Hematite, chromium green black

Acute Toxicity: other routes

Administrative data

Endpoint:

acute toxicity: other routes

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020-02-24 to 2020-03-02

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

This study was conducted as a dose range finding study, with limited study design focussing on assessing local effects of five different inorganic pigment substances. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess acute inhalation toxicity. The investigated mechanistic parameters (bronchoalveolar lavage (BAL) fluid analyses have no direct value for fullfilling data requirements under the REACH regulation.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 412 (28-day (subacute) inhalation toxicity study)

Version / remarks:

2018-06-15

Deviations:

yes

Remarks:

The study was conducted as range-finding study. Therefore, it has a limited study design.

Principles of method if other than guideline:

Female Wistar rats were exposed to chromium iron oxide at doses of 0.2, 0.8 or 3.2 mg by intratracheal instillation (total dose was instilled on two consecutive days) to investigate the lung toxicity potential with a bronchoalveolar lavage (BAL) analysis.

GLP compliance:

no

Remarks:

The investigations in this study were conducted under non-GLP conditions because of the screening, dose-range finding nature of the experiments. However, the experimental procedures followed the GLP rules (e.g. use of SOPs and documentation/archiving).

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chromium iron oxide

EC Number:

235-790-8

EC Name:

Chromium iron oxide

Cas Number:

12737-27-8

Molecular formula:

Fe(x)Cr(2-x)O3 0,65≤x≤1,75

IUPAC Name:

Chromium iron hematite

Test material form:

solid: particulate/powder

Details on test material:

- Chemical description: Chromium iron oxide
- Substance type: inorganic pigment
- State of aggregation: solid, black powder, odourless, Hematite-corundum structure
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry, protected from light

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 8 weeks old
- Weight at study initiation: approx. 182 g
- Housing: housed in Makrolon® (polycarbonate) cages type Ill, four rats per cage in the treated groups and three rats per cage in the vehicle control group; absorbing softwood ('ssn BK 8-15') bedding material.
- Diet: commercial chow in pellet form (ssniff”V1534; supplier: ssniff Spezialdiäten GmbH, Soest, Germany)
- Water: tap water
- Acclimation: approximately one week the animals were allowed to adjust and become acclimatized to the Fraunhofer ITEM environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

other: intratracheal instillation

Vehicle:

other: saline solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in vehicle, each aliquot in a volume of 0.3 mL. After gentle shaking all samples were sonicated for 2 minutes to guarantee a homogeneous suspension. After sonication samples were shaken again to perpetuate the homogeneity until administration to the animals.
The total dose was instilled in two aliquots on two consecutive days to achieve a homogeneous distribution of the test material in lungs.

Doses:

0.2, 0.8 and 3.2 mg

No. of animals per sex per dose:

4 females intreated group; 6 females in control group

Control animals:

yes

Remarks:

vehicle control

Details on study design:

- Duration of observation period following administration: 3 days
- Frequency of observations and weighing: all animals were clinically observed in their cages at least twice a day. Before sacrifice, they were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition. On the treatment days, the animals were clinically observed before and after treatment.
Individual body weight was recorded on day -1 before treatment and on day 3 at sacrifice for all animals.
- Necropsy of survivors performed: yes, all animals were subjected to a complete necropsy.

ORGAN WEIGHT:
the lungs were weighed, and the relative lung weight was calculated.

BRONCHOALVEOLAR FLUID (BALF) ANALYSIS:
analysis was performed in all rats 3 days after the last instillation. The total cell count and differential cell count were determined. The method of Henderson et al. (1987) was used with minor modifications.*
Following preparation, the lungs were lavaged with saline using two lavages of 4 ml (if half lung will be used only: 2 ml). The pooled lavage fluid was collected in calibrated tubes and the harvested volume was recorded. Until processing the lavage fluid was kept on ice. Leukocyte concentration of the lavagate was determined using a counting chamber and two cytoslides were prepared with a cytocentrifuge for differential cell count (macrophages, neutrophils, eosinophils, lymphocytes).

*Reference:
Henderson, R.F., Mauderly, J.L. Pickrell, J.A., Hahn, R.F., Muhle, H., and Rebar, A.H. (1987): Comparative study of bronchoalveolar lavage fluid: Effect of species, age and method of lavage. Exp. Lung Res. 13, 329 342.

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Body weight and lung wet weight data were calculated using a two-sample t-test assuming equal variances. BAL data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test.

Results and discussion

Effect levels

Remarks on result:

not determinable because of methodological limitations

Mortality:

All animals survived the study.

Clinical signs:

All animals tolerated well the exposure to the test item at all concentrations. No clinical observations outside the normal range were recorded.

Body weight:

Body weight development did not show any statistically significant changes as compared to concurrent controls.

Gross pathology:

Upon necropsy, test item- or dose-related macroscopical findings were observed. The lung associated lymph nodes (LALN) were moderately enlarged in one animal of the mid-dose group, and slightly to moderately enlarged in two animals of the high-dose group. There were some dark grey areas on whole lung in all animals of the mid- and high-dose group.The animals in the low-dose group, were without any special findings.

Other findings:

ORGAN WEIGHT:
The absolute lung weight was statistically significant increased in the low- and high-dose group (1.065 ± 0.028 g and 1.070 ± 0.046; P< 0.05) compared to the controls (0.985 ± 0.051 g), but the relative lung weight was not increased.

BRONCHOALVEOLAR LAVAGE FLUID ANALYSIS:
At day 3 post-treatment, a statistically significant increase in the percentage of lymphocytes were observed in the animals of the high-dose group (1.31 ± 0.55 %; P < 0.05) compared to the controls (0.08 ± 0.20 %). The low- and mid-dose animals showed %lymphocyte values in the range of the control animals (1.13 ± 1.27 and 0.31 ± 0.38 %).

Applicant's summary and conclusion

Conclusions:

Female Wistar rats were exposed to chromium iron oxide at doses of 0.2, 0.8 or 3.2 mg by intratracheal instillation (total dose was instilled on two consecutive days) to investigate the lung toxicity potential with a bronchoalveolar lavage (BAL) analysis. A vehicle control group was run concurrently.

This study was conducted as a dose range finding study, with limited study design focussing on assessing local effects of five different inorganic pigment substances.
The highest dose in this instillation experiment was assumed to lead to an overload condition (by way of read-across from other PSLT substances), which leads to an inflammatory response inter alia characterised by an increase of granulocytic cells.

Upon necropsy, test item- or dose-related macroscopical findings were observed, like enlarged lung associated lymph nodes (LALN), and some dark grey areas on whole lung in all animals of the mid- and high-dose group. The absolute but not the relative lung weights were increased in the animals of the low- and high-dose groups compared to controls.

At day 3 post-treatment, a statistically significant increase in the percentage of lymphocytes were observed in the animals of the high-dose group (1.31 %; P < 0.05) compared to the controls (0.08 %). The low- and mid-dose animals showed %lymphocyte values in the range of the control animals (1.13 and 0.31%).

On the basis of the results obtained in the BALF analysis after in vivo instillation of five different inorganic pigments combined with deposition modelling (using the MPPD model), the pigment with the highest reactivity in the respiratory tract was used for a subsequent 14-day inhalation dose-range finding study.
For the other four inorganic pigments, the concentrations for the main 90-day study was based on the results obtained in this instillation experiments combined with deposition modelling (using the MPPD model).