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Repeated dose toxicity: inhalation

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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication meeting basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Studies of the Chronic Inhalation of Coal Fly Ash by Rats
Author:
Raabe, O.G. et al.
Year:
1982
Bibliographic source:
Ann Occup Hyg. 1982;26(1-4):189-211.

Materials and methods

Principles of method if other than guideline:
Inhalation toxicity study in male rats whole body exposed at two dose levels for 7, 50, 90 and 180 days.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): aerosols of size-classified power plant fly ash

The fly ash was obtained from the electrostatic precipitator hoppers of a power plant burning western U.S., low-sulphur, high-ash coal,
and size-classified to remove most particles larger than 3 µm in aerodynamic diameter.
The electrostatic precipitator at the power plant operated at about 110 °C.
Respirable aerosols of fly ash were generated and transported to large (about 4 m3) exposure chambers.
The fly ash aerosols were generated using a Wright Dust Feed (WDF) mechanism (WRIGHT, 1950) by passing the dust flow from the WDF through a miniature cyclone designed to remove most particles and aggregates bigger than 2.5 pm in aerodynamic diameter. The cyclone and WDF cutter blade were constructed of mild steel (1.03% C, 0.5% Mn, 0.04% P and 0.05% S) to preclude nickel and chromium contamination of the aerosols.
The standard WDF cutter blade is composed of 95% copper and contains less than 0.05% Al, Be, Cr, Fe, Mn, Ni and Zn. This standard blade was rapidly abraded during pilot studies of fly ash re-aerosolisation while the use of the tempered, mild steel blade resulted in minimal abrasion during the chronic inhalation studies.


Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hilltop chronic respiratory disease (CRD)-free Sprague-Dawley rats
- Age at study initiation: 70 d
- Weight at study initiation: 334 - 372 g
- Housing: in pairs in stainless steel wire mesh cages, placed in a monolayer planar array perpendicular to the direction of aerosol flow


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: MMAD about 2 µm
Volume median diameter (VMD) of 1.77 µm
Geometric standard deviation of 1.52
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Large (about 4 m³) exposure chambers

- Method of conditioning air: Air supplied to each chamber was filtered for removal of particulate, chemical and bacteriological contaminants.

- System of generating particulates/aerosols: The fly ash aerosols were generated using a Wright Dust Feed (WDF) mechanism by passing the dust flow from the WDF through a miniature cyclone designed to remove most particles and aggregates bigger than 2.5 µm in aerodynamic diameter. The cyclone and WDF cutter blade were constructed of mild steel (1.03% C, 0.5% Mn, 0.04% P and 0.05% S) to preclude nickel and chromium contamination of the aerosols. The standard WDF cutter blade is composed of 95% copper and contains less than 0.05% Al, Be, Cr, Fe, Mn, Ni and Zn. This standard blade was rapidly abraded during pilot studies of fly ash re-aerosolization while the use of the tempered, mild steel blade resulted in minimal abrasion during the chronic inhalation studies.

- Temperature, humidity, pressure in air chamber: about 21 +/- 2 °C and 50 +/- 20% relative humidity

- Air flow rate: The resulting aerosols were then passed through a 85Kr-discharger to reduce the particle electrostatic charge to Boltzmann equilibrium, and then mixed with the main air flow of about 2000 L/min entering the exposure chambers.

- Air change rate: 30 air-volume changes per h were passed through each chamber after passage through a Mine Safety Appliance Model 15-86475 CBR filter assembly.

- Method of particle size determination: During exposure of rats, the fly ash aerosols were continuously monitored using a Royco 225 light-scattering particle counter, providing a strip chart recording of particle number concentration. Comparison measurements were made using a Climet 208 light-scattering particle counter. Particle size distribution data were collected with these instruments operated in conjunction with a multichannel pulse height analyser.

TEST ATMOSPHERE
Samples were collected periodically with a small point-to-plane electrostatic precipitator designed to collect representative samples for evaluation by electron microscopy. These samples were studied using a Zeiss EM 10A transmission electron microscope and an ETEC Autoscan VI scanning electron microscope.
Size data from electron micrographs were collected using a Zeiss particle size analyser and log-normal functions were fit to the size distribution data using the grouped data of projected-area diameters and the maximum likelihood method described by RAABE (1971).
Ninety-min samples were collected at 21 L/ min using a Sierra 216 AL radial slot impactor in order to evaluate the aerodynamic size distributions of the aerosols. Size-separated fractions were collected on pre-weighed glass fibre filter substrates placed on the impactor stages. Collected masses were determined by weighing. The mass distribution data collected with the cascade impactor were analysed by fitting log-normal functions with respect to aerodynamic (resistance) diameter as defined by RAABE (1976). Mass concentrations were measured by collection of samples on pre-weighed glass fibre and membrane filters by weighing the samples collected for 90 min at 25 L/min. Chemical analyses were conducted using samples collected on Nuclepore filters by atomic absorption and instrumental neutron activation (COLES et al., 1979).
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
8 h/day for up to 180 consecutive days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
between 0.57 and 0.67 mg/m³
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
between 4.2 and 4.3 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
(See Table 1 for details)
Series I: 9, 9 and 32 animals per dose exposed for 7, 50 and 90 days, respectively
Series II: 2 groups of 24 animals per dose exposed for 90 and 180 days, respectively
Control animals:
other: Controls were exposed to clean air under identical conditions
Details on study design:
In Series I (low dose), rats were removed and sacrificed for analysis after 7, 50 and 90 days of exposure.
Some of the 90-day animals were kept for a 23-day clearance period. Equal numbers of controls were also removed at the same time.

In Series II (high dose), exposed rats and controls were removed at 90 and 180 days for analysis.
Positive control:
no positive control used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, but no further details given

BODY WEIGHT: Yes
- Time schedule for examinations: at the beginning of the study and termination after 7, 50, 90 or 180 days.

LUNG BURDEN MEASUREMENTS:
Lungs were digested for analysis of aluminium content by atomic absorption spectroscopy

CALCULATION OF DEPOSITED FRACTION IN LUNG:
The total volumes of air inhaled during the exposure periods were multiplied by the appropriate average fly ash concentrations to yield the total mass of inhaled fly ash.

BIOCHEMICAL LUNG EVALUATIONS:
DNA, RNA and protein contents of homogenized lungs were determined.
Tracheal tissue culture techniques were used for determinations of mucus glycoprotein synthesis and secretion rates.

CELLULAR STUDIES:
Pulmonary alveolar macrophages and haematopoietic progenitor cell kinetics were analyzed.


Sacrifice and pathology:
PATHOLOGY:
Series I: 9, each of exposed and control rats were evaluated morphologically at 7, 50 and 90 days of exposure
Series II: 9, each of exposed and control rats were evaluated morphologically at 90 and 180 days of exposure

GROSS PATHOLOGY:
Lungs were collected, thoracic viscera were removed, the trachea was cannulated and the lungs were fixed by airway perfusion with Karnovsky's fixative, lung volumes were determined.

HISTOPATHOLOGY:
Lungs were analyzed by light microscopy (LM) and scanning electron microscopy (SEM).
Because intratracheal instillation of fixative might translocate either free inhaled particles or phagocytized particles, some of the animals were rapidly frozen for LM and SEM analyses.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities occured. No clinical signs of toxicity were observed in the animals of both dose groups.

BODY WEIGHT AND WEIGHT GAIN
No gross effects of exposure to fly ash on rat body weight were observed.

ORGAN WEIGHTS:
A small, but significant, increase in lung water content was observed after 7 days of exposure suggesting the presence of a mild oedema in these acutely exposed rats; longer periods of exposure did not seem to be associated with continued increased levels of lung water.

GROSS PATHOLOGY and HISTOPATHOLOGY: NON-NEOPLASTIC
No remarkable responses were observed in Series I. The longer and higher concentration Series II was conducted because of the negative results of Series I. The microbiological test before, during and after exposure for Series II showed no unusual or major bacterial invasion of the respiratory tract and it was completely free of any growth in culture for both controls and exposed animals at the end of the 180-day exposure.

Light microscope evaluation of large airway and pulmonary parenchyma showed exposed and control animals to be distinguished by only one histological feature; namely, exposed animals consistently had higher concentrations of pulmonary alveolar macrophages in the alveolar lumens and refractile brownish pigment was visible within these cells. The brown granular material within the macrophages was presumed to be fly ash. Also, the pulmonary alveolar macrophages in the lungs of exposed rats were significantly larger (p < 0.001) in observed diameter than those of control lungs (17.0µm for exposed versus 13.3 µm for controls). Alveolar septal walls occasionally were observed to contain small cellular aggregates consisting of some mononuclear leukocytes admixed with brownish particulate material (ash). The observed differences between exposed and control groups appeared to have minimal impact, if any, on the health status of the rats.

Scanning electron microscope evaluation of the lung airways focused on the centriacinar region, since this anatomical site is especially sensitive to damage by a variety of inhaled irritants. Morphological differences were not apparent between lungs of control and exposed rats. Back-scatter electron studies showed clumping and accumulation of particles in pulmonary alveolar macrophages.

LUNG BURDENS:
Rat lung homogenates were prepared from animals exposed to fly ash for 7, 50 or 90 days without clearance, or 90 days followed by 23 days clearance in Series I, and 90 or 180 days in Series II. The exposed rats had progressively increasing aluminium contents in their lungs at 7, 50 and 90 days of exposure. After the 23-day clearance period, the exposed rats contained essentially the same level of aluminium per lung as they did after 90 days of exposure.

CALCULATION OF DEPOSITION:
The body weights of the rats taken during exposure were used to estimate the total volume of fly ash aerosol breathed during the exposure periods.
Minute volumes were used in conjunction with the average measured aerosol mass concentrations to estimate the total mass of fly ash inhaled.
The lung burden of fly ash determined from the measured masses of Al in the lungs (assuming 12.6% Al in fly ash) were divided by the calculated total mass of inhaled fly ash to yield the apparent deposition fraction. For purposes of this calculation, clearance was assumed to be negligible. Hence, any clearance that did occur during exposure is reflected by a lower apparent deposition fraction. See Table 2 for details.

BIOCHEMICAL ANALYSES:
No significant changes were observed in the lung DNA contents.

MUCUS GLYCOPROTEIN SYNTHESIS AND SECRETION RATES:
Tracheal mucus glycoprotein secretion was decreased at 7, 50 and 90 days of exposure and increased at 180 days:
In Series I (low dose) a progressively decreasing rate of glycoprotein secretion by cultured rat trachea was observed after 50 and 90 days of exposure; the decrease after 90 days was significant. Since the tissue levels of glycoprotein in exposed rats (as % of control) remained approx. constant over this interval, the decreased rate of secretion suggests functional impairment of the secretory apparatus of the tracheal epithelial cells.
In Series II (high dose) glycoprotein secretion rates of exposed rats were increased above control values for rats exposed for 6 months.

CELLULAR STUDIES
Macrophages lavaged from lungs of exposed rats were more numerous and yielded more progenitor cell colonies in culture (at the 10% confidence level based on the Mann-Whitney rank-sum test) than from controls.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
4.2 mg/m³ air (analytical)
Based on:
other: test. mat. (respirable fraction of ashes)
Sex:
male
Basis for effect level:
other: no systemic effects
Dose descriptor:
LOEC
Remarks:
local
Effect level:
4.2 mg/m³ air (analytical)
Based on:
other: test. mat. (respirable fraction of ashes)
Sex:
male
Basis for effect level:
other: higher cellularity in alveolar septal walls, larger pulmonary alveolar macrophages and higher concentrations of pulmonary alveolar macrophages in the alveolar lumens

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 2

Deposition of inhaled fly ash in rats

Exposure time

(days)

Number of animals

Lung aluminiuma

(μg)

Minute volumeb(mL)

Inhaled fly ashb

(μg)

Lung burdenc

(μg)

Apparent deposition fraction (%)

Series I

 

 

 

 

 

 

7

4

6.8

184

455

54

11.8

50

4

34.1

207

3260

271

8.3

90

4

54.2

207

6180

430

6.9

90 + 23 days of clearance

6

40.0

213

6360

317

5.0

Series II

 

 

 

 

 

 

90

6

218

217

40750

1730

4.2

180

6

545

230

83300

4330

5.2

aNet after subtraction of control values; measured by atomic absorption spectroscopy

bCalculated values

cBased on 12.6 % Aluminium concentration

Applicant's summary and conclusion

Conclusions:
Using light microscopy and scanning electron microscopy, histological and cellular observations showed large numbers of small fly ash particles in the lung. However, there was no evidence of spontaneous lung disease and the animals were in good health at the end of the 180 days of exposure to 4.2 mg/m³. No major adverse effects were observed.
The only effects observed included small changes in some biochemical parameters and increased numbers of macrophages in the lung lumens. Additionally, the observed increase in colony-forming units of alveolar macrophages in culture from exposed animals without increases in activity of haematopoietic progenitor cells was indicative of recruitment of macrophages within the lung and activation of lung reserve progenitors as a direct result of deposition of fly ash. This response was considered an important natural response to inhaled particles and not being unique to coal fly ash.
Based on these findings, 4.2 mg/m³ of coal fly ash was considered a NOAEC for systemic effects and a LOEC for local effects.
The respirable fraction of Ashes (residues), cenospheres typically accounts for < 1.5% of the total mass (s. Particle size distribution). Accordingly, the systemic NOAEC/local LOEC value mentioned above correspond to a systemic NOAEC/local LOEC of ca. 280 mg/m³ based on total Ashes (residues), cenospheres.