Pulmonary Bioassay Studies with Terracess JS

Particles in Rats

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

Otsuka 540-0021 Japan

Running title: Pulmonary Toxicity of Terracess JS particle-types

Keywords: particle toxicity, potassium titanate particles, pulmonary toxicity, pulmonary

bioassay

Summary

The aim of this study was to assess lung toxicity in rats exposed to 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 1 or 5 mg/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 postSANITIZED Page 1 of 26
instillation time points of 1 week, 1 month, and 3 months. In addition, lung tissue from

PBS or 5 mg/kg particle-exposed (Terracess JS or a-quartz) rats were evaluated at

postexposure time points at 1 month and 3 months.

The BAL fluid results demonstrated that pulmonary instillation exposures in rats

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 effects when 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 in the

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 in rats following particle exposures to 1 or 5 mg/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, it is concluded that inhaled Terracess JS particles are expected to have a

low risk potential for producing adverse pulmonary health effects in exposed 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

2

SANITIZED Page 2 of 26
particle-types, such as Terracess TF, have demonstrated low toxicity in subchronic

inhalation studies in rats.

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 is an 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 were to assess and compare in rats, 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 in the applications envisioned by the

manufacturer, Otsuka Chemical Company, Ltd.

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SANITIZED Page 3 of 26
Experiment 1 - Rats were exposed via intratracheal instillation to two different

particulate-type samples with two doses/particu late (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. A variety 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 (in the lungs of a 250 mg rat). This is the lower limit of a particle

overload dose to ensure that there is a sufficient lung burden dose in order 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 a lung burden from which to conduct a dose response assessment.

Experiment 2 - Rats were exposed via intratracheal instillation to two different

particulate-type samples with two doses/particu late, 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

endpoints were assessed using lower respiratory tract histopathology assessments.

Animals

Groups of male Crl:CD(SD) rats (Charles River Laboratories, Inc., Raleigh, North

Carolina) were used in this study. The rats were approximately 8 weeks 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 (see Table 1 and Figure 1)

a-Quartz particles (crystalline silica, Min-U-SiI 5) ranging in particle size from

500 nm to 3 I.im were obtained from Pittsburgh Glass and Sand Corporation. Terracess

JS particles were obtained from the sponsor - Otsuka Chemical Company, Ltd. The

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SANITIZED Page 4 of 26
sponsor also provided characterization data for Terracess JS shown in Tablel and

Figure 1.

Sieved sample of TERRACESS JS (x 1000)

Figure la

Sieved sample of TERRACESS JS (x 1000)

Sieved sample of TERRACESS JS (x 3000)

Figure lb

Sieved sample of TERRACESS JS (x 3000)

5

SANITIZED Page 5 of 26
Table 1

Particle Physicochemical Characteristics and Analyses

Crystal structure - potassium titanium oxide CAS 12056-51-8

Particle size distribution in phosphate buffered saline

d0= 3.48 pm; d10 = 2.O6pm; d9() = 5.8 pm - laser diffraction analysis

Surface area = 5.52 m2/g

Composition K2Ti6O13

Purity - 100%

pH in deionized water = 10.4; pH in PBS = 7.5

General Experimental Protocol

Proposed Numbers of Animals for Terracess JS Experiments

Instillation 1 week 1 month 3 months

Groups August 20 August 27 September 22 November 18

1 PBS 5A 4*+5 A 4* +5A

2A Quartz-i mg/kg 5 A 5 A 5 A

2B Quartz-5 mg/kg 5 A 4* + 5A 4* + 5

3A Terracess JS -1 mg/kg 5A 5A 5A

3B Terracess JS- 5 mg/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 istopatho logical evaluation of lower respiratory tract (lung) tissue.

The bronchoalveolar lavage endpoints were obtained at 1 week, 1 month and

3 months 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 1 or 5 mg/kg Terracess JS particle-types or Min-U-Sil

quartz (crystalline silica) particles (see Table 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 a volume of phosphate-buffered saline

6

SANITIZED Page 6 of 26
(PBS) solution and subjected to vortexing for 5 minutes. Groups of PBS-instilled rats

served as vehicle controls. The lungs of PBS and particle-exposed rats were evaluated

by BAL fluid analyses at 1 week, 1 month and 3 months postexposure (pe).

For the lung histopathology evaluations (i.e., high doses (5 mg/kg) only at

1 month and 3 months postexposure), additional groups of animals (4 rats/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 a phosphatebuffered 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 a breakdown in the integrity of the 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

7

SANITIZED Page 7 of 26
microscopy (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. A one-way analysis of variance (ANOVA) and a Bartlett's test were

calculated for each sampling time. When the F test from ANOVA was significant, the

Dunnett's test was used to compare means from the control group to each 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 in the 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 (5 mg/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 1 week, 1 month or 3 months

post-instillation exposures (Figures 4-6). in contrast, intratracheal instillation exposures

to quartz particles (1 and 5 mg/kg) produced sustained pulmonary inflammatory

responses, as measured through 3 months postexposure (Figures 7-9).

BAL Fluid Parameters

No significant increases vs. controls in BAL fluid lactate dehydrogenase values

were measured in the lungs of rats exposed to low or high doses (5 mg/kg) of Terracess

JS particles at 1 week, 1 month or 3 month postexposure time periods. In contrast,

exposures to 1 or 5 mg/kg quartz particles produced a significant and persistent

8

SANITIZED Page 8 of 26
increase 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 in the

lungs of low or high dose (5 mg/kg) Terracess JS-exposed rats. In contrast, 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 in ALP values were measured in low-dose quartz-exposed (1 month 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 in this

study (i.e., 1 week, 1 month or 3 months). In addition, no significant increases vs.

controls were measured for any of the BAL fluid parameters in any of the Terracess JSexposed particle groups. However, quartz exposures produced sustained and

progressive lung inflammatory and cytotoxic responses.

Lung Tissue Studies

Lung Weights

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 rats were increased vs. controls or Terracess JS particle exposed

rats at 1 month and 3 months postexposure (Figure 13).

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SANITIZED Page 9 of 26
Histopathological 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 in the exposed animals at post-instillation exposure time periods ranging from

1 month to 3 months. Alveolar macrophages which had phagocytized particles were

evident in alveolar regions adjacent to terminal bronchioles, and this represents normal

physiological responses to 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. In addition, lung tissue thickening as a prelude to the development of

fibrosis was evident and progressive (Table 3, Figure 15).

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 a positive 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.

10

SANITIZED Page 10 of 26
Exposures 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 a 2-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 m2Ig).

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/in 3 to Terracess TF particles.

Exposures to 2 and 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 et a, 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 in rats to pigment-grade titanium dioxide particle-types (Bermudez et a!.,

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, in this 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.

SANITIZED Page 11 of 26
Acknowledgments

The following individuals made significant technical contributions to 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

on this study.

References

Bermudez E, Mangum JB, Asgharian B, Wong BA, Reverdy EE, Janszen DB, Hext PM,

Warheit DB, and Everitt JI. Long-term pulmonary responses of three laboratory

rodent species to 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. Two week inhalation

studies with disk-shaped particles of potassium titanate (Terracess TF) in rats.

SOT 45 1h Annual 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 (Terracess TF) in rats

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 toxicity dusts in rats results in pulmonary and

macrophage clearance impairments. Toxicol AppI Pharmacol, 145: 10-22, 1997.

Warheit DB, Brock W, Lee KP, Webb TR, Reed KL Comparative Pulmonary Toxicity Inhalation and Instillation Studies with Different TiO2 Particle Formulations:

Impact of Surface Treatments on Particle Toxicity. Toxicol Sci. 88:51 4-524, 2005

Sep 21;

Warheit DB, Webb TR, Sayes CM, Colvin VL, and Reed KL. Pulmonary Instillation

Studies with Nanoscale TiO2 Rods and Dots in Rats: Toxicity is not dependent

upon Particle Size and Surface Area. Toxicol Sci. 91: 227-236, 2006 Feb 22;

Warheit DB, Webb TR, Reed KL, Frerichs S, and Sayes CM. Pulmonary Toxicity Study in Rats with Three Forms of ultrafine-TiO2 Particles: Differential Responses

related to Surface Properties. Toxicology 230:90-104, 2007; 2006 Nov 10;

12

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Table 2

Protocol for Terracess JS Particle Bioassay Study

BAL fluid analyses @ 1 wk, 1 month, and 3 months postexposure

Lung tissue evaluation @ 1 month and 3 months postexposure *

Exposure Groups

* PBS (vehicle control)

* Particle-types (1 and 5 mg/kg) for BALE analysis

" Particle-types (5 mg/kg) for histopathology*

o a-Quartz particles (positive control)

o Terracess JS particles

Instillation

Exposure

Postexposure (pe) Evaluation via BAL and Lung Tissue

13

SANITIZED Page 13 of 26
Table 3

Terracess JS- histopathological analyses at 1 and 3 months postexposure

Group 1 - PBS instilled

1 month pe

1038 normal lung architecture

1039 normal lung architecture

1040 normal lung architecture

1041 normal lung architecture

3 months pe

1042 normal lung architecture

1043 normal lung architecture

1044 normal lung architecture

1045 normal lung architecture

Group 2B - Quartz 5 mg/kg instilled

1 month pe

1076 foamy macrophage accumulation

1077 foamy macrophages + early tissue thickening

1078 foamy macrophages + early tissue thickening

1079 foamy macrophages + early tissue thickening

3 months pe

1080 foamy macrophage accumulation + tissue thickening

1077 foamy macrophage accumulation + tissue thickening

1078 foamy macrophage accumulation + tissue thickening

1079 foamy macrophage accumulation + tissue thickening

Group 3B - Terracess JS 5 mg/kg instilled

1 month 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

3 months 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-

SANITIZED Page 14 of 26
Body Weights of Male Rats exposed to

Terracess JS or Quartz Particulates

700

600-

500-

~j400-

CO 300 --

CD 200 --- 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 in the 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.

15

SANITIZED Page 15 of 26
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

1 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg

PBS Min-U-SiI Quartz Particles Terracess - JS

Exposure Groups

E1 Week 011 Month 03 Month

Figure 3

Numbers of cells recovered in BAL fluids from Terracess JS, Min-U-Sil quartz-exposed

rats or corresponding controls at 1 week, 1 and 3 months pe. Values given are means

± S.D. The numbers of cells recovered by bronchoalveolar lavage from the lungs of

high dose quartz-exposed (5 mg/kg) groups were higher than any of the other groups

for all postexposure time periods.

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SANITIZED Page 16 of 26
Figure 4

Cytocentrifuge preparation of lavaged cells recovered from a rat exposed to Phosphatebuffered saline (PBS) at 1 week postinstillation exposure (magnification = 20x). The

micrograph demonstrates the distribution of macrophages which account for > 98% of

the recovered cells.

"lo

FigureA

4117

SANITIZED Page 17 of 26
4A

Figure 6

Gytocentrifuge preparation of lavaged cells recovered from a rat exposed to Terracess

JS particles at 3 months postinstillation exposure (magnification = 20x). The

micrograph demonstrates the lack of pulmonary inflammation as evidenced by an

absence of inflammatory cells and the distribution of macrophages which account for

> 98% of the recovered cells.

18

SANITIZED Page 18 of 26
Percent Neutrophils in BAL Fluids of Rats exposed to

Terracess JS or Quartz Particulates

80%

70%/

60%-

50%-

40%-

30%

20%-

1 mg/kg 5 m/kg 1 mg/kg 5 mg/kg

PBS Min-U-SiI Quartz Particles Terracess - JS

Exposure Groups

El1 Week D01 Month E3 3Month

Figure 7

Pulmonary inflammation in particu late- exposed rats and controls as evidenced by %

neutrophils (PMN) in BAL fluids at 1 week, 1 month and 3 months postexposure (pe). Values given are means ± S.D. No inflammatory response in Terracess JS-exposed

rats or vehicle controls. However, the exposures to quartz particles (1 and 5 mg/kg)

produced sustained pulmonary inflammatory responses, as measured through 3 months

postexposure.*p < 0.05 vs. PBS controls.

19

SANITIZED Page 19 of 26
tgA ';.i

4

Figure 8

Cytocentrifuge preparation of lavaged cells recovered from a rat exposed to Min-U-Sil

quartz particles at 1 week 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.

20

SANITIZED Page 20 of 26
Figure 9

Cytocentrifuge preparation of lavaged cells recovered from a rat exposed to Mmn-U-Sit

quartz particles at 3 months postinstillation exposure (magnification = 20x). The

micrograph demonstrates the sustained pulmonary inflammatory response as

evidenced by the presence of neutrophilic inflammatory cells as well as foamy

binucleated alveolar macrophages and normal-sized alveolar macrophages.

21

SANITIZED Page 21 of 26
BAL Fluid LDH Values in Rats exposed to

Terracess JS or Quartz Particulates

500

450

U) 400-

350

300-

_j 250-

'5 200

_j 150

S 100

1 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg

PBS Min-U-Sil Quartz Particles Terracess - JS

Exposure Groups

El1 Week 0 1 month 0 3 Month]

Figure 10

BAL fluid LDH values for particulate-exposed rats and corresponding controls at 1

week, 1 month and 3 months postexposure (pe). Values given are means ± S.D. No

significant increases vs. controls in BAL fluid lactate dehydrogenase values were

measured in the lungs of rats exposed to low or high doses (5 mg/kg) of Terracess JS

particles at 1 week, 1 month or 3 month postexposure time periods. In contrast,

exposures to 1 or 5 mg/kg quartz particles produced a significant and persistent increase vs. PBS controls in BAL fluid LDH values throughout the 3-month

postexposure period. .*p < 0.05 vs. PBS controls.

22

SANITIZED Page 22 of 26
BAL Fluid MTP Values in Rats exposed to

Terracess JS or Quartz Particulates

80-

70-

> 60-

0 50-

0 40-

.2 E E 30u

.~20-

0-

1 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg

PBS Min-U-Sii Quartz Particles Terracess - JS

Exposure Groups

El1 Week 0 1 mnonthE ~3 Month]

Figure 11

BAL fluid protein (MTP) values for particulate-exposed rats and corresponding controls

at 24 hrs, 1 week, 1 month and 3 months postexposure. Values given are means

± S.D. No significant increases in BAL fluid microprotein values were measured in the

lungs of low or high dose (5 mg/kg) Terracess JS-exposed rats. In contrast, 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. .*p <

0.05 vs. PBS controls.

23

SANITIZED Page 23 of 26
BAL Fluid ALKP Values in Rats exposed to

Terracess JS or 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, 1 week, 1 month and 3 months postexposure. Values

given are means ± S.D. No significant increases in BAL fluid alkaline phosphatase (ALP) values vs. controls were measured in Terracess JS rats. In contrast, significant increases in ALP values were measured in low-dose quartz-exposed (1 month pe) and

in high-dose quartz-exposed (1 week and 1 -month) rats. .'*p < 0.05 vs. PBS controls.

24

SANITIZED Page 24 of 26
Lung Weights of Rats exposed to

Terracess JS or Quartz Particulates

14.0

12.0

10.0

0)

8I .0

2J .0

0.0

P.... .. ... urz atce eraes-J

.. Exposure.Groups

perodsfolowigPiSilto) Lun weiht inhgQsuartz-Prilsepsed rat wer

inredgshctos or clterrcsateexposed rats atd 1orsodn motonth and 34 monthseek

postexposu re.

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I%

Figure 14

Light micrograph of lung tissue of a rats exposed to Terracess JS particles at 5 mg/kg)

at 3 months post-instillation exposure. The micrograph illustrates the terminal

bronchiole and corresponding alveolar duct and demonstrates normal lung architecture,

indicating that exposures to Terracess JS particles produced no adverse pulmonary effects. (Magnification =x40)

4A

Figure 15

Light micrograph of lung tissue from a rat exposed to quartz particles (5 mg/kg) at 3

months post-instillation exposure. This micrograph illustrates the terminal bronchial and

corresponding alveolar ducts. Note the substantial accumulation of foamy quartzcontaining alveolar macrophages which fill many of the alveoli (arrows) concomitant

with the prominence of tissue thickening along the junctions at the terminal bronchiole

and alveolar ducts (arrowhead). (Magnification = x40).

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