52, 278 –288 (1999)
Copyright © 1999 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Thirteen-Week Subchronic Rat Inhalation Toxicity Study with a
Recovery Phase of Trivalent Chromium Compounds, Chromic Oxide,
and Basic Chromium Sulfate
Michael J. Derelanko,* William E. Rinehart,† ,1 Roger J. Hilaski,‡ Roger B. Thompson,‡ and Eckhard Löser§
*AlliedSignal, Inc., Morristown, New Jersey; †Industrial Health Foundation, Pittsburgh, Pennsylvania; ‡MPI Research, Mattawan, Michigan;
and §Bayer AG, Wuppertal, Germany
Received 22 January 1999; accepted 24 July 1999
The toxicity of trivalent chromium compounds, chromic oxide and basic chromium sulfate, was investigated in rats in a
13-week nose-only inhalation study that included a 13-week
recovery period. Nose-only exposures to insoluble chromic oxide dust at 4.4, 15, or 44 mg/m 3 or soluble basic chromium
sulfate dust at 17, 54, or 168 mg/ m 3 (trivalent chromium
equivalent concentrations of 3, 10, and 30 mg/m 3 ) were carried
out for 6 h/day, 5 days/week. No compound-related mortality
occurred. General toxic effects, only observed with high-exposure levels of basic chromium sulfate, included sporadic signs of
labored breathing and depressed body weights. No apparent
compound-related effects were noted for sperm motility or
morphology, for any concentration of either test material. Bronchoalveolar lavage fluid evaluations showed test material in
mononuclear cells with chromic oxide and increased neutrophils, protein, lactic dehydrogenase and cellular debris with
basic chromium sulfate. The principle effects for both materials
were primarily to the respiratory tract. Chromic oxide caused
pathological changes in the bronchial and mediastinal lymphatic tissue and lungs, consisting of the presence of pigmentladen macrophages, lymphoid and septal hyperplasia, and interstitial inflammation similar to that observed with other inert
dusts. Basic chromium sulfate produced more severe and widespread effects in the nasal cavity, larynx, lungs, and mediastinal lymph node. Effects were characterized by accumulation of
foreign material, infiltration of alveolar macrophages, septal
cell hyperplasia, and granulomatous and chronic inflammation.
Pigment was still present in chromic oxide and, to a lesser
extent, in basic chromium sulfate-treated animals after the
13-week recovery period, with partial recovery of the pathological lesions. A NOAEL was not established for either test material, but 4.4 mg/m 3 was thought to be near the NOAEL level
for subchronic exposure to chromic oxide. The results of this
study indicate significant differences in toxicity to the respiratory tract between trivalent chromium compounds chromic
oxide and basic chromium sulfate. These are likely related to
differences in acidity and water solubility, rather than chromium concentration per se. This conclusion is substantiated by
the lack of effect on other internal organs.
1
To whom correspondence should be addressed at Industrial Health Foundation, 34 Penn Circle West, Pittsburgh, PA 15206-3612. Fax: (412) 363-6605.
Key Words: trivalent chromium; subchronic inhalation toxicity;
chromic oxide; basic chromium sulfate; respiratory toxicity.
Trivalent chromium is found in nature as the mineral chromite, which has direct uses as a refractory in industrial furnaces
and for producing alloys and specific chemicals. The chemicals, in turn, find uses in metal finishing, pigment manufacture,
and leather tanning. In its trivalent form, chromium is an
essential nutrient for animals and humans. It plays an essential
role in the metabolism of glucose, fat, and protein by potentiating the action of insulin (Anderson et al. 1981). Occupational
exposures to trivalent chromium can occur from the aforementioned industries as well as to painters, dye makers, printers,
rubber makers, and cement workers. Sources of trivalent chromium as a nutrient include drinking water and food. The
recommended daily intake for adults for trivalent chromium is
between 50 and 200 mg/kg/day (National Academy of Sciences, 1989).
Data available on the toxicity of trivalent chromium compounds via the oral route suggest that these materials are much
less toxic than are the hexavalent compounds, and that the
toxicity varies with water solubility (Akatsuka and Fairhall,
1934; Ivankovic and Preussman, 1975; Mackenzie et al., 1958;
Schroeder et al., 1965). These studies suggest that insoluble
compounds are not absorbed systemically to any significant
degree, as compared to soluble compounds.
Investigations on the toxicological properties of chromium
compounds by the inhalation route have largely centered on
hexavalent chromium. Studies on trivalent chromium by the
inhalation route are limited both in number and scope (Akatsuka and Fairhall, 1934; Henderson et al. 1979; Johansson et
al, 1986), but suggest that the toxicity of trivalent chromium
compounds is limited to the lungs, producing some biochemical and functional changes but little or no morphological
alterations. Glaser et al. (1986, 1988) reported that exposure of
rats for 22 h/day, 7 days/week for 18 months to a mixture of
CrO 3 (a hexavalent compound) and Cr 2O 3 at 0.1 mg Cr/m 3
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TRIVALENT CHROMIUM SUBCHRONIC TOXICITY
produced interstitial fibrosis of the lungs. It is unclear to what
extent, if any, this effect was related to the trivalent compound.
Due to the limited animal data on repeated inhalation exposure to trivalent chromium, it was decided to investigate toxicity and dose-response relationships in animals in more detail,
using 2 compounds of commercial importance, i.e., insoluble
chromic oxide (Cr 2O 3) and soluble basic chromium sulfate
(Cr 2 (OH) X (SO 4) y NaSO 4 2H 20). The results of these studies
are presented in this paper.
MATERIALS AND METHODS
Test material. Chromic oxide (CAS No. 1308 –38 –9) and basic chromium
sulfate (CAS No. 12336 –95–7), both dark green powders, were supplied by
British Chrome Chemicals (Urlay Nook, Eaglescliffe, Cleveland, U.K.). Prior
to the study, test article purity was determined to be greater than 99% Cr(III)
as Cr 2O 3 and less than 0.0001% Cr(VI) (non-detectable) for chromic oxide,
and 25% Cr(III) as Cr 2O 3 and less than 0.0003% Cr(VI)) (non-detectable) for
basic chromium sulfate. The test articles were stored in separate, unused 1-m 3
chambers that were continuously purged with a low flow of dry compressed
air. The test articles were selected as representative of insoluble and relatively
soluble trivalent chromium compounds.
Test animals and housing conditions. Male and female CDFt (Fischer
344)/Crl BR VAF/Plust rats, approximately 5 weeks of age, were obtained
from Charles River Laboratories (Raleigh, NC). Following 3 days of group
housing, animals were individually housed in stainless steel, suspended wiremesh cages and given free access to commercial laboratory feed (Purina
Certified Rodent Chow No. 5002) and tap water during the non-exposure
periods. Animal rooms were maintained on a 12-h light/dark cycle; temperature range was maintained at 21 6 2°C, and the relative humidity range was
43 6 11%.
Inhalation exposure system. Rats were exposed in stainless steel and
acrylic nose-only inhalation chambers operated with at least 12 chamber air
changes per h. Generation of chromic oxide particles was accomplished with
a modified low-output dust generator, using spinning glass beads over a packed
cake of test material. However, basic chromium sulfate particles were generated using an auger dust feeder and an air micronizer. Chamber samples were
determined once per h by standard gravimetric methods, with periodic analysis
for Cr(III) and Cr(VI). Particle-size measurements were made from each
exposure level using a cascade impactor once per day for the first two weeks
and weekly thereafter.
Experimental design. For the main study seven groups were established,
each consisting of 15 rats of each sex. Animals were randomly assigned to
groups based on body weight. One group served as a control. Three of the
groups were exposed to 1 of 3 concentrations of chromic oxide (4.4, 15, or 44
mg/m 3) and the remaining 3 groups were exposed to 1 of 3 concentrations of
basic chromium sulfate (17, 54, or 168 mg/m 3). The desired exposure levels
were selected to be multiples of the threshold limit value (TLV) for trivalent
chromium and set at chromium equivalents of 3, 10, and 30 mg/m 3 for each test
article. Rats were 7 weeks of age at the start of the exposures. The animals
were exposed for 6 h per day, 5 days per week for 65 exposures over 13
consecutive weeks. At the end of the exposures, 10 males and 10 females from
each group were sacrificed. The remaining 5 males and 5 females from each
group were maintained for an additional 13-week recovery period during
which time they received no additional exposures.
In order to evaluate bronchoalveolar lavage parameters, additional rats
(5/sex/group) from those described above were exposed to the same concentrations of chromic oxide or basic chromium sulfate as the main study animals
or served as controls. Exposures were limited to only 5 consecutive days, after
which bonchoalveolar lavage analyses were performed on these animals and
the animals were removed from the study.
279
Clinical observations. Animals were observed daily prior to and following
each exposure for clinical signs of toxicity, and were observed twice daily for
morbidity and mortality during the recovery period and on weekends. Individual body weights were recorded weekly during the exposure and recovery
periods. All animals received an indirect ophthalmoscopic examination during
the acclimation period and prior to terminal necropsy.
Clinical pathology. Standard hematology, clinical biochemistry, and urinalysis determinations were conducted on animals, 10 per sex per group,
designated for necropsy at the end of exposures. Animals were fasted overnight
prior to blood sampling, with water available. Blood samples were obtained
from the orbital sinus plexus. At necropsy, bone marrow smears were prepared
and differential cell counts were evaluated. All clinical procedures were
performed using automated instrumentation except bone marrow smears,
which were examined microscopically. Urinalysis determinations were conducted on samples collected overnight in stainless-steel metabolism cages.
Urinalysis determinations were performed by gross observation, microscopy,
and automated clinical analyzer. Following urinalysis testing, aliquots of the
remaining urine from 5 animals per sex from the control group, and the
high-exposure level groups for both test articles were submitted for Beta 2
-microglobulin analysis.
Pathology. Animals found dead or euthanized by design at study termination were necropsied. At necropsy the heart, lungs, liver, spleen, kidneys,
brain, adrenal glands, thyroid/parathyroid glands, testes, and ovaries were
weighed. Tissues typically harvested for subchronic studies were also removed
and preserved. All tissues were placed in 10% neutral buffered formalin,
except eye tissue, which was fixed in Davidson’s fixative. Microscopic evaluation was conducted on all hematoxylin and eosin-stained tissues from the
control group and high-exposure-level groups of both test articles. The kidneys, livers, nasal tissues, trachea, lungs, larynx, mediastinal and mandibular
lymph nodes, and gross lesions from all animals in the low- and mid-exposure
level groups for both test articles were also examined. A formal peer review of
the histopathologic findings was performed.
Bronchoalveolar lavage evaluation. Bronchoalveolar lavage (BAL) analyses were conducted on 5 animals per sex per group exposed for 5 consecutive
days with the main study animals. Rats were anesthetized by intraperitoneal
injection of sodium pentobarbital. The lungs, heart, trachea, larynx, and tongue
were removed en-block. Following tracheal cannulation, the airways were
washed 3 successive times with warmed, physiological saline (30 ml per gram
of body weight) and the resulting BALF was pooled. Nucleated cell counts
were performed using a Neubauer hemocytometer, and cell differential counts
were performed on Wright-Giemsa-stained smears. Chemical analyses performed spectrophotometrically included for lactate dehydrogenase (LDH),
total protein, beta-glucuronidase, and glutathione reductase. LDH determination was based on the reduction of pyruvate to lactate with concurrent oxidation of NADH to NAD, measured by a decrease in absorbance. Total protein
was measured by increased absorbance associated with pyrogallol red-molybdate complex with basic amino acids. Beta-glucuronidase activity was determined by enzymatic conversion to alcohol, D-glucuronate, and p-nitrophenol.
Glutathione reductase activity was determined from the enzymatic reduction of
oxidized glutathione and concurrent oxidation of NADPH to NADH.
Sperm evaluation. At necropsy, sperm samples from the left caudal epididymis of 10 males per group were used for automated evaluation of sperm
motility, count, and morphology. The concentration and morphology of the
sperm were evaluated using visual methods. Two hundred intact sperm were
evaluated from each animal for morphology. Intact sperm were evaluated as
normal or abnormal. The number of disarticulated sperm in each field was also
assessed.
Statistics. Statistical analyses were performed of body weights, clinical
pathology laboratory tests, BALF data, and organ weights using one-way
analysis of variance. If the result was non-significant, no additional analysis
was performed. If the result was significant, Bartlett’s test for homogeneity of
variance was performed. If Bartlett’s test was non-significant, Dunnett’s t-test
was used for pairwise comparisons. If Bartlett’s test was significant, the Welch
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DERELANKO ET AL.
toxicity observed in any other animals from this group. Clinical
signs of toxicity were limited to sporadic labored breathing,
noted during two of the weekly observations, in females exposed to the high concentration of basic chromium sulfate. No
exposure-related effects were noted for either test article in the
ophthalmologic evaluations or for sperm motility, morphology,
or concentration.
Body Weights
FIG. 1. Mean body weights of male rats during 13 weeks of exposure to
basic chromium sulfate. Body weights of the rats exposed to basic chromium
sulfate at 17 mg/m 3 were not statistically different from controls at any time
during the 13 weeks of exposure. Body weights of the groups exposed at 54
and 168 mg/m 3 were statistically different from controls at the first week and
all subsequent weeks, with the exception of week 3 for the mid-exposure
group.
t-test with Bonferonni correction was used for pairwise comparisons. The
Kruskal-Wallis analysis of variance, followed where appropriate by the MannWhitney U test, was used for those parameters where parametric analysis was
inappropriate. The level for statistical significance was set at p # 0.05.
RESULTS
Chromic oxide. Male and female mean body weights during exposures to chromic oxide were not statistically different
from the control group9s mean body weights in any week.
Mean body weights of males exposed to the high concentration
of chromic oxide were slightly lower than controls during the
recovery period but weight gains for these animals were similar
to controls.
Basic chromium sulfate. Statistically significant, exposurerelated reduced mean body weights were observed in the males
of the mid- and high-exposure groups and the females of the
high-exposure group during the 13-week exposure period (Fig.
1). At the recovery sacrifice, the males from the same exposure
groups continued to exhibit mean body weights that were
significantly lower than the control group (Fig. 2) but body
weight gains between the chromium-exposed and control
groups were similar. The female mean body weights were
comparable to the control group females in all treatment groups
at the recovery sacrifice. Although quantitative food consumption data were not collected, no observable decreases in food
consumption were noted during the study.
Chamber Concentrations and Particle Sizes
The mean aerosol concentrations and standard deviations
over 13 weeks were 4.4 6 0.23, 15 6 1.2, and 44 6 3.7 mg/m 3
for chromic oxide and 17 6 4.3, 54 6 4.2 and 168 6 25.3
mg/m 3 for basic chromium sulfate. Mean particle size distribution data (MMAD in microns (GSD)) over 13 weeks (21
samples per test group) were 1.8 (1.93), 1.9 (1.84), and 1.9
(1.78) for chromic oxide and 4.2 (2.48), 4.2 (2.37), and 4.5
(2.50) for basic chromium sulfate for the low-, mid- and
high-exposure groups, respectively. No hexavalent chromium
was detected (detection limit 5 10 ng/ml).
Clinical Observations/Mortality/Ophthalmology/Sperm
Evaluation
No compound-related mortalities occurred for either test
article during the conduct of this study. Six animals died on
exposure day 1 as a direct result of the restraint tubes, and they
were replaced. One male from the basic chromium sulfate
high-exposure (168 mg/m 3) group that died on exposure day 4
was not replaced. Although the specific cause of death was not
identified, this death was not considered related to exposure to
basic chromium sulfate, since there were no significant signs of
FIG. 2. Mean body weights of basic chromium sulfate-exposed male rats
during the 13-week recovery period (no exposures). Body weights of rats
previously exposed to basic chromium sulfate at 17 mg/m 3 were not statistically different from controls. Body weights of the groups previously exposed
to basic chrome sulfate at 54 and 168 mg/m 3 were statistically different from
controls at all points from week 14 –26.
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TRIVALENT CHROMIUM SUBCHRONIC TOXICITY
TABLE 1
Bronchoalveolar Lavage Fluid Values for Rats following Five Days of Exposure to Basic Chromium Sulfate
Nucleated cell
Segmented Lymphocytes Mononuclear b-Glucuronidase Protein mg/ Lactic dehydrogenase Glutathione
count per mm 3 neutrophils %
%
cells %
mU/L
dl
U/L
reductase U/L
Males
Control (n 5 5)
157 6 41.4 a
17 mg/m 3 (n 5 5)
49 6 11.3 **
54 mg/m 3 (n 5 4)
76 6 13.7 **
168 mg/m 3 (n 5 5) 86 6 34.9 *
Females
Control (n 5 5)
142 6 38.1
17 mg/m 3 (n 5 5)
46 6 11.2 **
54 mg/m 3 (n 5 5)
51 6 14.1 **
168 mg/m 3 (n 5 5) 64 6 26.5 **
1 6 1.8
7 6 4.8
21 6 6.3
48 6 11.4 **
3 6 6.2
1 6 1.1
0
3 6 3.9
0
10 6 5.0
47 6 18.8
45 6 17.6
0
1 6 0.8
2 6 1.8
3 6 3.1
96 6 7.8
92 6 4.9
79 6 6.1
48 6 13.8 **
100 6 0.4
89 6 5.4
50 6 19.9
51 6 18.5
160
160
160
1 6 0.9
3.7 6 0.87
4.0 6 0.74
4.6 6 1.4
8.2 6 3.3
29 6 20.2
25 6 5.7
44 6 12.6
88 6 36.6
4 6 1.8
5 6 0.7
6 6 1.4
6 6 1.4
2 6 0.9
1 6 0.4
3 6 2.5
2 6 0.9
3.3 6 0.81
4.6 6 0.48
7.7 6 3.01
9.5 6 3.18
30 6 8.2
35 6 15.2
71 6 28.6
115 6 36.5
4 6 1.5
6 6 1.6
5 6 0.7
7 6 2.3
a
Mean 6 standard deviation
* Significantly different from control, p , 0.05.
** Significantly different from control, p , 0.01.
Clinical Pathology
Chromic oxide. After 13 weeks of exposure, none of the
exposure groups, for either sex, exhibited a statistically significant difference from the control group for any hematological,
serum biochemical, or urinalysis parameters. Beta- microglobulins were not detected in urine samples from any group.
Basic chromium sulfate. Most hematology, serum biochemistry, and urinalysis values from all exposure groups were
similar to the control group at the terminal sacrifice. Increased
leukocytes associated with increased number of neutrophils,
some statistically significant, were noted in the mid- and highexposure groups for males and females. Alkaline phosphatase
was statistically elevated in high-exposure group females and
serum cholesterol was statistically decreased in mid- and highexposure group females.
Bronchoalveolar Lavage Evaluation
Chromic oxide. None of the exposure groups demonstrated
a statistically significant difference from the control group for
any BAL parameter. A yellow intracytoplasmic, crystalline
material was present within the mononuclear cells from all
exposure groups. The relative amount of material and percentage of affected cells increased progressively with increasing
exposure concentration. Small amounts of crystals were
present in .90% of the cells observed from the low-exposuregroup animals and moderate to large amounts of crystalline
material were noted in .99% of the cells observed in all
high-exposure-group animals. The amount of crystals in the
mid-exposure group was intermediate to the other 2 groups.
Basic chromium sulfate. Evaluation of the BAL fluid from
animals exposed to basic chromium sulfate demonstrated several compound-related effects (Table 1). Males and females at
all exposure levels showed statistically reduced total nucleated
cell counts. Segmented neutrophils increased while mononu-
clear cells decreased, although not to a statistically significant
degree, at all concentration levels. Non-statistical increases in
protein and lactic dehydrogenase were also observed. Increased amounts of cell debris and lysed cells were noted at all
exposure levels.
Organ Weights
Chromic oxide. Slight, yet statistically significant increases in mean absolute and relative lung/trachea weights
occurred in high-exposure-level group males. (Table 2). Macroscopic and histologic changes were present to explain these
organ weight changes. Lung weights were not affected in
females. Other statistically significant increases were observed
in the mean absolute and relative thyroid/parathyroid weights
in the mid-exposure-group females and in the mean relative
thyroid/parathyroid/body weight ratios in the high-exposuregroup females. These organ weight changes were very small
and their biological importance could not be determined. At the
recovery sacrifice, organ weights of all the exposure groups
were comparable to the control group.
Basic chromium sulfate. At the terminal sacrifice, the
males and females in all treatment groups demonstrated compound-related, statistically significant increases in mean absolute and relative lung/trachea weights. These organ-weight
changes corresponded to changes seen microscopically. Statistically significant changes in absolute and/or relative weights
of some other organs, primarily in the high-level exposure
group, were noted for brain (males), kidney (males), and thyroid/parathyroid (male and female). No microscopic tissue
alterations were present to explain these organ weight changes.
Other statistically significant terminal-sacrifice organ-weight
changes were noted in the high-exposure-level group for liver
(males), spleen (males and females), and testes. These changes
were generally very small with no corresponding microscopic
282
DERELANKO ET AL.
TABLE 2
Selected Organ Weight Changes at Terminal Sacrifice of Rats Exposed to Chromic Oxide
Males
Lung/trachea
wt (g)
wt/bw (% 3 10)
Females
Lung/trachea
wt (g)
wt/bw (% 3 10)
Thyroid/parathyroid
wt (mg)
wt/bw (% 3 10 3)
Control
4.4 mg/m 3
15 mg/m 3
44 mg/m 3
0.99 6 0.07
4.42 6 0.187
0.98 6 0.055
4.52 6 0.273
1.0 6 0.077
4.49 6 0.396
1.11 6 0.050 **
4.98 6 0.228 **
0.81 6 0.081
5.65 6 0.418
0.81 6 0.080
5.78 6 0.577
0.85 6 0.084
5.77 6 0.629
0.88 6 0.068
6.40 6 0.618
12 6 1.9
8.26 6 1.493
13 6 1.3
8.89 6 0.880
15 6 1.5 **
10.10 6 1.147 *
14 6 2.3
10.04 6 1.346 *
Note. Organ weight changes given as mean 6 SD; bw 5 body weight. Organ weights of exposed animals were not significantly different from control animals
at recovery sacrifice.
* p , 0.05; ** p , 0.01.
pathology. Weight changes in these organs are typically observed with depressed body weights as had occurred in animals
in the Auletta study (1995). At the recovery sacrifice, statistically significant increases in male and female mean absolute
and relative lung weights continued for the mid- and highexposure level groups. Microscopic changes were present to
explain the pulmonary weight increases. Other statistically
significant organ weight changes not associated with microscopic changes included decreases in male mean absolute
kidney weights and increased male mean relative brain/body
weight ratios in the mid- and high-exposure level. Table 3
summarizes the organ weight changes.
Gross anatomic pathology. Exposure-related macroscopic
findings at the terminal and recovery sacrifices were observed
in the lungs and mediastinal lymph nodes of most animals in
this study. Green lung discoloration was observed in animals
exposed to chromic oxide at all exposure levels. Gray lung
discoloration was commonly observed in animals exposed to
basic chromium sulfate at the mid- and high-exposure levels.
The degree of discoloration with both materials increased with
exposure level and was present both at the terminal and recovery sacrifices. Similar discoloration was observed in the mediastinal lymph nodes of animals exposed to chromic oxide
(terminal and recovery sacrifices) or basic chromium sulfate
(recovery sacrifice only). Mediastinal lymph-node enlargement
was observed at the recovery sacrifice in animals exposed to
chromic oxide (high exposure only) or basic chromium sulfate
(all exposure levels). Tan focus/foci were observed in the lungs
at the recovery sacrifice of a high percentage of males exposed
to the high level of basic chromium sulfate. The tan focus/foci
correlated well with the presence of alveolar macrophages, and
mediastinal lymph node enlargement correlated well with histiocytosis described below. Additional macroscopic observations were few in number and considered incidental.
Microscopic Pathology at Terminal sacrifice
Chromic oxide. Randomly distributed foci or aggregates of
pigmented macrophages filled with dense black pigment were
observed within alveolar spaces adjacent to the junctions of
terminal bronchioles and alveolar ducts and subjacent to the
pleura in males and females from all chromic-oxide treatment
groups. Similar black pigment was also observed at the tracheal bifurcation, in the peribronchial lymphoid tissue, and
within the mediastinal lymph node. The pigment stained black
with hematoxylin and eosin stain and was presumed to represent the test article. The presence of the pigment corresponded
to the green discoloration seen macroscopically. Trace to mild
chronic interstitial inflammation of the lung, characterized by
an infiltration of inflammatory cells, was observed in alveolar
septa surrounding aggregates of pigmented macrophages in
some mid-exposure and high-exposure level males and females. Chronic interstitial inflammation was accompanied by
septal cell hyperplasia (Type II pneumocytes) in some midand high-exposure level males. The microscopic changes were
generally associated with the pigment and corresponded to the
increased lung weight observed for the males in the highexposure-level group. Lymphoid hyperplasia of the node was
also present in all exposure groups. No test article-related
lesions were seen in the nasal cavities of animals exposed to
chromic oxide at any exposure level. Figure 3 displays photomicrographs of selected respiratory tissues.
Basic chromium sulfate. Chronic inflammation was observed involving the alveoli of all exposure-level groups, consisting of alveolar spaces filled with macrophages, neutrophils,
lymphocytes, and cellular debris. Some foci exhibited quite
intense inflammation and thickening of alveolar walls. Chronic
interstitial inflammation was usually multifocally distributed
and consisted of thickened alveolar septa caused by inflammatory cell infiltration and hyperplasia of alveolar septal cells
283
TRIVALENT CHROMIUM SUBCHRONIC TOXICITY
TABLE 3
Selected Organ Weight Changes at Terminal and Recovery Sacrifices of Rats Exposed to Basic Chromium Sulfate
Males
Lung/trachea
wt (g)
wt/bw (% 3 10)
Brain
wt (g)
wt/bw (% 3 10)
Kidney
wt (g)
wt/bw (% 3 10)
Liver
wt (g)
wt/bw (% 3 10)
Thyroid/parathyroid
wt (mg)
wt/bw (% 3 10 3)
Spleen
wt/(g)
wt/bw (% 3 10)
Testes
wt/(g)
wt/bw (% 3 10)
Females
Lung/trachea
wt (g)
wt/bw (% 3 10)
Thyroid/parathyroid
wt (mg)
wt/bw (% 3 10 3)
Spleen
wt (g)
wt/bw (% 3 10)
Control
17 mg/m 3
54 mg/m 3
168 mg/m 3
0.99 6 0.70
(1.32 6 0.113)
4.42 6 0.187
(3.89 6 0.214)
1.26 6 0.071 **
(1.52 6 0.132)
5.60 6 0.271 **
(4.66 6 0.373 **)
1.51 6 0.088 **
(1.95 6 0.068 **)
7.15 6 0.252**
(6.37 6 0.298 **)
1.86 6 0.89 **
(2.67 6 0.144 **)
10.69 6 0.688 **
(8.77 6 .274 **)
1.79 6 0.087
8.02 6 0.380
1.82 6 0.055
8.12 6 0.374
1.76 6 0.061
8.38 6 0.473
1.71 6 0.069 *
9.83 6 0.518 **
1.54 6 0.106
7.62 6 0.300
1.35 6 0.049 **
7.28 6 0.207
1.62 6 0.085
7.30 6 0.283
1.64 6 0.082
7.78 6 0.350 **
5.48 6 0.367
2.45 6 0.070
5.63 6 0.271
2.50 6 0.050
5.17 6 0.459
2.45 6 0.091
4.39 6 0.146 **
2.53 6 0.120
14 6 2.5
6.21 6 1.052
15 6 2.9
6.64 6 1.475
14 6 1.8
6.74 6 1.021
15 6 3.5
8.76 6 2.074 *
0.45 6 0.038
1.99 6 0.149
0.48 6 0.036
1.91 6 0.132
0.40 6 0.040 *
1.89 6 0.125
0.32 6 0.035 **
1.84 6 0.151
2.36 6 0.356
10.54 6 1.315
2.39 6 0.261
10.65 6 1.098
2.22 6 0.286
10.52 6 1.049
0.81 6 0.081
(0.93 6 0.079)
5.65 6 0.418)
(4.74 6 0.384)
0.98 6 0.094 **
(1.08 6 0.120)
6.99 6 0.619 **
(5.75 6 0.315*)
1.29 6 0.164 **
(1.59 6 0.120 **)
9.24 6 1.036 **
(8.02 6 0.750 **)
2.18 6 0.215
12.53 6 1.238 **
1.66 6 0.084 **
(2.45 6 0.120 **)
12.89 6 1.134 **
(13.34 6 0.614 **)
12 6 1.9
8.26 6 1.493
11 6 1.3
7.96 6 1.154
12 6 1.8
8.63 6 1.265
14 6 2.1 *
10.77 6 1.522 **
0.33 6 0.037
2.32 6 0.268
0.31 6 0.033
2.19 6 0.212
0.30 6 0.033
2.17 6 0.162
0.28 6 0.033 **
2.19 6 0.273
Note. Organ weight changes, values given as mean 6 SD; bw 5 body weight. Non-bracketed values 5 terminal sacrifice, bracketed values 5 recovery
sacrifice.
* p , 0.05; ** p , 0.01.
(type II pneumocytes). Multifocal areas of granulomatous inflammation, characterized by infiltration of macrophages and
multinucleated giant cells, was observed at all exposure levels
and was closely associated with foreign material seen in the
lung and presumed to be the test article. Trace to severe,
multifocal to diffuse pulmonary infiltration of alveolar macrophages with foamy or granular appearing acidophilic cytoplasm was observed in the alveolar lumens and correlated with
the gray discoloration of the lungs that was observed at necropsy.
These changes corresponded to the increased lung weights
observed in all the exposure groups. Green refractile foreign
material was present in the lamina propria and submucosa of
the larynx of animals in all treatment groups and was associ-
ated with an infiltration of macrophages and multinucleated
giant cells (granulomatous inflammation). Histiocytosis consisting of macrophages or histiocytes with abundant foamy
cytoplasm and lymphoid hyperplasia observed in the peribronchial lymphoid tissue and mediastinal lymph node correlated
with lymph node enlargement observed at necropsy. Changes
in nasal tissues considered to be test article-related, were
observed in males and females, and included acute inflammation, suppurative exudate, and mucoid exudate. One or more of
these changes were seen in all 4 examined levels of the nasal
cavity but were slightly more prevalent in the mid-posterior
portion. All other microscopic findings were incidental and/or
spontaneous and considered unrelated to the test article. Figure
4 displays photomicrographs of selected respiratory tissues.
284
DERELANKO ET AL.
FIG. 3. Photomicrographs (5103) of selected respiratory tissues from male rats exposed to chromic oxide for 13 weeks at 44 mg/m 3 : (a) particle-laden
macrophages on peribronchial alveoli, (b) accumulation of black pigment in the mediastinal lymph node, and (c) particle-laden macrophages in alveolar spaces
with early septal cell hyperplasia.
Recovery Sacrifice
Chromic oxide. In the lung, trace to mild pigmented macrophages and black pigment in the peribronchial lymphoid
tissue persisted in all treatment groups, males and females, at
approximately equal incidence and severity, as seen in the
terminal-sacrifice animals. Trace to mild septal cell hyperplasia
and trace to mild chronic interstitial inflammation persisted in
males of all treatment groups and females in the mid- and
FIG. 4. Photomicrographs (5103) of selected respiratory tissues from male rats exposed to basic chromium sulfate for 13 weeks at 168 mg/m 3 : (a)
granulomatous inflammation and foreign material in the lamina propria of the larynx, (b) granular macrophages in alveolar spaces with septal cell hyperplasia,
and (3) histiocytes (macrophages) with granular cytoplasm in the mediastinal lymph node.
TRIVALENT CHROMIUM SUBCHRONIC TOXICITY
high-exposure-level groups. These lesions were the same or
slightly increased in severity as compared to the terminalsacrifice groups. Trace to mild black pigment also persisted in
mediastinal lymph nodes in all exposure groups, with an apparent increase in incidence in some males in the two lowestexposure groups as compared to the terminal-sacrifice group,
suggesting pulmonary clearance via the lymphatic system.
Most of the pathologic changes observed at the recovery sacrifice were of minimal severity.
Basic chromium sulfate. Foreign material persisted in the
lungs of some animals in all exposure groups, but with decreased incidence in most groups. Trace to moderate chronic
inflammation of the alveoli, trace to moderate chronic interstitial inflammation, septal cell hyperplasia, and alveolar macrophages were approximately equal in incidence and severity at
the mid- and high-exposure levels as compared to the terminalsacrifice animals, with severity slightly reduced at the lowexposure level. Peribronchial histiocytosis continued to be
observed only in males and females of the mid- and highexposure groups. The incidence and severity in the mid-exposure group was approximately equal to the terminal sacrifice
animals while the incidence in the high-exposure group increased in both males and females, suggesting clearing of the
basic chromium sulfate via the reticuloendothelial system.
Granulomatous inflammation of the lung decreased in incidence except in the males and females of the high-exposure
group where the incidence was approximately equal to that of
the terminal sacrifice animals of this group. In the larynx,
foreign material and granulomatous inflammation was either
not detected at the low-exposure (both sexes) or mid-exposure
(males) or markedly decreased in incidence and severity in
mid-exposure females and high-exposure groups (both sexes)
as compared to the terminal-sacrifice animals. The incidence
and severity of histiocytosis and lymphoid hyperplasia of the
mediastinal lymph node generally remained at similar levels in
both males and females of all treated groups in the terminal and
recovery sacrifices. Nasal cavity findings were not detected
except for trace suppurative exudate seen in the most anterior
section in one or two animals from each treatment group. All
other microscopic findings were incidental and/or spontaneous
and considered unrelated to the test article.
DISCUSSION
The results of this study demonstrate that there are significant differences in the toxicity of trivalent chromium compounds, chromic oxide and basic chromium sulfate, to the
respiratory tract following a 3-month subchronic inhalation
exposure in rats. While for both materials the principle effects
observed after 90 days of exposure were primarily noted in the
respiratory tract, the type and location of effects observed were
different between the two trivalent chromium compounds despite the animals being exposed to equivalent concentrations of
trivalent chromium of 3, 10, or 30 mg/m 3.
285
Rats exposed to the insoluble chromic oxide developed
changes in the bronchial and mediastinal lymphatic tissue and
lung. The changes appeared to be directly associated with the
presence of pigment, observed both macroscopically and microscopically, in the affected tissues. They are believed to have
been a non-specific response to the physical presence of deposits of test material and not a direct toxic effect of the
chromic oxide. Similar effects have been reported to occur
with other inert, respirable particles of low toxicity. (Lee et al.,
1986, 1989; Muhle et al., 1991; Warheit et al., 1997). The
trachea and nasal cavities were generally unaffected following
exposure to chromic oxide.
The effects on the respiratory tract following 13 weeks of
exposure to basic chromium sulfate were of a more severe
nature and more widespread (including the nasal cavity and
larynx in addition to the lungs and mediastinal lymph node)
than observed for chromic oxide. Some of these effects could
have been related to the acidity of the basic chrome sulfate,
which readily forms acidic solutions (pH ' 2.8), presumably
with the sulfate group. Waters and Gardner (1975) have previously demonstrated the effect of chromic chloride on rabbit
macrophages exposed in vitro for 20 h to concentrations of
1–500 mg Cr/mL. The net number of viable cells was reduced
to 50% of control at concentrations of about 200 –250 mg
Cr/mL and the specific activity of acid phosphatase, a lysosomal enzyme, was proportionately decreased at similar concentrations. Important to their study was the effect of pH of the
culture medium. Using hydrochloric acid as a positive control,
no alterations were noted unless the pH was less than 6.03
versus the control value of 7.2. At lesser pH values, the effects
from hydrochloric acid alone on macrophages were not significantly different from those produced by chromic chloride at
the same pH level suggesting the effect was due to acid rather
than chromium ion, per se. The more widespread distribution
of lesions observed with basic chromium sulfate than seen with
chromic oxide may have been due to the increased water
solubility of basic chromium sulfate. Deposition in the respiratory tract is dependent on water solubility with the more
water-soluble materials depositing higher in the respiratory
tract (Newman, 1992). This may account for the concentrationrelated severity of effects seen in the nasal cavity and larynx
with basic chromium sulfate.
The difference in tissue reactions between chromic oxide
and basic chromium sulfate are quite apparent when one considers the BALF results. Despite the presence of crystalline
material within the mononuclear cells, no changes in BALF
parameters were noted following exposure to chromic oxide. In
contrast, basic chromium sulfate showed several treatmentrelated effects in BALF parameters.
The National Toxicology Program (1996a,b) has shown
similar differences for nickel sulfate (soluble) and nickel oxide
(insoluble) in 13-week exposures of rats and mice. Whereas
inflammatory lung lesions were noted in all exposed groups of
nickel sulfate as low as 0.12 mg compound/m 3 or 0.03 mg
286
DERELANKO ET AL.
Ni/m 3, similar inflammatory effects were not noted with nickel
oxide at concentrations below 2.5 mg compound/m 3, although
accumulation of black granular pigment was noted in macrophages.
A significant amount of pigment was still present in the
respiratory tract of chromic oxide-exposed animals after the
13-week recovery period along with the earlier observed pathological effects. Increased pigment in the lymphatic tissue suggests pulmonary clearance of the chromic oxide via the lymphatic system was occurring, albeit rather slowly. With basic
chromium sulfate, foreign material was absent or decreased
after the recovery period in the lungs, primarily at the low- and
mid-exposure levels, and was no longer found in the larynx of
low-exposure-level rats and mid-exposure level males. This
suggests that basic chromium sulfate is most likely more rapidly cleared from the respiratory tract than chromic oxide, due
to its increased water solubility. In a study of the distribution of
trivalent chromium in guinea pigs after intratracheal instillation
of chromium trichloride, 30% of the administered dose was
still present in the lungs 30 days after instillation and 12% after
60 days (Baetjer et al, 1959). Qualitatively, this appears similar
to what we observed with basic chromium sulfate, which, like
chromium trichloride, is relatively soluble. The much slower
clearance of chromic oxide from the lungs may have been due
to its insolubility resulting in decreased systemic absorption
and/or reduced clearance from the lung by normal clearance
mechanisms. Many of the exposure-related changes observed
with basic chromium sulfate continued after the 13-week recovery period, with approximately the same incidence and
severity as observed immediately after the exposure period.
However, granulomatous inflammation in the lungs was decreased or resolved in low- and mid-exposure-level rats and
nasal cavity effects were resolved in all animals except for
trace suppurative exudate randomly seen in a few rats. These
findings suggest that some of the effects of basic chromium
sulfate are reversible but the length of the recovery period was
not sufficient to determine if complete recovery would be
likely to occur with time.
It is of interest that other than the localized effects on the
respiratory tract, no evidence of systemic toxicity was observed with exposure to chromic oxide. Evidence of systemic
toxicity observed with basic chromium sulfate was primarily
limited to reductions in body weight not related to reduced
food consumption during the 13 weeks of exposure. While
there are no animal inhalation studies of similar exposure
length available in the literature for comparison, the results of
the current study tends to be in agreement with epidemiological
studies of Korallus (1974a,b). This study reported no changes
in hematology parameters or stomach or liver disorders in
workers employed in two factories that produced trivalent
chromium oxide or chromium sulfate. Foa et al. (1988) reported the absence of kidney effects in a population exposed to
chromic oxide. Moreover, systemic effects have not been generally observed with oral administration of trivalent chromium
compounds in several long-term drinking water and food studies in rats (Ivankovic and Preussmann, 1975; MacKenzie et al.,
1958; Schroeder, 1965). In the Ivankovic and Preussmann
study, no evidence of cardiotoxicity, hepatotoxicity, gastrointestinal toxicity or renal toxicity was found in rats given 1806
mg/kg trivalent chromium as chromium oxide 5 days per week
for 90 days or 2 years. In addition, there is no evidence of
systemic toxic effects from oral intake of trivalent chromium
compounds in mineral supplements. It is of interest that females exposed to the mid- and high levels of basic chromium
sulfate in our study showed a significant decrease in cholesterol
after 13 weeks of exposure. In addition, chromium supplements have been reported to reduce cholesterol levels (Evans,
1989; Offenbacher and Pi-Sunyer, 1980).
The absence of effects on sperm motility, morphology, and
concentration in the current study lend support to the findings
of Ivankovic and Preussman (1975), who saw no reproductive
effects to male rats administered 1806 mg of trivalent chromium/kg/day as chromium oxide, 5 days/week for 60 days before
breeding. Two studies have reported degeneration of seminiferous tubules and reduced sperm counts: one on trivalent
chromium in a 7-week mouse-diet study of chromium sulfate
(Zahid et al., 1990), and the other following intraperitoneal
injection of chromium nitrate to rabbits for 6 weeks (Behari,
1978). Both have been criticized: the Zahid study on technical
merit (Finley et al., 1993) and the Behari study because the ip
route is not toxicokinetically relevant to routes of chromium
exposure typically encountered.
The lack of systemic toxicity, particularly with the soluble
basic chromium sulfate, may be due to an inability of absorbed
trivalent chromium to penetrate cell membranes (ATSDR,
1993). In addition, oral absorption of trivalent chromium compounds is known to be poor both in animals (Akatsuka and
Fairhall, 1934) and humans (Anderson et al., 1993). Even
when administered as chromium piccolinate to enhance absorption for dietary supplementation, trivalent chromium absorption has been estimated at only approximately 3% (Gargas
et al., 1994). The identification of chromium in urine and
serum of humans occupationally exposed to soluble trivalent
compounds in air indicates that chromium can be absorbed
from the lungs (ATSDR, 1993). However, absorption by the
blood stream and mucociliary clearance is only 5–30% for
trivalent chromium compounds following intratracheal injection (ATSDR [Agency for Toxic Substances and Disease Registry], 1993).
Because of the microscopic effects observed in the respiratory tracts of some animals exposed to the low level of chromic
oxide, a NOAEL was not established in this study. However,
the low incidence and minimal severity of the pathological
effects in the low-level animals suggests that 4.4 mg/m 3 is very
near a NOAEL for subchronic exposure to chromic oxide. A
NOAEL was not established for basic chromium sulfate in this
study based on the pathological findings in the respiratory tract.
The low level of chromic oxide and basic chromium sulfate
TRIVALENT CHROMIUM SUBCHRONIC TOXICITY
corresponds to 3 mg/m 3 of trivalent chromium. The current
TLV is 0.5 mg/m 3 for trivalent chromium (ACGIH, 1996).
Korallus et al. (1974b) found no increase in the prevalence of
respiratory illness in a study of 128 workers from 2 factories
producing chromic oxide or chromium sulfate. The same result
was found in 106 workers at a factory that produced these
compounds under workroom levels less than or equal to 1.99
mg trivalent chromium/m 3 (Korallus et al. 1974a). This suggests that trivalent chromium compounds may be more toxic to
the rodent respiratory tract than to that of humans.
In conclusion, the results of this study indicate that there are
significant differences in toxicity to the respiratory tract between chromic oxide and basic chromium sulfate. These differences appear to be most likely related to the physical properties of the compounds, such as acidity and water-solubility,
which affected deposition, tissue reactions and clearance. The
acidic, water-soluble basic chromium sulfate cleared more
quickly but produced more severe and widespread tissue reactions, while the insoluble chromic oxide was less reactive but
cleared more slowly. This indicates that the toxicity of trivalent
chromium compounds cannot be predicted based simply on the
presence of trivalent chromium. Physical-chemical properties
such as solubility and pH must be considered.
Different TLVs have been established to account for differences in the toxicity of soluble versus insoluble hexavalent chromium compounds (ACGIH), as reviewed by Katz and Salem
(1993). An oral reference dose (RfD) has been established specifically for insoluble chromium (Finley et al. 1996). In this
regard, our study suggests that solubility of trivalent chromium
compounds should also be considered in establishing permissible
exposure levels for inhalation of trivalent chromium compounds.
ACKNOWLEDGMENTS
This study was supported by the Industrial Health Foundation Chromium
Chemicals Health and Environmental Committee. The Authors wish to acknowledge the following individuals for their contributions to this study: C.
Ulrich, B. Culp, E. Freytag, Dr. G. Walter of MPI Research for the technical
conduct of the study; Dr. J. Hardisty of Experimental Pathology Labs for
pathology consultation; and Mrs. R. Brown and R. Levy for help in preparing
the manuscript.
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