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Repeated dose toxicity: inhalation

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sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Thirteen-week subchronic rat inhalation toxicity study with a recovery phase of trivalent chromium compounds, chromic oxide, and basic chromium sulfate.
Derelanko, M.J., Rinehart, W.E., Hilaski, R.J., Thompson, R.B. &, Löser, E.
Bibliographic source:
Toxicological Sciences 52: 278-288

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
please refer to the remarks field of the field "Rationale reliability incl. deficienceis " above
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Chromium (III) oxide
EC Number:
EC Name:
Chromium (III) oxide
Cas Number:
Molecular formula:
chromium (III) oxide
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: dark green powders
Specific details on test material used for the study:
- Source of test material: British Chrome Chemicals (Orlay Nook, Eaglescliffe, Cleveland, U.K.)

- Storage condition of test material: test article was stored in separate, unused 1-m³ chambers that were continuously purged with a low flow of dry compressed air.

Test animals

other: CDF (Fischer 344)/Crl BR VAF/Plus
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 7 weeks of age
- Housing: housed in groups for three days and then individually housed in stainless steel, suspended wire-mesh cages
- Diet (ad libitum): commercial laboratory feed (Purina Certified Rodent Chow No. 5002)
- Water (ad libitum): tap water

- Temperature: 21 ± 2 °C
- Relative humidity: 43 ± 11%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Mass median aerodynamic diameter (MMAD):
>= 1.8 - <= 1.9 µm
Remarks on MMAD:
GSD (range): 1.78 - 1.93
Details on inhalation exposure:
- Exposure apparatus: rats were exposed in stainless steel and acrylic nose-only inhalation chambers operated with at least 12 chamber air changes per hour.

- System of generating particulates/aerosols: 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.

- Method of particle size determination: particle-size measurements were made from each exposure level using a cascade impactor once per day for the first two weeks and weekly thereafter.

- Brief description of analytical method used: chamber samples were determined once per hour by standard gravimetric methods, with periodic analysis for Cr(III) and Cr(VI).

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Please refer to the field "Details on inhalation exposure" above.
Duration of treatment / exposure:
Main study: 13 consecutive weeks
Bronchoalveolar lavage parameters: 5 consecutive days
Frequency of treatment:
Main study: 6 hours/day, 5 days/week for 65 exposure days
Doses / concentrationsopen allclose all
Dose / conc.:
4.4 mg/m³ air (analytical)
SD: 0.23
Dose / conc.:
15 mg/m³ air (analytical)
SD: 1.2
Dose / conc.:
44 mg/m³ air (analytical)
SD: 3.7
No. of animals per sex per dose:
Main study: 15 animals/sex/dose
Bronchoalveolar lavage evaluation: 5 animals/sex/dose
Control animals:
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.
- Post-exposure recovery period (main study): 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.
Positive control:
not specified


Observations and examinations performed and frequency:
- Time schedule: daily prior to and following each exposure
- Cage side observations checked: clinical signs

- Time schedule: twice daily during the recovery period and on weekends
- Cage side observations checked: morbidity and mortality


- Time schedule for examinations: weekly during the exposure and recovery periods

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified


- Time schedule for examinations: during the acclimation period and prior to terminal necropsy

- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, water available
- How many animals: 10 per sex per group, designated for necropsy at the end of exposures
At necropsy, bone marrow smears were prepared and differential cell counts were evaluated.

- Animals fasted: Yes, water available
- How many animals: 10 per sex per group, designated for necropsy at the end of exposures

- Metabolism cages used for collection of urine: Yes, overnight
- Animals fasted: Not specified
- How many animals: 10 per sex per group, designated for necropsy at the end of exposures
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 Beta2-microglobulin analysis.

IMMUNOLOGY: Not specified
Sacrifice and 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. Microscopic evaluation was conducted on all hematoxylin 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:
Rats were anesthetized and the lungs, heart, trachea, larynx, and tongue were removed en-block. Following tracheal cannulation, the airways were washed with warmed, physiological saline 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.
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:
no effects observed
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Details on results:
- 4.4, 15 and 44 mg/m³: no compound-related mortalities occurred during the conduct of this study. Some animals died on exposure day 1 as a direct result of the restraint tubes, and they were replaced.

- 4.4, 15 and 44 mg/m³: male and female mean body weights during exposures to chromic oxide were not statistically different from the control group's 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.

- 4.4, 15 and 44 mg/m³: no exposure-related effects were noted in the ophthalmologic evaluations.

- 4.4, 15 and 44 mg/m³: 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 parameters.

- 4.4, 15 and 44 mg/m³: after 13 weeks of exposure, none of the exposure groups, for either sex, exhibited a statistically significant difference from the control group for any serum biochemical parameters.

- 4.4, 15 and 44 mg/m³: after 13 weeks of exposure, none of the exposure groups, for either sex, exhibited a statistically significant difference from the control group for any urinalysis parameters. Beta- microglobulins were not detected in urine samples from any group.

- 4.4, 15 and 44 mg/m³: Slight, yet statistically significant increases in mean absolute and relative lung/trachea weights occurred in high-exposure-level group males. 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-exposure-group females. These organ weight changes were very small. At the recovery sacrifice, organ weights of all the exposure groups were comparable to the control group.
Please also refer to the field "Any other information on results incl. tables" below.

- 4.4, 15 and 44 mg/m³: 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.
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 chromic oxide (terminal and recovery sacrifices).
Mediastinal lymph-node enlargement was observed at the recovery sacrifice in animals exposed to chromic oxide (high exposure only).
Additional macroscopic observations were few in number and considered incidental.

- 4.4, 15 and 44 mg/m³:
1) Terminal sacrifice: 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 mid- and 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 high-exposure-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.

2) Recovery sacrifice: 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 high-exposure-level groups. These lesions were the same or slightly increased in severity as compared to the terminal-sacrifice 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 lowest-exposure 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.

- 4.4, 15 and 44 mg/m³: 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-exposure-group 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.

Effect levels

Dose descriptor:
Effect level:
15 mg/m³ air (analytical)
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Selected organ weight changes at terminal sacrifice of rats exposed to chromic oxide

   Control  4.4 mg/m3  15 mg/m3             44 mg/m3 
 Males Lung/trachea           
wt (g)  0.99 + 0.07  0.98 + 0.055  1.0 + 0.077  1.11 + 0.050**
wt/bw (% x 10)   4.42 + 0.187  4.52 + 0.273  4.49 + 0.396  4.98 + 0.228**
 Females Lung/trachea           
 wt (g)  0.81 + 0.081  0.81 + 0.080 0.85 + 0.084  0.88 + 0.068 
 wt/bw (% x 10)  5.65 + 0.418 5.78 + 0.577  5.77 + 0.629  6.40 + 0.618 
 wt (mg)  12 + 1.9 13 + 1.3  15 + 1.5**   14 + 2.3
 wt/bw (% x 103)  8.26 + 1.493  8.89 + 0.880  10.10 + 1.147*  10.04 + 1.346*
Note: Organ weight changes given as mean + SD; bw = body weight. Organ weights of exposed animals were not statistically different from control animals at recovery sacrifice. * p </= 0.05; ** p </= 0.01            

Applicant's summary and conclusion

Derelanko et al. (1999) investigated the repeated dose inhalation toxicitiy of chromium oxide in groups of 15 male and 15 female CDF (Fischer 344/Crl BR VAF/Plus rats, which received the substance via nose-only inhalation at concentrations of 4.4, 15 and 44 mg/m³ air (actual concentration). The animals received the substance 6 hours/day, 5 days/week for 13 consecutive days. An control group was run concurrently. Five animals/sex of the treatment groups were used as recovery group at the end of the treatment period for a duration of 13 weeks. In addition, separate groups of 5 animals/sex/dose were investigated for bronchoalveolar lavage parameters. No test item-related effects were observed for clinical signs, mortality, body weights,ophthalmological examination, haematology, clinical chemistry and urinalysis. In addition, no test item-related effects were found during the bronchoalveolar lavage evaluation.

A NOAEC of 15 mg Cr2O3/m³ is derived on the basis of a dose-dependent mild inflammatory response characterised by an increase of phagocytotic cells and a subsequent increase of lung weights being significant in the male animals of the high dose group. None of the BALF parameters (lactate dehydrogenase (LDH), total protein, beta-glucuronidase, and glutathione reductase) was affected after 90-day inhalation exposure, indicating a lack of tissue damage. No other adverse effects were observed and the inflammatory response was completely reversible within the recovery period.

The study is generally well documented and meets generally accepted scientific standards. A number of methodological or reporting deficiencies when comparing the publications with the relevant OECD TG 413 (1981): the guideline foresees that if recovery from test item-related effects is investigated, this may be done in a separate group of animals (10 animals/sex) given the highest dose and observing these animals during a post-treatment period of normally 28 days. In this study five animals of the regular high dose group were kept for a post-exposure group lasting 13 weeks; exposure parameters were not recorded( oxygen content, air flow, temperature, humidity); uncertainty, if equilibration of the chamber concentration was checked; clinical observations during exposure were not recorded; food consumption measurements were missing; haematological, clinical chemistry, and urinalysis parameters were not defined (only statement that these investigations were carried out); histopathological examination was not defined, but it was stated by the author that tissues harvested for this type of study were examined; historical and individual data were missing; exposure apparatus was not described in detail; unclear if actual concentration was measured in the animal's breathing zone