Inhalation Tolerance Study for p-Aramid Respirable Fiber-Shaped Particulates (RFP) in Rats

B. Bellmann1, O. Creutzenberg, C. Dasenbrock, H. Ernst, G. Pohlmann and H. Muhle

Fraunhofer Institute of Toxicology and Aerosol Research, D-30625 Hannover, Germany

Received July 1, 1999; accepted November 11, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was designed to assess the lung clearance function in rats after subchronic exposure to p-aramid respirable fiber-shaped particulates (RFP). Male Wistar rats were exposed 6 hrs/day, 5 days/week for 3 months to 50, 200, and 800 RFP/ml measured by scanning electron microscopy (SEM). Recovery effects were followed up through 9 months postexposure. The retention of RFP (length > 5 µm) was about 25 x 106 RFPs per lung in the low dose group after 3 months of exposure. The corresponding values in the medium and high dose groups amounted to overproportionally higher values of 122 x 106 and 576 x 106 RFPs per lung, respectively. A decrease in the length of the retained RFPs over the 9-month recovery period was observed, indicating a breakage of long fibrils. Alveolar clearance half-times measured by {gamma} tracers indicated a dust overloading of lungs for the high dose group at 0 and 3 months postexposure. Bronchoalveolar lavage parameters revealed that p-aramid RFPs induced pronounced inflammatory effects in the high and medium dose groups. Histopathologically, slight fibrotic and hyperplastic lesions were observed in the medium and high dose groups directly after the end of exposure. The findings at the 3-month postexposure interval resulted in a reduction of inflammatory changes in the medium and high dose groups compared to the sacrifices upon cessation of exposure. No histopathologic effects were detected in the low dose group. In the high dose group the maximum functionally tolerated dose was exceeded. The No Observed Adverse Effect Level (NOAEL) of RFP was 50 RFP/ml as measured by SEM.

Key Words: p-aramid; fiber-shaped; particulates; inhalation; rat; lung; dust overloading..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
p-Aramid fibers, as spun, consist of continuous filaments with a diameter of 10–20 µm. Abrasion due to mechanical processes can generate respirable-sized fibrils. Free, broken-off p-aramid fibrils have a strong tendency to agglomerate and to form nonrespirable clumps. Free p-aramid respirable-sized fiber-shaped particulates (RFP) have a typical ribbon shape (Lee et al., 1988Go; Searl, 1997Go).

Exposure to airborne p-aramid RFP in critical dimensions during manufacturing and at the workplace ranges in concentrations from 0.005 to 0.4 RFP/ml (8-h time weighted average), measured by optical microscopy (Cherrie et al., 1995Go). A substantial fraction of RFP have critical dimensions of length > 5 µm, diameter < 3 µm, and aspect ratio >= 3:1 (WHO, 1985). This WHO criterion was used consistently throughout this study.

A 2-year exposure to p-aramid RFP at concentrations (measured by optical microscopy) of 100 RFP/ml and a 1-year exposure of 400 RFP/ml followed by a 1-year recovery period caused an increased incidence of proliferative lesions in rats (Lee et al., 1988Go). Similar proliferative lesions have been observed in rat lungs under particle overload situations in other studies (Lee et al., 1985Go; Mermelstein et al., 1991Go; Muhle et al., 1991Go). If particle overload could be demonstrated at the above-mentioned concentrations, then the effects observed in the Lee study (1988) might be considered to be nonspecific to the test substance and related primarily to dust overload conditions.

The definition of the maximum tolerated dose (MTD) for solid particles is a subject of controversy (Muhle et al., 1990aGo; Oberdörster, 1997Go). The MTD, as defined by Sontag et al. (1976), is the highest dose of a test agent that can be predicted not to alter the animals' normal longevity from effects other than carcinogenicity during a chronic study. An additional provisional guideline of the U. S. Environmental Protection Agency indicates that a 10% increase in target organ weight also satisfies the MTD criterion (Environmental Protection Agency, 1986Go).

For inhalation studies with solid particles, a modification of the MTD definition was suggested to include a maximum functionally tolerated dose (MFTD), which is the maximum lung burden above which macrophage-mediated lung clearance is significantly impaired. However, no generally accepted definitions exist either for the MTD or the MFTD for solid particles (Oberdörster, 1997Go). As a reasonable criterion, a persistent 2- to 4-fold increase in macrophage-mediated clearance half-time as measured by the test material or suitable surrogate has been proposed (Muhle et al., 1990aGo). The objectives of the study were:


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
The test substance was a special batch of p-aramid fibrils. The preparation of p-aramid respirable-sized fiber-shaped particulates (RFP) has been described in the paper of Lee et al. (1988). For the purpose of this study, the test substance was characterized by analysis of the RFP size distribution of the aerosol using a scanning electron microscopy (SEM) technique (Bellmann and Muhle, 1994Go) with minor modifications.

Size distribution.
For sizing, videoprints of the SEM screen with different magnifications were used (3000 x for RFPs with length > 20 µm, 6000 x for RFPs with length < 20 µm). Only RFPs with length > 1 µm were measured. For each of the aerosol samples, the length and the diameter of at least 200 RFPs (length:diameter > 3:1, length > 1 µm) including at least 100 RFP longer than 5 µm were measured. The different areas used for analysis of the different length fractions were taken into account by using weighting factors. The arithmetic and geometric means and standard deviations of RFP length and diameter are given in Table 1Go. In Table 2Go the 10, 50, and 90 percentiles of the length, diameter, and calculated aerodynamic diameter distribution are summarized.


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TABLE 1 Size Distribution of Aerosol Samples of RFPs
 

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TABLE 2 Size Distribution of Aerosol Samples of RFPs: 10, 50, and 90 Percentiles
 
Animal model.
Male Wistar rats (strain Crl:WI(WU)BR) were purchased from Charles River Deutschland, Sulzfeld, Germany. The age of the animals at the start of exposure was 10 weeks. Animals were housed under conventional laboratory conditions: two rats per polycarbonate cage (800 cm2), tap water and pelleted food (1324N spec. prepared, Altromin International, Lage, Germany) ad libitum, softwood bedding (altromin 3/4, Altromin International, Lage, Germany), room temperature 22 ± 2°C, relative humidity: 55 ±15%, 12-h dark/light cycling. During exposure the rats were fixed in nose-only tubes (C. R. Equipments, Tannay, Switzerland) with no access to food and water. All animals were observed in their cages on a daily basis. Individual body weights were recorded once per week up to 3 months and thereafter every 2 weeks throughout the study.

General study outline.
A 3-month subchronic inhalation study with a 9-month postexposure observation period was performed. The study design is shown in Table 3Go. The presented RFP concentrations are SEM values. The justification for selecting the aerosol concentrations used in this study was to adjust to exposure conditions similar to those reported in the study of Lee et al. (1988).


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TABLE 3 Study Design
 
The duration of exposure was 6 h/day, 5 days/week for 3 months (total number of exposure days: 65). Subsequent to this period, there was a 9 month postexposure observation period.

Aerosol generation and exposure.
The aerosol generation was performed using the set-up of DuPont Haskell Laboratory (Warheit et al., 1992Go). Atmospheres of ultrafine RFPs were generated with a K-tron bin feeder (K-tron Co., Glassboro, NJ) equipped with twin screws. RFPs were metered into a plastic funnel connected to a micro-jet apparatus (Micro-jet, Fluid Energy Co., Hatfield, PA). This air-impingement device was utilized to separate fine RFPs from the larger fiber clumps. RFPs were then drawn through a cyclone and into a settling chamber. The aerosol for the medium and low dose group was obtained by diluting the aerosol for the high dose group. The electrical charge of the test aerosol was neutralized by a 85Kr ß-radiation source to reduce the charge on the RFP. The RFP aerosol was given to the rats by a flow-past nose/snout-only inhalation exposure system. In this system, the RFP aerosol was supplied to each animal individually, and exhaled air was immediately exhausted. The airflow to each animal was approximately 1 l/min, which was calculated to be laminar flow.

Monitoring aerosol concentration and RFP dimensions.
The signals from particle counters (aerosol photometers) were continuously registered for each chamber and 20-min average values were stored for documentation (i.e., 18 average values per 6-h exposure period). The aerosols were sampled for the full duration of the daily exposure time on one Nuclepore7 filter (25 mm, pore size 0.8 µm) per chamber per day, which was used for SEM analysis. Samples were taken through special sampling ports of the inhalation chambers. Twice a week one sample from each chamber was analyzed for total RFP numbers for the calibration of the particle counters. For this analysis, the RFPs of the original filter were resuspended in methanol and a part of the suspension was filtered onto another Nuclepore® filter. During the first 9 days of exposure, additional samples for phase contrast optical microscopy (PCOM) were prepared from the same suspensions. The same SEM samples were used for analyzing the RFP size distribution. The recovery of fibrils by this procedure was validated. The calculated recovery rate of RFPs by this procedure was above 98%.

During the first 9 days of exposure, a comparison was made between the counting of aerosol samples by the SEM method and by the PCOM method that was used in the Lee study (1988) and the IOM study (1995). From this comparison a correction factor of 0.488 between SEM and PCOM was calculated. The aerosol concentrations of 25, 100, and 400 RFP per ml were based upon PCOM values that were obtained by conversion of SEM analysis data.

Gross pathology/necropsy.
For lung retention and bronchoalveolar lavage studies, the rats were anesthetized with an overdose of sodium pentobarbital (Nembutal®, 0.2 ml/100 g body weight) and exsanguinated by cutting the vena cava caudalis after opening the abdominal cavity. The diaphragm was cut, allowing the lungs to collapse. After careful preparation of the lungs and lung-associated lymph nodes (LALN), the wet weights of the organs were recorded. For histopathology assessments, an overdose of CO2 was used for euthanasia.

Digestion of lung tissue and organ burden measurement.
For a reliable quantification of the test material content in the lungs and LALN, a special method had to be used, considering the sensitivity of p-aramid RFPs to acid and alkaline chemical media. Lung digestion methods reported by Kelly et al. (1985) and by Searl (1997) were used and optimized in an adaptation and refinement process. Because the -CO-NH- bonds (peptide type) of p-aramid fibrils are degradable by acid or alkaline media, the incubation time was reduced to a minimum (to minimize the changes on RFP dimensions by the procedure), and to guarantee at the same time complete digestion of the lung tissue.

The lung tissue was dissolved by a two-step digestion method using ethanolic KOH and sodium hypochlorite. As a result, a reliable analysis procedure of the actual RFP retention in the lungs was established and validated.

Approximately one-sixth of the whole lung tissue was taken for digestion. At sacrifice (as described above), lungs of controls and exposed animals were divided into left lobe and right lobes. The right lobes were minced by scissors into pieces smaller than 5 mm. After mixing, about 15% (but not more than 0.3 g) of total lung tissue was utilized for digestion. This tissue sample was incubated in 20 ml of preheated ethanolic KOH (11 g KOH/82 ml EtOH/18 ml H2O) at 60°C for 30 min, which was shaken once manually after 15 min. Thereafter, 10 ml of chilled (4°C) NaOCl:water 1:9 v/v (NaOCl stock solution containing 12–13% active chlorine, Riedel-de Haen Co., Seelze/Hanover, Germany) was added and the mixture was incubated for another 5 min on ice. After centrifugation for 25 min at 26,900 x g at 4°C, the supernatant was removed. The RFP pellet with about 3 ml supernatant was resuspended in 40 ml H2O (containing 1% TWEEN). Suitable aliquots of the total RFP suspension volume, depending on dose group and sacrifice date, were taken to prepare filters (Nuclepore® filter, 47 mm or 25 mm diameter; pore size: 0.4 µm) for RFP analysis.

Analysis of number of RFPs in the lungs.
A part of the filter containing RFP of the dissolved lung tissue was prepared for and analyzed by SEM as described for the characterization of the test material. For each digested lung sample, at least 200 RFPs (length:diameter > 3:1, length > 1 µm), at least 100 RFPs longer than 5 µm and at least 50 RFPs longer than 20 µm were measured on SEM videoprints. For lower RFP counts (e.g., for controls), a minimum of 50 object fields was assessed for RFPs on the SEM screen. In this case, the number of analyzed RFPs per animal could be below 200. The magnification, the number of object fields, and the length and diameter of each RFP were recorded.

From these results the kinetics of the elimination of the RFPs from the lung was calculated by regression analysis of the logarithm of the number of RFPs versus time after inhalation exposure end. The half-time T1/2 was calculated from the regression coefficient k by: .

In addition, the size distribution of the RFPs was analyzed and bivariate analysis was made for all sacrifice dates.

Analysis of number of RFP in the LALN.
The lung-associated lymph nodes (LALN) were also used for RFP analysis. Lymph nodes of two animals from each group and each sacrifice date were digested and analyzed for translocated RFPs of p-aramid using the method described for lung tissue.

Calculation of RFP mass from RFP length and diameter data.
Mass of RFPs was calculated taking into account their ribbon morphology (Searl, 1997Go) and assuming a ribbon thickness of 0.1 µm. Then the mass is given by length x diameter x 0.1 x 1.4 (density of p-aramid).

Tracer inhalation and clearance measurement.
After the end of exposure, as well as 3 and 6 months postexposure, 60-min tracer inhalation tests with poorly soluble 46Sc2O3 particles were conducted. The thoracic {gamma}-activity of the tracer-exposed animals was measured twice a week for up to 90 days post tracer exposure using two sodium iodide detectors as described by Muhle et al. (1988). The rate coefficient for the long-term clearance was determined by logarithmic regression of the decay-corrected activity values on days 15–90 after tracer exposure for each animal. These data were used for statistical analysis.

The clearance rate coefficients k with their 95% confidence intervals were transformed to the corresponding retention half-times T1/2 for each treatment group by .

Bronchoalveolar lavage (BAL).
A bronchoalveolar lavage study was performed using the method of Henderson et al. (1987) with minor modifications (Muhle et al., 1990aGo). The lungs were lavaged with 2 x 5 ml saline without massaging the lung tissue. The leukocyte concentration was determined using a counting chamber. Cytoslides were prepared for differential cell counts. The lavagate was centrifuged at about 160 x g and the supernatant was used for the determination of some biochemical parameters [lactic dehydrogenase (LDH), ß-glucuronidase, total protein].

Histopathology.
Histopathology was performed on lung tissues of animals listed in Table 3Go. Lungs were fixed by airway infusion with buffered formalin (10%), trimmed according to Bahnemann et al. (1995), embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E).

Proliferation assay.
Formalin-fixed tissue of the terminal bronchioles and lung parenchyma was examined for cell proliferation using the sensitive S-phase response method (BrdU, see Warheit et al., 1992). The evaluation of the slides was done by analyzing 2000 airway cells/rat (terminal bronchioles) and 2000 cells per rat of the pulmonary parenchyma. Cells with distinct red nuclei were counted as labeled cells and the labeling index was expressed for both compartments as the percentage of labeled cells per total number of cells counted.

Statistical methods.
Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance, the means of the treated groups were compared with the means of the control groups based on the Dunnett's test. The statistical evaluation of the histopathologic findings was done with the two-tailed Fisher test by the P.L.A.C.E.S. system (version 2000.1), Instem Life Science Systems, UK.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aerosol Concentration and RFP Dimensions
As described previously, the aerosol concentrations were measured by SEM methods. For comparison to the chronic inhalation study of Lee et al. (1988), the aerosol concentration was additionally measured during the first 9 days by the PCOM method for the same samples. From this analysis, a converting factor (CF) of 0.488 was calculated by dividing the concentration of WHO-RFP by PCOM by the concentration of WHO-RFP by SEM.

The means and standard deviations of the RFP concentration in the aerosol samples of the three exposure chambers are summarized in Table 3Go. The deviation of the mean actual concentration to the target concentration was below 10% for all exposure chambers.

Body Weights, Weights of Lungs and Lung-Associated Lymph Nodes (LALN)
Body weight development during the exposure and posttreatment period did not show any evident differences between treatment groups and control group (data not shown).

The lung wet weights were increased 3 days after exposure to the test material in the medium and high dose groups. For the high dose group this effect was significant (1.604 vs. 1.328 g of the control group). Up to the 6-month postexposure sacrifice date, this significant effect persisted before returning to normal (after 9 months).

The weights of the LALN showed a significant increase in the high dose group compared to controls (40.3 vs. 28.8 mg) 3 days upon cessation of exposure. This effect was transient and disappeared within 9 months postexposure (data not shown).

Retention and Clearance of RFP
The results of RFP retention measurements are summarized in Table 4Go and in Figure 1Go. In Table 5Go the RFP concentrations of different length fractions are presented. In the medium RFP dose group (100 RFP/ml), an additional sacrifice date of 1-month postexposure was used to determine whether there was an increase in RFP during the first weeks. The number of RFPs < 10 µm was increased at this sacrifice date compared to the 3 days postexposure date, whereas for the RFP fraction with lengths above 10 µm the number of RFPs per lung decreased. This effect could be explained by breakage of long RFPs (length > 10 µm).


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TABLE 4 Analysis of Respirable Fiber-Shaped Particulates in the Digested Lungs
 


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FIG. 1. Decrease in number of respirable fiber-shaped particulates (WHO; > 10 µm; > 20 µm) in lungs after 90-day inhalation (geometric means with standard deviation).

 

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TABLE 5 Fiber Concentrations in Different Length Fractions
 
The size distribution of RFPs for the different sacrifice dates showed the following characteristics with time. The mean lengths of retained fibrils were progressively reduced during the postexposure period of 3 days to 3 months. The diameter of RFP did not change within this period (data not shown).

From the retention data, the half-time for the decrease in the number of RFPs, number of WHO RFPs, number of RFPs (length > 10 µm), number of RFPs (length > 20 µm), mass of RFPs, and cumulative length of RFPs was calculated (see Table 6Go). These half-times were calculated using lung retention data up to sacrifice date 9 months postexposure (see Table 4Go). Therefore, these half-times are mean values for the 9-month postexposure period. In the low and medium dose groups, all half-times (range of 46–76 days) were close to control group values for tracer particle clearance (range of 34–48 days; see Table 7Go). In the high dose group, a doubling of half-times was observed (range of 127–173 days). The only exception is the class of RFPs with a length > 20 µm (56 days), which is close to the range of tracer control values.


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TABLE 6 Half-Time of Alveolar Clearance for Different Respirable Fiber-Shaped Particulate Fractions
 

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TABLE 7 Half-Time of Alveolar Clearance of Labeled Particles
 
Tracer Inhalation and Clearance Measurement
The results of clearance measurements of labeled particles are presented in Table 7Go. Plots of the measured activity of individual rats versus time after inhalation of labeled 46Sc2O3 particles are presented in Figure 2Go. Comparing these half-times to the data of RFP clearance, a consistence is seen, i.e., a significant increase was found for the high dose group.



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FIG. 2. Alveolar clearance of labeled particles. Rats (eight per group and date) were exposed to labeled particles 2 days, 3 months, or 6 months after the end of RFP exposure and measured over a period of 90 days.

 
Bronchoalveolar Lavage (BAL) Data
The data of biochemical parameters are summarized in Table 8Go.


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TABLE 8 Biochemical Parameters in the Bronchoalveolar Lavage Fluid
 
Three days after the end of exposure, significant increases in BAL fluid levels compared to controls were measured in the medium (LDH, TP) and in the high dose group (LDH, ß-GLU, TP). In the medium dose group levels were elevated about 2-fold and in the high dose group about 3- to 5-fold compared to controls.

Recovery effects were evident 3 months postexposure in the medium dose group (no significant alterations for all parameters). In the high dose group, all three parameters were still significantly enhanced over controls at 3 months postexposure. Only for ß-GLU was there a decrease observed (about 50%; see Table 8Go) compared to the 3 days postexposure data.

At 9 months postexposure, a full recovery was also detected in the high dose group.

Differential Cell Count
The results of the differential cell count are presented in Figure 3Go. The bar charts are subdivided by the cell concentrations of macrophages, PMNs and lymphocytes. Three days after the end of exposure, the concentration of leukocytes was significantly increased in the high dose group. At 3 months postexposure, the cell concentration in the high dose group returned to normal values.



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FIG. 3. Differential cell counts in bronchoalveolar lavage fluids.

 
Three days after the end of exposure, the percentages of PMNs and lymphocytes (absolute percentage of leukocytes) were significantly increased in the medium and high dose group (about 20% and 25–30%, respectively). Three months postexposure, the PMN and lymphocyte percentages in the medium dose group returned to levels that were not significantly higher than in the control group. In the high dose group, these values were still significantly increased 3 months postexposure but were lower by a factor of about 2 compared to the 3-day postexposure sacrifice date.

At 9 months postexposure, the cell concentration was increased in all groups including controls, which could be explained as an age effect. In the high dose group, the percentage of lymphocytes was still significantly elevated but had retained close to control levels.

Histopathology
The time course of the main histopathologic findings is given in Table 9Go.


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TABLE 9 Summary of Histopathological Findings
 
In the medium and high dose groups, the incidences of accumulation of particle-laden macrophages and interstitial/peribronchiolar fibrosis were 5 out of 5 animals at all postexposure intervals (except interstitial/peribronchiolar fibrosis: four out of five rats of the medium dose group at the 3 month postexposure interval). For both changes, the severity grades decreased towards the 9-month postexposure interval from mainly slight to very slight. The particle-(RFP-) laden macrophages were preferentially located in the alveolar duct region and often formed granuloma-like aggregates. At the 3-, 6-, and 9-month postexposure interval, macrophages containing birefringent RFPs to a large extent were also located within the interstitium. Some RFP-laden macrophages were usually also present within the bronchus-associated lymphoid tissue (BALT). Occasionally some multinucleated giant cells associated with inhaled RFPs occurred in the lungs of RFP exposure groups.

Fibrotic changes mainly affected the alveolar duct regions and were not observed in the low dose p-aramid group. The fibrotic foci consisted of proliferated fibroblasts with slight deposition of collagen fibers and were closely associated with aggregates of particle-laden macrophages and interstitial mononuclear cell infiltrations.

Bronchiolo-alveolar hyperplasia mainly of the bronchiolar type (alveolar bronchiolization) was observed at a predominantly very slight (minimal) degree in 3 out of 5 to 5 out of 5 rats of the medium and high dose groups at each postexposure interval. At the 9-month postexposure interval, the incidence was 4 out of 5 and 3 out of 5 in the medium and high dose groups, respectively. The hyperplastic epithelium often consisted of only a few cells and usually developed in association with inflammatory and fibrotic foci. Alveolar bronchiolization is considered to be an adaptive response to dust exposure to facilitate increased particulate removal via the mucociliary escalator.

Inflammatory/mononuclear cell infiltration was observed at a significantly increased incidence only in the high dose group at the 3-day postexposure interval. Thereafter, this change was seen at incidences between 1 out of 5 in the medium and 3 out of 5 rats in the high dose group.

In the low dose group, the incidence of accumulation of particle-laden macrophages decreased from 5 out of 5 at the 3-day postexposure interval to 3 out of 5 at the 9-month postexposure interval. Interstitial/peribronchiolar fibrosis was observed at a very slight degree in 2 of 5 rats at both the 3- and 6-month postexposure intervals. The difference when compared to the control group was not statistically significant. Bronchiolo-alveolar hyperplasia and inflammatory/mononuclear cell infiltration were present in only 2 out of 5 and 1 out of 5 rats, respectively, at the 3-day postexposure interval and were absent thereafter.

In summary, the RFP-related inflammatory changes had continuously declined over the recovery period as reflected by lower incidences and degree of severity of most lesions. However, there were still RFP-laden macrophages present either within the alveoli or within the interstitial compartment at the end of the study.

Cell Proliferation Test
The results of the BrdU proliferation test are summarized in a bar plot (Fig. 4Go).



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FIG. 4. Labeling index of lung tissue cells (terminal bronchiolus and parenchyma).

 
The group-based mean labeling index for both the bronchiolar epithelial and the parenchymal (alveolar) cells of the treatment groups showed a dose-dependent increase at each time point investigated compared to the control group.

For the bronchiolar epithelial cells, a steady decrease of the labeling index from the 3-day towards the 9-month postexposure interval was observed in the medium and high dose groups. There was no dose-response relationship for the bronchiolar epithelial cells at the 9-month postexposure interval. In contrast, the parenchyma showed a steady increase of labeled cells up to the 6-month postexposure interval followed by a slight decrease towards the 9-month postexposure interval in all treated groups. At the 9-month postexposure interval, the parenchymal labeling index was still much higher than at the end of the 3-day postexposure interval, but there was no dose-response relationship for the parenchymal cells at the 6- and 9-month postexposure interval.

The decrease of the labeling index over time in the bronchiolar epithelium as opposed to an increase in the parenchymal labeling index may have only limited biological significance in view of the low number of labeled cells, thus not allowing a statistically well-grounded interpretation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main results of the study are summarized in an overview table (Table 10Go) together with a ranking of effects. This table demonstrates that clearance retardation of WHO RFPs and labeled particles, pulmonary inflammation, and interstitial/peribronchiolar fibrosis in the lungs of the medium and high dose group were significant findings. Some effects persisted over the 9-month recovery period. A No Observed Effect Level (NOEL) was found for the low dose group (i.e., 50 RFP/ml). In the following these results are discussed in detail.


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TABLE 10 Summary of the Main Findings after Subchronic Exposure to p-Aramid Respirable Fiber-Shaped Particulates
 
General Findings
Exposure of rats to RFPs did not induce signs of systemic effects. Throughout the study no unscheduled death occurred.

No effects on weights of lungs and lymph nodes were observed in the low dose group. Three days after the end of exposure, lung weights were increased in the medium dose group (not significant); in the high dose group the 1.2-fold increase was significantly different compared to concurrent controls. After 3 months of recovery, lung weights in the medium dose group returned to normal. Lung weights of the high dose group were elevated up to 6 months postexposure but returned to control levels at 9 months postexposure. Lymph node weights showed a significant increase in the high dose group 3 days upon cessation of p-aramid RFP inhalation. After an additional 3-month clean air period, this effect returned to normal. In the low and medium dose group no significant changes were observed.

Retention of Test Material in the Lungs
After 3 months of exposure, the retention of RFP (WHO fraction) was about 25 x 106 RFPs per lung in the low dose group (Table 4Go). The corresponding values in the medium and high dose group were 122 x 106 and 576 x 106 RFPs per lung, respectively. After 9 months of recovery, the RFP concentration in the lungs was decreased to 4% (low dose), 14% (medium dose), and 33% (high dose) of the original lung burden. For the RFP fraction with a length > 20 µm, the corresponding data after the 9-month recovery period were 0.3%, 1.4%, and 2.7% for the low, medium, and high dose group.

Clearance of Tracer Particles
The alveolar clearance (measured by using short-term exposure to {gamma}-labeled scandium oxide particles) was significantly retarded in the high dose group (Fig. 2Go and Table 7Go). The clearance half-time of labeled particles was increased 7.5-fold after the end of exposure, 3-fold at 3 months postexposure, and 2-fold at 6 months postexposure when compared to the control group. This retardation of lung clearance resembles the effect observed in various inhalation studies with poorly soluble particles and is caused by an overwhelming of the alveolar clearance capacities (lung overload effect; Morrow et al., 1991; Muhle et al., 1991). For the low dose group, no change in tracer clearance was detected after the end of RFP exposure when compared to controls.

Toxicokinetics of RFP in the Lungs
The toxicokinetics of RFP in lungs is one of the key issues in this study. The kinetics were investigated previously after an exposure of a few weeks in rats and hamsters (Kelly et al., 1993Go, Searl, 1997Go, Warheit et al., 1992Go, Warheit et al. 1996Go, Warheit et al., 1997aGo, Warheit et al., 1997bGo). These results indicated a biodegradibility of RFP in lungs as evidenced by shortening of the RFP retained in the lungs of exposed animals. Similar conclusions were drawn in a paper of Kelly et al. (1993) after a 2-year inhalation period. The authors concluded that the time required for fibrils to be reduced to < 5-µm fibrils in the lung was markedly less at lower exposure concentrations and shorter exposure time periods. The authors reported an increase in the number of fibrils < 5 µm retained in rat lungs after a 6-h exposure to 400 RFP/ml. Searl (1997) has reported a rapid clearance of the longer p-aramid fibrils during the first months after cessation of a 2-week exposure. She noticed an initial increase in the number of shorter fibrils. This observation was confirmed in the present study in the group exposed to 200 RFP/ml in which an additional sacrifice was conducted 1 month postexposure. The results of the lung burden measurements are shown in Figure 1Go and in Table 4Go.

Important findings are:

Searl (1997) stated that the clearance of p-aramid RFPs is poorly described by a first-order kinetic model. As can be seen from Figure 1Go, there is indeed a considerable scattering. However, more than 80% of the variance can be explained by a linear regression model (for all parameters and exposure groups in Table 6Go).

The clearance of labeled particles correlates with the clearance of the number of RFP, number of WHO RFP, and cumulative length of RFPs (see Table 6Go, Fig. 2Go, and Table 7Go). Differences were seen for RFP that are longer than 10 or 20 µm. There are various possibilities to explain these observations:

The clearance kinetics of RFPs longer than 20 µm show the fastest elimination, i.e., in terms of clearance velocity relative to actual fiber burden, in the period between 6 and 9 months postexposure. This observation cannot be explained by an enhanced mucociliary clearance of the long RFPs due to the deposition pattern during exposure. Therefore breakage of long fibrils best explains the experimental observations.

BAL Fluid Analysis: Differential Cell Count and Biochemical Parameters in the Supernatant
Investigations of the BAL fluid showed no effects in the low dose group; transient effects were observed in the medium dose group. In the high dose group, the recovery from inflammation appeared at a lower degree and at a later state. These results are in accordance with an overload effect (Morrow et al., 1988Go; Muhle et al., 1991Go).

Histopathology
Compared to the findings observed in the rats killed immediately after the exposure period, the 3-, 6-, and 9-month postexposure intervals had resulted in a decrease of (acute) inflammatory changes in the medium and high dose groups and in a shift of the macrophage accumulations from the alveolar spaces into the interstitium in all dose groups. A persistence of the very slight to slight fibrotic and hyperplastic lesions was observed in the medium and high dose groups at the end of the study.

The decline of inflammatory changes is reflected by lower incidences and the degree of severity of most lesions. After 9 months of recovery time, there were still alveolar macrophage accumulations present, although the majority of particle-laden macrophages were lodged within the interstitium.

Maximum Tolerated Dose (MTD) of RFPs
An increased incidence of proliferative lesions was reported after 2 years of exposure to 200 RFP/ml p-aramid and after 1 year exposure to 800 RFP/ml followed by a 1-year recovery period in rats (Lee et al., 1988Go). Similar lesions are observed in rat lungs at overload situations (Morrow et al., 1991Go) characterized by an immotility and dysfunction of alveolar macrophages due to an excessive uptake of materials. The question arises as to what extent nonspecific effects due to dust overloading of lungs may have influenced the results found by Lee et al. (1988).

An approach to assess the effects of p-aramid RFP on the clearance of labeled particles is to compare the results with previous studies after exposures to poorly soluble particles or mineral fibers. These studies were carried out at the Fraunhofer ITA (Heinrich et al., 1995Go; Muhle et al., 1990bGo; Muhle et al., 1991Go) using similar experimental methods with labeled particles. Examples are plastic spheres (toner for copy machines), TiO2 dust (pigment grade), stonewool MMVF21, or ceramic fibers RCF1.

In these previous studies, the retained mass of particles was determined by direct analytical chemistry methods. In the present study the number and dimensions of RFP in the lung were measured and the mass was calculated using a ribbon-shaped model (see Table 4Go). For the mineral fibers the retained mass was calculated from fiber number and dimensions using cylindrical geometry.

A similar approach as reported previously with other particles (Muhle et al., 1990bGo) is shown in Figure 5Go.



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FIG. 5. Normalized clearance rate coefficient of labeled particles vs. retained dust volume of various test materials. Other materials used for comparison originated from previous studies (see text for references).

 
In this figure the clearance rate of treatment groups was normalized to the control group value. The clearance rate coefficient data of inhaled tracer aerosols (85Sr-polystyrene particles) of these former studies were supplemented by data from studies with RCF 1 fibers, MMVF 21 fibers, and p-aramid RFPs (46Sc2O3 tracer particles).

These data show that, when compared on a volumetric basis (because the volume is the more adequate parameter reflecting macrophage function), in the high dose group and for the period starting 3 days postexposure in the medium dose group, p-aramid RFPs are more effective than isometric particles in reducing the macrophage-mediated clearance. The ceramic fiber type RCF1 and quartz are much more potent in the inhibition of clearance than the p-aramid RFPs.

Currently, there is only limited information available on systematic studies investigating whether the shape of fibrous particles influences the macrophage-mediated clearance kinetics and subsequent effects more than isometric particles (Yu et al., 1994Go).

On the basis of labeled particle clearance, in the first 3 months postexposure, the clearance was delayed in the highest exposure group from 34 days (controls) to 254 days (RFP 800; see Fig. 2Go and Table 7Go). This corresponds to about a 7-fold retardation of tracer particle clearance. This coincides with the BALF data, showing the highest parameter for inflammation (Table 8Go) and the highest proliferation of epithelial cells of the terminal bronchiole (Table 10Go). At this period the MFTD according to the above mentioned criteria of a 2–4 times retardation of alveolar clearance was reached. However, these data show that clearance retardation was transient and recovered thereafter.

In a recent paper, Oberdörster (1997) addressed the question of pulmonary carcinogenicity of inhaled particles and with regard to the MTD. He reported that there is no general agreement about a quantification of end points to define the MTD (pulmonary inflammation, increased epithelial cell proliferation, increased lung weight, impairment of particle clearance function, and significant histopathologic findings). Oberdörster (1997) suggested the need to perform multidose subchronic and chronic inhalation studies with known human particulate carcinogens (asbestos or crystalline silica). Quantification of effects in such studies using the noncancer end points listed above would identify a dose level without significant signs of toxicity at the end of the subchronic study. However, these multidose studies in rats with asbestos and crystalline silica have not been conducted.

In the study of Lee et al. (1988), 29 males and 14 females out of a total of 100 rats of the high dose group (400 RFP/ml) died of the consequences of dense, obstructing accumulations of p-aramid RFP in the ridges of alveolar duct bifurcations in the course of 1 year of exposure. This is a sure sign of overloading in the physical sense. Moreover, the surviving animals, allowed to recover for 1 more year, showed all the signs of overloading as defined by Morrow et al. (1991): pulmonary inflammation, increased epithelial proliferation, increased lung weight, impairment of particle clearance, fibrosis, and other significant histopathologic findings. At 100 RFP/ml, survival was equivalent to controls; there was no emphysema due to dust deposition and fewer proliferative epithelial lesions. In the current study, we demonstrated a 7-fold retardation of the alveolar clearance as measured by radiolabeled particles, and a retardation of RFP breakdown and clearance, concomitant with pulmonary inflammation and fibrosis at the end of three months exposure to 800 RFP/ml (400 RFP/ml as measured by light microscopy). We have found a 4-fold but reversible retardation of clearance at 200 RFP/ml (100 RFP/ml by PCOM).

These findings can be taken as confirmation that in the chronic study reported by Lee et al. (1988), the MFTD was exceeded at 400 RFP/ml, whereas 200 RFP/ml (100 RFP/ml by PCOM) may have been a borderline situation. Conversely, one may also conclude that a 7-fold increase in the clearance of radiolabeled particles after 3 months corresponds to an obvious overloading in a chronic study, whereas the reversible 4-fold increase and less pronounced effects at 100 RFP/ml correspond to the much better survival and less pronounced histopathologic findings (specifically epithelial proliferation) found in the chronic study at that concentration. In other words, the method described here represents a step forward towards resolving the challenge of quantification of end points to define the MFTD (Oberdörster, 1997Go).

In summary, the results from the end points of lung weights, pulmonary inflammation, particle clearance, and histopathology show consistency. The low dose resulted in no significant effects. In the medium dose exposure group, transient effects were measured. In the high dose group, effects were partly reduced after a 9-month postexposure period, but recovery was not complete. A summary of the main data is presented in Table 10Go. Additional findings were a decrease in the length of the lung-retained RFP over the 9-month recovery period.

The results indicate a dust overloading of lungs for the high dose group. For the medium dose group, a partial lung overloading was observed; the retardation of tracer clearance was reversible within 3 months after the end of exposure.

Based on findings in the low dose group, with the exception of accumulation of particle-laden macrophages, which represents a physiologic response rather than a pathologic one, the NOAEL for p-aramid respirable fiber-shaped particulates (RFP) was 25 RFP/ml (measured by optical microscopy) and 50 RFP/ml (measured by SEM) under the above-described experimental conditions.


    ACKNOWLEDGMENTS
 
This project was sponsored by DuPont de Nemours (Deutschland) GmbH, Bad Homburg, Germany and Akzo Nobel, Arnhem, The Netherlands. The authors acknowledge numerous fruitful discussions with D. B. Warheit (DuPont) and Ch. Braun (Akzo Nobel).


    NOTES
 
1 To whom correspondence should be addressed at Nikolai-Fuchs-Straße 1, D-30625 Hannover, Germany. Fax: (+49) 511-5350-155. E-mail: bellmann{at}ita.fhg.de. Back


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