Trichromium dicarbide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Read-across to chromium(III) oxide. The release of chromium from chromium carbide is very similar to the release from chromium metal and chromium(III) oxide and therefore the results obtained with these substances can readily be used in the assessment of trichromium dicarbide. Comparable to guideline study. The reliability score 1 is given, although read-across is used. This is motivated due to the similarity in the release of chromium from chromium(II) oxide and chromium carbide.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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., W. E. Rinehart, et al.

Year:

1999

Bibliographic source:

Toxicol Sci.52: 278-288.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Principles of method if other than guideline:

Nose-only exposures of chromium(III) oxide dust (4.4, 15 or 44 mg/m3) was carried out for 6 h/day 5 days/week during 13 weeks on 15 male and 15 female rats per concentration. 10 rats/sex were sacrificed at the end of the exposure and 5 rats/sex were maintained for a 13 week recovery period. An additional satellite group of 5 rats/sex/concentration was exposed to the same concentrations of chromium(III) oxide for 5 consecutive days in order to evaluate bronchoalveolar lavage parameters.
Clinical signs of toxicity were observed during the whole study period. At the study termination, standars haematology, clinical chemistry and urinalysis were conducted. Bone marrow smears were prepared, organs were weighed and tissues typically harvested in subchronic toxicity studies were preserved and evaluated microscopically.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chromium(III) oxide

IUPAC Name:

Chromium(III) oxide

Details on test material:

- Name of test material (as cited in study report): Chromic oxide (CAS No. 1308-38-9)
- Molecular formula (if other than submission substance): Cr2O3
- Molecular weight (if other than submission substance): 152
- Physical state: dark green powder
- Analytical purity: >99% Cr(III) as Cr2O3
- Impurities (identity and concentrations): < 0.0002% Cr(VI)
- Composition of test material, percentage of components: >99% Cr(III) as Cr2O3
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: in a separate, unused 1 m3 chamber that was continuously purged with a low flow of dry compressed air.
- Other:

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CDF (Fischer 344)Crl BR VAF/Plus rats from Charles River Laboratories (Raleigh, NC, USA)
- Age at study initiation: 7 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individual housing in stainless steel, suspended wire-mesh cages
- Diet (e.g. ad libitum): commercial laboratory feed Purina Certified Rodent Chow 5002 ad libitum
- Water (e.g. ad libitum): yes ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 degrees Celsius
- Humidity (%): 43±11% relative humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: week 13 To: week 26 (5 animals/sex/group)

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The MMAD in micrometers (GSD) over 13 weeks (21 samples per test group) were 1.8 (1.93), 1.9 (1.84) and 1.9 (1.78) for the 4.4, 15 and 44 mg chromium oxide/m3 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: no data
- Source and rate of air: no data
- Method of conditioning air: no data
- System of generating particulates/aerosols: accomplished with a low-output dust generator, using spinning glass beads over a packed cake of test material
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: at least 12 chamber air changes per hour
- Method of particle size determination: made from each exposure level using a cascade impactor once per day for the first two weeks, and weekly thereafter
- Treatment of exhaust air: no data


TEST ATMOSPHERE
- 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)
- Samples taken from breathing zone: no data on where the samples were taken from

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). The mean aerosol concentrations over 13 week were 4.4±0.23, 15±1.2 and 44 ±3.7 mg/m3 for the three chromium(III) oxide groups, corresponding to ca 3, 10 and 30 mg Cr(III)/m3..

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week, 6 hours/day

Doses / concentrations

Remarks:

Doses / Concentrations:
mean conc. 4.4, 15, 44 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

15

Control animals:

yes, sham-exposed

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: randomly assigned to groups based on body weight.
- Rationale for selecting satellite groups: additional rats (5/sex/dose) from those described above were randomly selected
- Post-exposure recovery period in satellite groups: no (bronchoalveolar lavage analyses were performed immediately after the 5 day exposure, after which the animals were removed from the study)

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- daily observations prior to and following each exposure for clinical signs of toxicity
- twice daily for morbidity and mortality during the recovery period and on weekends


BODY WEIGHT: Yes
- Time schedule for examinations: Recorded weekly during the exposure and recovery period


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data


FOOD EFFICIENCY:
- 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: No data


WATER CONSUMPTION: No data


OPHTHALMOSCOPIC EXAMINATION: Yes (indirect ophthalmoscopic examination)
- Time schedule for examinations: during the acclimation period and prior to terminal necropsy
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of exposure
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10/sex/group
- No data on which parameters were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of exposure
- Animals fasted: No data
- How many animals:10/sex/group
- No data on which parameters were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: at the end of exposure
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Determinations were performed by gross observation, microscopy and automated clinical analyzer. No data on parameters examined. Aliquots of the remaining urine from 5 animals/sex from the control group and the high exposure group were submitted for Beta2-microglobulin analysis.


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
- sperm evaluation (at necropsy, sperm samples from the left caudal epididymis of 10 males per group)
- bronchoalveolar lavage fluid (BALF) analysis (on additional rats 5/sex/dose, exposure period 5 days)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

Statistical analyses were performed on body weights, clinical pathology laboratory tests, organ weights and BALF data using one-way analysis of variance. If the result was non-significant, no further tests were 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. Bartlett's test was significant, the Welch t-test with Bobferroni correction was used for pairwise comparison.
The Kruskal-Wallis analysis of variance, followed where appropriate by the Mann-Whitney U test, was used for those parameters where parametric analysis was inappropriate. p<0.05 was set as the level for statistical significance.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

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 examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Macroscopic examination showed green discoloration of the lungs in all exposure groups. Increasing the chromium oxide dose also increased the intensity of the colour. Microscopic examination showed accumulation of black pigment in alveolar spaces, at the tracheal bifurcation, in the peribronchial lymphoid tissue and the mediastinal lymph node. In some animals exposed to medium or high concentrations, trace to mild chronic interstitial inflammation was observed in the alveolar septa surrounding aggregates of pigmented macrophages. This was accompanied by hyperplasia in some animals.
CLINICAL SIGNS AND MORTALITY
No chromium(III) oxide related mortality or clinical signs of toxicity were observed at any exposure levels.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of male and female rats during exposures did not show any statistically significant differences as compared to the control groups. Mean body weights of males exposed to 44 mg/m3 chromium(III) oxide were slightly lower than controls during the recovery period, but weight gains for these animals did not differ from the control animals.

FOOD CONSUMPTION
No data

FOOD EFFICIENCY
No data

WATER CONSUMPTION
No data

OPHTHALMOSCOPIC EXAMINATION
No exposure-related effects

HAEMATOLOGY
No exposure-related effects

CLINICAL CHEMISTRY
No exposure-related effects

URINALYSIS
No exposure-related effects

NEUROBEHAVIOUR
No data

ORGAN WEIGHTS
Small, statistically significant increases in mean absolute and relative lung/trachea weights were observed among high-exposure (44 mg/m3) male rats, but not in females. This increase was explained by macroscopic and histologic changes.
Mean absolute and relative thyroid/parathyroid weights were significantly increased in the mid-exposure-group (15 mg/m3) females. Mean relative thyroid/parathyroid/body weight ratios in the 44 mg/m3 group of female rats were also significantly increased. The weight changes in these organs were very small, and their biological importance could not be determined.
At the recovery sacrifice (13 week recovery after exposures), the differences in organ weights of all the exposure groups compared with the controls were unsignificant.

GROSS PATHOLOGY
Most of the animals (both at the terminal and rcovery sacrifices) showed exposure-related macroscopic changes in the lungs and mediastinal lymph nodes. Green dicoloration was observed at all exposure levels, and increased with exposure concentration. Mediastinal lymph node enlargement was also observed in the 44 mg/m3 groups after the recovery period. In addition, a few other macroscopic observations were made, but these were considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Aggregates or foci of macrophages filled with dense black pigment were randomly distributed adjacent to the junctions of terminal bronchioles and alveolar ducts, and subjacent to the pleura in male and female rats exposed to all concentrations of chromium(III) oxide. Black pigment was also seen in the tracheal bifurcation, peribronchial lymphoid tissue, and within the mediastinal lymph node.
The pigment was assumed to represent the test substance and it stained black with hematoxylin and eosin stain. It corresponded to the green discoloration observed macroscopically.
In some of the 15 and 44 mg/m3 exposure group rats (males and females), infiltration of inflammatory cells in the alveolar septa surrounding aggregates of pigmented macrophages, indicated trace to mild chronic interstitial inflammation of the lungs. Chronic interstitial inflammation and septal cell hyperplasia (Type II pneumocytes) was furthermore observed in some males exposed to 15 or 44 mg/m3.
Lymphoid hyperplasia was observed in all test groups. No exposure-related lesions were observed in the nasal cavities of the exposed groups.
The microscopic changes observed were generally associated with the pigment and corresponded to the increased lung weight observed among males in the high-exposure group.
In animals sacrificed after the 13-week recovery period, trace to mild pigmented macrophages and black pigment were still found in the peribronchial lymphoid tissue, at about the same incidence and severity as in animals sacrificed immediately after the exposures. In all male treatment groups, and in the 15 and 44 mg/m3 female groups, trace to mild septal hyperplasia and trace to mild chronic interstitial inflammation persisted. The severity of these lesions were similar, or slightly increased, as compared with the terminal-sacrifice groups. Accumulation of black pigment in the mediastinal lymph nodes of all groups persisted. Only in some males of the 4.4 and 15 mg/m3 groups was there an increase in the incidence, indicating a pulmonary clearance via the lymphatic system.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data




OTHER FINDINGS
- No exposure related effects were observed in the evaluations on sperm motility, morphology, or concentration.
- No statistically significant differences in any of the BAL parameters where obseved when comparing exposure groups with controls. A yellow intracytoplasmic, crystalline material was present within the mononuclear cells from all exposure groups.

Effect levels

Dose descriptor:

LOAEC

Remarks:

(chromium(III) oxide)

Effect level:

>= 4.4 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Mild inflammatory reactions observed already at the lowest concentration (4.4 mg/m3, corresponding to 3 mg Cr(III)/m3.)

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Mild inflammatory reactions were observed in male and female rats already at the lowest exposure level. According to these studies the LOAEC for chromium(III) oxide is 4.4 mg/m3 (corresponding to 3 mg Cr(III)/m3), which is in fact most likely not much higher than the NOAEL for chromium(III) oxide in rats, as the severity and frequency of inflammatory changes in the lungs were minimal.

Executive summary:

A thirteen-week subchronic inhalation toxicity (nose only) study was performed by Derelanko et al(1999). This rat study meets the requirements of the OECD/EU guidelines for repeated dose toxicity testing. 15 male and 15 female rats were exposed to graded doses of chromium oxide (mean concentrations 4.4, 15 and 44 mg/m³) for 5 days/week, 6 hours per day, followed by two days of rest during 13 weeks. No mortality or clinical signs of toxicity were observed during the study. Macroscopic examination showed green discoloration of the lungs in all exposure groups. Increasing the chromium oxide dose also increased the intensity of the colour. Microscopic examination showed accumulation of black pigment in alveolar spaces, at the tracheal bifurcation, in the peribronchial lymphoid tissue and the mediastinal lymph node. In some animals exposed to medium or high concentrations, trace to mild chronic interstitial inflammation was observed in the alveolar septa surrounding aggregates of pigmented macrophages. This was accompanied by hyperplasia in some animals. Haematology, clinical biochemistry and urinalysis did not show any differences as compared with the control group, and thus this study demonstrates that the effects of chromium oxide inhalation are restricted to the lungs. Studies with a satellite group, which recovered for 13 weeks after the exposure before being sacrificed, showed a very slow lung clearance of the material, as pigment was present also after a recovery period. Similar types of effects have also been observed with other types of insoluble dusts, for instance titanium dioxide, carbonyl iron or carbon black.

 Therefore, the effects seen in rat lungs may mostly occur due to the particle effect, and not due to the toxic properties of the substance. However, possible intrinsic toxicity of the chromium ion cannot completely be disclosed. No NOAEL can be set based on these results, as mild inflammatory reactions were observed already at the lowest exposure level. According to these studies the LOAEL is 4.4 mg/m³, which is in fact most likely not much higher than the NOAEL for chromium(III) oxide in rats, as the severity and frequency of inflammatory changes in the lungs were minimal.