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EC number: 914-129-3 | CAS number: 12336-95-7
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Repeated dose toxicity: inhalation
Administrative data
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline-comparable study published in a peer-reviewed journal
Data source
Reference
- Reference Type:
- publication
- Title:
- Thirteen-week subchronic rat inhalation toxicity study with a recovery phase of trivalent chromium compounds, chromic oxide, and basic chromium sulfate.
- Author:
- Derelanko, M.J., Rinehart, W.E., Hilaski, R.J., Thompson, R.B. &, Löser, E.
- Year:
- 1 999
- Bibliographic source:
- Toxicological Sciences 52: 278-288
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- GLP compliance:
- not specified
- Limit test:
- no
Test material
- Reference substance name:
- Chromium hydroxide sulphate
- EC Number:
- 235-595-8
- EC Name:
- Chromium hydroxide sulphate
- Cas Number:
- 12336-95-7
- Molecular formula:
- CrHO5S
- IUPAC Name:
- chromium hydroxide sulphate
- Details on test material:
- No further details
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: British Chrome Chemicals (Urlay Nook, Eaglescliff, Cleveland, U.K.)
- Purity, including information on contaminants, isomers, etc.: 25 % Cr(III) as Cr2O3 and less than 0.0003 % Cr(VI) (non-detectable)
Test animals
- Species:
- rat
- Strain:
- Fischer 344
- Details on species / strain selection:
- CDF (Fischer 344)/Crl BR VAF/Plus
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 5 weeks, 7 weeks at strat of exposures
- Housing: Following three days of group housing, animals were individually housed in stainless steel, suspended wiremesh cages.
- Diet (e.g. ad libitum): ad libitum during the non-exposure periods
- Water (e.g. ad libitum): ad libitum during the non-exposure periods
- Acclimation period: 2 weeks
DETAILS OF FOOD AND WATER QUALITY:
Purina Certified Chow No. 5002, tap water
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 °C
- Humidity (%): 43 +/- 11%
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- inhalation: dust
- Type of inhalation exposure:
- nose only
- Vehicle:
- other: unchanged (no vehicle)
- Mass median aerodynamic diameter (MMAD):
- >= 4.2 - <= 4.5 µm
- Geometric standard deviation (GSD):
- 2.45
- Remarks on MMAD:
- MMAD / GSD: Mean particle size distribution in microns (GSD) for three test groups over 13 weeks was 4.2 (2.48), 4.2 (2.37), and 4.5 (2.50) for the low-, mid-, and high-exposure groups, respectively.
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and acrylic nose-only inhalation chambers
- Method of holding animals in test chamber: restraint tubes
- System of generating particulates/aerosols: Generation of particles was accomplished using an auger dust feeder and an air micronizer.
- Air change rate: 12 chamber air changes per hour
TEST ATMOSPHERE
- Brief description of analytical method used: standard gravimetric methods with periodic analysis for Cr(III) and Cr(VI)
- Samples taken from breathing zone: yes, once per hour - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Chamber samples were determined once per hour by standard gravimetric methods with periodic analysis for Cr(III) and Cr(VI).
- Duration of treatment / exposure:
- Main study: 13 weeks plus an additional 13-week recovery period for some animals.
Bronchoalveolar lavage evaluation: 5 consecutive days - Frequency of treatment:
- Main study: 6 hours per day, 5 days per week for 65 exposures
Doses / concentrationsopen allclose all
- Dose / conc.:
- 17 mg/m³ air (nominal)
- Dose / conc.:
- 54 mg/m³ air (nominal)
- Dose / conc.:
- 168 mg/m³ air (nominal)
- Dose / conc.:
- 3 mg/m³ air
- Remarks:
- chromium (III) equivalents
- Dose / conc.:
- 10 mg/m³ air
- Remarks:
- chromium (III) equivalents
- Dose / conc.:
- 30 mg/m³ air
- Remarks:
- chromium (III) equivalents
- No. of animals per sex per dose:
- 15 animals/sex/dose in the main study
5 animals/sex/dose for the bronchoalveolar lavage evaluation - Control animals:
- yes
- Details on study design:
- - Dose selection rationale:
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/m3.
- Rationale for animal assignment (if not random): Animals were randomly assigned to groups based on body weight.
- Fasting period before blood sampling for clinical biochemistry: fasted overnight
- Rationale for selecting satellite groups: 5 males and 5 females
- Post-exposure recovery period in satellite groups: 13-week recovery period without exposures - Positive control:
- No
Examinations
- Observations and examinations performed and frequency:
- 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. Standard haematology, clinical biochemistry, and urinalysis determinations were conducted on animals (10/sex/group) at the end of exposures.
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (prior to and following each exposure)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (prior to and following each exposure)
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimation and prior to necropsy
- Dose groups that were examined: all animals
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of exposures
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes
- How many animals: 10/sex/dose
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of exposures
- Animals fasted: Yes
- How many animals: 10/sex/dose
URINALYSIS: Yes
- Time schedule for collection of urine: end of exposures
- Metabolism cages used for collection of urine: Yes
BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: after 5 exposures on 5 consecutive days
- Dose groups that were examined: all
- Number of animals: 5/sex - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
At necropsy the heart, lungs, liver, spleen, kidneys, brain, adrenal glands, thyroid/parathyroid glands, testes, and ovaries were weighed. Microscopic evaluation was conducted on all haematoxylin and eosin-stained tissues from the control and high-exposure level groups. The kidneys, liver, nasal tissue, trachea, lungs, larynx, mediastinal and mandibular lymph nodes, and gross lesions from all animals in the low- and mid-exposure level groups were also examined. - Other examinations:
- Main study: Sperm motility, count and morphology of 10 males/group
Bronchoalveolar lavage evaluation: Nucleated cell counts and cell differential counts were performed on pooled BALF. Chemical analyses for lactate dehydrogenase, total protein, beta-glucuronidase and glutathione reductase were performed. - Statistics:
- One-way analysis of variance on body weights, clinical pathology laboratory tests, BALF data and organ weights. 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 Barlett's test was significant, the Welch t-test with Bonferroni correction was used for pairwise comparisons. The Kruskal-Wallis analysis of variance, followed where appropriate by Mann-Whitney test was used for those parameters where parametric analysis was inappropriate. The level for statistical significance was set at p = 0.05.
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Sporadic laboured breathing in females of the high exposure group.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Six animals died on exposure day 1 as a direct result of the restraint tubes. They were replaced. One male of the high exposure group died on day 4. This was not considered to be compound-related.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Statistically significant exposure-related reduced mean body weights were observed in the males of the mid- and high exposure groups and the females of the high-exposure group. 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, but body weight gains were similar between the exposure groups and the control group. The female mean body weights in all treatment groups were comparable to the control group females at the recovery sacrifice
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No observable decreases in food consumption were noted during the study.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased leukocytes associated with increased number of neutrophils, some statistically significant, were noted in the mid- and high-exposure groups for males and females.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Alkaline phosphatase was statistically elevated in high-exposure group females and serum cholesterol was statistically decreased in mid- and high-exposure group females.
- Endocrine findings:
- not examined
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- At terminal sacrifice, males and females in all treatment groups demonstrated compound-related, statistically significant increases in mean absolute and relative lung/trachea weights. Statistically significant changes in absolute and/or relative weights of some other organs are listed below (See Table 1); changes in the weights of some organs are secondary to bodyweight effects and, with the exception of the lungs, effects occurred in the absence of microscopic correlates.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Gray lung discoloration was commonly observed in animals of the mid- and high-exposure levels. The degree of discoloration increased with exposure level and was present both at the terminal and recovery sacrifices. Similar discoloration was observed in the mediastinal lymph nodes of exposed animals (recovery sacrifice only) while mediastinal lymph node enlargement was seen at 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.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Chronic inflammation was observed involving the alveoli of all exposure-level groups consisting of alveolar spaces filled with macrophages, neutrophils, lymphocytes, and cellular debris. 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 the test article. These changes corresponded to the increased lung weights observed in all exposure groups. Green refractile foreign material was present in the larynx of animals in all treatment groups. Histiocytosis and lymphoid hyperplasia 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.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- No apparent compound-related effects were noted for sperm motility or morphology for any concentration.
At recovery sacrifice, foreign material persisted in the lungs of some animals in all exposure groups, but with decreased incidence in most groups. 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.
Bronchoalveolar lavage evaluation
Males and females at all exposure levels showed statistically reduced total nucleated cell counts. Segmented neutrophils increased while mononuclear 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. - Details on results:
- Main 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 sulphate high-exposure (168 mg/m3) group died on exposure day 4 and was not replaced. Although the specific cause of death was not identified, this death was not considered related to exposure to basic chromium sulphate, since there were no significant signs of 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 sulphate.
Statistically significant, exposure-related 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. At the recovery sacrifice, males from the same exposure groups continued to exhibit mean body weights that were significantly lower than the control group but body weight gains between treated and control groups were similar.
Most haematology, serum biochemistry, and urinalysis values were similar to the control group. Increased leukocytes associated with increased number of neutrophils, some statistically significant, were noted in mid-and high-exposure groups for males and females. Alkaline phosphatase was statistically elevated in high-exposure group females and serum cholesterol was statistically decreased in mid- and high- exposure group females.
At terminal sacrifice, males and females in all treatment groups demonstrated compound-related, statistically significant increases in mean absolute and relative lung/trachea weights. Statistically significant changes in absolute and/or relative weights of some other organs are listed below (See Table 1); changes in the weights of some organs are secondary to bodyweight effects and, with the exception of the lungs, effects occurred in the absence of microscopic correlates.
Gray lung discoloration was commonly observed in animals exposed to basic chromium sulphate at the mid- and high-exposure levels. The degree of discoloration 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 basic chromium sulphate (recovery sacrifice only) while mediastinal lymph node enlargement was seen at 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 sulphate.
Chronic inflammation was observed involving the alveoli of all exposure-level groups consisting of alveolar spaces filled with macrophages, neutrophils, lymphocytes, and cellular debris. 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 the test article. These changes corresponded to the increased lung weights observed in all exposure groups. Green refractile foreign material was present in the larynx of animals in all treatment groups. Histiocytosis and lymphoid hyperplasia 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.
No apparent compound-related effects were noted for sperm motility or morphology for any concentration of basic chromium sulphate.
At recovery sacrifice, foreign material persisted in the lungs of some animals in all exposure groups, but with decreased incidence in most groups. 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.
Bronchoalveolar lavage evaluation
Males and females at all exposure levels showed statistically reduced total nucleated cell counts. Segmented neutrophils increased while mononuclear 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.
Effect levels
open allclose all
- Dose descriptor:
- LOAEC
- Remarks:
- [chromium hydroxide sulphate]
- Effect level:
- 17 mg/m³ air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: pathological findings in the respiratory tract
- Remarks on result:
- other: A NOAEC was not established in this study.
- Dose descriptor:
- LOAEC
- Remarks:
- [Cr3+ equivalents]
- Effect level:
- 3 mg/m³ air
- Based on:
- element
- Sex:
- male/female
- Basis for effect level:
- other: pathological findings in the respiratory tract
- Remarks on result:
- other: A NOAEC was not established in this study.
Target system / organ toxicity
- Critical effects observed:
- not specified
Any other information on results incl. tables
Table 1: Selected organ weight changes at terminal and recovery sacrifices of rats exposed to chromium hydroxide sulphate
Control | 17 mg/m3 | 54 mg/m3 | 168 mg/m3 | |||
Males Lung/trachea | ||||||
wt (g) | 0.99 + 0.70(1.32 + 0.113) | 1.26 + 0.071**(1.52 + 0.132) | 1.51 + 0.088**(1.95 + 0.068**) | 1.86 + 0.89**(2.67 + 0.144**) | ||
wt/bw (% x 10) | 4.42 + 0.187(3.89 + 0.214) | 5.60 + 0.271**(4.66 + 0.373**) | 7.15 + 0.252**(6.37 + 0.298**) | 10.69 + 0.688**(8.77 + 0.274**) | ||
Brain | ||||||
wt (g) | 1.79 + 0.087 | 1.82 + 0.055 | 1.76 + 0.061 | 1.71 + 0.069* | ||
wt/bw (% x 10) | 8.02 + 0.380 | 8.12 + 0.374 | 8.38 + 0.473 | 9.83 + 0.518** | ||
Kidney | ||||||
wt (g) | 1.54 + 0.106 | 1.35 + 0.049** | 1.62 + 0.085 | 1.64 + 0.082 | ||
wt/bw (% x 10) | 7.62 + 0.300 | 7.28 + 0.207 | 7.30 + 0.283 | 7.78 + 0.350** | ||
Liver | ||||||
wt (g) | 5.48 + 0.367 | 5.63 + 0.271 | 5.17 + 0.459 | 4.39 + 0.146** | ||
wt/bw (% x 10) | 2.45 + 0.070 | 2.50 + 0.050 | 2.45 + 0.091 | 2.53 + 0.120 | ||
Thyroid/parathyroid | ||||||
wt (mg) | 14 + 2.5 | 15 + 2.9 | 14 + 1.8 | 15 + 3.5 | ||
wt/bw (% x 10) | 6.21 + 1.052 | 6.64 + 1.475 | 6.74 + 1.021 | 8.76 + 2.074* | ||
Spleen | ||||||
wt (g) | 0.45 + 0.038 | 0.48 + 0.036 | 0.40 + 0.040* | 0.32 + 0.035** | ||
wt/bw (% x 10) | 1.99 + 0.149 | 1.91 + 0.132 | 1.89 + 0.125 | 1.84 + 0.151 | ||
Testes | ||||||
wt (g) | 2.36 + 0.356 | 2.39 + 0.261 | 2.22 + 0.286 | 2.18 + 0.215 | ||
wt/bw (% x 10) | 10.54 + 1.315 | 10.65 + 1.098 | 10.52 + 1.049 | 12.53 + 1.238** | ||
Females Lung/trachea | ||||||
wt (g) | 0.81 + 0.081(0.93 + 0.079) | 0.98 + 0.094**(1.08 + 0.120) | 1.29 + 0.164**(1.59 + 0.120**) | 1.66 + 0.084**(2.45 + 0.120**) | ||
wt/bw (% x 10) | 5.65 + 0.418(4.74 + 0.384) | 6.99 + 0.619**(5.75 + 0.315*) | 9.24 + 1.036**(8.02 + 0.750**) | 12.89 + 1.134**(13.34 + 0.614**) | ||
Thyroid/parathyroid | ||||||
wt (mg) | 12 + 1.9 | 11 + 1.3 | 12 + 1.8 | 14 + 2.1* | ||
wt/bw (% x 10) | 8.26 + 1.493 | 7.96 + 1.154 | 8.63 + 1.265 | 10.77 + 1.522** | ||
Spleen | ||||||
wt (g) | 0.33 + 0.037 | 0.31 + 0.033 | 0.30 + 0.033 | 0.28 + 0.033** | ||
wt/bw (% x 10) | 2.32 + 0.268 | 2.19 + 0.212 | 2.17 + 0.162 | 2.19 + 0.273 | ||
Note: Organ weight changes, values given as mean + SD: bw = body weight. Non-bracketed values = terminal sacrifice, bracketed values = recovery sacrifice. * p < 0.05; ** p < 0.01 |
Applicant's summary and conclusion
- Conclusions:
- No systemic toxicity was observed in rats following a 13-week nose-only inhalation study of chromium hydroxide sulphate. Pathological findings were observed in the respiratory tract and are associated with the deposition of inhaled particulate material.
- Executive summary:
Chromium hydroxide sulphate was tested in a guideline-compliant 3 -month subchronic inhalation study in rats at doses of 17 mg/m³, 54 mg/m³ and 168 mg/m³ The principle effects observed were noted in the respiratory tract. Exposed rats developed changes in the mediastinal lymphatic tissue, lung, larynx and nasal cavity. Foreign material was absent or decreased after the recovery period in the lungs and was no longer found in the larynx of low- and mid-exposure level rats. This suggests that basic chromium sulphate is most likely rapidly cleared from the respiratory tract due to its high water solubility. Evidence of systemic toxicity was primarily limited to reductions in body weight not related to reduced food consumption. A NOAEC was not established in this study based on the pathological findings in the respiratory tract. The LOAEC for this study was 17 mg/m³ chromium hydroxide sulphate, equivalent to 3 mg/m³ Cr³+.
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