-------
point (sic) and elementary analysis." From this information we conclude that
the consistent positive responses in Salmonella can be attributed to DCM.
Barber et al. (1980) conducted their tests in a chemically inert, closed incu-
bation system and analyzed the concentrations of DCM in the vapor-phase head
space and in the aqueous phase of a test plate by gas-liquid chromatography
(Barber et al., 1981). Based on this information, the mutagenic responses at
the highest dose (i.e., 115 umoles/plate) for TA1535, TA98, and TA100 were
0.0006, 0.006, and 0.03 revertants per pmol, respectively, indicating DCM is a
weak mutagen for Salmonella under the conditions of the test.
The results discussed above clearly show that DCM is mutagenic in
Salmonella. However, questions have been raised about the applicability of
these results to predicting mutagenicity in other species, especially mammals.
DCM is metabolized, apparently via mutagenic intermediates, to CO and COp in
both rodents and humans (see Chapter 4). CO is produced by oxidative dechlo-
rination of DCM by the microsomal P* mixed function oxidase system. Formyl
chloride is believed to be an intermediate in this pathway. A second cytosolic
glutathione transferase system dehalogenates DCM to produce formaldehyde, which
is further oxidized to CO^. This pathway is thought to proceed via an S-
chloromethyl glutathione intermediate (Ahmed and Anders, 1978; Kubic and Anders,
1975). Formyl chloride and S-chloromethyl glutathione are highly reactive
alkylating agents. Salmonella also metabolizes DCM to COp and CO apparently
by reaction pathways similar to those occurring in mammals (Green, 1980, 1981).
Because of the reactivity of formaldehyde, formyl chloride, and S-chloromethyl
glutathione, and the proximity of bacterial DNA to bacterial cytoplasmic enzymes,
it has been hypothesized that these chemical substances are more effective as
mutagens when they are formed by bacterial metabolism than when they are formed
outside the bacterial cell by rat liver fractions (Green, 1980, 1981). The
basis for this hypothesis is that rat liver fractions used for metabolic acti-
vation have little effect on increasing the mutagenicity of methylene chloride
in the Ames test. The implication is made that as organismic complexity is
increased, there is less likelihood that DCM will cause mutations. It is argued
that compartmentalization of DNA into the nucleus protects the genetic material
from exposure to the mutagenic metabolites of DCM (i.e., they would react with
other cellular constituents first) and thus there is little or no mutagenic
risk. The positive results using eukaryotes, discussed in the following sec-
tions, argue against this hypothesis.
5-31
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5.3.2.1.2 Yeast. Call en et al. (1980) studied the ability of DCM obtained'
from Fisher Scientific Company (purity not reported) and six other halogenatJSS
hydrocarbons to cause gene conversion, mitotic recombination, and reverse-
mutations in Saccharomyces cerevisiae (Table 5-5). Strain D7 log phase celjl
were incubated for 1 hour in culture medium containing 0, 104, 157, and 209 Ifflf
DCM. The percent survival for these doses were 100, 77, 42, and <0.1, respec-
tively. Due to the toxicity of the compound, the genetic end points were not
measured at the highest dose. The response for the other doses (0, 104, and
157 mM) expressed per 10 survivors were: gene conversion at the trp-5 locus
(18, 28, and 107); mitotic recombination for ade-2 (310, 190, and 4,490); total
genetic alterations for ade-2 (3,300, 3,900, and 14,000); and reverse mutations
for ilv-1 (2.7, 4.4, and 5.8). A greater than twofold dose-related increase
over negative controls was observed for each endpoint measured. The magnitude
of recombinogenic response at the ade locus may have been overestimated in this
study because the treatment regime used for estimating the recombinants overlaps
that used for estimating the number of trp-5 convertants. No exogenously
applied metabolic activation was used in these experiments, which indicates
that yeast metabolizes DCM intracellularly to a mutagenic intermediate(s) thaf
reaches nuclear DNA. In another genetic study employing yeast, Simmon et al.
(1977) reported that DCM (source and purity not given, but stated to be the
highest available purity) did not increase mitotic recombination in strain D3
Q
of Saccharomyces cerevisiae when cells (1 x 10 ) in suspension culture .were
exposed for 4 hours at 30ฐC (Table 5-5). The doses used and the actual expeff-
mental values obtained for mitotic recombination were not reported. The dis-
crepancies between the work by Call en et al. (1980) and Simmon et al. (1977)
may be due to a number of factors including the different strains used (D3
versus D7), exposure time differences (4 hours versus 1 hour), or differences
in the incubation temperature (30ฐC versus 37ฐC). Callen et al. (1980) repfrtecj
that an increase in the treatment time of D7 cells with DCM from 1 hour to 4
hours significantly reduced the level of genetic activity. Other variables,
such as a lower level of P.5Q enzymes in strain D3, could conceivably account
for the discrepancy in the results. At this time, DCM is considered to be a
positive mutagen in yeast.
5.3.2.1.3 Drosophila. Two reports are available concerning the ability of
DCM to induce sex-linked recessive lethal mutations in Drosophila melanogaster
(Table 5-6). Abrahamson and Valencia (1980) reported negative results, while
5-32
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5-35
-------
a positive response was reported by Gocke et al. (1981). Abrahamson and Valencia
(1980) conducted their sex-linked recessive lethal tests using two routes of
administration: adult feeding and injection. Due to the low solubility of
DCM in aqueous solutions, high concentrations of the test substance were not
used in these experiments, which may account for the negative response observed.
In the feeding study, male Canton S flies were placed in culture vials contain-
ing glass microfiber paper soaked with a saturated solution of 1.9% DCM in a
sugar solution (224 mM DCM) for 3 days. The feeding solution was added twice
daily to compensate for evaporation of the compound. At this dose, there was
no evidence of toxicity. After mating, chromosomes from the 14,682 offspring
of treated parents and chromosomes from the 12,450 offspring of concurrent
control parents were assessed for recessive lethal mutations. No evidence of
mutagenicity was observed. DCM gave a level of 0.204% lethal mutations com-
pared to 0.215% for controls. However, because of the volatility and insolu-
bility of DCM, the actual dosing to the animals may have been much less than
expected.
In the injection study, 0.3 |jl of an isotonic solution containing 0.2%
DCM was administered to male flies. This exposure level resulted in 30% post-
injection mortality. However, the post-injection mortality observed for the
controls was not reported. Because the mortality observed in studies such as
this is due not only to the test chemical administered, but also to the damage
caused by injection, concurrent negative controls upon which to base conclusions
concerning toxicity of the test chemical are necessary.- After mating, 8,262
chromosomes from the offspring of treated parents and 8,723 chromosomes from ,
the offspring of control parents were assessed for recessive lethal mutations.
No evidence of mutagenicity was observed by this route of administration.
Flies injected with 0.2% DCM had 0.157% lethals compared to 0.206% for controls.
Gocke et al. (1981) also tested DCM (Merck, Darmstadt, FRG, purity not
given) for its ability to induce sex-linked recessive lethal mutations in
Drosophila. Two solutions, 125 mM and 625 mM in 2% DMSO and 5% saccharose,
were fed to wild-type Berlin K male flies for an unreported period of time.
The higher dose (625 mM) is reportedly close to the LDcn- These males were
then mated to Base females. Three broods were scored (i.e., offspring from
virgin females mated to treated males on days 1 through 3, 4 through 6, and 7
through 10 after exposure). There were significantly more lethals (24 out of
5-36
-------
4,845 chromosomes scored) for brood 1 (0.50% lethal mutations) compared to the
negative controls, (19 lethals out of 7,130 chromosomes, 0.27% lethal mutations),
P <0.05. Elevated, but not statistically significant increases in lethal
mutations were noted in broods 2 (0.16 percent compared to 0.14% lethals) and
3 (0.47 percent compared to 0.39% lethals) of the treated flies compared to
the controls. As noted in Table 5-6 the incidence of lethals is dose-related.
The incidence of lethals for the high dose in brood 2 is elevated nearly three-
fold over th;e corresponding negative control value (0.41 versus 0.14 lethals,
. respectively), but this may not be a significant increase because of the small
sample size (735 vials). This test indicates DCM is mutagenic to sperm in
Drosophila (a multicellular eukaryote). The discrepancies in results between
Abrahamson and Valencia (1980) and Gocke et al. (1981) may be due to stock-
specific differences (Canton S. versus Berlin K) in the metabolic activation
of DCM or more likely to the larger doses of DCM employed by Gocke et al. (1981).
5.3.2.1.4 Nematodes. In another sex-linked recessive lethal test, Samoiloff
et al. (1980) tested DCM for its ability to mutate the nematode Panagrellus
redivivus (Table 5-6). Individual females homozygous for the X-linked mutation
b7 (coiled phenotype in liquid medium) were grown for 120 hours in the presence
of several concentrations of DCM ranging from 10~3M to 10~8M. They were then
washed and mated to S-15 males who carry an X-chromosome crossover suppressor
extending at least 15 recombination units to either side of b7. One hundred
female progeny were collected and mated to wild-type (C-15) males and their
progeny scored for the presence of the b7 phenotype. The absence of b7 male
progeny indicates lethality of the X-chromosome marked with b7 derived from a
female grown on DCM. Three replicate experiments were performed. A non-dose-
related increase in the level of lethals was observed in the progeny of DCM-
treated worms compared to the negative controls. For worms treated with 10~8
"6-4 c. '
10 , and 10 M DCM, the corresponding lethal mutations/10 loci were 6.0, 10.1,
and 9.8, respectively, compared to an estimated spontaneous mutation frequency
of 2.2 x 10 mutations/locus. Some of the positive controls tested concurrent-
ly, such as proflavine, yielded a positive response (12.5, 10.0, and 28.6
lethals/10 loci at 10~8, 10~6, and 10~4M, respectively). But others, such as
aflatoxin B and ethyl methanesulfonate (EMS), did not cause an increase in
lethal mutations. The investigators suggest that DCM is mutagenic in nema-
todes, but firm conclusions cannot be made because the assay is not validated
5-37
-------
and, more importantly, because of the negative responses obtained with some of
the positive controls.
5.3.2.1.5 Mammalian cells in culture. Jongen et al. (1981) tested DCM for
its mutagenic potential in several mammalian cell culture tests. Testing for
the induction of forward mutations at the HGPRT locus is described here (Table
5-7). Testing for the ability of DCM to cause sister chromatid exchanges
(SCE), unscheduled DNA synthesis (UDS), and inhibition of DMA synthesis (IDS)
will be discussed later in the section on other indicators of DNA damage.
In their testing of the ability of DCM to cause forward mutations, Jongen
et al. (1981) incubated log phase CHO and V79 cells with 1, 2, 3, 4, and 5%
DCM or 1, 2, 3, and 4% DCM, respectively, at 37ฐC for 1 hour in a closed glass
container without exogenous S9 mix. DCM was obtained from Merck (analytical
grade). The cells were exposed to gaseous DCM and then DCM in solution for
15-minute intervals each by alternately tilting the plates then placing them
horizontally. After growth to allow for an 8-day (CHO cells) or 6-day (V79
cells) expression period, mutant cells were selected in thioguaninecontaining
medium. DCM failed to increase the mutation frequency of either cell line at
any dose compared to controls. However, DCM was not very cytotoxic to either
cell line. At the highest dose, survival decreased only 20 percent. It would
be appropriate to repeat the experiment using higher doses of DCM. EMS yielded
a positive, dose-dependent increase in mutation induction in V79 cells, but it
was not tested in CHO cells.
Based on the positive responses in bacteria, yeast, and Drosophila, and
the suggestive positive response in the nematode, DCM is judged to be capable
of causing gene mutations. Metabolic activation to highly reactive mutagenic
metabolites apparently accounts for this response, and although these are
thought to be unstable, they seem to be capable of interacting with genetic
material of both prokaryotes and eukaryotes.
5.3.2.2 Chromosomal AberrationsThree studies on the ability of DCM to cause
chromosomal aberrations were evaluated. Burek et al. (1984) subjected 4 groups
of 10 Sprague-Dawley albino rats (Spartan substrain, SPF-derived, 5 males and
5 females) to 0, 500, 1,500, or 3,500 ppm (0, 1735, 5205, 12,145 mg/m3) DCM by
inhalation 6 hr/day, 5 days/week for 6 months. The animals were then
sacrificed, bone marrow cells were collected, chromosome preparations were
made, and slides were coded and analyzed. Two hundred metaphases per animal
were scored and aberrations were tabulated (See Table 5-8). No increase
5-38
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5-39
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In the total frequency of abnormal cells or in the frequency of any specific
type of aberration was noted in the treated animals compared to the controls.
There were 1.1 ฑ 1.3, 0.6 ฑ 0.7, 0.8 ฑ 1.2, and 1.1 ฑ 0.9% cells with aberra-
tions in animals treated with 0, 500, 1,500, and 3,500 ppm (0, 1735, 5205,
12,145 mg/m ) DCM, respectively.
Thilagar and Kumaroo (1983) treated CHO cells grown in either plastic or
glass culture flasks with 0, 2, 5, 10 and in one experiment 15 pi/ml (i.e., 0,
31, 78, 156, and 234 mM) DCM for 2 hours with or 12 hours without 59 mix derived
from Aroclor-induced rat livers. DCM was obtained from Fisher Scientific (cer-
tified A.C.S., lot no. 713580). After the exposure period, the cells were washed,
refed, and allowed to grow before being arrested at metaphase with colcemid
and harvested for chromosome preparation. Slides were coded and read "blind;"
100 cells were scored for each dose level (50 cells/duplicate flask). DCM in-
duced a dose-related increase in chromosome aberrations (see Table 5-8) ranging
from 0.02 aberration/cell in the negative controls to 1.44 aberrations/ cell
at 15 ul/ml(234 mM). The response was not dependent on the presence of the
exogenous metabolic activation system.
Gocke et al. (1981) assessed the ability of DCM (Merck, Darmstadt; purity
not given) to cause micronuclei in polychromatic erythrocytes (PCE). Two male
and two female NMRI mice were used for each of three dose levels (425, 850,
and 1,700 mg/kg per intraperitoneal injection). The highest dose approximated
the LD,-n for mice. Intraperitoneal injections of each dose v/ere given at 0
ou
and 24 hours, the animals were sacrificed at 30 hours, bone marrow smears were
made, and 1,000 PCEs per animal were scored for the presence of micronuclei.
An increase in PCEs with micronuclei was observed at the two highest doses,
but the response was not dose-related and was not double the control value.
Thus, the results are considered suggestive of a positive response but are not
conclusive. (There were 0.19% micronuclei in the untreated controls compared
to 0.35% micronuclei in the animals receiving two injections of 850 mg/kg, and
0.28% micronuclei at the highest dose).
Based on the positive response reported by Thilagar and Kumaroo (1983),
DCM is tentatively judged to be capable of causing chromosomal aberrations.
The rn vivo negative responses reported by Dow Chemical Company (1980) and
Gocke et al. (1981) are not readily comparable with the j_n vitro results.
5.3.2.3 Other Indicators of DNA Damage
5.3.2.3.1 Sister chromatid exchange (SCE). Two papers have been published on
the ability of DCM to induce SCEs (Table 5-9). Jongen et al. (1981) tested
5-40
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the ability of 0.5, 1.0, 2.0, 3.0, and 4% DCM (i.e., 58, 118, 235, 353, and
471 mM) to induce SCEs in V79 cells. Log phase cells were incubated at 37ฐC
for 1 hour in a closed glass container. The cells were exposed to DCM in the
gaseous phase and in the medium by tilting the plates for 15 minutes, then
placing them horizontally. The experiment was conducted seven times and each
yielded a dose-related increase in SCEs/cell, which approached but did not
exceed a twofold increase above the control level. An analysis of variance of
effects of different doses within experiments showed the increases in frequency
of SCEs to be statistically significant (p <0.001). Increasing the exposure
time to 2 hours or 4 hours or using S9 from rat liver did not alter the shape
of the dose-response curve, which plateaued at 1% DCM. The authors suggest
that this phenomenon is due to a saturation of the metabolic activation system
of V79 cells.
Thilagar and Kumaroo (1983) exposed CHO cells to 0, 2, 5, 10, and in one
experiment 15 ul DCM/ml of medium (0, 31, 78, 156, and 234 mM DCM) for 2 hours
with and 24 hours without metabolic activation. The cells were grown for 24
hours in BrdUrd followed by a mitotic shake off, fixing, and staining by a
fluorescence-pius-Giemsa technique, and then the coded slides were scored
"blind." Slight dose-related elevations in SCE values were noted (see Table
5-9), but they never exceeded a 50% increase at the highest dose. The authors
judged their test to be negative, but the concentrations were lower than used
by Jongen et al. (1981).
McCarroll et al. (1983) reported in an abstract that consistent and dose-
related increases were observed in SCEs in CHO cells following 24-hour exposures
to 1, 3.6, 5.4, and 7.0% atmospheres of DCM. A 7% atmosphere was required to
elicit a statistically significant increase. Based on the reports of Jongen
et al. (1981), Thilagar and Kumaroo (1983), and McCarroll et al. (1983), DCM
is capable of causing DNA damage, that results in SCEs.
5.3.2.3.2 DNA repair assays. In their study of the genotoxic potential of
DCM, Jongen et al. (1981) also measured UDS and IDS in V79 cells and primary
human fibroblasts (AH cells). These experiments were conducted by exposing
105 cells attached to glass covers!ips (UDS assay) or to glass petri plates
(IDS assay) to 0.5, 1.0, 2.0, 3.0, and 5.0% DCM (58, 118, 235, 353, and 471
mM, respectively) without metabolic activation. UDS experiments were done in
duplicate, and at least 25 nuclei of non-S phase cells were scored for the
number of silver grains/nucleus at each dose level. DCM had no detectable
effect on UDS in either cell line. In the IDS assays, the relative rate of
5-42
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5-43
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DNA synthesis was determined radioisotopically immediately after DCM exposure
and 0.5, 1.5, and 3.5 hours later. The average of duplicate samples revealed
that DCM inhibited DNA synthesis in V79 and AH cells at all dose levels com-
pared to controls but that synthesis recovered with time after exposure in all
cases. This is unlike the persistent inhibition of DNA synthesis by the posi-
tive control 4-nitroquinoline-l-oxide. The authors conclude that DCM was not
inducing genetic damage in cells but was inhibiting DNA synthesis by an effect
on cell metabolism.
Perocco and Prodi (1981) also performed a UDS assay using DCM. They col-
lected blood samples from healthy individuals for their studies, separated the
lymphocytes, and cultured 5 x 10 cells in 0.2-ml medium for 4 hours at 37ฐC
in the presence or absence of DCM (Carlo Erban, Milan, Italy or MerckSchuchardt,
Darmstadt, FRG, 97 to 99 percent pure). The tests were conducted both in the
presence and in the absence of PCB-induced rat liver S9 mix. A comparison was
made between treated and untreated cells for scheduled DNA synthesis (i.e.,
DNA replication) and UDS. No difference was noted between the groups with
3
respect to scheduled DNA synthesis measured as dpm of [ H] deoxythymidylic
acid (TdR) after 4 hours of culture (2,661 ฑ 57 dpm in untreated cells com-
pared to 2,356 ฑ 111 dpm in cells treated with 5 ul/ml [78 mM] DCM). Subse-
quently, 2.5, 5, and 10 ul/ml (39, 78, and 156 mM) DCM was added to cells
cultured in 10 mM hydroxyurea to suppress scheduled DNA synthesis. The amount
of unscheduled DNA synthesis was estimated by measuring dpm from incorporated
[3H]TdR 4 hr later. At 10 ul/ml DCM, 532 ฑ 31 and 537 ฑ 39 dpm were counted
without and with exogenous metabolic activation, respectively. Both values
were lower than corresponding negative controls of 715 ฑ 24 and 612 ฑ 26 dpm,
respectively. No positive controls were run to ensure that the system was
working properly, although testing of chloromethyl methyl ether (CMME) with
activation resulted in a doubling of dpms over the corresponding negative
control values (1,320 ฑ 57 at 5 ul/ml CMME versus 612 ฑ 26 untreated). The
authors calculated an effective DNA repair value (r) for each chemical based
on the control and experimental values with and without metabolic activation.
DCM was evaluated by the authors as negative in the test, but they did not
state their criteria for classifying a chemical as positive. None of the
experimental values from cells treated with DCM had higher dpm values than the
controls.
Based on these experiments there is no evidence that DCM specifically in-
hibits DNA synthesis or causes UDS.
5-44
-------
5.3.2.4 Summary and Conclusions. Dichloromethane has been tested for its
ability to cause gene mutations (in Salmonella, yeast, Drosophila, Panagrellus,
and cultured mammalian cells), chromosomal aberrations (in rats, mice, and
cultured mammalian cells), and other indicators of DNA damage in cultured cells
(sister chromatid exchange, unscheduled DNA synthesis, and inhibition of DNA
synthesis).
Commercially available samples of DCM gave.positive results in Salmonella.
yeast, and Drosophila. The responses were weak under treatment conditions used
and were obtained without the addition of metabolic activation systems (e.g.,
S9 mix). The data suggest that DCM may be metabolized j[n vivo to a^mutagenic
metabolite(s). Some negative results have been reported for gene mutation tests
in fungi (Saccharomyces) and mammalian cells in culture, but these may represent
false negative results because of the treatment conditions used. DCM has also
been reported to induce chromosomal aberrations in cultured mammalian cells
but not in bone marrow cells from animals exposed jji vivo, perhaps because a
sufficient dose of DCM did not reach the bone marrow. DCM causes a weak in-
crease in SCEs but has not been shown to cause UDS or inhibit DNA synthesis.
Mutagenicity tests of DCM have given positive responses in four different
organisms based on the weight of available evidence. DCM is judged to be a
mutagen with the potential of inducing gene mutations in exposed human cells.
A positive response in cultured mammalian cells indicates that DCM also causes
chromosomal aberrations, but additional testing in another j_n vivo or in vitro
chromosomal aberration assay is needed to confirm the available data. If such
tests are conducted, care should be taken to ensure that the test cells are
exposed to sufficiently high doses of DCM, otherwise false negative responses
may be obtained.
5.3.3 Evaluation of the Carcinogenicity of DCM
The purpose of this section is to provide an evaluation of the likelihood
that DCM is a human carcinogen and, on the assumption that it is a human
carcinogen, to provide a basis for estimating its public health impact, includ-
ing a potency evaluation in relation to other carcinogens. The evaluation of
carcinogenicity depends heavily on animal bioassays and epidemiologic evidence.
However, other factors, including mutagenicity, metabolism (particularly in
relation to interaction with DNA), and pharmacokinetic behavior, are important
to the qualitative and quantitative assessment of carcinogenicity. The avail-
able information on these subjects is reviewed in other sections of this
; 5-45
-------
document. This section presents an evaluation of the animal bioassays, the
human epidemiologic evidence, the quantitative aspects of assessment, and
finally, a summary and conclusions section dealing with all of the relevant
aspects of the carcinogenicity of DCM. Further, the National Toxicology
Program (NTP) rat and mouse gavage bioassay draft technical report (1982) on
DCM was cancelled because of data discrepancies at their contract laboratory
(memo from John A. Moore dated July 25, 1983).
5.3.3.1 Animal Studies
5.3.3.1.1 Dow Chemical Company (1980) inhalation study in rats. A total of
1,032 male and female Sprague-Dawley rats (129/sex for each exposure concentra-
tion) were exposed by inhalation to DCM at 0, 500, 1,500, or 3,500 ppm (0,
o
1,735, 5,205, or 12,145 mg/m ) for 6 hr/day, 5 days/week (excluding holidays),
in a 2-year toxicity and oncogenicity study. Approximately 95 rats of each
sex for each exposure concentration were part of the chronic toxicity and
oncogenicity portion of the study. This number also included those animals
that died spontaneously, were killed moribund during the study, or were killed
at the end of the 2-year exposure. The remaining animals were sacrificed as
part of the cytogenetic studies or for one of the interim kills at either 6,
12, 15, or 18 mo of exposure. The rats were received at 6 to 7 weeks of age
(males weighed 220 to 250 g; females weighed 170 to 200 g) from Spartan Research
Animals, Inc., Haslett, Michigan, and were individually marked for identifica-
tion with metal ear tags. All rats were maintained on a 12-hour light/dark
cycle. They were observed daily, including weekends and holidays, for general
health status and signs of possible toxicity.
Dichloromethane representative of technical grade material was obtained
from Dow Chemical Company, Plaquemine, Louisiana, and was used throughout the
exposure. Fourteen different samples of DCM were analyzed during the 2 years
of animal exposure; each sample showed 99% pure DCM, with a few trace chemical
contaminants that varied slightly from sample to sample, as shown in Table
5-10. The concentration of DCM vapor in chambers was considered well within
the range of expected variability. Hematologic determinations, serum clinical
chemistry, urinalysis, bone marrow collection, and blood carboxyhemoglobin
(COHb) determination were done in animals sacrificed at 6, 12, 15, and 18 mo
(interim kills). Plasma estradiol determination was done at the 12- and 18-mo
interim kills. This included samples from six controls/sex and four high-
exposure animals (3,500 ppm)/sex from the 12-mo kill, which were pooled
5-46
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together (two animals/sample) to give three control samples and two high
exposure (3,500 ppm) samples/sex. Ten individual samples/sex (not pooled)
from the high exposure and control groups were also sent from the 18-mo kill.
All animals that either died spontaneously, were killed in moribund con-
dition, or were killed at the interim or terminal kills were subjected to
complete gross and microscopic pathological examinations by a veterinary path-
ologist. Liver samples for possible electron microscopic evaluation were
collected.
In females exposed to 3,500 ppm, there was a statistically significant
increase of mortality from the 18th through the 24th mo that may be exposure
related. The remaining treated groups in males or females did not differ
significantly from the controls (Table 5-11). There was no exposure-related
difference in body weights of either male or female rats exposed to 500,
1,500, and 3,500 ppm DCM.
Although some hematologic values were increased and others were decreased,
the mean values were within the normal range of biological variability. Serum
glutamic pyruvic transaminase (SGPT), blood urea nitrogen (BUN), and serum
alkaline phosphatase (AP) values were in the normal range. It is noted that
the females had significantly increased (P <0.025) plasma estradiol levels at
18 mo, which may be related to the higher incidence of mammary tumors in the
exposed (3,500 ppm) group. Urinalysis findings were in the normal range, with
the exception of a few statistically significant values in specific gravity in
males exposed to 1,500 ppm at 6 mo and males and females exposed to 3,500 ppm
at 12 mo. Rats exposed to 500, 1,500, or 3,500 ppm had elevated COHb values
but with no evidence of either dose-response or increased values with prolonged
exposure.
5.3.3.1.1.1 Gross and histopathologic observations of rats from the 6-,
12-. 15-, and 18-month interim kills. Numerous gross and histopathologic
observations were recorded for control and DCM-exposed rats at each time
period, and most were typical of spontaneous or naturally-occurring lesions
normally seen in rats of this strain. There were many palpable masses in
males and females. Some palpable masses appeared to be abscesses of the prepu-
tial or clitoral glands, while others were cyst-like lesions of the skin. The
3
total number of masses in the 3,500 ppm (12,145 mg/m ) group of males was
significantly increased over the controls at 15, IB, and 21 mo, but not at
5-48
-------
TABLE 5-11. CUMULATIVE PERCENT MORTALITY OF RATS
2-YEAR DICHLOROMETHANE INHALATION STUDY
DCM concentration, ppm
Month of
study
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
0.8
0.8
0.8
0.8
0.8
0.8
0.9
0.9
1.7
1.7
1.7
2.6
2.7
3.6
10.9
15.2
23.8
32.4
44.2
48.4
56.8
63.2
80.0
85.3
500
0
0
0
0.8
0.8
1.6
2.6
3.4
3.4
4.3
6.0
6.8
10.9
10.9
11.8
16.2
20.0
36.2
45.3
52.6
56.8
65.3
73.7
85.3
Males
1,500
0
0
0
0
0
0.8
0.9
0.9
0.9
1.7
3.4
4.3
5.5
10.0
13.6
20.0
31.4
37.1
49.5
63.2
74.7*
83.2*
89.5
93.7
Females
3,500
0
0.8
2.3
3.9
4.7
4.7
6.0
6.0
6.0
6.0
6.8
8.5
9.8
15.2*
21.4
29.0*
33.6
43.0
54.6
58.8
72.2*
80.4*
87.6
92.8
0
0
0
0.8
0.8
0.8
0.8
0.9
0.9
3.4
5.1
5.1
6.0
7.2
9.0
13.5
14.2
23.6
26.4
36.5
42.7
49.0
62.5
70.8
78.1
500
0
0
0
0.8
0.8
0.8
1.7
2.6
3.4
5.1
6.0
6.8
8.2
8.2
10.9
15.2
20.0
29.5
42.1
50.5
58.9
64.2
65.3
74.7
1,500
0
0
0
0.8
0.8
0.8
1.7
2.6
1.7
2.6
4.3
5.1
8.2
8.2
10.0
13.3
20.0
29.5
46.3
55.8
65.3*
73.7
81.1
86.3
3,500
0
0
0
0
0.8
0.8
0.9
0.9
2.6
2.6
4.3
6.0
12.5
12.5
19.6
20.6
29.9
42.1*
57.7*
68.0*
81.4*
86.6*
90.7*
95.9*
*Significantly different from control by Fisher'.s Exact Test, p <0.05.
Source: Dow Chemical Company, 1980.
5-49
-------
23 months. Female rats exposed to 500, 1,500, and 3,500 ppm showed an exposure-
related increase in total number of masses. There was also a trend of increased
benign mammary tumors in females exposed to 1,500 and 3,500 ppm. The total
numbers of animals with benign mammary gland tumors were 9/28 (0 ppm), 10/29
(500 ppm), 11/29 (1,500 ppm), and 14/27 (3,500 ppm), whereas the total numbers
of benign mammary gland tumors were 17/28 (0 ppm), 17/29 (500 ppm), 28/29
-A. -~\ n
(1,500 ppm, p = 9.23 x 10 ^), and 37/27 (3,500 ppm, p = 2.33 x 10 1U). These
observations were apparent only when the cumulative results of the 6-, 12-,
15-, and 18-mo kills were evaluated.
A few other observations appeared to reflect exposure-related lesions.
The liver was the only organ that exhibited definite exposure-related non-
neoplastic effects in both males and females at all exposure concentrations.
Grossly, the effect was most prominent in females exposed to 3,500 ppm and
consisted of increased numbers of dark or pale foci. The control group had an
incidence of 0/28, while the 3,500 ppm DCM-exposed female rats had a signifi-
cantly greater number of foci (11/27). Some rats from the 3,500 ppm exposure
group had mottled livers or had an accentuated lobular pattern to the liver
(0/29 control males compared to 6/27 exposed males). Because of the limited
number of rats at each interim kill, this latter change may be related to
exposure but may also be due to biological variability.
Histologically observed, exposure-related lesions were present in the
livers of both males and females exposed to 500, 1,500, or 3,500 ppm. In
males, the total number of animals with any degree of vacuolization consistent
with fatty changes were 5/29 (0 ppm), 19/29 (500 ppm, p = 2.05 x 10~4), 21/29
(1,500 ppm, p = 2.47 x 10~5), and 23/27 (3,500 ppm, p = 2.81 x 10"7). The
livers of female rats also had alterations considered to be related to DCM
exposure. The total numbers of females with any degree of vacuolization
consistent with fatty changes were 13/28 (0 ppm), 20/29 (500 ppm, p = 7.16 x
10~2), 20/29 (1,500 ppm, p = 7.16 x 10"2), and 22/27 (3,500 ppm, p = 7.16 x
10 ). Because of these effects, it may be considered that this experiment
was performed at the maximum tolerated dose (MTD).
5.3.3.1.1.2 Gross and histopathologic observations of rats killed
moribund or dying spontaneously during the study and those from terminal
sacrifice (24 months).
Non-neoplastic observationsThe liver was affected in both males and
females exposed to 500, 1,500, or 3,500 ppm. The percentage of total rats
5-50
-------
with any degree of vacuolization was 17, 38, 45, and 54% in the males of the
0, 500, 1,500 and 3,500 ppm exposure groups, respectively, and 34, 52, 59, and
65% in the females, respectively. Also, the degree of severity tended to
increase with the dose. The males in all exposure groups had fewer cases of
grossly observed mottled and/or enlarged adrenals. These gross alterations
appeared to correspond to the histologically observed decrease in the number
of cases of adrenal cortical necrosis, but the nodular hyperplasia incidence
(unilateral or bilateral) was increased: 18/95 (0 ppm), 30/95 (500 ppm, p =
3.07 x 10~2), 31/95 (1,500 ppm, p = 2.2 x 10"2), and 24/97 (3,500 ppm).
Tumor or tumor-like lesionsThe Sprague-Dawley strain of rats used in
this study historically has had a high spontaneous incidence of benign mammary
tumors. The incidence varies slightly from study to study, but normally
exceeds 80% in females and about 10% in males by the end of a 2-yr study. The
mammary gland tumors have been classified, based on their predominant morpho-
logical cellular pattern, as fibromas, fibroadenomas, or adenomas.
The benign mammary tumor response was present in males and, to a lesser
extent, in females. There was a non-statistically significant increase in the
number of male rats with a benign mammary tumor exposed to 3,500 ppm (14/95 as
compared to 7/95, 3/95, and 7/95 in the 0, 500, or 1,500 exposure groups).
There was a slight increase in the total number of benign mammary tumors in
males exposed to 0 ppm (8/95), 500 ppm (6/95), 1,500 ppm (11/95), or 3,500 ppm
(17/97, P = 4.6 x 10"2).
The total number of female rats with a benign mammary tumor did not
increase in any exposure group (0, 500, 1,500, or 3,500 ppm groups had totals
of 79/96, 81/95, 80/95, and 83/97 benign mammary tumors, respectively). How-
ever, the total number of benign mammary tumors increased in an exposure-
related manner, with 165/96 in the controls, and 218/95, 245/95, and 287/97 in
the females exposed to 500, 1,500, and 3,500 ppm, respectively. Expressed
another way, the average number of benign mammary tumors per tumor-bearing
female rat increased from 1.7 in the control rats, to 2.3 in rats exposed to
500 ppm, to 2.6 in those exposed to 1,500 ppm, and to 3.0 in those exposed to
3,500 ppm. This effect is exposure related, and a dose-response relationship
was apparent. There was no indication of an increased number or incidence of
malignant mammary tumors in either males or females.
5-51
-------
The number of malignant tumors (Table 5-12) increased in male rats exposed
to 3,500 ppm. This increase did not appear to correlate clearly with an in-
creased number of any one tumor type or location. However, this observation
led Dow Chemical Co. (1980) to re-evaluate the gross and histopathologic data
on all tumors arising in or around the salivary glands. Table 5-13 lists the
specific individual animal data for these salivary gland area tumors, showing
the palpable mass data, specific histopathologic diagnoses, and the number of
sarcomas with metastases. Table 5-14 summarizes the incidence of salivary
gland region sarcomas in male rats.
Grossly, these tumors were large (several centimeters in diameter),
cystic, necrotic, or hemorrhagic. They appeared to invade all adjacent
tissues in the neck region, and often completely replaced the normal salivary
gland tissue. Histologically, all were sarcomas. They were composed of cells
that varied from round to spindle-shaped, but that appeared to be of mesenchymal
cell origin. Mitotic figures were frequently observed, as were necrosis and
local invasion into adjacent tissues. Most tumors had remnants of normal
salivary acini or ducts that were caught up in the cellular proliferation.
Two were relatively small masses, and appeared to be arising in the intersti-
tial and capsular tissue of the salivary glands.
One tumor of this type was found in the controls (1/93) compared to 0/94
in the 500 ppm exposure group, 5/91 in males from the 1,500 ppm exposure
group, and 11/88 in males from the 3,500 ppm exposure group (P = 0.002).
Historically, a spontaneous incidence (0 to 2%) of this tumor type has been
observed in Dow's laboratory. Therefore, the 12.5% incidence (11 of the 88
rats, Table 5-14) found in the males from the 3,500 ppm group was higher than
the corresponding controls of this study, and was higher than expected based
on historical control data for male rats of this strain. Also, the males
exposed to 1,500 ppm had five of these tumors, which was also slightly higher
than expected, but was not statistically significant. Therefore, this effort
appeared to be exposure related in the males exposed to 3,500 ppm.
The total number of male rats with malignant tumors was similar in the
control, 500 ppm, and 1,500 ppm exposure groups. Males exposed to 3,500 ppm
had an increase in this category, since 69 of the 124 rats had malignant
tumors compared to 57, 59, and 57 in the 0, 500, and 1,500 ppm exposure groups,
respectively.
In 1984, Burek et al. published the results of the Dow Chemical Company
(1980) study.
5-52
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TABLE 5-14. SUMMARY OF SALIVARY GLAND REGION SARCOMA INCIDENCE IN MALE
RATS IN A 2-YEAR INHALATION STUDY WITH DICHLOROMETHANE
Dose
0 ppm
500 ppm
1500 ppm
3500 ppm
Incidence*
1/93
0/94
5/91
11/88
(1%)
(0%)
(5.5%)
(12.5%)
Fisher1
(P =
(P =
s exact
0.10, N.
0.002)
test
s.)
*Cochran-Armitage test for linear trend, p <0.0001.
N.S. = Not significant.
Source: Dow Chemical Company, 1980.
5.3.3.1.2 Dow Chemical Company (1980) inhalation study in hamsters. A total
of 866 Golden Syrian hamsters [Ela: Eng (syr) strain; Engle Laboratory Animals,
Inc., Farmersburg, Indiana] (107 to 109/sex per exposure concentration) were
exposed by inhalation to 0, 500, 1,500, and 3,500 ppm (0, 1735, 5205, and
o
12145 mg/m ) of DCM. The materials and methods for experimental design are
the same as mentioned previously in the rat portion of the Dow Chemical Company
(1980) study. The body weights of the hamsters were 61 to 70 g when they were
received. The hamsters were marked by unique toe clips for group identification
and by ear punch for individual identification within the cages.
The mortality data for males and females are presented in Table 5-15.
Female hamsters exposed to DCM at 3,500 ppm had a statistically significant
decreased mortality from the 13th through the 24th mo of the study. Females
exposed to 1,500 ppm also had statistically significant decreased mortality
from the 20th through the 24th mo. This decreased mortality in females exposed
to 3,500 and 1,500 ppm was considered to be exposure related. The remaining
exposure groups of male (500, 1,500, and 3,500 ppm) and female (500 ppm)
hamsters had no exposure-related differences in mortality. Some hamsters in
each group had alopecia at 5.5 mo into the study, but this alopecia was
secondary to a mange mite (Demodex species) infection. This parasite did not
result in increased mortality or morbidity. No treatment-related differences
were observed in the body weights of either males or females exposed to 500,
1,500, or 3,500 ppm of DCM.
5-56
-------
TABLE 5-15. CUMULATIVE PERCENT MORTALITY OF HAMSTERS
2-YEAR DICHLOROMETHANE INHALATION STUDY
DCM concentration, ppm
Month of
study
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Male
0
0
0
0
0
0
0
1.0
1.9
1.9
2.9
3.8
5.1*
6.4
10.6
14.9
23.4
24.5
31.9
36.0
37.1
41.6
56.2
61.8
82.0
500
0
0
0
0
0.9
1.9
1.9
3.8
5.8
5.8
5.8
. 6.7
10.1
14.1
17.2
18.2
19.2
24.2
29.8
43.6
51.1
61.7
63.8
78.7
1,500
0
0
0
0
0
0
1.0
1.9
1.9
2.9
2.9
3.9
10.2
13.3
14.3
17.3
19.4
24.5
31.2
41.9
54.8
68.8
75.3
88.2
3,500
0
0
0
0.9
3.7
3.7
5.8
5.8
6.7
9.6
11.5
12.5
17. 2f
24.2
26.3
33.3
34.3
37.4
42.6
55. 3f
60.6
69.1
74.5
85.1
0
0.9
0.9
1.9
1.9
2.8
3.7
3.9
4.9
4.9
5.8
9.7
13.3*
22.6
26.9
32.3
36.6
41.9
52.7
63.6
71.6
80.7
88.6
94.3
100.0
Females
500
0
0
0.9
1.9
1.9
2.8
4.9
4.9
5.8
9.7
10.7
11.7
16.3
24.5
26.5
30.6
32.7
38.8
59.1
63.4
70.0
82.8
91.4
95.7
1,500
0.9
0.9
1.9
1.9
2.8
3.7
4.8
7.6
8.6
8.6
9.5
13.3
15.0
20.0
24.0
29.0
32.0
40.0
50.5
55. 8t
61.lt
68. 4t
75. 8t
89. 5t
3,500
0
0
0.9
0.9
0.9
0.9
2.9
3.9
3.9
4.9
4.9
4.9
7. It
11. 2t
15. 3t
23. 5f
27. 6t
30. 6t
40. 9t
45. 2t
53. 8t
72. Ot
80. 6t
90. 3t
*Five males and five females died due to food deprivation. These animals were
subsequently deleted from mortality calculations.
tSignificantly different from controls by Fisher's Exact Test, P <0.05.
Source: Dow Chemical Company, 1980.
Based on the information available to the Carcinogen Assessment Group
(CAG), it is very difficult to conclude whether the MTD was used. Dow Chemical
Company has not submitted a 90-day dose-finding study, but a 30-day inhalation
study has been reported in a letter from Dr. J. Burek to Dr. D. Singh, dated
May 1, 1981.
"The study was conducted prior to the two-yr study, but has not been
reported. CD-I mice, Golden Syrian hamsters, Sprague-Dawley and CDF (F-344)
5-57
-------
rats were exposed to 0, 2,500, 5,000, or 8,000 ppm DCM vapor 6 hr/day, 5
days/week, for a total of 20, 19, 20 or 6-2/3 exposures, respectively, in
21-29 days. Body weight data was obtained throughout the study. Clinical
chemistry parameters were measured. All animals underwent gross pathological
examination at the termination of the experiment. The weights of the liver
and kidneys were recorded from animals in the 0, 2,500, and 5,000 ppm groups,
and organ/body weight ratios were calculated. Animals exposed to 8,000 ppm
DCM exhibited anesthetic effects, increased blood urea nitrogen levels in
Sprague-Dawley rats, and decreased body weights in rats. The animals exposed
to 5,000 ppm showed slight anesthesia, decreased body weight in male rats,
increased SGPT values in female mice and Sprague-Dawley rats and increased
liver weights in female mice, hamsters and rats. Animals exposed to 2,500 ppm
DCM appeared to scratch more than controls and therefore appeared to be affected,
but showed no other effect attributable to exposure. The target organ in this
study was the liver. Because of the results obtained, 8,000 and 5,000 ppm DCM
were considered to be too high a dose level for the two-year study, and 2,500
ppm did not appear to have produced a severe enough response over the 30-day
period. Therefore, concentrations of 3,500 ppm of DCM was [sic] chosen as the
top dose for the two-year study."
Dow Chemical Company (1980) conducted a subchronic dose-finding study for
a perfeid of only 30 days rather than for the 90-day period that is usual in
most animal bioassays. Further, body weight or mortality rate in the experi-
mental group of hamsters does not decrease as compared to the controls.
Hematologic determinations, serum clinical chemistry, urinalysis, bone marrow
collection, and blood COHb determination were done in animals at the 6-, 12-,
18-, and 24-mo interim kills. No treatment-related effects were observed in
any of the parameters evaluated in male or female hamsters after 6, 12, 18, or
24 mo of exposure to 500, 1,500, or 3,500 ppm of DCM, respectively.
Carboxyhemoglobin determinations were performed on the blood of male and
female hamsters following 22 mo on test. Males and females exposed to DCM at
500, 1,500, or 3,500 ppm all had significantly elevated COHb values. There
was a slight trend in a dose-response relationship in females, since the mean
i
CpHb value for those exposed to 500 ppm was 23.6%, while the values for females
exposed to 1,500 or 3,500 ppm were 30.2% and 34.6%, respectively. However, an
apparent dose-response relationship was not observed in males. Dow Chemical
Company (1980) indicated that these data, when compared to those in the rat
5-58
-------
study, suggested that hamsters had a greater degree of metabolism of DCM to
carbon monoxide. Furthermore, the apparent dose-response in females was
surprising. As a result, additional hamsters were exposed to a single 6-hr
exposure, and their COHb values were determined. There was no apparent sex
difference, and the dose-response relationship observed in females after 22
months could not be verified. Since there was no dose-response relationship
in male and female rats or male hamsters, and since the female hamsters
exposed to a single 6-hr exposure did not show a dose-response relationship,
the apparent trend for an exposure-related increase in female hamsters at 22
months may be a cumulative effect.
5.3.3.1.2.1 Gross and histopathologic observations of hamsters from the
6-, 12-, and 18-month interim kills. A variety of gross and histopathologic
observations were recorded for hamsters that were sacrificed at the 6-, 12-,
or 18-mo interim kills. Histopathologically, exposure-related differences
were present, and consisted of decreased numbers of hamsters with amyloidosis
of the liver, kidney, adrenals, thyroid, and spleen. A few animals in each
group may or may not represent the trend of amyloidosis in males.
5.3.3.1.2.2 Gross and histopathologic observations of hamsters killed
moribund or dying spontaneously during the study and those from terminal
sacrifice (24-months).
Neoplastic and non-neoplastic observationsGross and histopathologic
examinations were conducted on all hamsters that died or were killed moribund
during the study and on all surviving hamsters at the end of the study. The
histopathologic observations for males and females are presented in the Dow
Chemical Company report (1980, tables 124 and 127). The observations shown
include all the neoplastic and non-neoplastic lesions recorded for these
hamsters. Most observations were within the normal or expected range for
Golden Syrian hamsters, as indicated by Dow Chemical Company. The female
hamsters had increased incidences of lymphosarcoma in the experimental group.
The incidences were 1/96, 6/95, 3/95, and 7/97 (p = 0.033) in the 0, 500,
1,500, and 3,500 ppm groups, respectively. A re-evaluation of lymphosarcoma
data of female hamsters by the Carcinogen Assessment Group (CAG) resulted in
the following incidences: 1/91, 6/92, 3/91, and 7/91 (P =0.032) in the 0,
500, 1,500, and 3,500 ppm groups, respectively. The differences between the
denominators above reflect the CAG's use of actual numbers of animals examined
(which did not include animals that were severely cannibalized, autolyzed, or
5-59
-------
missing), whereas the Dow denominators included the total number of animals.
Also, only a small number of mammary gland tissues were examined, and no
lesions were found. Table 5-16 summarizes the total tumor data. The total
number of hamsters with benign tumors increased significantly in females at
3,500 ppm; the total number of hamsters with malignant tumors increased
significantly in males at 1,500 ppm.
In 1984, Burek et al. published the results of the Dow Chemical Company
(1980) study.
5.3.3.1.3 Summary of the Dow Chemical Company (1980) rat and hamster inhala-
tion studies. Based on all the data evaluated, the four following points are
considered to be major findings in the rat and hamster studies:
1. Male rats exposed to 1,500 or 3,500 ppm appeared to have an increased
number of sarcomas in the ventral midcervical area near the salivary glands.
There were 1/93, 0/94, 5/91, and 11/88 (P = 0.002) sarcomas in male rats
exposed to 0, 500, 1,500, or 3,500 ppm, respectively. Based on routine
sections, special stains, and ultrastructural evaluations, these tumors
appeared to be of mesenchymal cell origin; however, a myoepithelial cell
origin of these cells could not be ruled out. These tumors had some areas
that morphologically resembled one cell type (i.e., neurofibrosarcoma,
fibrosarcoma), and still other tumors had cell types that were undif-
ferentiated or pleomorphic. In some, one cell type was predominant, while in
others, areas of all of the above cell types were present, depending on the
area of the tumor examined. Furthermore, the origin of each of these tumors
remains questionable. All appeared to be arising in the midcervical region,
and all involved the salivary glands. Only two tumors were small enough to be
localized within the salivary gland. The rest were larger tumors that clearly
involved the salivary glands as well as adjacent tissues, and could have been
growing either into or out of the salivary glands. However, they probably
arose within the salivary glands based on the two localized small tumors
described above.
Therefore, there was an apparent association between the increased inci-
dence of sarcomas in the salivary gland region of male rats and prolonged
exposure via inhalation to 1,500 or 3,500 ppm DCM. There were no salivary
gland sarcomas in female rats or in hamsters of either sex. Further, it will
be of interest to find out what kind of lesions are present or absent in the
ongoing National Toxicology Program inhalation study.
5-60
-------
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5-61
-------
2. Male and female rats exposed to DCM had increased numbers of benign
mammary tumors as compared to control values. Female rats exposed to 500,
1,500, or 3,500 ppm of DCM had increased numbers of benign mammary tumors per
tumor-bearing rat in comparison with the controls. The increase was evident
in the palpable mass data and the gross necropsy findings, which were con-
firmed by the histopathologic examination. The total number of female rats
with benign mammary tumors was not statistically increased in any exposure
group (0, 500, 1,500, and 3,500 ppm groups had a total of 79, 81, 80, and 8-3
animals with benign mammary tumors, respectively). Sprague-Dawley rats have
very high incidences of spontaneous mammary tumors. However, the total number
of benign mammary tumors has increased in an exposure-related manner with
165/92 in the controls and 218/90, 245/92, and 287/95 in the females exposed
to 500, 1,500, or 3,500 ppm, respectively. Expressed another way, the average
number of benign mammary tumors per female rat increased from 1.7 in the
controls, to 2.3 in those exposed to 500 ppm, to 2.6 in those exposed to 1,500
ppm, and to 3.0 in those exposed to 3,500 ppm. This increase was considered
to be exposure-related and dose dependent.
A mammary tumor response was present in male rats also, but to a lesser
extent than in females. The number of rats with benign mammary tumors in
males exposed to 3,500 ppm increased but this increase was not statistically
significant. The total number of benign mamary tumors in males exposed to
1,500 or 3,500 ppm increased slightly. As was the case in females, these
effects in males exposed to 1,500 or 3,500 ppm were considered to be exposure-
related.
There were no mammary gland tumors in male or female hamsters. Also only
28/92, 44/93, 30/94, and 27/93 mammary gland tissues were examined in the 0,
500, 1,500, and 3,500 ppm groups, respectively. Not a single lesion was
recognized in the mammary gland tissues examined. The CAG feels that a greater
number of hamster mammary gland tissues should have been examined to better
evaluate the true incidence of mammary tumors.
3. There was an increased incidence of lymphosarcoma in female hamsters.
The incidence was 1/96, 6/95, 3/95, and 7/97 (p = 0.033) in the 0-, 500-,
1,500-and 3,500-ppm groups, respectively Dow Chemical Company (1980). A
re-evaluation of lymphosarcoma data of female hamsters by the CAG resulted in
the following incidences: 1/91, 6/92, 3/91, and 7/91 (P = 0.032) in the 0,
500, 1,500, and 3,500 ppm groups, respectively. The differences between the
5-62
-------
denominators above reflect the CAG's use of actual numbers of animals examined
(which did not include animals that were severely cannibalized, autolyzed, or
missing), whereas the Dow Chemical Company (1980) denominators included the
total number of animals. Dow Chemical Company (1980) reported that the females
exposed to 3,500 ppm had better survival (statistically significant) than the
controls and thereby had a greater chance to develop these tumors. After
correction for survival (1/39 versus 7/63) by the CAG, these data are not
statistically significant (P = 0.12).
4. There appears to be a question as to whether the doses given the rat
and hamster were at or near the MTD. The body weights of male rats increased
particularly toward the latter part of the experiment, whereas the body weights
of female rats were unaffected in any experimental group. Exposure to 3,500
ppm resulted in an increased mortality rate in female rats during the last 6
months, but the male rats were unaffected at any concentration. On the other
hand, decreased mortality was observed in female hamsters exposed to 1,500 and
3,500 ppm, while mortality in male hamsters was unaffected at 500, 1,500, and
3,500 ppm based on only a 30-day rat and hamster inhalation (dose-finding)
study. Based on this information, it is difficult to judge whether the animals
were given a dose equal to the MTD.
5.3.3.1.4 Dow Chemical Company (1982) inhalation toxicity and oncogenicity
study in rats. A 2-yr inhalation study of DCM with Sprague Dawley rats and
Golden Syrian hamsters by Dow Chemical Company (1980) has been described
earlier in this document. In that study, animals were exposed 6 hr/day, 5
days/week for 2 yr to DCM at 0, 500, 1,500, and 3,500 ppm. The carcinogenic
response was positive in rats but negative in hamsters. In rats, the liver
appeared to be the target organ affected by exposure. Hepatocellular vacuoli-
zation, consistent with fatty change, was observed in male and female rats
inhaling 500, 1,500, or 3,500 ppm DCM. There was an increased incidence-of
multinucleated hepatocytes upon exposure to. 500, 1,500, or 3,500 ppm, and an
increased number of foci and areas of altered hepatocytes at 3,500 ppm in
female rats. Benign mammary tumors were increased in male rats inhaling 1,500
or 3,500 ppm, and in female rats inhaling 500, 1,500, or 3,500 ppm DCM. Male
rats exposed to 1,500 or 3,500 ppm DCM had an increased number of sarcomas in
the region of the salivary gland. Female rats inhaling 3,500 ppm DCM had an
increased mortality rate. Carboxyhemoglobin levels in the blood of rats
exposed to DCM were higher than control levels; however, no differences were
5-63
-------
observed in COHb levels of animals inhaling DCM at 500 ppm versus animals
inhaling it at 3,500 ppm. The objective of this second study was to further
investigate the toxicity of DCM at concentrations far below those that may
cause saturation of the metabolic processes in rats.
A total of 360 male and 492 female Sprague-Dawley rats (Spartan substrain,
6 to 8 weeks old) were used in this study. Groups of 90 rats/sex were exposed
by inhalation to 0 (control), 50, 200, and 500 ppm (0, 173, 692, and 1,735
mg/m3) DCM (technical grade, lot #TA 05038, with purity of at least 99.5
percent) 6 hr/day, 5 days/week, for 20 (males) or 24 mo (females). Occasional-
ly an exposure was shorter than 6 hr, due to vapor generation or mechanical
problem. In addition, 30 extra female rats, identified as 500/0, were exposed
to 500 ppm DCM for the first 12 mo of the study and were housed as control
rats for the duration of the study (last 12 mo). Another 30 female rats,
identified as 0/500, were housed in the same manner as control rats for the
first 12 mo of the study and were exposed to 500 ppm DCM for the remaining
12 mo of the study. To determine the rate of DNA synthesis in the liver, 18
female rats were included in each group. After 6, 12, 15, and 18 mo of
exposure, five rats of each sex at each exposure level were sacrificed. In
addition, five female rats from each of the 500/0 and 0/500 groups were sacri-
ficed at the 18-mo interim necropsy.
Clinical laboratory tests for chemistry, plasma hormone levels, and DNA
were made on interim sacrificed rats. Gross and microscopic examinations were
made of animals at interim and terminal sacrifice, as well as of animals dying
spontaneously and those that were killed moribund during the study.
As reported by the authors, the nominal and analytical concentrations of
DCM in the chambers were in close agreement, indicating no detectable loss or
decomposition of test material during vaporization. Approximately 2 mo after
the initial exposure to DCM, symptoms consistent with sialodacryoadenitis (SDA
virus) were observed in male and female rats in each experimental group,
including control groups. The rats from all exposure groups appeared to be
equally affected, and the symptoms were not apparent 3 weeks after the initial
observation.
No significant difference in body weight gain was noticed in male rats,
but the mean body weights of female rats at 50, 200, or 500 ppm were signifi-
cantly higher throughout the study period in comparison with controls. Although
the authors of this study consider this to be a reflection of biological
5-64
-------
variability, the CAG considered that the highest dose was not the MTD because
the same strain of rat tolerated a dose of 3,500 ppm by inhalation in the
previous study at the same laboratory (Dow Chemical Company, 1980).
No increase in mortality rate from that of the control group was observed
in male or female rats. According to the authors, "...due to the high mortality
rate in all groups of male rats, the terminal necropsy for male rats occurred
during the 21st month of exposure to methylene chloride to ensure adequate
numbers of surviving animals for pathologic evaluation." This is not consistent
with the findings in Tables 5-17 and 5-18.
No significant effect on absolute or relative organ weight was seen in
male or female rats. Blood COHb levels were significantly elevated (P <0.05)
above controls in all experimental groups of rats. Incorporation of 3H-
thymidine into hepatic DNA was unaffected in female rats at 6 and 12 mo. DNA
synthesis in rats was not determined at 15 mo, as originally scheduled in the
protocol, due to the number of mammary tumors observed in female rats at 200
and 500 ppm.
Results from the 6-, 12-, 15-, and 18-mo interim necropsies showed no
definite exposure-related gross or histopathologic findings in male and female
rats from any of the interim sacrifices. An exception was the interim
sacrifice of female rats at 15 mo, where 1, 3, 4, and 5 females inhaling DCM
at 0, 50, 200, or 500 ppm, respectively, had a focus or foci of altered cells
in the liver. This effect was not apparent in female rats from 6-, 12-, or
18-mo interim sacrifices, nor was it apparent from rats dying spontaneously,
killed moribund during the study, or terminally sacrificed.
There were no significant histological lesions observed in other organs,
with the exception of the liver. Data on the liver lesions are given in
Tables 5-19 and 5-20. In males, the incidence of hepatocellular vacuolization
increased slightly (Table 5-19). The liver lesions in female rats were signi-
ficantly increased for foci of altered cell, hepatocellular vacuolization, and
multinucleated hepatocytes at 500 ppm (Table 5-20) as compared to controls.
The significance of these alterations in the liver is not known. Further, the
number of liver lesions appeared to be increased in the 500/0 group at terminal
sacrifice, combined sacrifice, and death as compared to controls, but this was
not the case in the 0/500 ppm group. There were no significant differences in
any tumor type for liver, kidney, spleen, brain, salivary gland, lung, skin,
pancreas, and mammary gland in male and female rats, with the exception of
mammary gland tumors, which were significantly higher in females (Table 5-21).
5-65
-------
TABLE 5-17. MONTHLY MORTALITY DATA FOR MALE RATS IN
A 2-YEAR DICHLOROMETHANE INHALATION TOXICITY AND ONCOGENICITY STUDY (%)
DCM concentration (ppm)
Month
of
study
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Terminal sacrifice
0
0*
0
0
0
0
0
0
0
1(1/85)
1(1/85)
4(3/85)
4(3/85)
5(4/80)
15(12/80)
20(16/80)
33(25/75)
37(28/75)
47(35/75)
59(41/70)
70(49/70)
74(52/70)
50
0
0
1(1/90)
1(1/90)
1(1/90)
1(1/90)
2(2/85)
2(2/85)
4(3/85)
4(3/85)
4(3/85)
5(4/85)
9(7/80)
13(10/80)
18(14/80)
20(15/75)t
37(28/75)
45(34/75)
64(45/70)
70(49/70)
73(51/70)
200
0
0
0
1(1/90)
1(1/90)
1(1/90)
2(2/85)
4(3/85)
4(3/85)
4(3/85)
5(4/85)
7(6/85)
11(9/80)
14(11/80)
23(18/80)
31(23/75)
37(28/75)
53(40/75)
69(48/70)
79(55/70)
81(57/70)
500
0
0
0
0
0
0
0
0
1(1/85)
1(1/85)
1(1/85)
1(1/85)
4(3/80)
8(6/80)
13(10/80)
23(17/75)
25(19/75)
41(31/75)
50(35/70)
66(46/70)
73(51/70)
^Percent mortality (number dead/original number of animals minus animals
sacrificed for an interim necropsy).
tSignificantly different from control value by Fisher's Exact Test.
Source: Dow Chemical Company, 1982.
5-66
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TABLE 5-18. MONTHLY MORTALITY DATA FOR FEMALE RATS IN
A 2-YEAR DICHLOROMETHANE INHALATION TOXICITY AND ONCOGENICITY STUDY (%)
PCM concentration (ppm)
Month
of study
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Terminal
sacrifice
(after 24
months)
0
0*
0
0
0
0
0
0
0
1(1/85)
1(1/85)
2(2/85)
4(3/85)
4(3/80)
5(4/80)
9(7/80)
12(9/75)
15(11/75)
19(14/75)
30(21/70)
36(25/70)
43(30/70)
53(37/70)
60(42/70)
64(45/70)
50
0
0
0
0
0
0
0
0
0
0
0
2(2/85)
5(4/80)
5(4/80)
9(7/80)
15(11/75)
15(11/75)
17(13/75)
27(19/70)
36(25/70)
46(32/70)
53(37/70)
63(44/70)
76(35/70)
200
1(1/90)
1(1/90)
1(1/90)
1(1/90)
1(1/90)
1(1/85)
1(1/85)
2(2/85)
2(2/85)
2(2/85)
2(2/85)
2(2/85)
5(4/80)
5(4/80)
10(8/80)
13(10/75)
15(11/75)
21(16/75)
30(21/70)
34(24/70)
49(34/70)
54(38/70)
64(45/70)
67(47/70)
500
0
0
0
0
0
1(1/90)
1(1/85)
1(1/85)
1(1/85)
1(1/85)
1(1/85)
4(3/85)
6(5/80)
9(7/80)
15(12/80)
17(13/75)
19(14/75)
28(21/75)
37(26/70)
39(27/70)
47(33/70)
50(35/70)
54(38/70)
61(43/70)
0/500
0
0
0
0
0
0
0
0
0
0
0
0
3(1/30)
3(1/30)
3(1/30)
7(2/30)
7(2/30)
17(5/30)
24(6/25)
32(8/25)
36(9/25)
48(12/25)
60(15/25)
72(18/25)
500/0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ot
Of
3(l/30)f
8(l/25)t
8(2/25)t
16(4/25)f
36(9/25)f
48(12/25)t
52(13/25)
^Percent mortality (number dead/original number of animals minus animals sacrificed
for an interim necropsy).
tSignificantly different from control value by Fisher's Exact Test.
Source: Dow Chemical Company, 1982.
5-67
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TABLE 5-19. NON-NEOPLASTIC LIVER LESIONS IN MALE RATS
Exposure level
Cppm)
Foci of
altered cells
Hepatocellular
vacuolization
Terminal kill
Moribund and
0
50
200
500
1/18
3/19
5/13*
7/19*
3/18
9/19*
7/13*
8/19
death
Combi ned
0
50
200
500
0
50
200
500
6/52
6/51
6/57
4/51
7/70
9/70
11/70
11/70
19/52
9/51
14/57
20/51
22/70
18/70
21/70
28/70
*Fisher's Exact Test, P <0.05:
Source: Dow Chemical Company, 1982.
5-68
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TABLE 5-20. NON-NEOPLASTIC LIVER LESIONS IN FEMALE RATS
Exposure level
(ppm)
Moribund and
spontaneous
500
0/500
500/0
Foci of
altered cells
Hepatocellular
vacuolization
Multinucleated
hepatocytes
Terminal kill
0
50
200
9/25
5/17
10/22
23/25
15/17
21/22
4/25
4/17
5/22
17/27*
5/7
8/12
23/27
6/7
12/12
16/27*
1/7
6/12*
death
Combined
0
50
200
500
0/500
500/0
0
50
200
500
0/500
500/0
12/45
12/53
17/48
14/43
7/18
5/13
21/70
17/70
27/70
31/70
12/25
13/25
i 18/45
27/53
23/48
30/43*
9/18
4/13
41/70
42/70
44/70
53/70*
15/25
16/25
4/45
2/53
7/48
11/42*
2/18
3/13
8/70
6/70
12/70
27/70*
3/25
9/25*
*Fisher's Exact Test, P <0.05.
0/500 = rats exposed to 500 ppm DCM for first 12 months.
500/0 = rats exposed to 500 ppm DCM for last 12 months.
Source: Dow Chemical Company, 1982.
5-69
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TABLE 5-21. SUMMARY OF MAMMARY GLAND TUMORS IN FEMALE RATS
Rats with a
benign mammary
tumor
(adenoma,
fibroadenoma,
or fibroma)
Total number
of benign
mammary tumors
(adenomas,
fibroadenomas,
and fibromas)
Exposure
level
(ppm)
0
50
200
500
0/500
500/0
0
50
200
500
0/500
500/0
Number
of rats
70
70
70
70
25
25
70
70
70
70
25
25
Moribund and
spontaneous
35
43
41
32
17
11
69
97
91
68
35
27
Terminal
kill
17
15
20
23
6
12*
36
36
44
79
15
33
Cumulative
52
58
61*
55
o o
23
23
105
133
135
147
ป- f\
50
60
^Significantly different from control when analyzed by Fisher's Exact
Test, P <0.05.
tData could not be analyzed by Fisher's Exact Test.
0/500 = rats exposed to 500 ppm DCM for first 12 months.
500/0 = rats exposed to 500 ppm DCM for last 12 months.
Source: Dow Chemical Company, 1982.
5-70
-------
In summary, there were significant increases in non-neoplastic liver
lesions (i.e., hepatocellular vacuolization and multinucleated hepatocytes) in
female rats at 500 ppm. There was an increase in benign mammary tumors
(adenoma, fibroma, and fibroadenoma) in female rats. The number of benign
mammary tumors/tumor-bearing rats observed in female rats was 2.0, 2.3, 2.2,
and 2.7 in rats inhaling DCM at 0, 50, 200, and 500 ppm, respectively. Female
rats of group 500/0 showed effects that were similar to rats exposed to 500
ppm for 24 months, but the 0/500 group did not differ from the control group.
In conclusion, the results of this study offer very limited evidence of
the carcinogenicity of DCM. However, the highest dose in this study is half
of the lowest doses in both the NTP gavage study (1982 draft) and the ongoing
NTP inhalation study.
5-3.3.1.5 National Coffee Association (1982a.b) study in rats. On August 11,
1982, Hazelton Laboratories America, Inc., reported on a chronic study in
Fischer 344 rats administered DCM in deionized drinking water for 24 mo. This
study was sponsored by the National Coffee Association (NCA), and utilized
1,000 animals in 7 different dose groups or regimens (Table 5-22). The actual
mean daily consumption levels of DCM in the drinking water were similar to the
expected target levels (Table 5-23).
Interim sacrifices were performed at 26, 52, and 78 weeks of treatment
with 5, 10, or 20 rats of each sex from each dose group, respectively, with
the exception of the animals in Group 7 (the recovery group, which were only
sacrificed with the remaining terminal animals at 104 weeks). The effects of
compound administration were evaluated using the following criteria: survival,
body weight gains, total food consumption, water consumption, clinical observa-
tions, ophthalmoscopic findings, clinical pathology, organ and tissue weights,
and gross and microscopic pathology.
No compound-related findings were reported for either survival, clinical
observations, opthalmoscopic findings, gross necropsy findings, or organ
weight data. Throughout the study, small but significantly decreased body
weight gains and water consumption were reported for both male and female rats
in Groups 5, 6, and 7. Food consumption also decreased, but this criterion
was only monitored for the first 13 weeks. These decreased effects were
attributed to DCM administration. Low-magnitude statistical increases in mean
hemotocrit, hemoglobin, and red blood cell counts were noted in both male and
female animals of Groups 4, 5, and 6. In most cases, these were within the
range of historical control values of Fischer 344 rats.
5-71
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TABLE 5-22. GROUP ASSIGNMENT OF FISCHER 344 RATS ADMINISTERED
DICHLOROMETHANE IN DEIONIZED DRINKING WATER FOR 24 MONTHS
1.
2.
3.
4.
5.
6.
7.
Group number
Control
Control
Low-dose
Mid-dose 1
Mid-dose 2
High-dose
High-dose/recovery
(78 weeks/26 weeks)
Number
Males
85
50
85
85
85
85
25
of animals
Females
85
50
85
85
85
85
25
Target dose,
mg/kg/day
0
0
5
50
125
250
250
Source: National Coffee Association, 1982a.
TABLE 5-23. MEAN DAILY CONSUMPTION OF DICHLOROMETHANE IN A 24-MONTH
CHRONIC TOXICITY AND ONCOGENICITY STUDY IN FISCHER 344 RATS
Group
3
4
5
6
7
Target level,
mg/kg/day
5
50
125
250
250*
Males
5.85
52.28
125.04
235.00
232.13
Females
6.47
58.32
135.59
262.81
268.72
*The designated recovery group (Group 7) mean is for the first 78 weeks only.
Source: National Coffee Association, 1982a.
5-72
-------
Histopathological alterations were described in the livers of rats of
both sexes in Groups 4, 5, 6, and 7. These changes consisted of an increased
incidence of foci/areas of cellular alteration in Groups 4, 5, 6, and 7 and of
fatty changes in Groups 5 and 6 after 78 and 104 weeks of treatment. The
incidence of neoplastic nodules and/or hepatocellular carcinomas in female
Fischer 344 rats (Table 5-24) was derived from the data presented in Volumes
I-IV of the August 11, 1982, NCA report (National Coffee Association 1982a)
and in the Addition to the Final NCA Report of November 5, 1982 (National
Coffee Association 1982b). Male rats did not show an increased incidence of
liver tumors in treated animals versus controls (Table 5-24). These statist-
ically significant increases in the incidences of liver tumors in female rats
were within the range of historical control values at this laboratory (Table
5-25), as presented in a letter from Dr. John Kirschman of General Foods to
Dr. Dharm Singh of the CAG, dated February 17, 1983. Therefore, based on a
review of the NCA study, DCM administered in deionized water at doses up to
250 mg/kg/day was borderline for carcinogenicity to Fischer 344 rats.
5.3.3.1.6 National Coffee Association (1983) study in mice. In December
1983, Hazelton Laboratories of America, Inc. reported on a chronic study in
B6C3F1 mice administered DCM in deionized water for 24 mo. This study,
sponsored by the National Coffee Association (NCA), utilized 1,000 mice (650
males and 350 females) in six different dose groups or regimens (Table 5-26).
The actual mean daily consumption levels of DCM in drinking water were similar
to the expected target levels (Table 5-27). Each animal was housed individual-
ly and identified by a unique number.
Adjusted survival for male mice at 104 weeks was 88.3% for Group 1, 76.6%
for Group 2, 81.5% for Group 3, 81.0% for Group 4, 82.8% for Group 5, and
81.5% for Group 6. For female mice, adjusted survival at 104 weeks was 69.4%
for Group 1, 78.0% for Group 2, 73.0% for Group 3, 84.0% for Group 4, 76.0%
for Group 5, and 91.8% for Group 6. No compound-related significant differ-
ences between groups were found, but survival was slightly increased in Groups
4 and 6.
There were some exceptional clinical findings, i.e., convulsions charac-
terized by quaking, extension of the head, and lowering of the pinnae of the
ears (peyer reaction). In some cases the behavior progressed to full seizure.
There was no correlation to any increased mortality. These convulsions were
5-73
-------
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TABLE 5-26. GROUP ASSIGNMENT OF B6C3F1 MICE ADMINISTERED DICHLOROMETHANE
IN DEIONIZED DRINKING WATER FOR 24 MONTHS
Group number
Control
Control
Low-dose
Mid-dose 1
Mid-dose 2
High-dose
Number
Males
60
65
200
100
100
125
of animals
Females
50
50
50
50
50
50
Target dose
(mg/kg/day)
0
0
60
125
185
250
Source: National Coffee Association, 1983.
TABLE 5-27. MEAN DAILY CONSUMPTION OF DICHLOROMETHANE IN A 24-MONTH
CHRONIC TOXICITY AND ONCOGENICITY STUDY IN B6C3F1 MICE
Group
3
4
5
6
Target level
(mg/kg/day)
60
125
185
250
Males
mg/kg/day ฑ S.D.
60.55 ฑ 7.680
123.61 ฑ 14.356
177.48 ฑ 19.214
234.29 ฑ 26.495
Females
mg/kg/day ฑ S.D.
59.46 ฑ 8.413
118.19 ฑ 16.799
172.41 ฑ 22.950
237.76 ฑ 29.329
Source: National Coffee Association, 1983.
5-76
-------
demonstrated in each group including the controls, at least once, and were
noted during body weight procedure when animals were placed in pans. The
occurrence of convulsions in B6C3F1 mice is presented in Table 5-28.
An increased number of Harden'an gland neoplasms were also observed in
males in Groups 4 and 6. The other groups were similar to the controls (Table
5-29). The increase in Groups 4 and 6 may be a result of normal biological
variation as suggested by the sponsors.
No compound-related findings were observed for either survival, body
weight changes, water consumption, clinical observation, leukocyte counts, and
gross necropsy findings. Food consumption was not done in this experiment.
Histomorphologic alterations of the liver were observed in both male and
female mice in the high-dose group (containing greater amounts of oil Red 0
positive material). Hepatocellular lesions were observed in all groups of
both sexes, including controls. The lesions in the treated female group were
comparable to controls (Table 5-30). In males, there was also a statistically
significant increase in the incidence of hepatocellular adenomas and/or car-
cinomas in Groups 4 and 5 and a borderline significant increase in Group 6
(the high-dose group).
Based on the results of this study, DCM administered in deionized water
at doses up to 250 mg/kg/day can be considered borderline for carcinogen!city
to B6C3F1 male mice.
5.3.3.1.7 National Toxicology Program (1982 draft) gavage study in rats and
mice. The National Toxicology Program (NTP) conducted a 2-yr carcinogenesis
bioassay of food-grade DCM and reported on their results in a draft technical
report dated September 22, 1982 (National Toxicology Program, 1982). Dichloro-
methane was administered in corn oil by gavage to male and female Fischer
344/N rats and B6C3F1 mice. The DCM was more than 99.5% pure, with vapor
phase chromatographic analysis detecting the presence of vinylidene chloride
and trans-dichloroethylene up to 0.4%. A 13-week dose finding study was
conducted to evaluate the toxicity of the compound. Based on survival, body
weight gain, and histopathological examination, doses of 500 and 1,000 mg/kg
by gavage were selected for male and female rats and mice for the 2-yr study.
The NTP announced on July 25, 1983 that the draft NTP report would not be
issued as a final report due to discrepancies in experimental data that com-
promise a clear interpretation. NTP further allowed that pending the results
5-77
-------
TABLE 5-28. OCCURRENCE OF CONVULSIONS IN MALE AND FEMALE B6C3F1 MICE
Males
Females
4
86.8 89.8
5.4 6.3
Percent with convulsions at least once
80.4 74.1 76.8 77.2 100.0 89.7 90.7 88.1
Mean frequency of occurrence
4.1 4.3 5.2 5.0 8.6 7.6 8.0 8.0
94.7 91.1
7.1 8.3
Source: National Coffee Association, 1983.
TABLE 5-29. , NUMBER OF MALE MICE WITH HARDERIAN GLAND NEOPLASMS
^Harderian gland
Adenoma
Carcinoma
Percent
Adenoma
Carcinoma
Combined
Group: 1
Number examined: 60
3
, 0
5
0
5
2
65
2
2
3
3
6
3
200
13
0
7
0
7
4
100
9
0
9
0
9
5
99
5
0
5
0
5
6
121
11
0
9
0
9
Source: National Coffee Association, 1983.
5-78
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of an in-depth audit, select and relevant information from these gavage studies
might be incorporated into the future draft technical report for DCM inhalation
studies.
5.3.3.1.8 Theiss et al. (1977). A pulmonary tumor bioassay in mice was
reported by Theiss et al. (1977). Groups of 20 male strain A mice were injected
intraperitoneally three times a week with 0, 160, 400, or 800 mg/kg DCM for a
total of 16 or 17 injections. Mice were sacrificed 24 weeks after the first
injection, and the lungs were examined under a dissecting microscope for
surface adenomas. Some adenomas were confirmed by histology.
Tumors were found at all three dose levels; however, due to poor survival
and the small number of animals, the increase in tumors did not reach statis-
tical significance at the two highest dose levels (Table 5-31). At the lowest
dose, a highly significant increase in the number of tumors was observed (P =
0.013). Therefore, this study was marginally positive for carcinogenicity.
TABLE 5-31. PULMONARY TUMOR BIOASSAY IN STRAIN A MICE
Dose
(mg/kg)
0
60
400
800
Total dose
given
0
2,720
6,800
12,800
No. of mice
at beginning
20
20
20
20
No. of mice
examined
for tumors
15
18
5
12
Tumors/
mouse
0.27
0.94
0.80
0.50
Significance*
<0.013
>0.1
>0.1
Source: Adapted from Thiess et al., 1977.
*The test of significance used is the exact test of ratio of two
Poisson parameters.
5.3.3.1.9 Heppel et al. (1944). Heppel et al. (1944) exposed dogs, rabbits,
guinea pigs, and rats to DCM by inhalation at levels of 5,000 ppm (17,350
3 3
mg/m) for 7 hr/day and 10,000 ppm (34,700 mg/m ) for 4 hr/day, 5 days/week,
for 6 months. No tumors developed in any animals.
5.3.3.1.10 MacEwen et al. (1972). MacEwen et al. (1972) exposed dogs to DCM
by inhalation at 500 ppm (1,735 mg/m ) for 14 weeks; no tumors were reported,
but edema of the meninges of the brain occurred. Neither this study nor the
5-80
-------
Heppel et al. (1944) study could have detected a carcinogenic response because
of the shortness of the observation times and the fact that these studies were
not originally designed to test for carcinogenicity.
5.3.3.1.11 Other animal studies in progress. The National Toxicology Program
sponsored a 2-yr inhalation study in male and female Fischer 344/N rats and
B6C3F1 mice at exposure concentrations of 0, 1,000, 2,000, and 4,000 ppm (0,
3,470, 6,940, and 13,880 mg/m3) for rats and 0, 2,000, and 4,000 ppm (0,
6,940, and 13,880 mg/m ) for mice. The animals have been sacrificed (April
1983), but the pathology report is not yet available.
5.3.3.1.12 Cell transformation studies. Price et al. (1978) exposed Fischer
rat embryo cell cultures (F1706, subculture 108) to DCM liquid at concentrations
of 1.6 x 102 and 1.6 x 10 uM for 48 hr. Dichloromethane was diluted with
growth medium to yield the appropriate doses. The DCM sample, obtained from
the Fisher Scientific Company, was >_99.9% pure. The cells were grown in
Eagles minimum essential medium in Earle's salts supplemented with 10 percent
fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 ug
penicillin, and 100 ug streptomycin/ml. Quadruplicate cultures were treated
at 50 percent confluency with each dose. After treatment, cells were cultured
in growth medium alone at 37ฐC. Transformation of cells treated with either
dose level of DCM was observed by 23 and 30 days of incubation, and was charac-
terized by progressively growing foci composed of cells lacking contact inhibi-
tion and orientation. There was no transformation of cells grown in medium
alone or in the presence of a 1:1,000 acetone concentration, even after a
subculture. Twenty and 27 microscopic foci per three dishes with the low and
high DCM doses respectively, were found in dishes inoculated with 50,000 cells
from cultures treated four subcultures earlier and held for 4 weeks at 37ฐC in
a humidified C00 incubator before staining.
2
Subcutaneous injection of cells treated with 1.6 x 10 uM DCM five subcul-
tures earlier produced local fibrosarcomas in 5/5 newborn Fischer 344 rats
within 60 days following treatment. The ability of cells grown in growth
medium alone to induce local fibrosarcomas was not determined; however, negative
responses were obtained with cells grown in the presence of a 1:1,000 concentra-
tion of acetone. Exposure of cells to 3.7 x 10~ uM 3-methylcholanthrene
produced 124 microscopic foci per three dishes in the inoculation test described
above by 37 days of incubation, and local fibrosarcomas in 12/12 rats by 27
days following subcutaneous injection of cells. The exposure of 3-methylcho-
lanthrene was attained by initial dilution in acetone to 1 mg/ml followed by
5-81
-------
further dilution in growth medium to 0.1 pg/ml (personal communication from
Or. P. J. Price).
Dr. Price wrote a letter to the CAG dated Nov. 14, 1980, saying that "the
analysis of methylene chloride showed a purity of 99.9 percent. The original
study was done in quadruplicate and in each case the Fischer rat cells were
transformed. Since the publication, the same batch of methylene chloride was
sent to Andy Sivak at Arthur D. Little to be run against the Kakunago clone
A31 of BALB/c 3T3. It did not transform his cells. We then repeated the
study in Fischer rat cells and at the same time tested methylene chloride sent
to us by the National Coffee Producers Association. The test was run in
triplicate. The Fisher methylene chloride again transformed the cells, while
the National Coffee Producers' (supplied by Diamond Shamrock) was negative."
The differing responses in the two experiments may have been due to differences
in the levels of impurities present in the samples used. The chemical composi-
tions of DCM samples from different suppliers are given in Table 5-32.
The Fischer rat embryo cell line contains the genome of the Rauscher
leukemia virus, but there is no basis for minimizing the positive results.
Since the mode of action of DCM is not known, this transformation may be due
to activation of the virus.
5.3.3.2 Epidemiologic Studies
5.3.3.2.1 Friedlander et al. (1978), Hearne and Friedlander (1981). Fried!ander
et al. (1978) performed mortality analyses of male Eastman-Kodak employees
exposed to low levels of DCM. This study was updated by Hearne and Friedlander
(1981). Measurements from 1959 to 1975 ranged from 30 to 120 ppm (104 to
2
416 mg/m ). Both the original 1978 analysis and the 1981 update found no in-
crease in neoplasms, heart disease, or any other cause of death in comparison
with the two control groups, which were composed of other Kodak employees and
of New York State males. The population was relatively stable and the workers
were rotated throughout the work area, and thus exposure was averaged among all
the workers. Dichloromethane had been used for 30 years as the primary solvent
in this Eastman-Kodak operation.
Two separate mortality analyses were done. In the earlier paper, one
approach used the proportionate mortality ratio to examine 334 deaths of
DCM-exposed workers during 1956 to 1976. Seventy-one neoplasms were found; 73
were expected based on other Kodak employee mortality ratios. Furthermore, no
single site was over-represented.
5-82
-------
TABLE 5-32. CHEMICAL COMPOSITIONS OF DICHLOROMETHANE SAMPLES (ppm);
Fisher D-123
Lot 761542
Diamond Shamrock
Dow
Methyl chloride
Vinyl chloride
Ethyl chloride
Vinylidine chloride
Carbon tetrachloride
Chloroform
Trichloroethylene
1,2-Di chlorethy1ene
Methyl bromide
Cyclohexane
0.5
0.8
329
3.6
3
33
369
26
20
300
<5
86
11
305
Presented by Drs. Sivak and Kirschman at the Science Advisory Board
Meeting, Sept. 4-5, 1980.
bFisher sample used by Dr. P. Price in two series of cell transformation
studies.
cThis material is under test in NCA's chronic rat study on DCM in drinking
water. It was also used in Dr. Price's follow-up study.
dThis material, also analyzed by NCI, with the same results as given by
Drs. Sivak and Kirschman, was used in the following tests:
NCA's 90-day studies in rats and mice
Dr. Sivak's neoplastic transformation assay
Dr. P. Price's follow-up series of cell transformation tests
NCI's chronic bioassay studies in rats and mice presently under way
5-83
-------
A second approach, included in the first paper and used exclusively in
the update, was a cohort mortality study of all 751 employees who were in the
DCM work area in 1964. The results of the update are shown in Tables 5-33 and
5-34, taken from that paper. There were 110 deaths during the 16-year follow-
up (retrospective). Two control groups were used: other Kodak employees, and
New York State males. The expected numbers of deaths in the exposed groups
based upon the control group experiences were 105 and 168, respectively. The
differences between the observed and the expected deaths based on the controls
are either not statistically significant (other Kodak employees) or the expec-
ted deaths based on New York State males are significantly increased over the
observed numbers.
The results show that malignant neoplasms accounted for 24 of the 110
deaths in the study cohort, which was less than the 28.6 or 38.5 expected
malignancies based on the control data. Nine of these 24 deaths were from
respiratory cancer (7.6 and 13.6 were expected based on the control groups)
and seven were from cancers of the digestive organs (less than expected).
Only the two deaths associated with brain or nervous tissue represented a
higher than expected total (SMR = 169 and SMR = 227 versus two control groups),
but these SMRs (standard mortality ratios) were not statistically significant.
Further stratification of the cohort focused on the 252 males with 20
years or more of exposure who were employed in 1964. In this group 59 deaths
occurred: 13 were due to malignant neoplasms (17.8 and 24.7 expected based on
the two control groups) and 32 were due to circulatory diseases (37.9 and 59.7
expected).
A further analysis of the 252 males shows that this cohort had a median
age of approximately 54 years in 1964. With this group the more common cancers
would have to be markedly increased for there to be a reasonable probability
of detecting the increase. For example, following the cohort for 16 years,
cancer mortality at this age would require 10 deaths from respiratory cancer
to detect a significant result at the P = 0.05 level. This represents an
increase of at least 100 percent over that expected, the expected probability
!*
least 100 percent over that expected, the expected probability of lung cancer
death for this cohort being 0.018 over the 16 years, based on other Kodak
employees' rates. The statistical power to detect 100-percent increases (at a
= 0.05, one-sided) is about 95 percent for all malignancies and 45 percent for
respiratory cancer deaths. The remaining 499 males with less than 20 years
5-84
-------
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5-86
-------
exposure were significantly younger (median age about 36 years), and a follow-up
of this cohort for 16 years mortality might fail to detect even a moderate
effect, since expected cancer mortality in this age group is so low.
5.3.3.2.2 Ott et al. (1983a,b,c,d,e). Ott et al. (1983a,b,c,d,e) reported
the results of a health evaluation of employees of one fiber production plant
who were exposed to DCM as part of a solvent mixture consisting of this sub-
stance plus methanol and also acetone in a separate container. A second fiber
production plant utilizing only acetone, but similar to the first in other
respects, was selected as a referent population. The investigation focused
primarily on health effects occurring to the cardiovascular system stemming
from the metabolism of DCM to COHb in the body. In addition to a retrospective
cohort mortality study, the authors examined several other health end points
in the still-employed group. These involved clinical evaluations, electrocar-
diographic monitoring, metabolism tests, and evaluation of oxygen half-
'saturation pressures.
q
Environmental exposure in the plant varied from 140 ppm (486 mg/m ) in
3
areas of low DCM use to 475 ppm (1,748 mg/m ) in areas of high DCM use, based
on an 8-hr time-weighted average (TWA). Methanol was present in smaller
quantities by a factor of ten, while acetone ranged from 100 to 1,000 ppm (347
to 3,470 mg/m3) TWA in both plants (Ott et al., 1983a). Industrial hygiene
surveys were conducted from September 1977 to February 1978.
To qualify for inclusion in the cohort, production employees (both men
and women) had to have worked a minimum of 3 months in the preparation or
extrusion areas of either plant during January 1, 1954, to January 1, 1977.
In the DCM plant, both cellulose diacetate (acetate) and cellulose triacetate
(CTA) fibers were made side by side. Although acetone was present in both
plants, it was the solvent of choice for making acetate fibers in the referent
plant. The exposed cohort consisted of 1,271 persons versus 948 persons in
the referent plant, as follows:
White men
women
PCM plant
487
615
plant
696
248
Nonwhite men
women
Total
64
105
1271
1
3
948
5-87
-------
Persons were followed through June of 1977. The authors noted that vital
status was not available for 226 (18 percent) of the DCM-exposed cohort and
112 (12 percent) of the companion plant. The authors commented that few
deaths would be added from this last group based on "previous experience with
the social security follow-up mechanism." Such a statement is subject to
question, however, without knowing the age distribution of this group.
The authors found no excessive mortality from any cause in the "exposed"
or the "referent" population, either when the group with unknown vital status
was presumed lost to follow-up at the time lost, or was presumed alive until
June of 1977; i.e., person-years would cease accumulating at the time the
person was lost to follow-up in the former instance, but in the latter case,
person-years would continue to accumulate until the end of the follow-up
period, as if the individual were still alive. Altogether in the exposed
category for white men, 37 observed deaths out of 487 were seen versus 34.9
expected based on the latter definition, or 30.4 expected based on the former
definition regarding the group with unknown vital status. For white women,
only 11 deaths were observed versus either 15.9 or 13.5, depending upon the
choice of the first or second definition above. For malignant neoplasms in
white men, there were five observed deaths versus either 6.3 or 5.6 expected.
In white women, two observed deaths were seen, versus either 5.2 or 4.5 expec-
ted. No cancer deaths were reported in nonwhite males and females, probably
due to the small size of these select subgroups. Since the authors were
mainly interested in cardiovascular effects, they examined further only
ischemic heart disease in terms of duration of exposure and length of follow-
up. Even when one considers only individuals with a minimum of 10 years of
exposure who .were followed for a minimum of 10 years, only 2 male deaths were
observed versus 1.7 expected based On this cause. Ischemic heart disease was
not observed as a cause of death in women. No corresponding data are avail-
able for any form of cancer by latency or duration of employment.
Several qualifications limit-the possible use of this study as a sensitive
indicator of mortality. Foremost among these is the relatively low and unusual
distribution of mortality among the members of the cohort. Because of the
excessive numbers of observed and expected deaths from external causes compared
to the numbers of observed and expected deaths from malignant neoplasms, and
the fact that deaths from accidents are the leading cause of death in young
males nationwide, it is quite likely that the cohort is a relatively youthful
5-i
-------
group. This is further evidenced by the surprisingly few deaths observed
overall compared to the size of the cohortless than 10 percent In all In-
stances (white men, nonwhite men, and white females). No age breakdown was
provided to evaluate this observation.
Additionally, only 310 of the exposed males have had a chance to be
followed at least 17.5 years. Some 241 exposed males entered the cohort after
1960 and could not possibly have been followed 17.5 years. Because of the 15-
to 20-year latency period involved with most human cancers, cancer effects
attributable to DCM exposure would probably not have been expected to manifest
themselves prior to the 17th year. Thus, the power of this study to detect a
statistically significant elevated risk of cancer (as well as ischemic heart
disease) is low.
Another possible problem deals with the extent of follow-up of the cohort.
Almost 18 percent (226) of the exposed cohort was lost to follow-up as of June
1977. Although the authors discount that as unimportant, it should be of
concern that if the ages of the lost-to-follow-up group are relatively advanced,
the likelihood is great that enhanced mortality will be in the higher age
groups. Whenever extraordinary means are employed to determine the vital
status of a subgroup of the cohort for which all other methods of follow-up
have failed, the residual deaths found as a result of this endeavor are usual-
ly overly represented by sudden deaths due to heart failure or accidents. The
opportunity for leaving a record of their deaths is minimized because of the
nature of the deaths; hence, it becomes less likely that the vital status can
be determined.
Still another problem with this study is the "healthy worker" effect. In
most studies of this kind, workers at the time of their employment and shortly
thereafter are generally somewhat healthier than the population from whence
they came. Ill or infirm persons do not usually choose jobs that may be
detrimental to their health. This tendency usually results in as much as a 20
percent deficit of mortality compared to that expected.
In summary, this study is inadequate to assess cancer mortality in the
described cohort for the reasons stated. The study results do not exclude the
possibility of increased health risk in the study population. The study
focuses mainly on heart disease as a consequence of DCM exposure.
5.3.3.3 Quantitative Estimation. This quantitative section deals with the
unit risk for DCM in air and water and the potency of DCM relative to other
5-89
-------
carcinogens that the CAG has evaluated. The unit risk estimate for an air or
water pollutant is defined as the incremental lifetime cancer risk occurring in
a hypothetical population in which all individuals are exposed continuously
o
from birth throughout their lifetimes to a concentration of 1 ug/m of the
agent in the air they breathe or to a concentration of 1 pg/l in the water
they drink. This calculation provides a quantitative estimation of the impact
of the agent as a carcinogen. Unit risk estimates are used to compare the
carcinogenic potency of several agents with each other and to give a crude
indication of the population risk that might be associated with air or water
exposure to these agents, if the actual exposures are known.
5.3.3.3.1 Procedures for the determination of unit risk for animals. The
data used for the quantitative estimate are taken from one or both of the
following: 1) lifetime animal studies, and 2) human studies where excess
cancer risk has been associated with exposure to the agent. In animal studies
it is assumed, unless evidence exists to the contrary, that if a carcinogenic
response occurs at the dose levels used in the study, then responses will also
occur at all lower doses, with an incidence determined by an extrapolation
model.
There is no solid scientific basis for any mathematical extrapolation
model that relates carcinogen exposure to cancer risks at the extremely low
concentrations that must be dealt with in evaluating environmental hazards.
For practical reasons, such low levels of risk cannot be measured directly
either by animal experiments or by epidemiologic studies. We must, therefore,
depend on our current understanding of the mechanisms of-carcinogenesis for
guidance as to which risk model to use. At the present time, the dominant
view of the carcinogenic process involves the concept that most cancer-causing
agents also cause irreversible damage to DNA and are mutagenic. There is
reason to expect that the quantal type of biological response, which is charac-
teristic of mutagenesis, is associated with a linear non-threshold dose-response
relationship. Indeed, there is substantial evidence from mutagenicity studies
with both ionizing radiation and a wide variety of chemicals that this type of
dose-response model is the appropriate one to use. This is particularly true
at the lower end of the dose-response curve; at higher doses, there can be an
upward curvature, probably reflecting the effects of multistage processes on
the mutagenic response. The linear non-threshold dose-response relationship
is also consistent with the relatively few epidemiologic studies of cancer
5-90
-------
responses to specific agents that contain enough information to make the
evaluation possible (e.g., radiation- induced leukemia, breast and thyroid
cancer, skin cancer induced by arsenic in drinking water, liver cancer induced
by aflatoxins in the diet). Also, some evidence from animal experiments is
consistent with the linear non-threshold model (e.g., liver tumors induced in
mice by 2-acetylaminofluorene in the large scale EDQ-, study at the National
Center for lexicological Research, and the initiation stage of the two- stage
carcinogenesis model in rat liver and mouse skin).
Because its scientific basis, although limited, is the best of any of the
current mathematical extrapolation models, the linear non-threshold model has
been adopted as the primary basis for risk extrapolation in the low-dose
region of the dose-response relationship. The risk estimates made with this
model should be regarded as conservative, representing the most plausible
upper limit for the risk; i.e., the true risk is not likely to be higher than
the estimate, but it could be lower.
The mathematical formulation chosen to describe the linear non- threshold
dose-response relationship at low doses is the linearized multistage model.
This model employs enough arbitrary constants to be able to fit almost any
monotonically increasing dose- response data, and it incorporates a procedure
for estimating the largest possible linear slope (in the 95 percent confidence
limit sense) at low extrapolated doses that is consistent with the data at all
dose levels of the experiment.
5.3.3.3.1.1 Description of the low-dose animal extrapolation model. Let
P(d) represent the lifetime risk (probability) of cancer at dose d. The
multistage model has the form
P(d) = 1 - exp [-(q
Q
where
Equivalently,
where
qd
Pt(d) = 1 - exp [-
- P(0)
qi > 0, i = 0, 1, 2, ..., k.
+ q2d2 + ... + qRdk)]
is the extra risk over background rate at dose d or the effect of treatment.
5-91
-------
The point estimate of the coefficients q.. , i = 0, 1, 2, .. . , k, and
consequently the extra risk function Pt(d) at any given dose d, is calculated
by maximizing the likelihood function of the data.
The point estimate and the 95% upper confidence limit of the extra risk,
P,(d), are calculated by using the computer program GLOBAL79, developed by
o
Crump 'and Watson (1979). At low doses, upper 95% confidence limits on the
extra risk and lower 95% confidence limits on the dose producing a given risk
are determined from a 95% upper confidence limit, q*, on parameter qr When-
ever q, > 0, at low doses the extra risk Pt(d) has the approximate form P^Cd)
= q, x d. Therefore, q| x d is a 95% upper confidence limit on the extra
risk, and R/q? is a 95% lower confidence limit on the dose producing an extra
risk of R. Let LQ be the maximum value of the log- likelihood function. The
upper limit, qฃ, is calculated by increasing qI to a value q| such that when
the log- likelihood is remaximized subject to this fixed value, qฃ, for the
linear coefficient, the resulting maximum value of the log- likelihood 1^
satisfies the equation
2 (l_0 - L^) = 2.70554
where 2.70554 is the cumulative 90% point of the chi -square distribution with
one degree of freedonij which corresponds to a 95% upper limit (one-sided).
This approach of computing the upper confidence limit for the extra risk,
P.(d), is an improvement on the Crump et al. (1977) model.. The upper con-
o
fidence limit for the extra risk calculated at low doses is always linear.
This is conceptually consistent with the linear non-threshold concept dis-
cussed earlier. The slope, q?, is taken as an upper bound of the potency of
the chemical in inducing cancer at low doses. (In the section calculating the
risk estimates, Pt(d) will be abbreviated as P.)
In fitting the dose-response model, the number of terms in the polynomial
is equal to (h-1), where h is the number of dose groups in the experiment,
including the control group.
Whenever the multistage model does not fit the data sufficiently, data at
the highest dose are deleted, and the model is refit to the rest of the data.
This is continued until an acceptable fit to the data is obtained. To determine
whether or not a fit is acceptable, the chi -square statistic
5-92
-------
X2 =
h
I
1=1
.th
is calculated where N. is the number of animals in the i dose group, X. is
f- h
the number of animals in the i dose group with a tumor response, P. is the
J_ L '
probability of a response in the i dose group estimated by fitting the
multistage model to the data, and h is the number of remaining groups. The
p
fit is unacceptable whenever X is larger than the cumulative 99% point of the
chi-square distribution with f degrees of freedom, where f equals the number
of dose groups minus the number of non-zero multistage coefficients.
5.3.3.3.1.2 Selection of data. For some chemicals, several studies
using different animal species, strains, and sexes and run at several doses
and different routes of exposure are available. A choice must be made as to
which of the data sets from several studies to use in the model. The pro-
cedures used in evaluating these data are consistent with the approach of
making a maximum likely-risk estimate. They are listed as follows:
1. The tumor incidence data are separated according to organ sites or
tumor types. The set of data (i.e., dose and tumor incidence) used in the
model is the set where the incidence is significantly higher statistically
than the control for at least one test dose level or where the tumor incidence
rate shows a statistically significant trend with respect to dose level. The
*
data set that gives the highest estimate of the lifetime carcinogenic risk, q-,
is selected in most cases. However, efforts are made to exclude data sets
that produce spuriously high risk estimates because of a small number of
animals. That is, if two sets of data show a similar dose-response relation-
ship and one has a very small sample size, the set of data having the larger
sample size is selected for calculating the carcinogenic potency.
2. If there are two or more data sets of comparable size that are
identical with respect to species, strain, sex, and tumor sites, the geometric
*
mean of q.., estimated from each of these data sets, is used for risk assess-
ment. The geometric mean of numbers A-,, A,,, ..., A is defined as
(A-, x
vl/m
5-93
-------
3. If two or more significant tumor sites are observed in the same
study, and if the data are available, the number of animals with at least one
of the specific tumor sites under consideration is used as incidence data in
the model.
5.3.3.3.1.3 Calculation of human equivalent dosages. It is appropriate
to correct for metabolism differences between species and absorption factors
via different routes of administration. Following the suggestion of Mantel
and Schneiderman (1975), it is assumed that mg/surface area/day is an equivalent
dose between species. Since the surface area is approximately proportional to
the two-thirds power of the weight, as would be the case for a perfect sphere,
the exposure in mg/day per two-thirds power of the weight is also considered
to be equivalent exposure. In an animal experiment, this equivalent dose is
computed in the following manner. Let
L. =
1 =
m
W =
duration of experiment
duration of exposure
average dose per day in mg during administration of the agent
(i.e., during 1 ), and
average weight of the experimental animal.
Then, the lifetime average exposure is
1 x m
d=
InhalationWhen exposure is via inhalation, the calculation of dose can
be considered for two cases where 1) the carcinogenic agent is either a com-
pletely water-soluble gas or an aerosol and is absorbed proportionally to the
amount of air breathed in, and 2) where the carcinogen is a poorly water-
soluble gas that reaches an equilibrium between the air breathed and the body
compartments. After equilibrium is reached, the rate of absorption of these
agents is expected to be proportional to the metabolic rate, which in turn is
proportional to the rate of oxygen consumption, which in turn is a function of
surface area.
Case 1~Agents that are in the form of particulate matter of virtually
completely absorbed gases, such as sulfur dioxide, can reasonably be expected
5-94
-------
to be absorbed proportionally to the breathing rate. In this case tne exposure
Jn ing/day may be expressed as
m = I x v x r
3 3
where. I = inhalation rate per day in m , v = mg/m of the agent in air, and
r = the absorption fraction.
The inhalation rates, I, for various species can be calculated from the
observations of the Federation of American Societies for Experimental Biology
(FASEB, 1974) that 25-g mice breathe 34.5 liters/day and 113 g-rats breathe
105 liters/day. For mice and rats of other weights W (in kilograms), the sur-
face area proportionality can be used to find breathing rates in m3/day, as
follows:
For mice, I - 0.0345 (W/0.025)2/3 m3/day
For rats, I = 0.105 (W/0.113)2/3 m3/day.
o
For humans, the value of 20 m /day* is adopted as a standard breathing rate
(International Commission on Radiological Protection, 1977).
2/o
The equivalent exposure in mg/W ' for these agents can be derived from
the air intake data in a way analogous to the food intake data. The empirical
factors for the air intake per kilogram per day, i = I/W, based upon the pre-
viously stated relationships, are tabulated as follows:
Species
Man
Rats
Mice
W
70
0.35
0.03
i = I/W
0.29
0.64
1.3
Therefore, for particulates or completely absorbed gases, the equivalent ex-
2/3
posure in mg/W is
*From "Recommendation of International Commission on Radiological Protec-
7 3
tion," page 9. The average breathing rate is 10 cm per 8-hour workday and
7 3
2 x 10 cm in 24 hours.
5-95
-------
- lWvr -
wr
vr.
In the absence of experimental information or a sound theoretical argument to
the contrary, the fraction absorbed, r, is assumed to be the same for all
species.
Case 2 The dose in mg/day of partially soluble vapors is proportional to
O /O
the 02 consumption, which in turn is proportional to W and is also propor-
tional to the solubility of the gas in body fluids, which can be expressed as
an absorption coefficient, r, for the gas. Therefore, expressing the 02
consumption as 02 = k W2/3, where k is a constant independent of species, it
follows that
m = k W2/3 x v x r
or
d = -JL- = kvr.
As with Case 1, in the absence of experimental information or a sound theoretical
argument to the contrary, the absorption fraction, r, is assumed to be the
same for all species. Therefore, for these substances a certain concentration
in ppm or ug/m3 in experimental animals is equivalent to the same concentration
in humans. This is supported by the observation that the minimum alveolar
concentration necessary to produce a given "stage" of anesthesia is similar in
man and animals (Dripps et al., 1977). When the animals are exposed via the
oral route and human exposure is via inhalation or vice versa, the assumption
is made, unless there is pharmacokinetic evidence to the contrary, that absorp-
tion is equal by either exposure route.
5.3.3.3.1.4 Calculation of the unit risk from animal studies. The 95
percent upper-limit risk associated with -d mg/kg ' /day is obtained from
6LOBAL79, and for most cases of interest to risk assessment, can be adequately
approximated by" P(d) = 1 - exp (-qjd). A "unit risk" in units X is the risk
corresponding to an exposure of X = 1. This value is estimated by finding the
number of mg/kg2/3/day that corresponds to one unit of X and substituting this
value into the relationship expressed above. Thus, for example, if X is in
5-96
-------
units of pg/m in the air, we have for case 1, d = 0.29 x 701'3 x 10~3 ug/kg
o
per day, and for case 2, d = 1, when pg/m is the unit used to compute para-
meters in animal experiments.
If exposures are given in terms of ppm in air, the following calculation
may be used:
1 ppm = 1.2 x
molecular weight (gas)
molecular weight (air)
mg/m .
Note that an equivalent method of calculating unit risk would be to use mg/kg/day
for the animal exposures, and then to increase the jth polynomial coefficient
by an amount
(Wh/Wa)j73 j = 1, 2, ..., k
and to use mg/kg/day equivalents for the unit risk values. In the section
calculating the unit risk for animals, the final q? will always be the upper-
limit potency estimate for human risk based on animal data.
5.3.3.3.1.5 Interpretation of quantitative estimates. For several reasons,
the unit risk estimate based on animal bioassays is only an approximate indica-
tion of the absolute risk in populations exposed to known carcinogen concentra-
tions. First, there are important species differences in uptake, metabolism,
and organ distribution of carcinogens, as well as species differences in
target site susceptibility, immunological responses, hormone function, dietary
factors, and disease. Second, the concept of equivalent doses for humans
compared to animals on a mg/surface area basis is virtually without experimental
verification regarding carcinogenic response. Finally, human populations are
variable with respect to genetic constitution, diet, living environment,
activity patterns, and other cultural factors.
The unit risk estimate can give a rough indication of the relative potency
of a given agent as compared with other carcinogens. The comparative potency
of different agents is more reliable when the comparison is based on studies
in the same test species, strain, and sex, and by the same route of exposure,
preferably inhalation.
The quantitative aspect of carcinogen risk assessment is included here
because it may be of use in the regulatory decision-making process, e.g.,
5-97
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setting regulatory priorities, or evaluating the adequacy of technology-based
controls. However, with present technology, only imprecise estimations are
possible concerning cancer risks to humans at low levels of exposure. At
best, the linear extrapolation model used here provides a rough but plausible
estimate of the upper limit of risk, and while the true risk is probably not
much more than the estimated risk, it could be considerably lower. The risk
estimates presented in subsequent sections should not be regarded, therefore,
as accurate representations of the true cancer risks even when the exposures
are accurately defined. The estimates presented may, however, be factored
into regulatory decisions to the extent that the concept of upper risk limits
is found to be useful.
5.3.3.3.1.6 Alternative methodological approaches. Methods used by the
CAG for quantitative assessment are consistently conservative, i.e., they tend
toward high estimates of risk. The most important part of the methodology
contributing to this conservatism is the linear non-threshold extrapolation
model. There are a variety of other extrapolation models that could be used,
all of which would give lower risk estimates. These alternative models, the
one-hit, Probit, and Weibull models, have not been used by the CAG in the
following analysis, but are included for comparison in the appendix. The
CAG's position is that, given the limited data available from these animal
bioassays, especially at the high-dose levels required for testing, almost
nothing is known about the true shape of the dose-response curve at low
environmental levels and that the risk estimates obtained by use of the linear
non-threshold model represent plausible upper limits only.
Extrapolation from animals to humans could also be done on the basis of
relative weight rather than on the basis of relative surface area. Although
the latter approach, used here, has more justification in terms of human
pharmacological responses, it is not yet clear which of the two approaches is
more appropriate for the assessment of carcinogenicity. In the absence of
information on this point, it seems appropriate to use the more conservative
basis for extrapolation, the relative surface area.
5.3.3.3.2 Unit risk potency estimates and relative potency. Neither of the
two available epidemiologic studies provided positive results from which to
derive a quantitative risk estimate for DCM exposure. With respect to animal
data, three chronic studies are relevant to this discussion: two inhalation
studies and one drinking water study. These are summarized in Table 5-35. In
5-98
-------
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5-99
-------
the two inhalation studies (Dow Chemical Company 1980, 1982) only the salivary
gland regions in male Sprague-Dawley rats in the 1980 study showed statistically
significant increased cancers. These results are discussed in Section 5.3.3.1.1.2
and are presented in Tables 5-12 through 5-14. In the drinking water study
(NCA 1982), the increase over untreated controls in combined neoplastic nodules
and hepatocellular carcinomas in female rats was the only statistically signi-
ficant finding (Table 5-24). However, when compared with historical controls,
these results lose their statistical significance.
5.3.3.3.2.1 Unit risk (ug/m3) for inhalation studies. The data used for
estimates of the unit risk for inhalation are presented in Table 5-36, which
shows the positive salivary gland region sarcomas for the inhalation bioassay.
Exposure was 6 hr/day, 5 days/week, for 2 years. Equivalent dosages were
determined for humans from the animal dosages utilizing the equivalent dosage
methodology presented previously. As described in the section on pulmonary
uptake and distribution, DCM is readily absorbed into the body following
inhalation and it equilibrates rapidly across the alveolar epithelium. There-
fore, the CAG considers it a virtually completely absorbed gas especially at
low doses and determines equivalent human exposure as explained under Case 1
of the inhalation section. As presented in Table 5-36, the nominal exposures
are nearly 15 times the human lifetime equivalent exposures. This difference,
24/6 x 7/5 = 5.6, is partly due to the use of a continuous equivalent dosage.
There is an additional factor of about 2.6, however, which is attributable to
the nature of the method used for determining human equivalent dosages for
inhalation studies. Put another way, if DCM had been determined to-be a
partially soluble vapor, the unit risk slope would be lower by a factor of
about 2.6.
When the incidence data given above were fitted with the continuous human
equivalent exposures, the linearized multistage model yielded the following
value for the 95 percent upper limit of risk:
q* = 1.8 x 10"7(|jg/m3)~1
5.3.3.3.2.2 Unit risk (mg/kg/day) and (ug/1) for oral studies. This
unit risk estimate should be used only under the assumption that DCM is a
potential human carcinogen. As discussed in the qualitative section, there is
only limited animal evidence and no human evidence to support that assumption.
5-100
-------
TABLE 5-36. INCIDENCE RATES OF SALIVARY GLAND REGION SARCOMAS IN
MALE SPRAGUE-DAWLEY RATS IN THE
DOW CHEMICAL COMPANY (1980) DICHLOROMETHANE INHALATION STUDY
Continuous human
(animal nominal)
ppm and
0 (0)
34 (500)
103 (1,500)
240 (3,500)
equivalent
exposures
pg/m
0 (0)
1.2 x 105 (1.8 x
3.6 x 105 (5.2 x
8.4 x 105 (1.2 x
106)
106)
107)
Incidence rates
Number of rats with tumors/
total rats examined (%)
1/93 (1%)
0/94 (0%)
5/91 (5%)
11/88 (12%)
* 1 ppm x 1.2 x 103 pg/m3 x 84.9 = 3.5 x 103 pg/m3
2875
o
An exposure of 500 ppm DCM in air expressed as pg/nr is
500 ppm x 3.5 x 103 pg/m3 = 1.8 x 106 [jg/m3
ppm
Since animal exposure was for 6 hr/day, 5 days/week, the animal continuous
lifetime exposure equivalent was
1.8 x 106 pg/m3 x 6 x 5 = 3.2 x 105 pg/m3
24 7
The human equivalent dose for DCM is calculated first by determining the amount
actually breathed by the rats. As presented in the inhalation dose equivalence
section, 350 g rats breathe
I = 0.105 (0.350/0.113)2/3 = 0.223 m3 (air)/day.
Thus continuous animal dose was 3.2 x 105 pg/m3 x 0.223 m3/day = 7.15 x 104 pg/
day, or, for a 350 g rat, 7.15 x 104 pg/day/0.350 kg = 2.0 x 105 pg/kg/day.
The human equivalent dose is
2.0 x 105 pg/kg/day -=- (70/0.350)173 = 3.5 x 104 pg/kg/day
or
3.5 x 104 pg/kg/day x 70 kg = 1.2 x 105 pg/m3.
20 m3
5-101
-------
Since publication of a final NTP report on gavage studies for rats and
mice on DCM has been cancelled, there is no suitable oral study from which to
estimate a unit risk.
5.3.3.3.2.3 Comparison of animal and human inhalation studies. The
study of Kodak employees, yielding negative cancer results, is compared with
the positive tumor results of the rat inhalation study (Dow Chemical Company,
1980). In the latter study, the salivary gland region tumors in male Sprague-
~7 3 "1
Dawley rats led to a 95% upper-limit slope estimate of qฃ=1.8 x 10 ((jg/m ) .
If this slope factor is applied to the human inhalation study, in which time-
weighted average exposure was estimated as ranging from approximately 30 to
120 ppm, the expected impact of exposure can be estimated. For this purpose,
1 ug/m3 of DCM is equivalent to 2.9 x 10 ppm. Thus, the upper-limit slope
in ppm is
q* = 1.8 x 10"7 (pg/m3)'1 x 3.45 x 103 ug/m3 = 6.2 x 10"4 ppm"1.
ppm
Since this upper-limit slope is based on continuous lifetime equivalent exposure,
the exposure range of 30 to 120 ppm time-weighted average must be adjusted to
lifetime equivalence as follows:
30 ppm x 20 years x 240 days x 8 hr = 1.88 ppm
70 365" 2?
and 7.52 ppm lifetime equivalence for the 120 ppm exposure group, assuming 20
years of exposure for the 252 long-term exposure workers. Based on this
upper-limit slope factor and the range of exposure, the group of 252 workers
could expect an additional lifetime risk of between
R = 6.2 x 10~4 ppm"1 x 1.88 ppm = 1.2 x 10"3 and 4.7 x 10~3.
For these 252 workers, this would translate to an upper limit of between 252 x
1.2 x 10~3 = 0.3 and 1.2 excess lifetime cancer deaths. Based on the total
expected Kodak employee deaths of 65.9 (Table 5-33) for this 20-yr minimally
exposed cohort, we would expect 26% of the cohort to die in the 17-yr follow-
up period. Transposing this 26% to the expected excess lifetime cancer deaths,
an upper limit of between 0.1 and 0.3 excess cancer deaths can be expected
5-102
-------
.during the 17-yr follow-up period. The power to detect this increase from
17.77 to 18.1 cancer deaths is less than 10%. If these excess cancer deaths
were from cancers of one specific site, the power would be greater, but not
great enough to declare this a negative study. Even for a rare cancer, such
as a liver cancer, the expected number of cases would be much less than 1.
Since only deaths can be observed, the power to observe one death from liver
cancer in this cohort is quite small.
Based on the analysis presented above, the study of Kodak workers exposed
to DCM, showing no increase in cancer, cannot be judged as having negative
results because of its low power, which is related to low exposure from a weak
animal carcinogen.
5.3.3.3.2.4 Relative potency. One use of the unit risk concept is to
compare the relative potencies of carcinogens. To estimate relative potency
on a per mole basis, the unit risk slope factor is multiplied by the molecular
weight and the resulting number is expressed in terms of (mmol/kg/day)"1.
This is called the relative potency index.
Figure 5-1 is a histogram representing the frequency distribution of the
potency indices of 53 chemicals evaluated by the CAG as suspect carcinogens.
The actual data summarized by the histogram are presented in Table 5-37.
Where human data are available for a compound, they have been used to calcu-
late the index. Where no human data are available, animal oral studies and
animal inhalation studies have been used, in that order. Animal oral studies
are selected over animal inhalation studies because most of the chemicals have
been subjected to animal oral studies; this allows potency comparisons by
route.
The potency index for DCM, based on salivary gland region tumors in male
Sprague-Dawley rats in the Dow inhalation study (Dow Chemical Company, 1980),
_p -T
is 5.3 x 10 (mmol/kg/day) . This is derived as follows: The slope esti-
in converted units of 6.3 x
mate from the Dow study, 1.8 x 10~7 (pg/m3)'1
10 (mg/kg/day) , is multiplied by the molecular weight of 84.9 to give a
potency index of 5.3 x 10 . Rounding off of the nearest order of magnitude
gives a value of 10 , which is the scale presented on the horizontal axis of
Figure 5-1. The index of 5.3 x 10~2 is the least potent of the 53 suspected
carcinogens. Ranking of the relative potency indices is subject to the uncer-
tainty of comparing estimates of potency of different chemicals based on
different routes of exposure to different species using studies of different
5-103
-------
20
4th 3rd 2nd 1st
QUARTILE QUARTILE QUARTILE QUARTILE
1 23456
LOG OF POTENCY INDEX
7
8
Figure 5-1. Histogram representing frequency distribution of the potency indices of 53 suspect
carcinogens evaluated by the Carcinogen Assessment Group.
5-104
-------
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quality. Furthermore, all the indices are based on estimates of low-dose risk
using linear extrapolation from the observational range. Thus these indices
are not valid for the comparison of potencies in the experimental or observa-
tional range if linearity does not exist there. The potency index for DCM,
furthermore, is valid only under the assumption that DCM is a potential human
carcinogen. The evidence for that is limited.
5.3.3.3.2.5 Summary of quantitative estimation. No positive epidemic-
logic studies exist from which to estimate a unit risk for exposure to DCM.
Only one animal data set has shown increased cancers from which a unit risk
assessment could be estimated. In the Dow Chemical Company (1980) rat inha-
lation study, there were increased sarcomas in the salivary gland region of
male rats; this yielded an upper 95% limit of the potency estimate of q? = 1.8
x 10"7 (Mg/m3)'1, equivalent to q* = 6.3 x 10"4 (mg/kg/day)'1 by the oral
route. However, no positive data exist with which to provide a direct oral
route estimate. In total, there is only limited evidence that DCM is a poten-
tial human carcinogen. The unit risk estimate is valid only if one accepts
that limited evidence. Further, if one chooses to express the potency of DCM
relative to that of 53 chemicals evaluated as suspect carcinogens, DCM is the
weakest, ranking last.
5.3.3.4 Summary. Six chronic studies of DCM administered to animals have
been reported: four in rats, one in mice, and one in hamsters. The Dow
Chemical Company (1980) reported the results of chronic inhalation studies in
rats and hamsters. The rat 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, as well as a statistically significant increased
incidence of ventral cervical sarcomas, probably of salivary gland origin, in
male rats. The response pattern of the salivary gland tumors is unusual,
consisting of sarcomas only and appearing in males but not in females. In
hamsters, there was an increased incidence of lymphosarcoma in females only,
which was not statistically significant after correction for survival. The
results of the Dow Chemical Company (1980) study have since been published by
Burek et al. (1984). The second inhalation study in rats by the Dow Chemical
Company (1982) reported that there were no compound-related increased inci-
dences of any tumor type, but that the highest dose was far below that of the
previous Dow inhalation study in rats. The National Coffee Association (1982a,b)
drinking water study in Fischer 344 rats reported that the incidence of neo-
5-109
-------
plastic nodules and/or hepatocellular carcinomas in female rats was increased
significantly with respect to matched controls, but their 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 carcino-
mas. The National Toxicology Program (1982) draft gavage study on rats and
mice will not be published as a final report due to data discrepancies.
Selected information from the gavage studies may be incorporated into the
future NTP inhalation bioassay, pending the results of the in-depth audit.
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 inadequate because they were not carried out
for a full lifetime (Heppel et al., 1944; MacEwen et al., 1972).
One carcinogenicity study of DCM in animals is currently in progress: an
NTP 2-year bioassay by inhalation in Fischer 344/N rats and B6C3F1 mice.
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. (1983 a,b,c,d,e).
Although neither study showed excessive risk, both showed sufficient defi-
ciencies to prevent them from being judged negative studies. The Friedlander
et al. study (1978) lacked great enough exposure (based on animal cancer
potency estimates) to provide sufficient statistical power to detect a poten-
tial carcinogenic effect. The Ott et al. study (1983 a,b,c,d,e), among other
deficiencies, lacked a sufficient latency period for site-specific cancer.
Only one animal data set has shown increased cancers from which a unit
risk assessment could be estimated. In the Dow Chemical Company (1980) rat
inhalation study, there were increased sarcomas in the salivary gland region
of male rats; this yielded an upper 95 percent limit of the potency estimate
-4 -1
equivalent to q* = 6.3 x 10 (mg/kg/day) by
of q* = 1.8 x 10"7 (ug/m3)'1
the oral route. This upper bound unit risk estimate should be used only under
the assumption that DCM is a potential human carcinogen. As discussed pre-
viously, there is only limited animal evidence and no human evidence to support
that assumption.
5-110
-------
5.3.3.5 Conclusions. Animal studies show a statistically positive salivary
gland sarcoma response in male rats (Dow Chemical Company, 1980) and a border-
line hepatocellular neoplastic nodule response in the rat (National Coffee
Association, 1982). There is also evidence that DCM is weakly mutagenic.
According to the criteria of the International Agency for Research on Cancer
(IARC), the weight of evidence for carcinogenicity in animals is limited.
There was an absence of epidemiologic evidence for the carcinogenicity of
DCM in a well-conducted epidemiologic study having long-term exposure. However,
on the basis of animal data, the level of exposure to the individuals in the
study was too low to produce an observable increase in cancer. The overall
evaluation of DCM, based on IARC criteria, is Group 3, meaning that the chemical
cannot be classified as to its carcinogenicity for humans.
The upper-limit unit risk for DCM, based on a rat inhalation study, is
estimated to be roughly 1.8 x 10~ for a lifetime exposure to 1 pg/m in air.
The above is true only under the assumption that DCM is a potential human car-
cinogen, although the likelihood of this is highly uncertain given the
currently assessed data base. Even under that assumption, the potency of DCM
is the lowest of the 53 chemicals that the CAG has evaluated as suspect carci-
nogens. The Environmental Health Committee of EPA's Science Advisory Board
(SAB) indicated that the unit risk estimation was "scientifically" unsup-
portable because of the questionable value of the animal study upon which the
unit risk estimation was based, and that consideration should be given to
deleting the analysis from this document. The SAB noted, however, that the
unit risk estimates may be useful for risk management purposes. In light of
the potential need for risk management analysis, the risk estimation section
has been retained.
Preliminary results of a new NTP study in mice indicate that DCM induces
a high incidence of lung and liver tumors in both sexes. When the study is
completed and available, the results will be evaluated, and the qualitative
and quantitative portions of this assessment updated. For this reason the
present report is regarded as interim.
5-111
-------
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5-122
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APPENDIX
COMPARISON OF RESULTS BY VARIOUS EXTRAPOLATION MODELS
The estimates of unit risk based on animal studies presented in the body
of this document are all calculated by the use of the linearized multistage
model. The reasons for its use have been detailed herein. Essentially, this
model is part of a methodology that estimates a conservative linear slope at
low extrapolation doses and is consistent with the data at all dose levels of
the experiment. It is a nonthreshold model which holds that the upper-limit of
risk predicted by a linear extrapolation to low levels of the dose-response
relationship is the most plausible upper limit for the risk.
Other models have also been used for extrapolation, and include the
three nonthreshold models presented here: the one-hit, the log-Probit, and
the Weibull. The one-hit model is characterized by a continuous downward
curvature, but is linear at low doses. It can be considered the linear form or
first stage of the multistage model because of its functional form. Because of
this and its downward curvature, the one-hit model will always yield estimates
of low-level risk that are at least as large as those of the multistage model.
Further, whenever the data can be fitted adequately by means of the one-hit
model, estimates from the two procedures will be comparable.
The other two models, the log-Probit and the Weibull, are often used to
fit toxicological data in the observable range, because of their general "S"
curvature. The low-dose upward curvatures of these two models usually yield
lower low-dose risk estimates than those of the one-hit or multistage model.
The log-Probit model was originally proposed for use in problems of
biological assay, such as the assessment of potency of toxicants and drugs,
and has usually been used to estimate such values as percentile lethal dose
or percentile effective dose. Its development was strictly empirical, i.e.,
it was observed that several log dose-response relationships followed the
cumulative normal probability distribution function. In fitting the cancer
bioassay data, assuming an independent background, this becomes:
P(D;a,b,c) = c + (1-c) * (a+blog10D) a,b < c < 1
A-l
-------
where P is the proportion responding at dose D, c is an estimate of the back-
ground rate, a is an estimate of the standarized mean of individual tolerances,
and b is an estimate of the log dose-Probit response slope.
The one-hit model arises from the theory that a single molecule of a
carcinogen has a probability of transforming a single noncarcinogenic cell
into a carcinogenic one. It has the probability distribution function:
P(D;a,b) = l-exp-(a+bd) a,b > 0
where a and b are the parameter estimates. The estimate a represents the
background or zero dose rate, and the parameter estimated by b represents the
linear component or slope of the dose-response model. In discussing the added
risk over background, incorporation of Abbott's correction leads to
P(D;b) = l-exp-(bd) b > 0
Finally, a model from the theory of carcinogenesis arises from the multihit
model applied to multiple target cells. This model has been termed here the
Weibull model. It is of the form
P(D;b,k) = l-exp-(bdk) b,k > 0
For the power of dose only, the restriction k > 0 has been placed on this
model. When k > 1, this model yields low-dose estimates of risks usually
significantly lower than either the multistage or one-hit models, which are
linear at low doses. All three of these models usually project risk estimates
significantly higher at the low exposure levels than those from the log-Probit.
The estimates of added risk for low doses for the above models are given
in Table A-l for the DCM inhalation study. Both maximum likelihood estimates
and 95% upper confidence limits are presented. All estimates incorporate
Abbott's correction for independent background rate.
A-2
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