An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials

Test material characterization

Table 1 presents the test materials’ primary particle size (PPS; determined by transmission
electron microscopy (TEM) or scanning electron microscopy (SEM)), BET surface area,
and their particle size distribution (mean or modal values, d
50
and d
90
) in water, KRPG buffer or F-12K medium. In general, particle sizes measured with
tracking analysis in H
2
O were larger than the corresponding PPS. This was assessed as being either an indication
for agglomeration and/or the material-dependent detection limit of the tracking analysis
method.

Table 1. Primary characterisation of the test materials and agglomeration in biological fluids

For most test materials, the numbers of particles dispersed in KRPG buffer or F-12K
medium after 16-h incubation underscored the detection limit for reliable tracking
analysis (indicated as ‘not detectable’ in Table 1). Only for SiO
2
.naked, SiO
2
.amino, SiO
2
.phosphate, SiO
2
NM-203, the inorganic pigment Fe
2
O
3
, and the two organic pigments DPP Orange N and Pigment Blue 15:1, measurable numbers
of dispersed particles were recorded in KRPG buffer or F-12K medium at the end of
the incubation period (Table 1):

Despite their small size and low light scattering properties, colloidal SiO
2
.naked and its negatively charged surface functionalized variants SiO
2
.amino and SiO
2
.phosphate were observable with tracking analysis in H
2
O, and particle sizes matched the upper PPS. Under testing conditions, i.e., in KRPG
buffer and in F-12K medium, particle sizes of these materials only slightly increased,
gravitational settling was minimal, and only few agglomerates became visible at the
bottom of the culture dish. Particle concentrations in F-12K medium ranged from 1.0 × 10
11
particles/mL for SiO
2
.phosphate to 2.9 × 10
11
particles/mL for SiO
2
.amino. By contrast, the neutral SiO
2
.PEG completely agglomerated in all media and no dispersed nanoparticles were detectable
in either KRPG buffer or F-12K medium.

Unlike the colloidal SiO
2
NMs, the dry-powder amorphous SiO
2
, i.e., precipitated SiO
2
NM-200 and pyrogenic SiO
2
NM-203, required extensive ultrasonication to destroy large agglomerates. Nevertheless,
dispersed SiO
2
NM-200 or NM-203 nanoparticles were not detectable by tracking analysis in F-12K medium
at the end of the incubation period (but in KRPG buffer for SiO
2
NM-203). For both dry-powder SiO
2
test items, gravitational settling occurred that was more pronounced than for the
colloidal SiO
2
NMs.

Within 16 h, low, but detectable amounts of dispersed particles of inorganic Fe
2
O
3
or organic DPP Orange N were observed in the F-12K supernatant with mean particle
sizes of 113 and 89 nm, respectively. By comparison, the PPSs were 15 nm for Fe
2
O
3
and 30–400 × 10–50 nm for DPP Orange N. Even though slight colourations remained in
the medium after agglomerate sedimentation, this could be corrected for using cell-free
controls and, therefore, did not affect the OD measurements. Particle concentrations
in F-12K medium were 2.7 × 10
8
for Fe
2
O
3
and 1.3 × 10
8
for DPP Orange N, i.e., by a factor of 10
3
lower than the concentrations recorded for the dispersed colloidal SiO
2
NMs.

Pigment Blue 15:1 could be dispersed in H
2
O by ultrasonication (180 ?g/mL resulted in 5 × 10
10
particles/mL; d
50
:191 nm as compared to a PPS of 17 nm). However, less than 5 % of that particle number
concentration were observed in F-12K medium after 16 h incubation (180 ?g/mL resulted
in 2.1 × 10
9
particles/mL; d
50
: 173 nm) in spite of the addition of 0.05 % (w/v) bovine serum albumin during ultrasonication.
The vast majority of this nanosized pigment formed blue agglomerates.

Cellular uptake of the test materials

Agglomerated particles settled to the bottom of the culture vessel where they were
visible with phase contrast optics. As a rule, the test materials agglomerated and
settled gravitationally within the 16-h incubation period. This settling occurred
within minutes for corundum, within less than 1 h for quartz DQ12, 3–4 h for ZrO
2
.TODA, 6 h for ZrO
2
.acrylate, or by the end of the 16 h incubation period for TiO
2
NM-105 and all four CeO
2
NMs. Further as a rule, up to the highest test material concentration of 180 ?g/mL,
all test materials were completely engulfed by the NR8383 AMs (that are present at
the bottom of the cell culture vessels) by the end of the 16-h incubation period (cf. Fig. 1 for phase-contrast images of settled particles and test material-laden NR8383 AMs
for corundum, quartz DQ12, TiO
2
NM-105, CeO
2
NM-212, SiO
2
.naked, BaSO
4
NM-220, DPP Orange N, and graphite nanoplatelets).

Fig. 1. Test material sedimentation and uptake by NR8383 rat alveolar macrophages. A Corundum;
B Quartz DQ12; C TiO
2
NM-105; D CeO
2
NM-212; E SiO
2
.naked; F BaSO
4
NM-220; G DPP Orange N; H Graphite nanoplatelets. Phase contrast micrographs show
settled particles at the bottom of cell-free wells in 96-well plates (a1–h1 on the left side of the individual images) and corresponding particle-laden NR8383 cells at the end
of the 16-h incubation period with the same particle concentration a2–h2 on the right side of the individual images). Note that the uptake of particles (loaded with 90 ?g/mL)
appears complete as viewed by light microscopy, except for SiO
2
.naked (loaded with 22.5 ?g/mL), where settled particles are hardly visible

As exceptions to this rule, additional and specific observations were made for the
following materials. Whereas complete cellular uptake was recorded for quartz DQ12
up to test concentrations of 90 ?g/mL, many particles were visible outside deteriorated
cells at 180 ?g/mL underlining the pronounced cytotoxic effect of this material. AlOOH
formed branched agglomerates under culture conditions which settled as a fluffy layer.
For the different types of CeO
2
NMs, cellular uptake was complete up to a concentration of 90 ?g/mL, whereas very
few agglomerates remained between the NR8383 cells at 180 ?g/mL. Graphite nanoplatelets
have a strong light absorbance. Nevertheless this carbonaceous NM could be measured
colourimetrically up to a concentration of 180 ?g/mL since the substance precipitated.
Although largely ingested by NR8383 macrophages, some large flocs of graphite nanoplatelets
were not fully internalised, but were contacted by surrounding cells.

In conclusion, for most test materials, complete cellular uptake was recorded by the
end of the incubation period at all tested concentrations. Only for SiO
2
.naked, SiO
2
.amino and SiO
2
.phosphate, relevant proportions had not sedimented within 16 h (and hence could not
be taken up by the AMs). Further, for graphene nanoplatelets, that did sediment, the
highest test substance concentration was not fully internalized by the cells.

In vitro studies and test material assignment as active or passive

In the following, for each test material, the in vitro data are presented and evaluated
to assign the material as either passive or active, and this in vitro assignment is
directly compared to the outcome of the corresponding available in vivo studies and
the resulting in vivo categorization as active or passive test materials.

Table 2 provides an overview of the data collected in the in vitro NR8383 AM assay determining
extracellular release of LDH, GLU, TNF-?, and H
2
O
2
listing all endpoint-specific data recorded at all test material concentrations (expressed
in mass per volume metrics; i.e. µg/mL). In further processing these data, Table 3 presents the endpoint-specific significant in vitro LOAECs recorded for each test
material, expressing these values both in mass per volume metrics and in relation
to the BET surface area (i.e., mm
2
/mL). Thereby, Table 3 reveals whether significant effects occurred below the threshold value of 6000 mm
2
/mL, and if so, how many parameters were significantly affected below the threshold
for a given test material. Based thereupon, Table 3 further provides the in vitro NM assignments as either active or passive with contrasting
juxtaposition to the STIS NOAECs, LOAECs and in vivo categorizations as active or
passive.

Table 2. Effects of the negative control corundum, the positive control quartz DQ12, and the
20 test materials on the NR8383 cells

Table 3. Comparison of significant in vitro LOAECs to NOAECs and LOAECs recorded in rat short-term
inhalation studies

For a better overview, Tables 2 and 3, just as the following subsections of “In vitro studies and test material assignment
as active or passive”, are subdivided into the following sections: First, the data
for the NC and PC are provided, i.e., corundum (Al
2
O
3
) and quartz DQ12. Next, the data for the seven metal oxide NMs that were identified
as active in vitro are presented, i.e., TiO
2
NM-105, ZnO NM-111, and all four tested CeO
2
NMs. This is followed by the data recorded for the amorphous SiO
2
NMs. The subsequent section presents the data for the four metal oxide and metal sulphate
NMs that were identified as passive in vitro, i.e., AlOOH, BaSO
4
NM-220, Fe
2
O
3
, and both surface-functionalized ZrO
2
. The final two sections present the data recorded for the two nanosized organic pigments
and graphite nanoplatelets.

Corundum

In vitro, corundum is assigned as passive

Corundum induced very slight dose-dependent increases of LDH, TNF-? and H
2
O
2
, all of which were not significantly different from the non-treated controls. This
lack of effects confirms the suitability of corundum as NC and as a negative benchmark
material.

In vivo categorization confirms corundum passivity

Previous rat STISs underlined the inert nature of respirable corundum particles (aerosol
concentration 20 mg/m
3
; 2-week exposure, 5 days/week; 5 h/day) 77]. Also in rat instillation studies, corundum proved to be a chemically inert particle
which hardly elicited any pulmonary inflammatory or fibrogenic effects 101], 102]. However, high intratracheal instillation doses of 7.5 mg/rat lung 102] or 5 mg/100 g body weight 103], which are both in the overload range, elicited BALF changes that mainly consisted
of elevated polymorphonuclear neutrophil (PMN) counts. In line with these findings,
the United States National Institute for Occupational Safety and Health (NIOSH) has
set an occupational exposure limit (OEL) of 5 mg/m
3
for respirable corundum (summarized by Krewski et al. 104]). Taken together, corundum is categorized as passive in vivo, which confirms in vitro
passivity.

Quartz DQ12

In vitro, quartz DQ12 is assigned as active

The in vitro macrophage toxicity of quartz DQ12 is well-known 54], 105]. Quartz DQ12 induced dose-dependent releases of LDH, GLU, and TNF-?, significant
well below the threshold value of 6000 mm
2
/mL. Even though quartz DQ12 hardly elicited any extracellular H
2
O
2
formation, the findings confirm its suitability as PC and results in quartz DQ12 assignment
as active material.

In vivo categorization confirms quartz DQ12 activity

In a rat 28-day sub-acute inhalation study, a NOAEC of 0.1 mg/m
3
was recorded for alpha-quartz (median particle size: 1.7 µm) 106]. Due to its progressive inflammatory, fibrogenic and genotoxic effects, micron-sized
quartz DQ12 is widely used as a PC for in vivo studies 102], 107]. As PC, it was tested at one (high) concentration, each, in two rat 5-day STISs.
At 25 mg/m
3
, quartz DQ12 produced progressively severe effects over the 3-month post-exposure
period 80] with similar findings recorded at 100 mg/m
381]. Generally, the results observed in the STIS at the end of the post-exposure observation
periods resembled those recorded in sub-chronic inhalation studies 108]: Macrophage, monocyte, PMN, and also lymphocyte counts were increased in the lung
parenchyma and BALF, which coincided with elevated levels of total protein and enzyme
activities [LDH, alkaline phosphatase (AP), ?-glutamyltransferase (GGT) and N-acetyl-glucosaminidase
(NAG)]. Upon intratracheal instillation in rats, pulmonary fibrosis was observed even
after bolus doses of only 0.15–0.3 mg 102], 109]. Such dosages may be reached under STIS conditions by aerosol concentrations that
are far lower than 10 mg/m
3
. In conclusion, quartz DQ12 is categorized as active material, which confirms the
in vitro assignment.

Active metal oxide NMs (TiO
2
, ZnO, CeO
2
)

In vitro, TiO
2
NM-105 is assigned as active

TiO
2
NM-105 elicited dose dependent increases of LDH, GLU, and TNF-? (significant in vitro
LOAECs at 4230 mm
2
/mL, each), whereas H
2
O
2
formation did not differ from the vehicle control. Since significant in vitro LOAECs
below the threshold value of 6000 mm
2
/mL were recorded for 3 of the 4 parameters, TiO
2
NM-105 is assigned as active NM.

In vivo categorization confirms TiO
2
NM-105 activity

In a number of different STISs assessing both coated and uncoated TiO
2
NMs, pulmonary inflammatory changes were recorded, with a NOAEC for TiO
2
NM-105 of 2 mg/m
3
and a LOAEC of 2 mg/m
3
. Most prominent findings were BALF increases in total cell count, PMNs, and AMs.
Additionally, total protein and the activities of LDH, ALP, GGT, NAG were increased
10], 11], 81]. Based upon these in vivo STIS data, TiO
2
NM-105 is categorized as active NM, which confirms the in vitro assignment.

ZnO NM-111 is assigned as in vitro active

ZnO NM-111, that is coated with triethoxycaprylylsilane, is a NM with a comparably
small BET surface area of 15.1 m
2
/g. This material dissolves in acidic environments thereby shedding zinc ions 79]. Maximum cytotoxicity (release of LDH) was already observed at 22.5 ?g/mL (significant
in vitro LOAEC at 5.6 ?g/mL, i.e., 84 mm
2
/mL), while the parameter GLU became significant at 90 ?g/mL (1350 mm
2
/mL) and TNF-? induction was at its maximum between 22.5 and 45 ?g/mL (in vitro LOAEC
338 mm
2
/mL). TNF-? formation was inhibited at higher concentrations due to progressive cell
degradation. Extracellular H
2
O
2
formation induced by ZnO NM-111 was in the same range as the concurrent vehicle control
value. In parallel studies, the non-coated core material ZnO NM-110 was even more
toxic than coated ZnO NM-111 for all parameters tested (data not shown). Since significant
in vitro LOAECs below the threshold value of 6000 mm
2
/mL were recorded for 3 of the 4 parameters, ZnO NM-111 (just as ZnO NM-110) is assigned
as active NM.

In vivo categorization confirms ZnO NM-111 activity

Coated ZnO NM-111 elicited extensive signs of inflammation in the BALF upon 5-day
inhalation exposure to 2.5 mg/m
3
(NOAEC: 0.5 mg/m
3
). The most prominent findings were increased total cell counts caused by invaded
PMNs, lymphocytes, and monocytes 11]. Total protein concentration and enzyme activities (GGT, LDH, ALP and NAG) were increased
as well, just as a number of inflammatory mediators, i.e., cytokine-induced neutrophil
chemoattractant 1 (CINC-1, the rat homologue to IL-8), clusterin, cystatin C, granulocyte
chemotactic protein 2 (GCP-2), and MCP-1. In a 14-day STIS, 8 mg/m
3
uncoated ZnO NM-110 (only this concentration tested) also elicited pulmonary inflammatory
effects so that a 14-day NOAEC  8 mg/m
3
was assigned to ZnO NM-110 110]. Based upon these in vivo STIS data, ZnO NM-111 (just as ZnO NM-110) is categorized
as active NM, which confirms the in vitro assignment.

All four CeO
2
NMs (Al-doped CeO
2
, nano-CeO
2
, CeO
2
NM-211, and CeO
2
NM-212) are assigned as in vitro active

All four types of CeO
2
NMs dose-dependently increased LDH and GLU release. The in vitro LOAECs recorded for
LDH were significant and below the threshold value of 6000 mm
2
/mL for all four CeO
2
NMs. Additionally, for Al-doped CeO
2
, the in vitro LOAEC recorded for GLU was significant and below the threshold value.
Evaluation of these two parameters that indicate cell membrane damage resulted in
the following ranking of cytotoxicity (when using the particle surface area-based
LOAECs): Al-doped CeO
2
  CeO
2
NM-212  nano-CeO
2
  CeO
2
NM-211. H
2
O
2
formation was far less pronounced (and never significant), but also headed by Al-doped
CeO
2
. Finally, the in vitro LOAECs recorded for TNF-? induction were significant and below
the 6000 mm
2
/mL threshold value for all four CeO
2
NMs, albeit with a slightly different ranking, i.e., CeO
2
NM-212  Al-doped CeO
2
  CeO
2
NM-211 = nano-CeO
2
. Accordingly, the findings indicate different biological activities and potencies
of in vitro cellular effects of the four tested CeO
2
NMs with Al-doped CeO
2
and CeO
2
NM-212 eliciting more pronounced effects than nano-CeO
2
or CeO
2
NM-211. Since significant in vitro LOAECs below the threshold value of 6000 mm
2
/mL were consistently recorded for the two parameters LDH and TNF-? (and additionally
for GLU in the case of Al-doped CeO
2
), all four CeO
2
NMs are assigned as active.

In vivo categorization confirms activity for all four CeO
2
NMs

All four CeO
2
NMs induced a pronounced transient inflammation at 0.5 mg/m
3
, and effects increased at higher doses up to 25 mg/m
311], 111]. Three days post-exposure, relative PMN fractions in the BALF increased to 76 and
79 % for Al-doped CeO
2
and nano-CeO
2
, respectively, and concomitantly, the BALF total protein levels were elevated 11]. For all four CeO
2
NMs, a NOAEC of 0.5 mg/m
3
was assigned 11], 111]. Accordingly, all four CeO
2
NMs are categorized as active, which confirms the in vitro assignment.

For an in-depth in vitro-in vivo comparison of the test results recorded for the four
CeO
2
NMs, the in vitro data were further compared to specific BALF findings that are directly
related to AM-induced alterations of the in vivo rat lung, i.e. absolute values of
total cells, AM, PMN as well as total protein concentration. By contrast to the above-mentioned
particle surface area-based in vitro ranking, the following ranking of cytotoxicity
is achieved when using the mass-based in vitro data: Al-doped CeO
2
  CeO
2
NM-211 = nano-CeO
2
  CeO
2
NM-212. As presented in further detail in the Additional file 1: Table S2, the BALF findings 11], 111] allow the following conclusions on in vivo AM-related pulmonary alterations: Al-doped
CeO
2
and nano-CeO
2
dose dependently decreased the number of AMs in the BALF and increased PMN counts
and total protein concentration alike, whereas the corresponding effects elicited
by CeO
2
NM-211 and CeO
2
NM-212 were approx. 60 % lower over the entire concentration range. Hence, even though
the four different CeO
2
NMs were assigned the same STIS NOAEC of 0.5 mg/m
3
, there were gradual differences in BALF parameters at nominally identical conditions
of NM in the inspired air. Also the lung burdens recorded for the four different CeO
2
NMs upon 5-day inhalation exposure differed 11], 111]. These differences are partly reflected by the in vitro tests in which Al-doped CeO
2
was clearly more bioactive than e.g., CeO
2
NM-212, at least in the low to middle dose range.

Amorphous SiO
2
NMs

Colloidal SiO
2
.naked is assigned as in vitro active and its surface-functionalized variants (SiO
2
.PEG, SiO
2
.amino, and SiO
2
.phosphate) as in vitro passive

For all four colloidal SiO
2
NMs, significant in vitro LOAECs were recorded for all four test parameters with the
exception of GLU for SiO
2
.phosphate. However, as a rule, these significant LOAECs by far exceeded the threshold
value of 6000 mm
2
/mL. Only for SiO
2
.naked, LDH and TNF-? attained 4500 mm
2
/mL, each, and for SiO
2
.amino, TNF-? attained this same value. Accordingly, only for the non-surface functionalized
SiO
2
.naked, two parameters with significant in vitro LOAECs below 6000 mm
2
/mL, i.e., the threshold for biologically relevant, particle-specific (i.e., non cellular
overload-induced) effects, were recorded. In conclusion, SiO
2
.naked is assigned as in vitro active, whereas its surface-functionalized variants
SiO
2
.PEG, SiO
2
.amino, and SiO
2
.phosphate are assigned as passive.

In vivo categorizations confirm both SiO
2
.naked activity and the passivity of the surface-functionalized SiO
2

At 10 and 50 mg/m
3
, SiO
2
.naked evoked dose-dependent signs of inflammation in the rat STIS 11]. Three days after the final exposure, the most predominant significant effect in
the BALF was an increased PMN count that was accompanied by slightly elevated BALF
lymphocyte counts and moderately increased numbers of blood PMNs. In histopathological
evaluation, multifocal macrophage aggregates were observed in the lung that exacerbated
towards a slight multi-focal pulmonary inflammation by the end of the 3-week exposure
free period. Accordingly, a NOAEC of 2.5 mg/m
3
was assigned to SiO
2
.naked 11]. By contrast, no adverse effects were observed after inhalation exposure to up to
50 mg/m
3
SiO
2
.PEG, SiO
2
.phosphate, or SiO
2
.amino, and the NOAEC for these materials was assessed as being ?50 mg/m
311]. Based upon the in vivo STIS data, SiO
2
.naked is categorized as active NM and SiO
2
.PEG, SiO
2
.phosphate, and SiO
2
.amino as passive NMs, which confirms the in vitro assignment.

SiO
2
NM-200 and NM-203 are assigned as in vitro active

Both precipitated SiO
2
NM-200 and pyrogenic SiO
2
NM-203 consistently elicited significant LDH, GLU and TNF-? release at 22.5 ?g/mL,
each. For all three parameters, this corresponds to significant in vitro LOAECs of
4253 and 4500 mm
2
/mL, for SiO
2
NM-200 and NM-203, respectively. Accordingly, for both dry-powder amorphous SiO
2
NMs, two parameters, each, ranged below the threshold value of 6000 mm
2
/mL, and both SiO
2
NM-200 and NM-203 are assigned as active.

In vivo categorization confirms SiO
2
NM-200 and NM-203 activity

For precipitated SiO
2
NM-200 and pyrogenic SiO
2
NM-203, STIS data were available for precipitated Zeosil
®
45 and pyrogenic Cab-O-Sil
®
M5. The equivalence of these materials to SiO
2
NM-200 and NM-203, respectively, has been established based upon concordance in production
process and minimum degree of material purity as well as comparability of specific
surface area and agglomerate size 80], 112], 113]. In the rat STIS published by Arts et al. 80], test material concentrations of 1, 5, and 25 mg/m
3
were applied, and 1 mg/m
3
was recorded as NOAEC for both SiO
2
NM-200 and NM-203, whereas 5 mg/m
3
was assessed as LOAEC. For SiO
2
NM-200, increased weights of the lungs and lung-associated lymph nodes (LALNs) as
well as an inflammatory response of the lung tissue were recorded at 5 and 25 mg/m
3
that were accompanied by dose-dependently increased PMN counts, enzyme activities,
and protein levels in the BALF. For SiO
2
NM-203, increased lung weights and hypertrophy of the bronchiolar epithelium were
significant in the 5 and 25 mg/m
3
test groups. The LALNs contained increased silica levels, and, again, dose-dependently
increased PMN and macrophage counts in the BALF indicated inflammatory reactions.
For both materials, all effects were fully reversible within 3 months post-exposure
80]. Based upon the in vivo STIS data, SiO
2
NM-200 and NM-203 are categorized as active, which confirms the in vitro assignment.

Passive metal oxide and metal sulphate NMs (AlOOH, BaSO
4
, Fe
2
O
3
, ZrO
2
)

AlOOH is assigned as in vitro passive

AlOOH (boehmite) elicited dose-dependent and significant increases of LDH and TNF-?
(significant in vitro LOAECs: 9450 and 18,900 mm
2
/mL, respectively). Hence, even though significant LOAECs were recorded for two parameters,
both values ranged well above the threshold value of 6000 mm
2
/mL indicating that the observed effects were elicited under in vitro cellular overload
conditions. Accordingly, these effects are assessed as not-particle specific, and
AlOOH is assigned as passive.

In vivo categorization confirms AlOOH passivity

The AlOOH NM included in the present study (PPS: 40 nm; BET surface area: 105 m
2
/g) and a smaller AlOOH variant (PPS: 10 nm; BET surface area: 182 m
2
/g) were submitted to a 28-day rat sub-acute inhalation study followed by a 3-month
post-exposure observation period 58]. In this study, no adverse effects were recorded at AlOOH aerosol concentrations
of 0.4 or 3 mg/m
3
. Pulmonary inflammation (recorded by significantly altered BALF parameters, increased
lung and LALN weights and histopathological findings) was observed at 28 mg/m
3
AlOOH. However, these effects were only elicited by cumulative doses exceeding approx.
1 mg AlOOH/g lung at the end of the 28-day exposure period 58]. Since the concentration dependence and time-course changes of aluminum lung burden
demonstrated a precipitous increase in elimination half-time at aerosol concentrations
of 28 mg/m
3
, Pauluhn assessed these findings as being consistent with pulmonary overload 58]. An earlier intratracheal instillation study confirmed these results indicating a
NOAEC of 0.6 mg/rat lung, whereas reversible increases in BALF PMN and total protein
levels were recorded at bolus doses of 1.2 mg/rat lung 84]. Since AlOOH was tested for 28 days (i.e., four consecutive 5-day exposure periods),
the NOAEC of 3 mg/m
3
that Pauluhn 58] recorded for this material was converted to a 5-day NOAEC by multiplying it by a
factor of four 94]. In accordance with this calculated 5-day NOAEC of 12 mg/m
3
, AlOOH is categorized as passive, which confirms the in vitro assignment.

BaSO
4
NM-220 is assigned as in vitro passive

BaSO
4
NM-220 did not elicit any cellular effects that differed from the vehicle or corundum
controls. Therefore, BaSO
4
NM-220 is assigned as passive.

In vivo categorization confirms BaSO
4
NM-220 passivity

No adverse effects were observed in a rat STIS after inhalation exposure to up to
50 mg/m
3
BaSO
4
NM-220 11]. Accordingly, BaSO
4
NM-220 is categorized as passive, which confirms the in vitro assignment.

Fe
2
O
3
is assigned as in vitro passive

For Fe
2
O
3
(hematite), the only significant LOAEC was recorded for TNF-? (8266 mm
2
/mL). This one in vitro LOAEC further exceeded the threshold value of 6000 mm
2
/mL. Of note, the cytotoxic effects elicited by this nanosized Fe
2
O
3
were equal to or lower than those of its bulk counterpart, whereas the amount of sedimented
test material appeared comparable (data not shown). Since only one parameter was affected,
nanosized Fe
2
O
3
is assigned as passive.

In vivo categorization confirms Fe
2
O
3
passivity

Inhalation exposure to up to 30 mg/m
3
Fe
2
O
3
(or its non-nanosized counterpart; data not shown) in a STIS did not cause any adverse
effects in the rat lung as was determined by BALF evaluation, hematology and histopathological
evaluation 79]. Based upon this in vivo study, Fe
2
O
3
(just as its non-nanosized counterpart) is categorized as passive, which confirms
the in vitro assignment.

ZrO
2
.TODA and ZrO
2
.acrylate are assigned as in vitro passive

ZrO
2
.TODA and ZrO
2
.acrylate dose-dependently and significantly increased LDH, TNF-?, and H
2
O
2
formation, and ZrO
2
.acrylate additionally GLU. However, apart from the LDH value recorded for ZrO
2
.TODA (in vitro LOAEC: 5265 mm
2
/mL), which laid just below the 6000 mm
2
/mL threshold, all other significant in vitro LOAECs recorded for either ZrO
2
.TODA or ZrO
2
.acrylate exceeded the threshold value. Accordingly, both ZrO
2
.TODA and ZrO
2
.acrylate are assigned as passive.

In vivo categorization confirms ZrO
2
.TODA and ZrO
2
.acrylate passivity

No adverse effects were observed in a rat STIS after inhalation exposure to up to
50 mg/m
3
ZrO
2
.acrylate or ZrO
2
.TODA 11]. In rat intratracheal instillation studies, bolus dose-NOAECs of 0.6 and 1.2 mg/rat
lung were recorded for ZrO
2
.TODA and ZrO
2
.acrylate, respectively 114]. Based upon these in vivo studies, both ZrO
2
.TODA and ZrO
2
.acrylate are categorized as passive, which confirms the in vitro assignment.

Nanosized organic pigments

DPP Orange N is assigned as in vitro passive

DPP Orange N elicited a significant increase of TNF-? (in vitro LOAEC 2880 mm
2
/mL) that ranged well below the threshold value of 6000 mm
2
/mL. However, since LDH, GLU, and H
2
O
2
formation were not significantly altered, the premise that at least two of the four
parameters had to be altered to indicate NM activity was not met. Of note, also the
bulk counterpart to DPP Orange N, DPP Orange B, did not elicit relevant cytotoxicity
in the in vitro NR8383 AM assay (data not shown). Accordingly, DPP Orange N (and DPP
Orange B) are assigned as passive.

In vivo categorization confirms DPP Orange N passivity

Inhalation exposure to up to 30 mg/m
3
DPP Orange N in a STIS did not cause any adverse effects in the rat lung as was determined
by BALF evaluation, hematology and histopathological evaluation 79]. For the respective bulk material DPP Orange B, high aerosol concentrations of 30 mg/m
3
slightly increased total cell count, PMN, MCP-1 and osteopontin in the BALF (data
not shown). Based upon these in vivo studies, DPP Orange N (just as DPP Orange B)
are categorized as passive, which confirms the in vitro assignment.

Pigment Blue 15:1 is assigned as in vitro active

Pigment Blue 15:1 elicited dose-dependent increases of LDH and GLU with significant
LOAECs of 4770 mm
2
/mL, each. (The slightly blue colouration of the supernatants could be corrected for
via cell-free controls.) Accordingly, two parameters had significant LOAECs ranging
below the threshold value of 6000 mm
2
/mL. Based upon these in vitro findings, Pigment Blue 15:1 is classified as active.

In vivo categorization indicates Pigment Blue 15:1 passivity, thereby refuting the
in vitro assignment as over-predictive

For Pigment Blue 15:1, a STIS NOAEC of 30 mg/m
3
was recorded 79]. At this aerosol concentration, blue pigment-laden AMs were observed in the lung
parenchyma and LALNs. Further, slight epithelial hypertrophy or hyperplasia was noted
in terminal bronchioles that however were assessed as rather reflecting the challenged
biological clearance mechanism than adverse reactions. All findings were fully reversible
within the 3-week post-exposure period. Based upon this in vivo study, Pigment Blue
15:1 is categorized as passive. Accordingly, for this pigment, the in vitro NR8383
AM assay over-predicted its toxic potential in the rat STIS.

Graphite nanoplatelets

Graphite nanoplatelets are assigned as in vitro passive

Graphite nanoplatelets elicited significantly increased GLU and TNF-? levels (in vitro
LOAECs, 3330 and 6660 mm
2
/mL, respectively. Accordingly, only one parameter (GLU) ranged below the threshold
value of 6000 mm
2
/mL. Based upon these findings, graphite nanoplatelets are assigned as passive.

In vivo categorization confirms passivity of graphite nanoplatelets

Inhalation exposure to up to 50 mg/m
3
graphite nanoplatelets in a STIS did not cause any adverse effects in the rat lung
as was determined by BALF evaluation, hematology and histopathological evaluation
82]. Based upon this in vivo study, graphite nanoplatelets are categorized as passive,
which confirms the in vitro assignment.

Summary of in vitro–in vivo comparisons

In summary, for 19 of the 20 test materials, the in vitro NR838 AM assay addressing
extracellular release of LDH, GLU, TNF-? and H
2
O
2
correctly predicted in vivo activity or passivity in the rat STIS. Pigment Blue 15:1
was the only material that tested false positive: Based upon the significant in vitro
LOAECs that ranged below the threshold value of 6000 mm
2
/mL for LDH and GLU (each: 4770 mm
2
/mL), 2 of the 4 in vitro parameters were positive. This resulted in Pigment Blue
15:1 assignment as active, whereas it had been categorized as passive based upon the
high STIS NOAEC of 30 mg/m
3
.

By contrast, for SiO
2
.amino, ZrO
2
.TODA, DPP Orange N, and graphite nanoplatelets, only one parameter each tested positive
with significant in vitro LOAECs ranging below the threshold value of 6000 mm
2
/mL. ZrO
2
.TODA only triggered LDH, graphite nanoplatelets only triggered GLU, and SiO
2
.amino and DPP Orange N each only triggered TNF-? release. By definition, this in
vitro outcome resulted in their assignment as passive, and this result was confirmed
by the in vivo data available for all four of these test materials.

Applying the Cooper statistics 93], the in vitro NR8383 AM assay performed under the conditions of the present study
had a specificity of 91 % and a sensitivity of 100 % with an overall accuracy of 95 %.
The rates for negative and positive prediction were 90 and 100 % (Table 4).

Table 4. Determination of the accuracy, sensitivity and specificity of the in vitro NR8383
alveolar macrophage assay

Testing against a particulate benchmark material as a NC is mandatory in an empirical
assay, since it provides information on the reliability and reproducibility of the
behaviour of the cells under loading conditions. When evaluating all test results
against the corresponding values recorded for the negative (albeit micron-sized) benchmark
material corundum (Additional file 1: Table S1), Pigment Blue 15:1 was again assessed false positive. Since the test results
obtained for corundum were slightly higher than those recorded for the particle-free
vehicle control, the corundum-based evaluation resulted in some minor deviations from
the vehicle control-based evaluation. This, however, resulted in a number of statistically
relevant differences. Using the corundum-based evaluation, SiO
2
NM-203 was assessed ‘false negative’ (only one significant LOAEC  6000 mm
2
/mL was recorded, i.e., for GLU). Furthermore, ZrO
2
.TODA and SiO
2
.phosphate exhibited one positive finding, each. A sharp demarcation line between
active and passive materials will be prone to erroneous decisions. Therefore, the
premise had been set that at least two of the four parameters had to be altered to
assign a material as active. Notwithstanding, false negative findings are detrimental
for regulatory toxicity testing, and for this reason the statistical evaluation against
untreated cells proved superior to the evaluation against the negative benchmark material
corundum. Accordingly, in the subsequent discussion, only the vehicle-control-based
evaluation is addressed.

You May Also Like

Total, saturated fat linked to increased risk of lung cancer

We’ve Got To Stop Using These Words To Talk About Cancer Drugs

‘I Started Losing All of My Hair When I Was 25’