was observed
equally in all treatment groups, including the air-exposed group (Treatment B), suggests strongly that it is
related to the exposure procedure (Table 10).
Significant alterations in serum chemistries were also observed (Table 11). Total protein, albumin
and cholesterol were lower in Treatment C than in Treatment B; however, Treatment C was not different
from the cage controls for these three measurements. On the other hand, these three serum chemistries
were significantly greater in Treatments B (air-exposed) and A than in the cage controls. One common
cause of increased serum proteins is dehydration, which certainly could have resulted from the exposure
procedure. Increased cholesterol is nonspedfically associated with more chronic changes in the
cardiovascular, hepatic, kidney, and pancreatic systems. Why Treatment C was less affected is uncertain;
however, the Treatment C-related decrease in liver weight noted above may correspond to the decreased
ability of this treatment group to express these particular proteins. What toxicity might be associated is
unclear, especially in light of the similarity in these measurements to the cage control mice.
Five additional clinical serum chemistries were analyzed (AST, BUN, glucose, ICD, and triglycerides)
and were found to be significantly different between the unrestrained, non-exposed cage controls and the
restrained and air-exposed controls (Treatment B). Increases in AST, BUN, and ICD were observed,
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whereas glucose and triglycerides were decreased. Although these differences were statistically significant,
they are difficult to ascribe to a specific toxicity because the magnitude of most of the changes would not be
considered of biological significance in human clinical medicine. Interestingly, Treatment C showed an even
more pronounced difference from the cage controls than did Treatment B (Table 11), possibly reflecting the
altered liver weights and serum chemistry results discussed above.
Increased AST, in humans, is typically associated with myocardial infarction, muscle and liver injury,
acute liver necrosis, or acute pancreatitis, but also may increase with acute stress. Increased BUN occurs
with impaired kidney function, salt and water depletion, hemorrhage, stress, and acute myocardial infarction.
Glucose is decreased with hypoglycemia and liver disease, but is typically increased with stress. Liver cell
injury is associated with an increase in ICD, for which this measurement is both sensitive and specific;
however, malnutrition has been shown to sometimes increase ICD. A decrease in triglycerides is
associated with stress and malnutrition. It is likely that all of the effects can be accounted for by food and
fluid restriction and restraint-induced stress. However, it is possible that the treatments may also have
some effect on liver and heart tissue. Table 15 shows the significant clinical chemistry and peripheral blood
responses observed in Treatment B as related to potential causes of these changes from the human clinical
literature (Wallach, 1978).
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Table 15. Potential Causes of Clinical Chemistry and Blood Neutrophil Responses
Measured
Parameter
AST
BUN
Glucose
Protein
ICD
Albumin
Triglycerides
Cholesterol
% Neutrophils
Treatment
B
t
t
4
t
t
t
4
t
T
Stress
t
t
t
t
4
t
Dehydration
t
t
Food Depri-
vation
4
4
t
4
4
T
Liver
Injury
T
4
4
t
4
t
Heart
Injury
t
t
t
T
EPA-73
-------
Potential Confounds The carpet study protocol was founded on the premise that two different
carpets previously shown by Anderson Laboratories to induce severe neurotoxicity or death would produce
similar effects when retested under analogous conditions in the laboratories of both Anderson and EPA. In
addition, it was anticipated that carpet-related responses in exposed mice would be distinguishable from
sham, dean-air exposed control mice. The biological end points evaluated by EPA included those used in
the standard Anderson Laboratories protocol and several additional biomarkers indicative of systemic organ
function or toxicity. However, following carefully conducted experiments and after thorough analysis of the
study data, no biologically significant effects attributable to carpet vapor exposure could be discerned. All
treatment groups, A, B, and C, appeared essentially unaffected; only when the restrained clean-air controls
were compared to unrestrained, non-exposed cohorts did there appear a consistent difference. The
occasional small, but statistically significant changes in one or another biomarker in the actual carpet-
exposed groups, although notable, could not be reasonably attributed to a toxic syndrome. Although this
lack of response would appear to support the contention that the carpets tested simply did not possess
inherent toxicity, it would seem reasonable that various factors, which could have influenced the response or
its detection, be examined carefully as potential confounds.
Animal Model The use of Swiss-Webster (SW) mice in these studies must be considered a potential
source of substantial variability. The carpet-effect in the SW, an outbred strain, is inherently variable from
mouse to mouse (as reported by Anderson Laboratories), and with regard to the nonlethal effects described,
appears to be quite transient and sporadic. The strain of mouse used in the EPA portion of the study
(including gender, age and size, and even vendor) was identical to that used by Anderson. A complete
microbiological evaluation of the mice, both upon receipt and in sentinels, verified the health status and lack
of infection in the animals. The mice were housed in an AAALAC-certified vivarium upon receipt and care
was taken to avoid an infectious outcome over the course of the study (about 10 days including the 7-day
acclimatization). The Anderson mice, otherwise the same, were housed in a facility ostensibly clean and
EPA - 74
-------
healthy, but actually of unknown microbiological status (e.g., ducts, air conditioning, etc.). Whether some
relatively passive organism residing in the Anderson mice could render them 'susceptible" as compared to
those of EPA seems unlikely, but is unknown. Moreover, by what mechanism a frequently rapid-onset,
acute response might result from such an interaction is also unknown.
Exposure Factors The large variability in animal response described previously by Anderson, even within a
given run of four animals, raises the question of exposure. The exposure system used in these studies by
EPA was designed after that in use at Anderson Laboratories. The aquarium source chamber and plumbing
were identical. However, there were some essential differences in the actual exposure method used in the
EPA study compared to the standard Anderson exposure protocol. These differences arose from the desire
to standardize and better control the quality of the exposure Itself. The EPA tests used zero-grade bottled
air that was certified "clean" by the air chemistry co-investigators working on this project This bottled air
was humidified through a distilled water trap and was provided to a manifold connected to the inlet port of
the chamber. The chamber air was drawn from this manifold at a flow rate similar to that used in
Anderson's tests (7 LPM). The excess air was exhausted to the room. Hence, the air passing through the
source (carpet) chamber was as dean as possible (as certified in test samples). This procedure contrasts
with the unfiltered "room air" drawn into the chamber system at Anderson Laboratories. The contaminant
status of room air is probably quite variable, but previous spot samples that EPA chemists collected at
Anderson Laboratories did noi indicate unique components that would lead to the conclusion that this
difference in source air is the origin of the biologic differences. Similarly, it would seem unlikely that
streaming of air through the EPA chamber would somehow avoid entraining the diffusion-distributed vapors
that would have emanated from the carpet during the one-hour incubation or during the test run. However,
this possibility merits direct testing and validation because the inlet and exit outlets on the exposure
chamber are on the same face and no provision is made to mix air within the chamber.
EPA - 75
-------
After the carpet test series was completed, quality assurance checks revealed that the humidity
sensor used in the EPA test system had failed over the course of the study. This failure was not abrupt, but
was a slow loss of accuracy; previous experience with these sensors had never encountered a failure and
the effect on the dilution air adjustments was minor and not noticed. The result of the failure was that the
humidity, rather than being standardized at 50%, ranged from 18.7% to 29.2%. The effect of this on the
carpet emission is uncertain. Similarly, how this compares to the earlier runs with these carpets by
Anderson Laboratories is uncertain because Anderson Laboratories does not control humidity in the
laboratory and records room humidity with a simple "home-style" hygrometer. Given that Anderson
Laboratories has tested many of the sample carpets during the winter season, it is likely that the humidity
range of the EPA test was not unusually deviant from the typical conditions in Dedham, MA, though this
difference needs further investigation.
Each animal was sealed and secured into individual plethysmographs with its head protruding into
the glass exposure unit by a thin rubber dam with a sized hole. The standard head hole used in the EPA
tests was slightly smaller than that used in the Anderson Laboratories system, and the gauge of the rubber
dam was somewhat thinner. The hole size adopted was determined as a balance between the restraint
provided by the dam and the comfort of the animal. Ancillary tests conducted by EPA suggest that the
slightly smaller hole size may reduce breathing frequency slightly, but does not appear to affect the form of
the tidal breath trace, indicating no exogenous bronchoconstrictive stress on the animal. Because tidal
volume depth cannot be accurately measured in this plethysmographic arrangement, it is possible that there
was a slight difference in the depth of each breath between the laboratories involved, but it would seem
unlikely to be of such magnitude as to affect the dose to the animals and, hence, their response.
Carpet Factors The lack of response may reside in the heterogeneity of the carpets and the samples
thereof. The CPSC samples were selected randomly from various locations of the test carpets without
EPA • 76
-------
regard for use areas or other potential variables. With "carpet" being the toxicant as best as can be
currently defined, it seems quite likely that regions of the total carpet area would have less or none of the
factors contributing to the observations of toxitity by Anderson Laboratories. Even Anderson Laboratories
reports that some test results of carpet from the same source are not readily reproduced for reasons
unknown. Such a possibility suggests that retests of these carpets or parallel tests as are being conducted
at Anderson Laboratories as part of the overall test plan for the ORD Carpet Study could result in disparate
findings. At this point in time, there is no way of establishing a probability that carpet heterogeneity and
sample distribution events could occur. Additional potential confounding factors might arise from differences
in storage and handling of the original test samples as compared to the samples used in the ORD study,
but in light of the wide range of handling conditions prior to testing in Anderson Laboratories, this variable
would appear to not be a major confounder if reasonable care in storage of the samples is followed.
Conclusion There appeared to be no severe toxic effects associated with exposure to the off-gassing of
the two tested complaint carpets. Incidental findings, of statistical significance, but unlikely to be of
biological significance, were observed. Of all the effects that were observed, only the number of falls in the
functional observational battery would be suggestive of an "adverse" effect (Table 16).
EPA-77
-------
Table 16. Summary of lexicological Findings
Treatment A
TBAA*«MAM* D
reatment o
Treatment C
Adverse
Postexposure
Irritation
Irritation
Postexposure
Postexposure
Postmortem
Postmortem
Postmortem
Postmortem
Postmortem
Postmortem
Postexposure
Postexposure
Postexposure
Postmortem
Postmortem
Postmortem
Postmortem
Postmortem
t Falls off Jar
Possibly Adverse
i Frequency (ASTM)
t Pulmonary Irritation
A Hemoglobin
t BAL Lymphocytes
t Liver Necrosis
t Thymlc Necrosis
T Sensory Irritation
t Lacrtmation
t Hyper Alertness
t Heart Necrosis
t Heart Vessel Necrosis
t Thymlc Necrosis
Adversity Unknown
t Disruption Index
T BAL Protein
t Tall Pinch Response
t Click Response
T Pinna Hemorrhage
t Pituitary Hemorrhage
4- Frequency (ASTM)
4- Liver Weight
i Hemoglobin
t Heart Necrosis
t Heart Vessel Necrosis
T Dilated Pinna
t BAL Protein
4- Serum Protein/Albumin
4- Serum Cholesterol
t Pinna Hemorrhage
t Pituitary Hemorrhage
EPA - 78
-------
Approximately the same number of "possibly adverse* effects occurred in the three treatment
groups, with almost all treatment groups showing an effect in each of the three categories of measurements:
irritation, postexposure (including general appearance and the functional observational battery), and
postmortem evaluation (Table 16). Most likely, this indicates spurious findings relative to the number of
unprotected multiple comparisons performed. It is interesting that Treatment A, which showed some
evidence of pulmonary irritation (not significant) without a change in rate, also had an increase in BAL
protein and lymphocytes, whereas Treatment B, which showed more sensory irritant patterns, also had
more lacrimation. Treatment C had more postmortem signs of systemic toxicity (decreased liver weight and
small changes in serum chemistry), but had less histopathological signs of liver compared to Treatments A
and B. In conclusion, based on this assessment of irritation, neurobehavioral effects and a general toxicity
screen, there is no indication that exposure to off-gassing from these two carpets poses a toxicological
threat
EPA - 79
-------
References
Alarie, Y. (1973) Sensory irritation by airborne chemicals. CRC Critical Reviews in Toxicology, pp. 299-363.
Alarie, Y. (1981) Bioassay for evaluating the potency of airborne sensory irritants and predicting acceptable
levels of exposure in man. Fd. Cosmet Toxicol. 19:623-626.
Beaton, J.M., J.D. Prejean, R.D. Irwin, J.K. Dunnick, D.S. Baal, H.D. Giles, and AA Bartolucci (1990) The
neurobehavioral assessment of the effects of 28-day administration of acrylamide on B6C3F1 mice." Paper
presented at the 8th International Neurotoxicology Conference: Role of Toxicants in Neurological Disorders,
Little Rock, AK, October.
Bos, P.M.J., A. Zwart, P.G.J. Reuzel, and P.C. Bragt (1992) Evaluation of the sensory irritation test for the
assessment of occupational health risk. Critical Reviews in Toxicology 21(6):423-450.
Brown, LJ. (1980) A new instrument for the simultaneous measurement of total hemoglobin, %
oxyhemoglobin, % carboxyhemoglobin, % methemoglobin and oxygen in whole blood. IEEE Transactions
On Biomedical Engineering, V BME—E-27, 3, pp. 132-138
Creason, J.P. (1989) Data evaluation and statistical analysis of functional observational battery data using a
linear models approach. J. Am. Coll. Toxicol. 8:157-169.
Feldman, D.B. and J.C. Seely (1988) Necropsy Guide: Rodents and the Rabbit CRC Press, Inc. Boca
Raton, FL; pp 51-76
EPA-80
-------
Ohio, AJ., T.P. Kennedy, G.E. Hatch, and J.S. Tepper (1991) Reduction of neutrophil influx diminishes
lung injury and mortality following phosgene inhalation. J. Appl. Physiol. 71(2) 657-665
Irwin, S. (1968) Comprehensive observational assessment: la. A systematic, quantitative procedure for
assessing the behavioral and physiologic state of the mouse. Psychopharmacologia (Berlin) 13:222-257.
Moser, V.C., J.P. McCormick, J.P. Creason, and R.C. MacPhail (1988) Comparison of chlordimeform and
carbaryl using a functional observational battery. Fund. Appl. Toxicol. 11:189-206.
Moser, V.C. (1989) Screening approaches for neurotoxictty - a functional observational battery. J. Am. Coll.
Toxicol. 8:85-93.
Moser, V.C. (1990) Approaches to assessing the validity of a functional observational battery. Neurotoxicol.
Teratol. 12:483-488.
Moser, V.C. (1991) Applications of a neurobehavioral screening battery. J. Am. Coll. Toxicol. 10:661-669.
Moser, V.C. (1990) Application and assessment of neurobehavioral screening methods in rats. In Advances
in Neurobehavioral Toxicology: Applications in Environmental and Occupational Health, edited by B.L
Johnson. Chelsea, Ml: Lewis Publishers; pp. 419-432.
Operator's Manual IL282 CO-Oximeter, Instrumentation Laboratory, inc. (1977)
Lexington, MA
EPA - 81
-------
Smialowicz, R.J., J.E Simmons, R.W. Luebke, and J.W. Allis (1991) Immunotoxicological assessment of
subacute exposure of rats to cartx>n tetrachloride with comparison to hepatotoxicity and nephrotoxicity.
Fund. Appl. Toxicol. 17: 186-196.
SAS Institute Inc. (1990) SAS/STAT User's Guide, Version 6. Gary, NC/ SAS Institute. 1990.
Standard Test Method for Estimating Sensory Irritancy of Airborne Chemicals. American Society for Testing
and Materials, ASTM Designation E-981-84 (also appendix A)
Tegeris, J.S. and R.L Balster (submitted) Comparison of the acute behavioral effects of alkylbenzenes
using a functional observational battery in mice. Fund. Appl. Toxicol.
Tegeris, J.S. (1991) "Acute Behavioral Effects of Alkylbenzenes Evaluated Utilizing a Functional
Observational Battery and Schedule-Controlled Operant Behavior.* Ph.D. dissertation, Medical College of
Virginia (Richmond, VA).
Tepper, J.S. and D.L Costa (1992) Will the mouse bioassay for estimating sensory irritation (ASTM E-981-
84) be useful for evaluation of indoor air contaminants. Indoor Environ. (1992)
Tilson, H.A. and V.C. Moser (1992) Comparison of screening approaches. NeuroToxicology 13:1-14.
U.S. Environmental Protection Agency (EPA) (1991) Neurotoxidty Test Guidelines Addendum 10, Pesticides
Assessment Guidelines Subdivision F. Publication Number PB91-154617, Springfield Va. National
Technical Information Services.
EPA-82
-------
Wallach, J. (1978) Interpretation of Diagnostic Test: A Handbook Synopsis of Laboratory Medicine. Little,
Brown and Company, Boston, MA.
EPA -83
-------
Figure Legends
Figure 1. Diagram of the exposure system for evaluating carpet emissions.
Figure 2. Mean body weight of mice in each treatment group, recorded before each of the four exposures.
Figure 3. Mean frequency of breathing of mice during the control period: (a) collapsed over all four
exposures for each treatment, and (b) collapsed across treatments for each of the four exposures.
Figure 4. Percent decrease in frequency of breathing of mice for each of the four exposures during the
exposure period: (a) individual mouse data for each treatment for each exposure, and (b) mean percent
decrease collapsed across treatments for each of the four exposures.
Figure 5. Mean percent change in frequency of breathing of mice for each of the four exposures during the
recovery period: (a) collapsed over all four exposures for each treatment, and (b) collapsed across
treatments for each of the four exposures.
Figure 6. Percent incidence of mice in each treatment group receiving a rating of none, slight, moderate, or
severe sensory irritation during the exposure periods. Data are collapsed across all four exposures.
Figure 7. Percent incidence of mice in each treatment group receiving a rating of none, slight, moderate, or
severe pulmonary irritation during the exposure periods. Data are collapsed across all four exposures.
Figure 8. Percent incidence of mice in each treatment group receiving a rating of none, slight, moderate, or
severe disruption index. Data are collapsed across all periods of all four exposures.
EPA-84
-------
Figure 9. Percent incidence of mice in each treatment group showing lacrimation following each of the four
exposures.
Figure 10. Percent incidence of mice in each treatment group showing dilated pinna vessels following each
of the four exposures.
Figure 11. Incidence of mice in each treatment group falling off the jar 0,1,2, or 3 times, after the second
exposure (day 1) and the fourth exposure (day 2).
Figure 12. Incidence of mice in each treatment group showing decreased, the same, or increased scores in
response to the tail-pinch stimulus. The score for each mouse was calculated as the difference from that
mouse's preexposure value, and ranged from -1 ("
-------
Figure 16. Mean serum chemistry values: (a) cholesterol, (b) protein, and (c) albumin. Data are presented
for mice in each treatment group and the non-exposed cage-control mice.
Figure 17. Mean lavage values: (a) glutathione in nasal lavage fluid, and (b) protein In lung lavage fluid.
Data are presented for mice in each treatment group and the non-exposed cage-control mice.
EPA-86
-------
HIGURE 1
Zero Grade
Flow Control
System
£
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-------
30
FIG. 2 Mean Postexpo^ure Body Weight Treatment
28-
05
D)
g>26^
'(D
24-
22
2 3
Exposure Number
A
B
C
ui
-------
.? 250
Fig. 3 Control Period
B
Treatment
2 3
Exposure Number
EPA-89
-------
Fig. 4 Exposure Period
>s
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EPA - 90
-------
Fig. 5 Recovery Period
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i
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-5
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Exposure Number
EPA - 91
-------
FIG. 6 SENSORY IRRITATION - EXPOSURE
LU
O
z
UJ
9
o
100 {
80 -
60 -
40 -
.20 -
0
B C
TREATMENT
8!
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MONE
SLIGHT
MODERATE
SEVERE
-------
FIG. 7 PULMONARY IRRITATION - EXPOSURE
in
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80 -
60 -
40 -
20 -
0
s
£
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SLIGHT
MODERATE
SEVERE
B C
TREATMENT
-------
FIG. 8 DISRUPTION INDEX - COLLAPSED
UJ
O
z
HI
Q
o
100 f
80 -
60 -
40 -
20 -
0
A
B C
TREATMENT
£
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NONE
SLIGHT
MODERATE
SEVERE
-------
FIG. 9 LACRIMATION
?
0.
Ul
EXPOSURE 4
EXPOSURE 3
EXPOSURE 2
EXPOSURE 1
B C
TREATMENT
-------
UJ
o
UJ
g
o
FIG. 10 DILATED PINNA VESSELS
120 (-
100 -
9 80 -
- 60 -
40 -
20 '-
0
UJ
EXPOSURE 4
EXPOSURE 3
EXPOSURE 2
EXPOSURE 1
B C
TREATMENT
-------
FIG. 11 JAR TASK PERFORMANCE - DAY 1
B C
TREATMENT
JAR TASK PERFORMANCE - DAY 2
B C
TREATMENT
EPA - 97
-------
FIG. 12 TAIL PINCH RESPONSE
DAY 1 & 2 COMBINED
UJ
O
z
HI
o
o
16
14
12
10
8
.6
4
2
0
B C
TREATMENT
2
UJ
» DAY 0
>DAYO
= DAYO
-------
FIG. 13 CLICK RESPONSE
UJ
o
z
UJ
9
o
DAYS 1 & 2 COMBINED
UJ
EXTREME
CLEAR
SLIGHT
NONE
B C
TREA JIENT
-------
. 14 ALERTNESS ON DAY 2
B C
TREATMENT
EXCITED
ALERT
LOW
STUPOR
g
-------
Asbestos Tozlclty and GSH-dependent Protection 14
20. Gulumian, M.; Sardianos, F.; Kllroe-Smlth, T.; Ockerse, G. Llpid
peroxidation in microsomes induced by crocidolite fibers. Chem. Biol.
Interact. 44:111-118; 1983.
21. Bonneau, L.; Pezerat, H. Etudes des sites donneurs et acceptures d'an
election en surface des andantes. J. Chem. Phys. 80:273-280; 1983.
22. Kamp, D. W.; Graceffa, P.; Pryor, W.A.; Weitzman, S.A. The role of free
radicals in asbestos induced diseases. Free Rad. Biol. Med.12:293-315;1992.
23. Fontecave, M.; Mansuy, D.; Jaquen, M.; Pezerat, H. The stimulatory effects
of asbestos as NADPH-dependent lipid peroxidation in rat liver microsomes.
Biochem. Jt 241:561-565; 1987.
24. Morehouse, L.A.; Thomas, C.E.; Aust, S.D. Superozide generation by NADPH-
cytochrome P-450 reductase: The effect of iron chelators and the role of
superozlde in microsomal lipid peroxidation. Arch. Biochem. Biophys. 232:
366-377; 1984.
25. Aust, S.D.; Morehouse, L.A.; Thomas, C.E. Role of metals in oxygen radical
reactions. Free Rad. Biol. Med. 1:3-25; 1985.
26. Diplock, A.T. Vitamin E, In: Fat soluble vitamins, their biochemistry and
their applications; Diplock, A.T., ed, Technonic Publishing Co. .Inc.,
Lancaster-Basal; 1985:154-224.
27. Kornbrust, D.J.; Mavis, R.D. Relative susceptibility of microsomes from
lung, heart, liver, kidney, brain and testes to lipid peroxidation: Correl-
ation with vitamin E content. Lipid 15:315-322; 1980.
28. Burk, R.F. Glutathione-dependent protection by rat liver microsomal protein
against lipid peroxidation. Biochim. Biophys. Acta, 757:21-28; 1983.
-------
Table:!
Chrysotile-mediated enzymatic llpid peroxidation in rat lung microsomes supplemented with vitamin E in presence of
GSH and other quenchers of reactive oxygen species.
Incubation system
Microsomes
4 NADPH (0.4 mM)
4 Chrysotile (500 ug)
Complete
+ GSH (1 mM)
4SOD (100 units)
+ Mannitol (1 mM)
4Catalase (ISO units)
4 B-Carotene (0.5 mM)
Control
group
0
0.56+0.04
0.4840.01
2.5040.35
1.2740.17C
1.8340.21NS
1.7740.23NS
1.804p.22HS
1.9840.31NS
4 Vitamin E
group
0
0.1940.06
0.1640.04
1.2340.17
0.3040.06a'(tt)
0.6740.04a'(a)
0.7040.05C>(b)
0.7440.04C'(a)
0.7240.08°' (b)
% Protection by
vitamin E
-
-
-
51
76
63
55
59
64
Values are mean 4 SE (n-3) and expressedas nmol MDA formed /ag protein. Complete " Microsomes 4 NADPH 4 Chrysotile
•WLst»*t*Iv*-
Letters without parentheses - Compared with icomplete systems* Let
h
trol groups. ap< 0.01; p< 0.02; °p< 0.05; NS * Not significant.
Letters without parentheses - Compared with icomplete systems* Letters in parentheses " Compared with respective con-
h
-------
Tablet2
Effect of chrysotile on the activity of vitamin E regeneration factor in rat lung mlcrosomes supplemented with
vitamin E in presence of GSH and other quenchers of reactive oxygen species.
Incubation system
Mlcrosomes + Ascorbate
Mlcrosomes + Ascorbate + GSH (ImM)
Complete
+ GSH (ImM)
+ SOD (100 units)
+ Mannitol (1 mM)
+ Catalase (150 units)
+ B-Carotene (0*5 mM)
w ^ «_*•>••• MW ^ «» jf fiiv£ £ IK •••
20 min.
0.096+0.006
0.032+0.002
0.164+0.009
0.100+0.005b
0.117+0.004a
0.120+0.005b
0.115+0.009a
0.110+p.010b
.grfiup.
40 mln.
0.108+0.004
0.036+0.003
0.194+0.016
0.115+0.005a
O.130+J0.006a
0. 129+0. 003a
0.120+p.015b
0.124+0.001a
+.Yltaml
20 mln.
0.080+0.006
0.015+0.001
0.120+0.009
0. 046+0. 002a
0.079+0.006b
0.078+0.010°
0. 081+0. 006b
0. 076+0. 005b
£.E_grouB_
40 mln.
0.089+0.007
0.025+0.002
0.146+0.008
0.058+0.006a
0. 085+0. 009a
0.088+0.008a
0.090+0. 01 lb
0.085+0.012b
Values are mean + SE (n-3) and expressed as ^^35-500* ComPlete " Mlcrosomes + Ascorbate + Chrysotile,
Letters - Compared with respective complete systems. p<0.01; p<0.02; p<0.05.
-------
Table:3
Effect of chrysotlle on the vitamin E
of GSH and other quenchers of reactive
Incubation system
Microsomes
+ NADPH (0.4 mM)
+ Chrysotile (500 ug)
Complete
+ GSH (1 mM)
+ SOD (100 units)
+ Mannitol (1 mM)
+ Catalase (150 units)
+ B-Carotene (0.5 mM)
content in rat lung microsomes
oxygen species.
Control
group
0.70+0.08
0.38+0.08(d)
0.404<).07(d)
0.26+0.05(b)
0.6440.11d
«c
0.31+p.lOws
Q.3W.QB*S
0.3640.07NS
0. 33^.08^
supplemented with vitamin E in the presence
+ Vitamin E
group
1.92+0.06
1.6l40.06(d)
(t>)
1 .-5040. 08 v
1.06:M).08(a)
1.99+0.09b
1.49+0.04b
1.454p.02b
1.5240.04b
1.5540.07b
Values are mean + SE (n-3) and expressed as n moles/mg protein. Complete - Microsomes + NADPH + Chrysotile.
Letters in parenthesis • Compared with microsomes only. Letters without parenthesis - Compared with respective
complete systems. ap<0.001; bp<0.01; Cp<0.02; dp<0.05; NS • Not significant.
-------
Tablet4
Effect of chrysotile on the glutathione-S-transferase activity profiles in rat lung microsome8 supplemented with
vitamin E in presence of GSH.
Incubation system
Microsomes
+ NADPH (0.4 mM)
+ Chrysotile (500 ug)
Complete
+ GSH (1 mM)
Control
group
+ Vitamin E
group
50.42+4.79
150.89+10.11
149.71+11.75
181.35+10.21
79.43+9.24*
49.33+3.88
121.01+9.25
115.60+10.40
143.34+9.75(b)
54.30+8.29a'(NS)
% Protection
by vitamin E
21
32
Enzyme activity is mean+SE (n - 3) and expressed as nmol CDNB conjugated/mg protein/mln.
Complete " Microsomes + HADPH + Chrysotile. Letters without parentheses - Compared with respective complete system.
Letters in parentheses - Compared with respective control group. ap<0.02; p<0.05; NS - Not significant.
-------
16
14
Sho-
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0)
4
2
. O-1-
T
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. S t .' ft •
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•v/u
' t\
Control
FIG. 15 Hemoglobin
UJ
A B
Treatment
-------
— 1 I /on
5
ri25-
o
v 100-
Q) 7c
o 75~
0 50-
1 25-
£ n
'"T .
•"
I 3
I2
B1-
Q)
co o-
4-
*
1
0)
CO
Control
Control
Control
hig.1t> berum uhemistries
T
B
T
B
A B
Treatment
EPA -102
-------
0.6
0.5-
E
§> 0.4-
0 n •*
c U.o-
0.2-
0.1-
Fig. 17
Nasal Lavage
I
Control
A B
Treatment
200
180-
I 160-
.£ MO-
TS 120:
100-
80:
60-
40-
20-
0)
o>
CD
CO
Lung Lavage
Control
A B
Treatment
i
EPA -103
-------
APPENDIX A
SUMMARY OF FOB DATA FOR EPA FORMAL REPLICATION STUDY
EPA -104
-------
SUMMARY OF FOB DATA FOR EPA FORMAL REPLICATION STUDY
Neuromuscular Function:
Jar Task
fell Ox
1x
2x
3x
Grip Strength
dropped Ox
1x
2x
3x
Body Tone
1 hypotonia
2 slightly flaccid
3 normal
4 hypertonia
CARPET A
samples 1,6
Test Day
0
1
2
CARPET B
samples 2,3
Test Day
0
1
2
CARPET C
samples 4,5
Test Day
0
1
2
4
2
1
1
8
0
0
0
0
1
7
0
2
1
1
4
8
0
0
0
0
2
6
0
1
3
3
1
8
0
0
0
0
4
4
0
6
1
1
0
8
0
0
0
0
0
8
0
5
3
0
0
7
0
1
0
0
2
6
0
3
4
1
0
7
0
1
0
0
2
6
0
4
2
2
0
8
0
0
0
0
0
8
0
4
2
2
0
8
0
0
0
0
1
7
0
7
1
0
0
8
0
0
0
0
1
7
0
EPA -105
-------
Righting Reflex
1 normal
2 slightly slow
3 difficult
4 not present
Body Posture
1 lying on side
2 pelvis flat
3 upright
4 hunched
Body Tift
1 normal
2 head tilts
3 shoulder leans
4 body lists
CARPET A
samples 1,6
Test Day
0
8
0
0
0
0
0
8
0
8
0
0
0
1
8
0
0
0
0
0
8
0
8
0
0
0
2
8
0
0
0
0
0
8
0
8
0
0
0
CARPET B
samples 2,3
Test Day
0
8
0
0
0
0
0
8
0
8
0
0
0
1
7
1
0
0
0
0
8
0
8
0
0
0
2
7
1
0
0
0
0
7
1
8
0
0
0
CARPET C
samples 4,5
Test Day
0
8
0
0
0
0
0
8
0
8
0
0
0
1
7
1
0
0
0
0
8
0
8
0
0
0
2
8
0
0
0
0
0
8
0
8
0
0
0
EPA-106
-------
Ataxic Gait
1 none
2 slight
3 somewhat
4 marked
5 severe
Abnormal Gait Score 1 none
2 slight
3 somewhat
4 marked
5 severe
Inverted Screen
fast <30 sec
slow £30 sec
hanging
dropped
CARPET A
samples 1,6
Test Day
0
8
0
0
0
0
5
3
0
0
0
1
6
2
0
0
0
1
3
4
0
0
5
2
1
0
2
7
1
0
0
0
1
4
3
0
0
5
2
1
0
CARPET B
samples 2,3
Test Day
0
8
0
0
0
0
5
3
0
0
0
1
8
0
0
0
0
0
2
5
1
0
4
2
1
1
2
6
2
0
0
0
1
0
6
1
0
5
0
2
1
CARPET C
samples 4,5
Test Day
0
8
0
0
0
0
3
5
0
0
0
1
8
0
0
0
0
1
3
3
1
0
3
4
1
0
2
8
0
0
0
0
1
2
5
0
0
5
3
0
0
EPA -107
-------
Missteps
1 none
2 few
3 some
4 legs hanging
Sensorimotor
Measures:
Forelimb Placing
present
absent
Air Puff Response
1 none
2 slight
3 dear
4 extreme
CARPET A
samples 1,6
Test Day
0
7
1
0
0
1
5
3
0
0
2
3
5
0
0
CARPET B
samples 2,3
Test Day
0
5
3
0
0
1
3
4
1
0
2
4
2
2
0
CARPET C
samples 4,5
Test Day
0
4
4
0
0
1
5
3
0
0
2
6
2
0
0
8
0
0
2
6
0
8
0
0
0
8
0
8
0
0
2
5
1
8
0
0
2
5
1
8
0
0
0
5
3
8
0
0
2
4
2
8
0
0
0
6
2
8
0
0
1
5
2
8
0
0
1
4
3
EPA-108
-------
Click Response
1 none
2 slight
3 clear
4 extreme
Tail Pinch Response
1 none
2 slight
3 dear
4 extreme
CARPET A
samples 1 ,6
Test Day
0
0
4
4
0
0
2
6
0
1
0
7
1
0
0
2
6
0
2
0
7
1
0
0
2
5
1
CARPET B
samples 2,3
Test Day
0
0
4
3
1
1
3
4
0
1
0
3
5
0
0
1
5
2
2
0
4
4
0
0
1
5
2
CARPET C
samples 4,5
Test Day
0
0
6
2
0
0
0
7
1
1
0
4
4
0
0
1
5
2
2
0
7
1
0
0
1
4
3
EPA -109
-------
Activity and
Excitability
Measures:
Alertness
1 stupor
2 low
3 alert
4 hyperalert
Handling Reactivity
1 low
2 slight
3 active
4 moderate
5 high
CARPET A
samples 1,6
Test Day
0
1
2
CARPET B
samples 2,3
Test Day
0
1
2
CARPET C
samples 4,5
Test Day
0
1
2
.
0
0
8
0
1
5
2
0
0
0
0
8
0
0
4
4
0
0
0
2
6
0
0
5
3
0
0
0
0
7
1
1
3
4
0
0
0
1
6
1
0
5
2
1
0
0
0
5
3
1
2
4
1
0
0
0
8
0
1
4
2
1
0
0
0
8
0
2
3
2
1
0
0
0
8
0
1
1
6
0
0
EPA-110
-------
Activity Level
1 none
2 low
3 somewhat low
4 active
5 clear
6 hyperactive
Rears
X
SEM
Clonic Movements
1 none
2 smacking
3 quivers
4 mild tremors
5 severe tremors
6 myodonus
7 convulsions
CARPET A
samples 1,6
Test Day
0
0
0
2
3
3
0
10.0
2.4
8
0
0
0
0
0
0
1
0
1
3
2
0
2
11.5
3.0
8
0
0
0
0
0
0
2
1
1
3
1
1
1
8.6
2.7
7
0
1
0
0
0
0
CARPET B
samples 2,3
Test Day
0
0
1
2
2
3
0
7.4
2.4
8
0
0
0
0
0
0
1
0
0
2
3
2
1
7.6
2.7
8
0
0
0
0
0
0
2
0
0
2
1
2
3
13.3
32
7
0
1
0
0
0
0
CARPET C
samples 4,5
Test Day
0
0
0
1
3
4
0
9.5
2.4
8
0
0
0
0
0
0
1
0
0
2
3
3
0
9.1
2.5
8
0
0
0
0
0
0
2
0
0
1
1
6
0
7.3
1.8
8
0
0
0
0
0
0
EPA-111
-------
Tonic Movements
1 none
2 extension
3 opisthotonus
4 emprosthotonus
5 popcorn
6 convulsion
CARPET A
samples 1,6
Test Day
0
8
0
0
0
0
0
1
8
0
0
0
0
0
2
8
0
0
0
0
0
CARPET B
samples 2,3
Test Day
0
8
0
0
0
0
0
1
8
0
0
0
0
0
2
8
0
0
0
0
0
CARPET C
samples 4,5
Test Day
0
8
0
0
0
0
0
1
8
0
0
0
0
0
2
8
0
0
0
0
0
EPA-112
-------
Other Measures:
Tilted Screen
up>2x
even>2x
down>2x
1,1,1
Vocalizations
present
none
Bizarre Behaviors
present
none
Stereotypies
present
none
Diarrhea
present
none
CARPET A
samples 1,6
Test Day
0
1
2
CARPET B
samples 2,3
Test Day
0
1
2
CARPET C
samples 4,5
Test Day
0
1
2
4
1
3
0
0
8
0
8
0
8
0
8
1
2
3
2
0
8
0
8
0
8
0
8
1
2
3
2
0
8
0
8
0
8
0
8
6
0
2
0
0
8
0
8
0
8
0
8
4
1
3
0
0
8
0
8
0
8
0
8
4
2
2
0
0
8
0
8
0
8
0
8
5
1
2
0
0
8
0
8
0
8
0
8
2
1
3
2
0
8
0
8
0
8
0
8
2
0
6
0
0
8
0
8
0
8
.0
8
EPA-113
-------
General Appearance
Measures:
Facial Swelling
present
absent
Chromodacryorrhea
present
absent
Lacrimation
1 none
2 mild
3 severe
Gasping
present
absent
Cyanosis
present
absent
CARPET A
samples 1,6
Test Day
0
1
2
CARPET B
samples 2,3
Test Day
0
1
2
CARPET C
samples 4,5
Test Day
0
1
2
0
8
0
8
8
0
0
0
8
0
8
8
0
2
6
0
7
1
0
8
0
8
3
5
1
7
1
4
3
0
8
0
8
0
8
0
8
8
0
0
0
8
0
8
8
0
4
4
0
6
2
0
8
0
8
7
1
3
5
0
6
2
0
8
0
8
0
8
0
8
8
0
0
0
8
0
8
8
0
3
5
0
8
0
0
8
0
8
4
4
0
8
4
4
0
0
8
0
8
EPA-114
-------
Exophthalmus
present
absent
Palpebral Closure
1 eyes open
2 slight droop
3ptosis
4 eyes shut
Piloerection
present
absent
Salivation
1 none
2 mild
3 severe
Splotchy ears
present
absent
CARPET A
samples 1 ,6
Test Day
0
0
8
8
0
0
0
0
8
8
0
0
0
8
1
0
8
8
0
0
0
1
7
8
0
0
6
2
2
0
8
8
0
0
0
0
8
8
0
0
1
7
CARPET B
samples 2,3
Test Day
0
0
8
8
0
0
0
0
8
8
0
0
0
8
1
0
8
8
0
0
0
2
6
8
0
0
7
1
2
0
8
8
0
0
0
0
8
8
0
0
5
3
CARPET C
samples 4,5
Test Day
0
0
8
8
0
0
0
0
8
8
0
0
0
8
1
0
8
7
0
1
0
0
8
8
0
0
5
3
2
0
8
8
0
0
0
0
8
8
0
0
3
5
EPA-115
-------
DRAFT REPORT
Double Blind Study
Biological Effects of Carpets
Anderson Laboratories, Inc.
Dedliam, Massachusetts
KL-i
-------
CAP.PET RTTTTW PFPftPT
DATE OF REPORT: A
CXHMT BTOPY REPORT
Z. V00UW1UI •OOULBY
This study is a double blind evaluation of biological potency of
•nice ions fron two earpAt.R (Experiment A, samples 1, 6,
Experiment C, samples 4, 5) and one control (Experiment B,
samples 2, 7). Each is tested two times in the randomized order
determined by Consumer Product Safety Commission, (CPSC) .
The questions being addressed ask:
1. Is there a measurable biological effect in animals following
•xpocuro to th* t««t •unplec?
2. uo The rest data reveal airter«nce» I>«LW««H ui« errectb or
the carpet sample* and the eontx-el oanplo?
3. How do the findings from Anderson Laboratories, Inc. relate
m < T».-1 i r»o Pr-^r-.ji n^ S rmc
The sample custodian receives the sample, transcribes
identification and places the sample into a chamber which he then
seals. The contents, if any are, not visible. He positions the
vdiuple chamber in the test laboratory. At the completion of the
test the sample custodian repackages the sample to be returned to
the CPSC.
Sample Preparation
Three one square foot pieces of a test sample formed into
cylindrical ehape with face fiber euteido rn.ro plaood in the
wliauibvj. . Hume lieeliuy paU» ait; u»cJ Lw waiui Lite Ail temperature
to 37°C.
Animals
For the test, male Swiss Webster mice are positioned in the glass
• iiluml vliojiiU«A • AUv KtsoU «Ai.«*ia» XtiUw Ulm w«iiCxoa. wjl.X«iav^. A
flexible seal around the neck of the animal allows the animals to
. .
side arm wnich serves as a whole body plethy»nograph. Itooa air
(7 liters/minute) Is used for ventilation of the chamber. During
baseline and recovery periods the air is taken directly into the
animal exposure chamber. During the animal exposure, the air is
Cf>A:EU-W.VPS 5/17/93
Page 1 of 16
-------
CARPET STUDY REPORT
DATE OF REPORT: *
drawn through the sample chamber and then to the animal exposure
chamber.
Respiratory activity is detected and recorded by microphones
attached to the plethysmograph and connected to a high speed
recorder and computer.
Respiratory Effects
These data provide information concerning irritant chemical
presence and potency in the test atmosphere. The upper and lower
airway reflexes are studied by ASTM E 981 "Estimating Potency of
Air Borne Irritant Chemicals.*1 The graphic indication of sensory
irritation [(51), upper airways] is a diagnostic pause or slowing
at me completion or liutplxaUloii a* »««ii wu liawingt* of
individual animal respiratory movements. The respiratory rate
decreases proportionally.
Pulmonary irritation [ (PI) , lower airways] is detected as a
change in the slope of the respiratory tracing at the end of
expiration which may not be accompanied by a rate change. The
moderate or severe effects are reported.
Animal Evaluation
Acute toxic effects of the nervous system are studied by an EPA
protocol (Functional Observational Battery (FOB) by Virginia
Moser) designed to detect neurological changes through systematic
observation of animals. One day prior to study and following
•ach «xp&*uL-«, animal* are *Lberv*<2 and the status c.f each is
recorded using this FOB.
T«»t Cliaiubex. Muni Luring
Temperature and humidity arc measured in specified locations
throughout the testing. Total volatile organic compounds (TVOC)
is measured following each animal exposure.
Results and Discussion
The summary of results is presented on Table 1.
Tlie sLudy »lii»«*«O LtoLli w«tiwrjf «iii«3 £>olMiuim«y l&c J La Llun In
animal » VApuaeO tw wai.pvL «iui»vluit» (CApviituviiL* A and C) .
sensory effect was slight or moderate never severe. The
pulmonary effect was severe for two animals in each experiment.
Hcithcr ocnaory nor nodcrate/acvcre pulnonary irritation io oeen
on records of Experiment B (control samples 2 and 7) . FOB data
EP»:EPA-lt>T.VPI
Page 2 of 16
-------
Summary of Findings
Double Blind Study,
Anderson Laboratories, Inc.
ir
o
CARPET
Sample 1,6
CARPET
Sample 4,5
AIR
Control 2,7
end point
Sensory Irritation
Pulmonary Irritatian
FOB Extreme changes*
appearance (
»
activity excitability
neuromuscular ,
Death from Toxicit/
during exposure
after exposure
slight
severe, two animals
9/9
7/9
12/13
0/8
2/8
* # test categories showing extreme change
unblinded 5/12/93 ar*J 5/13/93
study in conjunction with EPA, CPSC
slight/ moderate
severe, two animals
8/9
9/9
13/13
0/8
3/8
Table 1
no
no
2/9
1/9
0/13
0/8
0/8
-------
CARPET STUDY REPORT
DATE OF REPORT: *
•hows that the emissions studied in Experiments A and C caused
clear changes of animal behavior and appearance.
There were no deaths due to toxicity during the exposure period
for any experiment. A total of 5 animals died following the
fourth exposure (day 2) on days 3, 5, 6 end 7. Two of these
»»le were
-------
MfiY 2B '93 14:55 flNDERSON no*
CARPET STUDY REPORT
DATE OF REPORT: A
II. IKTRODUCTIOH
This study is a double blind evaluation of biological potency of
•HuXvoAwiio r«-wM« ua*«-wu w«»w«.ea eunpxea* vrwo oz 'CAC aa&pxea ore
carpet, one is a control. Each cample is tested two tines in a
randomized order.
The questions being addressed ask:
1. Is there a measurable biological effect in animals following
exposure to the test samples?
2. Do the test data reveal differences between the effects of
the carpet samples and the control sample?
3. How do the findings from Anderson Laboratories, Inc. relate
to the findings of the EPA team?
A similar study is being conducted by EPA staff and contractors
at Research Triangle Park. The protocols of the Anderson
Laboratories and EPA (RTP) differ in certain details. In
particular, the poet exposure treatment of animals will introduce
variation into the data collected.
III. METHODS
A. Exposure System
1. Air and Humidity
a. Air Source
The source of the air for all studies is the
laboratory. During baseline and recovery periods, the
air is pulled directly from the room into the glass
animal exposure chamber. During animal exposure, the
air is drawn from the room through the sample chamber
and through a 4 inch glass connection (3/4" ID) to the
animal exposure chamber.
b. Air Flow Control - Calibration
Air flow is 7 liters/minute, moved by a peristaltic
pump (Cole Panner) downstream of the animal exposure
system. The air flow is controlled by a flow meter
(Gilmont) which is calibrated by comparison to a soap
bubble meter (SKC) each day before animal exposures.
The dry gas meter is used to check air flow at the
EPA:E>A-«PT.W>S 5/17/93
Page 4 of 16
AL-fc
-------
CARPET STUDY REPORT
DATE or REPORT:
ef the •oanple chamber before— and a-fter each
animal exposure. This information is recorded in
laboratory record books.
c. Humidity
The humidity of the ambient air is recorded at 15
minute intervals throughout each animal exposure
interval. A sling psychrometer (Bacharach, Inc., PGK,
PA) is used to determine room air humidity according to
the prescribed schedule.
2. Exposure System/Aquarium
a. Source and Preparation
Two jiew glatii* aquaria were obtained Izoiii a local pet
supply store. The sealant adhesive in the interior of
the chamber was removed as far as possible by means of
a sharp blade. A plastic reinforcing band and lip
around the open edge of the chamber is not removed.
After removal of the sealant, the chamber is vashed
with vater and Alconox and wiped dry with 95% alcohol.
At the completion of each set of four exposures with a
single sample , the chamber was again cleaned with
water, Alconox and alcohol.
b. Chamber Pace and Fittings
The open side of the chamber is covered by a three
panel assembly. Two side panels (10.25 by 2.5 inches)
are fashioned from plastic faced on the inside by
Teflon film. The central panel (10.25 by 14.75 inches)
is glass. Duct tape is used to hold the composite
panel together. Nylon fittings are inserted into the
side panels. During use, the front panel is attached
to the aquarium with duct tape.
c. Heating
Home heating pads are used to warm the system. One pad
measures 11.5 by 15 inches and is placed under the
bottom of the chamber, the second measures 20 by 30
inches and is positioned on the top, back and sides.
Th« air tanporatur* ie hotted to 37«C by turning both
exposure begins. The temperature is adjusted by
lP*:tPA-MT.Wf| 5/1T/W
Page 5 of 16
-------
fWY 20 *93 14:57 flNDERSON 703 P23
CARPET STUDY REPORT
DATE OF REPORT: *
changing settings of each switch. During exposures,
the settings are typically on high.
d. Insulation
A double layer of metal faced fiberglass insulation is
used to cover all of the sample holding chamber during
the experiment.
e. Test Sample Source and Placement
Each test sample is delivered to the laboratory on the
day preceeding its use. The samples are to have been
provided as three one square foot pieces of a test
material. The diagonal corners of each piece are
fastened together to form a cylinder. The face fiber
is the external surface of the cylinder. Two of the
cylinders are placed directly on the floor of the
chamber parallel to the long dimension of the aquarium.
The third cylinder is positioned on top of the first
two canplac. Th« chamber ie fehen e«al«d until «tudy
the following day.
f. Blinding Precautions
The samples are received in our laboratory in a Federal
Express delivery box. After working hours of the day
of receipt, the sample custodian opens the box,
transcribes all identification into a sample book
(which is kept in his posession) and places the sample
pieces into o chamber, which he -then eeale. The
ehambor ic entirely oovorod by a layer of duot ^ape oe
that the contents, if any, are not visible. The sample
custodian places the sample chamber in the test
laboratory. At the completion of the test, the sample
custodian opens the chamber/ removes and repackages the
sample in the original delivery box. The sample is
returned to the sender.
3. Exposure Chamber with Plethysmograph
a. Source and Preparation
The animal exposure chamber is custom made by a glass
blower according to the drawings of the ASTM E 981
publication (Appendix I). Between samples, it is
washed with Alconox, dried with 95% alcohol and then
£PA:!PA-m.WP$ 5/17/93
Page 6 of 16
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CARPET STUDY REPORT
DATE OF REPORT: *
air dried. Between trials with a single sample the
glass chamber is wiped out with 95% alcohol.
b. The Flethysmograph
nr« positioned In the sidearms of the qlass
aninal chamber. The bead extends through a hole into
the central cylinder. A flexible seal around the neck
of the animal allows the test animals to breathe the
test atmosphere while the body is enclosed within the
sideann (closed with a hard rubber stopper) . The neck
seal is made of a latex dental dan (medium gage)
reinforced and held in place by duct tape. A central
hole in the latex dam is of a size appropriate for the
animal weights and is made with a cork borer.
Typically, the hole through the reinforcing duct tape
is size 8, through the latex, size 6. The reinforced
dan is carefully positioned in the chamber and the duct
tape seal checked for smooth fit.
c. Transducers and Attachment
The four transducers are miniature microphones
manufactured by Fafcuda Denshi (Japan) . Each microphone
is *tt-»ff>»»rt hy a short: piece of flexible PFC tubinq to
the sample port on a sideann. The cable is directly
connected to the Could R5 3400 recorder.
4. Exposure Measurement System
a. Temperature System and Calibration
The temperature measurement* ar* made hy means of a
coie Farmer •rnermister/tfiermoineter. Tim Ui«i.ini»tcA«,,
range -40 to 100°C, are manufactured by YSI, Inc. and
are NIST traceable. The device has been calibrated by
comparison of digital output to thermometer readings
using hot and cold water as convenient test conditions.
The thermometer will be provided to EPA for reference
measurements at a later time.
During testing, three temperature probes are used.
They are positioned at sites identified by the EPA in
the protocol, one is outside the sample chamber between
the heating pad and the bottom glass surface, two is
outside the sample chamber between the top glass
.surface and the heating pad, and the third is inside
the sample chamber in air.
EPA:E»A>KP7.M>S 5/17/93
Page 7 of 16
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DRY 20."93 14:59 WitrouN 7^
CARPET STUDY REPORT
DATE OF REPORT: *
b. Other Monitoring
Oxygen, carbon dioxide and chamber pressure are not
measured. Total volatile organic chemicals (TVOC) are
measured by flane ionitation detector (Rosemont )
calibrated against a methane standard before each use.
This measurement is made folloving each animal exposure
cession by attaching the sample chamber outlet to the
inlet of the FID device.
B. . Animals
1. Procurement Specification
Swiss Webster outbred male mice are obtained from
Taconic Farms, Germantown, New York. They are
delivered by truck and arrive on Tuesday of each week.
The purchase weight specified is 13 to 15 grams. Ten
percent of the animals are weighted at delivery to
confirm the animal weights.
2. Housing Conditions
For one week before testing, animals are housed in an
animal room with controlled heat, ventilation and light
(12/12). No other species is kept in this facility.
The food (Formulab Chow 5008, Purina Kills) is
available ad lib and is used within its marked shelf
life. Dedham tap water is available except during
actual experimental procedures. The bedding is Bed O
Cobs (The Andersons) changed 2 times/week. Animals are
housed in groups of 10 for a minimum of 7 days prior to
test.
After testing has begun, animals are housed in groups
of four. The exposed animals are kept in a separately
ventilated compartment of the same animal room.
3. Health Evaluation
Animals are evaluated by brief visual examination and
by body weight measurements at receipt and before
selection for testing. The animals are between 25.5
and 28 grams at the initiation of the study.
C. Respiratory Frequency Measurements
EPA:£PA-RPT.W»»« 5/17/93
Page 8 of 16
flL-10
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CARPET STUDY REPORT
DATE OF REPORT: A
1. Data Acquisition System
The data acquisition system is described in the ASTK E
981 protocol. The computer hardware was selected by
Dr. Yves Alarie, author of the protocol because of its
suitability for the intended uses.
The software was developed for this use at the
University of Pittsburgh vhere the validation was
conducted .
In addition, at intervals, the program output is
directly compared against results of a hand counted
recorder tracing.
2. Subjective Evaluation of Waveforms
Scoring Criteria
a. Sensory Irritation
The graphic indication of sensory irritation is a pause
or slowing at the completion of inspiration as seen on
tracings of individual animal respiratory movements.
As the pause becomes longer with increased potency of
the irritant exposure, the respiratory rate decreases
proportionally. Copies of tracings showing the
examples of SI and FI are included on Appendix II.
Example 1 is a classic example of a response to a
chemical having strong sensory irritant properties.
Example 2 is a slight SI response but a recognizable
change from normal. In the presence of a detectable
pattern change (example 2) the calculated mean group
rate change of baseline is used to quantify the
response. Rate change is summarized as maximum %
deviation (three minute duration) from normal baseline
value for each exposure. If no pattern change is found
a rate change is not an indication of sensory
irritation.
b. Pulmonary Irritation
This is detected as a change in the slope of the
respiratory tracing at the end of expiration or
initiation of the next inspiration. The pattern change
may not be accompanied by a rate change. The severity
of the effect is categorized as slight, moderate or
severe as defined in the ASTM £981 protocol. The
Page 9 of 16
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28 '93 J5:00 flKDERSON 703 PJ.3
CARPET STUDY REPORT
DATE OF REPORT: A
effect is described as slight if the pattern tracing
shows only a rounding of the expiratory peak. A
tracing resulting from exposure to a pulmonary irritant
of moderate potency is described as a flattening of the
expiratory pattern with indications of a hook or tail.
During expiration the severe effect shows a more
pronounced change of slope vith an accentuated tail.
These patterns are printed in the ASTM £981 protocol
vith examples in Appendix II.
In our laboratory only moderate or severe pattern changes
are reported.
D. Functional Observational Battery (FOB)
The description of this evaluation procedure and the scoring
form are included as Appendix III.
Test Procedure
One day prior to study, animals are observed and scored
using the EPA FOB.
At the conclusion of each animal exposure period, the
animals are removed from the exposure chamber and placed in
a prepared cage vith food vater and bedding. After 15
minutes, the animals are observed and scored according to
the written description of the EPA FOB.
As an exception to the EPA procedure, animals are placed on
a three sided elevated shelf (as contrasted to a low
laboratory cart having 4 sides} for observation of gait,
activity and posture. The shelf is cleaned after each
animal is observed.
E. Study Design
1. Exposure Protocol - Sequence of Events
Check and adjust air flov.
Measure room humidity. Start to heat chamber
containing sample.
Position thermisters to determine temperatures, record
findings at tc and at 15 minute intervals throughout
the study.
5/17/W
Page 10 of 16
flL-13,
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CARPET STUDY REPORT
DATE OF REPORT: *
Weigh and record animal weights and Bark tails for ZD.
Position each mouse in plethysmograph with haad
extending into the central core of the exposure
chamber.
Close open-end of each animal chamber sidearm with a
hard rubber stopper.
Ventilate animal exposure chamber using room air at a
rate of 7 liters/minute.
Fix microphone to the sampling port on each sidearm.
Insert thermometer into central sampling port of animal
chamber .
Allow 15 to 20 minutes for animals to adjust to the
chamber.
Collect 15 minute baseline respiratory rate data on
recorder and computer.
Observe record for signs of pulmonary and sensory
irritation.
Connect sample chamber to the animal exposure chamber.
Collect respiratory rate data on recorder and computer
for 60 minutes.
Observe record for signs of pulmonary and sensory
irritation.
Disconnect sample chamber from animal exposure chamber.
Mark chart. Continue ventilation of animal chamber
vith room air.
Determine TVOC in test atmosphere.
Collect respiratory rate data on recorder and computer
during IS minute recovery period.
Observe record for signs of pulmonary and sensory
irritation.
Remove animals from exposure chamber, place in
container vith food and water.
Page 11 of 16
-------
CARPET STUDY REPORT
DATE OF REPORT: *
Evaluate each animal using fOB after a minimum of 15
minutes in container.
Return animals to animal room for desired internal.
2. Experimental Protocol
For each experiment (A,B,C) there is a sequence of four
animal exposures. Each group of animals is exposed two
times on day one followed by two times on day two.
There is a minimum of two hours between the end of the
first exposure of the day and the start of the eecond.
Body weights are determined and recorded before each
exposure.
3. Study Protocol
A total of six experiments were planned for the study,
two experimental samples and one control sample. Two
examples of each were to have been provided to each
test lab in a random order by the CPSC. Because of an
error, one sample was incorrectly provided to Anderson
Laboratories, Inc. but not to EPA. The data from the
incorrect sample (number 3) have been discarded. A new
sample has been provided by CPSC and has been tested at
Anderson Laboratories as sample 7 (Appendix 4).
F. Data Analysis and Statistics
The data are summarized (as we have been advised by M. Mason
of EPA) by combining samples 1 and 6, samples 4 and 5 and
samples 2 and 7. No statistical analysis is offered.
XV. RE8ULT8
A. Experiment A (Samples 1 and 6)
2. Exposure System
The temperature and humidity are reported on Tables 1
through 8, Appendix V.
TVOC's measured by flame ionization detector
(calibrated against methane standard) ranged from 4.4
ppn, high to 2.4 ppm, low.
2. Body Weight Data
e»A:EM>WT.*S
Page 12 of 16
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CARPET STUDY REPORT
DATE OP REPORT: *
Day one, the mean starting weight of the 8 animals was
26.3 grains. Day 2, the mean starting weight was 23.7
grans. This represents an approximate 10% body weight
loss over the course of one day. The weights following
the exposures are not used for this calculation due to
the possibility of dehydration effects.
3. Irritation Measurement
a. Sensory—Irritation f£I)
Examination of the record shoved a change in the
r*«pir»t.r>ry p»t:t:«rn which i* defined as SI. No
irritation pattern was noted in two exposures and in a
third exposure the numerical change was at the no
effect level (<12%). For the remaining 5 exposures,
fha thT-*« mint!*.* VA!UAK WArA 14, 1I>, 17. 20 and 23%.
This is at the borderline between slight and moderate
effect.
b. Pulmonary Irritation (PI)
The tracing of each animal is examined and scored
4uri.ng fi.v* i.rtt*x-vr*l* in •»«>* *wpA*uv«. Thie reeulfee
in a maximum ot 160 observations, examination ot tne
record shows a change in the respiratory pattern
defined as PI. In the first two exposures (day 1,
samples 1 and 6), the PI was classed as moderate in
only 3 of 80 observations. During day two (exposures 3
and 4) of OO obaervoti,on» fehc TZ wao ooered ae moderate
once and severe 20 times. Two animals reacted
strongly, the others did not.
4. Functional Observational Battery (FOB)
The observations have been grouped as neuromuscular
function, activity and excitability and general
appearance. For eaoh obe*rvation, each individual
score is compared against the preseore value for the
group of animals being studied. The number of
observations outside the preseore range for the group
have been presented in Appendix VI. In addition, the
observations showing a severe effect (maximal deviation
from normal) are listed on Table 2.
Deaths
CPA:EPA-*»T.WPS 5/17/93
Page 13 of 16
0L-IS
-------
fifty 20 '93 15:04 flNDERSON 703 Pl7
CARPET STUDY REPORT
DATE OF REPORT: *
There were no toxieity related death* during Uits
exposures. There were, however, 2 deaths which
on day* 9 and 6 o£ the experiment.
6. Experiment B (Examples 2 and 7)
1. Exposure System
V.
TVOC iiivakuxviucnU* xcui^c £AWIU 2.1 ppu, high to l.C ppn,
low.
2. Body Weight Data
Day one, the mean starting weight of the 8 animals was
25*5 grano. Day two, the atoan charting waight vac 3E.O
grava. Thi« represents an approaii»»t» 9% Krw
-------
CARPET STUDY REPORT
DATE OF REPORT: *
which are outside the prescore range for the group have
been presented in Appendix VI. The only observations
shoving a change distinct from normal are vocalization,
piloerection, and ear petechiae. These are presented
in Appendix on Tables 3.
Deaths
There were no toxicity related deaths.
Experiment C (Samples 4 and 5)
1. Exposure System
The temperature and humidity are reported on Tables 17
through 24, Appendix V.
TVOC measurements range from 5.4 ppm, high to 3.7 ppm,
low.
2. Body Weight Data
Day one, the mean starting weight of the 8 animals was
26.0 grams. Day two, the mean starting weight was 23.8
grams. This represents an approximately 9% body weight
loss over the course of one day. The weights following
the exposures are not used for this calculation due to
the possibility of dehydration effects.
3. Irritation Measurement
a. Sensory Irritation
Examination of the record showed a change in the
respiratory pattern which is defined as SI. An
irritation pattern is noted in all exposure records.
The maximum mean group % decrease from baseline rate
(three minute duration) are 12, 12, 12, 13, 14, 14, 15
and 18%. This describes a consistent effect reflecting
sensory irritants having slight potency.
b. Pulmonary Irritation
The tracing of each animal was examined and scored
during five intervals in each exposure. This results
in a maximum of 151 observations (two animals died from
accidental eauc«c during •xpecura four). Examination
of the record showed no detectable change in the
n.U»S S/17/W
Page 15 of 16
flL-ll-
-------
CARPET STUDY REPORT
DATE OF REPORT: A
respiratory pattern defined as PI during exposures 1
and 2. During day two (exposures 3 and 4) of 71
observations the PZ was scored as moderate 2 tines and
severe 24 tines. The effect was strong in three
animals and absent in others.
4. Functional Observational Battery
The observations have been grouped as neuromuscular
function, activity and excitability and general
appearance. For each observation, the individual
animal score is compared against the prescore value for
the group of animals being studied. The number of
observations which are outside the prescore range for
the group have been presented in Appendix VI. The
observations shoving a severe effect (a maximal
deviation from normal) are listed on Table 4.
Deaths
There were no toxicity related deaths during the
exposures. There were, however, 3 deaths which
occurred on days 5 (two deaths) and 7 of the
experiment.
DISCUSSION
Information was received by phone from Mark Mason that the
samples were grouped as 1/6, 2/3 and 4/5. Because the records of
our sample custodian did not confirm the grouping of similar
elements, Hark Mason was contacted for discussion and
instructions. A replacement sample was received from CPSC on May
5th. All usual blinding procedures were followed. The sample
was tested on Kay 10th and llth. For this report, therefore,
Experiment B includes samples 2 and 7. On May 12th, Anderson
Laboratories, Inc. was informed again by phone that samples 2 and
7 are the control materials. May 13th a letter was received from
CPSC which identifies 1, 6 (Experiment A) and 4, 5 (Experiment C)
as carpet samples. Experiment A and Experiment C were carpets.
The data from Experiment A indicates the presence of sensory
J.... J. «-~..k ..*.__. £ ....»._ 1 .. *.»._ k._..l. _*._.._*,*._.._. «1._ _C*»VC ___
changes ot the sample as a result of heating, instability of the
•>tijin«*l ( »>M^H.iiiH«i> •.»*• etui*** i.M.«ntJil«i*X wlcllll** iiP lnl»r««itl IIMIM «»T !.!«»-
test sample with its absorbent layers resulting in emission and
remission of the chemicals biologically potent.
EM:EP*-IPT.WPS S/17/93
' Page 16 of 16
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CARPET STUDY REPORT
DATE OF REPORT: A
The pulmonary irritation response was primarily shown by two
individual animals (one with cample 1 and one with sample 6) and
vas pronounced on the second day. This increased response on the
second day is fairly typical of the effects noted with previous
carpet samples tested in this laboratory. The effects which were
identified by way of the FOB showed several changes compared to
prescore which were not mirrored in the sample results. In the
observations grouped as alertness/excitability the greatest
change from normal included alertness, handling and reactivity,
activity and rearing. Both increased and decreased activity
scores were recorded. Although the vocalization scores were
rather different from prescore the comparison with the control
I »wl 1 1 -.<• I *M! lit*. I 111*. «>rr«>i:l mlijlil IM. i »1«t !.*>>! l.ti t.lm £>VOt1Mi3uv* ttm
well as to the test exposure.
In the group "general appearance*1, facial swelling, lacrimation,
gasping, palpegbral closure and ear petechiae are the findings
wnicn were nor consistent wirn prescore or control, u-ne general
appearance group is primarily scored as present or absent and
T-»pT-»«»T»t' » r»1»»r rtif f»r*»tm« from t.h« «t.»irt:
-------
Appearance Data Summary
Extreme deviation from pre test values
test
facial swelling
bleeding
lecrimation
gasping
cyanosis
exophthalmus
palpebral closure
piloerection
ear petechiae
% observations showing
sample 1 ,6
34
3
13
52
6
6
13
9
63
j extreme deviation from
sample 4,5
25
13
13
37
17
13
7
0
50
pre test
sample 2,7
0
0
0
0
0
0
0
3
32
Aftderson Laboratories,-Inc,
-------
Extreme deviation from pre test values
3
3 test
alertness
handling reactivity
activity
vocalization
air puff
| click
tail pinch
jjj clonic movements
* tonic movements
«
% observations she
sample 1,6
25
28
22
40
3
19
3
0
0
wing extreme deviation
sample 4,5
17
37
10
33
3
3
3
3
3
from pre test
sample 2,7
0
0
0
19
0
0
0
0
0
-------
8
s
in
8
Neuromuscular Data Summary
Extreme Deviation from Pre test values
% 0
test
jar
grip strength
body tone
righting reflex
body posture
ataxic gait
gait score
impaired gait
inverted screen
mis steps
reach reflex
tilted screen
body tilt
bservations showii
sample 1,6
19
9
22
13
56
6
13
25
19
16
31
19
0
ig extreme deviati
sample 4,5
27
27
20
13
5O
7
17
27
23
3
50
2O
17
on from pre study
sample 2,7
0
0
0
o
0
o *
o ^
o
o
0
0
o
0
; Anderson Laboratories, Inc.
-------
APPENDIX I
ASTM E 981
Anderson Laboratories, Inc.
-------
Due study
MOUNB number
Mf USk
MM** HIM fcllOtrfcit
Tilted ICreeD lest
mmf* 9 rtmr« UP.TTVEN.noWN O trit«)
Mis-Heps
mk:M
^oreunib placing
MlflUl: 1 J
Onp strength
Handling reactivity
rwA 1-3
Lacrunation
nnk- 1-3
Filpebral closure
I«\k: M
Salivauon
mk: 1-3
pyoereciion
MMMl: U
ExophihBlmus
ninul: 1 J
Cyanosis
gtntal- U (indictu when I
aspmg
quintal: 1.2
racial swelling
Stnul: 1.2
leeding (eye.ear.nosc i
ouinul- 1.2 (indicate wliicln
Body lone
raid: M
-
Test period
•
i
Ohser
i
nn
. i
.
* • *•
*
1 •
i
!
1
I
:'i
Righting renex
Rears
ce»nt- * mn (3 min>
Body posture
rfcicripiive- M
Body tilt
rwik-M
Qonic movements
I i : . !
i i :
I i
Tonic movements :
detcrtpttve- l-f >
Gait score
rink- 1-5
Impaired gan
Ataxic gait
rtnk 1-5
\lenness •
ink M
Activtry
»nk:l-«
Di&rrnea
ninul- 1.2
kir pun response
r»k- M
Click response
rwik: M
Tail pinch response
rwk: M
invened screen test
• KC ie c1imb.HANO.DKOP
vocalizations
qn.nul- l.2«lf*S)
dttcripllvtr icpclilivt bch«v«on
ittieriplivf unuiuit bchivien
comments
i
:
t
*
i
i
i
t
i
i
i
i
'
i
I
i
-------
Appendix II
EXAMPLES OF
RESPIRATORY TRACINGS
Anderson Laboratories, Inc.
-------
I !
ii
-------
t>coce
-------
Appendix
FUNCTIONAL OBSERVATIONAL
BATTERY
Anderson Laboratories, Inc.
-------
HYBRID FUNCTIONAL OBSERVATIONAL BATTERY VCMoser
FOR USE WITH MICE EXPOSED TO CARPET SAMPLES ManTech Environ. Tech.
reviled 1/14/93
Mice will be individually tested A pre-exposure. or baseline, test will be conducted (preferably the day before exposures
begin, or else immediately before the first exposure). Post-exposure tests will be initiated 10-15 minutes after removal
from the exposure chamber. At a minimum, tests will be conducted after the second and fourth exposures, and more
often as necessary to document behavioral changes. Tests are performed in the order listed below.
•
Type of data:
Dadescriptive data. Note-more than one description is possible.
R«nnk order data
Q»quaTital, or yes/no data
Mnterval or continuous data, including count data
Jar task (I) - Place mouse on 1-gallon glass jar. hanging from the rim of the jar by only one hind leg. Count how many times,
out of three tries, the mouse falls off the jar (usually the mouse pulls up to stand upright on the jar rim).
»
Tilted screen test (I) - Holding the screen at a 45* angle, place the mouse sideways (horizontal). Count how many times the
mouse either rotates and walks up the screen (UP), walks straight across the screen (EVEN), or rotates downward and
walks down (DOWN). This is repeated three times, with a maximum of 20 sec on each try.
Mis-steps (R) - How often the mouse slips a paw down between the wire mesh while on the tilted screen.
1) none, paws are placed correctly on wire mesh
2) legs slip down only few times
3) legs slip down about half of the steps
4) legs are hanging between mesh with no attempt to pull them up
Hold mouse by tail and lower it towards edge of wire mesh screen. Watch for forelimb placing response as mouse reaches for
screen, then allow mouse to grab edge of screen and test grip strength.
Forelimb placing (Q) - Note extension and reaching with forelimbs as mouse reaches towards screen. Allow up to 5 seconds
for response to take place.
1) placing response is present
2) no response
Grip strength (I) - Raise up the mouse until the screen is about perpendicular. Count the number of times, out of three tries, the
mouse drops the screen before it reaches the 90* point
Holding the mouse in hand, assess the following measures.
Reactivity to'being handled (R) - The extent of activity and resistance to being held.
1) low. no resistance, mouse does not move
2) moderately low, slight resistance, some movement
3) active, constantly moving around in hand
4) moderately high, frantic movement, may be tense or rigid in hand
5) high, squirming, or twisting, or attempting to bite
Lacrimation (R) - evidenced by wetness around eyes
l)none
2) slight
3) severe
Palpebral closure (R)
1) eyelids wide open
2) eyelids slightly drooping
3) eyelids drooping approximately half-way
4) eyes completely shut
-------
Involuntary motor movements (D)
Qonie » Repetitive contractions and relaxations of muscles
Dnone
2) repetitive movements of mouth and jaws, smacking
3) fine quivers of limbs, ears, head, or skin
4) mild tremors, moderately coarse
5) arvere or whole body tremors, extremely coarse
6)myoclonicjerks
7) clonic convulsions
YOGJC • Prolonged contractions of muscles
Dnone
2) contraction of extensors such that limbs are rigid and extended
3) opisthotonus - head and body rigidly arched backwards
4) emprosthotonus • bead and body rigidly extended forward
5) explosive jumps into the air with all feet leaving the surface
6) severe clonic and/or tonic convulsions resulting in dyspnea, postictal depression, or death
Gait acore (R) • Degree of any abnormality of gait excluding ataxia (see below). If only ataxia present, then gait score * 1.
ataxic score > 1
l)none
2) slightly abnormal
3) somewhat abnormal
4) markedly abnormal
5) severely abnormal
•impaired gait (D) • Note, if mouse did not move during the 2-min observation period, it may be gently prodded in order to
observe the gait. If gait score is 2 or higher, the type of impairment should be indicated here.
l)none
2) hindlimbs show uncoordinated placement, exaggerated or overcompensated movements, or are splayed
3) walks on tiptoes, hindlegs perpendicular to surface
4) flat foot walk, teg(s) flat on surface, crawling
Ataxic gait (R) - Swaying, lurching, rocking, stumbling
l)none
2) slight but definite
3) somewhat, can locomote without falling
4) marked, falls over occasionally
S) severe, cannot locomote without falling
Alertness (R) • level of arousal in the open field
1) very low. stupor, coma, slight or no vibrissae movement
2) tow. some head or body movement, occasionally attends to surroundings
3) alert,'interested in surroundings, exploration, sniffing
4) high, tense, excited, sudden darting or freezing
Activity (R) - amount of activity in the open field
1) lethargy, no body movement
2) low, somewhat sluggish, little movement
3) somewhat tow. some exploratory movements
4) tow but active, exploratory movements but mostly walking with very little or no rumfing
S) clearly active, exploratory movements, includes walking and running
6) high, very active, darting or running
Varrhea (Q)
l)none
2) present
-------
Salivation (R) • evidenced by wetness around mouth and chin
Doone
2) slight
3)aevere
Piloerection (Q)
1) no piloerecnon
2) indicates presence of piloenxtion. coal does not lie down after stroking
Exophthalnras (QT
Dnone
2) indicates presence of bulging eyes
Cyanosis (Q) • evidenced by blueness of skin, paws, ears, or tail
l)none
2) present, indicate where
Gasping (Q) - evidenced by laborious and/or convulsive breathing
1) none
2) present
Facial swelling (Q)
l)none
2) present
Bleeding of eyes, ears, or nose (Q)
1) none
2) present, describe where
Body lone (R) - Assess the resistance of abdominal muscle.
1) hypotonia, completely flaccid, limp
2) slightly flaccid, abdomen shows slow return when compressed
3) body tone present, abdomen has resistance when compressed
4) hypenonia, body stiff, abdomen displays great resistance
Righting reflex (R) • Holding mouse between palms of the hands, flip hands over so that mouse is on its back. Do this several
times, rating overall ease with which it turns over and regains normal posture
1) flips over immediately
2) slightly uncoordinated, but flips over within 1-2 sec
3) difficulty turning over, takes several seconds
4) cannot turn over within 10 sec
Place mouse in open-field (can-top with 3-inch lip around the perimeter) for exactly 2 minutes. During this time, the number
of rears is counted and other observations are made.
Rearing (I) - Defined as each time the front legs of the mouse come completely off the surface, although the mouse does not
necessarily have to raise itself up. Includes when the mouse uses the side or lip of a can top as support, also includes
instances when the mouse lifts front paws for grooming.
Body posture (D)
1) lying on side
2) completely flattened, pelvis flat on surface
i 3) sitting or walking upright, pelvis off surface
4) hunched, back raised up
Body tilt (R)
1) typical posture, with head straight forward
2) head tilts to one side .
3) hand and shoulders lean or tilt to one side
4) whole body leans or tilts to one side, including falling over
-------
Stimulus reactivity • performed while mouse is sluing on open field
Air puff response (R) • Blow into face of mouse.
1) no motion or response
2) flight or sluggish reaction, e.g., blink or flinch
3) elev faction, visible startle response
•t 4) exaggerated reaction, e.g.. jumps or flips into air
Cfick response (R)* position clicker approximately 3 cm above the back of the mouse and make sudden sound.
1) no reaction or response
2) slight or sluggish reaction, some evidence that noise was heard, e.g.. ear flick
3) clear reaction, visible startle response, quick darting
4) exaggerated reaction, e.g., jumps, bites, or attacks
Tail pinch response (R) - metal tweezers are used to squeeze the tail approximately 1-2 cm from the tip
1) no reaction or response
2) slight or sluggish reaction, some evidence that pinch was felt, e.g., tail flick
3) clear reaction, visible response or jump, quick darting
4) exaggerated reaction, e.g.. jumps, bites, or attacks
Inverted screen tests 0) • Place mouse on screen and invert. Measure seconds necessary for mouse to climb to the top. Note
"D" if mouse drops off. or "H" if mouse remains hanging, to a maximum of 60 sec. During the time-0 test, mice will be
given a total of 3 chances in order to "train" the mouse to climb to the top within 20 sec. A notation will be made as to
how many trials were required or if a mouse is unable to invert after 3 trials.
Vocalizations (Q) • Note number of vocalizations, whether or not provoked by handling, testing, etc.
1) none, or only 1 or 2 instances
2) 3 or more instances
Siereorypy • record any behaviors thai are excessive or highly repetitive such as circling, stereotypic grooming, pacing.
repetitive sniffing, or head weaving.
Bizarre behavior • record any unusual behaviors such as self-mutilalion. Straub tail (tail straight up), retropulsion. writhing,
flopping, spinning, or attacking.
Other - includes soiled fur. fur discoloration, crustiness around face or eyes, emaciation, or any other findings of interest.
-------
APPENDIX IV
COMMUNICATION OF UNBLINDING
AND SAMPLE REPLACEMENT
Anderson Laboratories, Inc.
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
AIR AND ENERGY ENGINEERING RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
May 4, 1993
Dr. Rosiland Anderson
Anderson Laboratories Inc.
30 River Street
Dedham, MA 02026
Fax: 617-364-6709
Dear Dr. Anderson,
After our conversation last Friday, I spoke with Dr. Schaeffer at
CPSC and with Mr. Ezra Chan, your sample custodian for the split
sample test phase. It is clear to me that an inadvertent mixup
occurred in preparation of the third sample that was sent to
Anderson Labs. I cannot reveal the details of the mixup without
foiling the blinding procedure so please bear with me until after
we have swapped results, at which time I'll explain what
happened. Please repeat the exposure sequence with the sample
that CPSC is sending out today. You should receive the package
by 10 AM Wednesday, May 5th. Please follow the established
blinded procedure for sample loading and testing. Pair the new
data with exposure series number two and disregard the original
data, it is not relevant to the test plan.
Please accept my apologies for the delay and extra effort this
mistake has caused. I realize that this will make it necessary
to move the date for exchanging preliminary reports to Monday,
May 10th. Please call me later this week and we will discuss
details regarding exchange of preliminary reports.
Sincerely,
Mark Mason
Project Coordinator
cc: Dan Costa
Robert Dyer
-------
MAY 13 £93
U.S. CONSUMER PRODUCT SAFETY COMMISSION
WASHINGTON. D.C. 2OSO7
Rosalind Anderson, Ph.D.
Anderson Laboratories Inc.
30 River Street
Dtdhll, HA 02026
Dear Dr. Anderson:
This letter serves to officially reveal the order in which
carpet aubsamples and their sham controls were shipped to an
Environmental Protection Agency (EPA) contractor, Acurex
Environmental, and Anderson Laboratories during the phase Z
evaluation of the carpet toxicity bioassay. Six shipments were
sent by the Consumer Product Safety Commission (CPSC) staff to
the sample custodians at each testing laboratory at staggered
intervals during March, 1993. Four shipments were Federal
Express packages that contained two subsamples (1 and 2) each of
two different carpets (A and 6) sealed in labeled Tedlar bags.
Two packages containing labeled bags without carpet were sent to
indicate that control tests should be run with no carpet in the
source chamber. The testing was intended to be "blinded" so that
the individuals conducting the animal experiments did not know
the identity of the samples being tested. The order in which the
six packages were shipped are tabulated below along with the
dates the shipments were made, the source identification, and the
contents of the labeled bags.
Shipment
1
2
3
4
5
6
Date
Karch 4, 1993
March B, 1993
March 16, 1993
March 22, 1993
March 25, 1993
March 29, 1993
Source ID
Carpet Al
Control 1
Control 2
Carpet Bl
Carpet B2
Carpet A2
Contents
Pink C&rpet
Empty
Paper
Blue Carpet
Blue Carpet
Pink Carpet
Some explanation is needed in regard to the control
shipments 2 and 3, Prigr tG the initiation Of the BtUCty; it WU
agreed that a package solely containing an empty bag would serve
-------
to signal that a control experiment was to be performed at the
testing laboratories. As a result, shipment 2 contained an empty
bag. However, ^flt,irir1nr^cJl^fjffihj^r\s
-------
APPENDIX V
TEMPERATURE AND HUMIDITY VALUES
Anderson Laboratories, Inc.
-------
TEST NUMBER: |921
SAMPLE SOURCE: CPSC SAMPLE 1
TABLE 1» TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM!
BOTTOM OUT-
SIDE S2
(°C)
72.6
73.7
74.6
75.4
76.0
75.9
75.7
?LE CHAMBER
TOP OUT-
SIDE S7
(°C)
42.3
43.4
45.0
47.1
47.4
47.8
47.9
AIR Al
(°C)
35.3
36.4
32.8
33.0
33.4
33.3
33.2
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.0
23.0
24.0
24.0
24.0
24.0
24.2
ROOM
HUMIDITY
(%)
38.0
38.0
37.0
38.0
38.5
38.5
38.5
ROOM
TEMPERATURE
<°F)
71
71
71
71
71
72
71
0°
1^
I
-J
ct
-------
TEST NUMBER: |922
SAMPLE SOURCE: CPSC SAMPLE 1
EXPERIMENT A
T&BLB 2S TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
(°C)
75.4
75.7
76.0
76.7
76.8
77.2
76.3
>LE CHAMBER
TOP OUT-
SIDE S7
(°C)
48.0
48.1
47.8
47.5
47.1
47.0
47.1
AIR Al
(°C)
37.9
38.2
38.0
37.1
36.6
36.7
37.6
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23
23.5
24.5
24.5
24.0
24.0
24.0
ROOM
HUMIDITY
(*)
38.0
38.0
37.5
38.0
36.5
36.5
36.5
ROOM
TEMPERATURE
(OF)
71
71
71
71
71
71
71
-------
TEST NUMBER: |923
SAMPLE SOURCE: CPSC SAMPLE 1
EXPERIMENT A
TABLE 31 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM1
BOTTOM OUT-
SIDE S2
(°C)
73.9
74.4
75.6
76.1
76.0
76.5
76.4
?LE CHAMBER
TOP OUT-
SIDE S7
(°C)
39.2
40.4
41.3
42.3
42.7
42.3
43.8
AIR Al
<°C)
35.7
35.6
34.0
33.5
33.7
33.6
33.6
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.5
24.0
24.0
24.0
24.5
24.5
24.0
ROOM
HUMIDITY
(%)
37.0
36.5
36.1
36.0
36.0
36.0
36.0
ROOM
TEMPERATURE
<°F)
70
70
70
70
70
70
70
o
IT
-------
TEST NUMBER: |924
SAMPLE SOURCE: CPSC SAMPLE 1
EXPERIMENT A
TABLE 4S TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
<°C)
76.3
76.9
76.6
77.0
77.4
77.8
78.0
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
46.8
47.0
47.0
46.8
46.6
46.6
46.6
AIR Al
(°C)
35.2
35.3
34.0
33.9
33.8
33.8
34.0
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.0
23.0
24.0
24.5
25.0
25.0
25.0
ROOM
HUMIDITY
(%)
36.0
36.0
35.5
35.5
36.0
36.5
36.5
ROOM
TEMPERATURE
(°F)
70
70
70
70
70
70
70
T
t
-J
-------
TEST NUMBER: |962
SAMPLE SOURCE: CPSC SAMPLE 6
EXPERIMENT A
TABLE 5s TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM1
BOTTOM OUT-
SIDE S2
<°C)
74.3
75.2
75.0
75.6
76.2
75.9
76.4
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
38.0
37.8
39.0
39.0
39.9
40.5
40.7
AIR Al
(°C)
34.2
35.2
33.5
34.1
34.4
34.2
34.9
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.0
23.5
24.0
24.0
24.0
24.0
ROOM
HUMIDITY
(%)
45
45
46
46
46
45
46
ROOM
TEMPERATURE
(°F)
68
69
70
70
70
70
70
ci
-------
TEST NUMBER: |963
SAMPLE SOURCE: CPSC SAMPLE 6
EXPERIMENT A
TABLE 61 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
(°C)
72.6
74.8
77.5
76.5
76.0
76.0
75.8
>LE CHAMBER
TOP OUT-
SIDE S7
(°C)
45.6
45.6
44.0
43.3
43.2
43.1
43.1
AIR Al
<°C)
36.6
36.9
36.1
37.0
37.4
36.4
37.5
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.5
23.5
24.0
24.0
24.0
24.0
24.0
ROOM
HUMIDITY
(%)
45
46
46
46
46
46
46
ROOM
TEMPERATURE
<°F)
70
70
70
70
70
70
70
I
-------
TEST NUMBER: I964
SAMPLE SOURCE: CPSC SAMPLE 6
EXPERIMENT A
TABLE 71 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM!
BOTTOM OUT-
SIDE S2
(°C)
78.4
78.8
78.9
78.9
78.8
78.7
79.1
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
36.2
37.7
38.9
39.9
40.3
41.1
41.5
AIR Al
(°C)
32.0
34.6
33.8
34.1
34.2
34.0
35.6
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.0
24.0
24.0
24.0
24.0
24.0
ROOM
HUMIDITY
(*)
47
46
45
45
45
45
45
ROOM
TEMPERATURE
<°F)
70
70
70
70
70
70
70
i
-J
-------
TEST NUMBER: *965
SAMPLE SOURCE: CPSC SAMPLE 6
EXPERIMENT A
TABLE 8: TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM!
BOTTOM OUT-
SIDE S2
<°C)
70.2
73.4
73.6
73.3
72.9
72.7
72.0
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
43.5
42.8
41.4
39.8
39.3
39.1
39.0
AIR Al
(°C)
34.0
35.3
34.8
34.0
33.7
33.6
34.3
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.5
24.0
24.0
24.0
24.0
24.0
24.0
ROOM
HUMIDITY
(*)
44
44
44
44
44
44
44
ROOM
TEMPERATURE
(°F)
70
70
70
71
71
71
70
i
-j
Q
-------
TEST NUMBER: |925
SAMPLE SOURCE: CPSC SAMPLE 2
B
TABLE 91 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM!
BOTTOM OUT-
SIDE S2
(°C)
65.4
67.0
66.8
66.4
66.1
66.6
67.5
>LE CHAMBER
TOP OUT-
SIDE S7
(°C)
43.5
44.8
45.7
46.0
46.4
46.5
47.1
AIR Al
(°C)
37.1
38.7
37.5
37.5
37.3
37.3
39.1
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.5
24.0
24.5
24.5
25.0
25.0
24.0
ROOM
HUMIDITY
(%)
36.0
36.0
36.0
36.0
36.0
36.0
36.0
ROOM
TEMPERATURE
(°F)
71
71
71
71
71
71
?1
-------
TEST NUMBER: 1926
SAMPLE SOURCE: CPSC SAMPLE 2
EXPERIMENT B
TABLE 101 TBMPERATORB, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM!
BOTTOM OUT-
SIDE S2
(°C)
66.5
65.8
65.4
65.7
65.9
65.6
65.7
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
42.0
41.9
41.6
41.2
40.9
40.8
41.2
AIR Al
(°C)
37.2
37.2
35.1
34.6
34.6
34.6
36.0
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.5
23.5
24.0
24.5
25.0
25.0
24.5
ROOM
HUMIDITY
(*)
36.0
36.0
35.0
35.0
35.0
35.0
35.0
ROOM
TEMPERATURE
(OP)
71
71
70
70
70
70
70
-------
TEST NUMBER: |927
SAMPLE SOURCE: CPSC SAMPLE 2
EXPERIMENT B
TABLE lls TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
(°C)
66.0
66.0
65.0
64.4
64.9
64.7
65.6
PLE CHAMBER
TOP OUT-
SIDE S7
<°C)
37.5
39.0
39.4
39.7
39.9
40.0
40.5
AIR Al
(°C)
34.2
35.5
33.8
33.7
33.9
33.8
35.6
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.5
24.0
24.5
25.0
25.0
24.5
ROOM
HUMIDITY
(«)
29.0
29.0
29.0
29.0
27.0
27.0
27.0
ROOM
TEMPERATURE
(°F)
70
71
71
71
71
71
71
-------
TEST NUMBER: ft928
SAMPLE SOURCE: CPSC SAMPLE 2
EXPERIMENT B
TABLE 121 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
<°C)
67.2
67.9
66.8
66.4
66.6
66.2
66.1
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
43.0
43.1
42.6
42.2
41.9
42.0
42.2
AIR Al
<°C)
38.8
39.1
36.8
35.5
35.2
35.8
37.2
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.5
23.5
24.0
25.0
25.0
25.0
24.5
ROOM
HUMIDITY
(%)
28.0
28.0
27.0
27.0
27.0
27.0
27.0
ROOM
TEMPERATURE
(OF)
70
70
70
70
70
70
70
cr
3-
Jl
cr
-------
TEST NUMBER: |1034
SAMPLE SOURCE: 7
EXPERIMENT B
TABLE 13| TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
(°C)
66.6
66.8
65.0
65.0
65.4
65.8
65.9
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
39.8
39.9
41.8
43.5
44.7
44.9
45.5
AIR Al
(°C)
35.1
35.4
35.8
36.8
37.5
38.0
38.2
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
22.0
22.0
23.0
23.5
23.5
23.5
23.0
ROOM
HUMIDITY
(%)
55
55
55
55
55
55
54
ROOM
TEMPERATURE
(°P)
70
70
70
71
71
72
72
LP
^
ct
-------
TEST NUMBER: 11035
SAMPLE SOURCE: 7
EXPERIMENT B
TABLE 14s TBMPBRATOBB, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM1
BOTTOM OUT-
SIDE S2
(°C)
68.0
67.7
67.1
67.1
67.0
67.0
67.1
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
43.3
43.4
44.2
44.3
45.1
45.0
45.0
AIR Al
(°C)
39.1
39.4
39.1
39.2
39.3
39.2
39.3
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.0
23.0
23.5
24.0
24.0
24.0
ROOM
HUMIDITY
(%)
54
54
54
54
54
54
55
ROOM
TEMPERATURE
(OF)
72
72
72
73
74
74
74
-------
TEST NUMBER: 11036
SAMPLE SOURCE: 7
EXPERIMENT B
TABLE 151 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
IS MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
(°C)
65.3
65.6
65.5
65.0
65.2
65.5
65.3
>LE CHAMBER
TOP OUT-
SIDE S7
(°C)
41.7
41.4
40.9
40.4
40.2
40.2
40.3
AIR Al
<°C)
38.6
35.9
35.4
35.5
35.5
35.1
35.6
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
21.5
22.0
22.0
23.0
23.5
23.5
23.5
ROOM
HUMIDITY
(%)
55
55
55
55
54
54
54
ROOM
TEMPERATURE
<°F)
71
71
71
71
72
72
72
10
cr
-------
TEST NUMBER: |1037
SAMPLE SOURCE: 7
EXPERIMENT B
TABLE 16» TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
(°C)
70.2
70.1
70.1
69.9
69.9
69.6
70.1
>LE CHAMBER
TOP OUT-
SIDE S7
(°C)
43.1
43.3
43.7
43.2
43.0
43.1
43.8
AIR Al
<°C)
39.7
39.6
38.8
38.2
38.5
38.5
39.6
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.5
24.0
24.0
24.5
24.5
24.5
24.0
ROOM
HUMIDITY
(%)
56
56
56
56
58
58
58
ROOM
TEMPERATURE
(OF)
73
73
72
72
72
72
71
in
-------
TEST NUMBER: I944
SAMPLE SOURCE: CPSC SAMPLE 4
EXPERIMENT C
TABLE 17 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM!
BOTTOM OUT-
SIDE S2
<°C)
68.1
68.4
69.8
71.0
71.3
71.4
71.5
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
44.2
44.8
45.1
45.1
45.3
45.6
45.7
AIR Al
(°C)
36.2
36.4
35.1
35.2
35.3
35.0
35.0
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.5
23.5
23.5
24.0
24.0
24.0
ROOM
HUMIDITY
(%)
36
36
36
37
37
37
37
ROOM
TEMPERATURE
(OF)
70
70
70
70
70
70
70
LO
-------
TEST NUMBER: 1945
SAMPLE SOURCE: CPSC SAMPLE 4
EXPERIMENT C
TABLE 16s TBMPBBATORB, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM1
BOTTOM OUT-
SIDE S2
<°C)
65.3
65.9
67.6
68.0
68.2
69.4
69.9
>LE CHAMBER
TOP OUT-
SIDE S7
(°C)
48.0
47.8
47.6
47.0
46.8
46.6
46.8
AIR Al
<°C)
34.6
35.0
34.8
34.5
34.5
36.0
36.0
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.0
23.5
24.0
24.0
24.0
24.0
24.0
ROOM
HUMIDITY
(%)
37
37
37
37
37
37
37
ROOM
TEMPERATURE
(°F)
71
71
70
70
70
71
71
I/
4
-------
TEST NUMBER: |946
SAMPLE SOURCE: CPSC SAMPLE 4
EXPERIMENT C
TABLE 19S TBMPBRATORB, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
(°C)
68.5
69.6
71.3
72.3
73.6
73.8
72.5
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
43.1
45.3
46.1
46.7
46.4
46.3
46.5
AIR Al
(°C)
37.0
37.7
33.0
33.1
33.1
33.4
35.0
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.0
23.5
23.5
23.5
23.5
23.0
ROOM
HUMIDITY
(%)
39
39
39
39
39
38
39
ROOM
TEMPERATURE
<°F)
70
70
70
70
70
60
70
I
-J
-------
TEST NUMBER: |947
SAMPLE SOURCE: CPSC SAMPLE 4
EXPERIMENT C
TABLE 20X TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM!
BOTTOM OUT-
SIDE S2
(°C)
72.9
72.8
73.8
74.2
74.7
74.0
74.1
>LE CHAMBER
TOP OUT-
SIDE S7
(°C)
48.1
48.3
48.0
47.9
47.8
47.6
47.7
AIR Al
(°C)
36.5
35.8
37.6
38.4
39.0
39.7
39.9
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.0
23.5
24.0
24.0
24.0
24.0
ROOM
HUMIDITY
(%)
39
40
40
39
39
41
41
ROOM
TEMPERATURE
(°F)
70
70
70
69
70
70
70
r*-
i
-------
TEST NUMBER: |958
SAMPLE SOURCE: CPSC SAMPLE 5
EXPERIMENT c
TABLE 211 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM1
BOTTOM OUT-
SIDE S2
(°C)
69.7
70.3
71.5
71.5
71.8
71.9
71.2
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
44.6
45.9
46.5
47.2
47.6
48.1
48.5
AIR Al
<°C)
35.8
36.4
36.1
36.5
36.7
37.1
38.0
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.0
23.5
23.5
24.0
24.0
23.5
ROOM
HUMIDITY
(%)
48
48
47
47
43
48
48
ROOM
TEMPERATURE
<°F)
70
70
70
70
70
70
70
QO
in
-------
TEST NUMBER: |959
SAMPLE SOURCE: CPSC SAMPLE 5
EXPERIMENT C
TABLE 221 TBMPBRATORB, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAM1
BOTTOM OUT-
SIDE S2
<°C)
72.2
72.6
72.5
72.4
72.3
72.7
71.6
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
42.6
44.4
43.5
42.9
41.5
41.4
41.5
AIR Al
<°C)
39.5
39.6
38.5
38.2
37.6
37.4
37.8
MOUSE
CHAMBER
SAMPLING
PORT
(°C)
23.0
23.0
23.5
24.0
24.5
25.0
24.5
ROOM
HUMIDITY
(%)
48
47
47
48
48
48
48
ROOM
TEMPERATURE
(OF)
71
71
70
70
70
71
71
cr
tf
i
-------
TEST NUMBER: I960
SAMPLE SOURCE: CPSC SAMPLE 5
EXPERIMENT C
TABLE 231 TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
<°C)
72.8
72.1
74.5
74.4
74.3
74.4
73.8
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
38.0
43.8
45.6
45.9
46.7
47.1
47.6
AIR Al
(°C)
37.1
38.1
38.0
38.6
39.2
39.2
39.4
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.0
23.0
24.0
24.5
24.0
24.0
24.0
ROOM
HUMIDITY
(%)
47
47
48
48
48
48
48
ROOM
TEMPERATURE
(°F)
70
70
70
70
70
70
70
o
>o
-J
-------
TEST NUMBER: |961
SAMPLE SOURCE: CPSC SAMPLE 5
EXPERIMENT C
TABLE 24S TEMPERATURE, HUMIDITY RECORD
TIME
START BASELINE
TIME 0 (START EXPOSURE)
15 MIN
30 MIN
45 MIN
60 MIN
75 MIN
SAMI
BOTTOM OUT-
SIDE S2
(°C)
71.2
71.3
70.8
71.4
68.6
68.3
68.5
>LE CHAMBER
TOP OUT-
SIDE S7
<°C)
44.6
44.9
45.5
45.5
45.6
45.6
45.8
AIR Al
(°C)
38.5
38.9
38.2
38.7
38.8
38.9
40.0
MOUSE
CHAMBER
SAMPLING
PORT
<°C)
23.5
23.0
23.5
24.0
24.0
24.0
24.0
ROOM
HUMIDITY
(%)
46
46
46
46
45
45
45
ROOM
TEMPERATURE
(°F)
70
70
70
70
70
70
.70
0
-J
-------
APPENDIX VI
FOB, EXPERIMENT A
Anderson Laboratories, Inc.
AL-kl
-------
ACTIVITY / EXCITABILITY
Sample 1, 6
DAYS 1,2
OBSERVATION
alert
handling reactive
activity
vocalization
air puff
click
tail pinch
clonic
tonic
rears
PRE SCORE
#OBS X SCORE
7X3, 1X2
7X3, 1X2
5X4, 3X5
8XA
8X2
7X2, 1X3
8X2
8X1
8X1
3-11
# OBS OUTSIDE
PRE SCORE RANGE
15
11
17
10
5
7
8
31
1
20
#
OBS
32
32
32
32
32
32
32
32
32
32
%OUT
OF RANGE
47
34
53
31
16
22
25
97
3
63
r-
P/A = present/absent
ep17
ANDERSON LABORATORIES, INC.
-------
ACTIVITY/ EXCITABILI
EXTREME CHANGES, Samples 1,6
DAYS 1,2
OBSERVATION
alert
handling reactive
activity
vocalization
air puff
click
tail pinch
REARS
PRE SCORE
#OBS X SCORE
7X3,1X2
7X3,1X2
5X4, 3X5
8XA
8X2
7X2, 1 X3
8X2
45/8
5.6
OBSERVATIONS SHOWING
EXTREME SCORE
6X STUPOR, 2X HYPER
9X LOW,
6XNONE, 1X HYPER
13X PRESENT
1X EXTREME
6X NONE
1X EXTREME
84/32
2.6
11X NO REARS
TOTAL #
OBS
32
32
32
32
32
32
32
p/A = present/absent
epactiv16
-------
Neuromuscular Function
Samples 1, 6
DAYS 1,2
OBSERVATION
jar
grip strength
body tone
righting reflex
body posture
body tilt
ataxic gate
gait score
impaired gait
inverted screen
mis-steps
reach reflex
tilt screen
PRE SCORE
# OBS X SCORE
7X0,1X1
7X0,1X1
8X3
8X1
8X3
8X1
8X1
8X1
8X1
7XH, 1XFAST
8X1
8X1 (PRESENT)
8X3 up
# OBS OUTSIDE
PRE SCORE RANGE
13
8
30
23
20
11
4
31
29
14
15
10
17
TOTAL #
OBS
32
32
32
32
32
32
30
31
29
32
31
32
32
% OUT
OF RANGE
41
25
94
72
63
34
13
100
100
44
48
31
53
IF*
*£.
EPN16A
-------
iNeuromuscuiar i-unction
EXTREME CHANGES Samples 1,6
DAYS 1,2
OBSERVATION
jar
grip strength
body tone
righting reflex
body posture
ataxic gate
gait score
impaired gait
inverted screen
mis-steps
reach reflex
tilt screen
PRE SCORE
# OBS X SCORE
7X0,1X1
7X0,1X1
8X3
8X1
8X3
8X1
8X1
8X1
7XH, 1XFAST
8X1
8X1 (PRESENT)
8X3 up
OBSERVATIONS
EXTREME SCORE
6X 3 FALLS
3X 3 DROPS
7X HYPOTONIA
4X NOT PRESENT
19X HUNCHED
2X MARKED
4X SEVERE
8X SCORE 4
6X DROP
5X LEGS HANGING
1 0X ABSENT
6X 3 DOWN
#
OBS
32
32
32
32
32
30
31
29
32
31
32
32
%
EXTREME
19
9
22
13
59
7
13
28
19
16
31
19
r
e-
\n
EP9
ANDERSON LABORATORIES, INC.
-------
GENERAL APPEARANCE
Samples 1, 6
DAYS 1,2
OBSERVATION
face swelling
bleeding
lacrimation
salivation
diarrhea
gasping
cyanosis
exophthalmus
eye closure
piloerection
ear petechiae
PRE SCORE
8XA
8XA
8X1
8X1
8XA
8XA
8XA
8XA
8X1
8XA
8XA
# OBS OUTSIDE
PRE SCORE RANGE
11
1
20
0
0
16
2
2
14
3
20
Total #
OBS
32
32
32
32
32
31
32
32
32
32
32
%OUT
OF RANGE
34
3
63
0
0
52
6
6
41
9
63
r
P/A = present/absent
EPAPEAR3
-------
GHRERAL APPEARANCE
EXTREME CHANGES, Samples 1, 6
DAYS 1,2
OBSERVATION
face swelling
bleeding
lacrimation
gasping
cyanosis
exophthalmus
eye closure
piloerection
ear petechiae
PRE SCORE
8XA
8XA
8X1
8XA
8XA
8XA
8X1
8XA
8XA
OBSERVATIONS
EXTREME SCORE
11X PRESENT
1X PRESENT
4X SEVERE
1 6X PRESENT
2X PRESENT
2X PRESENT
4X EYES SHUT
3X PRESENT
20X PRESENT
#
OBS
32
32
32
31
32
32
32
32
32
%
EXTREME
34
3
13
52
6
6
13
9
62
r-
ts
>I
EPA16 MAX
P/A =PRESENT/ ABSENT
ANDERSON LABORATORIES, INC.
-------
OTHER OBSERVATIONS
SAMPLES 1, 6
OBSERVATION FREQUENCY
POPCORN 2
FALL OVER 1
NO ACTIVITY 12
BLEEDING EYE 1
REPETITIVE MOTION
FOOT 2
CIRCLE 2
NOSE BUMP 4
BUMP INTO WALL 1
ANDERSON LABORATORIES, INC.
-------
APPENDIX VII
FOB, EXPERIMENT B
Anderson Laboratories, Inc.
-------
ACTIVITY / EXCITABILITY
Samples 2,7
DAYS 1,2
OBSERVATION
alert
handling reactive
activity
vocalization
air puff
click
tail pinch
clonic
tonic
rears
PRE SCORE
#OBS X SCORE
8X3
7X3,1X2
4X4, 4X5
8XA
7X2,1X3
8X2
4X1 , 4X2
8X1
8X1
1-10
# OBS OUTSIDE
PRE SCORE RANGE
0
0
0
6
2
4
9
8
0
5
TOTAL #
OBS
31
31
31
31
31
31
31
31
31
31
%OUT
OF RANGE
0
0
0
19
6
13
29
26
0
16
P/A = present/absent
ep11
ANDERSON LABORATORIES. INC.
-------
ACTIVITY / EXCITABILITY
EXTREME CHANGES, Sample 2,7
DAYS 1,2
r-
OBSERVATION
vocalization
PRE SCORE
#OBS X SCORE
8XA
# OBSERVATIONS SHOWING
EXTREME SCORE
6X PRESENT
OBS
31
EXTREME
19
p/A = present/absent
ep13
ANDERSON UBORATORIES, INC.
-------
Neuromuscular Function
Samples 2,7
DAYS 1,2
OBSERVATION
jar
grip strength
body tone
righting reflex
body posture
body tilt
ataxic gate
abnormal gait score
impaired gait
inverted screen
mis-steps
reach reflex
tilted screen
PRE SCORE
# OBS X SCORE
3X0, 1X1
4X0
4X3
4X1
4X3
4X1
4X1
4X1
4X1
4XH
4X1
4X1 (PRESENT)
4X0 (DOWN)
# OBS OUTSIDE
PRE SCORE RANGE
0
0
2
1
5
0
0
9
9
8
0
0
3
#
OBS
31
31
31
31
31
31
31
31
31
31
31
31
31
%OUT
OF RANGE
0
0
6
3
16
0
0
29
29
26
0
0
10
V
EPAN2A
ANDERSON IABORATORIES INC.
-------
iwuromuscular Function
EXTREME CHANGES, Samples 2,7
DAYS 1,2
OBSERVATION
PRE SCORE
# OBS X SCORE
OBSERVATIONS SHOWING
EXTREME SCORE
TOTAL #
OBS
NONE
l/J
EPAN2A MAX
ANDERSON LABORATORIES. INC.
-------
GENERAL APPEARANCE
Samples 2,7
DAYS 1,2
OBSERVATION
face swelling
bleeding
lacrimation
salivation
diarrhea
gasping
cyanosis
exophthalmus
eye closure
piloerection
ear petechiae
PRE SCORE
8XA
8XA
8X1
8X1
8XA
8XA
8XA
8XA
8X1
8XA
8XA
# OBS OUTSIDE
PRE SCORE RANGE
0
0
2
0
0
0
0
0
0
1
10
Total #
OBS
31
31
31
31
31
31
31
31
31
31
31
% OUT
OF RANGE
0
0
6
0
0
0
0
0
0
3
32
44
p/A = present/absent
EP15
ANDERSON LABORATORIES, INC.
-------
UhNhHAL APPEARANCE
EXTREME CHANGES, Samples 2,7
DAYS 1,2
OBSERVATION
PRE SCORE
# OBSERVATIONS
EXTREME SCORE
Total
#OBS
EXTREME
ear petechiae
4XA
10X PRESENT
31
32
EPAR 2,7 max
P/A ^PRESENT/ ABSENT
ANDERSON LABORATORIES, INC.
-------
OTHER OBSERVATIONS
SAMPLES 2, 7
OBSERVATION FREQUENCY
REPETITIVE MOTIONS
FOOT 1
CIRCLE 4
ANDERSON LABORATORIES. INC.
AL-7-6
-------
APPENDIX VIII
FOB, EXPERIMENT C
Anderson Laboratories, Inc.
AL-7?
-------
ACTIVITY / EXCITABILITY
Sample 4, 5
DAYS 1,2
OBSERVATION
alert
handling reactive
T activity
• _ .,. ,.
•^j
oO vocalization
air puff
click
tail pinch
clonic
tonic
rears
PRE SCORE
#OBS X SCORE
8X3
6X3, 2X2
7X4, 1X3
8XA
8X2
8X2
7X2, 1X1
8X1
8X1
1-8
# OBS OUTSIDE
PRE SCORE RANGE
15
11
17
10
5
7
8
30
1
16 (3 UP, 13 DOWN)
#
OBS
30
30
30
30
30
30
30
30
30
30
%OUT
OF RANGE
50
37
57
33
20
23
27
100
3
53
P/A = present/absent
ep14
ANDERSON LABORATORIES, INC.
-------
ACTIVITY / EXCITABILITY
EXTREME CHANGES, Sample 4, 5
DAYS 1,2
OBSERVATION
alert
handling reactive
activity
vocalization
air puff
click
tail pinch
clonic
tonic
PRE SCORE
#OBS X SCORE
8X3
6X3, 2X2
7X4, 1X3
8XA
8X2
8X2
7X2, 1X1
8X1
8X1
OBSERVATIONS SHOWING
EXTREME SCORE
5X STUPOR
1OX LOW, 1X HIGH
2X NONE, 1X HYPER
10X PRESENT
1X EXTREME
1X EXTREME
1X EXTREME
1X MYOCLONUS
1X EMPROSTHOTONUS
#
OBS
30
30
30
30
30
30
30
30
30
%
EXTREME
17
37
10
37
3
3
3
3
3
P/A present/absent
epio
ANDERSON LABORATORIES, INC.
-------
Neuromuscular Function
Samples 4, 5
DAYS 1,2
OBSERVATION
jar
grip strength
body tone
righting reflex
body posture
body tilt
ataxic gate
gait score
impaired gait
inverted screen
mis-steps
reach reflex
tilt screen
PRE SCORE
# OBS X SCORE
6X0, 2X1
7X0,1X1
8X3
8X1
8X3
8X1
8X1
8X1
8X1
7XH, 1XFAST
8X1
8X1 (PRESENT)
8Xup
# OBS OUTSIDE
PRE SCORE RANGE
9
11
29
20
17
13
2
21
21
10
16
15
15
TOTAL #
OBS
30
29
30
30
29
30
28
30
30
30
30
30
30
% OUT
OF RANGE
30
38
97
67
59
43
7
70
70
33
53
50
50
oO
0
EPAN4,5
-------
Neuromuscular Function
EXTREME CHANGES, SAMPLES 4,5
DAYS 1,2
OBSERVATION
jar
grip strength
body tone
righting reflex
body posture
body tilt
ataxic gate
gait score
impaired gait
inverted screen
mis-steps
reach reflex
tilt screen
PRE SCORE
# OBS X SCORE
6X0, 2X1
7X0, 1X1
8X3
8X1
8X3
8X1
8X1
8X1
8X1
7XH, 1XFAST
8X1
8X1 (PRESENT)
8X3UP
OBSERVATIONS
EXTREME CHANGE
8X3 FALLS
8X3 DROPS
6X HYPOTINIA
4X NOT PRESENT
1X ON SIDE, 14X HUNCHED
5X SHOULDER OR BODY LIST
2X SEVERE
5X SEVERE
8X4
7X DROP
1X LEGS HANGING
15X ABSENT
6X 3DOWN
TOTAL
#OBS
30
29
30
30
29
30
28
30
30
30
30
30
30
%
EXTREME
27
28
20
13
3
17
7
17
27
23
3
50
20
r-
i
oO
EPAN4.5 max
ANDERSON LABORATORIES, INC.
-------
GENERAL APPEARANCE
Samples 4, 5
DAYS 1,2
OBSERVATION
face swelling
bleeding
lacrimation
salivation
diarrhea
gasping
cyanosis
exophthalmus
eye closure
piloerection
ear petechiae
PRE SCORE
8XA
8XA
8X1
8X1
8XA
8XA
8XA
8XA
8X1
8XA
8XA
# OBS OUTSIDE
PRE SCORE RANGE
7
4
16
2
0
11
5
4
9
0
15
Total #
OBS
30
29
30
30
30
30
30
30
30
30
30
%OUT
OF RANGE
23
14
53
7
0
37
17
13
30
0
50
oC,
p/A = present / absent
EPS
ANDERSON LABORATORIES, INC.
-------
UtNLKAL APPEARANCE
EXTREME CHANGES, SAMPLES 4,5
DAYS 1,2
OBSERVATION
face swelling
bleeding
lacrimation
gasping
cyanosis
exophthalmus
eye closure
ear petechlae
PRE SCORE
8XA
8XA
8X1
8XA
8XA
8XA
8X1
8XA
OBSERVATIONS
EXTREME SCORE
7X PRESENT
4X PRESENT
4X SEVERE
11X PRESENT
5X PRESENT
4X PRESENT
2X EYES SHUT
15X PRESENT
#
OBS
30
29
30
30
30
30
30
30
%
EXTREME
23
14
13
37
17
13
7
50
r-
ep6
P/A = PRESENT/ABSENT
ANDERSON LABORATORIES, INC.
-------
OTHER OBSERVATIONS
SAMPLES 4, 5
OBSERVATION FREQUENCY
POPCORN 3
NO ACTIVITY 5
BLEEDING NOSE 2
BLEEDING EYE -1
REACHING WITH ONE ARM 1
SPASMODIC CHIRPING 1
REPETITIVE MOTION
CIRCLE 3
WALK OFF EDGE 3
ANDERSON LABORATORIES. INC.
-------
REVIEW OF PHASE I OF EPA/ORD CARPET STUDY
Prepared for the U.S.E.P.A.
Richard B. Schlesinger, Ph.D.
Department of Environmental Medicine
New York University Medical Center
New York, NY
June 1,1993
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This report provides my views of the Phase I component of the
EPA/ORD carpet study as described in the written reports and in oral
presentations at EPA/HERL on May 26 and 27th, 1993.
GENERAL CONCLUSION
The goal of Phase I was to perform, to the extent possible, replicate
studies at EPA/HERL and at Anderson Laboratories. The study design was
originally provided by EPA and was subsequently modified by a peer review
panel. The experiments at both sites were conducted within the constraints
imposed by the protocol. With this in mind, the study as performed by EPA
clearly conformed to GLP, and was conducted with appropriate and adequate
QA in all components and for both the bioassays and the chemical/physical
characterization of carpet emissions. Some concern about data analysis for
the FOB is expressed, but this is not a major problem. Accordingly, it is my
opinion that the EPA did perform a scientifically valid study of the effect of
carpet emissions on mice, and was not able to find any toxic effects which
could be ascribed to the carpets tested under the conditions imposed by the
experimental design. Furthermore, the levels of emissions of any potentially
toxic chemicals from these carpets was very low, and unless there was some
exotic interaction between these chemicals, the lack of biological response is
not surprising.
On the other hand, the study as performed by Anderson Laboratories
did not conform to GLP, inasmuch as the double blind condition was not
maintained, the animal housing was substandard (possibly resulting in
compromised mice), and the data presentation and analysis for the FOB and
analysis of the respiratory waveforms are of major concern. The lack of
double blind conditions at Anderson Laboratories could have resulted in
exaggeration of the severity of response for the FOB and the respiratory
waveform analyses, which tend to be somewhat subjective in nature under
ideal conditions. Fu therm ore, the inadequate housing of the mice at
Anderson Laboratories could have resulted in some change in their health
status, perhaps increasing susceptibility to even low levels of chemicals
emitted by the heated carpets or, more likely, to some combination of these
emissions and stress /restraint. However, this conclusion cannot be supported
with hard evidence, since no necropsy was performed after exposure nor was
-------
there any provision for sentinel (colony control) animals in the Anderson
facility protocol.
SPECIFIC CONCERNS
Double Blind Nature of the Study
One of the most important conditions of the study that must be
maintained is its double blind nature, especially since some of the bioassays
tend to be subjective. To this end, and in both laboratories, a sample custodian
received the carpet samples and loaded them into the source chamber. The
chamber was then taped so the carpet was not visible, and this source
chamber was transported to another area for connection to the exposure
chamber. Thus, theoretically, the technician actually perfoming the exposures
should not be aware of whether the exposure was to any carpet emissions or
was a sham control.
At EPA, the individual performing the exposures was not the same
individual who performed the bioassays. Furthermore, the technician who
attached the source chamber to the exposure chamber was not the same
individual who performed the actual exposures. Thus, the individual
analyzing the data was unaware of the exposure being performed, except for
any suspicion based upon response. However, in a number of cases, the sham
exposure resulted in greater responses than those noted with carpet samples
in the source chamber, which would serve to suggest a lack of bias towards
exaggeration of responses to carpet samples.
At Anderson Laboratories/ as at EPA, samples were received and loaded
by the sample custodian. However, only one other technician was responsible
for performing pre-exposure screening, for connecting the source chamber to
the exposure chamber, for conducting the exposures, for obtaining the
biological data, and generally also for rating the FOB and scoring the
waveform respiratory data. There was, therefore, opportunity for this
individual to know whether the source chamber was loaded with carpet or
was a sham exposure. This had the potential to bias the data analysis.
Furthemore, a TVOC monitor was used after the exposures by the same
technican that conducted the exposure, which provided this individual with
additional opportunity to know whether the exposure was sham or carpet,
since the carpet exposures were characterized by higher emissions than the
PR-
-------
sham exposures. No on-line monitors were used by EPA during or after the
exposures; all such measures were performed separately. Based upon the
above, it is my conclusion that the study as performed at Anderson
Laboratories was dearly not double blinded.
Measurements of Sensory and Pulmonary Irritation
Changes in respiratory rate were monitored by computerized systems
in both laboratories. However, the waveform analysis is somewhat
subjective. From discussions, it was noted that the technician at Anderson
Laboratories would occasionally adjust the gain or sensitivity of the recording
device, which would serve to change the apparent scale of the waveform
tracing. This change in gain was not always noted on the recordings, and had
the potential to result in conclusions of exaggerated responses. Futhermore,
the technician would stop and start the recorder during the measurement
period; any shift in baseline would not be known with such a procedure.
Ideally, all respiratory tracings obtained at both EPA and Anderson
Laboratories should have been read by readers at both sites, so as to provide
some inter-laboratory comparison. From discussions, it was concluded that
the reader from EPA did look at some tracings obtained at Anderson
Laboratories and found reasonable agreement when tracing scales were not
changed during recording. However, it was also noted that when both readers
did not agree, that the Anderson Laboratory reader tended to rank the effect as
more severe than did the EPA reader. As indicated above, the lack of double
blind conditions may have affected conclusion as to the degree of severity of
response.
At EPA, respiratory waveforms and rates were recorded during the
entire period of the exposure, as well as in pre- and post-exposure periods. At
Anderson Laboratories, tracings were recorded only during discrete intervals
during these times. There were, for example, three such periods during each
animal exposure period. Furthermore at EPA, all breaths were analyzed. On
the other hand, at Anderson Laboratories, the tracings were scanned and only
series of breaths that met some criterion, which is not dear to me, for
moderate to severe changes were analyzed. Thus, the database at EPA was
larger, and provided a better basis for determination of any change due to
toxicant exposure.
-------
Presentations by Anderson Laboratories indicated that sham control
exposed animals consistently had scores of zero for all parameters, indicating
no effect compared to pre-exposure levels. On the other hand, control
animals at EPA did show changes from pre-exposure levels, which in some
cases were greater than those following carpet emission exposures. The low
control levels for Anderson would further serve to exaggerate any response
seen following carpet expousre. Furthermore, in my experience, there are
always some control animals that show some response. If such animals are
removed from the study, it would further serve to exaggerate effects in
toxicant exposed animals.
Animal Housing Facility/Animal Health Status
The animal housing facility at EPA met appropriate standards. The
facility at Anderson Laboratories did not. This latter consisted of a separate
smaller room (about 12 ft x 12 ft) within a larger laboratory room in which
exposures were performed. The relative humidity and temperature within
the housing room was not rigidly controlled.; a through the wall air
conditioner was apparently used for some ventilation. Airflow was from the
laboratory into the animal room, and then out an exhaust This is opposite
from what should be maintained, namely positive pressure within the
animal housing room driving air from the animal quarters into some other
space. Under the conditions at Anderson Laboratories, any contaminants,
chemical or biological, present in the larger laboratory room would be drawn
into the animal quarters. Since the mice were housed for at least one week in
this facility prior to their use, it is quite possible that their condition was not
the same as that of the mice properly housed at EPA. But since no sentinel
animals were necropsied at Anderson Laboratories, any adverse effect of
housing in their facility is not known. However, it is possible that the mice
may have become hypersensitive to any low level carpet emissions, or more
likely to a combination of stress plus these emissions, due to some pathogen
or other airborne contaminant entering the housing quarters.
All animals exposed at EPA underwent a necropsy. However, those
exposed at Anderson Laboratories were not subjected to a valid necropsy.
Thus, in the latter case, the cause of death following exposure to carpet
emissions is unknown, and the health status of the animals compared to
those at EPA is unknown. This lack of information is a major problem in
PR.-B
-------
attempting to understand the differences in responses obtained at the two
study sites.
Room Air v. Zero Air/Carpet Water Content
Air entering the source chamber, and eventually the exposure
chamber, was room air at Anderson Laboratories and humidified zero air at
EPA. From discussions at EPA, it was determined that the air at Anderson
Laboratories had an RH of 30-50%. This is comparable to that used at EPA,
which was actually lower than expected. Thus, differences in humidity of air
entering the experimental system is likely not a factor accounting for
differences in biological response.
During discussions at EPA, a suggested possibility for such differences
was differences in water content of the carpet samples. In order to account for
biological response differences, all of the samples used at Anderson
Laboratories would have had to have a greater water content than the
samples used at EPA. Based upon the sampling scheme used to obtain carpets
for testing, the probability of this occuring is essentially nil. Thus, it is
unlikely that differences in moisture content account for differences in
biological responses between the two laboratories.
RECOMMENDATIONS
There are various routes which may be taken to attempt to resolve the
apparant discrepancy between results in the two laboratories. One approach
would be to have the study repeated by another, independent laboratory, but
this would be quite difficult, since it would also have to be repeated at EPA to
obtain some baseline for comparison. It may be better to attempt to determine
the reasons for differences in response. This could be done by having EPA
personnel together with Anderson personnel repeat the study at Anderson
Laboratories with EPA exposure equiptment, EPA double blind study
procedure and Anderson mice, which would under complete necropsy
following each exposure series. This would go a long way to help resolve the
apparent "disprepancy" in results of the two laboratories.
-------
Review of EPA and Anderson carpet experiment*
Harriet A. Burge
Based strictly on the quality of the report* (including description of
tests, results, discussion, and quality control plans) and on the post
exposure pathology data from EPA, one would have to place more
confidence in the EPA results than in those of Dr. Anderson's lab.
However, the negative case is clearly difficult to prove, and positive
results tend to be more persuasive even when the study designs are less
rigorous (or less rigorously described).
The data presented by the EPA appear to Bake a good case for little or
no effect in spite of the fact that total VOCs were elevated in the
presence of carpet, and eome recognized toxins were measured (although
in low levels). It should be noted that we already knew that carpeting
emits VOCs. Zt would have been useful for the EPA report to contain
examples of actual tracings that demonstrated slight, moderate or
severe respiratory effects (these were included in Anderson's report).
Without these, it is not possible to determine whether or not both
groups interpreted the tracings in the same way.
Since Anderson's results differ from those of the EPA,-and no obvious
reason is apparent, the case continues to be unresolved (i.e., we
don't know whether or not the carpet causes mouse toxicity). Zt is
still possible that some difference between the systems that controls
the nature of exposure to the carpet is responsible for the disparate
results. Zt is also possible that some inadvertent action on the part
of one (or both) of the investigative groups is controlling the outcome
of these experiments independent of the carpet exposures. Z suggest
that during the next phase, at least 1 EPA observer attends Anderson's
experiments, and that Dr. Anderson and/or a person she designates
attend the EPA experiments. Z would also suggest that an outside
observer attend both sets of experiments (a panelist would be a likely
choice, especially if one of the panel feels especially capable of
evaluating the respiratory function tests). Z would also suggest that
the person (at both sites) responsible for observing the nice and
recording observations be blinded to the concurrently collected ambient
measurements, since the temperatures at the bottom of the tank appear
to provide a clear indication of absence of carpeting in the chamber.
An additional control should be a "no complaint* carpet that did not
produce effects in Dr. Anderson's lab.
Zf, following the next series of experiments, a clear indication should
exist that exposure to the carpet is causing toxicity in mice, Z would
suggest that the experiments be run under more normal temperature
conditions. It is true that offgassing can be stimulated by heat.
However, in the real world of carpeting, it is probably quite unusual
for carpet materials to reach these temperatures in occupied
residential or office interiors.
With respect to public release of this data, my first choice would be
not to release information until the discrepancy problem has been
solved. However, since some release will probably be necessary, Z
-------
would suggaat a carefully wordad atataaant atraaaing that tha EPA
•xpariaanta did not daaonatrata an affact uaing aathoda that tha •xpart
panal conaidarad appropriata, but that tha EPA faala that tha iasua ia
important anough to continua taating.
Spaoific Coaaanta:
Dr. Andaraon atraaaaa that tha EPA poat axpoaura traataant ia diffarant
and that thia introducad variation' into tha data collactad. Sha
doaan't diacuaa thia furthar. Killing tha mica for pathology (aaeuaing
thia ia tha diffaranca aha aaani) obvioualy. would pravant obaarvation
of lata daatha dua to traatnant, but would not affaet diffaraneaa
obaarvad during tha axpoaura axpariaanta.
Zt ia difficult (without actually aaaing tha procadura) to iaagina why
a "low laboratory cart having 4 aidaa" would produca diffarant raaulta
froa a thraa-sidad alevatad ahelf in tha FOB atudias
Microbiological data:
A* axpactad, thaaa raaultc ara unraaarkabla and provida no inaight as
to poaaibla aadiatora of aithar tha "major" affacta aaan by Dr.
Andaraon or tha Binimal affacta of carpat axpoaura aaan by tha EPA. Aa
•tatad in tha raport, aicrobial analyaia on aaaplaa collactad long
aftar allagad axpoaura/conplaint apiaodaa nay not (and, in fact, ara
probably not) rapraaentativa of that axpoaura. At tha tiaa of taating,
unlaaa tha carpating was supporting activa aicrobial growth that would
ralaaaa VOC'a, it ia unlikaly that, in a atatic ayataa of thia aort/
particulata axpoaura to biological aganta would occur.
-------
Specific comments on the aicrobiology report:
Pg 5 (also on Pg 8): The use of the word "significantly" implies that
•oae statistical tact vac used. It is antiraly possible for vary email
consiatant diffarancaa to ba atatiatieally significant, and aultiple
tasts on thasa sample* could vail hava producad auch significance.
Actually, all you can say la that tha lava Is appaar to ba siailar.
Pg 7: Othar raaaona for the variability batvaan cultura media include
tha overgrovth by members of tha aucorales on HEX and 2% KEA.
Z diaagraa that tha cultura raaulta from R-800-3516 did not changa
aftar storage. Gliocladiua positiva plataa vant from .12 to 1 and
Trichoderaa positiva plataa vant froa 2 to 16. This indicataa a claar
changa that is not rafloctad in nuabers and points up the fact that
number* alona ara of little uaa in this kind of study. Note that
Asperglllua glaueua ia no longer a valid species nane, and that either
the actual species should be determined, or the taxon listed as
Aspergillus glaueua group.
The slightly higher levels and larger diveraity of taxa recovered froa
R-B00-3516 aay also ba due to noraal variance in fungal populations in
carpet, or to variance in tha aathods uaed. Actually, X vould consider
R-BOD-3S16 to be considerably different than tha other tvo samples.
Only Cladosporiua is consistently frequent across all three carpet
types. R-800-3516 has higher frequencies (by a factor of at laaat tvo)
for Panicilliua, Aapargillus, yeast, Alternaria, Gliocladiua, and
Trichodaraa. Z developed the following chart from your data to aafce
this evaluation.
Panicilliua
Cladoaporiua
Aspergillus
yeast
Rhitopus
Neurospora
Alternaria
Gliocladiua
Trichodaraa
R872-72
24
33
18
4
7
0
4
R-800-1205
Before After
32
26
15
4
0
2
0
22
32
9
0
4
2
0
R-800-3516
Before After (Storage)
72
37
44
11
4
2
6
22
4
74
44
37
7
0
4
9
2
30
Coaparing fungal counts per graa of carpet and par gran of duet is
irrelevant, and gives the (possibly) erroneous impression that the
current samples contained fever colony foraing units than thoaa in the
listed literature citations. Zn fact, tha frequency of Penicillium,
Aspergillus, Gliocladiua, and Trlchoderaa is an indication that R-800-
3516 is probably "contaminated."
pg 9: There is at least one paper in the literature that related dust
recoveries to airborne recoveries (Cravesen 1978*). A bigger problea
-------
here ia the fact that you measured CPU/gram of carpet rather than par
gran of duat. Carpat ia unlikely to become airborne, and you hava no
idea of tha fraction of recoveries that vara from duat in tha carpet or
were from fungi and bactaria adharing tightly to tha earpat fibara that
ara unlikaly avar to become airborna. Thia is why va uaa duat rather
than earpat.
•Grevesen S. 1978. Identification and prevalence of eulturable
aesophilic aicrofungi in houaa duat fro* 100 Danish homes. Comparison
between airborne and duet-bound fungi. Allergy 33(5):268-72
Tha endotoxin part of thia report ia weak. For example, how was
endotoxin extracted from tha carpet? Extraction method controla how
auch endotoxin you get even fron leaa conplex substrates than carpet.
Haa the aaaay uaed been checked for interference by other carpet/dust
components? Note that the endotoxin aaaay ia a bioaaaay that ia easily
perturbed by extraneoua materials in environmental samples. Also, a*
for bacteria and fungi, it ia irrelevant to compare levela recovered
from carpet to level* recovered from duat. Endotoxin ia well-known to
•tick quite tightly to eurfacea in way* that would be unlikely to allow
aarosolization, but would allow releaae into waahing aolutiona.
There ie conaidarably more literature on the microbiology of carpet
duat than ia lieted in the referencea. Considering the "volatility"
and visibility of thia project, the EPA would be wise to conduct an in-
depth and critical literature review on carpet research.
r
Tables: What ia the minimum level of aensitivity? i.e., vhat does
"below detection limit mean? Zf thia ia different for each table or
entry, then it would be better to use
-------
Lawrence Berkeley Laborator
1 Cyclotron Road Berkeley. California 94720
Energy & Environment Division (510) 486-4000 • FTS 451 -4000
June 1. 1993
Dr. Robert Dyer
Associate Director (MD-51)
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
RE: Review of Phase 1 of the EPA/ORD Carpet Study
Dear Dr. Dyer:
This letter report contains my review comments on the results of Phase 1 of the
EPA/ORD carpet study. These comments are based primarily on the reports provided
by the EPA study team and Anderson Laboratories, Inc. (AL) and on the oral
presentations made at HERL on May 26, 1993. In addition, the group of reviewers
separately interviewed the EPA study team and Dr. Anderson on May 27.
It is my opinion that the EPA team adequately reproduced in their laboratory the
apparatus and procedures developed at AL to measure the respiratory and neurotoxic
effects of carpets on mice. This was accomplished through numerous discussions with
Dr. Anderson and by reciprocal visits, including one week at AL in which the EPA team
worked side-by side with the AL technician. The comparability of the apparatus is, in
part, demonstrated by the temperature data. The temperature range in the source
chamber (location Ai) at AL was 32-40° C; at EPA the range was 36-39* C, The
temperature range in the mouse exposure chamber at AL was 22-25° C; at EPA the
range was 22-26° C with one value tt 30° C. EPA made one significant modification to
the apparatus which was to use humidified zero-grade air as the inlet air for the system.
This is a justified improvement resulting in a more controlled test environment As a
result, the humidities of the inlet air were somewhat different At AL the range was
27-58 % RH; at EPA the range was 19-29 % RH. Other differences between the test
systems were the use of Swiss-Webster mice from different suppliers; behavioral
examinations after every exposure at AL and only after the second and fourth exposures
at EPA; and the use of thinner mouse collars with somewhat smaller holes at EPA.
None of these differences would be expected to have an obvious impact on the ability of
the systems to detect the toxic effects of emissions from carpets.
The focus of Phase I of the study was to attempt to replicate the previously
reported AL results by conducting simultaneous experiments at both laboratories using
carpets that had been shown by AL to have severe adverse effects on mice. The
experiments were to be replicated to provide a larger sample size and the possibility of
-------
statistical analyses. Blinding procedures were to be used at both laboratories to ensure
that the individuals collecting the data did not know the contents of the source
chambers.
In my opinion, the research conducted by EPA was of very high quality.
Experts from various laboratories within EPA, as well as outside contractors, were
brought together to participate in the study. The team consisted of nationally and
internationally recognized experts in emissions testing, chemical characterization,
microbiology, and toxicology. The animals were properly housed and cared for.
Sentinel animals were used to assess the colonies for sub-chronic disease. The blinding
procedures built into the study were effective and strictly adhered to. These blinding
procedures included receipt of the samples by an offsite contractor, loading of the
source chambers by this contractor, use of separate observers for both the irritation tests
and the Functional Observational Battery (FOB) that were not present when the mice
were loaded into the chambers or when the mouse exposure chambers were connected
and disconnected from the source chambers, and chemical analyses conducted by another
team using a separate but equivalent apparatus with no mice. The data for the
pulmonary irritancy (PI) and FOB tests were rigorously analyzed in a manner that would
tend to increase the chance of finding false positive results (/.«., the analyses were
conservative). Extensive quality assurance procedures were used'throughout all aspects
of the study, including the use of another offsite contractor to review procedures.
The same cannot be said about the quality of the research conducted at AL. The
most serious problem, which could have had a significant impact on the results of the PI
and FOB tests, was the likely breach of the blinding procedures at AL. This breach may
have started from the time the samples first arrived at AL by Federal Express. The
packages were delivered in the afternoon at approximately 3 pm and sat on the
secretary's desk until the sample custodian arrived at 7 pm. The package for Test 2, the
first blank, was marked •empty* on the outside by CPSC and not weighted to compensate
for the weight of carpet. This package would have been visible and accessible to all
personnel since the entire facility is quite small. For each sample, the sample custodian
loaded the contents of the package into the source chamber, taped the chamber to
conceal the contents, placed the chamber on t cart, wheeled the cart into the testing
laboratory, and left The next morning the laboratory technician started the test. This
technician performed all aspects of the test His functions included making the baseline
sensory irritancy (SI) and PI measurements and then hooking the mouse exposure
chamber up to the source chamber. At this point, it is quite possible that the contents
of the source chamber could be viewed through the connecting port on the source
chamber. After the one hour exposure period, the technician removed the exposure
chamber from the source chamber and measured the concentration of TVOC an the
atmosphere of the source chamber using a simple hydrocarbon analyzer which
presumably produces a result within a few minutes and provides a readout. The results
presented by AL. show a distinct difference between the TVOC concentrations of empty
and carpet-loaded source chambers. The TVOC concentrations for Experiment B, the
blank, are reported to be 1.6-2.1 ppm as methane. The TVOC concentrations for
Experiment A were 2.4-4.4. For Experiment C, the concentrations were 3.7-5.4 ppm.
With carpets, the highest concentrations would occur at the end of the first exposure.
Based on all of their previous data using the same system (i.f., the analysis of hundreds
PC. - 12
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— » .*•
s =» i lr.c*oor-
of samples), the technician would probably know it that point whether the source
chamber was empty or contained carpet, Next, the technician performed the FOB.
Another possible clue that a blank was being tested would be the clean SI and PI tracing
reported for the blanks. Test 3, another blank, was an inadvertent test of computer
paper. The results of this test have not been reported by AL. When AL was informed
of the sample pairings at the end of the study, AL indicated that their records showed
that Tests 2 and 3 were not pairs. Another sample was sent, this time an empty bag
with the box weighted with paper. However, since AL had been informed of the
pairings and undoubtedly knew that two pairs of carpet samples had already been tested,
it would be easy for them to assume that this final sample was indeed another blank.
It is surprising and of some concern that Dr. Anderson did not recognize the
relatively easy ways in which the blind could be broken at AL and did not take any
steps to eliminate the potential problems.
The extent to which breaking the blind might have had on the results produced
by AL is impossible to assess. However, experience shows that the anticipation of
results can introduce significant biases into subjective measurements, such as the PI and
FOB tests. This as why blinding procedures are used in scientific investigations.
Such biases cannot explain the extreme behavior effects or the deaths reported by
AL for Experiments A and C with carpets. Some other factor may be involved. One of
the other reviewers (R. Schelsinger) raised the possibility of sub-chronic disease making
the animals more susceptible to stress and toxic exposure. Another possibility is that
stress induced by the collars and restraint in the plethsmographs might have made the
mice more susceptible. J. Tepper told the reviewers that mice can die as the result of
having their head shoved through the hole in the collar or from forcing their way out of
a collar. Interestingly, Dr. Anderson told the reviewers that unrestrained mice exposed
to emissions from carpets exhibit severe behavioral effects but not death. AL has not
attempted to determine the cause of death of the mice from their tests.
It is possible that there is some subtle phenomena occurring that is not directly
related to exposures to emissions of toxic chemicals from carpets. Other than factors
involving the source and health of the mice, the major difference between the apparatus
at the two laboratories is EPA's use of humidified zero air. The humidities of inlet air
at AL were higher than those at EPA, but the typical difference is only about 10 % RH.
It is difficult to postulate a mechanism, either biological or chemical, whereby this small
difference could have such a dramatic effect
I reviewed all of the chemical emissions data provided in the reports from
Acurex Environmental Consultants and Research Triangle Institute (RTI). The multi-
sorbent method used by both laboratories for the analysis of volatile organic compounds
(VOCs) is appropriate for this application as it measures a broad spectrum of compounds
with high sensitivity. The DNPH method used for the carbonyl compounds is the best
available method for this class of compounds. An intercalibration was performed
between Acurex and RTI to ensure comparability of the data. In addition, numerous
calibrations and other quality assurance procedures were used by both laboratories for
the chemical analyses.
-13 3
-------
The TVOC emissions from both carpet samples were elevated compared to what
would be expected at room temperature, but still quite low compared to emissions of
TVOC from "wet* sources, such as adhesives, paints, and other coatings. During the
four exposure periods (4 hours total) the total emissions of TVOC were only 4-5 mg.
This is equivalent to 14-17 mg nv* , which compares to total emissions of TVOC from
new styrene-butadiene rubber latex carpets over 24 hours of 1-26 mg m-> (Hodgson,
AT., J.D. Wooley, and J.M. Daisey, 1993, Emissions of volatile organic compounds from
new carpets measured in a large-scale environmental chamber, J. Air Waste Manage.
Assoc. 43: 316-324). The composition of the VOC samples wts also unremarkable. Both
carpet samples were dominated by Cn • Cu alkene hydrocarbons and siloxane
compounds. The siloxanes were also emitted by the aquarium. Carpet A had higher
emissions of butylated hydroxy toluene and formaldehyde. The alkene hydrocarbons
would be expected to have relatively low toxicity. Formaldehyde might play a role in
sensory irritation, but would not be expected to cause behavioral effects or death in
mice. In summary, the chemical emissions data do not seem to provide any clues about
the cause of the severe effects reported by AL.
f
Additional work as required to resolve the differences between the two
laboratories regarding the PI and FOB results and the deaths. Firstly, I recommend that
the raw data be exchanged between the two laboratories. The investigators may
interpret the other laboratory's respiratory tracings differently and, thus, account for
some of the differences in the PI results that were observed. The FOB results presented
by AL could benefit from the statistical analyses available at EPA. It is impossible to
fully evaluate these results as they are now presented because of their highly condensed
summary form. For example, differences among exposures for a test or between
replicates for a treatment are obscured. Secondly, additional data obtained by AL should
be examined. The issue of reproducibflity could be assessed by comparing the results
obtained by AL during this study to their previous results for the same carpets. The
results of the experiment with paper would provide another control against which to
evaluate the carpet results. Thirdly, an attempt should be made to determine the cause
of death of mice at AL. Complete autopsies art needed. Relatively simple experiments
could be conducted to evaluate the potential roll of the hole size and thickness of the
mouse collars in causing death. Other experiments using known mixtures of VOCs could
be conducted. Finally, additional tests with carpets and controls could be performed in
which the blinding procedures were strictly adhered to by both parties. This might
involve both AL and EPA teams making simultaneous observations at AL.
I would focus on efforts to understand the disparity in the Phase I results before
proceeding with additional experiments. If additional experiments still seem warranted,
they could be conducted with a limited number of carpet samples in the new all-glass
chamber as suggested by D. Costa. It would be of particular value to establish dose-
response curves for any observed effects. The SI and PI tests should definitely be
separated from the FOB test. The SI and PI tests should follow ASTM £981 in which •
single exposure is used since the EPA data suggest that the test procedure of multiple
restrained exposures produces measurable stress in the mice. The FOB test should be
conducted with mice not restrained by collars to eliminate this confounding factor.
PC.-I4
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I recommend that the summary report, "Evaluation of Carpet Toxicity - Phase I,*
by R. Dyer and D. Costa be amended to specifically address the comparison of the
results obtained by EPA and those obtained by AL. The high quality and the
thoroughness of the research conducted at EPA should be emphasized. It is justified to
conclude that even when all reasonable efforts were made to replicate the apparatus and
procedures used by AL, it was not possible to reproduce any of the results of AL
showing the study carpets to be toxic to mice.
I further recommend that EPA sponsor a scientific forum on the issue of carpet
toxicity to mice to include other researchers and organizations who have either tested the
same carpets using similar methods or have experience with these types of tests. Such a
meeting might generate additional insight into the potential causes of the toxic effects
reported by AL.
Please contact me if you have any questions regarding these review comments.
Thank you again for the opportunity to participate in an interesting and important
research effort
Sincerely,
Alfred T. Hodgson
MS 70-193A
510-4S6-5301
FAX 486-6653
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REPORT TO EPA ON EPA/ORD CARPET STUDY
D. E. McMillan
University of Arkansas for Medical Sciences
This report is an independent report written by the
author listed above. The report will review the data
provided to him in written form as well as the data
presented at the meeting held at EPA on May 26-27, 1993.
The focus of this report will be on the Functional
Observational Battery (FOB) data provided by Anderson
Laboratories (AL) and the EPA; however, some general issues
regarding experimental design and conduct of the experiments
will be included.
I. Quality of the research that was conducted
A. Carpet Samples
The decision was made to test samples from two
•
different carpets about which complaints had been received.
Although it makes sense to begin testing with carpets
presumed to be toxic, the study of used carpets is a
complicating factor. Even if both AL and EPA had confirmed
a toxic effect from carpet emissions, it would not have been
possible to establish whether the toxicity resulted from the
manufactured carpet, or from contamination during use and
subsequent handling of the carpet. Further testing would be
required to answer this question. Since the EPA was not
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able to replicate the AL findings this has become a moot
point; however, in the future it might be appropriate to
conduct some tests with new samples of the carpets listed as
suspect to avoid this problem.
B. AL study
AL reported large effects in almost all treatment
groups including the death of 5 exposed animals. Sham
exposed animals showed remarkably few effects, thereby
establishing a large treatment effect. There are a number
of problems with the experiments conducted at AL that
somewhat limit the interpretation of their data. The first
problem concerns the double blind design of the study.
Samples were received at AL in the late afternoon and were
placed by the courier on the desk of a secretary. It is not
clear who signed for the delivery, but apparently the sample
container sat on the desk with no security until
approximately 7:00 PM at which time the cample custodian
arrived, removed the sample, placed it in the source
chamber and sealed the chamber. The sample custodian then
moved the chamber to the test room where it remained until
the next morning. The lack of security of the sample to
this point allowed a number of opportunities to break the
blind. However, assuming that there were no deliberate
attempts to break the blind, the testing procedure almost
certainly did so.
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The next morning the technician who conducted all
subsequent testing initiated heating of the carpet samples
and placed the animals in the exposure chamber. During
exposure the technician measured the rate and patterns of
respiratory responding of the animals. During this period
the technician constantly monitored the animals. Dr.
Anderson stated on several occasions how clean (lacking in
effect) the data were from the control animals, which
certainly is consistent with the data in the written report.
Since the same technician conducted the FOB, after
completing the respiratory measurements, it would be almost
impossible for this technician not to know at the time of
FOB testing whether an animal had been exposed to carpet
emissions or sham exposure. If there was any doubt about
group identity at this point, the measurement of total
volatile organic chemicals by flame ionization detection by
the same technician, would eliminate that doubt. Thus, it
seems unlikely that the AL observations for the FOB were
done according to a blind procedure as specified.
Another problem with the experiments done at AL is the
data analysis. As I understand their procedure for the FOB,
on the day prior to exposure to carpet emissions the 8 test
animals are observed and scored on the FOB. On the basis of
these pre-exposure observations, a range is established for
each group. Subsequently, the FOB is repeated four times
after sham or carpet-emission exposure. Data are reported
as number of animals falling outside the range. Based on
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the relatively small number of control observations relative
to the number of treatment observations, it would be
expected that many experimental values would fall outside
the control range. This was clearly the case for the
treatment groups, but not for the sham exposure groups.
Reasons that might account for the difference between
sham and exposure groups include an effect of the carpet
emission exposure, bias due to loss of the blind, or other
yet to be determined factors. The manner in which the AL
data are presented makes further detailed analysis of the
data very difficult. It is unclear, for example, whether or
not the effects reported by AL are seen repeatedly in only a
few animals, or are seen sporadically in other animals. All
of the data are heavily derived so that the absolute
comparability of groups before, during and after exposure
becomes very difficult. Dr. Anderson indicated that she
would be willing to supply less derived data for further
examination of this issue.
C. EPA Study
The EPA study appears to have been carefully conducted.
The chambers at EPA were loaded by outside personnel. The
FOB was conducted by individuals very well trained in
conducting this battery and they were not the same personnel
that conducted the other tests. There does not appear to
have been much opportunity for those conducting the testing
at EPA to have broken the blind (barring the possibility of
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fraudulent conduct of the scientists, which seems most
unlikely).
Relative to the rather simple analysis conducted by AL,
the statistical analysis applied to the data developed by
the EPA was sophisticated. The statistical analysis of the
EPA data gives this reviewer greater confidence in the EPA
findings than is possible from the AL data analysis.
However, some of the EPA data also were presented in a
derived form (percentage incidence, percent change from
control, etc.) that made it very difficult to determine the
comparability of control and treatment groups before
exposure and other details of what actually happened. I
also found the 3-dimensional bar graphs that they used for
the presentation of some data difficult to follow, but this
may be a personal reaction against this form of graphics.
Clearly, the EPA data could be presented in a form that
would provide more information than the present version
does.
In summary, the general design and conduct of the EPA
studies appears to be more rigorous than that done by AL and
from a scientific viewpoint I am inclined to place much
greater confidence in their data than that from AL.
Nevertheless, there other possible explanations for the
differences between EPA and AL findings and since the
welfare of the public exposed to carpets is at issue it
seems clear that further studies should be done to determine
the reasons why AL finds effects that EPA cannot replicate
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and to further determine whether or not carpet emissions
constitute a public health problem.
II. INTERPRETATION OF THE DATA
Using their criterion of treatment values falling
outside the range of control values, AL finds evidence of
sensory irritation and changes in the FOB including changes
in their groupings of altertness/excitability, neuromuscular
function and appearance. It should be noted that the data
analysis done by AL (proportion of animals that fall outside
the control range established by pre-exposure observations
in the same animals) gives equal weight to both increases
and decreases in the magnitude of the dependent variables.
In some instances the changes that they measured
consistently moved in the same direction. In other
instances both increases and decreases occurred, so that
their treatment effect was to observe an increase in
variability of the treatment group relative to the control
group. It is possible that some of these differences would
not be significant when analyzed by more rigorous
statistics. Although the interpretation of the AL data are
limited by the presumption of the broken blind and the
minimal statistical evaluation, it seems unlikely that the
AL effects would disappear with more rigorous attention to
these problems. This is particularly true with respect to
the deaths that they observed in the treatment groups.
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The EPA investigators found few significant
differences. In some instances the significant differences
they found were due to changes in the control group over
tine, or were found in only one of the two treatment groups.
On the basis of this analysis the EPA's conclusion that they
found no differences seems appropriate. In fact, based on
the analysis of the data presented, both AL's conclusion
that there clear differences between sham exposure and
actual exposure to carpet emissions and EPA's conclusion
that there were no differences, are appropriate. At the
review, Dr. Virginia Moser presented a preliminary analysis
of the EPA data using the AL» method of analysis. This
preliminary analysis did not account for the differences
between laboratories. Obviously, differences in statistical
rigor cannot account for the significant death rate at AL
which was not observed at EPA.
Although I have little expertise in the analysis of
source materials, the chemistry data appear to support the
EPA findings. There is no toxic chemical identified in the
carpet emissions in an amount sufficient to produce the
observed toxicity. Although the subject of toxic effects of
mixtures has not been well studied by toxicologists, the
very low levels of toxic chemicals make any powerful
synergistic effects unlikely, although impossible to
eliminate entirely.
III. FOCUS OF THE REPORTS
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Both the draft report suppled by AL and that suppled by
EPA are appropriately focused on the fundamental issues.
The AL includes an executive summary, an adequate
description of the methods and procedures, and a rather
brief results section. Most of the relevant data a
presented in accompanying appendices. After brief mention
of the problem that occurred with sample submission, the AL
report concludes with the obvious, namely that exposure to
carpet emissions produced large effects in the exposed
animals relative to sham exposure.
The draft report from the EPA is broader, but this is
not inappropriate. The EPA study included a great deal of
additional testing including tests on the identification of
source emissions, postmortem evaluations, quality assurance,
etc. The additional data required the expansion of the
results section of the report as well. The discussion
section of the EPA draft also includes a section that
speculates on some of the reasons that might relate to the
reasons why EPA was not able to replicate the results
obtained by AL.
In summary, both draft reports are appropriately
focused, despite some dissatisfaction of this reviewer with
the details of the data presentation in both draft reports.
The bottom line is that AL has replicated their previous
results. EPA, despite an intensive effort, has not been
able to replicate the AL findings. The studies conducted by
EPA are superior scientifically to those conducted by AL;
8
D O -^ *
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however, it does not seen likely that any deficiencies in
the AL procedures could account for all of the large
differences that they obtained.
Because of the publicity surrounding putative problems
with carpet emissions and the economic issues associated
with these studies the issue of possible fraud on the part
of scientists at AL, or EPA must be raised. Fortunately,
there is no evidence that scientists at either institution
have in any way acted inappropriately. Visits between
laboratories have occurred. Both laboratories appear quite
willing to allow others to examine their data in detail.
The presumed loss of the blind at AL relates to the small
size of their operation and lack of sufficient personnel to
permit the isolation of experiments from each other and not
to any intentional attempt to violate the blind conditions
of experimentation.
IV. APPROPRIATE NEXT STEPS
It is the opinion of this reviewer that EPA cannot
allow this matter to drop at this time. Although their
attempt to replicate the AL results has failed, the issue of
whether or not carpet emissions constitute a public health
problem is not resolved and further testing is required.
The following recommendations and the reasons for making
them are offered:
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A. Replication
An independent laboratory should conduct an independent
systematic replication of the data. Because of the
political sensitivity of the issue this laboratory should
have no association with EPA, AL, the carpet industry, or
anyone else already associated with the controversy.
Alternatively, EPA and AL night conduct a joint study. For
example, a replication might be done at EPA and supervised
by EPA personnel using AL equipment, staff and animals, or
vice versa.
B. Systematic replication
Any replication should not be an exact replication in
the opinion of this reviewer. A better exposure system
should be used to study carpet emissions. New carpets of
the type previously implicated as problem carpets should be
investigated. At least one increased emission exposure
level should be studied. If EPA failed to replicate the AL
result because their methods lacked sensitivity, effects may
be seen at a higher exposure level. Great care should be
taken to separate respiratory testing from FOB testing so
that the blind cannot be accidentally broken. Perhaps
consideration should be given to the use of different
animals for respiratory and FOB testing with the FOB animals
receiving whole body exposure, since the EPA's data seem to
indicate that the restraint is very stressful and could
contribute to the FOB effects. The statistical analysis to
be applied to the data should be specified as part of the
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replication attempt. Consideration should also be given to
study of emissions from unheated carpet, since the heated
carpet may not model most real world exposure. An
completely objective behavioral measure should be added to
the test procedure. Since experimental bias is an issue in
these experiments, the addition of a behavioral procedure
that does not require observer judgments would be a welcome
addition. Measurements of spontaneous locomotor activity or
schedule-controlled responding are suggested.
C. Laboratory Differences
Attempts to determine reasons for the differences in
laboratory findings should continue to be pursued. Current
methods of risk assessment often place emphasis on the
extreme result, even when conflicting studies are
scientifically stronger. Once a report of toxicity enters
the literature, several negative reports may be required to
decrease the perception of toxicity. Therefore, it remains
important to continue to explore reasons for the
differences between labs. If it could be established that
something is causing effects at AL that is only remotely
related to exposure to carpet emissions, or that something
is happening at EPA to prevent them from obtaining what are
real effects of emissions, the entire issue would be
resolved. Some of the hypotheses to explain reasons why
laboratory results differed evolved from discussions at the
review. These hypotheses include a role for water vapor in
the carpet samples affecting the toxicity of the emissions,
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a role for conditioned stress during multiple carpet
exposures, differences between laboratories in animals in
the outbred strain that was used, possible pathogens in the
laboratory, variability in the samples submitted to the
testing laboratories and others. These and other hypotheses
should be tested.
D. Cost-Effectiveness Considerations
The reviewer realizes that the suggestions for
additional action may not be very helpful to EPA because
they are broad, expensive and time consuming.
Unfortunately, for the reasons outlined, I see few
alternatives. If forced to prioritize among these choices I
would suggest that EPA simultaneously pursue as extensive a
systematic replication as funding and time permit with the
replication to be conducted at an independent laboratory.
At the same time investigations into the reasons for the
different results should continue, beginning with a complete
data exchange. EPA and AL staff should meet to discuss
possible reasons for the differences, prioritize the order
in which the hypotheses about testing are scheduled for
testing and begin to do the appropriate tests. It is in the
best interests of both EPA and AL, as veil as the health of
the public and the carpet industry, that these issues be
resolved as soon as possible.
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