Silicon dioxide

Specific investigations: other studies

Administrative data

Endpoint:

biochemical or cellular interactions

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

NR8383 alveolar macrophage (AM) assay (in vitro)
in vivo rat intratracheal instillation study (in vivo, single administration)

GLP compliance:

not specified

Type of method:

other: main study in vitro, but also in vivo supporting test

Endpoint addressed:

respiratory irritation

Test material

Test material information

Constituent 1

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

O2Si

IUPAC Name:

dioxosilane

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid - liquid: suspension

Details on test material:

different grades with SSA between 30 and 360 m2/g; sodium stabilised or alumina- or silane-modified

Specific details on test material used for the study:

four colloidal amorphous SiO2 nanomaterials that differed only by their primary particle size (9, 15, 30, and 55 nm):
Levasil 300/30% (9 nm SiO2), Levasil 200/40% (15 nm SiO2), Levasil 100/45% (30 nm SiO2), and Levasil 50/50% (55 nm SiO2)

Test animals

Species:

rat

Strain:

Wistar

Administration / exposure

Route of administration:

intratracheal

Details on exposure:

The highest test material concentration applied in the NR8383 AM assay and dose applied in the rat intratracheal instillation study were calculated to reflect an aerosol concentration of 50 mg/m3 in the rat STIS. In the rat STIS, inhalation exposure to 50 mg/m3 15 nm SiO2 for 6 h a day on 5 consecutive
days resulted in a lung burden of 342 µg]. Accordingly, for the intratracheal instillation study, a bolus dose of 360 mg/lung was selected.
Bolus doses of 360 µg 55 nm and 15 nm SiO2 in 500 µL 0.9% NaCl (i.e., 720 mg/mL) were administered toWistar rats (Crl:WI; Charles River, Germany), 8 weeks old on the day of instillation, by single intratracheal instillation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5.6 other: µg/ml

Remarks:

NR8383 AM assay (main study)
cf. Table 2 (below)

Doses / concentrations 2

Dose / conc.:

11.2 other: µg/ml

Remarks:

NR8383 AM assay (main study)
cf. Table 2 (below)

Doses / concentrations 3

Dose / conc.:

22.5 other: µg/ml

Remarks:

NR8383 AM assay (main study)
cf. Table 2 (below)

Doses / concentrations 4

Dose / conc.:

45 other: µg/ml

Remarks:

NR8383 AM assay (main study)
cf. Table 2 (below)

Doses / concentrations 5

Dose / conc.:

360 other: µg/lung

Remarks:

Rat intratracheal instillation study: Bolus dose of 360 µg/lung was set to reflect the rat lung burden of 342 µg immediately after 6-day exposure to an aerosol concentration of 50 mg/m3 in the STIS.

No. of animals per sex per dose:

8 in total (in vivo)

Control animals:

yes, concurrent vehicle

Results and discussion

Any other information on results incl. tables

In Vitro NR8383 AM Assay

H2O2 Synthesis

In the Amplex Red assay, 90 min incubation with 5.6–45 μg/mL 55 nm SiO2 or 30 nm SiO2 did not result in significant H2O2 synthesis. For the smaller 9 and 15 nm SiO2, moderately significant responses were observed at the highest concentration (45 μg/mL; p-values ≤ 0.01 and ≤0.05, respectively) (Figure 1).

LDH and GLU Release

For all four test materials, LDH release increased with increasing concentration (Figure 1). At the highest nominal concentration (45 μg/mL), all test materials elicited highly significant LDH release (p-value ≤ 0.001). Additionally, 9 nm and 15 nm SiO2 elicited moderately significant effects at 22.5 μg/mL (p-values ≤ 0.05 and ≤0.01, respectively). Hence, the two smaller test materials induced significant LDH release at lower concentrations than the two larger test materials.

GLU release upon treatment with the four test materials largely reflected LDH release (Figure 1). However, it was less pronounced and the maximum values did not exceed 16% of GLU release elicited by the positive control 0.1% Triton X.

As determined by TNFa-specific enzyme-linked immunosorbent assay (ELISA), TNFa content in the supernatant of the treated NR8383 AMs generally increased with increasing test material concentration. Further, at equal nominal concentrations of 22.5 and 45 µg/mL, TNFa release increased with decreasing particle size of the test materials. The 55 nm SiO2 did not elicit significant TNFa release at any concentration (5.6–45 µg/mL); 30 nm SiO2, 15 nm SiO2, and 9 nm SiO2 induced significant TNFa release at 45 µg/mL (p-values <= 0.01, <=0.001, and <=0.001, respectively). Furthermore, the 9 nm SiO2 induced significant TNFa release at 22.5 µg/mL (p-value <= 0.001) (Figure 1).

Plotting the TNFa release data for all four test materials as surface area–based concentrations yielded sigmoidal curves (Figure 2a,b), with R2 values of 0.93 (ISDD-calculated effective concentrations) and 0.94 (nominal concentrations). EC50 values were 4873 mm2/mL and 6702 mm2/mL, respectively (Figure 2a,b). When the data were plotted as particle mass–based nominal concentrations, no such regularity was observed (Figure 2c). These plots indicate that effects were surface area–dependent (and hence also size-dependent).

Overall Evaluation of In Vitro Test Results to Distinguish between Passive and Active Test

Table 3 provides an overview of the in vitro lowest observed adverse effect concentrations (LOAECs, defined as the lowest test material concentration eliciting a significant cellular effect) recorded for 9 nm SiO2, 15 nm SiO2, 30 nm SiO2, and 55 nm SiO2 in the NR8383 AM assay. For each test material, the parameters (H2O2, LDH, GLU, or TNFα release) for which the in vitro LOAEC undercut the previously set threshold of 6000 mm2/mL were recorded as positive. Test materials

were assessed as active (MG4) if at least two parameters were positive, and passive (MG3) if no or only one parameter was positive.

In Vivo Rat Intratracheal Instillation Study

Before and after instillation, rats treated with single bolus doses of 360 µg 15 nm or 55 nm SiO2 per lung showed no clinical signs that differed from the control group. Mean body weights measured before instillation, on the first day after instillation, and on day three just before necropsy were also comparable to the control animals (Table 4). The hematological examination revealed a small but significant decrease in platelet counts for 55 nm SiO2 and a significant increase in absolute neutrophil

counts for 15 nm SiO2 (Table 5 and Figure 3). In the bronchoalveolar lavage fluid (BALF) of rats treated with 55 nm SiO2, significant increases in absolute lymphocytes, polymorphonuclear neutrophils (PMNs), atypical cells, and eosinophils were recorded. In the BALF of rats treated with 15 nm SiO2, the absolute counts of all examined cell types (except for macrophages) were significantly increased. The increased total cell count was mainly attributed to the influx of PMNs and lymphocytes. Consistently, the cytological alterations recorded in the BALF of rats treated with 15 nm SiO2 were qualitatively similar but more pronounced than those induced by 55 nm SiO2. Further, the total protein level in the BALF of rats treated with 15 nm SiO2 was elevated 4.7-fold compared to the control animals, and all measured enzyme activity

(y-glutamyltransferase (GGT), LDH, alkaline phosphatase (ALP), and N-acetyl-ß-glucosaminidase (NAG)) was increased two- to fivefold. In the BALF of rats treated with 55 nm SiO2, the total protein level increased 1.9-fold, and LDH and ALP were the only enzymes with significantly increased activity (2.7 and 1.8-fold, respectively) (Table 5 and Figure 3).

The observation that 15 nm SiO2 elicited more pronounced effects than 55 nm SiO2 was further underrlined by the pathological findings (Table 4). Rats treated with 15 nm SiO2 showed noticeable increases in lung weight (+38% compared to the control group) and spleen weight (+19%). Furthermore, they exhibited enlarged mediastinal lymph nodes, most likely due to inflammatory activation. By comparison, treatment with 55 nm SiO2 led to no such abnormalities. For both treatment groups, the increased lung weight was assessed as being caused by an influx of inflammatory cells and the resulting swelling of the tissue due to overall inflammation, and they were consistent with the histopathological examination: in all lung lobes of two of the three animals treated with 55 nm SiO2, mild multifocal granulomatous inflammation with thickening of the alveolar walls was observed, caused by infiltrated macrophages and granulocytes. Again, the lungs of all three rats from the 15 nm SiO2 treatment group were more severely affected, and mild to severe lympho-reticular hyperplasia of the mediastinal lymph nodes was recorded (data not shown). This microscopic finding is common when lymph nodes are activated by an inflammatory process in the area of drainage, and it was consistent with the macroscopically diagnosed lymph node enlargement.

Applicant's summary and conclusion

Conclusions:

In vitro, all four nanomaterials elicited the release of concentration-dependent lactate dehydrogenase, ß-glucuronidase, and tumor necrosis factor alpha, and the two smaller materials also released H2O2. All effects were size-dependent. Since the colloidal SiO2 remained well-dispersed in serum-free in vitro conditions, effective particle concentrations reaching the cells were estimated using different models. Evaluating the effective concentration–based in vitro effects
using the Decision-making framework for the grouping and testing of nanomaterials, all four nanomaterials were assigned as “active.” This assignment and the size dependency of effects were consistent with the outcomes of intratracheal instillation studies and available short-term rat inhalation data for 15 nm SiO2.

Executive summary:

The in vitro effects of four colloidal amorphous SiO2 nanomaterials that differed only by their primary particle size (9, 15, 30, and 55 nm) were analyzed using the rat NR8383 alveolar macrophage (AM) assay. Data were compared to effects of single doses of 15 nm and 55 nm SiO2 intratracheally instilled in rat lungs.