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DRAFT                                                        EPA-600/8-82-004FA
DO NOT QUOTE OR CITE
                                                             Review Draft
                   ADDENDUM TO THE HEALTH ASSESSMENT DOCUMENT

                    FOR DICHLOROMETHANE (METHYLENE CHLORIDE)

                         Updated Carcinogen Assessment

                    of Dichloromethane (Methylene Chloride)
                                     NOTICE
THIS DOCUMENT IS A PRELIMINARY DRAFT.  It has not been formally released by
the U.S. Environmental Protection Agency and should not at this stage be
construed to represent Agency policy.  It is being circulated for comment
on its technical accuracy and policy implications.
                 Office of Health and Environmental Assessment
                       Office of Research and Development
                      U.S. Environmental Protection Agency
                                Washington, D.C.

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                                    CONTENTS



Authors, Contributors, and Reviewers	••  ...  •	,iv

1.  SUMMARY AND CONCLUSIONS.  .  .  .  .....   .   .	1

    1.1.  SUMMARY.  ..  ....  .   .   .   .   .   .   .   .   .   .   .   .   .    1

          1.1.1.  Qualitative Assessment   ...   	    1
          1.1.2.  Pharmacokinetics/Metabolism.   .   	    4
          1.1.3.  Quantitative Assessment	    5

    1,2.  CONCLUSIONS.  .  .	   .   .   .   .   .   .   .   .   .    8

2.  INTRODUCTION	   ........   10

3.  CARCINOGENICITY	   .11

     3.1.  NATIONAL TOXICOLOGY PROGRAM INHALATION  BIOASSAY  (1985,  DRAFT).   .   11

           3.1.1.  Rat Study  .  .  .  .   .   .   .   .   .   .	12
           3.1.2.  Mouse Study.  .  .  .   .  ;.   .	   .   .   23
           3.1.3.  Summary .  .  .  .  .   .   ...	32

     3.2.  PHARMACOKINETICS/METABOLISM .   .   .   .   .   .   .......   35

           3.2.1.  In Vitro Metabolism/Pathways .   .   ...   ...   .   .35
           3.2.2.  Tissue Distribution .	   38
           3.2.3.  In Vivo Metabolism/Effect of Dose	39
         .3.2.4.  Human Studies .  .  .   .   ...   .   .   ...   .   .   .48
           3.2.5.  Summary	51

4. .QUANTITATIVE ESTIMATION (USING THE NTP INHALATION BIOASSAY)	57

     4.1.  SUMMARY OF NTP FINDINGS USED FOR  QUANTITATIVE ANALYSIS  ....   57
     4.2.  DOSE-RESPONSE MODEL SELECTION	   60
     4.3.  APPLICATION OF THE MULTISTAGE  MODEL  TO  NTP BIOASSAY DATA   ...   61
     4.4.  RISK ANALYSIS CONSIDERING TIME-TO-TUMOR INFORMATION 	   67
     4.5.  COMPARISON OF RISKS ESTIMATED  WITH OTHER DOSE-RESPONSE
           MODELS	V	73
     4.6.  COMPARISON OF NTP (1985) RESULTS  WITH OTHER BIOASSAYS   .   .   .   .81
     4.7.  DERIVATION OF HUMAN UNIT RISK  ESTIMATES FOR INHALATION
           OF DCM .  .  .  .  .  .  /	85
     4.8.  HUMAN UNIT RISK ESTIMATE FOR INGESTION  OF  DCM	.89
     4.9.  COMPARISON OF ANIMAL AND HUMAN DATA  RELEVANT  TO  CANCER  RISK   .   .   92

REFERENCES  .  .  .  .  .  .  .  .  .	95
                                      iii

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                      AUTHORS,  CONTRIBUTORS,  AND  REVIEWERS



     The Carcinogen Assessment  Group  within the Office  of Health and Environ-

mental Assessment was responsible for preparing this  document.


PRINCIPAL AUTHORS

          Dharm V. Singh, Ph.D.                    Chapters  1 and 3
          Hugh L. Spitzer, B.S.                    Chapters  1,  2, and 3
          Paul D. White, B.A.1                      Chapters  1 and 4


PARTICIPATING MEMBERS

Roy E. Albert, M.U., Chairman
Steven Bayard, Ph.D.
David L. Bayliss, M.S.
Robert P. Beliles, Ph.D.
Chao W. Chen, Ph.D.
Arthur Chiu, Ph.D., M.D.
Margaret M.L. Chu, Ph.D.
Herman J. Gibb, B.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charalingayya B. Hiremath, Ph.D.
James W. Holder, Ph.D.
Robert E. McGaughy, Ph.D., Acting Technical Director
Jean C. Parker, Ph.D.
William E. Pepelko, Ph.D.
Charles H. Ris, P.E., Acting Executive Director
Todd W. Thorslund, Ph.D.
^•Exposure Assessment Group, Office of Health and Environmental  Assessment,
                                       IV

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                   .1.  SUMMARY AND CONCLUSIONS

1.1.  SUMMARY
1.1.1.  Qualitative Assessment
     There have been eight chronic studies in which dichloromethane  (methy-
lene chloride, DCM) was administered to animals:   five in  rats,  two  in  mice,
and one in hamsters.  The Dow Chemical Company (1980) reported the results of
chronic inhalation studies in rats and hamsters.   There was a statistically
significant increased incidence of ventral cervical sarcomas, probably  of the
salivary gland, consisting of sarcomas only, and  appearing in males  but not in
females.  In addition, the study showed a small  increase in the number  of
benign mammary tumors compared to controls in female rats  at all  doses  and in
male rats at the highest dose.  In hamsters, there was an  increased  incidence
of lymphosarcoma in females which was not statistically significant  after
correction for survival.  In a second inhalation  study, the Dow Chemical Com-
pany (1982) reported that there was no increase  in compound-related  tumors in
rats; however, the highest dose used in this study was far below that of the
previous study.  The National Coffee Association  (1982a, b) conducted a study
in which Fischer 344 rats and B6C3F1 mice were exposed to  DCM in drinking
water.  The results indicated that Fischer 344 rats had an increased incidence
of neoplastic nodules and/or hepatocellular carcinomas in  female rats,  which
was significant with respect to matched controls; however, the incidence was
within the range of historical control values at  that laboratory.  The  National
Coffee Association  (1983) drinking water study in B6C3F1 mice also showed  a
borderline response of combined neoplastic nodules and hepatocellular carci-
nomas.  The National Toxicology Program (1982) draft gavage study on rats  and
mice has not been published due to data discrepanci.es; however, usable  infor-

                                       1

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mation from the gavage studies has been  incorporated  by  the  NTP  into  the  inha-
lation bioassay (1985, draft).
     The recently released NTP (1985)  inhalation  bioassay  concluded that  "there
was some evidence of carcinogenicity of  dichloromethane  for  male F344/N rats
as shown by increased incidence of benign neoplasms  of the mammary gland.
There was clear evidence of carcinogenicity of dichloromethane  for female
F344/N rats as shown by increased incidence of benign neoplasms  of the mammary
gland.  There was clear evidence of carcinogenicity  of dichloromethane for
male and female B6C3F1 mice, as shown by increased incidences of alveolar/
bronchiolar neoplasms and of hepatocellular neoplasms."
     There are some other inadequate animal studies  in the literature.  One
study (Theiss et al., 1977) reported a marginally positive pulmonary  adenoma
response in strain A mice injected intraperitoneally  with  DCM.   Two  negative
animal inhalation studies were judged to be inadequate because  they were  not
carried out for the full lifetime of the animals  (Heppel et  al., 1944; MacEwen
et al., 1972).
     Positive results in a rat embryo cell transformation  study were  reported
by Price et al. (1978).  The significance of their findings  with regard to
carcinogenicity is not well understood at the present time.
     The epidemiologic data consist of two studies:   Friedlander et  al.  (1978),
updated by Hearne and Friedlander (1981), and Ott.et al. (1983a, b,  c,  d, e).
Although neither study showed excessive risk, both showed  sufficient  deficien-
cies to prevent them from being judged negative studies.  The Friedlander et
al. study  (1978) lacked a large enough exposure (based on  animal cancer  potency
estimates) to provide sufficient statistical power to detect a potential  carci-
nogenic effect.  The Ott et al. (1983a, b, c, d,  e)  study, among other defici-
encies, lacked a sufficient latency period for site-specific cancer.

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     The NTP (1985) inhalation bioassay of DCM was  conducted  in  male  and
female F344/N rats and B6C3F1 mice.   The animals  were  exposed at concentra-
tions of 0, 1000, 2000, and 4000 ppm for rats and 0,  2000,  and 4000 ppm for
mice, 6 hours/day, 5 days/week, for  102 weeks.  There  was  an  increased inci-
dence of benign mammary gland neoplasms, primarily  fibroadenomas, in  both  male
and female rats.  In female rats there was a significant  increase in  hepato-
cellular neoplastic nodules and hepatocellular carcinomas  (combined)  by the
trend test only.  There was also a statistically  significant  increase of mono-
nuclear cell leukemias in female rats by age adjustment.   In  male rats there
was a significant increase in mesotheliomas, primarily in  the tunica  vagina-
lis.  Lastly, a marginally significant increase was noted  in  adrenal  pheo-
chromocytomas and interstitial cell  tumors in male rats and pituitary gland
adenomas and carcinomas combined, in male and female rats  by  the trend test
only.
     In the study using B6C3F1 mice, there was a highly significant  increase
in alveolar/bronchiolar adenoma and/or carcinoma in both  sexes of mice.  The
incidence of hepatocellular adenoma  and hepatocellular carcinoma combined  was
increased in the high-dose male group and in both dosed groups of female mice.
It should be noted that there was also a dose-related increase in the number  of
mice bearing multiple lung and liver tumors.  The control  mice had no more than
one lung tumor per mouse, whereas 38% of all dosed males  and 42% of  all  dosed
females had multiple lung tumors.  The incidence of multiple hepatocellular
tumors in the exposed groups increased in both sexes in a dose-related manner.
Multiple hepatocellular tumors were  found in only 4% of the male controls, and
none were found in the female controls.  In contrast,  28% of the males  and 32%
of the exposed females exhibited multiple liver tumors.
     The NTP concluded that, under the conditions of this  bioassay,  there  was

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some evidence of the carcinogenicity of DCM  for  male  F344/N  rats  as  shown by  an
increased incidence of benign neoplasms of the mammary  gland.   There was suffi-
cient or clear evidence of the carcinogenicity of  DCM for  female  F344/N  rats  as
shown by an increased incidence of benign neoplasms of  the mammary gland.
There was sufficient or clear evidence of carcinogenicity  in male and  female
B6C3F1 mice as shown by increased incidences  of  lung  and  liver  tumors.
1.1.2.  Pharmacoki neti cs/Metabo.1 i sm
     The available data have been analyzed to determine if there  are quali-
tative or quantitative metabolic differences  or  similarities between species
which may alter the assumptions '.used in estimating the  carcinogenic  risk
arising from exposure to DCM.
     The results of both ui vitro and in vivo studies indicate  that  DCM  is
metabolized via two pathways.  One pathway yields  carbon  monoxide as an  end
product, and the other pathway yields carbon  dioxide  as an end  product with
formaldehyde and formic acid as metabolic intermediates.   Each  pathway involves
formation of a metabolically active intermediate that is  theoretically capable
of irreversibly binding to cellular macromolecules.   A comparative analysis  of
the capability of various tissues to metabolize  DCM  indicates that the liver
is the primary site of metabolism, with some  metabolism taking  place in  the
lung and kidney.  An analysis of the available in  vivo  data  suggests that when
rats or mice are exposed to high concentrations  of DCM  they  exhale more  carbon
dioxide and excrete more formic acid than carbon monoxide.  At  exposure  to  low
concentrations of DCM, both pathways appear  to be  utilized about  equally.   At
the present time the implications of these  observations in assessing the car-
cinogenic potency of DCM are unclear.
     A comparative analysis of the data from in  vivo  studies in mice,  rats,
and humans indicates that all three species  metabolize DCM to carbon monoxide.

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Both mice and rats metabolize DCM to carbon  dioxide.   There  are  no  human  data
on the metabolism of DCM to carbon dioxide.   However,  based  on uptake  data,
some investigators have speculated that  this pathway  is  functional  in  humans.
     At present, the available data are  insufficient  for the purpose of esti-
mating doses at which metabolism is saturated.   The  data indicate that, at
low doses, little unmetabolized DCM is exhaled.   At  high doses there is a
significant exhalation of DCM immediately post-exposure.  The available data
do suggest that at high doses more DCM is taken  up into  the  body.   Currently,
the data are insufficient to determine the relationship  between  exposure  con-
centration and uptake.  Based on this analysis  it is  concluded that the avail-
able data do not offer useful parameters for modifying the assumptions used in
the calculation of the carcinogenic unit risk of DCM.
1.1.3.  Quantitative Estimation
     In the previous carcinogenicity evaluation  of DCM,  a quantitative estimate
for the upper-bound incremental unit risk was developed  on the basis of sali-
vary gland region tumors seen in an inhalation  study  with male  rats (U.S. EPA,
1985).
     The upper-bound estimate of incremental unit risk has been  re-evaluated
using the results of the NTP inhalation  bioassay (NTP, 1985). The  risk calcu-
lations presented here are based primarily on the NTP findings  of  carcinogeni-
city to the liver and lung in male and female mice.   The elevated  mammary tumor
incidence in female rats and mammary and subcutaneous tumor  incidence  in  male
rats were also used for risk analysis.  In mice, both separate  and  combined
analyses were conducted for benign and.malignant tumor types.  Risk calcula-
tions were made for mice developing either the  lung or liver tumors in order to
indicate the total risk associated with  tumors  of these  two  organs. There were
no adequate metabolic or pharmacokinetic data to support any modifications to

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the experimentally applied doses.   Thus,  risk  calculations were based on the
experimentally applied doses (in ppm using  the study  dose schedule), with sub-
sequent adjustment to estimate human equivalent doses and risks.  The multi-
stage dose-response model, as incorporated  in  the GLOBAL83 computer program
(with the number of terms restricted to the number  of experimental dose groups
minus one), is the primary model utilized in the analysis.  Both maximum like-
lihood estimates (MLE) and 95% upper confidence limit (UCL) values for risk
are given.
     The multistage model was found to provide an adequate fit to the experi-
mental data for the tumor sites, tumor pathology types,  sexes, and species
groups examined.  The highest estimate of risk was  obtained from the UCL value
for combined adenoma and carcinoma response in the  lung  and/or liver of female
mice.  To provide comparison with the basic multistage risk estimates, addi-
tional calculations were made with other  risk  estimation approaches and models
using the data on mice having lung or liver tumors.
     An analysis which excluded animals that died before the  first tumors
developed produced similar risk estimates (results  were  within 10% for female
mice with lung and/or liver adenomas and  carcinomas combined).
     A time-to-tumor analysis using the multistage  model,  as  formulated in the
WEIBULL82 computer program, was also applied to the data to determine  if the
inclusion of a time term would influence  risk  estimates.  The time-to-tumor
estimates for the UCL of risk at low dose were generally in good  agreement
with the multistage model.
     The probit and dichotomous Weibull models, in  both  background-independent
and background-additive formulations (using the RISK81 computer  program), were
applied to the mouse data for comparison  with  the  multistage  model.   For
combined lung and liver tumors in female  mice, the  background-additive formula-

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tions of both the probit and Weibull  models  are in good agreement with  the



multistage model.  The background-independent  formulations  of  the



probit and Weibull models lead to much lower risk  estimates.



     The quantitative risk estimates  developed from the NTP inhalation  bio-



assay data were compared for consistency with  findings  in earlier  long-term



bioassays of DCM conducted by the Dow Chemical Company  and  the National  Coffee



Association.  These studies provide some evidence  of DCM-induced tumors  con-



sistent with the NTP findings.  Multistage model  UCL calculations  using  the



results from these studies are comparable to,  and  in some cases exceed,



estimates for respective tumor sites  in the NTP study.   In  addition,  the Dow



inhalation study in rats showed an increase in tumors of the salivary gland



region; the multistage UCL risk estimates for  mammary tumors in the NTP female



rats (the highest risk finding for the NTP rats)  exceed the corresponding risk



estimate based on the salivary tumors by a factor  of three.



     Equivalent human dose and upper-bound incremental  unit risk  estimates were



developed using the standard assumptions of the Carcinogen  Assessment Group on



the inhalation rates of rodents and humans,  and use of the  body weight,  to the



two-thirds power interspecies extrapolation factor.



     Using the multistage UCL estimates for female mice with either adenomas  or



carcinomas of the lung and/or liver,  the upper-limit incremental  unit risk from



exposure over a lifetime to 1 mg/kg/day DCM is 1.4.x 10~2.   Equivalently, the



unit risk for inhaling 1'yg/m^ DCM over a lifetime, is 4.1 x 10"^;  the unit



risk for exposure to 1 ppm DCM is 1.4 x 10~2.



     Estimates of the incremental unit risk from exposure to DCM in drinking



water were made using two approaches:  first,  based on the  findings of liver,



but not lung, tumors in the NTP inhalation bioassay with mice, and secondly,



using.the suggestively positive finding of liver tumors in  the National  Coffee





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Association (1983) ingestion study in  mice.   Since  the  risk  estimates  from
these two studies are roughly comparable,  the mean  of the  derived  risk  values
is chosen for the unit risk estimate via  ingestion.  Using the  mean  of  the UCL
risk calculations from these two studies,  consumption of drinking  water con-
taining 1 \i g/L DCM over a lifetime has an  associated upper-bound incremental
unit risk estimate of 2.1 x 10'?.
     The upper-bound incremental unit  risk for inhalation  exposure estimated
using the NTP bioassay was compared with  the  findings of the strongest  epide-
miologic study of workers exposed to DCM.   While  the epidemiologic study did
not show any evidence for the carcinogenicity of  DCM, power  calculations showed
that the study also did not have the power to detect the estimated increase
with any degree of confidence.
1.2.  CONCLUSIONS
     Animal studies showed a statistically positive salivary gland sarcoma  re-
sponse in male rats (Dow Chemical Company, 1980)  and a  borderline  hepatocell-
ular neoplastic nodule response in female rats (National Coffee Association,
1982a, b).  There is some evidence of the carcinogenicity  of DCM in  male rats,
as shown by an increased incidence of benign  mammary gland neoplasms;  and
clear evidence in female rats (Dow Chemical Company,  1980; Burek et  al., 1984;
NTP, 1985).  There is clear evidence for the  carcinogenicity of DCM  in male
and female mice, as shown by statistically significant  increased incidences
of alveolar/bronchiolar neoplasms and hepatocellular neoplasms  (NTP, 1985
draft).  There is also evidence that DCM is weakly  mutagenic.  Using the pro-
posed EPA guidelines for carcinogen risk assessment (U.S.  EPA,  1984),  the
weight-of-evidence ranking for the carcinogenicity  of  DCM  in experimental
animals is "sufficient," and for human evidence the ranking is  "inadequate."
Overall, an EPA category of B2 is assigned to DCM,  meaning that DCM  is to be

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considered a "probable" human carcinogen.   According to the criteria of the
International  Agency for Research on  Cancer (IARC), the weight-of-evidence for
the carcinogenicity of DCM in animals is  "sufficient," placing  it  in Group 2B.
The upper-bound incremental  unit  risk for  the  inhalation  of air contaminated
with DCM is 4.1 x 10~6 (u g/m3)"1 -[B2].   The  upper-bound incremental
unit risk for drinking water is 0.21  x 10~6 (ug/L)"1  -[B2].  The  CA6
potency index for DCM is 1.2 (mmol/kg/day)"1,  which places DCM  in  the lowest
quartile of the ranking of the chemicals  that  the  CAG  has evaluated as suspect
carcinogens.

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                                2.   INTRODUCTION

     In February of 1985 the Office of Health  and  Environmental Assessment
published a Health Assessment Document on  Dichloromethane  (Methylene Chloride).
The document, which contained an analysis  of evidence  for  the  carcinogenic
potential of dichloromethane (DCM), concluded:   "Using the criteria of  the
International Agency for Research on Cancer (IARC),  the weight of  evidence  for
carcinogenicity in animals is judged to be limited.  .  . .  based upon the
statistically positive salivary gland sarcoma  response in  male rats  (Dow
Chemical Company, 1980) and the borderline hepatocell.ular  neoplastic nodule
response in the rat and hepatocellular adenoma and/or  carcinoma in male mice
(National Coffee Association, 1982-1983)."  It was further concluded:   "When
the absence of epidemiological evidence is considered  along with the limited
animal evidence, as well as the potential  for  DCM  to cause gene mutations  in
mammalian systems, DCM is judged to be in  IARC Group 3 ... ." Because the
National Toxicology Program  (NTP) inhalation  bioassay  has  been completed  (NTP,
1985, draft) and the report has been reviewed  and  approved by  the  NTP  Board of
Scientific Councillors, it is necessary to update  the  February 1985  Health
Assessment Document for Dichloromethane (Methylene Chloride).
     The purpose of this addendum is to:
     o  Review and integrate the data obtained  in the NTP inhalation  bioassay,
     »  Analyze the pharmacokinetic/metabolic  data  presented in Chapter 4 of
       the Health Assessment Document and  determine its usefulness in  the
       quantitative estimation of carcinogenic risk, and
     0  Revise the estimated carcinogenic potency for DCM using the data from
       the NTP bioassay and pharmacokinetic data if appropriate.
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                              3.  CARCINOGENICITY







3.1  NATIONAL TOXICOLOGY PROGRAM INHALATION BIOASSAY (1985,  DRAFT)



     A 2-year carcinogenesis study of DCM (99% pure) was  conducted  at  Battelle



Pacific Northwest Laboratories by inhalation exposure to  groups  of  50  male  and



female F344/N rats and B6C3F1 mice (6 hours/day,  5 days/week)  for 102  weeks.



The exposure concentrations used were 0, 1000, 2000, or 4000 ppm for  rats and



0, 2000, or 4000 ppm for mice.  These doses were  selected on the basis of re-



sults obtained from a 13-week subchronic inhalation study in which  animals



were exposed to concentrations of 525 to 8400 ppm 6 hours/day, 5 days/week.



The maximum exposure concentration of 4000 ppm was selected  because mimimal



histopathologic changes were found after exposure to 4000 ppm.  The second



dose was 2000 ppm for both species.  The third dose, 1000 ppm, was  added for



rats because in an earlier inhalation study in male and female Sprague-Dawley



rats (Dow Chemical Company, 1980; Burek et al., 1984) reduced survival was



observed in the highest exposure group, 3500 ppm.



     All animals used in this experiment were produced under strict barrier



conditions at Charles River Breeding Laboratories under a contract  to  the car-



cinogenesis program of the National Toxicology Program (NTP).   The  rats were



placed in the study at 7 to 8 weeks of age and mice at 8 to  9 weeks.   All



animals were housed individually.  Food and water were available ad libitum



except during exposure periods, when only water was available.  All animals



were observed twice a day for signs of moribundity or mortality.  Clinical



signs were recorded every week.  Body weight was  recorded once a week.  A



comp.lete quality-controlled environment was maintained during the experiment.



The probability of survival was estimated by the  product-limit procedure of



Kaplan and Meier  (1958).  Tests of significance included pair-wise  comparisons





                                       11

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of high-dose and low-dose groups  with  controls  and  tests  for  overall  dose-



response trends.  Life table analysis,  incidental tumor analysis,  and the



Fisher Exact Test were used to evaluate tumor incidence.



3.1.1.  Rat Study



     The mean body weights of experimental  and  control rats of  each  sex were



similar throughout the studies (Figure  1).   Rats  exposed  to 4000 ppm, the



highest dose, were restless and pawed  at the eyes and  muzzle  during  the expo-



sure period.  The survival of male and  female rats  exposed to DCM  is  shown  in



Figure 2.  The survival of female rats  was  significantly  lower  than  that of the



controls after week 100, and the  survival  in all  groups of male rats  at the



termination of the study was low  (Table 1). There  were many  deaths  of males in



the final 16 weeks of the study.   The  decreased survival  is believed  to be



related to high incidence of leukemias.



     There was. a significant positive  trend for mammary gland fibroadenoma  and



adenomas or fibroma (combined) in male  and  female rats.   The  incidence in high-



dosed males (0/50, 0/50, 2/50, and 5/50) and in females  (7/50,  13/50, 14/50,



and 23/50) were significantly (p  < 0.001) higher than  the controls (Tables  2



and 3).  Also, subcutaneous fibroma or sarcoma  (combined),  located in the mam-



mary area in male rats, occurred with  a significant positive  trend (p = 0.008),



and the incidence in the high-dose group was significantly  (p < 0.05) greater



than in the controls (Table 2).  The subcutaneous tumors  all  occurred in the



area of the mammary chain; therefore,  the subcutaneous tumors were combined by



the NTP for comparative purposes  (male rats 1/50, 1/50,  4/50, and  9/50).  The



incidence of subcutaneous tumors in the highest dose group was  significantly



(p = 0.002) higher in than the controls.  The  historical  incidence of mammary



gland tumors at the same laboratory is 0% in males  and 16% in females,  and  the



NTP historical control incidence is 3% in males and 28%  in females for the





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                             4t       M
                             WOKS ON STUDY
                                 §
                         -8-
                            *
                              WOKS ON STUDY
Figure 1.   Growth  curves for rats exposed  to dichloromethane
by inhalation  for  2 years.
SOURCE:  NTP,  1985.
                                13

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H a 2.000 PPU
A -4,000 PPU













                               WOKS ON STUDY
           FEMALE RATS
              UNTREATED
           O-1.000 PPVI
              2.000 PPU
              4,000 PPM
                              WEEKS ON STUDY
Figure  2.   Kaplan-Meier survival  curves  for rats exposed to
dicfiloromethane  by  inhalation  for 2 years.

SOURCE:   NTP, 1985.
                                  14

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                   TABLE 1.  SURVIVAL OF RATS IN THE 2-YEAR INHALATION STUDY OF DICHLOROMETHANE
                                                     Control.
      1,000 ppm
2,000 ppm
4,000 ppm
\
Male*
Animals initially in study
Nonacci dental deaths before termination15
Killed at termination
Survival p values0
Female3
Animals initially in study
Nonacci dental deaths before termination^5
Killed at termination
Survival p values0


50
34
16
0.116

50
20
30
0.006


50
34
16
0.945

50
28
22
0.223


. 50
33
17
0.935

- . 50
28
22
0.118


50
41
9
0.163

50
35
15
0.006
aTerminal  kill  period:  week 104.
^Includes  animals killed in a moribund condition.
cThe results of the life table trend test are in the control
 comparisons with the controls are in the dosed columns.

SOURCE:  NTP, 1985.
column, and those of the life table pairwise

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TABLE
ANALYSIS OF PRIMARY TUMORS IN MALE RATS IN THE TWO-YEAR INHALATION
                STUDY OF  OICHLOROMETHANE
                                Control
                                              1,000 ppm
                                                    2,000 ppm
4,000 ppm
Subcutaneous tissue: Fibroma
Overall rates8
Adjusted rates"
Terminal rates0
Week of first observation
Life table testsd.
Incidental tumor tests'1
Cochran-Armitage Trend Testd
Fisher Exact Testd
Subcutaneous tissue: Fibroma or sarcoma
Overall rates8
Adjusted rates0
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor tests'1
Cochran-Armitage Trend Testd
Fisher Exact Testd
Hematopoietic system: Hononuclear cell leukemia
Overall rates*
Adjusted rates0
Terminal rates0
Week of first observation
Life table tests'1
Incidental tumor testsd
Cochran-Armitage Trend Test"1
Fisher Exact Testd
Adrenal : Pheochromoeytoma
Overall rates8
Adjusted ratesb
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor tests'1
Cocnran-Armltage Trend Testd
Fisher Exact Testd
Adrenal: Pheochromoeytoma or pheochromocytoma,
Overall rates8
Adjusted rates0
Terminal rates0
Week of first observation
Life table tests'1
Incidental tumor tests'1
Cochran-Armltage Trend Testd
Fisher Exact Testd

1/50(2%)
6.3%
1/16(6%)
104
p- 0.024
p=0.064
p=0.072


1/50(2%)
6.3%
1/16(6%)
104
P'0.008
p-0.026
p-0.029


34/50(68%)
80.31
8/16(50%)
57
p-0.045
p-0.399
p-0.251


. 5/50(10%)
23.5%
2/16(13%)
75
p-0.035
p-0.131
p-0.192

malignant
5/50(10%)
23.5%
2/16(13%)
75
p-0.034
' p-0.134
p-0.186


1/50(2%)
6.3%
1/16(6%)
104
p=0.764
p=0.764

p-0.753

1/50(2%)
6.3%
1/16(6%)
104
p=0.764
p»0.764

p-0.753

26/50(52%)
77.0%
9/16(56%)
82
P-0.147N
p*0.049N

p=0.076N

11/50(22%)
46.4%
5/16(31%)
89
p=0.094
p-0.093

p-0.086

11/50(22%)
46.4%
5/16(31%)
89
p- 0.094
p-0.093

P'0.086

2/50(4%)
9.2%
1/17(6%)
96
p-0.523
p-0.505

p-0.500

2/50(4%)
9.2%
1/17(6%)
96
p=0.523
p=0.505

p-0.500

32/50(64%)
80.2%
10/17(59%)
71 '
P-0.400N
p=0,434N

p=0.417N

10/50(20%)
45.4%
6/17(35%)
89
p-0.149'
p»0.131

p»0.131

11/50(20%)
47.0%
6/17(35%)
89
p«0.104
p«0.087

p-0.086

4/50(8%)
19.5%
0/9(0%)
89
p-0.095
P'0.204

p=0.181

5/50(10%)
22.7%
0/9(0%)
89
p=0.050
p=0.125

p=0.102

35/50(70%)
89.4%
6/9(67%)
75
p=0.134
p=0.487N

P"0.500

10/50(20%)
52.9%
3/9(33%)
80
p«0.039
p«0.108

p»0.131

10/50(20%)
52.9%
3/9(33%)
80
p* 0.039
p-0.108

p=0.131
                                                               ('coh'f 1 nued  on  the  following page)
                                     16

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TABLE 2.  (continued)

Mammary gland: Fibroadenoma
Overall rates3
Adjusted rates6
Terminal ratesc
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Mammary gland: Adenoma or f ibroadenoma
Overall rates3
Adjusted ratesb
Terminal rates0
Week of first observation
Life table tests'1
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Mammary gland or subcutaneous tissue: Adenoma,
Overall rates3
Adjusted ratesb
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Testis: Interstitial cell tumor
Overall rates3
Adjusted rates'5
Terminal ratesc
Week of first observation
Life table testsd
Incidental tumor tests'1
Cochran-Armitage Trend Testd
Fisher Exact Testd
Tunica vaginalis: Malignant mesothelioma
Overall rates3
Adjusted ratesb
Terminal rates0
Week of first observation
Life table tests"
Incidental tumor tests'1
Cochran-Armitage Trend Testd
Fisher Exact Testd
Control

0/50(0%)
0.0%
0/16(0%)

p<0.001
p<0.003
p-0.009


0/50(0%)
0.0%
0/16(0%)

p<0.001
p<0.001
p=0.003

f ibroadenoma, or
1/50(2%)
6.3%
1/16(6%)
104
p<0.001
p=0.003
p<0.001


39/50(78%)
94.9%
14/16(88%)
65
p=0.009
p=0.114
p=0.129


0/50(0%)
0.0%
0/16(0%)

p-0.025
p»0.060
p=0.044

1,000 ppm

0/50(0%)
0.0%
6/16(0%)

e
e

e

0/50(0%)
0.0%
0/16(0%)

e
e

e
fibroma
1/50(2%)
6.3%
1/16(6%)
104
p=0.764
P'0.764

p=0.753N

37/49(76%)
97.3%
15/16(94%)
69
p*0.420N
p«0.385N '

p=0.478N

1/50(2%)
2.2%
0/16(0%)
. 69
p=0.496
p*0.473

p=0.500
2,000 ppm

2/50(4%)
11.8%
2/17(12%)
104
p<=0.250
p=0.250

p=0.247

2/50(4%)
11.8%
2/17(12%)
104
p=0.250
p*0.250

p=0.247

4/50(8%)
20.6%
3/17(18%)
96
p=0.196
p=0.186

p-0.181

41/60(82%)
95.2%
15/17(88%)
75
p*0.512
p=0.387

p=0.401

0/50(0%)
0.0%
0/17(0%)

e
e

e
4,000 ppp

4/50(8%)
34.0%
2/9(221)
101
p=0.020
p=0.040

p=0.059

5/50(10%)
36.6%
2/9(22%)
93
p=0.010
p=0.023

p=0.028

9/50(18%)
49.0'*
2/9(22%)
89
p=0.002
p=0.008

p= 0.008

43/50(86%)
97.7%
8/9(89%)
75
p=0.029
p=0.253

p=0.218

3/50(6%)
19.6%
0/9(0%)
92
p= 0.068
p=0.172

p=0.121
                                  (continued on the following page)
        17

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                                               TABLE  2.   (continued)
                                                   Control
                                                                 1,000  ppm
2,000 ppm
4,000 ppm
Tunica vaginalis: Mesothelioma (all  types)
  Overall  rates*                                   0/50(0%)       1/50(2%)         4/50(8%)         4/50(8%)'
  Adjusted rates1*                                  0.0%           2.2%             19.2%            24.4%
  Terminal ratesc                                  0/16(0%)       0/16(0%)         2/17(12%)        0/9(0%)
  Week of  first observation   '                                   69             .> 96               92
  Life table testsd            .                    p=0.009        p=0.496         p=0.070          p=0.031
  Incidental tumor testsd                          p«0.030        p=0.473         p=0.062          p=0.097
  Cochran-Armitage Trend Testd                     p=0.029
  Fisher Exact Testd                                             p=0.500         p=0.059          p=0.059

All  sites: Malignant mesothelioma
  Overall  rates9                                   0/50(0%)       2/50(4%)         0/50(0%)         3/50(6%)
  Adjusted ratesb                                  0.0%           4.4%             0.0%            19.6%
  Terminal ratesc                                  0/16(0%)       0/16(0%)         0/17(0%)         0/9(0%)
  Week of  first observation                                      69                               92
  Life table tests"                                p=0.066        p*0.243         e               p=0.068
  Incidental tumor testsd                          p=0.136        p=0.225         e               p=0.172
  Cochran-Armitage Trend Testd                     p=0.097
  Fisher Exact Testd                                             p=0.247         e               p=0.121

All  sites: Mesothelioma (all types)
  Overall  rates9                                   0/50(0%)       2/50(4%)         5/50(10%)        4/50(8%)
  Adjusted ratesb                                  0.0%           4.4%             22.8%            24.4%
  Terminal rates^                                  0/16(0%)       0/16(0%)         2/17(12%)        0/9(0%)
  Week of first observation                                      69               96               92
  Life table testsd                                p=0.020        p=0.243         p=0.038          p=0.031
  Incidental tumor testsd                          p=0.063        p=0.225         p=0.030          p=0.097
  Cochran-Armitage Trend Testd                     p=0.052
  Fisher Exact Testd                                             p=0.247      '   p=0.028          p=0.059
^Number of tumor-bearing animals/number of animals examined at the site.
^Kaplan-Meier estimated tumor incidences at the end of the study after adjusting for intercurrent mortality.
C0bserved tumor incidence at terminal kill.
dBeneath the control incidence are the p values associated with the trend test.  Beneath the dosed group
 incidence are the p values corresponding to pairwise comparisons between the dosed group and the controls.
 The life table analysis regards tumors in animals dying prior to terminal  kill as being (directly or indirectly)
 the cause of death.  The incidental tumor test regards these lesions as nonfatal.  The Cochran-Armitage Trend
 Test and the Fisher Exact Test directly compare the overall  incidence rates.  A negative trend or lower
 incidence in a dose group is indicated by the letter N.
eNo p value is presented because no tumors were observed in the dosed and control  groups.

Mammary gland fibroadenoma:  Historical incidence at testing laboratory 0/100 (0%); historical  incidence in NTP
 studies 51/1,727 (3%) t 3%.
Mesothelioma--all sites:  Historical incidence at testing laboratory 4/100 (4%); historical  incidence in NTP
 studies 44/1,727 (3%) ±2%.
Mononuclear cell leukemia: Historical incidence at testing laboratory 36/100 (36%); historical  incidence in NTP
 studies 458/1,727 (27%) ± 9%.

SOURCE:  NTP, 1985.
                                                         18

-------
TABLE 3.
ANALYSIS OF PRIMARY TUMORS  IN FEMALE  RATS  IN  THE TWO-YEAK  INHALATION
                 STUDY  OF DICHLOROMETHANE
                                 Control'
                                     1,000  ppm
2,000 ppm
4,000 ppm
Hematopo1et1c system: Mononuclear cell leukemia
Overall rates8
Adjusted ratesb
Terminal ratesc
Week of first observation
Life table tests'1
Incidental tumor tests'1
Cochran-Armitage Trend Testd
Fisher Exact Testd
Liver: Neoplastic nodule
Overall rates3
Adjusted rates'"
Terminal rates'
Meek of first observation
Life table testsd
Incidental tumor tests'1
Cochran-Armitage Trend Testd
Fisher Exact Testd
Liver: Neoplastic nodule or hepatocellular
Overall rates'
Adjusted ratesb
Terminal rates0
Week of first observation
Life table tests'1
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Mammary gland: Flbroadenoraa
Overall rates8
Adjusted ratesb
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Mammary gland: Adenoma or fibroadenoma
Overall rates8
Adjusted ratesb
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Test"3
17/50(34%)
41.11
8/30(271)
73
p= 0.009
p-0.273
p«0.086


2/50(41)
6.71
2/30(71)
104
p-0.030
p=0.097
p-0.078

carcinoma
2/50(4%)
6.71
2/30(71)
104
p-0.027
p-0.086
P'0.079


5/50(101)
15.71
4/30(131)
96
p<0.001
p<0.001
p<0.001


5/50(101)
15.71
4/30(131)
96
p<0.001
p<0.001
p<0.001

17/50(341)
44.41
4/22(181)
76
p=0.402
P-0.425N

P-O.S84N

1/50(2%)
2.4%
0/22(0%)
61
p»0.5'69N
P-0.494N

P-0.500N

1/50(2%)
2.0%
0/22(0%)
61
P-0.569N
P-0.494N

p-O.SOON

11/50(221)
41.21
8/22(36%)
74
p- 0.028
p»0.04Q

p*0.086

11/50(22%)
41.2%
8/22(36%)
74
p- 0.028
p- 0.049

p-0.086
23/50(46%)
63.6%
10/22(45%)
73
p-0.049
p-0.189

p*0.154

3/50(6%)
10.2%
1/22(51)
85
p= 0.382
p-0.482

p=0.500

4/50(8%)
14.41
2/22(91)
85
P'0.223
p=0.297

p-0.339

13/50(26%)
43.6%
7/22(32%)
65
p=0.009
p=0.025

p'0.033

13/50(26%)
43.6%
7/22(32%)
65
p»0.009
p«0.025

p-0.033
23/50(46%)
53.1%
1/15(71)
63
p=0.028
p«0.579

P'0.154

5/50(10%)
19.6%
1/15(7%)
73
p= 0.080
p=0.229

?'0.218

5/50(10%)
19.6%
1/15(7%)
73
p=0.080
p«0.229

P'0.218

22/50(44%)
79.4%
10/15(67%)
73
p<0.001
p<0.001

p<0.001

23/50(46%)
83.51
11/15(731)
73
p<0.001
p<0.001

p<0.001
                                                               (continued  on  the following  page)
                                    19

-------
                                               TABLE 3.  (continued)
                                                   Control
                                                                 1,000 ppm
                                           2,000 ppm
                                4,000 ppm
Mammary gland: Adenoma, fibroadenoma.
  Overall ratesa
  Adjusted rates'5
  Terminal ratesc
  Week of first observation
  Life table testsd
  Incidental tumor tests'1
  Cochran-Armitage Trend Test'1
  Fisher Exact Testd

Mammary gland: Adenoma, fibroadenoma.
  Overall rates3
  Adjusted rates'5
  Terminal ratesc
  Week of first observation
  Life table tests'1
  Incidental tumor tests'1
  Cochran-Armitage Trend Testd
  Fisher Exact Testd
or adenocarcinoma
             6/50(12%)
             17.8%
             4/30(13%)
             92
             p<0.001
             p<0.001
             p<0.001
13/50(26%)
44.4%
8/22(36%)
74
p=0.023
p=0.053

p=0.062
adenocarcinoma,  or mixed  tumor,  malignant
             7/50(14%)
             20.0%
             4/30(13%)
             92
             p<0.001
             p<0.001
             p<0.001
13/50(26%)
44.4%
8/22(36%)
74
p=0.045
p=0.092

p*0.105
14/50(28%)
44.9%
7/22(32%)
65
p=0.012
p=0.043

p=0.039
e!4/50(28%)
 44.9%
 7/22(32%)
 65
 p=0.022  •
 p=0.083

 p=0.070
                 23/50(46%)
                 83.5%
                 11/15(73%)
                 73
                 p<0.001
                 p<0.001

                 p<0.001
                23/50(46%)
                83.5%
                11/15(73%)
                73
                p<0.001
                p<0.001

                p<0.001
aNunber of tumor-bearing animals/number of animals examined at the site.
bKaplan-Meier estimated tumor incidences at the end of the study after adjusting for intercurrent mortality.
C0bserved tumor incidence at terminal kill.
^Beneath the control incidence are the p .values associated with the trend test.  Beneath the dosed group
 incidence are the p values corresponding to pairwise comparisons between the dosed group and the controls.
 The life table analysis regards tumors in animals dying prior to terminal kill as being (directly or indirectly)
 the cause of death.  The incidental tumor test regards these lesions as nonfatal.  The Cochran-Armitage Trend
 Test and the Fisher Exact Test directly compare the overall incidence rates..  A negative trend or lower
 incidence in a dose group is indicated by the letter N.
eA carcinoma was also present in one of the animals that had a fibroadenoma.

Mammary gland fibroadenoma:  Historical incidence at testing laboratory 16/99 (16%); historical incidence in NTP
 studies 492/1,772 (28%) * 10%.
Mononuclear cell leukemia:  Historical incidence at testing laboratory 27/99 (27%); historical incidence in NTP
 studies 307/1,772 (17%) ± 6.

SOURCE:  NTP, 1985.
                                                        20

-------
same strain of rats.  The increased incidence of mammary  gland tumors  is
consistent with the results reported by Dow Chemical  Company (1980)  and
Burek et al. (1984) in Sprague-Dawley rats (U.S. EPA, 1985).  This study  has
been reviewed previously.  Sprague-Dawley rats have a spontaneous  incidence of
mammary gland tumors, about 80% in female and 10% in  males.   In males  (Burek
et al., 1984) the mammary tumors increased in the highest dose group to 14/97,
as compared to 7/92 in the controls.  In females the  number  of tumors  per rat
increased with dose.  The increased incidence of benign mammary gland  tumors
in males and females provides some supportive evidence for mammary gland
carcinogenesis.  Maltoni (1984) also reported at the  Food Solvent  Workshop
(March 8-9, 1984) on a study in which DCM was administered by gavage at 500
mg/kg/day, 5 days/week for 64 weeks, followed by an observation period until
spontaneous death.  Maltoni (1984) observed an increased incidence of  mammary
gland tumors in Sprague-Dawley rats.  Thus, the incidence of mammary gland
benign tumors in female rats in the present NTP study is consistent  with  the
reports of Burek et al.  (1984), Maltoni (1984), and  Nitschke et al. (1982).
     The incidence of liver neoplastic nodules or hepatocellular carcinomas
(combined) in female rats  (2/50, 1/50, 4/50, and 5/50) occurred with positive
trends (p = 0.030) by life table analysis only (Table 3).  The incidence  in
the high-dose group was not significantly greater than in the controls; this
result is consistent with observations made by other  investigators (Maltoni,
1984; NTP, 1982, unpublished; National Coffee Association, 1982a,  b).   Maltoni
(1984) reported that gavage administration of 100 or  500 mg/kg/day for 64
weeks induced a dose-related increase in the incidence of nodular  hyperplasia
of the liver in Sprague-Dawley rats.  The earlier gavage study (NTP, 1982,
unpublished) indicated that administration of 500 or  1000 mg/kg/day  increased
the incidence of hepatocellular nodules in both male  and female F344/N rats.

                                       21

-------
Furthermore, there was a significant increase  in  liver  tumors  in  female  rats



dosed at 250 mg/kg/day in the drinking water study  (National Coffee  Associa-



tion, 1982), although the number of tumors  were within  the  range  of  the  his-



torical control values of the laboratory.   The pharmacokinetic data  presented



by Dr. Kirshman at the Food Solvent Workshop (1984,  page 41) indicated that 250



my/kg/day in the drinking water study was  equivalent to a 750  ppm inhalation



level, which is 1/5 of the MTD used in the  Burek  et  al. (1984) study and in the



NTP (1985) study.  The NTP reported that the highest exposure  concentration



(4000 ppm) in the inhalation study has been estimated to be equivalent to 1300



mg/kg/day from an oral dose.



      In male rats the incidence of mesothelioma  arising from tunica  vaginalis



(0/5, 2/50, 5/50, and 4/50) occurred with  a significant positive  trend  (p =



0.02); the incidence in the mid- and high-dose groups was significantly  higher



(p =  0.038, p = 0.30) than in the controls  (Table 2).  This increased incidence



may not be due to administration of DCM because  the  concurrent controls  in the



same  laboratory were low in comparison with earlier  inhalation studies  (4/100).



Mononuclear cell leukemia in male (Table 2) and  female (Table  3)  rats occurred



with  a significant positive trend by life table  analysis only.  The  incidence



(17/50, 17/50, 23/50, 23/50) in females was significantly greater than  in the



controls at the mid-dose (p = 0.049) and high-dose (p = 0.028) levels.



      Some other tumor incidences were increased marginally in  experimental



groups as compared to tnc controls.  These increases were characterized  by a



significant trend only.  These tumors included adrenal  gland pheochromocytoma



and interstitial cell tumors in males (Table 2)  and pituitary  gland  adenoma



or carcinoma (combined) in males and females (Tables 2 and 3).  The  squamous



cell  metaplasia of the nasal cavity in female rats (1/50, 2/50, 3/50, and 9/50)



was increased significantly in the high-dose group,  but no nasal  tumors  were





                                       22

-------
found in this group.



3.1.2.  Mouse Study



     The mean initial body weight of males in the 4000 ppm group  was  15%  lower



than that of controls (Figure 3).  The mean body weights were comparable  in



the high-dose and control groups until week 90, but after week 90 the body



weights were 8% to 11% lower than those of controls.   During the  exposure



period, the mice were hyperactive.  The probabilities of survival  of  male and



female mice are shown in Figure 4.  The survival in both male and female  high-



dose groups decreased significantly compared with controls (Table 4).  The



reduced survival may have been due to chemically-induced lung and liver tumors



in both male and female mice.



     The incidences of lung tumors increased significantly (p = 0.0001) in  both



males  (Table 5) and females (Table 6).  The latent period for tumor induction



was significantly (trend analysis) decreased in the high-dose groups  as compared



to controls.  The tumors observed were alveolar/bronchiolar adenomas  (males,



3/50,  19/50, and 24/50; females, 2/50, 23/48, and 28/48) and alveolar/bronchiolar



carcinomas  (males, 2/50, 10/50, and 28/50; females, 1/50, 13/48,  29/48, and



29/48).  In addition to the dose-related increase in lung tumors  in male  and



female mice, there were dose-related increases in multiple lung tumor-bearing



mice  (Table 7).  The multiplicity of tumors included both alveolar/bronchiolar



adenoma and carcinoma.  Only one lung tumor per mouse was found in the controls,



whereas 70% of the high-dose males and females had multiple tumors (males,



0/50,  10/50, and 28/bO: females, 0/50, 11/48, and 29/48).  In the experimental



groups, 38% of the dosed male mice and 42% of the dosed female mice had multiple



lung tumors.  These results are consistent with the data obtained in  other



studies.  In the earlier NTP (1982, unpublished) gavage study, DCM produced a



significant increase in lung tumors in female mice.   Maltoni (1984)  also





                                       23

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> a M 4i M n M K>
                             WEEKS ON 51UDY
Figure  3.   Growth  curves for mice  exposed to  dichloromethane
by inhalation for  2  years.


SOURCE:   NTP, 1985.
                               24

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M
I
           JALE UICE
           = UNTREATED I
         IO=2.000 PPUI
                              WEEKS ON STUDY
          FEMALE UICE
          • a UNTREATED
          O» 2.000 PPM
            4.000 PPM
                              WEEKS ON STUDY
 Figure  4.   Kaplan-Meier  survival curves for mice exposed to
 dichloromethane  by inhalation for  2  years.

 SOURCE:   NTP, 1985.
                                 25

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                           TABLE  4.   SURVIVAL  OF  MICE  IN  THE  2-YEAR  INHALATION  STUDY
                                               OF DICHLOROMETHANE
                                                              Control
                                                           2,000 ppm
           4,000 ppm
ro
CTl
Male3

Animals initially in study
Nonaccidental deaths before termination^
Accidentally killed
Killed at termination
Died during termination period
Survival p values0

Female9

Animals initially in study
Nonaccidental deaths before termination'3
Accidentally killed
Killed.at termination
Died during termination period
Survival p values0
                                                                 50
                                                                 11
                                                                 0
                                                                 39
                                                                 0
                                                                 <0.001
                                                                 50
                                                                 24
                                                                 1
                                                                 0
                                                                 25
                                                                 0.002
50
24
 2
24
 0
<0.010
50
22
 2
 1
25
 0.678
 50
 38
  1
  9
.  2
 <0.001
 50
 40
  1
  1
  8
  0.004
               aTerminal kill period: week 104.
               ^Includes animals killed in a moribund condition.
               cThe results of the life table trend test are in the control column, and those of
                the life table pairwise comparisons with the controls are in the dosed columns.
               SOURCE:  NTP, 1985.

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TABLE 5.
          ANALYSIS OF  PRIMARY  TUMORS  IN  MALE MICE  IN THE TWO-YEAR INHALATION
                          STUDY  OF  D1CHLOROMETHANE

Lung: Al veolar/bronchlolar adenoma
Overall rates8
Adjusted rates6
Terminal rates0
Week of first observation
Life table tests'1
Incidental tumor testbd
Cochran-Annitaye Trend Testd
Fisher Exact Testd
Luny: Al veolar/bronchfolar carcinoma
Overall rates8
Adjusted ratesb
Tenninal ratesc
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Lung: Al veolar/bronchlolar adenoma or
Overall rates3
Adjusted rates'3
Tenninal rates0
Week of first observation
Life table tests'1
Incidental tumor tests'1
Cochran-Armitaye Trend Testd
Fisher Exact Testd
Circulatory system: Hemangiosarcoma
Overall rates8
Adjusted rates6
Terminal ratesc
We»K of first observation
Life table tests'1
Incidental tumor tests'1
Cochran-Armitaye Trend Testd
Fisher Exact Testd
Control

3/50(6%)
7.7%
3/39(8%)
104
p<0.001
p<0.001
p<0.001


2/50(,«)
4.9%
1/39(3%)
94
p<0.001
p<0.001
p<0.001

carcinoma
5/50(10%)
12.4%
4/39(10%)
94
p<0.001
p<0.001
p<0.001


1/50(2%)
2.6%
1/39(3%)
104
p-0.007
p-0.083
p=0.06Q

2,000 ppm

19/50(38%)
55.6%
10/24(42%)
71
p<0.001
p<0.001

p<0.001

10/50(20%)
34.0%
6/24(25%)
78
p<0.002
p<0.016

p<0.014

27/50(54%)
74.2%
15/24(63%)
71
p<0.001
p<0.001

p<0.001

2/50(4%)
7.6%
1/24(4%)
101
p=0.352
p=0.495

p=0.500
4,000 ppm

24/50(48i)
78.5%
6/11(55%)
70
p<0.001
p<0.001

p<0.001

28/50(56%)
92.9%
9/11(82%)
72
p<0.001
p<0.001

p<0.001

40/50(80%)
. 100.0%
11/11(100%)
70
p<0.001
p<0.001

p<0.001

5/50(10%)
21.4%
1/11(9%)
70
p=0.017
p*0.142

p=0.102
Circulatory system: Hemangioma or hemangiosarcoma
Overall rates*
Adjusted rates6
Terminal rates0
Week of first observation
Life table tests'1
Incidental tumor tests'1
Cochran-Armitage Trend Test11
Fisher Exact Testd
2/50(4%)
4.8%
1/39(3%)
87
p-0.010
p<=0.170
p«0.080

2/50(4%)
7.6%
1/24(4%)
101
p=0.558
p*0.643N

p=0.691
6/50(12%)
25.8%
1/11(9<)
70
p-0.022
p='!.^U

p=0.134
                                  27

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                                               TABLE  5.   (continued)
                                                 Control
                                                                         2,000  ppm
                        4,000 ppm
Liver: Hepatocellular adenoma
Overall rates3
Adjusted ratesb
Terminal ratesc
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd

10/50(20%)
23.0%
7/39(181)
73
p<0.001
p<0.075
p-0.194


14/49(29%)
46.9%
9/24(38%)
71
p=0.041
p«0.161

p=0.224

14/49(29%)
68.3%
6/11(55%)
80
p=0.001
p=0.095

p=0.224
Liver: Hepatocellular carcinoma
  Overall rates3                                13/50(26%)
  Adjusted ratesb                               29.7%
  Terminal rates^                               9/39(23%)
  Week of first observation                     73
  Life table tests'1                             p<0.001
  Incidental tumor tests'1                       p=0.016
  Cochran-Armitage Trend Testd                  p=0.004
  Fisher Exact Testd

Liver: Hepatocellular adenoma or carcinoma
  Overall rates3                                22/50(44%)
  Adjusted ratesb                               48.3%
  Terminal rates^                               16/39(41%)
  Week of first observation                     73
  Life table testsd                             p<0.001
  Incidental tumor tests'1                       p=0.010
  Cochran-Armitage Trend Testd                  p=0.013
  Fisher Exact Testd
15/49(31%)
43.7%
7/24(29%)
72
p-=0.111
p=0.422

p«0.387
22/49(49%)
66.8%
13/24(54%)
71
p=0.048
p=0.305

p=0.384
26/49(53%)
76.4%
5/11(45%)
61
p<0.001
p=0.042

p=0.005
33/49(67%)
93.0%
9/11(82%)
61
p<0.001
p=0.020

p=0.016
aNumber of tumor-bearing animals/number of animals examined at the site.
bKaplan-Meier estimated tumor incidences at the end of the study  after  adjusting  for intercurrent  mortality.
'Observed tumor incidence at terminal kill.
^Beneath the control incidence are the p values associated with the trend test.   Beneath the dosed group
 incidence are the p values corresponding to pairwise comparisons between the dosed group and the  controls.
 The life table analysis regards tumors in animals dying prior to terminal  kill as  being (directly or indirectly)
 the cause of death.  The incidental tumor test regards these lesions  as  nonfatal.   The Cochran-Armitage Trend
 Test and the Fisher Exact Test directly compare the overall  incidence  rates.  A  negative trend or lower
 incidence in a dose group is indicated by the letter N.

Alveolar/bronchiolar adenoma or carcinoma:  Historical incidence  at testing laboratory 31/100 (31%);  historical
 incidence in NTP studies 296/1,780 (17%) t 8%.
Hepatocellular adenoma or carcinoma:  Historical incidence at testing  laboratory  28/100 (28%); historical inci-
 dence in NTP studies 540/1,784 (30%) ± 8%.
Hemangioma or hemangiosarcoma:  Historical incidence at testing laboratory 2/100  (2%); historical  incidence  in NTP
 studies 78/1,791 (4%) ± 4%.

SOURCE:  NTP, 1985.
                                                      28

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TABLE 6.
          ANALYSIS OF  PRIMARY  TUMORS  IN FEMALE  MICE  IN  THE TWO-YEAR  INHALATION
                           STUDY  OF IHCHLOKOMETHANE

Lung: Al veolar/bronchiolar adenoma
Overall rates3
Adjusted ratesh
Terminal ratesc
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Lung: Al veolar/bronchiolar carcinoma
Overall rates3
Adjusted rates6
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor tests'1
Cochran-Armitage Trend Testd
Fisher Exact Testd
Lung: Al veolar/bronchiolar adenoma or
Overall rates9
Adjusted rates'"
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Liver: Hepatocel lular adenoma
Overall rates3
Adjusted ratesb
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitaije Trend Testd '
Fisher Exact Testd
Liver: Hepatocel lular carcinoma
Overall rates3
Adjusted rates'5
Terminal rates0
Week of first observation
Life table testsd
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd
Control

2/50(4%)
6.7%
1/25(4%)
87'
p<0.001
p<0.001
p<0.001


1/50(4%)
4.0%
1/25(4%)
104
p<0.001
p<0.001
p<0.001

carcinoma
3/50(6%)
10.6%
2/25(8%)
87
p<0.001
p<0.001
p<0.001


2/50(4%)
6.5%
1/25(4%)
84
p<0.001
p<0.001
p<0.001


1/50(2%)
4.0%
1/25(4%)
104
p<0.001
p<0.001
p<0.001

2,000 ppro

23/48(48%)
66.5%
14/25(56%)
83
p<0.001
' p<0.001

p<0.001

13/48(27%)
45.9%
10/25(40%)
89
p<0.001
p<0.001

p<0.001

30/48(63%)
82.9%
19/25(76%)
83
p<0.001
p<0.001

p<0.001

6/48(13%) ••
21.3%
4/25(16%)
96
p=0.151
p=0.155

p=0.121

11/48(23%)
34.0%
.6/25(24%)
83
p=0.005
p=0.004

p=0.001
4,000 ppm

28/48(58%)
91.1%
6/a'v7S'il
68
p<0.001
p<0.001

p<0.001

29/48(60%)
92.2%
6/8(75S)
68
p<0.001
p<0.001

p<0.001

41/48(85%)
100.0%
8/8(100%)
63
p<0.001
p<0.001

p<0.001

22/48(46%)
83.0%
5/3(63%)
63
p<0.001
p<0.001

p<0.001

32/48(67%)
96.5%
7/8(88%)
68
p<0.001
p<0.001

p<0.001
                                                               (continued  on  the  following page)
                                  29

-------
                                               TABLE  6.   (continued)
                                                 Control
Thyroid gland:  Follicular cell  adenoma
  Overall  rates8                                1/48(21)
  Adjusted ratesb                               4.2%
  Terminal rates0                               1/24(4%)
  Week of  first observation                     104
  Life table testsd                   '          p*0.012
  Incidental tumor testsd                       p«0.040
  Cochran-Armitaye Trend Testd                   p-0.093
  Fisher Exact Testd
                                                                         2,000 ppm
1/47(2%)
4.0%
1/25(4%)
104
p=0.754N
p=0.754N

p=0.747
                        4,000 ppm
Liver: Hepatocellular adenoma or carcinoma
Overall rates9
Adjusted ratesb
Terminal rates0
Week of first observation
Life table tests'1
Incidental tumor testsd
Cochran-Armitage Trend Testd
Fisher Exact Testd

3/50(6%)
10.4%
2/25(8%)
84
p<0.001
p<0.001
p<0.001


16/48(33%)
48.0%
9/25(36%)
83
p=0.002
p=0.002

p<0.001

40/48(83%)
100.0%
8/8(100%)
68
p<0.001
p<0.001

p<0.001
4/46(9%)
35.0%
2/8(25%)
77
p=0.022
p=0.069

p=0.168
aNumber of tumor-bearing animals/number of animals  examined  at  the  site.
bKaplan-Meier estimated tumor incidences at the  end of  the study  after  adjusting  for  intercurrent  mortality.
C0bserved tumor incidence at terminal  kill.
dBeneath the control  incidence are the p values  associated with the trend  test.   Beneath  the  dosed group
 incidence are the p values corresponding to pairwise comparisons between  the  dosed group and the  controls.
 The life table analysis regards tumors in animals  dying  prior  to terminal  kill as being  (directly or  indirectly)
 the cause of death.  The incidental  tumor test  regards these  lesions as nonfatal.  The Cochran-Armitage  Trend
 Test and the Fisher Exact Test directly compare the overall incidence  rates.  A  negative trend  or lower
 incidence in a dose group is indicated by the letter N.

Alveolar/bronchiolar adenoma or carcinoma:  Historical  incidence  at testing laboratory  10/100 (10%); historical
 incidence in NTP studies 122/1,777 (7%) ± 4%.
Hepatocellular adenoma or carcinoma:   Historical incidence at  testing laboratory  5/100  (5%);  historical  incidence
 in NTP studies 147/1,781 (8%) ±5%.

SOURCE:  NTP, 1985.
                                                       30

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                TABLE 7.   MULTIPLICITY OF PULMONARY TUMORS IN MICE
                            EXPOSED TO DICHLOROMETHANE
                                                 Exposure groups (ppm)
Diagnoses
                2,000
                 4,000
Male

One adenoma and
  one carcinoma
Multiple adenomas
Multiple carcinomas
Multiple adenomas
  and multiple carcinomas
One adenoma and
  multiple carcinomas
Multiple adenomas and
  one carcinoma

Incidence of mice with
  multiple tumors

No. of mice with multiple
  tumors/no, of mice with
  pulmonary tumors

Female

One adenoma and
  one carcinoma
Multiple adenomas
Multiple carcinomas
Multiple adenomas
  and multiple carcinomas
One adenoma and
  multiple carcinomas
Multiple adenomas and
  one carcinoma

Incidence of mice with
  multiple tumors

No. of mice with multiple
  tumors/no, of mice with
  pulmonary tumors
0/50
0/50
0/50

0/50

0/50

0/50


0/50(0%)



0/5(0%)
0/50
0/50
0/50

0/50

0/50

0/50


0/50(0%)



0/3(0%)
 1/50
 5/50
,3/50

 0/50

 1/50

 0/50


10/50(20%)



10/27(37%)
 2/48
 4/48
 1/48

 0/48

 2/48

 2/48


11/48(23%)



11/30(37%)
 3/50
 4/50
12/50

 3/50

 3/50

 3/50


28/50(56%)



28/40(70%)




 4/48
 5/48
 8/48

 2/48

 7/48

 3/48


29/48(60%)



29/41(71%)
SOURCE:  NTP, 1985.
                                       31

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reported an increased incidence of lung tumors  (one and one-half times)  in



male mice dosed with 100 to 500 mg/kg/day DCM by gavage.



     In male mice (Table 5) the hepatocellular  adenoma  or carcinoma  (combined)



(22/50, 24/49, and 33/49) and hepatocellular carcinoma  (13/50,  15/49,  and  26/49)



were increased significantly (p < 0.001), especially at 4000 ppm.  In  female



mice (Table 6), DCM produced dose-related increases in  both hepatocellular



adenoma (2/50, 6/48, and 22/48) and hepatocellular carcinoma (1/50,  11/48,  and



32/48), which were highly significant by any statistical  test.   The  incidence



of these tumors in the controls was consistent  with the historical  control



values of this laboratory.  The multiplicity of the hepatocellular  neoplasms



was common in the male and female dosed mice (Table 8).  It should  be  noted



that only 4% of the male control mice and none  of the female control mice  had



multiplicity of liver tumors.  The multiplicity of hepatocellular tumors in



both male and female mice increased significantly in a  dose-related  manner



(males, 2/50, 11/49, and 16/46; females, 0/50,  3/48, and 28/48).  There  were



27/57  (47%) males and 31/56  (55%) females with  multiple tumors.  The increased



incidence of hepatocellular tumors is consistent with the results of the



previous NTP  (1982, unpublished) gavage study,  the Maltoni  (1984) study, and



the National Coffee Association (1983) drinking water study.  The National



Coffee Association study produced a borderline significant increase  in liver



tumors at a dose significantly less than the maximum tolerated dose.  There



was also an increase in hemangiosarcoma  (1/50, 2/50, and 5/50) or hemangioma



and hemangiosarcoma  (2/50, 2/50, and 6/50) in male mice  (Table 7),  which



occurred with a positive trend by life table analysis.



3.1.3.  Summary



     The results of the NTP  inhalation bioassay  (1985,  draft) using F344/N



rats showed an increased incidence of benign mammary gland  neoplasms,  primarily





                                       32

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                  TABLE 8.  MULTIPLICITY OF LIVER TUMORS IN MICE
                            EXPOSED TO DICHLOROMETHANE
                                                 Exposure groups (ppm)
Diagnoses
                2,000
                 4,000
Male

One adenoma and
  one carcinoma
Multiple adenomas
Multiple carcinomas
Multiple adenomas
  and multiple carcinomas
One adenoma and
  multiple carcinomas
Multiple adenomas and
  one carcinoma

Incidence of mice with
  multiple tumors

No. of mice with multiple
  tumors/no, of mice with
  pulmonary tumors

Female

One adenoma and
  one carcinoma
Multiple adenomas
Multiple carcinomas
Multiple adenomas
  and multiple carcinomas
One adenoma and
  multiple carcinomas
Multiple adenomas and
  one carcinoma

Incidence of mice with
  multiple tumors

No. of mice with multiple
  tumors/no, of mice with
  pulmonary tumors
1/50
0/50
1/50

0/50

0/50

0/50


2/50(4%)



2/22(9%)
0/50
0/50
0/50

0/50

0/50

0/50


0/5(0%)



0/3(0%)
 2/49
 3/49
 3/49

 0/49

 0/49

 3/49


11/49(22%)



11/24(46%)
 1/48
 0/48
 2/48

 0/48

 0/48

 0/48


 3/48(6%)



 3/16(19%)
 3/49
 3/49
 6/49

 1/49

 2/49

 1/49


16/49(33%)



16/33(48%)
 6/48
 4/48
10/48

 3/48  •

 1/48

 4/48


28/48(58%)



28/40(70%)
SOURCE:  NTP, 1985.
                                       33

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fibroadenomas, in both male and female rats.   In  female  rats,  there was  also  a
significant increase in hepatocellular neoplastic nodules  and  hepatocellular
carcinomas (combined) by the trend test only  and  a statistically  significant
increase of mononuclear cell leukemias by  age adjustment.   In  male rats, there
was a significant increase in mesotheliomas,  primarily  from the tunica vagina-
lis.  Lastly, there was a marginally  significant  increase  in adrenal  pheochro-
mocytomas and interstitial cell tumors in  males  and pituitary  gland adenomas
and carcinomas (combined) in male and female  rats by the trend test only.
     In the NTP inhalation bioassay using  B6C3F1  mice,  a highly significant
increase in alveolar/bronchiolar adenoma and/or  carcinoma  was  observed in  both
sexes.  The incidence of hepatocellular adenoma  and hepatocellular carcinoma
(combined) was increased in the high-dose  males  and in  both dosed groups of
females.  There was also a dose-related increase in the number of mice bearing
multiple lung and liver tumors.  Only one  lung tumor per mouse was found,
whereas 38% of all dosed male mice and 42% of all dosed female mice had  mul-
tiple lung tumors.  The incidence of multiple hepatocellular tumors in the
exposed groups increased in both sexes in  a dose-related manner.  Multiple
hepatocellular tumors were found in only 4% of the male controls, but none were
found in the female controls; in contrast, 28% of the exposed  males and  32% of
the exposed females exhibited multiple liver tumors.
     The NTP concluded that, under the conditions of this  bioassay, there
was: some evidence of DCM carcinogenicity  for male F344/N  rats as shown  by an
increased incidence of benign neoplasms of the mammary gland,  sufficient or
clear evidence of DCM carcinogenicity for  female F344/N rats as  shown by an
increased incidence of benign neoplasms of the mammary gland,  and sufficient  or
clear evidence of DCM carcinogenicity in male and female B6C3F1  mice  as  shown
by  increased  incidences of  lung and liver tumors.

                                       34

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3.2.  PHARMACOKINETICS/METABOLISM

     The purpose of this analysis is to determine  if  there  are  available

pharmacokinetic/metabolism data that can be useful  in the assessment  of the

carcinogenic risk arising from exposure to methyl ene  chloride (DCM).   Most of

the data used in this analysis have previously been reviewed  (U.S.  EPA, 1985)

and therefore will not be discussed in detail.  Data  on  routes  of  exposure,

rates of ingestion, metabolism, and administered versus  effective  dose in

the NTP bioassay have been reviewed in order to determine if  there are

qualitative or quantitative differences or similarities  between species

which may alter the assumptions used in estimating risks.   Relevant new data

have been incorporated as appropriate.

3.2.1.  In Vitro Metabolism/Pathways

     The preponderance of data obtained from both  in  vivo and in vitro

experiments indicate that DCM and .other dihalomethanes are  biotransformed to

both carbon monoxide and carbon dioxide.  Carbon monoxide is  the end product

of microsomal oxidation, and carbon dioxide is an  end product of cytosolic

metabolism.

     A number of investigators (Kubic and Anders,  1975,  1978; Hogan et al.,

1976; Stevens and Anders, 1978, 1979; and Ahmed and Anders, 1978)  have studied

the metabolism of DCM and other dihalomethanes in experiments using rat  liver

microsomes.  These studies have resulted in the following observations:

     o   NADPH and molecular oxygen are required for maximal activity;

     «   Anaerobic conditions reduce the rate of DCM conversion  to

        carbon monoxide by 80 percent;

     o   There is a high correlation betw°en the in vitro production of
        carbon monoxide and microsomal Pg content;
    o   Pretreatment of test animals with P    inducers resulted in
                                      35

-------
        increased conversion of DCM to  carbon  monoxide  by  rat  liver



        microsomes;



     •  Pretreatment of test animals with  P^Q inhibitors  resulted in



        decreased conversion of DCM to  carbon  monoxide  by  rat  liver



        microsomes;  and



     •  Cleavage of  the carbon-hydrogen bond  is the rate-limiting



        step in dihalomethane metabolism.



Based on these observations, Anders et  al. (1977)  postulated that DCM  is



biotransformed to carbon monoxide by the microsomal  mixed-function oxidases



via formation of a formyl  halide intermediate.  The proposed mechanism for



the metabolism of dihalomethanes by liver  microsomes is summarized in



Figure 5.



     A number of investigators (Heppel  and Porterfield, 1948;  Kubic  and



Anders, 1975; Ahmed  and Anders, 1976, 1978) have studied the metabolism of



DCM and other dihalomethanes to carbon  dioxide, formaldehyde,  and  formic  acid



using a rat liver cytosolic fraction.  They have made the following  observa-



tions:



     •  The reaction does not require molecular oxygen;



     •  The reaction is glutathione-dependeht;



     •  Chemicals known to complex with glutathione inhibit



        the reaction; and



     •  Removal of formaldehyde dehydrogenase by ammonium hydroxide



        fractionation results in the formation of formaldehyde only,



        and not formic acid.



Based on these observations, Ahmed et al.  (1980) postulated that DCM is bio-



transformed to carbon dioxide by the liver cytosolic fraction  via  formation



of a  reactive formyl intermediate.  The proposed mechanism for the metabolism






                                      36

-------
         Microsomal  Pathway
                        MFO
X«CH~ X/.
NADPH
-H
-X
>
,c
\
OH
Nonenzymatic
rearrangement
f
Covalent ^ H Spontaneous
lipid 0 Decomposition
protein "H, -X
         Cytosolic Pathway
                             formyl halide
                                                                CO
             X - CH
GSH   	
transferase
                                     NAD
                                         GS - CH2X
                                        S-halomethylglutathione
                                                   HOH
                            Nonenzymatic
                            hydrolysis
                                                   GS  -  CH2OH
                                                  S-hydroxymethyl glutathione
GS - C

        0

S-formyl glutathione

  HOH
                 S-formyl  glutathione
                    hydrolase
           HC "     •»• GSH
             ^ OH
             formic  acid
                                           formaldehyde
                                          dehydrogenase
                                              metabolic
                                            incorporation
                                H2C=0 + GSH
                               formaldehyde
                                    v
                                   co2
Figure 5.  Proposed reaction mechanisms  for  the metabolism  of dihalomethanes to
carbon monoxide, carbon dioxide,  formaldehyde, formic  acid,  and  inorganic halide.

SOURCE:  Ahmed et al.,  1980.
                                        37

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of dihalomethanes by liver cytosol  is summarized  in  Figure  5.



3.2.2.  Tissue Distribution



     There have been a number of studies  which  have  compared DCM metabolism



by means of microsomes and/or cytosol prepared  from  various tissues.   Kubic



and Anders (1975) compared the biotransformation  of  DCM to  carbon monoxide by



liver, kidney, and lung microsomes.  They found liver microsomes to be five



times more active than lung microsomes,  and 30  times more active than kidney



microsomes.  Ahmed and Anders (1976)  compared the biotransformation of DCM to



carbon dioxide by cytosolic fractions prepared  from  various tissues of the



rat.  The-highest activities were found  in liver, lung, and kidney cytosol.



Liver cytosol was found to be 15 times more active than lung cytosol  and 12.5



times more active than kidney cytosol.  Ahmed and Anders (1978)  measured the



metabolism of dibromomethane to bromide,  formaldehyde, and  formic acid using



rat liver cytosol, and observed that  free bromide was formed at  the rate of



27.7 * 3.8 nmoles/mg protein/minute,  and  that the amount of formaldehyde plus



formic acid formed was 12.6 * 0.8 nmoles/mg protein/minute.  Kubic and Anders



(1975), using rat liver microsomes or cytosol from the same liver preparation,



measured dibromomethane metabolism and found that the microsomes yielded 3.9



nmoles carbon monoxide/mg protein/minute and 14.0 nmoles free bromide/mg



protein/minute.  The cytosol fraction from the  same  liver preparation con-



verted dibromomethane to bromide at a rate of 9.1 nmoles/mg protein/minute.



Based on these data, it appears that  similar amounts of DCM/mg protein/unit



time are converted to carbon monoxide or carbon dioxide by  microsomes and by



cytosol.  Since microsomes comprise 2% to 5% of liver and cytosol comprises



about 10% of liver protein (Estabrook et al., 1971;  Hogeboom et  al., 1953),



it would be anticipated, on a mass basis, that  the cytosol  would metabolize



more DCM than would microsomes.  It has  been postulated that each pathway





                                      38

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involves the formation of an active intermediate,  and  each  intermediate  can
be a potential alkylating agent.   The available  data also indicate  that  most
metabolism of DCM occurs in the liver, although  small  amounts  of  activity
have been detected in the lung and kidney.   Analysis of  rat tissues 48 hours
post-exposure to ^C-DCM showed that liver,  kidney,  and  lung contain the most
radioactivity (McKenna et al., 1982).  At the present  time, the implications
of these observations in assessing the carcinogenic  potency of DCM  are unclear.
3.2.3.  In Vivo Metabolism/Effect of Dose
     There have been a number of in vivo studies in  which investigators  have
given animals l^C-DCM an(j tnen measured the amount of  exhaled l^C-carbon monox-
ide and l^C-carbon dioxide.  Yesair et al.  (1977)  and  Angelo (1985) assessed
the metabolism of DCM by mice.  Yesair et al. (1977)  gave  groups  of mice 1 mg/
kg or 100 mg/kg l^C-DCM in corn oil by intraperitoneal injection, and then
collected metabolic by-products for 96 hours.  (The  authors stated, without
giving data, that most of the observed metabolism took place during the  first
12 hours post-exposure.)  Angelo (1985) gave groups  of mice 10 mg/kg or  50 mg/
kg l^C-DCM in 25% polyethylene glycol by tail vein injection, and then collec-
ted metabolic by-products for 4 hours.  The data collected  by these investiga-
tors are summarized in Table 9.  The data show that  the mouse is  able to
metabolize DCM to carbon monoxide and carbon dioxide,  and  that over a large
dose range, equal amounts of DCM are converted to carbon monoxide and carbon
dioxide.  However, because of different experimental  designs used by Yesair
et al.  (1977) and Angelo (1985), it is not possible  to assess whether there
is a dose-response relationship.  Indeed, the greater conversion of DCM  to
carbon monoxide and carbon dioxide in mice given 100 mg/kg  compared to those
given smaller doses is probably an artifact of experimental design, namely
the use of corn oil carrier in the intraperitoneal injection of DCM.
                                      39

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                 TABLE 9.   IN VIVO METABOLISM OF DCM BY MICE
Dose
1 mg/kgb
10 mg/kgc
50 mg/kgc
100 mg/kgb

(11.76 umoles/kg)
(117.6 umoles/kg)
(588 umoles/kg)
(1,176 umoles/kg)
DCM
exhaled3
'
56.9
382.2
470.0
% of dose
exhaled

48.4
65.0
40.0
C02a
5.9
23.8
80.6
294.0
C0a
5.3
17.5
29.4
235.0
aValues are umoles/kg.
bYesair et al.  (1977).
cAngelo (1985).
     There has been one animal  study  reported  on  the.metabolism  of  inhaled

14C-DCM.  McKenna et al. (1982)  gave  groups  of rats  a  single  6-hour inhalation

exposure to 50, 500, or 1500 ppm l^C-DCM.  At  the end  of  the  exposure  period,

exhaled DCM, carbon dioxide, carbon monoxide,  and urine were  collected for

48 hours.  At the end .of the 48-hour  collection period, the rats were  sacri-

ficed and tissue levels of radioactivity were  determined.  The data obtained

from this study are summarized  in Tables  10  and 11 and in  Figures 6 and 7.  The

authors interpreted these data  to indicate saturability of metabolism  because

of the following observations:

     •  The percent of administered DCM metabolized  to carbon dioxide  and

        carbon monoxide declined with increasing  dose; and

     •  There was less than a proportional increase  in tissue levels

        of radioactivity.

The key assumption made by McKenna et al.  (1982)  was that the disproportion-

ate exhalation of DCM indicated saturated  metabolism.   However,  a review of
                                     40

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                TABLE 10.   BODY BURDENS AND METABOLIZED ^C-DCM
                  IN RATS  AFTER INHALATION EXPOSURE TO 11+C-DCM
Exposure
concentration
50 ppm
500 ppm
1,500 ppm
Total body burden ,a
mgEq ll*DCM/kg
5.53 ±
48.41 ±
109.14 ±
0.18
4.33
3.15
Metabolized 1£*DCMa
mgEq 11+DCM/kg
5.23 ±
33.49 ±
49.08 ±
0.32
0.33
1.37
Metabol ized
94.6
69.2
45.0
aValues are mean ± standard deviation; number of animals in each group = 3.

SOURCE:  McKenna et al., 1982.
            TABLE 11.  FATE OF i^C-DCM IN RATS AFTER A SINGLE 6-HOUR
                             INHALATION EXPOSURE
                                % body burden (x   S.D., n = 3)
Parameter
measured
Expi red
Expired
Expi red
Urine
Feces
Carcass
Skin
CH2C12
C02
CO




Cage wash
5
26
26
8
1
23
6
0
50 ppm
.42
.20
.67
.90
.94
.26
.85
.75
0.73
1.21
3.00
0.39
0.19
1.62
1.62
0.33
30
22
18
8
1
11
6
0
500 ppm
.40
.53
.09
.41
.85
.65
.72
.24
7
4
0
0
0
1
0
0
.10
.57
.81
.90
.68
.87
.13
.23
1500 ppm
55
13
10
7
2
7
3
0
.00
.61
.23
.20
.33
.24
.97
.43
1.92
1.20
1.68
0.74
0.05
0.65
0.15
0.15
SOURCE:  McKenna et al., 1982.
                                     41

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         10.0 r-
     "&

      •V
     U
     X
     U


     5
     U)
          0.01
         0.001
                                                             50 ppm

                                                             500 ppm
                                                             1500 ppm
                              EXPOSURE

                               I   I   I
                                        4      5


                                       TIME, hours
6/0
Figure 6.  Plasma levels of  DCM  in  rats  during  and after DCM exposure for 6
hours.  Data points represent mean  ±  standard deviation for two to four rats.

SOURCE:  McKenna et al., 1982.
                                         42

-------
       100.0
        10.0
    0
    o
    o
    a
    O
    O
    XI
    X
1.0
         0.1
                                  i   '   i  '   i   '   M  i   y
                           EXPOSURE-
               I      I
                                                        50 ppm
                                                        500 ppm
                                                        1500 ppm
                I   I   I   I   I    I   I   I   I   I   I  I    I   I   I
                                     4      5     6/0

                                       TIME, hours
Figure 7.  Blood COHb concentrations in  rats during and  after  a  6-hour  inhala-
tion exposure to DCM.   Each data point  is the mean ±  standard error  for two to
four rats.

SOURCE:  McKenna et al., 1982.
                                        43

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the study indicates that the data do not support  this  assumption  since  the



measurements were made when the DCM concentration within  the animal  was rapidly



changing.  For the purpose of analysis we selected a three-compartment  model



(lungs, blood, and tissues).  In this model  system the DCM concentration within



the animal is dependent upon three components:   1) uptake by the  lungs  via



passive diffusion, 2) diffusion into the blood,  and 3) uptake by  the tissues,



metabolism, and exhalation.  First, given the volatility  of DCM,  it  is  reason-



able to expect that exhalation would be the  major contributing component in



modulating the post-exposure concentration within the  animal.  Indeed,  the  very



rapid decline in the blood concentration of  DCM  (10 pg/mL to 0.1  pg/mL  in



less than 30 minutes) supports this assumption.   Thus, the amount of DCM



available for metabolism becomes a function  of the amount remaining  in  the



tissues post-exposure, and therefore is governed by the air/blood partition



coefficient of DCM.  While the data reported by  McKenna et al. (1982) were  not



obtained under the steady-state conditions necessary for  the comparison of



different doses, the study showed that during the exposure period the carboxy-



hemoglobin (COHb) levels in rats exposed to  500  and 1500  ppm DCM  were the



same (Figure 7).  These data would support the concept that the carbon  monox-



ide pathway was saturated during the period  of exposure.   However, since



similar data on the carbon dioxide pathway were  not obtained, it  is  not



possible to conclude that saturated metabolism occurred during the exposure



period.



     One laboratory has reported results from similar  experiments using mice



and rats.  Angelo (1985) gave 10 mg/kg or 50 mg/kg 14C-DCM intravenously to



groups of mice or rats and then measured the amount of exhaled DCM,  carbon



dioxide, and carbon monoxide for 4 hours post-exposure.  The data from  these



experiments are summarized in Tables 12 and 13.   Rats  given 10 mg/kg or 50






                                      44

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                    TABLE 12.  METABOLISM OF DCM FOLLOWING
              INTRAVENOUS ADMINISTRATION OF 10 mg/kg OR 50 mg/kg
                                 Mice                          Rats
     Exhaled            10 mg/kg      50 mg/kg       10 mg/kg      50 mg/kg


DCM                     48.4 ±6.1    65.0+4.2     44.5 ± 3.7    57.6+6.1

Carbon dioxide          20.2* 2.0    13.71 1.5     16.0 ± 1.0     9.9 ± 0.7

Carbon monoxide         14.9 ± 4.0     8.4 ± 2.9     12.7 ± 2.2     8.7 ± 2.6


aValues are percent of administered dose + standard deviation.

SOURCE:  Angelo, 1985.
                    TABLE 13.  METABOLISM OF DCM FOLLOWING
       INTRAVENOUS ADMINISTRATION TO CARBON MONOXIDE AND CARBON DIOXIDE
Time
post-
exposure
(min.)
0-60
60-240
Total
0-60
60-240
Total
Mice

Carbon
Dose monoxide
10 mg/kg 0.7a
0.7
1.4
50 mg/kg 2.1
2.2
4.3
Rats

Carbon
dioxide
1.7
0.3
2.0
5.7
1.2
6.9

Carbon
monoxide
0.2
1.1
1.3
0.7
3.7
4.4

Carbon
dioxide
0.8
0.7
1.5
1.9
3.1
5.0
aValues are mg/kg.

SOURCE:  Angelo, 1985.
                                     45

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mg/kg immediately (less than 20 minutes)  exhaled about 35% of the administered

dose, while mice immediately exhaled 45%  of the low dose and 60% of the high

dose (Table 14).  The data, as shown in Table 14, indicate that in both

species the amount of DCM metabolized to  carbon monoxide and carbon dioxide

is not proportional to the increase in administered dose.  However, if the

administered dose is adjusted by subtracting the amount of DCM immediately

exhaled, there is a proportional relationship between dose and metabolites

formed in the mouse and the rat.

     The adjusted data indicate that on a mg/kg basis the rat and mouse appear

to be capable of metabolizing the same amount of DCM over a 4-hour period.

This conclusion is supported by the findings of Yesair et al. (1977) and

McKenna and Zempel (1981), who assessed the metabolism of small amounts of

DCM  in mice or rats.  Both of the latter studies showed that most of the

administered dose was metabolized to carbon monoxide and carbon dioxide.
                TABLE 14.  METABOLISM OF DCM BY RATS AND MICE
               EFFECT OF DOSE CORRECTION FOR EXHALED SUBSTRATE
Species
Mouse

Rat

Dose
10
50
10
50
Amount
exhaleda»b
4.6
29.8
3.2
17.6
Adjusted
dose3
5.4
20.2
6.8
32.4
Amount of
C0+C02 formed3
3.5
11.1
2.9
9.4
aValues  are mg/kg.
^Amount  exhaled  in  first  20 minutes.

SOURCE:   Adapted  from Angelo, 1985.
                                      46

-------
     Rodkey and Collison (1977a)  exposed a group of  4  rats  to  154 ymoles  of
DCM (1255 ppm) in a chamber having a closed rebreathing  system and  determined
the amount of carbon monoxide exhaled as a function  of time.   After a  short
lag period, the rats exhaled carbon monoxide at a rate of 30 ymoles/kg/hour.
The authors stated that they obtained similar results  after giving  animals
the same dose of DCM intraperitoneally.   In a second experiment, a  group  of 4
rats exposed to 793 ymoles of DCM (6462  ppm) exhaled 40  ymoles carbon
monoxide/kg/hour.  These data indicate that in the rat the  microsomal  pathway
is almost saturated at DCM exposures as  low as 1255  ppm  and that, therefore,
the Vmax equals about 30 to 40 ymoles/kg/hour.  Rodkey and  Collison (1977b)
also measured DCM biotransformation to carbon monoxide and  carbon dioxide.
Rats were exposed to 200 ymoles of DCM in a chamber  with a  closed  rebreath-
ing system, and the amount of carbon monoxide and carbon dioxide produced
were determined at 7 to 9 hours post-exposure.  The  amount  of  carbon monoxide
formed was about 90 ymoles, whereas the  amount of excess carbon dioxide
formed was only 56 ymoles.  These data appear to differ  significantly  from
those reported by McKenna et al.  (1982)  and DiVincenzo and  Hamilton (1975),
who found that more carbon dioxide than  carbon monoxide  was exhaled.  However,
since Rodkey and Collison (1977b) accounted for only about  75% of the  admin-
istered dose, these studies are not directly comparable.
     It has been estimated that.mice metabolize DCM to carbon  monoxide at a
rate of about 19 ymoles/kg/hr (EPA, 1985).  This value was  determined  by
assuming that the carbon monoxide formation was constant over  the  12-hour
post-exposure period in the experiments  performed by Yesair et al.  (1977).
Given the volatility of DCM, it is unlikely that the substrate concentra-
tion was saturating for 12 hours.  Thus  the rate constant  for  carbon monoxide
formation in the mouse could well exceed 19 ymoles/kg/hr.
                                     47

-------
     The in vivo metabolic studies  confirm the data obtained in vitro that DCM
is biotransformed to carbon monoxide  and carbon dioxide.  There are some data
which indicate that formation of  carbon monoxide in the rat reached Vmax at
exposures of less than 1200 ppm.  There are no data on the exposure concen-
tration required to saturate the  carbon dioxide pathway.  Comparative data on
species differences and similarities  are limited.  At low doses both rats and
mice metabolize similar amounts of  DCM to carbon monoxide and carbon dioxide.
3.2.4.  Human Studies
     There have been three studies  in which volunteers have been exposed to
DCM.  DiVincenzo and Kaplan (1981a, b) evaluated the conversion of DCM to
carbon monoxide in sedentary, non-smoking individuals and in individuals
engaged in physical activity.  Exposure, in a chamber, was to 50, 100, 150,
or 200 ppm DCM for 7.5 hours or for 7.5 hours/day  for 5 consecutive days.
The metabolism of DCM was also studied in men engaged in physical activity.
DiVincenzo and Kaplan (1981a) found that in sedentary individuals the pul-
monary uptake of DCM was linear over  the range studied (50-200 ppm), and that
excretion of carbon monoxide was  proportional to the pulmonary uptake of DCM.
The authors noted that only 25% to  34% of the DCM  taken up was converted to
carbon monoxide, and therefore hypothesized, based on data from animal studies,
that up to 70% was metabolized to carbon dioxide.
     Sedentary volunteers exposed once for 7.5 hours to 50, 100, 150, or 200
ppm of DCM had peak COHb concentrations of 1.9%, 3.4%, 5.3%, and 6.8%, respec-
tively.  When sedentary volunteers  were exposed to 100, 150, or 200 ppm DCM
for 7.5 hours/day for 5 consecutive days, the COHb of those exposed to 150
ppm or 200 ppm increased to levels  above that of the single exposure.  The
volunteer exposed to 200 ppm DCM  had  a COHb of about 5% on day 1 and a COHb
of about 6.5% on day 4.  The carbon monoxide concentrations in the breath of

                                     48

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the volunteers also increased each day during exposure to high  concentrations
(150-200 ppm) of DCM.  Conversely, the peak level  of expired DCM in  the  breath
of the volunteers remained constant (peak level  did not change) throughout
the exposure period.
     DiVincenzo and Kaplan (1981b) also measured DCM uptake and metabolism
during exercise.  The data show (see Table 15) that uptake of DCM is directly
related to work intensity, and that the amount of DCM metabolized to carbon
monoxide is directly proportional  to pulmonary uptake.  This is illustrated
by the finding that a sedentary volunteer exposed to 200 ppm DCM exhaled 6.1
mmoles of carbon monoxide (with a pulmonary uptake 21.1 mmoles  DCM), while an
exercising volunteer exposed to just 100 ppm DCM exhaled 11.8 mmoles of  carbon
monoxide (with a pulmonary uptake of 41.9 mmoles DCM).  The latter observation
suggests that the metabolic capacity of people exposed to 400 ppm DCM would
not be exceeded.
     McKenna et al. (1980) exposed volunteers to 100 or 350 ppm DCM for  6
hours and measured various parameters, including blood and exhaled air levels
of DCM, COHb, and exhaled carbon monoxide.  The data showed that the blood
level of DCM for both concentrations reached a steady-state in  about 2 hours.
At the end of the 6-hour exposure, the COHb concentration of the group exposed
to 350 ppm DCM was 1.4-fold higher than that of the group exposed to 100 ppm.
Likewise, the concentration of exhaled carbon monoxide in the group exposed
to 350 ppm DCM was 2.1-fold higher than that of the group exposed to 100 ppm.
The authors assumed that once steady-state is achieved, further uptake of DCM
would be proportional to the rate of metabolism of DCM.  Consistent with this
steady-state assumption, McKenna et al. (1980) interpreted these findings to
mean that the COHb and carbon monoxide levels between the high and low groups
were less than proportional as metabolism became saturated.

                                      49

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            TABLE 15.   EFFECT OF  EXERCISE ON THE PULMONARY UPTAKE
           AND METABOLISM OF  METHYLENE CHLORIDE DURING EXPERIMENTAL
                    EXPOSURES TO  METHYLENE CHLORIDE VAPOR
Work intensity
Volunteer (mL Q£ min~l kg"*)
1 4
2 14
3 15
19
4 16
28
Pulmonary
uptake of DCM
(mmoles)
10.7
19.4
30.7
36.4
28.8
41.9
Total pulmonary
excretion of CO
(mmoles)
2.7
5.1
10.2
14.3
10.4
11.8
SOURCE:  DiVincenzo and Kaplan,  1981b.
     A review of the McKenna et  al.  (1980) study indicates that the data do

not support the assumption that  a  steady-state blood level of DCM is a reflec-

tion of saturated metabolism. The DCM concentration in the blood in the sim-

plest model is a function of three components:  uptake by the tissues, metabo-

lism, and exhalation.  First, given  the  volatility of DCM, it is reasonable to

expect that exhalation is the major  contributing component that modulates the

blood level.  Indeed, the data from  the  study support this assumption, since

the amount of DCM exhaled increased  disproportionately with dose.   In addition,

the very rapid decline in the blood  level of DCM post-exposure argues very

strongly that exhalation is the  major component governing the observed blood

level.  Furthermore, the data reported by DiVincenzo and Kaplan (1981a) on

volunteers exposed to 50 to 200  ppm  DCM  clearly show a slight but distinct

disproportionate exhalation of DCM when  the body mechanism to metabolize DCM


                                     50

-------
has not been saturated.
     The finding by McKenna et al. (1980)  that  the  COHb  and  carbon monoxide
levels in the group exposed to 350 ppm DCM were less  than  3.5-fold higher than
in the group exposed to 100 ppm, and the subsequent interpretation that this
indicates saturated metabolism, ignores the limitations  of the design of the
experiment.  The initial  amount of product formed using  the  same  amount of
enzyme and two different  concentrations of substrate  appears to be the same,
and if at least one concentration of substrate  is less than  saturating, the
ratio of product formed will continue to change until a  steady-state or con-
stant rate of carbon monoxide formation is achieved.  The  McKenna et al.
(1980a) data clearly showed that both the COHb  and  the concentration of ex-
haled carbon monoxide concentration were increasing during the exposure
portion of the study (Figures 8 and 9), and thus, it  is  not  possible to
predict steady-state concentrations of either COHb  or exhaled carbon monoxide.
Lastly, DCM is metabolized via two pathways. Data  from  animal studies
indicate that both pathways are of equal importance,  and that perhaps the
carbon dioxide pathway is more important.   The  authors'  conclusion, based
on data from only one pathway, that metabolic saturation in  humans is
achieved at less than 350 ppm DCM is therefore  premature.
3.2.5.  Summary
     The results of both  in vitro and in vivo studies indicate that DCM is
metabolized via two pathways.  One pathway yields carbon monoxide as an end
product, and the other yields carbon dioxide as an  end product with formalde-
hyde and formic acid as metabolic intermediates. Each pathway involves
formation of a metabolically active intermediate which is  theoretically
capable of irreversibly binding to cellular macromolecules.  A comparative
analysis of the capability of various tissues to metabolize  DCM indicates

                                      51

-------
   100—i
    10—J
 •o
 o
 _

 O


 I
 O
 O
     1	
        -
          Exposure
        0246
                                 350 ppm
                  0  2  4   6   8  10 12 14 16  18  20  22 24


                                    Hours
Figure 8.  COHb level  in volunteers exposed to 100 ppm or 350 ppm DCM.



SOURCE:  McKenna et al., 1980.
                                  52

-------
       100 —,
CO IN EXPIRED AIR
        10 —
     c.


     O

     •3
     X
     tu
         1  __
              Exposure
                I   i    I
             0246
                      0  2   4   6.8  10  12 14 16  18  20  22 24

                                         Hours
Figure 9.  Exhaled carbon monoxide by volunteers exposed to 100 ppm or 350 ppm
OCM.


SOURCE:  McKenna et al., 1980.
                                      53

-------
that the liver is the primary site of  metabolism,  with  some metabolism taking
place in the lung and kidney.  An  analysis  of  the  available in vitro data
suggests that the carbon dioxide  pathway  may metabolize significantly more
DCM than the carbon monoxide pathway.   Consistent  with  this observation  is j[n_
vivo data which suggest that when  rats or mice are exposed to high  concentra-
tions of DCM they exhale more carbon dioxide and excrete more formic acid
than carbon monoxide.  At exposure to  low concentrations of DCM,  both pathways
are utilized about equally.  The  data  from  rat studies  also suggest that the
route of exposure, at low doses,  results  in similar metabolic profiles.
     A comparative analysis of the data from in vivo studies  in mice, rats,
and humans indicates that all three species metabolize  DCM to carbon monoxide
(Table 16).  Both mice and rats metabolize  DCM to  carbon dioxide.   There are
no human data on the metabolism of DCM to carbon dioxide.  However, based  on
uptake data, some investigators have speculated that this pathway is func-
tional in humans.
     Groups of rats exposed to 500 or  1500  ppm DCM have the same  COHb,  sug-
gesting that the carbon monoxide  (microsomal)  pathway has been  saturated.
There are no similar data on the  cytosolic  pathway.  One group  of investiga-
tors has suggested that the microsomal pathway is  saturated in  humans at less
than 350 ppm DCM.  However, an analysis of  the study indicates  that the  data
do not support this conclusion.
     At present, the available data are insufficient for the  purpose of  esti-
mating doses of DCM at which metabolism is  saturated.  The available data
indicate that at low doses little unmetabolized DCM is exhaled,  and that at
high doses there is a significant exhalation of DCM immediately  post-exposure.
The data suggest that at high doses more DCM  is taken up into the body.
Currently, there are insufficient data to determine the relationship between

                                      54

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               TABLE 16.   METABOLISM OF  DCM TO CARBON MONOXIDE,
          CARBON DIOXIDE,  AND FORMIC ACID  IN MICE,  RATS,  AND  HUMANS
Species
Exposure/uptake
Carbon monoxide
  (ymoles/kg)
Carbon dioxide
  (ymoles/kg)
Mice3
Miceb
Miceb
Mice3
Ratsc
Ratsb
Ratsc
Ratsb
Ratsd
Rats6
Rats6
Rats6
Humans^



11.8 ymoles/kg
117.6 ymoles/kg
588.0 ymoles/kg
1176.0 ymoles/kg
11.8 ymoles/kg
117.6 ymoles/kg
588.0 ymoles/kg
588.0 ymoles/kg
6009.4 ymoles/kg
50 ppm (65.0 ymoles/kg)
500 ppm (569.3 ymoles/kg)
1500 ppm (1,283.5 ymoles/kg)
50 ppm (79.1 ymoles/kg)
100 ppm (152.9 ymoles/kg)
150 ppm (219.7 ymoles/kg)
200 ppm (301.0 ymoles/kg)
5.3
16.5
50.6
235
3.6
15.2
70
51.7
129.2
17
103
131
18.6
28.6
71.4- •
87.4
5.9
22.3
81.1
294
4.1 (4.7)9
17.6
37 (48.8)
58.8
182.9 (242.9)
17.3 (23.1)
128 (175.8)
174 (266.4)
55.8
107.1
154.3
210
aYesair et al. (1977).  DCM given i.p.  in corn oil.   According to the authors,
 the metabolism of methylene was essentially complete 12 hours post-exposure.
bAngelo (1985).  DCM given i.v. in 25%  polyethylene  glycol.   Metabolism was
 monitored for 4 hours post-exposure.
GMcKenna and Zempel (1981).  DCM given  by gavage.  Metabolism was monitored
 for 48 hours post-exposure.
dDiVincenzo and Hamilton (1975).  DCM  given i.p.  in  corn oil.  Metabolism was
 monitored for 48 hours post-exposure.
6McKenna et al. (1982).  DCM given in  a single inhalation exposure for 6 hours,
 Metabolism was monitored  for 48 hours  post-exposure.
DDiVincenzo and Kaplan (1981).  DCM given in a single inhalation exposure for
 7.5 hours.  The amount of post-exposure carbon monoxide exhaled was  measured
 for 24 hours.  The amount of carbon dioxide exhaled was based on assumptions
 made by the authors.  For the purpose  of comparison, it was assumed  that the
 weight of each volunteer  was 70 kg.
9Carbon dioxide + radioactivity in urine (assumed  to be formic acid).
                                     55

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exposure concentration and uptake.   It  is  of  interest to note that the obser-
vation has been made that fat people take  up  more DCM than thin people, and
and that high levels of DCM are found in fat  tissues post-exposure (U.S. EPA,
1985).
     There is a paucity of data on  the  genotoxicity of DCM.  Commercially
available DCM gives weak but positive results in Salmonella, yeast, and
Drosophila without metabolic activation.   It  has been shown to induce chro-
mosomal aberrations in some cultured mammalian cell systems but not in
others.  DCM also causes a weak increase in sister chromatid exchange, but
it has not been shown to cause unscheduled DNA synthesis or to inhibit DNA
synthesis (U.S. EPA, 1985).  At the present time, based on positive mutagenic
studies and the likelihood that reactive intermediates are formed during
biotransformation to carbon monoxide and carbon dioxide, it seems reasonable
to assume that DCM exerts its carcinogenic effect via a genotoxic mechanism.
     Based on this analysis, it is  concluded  that the available pharmacokine-
tic/metabolism data do not offer useful parameters for making assumptions in
the calculation of quantitative carcinogenic  risk assessments for DCM.
                                      56

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                         4.   QUANTITATIVE  ESTIMATION
                     (USING  THE NTP INHALATION BIOASSAY)
     The EPA Office of Health and Environmental  Assessment  has  recently  pub-
lished a comprehensive document on the health  effects  of  dichloromethane
(methylene chloride, DCM)  (U.S. EPA,  1985).  After the completion  of  this
report, the NTP released the findings of an  inhalation toxicology  and carcino-
genesis study of DCM in F344/N rats and B6C3F1 mice (NTP, 1985, draft).  The
qualitative findings of this study are discussed in a  preceding section  of
this document; here, the NTP findings are used to develop estimates of unit
incremental cancer risks for humans exposed  to DCM.  Variations in extrapo-
lated risks using different cancer end points  from the NTP  study are  discussed,
as well as the influence of the dose-response  model selected.   The quantita-
tive findings are also compared with  earlier experimental carcinogenesis
studies of DCM and with the limited information available from  epidemiologic
studies.
4.1.  SUMMARY OF NTP FINDINGS USED FOR QUANTITATIVE ANALYSIS
     In an earlier section of this document, the end points of  mammary and
subcutaneous tumors in rats and lung  and liver tumors  in  mice were determined
to be the sites where the NTP study produced the strongest  findings of car-
cinogenicity for DCM.  Tables 17 and  18 present the NTP tumor  incidence  find-
ings for these sites.  The denominator for each data point  is the number of
animals that were examined at the specific tumor site.  The statistical
significance of the NTP findings has  been discussed earlier in  this  report,
and the findings are only summarized  in Tables 17 and  18.
     The data in these two tables will serve as the basis for the quantitative
risk estimates to be developed.  The  EPA draft guidelines for carcinogen risk
                                      57

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                                    TABLE 17.   SUMMARY  OF  NTP  INHALATION  STUDY  OF  DCM:
                                   FINDINGS FOR MAMMARY AND  SUBCUTANEOUS  TUMORS IN RATS
01
00
. Dose
Site/tumor Control
Males
Mammary gland: adenoma or fibroadenoma3 0/50
Subcutaneous tissue: fibroma0 1/50
Mammary gland or subcutaneous tissue: 1/50
adenoma, fibroadenoma or fibroma3
Females
Mammary gland: fibroadenoma3 5/50
Mammary gland: adenoma, 7/50
fibroadenoma, adenocarcinoma, or
mixed tumor, malignant3
1,000 ppm
0/50
1/50
; 1/50
ll/50b
13/50°
2,000 ppm
2/50
2/50
4/50
13/50b
14/50b
4,000 ppm
5/50b
4/50
9/50d
22/50d
23/50d
    3A11  trend  tests  for  tumor  incidence  positive  at  p  <  0.01  nominal  level.
    D0ne  or more  positive pairwise  comparison(s) with control  group  p  < 0.05 nominal  level.
    C0ne  or more  positive trend test(s) for  tumor  incidence  p  <  0.05 nominal level.
    dAll  pairwise comparisons with  control group positive p  <  0.01 nominal  level.
  1
  '  Note:   Tumor  trend  tests  reported  by  NTP:   life table, incidental  tumor, and Cochran-Armitage tests;
           pairwise tests:  life table, incidental tumor,  and  Fisher Exact  tests.

    SOURCE:  NTP, 1985.

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                               TABLE 18.  SUMMARY OF NTP INHALATION STUDY OF DCM FINDINGS FOR LUNG AND LIVER TUMORS IN MICE
                                                                                                           Dose
                    Tumor                                                              Control            2,000 ppm       4,000 ppm


                   Males

                   Alveolar/bronchiolar adenoma3                                         3/50             19/50b           24/50b
                   Alveolar/bronchiolar carcinoma8                                       2/50             10/50C           28/50b
                   Alveolar/bronchiolar adenoma or carcinoma3                         .   5/50             27/50b           40/50b

                   Hepatocellular adenoma^                                              10/50             14/49C           14/49C
                   Hepatocellular carcinomad                                            13/50             15/49         .  26/49c
                   Hepatocellular adenoma or carcinoma3                                 22/50             24/49           33/49c

                   Alveolar/bronchiolar or hepatocellular carcinoma6^                  15/50             21/49           39/49b
                   Alveolar/bronchiolar or hepatocellular adenoma or carcinoma6'^       27/50             34/49d    .       45/49b
01
10
                   Females
                   Alveolar/bronchiolar adenoma3                                         2/50             23/48b           28/48b
                   Alveolar/bronchiolar carcinoma3                .                       1/50             13/48b           29/48h
                   Alveolar/bronchiolar adenoma or carcinoma3                            3/50             30/48b           41/48b

                   Hepatocellular adenoma3                         .                      2/50              6/48            22/48b
                   Hepatocellular carcinoma3                                             1/50             ll/48b           32/48b
                   Hepatocellular adenoma or carcinoma3                                  3/50             16/48b           40/48b

                   Alveolar/bronchiolar or hepatocellular carcinoma6''                   1/50             21/48b           43/47b
                   Alveolar/bronchiolar or hepatocellular adenoma or carcinoma6*'        5/50             36/48b           46/47b


                   3A11  trend tests for tumor incidence positive at p < 0.01 nominal  level.
                   bAll  pairwise comparisons with control group positive p < 0.01 nominal  level.
                   C0ne  or more positive pairwise comparison(s) with control group p  < 0.05  nominal  level.
                   done  or more positive trend test(s)  for tumor incidence at p < 0-05 nominal  level.
                   eDenominators are number of animals  examined for tumors at both luny and  liver sites.
                   'Tumor grouping not presented in NTP report; significance determined using Fisher Exact  Test.

                   Note:  Tumor trend tests reported by NTP:   life table, incidental  tumor,  and Cochran-Armitage  tests;
                          pairwise tests:  life table,  incidental tumor, and Fisher Exact  tests.

                   SOURCE:  NTP, 1985.

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assessment (U.S. EPA, 1984) call  for risk  estimation  using  the  combined  inci-



dence of statistically elevated tumors.  The  combined incidence of  lung  and



liver tumors in mice is given for quantitative  analysis,  and  does not  imply



that the tumors are biologically related.



4.2.  DOSE-RESPONSE MODEL SELECTION



     The EPA draft guidelines for carcinogen  risk  assessment  (U.S.  EPA,  1984)



express the fact that there is no rigorously  established  scientific basis



for the selection of a dose-response model  to predict carcinogen risks at  low



doses.  In the typical situation where there  is limited information on which to



base the selection of a model, the draft EPA  guidelines express a preference



for the multistage dose-response model.  The  guidelines place emphasis on  the



upper confidence limit (UCL) risk estimates derived from  this model.  The  bases



for the preference include the following:



     1)   The multistage model incorporates the current scientific  opinion



          that multiple steps are involved in the  process of  cancer develop-



          ment, and that a chemical carcinogen  can contribute to one or  more



          of these steps.



     2)   The UCL of the multistage model  produces a risk estimate  that  is



          linear at low dose (LDL).  An LDL dose-response is  expected  when



          a carcinogen accelerates stages  of  the carcinogenic process  that



          lead to the background occurrence of  cancer in  unexposed  members



          of the population.



     3)   The UCL of the multistage model  produces a "plausible upper bound"



          estimate of risk, i.e., an estimate that is reasonable but is  usu-



          ally as high or higher than estimates derived from  other  models;



          models that are not linear at low dose will generally lead to  sub-
                                       60

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          stantially lower risk estimates.
     4)   The multistage UCL is stable under small  changes  in the input
          values for tumor incidence; in contrast,  the  maximum  likelihood
          estimate (MLE) can be unstable if the  results in  one  or a few
          animals are changed.
     Because of the role of genetic and mutational  factors  in the development
of many cancers, a supportive biological argument for LDL can be made strongly
for chemicals that are known to cause genetic damage.   The  EPA  Health Assess-
ment Document for Dichloromethane (U.S. EPA, 1985)  concluded that the weight
of evidence shows that DCM is capable of causing gene mutations and has the
potential to cause such effects in exposed  human cells. Further testing to
determine the strength of mammalian evidence was recommended.   These findings
provide additional support for the application of an LDL dose-reponse model
in estimating DCM cancer risks.
     Considering both EPA's policy for carcinogen evaluation and the biological
information available specifically for DCM, the  multistage  model was selected
as the primary model to be applied in this  risk  assessment. Versions of the
multistage model which incorporate time-to-tumor information have been devel-
oped.  While time-to-tumor models generally do not  produce  risk estimates that
differ greatly from similar dichotomous models,  the results are presented for
comparison.  Several other models are also  presented for comparison with the
multistage model.
4.3.  APPLICATION OF THE MULTISTAGE MODEL TO NTP BIOASSAY DATA
     The multistage dose-response model, as incorporated in the GLOBAL83
computer program developed by Howe (1983),  has been applied to  the NTP bio-
assay data given in Section 4.2.  The model is applied  to the experimentally
                                       61

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rodents exposed to DCM following the same  time  pattern  as  the  NTP  bioassay.



     A separate section of this report  reviews  the  available pharmacokinetic



and metabolic data on DCM and concludes that  these  data do not provide  an



adequate basis for modifications to the experimental  doses for use in



quantitative risk assessment.



     The GLOBAL83 program enables the user to select  the highest degree of



polynomial that the program will allow in  the model.  GLOBAL83 runs were made



for a polynomial degree equal to the number of  doses  (counting the control)



minus one.  Tables 19, 20, 21, and 22 present the  results  of these computa-



tions.



     Several conclusions can be drawn from these data.



     1)  The multistage model provides  an  adequate  fit  for all tumor



         groupings analyzed in both rats and  mice.



     2)  In rats, the highest value for the UCL of  the  linear  multistage



         term was obtained for mammary tumors in female rats.   The highest



         value in males was lower by a factor of three.



     3)  In mice, the highest value for the UCL of  the  linear  term was



         obtained in females having either adenomas or  carcinomas  of the



         lung and/or liver.  The corresponding  value for males was lower



         by a factor of two.



     4)  In mice, the male and female high-dose groups  had a  high  percen-



         tage of tumors in both the lung and  liver.  As discussed  in



         Section 4.4., the high-dose mice also  showed elevated mortality



         in comparison to controls.  In these circumstances,  competing



         risks can lead to underestimates of  risk  attributable to  indivi-



         dual tumor types.



     5)  The NTP (1985) noted that all  male rat groups  (including  controls)






                                      62

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                                 TABLE 19.  GLOBAL83 MODEL PARAMETERS FOR NTP (1985)  RAT DCM DATA
OJ
3-stage model
Site q0 q^lO"3
(ppnr1)
Males
Mammary gland3 0.0 0.0'
Subcutaneous13 0.0191 0.00093
Mammary gland or 0.0197 0.00075
subcutaneous0
Females
Mammary glandd O.ill 0.107
Mammary gland6 0.161 0.0985
q2xlO'6
(ppnr2)
0.00696
0.00389
0.0117

0.0
0.0
q3xlO"10 q^xlO"3
(ppm-3) (ppm-1)
0.0 0.0311
0.0 0.0306
0.0 0.0540

0.00544 0.164
0.00846 0.164
        aAdenoma or fibroadenoma.
        ^Fibroma.
        cAdenoma, fibroma, or fibroadenoma.
        ^Fibroadenoma.
        eAdenoma, fibroadenoma, adenocarcinoma, or mixed tumor,  malignant.
        q^ = The itn power coefficient in the multistage model.

        q^ = The 95% upper confidence limit estimate of the linear coefficient.

        Values for the q^  apply for rats exposed under the NTP protocol  dose time  schedule.

        Calculations are based on an extra risk analysis.

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                          TABLE 20.  COMPARISON OF THREE-STAGE GLOBAL83 ESTIMATES WITH
                                         OBSERVED TUMOR RESPONSE - RATS
Tumor
Males
Mammary gland .
adenoma or
fibroadenoma
Subcutaneous
Mammary gland
or subcutaneous
Females
Mammary gland
fibroadenoma
Mammary gland,
Observed
1,000
Control ppm
0.0 0.0
2.0 2.0
2.0 2.0
10.0 22.0
14.0 26.0
response (%)
2,000 4,000
ppm ppm
4.0 10.0
4.0 8.0
8.0 18.0
26.0 44.0
28.0 46.0
Predicted
2,000
Control ppm
' 0.0 0.7
1.9 2.4
1.8 3.0
10.5 19.7
14.9 22.9
response (%)
4,000 4,000
ppm ppm
2.7 10.5
3.6 8.2
6.4 18.8
28.1 43.7
30.6 45.6
Chi
squared
0.66
0.06
0.42
0.30
0.46
all  tumors

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       TABLE 21.   GLOBAL83 MODEL PARAMETERS FOR NTP (1985)  MOUSE  DCM DATA
Two-stage model
Tumor
Males
Lung3 adenoma
Lung carcinoma
Lung adenoma or
carcinoma
Li verb adenoma
Liver carcinoma
Liver adenoma or
carcinoma
Lung or liver carcinoma
Lung or liver adenoma
or carcinoma
Females .
Lung adenoma
Lung carcinoma
Lung adenoma or
carcinoma
Liver adenoma
Liver carcinoma
Liver adenoma or
carcinoma
Lung or liver carcinoma
Lung or liver adenoma
or carcinoma
<0
0.0672
0.0398
0.105
0.237
0.285
0.565

0.333
0.771
0.0445
0.0202
0.0619
0.0356
0.0193
0.0576

0.0197
0.105
qi*io-3
(ppnr1)
0.166
0.0
0.295
0.0306
0.0
0.0

0.0
0.0
0.242
0.0690
0.453
0.0
0.0
0.0

0.0
0.345
q2xlO"6
(ppnr2)
0.0
0.0481
0.0202
0.0
0.0283
0.0338

0.0739
0.107
0.0
0.0394
0.0032
0.0336
0.0655
0.101

0.147
0.148
(ppnr1)
0.223
0.141
0.452
0.0810
0.142
0.195

0.190
0.429
0.310
0.233
0.579
0.0872
0.145
0.160

0.244
0.870
aLung = alveolar/bronchiolar.
^Liver = hepatocellular.
C0nly the linear parameters are given for the 4-stage model.
q.j = The ith power coefficient in the multistage model.

q-^ = The 95% upper confidence limit estimate of the linear coefficient.

Values for the q-j apply for mice exposed under the NTP protocol  dose time
schedule.

Calculations are based on an extra risk analysis.
                                       65

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                             TABLE 22.  COMPARISON OF TWO-STAGE GLOBAL83  ESTIMATES
                                      WITH OBSERVED TUMOR RESPONSE  -  MICE
CTi
Observed response (%)
Tumor
Females
Lung adenoma
Lung carcinoma
Lung carcinoma
or adenoma
Liver adenoma
Liver carcinoma
Liver carcinoma
or adenoma
Liver/lung carci-
noma
Liver/'lung carci-
noma or adenoma
Males
Lung adenoma
Lung carcinoma
Lung carcinoma
or adenoma
Liver adenoma
Liver carcinoma
Liver carcinoma
or adenoma
Lung/liver carci-
noma
Lung/liver carci-
noma or adenoma
Control

4.0
2.0
6.0

4.0
2.0
6.0

2.0

10.0


6.0
4.0
10.0

20.0
26.0
44.0

30.0

54.0

2,000
ppm

47.9
27.1
' 62.5

12.5
22.9
33.3

43.8

75.0


38.0
20.0
54.0

28.6
30.6
49.0

42.9

69.4

4,000
ppm

58.3
60.4
85.4

45.8
66.7
83.3

91.5

97.9


48.0
56.0
80.0

28.6
53.1
67.3

79.6

91.8

Two-stage predicti
Control

4.3
2.0
6.0

3.5
1.9
5.6

2.0

10.0


6.5
3.9
10.0

21.1
24.8
43.1

28.3

53.7

2,000
ppm

41.0
27.1
62.5

15.6
24.5
37.0

45.5

75.0


33.0
20.7
54.0

25.8
32.8
50.3

46.6

69.9

on (%)
4,000
ppm

63.7
60.4
85.4

43.6
65.6
81.3

90.6

97.9


52.0
55.5
80.0

30.2
52.2
66.9

78.0

91.7

Chi
square

1.54
<.01
<.01

0.49
0.93
0.43

0.10

<.01


0.90
0.02
<.01

0.30
0.16
0.06

0.42

0.08


-------
         experienced higher than usual  mortality  before  final  sacrifice.



         In female rats, there was  increased  mortality  in  the  high-dose



         group relative to controls.   Under these conditions,  competing



         risks may lead to underestimation  of the risk  attributable  to



         the tumors observed in the rats.   The NTP indicated that  the



         decreased rat survival is  likely to  be due to  the frequent



         occurrence of leukemia in  all  groups.



     The draft EPA carcinogen evaluation guidelines (U.S.  EPA, 1984) indicate



that weight should be placed on the analysis  of risks  using the animals  that



have tumors in any one of the sites found to  be statistically  elevated.   The



draft guidelines also indicate that weight  should be placed on the experimental



species and sex group showing the highest  risks.   On these grounds,  combined



carcinomas and adenomas of the lung and/or  liver  in female mice is the end



point of most weight.  Thus, additional analyses  were  conducted, with emphasis



on the data for mice having lung and/or liver tumors.   The following sections



describe these analyses.



4.4.  RISK ANALYSIS CONSIDERING TIME-TO-TUMOR INFORMATION



     In the NTP study, there was a  small number of deaths  in mice  before the



first lung or liver tumors were found.   The first lung  or  liver tumor was a



liver tumor in a high-dose male mouse at week 61.  Table 23 shows  the numbers



of male and female mice alive at 61 weeks  that were subsequently examined at



the lung and liver sites.



     While the number of animals lost to early mortality was small,  because



of the very high incidence of tumor responses seen in  the  high-dose  groups,



analyses were conducted to determine if the early deaths might have  an effect



on estimated risks.  Table 24 shows the results of applying the two-stage



multistage model to the mouse data  using the  denominators  from Table 23.






                                       67

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             TABLE 23.   MICE SURVIVING  TO  61  WEEKS  AND  RECEIVING
                      EXAMINATION  OF  THE LUNG AND LIVER

Response
Males
Females

Control
50
46
Dose
2,000 pptn
45
46

4,000 ppm
47
46
        Note:   In all, 13 mice were  excluded  from  counting  due to  early
               death.   The times  of  these  deaths were:  0-3  months, 5
               deaths; 3-6 months,  1 death;  6-9 months, 4 deaths;  9-12
               months, 1 death; 12 months-61  weeks,  2  deaths.

        SOURCE:  NTP,  1985.
     TABLE 24.  GLOBAL83 MODEL PARAMETERS FOR  MOUSE LUNG AND LIVER TUMORS
         COMBINED—NTP (1985)  DCM DATA ON ANIMALS  SURVIVING TO 61  WEEKS
                          (WHEN FIRST TUMOR OCCURRED)
Two-stage model parameters
Tumor
o c
qQ q-^xlO'0 q2x!0"°
(ppm"1) (ppm"2)
(ppm-1)
Males
Lung or liver
  carcinoma

Lung or liver carci-
  noma or adenoma

Females

Lung or liver carci-
  noma

Lung or liver carci-
  noma or adenoma
0.341
0.777
0.0211
0.114
0.0
0.0371
0.0
0.0
0.0854
0.139
0.159
0.363
0.235
0.582
0.245
0.785
Values of the q-j apply for the NTP protocol  dose schedule.

                                      68

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It should be noted that this simplified analysis  has  the  weakness  that  animals
dying before the first tumor was observed may in  reality  (as  would be seen  in
a large population) have experienced some risk of cancer, and secondly,  that
animals dying after the first tumor was observed  are  treated  as  having  been
at full lifetime risk of cancer independent of their  time of  death.
     The data in Table 24, in comparison with the data in Table  21,  show that
the GLOBAL q^ parameter estimates are consistent  with the earlier  analysis
and are not strongly influenced by the adjustment of  the  denominators.   In
contrast, for some of the analyses, the MLE q^ estimates  are  unstable:   for
male mice the qj estimate for combined adenomas or carcinomas of the lung or
liver in Table 21 is zero, whereas in Table 24 it is  positive.  For female
mice, for the same combination of tumors and sites, this  situation is reversed.
The variability of the MLE linear term is consistent  with the CAG's experience
that this parameter estimate is often unstable in response to small  changes
in the input data.
     To provide a comparison with the dichotomous multistage  model risk esti-
mates developed in the preceding section, a time-to-tumor formulation of the
multistage model, the WEIBULL82 time-to-tumor program, also developed by
Crump (19	), was applied to NTP cancer results in mice.   The WEIBULL82 pro-
gram is based on the following equation:
                Prob [effect] = 1 - exp C-Q (dose) x (time - T0)K]
where Q is a fitted polynomial of the same form utilized in GLOBAL83,  and T0
and K are fitted parameters for the time-dependence of the tumor response.
Risk calculations using this model can be made for two end points:   either
tumors are assumed to be "incidental" and unrelated to the cause of an animal's
                                       69

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death, or the tumors are "fatal"  and  are  assumed to produce death directly.
NTP studies do not attempt to identify the cause of death for animals in
cancer bioassays; therefore, a time-to-tumor analysis must hypothesize as to
the causes of deaths in study animals dying before final sacrifice.  The NTP
(1985) DCM bioassay report provides statistical analyses showing that in mice
both the male and female high-dose groups experienced elevated mortality in
the latter part of the 2-year study.  A simple comparison demonstrates that
the observed tumors may reasonably have produced this mortality.  Table 25
shows the study mortality divided into three time periods, deaths before 61
weeks (when the first lung or liver tumor was observed), deaths between 61
and 103 weeks, and deaths at the  final sacrifice at 104 weeks.  The table
also indicates the numbers of animals, in each death category, which were
observed to have carcinomas of either the lung or liver; these are the tumor
types that can be most strongly expected  to contribute to mortality.  Table
26 shows that mortality before the occurrence of the first lung or liver
tumor was small and comparable in all groups.   In the 61-103 week period,
where elevated treatment-associated mortality is seen, the number of animals
dying without carcinomas was relatively stable among the male dose groups.
In high-dose females, few animals died without carcinomas.  These data are
consistent with DCM-induced tumors leading to the increased mortality observed
in the NTP mice.
     In the following analysis, using the WEIBULL82 program, the effect of
the alternate assumptions of incidental or fatal tumors will be compared.
For completeness, both "fatal" and "incidental" analyses are presented for
adenoma response as well as for the other tumor groupings.  However, it is
noted that a "fatal" tumor analysis may be  less appropriate for adenomas.
The data from these analyses are  given in Table 26.

                                      70

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                TABLE 25.   MORTALITY  IN NTP  MOUSE  DCM  BIOASSAY
Dose
Males
Control
2,000 ppm '
4,000 ppm
Females
Control
2,000 ppm
4,000 ppm
Deaths before
61 weeks
0(0)a
4(0)
3(0)
4(0)
4(0)
4(0)
Deaths
61-103 weeks
11(5)
22(10)
36(29)
21(0)
21(8)
38(35)
Final
sacrifice deaths
39(10)
24(11)
11(10)
25(1)
25(13)
8(8)
aNumbers in parentheses indicate the number  of  animals  in  each  group  found to
 have lung or liver carcinomas.

SOURCE:   NTP, 1985.
                                     71

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             TABLE  26.   COMPARISON OF  HEIBULL82 AND GLOBAL83 PREDICTIONS FOR RISK AT 104 WEEKS--
                                         MICE LUNG AND LIVER TUMORS8
"Fatal" tumor
WEIBULL82 analysis
Tumor
Males
Lung adenoma
Lung carcinoma
Liver adenoma
Liver carcinoma
Lung or liver
carcinoma
Lung or liver
carcinoma
or adenoma
Females
Lung adenoma
Lung carcinoma
Liver adenoma
Liver carcinoma
Lung or liver
carcinoma
Lung or liver
carcinoma
or adenoma
q xlO-3
(ppm-1)
0.125
--
0.0344
0.0417
0.0573
0.106
0.0
<.001
0.0
0.002
0.0153
0.0769
q*xlO-3
(ppm-1)
0.173
—
0.0787
0.1502
0.199
0.252
0.148
0.0651
0.0440
0.0975
0.137
0.210
nb
5
-
5
5
4
4
2
4
4
4
4
4
"Incidental" tumor
WEIBULL82 analysis
(ppm-1)
0.232
0.0561
0.0687
0.0369
0.0526
0.178
0.352
0.109
0.0084
0.0526
0.119
0.0
q*xlO-3
(ppm-1)
0.299
0.199
0.131
0.159
0.304
0.644
0.447
0.248
0.105
0.198
0.392
0.822
n
5
5
5
5
4
4
4
4
4
4
4
4
GLOBAL83
Two-stage
(ppm-1)
0.166
0.0
0.0306
0.0
0.0
0.0
0.242
0.690
0.0
0.0918
0.0
0.345
q*xlO-3
(ppm-1)
0.223
0.141
0.0810
0.142
0.190
0.429
0.310
0.233
0.0872
0.145
0.244
0.870
aFor the WEIBULL82 model, q^ is defined aj the first-degree  polynomial  coefficient  multiplied  by  the
 time function evaluated at week 104.   q, is derived  from model  risk  predictions at  low  dose.
bn indicates the degree of the dose polynomial used  in the WEIBULL82 analysis.   Different  degrees  were
 used in this exploratory analysis.

Values of the qi apply for the NTP protocol  dose schedule.
                                                   72

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     The following observations can be drawn from these data:



     1)  The q1 and qj values for the "incidental" tumor analysis  from the



         WEIBULL83 program were generally higher than the values from the



         "fatal" tumor analysis.  However, the size of this  difference was



         moderate, constituting approximately a factor of two,  with a maximum



         difference of a factor of four for q^  Such differences  are



         expected because the "fatal" tumor analysis excludes  tumors found



         at final sacrifice, and these tumors provide important quantitative



         contributions to the NTP findings (see Table 25 for examples).



         While the qj estimates generally followed this same pattern, the



         deviations were greater in some cases.



     2)  q^ and q^ estimates from the two-stage GLOBAL83 analysis  agreed



         overall with the range of "incidental" and "fatal"  tumor  WEIBULL82



         analyses.  In all cases, the GLOBAL83 q^ values fell  either



         within or very close to the range of the two WEIBULL q^ values.



     From this exploratory analysis, it can be seen that use of the WEIBULL82



time-to-tumor analysis does not lead to risk estimates strongly different from



those derived from GLOBAL83.  The WEIBULL82 program has been less  widely



utilized than the GLOBAL83 program, and requires assumptions as to the cause



of animal death.  For these reasons, WEIBULL82 is less well  suited than



GLOBAL83 for formal use in the present risk assessment.



4.5.  COMPARISON OF RISKS ESTIMATED WITH OTHER DOSE-RESPONSE MODELS



     In order to provide comparison with the two-stage (restricted) multi-



stage estimates presented in Table 5, calculations for combined lung and



liver tumors were also made with the one-stage (one-hit) and four-stage



(dichotomous) formulations of the multistage model.  The one-stage or linear



model has been one of the most widely used models in carcinogen risk assess-
                                      73

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ment, and provides a simple formulation  of  the  hypothesis  that  many  fundamental



carcinogenic processes are linear in  nature.  The  four-stage  model shares  the



multistage rationale of the two-stage model,  but allows  a  sharper  upward cur-



vature in risk estimates.  Table 27 shows parameter estimates for  one-stage



and four-stage versions of the GLOBAL83  program for tumor  response data  from



Table 18.  The one-stage model fits the  experimental  data  acceptably for the



combined carcinomas and adenomas in both the  male  and female  groups.  For  the



females, the one-stage model  response estimates are within 4% of the observed



response for the three experimental doses;  for  males, the  estimates  are  within



6% of the observed response.   For carcinomas  alone, the  one-hit model  does not



provide a good fit to the data in either sex  (p <  0.05 males, p <  0.01 females,



by the chi square test).  As  can be seen from Tables 21  and 22, the  two-stage



model provides an acceptable  fit to the  data  for all  four  tumor end  points.



The four-stage model provides an exact fit  for  all  four  data  sets.



     These analyses demonstrate that  for combined  adenomas and  carcinomas  of



the lung and/or liver in both male and female mice, the  data  are compatible



with a linear dose-response,  and that the 95% UCL  linear term estimates  from



the three models are consistent within 20%  in males and  within  10% in females.



For the carcinoma response, where the one-stage model did  not fit  well,  the



four-stage model yielded positive MLE linear  term  estimates for both sexes,



while the two-stage model did not.  The  UCL four-stage risk estimates were



approximately 50% and 20% higher than the UCL two-stage  estimates  for combined



lung and liver carcinomas in  the males and  females, respectively.



     To provide comparisons with multistage risk model estimates,  two models



with different theoretical formulations, the  Weibull  (dichotomous) and probit



models, were used to analyze the combined lung  and liver tumor  data  in both the



male and female mice.  These models were fitted to the data using  the RISK81






                                       74

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                             TABLE  27.   ONE-STAGE  AND MULTISTAGE  MODEL  PARAMETERS  FOR TUMORS  IN MICE3
Tumor
                                    One-stage
                                             Chi
                                             square
                                         Two-stage
                                                    Cni
                                                   square
         Four-stage
                   Chi
                  square
in
Male mice

Lung or liver
  carcinoma

Lung or liver
  carcinoma
  or adenoma
                           0.238     0.329    4.44
                           0.348     0.505     1.94
                                  0.0      0.190    0.42
                                  0.0      0.424    <.01
0.058     0.223     <.01.
0.125     0.429     <.01
Female mice

Lung or liver
  carcinoma

Lung or liver
  carcinoma
  or adenoma
0.418    0.522    6.77


0.736    0.936    1.25
                                                         0.0
                                           0.244    <.01
                                                         0.345    0.870    <.01
0.120     0.368     <.01
                                                               0.472     0.870     <.01
      aUnits:  ppm~l
ID"3.

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computer program developed by Kovar and  Krewski  (1981).   The  RISK81  program



provides two formulations of both  models,  one  which  is  based  on  the  assumption



that the observed tumor incidence  is independent  of  the  background tumor  rates



observed in the controls, and a second  formulation which assumes that  the



carcinogen contributes a dose that is additive to the  background effects  seen



in the controls.  In the additive  case,  the  probit and  Weibull models  produce



risk estimates that are linear at  low dose.



     Tables 28 through 31 provide  the results  from these models  in comparison



to the two-stage GLOBAL83 results.  The  tables show  the  DCM doses that are



estimated to produce four given levels  of  risk under the different models, as



well as lower confidence limits (LCLs)  on  the  dose that  produces the specified



effect.  LCL dose estimates correspond  to  UCL  risk estimates.



     In all cases, the doses estimated  to  produce a  given risk by the  back-



ground-independent probit model are markedly higher  than those predicted  by  the



multistage model (four orders of magnitude difference  in the  female  mice  com-



bined tumor group).  While the independent Weibull MLE estimates of  dose  are



substantially below those obtained with  the  independent  probit model,  they are



substantially higher than those obtained with  the two-stage multistage model



in all four analyses.  The independent  Weibull MLE estimates  are broadly  com-



parable to the multistage MLE estimates  for  cases where the MLE  multistage



linear term is zero.  The LCL dose estimates of the  independent  Weibull model



are notably lower than the MLE estimates.



     The additive background formulation of  both  the Weibull  and probit models



converged to provide acceptable parameter  estimates  only for  the female mouse



data sets.  In these two cases, both models  produced estimates of dose for a



fixed risk that were linear at low dose and  were  markedly higher than  the



corresponding independent background models.  The MLE  and LCL dose estimates





                                       76

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                         TABLE 28.  MALE MICE CARCINOMAS OF LUNG OR LIVER:
                          DCM DOSES ESTIMATED TO PRODUCE GIVEN RISK LEVELS
                             (DOSES IN PPM UNDER NTP PROTOCOL SCHEDULE)
Risk level
10-2
10-4
10-6
10-8
GLOBAL83
two-stage
MLE LCL
369 52.4
36.8 0.524
3.68 5.24 x 10-3
0.368 5.24 x 10-5
Independent
probit
MLE
1,080
543
329
217
LCL
531
185
84.7
44.4
Independent
Wei bull
MLE
723
123
20.9
3.57
LCL
217
11.0
0.556
0.0281
Calculations are based on excess risk over background.
Probit and Weibull model estimates calculated using RISK81 computer program.
LCL estimates are the 95% confidence lower bound on dose (variance based on log dose for RISK81).

Note:  The additive probit and additive Weibull  models failed to converge for this data set.

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                             TABLE 29.  MALE MICE CARCINOMA OR ADENOMA OF LUNG OR LIVER:
                                   DCM DOSES ESTIMATED TO PRODUCE GIVEN RISK LEVELS
                                      (DOSES IN PPM UNDER NTP PROTOCOL SCHEDULE)
—I
00
Risk level
10-2
io-4
ID'6
10-8
GLOBAL83
two-stage
MLE LCL
306 23.4
30.6 0.233
3.06 2.33 x IO-3
0.306 2.33 x 10-5
Independent
probit
MLE
885
410
236
150
LCL
353
101
41.0
19.5
Independent
Weibull
MLE .
493
54.0
5.93
0.652
LCL
100
2.09
0.0439
9.16 x IO-4
         Calculations are based on excess risk over background.
         Probit and Weibull model estimates calculated using RISK81 computer program.
         LCL estimates are the 95% lower confidence limits on dose (variance based on log dose for RISK81),

         Note:  The additive probit and additive Weibull models failed to converge for this data set.

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                                     TABLE 30.  FEMALE MICE CARCINOMAS OF LUNG OR LIVER:
                                      DCM DOSES ESTIMATED TO PRODUCE GIVEN RISK LEVELS
                                         (DOSES IN PPM UNDER NTP PROTOCOL SCHEDULE)
Risk
level
10-2
10-^
ID'6
10-8
GLOBAL83
two-stage
MLE
262
26.1
2.61
0.261
LCL
40.7
0.410
4.10xlO-3
4*10x10-5
Independent
probit
MLE
769
412
260
176
LCL
503
218
117
70.1
MLE
163
1
0
1
Additive
probit
LCL
81.4
.92 0.78
.0192 0.0078
.92xlO-4 7.8x10-5
Independent Additive
Weibull Weibull
MLE
309
35.8
4.15
0.482
LCL MLE
142 134
8.03 1.52
0.452 0.0152
0.0255 1.52xlO-4
LCL
68.8
0.686
0.00686
6.86x10-5
Calculations are based on excess risk over background.
Probit and Weibull model estimates calculated using RISK81 computer program.
LCL estimates are the 95% lower bound on the dose (variance based on log dose for RISK81).

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                            TABLE 31.  FEMALE MICE CARCINOMA OR ADENOMA OF LUNG OR LIVER:
                                  DCM DOSES ESTIMATED TO PRODUCE GIVEN RISK LEVELS
                                     (DOSES IN PPM UNDER NTP PROTOCOL SCHEDULE)
CO
o
GLOBAL83
two-stage
Risk
level
10-2
10-4
ID'6
10-8
MLE
28.8
0.290
2.90x10-3
2.90x10-5
LCL
11.6
0.115
1.15x10-3
1.15x10-5
Independent
probit
MLE
478
238
141
92.5
LCL
198
67.4
30.2
15.6
Additive
probit
MLE
45.1
0.464
4.64x10-3
4.64x10-5
LCL
24.6
0.249
2.49x10-3
2.49x10-5
Independent
Wei bull
MLE
93.4
4.75
0.242
0.0124
LCL
17.2
0.196
2.23x10-3
2.53x10-5
Additive
Wei bull
MLE
36.3
0.369
3.69x10-3
3.69x10-5
LCL
15.7
0.156
1.56x10-3
1.56x10-5
     Calculations are based on excess risk over background.
     Probit and Weibull  model estimates calculated using RISK81 computer program.
     LCL estimates are the 95% lower bound on dose (variance based on log dose for RISK81).

-------
were quite comparable between the two models (maximum differences under 50% at



low dose).  For female mouse carcinomas, the MLE estimates from these models



lead to dose estimates that are markedly lower than the (non-linear)  MLE multi-



stage estimates.  For the combined carcinomas and adenomas, the MLE estimates



of additive Weibull, additive probit, and multistage models agreed within



approximately 50%.  For both groupings of tumors in female mice, the LCL dose



estimates of the additi.ve probit, additive Weibull, and multistage models were



within a factor of two of each other, with the two-stage multistage model



leading to the lower estimates of dose for a fixed risk.



4.6.  COMPARISON OF NTP (1985) RESULTS WITH OTHER BIOASSAYS



     There are several recent long-term animal studies of DCM in addition to



the NTP (1985) bioassay.  These studies are reviewed in detail  in the EPA



Health Assessment Document for DCM (U.S. EPA, 1985).  While there were signi-



ficant limitations in these studies, which often included maximum doses well



below what the animals could tolerate, some positive and suggestively positive



carcinogenic responses were obtained.  This section develops GLOBAL83 parameter



estimates (for a multistage model with the maximum polynomial degree equal to



the number of dose groups minus one) for the responses found in rats and mice,



using data taken from EPA (1985).



     1)  The Dow Chemical Company (1980) 2-year inhalation study in Sprague-



Dawley rats found some statistically positive elevations in mammary tumors in



this strain, which has a normally high spontaneous mammary tumor rate, making



positive results more difficult to obtain statistically.  The strongest find-



ing was the elevation of the total number of mammary tumors seen in female rats



(165/99 in controls vs. 287/97 in the high-dose groups).  However, the GLOBAL83



model cannot accommodate data of this form.  The percentages of mammary tumors



in the different female groups was not statistically elevated,  but can be used






                                       81

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in GLOBAL83 to calculate an upper-bound risk q^  on  the hypothesis  of  a  real
biological effect.  The male rat data,  which indicated an  elevated mammary
tumor response at the high-dose point (not significant) are also used in  this
manner (Table 32).
     For comparison, the combined mammary tumor  data from  the NTP  (1985)  study
in F344/N rats (using the same dosing schedule)  yielded values of  q^  =  0.0311
x 10"^ for males and q^ = 0.164 x 10"^ for females.  Thus, while the  mammary
tumor incidences were not statistically elevated in the Dow (1980) study, the
upper bounds on q^ that can be derived from the  Dow results are in close
agreement with the NTP (1985) study.
     At a second site, sarcomas in or around the salivary  gland in male rats,
the Dow (1980) study found statistically positive results  (Table 33).  The  NTP
study did not find an elevated sarcoma incidence in or around the salivary  gland.
     2)  The Dow Chemical Company (1982) inhalation study  found evidence  of an
elevated mammary tumor incidence in female rats  (the tumor percentage was sta-
tistically elevated at 200 ppm compared with controls).  The total number of
mammary tumors found also showed an increase with dose, as in the Dow (1980)
study.  GLOBAL83 is applied to the incidence data to determine the maximum
linear dose-response component that is compatible with these data (Table  34).
     The estimate of q^ derived from this study  is high compared with that  for
mammary tumor incidence in female F344/N rats in the NTP (1985) study (q^ =
0.164 x 10-3 ppm-1).
     3)  The National Coffee Association (NCA)(1982a, b) study found  evidence
of an increased occurrence of liver tumors in female F344 rats given  DCM in
drinking water.  The GLOBAL83 value for q^ is shown in Table 35, calculated
on the basis of the experimentally applied dose  units and also calculated
using the same dose units as in the NTP (1985) study.  (This second estimate
                                      82

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                         TABLE 32.   RAT MAMMARY  TUMORS
Dose3
Response
Females
Males
Control
179/96
7/95
500
ppm
81/96
3/95
1,500
ppm
80/95
7/95
3,500
ppm
83/97
14/95
GL.OBAL83
ql ppm-1
0.201 x 10-3
0.040 x 10~3
aDose administered 6 hrs/day,  5 days/wk,  for 2 years.

Value for q^ based on administered doses  and dose time schedule.

SOURCE:  Dow Chemical Co., 1980.
                   TABLE 33.  MALE RAT SALIVARY GLAND TUMORS
                           Dose3
                        500      1,500     3,500     ^   GLOBAL83
           Control       ppm       ppm       ppm     q  (x 10"3 ppm"1)
             1/93       0/94      5/91     11/88          0.043
           3Dose administered 6 hrs/day, 5 days/wk,  for.2 years.

           Value for q^ based on administered dose and  dose time
           schedule.

           Note:  These data show a statistically significant
                  (p < 0.001) test for a linear trend,  and the
                  high-dose group response is elevated  compared
                  to the controls (p = 0.002).

           SOURCE:  Dow Chemical Co., 1980.
                                       83

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                      TABLE 34.   FEMALE  RAT  MAMMARY  TUMORS
                           Dose3
           Control
            50
            ppm
200
ppm
500
ppm
52/70
                       58/70
                     61/70
         55/70
         1.22 x lO-3
           aDose administered 6 hrs/day,  5  days/wk,  for  2  years.

           Value for q^ based on administered  dose and dose  time
           schedule.

           SOURCE:   Dow Chemical Co.,  1982.
         '  TABLE 35.  NEOPLASTIC NODULES OR HEPATOCELLULAR CARCINOMA
                              IN FEMALE F344 RATS
Control
             Dose (mg/kg/day)a
                     	         *                  *
                                        ql                 ^1
         50     125      250      exp.  dose units      NTP  dose  units
 0/134
1/85    4/83    1/85    6/85
            0.470 x ID'3
                      0.22 x 10-3
aDose delivered in drinking water.

Note:  The tumor responses in the 50 and 250 mg/kg/day groups  were elevated in
       comparison with controls at the p < 0.05 level.

SOURCE:  NCA, 1982a, b.
                                      84

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was derived using the ppm to mg/kg/day conversion  factors  presented in sec-
tion 4.7. below.)  The NTP (1985)  report  noted that a positive, but marginal,
increase in female rat hepatocellular neoplastic nodules or hepatocellular
carcinomas was observed in that study (q^ approximately 0.03 x 10~3 ppm"1).
     4)  The NCA (1983) study in mice found  evidence of an increased incidence
of liver tumors in males.  As with rats,  a value of q^ using the dose units
of the NTP (1985) study was derived,  and  is  shown  in Table 36.  For comparison,
the NTP study, which found elevated rates for the  same tumors, yields a qt =
0.195 x 10-3 estimate.
     In conclusion, the studies discussed in this  section  provide some evi-
dence for DCM-induced tumors at sites where  the NTP  (1985) study found tumors;
in addition, the Dow (1980) study  showed  an  increase in salivary gland region
tumors in male rats.  Estimates of q£ have been derived for these sites to
indicate the maximum linear component of  a tumor dose-response that is consis-
tent with study findings.  These upper-bound risk  estimates are comparable to,
or in some cases, larger than, corresponding estimates derived from the NTP
(1985) study for the same tumor sites.
4.7.  DERIVATION OF HUMAN UNIT RISK ESTIMATES FOR  INHALATION OF DCM
     The EPA Health Assessment Document for  Dichloromethane  (U.S. EPA, 1985)
developed estimates of unit risks  to  humans  on the basis of the Dow (1980)
findings of salivary gland tumors  in  rats.  The same standard CAG assumptions
that were used in that document to convert between animal  and human doses are
also applied here.  It is noted that  in this assessment, the extrapolation
between high and low doses in humans  and  animals is done in a different order
than in the earlier CAG assessment; however, this  does not affect the results.
Table 37 summarizes the values of  q^  derived for tumors found in both
sexes of rats and mice in the NTP  study (data taken from Tables 19 and 20).

                                       85

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         TABLE 36.  HEPATOCELLULAR ADENOMAS OR  CARCINOMAS IN MALE  MICE
             Dose (mg/kg/day)a
Control    60
125     185     250      exp.  dose units       NTP dose units
24/125   51/200   30/100  31/99   35/125      0.995 x 10'3
                                               0.78 x 10-3
aDose administered in drinking water.

Note:  The response at the 125 and 185 mg/kg/day doses were elevated in com-
       parison with controls (p < 0.05).
SOURCE:  NCA, 1983.
              TABLE 37.  VALUES OF qT FOR NTP (1985) BIOASSAY USED
                           TO DERIVE HUMAN ql ESTIMATES
Male rat mammary
  or subcutaneous tumors

Female rat mammary
  tumors

Male mouse lung or liver
  adenoma or carcinoma

Female mouse lung or liver
  adenoma or carcinoma
                "Jt               "31
               :H = 0.0540 x 10" J (ppm"1  exp. protocol)
                  = 0.164  x 10"^ (ppm"1 exp. protocol)
                •fc               "51
               ^1 = 0.429  x 10~J (ppm"1  exp. protocol)
                  = 0.870  x 10~3 (ppm"1 exp. protocol)
                                      86

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     1)  These values are first converted to equivalent rodent values  for con-


tinuous exposure to DCM in units of mg/kg/day.  To make this conversion,  the
                                                            /

inhalation rates for the rodents must first be estimated.   This is done using


the following formulas (U.S. EPA, 1985):






                For mice:       I = 0.0345 (wt/0.025)2/3 m3/day


                For rats:       I = 0.105 (wt/0.113)2/3 m3/day






Inhalation rates are calculated using the NTP (1985) average weights for  male


and female mice and rats at the midpoint  of the bioassay (51-week data point;


the NTP report does not give the average  animal weight over the whole  study


period).  The data are given in Table 38.


     Dose conversion factors can now be calculated between the NTP schedule and


a continuous mg/kg/day exposure.  For example, in male rats:






             1 ppm NTP schedule = 10~6 x  3,478 g DCM/m3 x 1,000 mg/g


             x average exposure 4.29 hrs/day x 1 day/24 hrs x 0.268 m3/day


             /0.462 kg = .361 mg/kg/day




These data are given in Table 39.






     2)  Equivalent human DCM doses and q^ values are now calculated using


the CAG methodology for well-absorbed vapors (DCM in air is likely to  be


absorbed by the lungs to a high degree at low doses in both humans and rodents;


the interspecies conversion is being applied for the risks estimated at low


doses).  The CAG assumes (U.S. EPA, 1985) that humans and animals exposed to


equal doses of a carcinogen on a (mg/kg)2/3 basis over equivalent proportions
                                      87

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       TABLE 38.  ESTIMATED INHALATION RATES FOR NTP (1985)  TEST ANIMALS
                                Weight at                  Estimated
                           bioassay midpoint             inhalation rate
                                  (kg)                      (m3/day)
Male rat
Female rat
Male mouse
Female mouse
0.462
0.278
0.037
0.032
0.268
0.191
0.0448
0.0407
          TABLE 39.  DOSE CONVERSION FACTORS AND EQUIVALENT q? VALUES
                              FOR NTP (1985) STUDY
                                                 -
                          mg/kg/day equivalent            (mg/kg/day)-1
                           of 1 ppm exposure,              for continuous
                              NTP protocol                   exposure3
Male rat
Female rat .
Male mouse
Female mouse
0
0
0
0
.361
.467
.753
.791
0.
0.
0.
1.
149
383
570
10
X
X
X
X
lO-3
•ID'3
1.0-3
10-3
athese values of q^, which apply to the same^tumor types as are listed in
 Table 31, are obtained by multiplying the q^^ values in Table 31 by the
 reciprocals of the values in the first column of this table.
                                      88

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of a lifetime will  encounter the same degree  of  cancer  risk.



     This implies that a rodent with weight WR,  exposed to  a  dose  of  D mg/kg/



day and a human exposed to a dose of D^)"1/3 mg/kg/day encounter the



same lifetime cancer risks.  Table 40 contains human  dose equivalents and



values for q^.



     3)  To obtain an estimate of the unit risk  for a human inhaling  1 yg/m3



of DCM over a lifetime, the standard CAG assumption of  a human  inhalation  rate



of 20 m3/day (U.S. EPA, 1985) is applied.   A  continuous exposure to 1 yg/m3



of DCM is equal to an exposure of







         1 yg/m3 x 10~3 mg/yg x 20 m3/day  x  1/70 kg = 2.86  x  10"4  mg/kg/day







Using the value of q^ in female mice from  Table  34, an  upper-limit lifetime



cancer risk estimate of 4.1 x 10~6 is estimated  for this exposure. Alterna-



tively expressed, q^ = 4.1 x 10~6 (yg/m3)"1.   Using the relation that



1 yg/m3 DCM is equivalent to 2.88 x 10~4 ppm, qjj[ = 1.4  x 10~2 ppm"1



(continuous exposure).



     4)  The CAG's potency index is derived  by multiplying  q^ (mg/kg/day)"1



by the molecular weight of the compound (84.9 g/mole  for DCM) to obtain  q-^



(mmol/kg/ day)  .  For lung and liver tumors, combined, in  female  mice,  q-^



(mmol/kg/ day)~l =1.1.  This value is in  the fourth  quartile of the  CAG



histogram for the potency index distribution.



4.8.  HUMAN UNIT RISK ESTIMATE FOR INGESTION  OF  DCM



     Data both from the NTP inhalation study  and from the earlier  NCA studies



of DCM in drinking water will be considered  in connection with  a unit  risk



estimate for DCM ingestion.  All of the existing studies have limitations  for



estimating the risks from ingestion of DCM.   DCM is  rapidly absorbed  and sys-
                                       89

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temically distributed following either inhalation  or  ingestion  exposure;  thus

an inhalation study is relevant for assessing  hazards from ingestion  exposure.

Nonetheless, exposures via the two routes  are  likely  to  lead  to differing doses

reaching individual organs; in particular, an  inhalation study  may  result in a

higher degree of exposure to lung tissue  and a lesser exposure  to tissues in

the digestive system than an ingestion exposure.

     For this reason the NTP findings for  liver tumors,  but not lung  tumors,

in mice are used for quantitative estimation of risk  from DCM ingestion.   On

the other hand, it should be noted that an analysis based on  the NTP  study

may underestimate risks of liver tumors or other digestive system tumors.
          TABLE 40.  DOSE CONVERSION FACTORS AND EQUIVALENT qt VALUES
                 FOR RODENTS IN NTP (1985)  STUDY AND FOR HUMANS
                          Human mg/kg/day                    ^
                       equivalent for rodent                q^a
                           1 mg/kg/day                  (mg/kg/day)-1
                             exposure                       human


Male rat                       0.188                     0.793 x lO'3

Female rat                     0.158                     2.43 x 10"3

Male mouse                     0.0809                    7.05 x 10'3
Female mouse                   0.0770                   14.3  x 10"3


aThese values of q^, which apply to the same^tumor sites as those given in
 Table 31, are obtained by multiplying the q-^ values in Table 33 by the
 reciprocals of the values in the first column of this table.
                                      90

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     The NCA (1983) drinking water study in  mice  yielded  suggestive  but  not


conclusive evidence of a treatment-associated  increase  in hepatocellular carci-


nomas and/or adenomas in males (U.S.  EPA,  1985).   This  finding  can be directly


used to make an upper-bound risk estimate  from ingestion  exposure to DCM.


However, the NCA study utilized doses that were well  below the  maximum tolera-


ted dose (MTD) (U.S. EPA, 1985).  Thus,  it is  possible  that elevated tumor


incidences would have been found in other  tissues if  the  study  had been  conduc-


ted nearer to the MTD.


     a)  The unit risk from ingestion exposure is obtained using the q^  esti-


mate for liver tumors in female mice  (the  sex  with the  higher  risk estimate):


q^ = 0.160 (ppm"1).  The female mouse data are used for this calculation


because the stronger qualitative and  quantitative data  for carcinogenicity


in the liver were obtained for this sex  in the NTP (1985) study.   If the NTP


findings for male mice were used instead,  the  unit risk estimate given below


would be 20% higher.  Using the above dose-conversion procedures, this value

                                          *             O             1
leads to an equivalent human estimate of q^  =  2.6 x 10    (mg/kg/day)"1.


     If a 70-kg human drinks 2 L/day  of  water  containing  1 ug/L DCM, the


average daily exposure is





          1 yg/L x ID'3 mg/ug x 2 L/day  x  1/70 kg = 2.86  x 10~5 mg/kg/day





Using the above value for q^, the lifetime incremental  cancer  risk is estimated


at 7.5 x ID'8 (ug/L)'1.


     b)  Using the NCA (1983) study,  the value for q^ in  male  mice is 0.995 x


10~3 (mg/kg/day)-1, based on hepatocellular  carcinomas  and adenomas. The male


mouse data from the NCA study are used for this calculation because  only the


male mice showed evidence for carcinogenicity  in  the  liver in  this study, which
                                       91

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was conducted well  below the MTD.   Following  the  dose-conversion  procedure

given earlier in this section,  the  equivalent human  value  is  1.23 x  10~2  (mg/

kg/day)"1.  The corresponding UCL unit  risk estimate for drinking water is

3.5 x 10-7 (ug/L)-l.

     The unit risks calculated  on the basis of the two mouse  studies  are  com-

parable; therefore, the mean value  of 2.1  x 10~7  (yg/L)~l  is  used for the

unit risk estimate for drinking water exposure to DCM.

4.9.  COMPARISON OF ANIMAL AND  HUMAN DATA  RELEVANT TO CANCER  RISK

     The risk prediction for human  exposure to DCM can be  compared with the

observed cancer mortality in a  cohort of Eastman  Kodak employees  exposed  to  DCM

(Friedlander et al., 1978; Hearne and Friedlander, 1981).* Such  an  analysis

was included in U.S. EPA (1984) with reference to the finding of  salivary

tumors in rats, and a parallel  calculation will be shown here. The  continuous

lifetime equivalent DCM exposure of the Kodak employees was estimated to  be

between 1.88 and 7.52 ppm, based on a 20-year exposure for the 252 long-term

workers.  Based on the 95% upper-limit  slope  factor  for the preceding section

(q^ = 1.4 x 10"^ ppm~l for continuous human exposure), the upper  bound on the

lifetime cancer risk encountered by these  workers is estimated to be between
            •
0.026 and 0.105.  For the 252 workers,  this would translate to a  95% upper

limit of 6.6 to 26.5 excess cancer  deaths  over a  lifetime. However,  because

the study follow-up period was  17 years and most  workers were not observed

until death, it is probable that only a fraction  of  the estimated excess  cancer

deaths were seen.  In the absence of a  more  rigorous method,  it is estimated
*0tt et al. (1983) also reported an epidemiologic study of DCM workers;  how-
 ever, the limited follow-up in this study prevents any adequate comparison
 with estimated lifetime cancer risks.   In the Ott study, the overall  mortality
 was less than 10% for all subgroups during the study period.  See U.S.  EPA
 (1985) for further discussion of this  study.
                                       92

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that the fraction of cancer cases that would be observed in the follow-up
period is approximated by the overall  mortality expected in the follow-up:
65.9/252 deaths, or 26%.  Thus, a 95%  upper limit of between 1.7 and  6.8 cancer
deaths due to DCM exposure would have  been predicted for the cohort.   Using the
statistical methods presented by Beaumont and Breslow (1981), the power of  the
Friedlander study, with 17.8 expected  cancer deaths, to detect an excess of 1.7
deaths from total cancer (with 95% confidence) is 0.06; the power to  detect 6.8
cancer deaths is 0.31.
     Tumors of several types were found to be elevated in the NTP mouse and
rat bioassays.  Additionally, it cannot be generally expected that humans
and experimental animals will show a carcinogenic response at the same sites
when exposed to a chemical which is carcinogenic to both.  These factors pre-
vent rigorous comparison of the DCM cancer risk estimated from the NTP study
with findings of an epidemiologic study for particular cancer sites.   However,
as a tentative example of power comparisons that may be made, the ability of
the Friedlander study to detect excess lung cancer deaths is calculated.
     For this example, an estimate of  risk for lung cancer is obtained by ap-
plying the observed excess of lung tumors in female mice (the sex with the
stronger response) to estimate lung cancer deaths in the Friedlander  cohort.
Taking the q± value for female mouse lung carcinoma or adenoma from Table 5
(0.579 x 10~3) and applying the procedure of Section 4.7., the upper-bound  unit
risk estimate for humans is 9.5 x 10~3 (ppm~l) for continuous exposure. Using
this risk value with the same procedure followed above for total cancers leads
to the estimate of an upper bound of from 1.2 to 4.7 lung cancers due to DCM
exposure; the power of the Friedlander study, in which 4.6 lung cancer deaths
were expected, to detect such risk is  0.15 and 0.44, respectively.
     The preceding calculations show that the Friedlander study does  not have

                                       93

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the power to rule out  an  overall  cancer  risk, or  in the example presented, a



lung cancer risk, that is predicted  using the upper-bound  slope derived from



the NTP study.
                                      94

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                                  REFERENCES
Ahmed, A.E.; Anders, M.W.  (1976)  Metabolism of dihalomethanes  to  formalde-
     hyde and inorganic chloride.  Drug.  Metab.  Dispos.  4:356-361.

Ahmed, A.E.; Anders, M.W.  (1978)  Metabolism of dihalomethanes  to  formalde-
     hyde and inorganic halide.  II.  Studies on the mechanism of the
     reaction.  Biochem. Pharmacol. 27:2021-2025.

Ahmed, A.E.; Kubic, V.L.; Stevens,  J.L.;  Anders, M.W.  (1980)   Halogenated
     methanes: metabolism and toxicity.  Fed. Proc.  39(13):3150-3155.

Anders, M.W.; Kubic, V.L.; Ahmed, A.E.  (1977)  Metabolism  of  halogenated
     methanes and macromolecular binding.  J. Environ.  Pathol. Toxicol.
     1:117-121.

Angelo, M.J.  (1985)  Personal communication to  H.L. Spitzer,  U.S.  Environ-
     mental Protection Agency.

Beaumont, J.J.; Breslow, N.E.  (1981)  Power considerations in epidemiologic
     studies of vinyl chloride workers.  Am. J.  Epidemiol.  114:725-734.

Burek, J.D.; Nitschke, K.D.; Bell,  T.J.;  Wackerly, D.L.; Childs, R.C.;  Beyer,
     J.E:; Dittenber, D.A.; Rampy,  L.W.;  McKenna, M.J.   (1984)  Methylene
     chloride: a two-year inhalation toxicity and oncogenicity study  in  rats
     and hamsters.  Fund. appl. Toxicol.  4:30-47.

Crump, K.S.  (1982)  Weibull82
DiVincenzo, G.D.; Hamilton, M.L.  (1975)  Fate and disposition of l4C-methy-
     lene chloride in the rat.  Toxicol. Appl. Pharmacol. 32:385-393.

DiVincenzo, G.D.; Kaplan, C.J.  (1981a)  Uptake, metabolism, and elimination
     of methylene chloride vapor by humans.  Toxicol. Appl. Pharmacol.
     59:130-140.

DiVincenzo, G.D.; Kaplan, C.J.  (1981b)  Effect of exercise or smoking  on
     the uptake, metabolism, and excretion of methylene chloride vapor.
     Toxicol. Appl. Pharmacol. 59:141-148.

Dow Chemical Company.  (1980)  Methlene chloride: a two-year inhalation
     toxicity and oncogenicity study in rats and hamsters.  FYI-OTS-0281-0097,
     Follow-up response A.  U.S. Environmental Protection Agency, Office of
     Toxic Substances.

Dow Chemical Company.  (1980)  Methylene chloride: a two-year inhalation
     toxicity and oncogenicity study in rats.  Toxicology Research Labora-
     tory, Health and Environmental Sciences, Dow Chemical Company, Midland,
     MI.
                                      95

-------
Estabrook, R.W.;  Franklin,  M.;  Baron,  A.;  Shigematsu,  A.;  Hildebrandt, A.
     (1971)  Drugs and cell  regulation (E.  Minich,  ed.).   New  York:  Academic
     Press, pp. 227-254.

Friedlander, B.R.; Hearne,  F.T.;  Hall, S.   (1978)   Epidemiologic  investigation
     of employees chronically exposed  to methylene  chloride.   J.  Occup.  Med.
     20:657-666.

Hearne, F.T.; Friedlander,  B.R.  (1981)  Follow-up  of  methylene chloride
     study.  J. Occup. Med.  23:660.

Heppel, L.A.; Neal, P.A.   (1944)   Toxicology  of  dichloromethane  (methylene
     chloride).  II.  Its effect  on  running activity  in the male  rat.  J.  Ind.
     Hyg. Toxicol. 26:17-21.

Heppel, L.A.; Neal, P.A.; Perrin, T.L.;  Orr,  M.L.;  Porterfield, V.T.  (1944)
     Toxicology of dichloromethane (methylene chloride).   I.   Studies  on
     effects of daily inhalation.  J.  Ind.  Hyg.  Toxicol.  26:8-16.

Heppel, L.A.; Porterfield,  V.T.  (1948)  Enzymatic  dehalogenation of certain
     brominated and chlorinated compounds.  J.  Biol.  Chem. 176:763-769.

Hogan, G.K.; Smith, R.G.; Cornish, H.H.   (1976)   Studies  on the microsomal
     conversion of CH2C12 to CO.   Toxicol.  Appl. Pharmacol. 37(1):112.

Hogeboom, G.H.; Schneider,  W.C.;  Striebich, M.J.  (1953)   Localization and
     integration of cellular function.  Cancer Res. 13:617-632.

Howe, R.B.   (1983)  GLOBAL83: an  experimental program developed  for the  U.S.
     Environmental Protection Agency as an update to  GLOBAL82: a  computer
     program to extrapolate quantal  animal  toxicity data  to  low  doses  (May,
     1982).  K.S. Crump and Co.,  Inc., Ruston,  LA.   Unpublished.

Kirshman, J.   (1984)  Food solvents workshop  I:  methylene chloride.
     Proceedings of the workshop  sponsored by the Nutrition Foundation,
     Inc., Washington, D.C.  March 8-9, 1984, Bethesda,  MD, p. 41.

Kovar, J.; Krewski, D.   (1981)  RISK81: a  computer program for low-dose
     extrapolation of quantal response toxicity data.   Health  and Welfare,
     Canada.

Kubic, V.L.; Anders, M.W.   (1975)  Metabolism of dihalomethanes  to carbon
     monoxide.  II.   In vitro studies.  Drug  Metab. Dispos.  3:104-112.
        «,
Kubic, V.L.; Anders, M.W.   (1978)  Metabolism in dihalomethanes  to carbon
     monoxide.  III.  Studies on  the mechanism of the reaction.   Biochem.
     Pharmacol. 27:2349-2355.

MacEwen, J.D.; Vernot, E.H.; Haun, C.C.  (1972)  Continuous  animal exposure  to
     dichloromethane.  AMRL-TR-72-28,  Systems Corporation Report  No. W-71005.
     Wright-Patterson Air Force Base,  Ohio, Aerospace Medical  Research.
                                      96

-------
Maltoni, C.  (1984)  Food solvents workshop I:  methylene chloride.   Proceedings
     of the workshop sponsored by the Nutrition Foundation,  Inc.,  Washington,
     D.C.  March 8-9, 1984, Bethesda, MD.
McKenna, M.J.; Zempel , J.A.  (1981)  The dose-dependent metabolism of
     methylene chloride following oral administration to rats.  Food Cosmetics
     Toxi-.col. 19:73-78.

McKenna, M.J.; Zempel, J.A.; Braun, W.H.  (1982)  The pharmacokinetics of
     inhaled methylene chloride in rats.  Toxicol. Appl . Pharmacol . 65:1-10.

McKenna, J.J.; Saunders, J.H.; Boeckler, W.R.; Karbowski, R.J.; Nitschke,
     K.D.; Chenoweth, M.B.  (1980)  The pharmacokinetics of inhaled methylene
     chloride in human volunteers.  Paper #176, 19th Annual Meeting, Society
     of Toxicology, Washington, D.C., March 3-13.

National Coffee Association.   (1982a, Aug. 11)  Twenty-four month chronic
     toxicity and oncogenicity study of methylene chloride in rats.  Final
     report.  Prepared by Hazleton Laboratories America, Inc., Vienna, VA.
     Unpublished.

National Coffee Association.   (1982b, Nov. 5)  Twenty -four month chronic
     toxicity and oncogenicity study of methylene chloride in rats.  Addition
     to the final report.  Prepared by Hazleton Laboratories America, Inc.,
     Vienna, VA.  Unpublished.

National Coffee Association.   (1983, Nov. 30)  Twenty-four month oncogenicity
     study of methylene chloride in mice.  Prepared by Hazleton Laboratories
     America, Inc., Vienna, VA.  Unpublished.

National Toxicology Program (NTP).  (1982)  Draft technical report on the
     carcinogenesis bioassay of dichloromethane (methylene chloride), gavage
     study.  Research Triangle Park, NC, and Bethesda, MD.

National Toxicology Program (NTP).  (1985, Feb.)  NTP technical report on the
     toxicology and carcinogenesis studies of dichloromethane in F344/N rats
     and B6C3F1 mice (inhalation studies).  NTP TR 306.  Board draft.

Nitschke, K.; Burek, J.; Bell, T., Rampy, L., McKenna, M.  (1982)  Methylene
     chloride: a two-year inhalation toxicity and oncogenicity study.  Final
     report of studies conducted at the Toxicology Research Laboratory, Health
     and Environmental Sciences, Dow Chemical, Midland, MI, USA.  Cosponsored
     by Celanese Corporation, Dow Chemical, USA, Imperical Chemical Industry
     Ltd., Stauffer Chemical Company, and Vulcan Material Company.

Ott, M.G.; Skory, L.K.; Holder, B.B.; Bronson, J.M.; Williams, P.R.   (1983a)
     Health evaluation of employees occupational ly exposed to methylene
     chloride.  General study design ancl environmental considerations.  Scand.
     J. Health 9(Suppl. l):l-7.
                                      97

-------
Ott, M.G.; Skory ,'^L.K.; Holder,  B.B.;  Bronson,  J.M.;  Williams,  P.R.   (1983b)
     Health evaluation of employees  occupationally  exposed  to methylene
     chloride.  Mortality.  Scand.  J.  Work  Environ. Health  9:8-16.

Ott, M.G.; Skory, L.K.; Holder,  B.B.;  Bronson,  J.M.;  Williams,  P.R.   (1983c)
     Health evaluation of employees  occupationally  exposed  to methylene
     chloride.  Clinical laboratory  evaluation.  Scand.  J.  Work Environ.
     Health 9:17-25.

Ott, M.6.; Skory, L.K.; Holder,  B.B.;  Bronson,  J.M.;  Williams,  P.R.   (1983d)
     Health evaluation of employees  occupationally  exposed  to methylene
     chloride.  Twenty-four hour electrocardiographic monitoring.   Scand.  J.
     Work Environ. Health 9:26-30.

Ott, M.G.; Skory, L.K.; Holder,  B.B.;  Bronson,  J.M.;  Williams,  P.R.   (1983e)
     Health evaluation of employees  occupationally  exposed  to methylene
     chloride.  Metabolism data  and oxygen  half-saturation  pressures.  Scand.
     J. Work Environ. Health 9:31-38.

Price, P.O.; Hassett, C.M.; Mansfield, J.I.  (1978)  Transforming  activities
     of trichloroethylene and proposed industrial  alternatives.  In  Vitro
     14:290-293.

Rodkey, F.L.; Collison, H.A.  (1977a)   Biological  oxidation of  14C-methylene
     chloride to carbon monoxide and carbon dioxide by the  rat.  Toxicol.
     Appl. Pharmacol. 40:33-38.

Rodkey, F;L.; Collison, H.A.  (1977b)   Effect of dihalogenated  methanes  on the
     in vivo production of carbon monoxide  and  methane by rats.  Toxicol.
     A~p~pT7Tharmacol. 40:39-47.

Stevens, J.L.; Anders, M.W.  (1978)   Studies on the mechanisms  of  metabolism
     of haloforms to carbon monoxide.   Toxicol. Appl. Pharmacol. 45:297-298.

Stevens, J.L.; Anders, M.W.  (1979)   Metabolism of haloforms to carbon
     monoxide.  III.  Studies on the mechanism of the reaction.  Biochem.
     Pharmacol. 28:3189-3194.

Theiss, J.C.; Stoner, G.D.; Shimkin, M.B.;  Weisburger, E.K.  (1977)   Test  for
     carcinogenicity of organic  contaminants of United States  drinking waters
     by pulmonary tumor response in strain  A mice.   Cancer Res. 37:2717-2720.

U.S. Environmental Protection Agency.   (1984, Nov. 23)  Proposed guidelines
     for  carcinogen risk assessment.  Federal Register 49:46294-46301.

U.S. Environmental Protection Agency.   (1985, Feb.)  Health assessment docu-
     ment for dichloromethane (methylene chloride).  Final  report.  EPA-700/
     8-82-004F.  Prepared by the Office of  Health and Environmental  Assess-
     ment, Washington, D.C.

Yesair, D.W.; Jaques. P.; Shepis, P.;  Liss, R.H.   (1977)  Dose-related pharma-
     cokinetics of l^C-methylene chloride in mice.   Fed. Proc.  Am. Soc.
     Exp. Biol. 36:998  (abstract).


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