Silicic acid, aluminum magnesium sodium salt

Endpoint summary

Description of key information

Oral

No oral repeated dose toxicity studies available. Inhalation is the most appropriate route of administration.

 

Dermal

No dermal repeated dose toxicity studies.

 

Inhalation:

LOAEL (local): 1 mg/m³ (effective)

LOAEL (local): 3.23 mg/m³ (nominal)

No local NOAEL identified.

 

NOAEL (systemic): > 5 mg/m³ (effective)

NOAEL (systemic): > 16.13 mg/m³ (nominal)

 

Creutzenberg, 2014, OECD 413:

The target aerosol concentrations of 1, 2.5 and 5 mg NM-200/m³ were achieved to 104%, 100% and 101%, respectively. Calculation of the aerosol generation efficiency (actual vs. nominal concentration) resulted in 31%; this low value demonstrates that experimental conditions are different to any handling and use conditions of NM-200.

 - Due to the particle size distribution in the test medium the nominal concentration was 3.23 mg/m³, to achieve 1 mg/m³ aerosol test concentration.

 - Due to the particle size distribution in the test medium the nominal concentration was 8.06 mg/m³, to achieve 2.5 mg/m³ aerosol test concentration.

 - Due to the particle size distribution in the test medium the nominal concentration was 16.13 mg/m³, to achieve 5 mg/m³ aerosol test concentration.

 

This results in a NOAEL > 5 mg/m³ air corresponding to > 16.13 mg/m³ nominal concentration in air for NM-200 which is taken as a worst-case approach for SMAS. Worst case is justified based on the nine-times higher solubility of the SMAS in comparison to test item NM-200 (SAS). This suggests that a NOAEL for SMAS will be much higher (200 mg/m³). 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec. 11, 2011 - May 18, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Test material ist different from reference substance but comparable, see chapter 13, attachment 'Analogue Approach Justification'

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

2009-09-07

Deviations:

yes

Remarks:

additional endpoints; males only

Principles of method if other than guideline:

OECD guideline 413 with additional endpoints (bronchoalveolar lavage, cell proliferation, immunological parameters, oxidative stress analysis, electron microscope analysis, toxicokinetics) to address nanoparticle-specific aspects of toxicity

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

UPAC Name: Silicon Dioxide
Common Name, Synonyma: Synthetic Amorphous Silica, NM-200
CAS #: 12926-00-8
(CAS # for precipitated synthetic amorphous silica)

Information note:
CAS # 7631-86-9
(general CAS # for silica including synthetic amorphous silica)
Batch: NM-200 (Master-Batch: JRC, Ispra)
Expiry Date: 12/2012 (Stable particle sample)
Purity: 96.5% (value given by JRC, Ispra)
Molecular weight (g/mol): 60.74

Species:

rat

Strain:

Wistar

Remarks:

Wistar WU

Details on species / strain selection:

purchased from Charles River Deutschland (Sulzfeld, Germany)

Sex:

male

Details on test animals or test system and environmental conditions:

The age of the animals at the start of exposure was approx. 9-10 weeks and the weight approx. 280 gram, two rats housed per cage, tap water from the Hannover city water supplier was offered fresh weekly or more often, if necessary. As diet a commercial chow in pellet form was used, identified as Ssniff "V1534". 22 +/- 2 °C, 40 - 70% humidity, 12-hour light/dark.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 2.16 - ca. 3.12 µm

Remarks on MMAD:

The MMADs were determined by two methods: impactor and SEM analysis.
low dose: 2.16 µm, GSD: 0.09
mid dose: 2.94 µm, GSD: 0.2
high dose: 3.12 µm, GSD: 0.06

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The target aerosol concentrations of 1, 2.5 and 5 mg NM-200/m³ were achieved to 104%, 100% and 101%, respectively. Calculation of the aerosol generation efficiency (actual vs. nominal concentration) resulted in 31%; this low value demonstrates that experimental conditions are different to any handling and use conditions of NM-200.

- Due to the particle size distribution in the test medium the nominal concentration was
3.23 mg/m³, to achieve 1 mg/m³ aerosol test concentration.

- Due to the particle size distribution in the test medium the nominal concentration was
8.06 mg/m³, to achieve 2.5 mg/m³ aerosol test concentration.

- Due to the particle size distribution in the test medium the nominal concentration was
16.13 mg/m³, to achieve 5 mg/m³ aerosol test concentration.

Duration of treatment / exposure:

6 h / d

Frequency of treatment:

5 d / w for 90 days (= 65 exposure days)

Dose / conc.:

1 mg/m³ air

Remarks:

effective concentration (corresponds to 3.23 mg/m³ nominal comcentration)

Dose / conc.:

2.5 mg/m³ air

Remarks:

effective concentration (corresponds to 8.06 mg/m³ nominal comcentration)

Dose / conc.:

5 mg/m³ air

Remarks:

effective concentration (corresponds to 16.13 mg/m³ nominal comcentration)

No. of animals per sex per dose:

55 (males only)

Control animals:

yes, concurrent vehicle

Details on study design:

90-day post-inhalation recovery period

Observations and examinations performed and frequency:

All animals were clinically observed in their cages at least once a day. Once a week, they were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition. This included inspection of skin, fur, eyes, visible mucous membranes, examination for pathomorphological changes (e.g. unusual breathing pattern, masses, nodules), abnormal behaviour and central nervous symptoms (e.g. changes in gait, posture or grooming activity, unusual response to handling, secretion/excretion abnormalities, clonic/tonic movements, stereotypies) and/or other clinical abnormalities.
Individual body weight was recorded to the nearest 0.1 g twice a week for the first month and once a week throughout the remainder of the study (including post-exposure observation period) for all animals.

Sacrifice and pathology:

All animals were subjected to a complete necropsy, which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The lung and the lower half of the trachea were weighed, and used for BAL or histopathology. The following organs were trimmed and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, lung, and heart. The respiratory tract was preserved as follows: Nasal passages (including nasal -associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD Guideline no. 413 excluding those given in brackets were prepared for histopathology.

Other examinations:

Cytological parameters
• total cell count (recruitment of lung leukocytes)
• viability test (giving percentage of alive leukocytes among the total number of cells)
• differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions)
Biochemical parameters
• lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis)
• β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages)
• total protein (marker of transsudation; damage of epithelial cells).
Oxidative Stress and Immunotoxicological Parameters:
TNF-a, IL-8 (CINC-1), IL-6, TGF-b

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test. The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the Provantis system.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

only 2 / 220
Effects indicating systemic toxicity were not observed.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase of food comsumption as compared to controls was observed on several dates in all NM-200 dose groups. Due to inconsistency in dose-dependency and only small absolute differences observed these findings are considered as incidental.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes were observed for hemoglobin and for segmented neutrophils (calculated) in mid dose group. This findings are considered as incidental, not-treatment related findings.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

• Statistically significant increases of polymorphonuclear neutrophils, lymphocytes and lactic dehydrogenase, ß-glucuronidase and total protein levels/activity and decrease of macrophages were detected in the mid and high dose groups 1 day after end of exposure. In the low dose group a statistically significant increase of polymorphonuclear neutrophils was detected. However, all these effects were reversible and had returned to control levels at the 3-month post-exposure sacrifice date. Thus, NM-200 dust showed a strong acute response, however, rapid recovery upon cessation of exposure.
• The analysis of the oxidative stress related secretion of reactive oxygen intermediates (ROI) showed a decrease with and without zymosan stimulation in the high dose group 14 days after end of exposure as compared to clean air controls but not at 1 day postexposure. A significantly increased concentration of the stimulatory cytokine CINC-1 was observed 14 days after end of exposure but not at 1 day postexposure. For tumour necrosis factor-a and interleukin-6 no significant changes in treatment groups compared to controls were detected.
• In the NM-200 high dose group SiO2 particles (confirmation by EDX) were found within the cytoplasm of intraalveolar macrophages. The agglomerates of particles seem to be more densely packed 90 days post-exposure than 29 days post-exposure.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute organ wet weights showed statistically significant changes as compared to controls for the absolute lung weights (high dose group only) 1 day after end of exposure and after 3 months of recovery (mid and high dose group). The relative lung weights were statistically significantly increased 1 day after exposure end (mid and high dose group).

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination 1 day after end of exposure revealed
- (multi)focal mucous (goblet) cell hyperplasia and epithelial hyaline droplets, accompanied with multifocal epithelial (mixed) inflammatory cell infiltration, were diagnosed dose-dependently in 10 out of 10 rats in the nasal cavity (statistically significantly increased) in all NM-200 treated groups.
- a significant increase of alveolar infiltration of granulocytes in lungs was detected in the mid and high dose groups /interstitial macrophage infiltration and of (multi)focal very slight alveolar granulocyte infiltration in the lungs of the high dose group.
Histopathological examination 3 months after end of exposure revealed a full recovery of all treatment-related effects in lungs. In nasal cavities the effects persisted.

Datailed results on nasal cavity
1. Animals sacrificed after 90 days of treatment
(Multi)focal mucous (goblet) cell hyperplasia was diagnosed in a single male (slight) of the control group and dose-dependently in 10/10 males each of the NM-200 low- (all slight) and mid-dose groups (8/10 slight, 2/10 moderate) as well as in 9/9 males of ther high-dose group (2/9 slight, 7/9 moderate). Mucous (goblet) cell hyperplasia was predominantly observed in the respiratory epithelial lining of the nasal septum and the ventral nasal meatus in levels I to III of the 4 nasal cavity sections. Two of 10, 5/10 and 9/9 males of the NM-200 low, mid- and high-dose group, respectively, showed (multi)focal slight hyperplasia of the respiratory epithelium in addition to mucous cell hyperplasia.
Another exposure-related effect was the dose-dependent occurrence of very slight (minimal) to moderate epithelial hyaline droplets (eosinophilic inclusions), mainly within the respiratory epithelium of the nasal septum und ventral nasal meatus at levels II - III of the nasal cavity sections. While in the clean air control group, 4/10 (3/10 very slight, 1/10 slight) males revealed very slight focal hyaline droplets, this change showed a multifocal distribution in the particle exposure groups at incidences of 10/10 each in the NM-200 low- (all slight) and mid-dose groups (9/10 slight, 1/10 moderate) as well as in 9/9 males of the high-dose group (all moderate). In a single male of the control group (very slight) and in 10/10 (all very slight), 10/10 (6/10 very slight, 4/10 slight) and 9/9 (2/9 very slight, 7/9 slight) males of the NM-200 low-, mid- and high-dose group, respectively, the epithelial hyaline droplets were associated with multifocal epithelial (mixed) inflammatory cell infiltration.
Incidental findings such as very slight to slight focal subepithelial corpora amylacea and very slight focal subepithelial mononuclear cell infiltration were unrelated to exposure and observed in single rats of different groups.

2. Animals sacrificed at the end of the recovery period (day 182)
Focal mucous (goblet) cell hyperplasia was observed at a slight degree in 3/10 males of the control group. In the particle-exposure groups, multifocal mucous cell hyperplasia occurred dose-dependently at incidences of 8/10 (3/10 very slight, 3/10 slight, 2/10 moderate), 10/10 (all slight) and 10/10 (6/10 slight, 3/10 moderate, 1/10 severe) in rats of the NM-200 low-, mid- and high-dose group, respectively. In comparison to the main subset, the overall degree of severity of this change had slightly decreased. This was not the case for (multi)focal respiratory epithelial hyperplasia, which was diagnosed in 5/10 (1/10 very slight, 4/10 slight), 7/10 (2/10 very slight, 5/10 slight) and 10/10 (4/10 very slight, 6/10 slight) rats of the NM-200 low-, mid- and high-dose group, respectively. The incidences were slightly higher than in the main subset, however, not the effect as such.
(Multi)focal epithelial hyaline droplets (eosinophilic inclusions) were observed in 7/10 control animals (all very slight) and in 10/10 males each of the NM-200 low- (9/10 slight, 1/10 moderate), mid- (all slight) and high-dose group (2/10 slight, 7/10 moderate, 1/10 severe), respectively. Incidence, degree of severity and distribution of this change were comparable to the ones seen in the main subset. (Multi)focal epithelial (mixed) inflammatory cell infiltration associated with the epithelial hyaline droplets was diagnosed in 2/10 (all very slight), 8/10 (6/10 very slight, 2/10 slight), 9/10 (8/10 very slight, 1/10 slight) and 10/10 (8/10 very slight, 2/10 slight) rats of the control-, NM-200 low-, mid- and high-dose group, respectively. Although the incidences were about the same as in the main subset, there was a remarkable decrease in the degree of severity of the inflammatory infiltrates in all particle exposure groups towards the end of the 90-day recovery period. As a further exposure-related finding, a single male of the NM-200 high-dose group revealed a slight multifocal (chronic) inflammation of the nasal submucosal glands.
Incidental findings included very slight to slight focal subepithelial mononuclear cell infiltration in 2/10 control rats and slight focal subepithelial corpora amylacea in a single rat of the NM-200 mid-dose group.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

In all treatment groups, on day 8 and 91 postexposure no statistically significant increases in the proliferation index were detected

Details on results:

Lung burdens of 91, 172 and 307 µg were analysed on day 1 after end of exposure in the low, mid and high dose groups, respectively. Data at 3 months postexposure revealed that, in addition to the approx. 70 days of physiological clearance, a dissolution effect is responsible for the low values of 12, 21 and 34 µg/lung, respectively.
All test animals but two survived treatment and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

< 1 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

due to adverse effects at lowest tested concentration level

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

< 3.23 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (3.23 mg/m³ air / 0.31 = 1 mg/m³ air

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

1 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

3.23 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (3.23 mg/m³ air / 0.31 = 1 mg/m³ air

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 5 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Remarks on result:

other: value mentioned in study report

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 16.13 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (16.13 mg/m³ air / 0.31 = 5 mg/m³ air

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 other: mg/m³ air effective concentration

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.23 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2.5 other: mg/m³ air effective concentration

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

8.06 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

The actual (=effective) systemic concentration applied was calculated by dividing the stated nominal concentration of 5 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%:

16.13 mg/m³ air / 0.31 = 5 mg/m³ air

Conclusions:

Conclusion on NOAEL: Under the conditions of this test an LOAEL = 1 mg/m³ was derived (decisive endpoint: histopathology: mucous cell hyperplasia in nasal cavities). An experimental local NOAEL could not be derived (i.e. NOAEL < 1 mg/m³). But all effects showed either full reversible or showed a tendency to full reversibility.
The systemic NOAEL was 5 mg/m³ effective. At this dose level no systemic effects were observed. The nominal systemic concentration applied higher, and can be calculated by dividing the stated nominal concentration of 16.13 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%:
16.13 mg/m³ air / 0.31 = 5 mg/m³ air.

Executive summary:

A 3-month inhalation study with a structurally related SAS (NM-200) followed by a 90-day recovery period was performed in male rats. Calculation of the aerosol generation efficiency (effective vs. nominal concentration) resulted in 31%. Therefore, two effect levels are stated each, the effective concentration stated in the study report and the nominal concentration applied to achieve the target concentration (=effective concentration).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

16.13 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

RL1

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec. 11, 2011 - May 18, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Test material ist different from reference substance but comparable, see chapter 13, attachment 'Analogue Approach Justification'

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

2009-09-07

Deviations:

yes

Remarks:

additional endpoints; males only

Principles of method if other than guideline:

OECD guideline 413 with additional endpoints (bronchoalveolar lavage, cell proliferation, immunological parameters, oxidative stress analysis, electron microscope analysis, toxicokinetics) to address nanoparticle-specific aspects of toxicity

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

UPAC Name: Silicon Dioxide
Common Name, Synonyma: Synthetic Amorphous Silica, NM-200
CAS #: 12926-00-8
(CAS # for precipitated synthetic amorphous silica)

Information note:
CAS # 7631-86-9
(general CAS # for silica including synthetic amorphous silica)
Batch: NM-200 (Master-Batch: JRC, Ispra)
Expiry Date: 12/2012 (Stable particle sample)
Purity: 96.5% (value given by JRC, Ispra)
Molecular weight (g/mol): 60.74

Species:

rat

Strain:

Wistar

Remarks:

Wistar WU

Details on species / strain selection:

purchased from Charles River Deutschland (Sulzfeld, Germany)

Sex:

male

Details on test animals or test system and environmental conditions:

The age of the animals at the start of exposure was approx. 9-10 weeks and the weight approx. 280 gram, two rats housed per cage, tap water from the Hannover city water supplier was offered fresh weekly or more often, if necessary. As diet a commercial chow in pellet form was used, identified as Ssniff "V1534". 22 +/- 2 °C, 40 - 70% humidity, 12-hour light/dark.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 2.16 - ca. 3.12 µm

Remarks on MMAD:

The MMADs were determined by two methods: impactor and SEM analysis.
low dose: 2.16 µm, GSD: 0.09
mid dose: 2.94 µm, GSD: 0.2
high dose: 3.12 µm, GSD: 0.06

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The target aerosol concentrations of 1, 2.5 and 5 mg NM-200/m³ were achieved to 104%, 100% and 101%, respectively. Calculation of the aerosol generation efficiency (actual vs. nominal concentration) resulted in 31%; this low value demonstrates that experimental conditions are different to any handling and use conditions of NM-200.

- Due to the particle size distribution in the test medium the nominal concentration was
3.23 mg/m³, to achieve 1 mg/m³ aerosol test concentration.

- Due to the particle size distribution in the test medium the nominal concentration was
8.06 mg/m³, to achieve 2.5 mg/m³ aerosol test concentration.

- Due to the particle size distribution in the test medium the nominal concentration was
16.13 mg/m³, to achieve 5 mg/m³ aerosol test concentration.

Duration of treatment / exposure:

6 h / d

Frequency of treatment:

5 d / w for 90 days (= 65 exposure days)

Dose / conc.:

1 mg/m³ air

Remarks:

effective concentration (corresponds to 3.23 mg/m³ nominal comcentration)

Dose / conc.:

2.5 mg/m³ air

Remarks:

effective concentration (corresponds to 8.06 mg/m³ nominal comcentration)

Dose / conc.:

5 mg/m³ air

Remarks:

effective concentration (corresponds to 16.13 mg/m³ nominal comcentration)

No. of animals per sex per dose:

55 (males only)

Control animals:

yes, concurrent vehicle

Details on study design:

90-day post-inhalation recovery period

Observations and examinations performed and frequency:

All animals were clinically observed in their cages at least once a day. Once a week, they were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition. This included inspection of skin, fur, eyes, visible mucous membranes, examination for pathomorphological changes (e.g. unusual breathing pattern, masses, nodules), abnormal behaviour and central nervous symptoms (e.g. changes in gait, posture or grooming activity, unusual response to handling, secretion/excretion abnormalities, clonic/tonic movements, stereotypies) and/or other clinical abnormalities.
Individual body weight was recorded to the nearest 0.1 g twice a week for the first month and once a week throughout the remainder of the study (including post-exposure observation period) for all animals.

Sacrifice and pathology:

All animals were subjected to a complete necropsy, which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The lung and the lower half of the trachea were weighed, and used for BAL or histopathology. The following organs were trimmed and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, lung, and heart. The respiratory tract was preserved as follows: Nasal passages (including nasal -associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD Guideline no. 413 excluding those given in brackets were prepared for histopathology.

Other examinations:

Cytological parameters
• total cell count (recruitment of lung leukocytes)
• viability test (giving percentage of alive leukocytes among the total number of cells)
• differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions)
Biochemical parameters
• lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis)
• β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages)
• total protein (marker of transsudation; damage of epithelial cells).
Oxidative Stress and Immunotoxicological Parameters:
TNF-a, IL-8 (CINC-1), IL-6, TGF-b

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test. The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the Provantis system.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

only 2 / 220
Effects indicating systemic toxicity were not observed.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase of food comsumption as compared to controls was observed on several dates in all NM-200 dose groups. Due to inconsistency in dose-dependency and only small absolute differences observed these findings are considered as incidental.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes were observed for hemoglobin and for segmented neutrophils (calculated) in mid dose group. This findings are considered as incidental, not-treatment related findings.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

• Statistically significant increases of polymorphonuclear neutrophils, lymphocytes and lactic dehydrogenase, ß-glucuronidase and total protein levels/activity and decrease of macrophages were detected in the mid and high dose groups 1 day after end of exposure. In the low dose group a statistically significant increase of polymorphonuclear neutrophils was detected. However, all these effects were reversible and had returned to control levels at the 3-month post-exposure sacrifice date. Thus, NM-200 dust showed a strong acute response, however, rapid recovery upon cessation of exposure.
• The analysis of the oxidative stress related secretion of reactive oxygen intermediates (ROI) showed a decrease with and without zymosan stimulation in the high dose group 14 days after end of exposure as compared to clean air controls but not at 1 day postexposure. A significantly increased concentration of the stimulatory cytokine CINC-1 was observed 14 days after end of exposure but not at 1 day postexposure. For tumour necrosis factor-a and interleukin-6 no significant changes in treatment groups compared to controls were detected.
• In the NM-200 high dose group SiO2 particles (confirmation by EDX) were found within the cytoplasm of intraalveolar macrophages. The agglomerates of particles seem to be more densely packed 90 days post-exposure than 29 days post-exposure.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute organ wet weights showed statistically significant changes as compared to controls for the absolute lung weights (high dose group only) 1 day after end of exposure and after 3 months of recovery (mid and high dose group). The relative lung weights were statistically significantly increased 1 day after exposure end (mid and high dose group).

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination 1 day after end of exposure revealed
- (multi)focal mucous (goblet) cell hyperplasia and epithelial hyaline droplets, accompanied with multifocal epithelial (mixed) inflammatory cell infiltration, were diagnosed dose-dependently in 10 out of 10 rats in the nasal cavity (statistically significantly increased) in all NM-200 treated groups.
- a significant increase of alveolar infiltration of granulocytes in lungs was detected in the mid and high dose groups /interstitial macrophage infiltration and of (multi)focal very slight alveolar granulocyte infiltration in the lungs of the high dose group.
Histopathological examination 3 months after end of exposure revealed a full recovery of all treatment-related effects in lungs. In nasal cavities the effects persisted.

Datailed results on nasal cavity
1. Animals sacrificed after 90 days of treatment
(Multi)focal mucous (goblet) cell hyperplasia was diagnosed in a single male (slight) of the control group and dose-dependently in 10/10 males each of the NM-200 low- (all slight) and mid-dose groups (8/10 slight, 2/10 moderate) as well as in 9/9 males of ther high-dose group (2/9 slight, 7/9 moderate). Mucous (goblet) cell hyperplasia was predominantly observed in the respiratory epithelial lining of the nasal septum and the ventral nasal meatus in levels I to III of the 4 nasal cavity sections. Two of 10, 5/10 and 9/9 males of the NM-200 low, mid- and high-dose group, respectively, showed (multi)focal slight hyperplasia of the respiratory epithelium in addition to mucous cell hyperplasia.
Another exposure-related effect was the dose-dependent occurrence of very slight (minimal) to moderate epithelial hyaline droplets (eosinophilic inclusions), mainly within the respiratory epithelium of the nasal septum und ventral nasal meatus at levels II - III of the nasal cavity sections. While in the clean air control group, 4/10 (3/10 very slight, 1/10 slight) males revealed very slight focal hyaline droplets, this change showed a multifocal distribution in the particle exposure groups at incidences of 10/10 each in the NM-200 low- (all slight) and mid-dose groups (9/10 slight, 1/10 moderate) as well as in 9/9 males of the high-dose group (all moderate). In a single male of the control group (very slight) and in 10/10 (all very slight), 10/10 (6/10 very slight, 4/10 slight) and 9/9 (2/9 very slight, 7/9 slight) males of the NM-200 low-, mid- and high-dose group, respectively, the epithelial hyaline droplets were associated with multifocal epithelial (mixed) inflammatory cell infiltration.
Incidental findings such as very slight to slight focal subepithelial corpora amylacea and very slight focal subepithelial mononuclear cell infiltration were unrelated to exposure and observed in single rats of different groups.

2. Animals sacrificed at the end of the recovery period (day 182)
Focal mucous (goblet) cell hyperplasia was observed at a slight degree in 3/10 males of the control group. In the particle-exposure groups, multifocal mucous cell hyperplasia occurred dose-dependently at incidences of 8/10 (3/10 very slight, 3/10 slight, 2/10 moderate), 10/10 (all slight) and 10/10 (6/10 slight, 3/10 moderate, 1/10 severe) in rats of the NM-200 low-, mid- and high-dose group, respectively. In comparison to the main subset, the overall degree of severity of this change had slightly decreased. This was not the case for (multi)focal respiratory epithelial hyperplasia, which was diagnosed in 5/10 (1/10 very slight, 4/10 slight), 7/10 (2/10 very slight, 5/10 slight) and 10/10 (4/10 very slight, 6/10 slight) rats of the NM-200 low-, mid- and high-dose group, respectively. The incidences were slightly higher than in the main subset, however, not the effect as such.
(Multi)focal epithelial hyaline droplets (eosinophilic inclusions) were observed in 7/10 control animals (all very slight) and in 10/10 males each of the NM-200 low- (9/10 slight, 1/10 moderate), mid- (all slight) and high-dose group (2/10 slight, 7/10 moderate, 1/10 severe), respectively. Incidence, degree of severity and distribution of this change were comparable to the ones seen in the main subset. (Multi)focal epithelial (mixed) inflammatory cell infiltration associated with the epithelial hyaline droplets was diagnosed in 2/10 (all very slight), 8/10 (6/10 very slight, 2/10 slight), 9/10 (8/10 very slight, 1/10 slight) and 10/10 (8/10 very slight, 2/10 slight) rats of the control-, NM-200 low-, mid- and high-dose group, respectively. Although the incidences were about the same as in the main subset, there was a remarkable decrease in the degree of severity of the inflammatory infiltrates in all particle exposure groups towards the end of the 90-day recovery period. As a further exposure-related finding, a single male of the NM-200 high-dose group revealed a slight multifocal (chronic) inflammation of the nasal submucosal glands.
Incidental findings included very slight to slight focal subepithelial mononuclear cell infiltration in 2/10 control rats and slight focal subepithelial corpora amylacea in a single rat of the NM-200 mid-dose group.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

In all treatment groups, on day 8 and 91 postexposure no statistically significant increases in the proliferation index were detected

Details on results:

Lung burdens of 91, 172 and 307 µg were analysed on day 1 after end of exposure in the low, mid and high dose groups, respectively. Data at 3 months postexposure revealed that, in addition to the approx. 70 days of physiological clearance, a dissolution effect is responsible for the low values of 12, 21 and 34 µg/lung, respectively.
All test animals but two survived treatment and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

< 1 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

due to adverse effects at lowest tested concentration level

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

< 3.23 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (3.23 mg/m³ air / 0.31 = 1 mg/m³ air

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

1 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

3.23 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (3.23 mg/m³ air / 0.31 = 1 mg/m³ air

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 5 other: mg/m³ effective concentration

Based on:

test mat.

Sex:

male

Remarks on result:

other: value mentioned in study report

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 16.13 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: nominal concentration: calculated by dividing the stated concentration with the aerosol generation efficiency (effective vs. nominal concentration) of 31% (16.13 mg/m³ air / 0.31 = 5 mg/m³ air

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 other: mg/m³ air effective concentration

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.23 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2.5 other: mg/m³ air effective concentration

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

8.06 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

The actual (=effective) systemic concentration applied was calculated by dividing the stated nominal concentration of 5 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%:

16.13 mg/m³ air / 0.31 = 5 mg/m³ air

Conclusions:

Conclusion on NOAEL: Under the conditions of this test an LOAEL = 1 mg/m³ was derived (decisive endpoint: histopathology: mucous cell hyperplasia in nasal cavities). An experimental local NOAEL could not be derived (i.e. NOAEL < 1 mg/m³). But all effects showed either full reversible or showed a tendency to full reversibility.
The systemic NOAEL was 5 mg/m³ effective. At this dose level no systemic effects were observed. The nominal systemic concentration applied higher, and can be calculated by dividing the stated nominal concentration of 16.13 mg/m³ air with the aerosol generation efficiency (effective vs. nominal concentration) of 31%:
16.13 mg/m³ air / 0.31 = 5 mg/m³ air.

Executive summary:

A 3-month inhalation study with a structurally related SAS (NM-200) followed by a 90-day recovery period was performed in male rats. Calculation of the aerosol generation efficiency (effective vs. nominal concentration) resulted in 31%. Therefore, two effect levels are stated each, the effective concentration stated in the study report and the nominal concentration applied to achieve the target concentration (=effective concentration).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

3.23 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

RL2

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

other justification

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

other justification

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

Similar to OECD 453 a combined chronic toxicity/carcinogenicity study was carried out. No particular adverse effects were observed in rats and mice treated with Syloid 244 lifelong at dietary level of 5%, corresponding to 1760 - 3000mg/kg bw/day and 1780 - 3210 mg/kg bw/day for male and female rats, respectively and 5270 – 7490 mg/kg bw/day and 3950 – 13310 mg/kg bw/day for male and female mice, respectively (Takizawa et al. 1988).

In a feeding study, conducted similar to OECD 408, male and female rats received Syloid 244 for 26 weeks. No treatment-related findings were noted up to and including 8980 mg/kg bw/day (Hackenberg 1975). Isolated pathological findings were unrelated to dosing and common in untreated rats. No histopathological changes were observed in the kidneys and reproductive organs. The effect level (NOAEL) was > 8980 and > 7950 mg/kg bw/day for females and males, respectively.

 

Dermal

Repeated dose toxicity studies are not available for the dermal route.

 

Inhalation

The repeated dose toxicity study by inhalation of synthetic amorphous silica (NM-200) showed no systemic effects (Creutzenberg, 2014, OECD 413). The inhalation of respirable particles of NM-200 produced treatment-related inflammatory effects in the lungs. No progressive process of lesions has been observed. All NM-200 induced effects are reversible. Full recovery of all treatment-related effects in lungs. In nasal cavities, the effects persisted after 3 months but showed a tendency to full reversibility.

The relatively rapid clearance of the test item was confirmed; nanomaterial detection as solid matter by morphology (TEM analysis) was limited to the cytoplasm of alveolar macrophages.

 

The target aerosol concentrations of 1, 2.5 and 5 mg NM-200/m3 were achieved to 104%, 100% and 101%, respectively. Calculation of the aerosol generation efficiency (actual vs. nominal concentration) resulted in 31%; this low value demonstrates that experimental conditions are different to any handling and use conditions of NM-200.

 - Due to the particle size distribution in the test medium the nominal concentration was

3.23 mg/m³, to achieve 1 mg/m³ aerosol test concentration.

 - Due to the particle size distribution in the test medium the nominal concentration was

8.06 mg/m³, to achieve 2.5 mg/m³ aerosol test concentration.

 - Due to the particle size distribution in the test medium the nominal concentration was

16.13 mg/m³, to achieve 5 mg/m³ aerosol test concentration.

 

This results in a NOAEL > 5 mg/m³ air corresponding to > 16.13 mg/m³ nominal concentration in air for NM-200 which is taken as a worst-case approach for SMAS. Worst case is justified based on the nine-times higher solubility of the SMAS in comparison to test item NM-200 (SAS). This suggests that a NOAEL for SMAS will be much higher (200 mg/m³).

Justification for classification or non-classification

Oral

No treatment-related adverse effects were assessed with structurally related substances via the oral route.There is no need for classification. No signal word and hazard statement are required.

Dermal

Silicic acid, aluminium magnesium sodium salt will not penetrate the skin, therefore no systemic effects are expected. There is no need for classification. No signal word and hazard statement are required.

Inhalation

Lack of specific target organ/systemic toxicity; No systemic effects were found in combination of the chronic studies.

In accordance with the Globally Harmonised System (Regulation (EC) No 1272/2008), the test item does not need to be classified. No signal word and hazard statement are required.

STOT RE

Oral:

At given doses no effects on mortality, body weight, food consumption, (food efficiency, water consumption and compound intake, ophthalmoscopic examination, haematology, clinical chemistry, urinalysis), gross pathology, organ weights, and histopathology could be observed.

 

Inhalation:

Adverse effects observed were only local and reversible but not systemic. The inhalation study was conducted on synthetic amorphous silica as worst case. No progressive process of lesions has been observed. All synthetic amorphous silica induced effects suggested reversibility.

 

As silicic acid aluminium magnesium sodium salt usually agglomerates to larger particles, most of the inhaled material will be retained in the upper airways and/or be swallowed in case large agglomerates will exist.

 

Qualitative arguments for this suggestion are a nine times higher water solubility equal to a faster lung clearance and a much higher density (tapped and bulk) what is equal to a lower tendency for a lung burden.

In accordance with the Globally Harmonised System (Regulation (EC) No 1272/2008), the test item does not need to be classified for STOT RE. No signal word and hazard statement are required