# Pulmonary Bioassay StudieswithTerracess JS Particles inRats

Haskell Project Number: WR 17969, SC 1679
David B.Warheit DuPont Haskell Global Centers for Health &Environmental Sciences Newark, DE Corresponding author: D.B. Warheit DuPont Haskell Global Centers for Health &Environmental Sciences 1090 Elkton Rd., P.O. Box 50 Newark, Delaware 19714 U.S.A. Tel: (302) 366-5322 Fax: (302) 366-5207 e-mail: david.b.warheit@usa.dupont.com Sponsor: Otsuka Chemical Company, Ltd. 3-2-27, Ote-Dori Chuo-Ku Otsuka540-0021 Japan Running title: Pulmonary Toxicity ofTerracess JSparticle-types Keywords: particletoxicity, potassium titanate particles, pulmonary toxicity, pulmonary bioassay Summary The aim ofthis study wasto assess lung toxicity inrats exposedto Terracess JS particle-types and compare the effects to vehicle controls and to Min-U-Sil a-quartz particles as a positive benchmark control particle. Groups of male rats were intratracheally instilled with doses of 1or 5mg/kg of Terracess JS particles or a-quartz particles. Phosphate-buffered saline (PBS) solution instilled rats served as vehicle controls. Following exposures, the lungs of PBS and particle-exposed rats were evaluated for bronchoalveolar lavage (BAL) fluid inflammatory biomarkers at postinstillation time points of 1week, 1month, and 3months. Inaddition, lung tissuefrom PBS or 5 mg/kg particle-exposed (Terracess JS or a-quartz) rats were evaluated at postexposure time points at 1month and 3 months. The BAL fluid results demonstrated that pulmonary instillation exposures inrats to quartz particles produced sustained pulmonary inflammation and significant cytotoxic effects measured at 1 week, 1 month and 3 months postexposure. In contrast, exposures to Terracess JS particle-types produced no significant lung inflammatory or cell injury effectswhen compared to PBS vehicle control exposed rats. With regard to histopathology of lung tissue, pulmonary exposures to quartz particles in rats produced a progressive, dose-dependent lung inflammatory response characterized by neutrophils and foamy lipid-containing alveolar macrophage accumulation as well as evidence of early lung tissue thickening consistent with the development of pulmonary fibrosis at the 3-month postexposure time period. In contrast, histopathological analyses of lung tissues revealed that pulmonary exposures to Terracess JS particulates produced no significant adverse effects when compared to PBS-exposed controls, as evidenced by the normal lung architecture observed inthe exposed animals at post- instilIlation exposure time periods ranging from 1 month to 3 months. The results described herein demonstrate the benign nature of the pulmonary instillation response inrats following particle exposures to 1or 5mg/kg (approximately 1.25 mg) of Terracess JS particle-types in these pulmonary bioassay studies, using appropriate benchmark control particles for comparative evaluations. Thus, based on these results, itisconcluded that inhaled Terracess JS particles are expected to have a low risk potential for producing adverse pulmonary health effects inexposed workers. Introduction Terracess JS particles are composed of nonfibrous potassium titanate (K2Ti6O1 3)- particle-types and have been proposed for friction and filling applications. Previous pulmonary toxicity studies with analogous nonfibrous potassium titanate

2particle-types, such as Terracess TF, have demonstrated low toxicity in subchronic inhalation studies inrats. The current study was designed to determine whether Terracess JS particles impart significant acute toxicity in the lungs of rats, when compared to benchmark positive control particulates such as Min-U-Sil a-quartz particles, using a welldeveloped, short-term pulmonary bioassay (Warheit et al, 2007). The pulmonary bioassay model isan effective screening tool for assessing the acute lung toxicity of particle-types and utilizes an eclectic approach in the experimental design, namely 1)dose response characteristics; 2) time course evaluations; and 3) utilization of benchmark particle control samples. The objectives of this study wereto assessand compare inrats, using this shortterm pulmonary bioassay the acute pulmonary toxicity effects of intratracheally instilled Terracess JS (nonfibrous potassium titanate (K2Ti6O13)- particle-types materials, utilizing Min-U-Sil a-quartz particles as positive benchmark control particulates for comparisons. Quartz particles are known to produce sustained lung inflammation, tissue thickening and the subsequent development of pulmonary fibrosis and tumors in rats. Methods Overview of Project Two complementary and interrelated experiments assessing the pulmonary toxicity of Terracess JS (nonfibrous potassium titanate (K2Ti6O1 3) particle-types were conducted using intratracheal particle instillation exposure regimens. This study assessed the lung toxicity effects of Terracess JS particle-types using 1)bronchoalveolar lavage evaluations; and 2) lower respiratory tract histopathological assessments. Moreover, the study assessed the hazards and therefore tested the safety for workers handling these components inthe applications envisioned by the manufacturer, Otsuka Chemical Company, Ltd.

3Experiment 1

- Rats were exposed via intratracheal instillation to two different particulate-type samples with two doses/particulate (i.e., 1 and 5 mg/kg), namely 1)Terracess JS particles; 2)Min-U-Sil c-quartz particles (our standard positive control particle-type); and 3)vehicle control phosphate-buffered saline. Avariety of pulmonary biochemical and cellular endpoints were measured using bronchoalveolar lavage assessments. With regard to the intratracheally instilled doses of 1 and 5 mg/kg

- basically the upper 

### in vivo dose of 5 mg/kg is equivalent to -~1.25 mg of the instilled particle being tested (inthe lungs of a250 mg rat). This isthe lower limit of aparticle overload dose to ensure that there is asufficient lung burden dose inorder to provoke a potential pulmonary inflammatory response. One of the major objectives of the study is to determine if exposure to the particle-type produces a sustained pulmonary inflammatory response

- leading to the development of pulmonary fibrosis. The lower dose provides alung burden from which to conduct adose response assessment. Experiment 2

- Rats were exposed via intratracheal instillation to two different particulate-type samples with two doses/particulate, namely 1)Terracess JS particles; 2) Min-U-Sil a-quartz particles (our standard positive control particle-type); and 3)vehicle control phosphate-buffered saline. Lung weights and pulmonary tissue endpointswere assessed using lower respiratory tract histopathology assessments. Animals Groups of male Crl:CD(SD) rats (Charles River Laboratories, Inc., Raleigh, North Carolina) were used inthis study. The rats were approximately 8weeks old at study start (mean weights in the range of 226

- 281 grams). All procedures using animals were reviewed and approved by the Institutional Animal Care and Use Committee and the animal program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Particle-types (seeTable 1and Figure 1) a-Quartz particles (crystalline silica, Min-U-SiI 5) ranging in particle size from 500 nm to 3I.im were obtained from Pittsburgh Glass and Sand Corporation. Terracess JS particles were obtained from the sponsor - Otsuka Chemical Company, Ltd. The

4sponsor also provided characterization data for Terracess JS shown in Tablel and Figure 1. Sieved sample of TERRACESS JS (x1000)

Figure la Sieved sample ofTERRACESS JS (x1000)

Sieved sample ofTERRACESS JS (x3000)

Figure lb Sieved sample of TERRACESS JS (x3000)

5Table 1 Particle Physicochemical Characteristics and Analyses Crystal structure

- potassiumtitanium oxide CAS 12056-51-8 Particle size distribution inphosphate buffered saline

d0= 3.48 pm; d 10 = 2.O6pm; d 9() = 5.8 pm
- laser diffraction analysis Surface area = 5.52 m 2 /g 

### Composition K 2 Ti6 O13 Purity

- 100% pH indeionizedwater = 10.4; pH inPBS = 7.5
General Experimental Protocol Proposed Numbers of Animals for TerracessJS Experiments Instillation 1week 1month 3months Groups August 20 August 27 September22 November 18 1 PBS 5A 4*+5 A 4* +5A 2A Quartz-i mg/kg 5A 5A 5A 2B Quartz-5 mg/kg 5A 4*+ 5A 4*+ 5 

### 3A Terracess JS-1mg/kg 5A 5A 5A 3B Terracess JS- 5mg/kg 5A 4*+ 5A 4*+ 5A

*lung histopathology A bronchoalveolar lavage General Experimental Design The fundamental features of this pulmonary bioassay are dose-response evaluations and time-course assessments to determine the sustainability of any observed effect. Thus, the major endpoints of this study are the following: 1)time course and dose/response intensity of pulmonary inflammation and cytotoxicity; and 2) h istopathological evaluation of lower respiratory tract (lung) tissue. The bronchoalveolar lavage endpoints were obtained at 1week, 1month and 3months postexposure according to the schedule listed in Table 2. For the bronchoalveolar lavage studies, groups of rats (5 rats/group/dose) were intratracheally instilled with single doses of either 1or 5mg/kg Terracess JS particle-types or Min-U-Sil quartz (crystalline silica) particles (seeTable 1). The intratracheal instillation method of exposure can be a reliable qualitative screen for assessing the pulmonary toxicity of inhaled particles. All particles were prepared in avolume of phosphate-buffered saline

6(PBS) solution and subjected to vortexing for 5 minutes. Groups of PBS-instilled rats served asvehicle controls. The lungs of PBS and particle-exposed rats were evaluated by BAL fluid analyses at 1week, 1month and 3months postexposure (pe). For the lung histopathology evaluations (i.e., high doses (5 mg/kg) only at 1month and 3 months postexposure), additional groups of animals (4rats/group/high dose (5 mg/kg)/time period) were instilled with the particle-types listed above plus the vehicle control, i.e., phosphate-buffered saline (PBS). The lung-only histopathology experiment consisted of histopathological evaluations of the lower respiratory tract. Pulmonary Lavage The lungs of PBS and particulate-exposed rats were lavaged with aphosphatebuffered saline (PBS) solution. Methodologies for cell counts, differentials and pulmonary biomarkers in lavaged fluids were conducted as previously described (Warheit et al., 1997). Briefly, 12 mL of lavaged fluids recovered from the lungs of PBS or particulate-exposed rats were centrifuged at 700 g,and -2 mL of the supernatant was removed for biochemical studies. All biochemical assays were performed on BAL fluids using a Roche Diagnostics (BMO)/Hitachi@ 717 clinical chemistry analyzer using Roche Diagnostics (BMC)/Hitachi® reagents. Lactate dehydrogenase (LDH) is a cytoplasmic enzyme and is used as an indicator of cell injury. Alkaline phosphatase (ALP) activity is a measure of Type il alveolar epithelial cell secretory activity, and increased ALP activity in BAL fluids is considered to be an indicator of Type 11 lung epithelial cell toxicity. Increases in BAL fluid micro protein (MTP) concentrations generally are consistent with enhanced permeability of vascular proteins into the alveolar regions, indicating abreakdown inthe integrity ofthe alveolar-capillary barrier. Lung Histopathology Studies The lungs of rats exposed to particulates or PBS controls were prepared for microscopy by airway infusion of formalin fixative under pressure (21 cm H20) at 1

-month and 3-month postexposure time periods. Sagittal sections of the left and right lungs were made with a razor blade or scalpel. Tissues were dissected from left, right upper, and right lower regions of the lung and were subsequently prepared for light

7microscopy (paraffin embedded, sectioned, and hematoxylin-eosin stained (Warheit et al., 2007). Statistical Analyses Experimental values were compared to corresponding sham control values for each time point. Aone-way analysis of variance (ANOVA) and a Bartlett's test were calculated for each sampling time. When the Ftest from ANOVA was significant, the Dunnett's test was used to compare means from the control group toeach of the groups exposed to particulates. Significance vs. PBS controls was judged at the 0.05 probability level. Results Body Weights There were no significant differences inthe in-life body weights of rats during the study. The male rats appeared to grow at a normal rate and were not affected by exposures to particles (Figure 2). Bronchoalveolar Lavage Fluid Results Pulmonary Inflammation The numbers of cells recovered by bronchoalveolar lavage from the lungs of high dose quartz-exposed (5mg/kg) groups were substantially higher than any of the other groups for all postexposure time periods (Figure 3). Intratracheal instillation exposures to Terracess JS particle-types did not produce pulmonary inflammatory responses when compared to vehicle control samples at 1week, 1month or 3months post-instillation exposures (Figures 4-6). incontrast, intratracheal instillation exposures to quartz particles (1 and 5 mg/kg) produced sustained pulmonary inflammatory responses, as measured through 3months postexposure (Figures 7-9). BAL Fluid Parameters No significant increases vs. controls in BAL fluid lactate dehydrogenase values were measured inthe lungs of rats exposed to low or high doses (5mg/kg) of Terracess JS particles at 1week, 1 month or 3 month postexposure time periods. Incontrast, exposures to 1 or 5 mg/kg quartz particles produced a significant and persistent 8increase vs. PBS controls in BAL fluid LDH values throughout the 3-month postexposure period (Figure 10). No significant increases in BAL fluid microprotein values were measured inthe lungs of low or high dose (5mg/kg) Terracess JS-exposed rats. Incontrast, exposures to 1 mg/kg (at 3 months) or 5 mg/kg quartz particles (1-wk, 1-month and 3-months) produced sustained and significant increases in BAL fluid microprotein values (Figure 11). No significant increases in BAL fluid alkaline phosphatase (ALP) values vs. controls were measured in Terracess JS-exposed rats. In contrast, significant increases inALP values were measured inlow-dose quartz-exposed (1month pe) and in high-dose quartz-exposed rats (i.e., at 1 week and 1-month postexposure) (Figure 12.) To summarize the results with BAL fluid biomarkers, pulmonary exposures to quartz particles produced a sustained, dose-dependent, lung inflammatory response, concomitant with cytotoxic effects, measured from 1 week through 3 months postexposure. Exposures to Terracess JS particles (1 or 5 mg/kg) produced no pulmonary inflammatory responses at the postexposure time periods measured inthis study (i.e., 1week, 1 month or 3 months). Inaddition, no significant increases vs. controls were measured for any of the BAL fluid parameters inanyof the Terracess JSexposed particle groups. However, quartz exposures produced sustained and progressive lung inflammatory and cytotoxic responses. LungTissue Studies LungWeights Lung weights of rats generally were elevated with increasing age on the study (i.e., increased postexposure time periods following instillation). Lung weights in high dose quartz-exposed ratswere increased vs. controls or Terracess JS particle exposed rats at 1month and 3months postexposure (Figure 13).

9Histopathological Evaluation Histopathological analyses of lung tissues revealed that pulmonary exposures to fine-sized Terracess JS particle-types produced no significant adverse effects when compared to PBS-exposed controls, as evidenced by the normal lung architecture observed inthe exposed animals at post-instillation exposure time periods ranging from 1month to 3 months. Alveolar macrophages which had phagocytized particles were evident inalveolar regions adjacentto terminal bronchioles, and this represents normal physiological responsesto dust exposures (Figure 14). Histopathological analyses of lung tissues revealed that pulmonary exposures to a-quartz particles in rats produced dose-dependent lung inflammatory responses characterized by neutrophils and foamy (lipid-containing) alveolar macrophage accumulation. Inaddition, lung tissue thickening as a prelude to the development of fibrosiswas evident and progressive (Table 3,Figure15). Discussion The objective of this pulmonary bioassay study was to assess the acute lung toxicity of intratracheally instilled, Terracess JS particles in rats. Using pulmonary bioassay methodology, the lung toxicity of instilled Terracess JS particles were compared with apositive control particle-type, a-quartz particles. The specific aim was to determine whether exposures to Terracess JS particle-types could produce enhanced and sustained pulmonary inflammation and cytotoxicity in rats when compared to Min-U-Sil quartz particles, a particle-type known to produce significant toxic effects to the lung. Exposures to Terracess JS produced no significant adverse pulmonary effects. Results from the bronchoalveolar lavage fluid and cell proliferation evaluations demonstrated that pulmonary exposures to a-quartz particles, particularly at the higher dose, produced significant adverse effects vs. controls in pulmonary inflammation and cytotoxic effects. In contrast, Terracess JS particle exposures produced no differences when compared to PBS vehicle control samples. Histopathological evaluations demonstrated that quartz particle exposures produced pulmonary inflammation, foamy macrophage accumulation and tissue thickening (i.e., fibrosis), primarily at the junctions of terminal bronchioles and alveolar duct regions.
10Exposures to Terracess JS particles produced minor tissue reactions in the lungs, including macrophage accumulations of phagocytic cells, which are normal responses to high dust concentrations. The results of pulmonary toxicity studies with titanium-based particulates similar to Terracess JS have recently been reported. Sakai et a!. (2006) conducted a2-week inhalation toxicity study in rats with Terracess TF particles (potassium octatitanate

- disk-shaped particulates

- median particle size = 3.3 pm and surface area = 3.68 m 2

Ig). Rats were exposed to 5, 25, or 100 mg/in3 for 2 weeks and no inflammatory cell infiltration or evidence of lung fibrosis was observed (Sakai et a!., 2006). In a subsequent, longer-term inhalation toxicity study with the same material, rats were exposed for 90 days to aerosols of 2, 10 or 50 mg/in3 to Terracess TF particles. Exposures to 2and 10 mg/in3 produced normal physiological macrophage accumulation responses in the distal lungs of exposed rats. Exposures to the highest exposure concentration, 50 mg/in3 produced macrophage accumulation and minimal alveolar Type 11 epithelial cell hyperplasia

- and this was associated with the absence of pulmonary inflammatory cell infiltrates or lung fibrotic responses. The authors characterized the pulmonary response to aerosol exposures to disk-shaped Terracess TF particles as "nuisance dust responses" (Sakai eta, 2009). The lack of potency with regard to pulmonary hazard responses for inhaled Terracess TF octatitanate particles appears to be similar to the minimal effects reported for exposures inrats to pigment-grade titanium dioxide particle-types (Bermudez eta!., 2002; Warheit et a!., 2005, 2006, 2007). This finding suggests that these materials produced rather benign pulmonary effects. Similarly, the results reported herein for Terracess JS, inthis pulmonary bioassay study, are similar to other low toxicity dusts that have been tested in this system, using appropriate benchmark controls. To summarize, based on these results, inhaled Terracess JS particles are expected to have a low risk potential for producing adverse pulmonary health effects in workers exposed to these particulates.Acknowledgments The following individuals made significanttechnical contributionsto the conduct and implementation of this study: Ken Reed, Morgan Golt, David Grinstead, Elizabeth Wilkinson, Lisa Lewis, Carolyn Lloyd, Steve Records, Don Hildabrant, Antidio Lorenzo, Jeff Holt, Melissa Fallers, and Tracey White. Dr. Steven R.Frame was the pathologist onthisstudy.

References Bermudez E,Mangum JB, Asgharian B,Wong BA, Reverdy EE, Janszen DB, Hext PM, Warheit DB, and Everitt JI. Long-term pulmonary responses ofthree laboratory rodent speciesto subchronic inhalation of pigmentary titanium dioxide particles. Toxicol Sci. 70: 86-97, 2002. Sakai S, Inada K,Tanaka AK, Kelly DP, Sykes GP and Lee KP. Twoweek inhalation studies with disk-shaped particles of potassium titanate (Terracess TF) inrats. SOT 45 1hAnnual Meeting and ToxExpo. Program, San Diego, California

- page 142#1055, 2006. Sakai S, Inada K,Tanaka AK, Kelly DP, Sykes GP and Lee KP. Inhalation toxicity study of disk-shaped potassium octatitanate particles (TerracessTF) inrats following 90-day of aerosol exposure. Submitted, 2009. Warheit DB, Hansen JF, Yuen IS,Kelly DP, Snajdr 5, and Hartsky MA. Inhalation of high concentrations of low toxicitydusts inrats results inpulmonary and macrophage clearance impairments. ToxicolAppI Pharmacol, 145: 10-22, 1997. Warheit DB, Brock W,Lee KP, Webb TR, Reed KL Comparative Pulmonary Toxicity Inhalation and Instillation Studies with Different TiO2Particle Formulations: Impact of Surface Treatments on ParticleToxicity. Toxicol Sci. 88:514-524, 2005 Sep21; Warheit DB, Webb TR, Sayes CM, ColvinVL, and Reed KL. Pulmonary Instillation Studies with Nanoscale TiO2 Rods and Dots inRats: Toxicityisnot dependent upon Particle Size and Surface Area. Toxicol Sci. 91: 227-236, 2006 Feb 22; Warheit DB, Webb TR, Reed KL, FrerichsS, and Sayes CM. Pulmonary Toxicity Study inRatswith Three Forms of ultrafine-TiO2 Particles: Differential Responses relatedto Surface Properties. Toxicology 230:90-104, 2007; 2006 Nov 10;

12Table 2 Protocol forTerracess JS Particle Bioassay Study BAL fluid analyses 

### @1wk, 1month, and 3months postexposure Lung tissue evaluation 

### @1month and 3months postexposure* Exposure Groups * PBS (vehicle control) * Particle-types (1and 5mg/kg) for BALE analysis " Particle-types (5mg/kg) for histopathology*

o a-Quartz particles (positive control) o Terracess JS particles Instillation Exposure Postexposure (pe) Evaluation via BALand Lung Tissue

13Table 3 Terracess JS- histopathological analyses at 1and3months postexposure Group 1

- PBS instilled 1month pe 1038 normal lung architecture 1039 normal lung architecture 1040 normal lung architecture 1041 normal lung architecture 3months pe 1042 normal lung architecture 1043 normal lung architecture 1044 normal lung architecture 1045 normal lung architecture Group 2B

- Quartz 5mg/kg instilled 1month pe 1076 foamy macrophage accumulation 1077 foamy macrophages +earlytissue thickening 1078 foamy macrophages +earlytissue thickening 1079 foamy macrophages +earlytissue thickening 3months pe 1080 foamy macrophage accumulation +tissue thickening 1077 foamy macrophage accumulation +tissue thickening 1078 foamy macrophage accumulation +tissue thickening 1079 foamy macrophage accumulation +tissuethickening Group 3B

- Terracess JS 5mg/kg instilled 1month Pe 1114 normal lung architecture + phagocytic macrophages 1115 normal lung architecture + phagocytic macrophages 1116 normal lung architecture + phagocytic macrophages 1117 normal lung architecture +phagocytic macrophages 3months pe 1118 normal lung architecture +phagocytic macrophages 1119 normal lung architecture +phagocytic macrophages 1120 normal lung architecture + phagocytic macrophages 1121 normal lung architecture + phagocytic macrophages

14 

# 93-Body Weights of Male Rats exposed to Terracess JS or Quartz Particulates 700

600-

500- ~j400-

CO 300

-- CD200

--- Qat gk &~Quartz 5mg/kg 100 X______________________ * Terracess -JS 1mg/kg X *-Terracess

-JS 5mg/kg

1 2 3 4 5 6 7 8 9 10 12 13 14 Weeks on Study Figure 2 Body weights of male rats exposed to Terracess JS or quartz particles. There were no significant differences inthe in-life bodyweights of rats during the study. The male rats appeared to grow at anormal rateand were not affected by exposuresto particles.

15

### Total Cell Numbers in BAL Fluids of Rats exposed to Terracess JS or Quartz Particulates

3.OOE+07
2.50E+07T
S2.OOE+07 

### E

- 1

.50E+07
1.OOEi-07
5

.OOE+06. . 0.00E+00 1mg/kg 5mg/kg 1mg/kg 5mg/kg PBS Min-U-SiI Quartz Particles Terracess

- JS Exposure Groups E1 Week 011 Month 03Month 

### Figure 3 Numbers of cells recovered inBAL fluids from Terracess JS, Min-U-Sil quartz-exposed rats orcorresponding controls at 1week, 1and 3 months pe. Valuesgiven are means ±S.D. The numbers ofcells recovered by bronchoalveolar lavage from the lungs of high dosequartz-exposed (5mg/kg) groupswere higherthan any of the othergroups for all postexposure time periods.

16Figure 4 Cytocentrifuge preparation of lavaged cells recovered from aratexposed to Phosphatebuffered saline (PBS) at 1week postinstillation exposure (magnification = 20x). The micrograph demonstrates the distribution of macrophages which accountfor>98% of the recovered cells.

"lo FigureA

41174A

Figure 6 Gytocentrifuge preparation of lavaged cells recovered from arat exposedto Terracess JS particles at 3months postinstillation exposure (magnification = 20x). The micrograph demonstrates the lackof pulmonary inflammation as evidenced by an absence ofinflammatory cells and the distribution of macrophages which account for > 98% of the recovered cells.

18

### Percent Neutrophils in BAL Fluids of Rats exposed to Terracess JSor Quartz Particulates 80% 70%/ 60%- 50%- 40%-

30%
20%-

1mg/kg 5 m/kg 1mg/kg 5 mg/kg PBS Min-U-SiI Quartz Particles Terracess

- JS Exposure Groups El1 Week D01 Month E33Month 

### Figure 7 Pulmonary inflammation inparticulate- exposed rats and controls as evidenced by % neutrophils (PMN) inBALfluids at 1week, 1month and 3months postexposure (pe). Values given are means ± S.D. No inflammatory response inTerracess JS-exposed rats orvehicle controls. However, the exposures to quartz particles (1and 5mg/kg) produced sustained pulmonary inflammatory responses, as measured through 3months postexposure.*p <0.05 vs. PBS controls.

19

# tgA';.i

4

Figure 8 Cytocentrifuge preparation of lavaged cells recovered from arat exposedto Min-U-Sil quartz particles at 1week postinstillation exposure (magnification = 20x). The micrograph demonstrates the development of pulmonary inflammation as evidenced by the presence of neutrophilic inflammatory cells as well as alveolar macrophages.

20Figure 9 Cytocentrifuge preparation of lavaged cells recovered from arat exposed to Mmn-U-Sit quartz particles at3months postinstillation exposure (magnification = 20x). The micrograph demonstrates the sustained pulmonary inflammatory response as evidenced bythe presence of neutrophilic inflammatory cells aswell as foamy binucleated alveolar macrophages and normal-sized alveolar macrophages.

21BAL Fluid LDH Values inRats exposed to Terracess JS or Quartz Particulates

500 450 U) 400- 350 300- _j 250- '5 200 _j 150 S 100

1mg/kg 5mg/kg 1mg/kg 5mg/kg PBS Min-U-Sil Quartz Particles Terracess

-JS Exposure Groups El1 Week 01 month 03 Month] Figure 10 BALfluid LDH values for particulate-exposed rats and corresponding controls at 1 week, 1month and 3months postexposure (pe). Values given are means ±S.D. No significant increasesvs. controls inBAL fluid lactate dehydrogenase values were measured inthe lungs of rats exposed to low or high doses (5mg/kg) ofTerracess JS particles at 1week, 1month or3month postexposuretime periods. Incontrast, exposures to 1or 5mg/kg quartz particles produced asignificant and persistent increase vs. PBS controls inBAL fluid LDH values throughout the 3-month postexposure period. .*p <0.05 vs. PBS controls.

22

### BAL Fluid MTP Values inRats exposed to Terracess JSor Quartz Particulates

80- 70-

> 60-

0 50- 0 40- .2 E E 30u

.~20-

0- 1mg/kg 5mg/kg 1mg/kg 5mg/kg PBS Min-U-Sii Quartz Particles Terracess

- JS Exposure Groups El1 Week 01mnonthE ~3 Month] 

### Figure 11 BAL fluid protein (MTP) values for particulate-exposed rats and corresponding controls at 24 hrs, 1week, 1month and 3months postexposure. Values given are means ± S.D. No significantincreases inBAL fluid microprotein valueswere measured inthe lungs of lowor high dose (5mg/kg) Terracess JS-exposed rats. Incontrast, exposures to 1mg/kg (at 3months) or5mg/kg quartz particles (1 -wk, 1 -month and 3-months) produced sustained and significant increases inBAL fluid microprotein values. .*p < 0.05 vs. PBS controls.

23

### BAL Fluid ALKPValues inRats exposed to Terracess JSor Quartz Particulates

200- 180- 160 S 140- 0. 120 * 100- 80-



## 1 m/g5 mg/kg 1 mg/kg 5 mg/kg PBS Min-U-Sil Quartz Particles Terracess

- JS Exposure Groups 11111 Week 01 month E33Month 

### Figure 12 BAL fluid alkaline phosphatase (ALP) values for particulate-exposed rats and corresponding controls at 24 hrs, 1week, 1month and 3months postexposure. Values given are means ±S.D. No significantincreases inBAL fluid alkaline phosphatase (ALP) valuesvs. controls were measured inTerracess JS rats. Incontrast, significant increases inALP values were measured inlow-dose quartz-exposed (1month pe) and inhigh-dose quartz-exposed (1week and 1 -month) rats. .'*p < 0.05 vs. PBS controls.

24Lung Weights of Rats exposed to Terracess JSor Quartz Particulates 14.0
12.0

10.0
0) 8I .0

2J .0
0.0 P.... .. ... urz atce eraes-J

.. Exposure.Groups

perodsfolowigPiSilto) Lunweiht inhgQsuartz-Prilsepsed rat wer inredgshctos orclterrcsateexposed rats atd 1orsodn motonthand 34 monthseek postexposure.

25I%

Figure 14 Light micrograph of lung tissue of arats exposed to Terracess JS particles at 5mg/kg) at 3months post-instillation exposure. The micrograph illustratesthe terminal bronchiole and corresponding alveolar duct and demonstrates normal lung architecture, indicating that exposuresto Terracess JS particles produced no adverse pulmonary effects. (Magnification =x40)

4A

Figure 15 Light micrograph of lung tissuefrom arat exposed to quartz particles (5mg/kg) at 3 months post-instillation exposure. This micrograph illustratesthe terminal bronchial and corresponding alveolar ducts. Note the substantial accumulation of foamyquartzcontaining alveolar macrophages which fill many ofthe alveoli (arrows) concomitant with the prominence of tissuethickening along the junctions atthe terminal bronchiole and alveolar ducts (arrowhead). (Magnification = x40).

26