factors that influence uptake into these cells. Large particulates (>10
[mu]m) are generally deposited in the upper nasopharygeal region of the
respiratory tract and do not reach the bronchoalveolar region of the
lungs. Smaller Cr(VI) particulates will increasingly reach these lower
regions and come into contact with target cells.
    Once deposited in the lower respiratory tract, solubility of Cr(VI)
particulates becomes a major influence on disposition. Highly water
soluble Cr(VI), such as sodium chromate and chromic acid, rapidly
dissolves in the fluids lining the lung epithelia and can be taken up
by lung cells via facilitated diffusion mediated by sulfate/phosphate
anion transport channels (Ex. 35-148). This is because Cr(VI) exists in
a tetrahedral configuration as a chromate oxyanion similar to the
physiological anions, sulfate and phosphate (Ex. 35-

231). Using cultured human epithelial cells, Liu et al. showed that
soluble Cr(VI) uptake was time- and dose-dependant over a range of 1 to
300 [mu]m in the medium with 30 percent of the Cr(VI) transported into
the cells within two hours and 67 percent at 16 hours at the lowest
concentration (Ex. 31-22-18).
    Water insoluble Cr(VI) particulates do not readily dissolve into
epithelial lining fluids of the bronchoalveolar region. This has led to
claims that insoluble chromates, such as lead chromate pigments, are
not bioavailable and, therefore, are unable to cause carcinogenesis
(Ex. 31-15). However, several scientific studies indicate that
insoluble Cr(VI) particulates can come in close contact with the
bronchoalveolar epithelial cell surface, allowing enhanced uptake into
cells. Wise et al. showed that respirable lead chromate particles
adhere to the surface of rodent cells in culture causing cell-enhanced
dissolution of the chromate ion as well as phagocytosis of lead
chromate particles (Exs. 35-68; 35-67). The intracellular accumulation
was both time- and dose-dependant. Cellular uptake resulted in damage
to DNA, apoptosis (i.e., form of programmed cell death), and neoplastic
transformation (Ex. 35-119). Singh et al. showed that treatment of
normal human lung epithelial cells with insoluble lead chromate
particulates (0.4 to 2.0 [mu]g/cm\2\) or soluble sodium chromate (10
[mu]M) for 24 hours caused Cr(VI) uptake, Cr-DNA adduct formation, and
apoptosis (Ex. 35-66). The proximate genotoxic agent in these cell
systems was determined to be the chromate rather than the lead ions
(Ex. 35-327). Elias et al. reported that cell-enhanced particle
dissolution and uptake was also responsible for the cytotoxicity and
neoplastic transformation in Syrian hamster embryo cells caused by
Cr(VI) pigments, including several complex industrial chrome yellow and
molybdate orange pigments (Ex. 125). These studies are key experimental
evidence in the determination that water-insoluble Cr(VI) compounds, as
well as water soluble Cr(VI) compounds, are to be regarded as
carcinogenic agents. This determination is further discussed in the
next section (see V.B.9).
    Reduction to the poorly permeable Cr(III) in the epithelial lining
fluid limits cellular uptake of Cr(VI). Ascorbic acid and glutathione
(GSH) are believed to be the key molecules responsible for the
extracellular reduction. Cantin et al. reported high levels of GSH in
human alveolar epithelial lining fluid and Susuki et al. reported
significant levels of ascorbic acid in rat lung lavage fluids (Exs. 35-
147; 35-143). Susuki and Fukuda studied the kinetics of soluble Cr(VI)
reduction with ascorbic acid and GSH in vitro and following
intratracheal instillation (Ex. 35-90). They reported that the rate of
reduction was proportional to Cr(VI) concentration with a half-life of
just under one minute to several hours. They found the greatest
reduction rates with higher levels of reductants. Ascorbic acid was
more active than GSH. Cr(VI) reduction was slower in vivo than
predicted from in vitro and principally involved ascorbic acid, not
GSH. This research indicates that extracellular Cr(VI) reduction to
Cr(III) is variable depending on the concentration and nature of the
reductant in the epithelial fluid lining regions of the respiratory
tract. De Flora et al. determined the amount of soluble Cr(VI) reduced
in vitro by human bronchiolar alveolar fluid and pulmonary alveolar
macrophage fractions over a short period and used these specific
activities to estimate an ``overall reducing capacity'' of 0.9-1.8 mg
Cr(VI) and 136 mg Cr(VI) per day per individual, respectively (Ex. 35-
140).
    De Flora, Jones, and others have interpreted the extracellular
reduction data to mean that very high levels of Cr(VI) are required to
``overwhelm'' the reductive defense mechanism before target cell uptake
can occur and, as such, impart a ``threshold'' character to the
exposure-response (Exs. 35-139; 31-22-7). However, the threshold
capacity concept does not consider that facilitated lung cell uptake
and extracellular reduction are dynamic and parallel processes that
happen concurrently. If their rates are comparable then some cellular
uptake of Cr(VI) would be expected, even at levels that do not
``overwhelm'' the reductive capacity. Based on the in vitro kinetic
data, it would appear that such situations are plausible, especially
when concentrations of ascorbic acid are low. Unfortunately, there has
been little systematic study of the dose-dependence of Cr(VI) uptake in
the presence of physiological levels of ascorbate and GSH using
experimental systems that possess active anion transport capability.
The implications of extracellular reduction on the shape of Cr(VI)
dose--lung cancer response curve is further discussed in Section
VI.G.2.c.
    Wise et al. did study uptake of a single concentration of insoluble
lead chromate particles (0.8 [mu]g/cm2) and soluble sodium
chromate (1.3 [mu]M) in Chinese hamster ovary cells co-treated with a
physiological concentration (1mM) of ascorbate (Ex. 35-68). They found
that the ascorbate substantially reduced, but did not eliminate,
chromate ion uptake over a 24 hour period. Interestingly, ascorbate did
not affect phagocytic uptake of lead chromate particles, although it
eliminated the Cr(VI)-induced clastogenesis (e.g., DNA strand breakage
and chromatid exchange) as measured under their experimental
conditions.
    Singh et al. suggested that cell surface interactions with
insoluble lead chromate particulates created a concentrated
microenvironment of chromate ions resulting in higher intracellular
levels of chromium than would occur from soluble Cr(VI) (Ex. 35-149).
Cell membrane-enhanced uptake of Cr(VI) is consistent with the
intratracheal and intrabronchial instillation studies in rodents that
show greater carcinogenicity with slightly soluble (e.g., calcium
chromate and strontium chromate) than with the highly water-soluble
chromates (e.g., sodium chromate and chromic acid) (Ex. 11-2).
    Finally, Cr(VI) deposited in the tracheobronchial and alveolar
regions of the respiratory tract is cleared by the mucocilliary
escalator (soluble and particulate Cr(VI)) and macrophage phagocytosis
(particulate Cr(VI) only). In most instances, these clearance processes
take hours to days to completely clear Cr(VI) from the lung, but it can
take considerably longer for particulates deposited at certain sites.
For example, Ishikawa et al. showed that some workers had substantial
amounts of chromium particulates at the bifurcations of the large
bronchii for more than two decades after cessation of exposure (Ex. 35-
81). Mancuso reported chromium in the lungs of six chromate production
workers who died from lung cancer (as cited in Ex. 35-47). The interval
between last exposure to Cr(VI) until autopsy ranged from 15 months to
16 years. Using hollow casts of the human tracheobronchial tree and
comparing particle deposition with reported occurrence of bronchogenic
tumors, Schlesinger and Lippman were able to show good correlations
between sites of greatest deposition and increased incidence of
bronchial tumors (Ex. 35-102).
    b. Intracellular Reduction of Cr(VI). Once inside the cell, the
hexavalent chromate ion is rapidly reduced to intermediate oxidation
states, Cr(V) and Cr(IV), and the more chemically stable Cr(III).
Unlike Cr(VI), these other chromium forms are able to react with DNA
and protein to generate a variety of adducts and complexes. In
addition, reactive oxygen species (ROS) are produced during the
intracellular reduction of Cr(VI) that are also capable of damaging
DNA. These reactive

intermediates, and not Cr(VI) itself, are considered to be the ultimate
genotoxic agents that initiate the carcinogenic process.
    After crossing the cell membrane, Cr(VI) compounds can be non-
enzymatically converted to Cr(III) by several intracellular reducing
factors (Ex. 35-184). The most plentiful electron donors in the cell
are GSH, and other thiols, such as cysteine, and ascorbate. Connett and
Wetterhahn showed that a Cr(VI)-thioester initially forms in the
presence of GSH (Ex. 35-206). A two-phase reduction then occurs with
rapid conversion to Cr(V) and glutathionyl radical followed by
relatively slower reduction to Cr(III) that requires additional
molecules of GSH. Depletion of cellular GSH and other thiols is
believed to retard complete reduction of Cr(VI) to Cr(III), allowing
buildup of intermediates Cr(V) and Cr(IV). The molecular kinetics of
the Cr(VI) to Cr(III) reduction with ascorbate is less well understood
but can also involve intermediate formation of Cr(V) and free radicals
(Ex. 35-184).
    Another important class of intracellular Cr(VI) reductions are
catalyzed by flavoenzymes, such as GSH reductase, lipoyl dehydrogenase,
and ferredoxin-NADP oxidoreductase. The most prominent among these is
GSH reductase that uses NADPH as a cofactor in the presence of
molecular oxygen (O2) to form Cr(V)-NADPH complexes. During
the reaction, O2 undergoes one electron reduction to the
superoxide radical (O2-) which produces hydrogen
peroxide (H2O2) through the action of the enzyme
superoxide dismutase. The Cr(V)-NADPH can then react with
H2O2 to regenerate Cr(VI) giving off hydroxyl
radicals, a highly reactive oxygen species, by a Fenton-like reaction.
It is, therefore, possible for a single molecule of Cr(VI) to produce
many molecules of potentially DNA damaging ROS through a repeated
reduction/oxidation cycling process. Shi and Dalal used electron spin
resonance (ESR) to establish formation of Cr(V)-NADPH and hydroxyl
radical in an in vitro system (Ex. 35-169; 35-171). Sugiyama et al.
reported Cr(V) formation in cultured Chinese hamster cells treated with
soluble Cr(VI) (Ex.35-133). Using a low frequency ESR, Liu et al.
provided evidence of Cr(V) formation in vivo in mice injected with
soluble Cr(VI) (Ex. 35-141-28).
    Several studies have documented that Cr(VI) can generate Cr(V) and
ROS in cultured human lung epithelial cells and that this reduction/
oxidation pathway leads to DNA damage, activation of the p53 tumor
suppressor gene and stress-induced transcription factor NF-[kappa]B,
cell growth arrest, and apptosis (Exs. 35-125; 35-142; 31-22-18; 35-
135). Leonard et al. used ESR spin trapping, catalase, metal chelators,
free radical scavengers, and O2-free atmospheres to show
that hydroxyl radical generation involves a Fenton-like reaction with
soluble potassium dichromate (Ex. 31-22-17) and insoluble lead chromate
(Ex.35-137) in vitro. Liu et al. showed that the Cr(IV)/Cr(V) compounds
are also able to generate ROS with H2O2 in a
Fenton reduction/oxidation cycle in vitro (Ex. 35-183).
    Although most intracellular reduction of Cr(VI) is believed to
occur in the cytoplasm, Cr(VI) reduction can also occur in mitochondria
and the endoplasmic reticulum. Cr(VI) reduction can occur in the
mitochondria through the action of the electron transport complex (Ex.
35-230). The microsomal cytochrome P-450 system in the endoplasmic
reticulum also enzymatically reduces Cr(VI) to Cr(V), producing ROS
through reduction/oxidation cycling as described above (Ex. 35-171).
    c. Genotoxicity and Damage to DNA. A large number of studies have
examined multiple types of genotoxicity in a wide range of experimental
test systems. Many of the specific investigations have been previously
reviewed by IARC (Ex. 35-43), Klein (Ex. 35-134), ATSDR (Ex. 35-41),
and the K.S. Crump Group (Ex. 35-47) and will only be briefly
summarized here. The body of evidence establishes that both soluble and
insoluble forms of Cr(VI) cause structural DNA damage that can lead to
genotoxic events such as mutagenisis, clastogenisis, inhibition of DNA
replication and transcription, and altered gene expression, all of
which probably play a role in neoplastic transformation. The reactive
intermediates and products that occur from intracellular reduction of
Cr(VI) cause a wide variety of DNA lesions. The type(s) of DNA damage
that are most critical to the carcinogenic process is an area of active
investigation.
    Many Cr(VI) compounds are mutagenic in bacterial and mammalian test
systems (Ex. 35-118). In the bacterial Salmonella typhimurium strains,
soluble Cr(VI) caused base pair substitutions at A-T sites as well as
frame shift mutations (Ex. 35-161). Nestmann et al. also reported
forward and frame shift mutations in Salmonella typhimurium with pre-
solubilized lead chromate (Ex. 35-162). Several Cr(VI) compounds have
produced mutagenic responses at various genetic loci in mammalian cells
(Ex. 12-7). Clastogenic damage, such as sister chromatid exchange and
chromosomal aberrations, have also been reported for insoluble Cr(VI)
and soluble Cr(VI) (Exs. 35-132; 35-115). Mammalian cells undergo
neoplastic transformation following treatment with soluble Cr(VI) or
insoluble Cr(VI), including a number of slightly soluble zinc and
insoluble lead chromate pigments (Exs. 12-5; 35-186).
    Genotoxicity has been reported from Cr(VI) administration to
animals in vivo. Soluble Cr(VI) induced micronucleated erythrocytes in
mice following intraperitoneal (IP) administration (Ex. 35-150). It
also increased the mutation frequency in liver and bone marrow
following IP administration to lacZ transgenic mice (Exs. 35-168; 35-
163). Izzotti et al. reported DNA damage in the lungs of rats exposed
to soluble Cr(VI) by intratracheal instillation (Ex. 35-170).
Intratracheal instillation of soluble Cr(VI) produced a time- and dose-
dependant elevation in mutant frequency in the lung of Big Blue
transgenic mice (Ex. 35-174). Oral administration of soluble Cr(VI) in
animals did not produce genotoxicity in several studies probably due to
route-specific differences in absorption. OSHA is not aware of
genotoxicity studies from in vivo administration of insoluble Cr(VI).
Studies of chromosomal and DNA damage in workers exposed to Cr(VI) vary
in their findings. Some studies reported higher levels of chromosomal
aberrations, sister chromatid exchanges, or DNA strand breaks in
peripheral lymphocytes of stainless steel welders (Exs. 35-265; 35-160)
and electroplaters (Ex. 35-164). Other studies were not able to find
excess damage in DNA from the blood lymphocytes of workers exposed to
Cr(VI) (Exs. 35-185; 35-167). These reports are difficult to interpret
since co-exposure to other genotoxic agents (e.g., other metals,
cigarette smoke) likely existed and the extent of Cr(VI) exposures were
not known.
    Because of the consistent positive response across multiple assays
in a wide range of experimental systems from prokaryotic organisms
(e.g., bacteria) to human cells in vitro and animals in vivo, OSHA
regards Cr(VI) as an agent able to induce carcinogenesis through a
genotoxic mode of action. Both soluble and insoluble forms of Cr(VI)
are reported to cause genotoxicity and neoplastic transformation. On
the other hand, Cr(III) compounds do not easily cause genotoxicity in
intact cellular systems, presumably due to the inability of Cr(III) to
penetrate cell membranes (Exs. 12-7; 35-186).
    There has been a great deal of research to identify the types of
damage to DNA caused by Cr(VI), the reactive

intermediates that are responsible for the damage, and the specific
genetic lesions critical to carcinogenesis. It was shown that Cr(VI)
was inactive in DNA binding assays with isolated nuclei or purified DNA
(Ex. 35-47). However, Cr(III) was able to produce DNA protein cross-
links, sister chromatid exchanges, and chromosomal aberrations in an
acellular system. Zhitkovich et al. showed that incubation of Chinese
hamster ovary cells with soluble Cr(VI) produced ternary complexes of
Cr(III) cross-linked to cysteine, other amino acids, or glutathione and
the DNA phosphate backbone (Ex. 312). Utilizing the pSP189 shuttle
vector plasmid, they showed these DNA-Cr(III)-amino acid cross-links
were mutagenic when introduced in human fibroblasts (Ex. 35-131).
    Another research group showed that plasmid DNA treated with Cr(III)
produced intrastrand crosslinks and the production of these lesions
correlated with DNA polymerase arrest (Ex. 35-126). The same
intrastrand crosslinks and DNA polymerase arrest could also be induced
by Cr(VI) in the presence of ascorbate as a reducing agent to form
Cr(III) (Ex. 35-263). These results were confirmed in a cell system by
treating human lung fibroblasts with soluble Cr(VI), isolating genomic
DNA, and demonstrating dose-dependent guanine-specific arrest in a DNA
polymerase assay (Ex. 35-188). Cr(V) may also form intrastrand
crosslinks since Cr(V) interacts with DNA in vitro (Ex. 35-178). The
Cr(V)-DNA crosslinks are probably readily reduced to Cr(III) in cell
systems. Intrastrand crosslinks have also been implicated in inhibition
of RNA polymerase and DNA topoisomerase, leading to cell cycle arrest,
apoptosis and possibly other disturbances in cell growth that
contribute to the carcinogenic pathway (Ex. 35-149).
    DNA strand breaks and oxidative damage result from the one electron
reduction/oxidation cycling of Cr(VI), Cr(V), and Cr(IV). Shi et al.
showed that soluble Cr(VI) in the presence of ascorbate and
H2O2 caused DNA double strand breaks and 8-
hydroxy deoxyguanine (8-OHdG, a marker for oxidative DNA damage) in
vitro (Ex. 35-129). Leonard et al. showed that the DNA strand breaks
were reduced by several experimental conditions including an
O2-free atmosphere, catabolism of H2O2
by catalase, ROS depletion by free radical scavengers, and chelation of
Cr(V). They concluded that the strand breaks and 8-OHdG resulted from
DNA damage caused by hydroxyl radicals from Cr(VI) reduction/oxidation
cycling (Ex. 31-22-17). Generation of ROS-dependant DNA damage could
also be shown with insoluble Cr(VI) (Ex. 35-137). DNA strand breaks and
related damage caused by soluble Cr(VI) have been reported in Chinese
hamster cells (Ex. 35-128), human fibroblasts (Ex. 311), and human
prostate cells (Ex. 35-255). Pretreatment of Chinese hamster cells with
a metal chelator suppressed Cr(V) formation from Cr(VI) and decreased
DNA strand breaks (Ex. 35-197). Chinese hamster cells that developed
resistance to H2O2 damage also had reduced DNA
strand breaks from Cr(VI) treatment compared to the normal phenotype
(Ex. 35-176).
    Several researchers have been able to modulate Cr(VI)-induced DNA
damage using cellular reductants such as ascorbate, GSH and the free
radical scavenger tocopherol (vitamin E). This has provided insight
into the relationships between DNA damage, reduced chromium forms and
ROS. Sugiyama et al. showed that Chinese hamster cells pretreated with
ascorbate decreased soluble Cr(VI)-induced DNA strand damage (e.g.,
alkali-labile sites), but enhanced DNA-amino acid crosslinks (Ex. 35-
133). Standeven and Wetterhahn reported that elimination of ascorbate
from rat lung cytosol prior to in vitro incubation with soluble Cr(VI)
completely inhibited Cr-DNA binding (Ex. 35-180). However, not all
types of Cr-DNA binding are enhanced by ascorbate. Bridgewater et al.
found that high ratios of ascorbate to Cr(VI) actually decreased
intrastrand crosslinks in vitro while low ratios induced their
formation (Ex. 35-263). This finding is consistent with research by
Stearns and Watterhahn who showed that excessive ascorbate relative to
Cr(VI) leads to two-electron reduction of Cr(III) and formation of
Cr(III)-DNA monoadducts and DNA-Cr(III)-amino acid crosslinks (Ex. 35-
166). Low amounts of ascorbate primarily cause one-electron reduction
to intermediates Cr(V) and Cr(IV) that form crosslinks with DNA and ROS
responsible for DNA strand breaks, alkali-labile sites, and clastogenic
damage. This explains the apparent paradox that extracellular Cr(VI)
reduction by ascorbate to Cr(III) reduces Cr(VI)-induced DNA binding
but intracellular Cr(VI) reduction by ascorbate to Cr(III) enhances Cr-
DNA binding. The aforementioned studies used soluble forms of Cr(VI),
but Blankenship et al. showed that ascorbate pretreatment inhibited
chromosomal aberrations in Chinese hamster ovary cells caused by both
insoluble lead chromate particles as well as soluble Cr(VI) (Ex. 35-
115). Pretreatment with the free radical scavenger tocopherol also
inhibits chromosomal aberrations and alkali-labile sites in Cr(VI)-
treated cells (Exs. 35-115; 35-128).
    Studies of the different types of DNA damage caused by Cr(VI) and
the modulation of that damage inside the cell demonstrate that Cr(VI)
itself is not biologically active. Cr(VI) must undergo intracellular
reduction to Cr(V), Cr(IV), and Cr(III) before the damage to DNA can
occur. The evidence suggests that Cr(III) can cause DNA-Cr-amino acid,
DNA-Cr-DNA crosslinks and Cr-DNA monoadducts. Cr(V) and possibly Cr(IV)
contribute to intrastrand crosslinks and perhaps other Cr-DNA binding.
ROS generated during intracellular reduction of Cr(VI) lead to lesions
such as chromosomal aberrations, DNA strand breaks, and oxidative DNA
damage. The specific DNA lesions responsible for neoplastic
transformation have yet to be firmly established so all forms of DNA
damage should, at this time, be regarded as potential contributors to
carcinogenicity.
    d. Cr(VI)-induced Disturbances in the Regulation of Cell
Replication. Recent research has begun to elucidate how Cr(VI)-induced
oxidative stress and DNA lesions trigger cell signaling pathways that
regulate the cell growth cycle. The complex regulation of the cell
growth cycle by Cr(VI) involves activation of the p53 protein and other
transcription factors that respond to oxidative stress and DNA damage.
The cellular response ranges from a temporary pause in the cell cycle
to terminal growth arrest (i.e., viable cells that have lost the
ability to replicate) and a programmed form of cell death, known as
apoptosis. Apoptosis involves alterations in mitochondrial
permeability, release of cytochrome c and the action of several kinases
and caspases. Less is known about the molecular basis of terminal
growth arrest. Terminal growth arrest and apoptosis serve to eliminate
further growth of cells with unrepaired Cr(VI)-induced genetic damage.
However, it is believed that cells which escape these protective
mechanisms and regain replicative competence eventually become
resistant to normal growth regulation and can transform to a neoplastic
phenotype (Exs. 35-121; 35-122; 35-120).
    Blankenship et al. first described apoptosis as the primary mode of
cell death following a two hour treatment of Chinese hamster ovary
cells with high concentrations (>150 [mu]M) of soluble Cr(VI) (Ex. 35-
144). Apoptosis also occurs in human lung cells following short-term
treatment with soluble Cr(VI)

(Ex. 35-125) as well as longer term treatment (e.g., 24 hours) with
lower concentrations of soluble Cr(VI) (e.g., 10 [mu]M) and insoluble
Cr(VI) in the form of lead chromate (Ex. 35-166). Ye et al. found that
the Cr(VI) treatment that caused apoptosis also activated expression of
p53 protein (Ex. 35-125). This apoptotic response was substantially
reduced in a p53-deficient cell line treated with Cr(VI), suggesting
that the p53 activation was required for apoptosis. Other studies using
p53 null cells from mice and humans confirmed that Cr(VI)-induced
apoptosis is p53-dependent (Ex. 35-225).
    The p53 protein is a transcription factor known to be activated by
DNA damage, lead to cell cycle arrest, and regulate genes responsible
for either DNA repair or apoptosis. Therefore, it is likely that the
p53 activation is a response to the Cr(VI)-induced DNA damage.
Apoptosis (i.e., programmed cell death) is triggered once the Cr(VI)-
induced DNA damage becomes too extensive to successfully repair. In
this manner, apoptosis serves to prevent replication of genetically
damaged cells.
    Several researchers have gone on to further elucidate the molecular
pathways involved in Cr(VI)-induced apoptosis. ROS produced by
intracellular Cr(VI) reduction/oxidation cycling have been implicated
in the activation of p53 and apoptosis (Exs. 35-255; 35-122). Using
specific inhibitors, Pritchard et al. showed that mitochondrial release
of cytochrome c is critical to apoptotic death from Cr(VI) (Ex. 35-
159). Cytochrome c release from mitochondria could potentially result
from either direct membrane damage caused by Cr(VI)-induced ROS or
indirectly by enhanced expression of the p53-dependent apoptotic
proteins, Bax and Nova, known to increase mitochondrial membrane
permeability.
    Cr(VI) causes cell cycle arrest and reduces clonogenic potential
(i.e., normal cell growth) at very low concentrations (e.g., 1 [mu]M)
where significant apoptosis is not evident. Xu et al. showed that human
lung fibroblasts treated with low doses of Cr(VI) caused guanine-
guanine intrastrand crosslinks, guanine-specific polymerase arrest, and
inhibited cell growth at the G1/S phase of the cell cycle
(Ex. 35-188). Zhang et al. described a dose-dependent increase in
growth arrest at the G2/M phase of the cell cycle in a human
lung epithelial cell line following 24 hour Cr(VI) treatment over a
concentration range of 1 to 10 [mu]M (Ex. 35-135). The cell cycle
arrest could be partially eliminated by reducing production of Cr(VI)-
induced ROS. Apoptosis was not detected in these cells until a
concentration of 25 [mu]M Cr(VI) had been reached. These data suggest
that low cellular levels of Cr(VI) are able to cause DNA damage and
disrupt the normal cell growth cycle.
    Pritchard et al. studied the clonogenicity over two weeks of human
fibroblasts treated 24 hours with soluble Cr(VI) concentrations from 1
to 10 [mu]M (Ex. 35-120). They reported a progressive decline in cell
growth with increasing Cr(VI) concentration. Terminal growth arrest
(i.e., viable cells that have lost the ability to replicate) was
primarily responsible for the decrease in clonogenic survival below 4
[mu]M Cr(VI). At higher Cr(VI) concentrations, apoptosis was
increasingly responsible for the loss in clonogenicity. Pritchard et
al. and other research groups have suggested that a subset of cells
that continue to replicate following Cr(VI) exposure could contain
unrepaired genetic damage or could have become intrinsically resistant
to processes (e.g., apoptosis, terminal growth arrest) that normally
control their growth (Exs. 35-121; 35-122; 35-120). These surviving
cells would then be more prone to neoplastic progression and have
greater carcinogenic potential.
    e. Summary. Respirable chromate particulates are taken up by target
cells in the bronchoalveolar region of the lung, become intracellularly
reduced to several reactive genotoxic species able to damage DNA,
disrupt normal regulation of cell division and cause neoplastic
transformation. Scientific studies indicate that both water soluble and
insoluble Cr(VI) can be transported into the cell. In fact, cell
surface interactions with slightly soluble and insoluble chromates may
create a concentrated microenvironment of chromate ion, especially in
the case of the slightly soluble Cr(VI) compounds that more readily
dissociate. The higher concentration of chromate ion in close proximity
to the lung cells will likely result in higher intracellular Cr(VI)
than would occur from the highly water-soluble chromates. This is
consistent with the studies of respiratory tract carcinogenesis in
animals that indicate the most tumorigenic chromates had low to
moderate water solubility. Once inside the cell, Cr(VI) is converted to
several lower oxidation forms able to bind to and crosslink DNA. ROS
are produced during intracellular reduction/oxidation of Cr(VI) that
further damage DNA. These structural lesions are functionally
translated into a impaired DNA replication, mutagenesis, and altered
gene expression that ultimately lead to neoplastic transformation.
9. Conclusion
    In the NRPM, OSHA preliminarily concluded that the weight of
evidence supports the determination that all Cr(VI) compounds should be
regarded as carcinogenic to workers (69 FR at 59351). This conclusion
included the highly water soluble chromates, such as sodium chromate,
sodium dichromate, and chromic acid; chromates of slight and
intermediate water solubility such as calcium chromate, strontium
chromates, and many zinc chromates (e.g. zinc yellow); and chromates
that have very low water solubility and are generally considered to be
water insoluble such as barium chromate and lead chromates. The
strongest evidence supporting this conclusion comes from the many
cohort studies reporting excess lung cancer mortality among workers
engaged in the production of soluble chromates (Exs. 7-14; 31-22-11;
23; 31-18-4), chromate pigments (Exs. 7-36; 7-42; 7-46), and chrome
plating (Exs. 35-62; 35-271). Chromate production workers were
principally exposed to the highly soluble sodium chromate and
dichromate (Ex. 35-61) although lesser exposure to other chromates,
such as highly soluble chromic acid and slightly soluble calcium
chromate probably occurred. Pigment production workers were principally
exposed Cr(VI) in the form of lead and zinc chromates. Significantly
elevated lung cancer mortality was found in two British chromium
electroplating cohorts (Exs. 35-62; 35-271). These workers were exposed
to Cr(VI) in the form of chromic acid mist. Therefore, significantly
elevated lung cancer rates have been observed in working populations
exposed to a broad range of Cr(VI) compounds.
    Cellular research has shown that both highly water soluble (e.g.
sodium chromate) Cr(VI) and water insoluble (e.g. lead chromate) Cr(VI)
enter lung cells (see Section V.8.a) and undergo intracellular
reduction to several lower oxidation forms able to bind to and
crosslink DNA as well as generate reactive oxygen species that can
further damage DNA (see Section V.8.b). Soluble and insoluble Cr(VI)
compounds are reported to cause mutagenesis, clastogenesis, and
neoplastic transformation across multiple assays in a wide range of
experimental systems from prokaryotic organisms to human cells in vitro
and animals in vivo (see Section V.8.c).
    The carcinogenicity of various Cr(VI) compounds was examined after
instillation in the respiratory tract of rodents. Slightly water
soluble Cr(VI)

compounds, strontium chromate, calcium chromate, and some zinc
chromates produced a greater incidence of respiratory tract tumors than
highly water soluble (e.g. sodium dichromate and chromic acid) and
water insoluble (e.g. barium chromate and lead chromates) Cr(VI)
compounds under similar experimental protocol and conditions (see
Section V.7). This likely reflects the greater tendency for chromates
of intermediate water solubility to provide a persistent high local
concentration of solubilized Cr(VI) in close proximity to the target
cell. Highly soluble chromates rapidly dissolve and diffuse in the
aqueous fluid lining the epithelia of the lung. Thus, these chromates
are less able to achieve the higher local concentrations within close
proximity of the lung cell surface than the slightly water soluble
chromates. However, it has been shown that water-soluble Cr(VI) can
still enter lung cells, damage DNA, and cause cellular effects
consistent with carcinogenesis (Ex. 31-22-18; 35-125; 35-135; 35-142).
Like the slightly water soluble chromates, water insoluble Cr(VI)
particulates are able to come in close contact with the lung cell
surface and slowly dissolve into readily absorbed chromate ion. For
example, water insoluble lead chromate has been shown to enter human
airway cells both through extracellular solubilization as chromate ion
(Exs. 35-66; 35-327; 47-12-3) as well as internalization as
unsolubilized particulate (Exs. 35-66; 47-19-7). However, the rate of
solubilization and uptake of water insoluble Cr(VI) is expected to be
more limited than chromates with moderate solubility. Once chromate ion
is inside lung cells, studies have shown that similar cellular events
believed critical to initiating neoplastic transformation occur
regardless of whether the source is a highly soluble or insoluble
Cr(VI) compound (Ex. 35-327).
a. Public Comment on the Carcinogenicity of Cr(VI) Compounds
    In the NRPM, OSHA requested comment on whether currently available
epidemiologic and experimental studies supported the determination that
all Cr(VI) compounds possess carcinogenic potential and solicited
additional information that should be considered in evaluating relative
carcinogenic potency of the different Cr(VI) compounds (69 FR 59307).
Several comments supported the view that sufficient scientific evidence
exists to regard all Cr(VI) compounds as potential occupational
carcinogens (Exs. 38-106-2; 38-222; 39-73-2; 40-10-2; 42-2). The AFL-
CIO stated that `` * * * the agency has fully demonstrated that Cr(VI)
is a human carcinogen and that exposed workers are at risk of
developing lung cancer'' (Ex. 38-222). NIOSH stated that ``the
epidemiologic and experimental studies cited by OSHA support the
carcinogenic potential of all Cr(VI) compounds (i.e. water soluble,
insoluble, and slightly soluble)'' (Ex. 40-10-2, p. 4). Peter Lurie of
Public Citizen testified:

    As we heard repeatedly in the course of this hearing, scientific
experts, in fact, agree. They agree that the most reasonable
approach to the regulation is to consider them all [Cr(VI)
compounds] to be carcinogenic (Tr. 710).

    Several commenters agreed that the evidence supported the
qualitative determination that Cr(VI) compounds were carcinogenic but
wished to make clear that the information was inadequate to support
quantitative statements about relative potency of the individual
chromates (Exs. 38-106-2; 40-10-2; 42-2). For example, the Boeing
Company in their technical comments stated:

    The available data does support the conclusion that the low
solubility hexavalent chromium compounds [e.g. strontium chromate]
can cause cancer but evidence to support a quantitative comparison
of carcinogenic potency based on differences in solubility is
lacking (Ex. 38-106-2, p. 18).

Pigment Manufacturers' Comments on Carcinogenicity of Lead Chromate--
One group that did not regard all Cr(VI) compounds as occupational
carcinogens was the color pigment manufacturers who manufacture and
market lead chromate pigments which are primarily used in industrial
coatings and colored plastic articles. The color pigment manufacturers
maintain that their lead chromate products are unreactive in biological
systems, are not absorbed into the systemic circulation by any route,
and can not enter lung cells (Ex. 38-205, p. 14). Their principal
rationale is that lead chromate is virtually insoluble in water, is
unable to release chromate ion into aqueous media, and therefore, is
incapable of interacting with biological systems (Exs. 38-205, p. 95;
38-201-1, p. 9). The color pigment manufacturers assert that their lead
chromate pigment products are double encapsulated in a resin/plastic
matrix surrounded by a silica coating and that the encapsulated pigment
becomes even less ``bioavailable'' than unencapsulated ``less
stabilized'' lead chromates. They believe the extreme stability and
non-bioavailable nature of their products makes them a non-carcinogenic
form of Cr(VI) (Ex. 38-205, p. 106).
    According to the Color Pigment Manufacturers Association (CPMA),
several pieces of scientific evidence support their position, namely,
the lack of a significant excess of lung cancer mortality in three
cohorts of pigment workers engaged in the production of water-insoluble
lead chromate (Ex. 38-205, pp. 88-91) and the lack of statistically
significant elevated tumor incidence following a single instillation of
lead chromate in the respiratory tract of rats (Ex. 38-205, pp. 88-92).
They dismiss as irrelevant other animal studies that produced
statistically significant increases in tumors when lead chromate was
repeatedly injected by other routes. In addition, CPMA claims that the
lead chromate used in cellular studies that report genotoxicity was
reagent grade, was contaminated with soluble chromate, and was
inappropriately solubilized using strong acids and bases prior to
treatment (Exs. 38-205, pp. 93-94; 47-31, pp. 9-13). They are
especially critical of studies conducted by the Environmental and
Genetic Toxicology group at the University of Southern Maine that
report lead chromate particulates to be clastogenic in human lung cells
(Exs. 34-6-1; 38-205, pp. 98-102 & appendix D; 47-22). Instead, they
rely on two in vitro studies of lead chromate pigments that report a
lack of genotoxicity in cultured bacterial and hamster ovary cells,
respectively (Exs. 47-3 Appendix C; 38-205, p. 94).
    OSHA addresses many of the CPMA claims in other sections of the
preamble. The bioavailability issue of encapsulated lead chromate is
addressed in Section V.A.2. The CPMA request to consider the lack of
excess lung cancer mortality among pigment workers exposed exclusively
to lead chromate is discussed in Section V.B.2. The CPMA assertions
that animal studies are evidence that lead chromates are not
carcinogenic to workers are addressed in Section V.B.7. The studies
documenting uptake of lead chromate into lung cells are described in
Section V.B.8.a. Section V.B.8.c describes evidence that lead chromate
is genotoxic. As requested by CPMA, OSHA will pull these responses
together and expand on their concerns below.
    Lung Cancer Mortality in Pigments Workers Exposed to Lead
Chromate--Comments and testimony from NIOSH and others cite evidence of
excess lung cancer among pigment workers and support the results of
OSHA's preliminary risk assessment for color pigments in general and
for lead chromate in particular (Tr. 135-146, 316, 337, Ex. 40-18-1, p.
2). However, comments submitted by the CPMA and

the Dominion Colour Corporation (DCC) attributed the excess lung cancer
risk observed in pigment worker studies to zinc chromate (Tr. 1707,
1747, Exs. 38-201-1, p. 13; 38-205, p. 90; 40-7, p. 92). For example,
the CPMA stated that:

    When lead chromate and zinc chromate exposures occur
simultaneously, there appears to be a significant cancer hazard.
However, when lead chromate pigments alone are the source of
chromium exposure, a significant carcinogenic response has never
been found (Ex. 40-7, p. 92).

The latter statement refers to the Davies et al. (1984) study of
British pigment workers, the Cooper et al. (1983) study of U.S. pigment
workers, and the Kano et al. (1993) study of pigment workers in Japan,
all of which calculated separate observed and expected lung cancer
deaths for workers exposed exclusively to lead chromate (Ex. 38-205, p.
89). DCC and the Small Business Administration's Office of Advocacy
similarly stated that the excess lung cancer risk observed among
workers exposed to both zinc chromate and lead chromate cannot
necessarily be attributed to lead chromate (Exs. 38-201-1, p. 13; 38-7,
p. 4).
    OSHA agrees with CPMA and DCC that the excess lung cancer observed
in most pigment worker studies taken alone cannot be considered
conclusive evidence that lead chromate is carcinogenic. Given that the
workers were exposed to both zinc chromate and lead chromate, it is not
possible to draw strong conclusions about the effects of either
individual compound using only these studies. However, based on the
overall weight of available evidence, OSHA believes that the excess
lung cancer found in these studies is most likely attributable to lead
chromate as well as zinc chromate exposure. Lead chromate was the
primary source of Cr(VI) for several worker cohorts with excess lung
cancer (e.g., Davies et al. (1984), Factory A; Hayes et al. (1989); and
Deschamps et al. (1995)) (Exs. 7-42; 7-46; 35-234), and as previously
discussed, there is evidence from animal and mechanistic studies
supporting the carcinogenicity of both zinc chromate and lead chromate.
Considered in this context, the elevated risk of lung cancer observed
in most chromate pigment workers is consistent with the Agency's
determination that all Cr(VI) compounds--including lead chromate--
should be regarded as carcinogenic.
    Moreover, OSHA disagrees with the CPMA and DCC interpretation of
the data on workers exposed exclusively to lead chromate. In the
Preamble to the Proposed Rule, OSHA stated that ``[t]he number of lung
cancer deaths [in the Davies, Cooper, and Kano studies] is too small to
be meaningful'' with respect to the Agency's determination regarding
the carcinogenicity of lead chromate (FR 69 at 59332). The CPMA
subsequently argued that:

    [b]y this rationale, OSHA could never conclude that a compound
such as lead chromate pigment exhibits no carcinogenic potential
because there can never be enough lung cancer deaths to produce a
``meaningful'' result. This is an arbitrary and obviously biased
assessment which creates an insurmountable barrier. Since the lead
chromate pigments did not create an excess of lung cancer, there
cannot be a significant enough mortality from lung cancer to be
meaningful (Ex. 38-205, p. 90).

OSHA believes that these comments reflect a misunderstanding of the
sense in which the Davies, Cooper, and Kano studies are too small to be
meaningful, and also a misunderstanding of the Agency's position.
    Contrary to CPMA's argument, a study with no excess in lung cancer
mortality can provide evidence of a lack of carcinogenic effect if the
confidence limits for the measurement of effect are close to the null
value. In other words, the measured effect must be close to the null
and the study must have a high level of precision. In the case of the
Davies, Cooper, and Kano studies, the standardized mortality ratio
(SMR) is the measurement of interest and the null value is an SMR of 1.
Table V.10 below shows that the SMRs for these study populations are
near or below 1; however, the 95% confidence intervals for the SMRs are
quite wide, indicating that the estimated SMRs are imprecise. The Kano
data, for example, are statistically consistent with a ``true'' SMR as
low as 0.01 or as high as 2.62. The results of these studies are too
imprecise to provide evidence for or against the hypothesis that lead
chromate is carcinogenic.
[GRAPHIC] [TIFF OMITTED] TR28FE06.013

    This lack of precision may be partly explained by the small size of
the studies, as reflected in the low numbers of expected lung cancers.
However, it is the issue of precision, and not the number of lung
cancer deaths per se, that led OSHA to state in the preamble to the
proposed rule that the Davies, Cooper, and Kano studies cannot serve as
the basis of a meaningful analysis of lead chromate carcinogenicity
(Exs. 7-42; 2-D-1; 7-118). In contrast, a study population that has
confidence limits close to or below 1 would provide evidence to support
the DCC claim that `` * * * if lead chromate pigments possess any
carcinogenic potential at all, it must be extremely small'' (Ex. 38-
201-1, p. 14) at the exposure levels experienced by that population.
While this standard of evidence has not been met in the epidemiological
literature for pigment workers exposed exclusively to lead chromate
(i.e., the Davies, Cooper, and Kano studies), it is hardly an
``insurmountable barrier'' that sets up an impossible standard of proof
for those who contend that lead chromate is not carcinogenic.
    Some comments suggested that the Davies, Cooper, and Kano studies
should be combined to derive a summary risk measure for exposure to
lead chromate (see e.g. Ex. 38-201-1, pp. 13-14). However, OSHA
believes that these studies do not provide a

suitable basis of meta-analysis. There is little information with which
to assess factors recognized by epidemiologists as key to meta-
analysis, for example sources of bias or confounding in the individual
studies and comparability of exposures and worker characteristics
across studies, and to verify certain conditions required for
comparability of SMRs across these studies (see e.g. Modern
Epidemiology, Rothman and Greenland, p. 655). In addition, the
inclusion criteria and length of follow-up differ across the three
studies. Finally, each of the studies is extremely small. Even if it
were appropriate to calculate a `summary' SMR based on them, the
precision of this SMR would not be much improved compared to those of
the original studies.
    In their written testimony, DCC suggested that OSHA should
aggregate the data from the Davies, Cooper, and Kano studies in order
to determine whether there is a discrepancy between the results of
these three studies, taken together, and OSHA's preliminary risk
assessment (Ex. 38-201-1, pp. 13-14). DCC performed a calculation to
compare OSHA's risk model with the observed lung cancer in the three
cohorts. DCC stated that:

    OSHA estimates a chromate worker's risk of dying from lung
cancer due to occupational exposure as about one chance in four * *
* [Assuming that there were about] 200 workers in the Kano study,
the total in the three studies would be 600. A calculation of one
quarter would be 150 deaths. To compensate for a working life of
less than OSHA's 45 years [an assumption of 20 years] provides * * *
a refined estimate of about 70 deaths. An observed number less than
this could be due either to exposures already in practice averaging
much less than the current PEL of 52, or to lead chromate having
much less potential (if any) for carcinogenicity than other
chromates. In any event the actual incidence of death from lung
cancer would appear to be no more than one tenth of OSHA's best
estimate (Ex. 38-201-1, pp. 15-16).

The method suggested by DCC is not an appropriate way to assess the
carcinogenicity of lead chromate, to identify a discrepancy between the
pigment cohort results and OSHA's risk estimates, or to determine an
exposure limit for lead chromate. Among other problems, DCC's
calculation does not make a valid comparison between OSHA's risk
estimates and the results of the Davies, Cooper, and Kano studies.
OSHA's `best estimate' of lung cancer risk for any given Cr(VI)-exposed
population depends strongly on factors including exposure levels,
exposure duration, population age, and length of follow-up. The `one in
four' prediction cited by DCC applies to one specific risk scenario
(lifetime risk from 45 years of occupational exposure at the previous
PEL of 52 [mu]g/m\3\). OSHA's best estimate of risk would be lower for
a population with lower exposures (as noted by DCC), shorter duration
of exposure, or less than a lifetime of follow-up. Without adequate
information to adjust for each of these factors, a valid comparison
cannot be drawn between OSHA's risk predictions and the results of the
lead chromate cohort studies.
    The importance of accounting for cohort age and follow-up time may
be illustrated using information provided in the Cooper et al. study.
As shown in Table V-11 below, approximately three-fourths of the Cooper
et al. Plant 1 cohort members were less than 60 years old at the end of
follow-up.
[GRAPHIC] [TIFF OMITTED] TR28FE06.014

    For a population of 600 with approximately the same distribution of
follow-up time as described in the Cooper et al. publication (e.g.,
0.4% of workers are followed to age 84, 2% to age 79, etc.), OSHA's
risk model predicts about 3-15 excess lung cancers (making the DCC
assumption that workers are exposed for 20 years at 52 [mu]g/m\3\),
rather than the 70 deaths calculated by the DCC. If the workers were
typically exposed for less than 20 years or at levels lower than 52
[mu]g/m\3\, OSHA s model would predict still lower risk. A precise
comparison between OSHA's risk model and the observed lung cancer risk
in the Davies, Cooper and Kano cohorts is not possible without
demographic, work history and exposure information on the lead chromate
workers. (In particular, note that year 2000 background lung cancer
rates were used in the calculation above, as it was not feasible to
reconstruct appropriate reference rates without work history
information on the cohorts.) However, this exercise illustrates that
DCC's assertion of a large discrepancy between OSHA's risk model and
the available data on workers exposed exclusively to lead chromate is
not well-founded. To make a valid comparison between the OSHA risk

model and the lung cancer observed in the lead chromate cohorts would
require more information on exposure and follow-up than is available
for these cohorts.
    OSHA received comments and testimony from NIOSH and others
supporting of the Agency's interpretation of the epidemiological
literature on Cr(VI) color pigments, including lead chromate (Tr. 135-
146, 316, 337, Ex. 40-18-1, p. 2). At the hearing, Mr. Robert Park of
NIOSH stated that the available studies of workers exposed to chromate
pigments show `` * * * a general pattern of excess [lung cancer] * * *
'' and pointed out that ``[i]n several of the studies, lead [chromate]
was by far the major component of production, like 90 percent * * * So
I don't think there is any epidemiological evidence at this point that
gets lead off the hook'' (Tr. 337). Regarding the lack of statistically
significant excess lung cancer in several pigment worker cohorts, Mr.
Park identified study attributes that may have obscured an excess in
lung cancer, such as the high percentage of workers lost to follow-up
among immigrant workers in the Davies et al. study (Tr. 337) or a
healthy worker effect in the Hayes et al. study (Tr. 316). Dr. Paul
Schulte of NIOSH explained that

    * * * a lot of these studies that appear to be negative were
either of low power or had [some] other kind of conflicting
situation [so] that we can't really consider them truly negative
studies (Tr. 338).

Dr. Herman Gibb testified that the epidemiological studies relied on by
CPMA and DCC to question the carcinogenicity of lead chromate have very
low expected numbers of lung cancer deaths, so they `` * * * really
don't have a lot of ability to be able to detect a risk'' (Tr. 135-
136). Public Citizen agreed with OSHA's preliminary conclusion that
lead chromate is carcinogenic. Based on the major pigment worker
cohorts identified by OSHA in the Preamble to the Proposed Rule, Public
Citizen's Health Research Group concluded that

    * * * inadequately-powered studies, the standardized mortality
ratios for exposed workers are significantly elevated (range 1.5-
4.4) and a relationship between extent of exposure (whether measured
by duration of exposure or factory) generally emerges; [moreover,]
[t]hese studies must be placed in the context * * * of the animal
carcinogenicity studies * * * and the mechanistic studies reviewed
by OSHA (Ex. 40-18-1, p. 2).

Tumor Incidence in Experimental Animals Administered Lead Chromate--
CPMA also claims that the absence of evidence for carcinogenicity found
among the three cited cohorts of lead chromate pigment workers `` * * *
is further confirmed by the rat implantation studies of Levy'' (Ex. 38-
205, p. 98). They argue that these studies which involved implantation
into rat lungs `` * * * indicated no increased incidence of tumors for
lead chromate pigment, although more soluble chromates exhibited
varying degrees of carcinogenicity'' (Ex. 38-205, p. 93). They
dismissed other animal studies involving intramuscular and subcutaneous
injection of lead chromate which did report increased incidence of
tumors because they believe these techniques

    * * * are of questionable relevance in relation to human
workplace exposure conditions in industry, whereas tests involving
implantation in rat lung * * * are relevant to inhalation in
industrial exposures (Ex. 38-205, p. 93).

In a more recent submission, CPMA remarked that the intramuscular and
subcutaneous injection studies with lead chromate were contradictory
and `` * * * problematic in that false positive results frequently
occur during the study procedure (Ex. 47-31, p. 13).
    The rat implantation studies of Levy involved the surgical
placement of a Cr(VI)-containing pellet in the left bronchus of an
anesthetized rat (Exs. 10-1; 11-12; 11-2). This pellet procedure was an
attempt to deliver Cr(VI) compounds directly to the bronchial
epithelium and mimic continuous chronic in vivo dosing at the tissue
target site in order to assess the relative ability of different Cr(VI)
compounds to induce bronchogenic carcinoma. Histopathological
evaluation of the rat lung was conducted after a two year exposure
time. In most cases, approximately 100 rats were implanted with a
single pellet for each Cr(VI) test compound. The total lifetime dose of
Cr(VI) received by the animal was generally between 0.2 and 1.0 mg
depending on the compound. The amount of Cr(VI) that actually leached
from the cholesterol pellet and remained near the lung tissue was never
determined. At least 20 different commercially relevant Cr(VI)
compounds ranging from water insoluble to highly water soluble were
tested using this intrabronchial implantation protocol.
    The results of these studies are described in preamble section
V.B.7 and tables V-7, V-8, and V-9. Reagent grade lead chromate and six
different lead chromate pigments were tested. The lead chromate
pigments were a variety of different chrome yellows, including a silica
encapsulated chrome yellow, and molybdenum orange. The incidence of
bronchogenic cancer in the rats under this set of experimental
conditions was one percent or less for all the lead chromates tested.
This incidence was not statistically different from the negative
controls (i.e. rats implanted with a cholesterol pellet containing no
test compound) or rats administered either the water-insoluble barium
chromate or the highly soluble chromic acid and sodium dichromate. The
percent incidence of bronchogenic cancer in lead chromate-treated rats
was substantially less than that of rats treated with slightly soluble
strontium chromates (about 52 percent) and calcium chromate (24
percent). The type of bronchogenic cancer induced in these experiments
was almost entirely squamous cell carcinomas.
    OSHA does not agree with the CPMA position that absence of a
significant tumor incidence in the intrabronchial implantation studies
confirms that lead chromates lack carcinogenic activity and, therefore,
should not be subject to the OSHA Cr(VI) standard. The bioassay
protocol used approximately 100 test animals per experimental group.
This small number of animals limits the power of the bioassay to detect
tumor incidence below three to four percent with an acceptable degree
of statistical confidence. Three of the lead chromates, in fact,
produced a tumor incidence of about one percent (e.g. 1 tumor in 100
rats examined) which was not statistically significant. The researchers
only applied a single 2 mg [approximately 0.3 mg Cr(VI)] dose of lead
chromate to the bronchus of the rats. Since it was not experimentally
confirmed that the lead chromate pigments were able to freely leach
from the cholesterol pellet, the amount of Cr(VI) actually available to
the lung tissue is not entirely clear. Therefore, OSHA believes a more
appropriate interpretation of the study findings is that lead chromates
delivered to the respiratory tract at a dose of about 0.3 mg Cr(VI)
(maybe lower) lead to a less than three percent tumor incidence.
    However, OSHA agrees that the intrabronchial implantation protocol
does provide useful information regarding the relative carcinogenicity
of different Cr(VI) compounds once they are delivered and deposited in
the respiratory tract. No other study examines the carcinogenicity of
such a broad range of commercial Cr(VI) compounds under the same
experimental conditions in the relevant target organ to humans (i.e.
respiratory tract) following in vivo administration. OSHA agrees with
CPMA that the results of this study provide credible

evidence that water insoluble lead chromates are less carcinogenic than
some of the more moderately soluble chromates. Specifically, this
includes the slightly soluble zinc chromates (e.g. zinc yellow, zinc
potassium chromates, basic zinc chromates) as well as strontium
chromate and calcium chromate. Intrabronchial implantation of chromic
acid and other highly soluble Cr(VI) salts, such as sodium chromates,
did not induce a significant number of tumors. Therefore, these
experiments do not indicate lead chromate are less carcinogenic than
the highly water soluble Cr(VI) compounds.
    If the histopathology data from the intrabronchial implantation is
examined more closely, all lead chromates increased the incidence of
squamous metaplasia relative to controls, and, for some lead chromates,
squamous dysplasia of the bronchial epithelium occurred (Table 2, Ex.
11-2). Squamous metaplasia and dysplasia are generally considered to be
transformed cellular states from which a neoplasm (e.g. carcinomas) can
arise (Ex. 11-12). Increased squamous metaplasia was common among all
tested Cr(VI) compounds but not among Cr(III)-containing materials or
the negative controls (Ex. 11-12). The increased metaplasia induced by
lead chromates is unlikely to be due to bronchial inflammation since
the degree of inflammation was no greater than that observed in the
cholesterol-implanted controls (Table 2, Ex. 11-2).
    The squamous metaplasia and dysplasia in the rat lung model
following low dose lead chromate administration is consistent with a
low carcinogenic response (e.g. incidence of one percent or less) not
able to be detected under the conditions of the animal bioassay. This
explanation is supported by studies (discussed later in the section)
that show lead chromate can enter lung cells, damage DNA, and cause
genotoxic events leading to neoplastic transformation.
    Lead chromate carcinogenicity is also supported by the animal
studies that CPMA dismisses as problematic and of questionable
relevance. These studies administered lead chromates to rodents by
either the subcutaneous (Exs. 8-25, 5-2, 8-37) or intramuscular routes
(Ex. 10-2). While OSHA agrees that these routes may be less relevant to
occupational inhalation than implantation in the respiratory tract, the
studies exposed rats to a larger dose of lead chromate. The higher
amounts of Cr(VI) produced a significant incidence of tumors at the
injection site (see section V.B.7.c).
    The lead chromate pigments, chrome yellow and chrome orange,
induced injection site rhabdomyosarcomas and fibrosarcomas in 65
percent of animals following a single 30 mg injection in a saline
suspension (Ex. 8-37). The rats received a roughly ten fold higher dose
of Cr(VI) than in the intrabronchial bioassay. Rats injected with
saline alone did not develop injection site tumors. Only two percent or
less of rats receiving equal quantities of the inorganic pigments iron
yellow and iron red developed these tumors. The iron oxides are not
considered to be carcinogenic and do not give a significant neoplastic
response in this bioassay. OSHA has no reason to believe the
experimental procedure was problematic or given to frequent false
positives.
    A similarly high incidence (i.e. 70 percent) of the same injection
site sarcomas were found in an independent study in which rats were
injected intramuscularly with reagent grade lead chromate once a month
for nine months (Ex. 10-2). Each injection contained approximately 1.3
mg of Cr(VI) and the total dose administered was over 30 times higher
than the intrabronchial implantation. The lead chromate was
administered in a glycerin vehicle. The vehicle produced less than a
two percent incidence of injection site sarcomas when administered
alone.
    Contrary to statements by Eurocolour (Ex. 44-3D), lead chromate did
produce a low incidence of site-of-contact tumors in rats in an earlier
study when administered by either intramuscular or intrapleural
implantation (Ex. 10-4). There was no tumor incidence in the control
animals. The dose of lead chromate in this early publication was not
stated.
    Based on the increase in pre-neoplastic changes from the single low
dose intrabronchial implantation and the high incidence of malignant
tumors resulting from larger doses administered by subcutaneous and
intramuscular injection, it is scientifically reasonable to expect that
larger doses of lead chromate may have produced a higher incidence of
tumors in the more relevant intrabronchial implantation procedure. The
highly soluble sodium dichromate produced a small (statistically
insignificant) incidence of squamous cell carcinoma (i.e. one percent)
upon single low dose intrabronchial implantation similar to the lead
chromates (Ex. 11-2). In another study, sodium dichromate caused a
significant 17 percent increase in the incidence of respiratory tract
tumors when instilled once a week for 30 months in the trachea of rats
(Ex. 11-7). The weekly-administered dose for this repeated instillation
was about \1/5\th the dose of that used in the intrabronchial
implantation assay but the total administered dose after 30 months was
about 25 times higher. Rats that received a lower total dose of sodium
dichromate or the same total dose in more numerous instillations (i.e.
lower dose rate) developed substantially fewer tumors that were
statistically indistinguishable from the saline controls. A third study
found a 15 percent increase (not statistically significant) in lung
tumor incidence when rats repeatedly inhaled aerosolized sodium
dichromate for 18 months at the highest air concentrations tested (Ex.
10-11). These sodium dichromate studies are further described in
section V.B.7.a. The findings suggest that the lack of significant
carcinogenic activity in the intrabronchial implantation study
reflects, in part, the low administered dose employed in the bioassay.
    In his written testimony to OSHA, Dr. Harvey Clewell directly
addressed the issue of interpreting the absence of carcinogenicity in
an animal study as it relates to significant risk.

    First, the ability to detect an effect depends on the power of
the study design. A statistically-based No Observed Adverse Effect
Level (NOAEL) in a toxicity study does not necessarily mean that
there is no risk of adverse effect. For example, it has been
estimated that a NOAEL in a typical animal study can actually be
associated with the presence of an effect in as many as 10% to 30%
of the animals. Thus the failure to observe a statistically
significant increase in tumor incidence at a particular exposure
does not rule out the presence of a substantial carcinogenic effect
at that exposure * * *. Similarly the failure of Levy et al. (1986)
to detect an increase in tumors following intrabronchial
instillation of lead chromate does not in itself demonstrate a lack
of carcinogenic activity for that compound. It only demonstrates a
lower activity than for other compounds that showed activity in the
same experimental design. Presumably this lower activity is
primarily due to its low solubility; evidence of solubilization,
cellular uptake, and carcinogenic activity of this compound [i.e.
lead chromate] is provided in other studies (Maltoni et al. 1974,
Furst et al., 1976, Blankenship et al., 1997; Singh et al., 1999;
Wise et al., 2004) (Ex. 44.5, p. 13-14).

    OSHA agrees with Dr. Clewell that the inability to detect a
statistically significant incidence of tumors in one study that
administers a single low dose of lead chromate to a limited number of
animals is not evidence that this Cr(VI) compound lacks carcinogenic
activity. This is especially true when there exists an elevation in
pre-neoplastic lesions and other studies document significant

tumor incidence in animals administered higher doses of lead chromate.
    Cellular Uptake and Genotoxicity of Lead Chromate--CPMA disputes
the many studies that report lead chromate to be genotoxic or
clastogenic in cellular test systems (Exs. 35-162; 12-5; 35-119; 35-
188; 35-132; 35-68; 35-67; 35-115; 35-66; 47-22-1; 47-12-3; 35-327; 35-
436). They claim that the studies inappropriately solubilized the lead
chromate `` * * * in non-biological conditions such as strong alkali or
strong acid that causes the chemical breakdown of the lead chromate
crystal'' (Ex. 38-205, p. 94) and the ``lead chromate had been
dissolved * * * using aggressive substances'' (Ex. 38-205, p. 99). In a
later submission, CPMA states state that some of the cellular studies
used reagent grade lead chromate that is only >=98 percent pure and may
contain up to 2 percent soluble chromate (Ex. 47-31, p. 11). They
speculate that the interactions (e.g. chromate ion uptake, chromosomal
aberrations, DNA adducts, etc.) described in studies using cell
cultures treated with lead chromate are either due to the presumed
contamination of soluble chromate or some other undefined ``reactive
nature'' of lead chromate. CPMA adds that `` * * * the studies
referenced by OSHA [that use reagent grade lead chromate] have no
relevance to occupational exposures to commercial lead chromate
pigments'' (Ex. 38-205, p. 11-12).
    OSHA agrees that studies involving lead chromate pre-solubilized in
solutions of hydrochloric acid, sodium hydroxide or other strong acids
and bases prior to treatment with cells are not particularly relevant
to the inhalation of commercial lead chromate particulates. However,
several relevant cellular studies have demonstrated that lead chromate
particulates suspended in biological media and not can enter lung
cells, damage DNA, and cause altered gene expression as described
below.
    Beginning in the late 1980s, there has been a consistent research
effort to characterize the genotoxic potential of lead chromate
particulate in mammalian cells. The lead chromate was not pre-
solubilized prior to cell treatment in any of these investigations. In
most of the studies, lead chromate particles were rinsed with water and
then acetone. The rinses cleansed the particles of water- and acetone-
soluble contaminants before cell treatment. This served to remove any
potential water-soluble Cr(VI) present that might confound the study
results. In most instances, the lead chromate particles were filtered,
stirred or sonicated in suspension to break up the aggregated particles
into monomeric lead chromate particulates. These lead chromate
particulates were primarily less than 5 [mu]m in diameter. This is
consistent with the inhaled particle size expected to deposit in the
bronchial and alveolar regions of the lung where lung cancer occurs.
Air-dried lead chromate particulates were introduced to the cell
cultures in a suspension of either saline-based media or acetone. Lead
chromate particulate is considered to be insoluble in both solvents so
significant solubilization is not expected during the process of
creating a homogenous suspension.
    The initial research showed that lead chromate particulate
morphologically transformed mouse and hamster embryo cells (Exs. 35-
119; 12-5). One study tested a variety of lead chromate pigments of
different types (e.g. chrome yellows, chrome oranges, molybdate
oranges) as well as reagent grade lead chromate (Ex. 12-5). The
transformed cells displayed neoplastic properties (e.g. growth in soft
agar) and were tumorigenic when injected into animals (Ex. 35-119; 12-
5). While lead chromate particulate transformed mouse embryo cells, it
is important to note that lead chromate particulate was not found to be
mutagenic in these cells suggesting that other types of genetic lesions
(e.g. clastogenicity) may be involved (Ex. 35-119).
    Follow-on research established that lead chromate particulate
caused DNA-protein crosslinks, DNA strand breaks, and chromosomal
aberrations (i.e. chromatid deletions and achromatic lesions combined)
in mammalian cells rather than DNA nucleotide binding often associated
with base substitution and frameshift mutations captured in a standard
Ames assay (Exs. 35-132; 35-188). This distinguishes lead chromate
particulate from high concentrations of soluble Cr(VI) compounds or
pre-solubilized lead chromate which can cause these mutations.
    Lead chromate particulate enters mammalian embryo cells by two
distinct pathways (Ex. 35-68). It partially dissolves in the culture
medium (i.e. biological saline solution) to form chromate ion, which is
then transported into the cell. The rate of particle dissolution was
shown to be time- and concentration-dependent. The measured chromate
ion concentration was consistent with that predicted from the lead
chromate solubility constant in water. Lead chromate particulates were
shown to adhere to the embryo cell surface enhancing chromate ion
solubilization leading to sustained intracellular chromium levels and
measurable chromosomal damage (Ex. 35-67).
    Lead chromate particulates are also internalized into embryo cells,
without dissolution, by a phagocytic process (Ex. 35-68). The lead
chromate particles appeared to remain undissolved in tight vacuoles
(i.e. phagosomes) within the cell over a 24 hour period. Treatment of
embryo cells with lead chromate particulates in the presence of a
reducing agent (i.e. ascorbate) substantially reduced cellular uptake
of dissolved chromate ions and the chromosomal damage, but did not
impact the internalization of lead chromate particulates (Ex. 35-68).
This suggests that chromosomal damage by lead chromate was the result
of extracellular particle dissolution and not internalization under the
particular experimental conditions. Embryo cell treatment with large
amounts of lead glutamate that produced high intracellular lead in the
absence of Cr(VI) did not cause chromosomal damage further implicating
intracellular chromium as the putative clastogenic agent (Ex. 35-67).
    As the ability to maintain human tissue cells in culture improved
in the 1990s, dissolution and internalization of lead chromate
particulates, uptake of chromate ion, and the resulting chromosomal
damage were verified in human lung cells (Exs. 35-66; 47-22-1; 47-12-3;
35-327; 35-436). Lead chromate particulates are internalized, form
chromium adducts with DNA, and trigger dose-dependent apoptosis in
human small airway epithelial cells (Ex. 35-66). They also cause dose-
dependent increases in intracellular chromium, internalized lead
chromate particulates and chromosomal damage in human lung fibroblasts
(Exs. 47-22-1; 47-12-3). The chromosomal damage from lead chromate in
these human lung cells is dependent on the extracellular dissolution
and cell uptake of the chromate, rather than lead, in a manner similar
to dilute concentrations of the highly soluble sodium chromate (Ex. 47-
12-3; 35-327). Another water insoluble Cr(VI) compound, barium chromate
particulate, produces very similar responses in human lung fibroblasts
(Ex. 35-328). Human lung macrophages can phagocytize lead chromate
particulates and trigger oxidation-reduction of Cr(VI) to produce
reactive oxygen species capable of damaging DNA and altering gene
expression (Ex. 35-436).
    OSHA finds these recent studies to be carefully conceived and
executed by reputable academic laboratories. The scientific findings
have been published in well-respected peer reviewed

molecular cancer and toxicology journals, such as Carcinogenesis (Exs.
12-5, 35-68), Cancer Research (Ex. 35-119), Toxicology and Applied
Pharmacology (Exs. 35-66; 25-115), and Mutation Research (Exs. 35-132;
47-22-1; 35-327). Contrary to statements by CPMA, the results indicate
that lead chromate particulates are able to dissociate in the presence
of biological media without the aid of aggressive substances. The
resulting chromate ion is bioavailable to enter lung cells, damage
genetic material and initiate events critical to carcinogenesis. These
effects can not be attributed to small amounts of soluble chromate
contaminants since these substances are usually removed as part of the
test compound preparation prior to cell treatment.
    As one of the study authors, Dr. John Wise of the University of
Southern Maine, stated in his post-hearing comments:

    At no time did we dissolve lead chromate particles prior to
administration. At the initial onset of the administration of lead
chromate particles in our studies, the cells encountered intact lead
chromate particles. Any dissolution that occurred was the natural
result of the fate of lead chromate particles in a biological
environment (Ex. 47-12, p. 3).

Other scientists concurred that the methods and findings of the
cellular research with lead chromate were reasonable. Dr. Kathleen
MacMahon, a biologist from NIOSH stated:

    NIOSH believes that the methods that were used in the [lead
chromate] studies were credible and we support the results and
conclusions from those studies (Tr. 342).

Dr. Clewell said:

    As I recall, it [lead chromate particles] was suspended in
acetone and ultrasonically shaken to reduce it to submicron
particles, which seems like a reasonably good thing to do. There are
actually a couple of studies besides the Wise studies that have
looked at the question of the uptake of lead chromate. I have looked
at those studies and I don't really see any basic flaws in what they
did. It is obviously a challenge to reproduce inhalation exposure in
vitro (Tr. 180-181).

Chromosal Aberrations and Lead Chromate--Several submissions contained
testimony from another researcher, Dr. Earle Nestmann of CANTOX Health
Sciences International, that criticized the methodology and findings of
a study published by the research group at the University of Southern
Maine (Exs. 34-6-1; 38-205D; 47-12-1; 47-22). Dr. Nestmann viewed as
inappropriate the practice of combining the chromatid deletions and
achromatic lesions together as chromosomal aberrations. He indicated
the standard practice was to score these two types of lesions
separately and that only the deletions had biological relevance.
According to Dr. Nestmann, achromatic lesions are chromatid gaps (i.e.
lesion smaller than the width of one chromatid) that have no
clastogenic significance and serve to inflate the percentage of cells
with chromosomal aberrations (i.e. chromatid deletions or breaks). Dr.
Nestmann criticized the studies for not including a positive control
group that shows the experimental system responds to a `true'
clastogenic effect (i.e. a compound that clearly increases chromosomal
deletions without contribution from chromatid gaps).
    Dr. John Wise, the Director of the research laboratory at the
University of Southern Maine, responded that distinguishing chromatid
gaps from breaks is a subjective distinction (e.g. requiring judgment
as to the width of a lesion relative to the width of a chromatid) and
pooling these lesions simply reduces this potential bias (Ex. 47-12;
47-12-1). He stated that there is no consensus on whether gaps should
or should not be scored as a chromosomal aberration and that gaps have
been included as chromosomal aberrations in other publications. Dr.
Wise also points out that achromatic lesions have not been shown to
lack biological significance and that the most recent research
indicates that they may be related to DNA strand breaks, a
scientifically accepted genotoxic endpoint. Dr. Wise further believed
that a positive control was unnecessary in his experiments since the
purpose was not to determine whether lead chromate was a clastogenic
agent, which had already been established by other research. Rather,
the purpose of his studies was to assess Cr(VI) uptake and chromosomal
damage caused by water-insoluble lead chromate compared to that of
highly water soluble sodium chromate using a relevant in vitro cell
model (i.e. human lung cells).
    OSHA is not in a position to judge whether achromatic lesions
should be scored as a chromosomal aberration. However, OSHA agrees with
Dr. Nestmann that combining gaps and breaks together serves to increase
the experimental response rate in the studies. Given the lack of
consensus on the issue, it would have been of value to record these
endpoints separately. OSHA is not aware of data that show achromatic
gaps to be of no biological significance. The experimental data cited
above indicate that soluble and insoluble Cr(VI) compounds clearly
increase achromatic gaps in a concentration-dependent manner. The
chromatid lesions (gaps and breaks) may be chromosomal biomarkers
indicative of genetic damage that is critical to neoplastic
transformation. Furthermore, OSHA agrees with Dr. Wise that other
evidence establishes lead chromate as an agent able to cause DNA damage
and transform cells. The Agency considers the use of sodium chromate-
treated cells in the above set of experiments to be the appropriate
comparison group and does not find the absence of an additional
positive control group to be a technical deficiency of the studies.
OSHA considers the research conducted at the University of Southern
Maine documenting chromosomal damage in human lung cells following
treatment with lead chromate particulates to be consistent with results
from other studies (see Section V.B.8) and, thus, contributes to the
evidence that water insoluble lead chromate, like other chromates, is
able to enter lung cells and damage DNA.
    In post-hearing comments, CPMA provided a Canadian research
laboratory report that tested the lead chromate Pigment Yellow 34 for
chromosomal aberrations in a hamster embryo cell system (Ex. 47-3,
appendix C). The research was sponsored by DCC and its representative
Dr. Nestmann. Lead chromate particles over the concentration range of
0.1 [mu]/cm2 to 10 [mu]/cm2 were reported to not
induce chromosomal aberrations under the experimental test conditions.
Chromatid structural and terminal gaps were not scored as aberrations
in this study, even though the percentage of cells with these lesions
increased in a dose-dependent manner from two percent in the absence of
lead chromate to over thirteen percent in cells treated with 1 [mu]/
cm2 lead chromate pigment particles.
    This result is consistent with other experimental data that show
lead chromate particulates cause chromosomal lesions when administered
to mammalian embryo cells (Exs. 35-188; 35-132; 35-68; 35-67). The key
difference is how the various researchers interpreted the data. The
George Washington University group (i.e. Pateirno, Wise, Blankenship et
al.) considered the dose-dependent achromatic lesions (i.e. chromatid
gaps) as a clastogenic event and included them as chromosomal damage.
The Canadian test laboratory (i.e. Nucrotechnics) reported achromatic
lesions but did not score them as chromosomal aberrations. Reporting
achromatic lesions but not scoring them as chromosomal aberrations is
consistent with regulatory test guidelines as currently recommended by
EPA and OECD. The Nucrotechnics

data suggest that the tested lead chromate pigment caused a similar
degree of chromosomal damage (i.e. dose-dependent achromatic lesions
and chromosomal aberrations combined) in mammalian cells. This result
was similar to results produced by reagent grade lead chromate in
previous studies.
    Mutagenicity and Lead Chromate--CPMA also relied on a study that
reported a lack of mutagenicity for lead chromate pigments in a
bacterial assay using Salmonella Typhimurium TA 100 (Ex. 11-6). As
previously mentioned, this assay specifically measures point and
frameshift mutations usually caused by DNA adduct formation. The assay
is not sensitive to chromosomal damage, DNA strand breaks, or DNA
crosslinks most commonly found with low concentrations of Cr(VI)
compounds. Large amounts (50 to 500 [mu]g/plate) of highly soluble
sodium dichromate and slightly soluble calcium, strontium, and zinc
chromates, were found to be mutagenic in the study, but not the water
insoluble barium chromate and lead chromate pigments. However,
mutagenicity was observed when the acidic chelating agent,
nitrilotriacetic acid (NTA), was added to the assay to help solubilize
the water insoluble Cr(VI) compounds. The chelating agent was unable to
solubilize sufficient amounts of lead chromate pigments to cause
bacterial mutagenicity, if these pigments were more than five percent
encapsulated (weight to weight) with amorphous silica.
    OSHA finds the results of this study to be consistent with the
published literature that shows Cr(VI) mutagenicity requires high
concentrations of solubilized chromate ion (Exs. 35-118; 35-161). Large
amounts of water-soluble and slightly soluble Cr(VI) compounds produce
a mutagenic response in most studies since these Cr(VI) compounds can
dissociate to achieve a high concentration of chromate ion. Insoluble
lead chromate usually needs to be pre-solubilized under acidic or
alkaline conditions to achieve sufficient chromate ion to cause
mutagenicity (Ex. 35-162). The above study found highly and slightly
soluble chromates to be mutagenic as well as water insoluble lead
chromate pigments pre-solubilized with NTA. The lack of mutagenicity
for silica encapsulated lead chromate pigments under these experimental
conditions is likely the result of their greater resistance to acidic
digestion than unencapsulated lead chromate pigment.
    Failure to elicit a mutagenic response in a bacterial assay, with
or without NTA, is not a convincing demonstration that chromate ion can
not partially dissociate from encapsulated lead chromate in biological
media, enter mammalian cells, and elicit other types of genotoxicity.
As described above, chromosomal damage, believed to result from DNA
strand breaks and crosslinks, appears to be the critical genotoxic
endpoint for low concentrations of Cr(VI) compounds. Research has shown
that lead chromate and lead chromate pigment particulates in biological
media can cause chromosomal lesions and cell transformation without the
aid of strongly acidic or basic substances (Exs. 12-5; 35-119; 35-188;
35-132; 35-68; 35-67; 47-12-3; 35-327). While silica-encapsulated lead
chromate pigments have not been as thoroughly investigated as the
unencapsulated pigments or reagent grade lead chromate, one study
reported that lead silicochromate particles did have low solubility in
biological culture media and transformed hamster embryo cells (Ex. 12-
5).
    Information is not available in the record to adequately
demonstrate the efficiency and stability of the encapsulation process,
despite OSHA statements that such information would be of value in its
health effects evaluation and its request for such information (69 FR
59315-59316, 10/4/2004; Ex. 2A). In the absence of data to the
contrary, OSHA believes it prudent and plausible that encapsulated lead
chromate pigments are able to partially dissociate into chromate ion
available for lung cell uptake and/or be internalized in a manner
similar to other lead chromate particulates. The resulting
intracellular Cr(VI) leads to genotoxic damage and cellular events
critical to carcinogenesis.
    Public Comments on Carcinogenicity of Slightly Water Soluble Cr(VI)
Compounds--In its written comments to the NPRM, Boeing Corporation
stated that ``there is no persuasive scientific evidence for OSHA's
repeated assertion that low solubility hexavalent chromium compounds
[e.g. strontium and zinc chromates] are more potent carcinogens than
[highly] soluble [Cr(VI)] compounds'' (Ex. 38-106, p. 2). Boeing and
others in the aerospace industry are users of certain slightly soluble
Cr(VI) compounds, particularly strontium chromate, found in the
protective coatings applied to commercial and military aircraft.
    Boeing argues that OSHA, along with IARC, ACGIH and others, have
exclusively relied on intrabronchial implantation studies in animals
that are both not representative of inhalation exposures in the
workplace and are not consistent with the available animal inhalation
data (Ex. 38-106-2, p. 26). Boeing asserts that there is no evidence
that slightly soluble chromates behave differently in terms of their
absorption kinetics than highly soluble chromates when instilled in the
lungs of rats (Ex. 38-106-2, p. 19). Boeing believes the OSHA position
that slightly soluble Cr(VI) compounds are retained in the lung,
associate with cells, and cause high uptake or high local
concentrations to be inconsistent with other data showing these Cr(VI)
compounds quickly disperse in water (Ex. 38-106-2, p. 26). Boeing
concludes:

    There is no basis for the conclusion that low solubility [i.e.
slightly soluble] chromates could be more potent than [highly]
soluble, and some evidence the opposite may be the case. As a worst
case OSHA should conclude that there is inadequate evidence to
conclude that [highly] soluble and low-solubility compounds differ
in carcinogenic potency. It is critical that OSHA maintain a
distinction between low-solubility chromates and highly insoluble
chromates based on this data. (Ex. 38-106-2, p. 26)

    As noted earlier, OSHA as well as other commenters agree with
Boeing that the animal intrabronchial and intratracheal instillation
studies are not appropriate for quantitatively predicting lung cancer
risk to a worker breathing Cr(VI) dust and aerosols. However, many
stakeholders disagreed with the Boeing view and believed these animal
studies can be relied upon as qualitative evidence of relative
carcinogenic potency. CPMA, which relies on the rat intrabronchial
implantation results as evidence that lead chromate is non-
carcinogenic, states ``tests involving implantation in rat lung, as
carried out by Levy et al. in 1986, are relevant to inhalation in
industrial exposures'' (Ex. 38-205, p. 93). In their opening statement
NIOSH agreed with the preliminary OSHA determination that ``the less
water soluble [Cr(VI)] compounds may be more potent than the more water
soluble [Cr(VI)] compounds'' (Tr. 299). NIOSH identified the rat
intrabronchial implantation findings as the basis for their position
that the slightly soluble Cr(VI) compounds appear to be more
carcinogenic than the more soluble and insoluble Cr(VI) compounds (Tr.
334). Dr. Clewell testified that:

    Some animal studies suggest the solubility of hexavalent
chromium compounds influences their carcinogenic potency with
slightly soluble compounds having the higher potencies than highly
soluble or insoluble compounds. However, the evidence is inadequate
to conclude that specific hexavalent chromium compounds are not
carcinogenic. Moreover the designs of the studies were not
sufficient to quantitatively

estimate comparative potencies (Ex. 44-5, p. 15).

Respiratory Tract Instillation of Slightly Soluble Cr(VI) Compounds in
Rats--OSHA agrees that animal intrabronchial and intratracheal
implantation studies provide persuasive evidence that slightly soluble
Cr(VI) are more carcinogenic than the highly soluble Cr(VI) compounds.
As mentioned previously, these studies provide useful information
regarding the relative carcinogenicity of different Cr(VI) compounds
once they are delivered and deposited in the respiratory tract. For
example, one study examined the carcinogenicity of over twenty
different Cr(VI) compounds in rats, spanning a broad range of
solubilities, under the same experimental conditions in the relevant
target organ to humans (i.e. respiratory tract) following in vivo
administration (Ex. 11-2). A single administration of each Cr(VI) test
compound was instilled in the lower left bronchus of approximately 100
rats. The results were dramatic. Roughly 50 and 25 percent of the rats
receiving the slightly soluble strontium and calcium chromates,
respectively, developed bronchogenic carcinoma. No other Cr(VI)
compounds produced more than five percent tumor incidence. The highly
soluble sodium dichromate under the same experimental conditions caused
bronchogenic carcinoma in only a single rat.
    The higher relative potency of the slightly soluble calcium
chromate compared to the highly soluble sodium dichromate was confirmed
in another study in which each test compound was instilled at a low
dose level (i.e., 0.25 mg/kg) in the trachea of 80 rats five times
weekly for 30 months (Ex. 11-7). Using this experimental protocol, 7.5
percent of the slightly soluble calcium chromate-treated animals
developed brochioalveolar adenomas while none of the highly soluble
sodium dichromate-treated rats developed tumors. The tumor incidence at
this lower dose level occurred in the absence of serious lung pathology
and is believed to reflect the tumorigenic potential of the two Cr(VI)
compounds at workplace exposures of interest to OSHA. On the other
hand, a five-fold higher dose level that caused severe damage and
chronic inflammation to the rat lungs produced a similar fifteen
percent lung tumor incidence in both calcium and sodium chromate
treated rats. OSHA, as well as the study authors, believe the later
tumor response with the higher dose level did not result from direct
Cr(VI) interaction with cellular genes, but, instead, was primarily
driven by the cellular hyperplasia secondary to the considerable damage
to the lung tissue. Boeing also seems to attribute this result to
tissue damage stating ``most of the tumors were found in areas of
chronic inflammation and scarring, suggesting an effect that is
secondary to tissue damage'' (Ex. 38-106-2, p. 21).
    OSHA does not agree with some study interpretations advanced by
Boeing in support of their position that slightly soluble Cr(VI)
compounds are no more carcinogenic than highly soluble Cr(VI). For
example, Boeing claims that the intrabronchial implantation experiments
cannot be relied upon because the results do not correspond to findings
from animal inhalation studies (Ex. 38-106-2, p. 24-25). The primary
basis for the Boeing comparison were two rodent bioassays that reported
tumor incidence from the inhalation of different Cr(VI) compounds (Exs.
10-8; 10-11). In one study over 200 mice inhaled slightly soluble
calcium chromate powder for five hours per day, five days per week for
roughly two years (Ex. 10-8). In the other study, 19 rats inhaled an
aqueous sodium dichromate liquid aerosol virtually around the clock for
22 hours a day, seven days a week for eighteen months (Ex. 10-11). The
two studies reported a similar tumor incidence despite the lower total
weekly Cr(VI) dose of sodium dichromate in the second study. OSHA
believes the vastly different experimental protocols employed in these
studies do not allow for a legitimate comparison of carcinogenic
potency between Cr(VI) compounds. First, mouse and rat strains can
differ in their susceptibility to chemical-induced lung tumors. Second,
the proportion of respirable Cr(VI) may differ between a liquid aerosol
of aqueous sodium dichromate mist and an aerosol solid calcium chromate
particles suspended in air. Third, the opportunity for Cr(VI) clearance
will undoubtedly differ between a Cr(VI) dose inhaled nearly
continuously (e.g., 22 hours per day, seven days a week) and inhaled
intermittently (e.g., five hours a day, five days a week) over the
course of a week. These experimental variables can be expected to have
a major influence on tumor response and, thus, will obscure a true
comparison of carcinogenic potency. Boeing acknowledges that ``these
[inhalation] studies used very different protocols and are not directly
comparable'' (Ex. 38-106-2, p.24). On the other hand, slightly soluble
Cr(VI) compounds were found to cause a greater incidence of lung tumors
than highly soluble Cr(VI) compounds in two independent studies in
which the test compounds were instilled under the same dosing regime in
the same rodent models in research specifically designed to assess
relative Cr(VI) carcinogenic potency (Exs. 11-2; 11-7). Therefore, OSHA
believes any apparent lack of correspondence between animal inhalation
and instillation studies is due to an inability to compare inhalation
data from vastly different experimental protocols and should not
diminish the relevance of the instillation findings.
    Epidemiological Studies of Slightly Soluble Cr(VI) Compounds--
Boeing further argues that the greater carcinogenic potency experienced
by rats intrabronchially instilled with slightly soluble chromates
compared to rats instilled with highly soluble and water-insoluble
Cr(VI) compounds ``do not correspond qualitatively to observed lung
cancer in occupational exposure'' (Ex. 38-106-2, p. 21). Several other
industry stakeholders disagree. In explaining the excess lung cancer
mortality among pigment production workers, CPMA commented:

    [water-insoluble] Lead chromate pigments must be differentiated
from [slightly soluble] zinc chromate corrosion inhibitor additives,
which are consistently shown to be carcinogenic in various studies.
When [water insoluble] lead chromate and [slightly soluble] zinc
chromate exposures occur simultaneously, there appears to be a
significant cancer hazard. However, when lead chromate pigments
alone are the source of chromium exposure, a significant cancer
response has never been found (Ex. 38-205, p. 91).

In explaining the excess lung cancer mortality among chromate
production workers in the Gibb and Luippold cohorts, the Electric Power
Research Institute states that:

    One important distinction is that workers of the historical
chromate production industry were exposed to sparingly soluble forms
of calcium chromate in the roast mix, which are recognized to have
greater carcinogenic potential as compared to soluble forms of
Cr(VI) based on animal implantation studies (Ex. 38-8, p. 12).

Deborah Proctor of Exponent also testified:

    Several studies of chromate production worker cohorts have
demonstrated that the excess cancer risk is reduced when less lime
is added to the roast mixture, reducing worker exposure to the
sparingly soluble calcium chromate compounds'' (Ex. 40-12-5).

    OSHA believes there is merit to the above comments that workplace
exposure to slightly soluble Cr(VI) compounds may have contributed to
the higher lung cancer mortality in both pigments workers producing
mixed zinc and lead chromate pigments as well as

chromate production workers exposed to calcium chromate from high lime
production processes in the 1930s and 1940s. Other factors, such as
greater Cr(VI) exposure, probably also contributed to the higher lung
cancer mortality observed in these cohorts. In any case, these
epidemiological findings support the Boeing contention that the
epidemiological findings are inconsistent with the results from animal
intrabronchial implantation studies (Ex. 38-106-2, p. 26).
    Clearance, Retention, and Dissolution of Slightly Soluble Cr(VI)
Compounds in the Lung--Boeing argues that animal experiments that
examined the absorption, distribution and excretion of Cr(VI) compounds
after intratracheal instillation of Cr(VI) compounds in rats do not
show that highly soluble Cr(VI) is cleared more rapidly or retained in
the lung for shorter periods than slightly soluble Cr(VI) compounds
(Ex. 38-106-2, p. 18-19). The results of one study found that larger
amounts of water-insoluble lead chromate were retained in the lungs of
rats at both 30 minutes and at 50 days after instillation than for
highly soluble sodium chromate or slightly soluble zinc chromate (Ex.
35-56). Although the authors concluded that slightly soluble zinc
chromate was more slowly absorbed from the lung than the highly soluble
sodium chromate, the excretion and distribution of the absorbed
chromium from the zinc and sodium chromate instillations was similar.
Furthermore, there was little difference in the amounts of zinc and
sodium chromate retained by the lung at the two extreme time points
(e.g., 30 minutes and 50 days) measured in the study. OSHA agrees with
Boeing that these findings indicate slower clearance and longer
retention in the lung of the water insoluble lead chromate relative to
highly soluble sodium chromate, but not in the case of the slightly
soluble zinc chromate. Slower clearance and longer residence time in
the lung will generally enhance carcinogenic potential assuming other
dosimetric variables such as lung deposition, Cr(VI) concentration at
the lung cell surface, and dissociation into chromate ion are
unchanged.
    Boeing asserts that a study of strontium chromate dissociation from
paint primer contradicts the notion that slightly soluble are more
likely than highly soluble Cr(VI) compounds to concentrate and
dissociate at the lung cell surface (Ex. 38-106-2, p. 25). This
experimental research found that roughly 75 and 85 percent of strontium
chromate contained in metal surface primer coating particles was
solubilized in water after one and 24 hours, respectively (Ex. 31-2-1).
The primer particles were generated using a high volume, low pressure
spray gun according to manufacturer specifications, and collected in
water impingers. The authors concluded that their study demonstrated
that chromate dissociation from primer particles into the aqueous fluid
lining lung cells would be modestly hindered relative to highly water
soluble Cr(VI) aerosols.
    The slower dissociation of the slightly soluble Cr(VI) compound,
strontium chromate, plausibly explains its higher carcinogenicity in
animal implantation studies. The `modest hindrance' allows the
undissociated chromate to achieve higher concentrations at the surface
of the lung cells facilitating chromate transport into the cell. The
unhindered, instantaneous dispersion of highly water soluble chromates
in aqueous fluid lining of the respiratory tract is less likely to
achieve a high chromate concentration at the lung cell membrane. OSHA
believes the results of the above study support, not contradict, that
slightly soluble Cr(VI) may lead to higher chromium uptake into lung
cells than highly soluble Cr(VI) compounds.
    In summary, slightly soluble Cr(VI) compounds have consistently
caused higher lung tumor incidence in animal instillation studies
specifically designed to examine comparative carcinogenic potency in
the respiratory tract. The higher carcinogenic activity of slightly
soluble Cr(VI) is consistent with cellular studies that indicate that
chromate dissociation in close proximity to the lung cell surface may
be a critical feature to efficient chromate ion uptake. This is
probably best achieved by Cr(VI) compounds that have intermediate water
solubility rather than by highly water-soluble Cr(VI) that rapidly
dissolves and diffuses in the aqueous fluid layers lining the
respiratory tract. The higher carcinogenicity of slightly soluble
Cr(VI) may contribute, along with elevated Cr(VI) workplace exposures,
to the greater lung cancer mortality in certain occupational cohorts
exposed to both slightly soluble and other forms of Cr(VI). The vastly
different study protocols employed in the few animal inhalation
bioassays do not allow a valid comparison of lung tumor incidence
between slightly soluble and highly soluble Cr(VI) compounds.
b. Summary of Cr(VI) Carcinogenicity
    After carefully considering all the epidemiological, animal and
mechanistic evidence presented in the rulemaking record, OSHA regards
all Cr(VI) compounds as agents able to induce carcinogenesis through a
genotoxic mode of action. This position is consistent with findings of
IARC, EPA, and ACGIH that classified Cr(VI) compounds as known or
confirmed human carcinogens. Based on the above animal and experimental
evidence, OSHA believes that slightly soluble Cr(VI) compounds are
likely to exhibit a greater degree of carcinogenicity than highly water
soluble or water insoluble Cr(VI) when the same dose is delivered to
critical target cells in the respiratory tract of the exposed worker.
In its evaluation of different Cr(VI) compounds, ACGIH recommended
lower occupational exposure limits for the slightly soluble strontium
chromate (TLV of 0.5 [mu]g/m\3\) and calcium chromate (TLV of 1 [mu]g/
m\3\) than either water insoluble (TLV of 10 [mu]g/m\3\) or water
soluble (TLV of 50 [mu]g/m\3\) forms of Cr(VI) based on the animal
instillation studies cited above. While these animal instillation
studies are useful for hazard identification and qualitative
determinations of relative potency, they cannot be used to determine a
reliable quantitative estimate of risk for human workers breathing
these chromates during occupational exposure. This was due to use of
inadequate number of dose levels (e.g., single dose level) or a less
appropriate route of administration (e.g., tracheal instillation).
    It is not clear from the animal or cellular studies whether the
carcinogenic potency of water insoluble Cr(VI) compounds would be
expected to be more or less than highly water soluble Cr(VI). However,
it was found that a greater percentage of water insoluble lead chromate
remains in the lungs of rats for longer periods than the highly water
soluble sodium chromate when instilled intratracheally at similar doses
(Ex. 35-56). Since water insoluble lead chromate can persist for long
periods in the lung and increase intracellular levels of Cr and damage
DNA in human lung cells at low doses (e.g., 0.1 [mu]g/cm\2\), OSHA
believes that based on the scientific evidence discussed above it is
reasonable to regard the water insoluble Cr(VI) to be of similar
carcinogenic potency to highly soluble Cr(VI) compounds. No convincing
scientific evidence was introduced into the record that shows lead
chromate to be less carcinogenic than highly soluble chromate
compounds.

C. Non-cancer Respiratory Effects

    The following sections describe the evidence from the literature on
nasal irritation, nasal ulcerations, nasal perforations, asthma, and
bronchitis following inhalation exposure to water

soluble Cr(VI) compounds. The evidence clearly demonstrates that
workers can develop impairment to the respiratory system (nasal
irritation, nasal ulceration, nasal perforation, and asthma) after
workplace exposure to Cr(VI) compounds below the previous PEL.
    It is very clear from the evidence that workers may develop nasal
irritation, nasal tissue ulcerations, and nasal septum perforations at
occupational exposures level at or below the current PEL of 52 [mu]g/
m\3\. However, it is not clear what occupational exposure levels lead
to the development of occupational asthma or bronchitis.
1. Nasal Irritation, Nasal Tissue Ulcerations and Nasal Septum
Perforations
    Occupational exposure to Cr(VI) can lead to nasal tissue
ulcerations and nasal septum perforations. The nasal septum separates
the nostrils and is composed of a thin strip of cartilage. The nostril
tissue consists of an overlying mucous membrane known as the mucosa.
The initial lesion after Cr(VI) exposure is characterized by localized
inflammation or a reddening of the affected mucosa, which can later
lead to atrophy. This may progress to an ulceration of the mucosa layer
upon continued exposure (Ex. 35-1; Ex. 7-3). If exposure is
discontinued, the ulcer progression will stop and a scar may form. If
the tissue damage is sufficiently severe, it can result in a
perforation of the nasal septum, sometimes referred to chrome hole.
Individuals with nasal perforations may experience a range of signs and
symptoms, such as a whistling sound, bleeding, nasal discharge, and
infection. Some individuals may experience no noticeable effects.
    Several cohort and cross-sectional studies have described nasal
lesions from airborne exposure to Cr(VI) at various electroplating and
chrome production facilities. Most of these studies have been reviewed
by the Center for Disease Control's Agency for Toxic Substances and
Disease Registry (ATSDR) toxicological profile for chromium (Ex. 35-
41). OSHA reviewed the studies summarized in the profile, conducted its
own literature search, and evaluated studies and comments submitted to
the rulemaking record. In its evaluation, OSHA took into consideration
the exposure regimen and experimental conditions under which the
studies were performed, including exposure levels, duration of
exposure, number of animals, and the inclusion of appropriate control
groups. Studies were not included if they did not contribute to the
weight of evidence either because of inadequate documentation or
because of poor quality. This section only covers some of the key
studies and reviews. OSHA has also identified two case reports
demonstrating the development of nasal irritation and nasal septum
perforations, and these case reports are summarized as well. One case
report shows how a worker can develop the nasal perforations from
direct contact (i.e., touching the inner surface of the nose with
contaminated fingers).
    Lindberg and Hedenstierna examined the respiratory symptoms and
effects of 104 Swedish electroplaters (Ex. 9-126). Of the 104
electroplaters, 43 were exposed to chromic acid by inhalation. The
remaining 61 were exposed to a mixture of chromic acid and nitric acid,
hydrochloric acid, boric acid, nickel, and copper salts. The workers
were evaluated for respiratory symptoms, alterations in the condition
of the nasal tissue, and lung function. All workers were asked to fill
out a detailed questionnaire on their history of respiratory symptoms
and function. Physicians performed inspections of the nasal passages of
each worker. Workers were given a pulmonary function test to assess
lung function. For those 43 workers exposed exclusively to chromic
acid, the median exposure time was 2.5 years, ranging from 0.2 to 23.6
years. The workers were divided into two groups, a low exposure group
(19 workers exposed to eight-hour time weighted average levels below 2
[mu]g/m\3\) and a high exposure group (24 workers exposed to eight-hour
time weighted average levels above 2 [mu]g/m\3\). Personal air sampling
was conducted on 11 workers for an entire week at stations close to the
chrome baths to evaluate peak exposures and variations in exposure on
different days over the week. Nineteen office employees who were not
exposed to Cr(VI) were used as controls for nose and throat symptoms,
and 119 auto mechanics (no car painters or welders) whose lung function
had been evaluated using similar techniques to those used on Cr(VI)
exposed workers were used as controls for lung function.
    The investigators reported nasal tissue ulcerations and septum
perforations in a group of workers exposed to chromic acid as Cr(VI) at
peak exposure ranging from 20 [mu]g/m\3\ to 46 [mu]g/m\3\. The
prevalence of ulceration/perforation was statistically higher than the
control group. Of the 14 individuals in the 20-46 [mu]g/m\3\ exposure
group, 7 developed nasal ulcerations. In addition to nasal ulcerations,
2 of the 7 also had nasal perforations. Three additional individuals in
this group developed nasal perforations in the absence of ulcerations.
None of the 14 workers in the 20-46 [mu]g/m\3\ exposure group were
reported to have nasal tissue atrophy in the absence of the more
serious ulceration or perforation.
    At average exposure levels from 2 [mu]g/m\3\ to 20 [mu]g/m\3\, half
of the workers complained of ``constantly running nose,'' ``stuffy
nose,'' or ``there was a lot to blow out.'' (Authors do not provide
details of each complaint). Nasal tissue atrophy, in the absence of
ulcerations or perforations, was observed in 66 percent of
occupationally exposed workers (8 of 12 subjects) at relatively low
peak levels ranging from 2.5 [mu]g/m\3\ to 11 [mu]g/m\3\. No one
exposed to levels below 1 [mu]g/m\3\ (time-weighted average, TWA)
complained of respiratory symptoms or developed lesions.
    The authors also reported that in the exposed workers, both forced
vital capacity and forced expiratory volume in one second were reduced
by 0.2 L, when compared to controls. The forced mid-expiratory flow
diminished by 0.4 L/second from Monday morning to Thursday afternoon in
workers exposed to chromic acid as Cr(VI) at daily TWA average levels
of 2 [mu]g/m\3\ or higher. The effects were small, not outside the
normal range and transient. Workers recovered from the effects after
two days. There was no difference between the control and exposed group
after the weekend. The workers exposed to lower levels (2 [mu]g/m\3\ or
lower, TWA) showed no significant changes.
    Kuo et al. evaluated nasal septum ulcerations and perforations in
189 electroplaters in 11 electroplating factories (three factories used
chromic acid, six factories used nickel-chromium, and two factories
used zinc) in Taiwan (Ex. 35-10). Of the 189 workers, 26 used Cr(VI),
129 used nickel-chromium, and 34 used zinc. The control group consisted
of electroplaters who used nickel and zinc. All workers were asked to
fill out a questionnaire and were given a nasal examination including a
lung function test by a certified otolaryngologist. The authors
determined that 30% of the workers (8/26) that used chromic acid
developed nasal septum perforations and ulcerations and 38% (10/26)
developed nasal septum ulcers. Using the Mantel Extension Test for
Trends, the authors also found that chromium electroplaters had an
increased likelihood of developing nasal ulcers and perforations
compared to electroplating workers using nickel-chromium and zinc.
Personal sampling of airborne Cr(VI) results indicated the highest
levels (32 [mu]g/m\3\  35 [mu]g/m\3\, ranging from 0.1
[mu]g/m\3\-119 [mu]g/m\3\) near the electroplating tanks of the Cr(VI)
electroplating

factories (Ex. 35-11). Much lower personal sampling levels were
reported in the ``other areas in the manufacturing area'' and in the
``administrative area'' (TWA 0.16  0.10 [mu]g/m\3\) of the
Cr(VI) electroplating plant. The duration of sampling was not
indicated. The lung function tests showed that Cr(VI) electroplaters
had significantly lower forced vital capacity and forced expiratory
volume when compared to other exposure groups.
    Cohen et al. examined respiratory symptoms of 37 electroplaters
following inhalation exposure to chromic acid (Ex. 9-18). The mean
length of employment for the 37 electroplaters was 26.9 months (range
from 0.3 to 132 months). Fifteen workers employed in other parts of the
plant were randomly chosen for the control group (mean length of
employment was 26.1 months; range from 0.1 to 96). All workers were
asked to fill out a questionnaire on their respiratory history and to
provide details about their symptoms. An otolaryngologist then examined
each individual's nasal passages and identified ulcerations and
perforations. Air samples to measure Cr(VI) were collected for
electroplaters. The air sampling results of chromic acid as Cr(VI)
concentrations for electroplaters was a mean of 2.9 [mu]g/m\3\ (range
from non-detectable to 9.1 [mu]g/m\3\). The authors found that 95% of
the electroplaters developed pathologic changes in nasal mucosa.
Thirty-five of the 37 workers who were employed for more than 1 year
had nasal tissue damage. None of these workers reported any previous
job experience involving Cr(VI) exposure. Four workers developed nasal
perforations, 12 workers developed ulcerations and crusting of the
septal mucosa, 11 workers developed discoloration of the septal mucosa,
and eight workers developed shallow erosion of septal mucosa. The
control group consisted of 15 workers who were not exposed to Cr(VI) at
the plant. All but one had normal nasal mucosa. The one individual with
an abnormal finding was discovered to have had a previous Cr(VI)
exposure while working in a garment manufacturing operation as a fabric
dyer for three years. In addition to airborne exposure, the authors
observed employees frequently wiping their faces and picking their
noses with contaminated hands and fingers. Many did not wear any
protective gear, such as gloves, glasses, or coveralls.
    Lucas and Kramkowsi conducted a Health Hazard Evaluation (HHE) on
11 chrome platers in an industrial electroplating facility (Ex. 3-84).
The electroplaters worked for about 7.5 years on average. Physicians
evaluated each worker for chrome hole scars, nasal septum ulceration,
mucosa infection, nasal redness, perforated nasal septum, and wheezing.
Seventeen air samples for Cr(VI) exposure were collected in the chrome
area. Cr(VI) air concentrations ranged from 1 to 20 [mu]g/m\3\, with an
average of 4 [mu]g/m\3\. In addition to airborne exposure, the authors
observed workers being exposed to Cr(VI) by direct ``hand to nose''
contact, such as touching the nose with contaminated hands. Five
workers had nasal mucosa that became infected, two workers had nasal
septum ulcerations, two workers had atrophic scarring (author did not
provide explanation), possibly indicative of presence of past
ulcerations, and four workers had nasal septum perforations.
    Gomes evaluated 303 employees from 81 electroplating operations in
Sao Paulo, Brazil (Ex. 9-31). Results showed that more than two-thirds
of the workers had nasal septum ulcerations and perforations following
exposure to chromic acid at levels greater than 100 [mu]g/m\3\, but
less than 600 [mu]g/m\3\ (precise duration of exposure was not stated).
These effects were observed within one year of employment.
    Lin et al. examined nasal septum perforations and ulcerations in 79
electroplating workers from seven different chromium electroplating
factories in Taipei, Taiwan (Ex.35-13). Results showed six cases of
nasal septum perforations, four having scar formations, and 38 cases of
nasal septum ulcerations following inhalation exposure to chromic acid.
Air sampling near the electroplating tanks had the highest range of
chromic acid as Cr(VI) (mean of 28 [mu]g/m\3\; range from 0.7 to 168.3
[mu]g/m\3\). In addition to airborne exposures, the authors also
observed direct ``hand to nose'' contact where workers placed
contaminated fingers in their nose. The authors attributed the high
number of cases to poor industrial hygiene practices in the facilities.
Five of the seven factories did not have adequate ventilation systems
in place. Workers did not wear any PPE, including respirators.
    Bloomfield and Blum evaluated nasal tissue damage and nasal septum
perforations in 23 workers employed at six chromium electroplating
plants (Ex. 9-13). They found that daily exposure to chromic acid as
Cr(VI) at levels of 52 [mu]g/m\3\ or higher can lead to nasal tissue
damage. Three workers developed nasal ulcerations, two workers had
nasal perforations, nine workers had nose bleeds, and nine workers had
inflamed mucosa.
    Kleinfeld and Rosso found that seven out of nine of chrome
electroplaters had nasal septum ulcerations (Ex. 9-41). The nine
workers were exposed to chromic acid as Cr(VI) by inhalation at levels
ranging from 93 [mu]g/m\3\ to 728 [mu]g/m\3\. Duration of exposure
varied from two weeks to one year. Nasal septum ulcerations were noted
in some workers who had been employed for only one month.
    Royle, using questionnaire responses from 997 British
electroplaters exposed to chromic acid, reported a significant increase
in the prevalence of nasal ulcerations. The prevalence increased the
longer the worker was exposed to chromic acid (e.g., from 14 cases with
exposure less than one year to 62 cases with exposure over five years)
(Ex. 7-50). In all but 2 cases, air samples revealed chromic acid
concentrations of 0.03 mg/m\3\ (i.e., 30 [mu]g/m\3\).
    Gibb et al. reported nasal irritations, nasal septum bleeding,
nasal septum ulcerations and perforations among a cohort of 2,350
chrome production workers in a Baltimore plant (Ex. 31-22-12). A
description of the cohort is provided in detail in the cancer health
effects section V.B. of this preamble. The authors found that more than
60% of the cohort had experienced nasal ulcerations and irritations,
and that the workers developed these effects for the first time within
the first three months of being hired (median). Gibb et al. found that
the median annual exposure to Cr(VI) during first diagnosis of
irritated and/or ulcerated nasal septum was 10 [mu]g/m\3\. About 17% of
the cohort reported nasal perforations. Based on historical data, the
authors believe that the nasal findings are attributable to Cr(VI)
exposure.
    Gibb et al. also used a Proportional Hazard Model to evaluate the
relationship between Cr(VI) exposure and the first occurrence of each
of the clinical findings. Cr(VI) data was entered into the model as a
time dependent variable. Other explanatory variables were calendar year
of hire and age of hire. Results of the model indicated that airborne
Cr(VI) exposure was associated with the occurrence of nasal septum
ulceration (p = 0.0001). The lack of an association between airborne
Cr(VI) exposure and nasal perforation and bleeding nasal septum may
reflect the fact that Cr(VI) concentrations used in the model represent
annual averages for the job, in which the worker was involved in at the
time of the findings, rather than a short-term average. Annual averages
do not factor in day-to-day fluctuations or extreme episodic
occurrences. Also, the author believed that poor housekeeping

and hygiene practices may have contributed to these health effects as
well as Cr(VI) air borne concentrations.
    Based on their hazard model, Gibb et al. estimated the relative
risks for nasal septum ulcerations would increase 1.2 for each 52 [mu]g
of Cr(VI)/m\3\ increase in Cr(VI) air levels. They found a reduction in
the incidence of nasal findings in the later years. They found workers
from the earlier years who did not wear any PPE had a greater risk of
developing respiratory problems. They believe that the reduction in
ulcerations was possibly due to an increased use of respirators and
protective clothing and improved industrial hygiene practices at the
facility.
    The U.S. Public Health Service conducted a study of 897 chrome
production workers in seven chromate producing plants in the early
1950s (Ex. 7-3). The findings of this study were used in part as
justification for the current OSHA PEL. Workers were exposed by
inhalation to various water soluble chromates and bichromate compounds.
The total mean exposure to the workers was a TWA of 68 [mu]g/m\3\. Of
the 897 workers, 57% (or 509 workers) were found to have nasal septum
perforations. Nasal septum perforations were even observed in workers
during their first year on the job.
    Case reports provide further evidence that airborne exposure and
direct ``hand to nose'' contact of Cr(VI) compounds lead to the
development of nasal irritation and nasal septum perforations.
    For example, a 70-year-old man developed nasal irritation,
incrustation, and perforation after continuous daily exposure by
inhalation to chromium trioxide (doses were not specified, but most
likely quite high given the nature of his duties). This individual
inhaled chromium trioxide daily by placing his face directly over an
electroplating vessel. He worked in this capacity from 1934 to 1982.
His symptoms continued to worsen after he stopped working. By 1991, he
developed large perforations of the nasal septum and stenosis (or
constriction) of both nostrils by incrustation (Ex. 35-8).
    Similarly, a 30-year-old female jigger (a worker who prepares the
items prior to electroplating by attaching the items to be plated onto
jigs or frames) developed nasal perforation in her septum following
continuous exposure (doses in this case were not provided) to chromic
acid mists. She worked adjacent to the automated Cr(VI) electroplating
shop. She was also exposed to chromic acid from direct contact when she
placed her contaminated fingers in her nose. Her hands became
contaminated by handling wet components in the jigging and de-jigging
processes (Ex. 35-24).
    Evidence of nasal septum perforations has also been demonstrated in
experimental animals. Adachi exposed 23 C57BL mice to chromic acid by
inhalation at concentrations of 1.81 mg Cr(VI)/m\3\ for 120 min per
day, twice a week and 3.63 mg Cr(VI)/m\3\ for 30 minutes per day, two
days per week for up to 12 months (Ex. 35-26). Three of the 23 mice
developed nasal septum perforations in the 12 month exposure group.
    Adachi et al. also exposed 50 ICR female mice to chromic acid by
inhalation at concentrations of 3.18 mg Cr(VI)/m\3\ for 30 minutes per
day, two days per week for 18 months (Ex. 35-26-1). The authors used a
miniaturized chromium electroplating system to mimic electroplating
processes and exposures similar to working experience. Nasal septum
perforations were found in six mice that were sacrificed after 10
months of exposure. Of those mice that were sacrificed after 18 months
of exposure, nasal septum perforations were found in three mice.
2. Occupational Asthma
    Occupational asthma is considered ``a disease characterized by
variable airflow limitation and/or airway hyperresponsiveness due to
causes and conditions attributable to a particular occupational
environment and not to stimuli encountered outside the workplace'' (Ex.
35-15). Asthma is a serious illness that can damage the lungs and in
some cases be life threatening. The common symptoms associated with
asthma include heavy coughing while exercising or when resting after
exercising, shortness of breath, wheezing sound, and tightness of chest
(Exs. 35-3; 35-6).
    Cr(VI) is considered to be an airway sensitizer. Airway sensitizers
cause asthma through an immune response. The sensitizing agent
initially causes production of specific antibodies that attach to cells
in the airways. Subsequent exposure to the sensitizing agent, such as
Cr(VI), can trigger an immune-mediated narrowing of the airways and
onset of bronchial inflammation. All exposed workers do not become
sensitized to Cr(VI) and the asthma only occurs in sensitized
individuals. It is not clear what occupational exposure levels of
Cr(VI) compounds lead to airway sensitization or the development of
occupational asthma.
    The strongest evidence of occupational asthma has been demonstrated
in four case reports. OSHA chose to focus on these four case reports
because the data from other occupational studies do not exclusively
implicate Cr(VI). The four case reports have the following in common:
(1) The worker has a history of occupational exposure exclusively to
Cr(VI); (2) a physician has confirmed a diagnosis that the worker has
symptoms consistent with occupational asthma; and (3) the worker
exhibits functional signs of air restriction (e.g., low forced
expiratory volume in one second or low peak expiratory flow rate) upon
bronchial challenge with Cr(VI) compounds. These case reports
demonstrate, through challenge tests, that exposure to Cr(VI) compounds
can cause asthmatic responses. The other general case reports below did
not use challenge tests to confirm that Cr(VI) was responsible for the
asthma; however, these reports came from workers similarly exposed to
Cr(VI) such that Cr(VI) is likely to have been a contributing factor in
the development of their asthmatic symptoms.
    DaReave reported the case of a 48-year-old cement floorer who
developed asthma from inhaling airborne Cr(VI) (Ex. 35-7). This worker
had been exposed to Cr(VI) as a result of performing cement flooring
activities for more than 20 years. The worker complained of dyspnea,
shortness of breath, and wheezing after work, especially after working
in enclosed spaces. The Cr(VI) content in the cement was about 12 ppm.
A bronchial challenge test with potassium dichromate produced a 50%
decrease in forced expiratory volume in one second. The occupational
physician concluded that the worker's asthmatic condition, triggered by
exposure to Cr(VI) caused the worker to develop bronchial constriction.
    LeRoyer reported a case of a 28-year-old roofer who developed
asthma from breathing dust while sawing material made of corrugated
fiber cement containing Cr(VI) for nine years (Ex. 35-12). This worker
demonstrated symptoms such as wheezing, shortness of breath, coughing,
rhinitis, and headaches while working. Skin prick tests were all
negative. Several inhalation challenges were performed by physicians
and immediate asthmatic reactions were observed after nebulization of
potassium dichromate. A reduction (by 20%) in the forced expiratory
volume in one second after exposure to fiber cement dust was noted.
    Novey et al. reported a case of a 32-year-old electroplating worker
who developed asthma from working with chromium sulfate and nickel
salts (Ex. 35-16). He began experiencing coughs,

wheezing, and dyspnea within the first week of exposure. Separate
inhalation challenge tests given by physicians using chromium sulfate
and nickel salts resulted in positive reactions. The worker immediately
had difficulty breathing and started wheezing. The challenges caused
the forced expiratory volume in 1 second to decrease by 22% and the
forced expiratory volume in 1 second/forced vital capacity ratio to
decrease from 74.5% to 60.4%. The author believes the worker's
bronchial asthma was induced from inhaling chromium sulfate and nickel
salts. Similar findings were reported in a different individual by
Sastre (Ex.35-20).
    Shirakawa and Morimoto reported a case of a 50-year-old worker who
developed asthma while working at a metal-electroplating plant (Ex. 35-
21). Bronchial challenge by physicians produced positive results when
using potassium bichromate, followed by a rapid recovery within 5
minutes, when given no exposures. The worker's forced expiratory volume
in one second dropped by 37% after inhalation of potassium bichromate.