Toxicity of a Quartz with Occluded Surfaces in a 90-Day Intratracheal Instillation Study in Rats

Figures & data

TABLE 1  Physicochemical properties of test materials used in 90-day study

	Material
Characteristic	Quartz isolate	Reference quartz	TiO2
Surface area
By BET method^a (m^2/g)	21.2	3.7	1.9
By EGME method^b (m^2/g)	213	5.6	21.7
XRD average domain size^c (μ m)	0.076	0.19	—
XRD mineralogy
Quartz (%) (per NIST 1878A standard reference)	60	87	0
Montmorillonite (%)	30	0	0
Undefined amorphous silica (%)	8	13	0
Analcime (%)	2	0	0
Rutile (%)	0	0	99.5
Major element composition^d (normalized)
SiO2 (%)	87.88	99.30	—
Al2O3 (%)	7.58	0.00	—
Fe2O3 (%)	1.39	0.04	—
MnO (%)	0.00	0.00	—
Na2O (%)	0.79	0.21	—
K2O (%)	0.19	0.05	—
MgO (%)	0.78	0.00	—
CaO (%)	0.75	0.00	—
TiO2 (%)	0.08	0.02	98.17
P2O5 (%)	0.04	0.02	—
F (%)	0.07	0.02	—
S (%)	0.01	0.01	—
Total C (%)	0.39	0.33	—
Organic carbon (%)	0.05	0.00	—
Particle size by scanning electron microscopy
Mass mean geometric diameter (μ m)	3.58	3.01	1.80
Geometric Standard deviation	1.30	1.53	1.90
Differential scanning calorimetry (DSC)
α Quartz → β quartz transition temperature (°C)	Not detected	573	—
β Quartz → α quartz transition	None	Yes	—
Thermal XRD Analysis
α Quartz → β quartz transition temperature (°C)	by 675	573	—
Zeta potential^e
At pH 4 (mV)	−20.49	−35.42	−11.47
At pH 6 (mV)	−25.32	−30.49	−35.08
At pH 8 (mV)	−31.14	−39.09	−43.03
Electron spin resonance (ESR)^f
Hydroxyl radical (• OH) relative peak height	54	65	—
Endotoxin content
ng/g (EU/g)	100 (1000)	1.78 (17.8)	< 0.562 (< 5.62)

^aBrunauer–Emmet–Teller method (Brunauer et al., 1938).

^bEthylene glycol monoethyl ether method (Carter et al., 1965; Bower and Goertzen, 1959).

^cUsing the Warren–Averbach procedure (Warren and Averbach, 1953).

^dUsing standard lithium metaborate/tetraborate fusion methods with inductively coupled plasma (ICP) analysis.

^eA measurement of the magnitude of surface charge.

^fA measurement of the presence of free radicals.

FIG. 1  Quartz isolate (5000×).

FIG. 2  Reference quartz (5000×).

FIG. 3  Titanium dioxide Bayertitan T (5000×).

TABLE 2  Study design

Group	Test material	Cumulative dose (mg/kg)^a	Cumulative dose: quartz (mg/kg)^a	Cumulative dose: other particles (mg/kg)^a	Number of animals per group
1	Saline	Vehicle only	Vehicle only	Vehicle only	30
2	Quartz isolate	15.2	9.1	6.1	30
3	Positive control = 69.1% reference quartz + 30.9% titanium dioxide	10.5 Reference quartz + 4.7 titanium dioxide	9.1	1.4 + 4.7	30

^aAll groups were dosed, over 4 days, with 4 aliquots of the test materials at a volume of 0.3 ml.

TABLE 3  Means of parameters measured in BAL fluid 3, 28, and 90 days after the last of 4 instillations

		Lactic dehydrogenase (U/L)
Group	Parameter	Day 3	Day 28	Day 90
Saline control	Mean	52	30	38
	SD	21	7	6
	n	6	6	7
Quartz isolate	Mean	166	107	128
	SD	39	25	18
	n	6	6	7
Positive control	Mean	284***	361**	734***
	SD	132	258^#	132^###
	n	6	6	7
		β − Glucuronidase (U/L)
Saline control	Mean	0.3	0.2	0.3
	SD	0.1	0.1	0.1
	n	6	6	7
Quartz isolate	Mean	1.1***	0.8	1.1
	SD	0.2	0.1	0.3
	n	6	6	7
Positive control	Mean	1.6***	2.7***	12.6***
	SD	0.5^#	1.0^##	4.6^###
	n	6	6	7
		Total protein (mg/L)
Saline control	Mean	73	101	83
	SD	14	27	8
	n	6	6	7
Quartz isolate	Mean	204***	185	209
	SD	17	26	23
	n	6	6	7
Positive control	Mean	346***	512***	875***
	SD	60^###	129^###	182^###
	n	6	6	7

Note. Statistics: ANOVA + Tukey test, *P ≤ 5%, ** P ≤ 1%, and *** P ≤ 0.1% (vs. control); ^# P ≤ 5%, ^## P ≤ 1%, and ^### P ≤ 0.1% (positive control vs. QI).

TABLE 4  Differential cell count 3, 28, and 90 days after intratracheal instillation (means)

			3 Days
Group	Dose (mg/kg)		Leukocyte number (cells)	Macrophages (%)	Eosinophil PMNs (%)	Neutrophil PMNs (%)	Lymphocytes (%)	Cell viability (%)
Saline control	0	Mean	4.73 × 10^6	99.7	0.0	0.0	0.3	99
		SD	0.71 × 10^6	0.5	0.0	0.0	0.3	1
Quartz isolate	15.2	Mean	12.42 × 10^6*	79.9***	0.0	18.1***	2.0**	100
		SD	2,17 × 10^6	2.7	0.1	2.6	0.8	1
Positive control	10.5 + 4.7	Mean	19.95 × 10^6***	58.0***	0.0	39.0***	2.9***	100
		SD	6.88 × 10^6#	7.3^###	0.1	7.3^###	1.2	1
			28 Days
Saline control	0	Mean	3.44 × 10^6	99.4	0.0	0.3	0.3	99
		SD	1.04 × 10^6	0.5	0.0	0.3	0.4	1
Quartz isolate	15.2	Mean	10.88 × 10^6	72.7***	0.3*	24.9***	2.3	99
		SD	5.16 × 10^6	7.3	0.2	7.1	1.1	1
Positive control	10.5 + 4.7	Mean	40.82 × 10^6 ***	49.5***	0.0	44.8***	5.8***	100
		SD	7.55 × 10^6 ^###	4.9^###	0.0#	6.2^###	2.0^##	0
			90 Days
Saline control	0	Mean	3.34 × 10^6	99.3	0.0	0.1	0.6	100
		SD	0.36 × 10^6	0.7	0.0	0.2	0.7	0
Quartz isolate	15.2	Mean	6.81 × 10^6	64.5***	0.0	31.6***	4.0***	99
		SD	2.07 × 10^6	11.6	0.0	11.3	0.7	1
Positive control	10.5 + 4.7	Mean	218.21 × 10^6 ***	51.2***	0.0	45.8***	3.0***	100
		SD	79.36 × 10^6 ^###	6.2^#	0.0	5.5^##	1.2	0

Note. SD, standard deviation; n = 6–7 = number of animals. Statistics: ANOVA + Tukey test, *P ≤ 5%, ** P ≤ 1%, and *** P ≤ 0.1% (vs. control); # P ≤ 5%, ^## P ≤ 1%, and ^### P ≤ 0.1% (positive control vs. QI).

FIG. 4  Quartz isolate before instillation (5000×).

FIG. 5  Quartz isolate recovered from rat lung after 90 days by low-temperature plasma ashing (5000×).

FIG. 6  Lactic dehydrogenase levels in bronchoalveolar lavage fluid measured on days 3, 28, and day 90 after end of treatment.

FIG. 7  β-Glucuronidase levels measured in bronchoalveolar lavage fluid on days 3, 28, and day 90 after end of treatment.

FIG. 8  Total protein levels in bronchoalveolar lavage fluid measured on days 3, 28, and day 90 after end of treatment.

FIG. 9  Absolute numbers of polymorphonuclear neutrophils (PMNs) in bronchoalveolar lavage fluid (note log scale).

TABLE 5  Terminal body and lung wet weights 3, 28, and 90 days after the last of 4 instillations—BAL rats

Group	Dose (mg/kg)		Body weight (g)	Lung weight (g)	Lung weight/body weight × 1000
	Day 3
Saline control	0	Mean	316.3	1.1007	3.479
		Std	12.7	0.0662	0.129
Quartz isolate	15.2	Mean	317.4	*1.3298	4.191
		Std	15.4	0.0759	0.136
Positive control	10.5 + 4.7	Mean	317.4	***1.5630	***4.950
		Std	17.7	# 0.2025	# 0.808
	Day 28
Saline control	0	Mean	360.2	1.2076	3.350
		Std	34.4	0.1707	0.307
Quartz isolate	15.2	Mean	363.3	1.3729	*3.787
		Std	34.6	0.1335	0.286
Positive control	10.5 + 4.7	Mean	372.8	***1.8735	***5.032
		Std	16.4	^###0.0771	^###0.254
	Day 90
Saline control	0	Mean	422.0	1.2215	2.892
		Std	14.8	0.1094	0.194
Quartz isolate	15.2	Mean	442.9	1.4174	3.208
		Std	29.9	0.0881	0.233
Positive control	10.5 + 4.7	Mean	415.8	***2.4602	***5.908
		Std	31.0	^###0.2948	^###0.420

Note. Statistics: ANOVA + Tukey test, *P ≤ 5%, ** P ≤ 1%, and *** P ≤ 0.1% (vs. control); #P ≤ 5%, ^## P ≤ 1%, and ^### P ≤ 0.1% (positive control vs. QI).

FIG. 10  Mean absolute lung wet weights, BAL rats.

TABLE 6  Histopathological findings on days 28 and 90 posttreatment

		Day 28		Day 90
Finding	Grade	Quartz isolate	Reference quartz/TiO2 positive control	Quartz isolate	Reference quartz/TiO2 positive control
Lungs
Multifocal alveolar accumulations of particle-laden macrophages	slight	3		5
	moderate	2	5		4
	severe				1
Interstitial accumulations of particle-laden macrophages	very slight	1		1
	slight	4	5	4	5
Multifocal interstitial fibro-histiocytic granulomata	slight		3		5
	moderate		1
Multifocal alveolar lipoproteinosis^a	slight		2		5
	moderate		3
Multifocal interstitial (predominantly perivascular) mononuclear cell infiltrations	very slight	4		2	1
	slight	1	2	1	2
	moderate		3		2
Alveolar granulocytic infiltration	very slight	3		4
	slight	2	1	1	2
	moderate		4		3
Multifocal bronchiolo-alveolar hyperplasia (bronchiolar type)	very slight	5	2	3
	slight		3	1	5
Multifocal mucus (goblet) cell hyperplasia of the bronchi/bronchioles	very slight	2		1	2
	slight	1	5		3
Multifocal very slight (minimal) interstitial fibrosis^b	very slight	5	2	4
	slight		3		5
LALN
Accumulation of particle-laden macrophages	(very) slight	5		3
	moderate		4	2
	severe		1		5
Reactive lymphoid hyperplasia	slight	1	1	3
	moderate	1	4	2	5

^aThe pulmonary macrophages of the quartz isolate-treated group showed a marked foamy appearance, but without significant signs of degeneration and necrosis. In contrast, in the positive control group, the majority of alveolar macrophages were degenerating or necrotic, as reflected by alveolar lipoproteinosis. Lipoproteinaceous intra-alveolar material appeared to be mainly derived from decaying macrophages.

^bInterstitial fibrosis developed in close association with accumulation of (particle-laden) macrophages and inflammatory cells (PMN, mononuclear cells) and was characterized by localized discrete thickening of intra-alveolar septa due to increased deposition of collagen. In the positive control group, the interstitial fibro-histiocytic granulomas, when present, were characterized by central foci of foamy macrophages which at the periphery were rimmed by minimal amounts of collagenous (fibrotic) tissue that was continuous with the interstitium.

TABLE 7  Summary of the toxicological effects of the quartz isolate and positive control

	Quartz isolate			Positive control
Parameter	3 days post treatment	28 days post treatment	90 days post treatment	3 days post treatment	28 days post treatment	90 days post treatment
BAL of lungs
Total protein (BAL) (ratio treatment/control)	***2.8	−1.8	–2.5	***4.7	***5.1	***10.5
Total cell number (BAL) (ratio treatment/control)	*2.6	−3.1	–2.0	***4.2	***11.9	***65.3
PMN (BAL) (% in differential cell count)	***18.1	***24.9	***31.6	***39.0	***44.8	***45.8
Lung wet weights (absolute)	*	—	—	***	***	***
Lung wet weights (relative)	—	*	—	***	***	***
Histopathology
Inflammation	(+) to +		(+) to +	+ to ++		+ to ++
Interstitial fibrosis	(+)		(+)	+		+
Alveolar lipoproteinosis	—		—	++		+

Note. Significant effects as compared to positive control group: *5% **1% *** 0.1% (numerical values: see Tables 7–13). Histopathology: (+) very slight effect, + slight effect, ++, moderate effect, +++ high effect, – not significant.

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