f iff Systems. Jt\
                         Submitted to:

           Office of Pesticides and Toxic Substances
                Environmental Protection Agency
                       A01 M Street, SW
                     Washington, DC  20460

Attention:  Project Officer, Glenn Williams (TS-796) (3 copies)
            Contracting Officer, Malcolm P. Huneycutt (MD-33)  (1  copy)
                            TR-835-20

             EXPERT REVIEW OF PHARMACOKINETIC DATA:
                          FORMALDEHYDE

                    Final Evaluation Report
                        Prepared Under


                       Program No. 1415


                              for


                    Work Assignment No. 07


                    Contract No. 68-02-4228
                         January 2, 1986

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                                  DISCLAIMER
This document  has not been peer and administratively reviewed within EPA and
is for internal Agency use/distribution only.

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                                TABLE OF CONTENTS

                                                                        PAGE

1.0   INTRODUCTION	1-1

2.0   BACKGROUND	2-1

      2.1   Technical	2-1
      2.2   Administrative	2-1

3.0   DISCUSSION	3-1

      3.1   Distinguishing between Metabolically Incorporated and
            Crossllnked CH.O	3-1

            3.1.1   Metabolic Incorporations Versus Adduct  Formation
                    of CH 0 . .  .	3-1
            3.1.2   Crossfinked  CH 0 Located Exclusively in the
                    Interface (IF) DNA	3-2

      3.2   Experimental Methodology Limitations	3t-2
      3.3   Identity of Labeled  Fractions 	  3-4
      3.4   Other Measures of Exposure  	  3-4
      3.5   Nonlinearity for Crosslinked DNA at Low Doses	3-5

            3.5.1   Documentation of Nonlinearity for Low Dose
                    Crosslinked  DNA	  .  3-5
            3.5.2   Alternative  Explanations for Nonlinearity of Low
                    Dose Crosslinked DNA	3-6

      3.6   Sensitivity of the Study Conclusions to Statistical
            Analysis	3-8
      3.7   Adequacy of the Measure of Exposure .	3-9
      3.8   Utility of the Study in the Quantitative Risk Assessment
            of CH20	3-9

4.0   CONCLUSIONS AND RECOMMENDATIONS 	  4-1
                                                            continued-

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Table of Contents - continued
APPENDIX                                                                 PAGE

   1        Documents Provided to Reviewers 	   Al-1
   2        Additional References Relating to Expert  Review of
            Phannacokinetic Data:  Formaldehyde 	   A2-1
   3        Questions/Issues on Formaldehyde to be Addressed by
            Expert Panel  	   A3-1
   4        Information/Clarification Requested from  CUT by Expert
            Panel   	AA-1
   5        List of Participants	A5-1
   6        Agenda	A6-1
FIGURE
                                LIST OF FIGURES
          Estimated Slopes for Metabolic Incorporation of
          Respiratory AQ-DNA and Olfactory IF-DNA ....
PAGE
3-7
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1.0
INTRODUCTION
This report provides a summary of the  discussions and conclusions from the
*eV £ mf,r  8 c°nducted on December 2 and 3,  1985 under Work Assignment (WA)
ll' S'o^Xr  £leW °1V?arnaC,°klnetlC Data:  Formaldehyde" of Collet
No. 68-02-4228.  The preliminary draft of this report was produced on-site by
    r  K 5 fartlfXMt« to meet the U.S. Environmental Protection Agency
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2.0       BACKGROUND

2.1       Technical

The Environmental Health Committee of EPA's Science Advisory Board (SAB)
reviewed the draft report "Preliminary Assessment of Health Risk to Garment
Workers and Certain Hotre Residents from Exposure to Formaldehyde," dated
May 31, 1985, prepared by the Office of Toxic Substances (OTS).  One outcome
of the review is the committee's finding that the formaldehyde (CH.O)
assessment "will not be scientifically adequate without an analysis of the
pharmacokinetlc information and appropriate modification (of the assessment)
based on this analysis."  The committee is concerned with both the broad
question:  "How can one realistically begin to incorporate relevant kinetic
information into quantitative cancer risk assessments?"  and the specific
issue of whether the pharmacokinetic data published In a study by Casanova-
Schmitz et al. (1984) can be used in EPA's assessment of CH 0.

The OTS agrees that appropriate pharmacokinetlc data should be presented and
considered in the risk assessment; however, in its draft risk assessment of
CH.O, OTS agreed with an analysis performed by Cohn et al. (1985) that the use
of the Casanova-Schtnitz data would be premature.  OTS found that the Casanova-
Schmltz data do not support a modification of the risk estimated for CH.O^and,
thus, the OTS did not utilize the data either qualitatively or quantitatively
in its risk assessment.  However, to address the concern of the Environmental
Health Committee, OTS desired an independent, objective, expert analysis of
the issue so that it may reconsider the appropriateness of using the data in
its risk assessment.

Consequently, OTF authorized assembly of a team of expert scientists to
conduct an evaluation of the Casanova-Schmitz study and any relevant underlying
data developed in the study and to prepare an expert report that answers the
question whether the study provides data that should/could be used in the CH.O
risk assessment.

To accomplish this effort each member of the team of expert scientists was
initially provided the documents listed in Appendix 1.  Additional references,
listed in Appendix 2 were provided subsequently to each expert.  The experts
were asked to evaluate independently the documents and determine the extent to
which the pharmacokinetlc data are appropriate for use in the CH.O cancer risk
assessment.  In conducting their evaluation, the experts were asRed to consider
specifically each of the questions presented in Appendix 3 insofar as their
expertise allows.  They were also asked to identify any underlying data (e.g.,
lab books, data tables, etc.) desired from the Chemical Industry Institute of
Toxicology (CUT) which would be of assistance in addressing the specific
questions.  The list of information/clarification requested from CUT by the
experts is presented in Appendix 4.

2.2       Administrative

Upon receipt of the VA from EPA, efforts were initiated to assemble a seven-
member team of experts in metabolism, DNA adducts and statistics.  The review
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tear members arc identified on the List of Participants at Appendix 5.
Or. Lemone Yielding agreed to serve as Che Team Leader and Review Meeting
Chairperson.  As such, Dr. Yielding served as the principal author/editor of
the technical portions of this report.  Other individuals representing  EPA and
ICAIR, Life Systems, Inc., at the review meeting are also listed in Appendix 5,

The review meeting was conducted on December 2 and 3, 1985 at the Sheraton
University Center, Durham, NC.  The Agenda prepared for the meeting is
provided at Appendix 6.  Although the Agenda was prepared for a three-day
meeting, all Agenda items were completed at an accelerated pace and the
meeting only required two days.  On-site clerical facilities were provided
during the meeting to prepare the draft report.
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3.0       DISCUSSION

The following provides a summary of the discussions and evaluations conducted
at the review meeting regarding the lists of questions/issues presented in
Appendix 3 and Appendix 4.

Prior to the review meeting, written answers, statements and/or opinions were
prepared by the expert reviewers on each of the questions/issues presented in
Appendix 3.  These responses were provided to EPA immediately following the
meeting.  They are not provided in this report because they have been
superceded by the consensus opinions developed at the meeting and provided
below.

The representatives from CUT, listed in Appendix 5, participated in informal
discussion with the expert reviewers.  This discussion was in response to the
expert's requests for information/clarification listed in Appendix 4 and
Included an expanded question and answer session during which further details
were provided on CIIT's prior, ongoing and planned studies of CH.O.


3.1       Distinguishing between Metabollcally Incorporated and Crossllnked
          CH,0                                                            i
           1 A^

Questions/issues Nos. I and 3 of Appendix 3 were addressed together and are
discussed below.

3.1.1     Metabolic Incorporations Versus Adduct Formation or Crossllnked
          CH2£

The experimental evidence for metabolic incorporation of,crosslinked CH.O is
largely based upon the relative incorporation of  H or   C-CH.O into DNA of
respiratory epithelium.  The evidence presented in the written documentation
was suggestive bur not definitive in regard to this assumption.  Additional
evidence, presented at the interview with the CUT staff, Indicated that the
 H/  C ratio of the purine deoxyribonucleosides Isolated from aqueous DNA by
high performance liquid chromatography  (HPLC) was in accordance with metabolic
incorporation and furthermore, no shifts in pattern suggesting adduct forma-
tion were observed.  It was not clear whether or not these types of experi-
ments had been performed at all CH.O levels.  Interaction of CH.O with
tetrahydrofollc acid (THFA) could yield N5-10 methylene THFA directly, or
following oxidation to formate could yield N-10 formyl THFA.  The occurrence
of these reactions, the equilibration of the various THFA derivatives and the
reaction of the THFA derivatives with various one carbon acceptors could
markedly influence the DNA  H/  C ratio in metabolically-lncorporated CH.O.
The pool sizes of the nonradioactlve acceptors and.their metabolic Inter-
mediates could also markedly influence  the DNA  H/  C ratio due to metabolic
CH.O.  The relative reaction rates and  pool sizes are likely to vary under
different conditions.  Thus, the interpretation of the  H/  C ratio is very
complex.  This complexity necessitates  the isolation of the DNA bases (or
deoxyribonucleosides) and amino acids and the comparison of the  H/  C
profiles with authentic standards under conditions which separate any adducts
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from the standards.  Since It is not clear that this was performed at  all CH 0
doses, doubt remains as to the assumption which formed the basis of
distinguishing metabollcally Incorporated and crosslinked (or adducted) CH.O.

3.1.2     Crosslinked CH^O located Exclusively in the Interface (IF)  DNA

It is stated by Casanova-Schmltz et al. that all the crosslinked CH.O was
present in the IF rather than the aqueous (AQ) ONA.  This assertion was not
borne out by the written or oral documentation.  Several control experiments
which would have solidified the experimental basis for this assertion were not
performed.  The relative efficiency of extraction of the DNA from respiratory
epithelium under conditions of CH.O dosing (at various levels) should have
been determined.  That is, what Is the recovery of the total DNA from the
respiratory epithelium by the phenol procedure?  Furthermore, the specific
distribution of crosslinked DNA-protein In the IF-DNA fraction should have
been affirmed.  There is no doubt that at least some of the crosslinked CH.O
(DNA-protein) is found in the IF-DNA fraction.  Whether all of this crosslinked
fraction was in fact initially extracted from the tissue or whether some might
be found in the AQ-DNA are questions which have not have been satisfactorily
addressed.  These experiments could have been performed by adding radio-
actively-labeled DNA (or crosslinked DNA-protein) to a tissue homogenate and
following its distribution into the various fractions of the phenol procedure,
i.e., a standard recovery experiment.  Consequently, we believe that sufficient
detail for characterization of the IF-DNA fraction has not been provided.
This detail is absolutely necessary in order to validate the use of IF-DNA as
a measure of crosslinked CH.O, and therefore as a biological dosimeter.

3.2       Experimental Methodology Limitations

The inhalation methodology used to administer CH.O was appropriate and the
control and monitoring of CH.O concentration as well as the isotopic composi-
tion of the gas mixtures employed were adequate.  Infrared monitoring was done
continuously during exposure and the instruments and methods crosschecked with
other instruments and other methods.  Variations in CH.O concentrations were
small compared to other variables and are not likely to have much impact on
the experimental results.  These factors are not considered to represent an
important source of error in the experimental protocol.  In considering the
interpretation of the data in relation to human risk assessment, it should be
borne in mind that Inhalation by rats is restricted to the nose.  In humans,
inhalation can be expected to occur through both the nose and the mouth.  The
implications of this distinction with respect to human risk are unclear but
always represent a source of uncertainty when rodents are employed as hucan
surrogates in inhalation studies.

The DNA extraction procedure employed requires further validation since It is
of central importance in distinguishing between the aqueous and Interfaclal
DNA fractions on which the measurements of metabolic Incorporation and crosslink-
Ing depend.  There is no indication of the percentage of the total DNA recovered
by this procedure, and it is likely that the amount of DNA associated with the
interfacial  fraction will vary to some extent with the extraction conditions
employed  (e.g. ionic strength, temperature, etc.).  A standard recovery experi-
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ment as outlined in 3.1.2 should have been conducted.  The adequacy of the
extraction procedure can be assessed only after a more complete characteriza-
tion of the nature of the DNA occurring In the Interfacial fraction.  Small
variations In the extraction conditions could constitute an Important source of
experimental error and could exert a profound Influence on the  H/  C ratios
obtained.  It would be reassuring If, after protelnase treatment and hydro-
xyapatite chromatography, a mild acid hydrolysis (with or without added carrier
CH.O) could be shown to release radio-labeled CH.O from this material.  This
would give positive evidence for the existence or chemically-incorporated CH.O
in the IF-DNA fraction.

Exposures to labeled CH.O were routinely conducted over a period of six hours
with rats that had been preexposed to the same concentration of the gas for
six hours the previous day.  In view of the established temporal changes in
cell proliferation as well as the replacement of respiratory epithelial cells
by squamous cells during chronic exposure, there is some question whether the
results of the acute labeling studies will accurately reflect events occurring
during longer-term exposures.

This is particularly important when it is considered that squamous cell
carcinoma does not develop until 11 or 12 months into the chronic study and
that a large percentage of DNA protein crosslinks are subject to relatively
rapid repair.  CUT investigators argue that the short-term studies are the
most appropriate models for human exposure since in humans there is no evidence
of the marked changes in epithelial cell structure that are observed during the
chronic rat studies.  On the other hand, it is not really clear as to whether
or to what extent CH-O-DNA interactions differ during the course of chronic
exposure.

Thus, the short-term conditions employed to evaluate CH.O binding may or may
not reflect those occurring during chronic exposures, and there is con-
siderable uncertainty in relating the acute binding data directly with the
carcinogenic lesions occurring as a result of chronic exposure.

While clearly it is not possible to conduct binding studies throughout the
entire period of the chronic test, it would be useful for comparative purposes
to have data from animals exposed to CH.O for a longer period of time.  The
primary uncertainty associated with the data is that they represent an acute
model of a chronic lesion.  The extent to which this model is valid remains
to be determined.

Information provided by CUT scientists at the meeting addressed satisfactorily
initial questions concerning methodology for determination of  H/  C ratios.
Samples of raw data were provided which clearly showed very good counting
statistics.  The problems, which could potentially arise from quenching, were
minimized and controlled through (1) routine use of external standardization
(built into the scintillation counter), (2) routine counting of quenched and
unquenched standards with samples, and (3) using a relatively constant ratio
of scintillation fluid.to.the aqueous samples.  For standardization between
experiments, observed  H/  C ratios were normalized to the  H/  C ratio of the
gas phase to which the animals were exposed.
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There are numerous points at Vn*(b tritium kinetic isotopr effects could
influence the disposition of  H/  C dual-labeled CH 0.  For example, there
could be a large primary Isotope effect on the enzyraic oxidation of CH 0 to
formate.  Smaller secondary isotope effects would influence the addition of
nucleophiles to monomeric CH.O and the reactions by which formate equivalents
are incorporated into purine bases.  It would, in fact, be amazing if these
kinds of isotope effects did not enter into the results.  The problems arise
because of the complexity of the overall disposition of CH.O (see
Section 3.1.1).  This affords the opportunity that under different conditions
of administration the relative importance of the various steps will vary.
Since the overall (observed) isotope effect will be a composite (a weighted
average) of the Isotope effects on these individual steps, there is great
opportunity for quantitative variation among the. individual steps even in the
face of an apparently constant value for the  H/  C ratio observed in a
particular fraction such as AQ-DNA.  This is not to suggest that there are
problems of this sort with the data presented* <"?Jy that there is reason for
concern that there might be.  Measurement of  H/  C ratios found in specific
bases obtained by hydrolyzing AQ-DNA and 1F-DNA after both high and low doses
of CH.O would help clarify this situation.

3.3       Identity of Labeled Fractions
                                                                         i
This issue/question is addressed under Section 3.1 and the last part of
Section 3.2.

3.4       Other Measures of Exposure

Questions/issues Nos. 4 and 7 of Appendix 3 were addressed together and are
dlscuFFed below.

While a measure of the effective concentration of a chemical at its target
site (i.e., the delivered dose) would be preferable to the use of administered
dose for purposes of risk assessment, it is questionable whether the DNA-
binding data generated by Casanova-Schmltz et al. provide a validated measure
of CH 0 concentration in the nasal epithelium of exposed rats.  Since the
relationship between DNA binding and carcinogenicity have not yet been
established for CH.O, the observations on binding may be of some mechanistic
significance.  However, while DKA-binding may constitute a satisfactory
measure of target site (delivered) dose, its measurement by means of
dual- labeled CH.O incorporation seems unnecessarily sophisticated and complex.

What is really needed- as a biochemical dosimeter is some measure of the
chemical interaction of CH.O with intracellular macror.olecules other than
through metabolic incorporation into nucleotides and/or amino acids.
DNA-protein crosslinks, if they could be shown unambiguously to involve
methylene links originating from administered CH.O, would be a valid measure
of this.  Alternatively, since the jln vitro experiments show that proteins are
far more reactive toward CH.O than nucleic acids, and since the metabolic
Incorporation of CH.O equivalents into proteins is likely to be much less than
its metabolic incorporation into nucleic acids, the measurement of CH.O
covalently bound to Intracellular proteins could provide a simpler index of
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exposure of the cell to CH«0.  Furthermore, validation of these assumptions
could easily be accomplished.

While the data provided by the Casanova-Schmitz et al. study may provide some
measure of delivered dose, It Is not yet clear whether the data are a great
deal more useful than measures of administered dose for the purposes of risk
assessment.  In the absence of DNA binding data obtained following longer
periods of exposure, there is no Justification for assuming that the short-
term data bear any relationship to target-site concentrations likely to be
encountered throughout the two-year bioassay.

3.5       Nonlinearlty for Crosslinked DNA at Low Doses

3.5.1     Documentation of Nonlinearlty for Low Dose Crosslinked DNA

Casanova-Schmitz et al. calculate the amount of covalent binding from
equations Nos. 8 and 9 in their Appendix 2.  The issue of whether the result
actually represents the extent of crosslinked DNA is addressed elsewhere in
this report and will not be discussed further here.  The authors have assessed
the nonlinearity of the dependence of crosslinking on administered dose in two
ways.
                                                                          j
     1.   In the discussion section in Casanova-Schnitz et al. there Is a
          comparison between:

          a.   The actual values calculated from data at 2 ppm.

          b.   The value predicted by interpolating between the results for
               6 ppTr and the origin.  The observed values are 0.022 +/-
               0.005 nmol/mg (mean +/- 1 ISE), while the interpolated values
               are stated to be 0.078 +/- 0.013 (one third of the 6 ppm values,
               0.233 +/- 0.023; we note that simple division gives 0.078 +/-
               0.0078).  The conflict between observed and predicted values is
               clear.

     2.   In Appendix 3 of Casanova-Schritz et al., a more systematic test of
          proportionality is described.  The basic idea is to convert the
          concentration at each administered dose into an estimated slope by
          dividing by the dose.  If the response were truly proportional to
          dose, these estimated slopes would all be the same.  Therefore, the
          hypothesis may be tested by examining the consistency of the slopes.
          Since the standard errors of the estimated slopes are more or less
          constant, and in particular show no systematic tendency either to
          increase or decrease with Increasing dose, the statistical method of
          one-way analysis of variance is appropriate and leads to rejection
          of the null hypothesis of proportionality.

It has been suggested by Cohn (1984) and Cohn et al. (1985) that olfactory
IF-DKA may give a better indication of metabolic incorporation than does
respiratory AQ-DNA, and that when calculated In this alternative way, the
extent of crosslinking becomes proportional to dose.  This suggestion has been
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disputed by the Casanova-Schmltz et al. authors.  However, they argue in their
rebuttal that even when calculated In this alternative way, there is still
strong evidence of nonllnearity.  The comparison between results at 2 pptn and
6 ppo is no longer sharp, and the one-way analysis of variance yields only
marginally significant results.  However, the linear regression against dose
is statistically significant, and the authors correctly point out that this is
a more powerful test against alternative hypotheses of the type expected here.

Figure 1 provides a graphical assessment of the proportionality hypothesis.
It shows the estimated slopes calculated in both ways with standard errors
attached, plotted against dose.  The left-hand bar of each pair uses respiratory
AQ-DNA as the baseline, while the right-hand bar is based on olfactory IF DNA.
Although the nature of the nonconsistency is different for the two alternatives,
it is strong in both.  From a purely statistical point of view, the weakness
of the use of olfactory IF-DNA is that weaker pairing of the data leads to
generally larger standard errors, and consequently lower power to detect
nonconsistency (nonlinearity of amount of crosslinking).

To summarize, in contrast to the claims of Cohn (1984) and Cohn et al. (1985),
we find that the nonlinearity of "crosslinked DNA" as a function of administered
dose is adequately documented by Casanova-Schaitz et al.

3.5.2     Alternative Explanations for Konlinearity of Low Dose
          Crosslinked DNA

Each determination of crosslinked DNA is based on three replications with each
replicate based on material from four animals.  With such small numbers of
measurements, it is always possible that spurious results may be obtained.
The calculation of .significance levels is the statistician's way of trying to
evaluate the weight of evidence in small samples and to avoid being misled by
spurious indications.  The Casanova-Schmitz et al. authors have been careful
to calculate these.  However, additional data, especially below 6 ppm, would
add considerable substance to the results.  There are other mechanisms that
might lead to apparent nonlinearity.  For instance, suppose that there were a
small but constant loss in the measurement of IF-DNA.  This would be increasingly
important at low doses, and hence, would Induce an apparent threshold in the
amount of crosslinked DNA.  As indicated in Section 3.2, the efficiency of
extraction of DNA and the verification of distribution are of prime importance
in excluding possibilities such as these.

The authors have suggested that physiological and biochemical defense mechanisms
could become less efficient with increasing CH.O concentrations.  We agree
that the processes of tnucociliary clearance ana DNA repair could become
saturated as the CH.O concentration increases, and DNA-protein crosslink
formation could therefore increase disproportionately.
                                              3  14
Furthermore, the disproportionate increase in  H/  C ratio with increase in
CH 0 concentration might be due to artifactual disturbances in the  H/  C
ratio rather than a true increase in crosslinked DNA-protein in the IF frac-
tion.  The effect of CH.O is somewhat complicated, since CH.O is known to
inhibit DNA synthesis, yet cause an increase in cell turnover in a small
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0.05-
0.04 -
0.03-
0.02-
0.01-
  0  «4
                                             A Respiratory AQ-DNA*

                                             • Olfactory IF-DNA**
              T
              2
]
6
10
15
                        Administered Level of Formaldehyde, ppm
(a) Casanova-Schmitz, M., Starr, T.B., and Heck, H. D'A. (1984) Differentiation
   Between Metabolic Incorporation and Covalent Binding on the Labeling of
   Macromolecules in the Rat Nasal Mucpsa and Bone Marrow Inhaled ("Q- and
   (3H) CH20. Toxicology and Applied Pharmacology 76,26-44.
(b) Memorandum to Peter W. Preuss from Murray S. Conn concerning "Health
   Sciences comments in response to the Environmental Protection Agency's
   request for information regarding CH2O..!' dated July 16,1984.


    FlttJRE  1   ESTIMATED SLOPES FOR METABOLIC  INCORPORATION
           OF  RESPIRATORY  AQ-DNA  AND OLFACTORY IF-DNA
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percentage of cells.  Inhibition of DNA synthesis would decrease metabolic
incorporation but might also decrease sites for adduct formation if these are
restricted to the replication fork.  More experimentation ia thus necessary to
determine how Inhibition of DNA synthesis and/or increase in cell turnover
influence the  H/  C ratio in IF-DNA.

Cell death might also be involved in the nonproportional response in the
low dose range.  Thus, if cell death occurs, nucleic acids are released and
degraded.  The subsequent increase in nucleotldes, nucleosides, purines and
pyrimidines could inhibit de_ novo nucleotide synthesis from CH.O by feedback
regulation as well as increase the deoxynucleotide pools, thus, diluting out
radioactivity Incorporated into DNA.  A decrease in DNA synthesis from labeled
CH.O. in.the absence of an effect on adduct formation, would tend to increase
the  H/  C ratio disproportionately at higher CH 0 concentrations.

3.6       Sensitivity of the Study Conclusions to Statistical Analysis

The statistical methods for the comparison of AQ- and IF-DNA in terms of
incorporated concentrations of (  C) CH.O were described as a two-way analysis
of variance followed by paired t-tests For Individual concentrations.  The two
factors in the analysis of variance were concentrations of CH.O and type of
DNA:  IF and AQ.  It is not clear If the pairing of IF- and AQ-DNA determina-
tions were taken into account In the two-way analysis of variance.  Moreover,
the lack of homogeneity of variance between the low and high concentrations
was apparently not taken into account in the analysis; neither were ordered
alternatives over the concentrations when the two-way analysis of variance led
to statistically significant tests.  Thus, more powerful procedures could have
been employed to examine the differences In concentrations of (  C) CH.O
between IF- and AQ-DNA over CH 0 concentrations.

An examination of the data on respiratory DNA reveals that there Is a statis-
tically verifiable increase in the concentration of bound (  C) CH.O per mg
DNA over CH.O concentration as concluded in the paper.  These comments also
apply to the comparisons of  H/  C ratios between IF- and AQ-DNA as a function
of CH 0 concentrations.  Thus, the overall conclusions on the comparisons of
IF- and AQ-DNA as a function of CH.O concentrations hold even though the
statistical analysis could be strengthened.

In their comparisons of IF- and AQ-DNA with paired t-tests at separate CH.O
concentrations, Casanova-Schnitz et al. concluded that the bound (  C) CH.O
concentrations did not differ significantly at 0.3 and 2 ppm.  The power of
these paired t-tests is low for these CH.O concentrations because of the small
sample sizes at individual concentrations relative to the coefficient of
variability.  Although this is not critical with respect to the pattern of the
IF- and AQ-DNA data over the CH.O concentrations used in the experiment, it
does limit the extent to which Inferences can be made about the responses to
low concentrations for purposes of identifying no-response levels or making
lov-dose extrapolations for risk assessment.
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3.7       Adequacy of the Measure of Exposure

This question/Issue was fully addressed along with No.  4 in Section 3.4 above.

3.8       Utility of the Study in the Quantitative Risk Assessment of
          CH2Q

The panel recognized this study as an important step toward attempting to
assess the intracellular dose delivery of externally applieded CH_0.   These
efforts should be continued toward the ultimate goal of improving the assess-
ment of risk.  At its present level of development and  validation, however, the
study does not represent an adequate basis for quantitative risk assessment.
First, the problem of proper validation of the experimental methodologies must
be accomplished to assure that CH.O-DNA-proteln complexes are  identified
properly, and that the experimental assumptions are valid.  The evidence is not
sufficiently strong at this time to reject the linear dose extrapolation model.
Second, the selection of a single intracellular target  is complicated by the
nature of binding processes with DNA and could be augmented appropriately by
the additional analysis of binding to Intracellular proteins.   Third, and
perhaps most important, the selection of the acute model may not be entirely
appropriate since it is the chronic dosimetry that is most relevant to risk
assessment.  The factors which account for nonlinearity of dose delivery may
veil vary considerably (in either direction) as a result of chronic treatment.

This study is an important first step toward the introduction  of Intracellular
dosimetry Into the risk assessment process.  The continuation  and extension of
these investigations should be encouraged.
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4.0       'CONCLUSIONS AND RECOMMENDATIONS

The following summarizes the conclusions and recommendations developed by the
review team during the December 2 and 3* 1985 meeting.  These conclusions and
recommendations have undergone final review by each of the experts participating
in the meeting.

     1.   Some doubt still remains as to the validity of the assumptions which
          form the basis for distinguishing metabolically incorporated and
          crosslinked (or adducted) CH20, i.e.,  H/  C in DNA.

     2.   Experimental methods and controls were adequate with respect to
          monitoring the CH.O administration and analysis of dual-labeled
          materials.  However, the chloroform/iso-amylaIcohol/phenol extrac-
          tion for DNA and DNA crosslinked to proteins was not validated in
          terms of the identities of materials separated nor the overall
          efficiency and consistency of extraction.  The occurrence of under-
          lying variability Incorporation due to kinetic isotope effects on
          the disposition of tritiated CH.O can neither be assessed nor
          discounted.                                                     t

     3.   Sufficient documentation is still unavailable to state unequlvocably
          that all the crosslinked DNA-proteln complexes occur in the IF-DNA
          fraction.

     4.   There remains a need for an effective biochemical dosimeter to
          measure the dose of CH.O delivered to the cells of the nasal epithe-
          lium.  The data provided by Casanova-Schnitz et al. are not
          considered a sufficiently well-validated measure of this parameter.

     5.   The nonproportionality of the calculated concentration of bound
            C (CH.O)-DNA as a function of the administered dose is documented
          adequately.  Vhether the nonproportlonality truly reflects crosslink
          formation or is due to the small sample size, to a constant loss in
          the recovery of IF-DNA, or to artifactual disturbances In the  H/  C
          ratio remains to be elucidated.

                                                 14
     6.   The Increase in concentration of bound   C with the concentration of
          CH.O.is veil documented, as Is the Increase in the difference In the
           H/  C ratio between IF- and AQ-DNA.  The power of separate compari-
          sons for the 0.3 and 2 ppm doses Is low because of small sample size
          relative to the coefficient of variation.  This limits the potential
          for inferences about no-response levels and lov-dose extrapolations.

     7.   The study of Casanova-Schmitz et al. is an important first step
          toward quantitative assessment of the intrace'llular level of CH.O in
          the nasal mucosa of the rat following inhalation exposure.  At Its
          present level of validation, however, It does not provide a basis
          for such quantitation.  Furthermore, the selection of an acute study
          model may not be appropriate to the assesscent of chronic toxicity.
                                       4-1

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                                                                 £tf< Systems, J>
                                   APPENDIX 1

                         DOCUMENTS PROVIDED TO REVIEWERS
1.   Preliminary Assessment of Health Risks to Garment Workers  and  Certain
     Home Residents Exposure to Formaldehyde.   EPA Draft Report.  May 31,  1985.

2.   Casanova-Schmitz, M., Starr, T.B., and Heck, H.  D'A. (1984)  Differentia-
     tion Between Metabolic Incorporation and  Covalent Binding  on the Labeling
     of Macromolecules in the Rat Nasal Mucosa and Bone Marrow  Inhaled (   C)-
     and ( H) CH?0.  Toxicology and Applied Pharmacology 76,  26-44.

3.   Cohn, M.S., DiCarlo,  F.J., and Turturro,  A. (1985) Letter  to the Editor.
     Toxicology and Applied Pharmacology.  77, 363-364.

4.   	. (1985) Letter to the Editor.  Toxicology and
     Applied Pharmacology.  77, 365-368.

5.   	. (1985) Letter to the Editor.  Toxicology and
     Applied Pharmacology.  77, 358-361.

6.   Selected comments pertaining to the use of the CIII "effective  dose"
     experiment. . .

7.   Memorandum to Peter W. Preuss from Murray S. Cohn concerning "Health
     Sciences comments in response to the Environmental Protection  Agency's
     request for information regarding CH.O. . ." dated July  16,  1984.
                                     Al-1

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                                                                 £ife Systems. Jn
                                   APPENDIX 2

               ADDITIONAL REFERENCES RELATING TO EXPERT REVIEW OF
                      PHARMACOKINETIC DATA:  FORMALDEHYDE


1.   Starr TB, Buck RD.   1984.   The Importance of delivered  dose  In  estimating
     low-dose cancer risk from inhalation exposure to formaldehyde.   Fund.
     Appl. Tox. 4:740-753.

2.   Comments to the EPA Science Advisory Board by CUT scientists regarding
     the EPA draft entitled "Preliminary Assessment of Health Risks  to Garment
     Workers and Certain Home Residents from Exposure to Formaldehyde."
     June 21, 1985.

3.   Comments to the EPA Science Advisory Board by Dr. James A. Svenberg
     regarding the EPA draft entitled "Preliminary Assessment of  Health Risks
     to Garment Workers and Certain Home Residents from Exposure  to  Formaldehyde."
     July 9, 1985.

4.   Comments to the EPA Administrator by the Environmental  Health Committee of
     EPA's Science Advisory Board regarding the EPA draft entitled "Preliminary
     Assessment of Health Risks to Garment Workers and Certain Home  Residents
     from Exposure to Formaldehyde."  Undated.
                                     A2-1

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                                                                 &fe Systems, Jn
                                   APPENDIX 3

               QUESTIONS/ISSUES ON FORMALDEHYDE TO BE ADDRESSED BY
                                  EXPERT PANEL
1.   Are Che assumptions which form the basis for distinguishing between
     metabolically incorporated and crosslinked formaldehyde  adequately
     supported?

2.   The appropriateness or limitations of the experimental methodology
     used.

3.   What do the measurements taken in the Casanova-Schmitz study represent-
     i.e., is there ambiguity in the identity of the various  labeled fractions?
     Do the data establish that the IF fraction consist of crosslinked DNA?

4.   Are there any other data in the study that could be used as a measure of
     exposure in addition to the crosslinked DNA?

5.   Is the nonlinearity for crosslinked DNA at low doses adequately docu-
     mented?  Are there alternative explanations for these observations?  ;

6.   What is the sensitivity of the conclusions of the study  to both experi-
     mental error and the statistical treatment of the data?

7.   Does the study give a better measure of exposure than the "applied dose"
     for the second day post exposure; and If so, does It also give a better
     measure of the dose durinp the two-year bionssay?

8.   Conclusions the experts can draw concerning the utility  of the study
     (and any underlying data) in the quantitative risk assessment of
     formaldehyde.
                                     A3-1

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                                                                £ifc Systems, Jut
                                   APPENDIX 4

                  INFORMATION/CLARIFICATION REQUESTED FROM CUT
                                 BY EXPERT PANEL
1.   Would like to see Information on the  methodology and  equipment  used  for
     scintillation counting.   Needs information on  quench  correction and  the
     methods used to calculate DPMs.

     Would also like to see an example of  the  raw data used  to  calculate  DPM.
2.   Would like to review raw data used to perform two-way  analysis  of
     variance between aqueous phase and.interfacial DNA with respect to  their
     incorporation concentrations of (   C) CH_0 equivalents or
     their  H/  C ratios at different concentrations of CH.O.
^ ~,, « —*„_,	— with respect to
3.   Request copies of the following references:

     a.   Svenberg, J. A., Gross,  E. A.,  Martin,  J.,  and Popp,  J.A.  (1983a).
          Mechanisms of formaldehyde toxlclty.   In Formaldehyde Toxicity
          (J. E. Gibson, ed.)»  pp. 132-147.   Hemisphere, Washington,  D.C.

     b.   Swenberg, J. A., Gross,  E. A.,  Randall, H.  W., and  Barrow,  C.  S.
          (1983b).  The effect  of  formaldehyde  exposure on  cytotoxicity  and
          cell proliferation.   In  Formaldehydet  Toxicology,  Epidemiology
          and Mechanisms (J. J. Clary,  J. E.  Gibson,  and R. S.  Waritz, eds.),
          pp. 225-236. Dekker.  New York,
                                     AA-1

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                                                                 JCifc Systems, Jnc.
                                   APPENT1X 5

                                   TR-835-19

                            LIST OF PARTICIPANTS

              Expert Review of Pharmacokinetic Data:
            Formaldehyde
Participants
Dr. Edward Bresnlck
Eppley Institute for Research
  in Cancer
University of Nebraska Medical
  Center
Omaha, NE  68105
(402) 559-4238

Dr. Peter Bloomfield
North Carolina State University
518 Cox Hall
Raleigh, NC  27695
(919) 737-2541

Dr. Richard Cornell
Department of Biostatistics
School of Public Health
University of Michigan
Ann Arbor, MI  48109
(313) 764-5450

Dr. Helen Evans
Department of Radiology
Case Western Reserve University
2065 Adelbert Road
Cleveland, OH  44106
(216) 844-3530

Dr. Robert P. Hanzlik
Department' of Medicinal Chemistry
University of Kansas
Lawrence, KS  66045
(913) 864-3750

Dr. Christopher F. Wilkinson
Cornell University
N202  MVR
Ithaca, NY  14853
(607) 256-8112
Dr. Lenone Yielding***
Department of Anatomy
University of South Alabama
2042 Medical Sciences Building
Mobile. AL  36688
(205) 460-6490
EPA Staff

Dr. William Farland
Health and Environmental Revlev
   Division
Office of Toxic Substances
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC  20460
(202) 382-4241
CUT Representative(s)

Dr. Mercedes Casanova-Schmitz
Dr. James Gibson
Dr. Henry D'A Heck
Dr. Thomas B. Starr

Chemical Industry Inst. of Toxicology
P.O. Box 12137
Research Triangle Park, NC  27709
(919) 541-3440

ICAIR, Life Systems, Inc.

Dr. John P. Glennon, Program Manager
Mr. Daniel Mecklcy, Task Manager

   24755 Highpoint Road
   Cleveland, OH  44122
   (216) 464-3291
(a) Chairperson.
                                     A5-1

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                                                                 £ife Systems, Jut.
  Time
                     APPENDIX 6

                      TR-835-18
                       AGENDA
EXPERT REVIEW OF PHARMACOKINETIC DATA:   FORMALDEHYDE

                 December 2-4,  1985
           Chamber C, Greenbrier Ballroom
             Sheraton University Center
                     Durham,  NC
      Meeting Chairperson:  Dr. Lemone  Yielding


 	Agenda Item	  Individual
Monday, December 2, 1985
                                             (a)
 9:00 a.m.     Informal Technical Discussions

12:00 noon     Break

 1:00 p.m.     Welcome

               1.  Administrative Announcements
               2.  Summary of EPA's Needs

 1:45 p.m.     Meeting Objectives

 2:00 p.m.     Chairman's Opening Comments

 2:30 p.m. .    Input From CUT

 4:30 p.m.     Finalize List of Questions and Issues

 5:30 p.m.     Break

 7:00 p.m.     Discussion of Questions and Issues

               1.  Discussion
               2.  Consensus
               3.  Assignment of Draft Report Authors

 9:00 p.m.     Adjourn for Day
                                       (b)
                                                    D.  Meckley
                                                    V.  Farland

                                                    J.  Glennon

                                                    L.  Yielding

                                                    Dr. Heck

                                                    L.  Yielding



                                                    L.  Yielding
                                                            contlnued-
(a) Optional for those Individuals arriving December 1, 1985.
(b) Selected authors of Draft Report sections may adjourn to prepare  rough
    draft or entire meeting may adjourn to prepare Draft Report  sections  as
    determined by the participants.
                                     A6-1

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                                                                 £ife Systems. JM
Ap.cnda - continued
  Time
Agenda Item
Tuesday, December 3, 1985

 8:00 a.m.     Administrative Announcements

 8:10 a.m.     Chairman's Comments

 8:20 a.m.     Discussion of Questions and Issues

               1.  Discussion
               2.  Consensus
               3.  Assignment of Draft Report Authors

12:00 noon     Break

 1:00 p.m.     Discussion of Questions and Issues - continued

 3:00 p.n.     Meeting Status Summary

               1.  Questions and Issues Resolved
               2.  Remaining Action Items
               3.  Revision of Agenda/Schedule

 4:00 p.n.     Discussion of Questions and. Issues - continued

 5:00 p.m.     Adjourn for Day


Wednesday, December 4, 1985

 8:00 a.m.     Administrative Announcements

 8:10 a.m.     Draft Report Status

 8:20 a.m.     Preparation of Draft Report

               1.  Complete Action Items
               2.  Review/Discuss Draft Report Sections
               3.  Revise Draft Report Sections

12:00 noon     Break

 1:00 p.m.     Preparation of Draft Report - continued

 4:00 p.m.     Final Review of Draft Report Status


 5:00 p.n.     Adjourn
Individual



D. Meckley

L. Yielding

L. Yielding
                                    L.  Yielding

                                    L.  Yielding
                                    J.  Glennon
                                    L. Yielding
                                    D.  Meckley

                                    L.  Yielding

                                    L.  Yielding
                                    L.  Yielding

                                    L.  Yielding
                                    J.  Glennon
                                     A6-2

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                                              m
APPENDIX 1:  EXPERT  PANEL REPORT ON HCHO
             PHARMACOKINETIC  DATA AND CUT RESPONSE
Cinci
                                .
                            nnati ,

-------
Chemical Industry Institute of Toxicology
President. Robert A. Neal. PhD.
Vice President. Director of Research. James E. Gibson. PhD.
Vice President. Administration and Secretary. Donald A. Hart. Ed.D.


                         February 4,  1986
  P. O. Box 12137
Research Triangle Park,
 North Carolina 27709
  (919)541-2070
  Dr.  William H.  Farland
  Deputy Director
  Health and Environmental
   Review Division
  Office of Pesticides and
   Toxic Substances
  U.  S.  Environmental Protection Agency
  401  M  Street,  SW
  Washington, DC   20460

  Dear Dr. Farland:

       Enclosed are detailed  comments  on the document "Expert
  Review of  Pharmacokinetic  Data:     Formaldehyde"  which was
  authored  at  the   meeting  in   Research  Triangle   Park  on
  December 2-4.   I have also  sent copies of these comments to
  the  other members  of the committee  inviting their individual
  or  collective comments.  We  would  also welcome  any comments
  you  personally  might have  concerning  points raised in this
  critique.

                               Sincerely,
                               Robert  A.  Neal
                               President
  RAN:ewb

  Enclosure

-------
COMMENTS ON THE FINAL REPORT OF THE PANEL REVIEWING THE CUT PHARMACOKINETIC
                            DATA ON FORMALDEHYDE

                 M. Casanova, T. B. Starr, and H.  d'A.  Heck
    We have carefully examined  the  final   report  of the Panel  reviewing  the
CUT pharmacokinetic data on formaldehyde.    We find many of their concerns to
be without merit, and  we  disagree  strongly  with  their conclusions.   A  de-
tailed justification for this assessment of the Panel's report is given  below.

3.1.1.  Metabolic Incorporation versus Adduet Formation or Cross I inked CH^O
                                                                         &~

    The Panel states that the "interpretation  of  the 3H/14C ratio of the  DMA
due to metabolic incorporation of [ H]-  and [  CJCHJ)] is very complex" owing
to the fact that 'relative reaction  rates  and  pool sizes are likely to vary
under different conditions'.  Hence, the  Panel argues that it is necessary to
isolate the DNA bases  by  HPLC  and  to  determine  the isotope ratios  of  the
isolated bases.  The Panel  did  not  mention  that we have already undertaken
such studies, although we informed the Panel of this on December 2, 1985, when
we had our meeting  with  then.    The  Panel  did  not request details of  our
studies either at  the  meeting  or  subsequent  to  the  meeting, although we
volunteered to provide any   information  requested.    It  is surprising to us,
therefore, that they called  in their report for HPLC studies to be done.
                                     -1-

-------
    For the record, we would like to provide the essential  results of  our  HPLC


analyses of DNA.  The DNA  samples  that were analyzed were obtained from  rats

                                                       3         14
that had been exposed (6 hr) to  0.3,  2, or 6 ppra of [ H]- and [  C]formalde-


hyde.  These were the same samples that had previously been used for determin-


ations of covalently bound CHgO in  DNA (Casanova-Schmitz et £L, 1984).   Suf-


ficient AQ DNA remained from  those  samples  for  analysis by HPLC.  However,


only one IF DNA sample remained  from the initial experiments, and that sample


was obtained from rats exposed at 6 ppm.






    The major UV-absorbing peaks from  respiratory mucosal  AQ DNA samples elu-


ted at the same positions  as authentic purine and pyrimidine deoxyribonucleo-


side standards.  Calculation of the  base  compositions of the DNA samples was


performed after calibration of the UV  monitor with nucleoside standards.   The


values obtained for the base compositions  of the DNA samples agreed well  with


one another and with the  base  compositions reported in the  literature; obs.:


deoxyadenosine  (dAdo), 28.9 * 0.3 X;  deoxycytidine (dCyd), 21.3 * 0.4 X;  lit.


values (Shapiro, 1968): dAdo, 28.8 * 0.7  X;  dCyd, 20.5 * 0.5 X.  This signi-


fies that the base composition of the AQ  DNA is the same as that of the total


rat DNA.  The   nucleosides,  deoxyguanosine  (dGuo) and thymidine  (dThd),  were


not completely  resolved  in  the  chromatography,  hence, their  individual base


compositions were not calculated.






    As expected (Casanova-Schmitz et aj_.,  1984), most of the radioactivity  in


the AQ DNA from the  respiratory  nucosa  eluted  at the positions of the normal


deoxyribonucleosides, dGuo, dThd, and dAdo,  implying that the  labeling of the


AQ DNA was primarily caused  by  normal  metabolic incorporation.   In three  AQ
                                     -2-

-------
 DNA samples from rats exposed to  6  ppm  of  CH-0,  the percentage of the total

   C that eluted at the positions of  the normal  nucleosides  was  100%, 96%,  and

 90%, respectively.  In two of  the three samples, small  amounts  of  radioactiv-

 ity eluted prior to the .major peaks,  but no radioactivity  eluted after dAdo in

•any sample.  A Iate-eluting radioactive  peak would be expected  for 6-hydroxy-
                                                *••
 methyl-deoxyadenosine, a  postulated adduct of formaldehyde with  DNA (BeI and et


 al., 1984).  If such  an   adduct  were  formed,   it  did not remain in our  DNA

 samples until the time of analysis.



         3  14
     The  H/  C ratios of  the major peaks were consistent with normal metabolic

 incorporation.  The isotope  ratios  of  the  deoxyribonucleosides did  not  vary

 measurably with concentration over the range   0.3  to 6  ppm.  We observed  that

 the 3H/14C ratio of dAdo (0.55) was higher than that of  dGuo (0.25).   A higher

 isotope ratio for dAdo than for  dGuo  is consistent with  the known pathway of

 formaldehyde incorporation via tetrahydrofolate into positions 2 and 8 of  ino-

 sine monophosphate (IMP), the precursor of  both  GMP and  AMP.  The conversion

 of IMP to GMP involves the introduction of an oxygen atom  at position  2 of the

 purine ring  (with consequent loss of  H  at this position).   The conversion of
                                                       3
 IMP to AMP does not involve a  corresponding  loss of H at position 2.  Thus,

 AMP should have a higher  H/  C ratio than GMP, as observed experimentally.




     We observed no labeling of dCyd   in  our chromatograms.   This  implies  that

 the labeling of dThd  (3H/14C • 0.4)  was  due only to labeling at the 5-methyl

 position, which results from transfer  of  the nethylene carbon atom from N ,-

 N  -methyIene-tetrahydrofolate to deoxyuridine monophosphate.
                                      -3-

-------
        3  14
    The  H/  C ratios of the minor  peaks  seen  in two of the three chromato-


grams were low (ranging from 0.3  to  0.4),  resembling the ratios seen in the


major peaks.  This suggests that the minor peaks of radioactivity were not due

                                                            3  14
to covalent adducts, which would be expected to have higher  H/  C ratios than


those seen in the normal bases.  It  is likely that at least some of the minor
                                               »•
                                               f.
radioactive peaks in the AQ  DNA  samples  were due to slight contamination of


the DNA with RNA.  This hypothesis  is supported by the observation that minor


peaks eluted at positions similar to those obtained using ribonucleoside stan-


dards, adenosine and guanosine.  It is  also possible that a minor peak resul-


ted from deamination of dAdo to deoxyinosine, which might have occurred if the


alkaline phosphatase used in the hydrolysis  of DNA had been contaminated with


adenosine deaminase (Gehrke et a_L, 1982).  (No loss of  H would occur in this

                         3  14
reaction, therefore, the  H/  C ratio would remain unchanged.)  Finally, a mi-


nor peak may have been due to 5-methyl-deoxycytidine (5-MedCyd), a normal con-


stituent of rat DNA accounting for about 1% of the bases (Shapiro, 1968).  The


presumed labeling of the 5-MedCyd would be expected to occur via transfer of a


methyl group from S-adenosylmethionine to dCyd  in DNA (Kornberg, 1980).  This


methyl group could be  labeled, since  methionine might be synthesized  in small


amounts from methyl-tetrahydrofolate,  although  methionine  is usually consi-


dered to be an 'essential' ami no acid.






    It should be noted that even  if one assumes the most extreme case that 10X


of the radioactivity in the AQ  DNA  were  due to RNA, the error  in estimating


the 3H/14C ratio of the AQ DNA  would  be  only IX, due to the similar  isotope


ratios of RNA and AQ DNA.   Moreover, since RNA has a higher specific activity


than DNA (Casanova-Schmitz et ja_L, 1984),  the actual contamination of the DNA
                                     -4-

-------
by RNA would be less than 4% in the most extreme case.   Errors  of  these magni-



tudes are completely negligible in our calculations of  covalent binding.





                                                            3         14
    A single IF DMA sample from rats  exposed  to 6 ppm of [ H]- and [ CJCH-0



was also analyzed by HPLC.  In  this sample,  one additional  peak was seen that



was not present in any AQ  DMA  sample.    This*' peak eluted very  early  in the
                                                     •

chromatogram and had an apparent  H/  C ratio  >  1.0.   Such a  peak could con-


ceivably be incompletely digested  DNA  containing  covalent I y  bound GO, the



hydrolysis of which  was  prevented  by ' DMA-ami no  acid or DNA-peptide  cross-



linking.  This interpretation is, of course,  tentative, and additional studies



are needed to test this hypothesis.    As  in  the AQ DNA, there were no peaks


eluting after dAdo.
    It is difficult to unequivocally identify  minor peaks seen in HPLC, owing


to the low levels of radioactivity and the  small amounts of DNA in the IF DNA


samples.  After enzymatic hydrolysis,  each of the major metabolicaIly-labeled


nucleoside peaks in the IF DNA  contained  only  about 100 to 200 dpm, and the


total radioactivity in the  unidentified  early-eluting  minor  peak in IF DNA


with the high  H/  C ratio was only  80  dpm.  Thus, owing to the small amount


of DNA obtainable from the rat nasal mucosa, the low level of radioactivity in


the IF DNA that was due to  cross-1 inking, and the present lack of information


concerning the detailed structures  of  the  DNA-protein cross-links, we doubt


that much more information  than  we  have  already obtained would result from


continuing or extending the  HPLC  studies,  despite the recommendation of the


Panel.  At least the qua Iitative  outcome  of such studies has already emerged


from our work.
                                     -5-

-------
    It should be emphasized that the results  of our HPLC analyses of DMA sam-

ples collected from exposed rats  are  in  full  agreement with our conclusions

that AQ DNA does not contain covalently bound ChLO,  j_..e., that the labeling of

AH DNA is due to metabolic  incorporation,  whereas  the labeling of IF DNA is

caused both by metabolic incorporation and covalent binding.  We are satisfied
                                               *•
that the concerns of the Panel concerning the s&urce of the label in AQ and IF

DNA has been adequately addressed by our research.



3.1.2 Cross I inked CH^O Located Exclusively in the Interface (IF) DNA




    As discussed above,  there  was  £o  HPLC  evidence  for either adducts or

cross-links in the AQ DNA at either 0.3, 2, or 6 ppm.  In contrast, the IF DNA

at 6 ppm did provide such evidence.




    The Panel states in this section that "the efficiency of extraction of the

DNA from respiratory epithelium  under  conditions  of CrLO dosing (at various

levels) should have  been  determined1.    Detailed information concerning our

extraction efficiencies had  not  been  requested  by  the  Panel prior to the

meeting, nor was it requested subsequent  to  the meeting.  For the record, it

should be noted that the average  DNA  yield  per mg wet weight of respiratory

nucosal tissue (4.20 * 0.10 ftg; mean *  SE, n a 30) did not vary significantly

with concentration  (£ = 0.473;  one-way  ANOVA) over an airborne concentration

range of 0.3 to 15 ppm and exposure times of either 3 or 6 hr.  Thus, the pos-

sibility of a dose-dependent change  in the recovery of DNA  is ruled out by our

data.
                                     -6-

-------
    Regarding the question of the  efficiency  of our extraction procedure for



DNA, it should be noted that we  used the same extraction procedure to isolate



DMA from rat liver nuclei treated  J_n  vitro  with formaldehyde.  The yield of



highly purified ONA (chromatographed on hydroxyapatite and washed by ultrafil-



tration) that we obtained was 0.82 * 0.02 mg/g of liver.   This result compares

                                               <»«

extremely well with the total amount  of  DMA (unpurified) reported to be pre-



sent in rat hepatic nuclei (0.83 *  0.03 mg/g) (Blobel and Potter, 1966).  The
                                   (


approximately five-fold higher yield of  DMA  that  we obtained from the nasal



respiratory epithelium (see preceding paragraph)  indicates that the percentage



of the total tissue weight that is  due  to DMA is significantly higher in the



nasal mucosa than in the liver.







3.2 Experimental Methodology Limitations







    The Panel again raises  questions  about  the extraction efficiency of the



DMA, and it asserts that the amount  of  ONA  in the IF fraction will vary with



the extraction conditions employed.   However, the extraction conditions (vol-



umes, buffers, pH, ionic  strength,  temperature) were carefully held constant



throughout the experiment.  We were  well  aware of the  importance of reprodu-



cibly recovering a constant amount  of  DNA in all experiments.  Consequently,



the same individuals did  all  of  the  experiments,  and the experiments were



always performed with great care.   From  the inhalation exposure to the final



extraction of DNA, the experiments were carried  out on the same day using the



same protocol.  No samples were ever  stored.   The constancy of the ONA yield



with concentration noted above  is clear  evidence that our concerns  (and those



of the Panel) were properly addressed  from the beginning.  Therefore, the as-
                                     -7-

-------
section of the Panel of a  possibly  varying DNA yield with "variations in the

extraction conditions* is contradicted by the evidence.



    The question of the  interpretation  of  acute  vs. long-term exposure has

been addressed numerous times.  The  essential  point is that at low formalde-
                                               »•
hyde concentrations, _[..<•., those  concentrations  to which humans are normally

exposed, the transition from  normal  respiratory  to squamous epithelium does

not occur.  Therefore, covalent  binding studies in normal respiratory epithe-

lial cells are highly relevant  to  risk  assessment, where risk is defined as

the possibility of covalent  reaction  with  DNA  (which  may  or may not lead

eventually to cancer) under normal  human  exposure situations.  The high-con-

centration, long-term exposure studies do  not reveal what occurs biologically

at low concentrations, since the cell structure and tissue morphology has been

radically altered.  Indeed, one  of  the  panelists, Dr. Wilkinson of Cornell,

remarked at our meeting on December  2  that such high-dose exposures can well

be considered to exceed the "max!mum-to Ierated-dose*.



    The final point raised by  the  Panel  in  this section is the question of

isotope effects in the oxidation of  [3H]-  and  [14C]CH20.  At the time of the

meeting on December 2 we had already begun studies of possible isotope effects

in either the covalent binding or oxidation of CH-0.  These studies were star-

ted for reasons other than  those  raised  by  the Panel, however, the isotope-

effect studies were only  in  preliminary  stages in December, and, consequent-

ly, the results that we have now obtained could not be given to the Panel.  We

would  like, therefore, to present these results in this document.
                                     -8-

-------
    First, with regard to covalent  binding  of  formaldehyde,  we have  examined
    914
the  H/  C ratio of the IF  DNA recovered from freshly isolated  hepatic  nuclei

incubated in vitro with [3H]- and  [14C]CH20.    The IF DNA isolated from such

nuclei was heavily labeled with  H and   C, whereas the AQ DNA had practically

no radioactivity, consistent with our interpretation  that the IF DNA, and not

the AH DNA, contains covalently bound  GO.  Furthermore, the  percent  IF DNA

as well as the specific activity of  the IF DNA  increased with increasing con-
    x^
centra t ions of OLD and with increasing  times of reaction. The isotope ratio

of the IF DNA  relative  to  that  of  the reaction solution was approximately

1.034 * 0.009 (mean * SE, n = 6), indicating an  extremely small  isotope  effect

favoring the binding of [3H]CH20 over  that  of   [UC]CH20 to  DNA. An isotope

effect of this small magnitude  is  negligible  insofar as our calculations of

covalently bound GO in DNA are concerned.

                                                                         •
                                                                  3  14
    Second, with regard to metabolic oxidation,  we determined  the  H/ C ratio
    «         « *
of [ H]- and [  C]GO at various  times during in vitro incubations  of  selec-

ted concentrations of labeled  formaldehyde  with freshly isolated homogenates

of the rat respiratory mucosa and  NAD   (1  mM) .  In most cases, the reaction

solutions also contained glutathione (GSH), since the principal  enzyme respon-

sible for OO oxidation, formaldehyde dehydrogenase (FDH), is a GSH-requiring

enzyme.  We observed that  the  oxidation  of [n]- and [  C]QO catalyzed by

FDH occurs with a significant isotope effect.  The rate of oxidation of  [  C]-
CHjO was approximately 1.82-fold faster than that of nfJOO, indicating that

the hydride transfer step  in  the  GSH-dependent  oxidation  of GO to HCOOH

catalyzed by FDH is at least  partially  rate-limiting in GO oxidation.  The

magnitude of the isotope effect was independent of the GO concentration over
                                     -9-

-------
a 100-fold concentration range (from  0.1  to  11  /*M).   Furthermore, we  found



that aldehyde dehydrogenase exhibits a  similar  isotope effect to  that of  FOH



in its oxidation of CHgO to HCOOH,  \vhich is GSH-independent.







    Since an isotope effect in  ChLO  oxidation has  been demonstrated to  occur



in vitro, it can be presumed that  it  also occurs in vivo.   An isotope effect



occurring in the oxidation of OLD  to  HCOOH in vivo would result in an effec-



tive "enrichment" of [3H]CH20  relative, to  [14C]CH20 in the residual  (unoxi-



dized) CH20 in the nasal  mucosa.    The  3H/14C  ratio of the unoxidized CHgO



would, therefore, be greater than that of the inhaled gas.  In the calculation



of covalently bound CrLO in ONA (Casanova-Schmitz £t aj^., 1984), it was  impli-


                       3  14
citly assumed that the  H/  C  ratio  of  QUO  in the nasal  mucosaI cells  was



identical to that of the gas.   It  is now recognized that this assumption  may



be incorrect.  The assumption was  made because of the practical impossibility



of directly measuring the  H/  C ratio  of  unmetabol ized GO in the  cells of



the nasal mucosa.
    It can be readily shown that  an enrichment of [ HjChLO relative to [  C]-



CH.O in the cells, resulting from an isotope effect in oxidation, leads invar-



iably to an overestimate of the  amount  of OLD covalently bound to ONA, when



the calculation of  covalent  binding  is  done  using our published equations



(Casanova-Schmitz et a_L, 1984).  Thus,  by implicitly assuming ho isotope ef-



fect in CrLO oxidation, our calculation  yielded  an upper limit on the amount



of OLD bound to DMA.  Conversely, by assuming the isotope effect in QLO oxi-



dation to be maximal, j..,e., that it  occurs with an isotope effect of 1.82 in-



dependent of the CrLO concentration,  we  can  calculate a Iower limit for co-
                                     -10-

-------
valent binding of GO to  DMA.    (The  reason that 1.82 represents a maximal

isotope effect is that the other  routes  of CH«0 elimination from cells,  such

as covalent binding, diffusion out of  the cells, or reduction to rr.ethanol,  do
                             g
not involve breakage of the   H-C  bond,  and,   therefore, they would all  show


smaller isotope effects than that observed in oxidation.   Thus, the assumption
                                               <-
of a maximal isotope effect is  equivalent  to assuming that metabolism is the

only route of elimination.)   The  results  of  such calculations are shown in

Figure 1.




    The upper curve in Figure 1 is the same curve as that previously published


by us (Casanova-Schmitz £t jaL, 1984), which assumed no isotope effect in CH«0


oxidation.  The lower curve is  that  which would result if the isotope effect

had its maximal value.  Clearly, an overestimate of covalent binding occurs at


all concentrations.  However, the fundamental shape of the curves, j..£.» their

significant departure from linearity at low GO concentrations, is unaffected


by the isotope effect in ChLO oxidation.




    In reality, by assuming that an  isotope  effect occurs in vivo in the oxi-


dation of GO, the curve becomes,   if  anything, even more nonlinear than was

the case before the isotope effect  was  recognized to occur.  This is because


the enrichment of 3H  relative  to  14C  in  the residual (unoxidized) OO is

greatest under conditions  in which  the  metabolisa  of  QO to HCOOH is nost


nearly complete, j.-.e., »t  low  airborne  concentrations, and  is smallest under

conditions tn which the metabolism of  GO  to HCOOH  is  least complete, i.e.,

at high airborne concentrations (Melander and Saunders, 1980).  Therefore, the

overestimate of the amount of GO bound  to DMA  is greatest at  low concentra-
                                     -11-

-------
      UPPER AND LOWER BOUNDS FOR FORMALDEHYDE BINDING TO DNA
  800-r
                        [FORMALDEHYDE], ppm

Amounts of CH20 covalently bound to rat nasal mucosal DNA.
Upper and lower curves represent yields of covalently bound CH20
assuming either no isotope effect or a maximal (1.82) isotope
effect in the oxidation of CH20 by FDH.
                               Pig. 1

-------
tions and is smallest at high concentrations, causing the curve to become even

more nonlinear than before.  We  would  expect,  therefore, that the true cova-


lent binding curve should  approximate  the  lower  curve  of the two shown in

Figure 1 at low GO  concentrations,  and  it should approach more closely to

the upper curve at high CHjO  concentrations.   We conclude that low-dose non-
                                               ^.
linearity in the binding of CH^O to  DMA is supported rather than disproved by

our finding of an isotope effect in CH^O oxidation.




3.3 Identity of Labeled Fractions




    This issue is addressed in section 3.1 and 3.2.




3.4 Other Measures of Exposure




    The Panel remarks that our methods appear to be "unnecessarily sophistica-

ed and complex".  We feel that  our methods, far from being complex, are rela-

tively simple.  DMA was  isolated  and purified using established (hydroxyapa-

tite) techniques, and the resulting  DMA  was counted for radioactivity.  Cer-

tainly, the use of dual isotopes for  metabolic studies is not new.  The crit-

icism of our methods by the  Panel  as being too 'sophisticated" is not justi-

fied in our opinion.




    The Panel suggests measuring covalent binding to intraceIlular proteins as'

a "simpler index" of molecular  dostmetry.    We would ask four questions: (1)

Which  intracellular proteins would they recommend?  (2) What evidence is there


that covalent binding to proteins is related to mutagenesis or to cancer?  (3)
                                     -12-

-------
How would they deal with the  issue  of  protein turnover?  (4)  How would they

correct for labeling caused by  metabolism?   Clearly,  their proposal  requires

selecting a protein, or a  group  of  proteins,  that would be used to monitor

CHJO binding.   Such proteins would have to be quantitatively purified from the

other proteins 5n the nasal mucosa,  and  their rate of turnover would have to
                                               <
be separately determined.  Labeling due to metabolism would have to be differ-

entiated from that due to covalent  binding.  Finally,  after solving these ex-

tremely challenging experimental problems,  it  would  have to be assumed that

covalent binding to these proteins (presuming that such binding could be shown

to occur) is somehow  related  to  the  initiation  of cancer.  These critical

problems and assumptions clearly  mean  that  intracellular protein binding is

not a "simpler index* than DMA binding for molecular dosimetry purposes.


                                                              "x\

    Furthermore, we now have strong evidence that protein binding in vivo pri-

marily involves the extracellular proteins.    We informed the Panel that when

rats were pretreated with phorone, a  GSH  depleting agent, there was a marked

increase in the amount of GO  covalently  bound to DNA, as would be expected

if oxidation of GO to HCOOH were a major defense mechanism  (Casanova-Schmitz

and Heck, 1985).   In contrast, there  was no detectable  increase in the amount

of GO covalently bound  to  proteins  as  a  result of phorone pretreatment.

Therefore, GSH depletion was ineffective  in  enhancing the binding of GO to

proteins, suggesting that the proteins to. which GO  is bound are not protec-*

ted by metabolism.  One  is  led  to  conclude that the proteins must be either

cell surface proteins or extracellular proteins, the latter of which are, pre-

sumably, mucus proteins.  Binding  to  mucus proteins is  irrelevant to dosimet-

ry, since the  intracellular concentration  of  the toxicant,  not the extracel-

 lular concentration of the toxicant,  is of primary concern.


                                     -13-

-------
    With regard to the final comments  made  by  the Panel  in this Section,  we


have never asserted that the amount  of  CrLO  bound to DMA following an acute


exposure is necessarily the same as that  following a chronic exposure.   We do


not know the amount of CrLO bound under chronic exposure conditions.   However,


we do claim that short-term  exposure  conditions,   which do not cause massive

                                               **"
changes in cell structure  and  morphology,  should  more nearly represent the


chronic exposure situation at low concentrations of CrLO, j..j».f those to which


humans are actually exposed.



            *                                   .            *


3.5 Nonlinearity for Cross I inked DMA at Low Doses





3.5.1 Documentation of Nonlinearity for Low Dose Cross I inked DNA





No comment. „_





3.5.2 Alternative Explanations for NonIinear Ity of Low Dose Cross!inked DNA





    The Panel  suggests  that,  because  each  experiment  involved only three


replicates, it is possible  that the  results were spurious.  They suggest that


additional experiments below 6  ppm  would  add  considerable substance to the


results.  We"are surprised  by  this  comment,  since  in our meeting with the


Panel on December 2, we did present  such  evidence  to then.  As we showed at


that meeting, we have carried out additional  studies  at 0.9, 2, 4, and 6 ppm


(three replicates at  each  concentration)  (Casanova-Schmitz and Heck, 1985).


The results were fully consistent with those published previously.
                                     -14-

-------
    The Panel  also suggests the possibility  of  a  'small but constant  loss  in
the measurement of IF DNA* as being responsible  for low-dose nonlinearity.   In
section 3.2, we showed that the Panel's earlier  hypothesis  of a  dose-dependent
loss of DNA was inconsistent  with  our  results.     The hypothesis  of  a dose-
independent loss of DNA from the  IF  DNA  fraction can  be  readily shown to  be
invalid.  If one assumes a constant loss of IF ONA  at all GO concentrations,
as suggested by the Panel, calculations  can  be made of the amount of IF DNA
that would have to be lost at 2  and  at 6 ppm in order  to  linearize the cova-
lent binding curve.  Such calculations  were  performed   by us:   the curve for
covalent binding of CH.O to DNA at  2  and at 6  ppm was  recalculated using the
equations in Appendix 3 of Casanova-Schmitz  ^t  a_L (1984), but  allowing for a
constant loss of IF DNA.  This  loss would affect the binding calculation only
by changing the fraction of DNA that is IF:

            Measured (KIF DNA)/100 = (IF DNA)/(AQ DNA +  IF  DNA);

      •True" (JJIF DNAJ/100 • (IF DNA + Loss)/(AQ DNA * IF DNA  *  Loss).
    Figure 2 shows the effect of loss  on  the ratio of the amount of covalent
binding at 6 ppm to that at 2 ppm.  While the ratio is reduced somewhat as the
amount of lost IF DNA increases, in  agreement with the suggestion of the Pan-
el, it cannot drop below 8.2 even  with arbitrarily large losses.  However, in
order to linearize the binding response,  this  ratio would have to drop to 3,
the ratio of  the  two  airborne  formaldehyde  concentrations.   Therefore, a
constant loss of IF DNA, no  matter  how  large, cannot be responsible for the
observed nonlinearity in ChLO binding to DNA.
                                     -15-

-------
        HT
         0.0
0.5
1.5
2.0
                          IF  LOSS,  MG
Fig.  2.  Effect of a  hypothetical  constant   loss of IF ONA on the calculated



ratio of covalently bound ChLO at 6  ppm  and  at 2 ppm.   For  linearity of the



concentration-response curve, this ratio  should  be  3,   the  ratio of the two



airborne GO concentrations.

-------
                                                                  3  14
    The Panel suggests  that  "the  disproportionate  increase in   H/ C ratio


with increase in CrLO concentration  might  be due to artifactual  disturbances


...rather than a true increase in  crosslinked DMA-protein".   What is meant by


such "artifactual disturbances" is not clear,  although the Panel appears to be


referring to increases in cell  turnover.    We have already  remarked that in-
                                               *

creases in cell turnover could well contribute to the low-dose nonlinearity in


CrLO binding to DMA  (Casanova-Schmitz  et  al.,  1984),  just as saturation of


metabolic defense mechanisms could also  be a contributing factor.  The point


is that a change in  the  cell  turnover  rate  or a change in CrLO metabolism


would result in a  change  in  the  amount of  label metabolically  incorporated


into DMA.  However, the AQ DMA  provides a control for such changes, since the


labeling of AQ DMA is only  due  to  metabolism.  It is the difference between


the 3H/14C ratios of IF and AQ DNA, not the absolute value of the 3H/14C ratio


of the IF ONA, that  is  directly  related  to  the amount of covalently bound


CH20.





    Thus, the comment of the Panel does not call  into question the validity of


our conclusion that the increase in the  H/  C ratio of the IF ONA relative to


that of the AQ DNA is due  to  covalent binding.  Rather,  it simply suggests a


mechanism for the disproportionate increase   in  covalent binding at high con-


centrations, J..J9., increased cell turnover, which has already been proposed by


us.





    The  last comments of the Panel  in this section concern the possibility of


cell death, which could (according to  the Panel), "inhibit de novo nucleotide


synthesis..., as well as   increase  the  deoxynucleotide pools, thus, diluting
                                     -16-

-------
out radioactivity incorporated into DNA*.    The  Panel  then states that a "de-


                                                       3  14
crease in DNA synthesis...would tend  to  increase the   H/  C ratio dispropor-



tionately at higher CH^O concentrations*.     We  repeat that any change in the



labeling of DNA due to metabolism  is already accounted for by our measurement



of the radioactivity in the AQ  DNA,  which  is only due to metabolism and is,

                                               <»

therefore, an internal control  for  all  such "effects.   Thus, the Panel has



merely suggested a possible  mechanism  for  low-dose  nonlinearity.  We doubt



that this mechanism is plausible,  however, because at  low CIO concentrations



(0.3, 2, and 6 ppm) where  the  nonlinearity  occurs, cell death is not an im-



portant consideration.  We know from the work of Dr. Kevin Morgan at CUT that



nasal mucociliary activity continues even after many days of exposure to 6 ppm



of CH20.







3.6 Sensitivity of the Study Conclusions to Statistical Analysts







    This section deals only with  our  statistical analyses of  labeling, j..£.,



the   C-specific activity and the  normalized   H/  C isotope ratios in the IF



and AQ DNA.  The two-way  analyses  of variance of these measurements reported



in Casanova-Schmitz et aJK (1984) did  not  account for pairing between the IF



and AQ DNA determinations or possible  inhomogeneity of variance as a function



of the exposure concentration.  Furthermore,  we did not specify ordered  (over



concentration) alternatives to  the  null  hypothesis  of no treatment effects



prior to our examination of the  data.    Consequently, as noted by the Panel,



these analyses had somewhat less than  optimal power to detect systematic dif-



ferences  in treatment effects.   Despite this  limitation, statistically signi-



ficant effects of exposure concentration  and DNA fraction  (including  interac-
                                     -17-

-------
tion between these two factors) were  detected in both the   C specific  activ-


                       3  14
ity and the normalized  H/  C  isotope  ratios.     As has been verified  subse-



quently, more sensitive statistical  analysis  procedures  do no more than con-



firm these findings.  Thus, as was noted by the Panel, our overall  conclusions



regarding these measurements are valid.
    In the second and third paragraphs  of  this section,  the   C specific ac-



tivity measurements are described by  the  Panel  as 'bound (  C) CH-O*.   This



confusing terminology can  easily  give  readers  the mistaken impression that



covalent binding to DNA, rather  than  total    C specific activity in the two



DNA fractions, is being  discussed.    We  therefore strongly suggest changing



this phrase to '  C specific activity" or 'total   C specific activity".
    The absence of  a  statistically  significant  difference  between the   C



specific activities of the IF and AQ  DNA  fractions  at 0.3 and 2 ppm is des-



cribed by the Panel as "limiting  the  extent  to which inferences can be made



about the responses  to  low  concentrations  for  purposes of identifying no-



response levels or making  low-dose  extrapolations  for risk assessment."  We



disagree with this statement.  Such  extrapolations should not be based solely



on the difference between the   C  specific  activities of IF and AQ ONA, when



it is the difference between the  3H/14C ratios (not the 14C specific activity



difference) that is the-primary indicator  of covalent binding to DNA.  Signi-


                                3  14
ficant differences between the   H/  C  ratios  of  the two DNA fractions were



observed at all concentrations equal to  or greater than 2 ppm.  Consequently,



the concentration of CH-O covalentIy bound  to  DNA was found to differ signi-



ficantly from zero at these concentrations as well.
                                     -18-

-------
3.7 Adequacy of the Measure of Exposure







    No comment.







3.8 Utility of the Study in the Quantitative Risk Assessment of CH00
 _. .                                                               




    The Panel concludes that our results  should  not be used for risk assess-



ment.  Their conclusion  is  based  on  their arguments that: (1) experimental



methodologies have not been  properly  'validated"; (2) intracellular proteins



rather than ONA should be used as  the target; and (3) acute exposures may not



be relevant to chronic exposures.  We disagree with the Panel on all points.







    First, the experimental methodologies  have  been  validated, both by HPLC



analysis and by repetition of the  experiments, as discussed above in Sections



3.1.1 and 3.5.2.  We demonstrated the  lack of variation of the DNA yield with



concentration in Section 3.1.2, and  we  have  determined the magnitude of the



 isotope effect in CH^O oxidation and discussed its implications in Section 3.2



 (the binding curve becomes more  nonlinear  rather  than  less).  We showed in



Section 3.5.2 that the assumption of  a  constant  loss of DNA from the IF DNA



fraction cannot account for low-dose nonlinearity no matter how large the loss



 is assumed to be.  Finally,  we  discussed  the fact that all experiments were



carefully controlled to maintain constant extraction conditions for DNA at all



exposure concentrations in Section 3.2.







    Second, the argument that  intracellular  proteins should be used for dosi-



metry fails to recognize:  (1) that  covalent binding to proteins is not widely
                                     -19-

-------
acepted as a mechanism of  mutagenesis,   (2)   that proteins have a  much higher



rate of turnover than DMA, and that individual  proteins vary in their turnover



rate, (3) that correcting for labeling  of the proteins due to metabolism pre-



sents major experimental problems, and (4)  that,  in any case, protein binding



occurs primarily on the extracellular  proteins,  not on the intracellular pro-



teins.  These points are discussed in detail  in*Section 3.4.







    Third, the rationale for using acute  exposure data for risk assessment at



low concentrations is presented in section  3.2.   These exposure conditions do



not cause massive toxicity to the  tissue, and, therefore,  more closely repre-



sent the actual human chronic  exposure  situation.  The results obtained pro-



vide information about the ability of  CH-O to react with DMA under realistic,



low-1 eveI exposure conditions.







    In summary, we find the  concerns  of  the Panel  to be without merit and



their conclusions to be unsubstantiated.
                                     -20-

-------
REFERENCES

Beland, F. A., Fuller-ton, N. F.,  and  Heflich, R. H. (1984). Rapid isolation,
hydrolysis, and chromatography  of  formaldehyde-modified  ONA. J. Chromatogr.
308, 121-131.
                                               *•
Blobel, G., and Potter, V. R.  (1966). Nuclei from rat liver: isolation method
that combines purity with high yield. Science 154, 1662-1665.


Casanova-Schmitz, M., and Heck, H.  d'A. (1985). DNA-protein cross-linking in-
duced by formaldehyde (FA)   in  the  rat  respiratory mucosa: dependence on FA
concentration in normal rats and  in  rats  depleted of glutathione (GSH). The
Toxicolegist S. 128 (Abst. |509).

Casanova-Schmitz, M., Starr, T. B.,  and Heck, H. d'A. (1984).-Differentiation
between metabolic incorporation and covalent binding in the  labeling of macro-
molecules  in the rat nasal mucosa and  bone marrow by inhaled  [  C]- and [n]-
formaldehyde. Tox i eoI. AppI. Pharmacol. 76, 26-44.

Gehrke, C. W., Kuo, K. C., McCune,  R. A., and Gerhardt, K. 0. (1982). Quanti-
tative enzymatic hydrolysis  of  tRNAs. Reversed-phase high-performance  liquid
chromatography of tRNA nucleosides. J. Chromatogr. 230. 297-308.

Kornberg,  A.  (1980). DMA Rep Iication. Freeman, San Francisco, pp. 644-646.


Melander,  L., and Saunders,  W. H., Jr.  (1980). Reaction Rates of  Isotopic Mol-
ecules. Wiley, New .York, p.  99.

                                     -21-

-------
Shapiro, H. S. (1968). Distribution  of  purines and pyrimidines in deoxyribo-
noclcic acids, in Handbook of Bioch«mS.f.rr ffi  A. Sober, ed.), Chemical Rubber
Co., Cleveland, Ohio, pp. H:30-H:51.
                                   -22-

-------
APPENDIX 2:  INDIVIDUAL SUMMARIES OF EPIDEMIOLOGIC
             STUDIES REVIEWED

-------
Matanoski (1982) of Johns Hopkins University examined



mortality patterns of male pathologists in 2



professional societies, American Associations of



Pathologists and Bacteriologists (AAPB) and the American



Society for Experimental Pathology (ASEP).  The results



of these analyses were reported in a March 30, 1982



letter to John Martonik, Deputy Director of the



Occupational Safety and Health Administration (OSHA).



In separate analyses of each group, Matanoski compared



the causes of death to the number of expected deaths



using U.S. white males and using psychiatrists as the



referent group.  Additionally, Matanoski combined the



two pathologist groups without overlap and compared



proportions of deaths to those expected proportions



using 1) psychiatrists and 2) internists,



otolaryngologists, and opthalmologists as the referent.



     In the SMR analysis, Matanoski observed the same



pattern of deaths when either referent group was used as



the standard.  The present review has focused on results



from comparisons with psychiatrists.   For ASEP members,




apparent excesses were observed for neoplasms of the



liver (SMR=399, 3 observed),  pancreas (SMR=277,



5 observed), and lymphomas (SMR=272,  3 observed).



Deficits were observed for all deaths, all neoplastic



deaths,  and neoplasms of the lung, kidney, and bladder,



brain,  and lymphopoietic system.  None were



significant.  For AAPB members, Matanoski observed

-------
elevated mortality (not statiscally significant) from



esophageal and small intestinal neoplasms (SMR=156,



2 observed), pancreas (SMR=263, 9 observed), brain



(SMR=296, 5 observed), and lymphoma/multiple myeloma



(SMR=174, 3 observed).  Deficits were seen for all



causes of deaths, all cancer deaths, and neoplasms of



the stomach, large intestine, prostate, and



lymphopoietic system.  These deficits were not



statistically significant.



     Findings from the PMR analysis support the above



observations.  Matanoski, in addition, observed a



statistically significant increase in the proportions of



deaths due to neoplasms of the hypopharynx (PMR = 3060,



p_<_0.005, 2 observed).



Harrington and Shannon (1975) of the London School of



Hygiene and Tropical Medicine conducted a SMR analysis



of 2,079 pathologists who were members of the Royal



College of Pathologists or the Pathological Society of.



Great Britain during the period of 1955 to 1973.  By the



end of 1973, 156 deaths occurred.   The authors reported



a significant excess in mortality from lymphopoietic



system cancers (SMR=200,  8 observed, p<0.05),



particularly from lymphatic and hematopoietic diseases •



not due to Hodgkin's disease or leukemia (SMR=353,



6 observed, p<0.01).  Additionally, increased mortality



from Hodgkin's disease appeared (SMR=167, 1 observed)



for male pathologists in England and Wales.








                     - 2 -

-------
     Harrington and Shannon also presented an analysis



of 12,944 laboratory technicians who were registered



with the Council for Professions Supplementary to



Medicine.  Between August, 1963 and December 31, 1973,



154 deaths occurred.  Deficits of deaths were observed



from all causes and all neoplastic causes, including



neoplasms of the digestive tract and peritoneum, lung,



lymphohematopoietic system, Hodgkin's disease,  and



leukemia.  These observations were not statistically



significant.  The most striking statistically



significant excess in mortality was observed from



suicides (SMR=243, 17 observed, p<0.001).  In addition,



Harrington and Shannon observed some excess in



lymphohematopoietic deaths (not including leukemia or



Hodgkin's disease) (SMR=118,  2 observed).  This



observation was not statistically significant.



Harrington and Oakes (1982) more recently followed the



Royal College of Pathologists'  cohort from 1974 to 1989



and performed an SMR analysis of 2,720 members (2,307



males and 413 females) in which 126 total deaths (110



males,  16 females) occurred.   Harrington and Oakes



observed increased mortality in males from cancers of



the brain (SMR=331, 4 observed, p<0.05) and bladder



(SMR=107, 2 observed), from accidents (SMR=170,



13 observed p<0.05),  and from suicides (SMR=353,



7 observed p<0.01).  Increased mortality from lymphatic



and hematopoietic neoplasms was not reported for male








                     - 3 -

-------
     pathologists (2 observed deaths) but was reported for



     female pathologists (SMR=370, 1 observed death).



     Deficits in mortality (statistically significant,



     p<0.05) were observed for males from all neoplasms



     (SMR=61, 32 observed) and from neoplasms of the lung



     (SMR=41), 9 observed) and digestive and peritoneal



     system  (SMR=51, 8 observed).  All the malignant brain



     tumors diagnosed in males were of the astrocytoma/glioma



     cell type.



4.   Levine et al.   (1984) of CUT in an SMR analysis found



     excess mortality among Ontario morticians, relative to



     Ontario white males, from lymphopoietic cancer  (SMR=124,



     8 observed), particularly, leukemias/aleukemias



     (SMR=160, 4 observed), and brain cancers (SMR=115, 3



     observed).  None of these malignancies was significantly



     elevated.  Only cirrhosis of the liver and rheumatic



     heart disease  (SMR=199,  8 observed, p<0.05) showed



     significant excesses (SMR=171, 18 observed, p<0.001).



     Decreases in mortality (not statistically significant)



     were observed for neoplasms of the lung, digestive



     system, and buccal cavity.



          In an earlier analysis of these deaths where U.S.



     white males were used as the standard population, Levine



     observed increasing SMR's with increasing time since



     first exposure for cancers of the brain, lymphopoietic



     system, and leukemia/aleukemia.
                          - 4 -

-------
          Levine et al.  did not report exposure levels to



     which Ontario undertakers may be exposed, but data from



     a survey of seven West Virginia funeral homes were



     presented.   Mean time-weighted averages for breathing



     zone samplers showed HCHO levels between 0.3 ppm and



     0.9 ppm (Williams et al., 1984).  It is not known how



     similiar the exposures are between these 2 groups.



5.    Stroup (1984) noted excesses,  when compared to U.S.



    .white males, in mortality due to brain cancers (SMR=271,



     10 observed, p<0.01) and leukemias (SMR=148, 10



     observed)  in her unpublished SMR study of 2,239



     anatomists  who were members of the American Association



     of Anatomists (AAA).  Stroup noted excesses of the cell



     types astrocytoma/glioblastoma (all ten brain tumors)



     and myeloid leukemia (5 of the 10 leukemia deaths).



     Deficits in mortality were observed for neoplasms of the



     lung (SMR=28, 12 observed, p<0.05), buccal cavity and



     pharynx (SMR - 15,  1 observed, p<0.05) and of the nasal



     cavity or sinuses (0 observed, 0.14 expected).



          Stroup further examined the relationship between



     exposure and mortality from brain cancer and leukemia.



     The number  of years of membership in the AAA was used as



     a surrogate for exposure.  In these analyses, only the



     SMR's for brain cancer increased as the membership years



     increased.   To further examine the relationship between



     mortality from the  above three neoplasms and exposure to



     formaldehyde, Stroup categorized the anatomists'
                          - 5 -

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     subspecialities by their potential usage of HCHO.  Gross

     anatomists were classified as having high HCHO exposure,

     anatomists who had specialities in both gross anatomy

     and microanatomy had medium exposure, and

     microanatomists had low HCHO exposure.  No trend with

     formaldehyde rank was observed.  This analysis,  however,

     may be limited by the small numbers of deaths (less than

     5 in each category) and may be biased due to exposure

     misclassification.

          To examine if social class differences or if

     ascertainment may have biased the observed excess brain

     cancer and leukemia mortality, Stroup used psychiatrists

     as a comparison group.  In this comparison, the excess

     brain cancer and leukemia mortality remained (brain

     cancer,  SMR=572, p<0.01; leukemia, SMR=212,

     p<0.05).  It can be concluded from these analyses that

     neither social class differences nor ascertainment bias

     accounts for the observed increased in brain cancer and

     leukemia mortality.

6.   Wong (1983) observed that among 2,026 workers employed

     by Celanese in a HCHO manufacturing plant,  as compared

     to U.S.  males, mortality was increased (not

     statistically significant) from neoplasms of the skin

     (SMR=109, 95% CI*:2-717, 1 observed), bone (SMR=430,  95%

     CI:6-2751, 1 observed),  prostate (SMR=305,  95% CI:84-

     797, 4 observed), bladder (SMR=122, 95% CI:2-705,

                                                            *
     * 95% Confidence Interval (95% CI)


                          - 6 -  •

-------
1 observed), kidney (SMR=102, 95% CI:1-613, 1 observed),



brain (SMR=186, 95% CI:43-623, 3 observed), and



lymphopoietic system (SMR=136, 95% CI:43-623, 3 ob-




served), and lymphopoietic system (SMR=136, 95% CI:57-



338, 6 observed), including Hodgkin's disease (SMR=240,



95% CI:33-1063, 2 observed) and leukemia/aleukemia



(SMR=118, 95% CI:15-487, 2 observed).  Lung cancer



mortality was decreased (SMR=82, 95% CI:37-156, 9 ob-



served) and no nasal cavity or sinus neoplastic deaths



were observed.  Accounting for a latency of 20 years,



Wong observed significantly increased mortality from



cancer of the prostate  (SMR=431, 4 observed, p<0.05) and



apparently increased mortality from lymphopoietic system



(SMRS=231, 95% 01:62-591,  4 observed), including



Hodgkin's disease (SMR=582, 95% CI:8-3236, 1 observed).



Again, lung cancer mortality was decreased (SMR=87, 95%



CI:32-190, 6 observed).  Besides HCHO, this cohort had



potential exposures to other oxygenated hydrocarbons,



benzene, asbestos, and inorganic and organic pigments.



Exposure to benzene is particularly important since the



literature reports a causal association between leukemia



and benzene exposures (Heath, 1982).



Tabershaw Associates (1982) studied the same cohort as



Wong, with 58 men added who had incorrectly been



excluded and with the HCHO-exposed workers identified.



An SMR analysis of the exposed and unexposed cohorts and



a case-control analysis using randomly-selected controls








                     - 7 -

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among the non-cancer cases were conducted.  In the SMR



analysis of 867 HCHO-exposed workers, increased



mortality (not statistically significant) from prostatic



(SMR=364, 2 observed), brain/CNS (SMR=135, 1 observed)



cancers and from all accidents (SMR=103, 11 observed)



was reported.  Interestingly, Tabershaw Associates base



the brain/CNS conclusion on one observed death, yet the



text indicates that 2 men who had 6.7 years and 24 years



of exposure died of this cause.  Decreased mortality



from lung cancer (SMR=54, 3 observed) was observed along



with no deaths from neoplasms of the digestive organs



and peritoneum, nasal cavity and sinuses, and bladder.



     In the case-control analysis, increased odds ratios



for cancers of the prostate (OR=2.67) and lymphopoietic



system (OR=3.0), and for all neoplasms  (OR=1.2) were



reported for cases with 5 to 15 years of HCHO



exposure.  These increases were not statistically



significant.  Risks did not appear to increase with



increasing years of exposure or increasing number of



years employed at the plant.  Note that Tabershaw



Associates did not use an unexposed group as a



comparison, but compared the exposed employees to those



with less than 5 years of exposure.  The use of this



group as the "controls" may diminish the ability of this



analyses to detect a small elevation in risk due to a



higher background prevalance of formaldehyde exposure.
                     - 8 -

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8.    Acheson (1984a) of MRC Environmental Epidemiology Unit,



     Southampton General Hospital,  in an ongoing study of



     7,716 workers in six plants which use or manufacture



     HCHO,  has observed significant decreases in overall



     mortality (SMR=87, 1,619 observed,  p<0.05) and



     nonsignificant increases from buccal cavity and



     pharyngeal (SMR=121, 5 observed), esophageal (SMR=103,



     13 observed), respiratory (SMR=102, 236 observed),  and



     lung (SMR=105, 205 observed) cancers.



          Acheson et al. subjectively categorized exposure on



     the basis of workers'  recall of acute irritation.  These



     categories were defined as:  nil/background, <0.1 ppm;



     low, 0.1-0.5 ppm;  moderate, 0.6-2.0 ppm; high, >2.0 ppm.



     In analyses based  on these categories,  Acheson et al.



     found a significant excess of bone cancer mortality and



     a significant dose-response relationship for .lung cancer



     mortality in one plant (BIP),  the cohort with the



     highest exposure.   In a comparison with local controls,



     the dose-response  relationship (marginally significant)



     was still observed.  Acheson lacked smoking histories



     for the entire cohort,  and the BIP plant is located in



     the West Midlands  area,  an industrially polluted area



     with high referent lung cancer rates.  The use of a



     local  comparison may have overestimated the number  of



     expected lung cancer deaths (Enterline, 1976)  since the



     local  lung cancer  rates may be influenced by the BIP



     lung cancer deaths; reducing the power of the analysis.
                          - 9 -

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          In subsequent analysis of the mortality data for



     lung cancer among individuals employed at this plant,



     Acheson et al.  (1984b) observed that the risks for lung



     cancer did not increase with duration of employment or



     with cumulative doses (as assessed by three measures).



          In a third analysis of all 120 lung cancer deaths



     and 640 controls in the BIP plant, Acheson et al.



     (1984c) examined smoking history and previous



     employment.  Acheson et. al. found no differences between



     the cases and their controls.  Only 11% of the cases and



     12% of the controls had adequate information on smoking,



     however.



9.   Marsh (1983a) of the University of Pittsburgh conducted



     an SMR analysis and a case-control study nested within



     the cohort study of Monsanto chemical workers.  This



     plant produced plastics and workers had potential



     exposures to HCHO,  vinyl chloride, styrene, and



     cellulose acetate.   Marsh compared the mortality



     experience of all workers to the white male populations



     of the U.S., of Massachusetts,  and of Hampden County,



     the county from which the workforce was drawn.  In the



     SMR study, the cohort consisted of 2,490 male workers



     with a minimum of one year employment.  Among the 2,490



     workers, 591 deaths were identified by the company or by



     death certificate searches.  Marsh reported increased



     mortality (not statistically significant) due to all



     neoplasms (SMR=107, 127 observed).  Among all neoplasms,








                          -  10  -

-------
     excess mortality was observed from cancer of the buccal



     cavity and pharynx (SMR=155,  6 observed),  digestive



     organs and peritoneum (SMR=126,  44 observed), prostate



     (SMR=178, 14 observed),  bladder (SMR=135,  5 observed),



     genitourinary tract (SMR=169, 26 observed, p<0.05),



     Hodgkin's disease (SMR=118,  2 observed), and all other



     lymphopoietic tissue (SMR=153, 4 observed).  No



     relationship was observed between genitourinary system



     neoplasms and length of employment.



          In the matched case-control study based on the



     cancer deaths,  Marsh presented odds ratios for digestive



     system, rectal,  genitourinary, and prostatic cancers and



     21 occupational  exposure categories.  Two of the



     21 categories had pertinent exposure to HCHO either as a



     chemical (resin  production)  or in a product (resins



     processing).  Marsh observed increased odds ratios for



     digestive system cancer in the resins processing



     category (OR=1.83) and for rectal cancer in both



     categories (resins production, OR=3.75; resins



     processing, OR=2.00).   These increases were not



     statistically significant.  All cases in the



     occupational categories  had from 1 month to 5 years



     exposure and increasing risk was not observed with



     increasing duration of exposure.



10.   Bertazzi et al.  (1984) of the Institute of Occupational



     Health, University of Milan,  presented at the 3rd



     International Conference on Epidemiology and







                         - 11  -

-------
Occupational Health findings of a cohort study of HCHO



resin manufacturing workers.  The mortality experience



of 1,332 male employees who had worked six (6) months or



more between 1959 and 1980 was compared to the expected



number of deaths using national and local rates.



Bertazzi et al. noted that ambient monitoring showed



many work areas were above the Threshold Limit Value.



Area sampling values between 1974 and 1979 ranged



between 0.2 and 3.8 mg/m^.



     For the entire cohort, Bertazzi et al. observed



significantly increased mortality for all neoplasms



(SMR = 154, 42 observed, p<0.05 national rates) and for



lung cancer when both national (SMR = 236, p<0.05 18



observed) and local (SMR = 186, p < 0.05) rates were



used as the referent.  Mortality from digestive (SMR =



156, 14 observed, national rates) and lymphopoietic (SMR



= 201, 5 observed, national rates) and esophageal-



stomach (SMR = 148, 70 observed,  national rates)



neoplasms were apparently elevated.



     Bertazzi et al. had work histories for all but



18 percent of the cohort.  Using local rates as the



standard, Bertazzi et al. examined mortality among



formaldehyde-exposed workers.  In this analysis,



mortality appeared elevated from all causes (SMR = 111,



51 observed), and all neoplasms (SMR = 128, 19



observed), particularly alimentary tract (SMR = 155,



8 observed), lung (SMR = 136, 5 observed) and








                     -  12  -

-------
     hematologic (SMR = 273,  3 observed) neoplasms.  In



     analyses examining cause-specific death by duration of



     exposure, only the SMR's for lung cancer increased as



     the number of years employed increased.  Statistical



     testing was not performed to see if this trend was



     significant.



11.   Blair et al.  (1986) of the National Cancer Institute and



     the Formaldehyde Institute conducted a SMR study of



     26,561 workers in 10 plants which manufacture or use



     formaldehyde;  7 plants produced resins, 2 plants



     photographic  films, and one plant produced plywood.  Two



     of the 10 plants were included in the studies of Wong



     (1983), Tabershaw Associates (1982), Marsh (1983a and



     1983b), and Liebling et al. (1984).  The Blair et al.



     study cohort  was the largest ever studied for



     formaldehyde  exposure.  Any worker who had ever been



     employed in any one of the 10 plants was included in the



     cohort.  Over 80 percent of the cohort were white males.



          Blair et al. examined the relationship between



     mortality and exposure as categorized 3 ways:



     1) time-weighted average (TWA), 2) peak exposure, and



     3) cumulative exposure in ppm-years.



          In the analysis of TWA exposure, Blair et al.



     examined the  mortality experiences of white males with



     TWA exposures of >0.1 ppm (exposed) and compared their



     experience to the mortality experience of white males



     with TWA exposure of jc_0.1 ppm (nonexposed) .  Elevations








                          - 13  -

-------
in the SMR over 100 for the exposed appeared present for



cancer of the prostate (SMR=115, 33 observed), liver



(SMR=102, 11 observed), lung  (SMR=111, 201 observed),



bone  (SMR=123, 40 observed) and kidney (SMR=123,



18 observed) and for Hodgkin's disease"(SMR=142, 14



observed, while the SMR's of the nonexposed were less



than  100.  None of the elevated SMR's was statistically



significant.  In the analysis of peak exposure, only



Hodgkin's disease among white males showed a significant



increasing trend with intensity of exposure.  This trend



also  appeared present, but was not statistically



significant, when cumulative exposure was examined.



      In analyses examining cumulative exposure, Blair et



al.  observed among white males a significant elevation



of neoplasms of the lung (SMR=122, 212 observed,



p_<_0.05) and nasopharynx (SMR=300, 6 observed, p_^0.05)



whose cumulative exposure to formaldehyde was greater



than  0 ppm-years.  A trend with increasing exposure was



not reported for either site.  When latency, defined as



>20 years, was accounted for, the lung cancer excess



increased and remained significant (SMR=135, 146 ob-



served, p<0.05), but the nasopharyngeal excess became



marginally significant (SMR=300, 3 observed, p = 0.08).



Again, no trend with increasing exposure was observed



for either site.



      Exposure characterization for early exposures may



be subject to recall and misclassification bias,







                     - 14  -

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although an elaborate exposure matrix was developed  for



this study.  Historic exposures were estimated from



sensory perception, previous monitoring, and current



levels with knowledge of plant process changes.  Current



levels were determined using three methods, NIOSH P&CAM



125 area monitoring, and DuPont and 3M passive



dosimeters.  The use of passive monitors for low level,



short term exposures may not be valid and their use,



even weighted, may not be appropriate.  Second,



formaldehyde exposure levels for several jobs were below



the analytical method's level of detection, with the  job



identified as having an exposure of 0.0 ppm.  Thus,



weighting the exposures associated with 0.0 ppm (i.e.,



below the analytical method's level of detection) may be



inappropriate.  Third, industrial hygiene data show wide



variation in formaldehyde levels for a single given



job.  Fourth, sensory perception to formaldehyde may be



influenced by recall.  All of these factors compound to



reduce the certainty of the exposure categorization.



     Blair et al. argue that this study provides little



support that formaldehyde exposure is associated with



cancer.  The authors based this conclusion on the lack



of a dose-response relationship between exposure and



lung and nasopharyngeal neoplastic mortality.  Apparent



lack of an exposure-dose trend cannot diminish the



importance of the 30% excess in lung cancer mortality



and the 200% excess in nasopharyngeal mortality, for







                     -  15  -

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these are statistically significant increases.  Smoking



may not account for the observed excesses in lung cancer



mortality; when the SMR's across exposure groups for



lung cancer are compared with another smoking-related



endpoint, emphysema, the SMR's for emphysema decrease



with increasing exposure whereas the SMR's for lung



cancer remain elevated.



     There may be several reasons for the observed lack



of a dose-response trend in this study.  Most



importantly, misclassification and recall bias may be



present.  Second, even though workers who began



employment in the 1930's and 1940's are included in this



cohort, 44% of the cohort entered the study between 1956



and 1965.  Since vital status was obtained in 1980, a



full latency period for these workers may not have been



obtained and may bias the results towards the null



hypothesis of no effect.



     Subsequent to the release of this study, OTS/EPA



has received notice that 4 of the 6 nasopharyngeal



cancer deaths occurred at 1 plant which manufactured



resins and molded compounds.  There is a. significantly



elevated SMR for this plant (SMR=920, 4 observed,



p<0.01).  All 4 workers died 15 or more years since



first exposure and all 4 deaths had worked in the early



part of their employment in the same position.  The only



other pertinent exposure besides formaldehyde was to



cellulose pulp dust (personal conversation with Dr. Jim.








                     -  16  -

-------
     Collins,  Manager of Epidemiology,  American Cyanamid



     Company).



          Blair et al.  (1987) performed analyses of the




     nasopharyngeal and oropharyngeal cancer deaths which



     examined particulate exposure.   In these analyses, Blair



     et al.  observed for those workers  with particulate



     exposure an apparent increasing trend between



     nasopharyngeal cancer mortality and cumulative



     formaldehyde exposure, however, this trend was not



     statistically significant at a  p=0.05 level.   No trend



     was seen between nasopharyngeal cancer mortality and



     cumulative formaldehyde for those  workers not exposed to



     particulates nor was there a trend for oropharyngeal



     cancer  by cumulative formaldehyde,  regardless of



     particulate-exposure status.  These analyses are limited



     by the  small number of deaths in the subcategories.  In



     addition,  it is possible that the  nasopharyngeal cancer-



     formaldehyde dose-gradient, is  a surrogate for an



     unmeasured particulate gradient.  Blair et al. (1987)



     believe,  however,  that formaldehyde and particulates



     together appear to be a risk factor for nasopharyngeal




     cancer.  Blair et  al. (1987) postulated that the



     delivered formaldehyde dose for these workers may be



     higher  than was estimated in Blair et al. (1986)  due to




     formaldehyde attachment to the  particulate matter.



12.   Stayner et al. (1986) of the National Institute of



     Occupational Safety and Health  (NIOSH) conducted a








                          -  17 -

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cohort mortality study of 11,030 workers  in  3 garment



facilities that used formaldehyde resins  in  the



production of permanent press garments.   Two of  the



three facilities were included in the proportionate



mortality analyses of Stayner et al. (1985).  In this



analysis, which is described in #17, only the



proportions of deaths in 3 plants were analyzed.   In the



present cohort study, 1 plant in the PMR was replaced



with another larger plant, in terms of the number  of



employees, from another company.



     Garment workers included in the study cohort  must



have worked for at least 3 months between the time when



formaldehyde fabrics were first introduced into  the



production process and December 31, 1977.  The cohort



was composed mainly of workers who were white women,



were from plant 1,  had been employed from 3  months to



4 years, and had first exposures before 1963.



     Free formaldehyde was extensively measured by NIOSH



investigators between 1981 and 1984 using both area



samplers and personal monitors.  The time-weighted-



average HCHO level for these years was 0.15  ppm  (a range



of 0.14-0.17).  Stayner et al. sampled for potential



confounding exposures such as phenol, organic solvents,



and dust.  These industrial hygiene surveys  did not



identify any chemical exposures which could  result in



substantial confounding.  Likewise, nuisance dust  levels



were minimal.








                     -  18  -

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     A total of 609 deaths (822 expected; SMR=74) were



observed among 188,025 person-years.  No significant



deficits in site-specific deaths were noted by the



authors.  Stayner et al. observed statistically elevated



excesses in deaths due to cancer of the- buccal cavity



(4 observed, SMR=343, p<0.05), tonsils (2 observed,



SMR=694, p<0.05), and connective tissue (4 observed,



SMR=364, p<0.05).  The statistical test employed by



Stayner et al. is one-sided since the investigators had



a_ priori planned to examine the relationship between



formaldehyde exposure and elevations in cancer mortality



(not whether formaldehyde exposure was related to change



in cancer mortality, use of a two-sided test



statistic).  All 4 of the buccal cavity deaths were



among females whose first exposure was between 1955 and



1962.  Two of the 4 buccal cavity cancer deaths were



parotid tumors, and the other 2 deaths were cancers of



the oral mucosa and soft palate.



     There appeared to be an increase in mortality from



neoplasms of the lung, trachea, and bronchus



(39 observed, SMR=114) and other lymphopoietic sites



(5 observed, SMR=170), from leukemia (9 observed,



SMR=114), and from bronchitis (SMR=190, n=4).  Mortality



from neoplasms of the brain appeared decreased (5 ob-



served, SMR=71).  No deaths from nasal cancer were



observed in this cohort.
                     -  19  -

-------
     Stayner et al. analyzed the data by plant, by



length of latency, and by duration of exposure.  In



these analyses, Stayner et al. observed elevated



mortality from cancers of the trachea, bronchus, and



lung (29 observed, SMR=149, p<0.05) and connective



tissue (3 observed, SMR=514, p<0.05) in plant 1 only.



Stayner et al. noted that the remaining 2 plants each



lacked sufficient power to detect small to moderate



elevations in lung cancer risks.  In the analyses which



examined duration of exposure and latency period,



Stayner et al. observed the highest excesses in



mortality from cancers of the buccal cavity, connective



tissue, and other lymphopoietic tissue among those



workers with the longest duration of exposure  (10+



years)  and with the greatest latency period  (20+



years).  In these analyses, the risk of lung cancer



appeared to decrease with increasing duration of



exposure.  An EPA analysis of the data in the paper



shows there is a statistically significant increasing



trend (p < 0.05) between buccal cavity cancer mortality



and duration of exposure, although such an analysis had



not been performed by Stayner et al.



     Stayner et al. concluded that the excesses in



mortality from buccal cavity neoplasms, leukemia, and



other lymphopoietic neoplasms were consistant with the



hypothesis of being formaldehyde related.  It is not



known how the excess in connective tissue cancer








                     -  20  -

-------
     mortality may relate to exposure.  It must be recognized



     that these findings are based on a small number of cases



     and that confounding with other exposures may exist.



     The investigators believe,  however,  indirect evidence



     suggests that cigarette and alcohol consumption were not



     confounders for the observed excess in buccal cavity



     cancer mortality.



13.   Walrath and Fraumeni (1983) of NCI conducted a PMR study



     of 1,132 funeral directors  or embalmers licensed in New



     York.  In this cohort,  Walrath and Fraumeni observed



     significantly elevated mortality from skin (PMR=221,



     8 observed, p<0.05) and colon (PMR=143, 29 observed,



     p<0.05) neoplasms.  Elevations also appeared present for



     cancer of the buccal cavity and pharynx (PMR=113,



     8 observed), digestive system (PMR=104, 68 observed),



     liver (PMR=106, 5 observed), pancreas (SMR=105, 13 ob-



     served), lung (PMR=108, 72  observed), brain/CNS



     (PMR=156, 9 observed),  kidney (PMR=150, 8 observed), and



     lymphatic/hematopoietic system (PMR=121, 25 observed).



     No nasal cavity and sinus neoplasms were observed.



     Among those licensed as embalmers only, Walrath and



     Fraumeni observed increases in mortality from buccal



     cavity and pharyngeal (PMR=201,  7 observed),  skin



     (PMR=326, 5 observed, p<0.05), and brain/CNS (PMR=234,



     6 observed, p<0.05) cancers.  In the analysis for



     latency, Walrath and Fraumeni observed, for the entire
                         - 21 -

-------
     cohort, increasing PMR's for skin (p<0.05) and brain/CNS



     neoplasms with increasing time since first licensed.



          Walrath and Fraumeni did not report actual exposure



     data for these embalmers, but data from previous



     industrial hygiene surveys were presented.  In a NIOSH



     survey of a mortuary science college, formaldehyde



     levels ranged between 0.2 and 0.9 ppm.  Another survey



     of 6 funeral homes reported formaldehyde levels ranging



     from 0.1 to 5.3 ppm.



14.   Walrath and Fraumemi (1984) conducted another PMR



     analysis of 1,050 embalmers in California and observed



     similar findings as from the analyses of N.Y.



     embalmers.  Walrath and Fraumeni reported significantly



     increased mortality from neoplasms of the brain



     (PMR=193,  9 observed,  p<0.05), leukemia (PMR=175, 12




     observed,  p<0.05), and prostate (PMR=176, 23 observed,



     p<0.05).  Increases that were not statistically



     significant were also reported for lymphatic and



     hematopoietic system (PMR=123, 19 observed) and buccal



     cavity and pharyngeal (PMR=131, 8' observed) cancers.



     Mortality from lung neoplasms was slightly decreased



     (SMR=96, 41 observed).  As in the previous Walrath and



     Fraumeni study, no neoplasms of the nasal cavity and



     sinuses were reported.



          Walrath and Fraumeni did not report actual exposure



     data for these embalmers, but the above-mentioned



     industrial hygiene data were included.








                         - 22  -

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15.   Marsh (1983b) of the University of Pittsburgh conducted



     a PMR analysis of HCHO-exposed workers at the Monsanto



     plant described previously.  Marsh found 136 deaths



     among male workers with exposure of 1 month or greater



     in a "formaldehyde related plant area".  Marsh compared



     their mortality experience to U.S. male, age-race



     adjusted,  proportionality mortality data.



          In the HCHO-exposed white males (115 deaths),  Marsh



     observed increased (not statistically significant)



     mortality  from cancers of the genitourinary system



     (PMR = 121, 3 observed), including the bladder (PMR =



     330, 2 observed) and of the digestive organs and



     peritoneum (PMR = 127, 8 observed), particularly the



     pancreas (PMR = 160,  2 observed).   Marsh observed



     decreased  mortality from all neoplasms (PMR = 90,



     20 observed), particularly the respiratory system



     (PMR = 80,  2 observed).



          Only  21 deaths occurred among non-white



     formaldehyde-exposed males, and 2  of these deaths were



     from neoplasms (1 respiratory, 1 genitourinary).   Marsh



     does not report PMR's for those sites where less than 2



     deaths occurred.



          In this study, Marsh additionally examined cause-



     specific mortality for those workers not exposed to



     formaldehyde.  The most striking observations were of



     significant excesses in deaths from genitourinary tract



     neoplasms  (white males), (PMR = 192.3, 22 observed,








                          -  23  -

-------
     p<0.05) and from all neoplasms (non-white males),
     (PMR = 251, 5 observed, p<0.01),  particularly other
     malignant neoplasms (PMR = 882, 3 observed, p<0.01).
     Marsh does not present exposure information for the
     formaldehyde-exposed workers, but white male neoplastic,
     respiratory cancer, and genitourinary cancer deaths were
     analyzed by duration of employment (since being exposed
     to formaldehyde).  Only the PMR's for respiratory cancer
     increased with increasing years of employment.
          Since Marsh published this study, Infante of OSHA
     has found 1 cancer of the nasal sinus and 1 naso-
     pharyngeal cancer.  Both men died 3 years after Marsh's
     follow-up period.  The worker who died of cancer of the
     nasopharynx was a member of Marsh's cohort, but had been
     counted as living since he had not died at that time.
16.   An overlapping study was conducted by Liebling et al.
     (1984).  Liebling et al. identified 24 male workers who
     died between January 1, 1976 and December 31, 1980
     through union records, reports of former co-workers, and
     a systematic review of obituaries in local newspapers.
     Work histories were obtained from seniority lists.
          Proportionate mortality ratios were calculated to
     examine cause-specific mortality using the age, sex,
     race and cause-specific mortality proportions of the
     U.S. and county in which the plant is located.  To
     adjust for the healthy worker effect, age, sex, and
     race-standardized PCMR's based on county comparisons

                         -  24  -

-------
     were also calculated.  Deaths among 18 white and 6 black



     males with known HCHO exposure were identified.  Race-



     age-sex adjusted PMR's were significantly elevated for



     cancer of the colon based on U.S., county, and county



     cancer mortality proportions (PMR = 702, 424, 333,



     p_<^0.05), as were PMR's for buccal and pharyngeal cancer



     (PMR = 870, 952, 833, p^O.05).  Liebling et al. stated



     that the occurrence of a significant increase in



     proportionate mortality from buccal and pharyngeal



     cancer in this investigation is in accord with the type



     of cancer found in HCHO-exposed rodents.  Furthermore,



     the authors postulated that besides nasopharyngeal



     cancer, an association between HCHO exposure and cancer



     of the buccal cavity and pharynx in humans is



     biologically feasible since humans breathe through both



     the nose and mouth, while rats and mice are obligatory



     nose-breathers.  Like many other studies, this study is



     limited by the inability to completely separate HCHO



     exposure from exposure to other chemicals.



17.   Stayner et al. (1985) of NIOSH conducted a PMR study of



     256 deaths among garment workers in 3 plants.  Two of



     these plants were included in the cohort study



     identified in f!2 (Stayner, 1986).  Stayner et al.



     identified the 256 deaths from a death benefit fund.  In



     this cohort Stayner et al. observed significantly



     elevated mortality from buccal cavity (PMR=750,



     3 observed, p<0.001), biliary passages and liver








                          -  25  -

-------
     (PMR=313, 4 observed, p<0.01), and other lymphatic and



     hematopoietic site (PMR=400, 4 observed, p<0.05)



     cancers.  In analyses examining only the cancer deaths,



     buccal cavity (PCMR=682) and other lymphatic and



     hematopoietic site (PCMR=342) cancers remained



     significantly elevated (p<0.01 and p<0.05,



     respectively).   Additionally, those workers with both



     latency and duration of exposure of 10 years or greater



     showed significantly elevated mortality from all



     malignancies (PMR=137, 51 observed, p<0.05), buccal



     cavity (PMR=925, 2 observed, p<0.05), biliary passages



     and liver (PMR=467, 3 observed, p<0.05), and all



     lymphatic/hematopoietic sites (PMR=283, 8 observed,



     p<0.05), particularly other lymphatic and hematopoietic



     (PMR=761, 4 observed, p<0.05) cancers.



          Nonsignificant elevations in mortality were



     reported for liver not specified (PMR=426,  2 observed),



     skin (PMR=179,  2 observed), and all lymphatic and



     hematopoietic sites (PMR=163, 10 observed), including



     leukemia (PMR=400, 4 observed).



18.   Delzell and Grufferman (1983) of Duke University



     examined the mortality experience of 4,462 deaths



     between 1976-1978 of white female textile workers.



     Deaths and occupation as recorded on the death



     certificates were identified from state computer



     files.   In this  study the textile worker occupational



     code included workers in industries that manufactured







                          - 26  -

-------
     textile mill products, apparel, or other fabricated



     textile products.  Delzell and Grufferman observed



     significant excesses in mortality from cancer of the



     larynx (PMR = 280, 5 observed, p<0.05), connective



     tissue (PMR = 260, 10 observed, p<0.05), cervix



     (PMR = 210, 59 observed,  p<0.05), other unspecified



     genital organs (PMR = 270, 16 observed, p<0.05), and all



     lymphopoietic sites (ICDA 200-207) (PMR = 130,



     121 observed, p<0.01),• particularly non-Hodgkin's



     lymphoma (PMR = 170, 51 observed, p<0.05).   Decreases in



     mortality that were not statistically significant were



     observed for neoplasms of the lung (PMR = 90, 106 ob-



     served) and of the brain (PMR = 90, 17 observed).



          This study is limited by the unknown exposure



     status of each death.  The occupational code, textile



     worker, was used as an indirect measure of formaldehyde



     exposure.  This study is unable to identify whether



     formaldehyde had actually been an exposure, and if so,



     in what concentrations.  A second limitation is the



     insensitivity of death certificate occupational code



     analyses.



19.   Fayerweather et al. (1982) of DuPont showed elevated



     odds ratios, after a 15 to 24 year latency, for cancers



     of the prostate (OR=4.8,  8 cases), lymphopoietic system



     (OR=1.91, 6 cases), bone (OR=1.25, 3 cases),  and bladder



     (OR=7.0,  6 cases) among workers eligible for pension



     were exposed to HCHO 5 or more years.   Decreases in







                          -  27  -

-------
     mortality were observed for those employees working 5 or



     more years from colorectal (OR=0.74, 8 cases) and lung



     (OR=0.79, 15 cases) neoplasms.  No difference in



     mortality was observed for buccal cavity neoplasms



     (OR=1.0, 1 case).



          Fayerweather et al. examined formaldehyde exposure



     by 3 ways:  by the number of years worked around



     formaldehyde (less than 5 years, 5 or more years),



     whether the employee had intermittent or continuous



     formaldehyde exposure, and by a cumulative exposure



     index.   In these analyses, only the odds of mortality



     from bladder and from prostatic cancer increased as the



     exposure indices increased.



          Fayerweather et al. did not follow those employees



     ineligible for pension or those who had transferred,



     potentially comprising 15 to 20% of the work group.



20.   Brinton et al. (1984a) of NCI conducted a case-control



     study for cancer of the nasal cavity and sinuses.  They



     observed nonsignificantly increased odds ratios among



     males employed in the leather or shoe, chemical



     manufacturing, and carpentry industries and for



     exposures to chromiurn/chromates, nickel, and



     insecticides/pesticides/herbicides.  Among females,



     increased odds ratios were observed with employment in



     the textile/clothing/hosiery and paper/pulp mill



     industries and for exposures to mineral oils and other



     mineral/chemical gases.  None of the increased odds







                          - 28  -

-------
ratios was significant in the presence of control for



confounding variables.  Brinton et al. additionally



assessed reported occupational HCHO exposure and found



an odds ratio less than 1.0.  This ratio was unstable



based on only one male and one female.



     To examine the relationship between employment in



the textile and apparel industries with the risk of



nasal cancer, Brinton et al.  (1984b) further analyzed



the data from their previously published case-control



study (1984a).  The industries included textile and



cotton mills, apparel manufacturing, and hosiery.



Brinton et al. found an elevated risk of nasal cancers



associated with employment in the textile or apparel



industries, but the increased relative risk was found



only among female workers.  When histologic types of



nasal cancer were evaluated, both males and females were



found to be at increased risk of nasal adenocarcinoma,



with further enhancement of risks with exposure to dusty



work conditions.  Few individuals in this analysis had



exposure to formaldehyde (2 cases) and the ratio was



below 1.0  (OR=0.4).  The authors considered this study



to provide further evidence of an association between



employment in the textile industry and risk of nasal



cancer.  This study had limited ability to evaluate



nasal cavity cancer and a direct assessment of



formaldehyde exposure.
                     - 29 -

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21.  Tola et al.  (1980) of the Institute of Occupational



     Health, Finland, conducted a case-control study for



     cancer of the nose and paranasal sinsuses.  Forty-five



     cases were collected from the Finnish Cancer Registry



     between 1970 and 1973 and were age-sex matched to



     nonrespiratory cancer controls.



          Analyses examining an occupational etiology showed



     no single occupation being more common among the cases



     than among the controls, but leisure time knitting and



     sewing was significantly more common among female cases



     than among female controls (OR=4.8, 19 cases).  Other



     factors significantly associated with the cases were



     histories of serious nasal trauma, chronic rhinitis, and



     sinusitis.  Smoking was not significantly associated



     with nasal cavity and sinus cancer.



          This study is limited in its ability to evaluate



     formaldehyde exposure.  Occupational histories were



     obtained for 69% of the cases from a next-of-kin,



     potentially biasing the study towards the null



     hypothesis of no-association.



22.  Hernberg et al. (1983) of the Institute of Occupational



     Health, Finland, conducted, with participation from



     Denmark and Sweden, a collaborative case-control study



     of nasal and sinonasal cancer and its possible



     occupational etiology.  One hundred seventy cases



     diagnosed between 1977 and 1980 and reported to the



     prospective cancer registries were selected.  Each case








                          -  30  -

-------
     was sex-country-age at diagnosis matched with colorectal

     cancer controls.

          Elevated odds ratios were observed among cabinet-

     makers (OR=9.0) and mechanical engineering shop workers

     (OR=2.13).  Analysis for exposures showed elevated risk

     with hardwood dust (OR=1.7)*, softwood dust (OR=3.4,

     p<0.05)*, hardwood and softwood dust (OR=67, p<0.05)*,

     welding-flame cutting-soldering (OR=2.0, 17:6,

     p<0.05)**, chromium (OR=2.7, 16:6, p<0.05)**, nickel

     (OR=2.4,  12:5)** electroplating (OR=1.5, 9:6)**, and

     paint-lacquer (OR=3.0, 18 cases).   HCHO exposures may

     occur in this last category.  However, wood dust

     exposure is common and confounds the observed elevation.

23.   Hardell et al.  (1982)  of Umea, Sweden conducted a case-

     control study of nasal and pharyngeal cancers.  Seventy-

     one cases, first diagnosed between 1970-1979, and 541

     referents were specifically studied for relationships

     with phenoxy acid or chlordane exposure.  Cases were

     selected from the Swedish Cancer Register and referents

     were utilized from earlier case-control studies of soft

     tissue sarcoma and lymphoma and of colon neoplasms.

     These referents were apparently representative of the

     population between 1970 and 1978.

          Hardell et al. observed increased risks with

     exposure to chlorophenol (OR=6.7,  95% CI:2.8-16.2) and


     *Adjusted for smoking.
     **0dds ratio based on discordant pairs, concordant:
       discordant pairs noted.


                         -  31 -

-------
     phenoxy acids (OR=2.1, 95% CI:0.9-4.7).  The odds ratio



     for chlorophenol remained significantly elevated



     (OR=6.7) when controlled for wood dust exposure.  Of



     interest to this review,  Hardell et al. observed a



     significantly increased odds ratio  (OR=5.80, p<0.05)



     between nasal cancer and work in particleboard



     production.  It is not known whether this observation is



     confounded by wood dust exposure, which can occur in



     particleboard production.



24.   Olsen et al.  (1984) of the Danish Cancer Registry



     conducted a case-control study of nasal cancers.  This



     study examined 839 cancer registry cases (560 males,



     279 females), diagnosed between the years 1970-1982, who



     were matched  with 2,467 controls with cancer of the



     colon, rectum, prostate,  and breast on age-sex-year of



     diagnosis.  The researchers used a nationwide data



     linkage system which has linked cancer cases and



     previous employment.  Occupational histories came from



     the National  Supplementary Pension fund, established in



     1964, and the Central Population Registry.   Use of these



     national data sets eliminated the potential for recall



     bias since cases and controls were not interviewed.  In



     this case-control study,  Olsen et al. tested for



     associations  between HCHO, wood dust, paint-lacquer-



     glue, and metal exposure and sino-nasal cancers.



     Significantly increased risks were found for nasal



     cavity cancer for exposure to HCHO  (OR=2.8, 95%








                          -  32  -

-------
     CI:1.8-4.3), wood dust (OR=2.5, 95% CI:1.8-3.9), and



     paint-lacquer-glue (OR=2.1, 95% CI:1.4-3.0).  Exposure



     to both wood dust and HCHO can occur simultaneously, and



     Olsen et al. performed a stratified analysis which



     controlled for wood dust exposure.  In this analysis,



     the elevated risk with HCHO exposure was reduced to 1.6



     and became nonsignificant.  In this stratified analysis,



     both HCHO and wood dust exposure together resulted in an



     additive risk (OR=4.1, p<0.05, 95% CI:2.3-7.3).



25.   Hayes et al. (1986),  formerly of the Erasmus University



     of Rotterdam, presented findings of a case-control study



     of nose and nasal sinus tumors at the 3rd International



     Conference on Epidemiology and Occupational Health in



     Dublin, Ireland.   In their published study, Hayes et al.



     (1986) identified factors associated with 144 cases of



     nasal and nasal neoplasms diagnosed between 1978 and



     1981.  Living and deceased population controls were used



     as the comparison group.   Hayes et al. observed



     significant associations between male adenocarcinoma



     cases and work in furniture and cabinet making (OR=120,



     90% CI:30.9-613.2) and joinery (OR=16, 90% CI:2.8-



     85.3).  Nonsignificantly elevated risks were reported



     for all cell-types and employment in leather (OR=2.3*),



     metal  (OR=2.1*),  and floriculture (OR=3.7, 90% CI:0.



     9-18.1) industries.  In addition, Hayes et al. noted



     significantly increased risks between nonadenocarcinomas





     *90% confidence intervals not reported.





                         -  33  -

-------
and paint (OR=4.1, 90% CI:1.5-11.2), benzene  (OR=2.3,



90% CI:1.4-3.8), and HCHO (OR=2.2, 90% Girl.2-4.0)



exposure.



     In analyses examining HCHO exposures among male



study subjects with no or low levels of wood dust



exposure, different results were observed using 2 inde-



pendent assessments, A and B, for HCHO exposure.  By



classification A, a significant excess risk (OR=2.5*,



90% CI:1.2 - 5.0, 15 cases) was observed with HCHO



exposure.  The authors stated there appeared to be a



dose response relationship with level of exposure.  By



classification B, the odds ratio was reduced to 1.6 and



was not statistically significant (90% CI:0.9 - 2.8,



24 cases).  The authors attempted analyses of this type



for males subjects with high level of wood dust exposure



using classification B data. In this analysis, the



authors observed an apparent elevation of the odds ratio



(OR=1.9, 90% CI:0.7 - 5.5, 15 cases).



     EPA has analyzed the data from Classification A for



male subjects with no or low levels of wood dust



exposure and with high levels of wood dust exposure.  In



this analysis, the weighted odds ratio for HCHO exposure



was significant elevated (OR=1.9, 95% CIrl.l - 3.8).



     Additionally., the authors derived HCHO risks for



those classified as high wood dust exposed on both



assessments with respect to the wood dust exposure.  A



statistically significant elevation (odds ratio) in








                     -  34  -

-------
     nasal cavity cancer risk (all tumors: 3.4, 90% CI: 1.1-



     10.4; squamous cell carcinoma:3.8,  90% CI: 1.1-13.0) was



     observed.  There was, also, a significant (p<0.05)



     association for trend with time since first exposed for



     all tumor types and for squamous cell carcinomas.



26.   Roush et al. (1985) reported at the 1985 Meetings of the



     Society for Epidemiologic Research results of a case-



     control study of nasal sinus and nasopharyngeal



     cancers.  This study is yet to be published.   At the



     time of the SER presentation, 198 sinonasal and



     173 nasopharyngeal cancer cases had been identified, for



     the past 41 years, from the Connecticut Tumor



     Registry.  Controls (n=608) were sampled from death



     certificates.   The authors state that occupational



     histories were derived from city directories and from



     death certificates.



          Apparent elevations in the odds ratio for combined



     sinonasal and nasopharyngeal cancer (OR=2.2,  95% CI:0.7-



     7.0) were reported for work in the rubber industry and



     printing (OR=1.2, 95% CI:0.4-3.7),  morticians (OR=2.1,



     95% CI:0.7-6.5), and for physicians and dentists



     (OR=1.5, 95% CI:0.5-5.1).  All 4 categories were



     identified as having potential formaldehyde exposure.



     In analyses that examined occupational -formaldehyde



     exposure, 20 or more years prior to death, no



     associations were observed for those cases and controls



     over 68 years of age between either sinonasal cancer or







                          -  35  -

-------
     nasopharyngeal cancer.  For those cases and controls



     less than 68 years of age, apparent elevations in the



     odds ratio were observed for both sinonasal cancer



     (OR=1.4, 95% CI:0.6-3.0) and nasopharyngeal cancer



     (OR=1.7, 95% CI:0.9-3.5).



          Use of city directories and death certificates to



     ascertain occupational histories lacks sensitivity and



     may potentially introduce  bias.



27.   Partenan et al. (1985) conducted a nested case-control



     study of 60 respiratory cancer cases among male



     production workers in the  wood industry and in



     formaldehyde glue manufacturing.  Respiratory cancers



     were defined as mouth (other), tongue, pharynx, nasal



     sinuses, larynx,  and lung  (trachea).  Three referents



     alive at the time of diagnosis of the corresponding case



     were selected as controls.  These referents were matched



     to each case by year of birth.  Smoking histories were



     obtained for both cases and controls by mailed



     questionnaires or by interview.



          Smoking and survival  status were controlled for in



     the analyses since more complete work histories were



     obtained for subjects who  were alive at the time of data



     collection.  In addition,  HCHO exposure was assessed



     through the use of a job exposure matrix.  Odds ratios



     were calculated for 1) cumulative HCHO exposure >3 ppm-



     months 2) cumulative HCHO exposure >_3 ppm-months, _^10



     year latency, 3) peak exposure >2 ppm, 4) peak exposure.







                          -  36  -

-------
     >2 ppm, >_10 year latency,  5) HCHO-wood dust exposure



     >JL month,  6) HCHO-wood dust exposure >_l month, _>.1° years



     latency, and 7) "ever exposed."



          Results of the analyses showed apparent elevations



     in the odds ratio with cumulative HCHO -exposure which



     accounted for a 10 year latency (OR = 1.6, 8 cases) and



     with "ever exposed" (OR = 1.52, 55 cases).  The



     exposure-response relation between HCHO exposure and



     respiratory cancer was analyzed through the



     classification of levels-of-exposure.  In these



     analyses,  only the duration of exposure to HCHO-



     containing wood dust suggested a positive exposure-



     response relationship (1 month - 5 years; OR = 0.78,



     4 cases; >5 years,  OR = 1.82, 6 cases).



          This study is limited by low power and a short



     follow-up period; having an additional effect of



     lowering the power.  Thus, only very large excesses in



     human risks can be ruled out.



28.   Vaughan et al. (1986a,b; and as reported in SAIC,  1986)



     of the Fred Hutchinson Cancer Research Center,



     University of Washington,  conducted a population-based



     case-control study of sinonasal and pharyngeal cancers



     and possible associations with HCHO.  This study was



     composed of 53 sinonasal cases, 27 nasopharyngeal  cases,



     and 205 oro-hypo-pharyngeal cases which were identified



     from a tumor registry.  Controls (n=557) were selected



     from the general population and were matched to cases on







                         -  37  -

-------
sex and age.  The cases were identified between 1979 and



1983, and interviews were conducted in 1983.  Due to a



short survival time between diagnosis and death from




these neoplasms, next-of-kin (NOK) interviews were



obtained for 50% of the cases.  Comparison of cases and



controls showed that the control population appeared to



have a higher educational level than the cases, but this



difference was not statistically significant.



     Vaughan et al. examined personal, occupational, and



environmental HCHO exposures using logistic regression



analyses.  Formaldehyde exposure was directly assessed



by three measures:  1) maximum exposure level, 2) number



of years in a formaldehyde job, and 3) weighted sum of



years in a formaldehyde job.  Formaldehyde exposure was



indirectly assessed by identifying occupational and




domestic environments (mobile home residency,



remodeling, occupational resin-glue-adhesive exposure,



etc.) where formaldehyde had been previously reported.



     Analyses showed that smoking and alcohol were



significantly associated with sinonasal cancer.  When



occupational and environmental exposures were examined,




after control for smoking and alcohol consumption, the



odds ratio for exposure (>10,000 hours) to resins,



glues, and adhesives (OR = 3.8, 4 cases p_<_0.05) was



significantly elevated and the odds ratio with sawdust



exposure appeared elevated (OR =2.4, 8 cases,



p_<_0.10).  The trends with increasing exposure were








                     -  38  -

-------
significant for both exposures.  No association was



observed with the direct assessment of formaldehyde



exposure; the odds ratios were below 1.0.



     In analyses of the nasopharyngeal cases, Vaughan et



al. observed significant associations with smoking and



race.  After controlling for these variables,



significant elevations in the odds ratio were observed



with occupational exposure to stains, varnishes, and



solvents (OR = 4.0, p<0.05) and with ever having lived



in a mobile home (OR = 3.0, 8 cases,  p<0.05), with the



highest odds ratio observed for living 10 or more years



in a mobile home (OR =5.5, 4 cases,  p<0.05).  Trends



for both exposures were statistically significant.  An



association with formaldehyde exposure (as assessed from



the job linkage system) appeared present since the odds



ratios for formaldehyde exposure were above 1.0 and they



increased with increasing exposure, but these



conclusions are based on a total of 11 cases and were



not statistically significant.



     Analyses of the oro-hypopharyngeal cases showed



significant associations with cigarette and' alcohol use,



and an alcohol-sex interaction.  After considering these



variables, significant elevations of the odds ratio were



observed with exposure (>10,000 hours) with resins,



glues, and adhesives (OR = 3.9, p<0.05), stains,



varnishes, and solvents (OR = 3.9, p<0.05), and asbestos



(OR = 4.0, p<0.05).  Formaldehyde exposure (as assessed







                     -  39  -

-------
from the job linkage system) was not significantly



associated with neoplasms of these sites.  Analyses



which relied on self-reporting by eliminating next-of-



kin interviews showed an elevated odds ratio between



formaldehyde exposure, greater than 20 years, and



oropharyngeal cancer (OR = 2.0, 95% C.I.:0.9-4.6).



     Mobile home residency and occupational resin, glue,



and adhesive exposure were among the exposure variables



a_ priori selected as surrogates for formaldehyde



exposure.  Urea-formaldehyde resins have been used in



hardwood plywood for over 35 years (HPMA, 1984) and in



particleboard.  Hardwood plywood used as prefinished



wall panels saw tremendous growth in the 1950's and



1960's, coinciding with the growth of the mobile home



industry (HPMA, 1984).



     Several of the nasopharyngeal cancer cases who were



identified in this study as living in a mobile home



lived in what is generally called a recreational



vehicle.  The interpretation of Vaughan's results



changes little because manufacturing practices for



mobile homes and for recreational vehicles were very



similar; formaldehyde-containing products were used in



both.  By its retrospective nature, case-control study



is limited in its ability to identify if the cases had a



specific exposure and, if so, at what level of exposure.



     A second limitation of this study is the accuracy



of the NOK interviews.  Fifty percent of the cases were







                     -  40  -

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dead at the time of the interview and next-of-kin may



not remember all occupational histories, although one



expects that residential history may be more clearly



remembered.  Inclusion of NOK interviews potentially



biases the results towards the null hypothesis of no



effect.  There is support for the presence of such a



bias from analyses which eliminated NOK interviews.  The



resultant odds ratios in these analyses were larger than



those odds ratios in analyses which included both live



and NOK interviews.  It must be noted that the results



from NOK-eliminated analyses were not statistically



significant; a possible reflection of reduced number of



cases and, hence, reduced power.  Findings which are not



statistically significant, therefore, may be due to the



third limitation of this study, reduced power to detect



very small elevations in risk.
                     - 41 -

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APPENDIX 3:   ESTIMATES  OF RISK USING VARIOUS
              EXTRAPOLATION MODELS

-------
                             Appendix 3






     Eleven models were used to extrapolate risks from the CUT rat



malignant tumor data.  All were dichotomous ("tumor/no tumor" or




quantal) models.  The formulation of each model to accommodate



quantal response data was preferred to one including time as a



variable.  Simulation studies conducted for the EPA indicated that



inclusion of time as a variable would not provide much improvement



in estimation (Howe et al., 1984).  Additionally, the lack of



information about causes of death of experimental animals and the



adjustments made for sacrifice data would have necessitated



assumptions that could have brought the validity of results based



on time into question.




     Table 1 shows the parameter estimates (with standard errors),



log-likelihood,  and X2 goodness-of-fit test statistics (with p-



values) for the eleven models: one-, three-, and five- stage models



and additive and independent background forms of the probit, logit,



Weibull, and gamma-multihit models.



     Tables 2 through 6 give the maximum likelihood estimates (MLE)



and upper bound estimates for selected occupational exposures.  The



shapes of most models' upper-bound estimates tend to parallel the



shapes of the models themselves, unless a procedure has been



devised to provide otherwise.  This is the case for the linearized



multistage procedure, which provides a linear upper bound estimate



for a multistage model at low doses.  The MLE,  which is the



estimate given by a fitted model, takes only the experiment to



which the model has been fitted into account.  The upper bound



estimate is intended to account for experiment to experiment



variability as well as extrapolation uncertainties.

-------
                    Table 1.   Extrapolation Model  Statistics
Model
Independent Background
Probit
Logit
Weibull

Gamma

Additive Background
Probit

Logit
Weibull

Gamma

Multistage oj qj
5-Stage 0.0 0.0
3-Stage 0.0 0.0
Parameters
(Standard Error) Loglikelihooda
a
-7.13
(0.80)
-13.61
(2.04)
-12.53
(2.00)
0.79
(0.17)
a
-14.62
(61.49)
-15.46
(25.51)
-22.67
(42.31)
0.79
(2.52)
q* i
0.0 0.
0.0 3.
P
2.85
(0.31)
5.38
(0.78)
4.75
(0.76)
10.19
(2.21)
P
4.96
(16.23)
5.95
(7.66)
7.66
(11.51)
10.19
(52.06)
» q4
7
0.00 -99.30
(0.00)
0.00 -99.31
(0.00)
0.00 -99.32
(0.00)
0.00 -99.31
(0.00)
6
6.59 -99.31
(51.33)
0.87 -99.32
(12.04)
5.31 -99.41
(20.74)
0.00 -99.30
(24.98)
qs
0 4.34E-6 1.56E-6 -99.32
46E-4 - - -104.30
1 -Stage 0.0 3.94E-2 - -
-154.17
jf* Goodness -
of-Fit
(Significance
Level)6
<0.001
(0.996)
0.008
(0.927)
0.016
(0.899)
0.001
(0.979)
0.006
(0.937)
0.019
(0.892)
0.109
(0.741)
0.001
(0.979)

0.020
(0.99)c
6.98
79.05
«.0001)c
1  The closer  the  loglikelihood  to  zero  the better the model fits the
  observed data.

b  Significance  levels  less  than 0.01  indicate an inadequate fit.

    proximately distributed as  a chi  square with degrees of freedom equal
     the number of doses minus the number of nonzero parameter estimates.

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     Table 2.  Risks to Mobile Home Residents (HUD Standard)
      Level of Exposure:   0.15 ppm, 112 hours/week, 10 years
                        Point  Estimate     95% Upper Confidence
        Model             of Added Risk      Limit on Added Risk
Independent Background
     Probit                  0.0                   0.0
     Logit                  2 x 10"n             2 x 10~1§
     Weibull                2 x 10~w             2 x 10~9
     Gamma                  3 x 10~17             2 x 10~16
Additive Background
     Probit                 3 x 10~s              2 x 10~e
     Logit                  7 x 10~8              4 x 10~a
     Weibull                6 x 10"6              5 x 10~8
     Gamma                  3 x 10~17              8 x 10~14

5-Stage Multistage          1 x 10~9              2 x 10~4
3-Stage Multistage          6 x 10~4              1 x 10~4
1-Stage Multistage          3 x 10~8              4 x 10~3

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   Table 3.  Risks to Manufacturers  of Apparel  (OSHA Standard)

       Level of Exposure:  3.0 ppm, 36 hours/week, 40 years

                         Point Estimate      95%  Upper Confidence
      Model              of Added  Risk      Limit on Added Risk

Independent Background
     Probit                2 x  10~»              9 x 10'5
     Logit                 3 x  10~4              9 x 10~4
     Weibull               5 x  10~4              1 x 10~8
     Gamma                 1 x  10~4              4 x 10~4
Additive Background
     Probit                2 x  10~4              3 x 10~8
     Logit                 4 x  10~4              3 x 10~8
     Weibull               1 x  10~3              3 x 10~3
     Gamma                 1 x  10~4              2 x 10~3

5-Stage Multistage         5 x  10"4              6 x 10~8
3-Stage Multistage         6 x  10~8              9 x 10~8
1-Stage Multistage         8 x  10~2              9 x 10~2

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         Table 4.  Risks  to Manufacturers of Apparel
                   (Personal  Sample)
      Level of Exposure:   0.64  ppm,  36 hours/week,  40 years
                         Point  Estimate     95%  Upper Confidence
       Model             of Added Risk      Limit on Added Risk
Independent Background
     Probit                 8 x  10~17             3 x 10~"
     Logit                  8 x  10"1              4 x 10"7
     Weibull                3 x  10~7              1 x 10~6
     Gamma                  7 x  10~"             6 x 10~lf
Additive Background
     Probit                 5 x  10~7              2 x 10~8
     Logit                  1 x  10~fl              6 x 10~5
     Weibull                5 x  10~5              3 x 10~4
     Gamma                  7 x  10~n             3 x 10"8

5-Stage Multistage          6 x  10~7              l x 10~s
3-Stage Multistage          6 x  10~s              7 x 10~4
1-Stage Multistage          2 x  10~2              2 x 10~2

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         Table  5.   Risks to Manufacturers of Apparel
                    (Area Sample)
      Level  of  Exposure:   0.23 ppro,  36 hours/week, 40 years
                         Point Estimate     95% Upper Confidence
        Model            of Added Risk      Limit on Added Risk
Independent Background
     Probit                  0.0                  1 x 10~2B
     Logit                  3 x. 10~1§              2 x 10~9
     Weibull                2 x 10~8               1 x 10~8
     Gamma                  3 x 10~18              4 x 10~14
Additive Background
     Probit                 7 x 10~fl               4 x  10~6
     Logit                  2 x 10~7               1 x 10"5
     Weibull                1 x 10~6               1 x 10~4
     Gamma                  3 x 10~15              4 x 10~12

5-Stage Multistage          9 x 10~9               4 x 10~4
3-Stage Multistage          3 x 10~6               2 x 10~4
1-Stage Multistage          6 x 10~3               7 x 10~s

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         Table 6.  Risks to Manufacturers  of Apparel
                   (NIOSH Data)

      Level of Exposure:  0.17 ppm/  36  hours/week,  40 years
                        Point Estimate      95%  Upper Confidence
         Model          of Added Risk      Limit on Added Risk
Independent Background
     Probit                  0.0                    0.0
     Logit                  6 X 10""             4 x 10~11
     Weibull                5 x 10"11             4 x 10"'
     Gamma                  1 x 10~16             2 x 10~«

Additive Background
     Probit                 4 x 10~8              3 x  10"6
     Logit                  1 x 10~7              7 x  10"fl
     Weibull                9 x 10~6              8 x  10~s
     Gamma                  1 x 10~16             3 x 10"13

5-Stage Multistage          3 x 10~'              3 x  10~4
3-Stage Multistage          1 x 10~6              2 x  10~4
1-Stage Multistage          5 x 10~*              5 x  10"8

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APPENDIX 4:  DOCUMENTATION OF HIGH TO LOW DOSE
             EXTRAPOLATION MODELS USED IN
             QUANTITATIVE RISK ASSESSMENT-CONCISE
             DESCRIPTION

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                        TABLE OP COHTKHTS

                                                               Page

1.0  INTRODUCTION  	    1
2.0  GENERAL INFORMATION	    2
     2.1  THE NEED FOR HIGH- TO LOW-DOSE EXTRAPOLATION  	    2
     2.2  TYPE OF DATA  	    2
      i
     2.3  TYPES OF MODELS FOR QUANTAL RESPONSE DATA  	    3
     2.4  SPONTANEOUS BACKGROUND RESPONSE  	    4
3.0  PROBIT MODEL (LOGNORMAL)  	    8
4.0  LOGIT MODEL (LOG LOGISTIC MODEL) 	   10
5.0  WEIBULL MODEL (EXTREME VALUE)  	   11
6.0  ONE-HIT MODEL (LINEAR MODEL)  	   12
7.0  GAMMA-MULTIHIT MODEL  	   14
8.0  MULTISTAGE MODEL  	   17
REFERENCES  	   18

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1.0  INTRODUCTION
          The Design and Development Branch  (DDE) of the Exposure
Evaluation Division (BED) of the U.S. Environmental Protection
Agency currently uses six extrapolation models to estimate car-
cinogenic risk in humans from animal test data.  This report pro-
vides general introductory material and a concise description of
each model suitable for insertion into the background and methods
section of a quantitative risk assessment.  No mathematical equa-
tions are included and technical terms are avoided as much as
possible.  The introductory material covers the reason for high-
to low-dose extrapolation, the type of data used, classes of
models and incorporation of spontaneous background response.  The
six models that are described are the probit, logit, Weibull,-
one-hit, gamma-multihit and multistage models.  Technical details
and a more comprehensive bibliography appear in a companion re-
port (Chesson and Zanetos, 1983).

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2.0  GENERAL INFORMATION
2.1  THE NEED FOR HIGH- TO LOW-DOSE EXTRAPOLATION
          The most readily available source of information for
determining the health effects of toxic agents comes from experi-
mental tests on laboratory animals.  In order to obtain observ-
able effects within a reasonable time the laboratory animals must
be exposed to concentrations, or doses, of the toxic substance
that are higher than those expected to be experienced by humans.
Therefore it is necessary to predict the effects at low doses
from the effects observed at higher doses.  This process is
called "high- to low-dose extrapolation" and is carried out by
fitting a mathematical model to the observed data and using the
model to estimate the effect of the substance at low doses.

2.2  TYPE OF DATA
          A typical experiment to collect data for high- to low-
dose extrapolation consists of several groups of animals.  Each
group is exposed to a different dose, or concentration d, of the
agent under test and the numbers of animals in each group that
show a particular response within a fixed time period are record-
ed.  This type of data is often called "quantal" response or "di-
chotomous" data.  The data provide the basis for fitting dose
response models and extrapolating to low doses.  As used here, a
dose response model is a mathematical relationship between the
applied dose d, and the proportion of animals in the group, P(d),
that show the response.  When additional information, such as the
time between the start of exposure to the agent and appearance of

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a tumour, is available then other types of dose response models
may also be used.

2.3  TYPES OF MODELS FOR QUARTAL RBSPOSSB DATA
          The mathematical models used for describing guantal
responses fall into two broad types:
          •  Tolerance distribution models
          •  stochastic or mechanistic models.
(Krewski and Van Ryzin, 1981; Brown, 1983).
          Tolerance distribution models are based on the idea
that each individual in a population has its own tolerance to the
test agent.  If a dose does not exceed the tolerance of an
individual then there will be no response by that individual.  If
the dose exceeds the tolerance then a response will be observed.
Tolerance models differ from each other in the particular
mathematical expression used to describe the distribution of
tolerances in the population (Chand and Hoel, 1974).  The dis-
tributions are generally chosen because of their descriptive
power rather than on the basis of biological processes.
          Stochastic, or mechanistic, models are derived from
plausible biological arguments which lead to an expression for
P(d), the expected proportion of animals that will show a
response at dose d.  Sometimes the mechanistic argument leads to
a tolerance distribution.  Thus the distinction between the two
types of model is not always very obvious.

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          The behavior of a model at low doses is of particular
importance since it is the low dose region about which predic-
tions are to be made.  In this region the shape of the dose
response curve predicted by a model may be convex, linear or con-
cave (Figure 1).  The usual procedure is to estimate the dose d*f
corresponding to a particular low level of risk having taken into
account spontaneous background response (see Section 2.4 below).
The excess risk is commonly set at 10-6, i.e. the level at which
the expected proportion of individuals that will show the re-
sponse as a result of exposure to dose d* is one in 1,000,000.
The dose d* is called the "virtually safe dose" (VSD).  Figure 1
shows how the estimate of the VSD depends on the shape of the
dose response curve.  A linear curve will give a lower VSD than a
comparable convex curve and a higher VSD than a concave curve.
These relationships are used in some extrapolation procedures
that attempt to place a lower bound on the VSD rather than obtain
an actual estimate of it.  The estimates obtained in this way are
considered 'conservative1 in that they are expected to
overestimate risk and underestimate the VSD.

2.4  SPONTANEOUS BACKGROUND RESPONSE
          In an experiment to determine a dose response curve it
is possible that some animals might show a response even though
they receive zero dose of the chemical.  This spontaneous back-
ground response may be a result of many factors including the

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FIGURE 1.  THE RELATIONSHIP BETWEEN THE SHAPE OF THE DOSE RESPONSE
           CURVE AT LOW DOSES AND THE VSD (d*) (ASSUMING NO
           SPONTANEOUS BACKGROUND RESPONSE)

                      (a)  Concave
                      (b)  Linear
                      (c)  Convex

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presence of another response causing chemical, the genetic make-
up of the strain of animal, or a background level of  the  toxic
substance which is present in the environment.  The method of
incorporating the spontaneous background response into the model
affects the shape of the dose response curve at low doses and
hence estimates of the VSD.
          If the background response is assumed to be totally
independent of the response to the experimental dose  then the
shape of the dose response curve remains qualitatively the same
as when no background response is included.  However, if  the
background response is assumed to be additive in the  sense that
the effective dose is taken to be the background dose plus the
experimental dose then in many cases the dose response curve be-
comes linear at low doses  (Crump e_t aJL, 1976).  This  is true for
most of the commonly used models, including the probit, logit,
Weibull, one-hit, gamma-multihit and multistage models.   A dose
response curve that is linear at low doses will result in a
smaller VSD than a comparable dose response curve that is convex
at low doses.  Thus, a model that includes additive background
effects is likely to give estimates of the VSD which  are  smaller
than those estimated by a model without additive background.
          A model may include both independent and additive back-
ground but as long as the additive component is non-zero  the dose
response curve at low doses will be linear (Crump et  al,  1976;
Peto, 1978).
          It is often difficult to decide from experimental data
whether background responses are independent, additive, or both

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(Brown, 1993).  Therefore, it has been suggested  that models  in-
corporating additive background be used unless there is good
evidence to assume otherwise (State of California, Health and
Welfare Agency, 1982).

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3.0  PROBIT MODEL (LOGNORNAL)
          The probit model is a tolerance distribution model in
which the distribution of individual tolerances is assumed to
follow a lognormal distribution.  The dose response curve is s-
shaped and symmetric about the 50 percent level.  Since experi-
mental data often show this sort of pattern, the probit
model has been used extensively in toxicological studies and a
      t
standard statistical theory has been developed around it (Finney,
1971).  The use of the probit model to extrapolate to low doses
is more recent.
          When the background response is independent of the in-
duced response the probit model approaches zero very rapidly as
dose decreases, more rapidly than other common models used in
low-dose extrapolation (Krewski and Van Ryzin, 1981).  This means
that the probit model with independent background cannot be
linear at low doses and tends to give lower estimates of risk and
higher VSD's than those obtained from other models, or from
linear extrapolation.
          The Mantel-Bryan procedure (Mantel and Bryan, 1961;
Mantel e_t al, 1975) uses the probit model to obtain a "conserva-
tive" estimate of the VSD.  By taking an arbitrary value of 1 for
the slope parameter of the model and extrapolating from the range
of observable responses it is assumed that the extrapolated curve
will lie above the true curve and therefore provide a conserva-
tive estimate of a VSD.  However the validity of this procedure
has been questioned (Crump, 1977).  In particular, it is not

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clear that taking the value of the slope parameter as 1 is neces-
sarily conservative (Connfield et al, 1978).

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                                10
4.0  LOGIT MODEL (LOG LOGISTIC MODEL)
          The logit model is a tolerance distribution model which
has a very similar appearance to  the probit model within the
range of observable responses.  It is S-shaped and symmetric
about the 50 percent response level.  However it differs from the
probit model in that at low doses it approaches zero much more
slowly.  The model can be derived from chemical kinetic theory
and wa's proposed as a dose response model by Worcester and Wilson
(1943) and Berkson (1944).
          With independent background the excess risk may be
linear, convex or concave at low doses.  Low-dose linearity
implies a concave dose response curve at higher doses.  With
additive background the excess risk will always be linear at low
doses (Peto, 1978).  Therefore, linear extrapolation procedures
will tend to give estimates of VSD that are close to or smaller
than those based on the model itself unless the dose response
curve is concave at low doses.  In fitting a variety of models to
20 data sets Krewski and Van Ryzin (1981) found that estimates of
VSD based on the logit model were smaller than those based on the
probit model and similar to those based on the gamma.-multihit
model.

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                                11
5.0  WEIBDLL MODEL (EXTREME VALUE)
          The Weibull model is a tolerance  distribution model
which is suggested by human cancer incidence patterns  (Cooke e_t
al, 1979).  Pike  (1966) showed that two quite general assump-
tions, (1) cancer begins in a single cell, and  (2) individual
cells behave independently, can lead to a Weibull distribution
for tolerances.  The model can also be derived  from a •time-to-
tumour* argument  (Peto et al, 1972) or from a model based on
critical cell clusters (Scott and Hahn, 1980).  Generalized forms
of the Weibull model are discussed by Carlborg  (1981a).
          With independent background the excess risk at low
doses predicted by the Weibull model behaves in the same way as
the logit model.  The excess risk may be linear, convex or con-
cave depending on the value of the shape parameter.  Low dose
linearity implies a concave dose response curve at moderate and
high doses,  with additive background, the excess risk will al-
ways be linear at low doses (Peto, 1978).
          Estimates of the VSD based on the Weibull model tend to
be less conservative than the multistage model  but more conserva-
tive than the probit, logit and gamma-multihit  models  (Krewski
and Van Ryzin, 1981).

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                                12
6.0  ONE-BIT MODEL (LINEAR MODEL)
          The one-hit model is derived from a mechanistic de-
scription of the carcinogenic process.  Suppose that there is a
response after a susceptible site has been •hit" by a single bio-
logically effective unit of dose within a fixed period of time.
By assuming that the number of hits over that time period follows
a Poisson distribution and the average number of hits is propor-
tional to the dose, a formula is easily obtained for the proba-
bility (P(d)) of obtaining a response at a given dose (d).  The
Poisson distribution assumption is appropriate when hits occur
randomly through time and the occurrence of a hit has no effect
on the occurrence of other hits.
          In the absence of spontaneous background responses the
one-hit model has only one unknown parameter and is always linear
at low doses and concave at moderate and high doses.  Because of
its low dose linearity, it is often referred to as the linear
model.  It is a special case of the gamma-multihit, multistage
and Weibull models.
          The one-hit model does not provide a good fit to many
sets of empirical data because the model is concave at higher
dose levels whereas many data sets are convex.  It appears to be
appropriate for only one (hexachlorobenzene) of the 20 data sets
considered by Krewski and Van Ryzin (1981).  However, it has been
used extensively in low dose extrapolation as a conservative
estimate of risk, assuming that the true dose response curve is
likely to be convex at low doses and hence lie below that of the
one-hit model (Hoel e_t al, 1975; BEIR Report, 1972).  In some

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                                13
situations the one-hit model is only fitted to the lowest dose
groups where the experimental data are consistent with the model
(Altshuler, 1976).  The model has been criticized as being unduly
conservative in some circumstances.  For example, Van Ryzin and
Rai (1980) found that for ethylene thiourea the VSD (for risk
level 10~6) estimated by the one-hit model was approximately
1/60,600th that of the multihit model and l/8050th that of the
multistage model.

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                                14
7.0  GAMMA-HOLTIHIT MODEL
          The gamma-milltihit model can be derived from a mechan-
istic description of the carcinogenic process.  Suppose the
response to a particular chemical is the result of k biological
precursor events or "hits11 at a susceptible site within a fixed
period of time.  By assuming that the number of hits over that
time period follows a Poisson distribution and the average number
of hits is proprotional to the dose, a formula can be obtained
for the probability (P(d)) of obtaining a response at a given
dose (d).  The Poisson distribution assumption is appropriate
when hits occur randomly through time and the occurrence of a hit
has no effect on the occurrence of other hits.  When only one hit
(k«l) is required to cause a response, the model is referred to
as the one-hit model.
          The gamma-multihit model can also be regarded as a
tolerance distribution model since the formula for P(d) is
identical to the dose response curve generated by assuming that
each individual in the population has a particular tolerance
level to the chemical and that the distribution of tolerance
levels follows a gamma distribution.  In this situation the gamma
distribution is merely used to describe the shape of the dose
response curve and has no mechanistic implications.
          For small doses the dose response curve is concave when
k < 1, linear if k-1 and convex if k > 1 (Figure 1).  Thus the
gamma-multihit model provides a greater variety of behavior at
low doses than models which can have only linear or convex dose
response curves at low doses.  However, k < 1 is not easily

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                                15
interpretable in terms of the mechanistic "hit" model, and one
has to resort to the descriptive tolerance distribution
interpretation in this case.
          Although the gamma-multihit model has been recommended
for use in risk assessment calculations  (Food Safety Council,
1978), this recommendation has been criticized by Baseman et aJL
(1981) because the model can produce estimates of the VSD which
are unrealistically high or unrealistically low in certain
situations.  These problems appear to be less likely to occur
when an additive background effect is included in the model
thereby causing the dose response curve to be linear at low
doses.
          The gamma-multihit model tends to produce estimates of
the VSD which are less conservative than the multistage and
Heibull models and more conservative than the logit and probit
models (Krewski and Van Ryzin, 1981).

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                                16
8.0  MULTISTAGE MODEL
          The multistage model was derived to account for the
observation that for many types of cancer, death rate is propor-
tional to some power of age (e.g. Nordling, 1953).  The model was
developed by Armitage and Doll (1961) and extended by Crump et al
(1976).  The Armitage and Doll model assumes that a cell line  -
goes through k distinct stages in a specific order before becom-
ing cancerous and that the rate at which it progresses through
the ith stage is a constant \±.  Different cell lines develop
independently and the time to cancer is determined by the most
rapidly developing line.  This model predicts that cancer inci-
dence will increase as (age)*'1.
          Crump et al (1976) extended the Armitage and Doll model
by assuming that Xi, the rate at which a cell goes through the
ith stage, is linearly related to dose.  From these assumptions,
the probability of a response, P(d), at dose d can be expressed
in terms of a polynomial in d.  The model is essentially
unchanged irrespective of whether indepenedent or addititve
background responses are assumed.  It is linear at low doses if
the polynomial in d has a linear term and convex otherwise.  It
cannot be concave at low doses, but it can be simultaneously
linear at low doses and convex at moderate doses.  When k«l,
i.e., when there is only one stage, the model reduces to the one-
hit model.
          The multistage model cannot describe concave dose
response curves such as those observed with DDT, vinyl chloride,
diethylstilbestrol and ethylene dibromide (Carlborg, 1981b).

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                                17
Also, the shape of the curve in the extrapolated low dose  region
is relatively insensitive to the shape of the dose response  func-
tion in the observable range (Carlborg, 1981a).
          The parameters of the multistage model are more  compli-
cated to estimate than for other models.  However, computer  pro-
grams are available to carry out the calculations (Crump and
Watson, 1979; Howe and Crump, 1982).
      »
          Crump (1982) proposed a method of linear extrapolation
based on the multistage model, which has been adopted by the EPA
in setting water quality criteria (USEPA, 1980) and in other
areas of risk assessment.  The method is an improvement of the
extrapolation procedure proposed by Crump e_t al (1977).  It
involves approximating the dose response curve by a straight line
with slope given by the estimated linear term of the multistage
model.  This modification has been referred to as the "lineariz-
ed* multistage model and has an advantage over some other  linear
extrapolation procedures in that information from all the experi-
mental dose groups is used, not just the lower groups.  The
6LOBAL79 computer program (Crump and Watson, 1979) calculates
confidence limits for the linearized multistage model.  A newer
program (GLOBAL82, Howe and Crump, 1982) uses a different method
to allow valid confidence limits for estimates at higher doses as
well as at low doses.  At low doses the results obtained from
GLOBAL?9 and GLOBAL82 should be very similar.

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                                 18


                            REFERENCES
Altshuler B.  1976.  A Bayesian approach to assessing population
risks from environmental carcinogens.  In: Environmental Health.
A. Whittemore, ed.  SIAM:  Philadelphia


Armitage P, Doll R. 1961.  Stochastic models for carcinogenesis.
Proceedings 4th Berkeley Symposium IV, pp. 19-38.


Berkson J.  1944.  Application of the logistic function to
bioassay.  J. American statistical Association 39:134-167.


BEIR Report.  1972.  The effects on populations of exposures to
low levels of ionizing radiation.  Report of the advisory
committee of the biological effects of ionizing radiations.
National Academy of Sciences.  National Research Council,
Washington, DC.  Government Printing Publication No. 0-489-797.


Brown CC.  1983.  Learning about toxicity in humans from studies
on animals.  Cheratech, pp. 350-358.


Carlborg FW.  1981a.  2-Acetylaminofluorene and the Weibull
model.  Fd Co sine t Toxicol 19:367-371.


Carlborg FW.  1981b.  Multistage dose-response models in carcino-
genesis.  Fd Cosmet Toxicol 19:361-365.


Chand N, Hoel DG.  1974.  A comparison of models for determining
safe levels of environmental agents.  In: Reliability and
Biometry; Statistical Analysis of Lifelength. SIAM:
Philadelphia, pp. 681-700.


Chesson J, Zanetos MA.  1983.  Documentation of high- to low-dose
extrapolation models used in quantitative risk assessment.  Draft
Report.  Washington, DC:  Office of pesticides and Toxic
Substances.  U.S. Environmental Protection Agency.  Contract 68-
01-6721.


Cook PJ, Doll R, Fellingham SA.  1969.  A mathematical model for
the age distribution of cancer in man.  Int J Cancer 4:93-112.

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                                 19
                                    BS
                            (Continued)
Cornfield J, Carlborg FW, Van Ryzin J.   1978.   Setting  tolerances
on  the  basis of mathematical treatment of dose-response data ex-
trapolated  to  low doses.  In:  proceedings of  the First Interna-
tional  Congress on Toxicology.  Plaa GL, Duncan WAM, eds.  New
York:   Academic, pp. 143-164.


Crump KS.   1977.  Theoretical problems in the modified Mantel-
Bryan procedure.  Biometrics 33x752-755.
      i

Crump RS.   1982.  An improved procedure  for low-dose carcinogenic
risk assessment from animal data.  J. Environmental Path
Toxicology  5(2):675-684.


Crump KS, Guess HA, Deal KL.  1977.  Confidence intervals and
tests of hypothesis concerning dose-response relations  inferred
from animal carcinogenicity data.  Biometrics  33:437-451.


Crump RSr Hoel DG, Langley CH, Peto R.   1976.   Fundamental car-
cinogenic processes and their implications for  low dose risk
assessment.  Cancer Research 36:2973-2979.


Crump RS, Watson WW.  1979.  GLOBAL79:   A FORTRAN program to
extrapolate dichotomous animal carcinogenicity  to low dose.


Finney  DJ.  1971.  Probit analysis (3rd  edition).  London:
Cambridge University Press.


Food Safety Council.  1978.  Proposed system _for food safety
assessment.  Fd Cosraet Toxicol 16 Supp 2:1-136.(Revised report
published June 1980, by the Food Safety  Council, Washington, DC.)


Baseman JR, Hoel DG, Jennrich RI.  1981.  Some  practical problems
arising from use of the gamma multi-hit  model  for risk estima-
tion.   J. Toxicol Environmental Health 8:379-386.


Hoel DG, Gaylor DW, Rirschstein RL, Saffiotti U, Schneiderman MA.
'1975.   Estimation of risks of irreversible, delayed toxicity.  J.
Toxicol and Environmental Health 1:133-151.

-------
                                 20
                            REFERENCES
                            (Continued)
Howe RB, Crump KS.  1982.  GLOBAL 82.  A computer program to ex-
trapolate guantal animal toxicity data to low doses.  Report to
Office of Carcinogen Standards Occupational Safety and Health
Administration, US Dept. of Labor, Contract 41 DSC 252C3.


Krewski D, Van Ryzin J.  1981.  Dose response models for quantal
response toxicity data.  In:  Current Topics in Probability and
Statistics.  Csargo M, Davson D, Rao JNK, Saleh E, eds.  North-
Holland:  New York, pp. 201-281.


Mantel N, Bryan WR.  1961.  "Safety" testing of carcinogenic
agents.  J National Cancer Institute 27(2):455-470.


Mantel N, Bohidar NR, Brown CC, Ciminera JL, Tukey JW.  1975.  An
improved Mantel-Bryan procedure for "safety" testing of carcino-
gen's.  Cancer Research 34:865-872.


Peto R.  1978.  Carcinogenic effects of chronic exposure to very
low levels of toxic substances.  Environmental Health Perspec-
tives 22:155-159.


Peto R, Lee PN, Paige WS.  1972.  Statistical analysis of the
bioassay of continuous carcinogens.  Br. J. Cancer 26:258-261.


Pike MC.  1966.  A method of analysis of a certain class of ex-
periments in carcinogenesis.  Biometrics 22:142-161.


Scott BR, Hahn pp.  1980.  A model that leads to the Weibull
distribution function to characterize early radiation
probabilities.  Health Physics 39:521-530.


State of California Health and Welfare Agency.  1982.  Carcinogen
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exposures to carcinogens.


US EPA.  1980.  (U.S. Environmental Protection Agency).  Hater
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45(231):79347-79357.

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                                  21


                            REFERENCES
                            (continued)
VanvRyzin J, Rai K.  1980.  The use of guantal response data  to
make predictions.  In: The Scientific Basis of Toxicity
Assessment.  Witschi H, ed.  Elsevier/North Holland:  Biomedical
Press.



Worcester J, Wilson EB.  1943.  The determination of LD50 and its
sampling error in biossay.  Proceedings of National Academy of
Sciences 29:79-85.

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APPENDIX 5:  SENSITIVITY ANALYSIS OF CUT RAT DATA
             USING THE LINEARIZED MULTISTAGE MODEL

-------
                             Appendix 5





     Ten perturbations of the final CUT formaldehyde combined male



and female Fischer 344 rat data were constructed in 3 ways: one, as



if a dose had not been run; two, as if the response had been



different at an intermediate dose; or, three, the response was



different at the highest dose or control (see Table 1).



Perturbations 1 and 2 removed one intermediate dose and also



increased the number of animals at risk by 1, thereby lowering the



response rate at the highest dose.  Perturbation 3 eliminated the



response and number of animals at risk at the high dose entirely.



Perturbation 4 gave the CUT data set a positive response at



control.  Pertubations 5 and 7 use the correct final denominator or



number of animals at risk at the highest dose, but vary the numbers



responding.   Perturbations 6 and 8 lower or raise the response at



the next to the highest dose.  Perturbation 9 eliminates the control



data entirely.   Perturbation 10 gives a positive response at the



lowest positive dose where one did not appear in the CUT data set.



     For all perturbations, with a five-stage model the fit was



adequate (see Table 2, first column).  While the individual maximum



likelihood coefficients varied from perturbation to perturbation,



the predicted upper bound risks varied by less than a.factor of 2



(see Table 3).



     A graphical representation of the 10 perturbations is given in



Figures 1 an 2 which,  upon inspection,.reveal just how similar the



"curves" are.  Note in Figure 2 that the curve for perturbation 2



has a considerably steeper slope and is rather an outline.  This is



because the lowest positive dose point was dropped, a section of the



curve where the most information is needed.

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                                 Table 1.  Sensitivity of the 5-Stage Model

                                            (Model Statistics)
                              Dose  (ppm)

                          Responding/Tested
                                      Parameter Estimates
Perturbation
 0.0
2.0
5.6
14.3
                           q»
qs
     1



     2



     3



     4



     5



     6



     7



     8



     9



    10
0/156



0/156



0/156



1/156



0/156



0/156



0/156



0/156







0/156
0/159     -     94/141



        2/150   94/141



0/159   2/150
                         0.0  0.0



                         0.0  1. 5E-S



                         0.0  0.0
0/159   2/153   94/140 3.16E-1  0.0



0/159   2/153   95/140    0.0  0.0



0/159   1/153   94/140



0/159   2/153   93/140
       i


0/159   3/153   94/140



0/159   2/153   94/140



1/159   2/153   94/140
                         0.0  0.0



                         0.0  0.0



                         0.0  0.0



                         0.0  0.0



                         0.0  1.68E-3
                               0.0     0.0



                               5.99E-7  8.00E-7



                               0.0     0.0



                               0.0     0.0



                               0.0     0.0



                               0.0     0.0



                               0.0     0.0



                              4.06E-1I  0.0



                               0.0     0.0



                               0.0     0.0
                                         0.0      1.84E-0



                                         5.16E-*  1.47E-6



                                         0.0      2.42E-8



                                         0.0      1.86E-8



                                         4.00E-6  1.62E-0



                                         0.0      1.85E-6




                                         4.67E-I  1.50E-6



                                         1.52E-S  8.0 IE-7



                                         4.34E-I  1.56E-6



                                         ,0.0      1.81E-6

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            Table 2.  Sensitivity of the 5-Stage Model
                     (Model Goodness-of-Fit)
Perturbation
1
2
3
4
5
6
7
8
9
10
X1 Goodness-of-Fit
(Significance Level)
9 . 35E-3
(7.995)«
1.69E-26
(1.000)
1.24E-2
(7.995)
1.04
(7.75)
1.96E-2
«.50)
0.21
(0.98)
2.07E-2
(0.99)
4 . 44E-2
(7.90)
2.0 IE- 2
(7.90)
0.69
(7.98)
Loglikelihood
-89.758
-100.37
-10.634
-106.065
-98.592
-94.794
-100.023
-103.452
-99.34
-105.697
Approximately distributed as a chi square  with degrees of
freedom equal  to the number of doses  minus the number  of
nonzero parameter estimates.

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Table 3.  Coefficients (q*'s) corresponding to the upper bound on risk for the 5-Stage
          Model for ten perturbations of the model.
Perturbation
Number q*c
1 0.0000
2 0.0000
3 0.0000
4 2.5652 X ID'3
5 0.0000
6 0.0000
7 0.0000
8 0.0000
9 0.0000
10 0.0000
q
4.2556
4.6685
3.6499
2.3843
2.6652
1.7512
2.7074
3.5135
2.6866
4.9145
*l
X 10'3
X lO-3
X lO'3
x 10'3
X lO'3
X lO'3
X lO'3
x lO"3
X lO'3
X 10'3

0
0
0
0
0
0
0
0
0
0
q*2
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000

0
0
0
0
0
0
0
0
0
0
q*3
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000

0
0
0
0
0
0
0
0
0
0
q*4
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000

1
1

1
1
1
1
1
1
\
q*5
.7-353 X
.7056 X
0.0000
.7832 x
.8176 X
.7966 x
.7450 X
.7660 X
.7809 X
.7220 X

10'6
10'a

lO'6
io-e
10-'
io-«
io-«
io-°
10-"

-------
Figure 1.  Sensitivity of the 5-Stage Model for all ten perturbations of the dose-response
           data for Fischer 344 rats.
B.<1F-04


7.6E-04


6.7FI-04-

A
O 5.9F-04
0
I
T 5.0E-04
I
0
N 4.2E-04-
A
L
3.4E-04-
R
I
S 2.5E-04-
K

1.7E-04


8.4E-05
O.OE-»00
0
0
0
0

0
0
0
0
o

0
0
0
0
o

0
0
0
0
o
0
0
0 l
0
0

0°
T 	 1 	 1 	 1 	 1 	 T 	 1 	 1 	 i 	 1 	 r
.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.6 O.9 1.
 Legend:   111 Perturbation  1
          222 Perturbation  2
          333 Perturbation  3
    F/POSUHF  I. rvFL  (PPMI
444 Perturbation 4     777 Perturbation 7
555 Perturbation 5     888 Perturbation 8
666 Perturbation 6     999 Perturbation 9
                       000 Perturbation 10

-------
 Figure 2.   Sensitivity of the 5-Stage Model rescaled to display perturbations  1-9 of the
            dose-response data for Fischer 344 rats.







A
0
0
I
T

O
N
A
L

R
I
S
K



.



1


9

8.


7.


B.


5.


4.
3.


2.



1 .
0.

OF-05


OE-06

OF-OB


OE-06


OE-06


OE-OB


OF-06
OE-06


OE-06-



OE-06
OF+OO
1
0

1
2' •
2
2 8
*" Q
2 8
2
2 8
2 a
2 B
9 7
« 2 8 -,9
2 Z5

2 8 Z5
22 8 7§
2 8 25
2 7 8
2 8 §5
2 8 2 4 D
22 8 §*

S— 9 -t
d9 33I
2 8 al 3.1
2 8 a!' ^3t*
2 B flB •« 3 • ^
2 88a a|JI^ 3ii**$
i 	 1 	 1 	 1 	 1 	 1 	 1 	 1 	 1 	 1 	 r
0 O.t 0.2 0.3 0./1 0.5 0.6 O.7 0.8 O.9 l.{
                                   Fxposunr  ir-vFL  (PPM)
Legend:  111 Perturbation 1     444 Perturbation 4     777 Perturbation 7
         222 Perturbation 2     555 Perturbation 5     888 Perturbation 8
         333 Perturbation 3     666 Perturbation 6     999 Perturbation 9

-------