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REACH

Silicon dioxide

EC number: 231-545-4 | CAS number: 7631-86-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

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Administrative data

Description of key information

Repeated dose toxicity oral

The dataset for the endpoint oral repeated dose toxicity of non-surface treated SAScontains 9 studies, carried out between 1958 and 2014. The different production types of synthetic amorphous silica are all represented. The BET surface range of the test materials was from 150 to 350 m²/g. All studies fulfil the quality criteria reliability 1 or 2, as set up by Klimisch et al. Most studies were conducted as feeding studies but also gavage studies are available.

Significant substance related specific toxicity was absent in all studies and an overall NOAEL > 1000 mg/kg bw/day can be derived. Effects observed at higher concentrations above the limit dose were attributed to malnutrition due to the high silica intake.

Repeated dose toxicity inhalation

The evaluation of the repeated dose toxicity of hydrophilic (i.e. untreated) SAS is based on 16 studies with a specific surface (BET) between 30 – 700 m²/g. Overall, the effects depicted through these studies show a similar picture of pathology. Differences in severity of the similar pathological effects might be caused by study design such as exposure conditions, rat strain and test substance differences (particle size, primary structure, surface area, number, density, solubility...). At the end of exposure dose-dependent unspecific particle related local inflammation in lung and lung associated tissues (lymph nodes) as part of the clearance mechanism were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m³. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung mediastinal lymph nodes show as result of inflammation morphological changes.

Further examinations of these lymph nodes were performed (90-day inhalation toxicity studies, Fraunhofer ITEM, 2019) to address the question of Si clearance in the lymph nodes. Lung clearance from Si-particles is deemed to be via lymph flow, i.e., Si-particles should be filtered out and deposited within the associated lymph nodes. Based on the results of these examinations it can be concluded that the Si-content decreases in lymph nodes over the time. Overall the results confirm biosolubility of SAS over time. At the end of recovery Si content was not above control group.

Therefore, in context with "lung overload" described long-term lung effects of low toxic PSPs are not relevant for SAS, confirmed by a Pathology working group (PWG) review of a sub-chronic (13-week) inhalation toxicity study of aerosols of untreated pyrogenic SAS, silanized (i.e. surface treated) pyrogenic SAS , untreated precipitated SAS and quartz in rats (Hardisty et al., 2016). Indications for lung fibrosis, which is an irreversible effect, have been published in literature and were based on study materials of one of the applicants' studies according to OECD TG 413 (Sub-chronic inhalation toxicity, Reuzel et al., 1987; 1991). A pathology working group reviewed this finding using the same (original) tissue slides according to the highest current standards and concluded the rapid clearance of the synthetic amorphous silica products will not lead to persistent inflammation and epithelial cell proliferation and therefore will not result in fibrosis/lung tumour induction (Weber et al. 2018, “Aerosols of synthetic amorphous silica do not induce fibrosis in lungs after inhalation: Pathology working group review of histopathological specimens from a sub-chronic 13-week inhalation toxicity study in rats”. Toxicology Research and Application 2: 1–17). Irreversible lung fibrosis has not been associated to any of the eight sub-chronic studies to SAS.

These results were confirmed by the recent studies requested by ECHA with high and low surface area pyrogenic SAS, regarded as the worst case of synthetic amorphous silica forms, which showed no lung fibrosis and accordingly no increase of collagen (90-day inhalation toxicity studies, Fraunhofer ITEM, 2019).

There are a number of repeated dose studies for hydrophilic SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).

Repeated dose toxicity dermal

In a limited 3-week study from 1958, no systemic toxicity and no gross pathological or microscopic change was reported. The Silicon content in blood, urine, spleen, liver and kidney of the exposed animals was comparable to those of control animals. The NOAEL was 10000 mg/kg bw/day.

Please find the additional information in chapter 13:

Expert Review: Dekant, W et al. 2012 (Synthetic Amorphous Silica (SAS); Hazard Evaluation after Oral or Inhalation Exposure).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: Jan, 20 - Oct. 13, 2011; experimental phase: Jan. 26 - May 3, 2011
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:: 2008
GLP compliance:: yes (incl. QA statement)
Specific details on test material used for the study:: NM-200, supplied by JRC, Ispra, on behalf of the sponsor, CEFIC, The European Chemical Industry Council
Species:: rat
Strain:: Wistar
Details on species / strain selection:: purchased from: Charles River Deutschland, Sulzfeld, Germany
Sex:: male
Details on test animals or test system and environmental conditions:: Animals were housed in groups of 5 per cage in Makrolon Type IV cages in animal room T1.044 in the conventional area. Absorbent softwood was used as bedding material in the cages (Lignocel BK 8-15, ssniff GmbH, Soest, Germany). Drinking water from the Hannover city water supplier was offered fresh weekly, in Makrolon bottles (approximately 300 ml), ad libitum. Food was offered ad libitum fresh weekly. The diet used (ssniff R/M-H) was supplied by ssniff GmbH, Soest, Germany. The temperature in the animal room was set at 22 ± 2°C and the rel. humidity at 30 - 70%. The animal room lighting was a 12-hour light/dark cycle controlled by an automatic timing device.
Route of administration:: oral: gavage
Vehicle:: other: a solution of methylhydroxypropylcellulose (0.5 %) in deionised water (Milli-Q, Millipore)
Duration of treatment / exposure:: 28 d
Frequency of treatment:: daily
Dose / conc.:: 100 mg/kg bw/day (nominal)
Dose / conc.:: 300 mg/kg bw/day (nominal)
Dose / conc.:: 1 000 mg/kg bw/day (nominal)
Dose / conc.:: 1 000 mg/kg bw/day (nominal)
Remarks:: recovery group
No. of animals per sex per dose:: 5 (males only)
Control animals:: yes, concurrent vehicle
Details on study design:: 14-day observation phase for some animals (5 treated and 5 control)
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.
Sacrifice and pathology:: Gross necropsy was performed on all rats.
Histopathological examination of the cerebrum, cerebellum, peripheral nerve, trachea, thyroid, lung, mandibular lymphnode, mesenteric lymphnode, thymus, heart, skeletal muscle, liver, spleen, spinal cord, adrenals, forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, urinary bladder, eyes with optic nerve, testes, epididymis, coagulation glands, seminal vesicles, prostate, femur with bone marrow and joint was performed in 5 males of the high dose group 4 (1000 mg/kg bw Synthetic Amorphous Silica) and the control group 1 (vehicle). Gross findings of other organs were examined in addition.
Other examinations:: Locomotor Activity
During the last week of treatment, spontaneous locomotor activity over 60 minutes using the „Motitest“ computerized light-beam system (TSE, Homburg/Ts., Germany) was determined. The data were analyzed in 15-minutes intervals. In addition, the total values for distance, time in rest, time in movement, rearing time, and number of rearings were determined. Additional testing for spontaneous locomotor activity in the two recovery groups was not performed in the second recovery week, because no significant statistical differences between the dose groups and the control group were determined in the first testing.

Hematological, clinical chemistry analyses (hematology and serum chemistry) were performed before final sacrifice for all animals:
- Erythrocyte count, hemoglobin, hematocrit, mean erythrocyte volume, mean erythrocyte hemoglobin mass, mean erythrocyte hemoglobin concentration, total and differential leukocyte count, platelet count, prothrombin time, and thromboplastin time were recorded.
- Aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), gammaglutamyl transferase (EC 2.3.2.2), alkaline phosphatase (EC 3.1.3.1), sorbitol dehydrogenase (EC 1.1.1.14), cholinesterase (EC 3.1.1.8), creatin kinase (EC 2.7.3.2), total bilirubin, urea, creatinine, total protein, albumin, cholesterol, glucose, sodium, potassium, calcium, chloride, inorganic phosphate and triglyceride were measured. Globulin and
albumin/globulin ratio were calculated from the albumin and total protein data.

Analysis of Silicon in tissue (blood, kidney and liver) was performed by ICP-MS.
Statistics:: Differences between groups were considered case by case as statistically significant for p<0.05. Data were analyzed using analysis of variance. If the group means differed significantly according to the analysis of variance, the means of the treatment groups were compared with the means of the control group, using DUNNETT's modification of the t-test. Kruskal-Wallis ANOVA and Mann-Whitney U-test were applied in the case of non-homogeneous data. The statistical evaluation of the histopathological findings was done with the two-tailed FISHER test using the PROVANTIS system. For comparisons of semiquantitative data, the Chi-square test was used.
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, non-treatment-related
Description (incidence and severity):: The only statistically significant difference in body weight gain was observed in the mid-dose group between day 7 and 14. It was not dose dependent and transient and therefore considered to be incidental and of no toxicological relevance.
Food consumption and compound intake (if feeding study):: no effects observed
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: A slight but significant decrease was found in the partial thromboplastin time (PTT) in the mid dose group. The white blood cell count (WBC) and the lymphocyte count (LYMC) were significantly decreased in the mid dose group. However, the relative lymphocyte count (LYM) was unchanged. As all values were within the normal range, these changes are considered to be of minor toxicological relevance.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: Cholinesterase (CHE) and glucose (GLUC) levels were marginally decreased in the mid dose group. However, all values were within the normal range and the changes reported above are thus considered to be incidental.
Behaviour (functional findings):: no effects observed
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: No substance-related findings were observed.

Two animals (one male of group 2 (low dose), one male of group 5 (recovery control)) displayed an unilateral exophthalmus, probably caused by the previous retro-boulbous blood sampling.
One male of group 3 (mid dose) displayed a slight reduction in size of the prostate and the seminal vesicle. This alteration is considered to be an incidental finding, occasionally occurring in this strain.
One male of group 6 (recovery high dose) displayed a slight unilateral reduction in size of the testes and epididymis. This alteration is considered to be an incidental finding, occasionally occurring in this strain.
Neuropathological findings:: no effects observed
Description (incidence and severity):: No influence on locomotor activity was observed.
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: Substance-related findings were not observed in the histologically examined organs of males of the control and high dose group.

Lungs: Findings in the lungs occurred only in single animals of the control group like very slight focal bronchiolo-alveolar hyperplasia, slight alveolar histiocytosis (1/5), very slight pulmonary hemorrhage (1/5) and very slight/slight focal inflammatory cell infiltration (2/5).
Kidney: The finding of unilateral focal very slight tubular basophilia with nuclear crowding was seen in 1/5 males of the control group. Other degenerative changes of the kidneys such as unilateral slight focal subcapsular mononuclear cell infiltration associated with focal very slight fibrosis occurred in another control male, which showed a bilateral multifocal slight tubular dilatation in addition.
Liver: (Multi)focal very slight microgranulomas were diagnosed in 3/5 males of the control and in 5/5 males of the high dose group.
Prostate: 4/5 of the control animals and 5/5 of the high dose group animals showed very slight/slight (multi)focal inflammatory lesions.
Testes, Epididymis: Very slight/slight/moderate multifocal degeneration and depletion of germ cells was seen in 4/5 males of the control, and 4/5 males of the high dose group. Very slight/slight multifocal tubular atrophy was seen in 1/5 males of the control and in 2/5 males of the high dose group in addition. Intraluminal exfoliated degenerated germ cells were detected in the corresponding epididymis of 1/5 male of the control and 1/5 of the high dose group. A slight focal spermatic granuloma was noted in the epididymis of one control male. Very slight (multi)focal interstitial mononuclear cell infiltration was recorded in the epididymis of 1/5 males of the control and in 1/5 males of the high dose group. These alterations are considered to be incidental findings.
Other organs: Several other findings in various organs of the examined experimental groups were noted which also occurred incidentally and were not unusual for this strain and age.
Histopathological findings: neoplastic:: no effects observed
Other effects:: effects observed, non-treatment-related
Description (incidence and severity):: Determination of silicon ion concentration: The ion concentrations in kidney, liver and blood were comparable in control animals (group 1) and high dose animals (group 4) and no treatment related changes were observed.
: Key result
Dose descriptor:: NOAEL
Effect level:: >= 1 000 mg/kg bw/day (nominal)
Based on:: test mat.
Sex:: male
Basis for effect level:: behaviour (functional findings); body weight and weight gain; clinical biochemistry; clinical signs; food consumption and compound intake; gross pathology; haematology; histopathology: neoplastic; histopathology: non-neoplastic; mortality; neuropathology; organ weights and organ / body weight ratios; other: determination of silicon ion concentration
: Key result
Critical effects observed:: no
Conclusions:: Synthetic Amorphous Silica did not cause any substance-related effects in doses of up to 1000 mg/kg bodyweight after oral exposure for 28 days in male Wistar (WU) rats. Therefore, the highest dose tested (1000 mg/kg BW) was determined as the NOAEL in this study.
Executive summary:: The objective of this study was to evaluate the possible toxicity of Synthetic Amorphous Silica (NM-200) after oral administration (gavage) in rats for 28 days and an additional 14-day recovery period.

Reference 2

Endpoint:: sub-chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: Jun. 1 - Dec. 3, 1962
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:: reference to same study
Qualifier:: no guideline available
Principles of method if other than guideline:: 6 months administration in feed, 1 dose level
GLP compliance:: not specified
Specific details on test material used for the study:: Aerosil
Species:: rat
Strain:: Wistar
Sex:: male/female
Details on test animals or test system and environmental conditions:: c. 90-110 g, feed: Altromin R and water ad libitum, 23 +/- 2 °C
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 6 months
Dose / conc.:: 500 mg/kg bw/day (nominal)
Remarks:: actual males: 497 +/-30 mg/kg bw/d
actual females: 495 +/-39 mg/kg bw/d
No. of animals per sex per dose:: 20
Control animals:: yes
Details on study design:: complete sub-chronic oral toxicity study with further fertility study after 4.5 months (but latter only with 5 females and 1 male each in test and control group)
Observations and examinations performed and frequency:: daily: clinical signs, behaviour, food consumption
weekly. body weight
monthly: blood (of 5 animals)
Sacrifice and pathology:: necropsy (pathology and histopatholgy) performed on all rats
Other examinations:: fertility study: maternal fertility; body weight and clinical signs of F1 generation
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, non-treatment-related
Description (incidence and severity):: only minimal variabilities to control
Food consumption and compound intake (if feeding study):: no effects observed
Haematological findings:: no effects observed
Behaviour (functional findings):: no effects observed
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: only 1/40 animals showed a hepatic abscess
Histopathological findings: non-neoplastic:: not specified
Histopathological findings: neoplastic:: not specified
Other effects:: no effects observed
Description (incidence and severity):: fertility study. no effects on fertility, litter size, body weight and appearance of offspring
: Key result
Dose descriptor:: NOEL
Effect level:: >= 500 mg/kg bw/day (nominal)
Sex:: male/female
Basis for effect level:: behaviour (functional findings); body weight and weight gain; clinical signs; food consumption and compound intake; gross pathology; haematology; mortality; organ weights and organ / body weight ratios
: Key result
Critical effects observed:: no
Conclusions:: No signs for toxicity to rats were seen after 6 months of oral administration. Also no effects to fertlity were observed in a very limited study.
Executive summary:: The sub-chronic oral toxicity of Aerosil was evaluted in rats. The additional fertility study performed was only very limited, as only 1 male rat was mated with 5 females.

Reference 3

Endpoint:: sub-chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 27 Oct. 1980 - 27 Jan. 1981
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: comparable to guideline study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:: yes
Specific details on test material used for the study:: Sipernat 22
Species:: rat
Strain:: Wistar
Remarks:: albino
Details on species / strain selection:: obtained from: Central Institute for Breeding of Laboratory Animals TNO, Zeist / NL
Sex:: male/female
Details on test animals or test system and environmental conditions:: 3.5 weeks old, (at study initiation) 35 - 50 g, 5 per cage, 23 +/-1 °C, >= 40% humidity, institute's stock diet ad libitum, bottled tap water, 12 h light/dark
Route of administration:: oral: feed
Details on oral exposure:: DIET PREPARATION
Treated feed: 6-kg batches mixed with the test material for 2 min, freshly prepared 5x/13 weeks and stored at 15 °C until use.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: variation CV (low / mid / high dose): 12.5%, 5%, 5%
Duration of treatment / exposure:: 13 weeks
Frequency of treatment:: daily, continuous
Dose / conc.:: 0.5 other: % in diet
Dose / conc.:: 2 other: % in diet
Dose / conc.:: 8 other: % in diet
No. of animals per sex per dose:: 10
Control animals:: yes, plain diet
Details on study design:: Post-exposure period: no
Statistics:: Body weight and organ weight data were subjected to analysis of variance followed by Dunnett's Multiple Comparison test. Haematological findings, clinical chemistry values and urinalysis results were analysed by the Mann/Whitney U-test. The incidence of histopathological changes was examined by the Chi-square test.
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: No signs of reaction to the treatment were seen in any of the test rats during the experimental period. A number of rats in different groups showed clinical signs. However, there was no evidence of a dose-related response to the treatment.
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Description (incidence and severity):: Mean body weights did not show statistically significant differences between the various test groups and the controls in either sex.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: slightly increased food intake in high dose group
Food efficiency:: effects observed, treatment-related
Description (incidence and severity):: Food efficiency was slightly diminished in females of the top-dose group.
Water consumption and compound intake (if drinking water study):: no effects observed
Ophthalmological findings:: not examined
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: There were some isolated changes in red blood cell count and mean corpuscular volume in males of the low- and mid-dose groups. However, there was no evidence of a dose-related response to the treatment.
The mean corpuscular haemoglobin concentration (MCHC) was relatively low in the 8% group in females.
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: Total protein levels were relatively low in males and relatively high in females of the top-dose group. Glucose levels were slightly increased in females of the top-dose group.
Urinalysis findings:: no effects observed
Description (incidence and severity):: The density and volume of the urine showed no statistically significant differences between the various test groups and the controls. There were no changes in the urine with respect to appearance, pH, protein, glucose, ketones, occult blood,urobilinogen, bilirubine or microscopy of the sediment, which could be attributed to the ingestion of the test substance. No casts were found in any of the groups (table 10).
Behaviour (functional findings):: no effects observed
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: The absolute as well as the relative weight of the caecum, both filled and empty, was increased in the top-dose group in both sexes. But this was not considered toxilogical significant
Gross pathological findings:: no effects observed
Description (incidence and severity):: Gross examination at autopsy did not reveal any abnormalities attributable to the feeding of Sipernat 22.
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: Microscopical examination did not reveal any histopathological changes that could be attributed to the feeding of Sipernat 22. The observed histopathological changes are common findings in the strain of rats used. Moreover, the lesions were about equally distributed amongst control and test group, or occurred in one or a few rats only.
Histopathological findings: neoplastic:: not examined
: Key result
Dose descriptor:: NOEL
Effect level:: 8 other: % in diet
Sex:: male/female
Basis for effect level:: behaviour (functional findings); body weight and weight gain; clinical biochemistry; clinical signs; gross pathology; haematology; histopathology: non-neoplastic; mortality; organ weights and organ / body weight ratios; urinalysis; water consumption and compound intake
Remarks on result:: other: equivalent to app. 4 g/kg bw/d
: Key result
Critical effects observed:: no
Conclusions:: The no-toxic effect level of Sipernat 22 was 8% in the diet of rats when the rats were fed the diet for 13 weeks. This level was equivalent to an intake of approximately 4 g Sipernat 22/kg body weight/day.
Executive summary:: The study examined into the subchronic toxic effects of Sipernat 22 administered orally in the diet to rats.

Reference 4

Endpoint:: sub-chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: Start 05 Aug. 1974
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study with acceptable restrictions
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:: yes
Remarks:: exposure 6 months
Principles of method if other than guideline:: in the diet, 2 dose levels
GLP compliance:: no
Specific details on test material used for the study:: Syloid 244
Species:: rat
Strain:: Crj: CD(SD)
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River France, S.A.
- Age at study initiation: no data
- Weight at study initiation: mean values - 121 (m); 124 (f)
- Housing: 2 animals/cage
- Diet: Altromin 1321 ad libitum - only interrupted during urine collection
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 20
- Humidity (%): approx. 65
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
Route of administration:: oral: feed
Analytical verification of doses or concentrations:: no
Duration of treatment / exposure:: 26 weeks
Frequency of treatment:: daily, 7 days per week
Dose / conc.:: 3.2 other: % in diet
Remarks:: males: approx. 2170 mg/kg bw/day; females: approx. 2420 mg/kg bw/day
Dose / conc.:: 10 other: % in diet
Remarks:: males: approx. 7950 mg/kg bw/day; females: approx. 8980 mg/kg bw/day
No. of animals per sex per dose:: 12
Control animals:: yes, plain diet
Details on study design:: Post-exposure period: no
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
at least 1x/d

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- at start, weekly, and end of study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, for 2 animals/cage
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: no data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 13, and 26 week
- How many animals: each time 4 animals per group and sex
- Animals fasted: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 6, 13, and 26 week
- How many animals: each time 4 animals per group and sex
- Animals fasted: No

URINALYSIS: Yes
- Time schedule for collection of urine: 6, 13, and 26 week
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No, feed withdrawal during urine collection, water ad libitum

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:: Organ weights
Bone-marrow histocytology
Statistics:: no data
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not specified
Water consumption and compound intake (if drinking water study):: not specified
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: no effects observed
Behaviour (functional findings):: no effects observed
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: only some common findings, in treated as well as in control rats
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Histopathological findings: neoplastic:: no effects observed
: Key result
Dose descriptor:: NOEL
Remarks:: highest dose
Effect level:: 7 950 mg/kg bw/day (nominal)
Sex:: male
Basis for effect level:: body weight and weight gain; clinical signs; food consumption and compound intake; gross pathology; haematology; histopathology: non-neoplastic; mortality; organ weights and organ / body weight ratios
: Key result
Dose descriptor:: NOEL
Remarks:: highest dose
Effect level:: 8 980 mg/kg bw/day (nominal)
Sex:: female
Basis for effect level:: body weight and weight gain; clinical signs; food consumption and compound intake; gross pathology; haematology; histopathology: non-neoplastic; mortality; organ weights and organ / body weight ratios
: Key result
Critical effects observed:: no
Conclusions:: Up to 10 % in the diet neither treatment-related changes to organ weights and hispathology nor toxic effects were observed.
Executive summary:: In a 6-month study the oral toxicity potential of Syloid 244 was assessed in rats.

Reference 5

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Qualifier:: according to guideline
Guideline:: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:: Additionally, the acute phase proteins haptoglobin and α2-macroglobulin as well as cardiac troponin I were determined, and metabolome analysis was performed in plasma samples.
GLP compliance:: not specified
Specific details on test material used for the study:: SiO2·naked: SiO2 without surface modification (Levasil 200)
Species:: rat
Strain:: Wistar
Details on species / strain selection:: Wistar Crl:WI(Han) rats (Charles River Laboratories, Sulzfeld, Germany)
Sex:: male/female
Details on test animals or test system and environmental conditions:: 41 - 43 days old, housed in group of 5
Route of administration:: oral: gavage
Vehicle:: other: phosphate buffered saline & bovine serum albumin
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 4 weeks
Frequency of treatment:: daily
Dose / conc.:: 1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:: 5
Control animals:: yes, concurrent vehicle
Observations and examinations performed and frequency:: Detailed clinical observations, regular health inspections, assessment of food and water consumption and the determination of the body weight were performed.
Hematological and clinical chemical examinations as well as urinalyses were performed toward the end of the administration period, and all standard parameters listed in OECD TG 407, paragraphs 32, 34, and 35, were evaluated for all animals. Additionally, the acute phase proteins haptoglobin and α2-macroglobulin were determined in serum samples from all animals.
Sacrifice and pathology:: The exsanguinated animals were subjected to a full, detailed gross necropsy assessing and weighing all organs listed in OECD TG 407, paragraph 40. Additionally, all organs listed in OECD TG 407, paragraph 43, were preserved in neutral-buffered 10 % formalin (NBF) or modified Davidson’s solution for histopathological examination.
Statistics:: • Body weight and body weight change: A comparison of each group with the control group using Dunnett’s test (two sided) for the hypothesis of equal means;
• Blood and urine parameters with uni- or bidirectional changes: One or two-sided Wilcoxon test for the hypothesis of equal medians;
• Blood and urine parameters with unidirectional changes: Pair-wise, one-sided Wilcoxon test for the hypothesis of equal medians;
• Organ weight parameters: A pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test for the hypothesis of equal medians.
In all tests, levels of significance of p ≤ 0.05 and p ≤ 0.01 were recorded.
Clinical signs:: no effects observed
Mortality:: mortality observed, non-treatment-related
Description (incidence):: One female animal of the SiO2·naked test group was found dead on study day 5. The animal died because of a gavage error.
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Water consumption and compound intake (if drinking water study):: no effects observed
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: No red blood cell or coagulation parameters were changed in the male rats. Regarding white blood cell parameters, in the male animals of the SiO2·
naked test group, relative eosinophil (Eos.%) counts were lower as compared to the control group (1.0 vs. 1.5 %). The recorded value, however, was only marginally below the historical control range (1.1–2.8 %), the absolute eosinophil counts were not altered, and none of the other differential blood cell fractions were changed. Furthermore, the eosinophil counts were not changed in the female animals of the same test group. Therefore, these alterations were regarded as incidental and not treatment-related in accordance with the ECETOC (2002) criteria.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: In the male animals of the SiO2·naked test group, haptoglobin values were higher than those recorded for the control group (413.6 ng/mL as compared to 234.0 ng/mL). However, these haptoglobin values were within the historical control range (haptoglobin: 265.2–1.074.0 ng/mL, corresponding to a range of fourfold between the lowest and the highest mean of control groups previously recorded in unpublished in-house studies).
In the female rats of the SiO2·naked test group, the mean haptoglobin values were also considerably higher than those recorded for the respective control group (469.5 ng/ mL as compared to 141.7 ng/mL). Assessment of the implications of these values is impaired by the circumstance that
historical control ranges for haptoglobin values in female rats were neither available in-house, nor could be found in the published literature. Additionally, one of these animals not only had an extremely high haptoglobin value (i.e., 1,610.2 ng/mL), but also a high α2-macroglobulin value (49.21 ng/mL as compared to the mean control value of 12.65 ng/mL) and increased total white blood cell (WBC) and (absolute and relative) neutrophil counts [WBC: 8.44 as compared to 3.97 giga/L; neutrophils: 5.36 giga/L (63.5 %) as compared to 0.66 giga/L (19.1 %)]. Upon necropsy, no macroscopic or histopathological correlates to these findings could be determined. Therefore, this individual animal was diagnosed as most likely having had a systemic inflammation. In addition, for the other three (remaining) animals of this test group (one animal of this test group had died due to gavage error), all mentioned parameters were within the normal ranges, and there were no histopathologically relevant findings upon necropsy. (When excluding the haptoglobin value of 1,610.2 ng/mL, the mean haptoglobin value of the remaining animals of the SiO2·naked test group amounted to 89.3 ng/mL, i.e., it was even lower than the mean control value). Therefore, the mean haptoglobin value of the female rats of the SiO2·
naked test group, just as the altered parameters of the individual female rat, was assessed as not being related to the test substance SiO2·naked in accordance with the ECETOC (2002) criteria.
Urinalysis findings:: no effects observed
Organ weight findings including organ / body weight ratios:: effects observed, non-treatment-related
Description (incidence and severity):: There were no significant increases or decreases of the mean absolute organ weights. In regard to mean relative organ weights, the weight of the liver of the male animals of the SiO2·naked test group was increased. All of these changes were assessed as not being treatment-related in accordance with the ECETOC (2002) criteria since there were no corresponding changes in absolute organ weights or histopathological findings that would explain the weight changes.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: One female animal of the SiO2·naked test group died ahead of schedule having a thoracic effusion that was assessed as having been caused by a gavage error. Therefore, this finding was regarded as related to the administration procedure, but not to the test substances. All other gross lesions noted were single observations, and they were regarded to have developed spontaneously and to be unrelated to the test substances or the treatments
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: All findings were either single observations or they were equally distributed between the control and treatment groups. Therefore, all observations were considered to be incidental or spontaneous in origin and unrelated to the treatment.
Other effects:: no effects observed
Description (incidence and severity):: At a dose level of 1,000 mg/kg, none of the tested NMs had a biologically relevant impact on the plasma metabolome pattern of rats. When compared against the control animals on a significance level of p < 0.05, in both male and female animals, the number of significantly changed endogenous metabolites was below or at the false positive rate for all particles tested and were assessed as “statistical variance” of the metabolome analysis.
Dose descriptor:: NOAEL
Effect level:: > 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Critical effects observed:: no
Conclusions:: There were no test substance-related adverse effects for SiO2 without surface modification (Levasil 200). Moreover, metabolomics changes were below the threshold of effects.
Executive summary:: The effects of seven nanomaterials (four amorphous silicon dioxides with or without surface functionalization, two surface-functionalized zirconium dioxides, and barium sulfate) upon 28-day oral exposure to male or female rats were investigated. Only the effects of "SiO2 naked" Levasil 200 are documented here.

Reference 6

Endpoint:: chronic toxicity: oral
Remarks:: combined repeated dose and carcinogenicity
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Qualifier:: according to guideline
Guideline:: OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:: yes
Remarks:: Group size was 10 animals/sex/dose at interim kills instead of 20 each. At terminal sacrifice 20/sex/dose as recommended. Urinanalysis not performed.
Principles of method if other than guideline:: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:: not specified
Species:: mouse
Strain:: B6C3F1
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Funabashifarm Animal Co. Ltd. Japan
- Age at study initiation: 4 weeks
- Weight at study initiation: 21 - 27.3 g (male mice); 16 - 19.9 g (female mice)
- Fasting period before study:
- Housing: 5 mice/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +-1
- Humidity (%): 50 +-10 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 10 dark / 14 light
Route of administration:: oral: feed
Details on oral exposure:: DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
Analytical verification of doses or concentrations:: no
Duration of treatment / exposure:: 93 weeks, interim kill after 6 and 12 months (10 animals each)
Frequency of treatment:: continuously
Remarks:: Doses / Concentrations:
1.25, 2.5 and 5 %
Basis:
nominal in diet
No. of animals per sex per dose:: 40
Control animals:: yes, concurrent no treatment
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for survival and clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 55 weeks, thereafter every two weeks

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (weekly)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages
from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12, and 21 months
- Parameters checked in table 4-1/4-2 were examined.

CLINICAL CHEMISTRY: yes (see Report p. 30)
- Time schedule for collection of blood: 6, 12, and 21 months

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:: Student´s t-analysis variance test / Chi square test of Mantel-Hanszel for survival
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, treatment-related
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not specified
Water consumption and compound intake (if drinking water study):: not specified
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: no effects observed
Histopathological findings: non-neoplastic:: no effects observed
Histopathological findings: neoplastic:: no effects observed
Details on results:: SUBSTANCE UPTAKE
The mean cumulative intake after 93 weeks was 38.45, 79.78 and 160 g/mouse in males
and 37.02, 72.46 and 157.59 g/mouse in females, respectively.

The average doses of the male and female 5%-groups were approx. 5800 and 4500 mg/(kg bw*d)
after week 15 - 20 of the study start, while they were distinctly higher in the initial phase of life,
in particular in the female group (comp. Report, Tab. 2 and 3).
Specific silica intake decreased over time during growth and aging in relation to the relative reduction
in food intake. A reasonable average of 5800 and 4500 mg/(kg bw*d) is estimated from the
experimental data (see Report, Tab. 2 and 3). These estimates largely relate to a mean body weight
of 42 g and 45 g for males and females, respectively, from week 15 through 93, which agrees fairly
well with the growth curves (comp. Report, Fig. 1 and 2).

BODY WEIGHT AND WEIGHT GAIN
In the 2.5 and 5 % groups the food consumption increased, but this was accompanied by a decreased body-weight gain
in the 5-% group from week 15 through 50 (males, <0.01) and from week 30 through 50 (females, p<0.05).
No differences in mean body weights were found in the second half of the investigation (see Report Fig. 1 and 2 / Tab. 2 and 3).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In the 2.5 and 5 % groups the food consumption transiently increased.

HISTORICAL CONTROL DATA (if applicable): no data
Dose descriptor:: NOAEL
Remarks:: (highest dose level tested)
Effect level:: 5 other: % in the diet
Sex:: male/female
Basis for effect level:: other: overall effects clinical signs; mortality; body weight; food consumption; haematology; blood chemistry; gross pathology; organ weights; histopathology
Dose descriptor:: NOAEL
Remarks:: (average doses, highest level tested)
Effect level:: ca. 4 500 - ca. 5 800
Sex:: male/female
Basis for effect level:: other: see 'Remark'
Dose descriptor:: NOAEL
Remarks:: (highest dose level tested: 5 % in diet)
Effect level:: ca. 5 000 - ca. 7 000 mg/kg bw/day (actual dose received)
Sex:: male
Basis for effect level:: other: Specific silica intake decreased over time during growth and aging in relation to the relative reduction in food intake (see Report, Tab. 2)
Dose descriptor:: NOAEL
Remarks:: (highest dose level tested: 5 % in diet)
Effect level:: ca. 4 000 - ca. 13 000 mg/kg bw/day (actual dose received)
Sex:: female
Basis for effect level:: other: Specific silica intake decreased over time during growth and aging in relation to the relative reduction in food intake (see Report, Tab. 3)
Critical effects observed:: not specified

Reference 7

Endpoint:: chronic toxicity: oral
Remarks:: combined repeated dose and carcinogenicity
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Qualifier:: according to guideline
Guideline:: OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:: yes
Remarks:: Group size was 10 animals/sex/dose at interim kills instead of 20 each. At terminal sacrifice 20/sex/dose as recommended. Urinanalysis not performed.
Principles of method if other than guideline:: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:: not specified
Specific details on test material used for the study:: synthetic amorphous silicon dioxide Syloid 244
Species:: rat
Strain:: Fischer 344
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Funabashifarm Animal Co. Ltd. Japan
- Age at study initiation: 3 weeks
- Weight at study initiation: 117 - 150 g (male rats); 92 - 126 g (female rats)
- Fasting period before study:
- Housing: 2 rats/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +-1
- Humidity (%): 50 +-10 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 10 dark / 14 light
Route of administration:: oral: feed
Details on oral exposure:: DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
Analytical verification of doses or concentrations:: no
Duration of treatment / exposure:: 103 weeks, interim kill after 6 and 12 months (10 animals each)
Frequency of treatment:: continuously
Remarks:: Doses / Concentrations:
1.25, 2.5 and 5 %
Basis:
nominal in diet
No. of animals per sex per dose:: 40
Control animals:: yes, concurrent no treatment
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for survival and clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 55 weeks, thereafter every two weeks

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (weekly)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages
from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12, and 24 months
- Parameters checked in table 9-1/9-2 were examined.

CLINICAL CHEMISTRY: yes (see Report p. 30)
- Time schedule for collection of blood: 6, 12 and 24 months

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:: Student´s t-analysis variance test / Chi square test of Mantel-Hanszel for survival
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not specified
Water consumption and compound intake (if drinking water study):: not specified
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Gross pathological findings:: no effects observed
Histopathological findings: non-neoplastic:: no effects observed
Histopathological findings: neoplastic:: no effects observed
Details on results:: SUBSTANCE UPTAKE
The mean cumulative intake after 103 weeks was 143.46, 279.55 and 581.18 g/rat in males
and 107.25, 205,02 and 435.33 g/rat in females, respectively.
(Note: Misprint for substance uptake by males, 2.5 %, in Tab. 7: 179.55 must read 279.55 g/rat)

The average doses of the male and female 5%-groups were approx. 1800 to 2000 mg/(kg bw*d) after week 15 of the study start, while they were distinctly higher in the juvenile phase of life (comp. Report, Tab. 7 and 8).
Specific silica intake decreased over time during growth and aging in relation to the relative reduction in food intake. A reasonable average of 2000 mg/(kg bw*d) is estimated from the experimental data. This estimate relates to a mean body weight of 400 g and 300 g for males and females, respectively (see Report, Tab. 7 and 8), which agrees fairly well with the growth curves (comp. Report, Fig. 5 and 6) .

ORGAN WEIGHTS
Lower liver weights were noted from 12 to 24 months in the female 2.5 and 5 % dose groups (p =<0.01) (see Report Tab. 10-2).

HISTORICAL CONTROL DATA (if applicable): no data
Dose descriptor:: NOAEL
Remarks:: (highest dose level tested)
Effect level:: 5 other: % in the diet
Sex:: male/female
Dose descriptor:: NOAEL
Remarks:: (average dose, highest level tested)
Effect level:: ca. 2 000 mg/kg bw/day (actual dose received)
Sex:: male/female

Reference 8

Endpoint:: short-term repeated dose toxicity: oral
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
Qualifier:: no guideline followed
Principles of method if other than guideline:: 1 dose level., 20 administrations within 1 month, 2-month post observation period
GLP compliance:: not specified
Specific details on test material used for the study:: HDK V 15
Species:: rat
Strain:: not specified
Details on species / strain selection:: 200g body weight
Sex:: not specified
Route of administration:: oral: gavage
Vehicle:: water
Duration of treatment / exposure:: 20 / 30 days
Frequency of treatment:: once daily
Dose / conc.:: 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:: 20 animals / dose level (sexes not specified)
Control animals:: yes
Observations and examinations performed and frequency:: behaviour, food consumption, body weight
Sacrifice and pathology:: SO2 content in liver, spleen, kidneys
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Behaviour (functional findings):: no effects observed
Organ weight findings including organ / body weight ratios:: not examined
Details on results:: silicon dioxide in
liver: 4.2 µg (control: 1.8 µg)
spleen: 5.5 µg (control: 7.2 µg)
kidneys: 14.2 µg (control: 7.8 µg)
Dose descriptor:: NOEL
Effect level:: >= 500 mg/kg bw/day (nominal)
Sex:: not specified
Basis for effect level:: behaviour (functional findings); body weight and weight gain; clinical signs; food consumption and compound intake; mortality
Remarks on result:: not determinable due to absence of adverse toxic effects
Critical effects observed:: no
Conclusions:: No mortality, no clinical signs nor body weight changes were observed, but resorption and storage in liver and kidneys.
Executive summary:: The oral toxicity potential of HDK V 15 was evaluated in a 1-month feeding study in rats.

Reference 9

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: test procedure in accordance with national standard methods with acceptable restrictions
Reason / purpose for cross-reference:: reference to other study
Qualifier:: no guideline followed
Principles of method if other than guideline:: in the diet, 2 dose levels (one of them increased during study); preliminary study to a 6-month one
GLP compliance:: no
Specific details on test material used for the study:: Syloid 244
Species:: rat
Strain:: Sprague-Dawley
Remarks:: albino
Details on species / strain selection:: obtained from: Willi Gassner Versuchstierzuchtanstalt, Sulzfeld, Germany
Sex:: male/female
Details on test animals or test system and environmental conditions:: 1 per cage, feed: Altromin R and tap water ad libitum, c. 18 °C, c. 81 % humidity
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Analytical verification of doses or concentrations:: not specified
Duration of treatment / exposure:: 14 days
Frequency of treatment:: once daily, 7 days / week
Dose / conc.:: 10 other: % in diet
Remarks:: 10% = ca. 16.6 (m) or 16.4 (f) g/kg/bw
Dose / conc.:: 5 other: % in diet
Remarks:: for the first 10 days; thereafter increased to 20% in the diet for the remaining 4 days
(5% = ca. 5.9 (m) or 5.7 (f) g/kg/bw; 20% = ca. 23.6 (m) or 24.8 (f) g/kg/bw)
No. of animals per sex per dose:: 5
Control animals:: yes, plain diet
Observations and examinations performed and frequency:: daily: clinical signs, behaviour
every third day: body weight, food consumption
Sacrifice and pathology:: no necropsy performed
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Description (incidence and severity):: practically no difference to control
Food consumption and compound intake (if feeding study):: no effects observed
Water consumption and compound intake (if drinking water study):: no effects observed
Behaviour (functional findings):: no effects observed
Conclusions:: no divergences to control (in mortality, body weight, behaviour, food / water consumption)
Executive summary:: This was a prelimainary study to a 6-month one. The oral toxicty of Syloid 244 was assessed in rats for 2 weeks, but only clinical signs, body weight and food consumption were concidered.

Reference 10

Endpoint:: sub-chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:: no guideline available
Principles of method if other than guideline:: 90-days oral study in rats, administered in the diet, 3 dose levels
GLP compliance:: no
Specific details on test material used for the study:: Cab-O-Sil
Species:: rat
Strain:: other: albino rats of the Charles River strain
Sex:: male/female
Details on test animals or test system and environmental conditions:: males: 53-70 g, females: 52-71 g
housed individually, food and water ad libitum
Route of administration:: oral: feed
Details on route of administration:: in food: Purina lab chow
Details on analytical verification of doses or concentrations:: Individual body weights and food consumaption and observations of the general physical appearance and behavior of each rat were recorded once a week.
Duration of treatment / exposure:: 90 days
Frequency of treatment:: in the feed, which was available ad libitum
Dose / conc.:: 10 000 ppm
Remarks:: 1 % in feed
Dose / conc.:: 30 000 ppm
Remarks:: 3 % in feed
Dose / conc.:: 50 000 ppm
Remarks:: 5 % in feed
No. of animals per sex per dose:: 15
Control animals:: yes, plain diet
Positive control:: yes: cosmetic talc
Sacrifice and pathology:: after 45 days: 3 per sex and group; all remaining after 90 days
urine, oral mocous membrane, lung, thyroid, heart, stomach, small and large intestine, pancreas, spleen, liver, kidney, adrenal, urinary bladder, gonads, femur
Statistics:: criteria for statistical evaluation:
mortality (chi-square test)
body weight gain, food consuption (variance at the 5% probability level)
Clinical signs:: no effects observed
Mortality:: mortality observed, non-treatment-related
Description (incidence):: all 3 deaths in negative or positve control groups
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: no effects observed
Behaviour (functional findings):: no effects observed
Gross pathological findings:: no effects observed
Histopathological findings: non-neoplastic:: no effects observed
: Key result
Dose descriptor:: NOEL
Effect level:: >= 50 000 ppm
Sex:: male/female
: Key result
Critical effects observed:: no
Conclusions:: There were no gross signs of systemic toxicity which could be associated with the dietary ingestion of Cab-O-Sil over a 90-day period.
Executive summary:: An oral subchronic study was performed with Cab-O-Sil.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

other information

Study period:

experimental phase: Feb. 12, 2018 - May 29, 2019; study completion date: September, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please see also attached communication resp:
ECHA decision dated 11 March 2015 [decision number: SEV-D- 2114297905-32-01/F],
as amended by the Board of Appeal in its decision dated 30 June 2017 (A-015-2015)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2017

Deviations:

yes

Principles of method if other than guideline:

Deviations from the guideline: This study was conducted according to an ECHA decision (dated June, 30 2017). This decision skipped the endpoints clinical pathology and ophthalmology. Gross pathology and histopathology shall be conducted on the lungs, trachea, naso-pharyngeal tissues, nasal-associated lymphoid tissue (NALT) and larynx; other organs and tissues were excluded from examination. As an addition, collagen was analysed.

GLP compliance:

yes

Specific details on test material used for the study:

Test material 1
Name: SAS1 - High BET; BET: Approx. 400 m²/g (high surface area); Purity: > 99.8%; Molecular formula: SiO2; Molecular weight: 60.08 g/mol;
Manufacturer: CABOT Corporation, Tuscola IL, USA; CAS number: 7631-86-9, 112945-52-5; CAS name: Silica, amorphous, fumed, crystalline-free
EC number: 231-545-4; Date of delivery: January 4, 2018; Expiration date: September 19, 2019

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Male and female Wistar rats [strain Crl:WI(Han)] were purchased from Charles River Deutschland (Sulzfeld, Germany).
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996) such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany).
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: approx. 8-9 weeks
- Weight at study initiation: approx. 250 gram for males and approx. 175 gram for females
- Fasting period before study:
- Housing: Makrolon (polycarbonate) cages type IV, two rats of the same sex per cage, and were maintained under conventional laboratory conditions. Cages and absorbing softwood bedding material (Lignocel BK8-15) were changed twice a week or more often, if necessary.
- Diet: commercial chow in pellet form was used, identified as Ssniff V1534, purchased from Ssniff-Spezialdiäten (Soest, Germany). The diet was stored in room T1.015 at a temperature of max. 18 °C and a relative humidity of max. 70 %. No batch of diet was used after 6 months have elapsed from manufacturing date. The diet was offered fresh weekly or more often, if necessary.
- Water: Tap water from the Hannover city water supplier was offered fresh weekly or more often, if necessary, in a Makrolon bottle fitted with a stainless steel nipple top with a hole approximately 0.5 mm in diameter.
- Acclimation period: for a period of at least 3 weeks prior to exposure animals were trained to become accustomed to nose-only tubes

ENVIRONMENTAL CONDITIONS
In the cages the rats find paperboard crinklets as nesting material and wooden blocks for nibbling. The temperature and the relative humidity of the animal room were monitored electronically and recorded on a continuous basis
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 % for relative humidity
- Air changes (per hr): at least 10 times per hour light/dark cycle
- Photoperiod (hrs dark / hrs light): A 12-hour light/dark cycle was used controlled by an automatic timing device

IN-LIFE DATES: From: To:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

<= 3 µm

Geometric standard deviation (GSD):

3.53

Remarks on MMAD:

The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant.
Using this system a mass median aerodynamic diameter (MMAD) of ≤ 3 µm was achieved as required by the ECHA decision.

Details on inhalation exposure:

The two test item aerosols were generated by dispersing the dry powder. Dispersion was achieved by a feeding system and pressurized air dispersion nozzle (dynamic system). For each nose-only exposure unit (total of 8 units for groups 2 - 9), the aerosol was generated by a pneumatic disperser. The disperser was fed with the test item under computerized control, i.e. with a feedback loop to a scattering signal from an aerosol photometer which reflects the actual aerosol concentration.
The aerosol was given to the rats by a flow-past nose-only inhalation exposure system which was used for previous particle and fiber inhalation studies at Fraunhofer ITEM. In this system, aerosols were supplied to each rat individually, and exhaled air is immediately exhausted. The airflow to each rat was approximately 1 L/min which is calculated to be laminar. Therefore measurement of the oxygen concentration is not necessary. Prior to the 90-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.
For exposure to the test item the rats were restrained in acrylic tubes with a flexible stopper. The exposure tubes are arranged around a cylinder capable to take up 16 animals per level on four levels. The rat nose is located at the front end of a tube connected to the inner cylinder of the exposure unit delivering the aerosol. Through thin pipes, the aerosol is supplied to each rat nose individually and exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of individual rat at the cylinder is changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (including the clean air control: 9 units) are located each under a separate hood to prevent cross contamination among the different dose groups.
The duration of exposure was 6 hours/day, 5 days/week for 13 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Filter samples of the aerosols were taken at least twice per week to control the aerosol concentrations and to adjust the aerosol photometer readings. These samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats are inhaling the aerosol and will be analysed gravimetrically.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h / d
5 d / w

Dose / conc.:

0.5 mg/m³ air (nominal)

Dose / conc.:

1 mg/m³ air (nominal)

Dose / conc.:

2.5 mg/m³ air (nominal)

Dose / conc.:

5 mg/m³ air (nominal)

No. of animals per sex per dose:

10 (sacrified 1 day after treatment);
5 each (sacrified 90, 180 or 360 days after treatment) [cf. table 3 below, for details on dose groups]

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered to the test animals by nose-only inhalation. This administration route is preferred compared to the intratracheal instillation route because it is the more physiological way of particle deposition. Nose-only inhalation is preferred to whole-body inhalation because it provides a specific model of inhalation exposure, restricting ingestion via grooming activity.
To specify adequate aerosol concentrations in this 90-day study, a preceding 90-day study (6 hrs/day on 5days/week) was used as a basis (Fraunhofer ITEM study # 02G11018; conducted with another SAS, i.e. NM-200; JRC code). Based on this study 0.5, 1.0, 2.5 and 5 mg/m³ were used for the test item in the very low, low, mid and high dose groups, respectively. Upon cessation of exposure, animals were investigated for various endpoints (see Table 3) on days 1, 90, 180 and 360 of a subsequent post-exposure observation period. Sacrifice dates at 180 and 360 days post-exposure would have been skipped if no adverse findings were observed after 90 or 180 days. - Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Other:

Observations and examinations performed and frequency:

Cf. also table 5 below for details on examinations!

DETAILED CLINICAL OBSERVATIONS: Yes
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 includes 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. The observations were recorded daily with the PROVANTIS system. On the exposure days, the animals were clinically observed before, during and after exposure.

BODY WEIGHT: Yes
Individual body weights were recorded to the nearest 0.1 g twice a week for the first month and reduced to once a week throughout the remainder of the study provided there is no significant body weight effect in the first month (including post-exposure observation period) for all animals.
All body weight data were collected using electronic balances, interfaced with a computer and programmed for direct on-line data acquisition (Provantis).

FOOD CONSUMPTION
Food consumption was recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group.

WATER CONSUMPTION: Yes
Water consumption was recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group. CAGE SIDE OBSERVATIONS: Yes / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: No
- skipped by ECHA decision (dated June, 30 2017)

HAEMATOLOGY: No
- skipped by ECHA decision (dated June, 30 2017)

CLINICAL CHEMISTRY: No
- skipped by ECHA decision (dated June, 30 2017)

URINALYSIS: No
- skipped by ECHA decision (dated June, 30 2017)

OTHER:
This decision skipped the endpoints clinical pathology and ophthalmology. Gross pathology and histopathology shall be conducted on the lungs, trachea, naso-pharyngeal tissues, nasal-associated lymphoid tissue (NALT) and larynx; other organs and tissues were excluded from examination. As an addition, collagen was analysed.

Sacrifice and pathology:

Cf. also tables 5 and 6 below for details on examinations!

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 rats were anesthetized with an overdose pentobarbital sodium (Narcoren) and killed by cutting the vena cava caudalis.
The abdominal cavity was opened and the diaphragm was cut carefully allowing the lungs to collapse. Heart, oesophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN; mediastinal and tracheobronchial) will be removed from the lung convolution.
The lung and the lower half of the trachea were weighed and used for BAL (right lobes # 1-4) and histopathology (left lobe #5). For histopathology the left lung lobe # 5 was inflated under a pressure of about 20 cm water with formalin and was fixed by immersion for a minimum of two hours, and used for histopathology.
The following organs were trimmed and wet weights will be recorded:
Liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, thyroid, 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 in brackets and the seminal vesicles were prepared for histopathology. Tissues in brackets were preserved only.

Other examinations:

Bronchoalveolar Lavage (BAL)
Bronchoalveolar lavage was performed in 5 rats per time point and group, i.e. after end of exposure (day 1 of post-exposure) and after 3, 6 and 12 months of the post-observation period. The method of Henderson et al. (1987) was used with minor modifications.
Cytokines (e.g. IL-6, IL-8) will be measured in the BAL of 5 animals per sex and group on day 1, 90, 180 and 360 post exposure.
Collagen will be determined in lung tissue following an acidic hydrolysis with 37 % HCl. A hydroxyproline analysis will be done according to the method of Creemers & Jansen (1997). Alternatively, the analysis could be done on lungs starting with a homogenisation of the lung tissue. The collagen analysis in BALF will be done under non-GLP conditions.

Histopathology
Lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E). The following histopathological examinations were performed in 10 or 5 animals per sex and group after end of exposure:
• Histopathology on the respiratory tract in all animals of the clean air control group (dose group 1), the Synthetic Amorphous Silica high dose groups (dose groups 5 and 9) and of all animals that died or were killed moribund during the study. In the very low, low and mid dose groups (dose groups 2-4; 6-8), the respiratory tract may be analysed after the results in the high dose groups will have been completed.
• The respiratory tract includes lung lobes, with bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of all groups (groups 1, 5, 9). Other organs than the respiratory tract were included only if macroscopical findings occurred.
• Trimming of lungs: 3 sections; nose 4 sections.
For the animals sacrificed 3, 6 and 12 months post exposure, all tissues were preserved but only those showing lung changes on day 1 were examined histopathologically.

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. If necessary, further statistical procedures were applied upon agreement with the sponsor.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All animals survived their scheduled study period.
In the 13-week recovery (R1) period one female in group 5 (5.0 mg/m3) and one male (group 2, 0.5 mg/m3) were found dead. The female died due to astrocytoma in the brain.The cause of death of the male was mesenchymal tumor of the kidneys.
In the 52-week recovery (R3) one female (group 4, 2.5 mg/m3) was sacrified due to poor condition by malignant lymphoma. One male (group 4, 5.0 mg/m3) was sacrified due to poor condition by malignant schwannoma in the body cavity.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Relevant statistically significant changes were not observed in the treatment groups as compared to controls.
The enlarged abdomen with increased body weight and enlarged spleen and foci in liver from one male (group 2, 0.5 mg/mg3) during the 13-week recovery period was due to yolk sac carcinoma.
CAB-O-SIL S-17D induced a statistically significant increase of the absolute and relative lung wet weights in the female high dose group at 1 day post-exposure only. This effect had disappeared at 3 months post-exposure.
All other statistically significant changes detected at other organs than lungs are considered as incidental findings.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no relevant statistically significant changes as compared to concurrent controls

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

no relevant statistically significant changes as compared to concurrent controls

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Lung weights: statistically significant increase of the absolute and relative lung wet weights in the female high dose group at 1 day post-exposure. This effect had disappeared at 3 months post-exposure.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Upon necropsy, enlarged lung-associated lymph nodes (LALN) were observed in the high dose group. This is a particle-specific finding at lung overload conditions. Other test item- or dose-related macroscopical findings were not detected.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In nasal cavities, the major lesions consisted of:
- Goblet cell proliferation in levels 1 and 2 and nasopharyngeal duct at the end of treatment and after 13 weeks recovery in all dose groups.
- Hyaline inclusions in olfactory mucosa at higher incidences and severity with increased incidences during the course of the study.
- Chitinase-positive crystals in olfactory mucosa in nasal cavity levels 2-4 up to 26-week recovery without any further injury in olfactory mucosa, mainly in CAB-O-SIL S-17D treated animals.

In lungs, the findings consisted of:
- End of treatment:
- increased perivascular infiltration in groups low dose to high dose ≤ 1.0 mg/m3 groups.
- increased alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia dose-dependently for CAB-O-SIL S-17D and granulomas at the bronchio-alveloar junctions, granulomatous inflammation at a minor severity was noted in single animals from groups very low dose and low dose, and in most animals from all other test item treated groups mid dose to high dose.
- bronchio-alveolar hyperplasia in single animals from groups low dose up to high dose group.
- minimal macrophage agglomeration in the BALT of a few animals at ≤ 1.0 mg/m3 but all animals at 5.0 mg/m3.
- reversible BALT fibrogenesis at an increased incidence at higher doses.
- 13 weeks recovery:
- macrophage aggregates in animals at ≤ 1.0 mg/m3.
- single cases of granulomas at the alveolar-bronchiolar junctions in CAB-O-SIL S-17D-treated groups.
- Fibrogenesis due to inflammatory processes in one animal per sex at 0.5 mg/m3.
- 26 weeks recovery: similar findings than observed after 13 weeks recovery.
- 52 weeks recovery:
- no findings except the presence of macrophage agglomeration.

In lymph nodes, the gross lesions consisted of:
- End of treatment: enlarged lymph nodes at increased incidences in animals at >0.5 mg/m3, whereby almost all males at 5.0 mg/m3 were affected. After 13-, 26- and 52 weeks recovery gross lesions were similar.
- Histologically, the findings consisted of:
- End of treatment:
- granulomas in lymph nodes >0.5 mg/m3
- related granulomatous inflammation at a minor severity in single males >1.0 mg/m3
- lymphoid hyperplasia in most affected lymph nodes
- 13 weeks recovery:
- granulomas and single cases of granulomatous inflammation in animals at 5.0 mg/m3
- Fibrogenesis in one female 1.0 mg/m3.
- 26 weeks recovery:
- CAB-O-SIL S-17D: only single cases of lymphoid hyperplasia and granulomas in a few animals at 2.5 and 5.0 mg/m3
- 52 weeks recovery:
- no findings due to reversibility of effects

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar Lavage (BAL)
- Cytological and Biochemical Parameters
Mean values are shown in Figures 10-17 and Tables 43-58 in attached document BALF_Cytological amd Biochemical Parameters.
At day 1 post-exposure statistically significant increases of polymorphonuclear neutrophils (PMN) were detected in the mid and high dose groups of both sexes. In both dose groups a full recovery was detected at 3 months post-exposure.
For lactic dehydrogenase (LDH), ß-glucuronidase (GLU) and total protein (TP) no statistically significant increases were detected in all groups at all 4 sacrifice dates (but for total protein in the female high dose group at day 1).

Key result

Dose descriptor:

NOAEC

Effect level:

5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: BALF, lymph nodes

Remarks on result:

other: end of recovery; local lung effect; not substance specific, particle-related effect; no systemic effects

Remarks:

Overall, the effects depicted through these studies show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m3. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).

Dose descriptor:

NOAEC

Remarks:

Local lung inflammation

Effect level:

1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: end of exposure; local lung effect; not substance specific, particle-related effect; no systemic effects

Remarks:

Dose descriptor:

NOAEC

Effect level:

0.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lymph nodes

Remarks on result:

other: end of exposure / not substance specific, particle-related effect; no systemic effects

Remarks:

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Conclusions:

Under the conditions of this test at the end of recovery the Now-Observed-Adverse-Effect-Concentration (NOAEC) for the lung is based on histopathology and inflammatory marker 5 mg/m³ for the high surface area and the Low-Observed-Adverse-Effect-Concentration (LOAEC) for the low surface area SAS is 2.5 mg/m³.
The determination of the LOAEC for the low surface area SAS is a conservative approach due to the fact that it is based on minor morphological inflammatory changes only. These morphological changes were not accompanied by significant changes of inflammatory marker or any systemic effects. Therefore, these morphological findings can be evaluated as non-adverse since they can be considered as local physiological adaptive response to foreign material. At the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose -dependently and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the biosolubility of SAS reversibility of effects are demonstrated for both SAS grades examined. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance.

Executive summary:

Discussion and conclusion

The aim of this present study was to investigate the toxicity of two Synthetic Amorphous Silicas (High surface (BET) and low surface SAS in a 90-day nose-only inhalation study and to analyse effects also after an additional post-inhalation recovery period (up to 12 months); to establish exposure-dose-response relationships of the inhaled test item in rats after sub-chronic exposure and to use an experimental design adapted from OECD guideline 413 with additional endpoints (bronchoalveolar lavage, collagen content in lungs and lymph nodes).

Two-hundred twenty-five male and 225 female Wistar rats [strain Crl:WI (Han)] were used for this study and allocated to 9 treatment groups each: Clean air control, very low (0.5 mg/m³), low (1 mg/m³), mid (2.5 mg/m³) and high (5 mg/m³). The two test item aerosols were generated by dispersing the dry powder. Dispersion was achieved by a feeding system and pressurized air dispersion nozzle (dynamic system) developed by Fraunhofer ITEM (Koch, 1998). Using this system a mass median aerodynamic diameter (MMAD) of≤3 µm could be achieved as required by the ECHA decision. In the test item high dose groups a very slight lung overload situation (no volumetric overload, however, toxic impact due to surface chemistry) will be induced whereas in the low and mid dose groups the physiological lung clearance will not be retarded.

The observed differences in severity of the similar pathological effects are most likely caused by test substance differences (particle size distribution, surface area, number of silanols, density, volume, agglomeration status and biosolubility characteristics). At the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) was observed accompanied by corresponding changes of inflammatory markers in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in the bronchus-associated lymphoid tissues (BALT) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the varying biosolubility of SAS, reversibility of effects was demonstrated for both SAS grades examined.

These new studies with and low surface area pyrogenic SAS showed no lung fibrosis and accordingly no increase of collagen.

In association with lung clearance SAS accumulates in the regional lymph nodes associated with increase of lymph node size and inflammatory changes at the end of exposure. High surface SAS-exposed animals showed reversibility of lymph node effects in all affected dose groups at the end of recovery. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance.

The results of the lymph nodes are indicating that solubility of low surface SAS requires a longer period of time compared to high surface SAS. Final assessment of reversibility of lung lymph node effects for low surface SAS was not possible in this study. Further examinations of the lymph nodes are running to examine the lung lymph nodes of low surface SAS-exposed animals. SAS content in lymph nodes at the end of exposure will be compared with the results at the end of recovery by ashing and EDX examination and will be attached as amendment to this study report.

Indications for irreversible effects (lung fibrosis) have been published in literature and were based on study materials of one of the applicants' studies according to OECD TG 413 (Sub-chronic inhalation toxicity, Reuzel et al., 1987). A pathology working group reviewed this finding using the same (original) tissue slides according to the highest current standards and concluded the rapid clearance of the synthetic amorphous silica products will not lead to persistent inflammation and epithelial cell proliferation and therefore will not result in fibrosis/lung tumour induction (PWG Publication, Weber et al. 2018).The new studies performed on request by ECHA with high and low surface area pyrogenic SAS confirmed the PWG results and showed also no lung fibrosis and accordingly no increase of collagen.

Conclusion

Under the conditions of this test at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose-dependently and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the biosolubility of SAS reversibility of effects are demonstrated for both SAS grades examined. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance.

At the end of recovery the Now-Observed-Adverse-Effect-Concentration (NOAEC) for the lung is based on histopathology and inflammatory marker 5 mg/m³ for the high surface area and the Low-Observed-Adverse-Effect-Concentration (LOAEC) for the low surface area SAS is 2.5 mg/m³.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

experimental phase: Feb. 12, 2018 - May 29, 2019; study completion date: September, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2017

Deviations:

yes

Remarks:

This study was conducted according to an ECHA decision (dated June, 30 2017).

Principles of method if other than guideline:

Deviations from the guideline: This study will be conducted according to an ECHA decision (dated June, 30 2017). This decision skipped the endpoints clinical pathology and ophthalmology. Gross pathology and histopathology shall be conducted on the lungs, trachea, naso-pharyngeal tissues, nasal-associated lymphoid tissue (NALT) and larynx; other organs and tissues will be excluded from examination. As an addition, collagen will be analysed.

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

Test material 2
Name: SAS2 - Low BET; BET: Approx. 40-50 m²/g (low surface area); Purity: > 99.8%; Molecular formula: SiO2; Molecular weight: 60.08 g/mol;
CAS number: 7631-86-9, 112945-52-5; CAS name: Silica, amorphous, fumed, crystalline-free
EC number: 231-545-4; Date of delivery: January 4, 2018; Expiration date: December 10, 2019

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Male and female Wistar rats [strain Crl:WI(Han)] purchased from Charles River Deutschland (Sulzfeld, Germany).
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996) such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.

Sex:

male/female

Details on test animals or test system and environmental conditions:

For a period of at least 3 weeks prior to exposure animals were trained to become accustomed to nose-only tubes. The age of the animals at the start of exposure was approx. 8-9 weeks and the body weight approx. 250 gram for males and approx. 175 gram for females. Rats will be exposed to the test item by nose-only inhalation.
Animals were housed in Makrolon (polycarbonate) cages type IV, two rats of the same sex per cage, and were maintained under conventional laboratory conditions. Cages and absorbing softwood bedding material (Lignocel BK8-15) were changed twice a week or more often, if necessary. Tap water from the Hannover city water supplier was offered fresh weekly or more often, if necessary, in a Makrolon bottle fitted with a stainless steel nipple top with a hole approximately 0.5 mm in diameter. As diet a commercial chow in pellet form was used, identified as Ssniff V1534, purchased from Ssniff-Spezialdiäten (Soest, Germany). The diet was stored in room T1.015 at a temperature of max. 18 °C and a relative humidity of max. 70 %. No batch of diet was used after 6 months have elapsed from manufacturing date. The diet was offered fresh weekly or more often, if necessary.
In the cages the rats find paperboard crinklets as nesting material and wooden blocks for nibbling. The temperature and the relative humidity of the animal room was monitored electronically and recorded on a continuous basis. The limits was set at 22 °C ± 2 °C for temperature and 55 % ± 15 % for relative humidity. A 12-hour light/dark cycle was used controlled by an automatic timing device. The air exchange rate was at least 10 times per hour light/dark cycle was used controlled by an automatic timing device.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

<= 3 µm

Geometric standard deviation (GSD):

3.53

Remarks on MMAD:

Details on inhalation exposure:

The two test item aerosols were generated by dispersing the dry powder. Dispersion is achieved by a feeding system and pressurized air dispersion nozzle (dynamic system). For each nose-only exposure unit (total of 8 units for groups 2 - 9), the aerosol was generated by a pneumatic disperser. The disperser was fed with the test item under computerized control, i.e. with a feedback loop to a scattering signal from an aerosol photometer which reflects the actual aerosol concentration.
The aerosol was given to the rats by a flow-past nose-only inhalation exposure system which was used for previous particle and fiber inhalation studies at Fraunhofer ITEM. In this system, aerosols was supplied to each rat individually, and exhaled air is immediately exhausted. The airflow to each rat was approximately 1 L/min which is calculated to be laminar. Therefore measurement of the oxygen concentration is not necessary. Prior to the 90-day exposure of rats, technical trials to adjust particle size distributions and exposure levels was conducted.
For exposure to the test item the rats were restrained in acrylic tubes with a flexible stopper. The exposure tubes are arranged around a cylinder capable to take up 16 animals per level on four levels. The rat nose is located at the front end of a tube connected to the inner cylinder of the exposure unit delivering the aerosol. Through thin pipes, the aerosol is supplied to each rat nose individually and exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of individual rat at the cylinder is changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (including the clean air control: 9 units) are located each under a separate hood to prevent cross contamination among the different dose groups.
The duration of exposure will be 6 hours/day, 5 days/week for 13 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h / d
5 d / w

Dose / conc.:

0.5 mg/m³ air (nominal)

Dose / conc.:

1 mg/m³ air (nominal)

Dose / conc.:

2.5 mg/m³ air (nominal)

Dose / conc.:

5 mg/m³ air (nominal)

No. of animals per sex per dose:

10 (sacrified 1 day after treatment);
5 each (sacrified 90, 180 or 360 days after treatment) [cf. table 3 below, for details on dose groups]

Control animals:

yes, concurrent vehicle

Details on study design:

The test item were administered to the test animals by nose-only inhalation. This administration route is preferred compared to the intratracheal instillation route because it is the more physiological way of particle deposition. Nose-only inhalation is preferred to whole-body inhalation because it provides a specific model of inhalation exposure, restricting ingestion via grooming activity.
To specify adequate aerosol concentrations in this 90-day study, a preceding 90-day study (6 hrs/day on 5days/week) was used as a basis (Fraunhofer ITEM study # 02G11018; conducted with another SAS, i.e. NM-200; JRC code). Based on this study 0.5, 1.0, 2.5 and 5 mg/m³ were used for the test item in the very low, low, mid and high dose groups, respectively. Upon cessation of exposure, animals were investigated for various endpoints (see Table 3) on days 1, 90, 180 and 360 of a subsequent post-exposure observation period. Sacrifice dates at 180 and 360 days post-exposure may be skipped if no adverse findings will be observed after 90 or 180 days.

Observations and examinations performed and frequency:

Cf. also table 5 below for details on examinations!

Clinical Observations
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 includes 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. The observations were recorded daily with the PROVANTIS system. On the exposure days, the animals were clinically observed before, during and after exposure.

Body Weight
Individual body weights were recorded to the nearest 0.1 g twice a week for the first month and reduced to once a week throughout the remainder of the study provided there is no significant body weight effect in the first month (including post-exposure observation period) for all animals.
All body weight data were collected using electronic balances, interfaced with a computer and programmed for direct on-line data acquisition (Provantis).

Food Consumption
Food consumption were recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group.

Water Consumption
Water consumption were recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group.

Sacrifice and pathology:

Cf. also tables 5 and 6 below for details on examinations.

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 rats were anesthetized with an overdose pentobarbital sodium (Narcoren) and killed by cutting the vena cava caudalis.
The abdominal cavity were opened and the diaphragm were cut carefully allowing the lungs to collapse. Heart, oesophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN; mediastinal and tracheobronchial) will be removed from the lung convolution.
The lung and the lower half of the trachea were weighed and used for BAL (right lobes # 1-4) and histopathology (left lobe #5). For histopathology the left lung lobe # 5 were inflated under a pressure of about 20 cm water with formalin and were fixed by immersion for a minimum of two hours, and used for histopathology.
The following organs were trimmed and wet weights will be recorded:
Liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, thyroid, lung and heart. The respiratory tract were 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 in brackets and the seminal vesicles were prepared for histopathology. Tissues in brackets were preserved only.

Other examinations:

Bronchoalveolar Lavage (BAL)
Bronchoalveolar lavage were performed in 5 rats per time point and group, i.e. after end of exposure (day 1 of post-exposure) and after 3, 6 and 12 months of the post-observation period. The method of Henderson et al. (1987) were used with minor modifications.
Cytokines (e.g. IL-6, IL-8) were measured in the BAL of 5 animals per sex and group on day 1, 90, 180 and 360 post exposure.
Collagen were determined in lung tissue following an acidic hydrolysis with 37 % HCl. A hydroxyproline analysis was done according to the method of Creemers & Jansen (1997). Alternatively, the analysis could be done on lungs starting with a homogenisation of the lung tissue. The collagen analysis in BALF was done under non-GLP conditions.

Histopathology
Lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E). The following histopathological examinations were performed in 10 or 5 animals per sex and group after end of exposure:
• Histopathology on the respiratory tract in all animals of the clean air control group (dose group 1), the Synthetic Amorphous Silica high dose groups (dose groups 5 and 9) and of all animals that died or were killed moribund during the study. In the very low, low and mid dose groups (dose groups 2-4; 6-8), the respiratory tract may be analysed after the results in the high dose groups will have been completed.
• The respiratory tract includes lung lobes, with bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of all groups (groups 1, 5, 9). Other organs than the respiratory tract were included only if macroscopical findings occurred.
• Trimming of lungs: 3 sections; nose 4 sections.
For the animals sacrificed 3, 6 and 12 months post exposure, all tissues were preserved but only those showing lung changes on day 1 were examined histopathologically.

Statistics:

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Relevant statistically significant changes were not observed in the treatment groups as compared to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no relevant statistically significant changes as compared to concurrent controls

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

no relevant statistically significant changes as compared to concurrent controls

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Bronchoalveolar Lavage (BAL)
- Cytological and Biochemical Parameters
Mean values are shown in Figures 10-17 and Tables 43-58 in attached document BALF_Cytological and Biochemical Parameters.
At day 1 post-exposure statistically significant increases of PMN were detected in all dose groups of both sexes. Full recovery was detected in the very low dose group at 3 months, in the low dose group at 6 months and in the mid and high dose groups at 12 months post-exposure.
In the mid and high dose groups, statistically significant increases of lactic dehydrogenase (LDH), ß-glucuronidase (GLU) and total protein (TP) were observed at 1 and 90 days post-exposure; these effects returned to normalisation mostly at 6 and 12 months post-exposure.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

AEROSIL® OX 50 induced statistically significant increases of the absolute and relative lung wet weights in the low, mid and high dose groups at 1 day post-exposure (both sexes). Lung weights recovered at 3 months post-exposure; the high dose group only showed a persistent statistically significant increase at 6 and 12 months post-exposure.
All other statistically significant changes detected at other organs than lungs are considered as incidental findings.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In nasal cavities, the major lesions consisted of:
- Goblet cell proliferation in levels 1 and 2 and nasopharyngeal duct at the end of treatment and after 13 weeks recovery in all high surface SAS CAB-O-SIL S-17D and low surface SAS AEROSIL® OX 50 groups.
- Hyaline inclusions in olfactory mucosa at higher incidences and severity with increased incidences during the course of the study.
- Chitinase-positive crystals in olfactory mucosa in nasal cavity levels 2-4 up to 26-week recovery without any further injury in olfactory mucosa, mainly in CAB-O-SIL S-17D treated animals.

In lungs, the findings consisted of:
- End of treatment: discoloration or discoloured foci in lungs from animals treated at ≤ 1.0 mg/m3 AEROSIL® OX 50 associated with inflammatory lesions that increased in incidence and/or severity in test item-treated groups.
- increased perivascular infiltration in all AEROSIL® OX 50-treated groups.
- increased alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia dose-dependently for AEROSIL® OX 50 associated with interstitial inflammation, granulomas at the bronchio-alveloar junctions, granulomatous inflammation at a minor severity was noted in most animals from all other test item treated groups very low dose up to high dose (AEROSIL® OX 50).
- bronchio-alveolar hyperplasia in single animals from groups very low dose up to high dose of AEROSIL® OX 50.
- hyperplasia in the BALT in one group 6 male and one group 8 female (0.5 and 2.5 mg/m3 AEROSIL® OX 50).
- minimal macrophage agglomeration in the BALT in almost all animals treated with AEROSIL® OX 50 and granulomatous inflammation in the BALT in animals treated with AEROSIL® OX 50.
- reversible BALT fibrogenesis in all doses of AEROSIL® OX 50 with increasing incidence.
- 13 weeks recovery: foci in the lungs mainly in groups treated with AEROSIL® OX 50.
- increased perivascular infiltration in AEROSIL® OX 50-treated groups.
- increased in incidence and severity of alveolar macrophages in AEROSIL® OX 50-treated groups and macrophage aggregates in all groups treated with AEROSIL® OX 50.
- Increased incidence of macrophage type II hyperplasia and interstitial inflammation in AEROSIL® OX 50.
- Increased alveolar-bronchiolar hyperplasia in AEROSIL® OX 50-treated groups without clear dose-dependency.
- BALT macrophage agglomeration was noted in a few animals from AEROSIL® OX 50 groups associated with some cases of granulomatous inflammation. The latter caused fibrogenesis in the BALT in single cases of AEROSIL® OX 50-treated animals.
- Fibrogenesis due to inflammatory processes in most animals treated with AEROSIL® OX 50.
- 26 weeks recovery: similar findings than observed after 13 weeks recovery.
- 52 weeks recovery: still increased discoloured foci in lungs from animals at ≤ 1.0 mg/m3 in AEROSIL® OX 50-treated groups.
- AEROSIL® OX 50-treated groups: still a few inflammatory lesions present, mainly in animals ≤ 1.0 mg/m3.
- AEROSIL® OX 50-treated groups: no BALT inflammation present any longer, however, in a few animals at >1.0 mg/m3, there was a minimal BALT macrophage agglomeration.
- AEROSIL® OX 50-treated groups: inflammatory changes in the lungs at increased incidence in both sexes >1.0 mg/m3 AEROSIL® OX 50 due to still ongoing inflammatory processes.

In lymph nodes, the gross lesions consisted of:
- End of treatment: enlarged lymph nodes at increased incidences in animals at ≥0.5 mg/m3. After 13-, 26- and 52 weeks recovery, gross lesions were similar.
Histologically, the findings consisted of:
- End of treatment:
- granulomas in lymph nodes in all AEROSIL® OX 50 groups
- related granulomatous inflammation in a high number of animals from all groups
- lymphoid hyperplasia in most affected lymph nodes
- fibrogenesis in the lymph nodes from several animals from all AEROSIL® OX 50-treated groups, and fibrosis in one female at 0.5 mg/m3 AEROSIL® OX 50, and in both sexes at ≤ 1.0 mg/m3 AEROSIL® OX 50.
- 13 weeks recovery:
- Fibrogenesis at high incidence at minor severities in all groups.
- 26 weeks recovery:
- increased in incidence and severity of lymphoid hyperplasia in all groups and granulomas in all groups
- granulomatous inflammation and dose-dependent increased severity of fibrogenesis and fibrosis.
- 52 weeks recovery:
- granulomas or granulomatous inflammation
- increased incidence of lymphoid hyperplasia in animals at >1.0 mg/m3
- fibrogenesis or fibrosis in a few animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar Lavage (BAL)
- Cytological and Biochemical Parameters
At day 1 post-exposure statistically significant increases of PMN were detected in all dose groups of both sexes. Full recovery was detected in the very low dose group at 3 months, in the low dose group at 6 months and in the mid and high dose groups at 12 months post-exposure.
In the mid and high dose groups, statistically significant increases of lactic dehydrogenase (LDH), ß-glucuronidase (GLU) and total protein (TP) were observed at 1 and 90 days post-exposure; these effects returned to normalisation mostly at 6 and 12 months post-exposure.

Key result

Dose descriptor:

LOAEC

Effect level:

2.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: end of recovery; local lung effect; not substance specific, particle-related effect; no systemic effects. Minor morphological inflammatory changes were not accompanied by significant changes of inflammatory marker.

Remarks:

The determination of the LOAEC is a conservative approach due to the fact that it is based on minor morphological inflammatory changes only. These morphological changes were not accompanied by significant changes of inflammatory marker or any systemic effects. Therefore, these morphological findings can be evaluated as non-adverse since they can be considered as local physiological adaptive response to foreign material.

Dose descriptor:

LOEC

Effect level:

0.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: end of recovery; not substance specific, particle-related effect; no systemic effects

Remarks:

Dose descriptor:

LOAEC

Effect level:

0.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: end of exposure; not substance specific, particle-related effect; no systemic effects

Remarks:

Dose descriptor:

LOEC

Effect level:

0.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: end of exposure; not substance specific, particle-related effect; no systemic effects

Remarks:

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2.5 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Under the conditions of this test at the end of recovery the Now-Observed-Adverse-Effect-Concentration (NOAEC) for the lung is based on histopathology and inflammatory marker 5 mg/m³ for the high surface area and the Low-Observed-Adverse-Effect-Concentration (LOAEC) for the low surface area SAS is 2.5 mg/m³.

The determination of the LOAEC for the low surface area SAS is a conservative approach due to the fact that it is based on minor morphological inflammatory changes only. These morphological changes were not accompanied by significant changes of inflammatory marker or any systemic effects. Therefore, these morphological findings can be evaluated as non-adverse since they can be considered as local physiological adaptive response to foreign material. At the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. Test item related changes in lungs are dose -dependently and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the similar response directly after exposure (day 1). Based on the biosolubility of SAS reversibility of effects are demonstrated for both SAS grades examined. Most likely based on the lower solubility, low surface SAS-exposed animals showed still morphological inflammatory changes after 1-year recovery in all dose groups. These lymph node findings were not accompanied by any other findings up to a dose of 2.5 mg/m³ at the end of recovery. The lower lung and lymph node effect concentration of low surface SAS compared to high surface SAS is most likely related to the lower biosolubility of this SAS grade resulting in an extension of the lung clearance.

Executive summary:

Discussion and conclusion

These new studies with and low surface area pyrogenic SAS showed no lung fibrosis and accordingly no increase of collagen.

Conclusion

Reference 3

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Study period:: Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: assessment report
Remarks:: Pathology Working Group Review
Reason / purpose for cross-reference:: assessment report
Remarks:: Publication of results of the Pathology Working Group Review
Reason / purpose for cross-reference:: assessment report
Remarks:: Re-evaluation of the results of the Reuzel study 1987
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Principles of method if other than guideline:: Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline).
GLP compliance:: yes
Limit test:: no
Specific details on test material used for the study:: synthetic amorphous silicon dioxide Aerosil 200
Species:: rat
Strain:: Wistar
Details on species / strain selection:: obtained from: Central Institute for Breeding of Laboratory Animals TNO, Zeist / NL
Sex:: male/female
Details on test animals or test system and environmental conditions:: 4 weeks old, (at study initiation) 50 - 70 g, individually housed, 21-23 °C, generally between 65-75% humidity, institute's stock diet and unflouridated tap water ad libitum except during exposure, 12 h light/dark
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: whole body
Vehicle:: air
Remarks on MMAD:: no monitoring data due to technical difficulties
Details on inhalation exposure:: - Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m³
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Air flow rate: approx. 40 m³/h
- Air change rate: 40 / 2.3 = ~17/h
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: daily mean concentrations are documented:
based on 250 - 253 measurements: 1.26 (SEM 0.08) mg/m³; 5.88 (SEM 0.88) mg/m³; 31.04 (SEM 0.87) mg/m³
Duration of treatment / exposure:: 13 weeks
Frequency of treatment:: 6 hours/day, 5 days/week
Dose / conc.:: 1.3 mg/m³ air (analytical)
Remarks:: target concentration: 1 mg/m³
Dose / conc.:: 5.9 mg/m³ air (analytical)
Remarks:: target concentration: 6 mg/m³
Dose / conc.:: 31 mg/m³ air (analytical)
Remarks:: target concentration: 30 mg/m³
No. of animals per sex per dose:: 70
Control animals:: yes
Details on study design:: - Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- 50 rats per sex were kept for a recovery period of up to 52 weeks. Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks
Positive control:: Quartz (crystalline silica, 58 mg/m³)
Observations and examinations performed and frequency:: daily: clinical signs, behaviour
body weight (at start and weekly; every 4 weeks in post observation)
Sacrifice and pathology:: in weeks 14, 27, 41, 53 and 66: gross pathological examination
histopathological examination: nose, larynx, trachea, hilus, mediastinal lymph notes, lungs
Other examinations:: haematology, blood chemistry, urine (before necropsy)
Statistics:: Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: dry coat due to animals not learning rapidly enough to drink from the automatic watering system
Mortality:: mortality observed, non-treatment-related
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: decrease affecting males of mid and high dose groups; males of low dose group were only affected during the first half of exposure;
isolated increases in females of all dose groups
Food consumption and compound intake (if feeding study):: not specified
Food efficiency:: not specified
Water consumption and compound intake (if drinking water study):: not specified
Ophthalmological findings:: not examined
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: - higher red blood cell counts in males of high dose group, but recovery after c. 13 weeks post observation
- higher white blood cell counts in males and females of mid and high dose groups and increased numbers of neutrophilic leucocytes in females of low dose group, but both effects disappeared after c. 13-39 weeks post observation
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: Males of high dose group exhibited lower glucose values at the end of exposure.
Urinalysis findings:: effects observed, non-treatment-related
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: increase of absolute and relative lung weights in mid and high dose groups (m,f), also weights tended to be higher in rats of low dose; effects subsided in low and mid dose groups after 13 and 26 weeks post observation, respectively
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: All test animals showed swollen and spotted lungs, having a spongy consistency and/or irregular surface, and enlarged mediastinal lymph nodes; these effects disappeared after 26 weeks post obervation
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: - accumulation of alveolar macrophages, subsided within 39 weeks in low dose group, mostly subsided within 52 weeks in mid dose group, but not subsided in males and most females of high dose group.
- accumulation of granular material, cellular debris and polymophonuclear leucocytes only in three animals of high dose group
- increased septal cellularity present in 70% of rats of high dose group after 52 weeks of recovery, not found in rats of mid and low dose groups after 39 and 26 weeks, respectively
- alveolar bronchiolization seen in mid and high dose groups, but completely subsided in mid dose group after 26 weeks and which was present only to a minimal degree in high dose group after 52 weeks recovery
- fibrogenesis was not observed in the 1 mg/m3 pyrogenic Aerosil 200 group. Fibrogenesis was noted directly after the exposure period, but decreased in severity and frequency parallel with the collagen content until the end of the study, clearly indicating that fibers observed are due to reversible fibrogenesis and not irreversible fibrosis. This is confirmed by re-examination of the lungs by pathology working group (PWG). See results [PWG-] and [publication-XXXXX2018]).
- mediastinal lymph nodes showed slight to servere accumulations of macrophages in all test groups, which had disappeared after 39 weeks
Histopathological findings: neoplastic:: no effects observed
Details on results:: BODY WEIGHT AND WEIGHT GAIN
No effect in females at all dose levels (Tab. 6)
Depressive effect in males:
1 mg/m3: slightly at day 14 of exposure only (~ -5%)
6 mg/m3: slightly from day 49 to day 77 of exposure (~ - 6 to <5 %)
no more significant by end of exposure (day 91)
30 mg/m3: significantly throughout exposure: ~ -7 - -10 %, day 91: -7 %
Recovery: no difference from control at day 455 (52 weeks post-exposure)

HAEMATOLOGY
1 mg/m3: no effects
6 mg/m3: White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
but concentration-response relationship was poor.
After 3 months recovery, these blood parameters normalized in males and females.
30 mg/m3: Red blood cell count and hemoglobin were statistically higher in males, but not in females.
White blood cell count elevated in both males and females due to increases in the numbers of neutrophilic leukocytes,
at 3 months of recovery (days 176/177, Table 8/Table 13), but concentration-response relationship was poor.
In females, a slight increase above the control group apparently still existed after 6 months of recovery (day 275, Table 14).

CLINICAL CHEMISTRY
no significant effects

URINALYSIS
no significant effects

ORGAN WEIGHTS
No changes in heart, thyroid, thymus, adrenals, testes, brain, spleen, kidney
Treatment-related degrees of severity: swollen lungs and enlarged mediastinal lypmph nodes at the end of exposure
LUNG
1 mg/m3: no significant increase
6 mg/m3: mean increase in relative weight 1.7x (males), 1.4x (females)
30 mg/m3: mean increase in relative weight 2.3x (males), 2.0x (females)
LYMPH NODE: no weight data

PATHOLOGY
Swollen and spotted lungs and enlarged mediastinal lymph nodes, the degree of severity being treatment-related.
Mean absolut collagen content in lungs of male animals showed no effects in the 1 mg/m3 group, whereas in association with inflammation in the 6mg/m3 and 30mg/m3 group a significant increase was noted in the first recovery periode up to day 370. However, at the end of recovery no significant differences to control values were observed. The same picture was observed in female animals regarding absolute collagen content in lungs. Only the 30 mg/m3 female group showed still at the end of the recovery time (day 263) significant increase of collagen content. However, the results in this group are not plausible due to high variation of the values. Between day 297 and day 370 there was a decrease of 43.251 mg to 38.915 mg but between day 370 and day 462 the value of the collagen content started to increase again to 46.315 mg. Looking at the mean collagen content in lungs expressed relative to body weights showed also in this females no significant difference to control. Mean collagen contents in lungs expressed relative to body weight of all male and female dose groups shows no significant difference to control. Whereas crystalline Quartz exposed animals showed no reversibility of absolute and relative collagen content.
These results clearly indicate that increase of collagen in SAS exposed animals is associated with inflammation and reversible fibrogenesis and not with irreversible fibrosis as observed in quartz exposed animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
Accumulation of alveolar macrophages and granular material, cellular debris, polymorphonuclear leucocytes, increased septal cellularity, alveolar bronchialisation, cholesterol clefts, granuloma-like lesions and focal interstitial fibrosis were described in the lung. The term interstitial fibrosis was used in the original report, but need to be replaced by fibrogenesis which is reversible and associated with inflammation. The above shown reversibility of the collagen content at the end of recovery to normal control values confirm reversible fibrogenesis. (Focal interstitial fibrosis was not confirmed by re-examination of the lungs by pathology working group (PWG). See results [PWG-XXXXX] and [publication-XXXXX2018]).
Fibrogenesis was not observed in the 1 mg/m3 pyrogenic Aerosil 200 group. Fibrogenesis was noted directly after the exposure period, but decreased in severity and frequency parallel with the collagen content until the end of the study, clearly indicating that fibers observed are due to reversible fibrogenesis and not irreversible fibrosis.

Granuloma-like lesions were seen in a few animals at the end of exposure period and after 13 weeks of recovery. They did not show fibroblastic activity and hyalinization and regressed during recovery [not progressive, i.e. no silicogenic nodules formed (no silicosis)].

Accumulation of macrophages were seen in the mediastinal lymph nodes (disappeared after wk 39 post-exposure).

Treatment-related microscopic changes in the nasal region were occasionally found at the end of exposure period, such as focal necrosis and slight atrophy of the olfactory epithelium.

All types of pulmonary lesions were more marked in males than in females.

The level of 1.3 mg/m3 induced only slight changes after 13-wk exposure (Table 68), which generally recovered quickly (no differences from control after 13-wk post-exposure: Table 69).

Morphological changes after 13-wk exposure, that were considered statistically significant at 1.3 mg/m3 (Table 68):

males females ! males females
treated ! untreated controls
------------------------------------------------------------------------------------------------------------------------------
Accumulation of alveolar macrophages: slight in 10/10 (very) slight in 10/10 ! (very) slight 4/10 slight in 1/10
Intra-alveolar polymorphonuclear !
leukocytes: (very) slight in 6/10 (very) slight in 8/10 ! 0/10 0/10
Increased septal cellularity: ( very) slight in 10/10 (very) slight in 9/10 ! very slight 1/10 very slight 1/10
Olfactory epithelial atrophy: (very) slight in 5/10 (very) slight in 8/10 ! 0/10 0/10
Intracytoplasmic proteinaceous droplets !
-respiratory epithelium: in 8/10 in 9/10 ! 1/10 0/10
Mediastinal lymph node !
-macrophage accumulation: (very) slight in 8/10 (very) slight in 8/10 ! 0/10 0/10
------------------------------------------------------------------------------------------------------------------------------

These morphological changes are physiological response to foreign materials (particles) in the lung.

HISTOPATHOLOGY: NEOPLASTIC
No particular findings

HISTORICAL CONTROL DATA (if applicable): no data

OTHER FINDINGS - SILICA DEPOSITION
Silica could be detected in lungs only in relatively small amounts at the end of the exposure period (Tables 59):
on the average 0.1 - 0.2 mg per lung of male animal groups (not dose-related), 0.05 - 0.21 mg per lung of female groups (dose-related).
Only one male exposed to 30 mg/m3 showed a small amount of silica in the regional lymph node.
90 days after termination of exposure (day 188), no silica could be recovered from any animal.
(see Chapter 7.1: Degussa 87-0004-DGT_ Aero200_inhal_Si-deposition, 13 wk, rat_key_RL2)
: Key result
Dose descriptor:: NOAEC
Effect level:: > 1.3 - < 5.9 mg/m³ air (analytical)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: respiratory system: lower respiratory tract
Remarks on result:: other: End of exposure: NOAEC ca. 1.3 mg/m3 based on physiological response/adaptation to foreign material (particles). Considering reversibility in recovery: NOAEC > 1.3 mg/m3 - < 5.9 mg/m3. Reversibility of lung effects demonstrated after one year recovery.
Remarks:: Recovery is clearly demonstrated after one year. The lung showed no differences compared with control indicating a NOAEC above 1.3 mg/m3. According to Reuzel et al. 1991: NOAEC < 17 mg/m3. No substance specific, particle-related effects; no systemic effects
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1.3 mg/m³ air (nominal)
System:: respiratory system: lower respiratory tract
Organ:: lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: Aerosil 200 at a level of 31 mg/m³ (nominal: 30 mg/m³) induced servere changes mainly in the lungs and lung associated tissues (lymph nodes), which recovered during the one-year observation period.
Aerosil 200 at a level of 5.9 mg/m³ (nominal: 6 mg/m³) induced similar, but less servere changes. Most of the changes recovered during the one-year observation period.
Aerosil 200 at a level of 1.3 mg/m³ (nominal: 1 mg/m³) induced only slight changes, which generally recovered quickly.
End of exposure: NOAEC ca. 1.3 mg/m³ (nominal: 1 mg/m³) based on physiological response/adaptation to foreign material (particles). Recovery is cleary demonstrated in the study indicating an NOAEC above 1.3 mg/m³ (nominal: 1 mg/m³).
Indications for irreversible effects (lung fibrosis) have been published in literature and were based on study materials of one of the applicants' studies according to OECD TG 413 (studies E-87-0004 (Aerosil 200), (R 974), and (Sipernat 22S)). A pathology working group reviewed this finding using the same (original) tissue slides according to the highest current standards and concluded the clearance of the synthetic amorphous silica products will not lead to persistent inflammation and epithelial cell proliferation and therefore will not result in fibrosis/lung tumor induction ( PWG Publication 2018).
Executive summary:: A 3-month inhalation experiment followed by a 52-week recovery period was conducted with pyrogenic (Aerosil 200), precipitated (Sipernat 22 S) and surface-treated pyrogenic (Aerosil R 974) synthetic amorphous silica. The overall picture of these SAS grades is similar. At the end of exposure particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed, in the higher dose group accompanied by corresponding changes of hematological parameter (Red blood cell count, hemoglobin and white blood cell count). These effects are not substance-specific, all respirable particles will show the same response. Based on the biosolubility of SAS reversibility of effects is demonstrated in recovery groups in contrast to e.g. quartz exposed animals were no recovery was observed. SAS is not a poorly soluble particle (PSP). Therefore, in context with "lung overload" described long-term lung effects of low toxic PSPs are not relevant for SAS (Pathology working group (PWG) review of a sub-chronic (13-week) inhalation toxicity study of aerosols of AEROSIL® 200, AEROSIL® R 974, SIPERNAT® 22 S and quartz in rats). Irreversible lung fibrosis is not associated with sub-chronic exposure to SAS (Weber et al. 2018, “Aerosols of synthetic amorphous silica do not induce fibrosis in lungs after inhalation: Pathology working group review of histopathological specimens from a sub-chronic 13-week inhalation toxicity study in rats”. Toxicology Research and Application 2: 1–17). These results were confirmed by the new studies requested by ECHA with high and low surface area pyrogenic SAS which showed no lung fibrosis and accordingly no increase of collagen (90-day inhalation toxicity study at Fraunhofer ITEM, 2019).

Reference 4

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Study period:: Exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: assessment report
Remarks:: Pathology Working Group Review
Reason / purpose for cross-reference:: assessment report
Remarks:: Publication of results of the Pathology Working Group Review
Reason / purpose for cross-reference:: assessment report
Remarks:: Re-evaluation of the results of the Reuzel study 1987
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Principles of method if other than guideline:: Comparative study including Aerosil 200, Aerosil R 974 (pyrogenic, hydrophobic), Sipernat 22S (precipitated, hydrophilic) as well as quartz (crystalline).
GLP compliance:: yes
Limit test:: yes
Specific details on test material used for the study:: Sipernat 22S
Species:: rat
Strain:: Wistar
Details on species / strain selection:: obtained from: Central Institute for Breeding of Laboratory Animals TNO, Zeist / NL
Sex:: male/female
Details on test animals or test system and environmental conditions:: 4 weeks old, (at study initiation) 50 - 70 g, individually housed, 21-23 °C, generally between 65-75% humidity, institute's stock diet and unflouridated tap water ad libitum except during exposure, 12 h light/dark
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: whole body
Vehicle:: air
Remarks on MMAD:: MMAD / GSD: no monitoring data due to technical difficulties
Details on inhalation exposure:: - Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m³
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Air flow rate: approx. 40 m³/h
- Air change rate: 40 / 2.3 = ~17/h
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Daily mean concentrations are documented:
based on 254 measurements: 34.91 (SEM 0.49) mg/m³
Duration of treatment / exposure:: 13 weeks
Frequency of treatment:: 6 hours/day, 5 days/week
Dose / conc.:: 34.9 mg/m³ air (analytical)
Remarks:: target concentration: 30 mg/m³
No. of animals per sex per dose:: 70
Control animals:: yes
Details on study design:: - Dose selection rationale: based on range findings (14 d)
- Rationale for selecting satellite groups: post-exposure recovery period for examination of reversibility of effects
- 50 rats per sex were kept for a recovery period of up to 52 weeks. Post-exposure recovery period in satellite groups: 13, 26, 39, and 52 wks
Positive control:: Quartz (crystalline silica, 58 mg/m³)
Observations and examinations performed and frequency:: daily: clinical signs, behaviour
body weight (at start and weekly; every 4 weeks in post observation)
Sacrifice and pathology:: in weeks 14, 27, 41, 53 and 66: gross pathological examination
histopathological examination: nose, larynx, trachea, hilus, mediastinal lymph notes, lungs
Other examinations:: haematology, blood chemistry, urine (before necropsy)
Statistics:: Body weights: analysis of co-variance followed by Dunnett´s test
Histopathological changes and mortality: Fisher´s exact probability test
Organ weights, blood parameter: analysis of variance and Dunnett´s test
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: Initially, a number of animals, scattered over the groups, did not rapidly learn to drink from the automatic watering system of the inhalation chamber. Due to dehydration these animals showed transiently a dry coat.
Mortality:: mortality observed, non-treatment-related
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: In females exposed to Sipernat 22S body weights were generally lower them those of the controls but not always to a statistically significant extent. During the observation period the differences with controls quickly disappeared.
Statistically significantly lower body weight figures were observed during the exposure period and first months of the observation period in males. After 13 weeks of recovery the differences with the controls were no longer statistically significant.
Food consumption and compound intake (if feeding study):: not specified
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not specified
Ophthalmological findings:: not examined
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: At the end of the exposure period, males exposed to Sipernat 22S exhibited lower prothrombin times than did the controls. Animals of this, group did not show clearly higher white blood cell counts, despite the increased number of neutrophilic leucocytes in both sexes at the end of the exposure period. This increase was still seen in males after an observation period of c. 39 weeks.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: High mean glutamic-oxalacetic transaminase activity figures were shown in females exposed to Sipernat 22S both after an observation period of c. 26 and 39 weeks. This was due to high values in a few individual rats, which is quite normal at this age.
After an observation period of c. 39 weeks the inorganic phosphate figure was statistically significantly higher in exposed females than in controls. Because the increase was rebound at the end of the observation period, no toxicological significance was attached to that high value.
Urinalysis findings:: effects observed, non-treatment-related
Description (incidence and severity):: Lower urine content at the end of exposure (f) attributed to lower water intake
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: Lung weights in both males and females exposed to Sipernat 22S were only slightly, but statistically significantly, increased at the end of the exposure period. After an observation period of 13 weeks the lung weights were comparable with the control values.
Relative thymus weights were increased in males only at the end of the exposure period.
The absolute thymus weights were higher in females than in controls at the end of the observation period. Since the relative thymus weight did not show any significant difference with the controls, no toxicological significance was attached to this higher absolute thymus weight.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: All test animals showed swollen and spotted lungs, having a spongy consistency and/or irregular surface, and enlarged mediastinal lymph nodes; these effects disappeared after 26 weeks post obervation.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: - Accumulation of alveolar macrophages was still present to a significant degree after an observation period of 26 weeks, but after an observation period of 39 weeks there was no longer a statistically significant difference in incidence between test and control group.
- Accumulation of intra-alveolar polymorphonuclear leucocytes and increased septal cellularity; disappeared during an observation period of 13 weeks.
- The mediastinal lymph node changes comprised accumulations of macrophages. This finding was still present after an observation period of 13 weeks but to a very slight degree and the difference with the controls was not statistically significant. One or two animals of this group demonstrated (very) slight accumulations of macrophages during the whole observation period of 52 weeks.
Histopathological findings: neoplastic:: no effects observed
Other effects:: effects observed, treatment-related
Description (incidence and severity):: Collagen figures were only slightly higher in males and females exposed to Sipernat 22S than in controls and not always to a statistically significant degree.
Dose descriptor:: NOAEC
Effect level:: < 46 mg/m³ air (analytical)
Sex:: male/female
Remarks on result:: other: NOAEC taken from publication Reuzel et al. 1991. Based on dose range finding study not confirmed in this sub-chronic study. Behind this background questionable.
Remarks:: not substance specific, particle-related effect; no systemic effects
Critical effects observed:: yes
Lowest effective dose / conc.:: 34.9 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: lungs
Treatment related:: yes
Dose response relationship:: not specified
Relevant for humans:: yes
Conclusions:: Sipernat 22S induced changes which were similar to those of Aerosil 200, but they were rather mild in comparison to those of 31 mg Aerosil 200. In addition the changes quickly recovered, though silicon could still be detected in the lungs after 26 weeks of recovery, and in the lymph nodes even at the end of the observation period.
Indications for irreversible effects (lung fibrosis) have been published in literature and were based on study materials of one of the applicants' studies according to OECD TG 413 (studies E-87-0004 (Aerosil 200), (R 974), and (Sipernat 22S). A pathology working group reviewed this finding using the same (original) tissue slides according to the highest current standards and concluded the rapid clearance of the synthetic amorphous silica products will not lead to persistent inflammation and epithelial cell proliferation and therefore will not result in fibrosis/lung tumor induction (PWG Publication 2018).
Executive summary:: A 3-month inhalation experiment followed by a 52-week recovery period was conducted with pyrogenic (Aerosil 200), precipitated (Sipernat 22 S) and surface-treated pyrogenic (Aerosil R 974) synthetic amorphous silica. The overall picture of these SAS grades is similar. At the end of exposure particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed, in the higher dose group accompanied by corresponding changes of hematological parameter (Red blood cell count, hemoglobin and white blood cell count). These effects are not substance-specific, all respirable particles will show the same response. Based on the biosolubility of SAS reversibility of effects is demonstrated in recovery groups in contrast to e.g. quartz exposed animals were no recovery was observed.SAS is not a poorly soluble particle (PSP). Therefore, in context with "lung overload" described long-term lung effects of low toxic PSPs are not relevant for SAS (Pathology working group (PWG) review of a sub-chronic (13-week) inhalation toxicity study of aerosols of AEROSIL® 200, AEROSIL® R 974, SIPERNAT® 22 S and quartz in rats). Irreversible lung fibrosis is not associated with sub-chronic exposure to SAS (Weber et al. 2018, “Aerosols of synthetic amorphous silica do not induce fibrosis in lungs after inhalation: Pathology working group review of histopathological specimens from a sub-chronic 13-week inhalation toxicity study in rats”. Toxicology Research and Application 2: 1–17). These results were confirmed by the new studies requested by ECHA with high and low surface area pyrogenic SAS which showed no lung fibrosis and accordingly no increase of collagen (90-day inhalation toxicity study at Fraunhofer ITEM, 2019).

Reference 5

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: other: expert's review of a study
Adequacy of study:: other information
Study period:: (original study) exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985; re-evaluation in 2016
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Qualifier:: no guideline required
Principles of method if other than guideline:: This is not a study, but a review by a pathology working group (PWG) of one. (cf. the endpoint of the study for futher details)
GLP compliance:: no
Remarks:: This review does not constitute a study. Current Good Laboratory Practice Standards do not apply to this report, but all appropriate documentation of the review process has been maintained. The original study complied with GLP Standards.
Specific details on test material used for the study:: Aerosil 200, Aerosil R 974, Sipernat 22 S
Species:: rat
Strain:: Wistar
Details on species / strain selection:: obtained from: Central Institute for Breeding of Laboratory Animals TNO, Zeist / NL
Sex:: male/female
Details on test animals or test system and environmental conditions:: 4 weeks old, (at study initiation) 50 - 70 g, individually housed, 21-23 °C, generally between 65-75% humidity, institute's stock diet and unflouridated tap water ad libitum except during exposure, 12 h light/dark
From the necropsy at the end of 13-week treatment, only sections from males were still available!
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: whole body
Vehicle:: air
Remarks on MMAD:: no monitoring data due to technical difficulties
Details on inhalation exposure:: - Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m³
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Air flow rate: approx. 40 m³/h
- Air change rate: 40 / 2.3 = ~17/h
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 13 weeks
Frequency of treatment:: 6 hours/day, 5 days/week
Dose / conc.:: 1.3 mg/m³ air (analytical)
Remarks:: Aerosil 200
Dose / conc.:: 5.9 mg/m³ air (analytical)
Remarks:: Aerosil 200
Dose / conc.:: 31 mg/m³ air (analytical)
Remarks:: Aerosil 200
Dose / conc.:: 34.7 mg/m³ air (analytical)
Remarks:: Aerosil R 974
Dose / conc.:: 34.9 mg/m³ air (analytical)
Remarks:: Sipernat 22S
Control animals:: yes, sham-exposed
Details on study design:: The original experimental design is summarized in the spaces above and attached Table 1. From the necropsy at the end of 13-week treatment, only sections from males were still available.
Positive control:: quartz (58.5 mg/m³)
Other examinations:: All non-neoplastic findings were graded on a severity scale from 1 to 5 (minimal, slight, moderate, marked, and severe).
Statistics:: For group comparison, mean grades on lesions were calculated by the PathData System as sum of grades in groups divided by the number of affected individuals. A Fisher’s exact test was performed on all data. The data were evaluated at a significance level of p < 0.05 and p < 0.1. The trend test for nonneoplastic lesions (Armitage test) was applied comparing the findings after the 52-week recovery sacrifice, between controls and AEROSIL® 200 groups, controls and AEROSIL® R 974 groups, and controls and SIPERNAT® 22 S groups.
Details on results:: Results are not presented here. This is only an experts' review on results of an older study and its re-evaluation
Conclusions:: The Pathology Working Group concluded that unlike the changes induced in the lungs with Quartz (crystalline silica), changes induced following 13 weeks of exposure to synthetic amorphous silica products (1.3 and 5.9 mg/m³ Aerosil® 200; 34.7 mg/m³ Aerosil® R 974 and 34.9 mg/m³ Sipernat® 22S) were reversible following 52 weeks of recovery. At the end of the recovery period at the highest concentration of 31.0 mg/m³ Aerosil® 200, a minimal increase in alveolar macrophages and interstitial inflammation was observed. There was no significant difference compared to control animals except for these reactive changes.
Executive summary:: This is an experts' review on results of an older study and its re-evaluation. It focuses on three questions and their answers provided here in sufficient detail. The original study and its re-evaluation together with their results are not documented here, but have their own endpoints.

Reference 6

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

other: published experts' review of a study

Adequacy of study:

supporting study

Study period:

(original study) exposure: 20 Jul. 1984 - 19 Oct. 1984 / end observation: 15 Oct. 1985; re-evaluation in 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline required

Principles of method if other than guideline:

This is not a study, but a review by a pathology working group (PWG) of one. (cf. the endpoint of the study for futher details)

GLP compliance:

Remarks:

This review does not constitute a study. Current Good Laboratory Practice Standards do not apply to this report, but all appropriate documentation of the review process has been maintained. The original study complied with GLP Standards.

Specific details on test material used for the study:

Aerosil 200, Aerosil R 974, Sipernat 22S

Species:

rat

Strain:

Wistar

Details on species / strain selection:

obtained from: Central Institute for Breeding of Laboratory Animals TNO, Zeist / NL

Sex:

male/female

Details on test animals or test system and environmental conditions:

4 weeks old, (at study initiation) 50 - 70 g, individually housed, 21-23 °C, generally between 65-75% humidity, institute's stock diet and unflouridated tap water ad libitum except during exposure, 12 h light/dark
From the necropsy at the end of 13-week treatment, only sections from males were still available!

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

no monitoring data due to technical difficulties

Details on inhalation exposure:

- Exposure apparatus: stainless steel exposure chamber, multitiered (manufactered by Hazelton)
- Exposure chamber volume: 2.3 m³
- Method of holding animals in test chamber: single
- Source and rate of air: Aerosol entrance at top of the chamber
- System of generating particulates/aerosols: Institute´s dust generator with compressed air operating atomizer
- Air flow rate: approx. 40 m³/h
- Air change rate: 40 / 2.3 = ~17/h

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

1.3 mg/m³ air (analytical)

Remarks:

Aerosil 200

Dose / conc.:

5.9 mg/m³ air (analytical)

Remarks:

Aerosil 200

Dose / conc.:

31 mg/m³ air (analytical)

Remarks:

Aerosil 200

Dose / conc.:

34.7 mg/m³ air (analytical)

Remarks:

Aerosil R 974

Dose / conc.:

34.9 mg/m³ air (analytical)

Remarks:

Sipernat 22 S

Control animals:

yes, sham-exposed

Details on study design:

The original experimental design is summarized in the spaces above and attached Table 1. From the necropsy at the end of 13-week treatment, only sections from males were still available.

Positive control:

quartz (58.5 mg/m³)

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

s. below for details ("any other information on results")

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

s. below for details ("any other information on results")

Fibrosis/fibrogenesis

Fibrosis was not observed after the 13-week main test period, and after the 13-week recovery period. At the latter

sacrifice time point, in two animals of the high-dose AEROSIL ® 200 group, a minimal fibrogenesis (not statistically

significant in the Fisher’s test) was noted. After the 52-week recovery period, a minimal focal fibrosis was detected only in one animal from the control group, one animal from each of the low- and mid-dose AEROSIL® 200–treated animals, and in three animals of the high-dose AEROSIL® 200–treated animals. No fibrosis was present in AEROSIL® R 974 and SIPERNAT® 22 S–treated groups. Hence, it appears that aside from somereactive changes including the presence of alveolar macrophages and macrophage aggregations in high-dose AEROSIL® 200 animals, there was no significant difference compared to the control animals.

In contrast, a high level of multifocal fibrosis was recorded in quartz-treated animals after the 52-week recovery period. In addition, the pleura was affected showing a multifocal pleural mesothelial hyperplasia in a high portion of the animals.

Thirteen-week terminal sacrifice

At the 13-week sacrifice (Table 3), the lesions did not differ in sections evaluated from males only, between all test

item–treated groups. In control animals, there was interstitial inflammation in controls which was lower in incidence

than in AEROSIL® 200-treated animals (Figure 1).

Induced lesions consisted mainly of the presence of foreign material appearing in a dose-dependent manner in

AEROSIL® 200-treated groups; increasing severity of alveolarmacrophages; macrophage aggregations (low severity

in the SIPERNAT® 22 S-treated group); reactive pneumocyte macrophage type II hyperplasia; and granulomatous

inflammation and the presence of alveolar/bronchiolar junctional granulomas (highest incidence and severity in the

AEROSIL® R 974-treated group), and cleft granulomas.

The incidence and severity between the AEROSIL® 200 high-dose and AEROSIL® R 974 and SIPERNAT® 22 S–

treated groups was similar, except for cleft granulomas and a lower incidence of junctional granulomas (lowest incidence

in SIPERNAT® 22 S-treated animals, highest incidence in AEROSIL® R 974-treated animals). Alveolar/bronchiolar

epithelial hyperplasia was not dose-related, but appeared in all test item–treated groups. Furthermore, there were isolated cases of hyperplasia and of reactive macrophages in the bronchiolar associated lymphoid tissue (BALT) in AEROSIL ® 200-treated animals only (Figures 2 to 7).

Thirteen-week recovery sacrifice

In the 13-week recovery animals (Tables 4 and 5), all findings noted after 13 weeks of treatment were still present

although mostly appearing at lower severities. From this sacrifice onward, there were also sections from quartztreated

animals available.

For induced findings, there were no distinctive differences between both sexes. Foreign material in the alveoli was almost not visible any longer except for quartz-treated animals. Perivascular inflammation appeared at similar incidences

and severities in all groups, besides the quartz group, where the incidence and severity was recorded at higher

levels. Alveolar macrophages increased in incidence and severity, and macrophage aggregations were recorded in all

test item–treated groups. Pneumocyte type II hyperplasia was present in the mid- and high-dose AEROSIL® 200 and

the SIPERNAT® 22 S groups, and also in the quartz group. Interstitial inflammation was distributed randomly throughout the groups at higher incidences than in controls. Granulomatous inflammation was recorded at low severity degrees in test item–treated groups only. There were single cases of cleft granulomas in animals of the mid- and high-dose AEROSIL® 200 groups. Granulomas at the bronchiolar/alveolar junction were present in the test item–treated

groups, except for the low-dose AEROSIL® 200 group. The latter finding was of minimal severity apart from the AEROSIL ® R 974 and quartz–treated animals where it was recorded as moderate or severe (Figures 7 to 13).

Fibrogenesis, accompanying inflammatory processes, was diagnosed in two males of the high-dose AEROSIL ® 200 group (Figure 10). In addition, a finding not observed after the 13-week terminal sacrifice was noted, that is, a brownish pigment in alveolar macrophages in the alveoli of some animals from both sexes accumulated in the mid- and high-dose AEROSIL® 200 and SIPERNAT® 22 S–treated groups.

In quartz-treated animals, findings present at a higher severity compared to other groups consisted of foreign material (Figure 13), perivascular inflammation, alveolar macrophages (females only), granulomas at the bronchial alveolar junction, and granulomatous inflammation. Moreover, there was a clear affection of the BALT, that is, hyperplasia and accumulation of reactive macrophages.

Fifty-two-week recovery sacrifice

After the planned 52-week recovery (Tables 6 and 7), in controls, the number of inflammatory and reactive lesions

increased compared to previous sacrifices probably owing to age-related changes (Figures 14 and 15).

In test item–treated groups, all findings recorded in AEROSIL® 200, AEROSIL® R 974, and SIPERNAT® 22 S–treated males were similar to control animals (Figure 16), including one case of focal alveolar/bronchiolar hyperplasia (Figures 17 and 18). In females of the high-dose AEROSIL® 200 group, there was still a higher incidence of reactive changes. However, these findings were similar to control lesions that may represent age-related changes (Figure 19). A focal minimal pleural fibrosis was recorded in one animal per sex of the mid- and high-dose AEROSIL ® 200 (Figure 20).

In contrast, the quartz-treated animals showed severe intra-alveolar foreign material, associated with a high incidence

and severity of reactive alveolar macrophages, macrophage aggregations, the formation of alveolar/ bronchiolar junctional granulomas and especially cleft granulomas, as well as granulomatous inflammation, pneumocyte type II hyperplasia, multifocal fibrosis, and macrophage aggregates affecting the BALT. In addition, a high number of males, but less of females was affected by mesothelial hyperplasia (Figures 21 to 24). Additionally, there was one cystic keratinizing epithelioma in the quartztreated animals after 52 weeks of recovery, an expansile nodule with central keratinization and necrosis located within the pulmonary parenchyma. It was characterized by a thick, irregular, and more complex cyst wall that lacked orderly maturation. Epithelial cells showed an increased number of mitotic figures.

Conclusions:

During the reevaluation of the original sections, there were a number of degenerative and inflammatory lesions recorded in all test item treated as well as in the control groups. The treatment of animals by daily inhalation of AEROSIL® 200 at a concentration of 1.3–31 mg/m³, AEROSIL® R 974 (34.7 mg/m³), and SIPERNAT® 22 S (34.9 mg/m³) did not cause any irreversible changes in the lungs as seen over the complete recovery period.

Executive summary:

In a subchronic (13-week) inhalation toxicity study with a terminal sacrifice (after 13 weeks inhalation) and several recovery period sacrifices (13, 26, 39, and 52 weeks), the effects of AEROSIL® 200 (pyrogenic synthetic amorphous silica (SAS)), AEROSIL® R 974 (surface-treated pyrogenic SAS), and SIPERNAT® 22 S (precipitated SAS) were tested in rats at multiple dose levels. The aforementioned materials are all SAS products. A comparative group of animals was exposed to quartz dust. This study attempts to re-examine the lung tissues originally evaluated in a study published by Reuzel et al. using the current standards. To reach a high level of credibility, the results of the reevaluation were subsequently examined by a pathology working group (PWG).

This is a summary of the PWG's report including a comparison to the original findings.

Reference 7

Endpoint:: chronic toxicity: inhalation
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
Qualifier:: no guideline followed
Principles of method if other than guideline:: 1 dose level, 1 year administration, 5-month recovery period for some animals
GLP compliance:: not specified
Specific details on test material used for the study:: Kieselsäure FK 700 (= Sipernat 700)
Species:: rat
Sex:: female
Details on test animals or test system and environmental conditions:: c. 160-185 g
Route of administration:: inhalation: dust
Type of inhalation exposure:: whole body
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: measured with Gravimetric Dust Sampler Type 113a (Casella)
Duration of treatment / exposure:: up to 1 year
Frequency of treatment:: 5 h/d
5 d/w
Dose / conc.:: 55 mg/m³ air (analytical)
No. of animals per sex per dose:: 110 (females only)
Details on study design:: 16 rats were kept untreated for 5 months after one year of exposure.
Sacrifice and pathology:: gross pathological and histopathological examination of lungs and lymph nodes
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: gray-whitish sub-pleural foci without induration increasing with duration of the study, most of them subsided after 5 months of recovery
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: some bronchial and alviolar foci; alveolar desquamation, mostly subsided after 5 months of recovery; no fibrosis
Details on results:: mean SiO2 content in lungs: 1.022 mg and 3.443 mg after 4 and 12 months, respectively; reduced to 0.457 mg after 5 month of recovery (= 87 % elimination)
mean SiO2 content in mediastinal lymph nodes: 0.033 mg and 0.069 mg after 4 and 12 months, respectively; reduced to 0.052 mg after 5 month of recovery
Conclusions:: Some bronchial effects were shown after 1 year of exposure, but they mostly subsided after 5 months of recovery. No fibrosis was detected.
Executive summary:: Long-term toxicity effects were assessed and SiO₂content in lungs and lymph nodes were measured in rats treated with Sipernat 700.

Reference 8

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec. 21, 1983 - Jan. 4, 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

other: dose range findings study for

Qualifier:

no guideline followed

Principles of method if other than guideline:

10/14 days exposure, 3 dose levels, no post-observation period

GLP compliance:

not specified

Specific details on test material used for the study:

Aerosil 200

Species:

rat

Strain:

Wistar

Details on species / strain selection:

obtained from: Central Institute for Breeding of Laboratory Animals TNO, Zeist / NL

Sex:

male/female

Details on test animals or test system and environmental conditions:

4 weeks old, (at study initiation) 50 - 70 g, 5 males or females per cage, 22 +/-1 °C, 30-70% humidity, institute's stock diet and unflouridated tap water ad libitum except during exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of Aerosil 200 in the test atmospheres were determined by gravimetry. Samples of the test atmospheres were drawn through glass fiber filters (Sartorius SM 13430). The filters were weighed just prior to and after sampling. From the increase in weight and the volume of the test atmosphere drawn through the filter the concentration could be calculated.
The actual overall mean concentrations and the standard error of the mean (given in brackets) of the test material in the different inhalation chambers during the study were 17 (1), 44 (2) and 164 (9) mg/m air.

Duration of treatment / exposure:

6 hours per day for 2 weeks

Frequency of treatment:

5 days / week

Dose / conc.:

17 mg/m³ air (analytical)

Remarks:

nominal concentration: 15 mg/m³

Dose / conc.:

44 mg/m³ air (analytical)

Remarks:

nominal concentration: 45 mg/m³

Dose / conc.:

164 mg/m³ air (analytical)

Remarks:

nominal concentration: 135 mg/m³

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

- All animals were visually inspected for clinical symptoms and behaviour before and after exposure. During the weekends the inspections were per¬formed only once a day.
- Body weights of the individual animals were recorded just prior to the start of the study and then once weekly.
- Food consumption was determined during the second week of the exposure period.
- Examinations were performed in all rats in blood samples collected from the tip of the tail in week 2.

Sacrifice and pathology:

- Animals were killed by exsanguination from the abdominal aorta under ether anaesthesia at day 15. They were autopsied and examined for gross pathological changes.
- Histopathological examination was done on liver, kidneys, trachea and larynx of all animals of the high-level group and of all controls, and on the lungs with regional lymph nodes and the nasal cavity of all animals.

Statistics:

Body weights and absolute organ weights were analysed by analysis of covariance followed by Dunnett's multiple comparison test.
Analysis of variance and the Dunnett test were applied to food consumption and the organ to body weight ratios and haematological data. Histopathological data were evaluated by the the Fisher exact probability test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the end of the exposure period animals exposed to 164 mg Aerosil 200 / m³ exhibited nasal discharge.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female exposed to 164 mg Aerosil 200 / m³ died during the exposure at day 11. Gross and microscopic examination did not provide any information to establish the cause of death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of male rats exposed to 44 or 164 mg Aerosil 200 / m³ air were statistically significantly lower than those of the controls. The differences with the controls showed a concentration-response relationship. In females exposed to 164 mg Aerosil 200 / m³ body weights tended to be lower than in controls; however, the differences were not statistically significant .

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

By mistake food consumption was not determined during the first week of the study. During the second week food intake was statistically significantly . lower in males exposed to 44 or 164 mg/m³ than in controls. The differences with the controls were concentration-related.

Haematological findings:

no effects observed

Description (incidence and severity):

The different haematological parameters showed the usual variation between groups. There were no indications of an adverse effect of the test material.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Animals exposed to Aerosil 200 showed a transient slightly restless behaviour.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females of all exposure groups had statistically significantly higher absolute and relative lung weights than controls had. The differences in relative lung weight between the test groups and the control group showed a concentration-response relationship in both sexes.
Statistically significantly lower liver weights were observed in males, but not in females, of each of the test groups. The differences in both absolute and relative liver weights being concentration-related.
In males exposed to 44 or 164 mg Aerosil 200 / m³ air absolute kidney weights were lower than in controls, whereas in females they were similar with those of the controls. When the kidney weights were expressed relative to body weight, they were comparable in all groups of males, but statistically significantly greater in females exposed to 44 or 164 mg / m³ air than in controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross examination at autopsy revealed treatment-related changes in the lungs and mediastinal lymph nodes of several male and female animals in each of the test groups. The lungs were pale, spotted and/or spongy and occasionally showed an irregular surface. The mediastinal lymph nodes of a large number of test animals were enlarged.
The incidence of several other gross changes varied considerably amongst the groups (enlarged/flabby heart) or the gross changes were seen in one or a few animals only (small surface lesion of the liver and swollen uterus). There was no indication that any of these gross lesions was related to the inhalation of Aerosil 200.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathological changes were found only in the lungs and the mediastinal lymph nodes of both, male and female rats.

Lungs
The treatment-related changes in the lungs comprised accumulation of particulate material (most probably Aerosil 200) in the alveolar spaces, accumulation of alveolar macrophages, increased septal cellularity, alveolar interstitial pneumonia, early granulomata (aggregates of macrophages and lymphocytes, occasionally accompanied by some fibroblasts) and alveolar consolidation. These treatment-related changes in the lungs were found in animals of all test groups, and showed a concentration-related increase in severity.

Mediastinal lymph nodes
Aggregates of macrophages and fibroblasts, occasionally accompanied by deposits of particulate material, were indicated as early granulomata and were observed in the mediastinal lymph nodes of most animals of the 164-mg group and in several animals of the 44-mg group. In the 17-mg group only one male and one female rat showed such granulomata.

Key result

Dose descriptor:

NOAEL

Effect level:

< 17 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Not substance specific, particle-related effect; no systemic effects.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

17 mg/m³ air (analytical)

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.:

17 mg/m³ air (analytical)

System:

immune system

Organ:

lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

From the results of the present study it was concluded that under its conditions the "no adverse effect" level in rats was lower than 17 mg Aerosil 200 / m³ air.

Executive summary:

This study assessed the sub-acute inhaltion toxicity of Aerosil 200 in rats.

This study served as a preliminary to a sub-chronic one (Reuzel et al. 1987).

Reference 9

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec. 11, 2011 - May 18, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Based on current knowledge the selected time for recovery was too short.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

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:

NM-200

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:

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 mean concentrations were very close to the target concentration for all dose groups of the test item, i.e. amounted to 104, 100 and 101% in the low, mid and high dose groups.

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 (nominal)

Dose / conc.:

2.5 mg/m³ air (nominal)

Dose / conc.:

5 mg/m³ air (nominal)

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

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

other: no systemic toxicity

Remarks on result:

other: end of recovery (90 days)

Remarks:

All lesions in the lung showed a marked decrease both in incidence and severity during the recovery period of 90 days – except mild adaptive changes such as histiocytosis in alveoli and in the BALT. Not substance specific, particle-related effect; no systemic effects.

Dose descriptor:

NOAEL

Effect level:

< 1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

At end of exposure due to adverse effects at lowest tested concentration level. The degree of severity of several findings was decreased after the recovery period of 90 days. However, due to persistence of particle-exposure related adverse effect in all dose-groups, a NOAEL could not be established for the nasal cavity. Not substance specific, particle-related effect; no systemic effects.

Dose descriptor:

LOAEL

Effect level:

1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: end of exposure

Remarks:

Not substance specific, particle-related effect; no systemic effects.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

other: end of recovery

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

1 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

other: end of exposure

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

1 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

other: end of exposure and recovery

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Effects indicating systemic toxicity were not observed. Reversibility of particle related lung effects was demonstrated after 90 days recovery.

For nasal cavities severity of several findings was also decreased after the recovery period of 90 days. However, due to persistence of particle-exposure related adverse effect in all dose-groups, after three months recovery, a NOAEL could not be established for the nasal cavity. 90-day inhalation studies with one year recovery demonstrated no persistence of effects in nasal cavities (TNO, 1987; Fraunhofer, 2019).

Conclusions:

Conclusion on NOAEL: for the lung a NOAEL of 5 mg/m³ at the end of 90-day recovery could be derived.
For nasal cavities severity of several findings was also decreased after the recovery period of 90 days. However, due to persistence of particle-exposure related adverse effect in all dose-groups, after three months recovery, a NOAEL could not be established for the nasal cavity. 90-day inhalation studies with one year recovery demonstrated no persistence of effects in nasal cavities (TNO, 1987; Fraunhofer, 2019).
Under the conditions of this test for nasal cavities a LOAEL = 1 mg/m³ was derived (decisive endpoint: histopathology: mucous cell hyperplasia in nasal cavities).

Executive summary:

A 3-month inhalation study with NM-200 followed by a 90-day recovery period was performed in male rats.

Overall, the effects depicted through this study with a precipitated synthetic amorphous silica (SAS) show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. Differences in severity of the similar pathological effects might be caused by study design such as exposure conditions, rat strain and test substance differences (particle size, primary structure, surface area, number, density, solubility...). In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m³. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).

SAS is not a poorly soluble particle (PSP) (Roelofs and Vogelsberger, 2004; ECETOC TR No. 122). The US-EPA confirmed that SAS is not a PSP in context with a registration of a hydrophobic precipitated synthetic amorphous silica, surface-modified with organosilane (TSCA Section 5(a)(3) Determination for Premanufacture Notice (PMN) P-16-0192). Based on the data submitted the EPA did not consider hydrophobic with organosilane surface-treated precipitated synthetic amorphous silica as poorly soluble particle and withdraw the request for additional repeated-dose inhalation data (https://www.epa.gov/ sites/production/files/2018-10/documents/p-16-0192_determination_non-cbi_final.pdf).

Therefore, in context with "lung overload" described long-term lung effects of low toxic PSPs are not relevant for SAS, confirmed by a Pathology working group (PWG) review of a sub-chronic (13-week) inhalation toxicity study of aerosols of AEROSIL® 200, AEROSIL® R 974, SIPERNAT® 22 S and quartz in rats (Hardisty et al., 2016). Indications for lung fibrosis, which is an irreversible effect, have been published in literature and were based on study materials of one of the applicants' studies according to OECD TG 413 (Sub-chronic inhalation toxicity, Reuzel et al., 1987; 1991). A pathology working group reviewed this finding using the same (original) tissue slides according to the highest current standards and concluded the rapid clearance of the synthetic amorphous silica products will not lead to persistent inflammation and epithelial cell proliferation and therefore will not result in fibrosis/lung tumour induction (Weber et al. 2018, “Aerosols of synthetic amorphous silica do not induce fibrosis in lungs after inhalation: Pathology working group review of histopathological specimens from a sub-chronic 13-week inhalation toxicity study in rats”. Toxicology Research and Application 2: 1–17). Irreversible lung fibrosis has not been associated to any of the eight sub-chronic studies to SAS. These results were confirmed by the recent studies requested by ECHA with high and low surface area pyrogenic SAS, regarded as the worst case of synthetic amorphous silica forms, which showed no lung fibrosis and accordingly no increase of collagen (90-day inhalation toxicity studies, Fraunhofer ITEM, 2019).

Reference 10

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Study period:: Jul. 8, 1988 - Mar. 27, 1990
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: comparable to guideline study
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:: 3 dose levels, 6 hrs/d, 5 d/wk, 3-month post observation
GLP compliance:: yes
Specific details on test material used for the study:: Ludox CL-X
Species:: rat
Strain:: other: CD BR
Details on species / strain selection:: from. Charles River, Raleigh, NC, USA
Sex:: male
Details on test animals or test system and environmental conditions:: at start of exposure: 9 weeks old, 185-248 g., rats housed by groups, water and feed (Purina Certified Rodent Chow 5002) ad libitum,. 23 +/- 2 °C, 50 +/-10% humidity
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: nose only
Vehicle:: air
Mass median aerodynamic diameter (MMAD):: ca. 2.9 - ca. 3.7 µm
Details on inhalation exposure:: diluted 4:1 with deionized, distilled water
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: 60-minute intervals by gravimetric analysis
Duration of treatment / exposure:: 4 weeks
Frequency of treatment:: 6 h/d
5 d/w
Dose / conc.:: 10.1 mg/m³ air (analytical)
Dose / conc.:: 50.4 mg/m³ air (analytical)
Dose / conc.:: 154 mg/m³ air (analytical)
No. of animals per sex per dose:: 25 (males only)
Control animals:: yes, concurrent vehicle
Details on study design:: up to 94-day (3-month) recovery period
Observations and examinations performed and frequency:: at end of exposure period and at end of observation: blood and urine samples from 10 rats per group
Sacrifice and pathology:: 10 rats per group after end of exposure, 5 rats per group after 10 days of recovery and the remaining 10 per group at the end of the observation period
Statistics:: 0.05 probability level
Clinical signs:: no effects observed
Body weight and weight changes:: no effects observed
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: increase in mean neutrophil count and globulin concentration and decrease in mean lymphocyte count with 150 mg/m³; the increase in neutrophil count and decrease in lymphocyte count were still present after 3 months.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: increased absolute and relative lungs weights after exposure (50 and 150 mg/m³ groups) and 10 days recovery (150 mg/m³ group); no difference from control after full recovery period
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: 50 and 150 mg/m³ groups: dust-laden aveolar macrophages, neutrophilic infiltration, type II pneumocyte hyperplasia. Pulmonary lesions were progressively decreased, but small numbers of minute silicotic nodule-like lesions were present after 3 months, which did not increase in number or size with time.
Details on results:: lung clerance half-time in 50 and 150 mg/m³ groups: app. 50 days
: Key result
Dose descriptor:: NOAEC
Effect level:: 10.1 mg/m³ air (analytical)
Sex:: male
Basis for effect level:: gross pathology; organ weights and organ / body weight ratios
Remarks on result:: other:
Remarks:: No substance specific, particle-related effects; no systemic effects.
: Key result
Dose descriptor:: LOAEC
Effect level:: 50.4 mg/m³ air (analytical)
Sex:: male/female
Basis for effect level:: gross pathology; organ weights and organ / body weight ratios
Remarks on result:: other:
Remarks:: No substance specific, particle-related effects; no systemic effects.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 50.4 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: NOAEL = 10.1 mg/m³; pulmanory response: dust-laden aveolar macrophages, neutrophilic infiltration, type II pneumocyte hyperplasia with 50.4(+) mg/m³; clearance half-time: ca. 50 days
Executive summary:: The inhaltative toxicity potential of Ludox CL-X was assessed in a 4-week study in rats.

Reference 11

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: test procedure in accordance with national standard methods with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: other: second publication of
Qualifier:: no guideline followed
Principles of method if other than guideline:: 3 dose levels, 6 hrs/d, 5 d/wk, up to 3m post obs.
GLP compliance:: not specified
Specific details on test material used for the study:: Ludox (CS = colloidal silica)
Species:: rat
Strain:: Crj: CD(SD)
Sex:: male
Details on test animals or test system and environmental conditions:: Seven-week-old male Crl:CD BR rats were used in these studies. The rats were monitored on a monthly basis for several viruses, mycoplasma, bacteria, and parasites. They were housed singly in suspended, stainless steel, wire-mesh cages and quarantined for 6 days during a 13-day pretest period. During the quarantine period, rats were grouped by computer randomization such that the group mean body weights 5 days prior to the start of exposures were similar. Upon grouping and for the remainder of the exposure phase of the study rats were housed two per cage. At the start of the exposures rats were approximately 9 weeks old and weighed between 185 and 248 g, with group mean weights of approximately 215 g. Rats were provided with Purina Certified Rodent Chow 5002 and water ad libitum between exposures. Animal rooms were maintained on a timer-controlled, 12-hr/12-hr light/dark cycle. Environmental conditions of the animal rooms were targeted for a temperature of 23 ± 2°C and a relative humidity of 50
± 10%.
Route of administration:: inhalation: aerosol
Vehicle:: air
Remarks:: and water (s. below)
Remarks on MMAD:: Analytical ranges: 10.1 mg/m³ (SD: 2.6), MMAD: 3.7 µm, GSD: 1.9; 50.5 mg/m³ (SD: 11.7), MMAD: 3.3 µm, GSD: 2.1; 154 mg/m³ (SD: 30.2), MMAD: 2.9 µm, GSD: 2.3
Details on inhalation exposure:: Rats were exposed to aerosols containing Ludox colloidal silica in 150-liter, Rochester-style, stainless steel and glass inhalation chambers. The Ludox used for test atmosphere generation was diluted 4:1 with filtered-deionized water prior to use in order to more closely simulate product-use conditions and to reduce the pH of the sprayed liquid. Atmospheres containing Ludox aerosol were generated by spraying the diluted liquid directly into the tangential-feed tops of the inhalation chambers with spraying systems nebulizers.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The atmospheric concentration of silica was determined at approximately 60-min intervals using Gelman glass-fiber filters which were weighed on a Cahn 26 automatic electrobalance.
Duration of treatment / exposure:: 4 weeks
Frequency of treatment:: 6 hours / day
5 days / week
Dose / conc.:: 10 mg/m³ air (nominal)
Remarks:: mean analytical concentration: 10.1 mg/m³
Dose / conc.:: 50 mg/m³ air (nominal)
Remarks:: mean analytical concentration: 50.5 mg/m³
Dose / conc.:: 150 mg/m³ air (nominal)
Remarks:: mean analytical concentration: 154 mg/m³
No. of animals per sex per dose:: 25 (males only)
Control animals:: yes, concurrent vehicle
Details on study design:: Three test groups of 25 rats each were exposed to 10, 50, or 150 mg/m³ (based on dried silica content) of Ludox colloidal silica in air for 6 hr/day, 5 days/week for 4 weeks. A similarly composed control group of rats was exposed simultaneously to water and air only. Prior to each exposure, each
rat was individually restrained in a stainless steel or plastic cylinder with a conical nose piece. Each restrainer was inserted into the exposure chamber
such that only the nose of each rat extended into the chamber. For histopathologic evaluation, 10 rats per group were killed at the end of the exposure
period, 5 rats per group were killed 10 days later, and the remaining 10 rats in each group were killed 3 months after the last exposure.
Observations and examinations performed and frequency:: Body weights and clinical observations
During the exposure period, rats were individually weighed and observed for clinical signs of toxicity
twice a week before exposure. During the exposure period, clinical signs were taken at least once during exposure and immediately following exposure
and recorded as group clinical signs. During the recovery period, all rats were weighed and observed for clinical signs of toxicity at least once a week.
Sacrifice and pathology:: Clinical pathology
Urine samples were collected overnight after the 19th exposure, and on the 93th day of recovery. Samples were analyzed for volume, osmolality. urobilinogen. pH, hemoglobin or occult blood, glucose, protein, bihrubin, and ketone. The color and transparency of each sample were noted, and the sediment from each sample was examined microscopically. Blood samples were taken from the orbital sinus of 10 rats per group after the 20th exposure, and from the same 10 rats per group on the 94th day of recovery. The orbital sinus blood samples were collected from rats under light carbon dioxide anesthesia. Blood samples were analyzed for erythrocyte count, hemoglobin concentration, hematocrit, platelet count, leukocyte count, and relative numbers of neutrophils, band neutrophils, lymphocytes, atypical lymphocytes, eosinophils, monocytes, and basophils The absolute values of various types of leukocytes were calculated from the leukocytic data, and mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration (Wintrobe Indices) were calculated from the erythrocyte data. Serum activities of alkaline phosphatase, alanine aminotransferase, aspanate aminotransferase, cholesterol, creatirune, glucose, phosphorous, potassium, total protein, sodium, and urea nitrogen were also measured. Serum globulin concentration was calculated from the total protein and albumin concentrations.

Histopathology and lung silica evaluations
At the end of the 4-week exposure period and at the end of the 94-day recovery period, the right caudal lung lobe from 5 rats in each group was instilled intratracheally with Karnovsky's fixative for electron microscopy and the remaining lobes from these rats were instilled intratracheally with Bouin's fixative for light microscopy. The right lung lobes (anterior, middle, and caudal) from another 5 rats per group at these times were weighed and analyzed for silicon content and the left lobes were instilled intratracheally with Bouin's fixative for light microscopy. In the 5 rats sacrificed after a 10-day recovery period, the right lobes were weighed and analyzed for silicon content and the left lobe was instilled with Karnovsky's fixative for light and electron microscopy analysis.
After gross examination of all vital organs and tissues, representative sections of the following tissues in addition to the lung were examined microscopically. The liver, spleen, kidneys, lungs, testes, and brain were weighed at the time of necropsy. Bones, eyes, testes, and epididymides were fixed in Bouin's fixative. All other organs and tissues were fixed in 10% formalin solution except where noted previously Paraffin sections were prepared according to routine histologic techniques. All sections were stained with hematoxylin and eosin. In addition, all lung sections were stained with trichrome
and silver stains, respectively.
Statistics:: Mean body weights, body weight gains, absolute organ weights, and organ-to-body weight ratios for exposed rats were compared to those of control rats during the exposure and recovery periods. Exposure group values were compared to controls by the least significant difference test and Dunnett's test when the ratio of variance indicated a significant among-to-within group variation. Significant differences were declared at the 0.05 probability level.
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Immunological findings:: effects observed, treatment-related
Description (incidence and severity):: Compared to controls, there was an increase in mean neutrophil count and globulin concentration and a decrease in mean lymphocyte count observed at the end of the exposure period in the 150 mg/m³ group. At the end of the 3-month recovery period, the increase in mean neutrophil count and the decrease in mean lymphocyte count were still present.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: After 4 weeks of exposure, dose-related increases in mean lung weights and lung-to-body weight ratios were observed in rats exposed to 50 or 150 mg/m³ compared to controls. After 10 days of recovery, the lung-to-body weight ratios of rats at 50 or 150 mg/m³ were still increased. After 3 months of recovery, the lung weights of rats at 50 or 150 mg/m³ were no longer different from control values. Lung-weight effects were not observed in the 10 mg/m³ group.
Gross pathological findings:: no effects observed
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Four week's exposure
Table 3 shows a summary of main pulmonary lesions in rats at 4 weeks exposure and 10 days and 3 months recovery. At 10 mg/m³, the lungs showed normal alveolar architecture with a few dust-laden alveolar macrophages (AMs) in the alveolar airspaces. There were no aggregates of dust-laden AMs in the mediastinal lymph nodes. At 50 mg/m³, inhaled particles were mostly found in the intraalveolar AMs. The particles were translucent, spherical, approximately 1.5 µm in diameter, and slightly reflective. Most inhaled particles were retained in the aggregated AMs in alveoli adjacent to alveolar ducts
and were sharply demarcated from the remaining normal alveoli. Polymorphonuclear leukocytic (PML) infiltration was present with aggregated particle-laden AMs in the alveoli. The alveolar walls enclosing particle-laden AMs were slightly thickened with hyperplastic Type II pneumocytes. Some particle-laden AMs infiltrated into peribronchial lymphoid tissue and apparently translocated to tracheobronchial lymph nodes, where they formed well-circumscribed, multiple foci consisting of particle-laden AMs and hyperplastic histiocytes. However, neither inflammatory nor proliferative tissue responses were found around the foci of particle-laden AMs. At 150 mg/m³, the pathological lesions in the lungs and thoracic lymph nodes were similar to those seen at 50 mg/m³, but the magnitude of the lesions was somewhat greater.
Ten days PE
At 10 mg/m³, the lungs were normal in appearance. At 50 and 150 mg/m³, particle-laden AMs and the PML infiltration in the alveoli were markedly reduced in number and the particle-laden AMs were more loosely aggregated. Type II pneumocyte hyperplasia had essentially disappeared; however, some alveolar walls adjacent to particle-laden AMs were slightly thickened with proliferated epithelioid cells and fibroblasts. There was no significant difference in accumulation of particle-laden AMs and histiocytes in the thoracic lymph nodes when compared to that seen after the 4-week exposure period.
Three months PE
At 10 mg/m³ the lungs were normal in appearance. At 50 mg/m³ the lungs were almost normal in appearance but contained a small number of tiny nodular aggregates of particle-laden WIs and PMI. in some alveoli in 1 of 10 rats. One of 10 rats had a few silicotic nodules in pervascular regions adjacent to alveolar ducts. The nodular lesions consisted of particle-laden AMs and epithelioid cells with iymphocytic infiltration along the periphery. Thoracic lymph nodes were slightly enlarged with nodular accumulations of particle-laden AMs and histiocytes. At 150 mg/m³ PMI and Type II pneumocyte hyperplasia had disappeared. Dust-laden AMs were markedly decreased in number and were sharphly circumscribed in the alveolar airspaces without any collagen fiber deposition in the alveolar walls. However, some particle-laden AM contacted with alveolar walls and transformed into nodular aggregates in 9 of 10 rats. The lesions were devoid of collagen fiber deposition and there was no lymphocytic infiltration along the periphery of the nodular aggregates. In contrast, 3 of 10 rats had silicotic nodules that were characterized by loosely interwoven reticular fiber networks with minimal collagen fiber deposition and lymphocytic infiltration along the periphery of the nodular aggregates. The silicotic nodules were located in the alveolar ducts and adjacent penvascular region. Peribronchial lymphoid tissue showed hyperplasia with aggregates of particle-laden AMs and histiocytes. Also, thoracic lymph nodes were enlarged with accumulation of isolated nodules or conglomerated nodular lesions consisting of translocated particle-laden AMs and histiocytes. There was no inflammatory response to the aggregates of particle-laden AMs and histiocytes in the thoracic lymph nodes.
Details on results:: The amount of silica measured in the lungs of exposed rats at the end of the 4-week exposure period was directly proportional to the exposure concentration. When fitted to a monoexponential clearance model, the deposited silica in the 50 and 150 mg/m³ groups was found to clear from the lung with half-times of approximately 40 and 50 days, respectively. The clearance rates in the 10 mg/m³ group and in the group exposed at 150 mg/m³ for 1 day were much slower and appeared to almost plateau. It is not known whether this was due to sequestering of the inhaled silica, to background sources of silica (as evidenced by variable levels in the controls, to limitations in analytical methods, or to a combination of these factors.
: Key result
Dose descriptor:: NOEL
Effect level:: 10.1 mg/m³ air (analytical)
Sex:: male
Basis for effect level:: histopathology: non-neoplastic; immunology; organ weights and organ / body weight ratios
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
: Key result
Dose descriptor:: LOAEL
Effect level:: 50.5 mg/m³ air (analytical)
Sex:: male
Basis for effect level:: histopathology: non-neoplastic; organ weights and organ / body weight ratios
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 50.5 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: alveoli; lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: The exposure concentration of 10 mg/m³ Ludox CS was considered to be the no-effect concentration. There were no exposure-related clinical signs in any group.
Executive summary:: The toxictiy of Ludox (collodial silica) was evaluated in rats after repeated inhalation over 4 weeks.

Reference 12

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: other: first publication of
Qualifier:: no guideline followed
Principles of method if other than guideline:: No guideline followed but similar OECD 412: (nose only exposure, 3 dose levels, 6 hrs/d, 5 d/wk, for 4 weeks, up to 3-month post observation)
Following exposure and/or recovery, fluids and cells were recovered from the lungs by bronchoalveolar lavage (BAL) and measured for cellular and biochemical parameters. Additional groups of animals were processed for cell labeling studies or lung deposition studies.
GLP compliance:: not specified
Specific details on test material used for the study:: Ludox
Species:: rat
Strain:: Crj: CD(SD)
Sex:: male
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: nose only
Vehicle:: air
Remarks on MMAD:: Analytical ranges: 10.1 mg/m³ (SD: 2.61), MMAD: 3.7 µm; 50.5 mg/m³ (SD: 11.7), MMAD: 3.3 µm; 154 mg/m³ (SD: 30.2), MMAD: 2.9 µm
Details on inhalation exposure:: Prior to each exposure, each rat was individually restrained in a stainless-steel or plastic cylinder with a conical nose piece. Each restrainer was inserted in the exposure chamber so that only the nose of each rat protruded into the chamber. Rochester-style, 150-liter stainless-steel and glass chambers were used in these studies.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The atmospheric aerosol concentration of Ludox colloidal silica was determined at approximately 60-min intervals by gravimetric analysis.
Duration of treatment / exposure:: 2 or 4 weeks
Frequency of treatment:: 6 hours / day
5 days / week
Dose / conc.:: 10 mg/m³ air (nominal)
Remarks:: analytical mean concentration: 10.1 mg/m³
Dose / conc.:: 50 mg/m³ air (nominal)
Remarks:: analytical mean concentration: 50.5 mg/m³
Dose / conc.:: 150 mg/m³ air (nominal)
Remarks:: analytical mean concentration: 154 mg/m³
No. of animals per sex per dose:: 6 (males only)
Control animals:: yes, concurrent vehicle
Details on study design:: Groups of six rats each were selected from each of the exposure concentrations (i.e., 0, 10, 50, and 150 mg/m³ Ludox) and were evaluated after 2 or 4 weeks of exposure and at 3 months after a 4-week exposure to Ludox. The lungs of three of the six rats per group were lavaged, and the remaining three rats were given a pulse of tritiated thymidine ([3H]TdR) and euthanized 4 hr later. For the purposes of this report, the 0 mg/m³ level is referred to as
sham and/or control, and the 10, 50, and 150 mg/m³ exposure levels are referred to as low-, mid-, and high-exposure groups, respectively. In addition, lung tissue samples from five rats per group were analyzed for silica content immediately after the 4-week exposure.
Sacrifice and pathology:: Bronchoalveolar lavage procedures were conducted according to methods previously described (Warheit et ai, 1983, 1984).
Statistics:: p < 0.05
Clinical signs:: not examined
Mortality:: not examined
Body weight and weight changes:: not examined
Ophthalmological findings:: not examined
Haematological findings:: not examined
Urinalysis findings:: not examined
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Cell differential analyses of BAL fluids recovered from the lungs of Ludox-exposed rats exposed to 50 or 150 mg/m³ demonstrated significant increases in the numbers of granulocytes (primarily polymorphonuclear leukocytes) after 2 or 4 weeks of exposure (Table 3). Pulmonary inflammatory responses were reduced by 3 months postexposure, although a small, but significant increase in the numbers of PMNs was measured in animals exposed to
150 mg/m³ Ludox. Taken together, the increased numbers of pulmonary cells recovered by lavage concomitant with the emigration of PMNs into the lung at 2 weeks and 4 weeks in the mid- and high-exposure levels signify the presence of ongoing pulmonary inflammatory responses during the exposure periods. Although the numbers of cells in BAL fluids were slightly increased at the low-exposure level (10 mg/m³), no significant inflammatory responses were measured in these animals.
Histopathological findings: neoplastic:: no effects observed
Other effects:: effects observed, treatment-related
Description (incidence and severity):: Extracellular LDH in lung lavage fluid is considered to be a sensitive indicator of pulmonary cytotoxicity. Lactate dehydrogenase values in BAL fluids of rats were significantly increased above controls at 2 and 4 weeks, as well as following a recovery period in rats exposed to 150 mg/m³ Ludox. No significant differences in BAL LDH values were measured at any time postexposure between rats exposed to 10 or 50 mg/m³ Ludox and sham controls.
Protein values in BAL fluids were significantly increased over controls at 2 and/or 4 weeks in rats exposed to 150 mg/m³ Ludox but returned to near control values following a 3-month recovery period. The increases in BAL protein values suggest that Ludox exposure induced alterations in the permeability of the alveolar/capillary barrier, thus accounting for the transudation of serum proteins from the vasculature into alveolar regions.
Protein values in lavage fluids from all exposure groups were not significantly different from sham control values following a 3-month recovery period.
Alkaline phosphatase values in BAL fluids of Ludox-exposed rats were increased only in rats exposed to 150 mg/m³ Ludox for 2 weeks. It was interesting to note, however, that unlike the pattern observed with BAL LDH and protein values, there was a downward trend in ALP values after 2 weeks of exposure.

The labeling indices for lung parenchymal cells in animals exposed to 50 or 150 mg/m³ Ludox were significantly different from controls at 2 and 4
weeks. These results demonstrated an increased turnover of epithelial and interstitial cells following Ludox exposure. The labeling index for the low-exposure group was not significantly different from sham controls at any time period. In addition, the labeling index of lung parenchymal cells for all groups returned to control values following a 3-month recovery period.
The labeling index of terminal bronchiolar cells was similarly quantified in Ludox and sham-exposed rats. Significant differences were counted in the high-exposure levels groups at 2 and 4 weeks. In addition, increased cellular turnover was measured in airway cells of animals exposed to 50 mg/m³
for 2 weeks and after a recovery period, but not after 4 weeks. No significant differences were observed between animals exposed to 10 mg/m³ Ludox and sham controls.
Details on results:: Inhaled doses of Ludox colloidal silica were measured after 4-week exposures and were found to be 489 µg/lung (10 mg/m³ group), 2418 µg/lung (50 mg/m³), and 7378 µg/lung (150 mg/m³), respectively.
: Key result
Dose descriptor:: NOEL
Effect level:: 10.1 mg/m³ air (analytical)
Sex:: male
Basis for effect level:: histopathology: non-neoplastic
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
: Key result
Dose descriptor:: LOAEL
Effect level:: 50.5 mg/m³ air (analytical)
Sex:: male
Basis for effect level:: histopathology: non-neoplastic
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 50.5 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: According to authors, the determination of a no-observable-effect level (NOEL) of 10 mg/m³ had been consistent with results obtained by conventional toxicology methods and affirmed the utility of these biochemical, cellular, and autoradiographic techniques for providing a predictive screen to assess the toxic-ity of inhaled partides.
Executive summary:: The toxicity potential of Ludox colloidal silica after repeated (4-week) inhalative adminstration was assessed in rats. The purpose of this study was to determine the biological effects in rats of repeated inhalation.

Reference 13

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:: according to guideline
Guideline:: other: STIS NanoSafe2
Version / remarks:: rat short-term inhalation study (STIS) protocol developed within the project NanoCare of Germany and the EU by NanoSafe2 (www.nanosafe.org)
Deviations:: not specified
Principles of method if other than guideline:: 5 days inhalation, subsequent 3 weeks recovery period
bronchoalveolar lavage fluids (BALF) and histopathology of the whole respiration tract examined
GLP compliance:: not specified
Specific details on test material used for the study:: 13 different metalloxide nanomaterials and micron-scale zinc oxide
various suspensions of colloidal Synthetic Amorphous Silica (SAS) in water: colloidal uncoated amorphous silica (SiO2naked) as well as coated forms: SiO2polyacrylate, SiO2polyethylenglycol (SiO2PEG), SiO2phosphate and SiO2amino
Species:: rat
Strain:: Wistar
Details on species / strain selection:: strain Crl:WI (Han), specific pathogen free, obtained by Charles River Laboratory (Sulzfeld, Germany)
Sex:: male
Details on test animals or test system and environmental conditions:: 7 weeks old
for the first studies individual housing, for the majority of the studies social housing in polysulfonate cages (floor area abaout 2065cm³),
animal rooms: temperature: 20-24°C, realative humidity: 30-70%, light/dark cycle 12h
all animals acclimatized for about 2 weeks before the start of the study
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: nose/head only
Mass median aerodynamic diameter (MMAD):: ca. 0.8 - ca. 4 µm
Remarks on MMAD:: lowest and highest measurement of all the 5 different Silicamaterials tested, 2 measurements, each 2 times per substance by a 8-stage cascade impactor
primary particle size (nm): SiO2naked: 15, SiO2acrylate: 20, SiO2PEG: 15, SiO2phosphate: 15, SiO2amino: 15
Details on inhalation exposure:: respirable for rats, particle size distribution confirmed by a Scanning Mobile Particle Sizer and finally measure by aa aerosol spectrometer
concentrations refer to the German nuisance dust limit at the workplace for granular respirable dust (1.25 mg/m³)
Analytical verification of doses or concentrations:: yes
Remarks:: near target concentrations
Details on analytical verification of doses or concentrations:: a sampling device (Millipore) with glass fibre filter was used
concentrations calculated by weighting the blank filter before use and the filter after use
2 samples were drawn from each exposure chamber and each exposure day (total of 10 samples)
Duration of treatment / exposure:: 6 h/day, 5 consecutive days, 3 weeks (ie.e. 21days) post-exposure recovery (2 weeks, i.e. 14 days, for SiO2acrylate)
Frequency of treatment:: 6h/day
Dose / conc.:: 0.5 mg/m³ air
Remarks:: SiO2naked
Dose / conc.:: 2.5 mg/m³ air
Remarks:: SiO2naked
Dose / conc.:: 10 mg/m³ air
Remarks:: SiO2naked
Dose / conc.:: 50 mg/m³ air
Remarks:: SiO2naked
Dose / conc.:: 0.5 mg/m³ air
Remarks:: SiO2acrylate
Dose / conc.:: 2 mg/m³ air
Remarks:: SiO2acrylate
Dose / conc.:: 10 mg/m³ air
Remarks:: SiO2acrylate
Dose / conc.:: 2 mg/m³ air
Remarks:: SiO2PEG
Dose / conc.:: 10 mg/m³ air
Remarks:: SiO2PEG
Dose / conc.:: 50 mg/m³ air
Remarks:: SiO2PEG
Dose / conc.:: 2 mg/m³ air
Remarks:: SiO2phosphate
Dose / conc.:: 10 mg/m³ air
Remarks:: SiO2phosphate
Dose / conc.:: 50 mg/m³ air
Remarks:: SiO2phosphate
Dose / conc.:: 2 mg/m³ air
Remarks:: SiO2amino
Dose / conc.:: 10 mg/m³ air
Remarks:: SiO2amino
Dose / conc.:: 50 mg/m³ air
Remarks:: SiO2amino
No. of animals per sex per dose:: 5 animals per test group (exposure / recovery), males only
Control animals:: yes, sham-exposed
Details on study design:: body weights, organ weights, necropsy and histology of the respiratory tract, histology of the brain, electron microscopy, BALF cytology protein and enzyme activities, BALF cytokines and chemokines, cytokines and chemokines in lavaged lung tissue, hematology (according to OECD TG 412) including c-reactive protein and haptoglobin, organ burden
Observations and examinations performed and frequency:: at the end of exposure (exposure groups) or after recovery (recovery groups)
Sacrifice and pathology:: exsanguinated under Narcoren anesthesia
body weights, organ weights, necropsy and histology of the respiratory tract, histology of the brain (not SiO2naked), electron microskopy of spleen (only SiO2acrylate)
Other examinations:: BALF cytology protein and enzyme activity, BALF cytokines and chemokines, IL-1aTNF-a in lavaged lung tissue (not SiO2acrylate), hematology (blood cell counts, hemoglobin content, hematokrit, mean corpuscular volume etc.), haptoglobin, c-reactive protein, a2-macroglobulin in blood
Statistics:: body weights: Dunnett`s test, BALF data: Kruskal-Wallis test, Wilcoxon test or Mann-Whitney U-test
Clinical signs:: not specified
Mortality:: not specified
Body weight and weight changes:: not specified
Food consumption and compound intake (if feeding study):: not specified
Food efficiency:: not examined
Ophthalmological findings:: not examined
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: s. below: immunological findings
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: effects observed, treatment-related
Description (incidence and severity):: SiO2.naked: Inhalation exposure to an aerosol concentration of 50 mg/m3 naked amorphous silica caused marginal inflammation, slight and transient increased PMN neutrophil and lymphocyte counts were present in the BALF.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: SiO2.acrylate: Absolute spleen weights were increased by 37% and 30% in animals exposed to 2 and 10 mg/m3 SiO2.acrylate, respectively, and their relative spleen weights were increased by 35% and 26%, respectively. By contrast, the absolute and relative spleen weights of the recovery groups were comparable to the corresponding control values.
Gross pathological findings:: not examined
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: SiO2.naked: Histologically, multifocal macrophage aggregates were observed in the lung shortly after exposure. This finding exacerbated towards a slight multifocal pulmonary inflammation by the end of the 3-week exposure free period. Recovery period of 3 weeks is relatively short, apparently not long enough to measure the reversibility of the effects. At concentrations up to 50 mg/m3, SiO2.naked did not induce any significant changes of BALF cytokines or chemokines in the exposure groups, i.e. those rats that were euthanized shortly after the final exposure, or in the recovery groups that were euthanized after the 3-week post-exposure period.

SiO2.acrylate: In following up the recorded alterations in spleen weights, the lungs and spleens from three animals, each, from the control and high concentration groups were investigated by transmission electron microscopy. In the lung, electron-dense aggregates consisting of small (approx. 20 nm) particles were recorded in the alveolar space of the exposed animal with no corresponding findings in the control animals. In the spleen of the animals treated with 10 mg/m3 SiO2.acrylate, thrombocyte accumulations were observed whereas the spleens of the control animals were unaffected. Additionally, the cytoplasm of the splenetic lymphocytes seemed to be less homogenous in the exposed animals than in the control animals, and small electron-dense aggregates were found within the lymphocytic cytoplasm of the treated animals. Since silicon particles were detected in the spleen, these morphological changes were assessed as being related to the test material, even though the physiological meaning of the findings in the spleen remains unclear. The effects showed full reversibility.
Histopathological findings: neoplastic:: no effects observed
Details on results:: SiO2.PEG, SiO2.phosphate, and SiO2.amino: No adverse effects were observed after inhalation exposure to the three silica particles surface-modified with polyethyleneglycol (SiO2.PEG), phosphate (SiO2.phosphate), or amino groups (SiO2.amino) at concentrations up to 50 mg/m3.
: Key result
Dose descriptor:: NOAEC
Remarks:: SiO2naked
Effect level:: 2.5 mg/m³ air
Sex:: male
Basis for effect level:: histopathology: non-neoplastic; immunology
Remarks on result:: other:
Remarks:: Not substance specific, particle-related effect; no systemic effects.
: Key result
Dose descriptor:: NOAEC
Remarks:: SiO2acrylate
Effect level:: >= 10 mg/m³ air
Sex:: male
Basis for effect level:: organ weights and organ / body weight ratios
Remarks on result:: other: end of recovery (3 weeks)
Remarks:: Full reversibility of spleenic effects; no pulmonary effects at any time point. Not substance specific, particle-related effect.
: Key result
Dose descriptor:: NOAEC
Remarks:: SiO2PEG
Effect level:: >= 50 mg/m³ air
Sex:: male
Basis for effect level:: histopathology: non-neoplastic; immunology; organ weights and organ / body weight ratios
Remarks on result:: other:
Remarks:: Not substance specific, particle-related effect; no systemic effects.
: Key result
Dose descriptor:: NOAEC
Remarks:: SiO2phosphate
Effect level:: >= 50 mg/m³ air
Sex:: male
Basis for effect level:: histopathology: non-neoplastic; immunology; organ weights and organ / body weight ratios
Remarks on result:: other:
Remarks:: Not substance specific, particle-related effect; no systemic effects.
: Key result
Dose descriptor:: NOAEC
Remarks:: SiO2amino
Effect level:: >= 50 mg/m³ air
Sex:: male
Basis for effect level:: histopathology: non-neoplastic; immunology; organ weights and organ / body weight ratios
Remarks on result:: other:
Remarks:: Not substance specific, particle-related effect; no systemic effects.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 2.5 mg/m³ air
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.:: 10 mg/m³ air
System:: respiratory system: lower respiratory tract
Organ:: lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
: Key result
Critical effects observed:: no
Conclusions:: NOAEC for SiO2naked: 2.5 mg/m³ air (effects on respiratory system), LOAEL for SiO2naked: 10 mg/m³
NOAEC for SiO2acrylate: >= 10 mg/m³ air
NOAEC for SiO2PEG,SiO2phosphate and SiO2amino: >= 50 mg/m³ (no effects observed)

Recovery period of 3 weeks is relatively short, apparently not long enough to measure the reversibility of the effects (as far as observed).
Executive summary:: A standard short-term inhalation study (STIS) was applied for hazard assessment of 13 metal oxide nanomaterials and micron-scale zinc oxide. Only the results of the 5 tested colloidal Synthetic Amorphous Silica (SAS) substances are documented here.

Reference 14

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: test procedure in accordance with national standard methods with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:: yes
Remarks:: one high exposure concentration only, target-organ effects: therefore, histopathology limited; clinical chemistry/haematology not included; instead specific investigations (lung lavage cytology and biochemistry) because focussed on pulmonary effects.
Principles of method if other than guideline:: The testing programme included cellular and biochemical Bronchoalveolar Lavage Fluid Analysis (BAL) on inflammatory markers and histopathology.
GLP compliance:: not specified
Limit test:: yes
Species:: rat
Strain:: Fischer 344
Sex:: male
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Age at study initiation: no data
- Weight at study initiation: 200 - 250 g
Route of administration:: inhalation: dust
Type of inhalation exposure:: whole body
Mass median aerodynamic diameter (MMAD):: 0.81 µm
Details on inhalation exposure:: TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 300-L horizontal laminar-flow chamber, compartmentalised
- System of generating particulates/aerosols: screw-feed mechanism (ACCURate, Whitewater) in combination with a Venturi-type dust feeder
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination: no data

TEST ATMOSPHERE
- Brief description of analytical method used: no data
- Samples taken from breathing zone: no data
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 13 weeks
Frequency of treatment:: 6h/d, 5d/wk
Dose / conc.:: 50.4 mg/m³ air (analytical)
Remarks:: 50.4 +/- 19.0 mg/m³
No. of animals per sex per dose:: no data, for single analytical endpoint: n =4
Control animals:: yes, concurrent no treatment
Details on study design:: Post-exposure period: 3 and 8 months
Positive control:: Quartz (crystalline silica): Cristobalite, 3 mg/m3
Observations and examinations performed and frequency:: The testing programme included cellular and biochemical Bronchoalveolar Lavage Fluid Analysis (BAL) on inflammatory markers and histopathology.
Statistics:: Analysis of variance, Dunnett´s test for determination of differences between control and treated groups.
Clinical signs:: not examined
Mortality:: not examined
Body weight and weight changes:: not examined
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: effects observed, treatment-related
Description (incidence and severity):: Table 2 presents the results of the BAL fluid analysis. Exposure to 50-mg/m3 amorphous silica produced significant changes in all BAL parameters examined through the end of exposure. The changes in BAL endpoints in the amorphous silica group returned to near sham-exposed levels by 3 months post-exposure, and values were not significantly different from controls by 8 months post-exposure.
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: The amorphous silica-exposed animals had elevated numbers of neutrophils and macrophages, but the elevation was first detectable at the first observation point (45 days of treatment), and tended to decrease in the post-exposure period.
The amorphous silica proliferative response was evident at the earliest time point (45 days of exposure), and decreased precipitously at the 8-month post-exposure point. Some fibrosis, as detected by Gor mor’s trichrome staining, was present in the alveolar septa of lungs from silica-treated animals. Fibrosis is questionable as the reversibility of these findings were not examined. It should be defined as fibrogenesis in the case of reversibility.
Histopathological findings: neoplastic:: no effects observed
Dose descriptor:: LOAEL
Effect level:: 50.4 mg/m³ air (analytical)
Sex:: male
Basis for effect level:: histopathology: non-neoplastic; immunology
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
Critical effects observed:: yes
Lowest effective dose / conc.:: 50.4 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: lungs
Treatment related:: yes
Dose response relationship:: not specified
Relevant for humans:: yes
Critical effects observed:: yes
Lowest effective dose / conc.:: 50.4 mg/m³ air (analytical)
System:: immune system
Organ:: lymph node
Treatment related:: yes
Dose response relationship:: not specified
Relevant for humans:: yes
Conclusions:: Exposure concentration was selected to induce high pulmonary inflammatory-cell responses by Aerosil 200 to examine possible mutagenic effects in the main study (s. chapter 7.6.2). The LOAEL was 50.4 mg/m³ (only dose level tested).
Executive summary:: This is part of a comparative study including synthetic amorphous and crystalline silica. Here only the sub-chronic effects of Aerosil 200 (amorphous) are documented.

Reference 15

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: disregarded due to major methodological deficiencies
Reliability:: 4 (not assignable)
Rationale for reliability incl. deficiencies:: documentation insufficient for assessment
Remarks:: No data on test substance available due to insufficient documentation the study is disregarded
Qualifier:: no guideline followed
Principles of method if other than guideline:: 1 dose level, 5h/d, 5d/wk, up to 3 m post observation
GLP compliance:: not specified
Specific details on test material used for the study:: BS 111
No data on test substance available due to insufficient documentation.
Species:: rat
Strain:: Wistar
Sex:: female
Details on test animals or test system and environmental conditions:: c. 120 g
Route of administration:: inhalation: dust
Type of inhalation exposure:: whole body
Vehicle:: air
Analytical verification of doses or concentrations:: not specified
Duration of treatment / exposure:: 5 h/d
Frequency of treatment:: 5 days per week fpr 6 weeks
Dose / conc.:: 30 mg/m³ air (nominal)
No. of animals per sex per dose:: not specified
Control animals:: yes
Sacrifice and pathology:: after 48 hours, 5 days and 3 months
Gross pathological findings:: no effects observed
Histopathological findings: non-neoplastic:: no effects observed
Details on results:: no hints for toxic effects to the lungs
Conclusions:: No hints for toxic effects to the lungs were detected.
Executive summary:: The inhaltive toxicity potential with repeated doses of BS 111 was analysed in rats.

Reference 16

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: Dec. 2001 - Aug. 2002
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:: 1981-05-12
Deviations:: yes
Remarks:: only 5-day study
Qualifier:: equivalent or similar to guideline
Guideline:: EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:: 1992-12-29
Deviations:: yes
Remarks:: only 5-day study
Principles of method if other than guideline:: Method: in accordance with OECD Guideline 412, 12 May 1981 and directive 92/69/EEC, 29 Dec. 1992, but focus on the respiratory tract (lung and lymph nodes)
Cab-O-Sil was examined only in males because they had proven to be more sensitive than females.
GLP compliance:: yes (incl. QA statement)
Specific details on test material used for the study:: Cab-O-Sil M5
Species:: rat
Strain:: Wistar
Remarks:: albino
Details on species / strain selection:: obtained from Charles River Deutschland, Sulzfeld, Germany
Sex:: male
Details on test animals or test system and environmental conditions:: 2-3 housed per cage, 12 h light/dark, ca. 19-25 °C, 30-70 % humidity, food: SDS Rat & Mouse No. 3 Breeding Diet RM3 and tap water ad libitum
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: nose only
Vehicle:: clean air
Mass median aerodynamic diameter (MMAD):: ca. 1.2 - ca. 1.3 µm
Remarks on MMAD:: 2.2 - 3.5 µm on days 4-5, due to the use of a very short tubing between the chamber and the measurement device
Details on inhalation exposure:: Aerosol generation: Miniature screw conveyor, a dust feeder, (Institute´s design) connected to a low-velocity eductor in which the test material was aerolised. The eductors were operated with compressed humidified air. The test material was aerosolised and diluted with a defined amount of humidified air at the entrance of each exposure unit.
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 6 h/d
Frequency of treatment:: 5 consecutive days
Dose / conc.:: 1.39 mg/m³ air (analytical)
Remarks:: +/- 0.15 mg/m³
Dose / conc.:: 5.41 mg/m³ air (analytical)
Remarks:: +/- 0.34 mg/m³
Dose / conc.:: 25.3 mg/m³ air (analytical)
Remarks:: +/- 0.9 mg/m³
No. of animals per sex per dose:: 10 (males only)
Control animals:: yes, concurrent vehicle
Details on study design:: post exposure: 1 and 3 months
Positive control:: One extra group was exposed to 25 mg/m³ crystalline silica as a positive control group.
Observations and examinations performed and frequency:: clinical signs (daily), food consumption (daily), body weight (before and after exposure, thereafter weekly)
Sacrifice and pathology:: one third after exposure, another after 1-month recovery, and the last third after 3-month recovery
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: during exposure period in all groups: sligh body weight loss
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: no effects observed
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: mid and high concentration group: increases in ALP, albumin, total protein content, LDH, NAG, TNFa
Immunological findings:: effects observed, treatment-related
Description (incidence and severity):: significant concentration related-increase in the absolute and relative number of neutrophils with a concomittant significant decrease in the relative number of macrophages in animals of the mid and high concentration group
no treatment-related changes 3 months after exposure
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: mid and high concentration group: increased lung weights
high concentration group: increased tracheobronchial lymph nodes weights
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: mid and high concentration group: alveolar macrophage accumulation, reactive epithelial hypertrophy
high concentration group: polymorphonuclear cell infiltration
Other effects:: effects observed, treatment-related
Description (incidence and severity):: Silicon content:
One day after exposure, 43 µg Si (average) were analysed in lungs of high-dose animals, which was below detection limit after 1 month recovery
(<15 µg). [note: no determinations carried out in the low and mid-dose groups]
No increased Si levels were observed in the lymph nodes (below detection limit (<15 µg).
: Key result
Dose descriptor:: NOAEL
Effect level:: ca. 1.39 mg/m³ air (analytical)
Sex:: male/female
Basis for effect level:: clinical biochemistry; histopathology: non-neoplastic; immunology; organ weights and organ / body weight ratios
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
: Key result
Dose descriptor:: LOAEL
Effect level:: ca. 5.41 mg/m³ air (analytical)
Sex:: male/female
Basis for effect level:: clinical biochemistry; histopathology: non-neoplastic; immunology; organ weights and organ / body weight ratios
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 5.41 mg/m³ air (analytical)
System:: immune system
Organ:: lungs; lymph node
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 5.41 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: NOAEL = 1 mg/m³ (nominal; actual: 1.39 mg/m³); differential cell count, changes to biochemical parameters, lung weight, hispathology with 5+ mg/m³ (nominal; actual: 5.41 mg/m³).
Executive summary:: The short-term inhalation toxicity potential of Cab-O-Sil M5 was evaluated in rats.

Reference 17

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 4 (not assignable)
Rationale for reliability incl. deficiencies:: documentation insufficient for assessment
Qualifier:: no guideline followed
Principles of method if other than guideline:: 1 dose level: 5 h/d, and another dose level: 1 h/d, both on 5 d/wk, up to 3 m post observation
GLP compliance:: not specified
Specific details on test material used for the study:: HDN 20 (= HDK N 20)
Species:: rat
Strain:: Wistar
Sex:: female
Details on test animals or test system and environmental conditions:: c. 120 g
Route of administration:: inhalation: dust
Type of inhalation exposure:: whole body
Vehicle:: air
Analytical verification of doses or concentrations:: not specified
Details on analytical verification of doses or concentrations:: stated maximum deviation of apparatus used: +/- 10%
Duration of treatment / exposure:: 8 mg/m³: 5 h/d
40 mg/m³: 1 h/d
Frequency of treatment:: 5 d/w for 6 weeks
Dose / conc.:: 8 mg/m³ air (nominal)
Dose / conc.:: 40 mg/m³ air (nominal)
No. of animals per sex per dose:: not specified
Control animals:: yes
Sacrifice and pathology:: after 48 hours, 7 days, 3 weeks and 3 months
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: no effects observed
Histopathological findings: non-neoplastic:: no effects observed
Other effects:: effects observed, treatment-related
Description (incidence and severity):: accumulation in lungs: more than 50% decrease of accumumulated SiO2 within 48 hrs., nearly unmeasurable within 3 months
Details on results:: "high cytotoxicity, but no pulmonic effects" (no details are provided)
Conclusions:: high cytotoxicity, but no pulmonic effects
Executive summary:: The inhaltive toxicity potential with repeated doses of HDK N20 (here called HDN 20) was analysed in rats.

Reference 18

Endpoint:: short-term repeated dose toxicity: inhalation
Remarks:: comparison of 2 substances
Type of information:: other: Expert assessment
Adequacy of study:: other information
Reliability:: 4 (not assignable)
Rationale for reliability incl. deficiencies:: secondary literature
Remarks:: No data on test substance available due to insufficient documentation the study is disregarded
Reason / purpose for cross-reference:: assessment report
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:: yes
Remarks:: only 5 days
Principles of method if other than guideline:: comparison of inhalation toxicity potential of standard pyrogenic SAS to nanoparticle-rich SAS
GLP compliance:: yes
Species:: rat
Strain:: Wistar
Sex:: male
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: nose only
Vehicle:: not specified
Mass median aerodynamic diameter (MMAD):: ca. 1.7 - ca. 2 µm
Geometric standard deviation (GSD):: 1.75
Remarks on MMAD:: Above data is for standart SAS.
values for nanoparticle-rich ones: 1.6-2.1 µm, GSD: 2.25
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 6 h/d
Frequency of treatment:: for 5 days
Dose / conc.:: 1.39 mg/m³ air (nominal)
Remarks:: standart SAS
Dose / conc.:: 5.41 mg/m³ air (analytical)
Remarks:: standart SAS
Dose / conc.:: 25.3 mg/m³ air (analytical)
Remarks:: standart SAS
Dose / conc.:: 1.2 mg/m³ air (analytical)
Remarks:: nanoparticle-rich SAS
Dose / conc.:: 4.97 mg/m³ air (analytical)
Remarks:: nanoparticle-rich SAS
Dose / conc.:: 25.43 mg/m³ air (analytical)
Remarks:: nanoparticle-rich SAS
No. of animals per sex per dose:: 30 (males only)
Control animals:: yes
Details on study design:: 1- and 3-month observation period
Observations and examinations performed and frequency:: clinical signs, behaviour, food consumption, body weight
Sacrifice and pathology:: gross pathology, organ weights, histopathology of kidneys, lungs and lymph nodes
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: inflammatory in the lungs (nanoparticle-rich SAS, high concentration)
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: slight reduction (standard SAS)
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: increases of ALP, albumin, total protein content, LDH, NAG (standard SAS, mid and high concentration) and of TNF (standard SAS, high concentration)
increases of ALP, GGT and TNF (nanoparticle-rich SAS, high concentration)
Behaviour (functional findings):: not specified
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: absolute and relative lung weights (standart SAS, mid and high concntrations)
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: cellular changes in BALF: increase in absolute and relative numbers of neutrophilis with a concomitant decrease in the relative numbers of macrophages; pulmonary changes: alveolar macrophage accumulation, reactive epithelial hypertrophy (standard SAS, mid and high concentration) and polymorphonuclear cell inflitration (standard SAS, high concentration)
decreased precentage of mononuclear macro-phages and increased percentage and absolute number of neutrophilis in BALF; pulmatory changes: intraepithelial and peribronchial infiltartion of polymorphonuclear inflammatory cells (nanoparticle-rich SAS, high concentration)
: Key result
Dose descriptor:: NOAEL
Effect level:: 1 mg/m³ air (nominal)
Sex:: male
Basis for effect level:: clinical biochemistry; histopathology: non-neoplastic; organ weights and organ / body weight ratios
Remarks on result:: other: standard SAS, analytical concentration: 1.39 mg/m³
Remarks:: Not substance specific, particle-related effect; no systemic effects.
: Key result
Dose descriptor:: NOAEL
Effect level:: 5 mg/m³ air (nominal)
Sex:: male
Basis for effect level:: clinical biochemistry; clinical signs; histopathology: non-neoplastic
Remarks on result:: other: nanoparticle-rich SAS, analytical concentration: 4.97 mg/m³
Remarks:: Not substance specific, particle-related effect; no systemic effects.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1.39 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: blood; lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 4.97 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: blood; lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: NOAEL of standard SAS = 1.39 mg/m³; NOAEL of particle-rich SAS = 4.97 mg/m³
But the authors conluded that no significant differences in the extent and type of toxicity should be expected (due to higher a higher deposition rate of nanoparticle-rich SAS).
Executive summary:: The effects of standard and nanoparticle-rich SAS were compared in a 5-day inhalation study in rats.

Reference 19

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: supporting study
Reliability:: 4 (not assignable)
Rationale for reliability incl. deficiencies:: documentation insufficient for assessment
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: other: results are published in
Reason / purpose for cross-reference:: other: results are published in
Principles of method if other than guideline:: complement study to a subchronic one: 3 dose levels, 2 administration periods, 3-month post observation
GLP compliance:: not specified
Specific details on test material used for the study:: Ludox (colloidal silica)
Species:: rat
Strain:: Crj: CD(SD)
Sex:: not specified
Route of administration:: inhalation: dust
Type of inhalation exposure:: nose only
Vehicle:: not specified
Analytical verification of doses or concentrations:: not specified
Duration of treatment / exposure:: 2 or 4 weeks
Frequency of treatment:: not specified
Dose / conc.:: 10 mg/m³ air (nominal)
Dose / conc.:: 50 mg/m³ air (nominal)
Dose / conc.:: 150 mg/m³ air (nominal)
No. of animals per sex per dose:: not specified
Control animals:: yes
Observations and examinations performed and frequency:: Fluids and cells were recovered from the lungs by lavage (BAL) and measured for cellular and biochemical parameters.
Statistics:: P < 0.05
Clinical biochemistry findings:: effects observed, treatment-related
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Details on results:: Studies showed that exposure to 150 mg/m³ Ludox for 2 or 4 weeks produced pulmonary inflammation along with increases in BAL protein and LDH, and alkaline phosphatase values and reduced marcophage phagocytosis. Inflammatora responses, portein and LDH levels were increased and macrophage function decreased in the 510 mg/m³ group following 4 weeks of exposure. Most biochemical parameters for all groups returned to control values following a 3-month recovery period. Cell labelling studys demonstrated that the labelling index of terminal bronchiolar and lung parenchymal cells was increased in the 50 and 150 mg/³ groups after 2 and 4 weeks exposure, but returned to normal levels following a 3-month postexposure period. No significance in any parameter was detected in rats exposed to 10 mg/m³ at any time postexposure.
Dose descriptor:: NOEL
Effect level:: 10 mg/m³ air (nominal)
Sex:: not specified
Basis for effect level:: clinical biochemistry; histopathology: non-neoplastic
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
Critical effects observed:: yes
Lowest effective dose / conc.:: 50 mg/m³ air (nominal)
System:: respiratory system: lower respiratory tract
Organ:: bronchi; lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: NOEL: 10 mg/m³; inflammatory responses, increased protein and LDH levels and decreased macrophage functions with higher dose levels
Executive summary:: This complement study to a subchronic one tested the repeated inhalative toxicity of Ludox colloidal silica in rats. It is only badly documented.

Reference 20

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: Nov. 2001 - Aug. 2002
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:: yes
Remarks:: only 5 exposure days; only one sex (male), untreated control: only 6 animals; histopathology and organotoxicology limited; no clinical chemistry + haematology, but lung lavage cytology + biochemistry instead
Principles of method if other than guideline:: Method: other: in accordance with OECD Guide-line 412, 12 May 1981 and directive 92/69/EEC, 29 Dec. 1992
Syloid 74 was examined only in males because they had proven to be more sensitive than females, as observed in the first study (see Part I: 03a_ASASP2003_ZEOSIL 45_inhalation, 5 d, rat)
GLP compliance:: yes (incl. QA statement)
Specific details on test material used for the study:: Syloid 74
Species:: rat
Strain:: Wistar
Details on species / strain selection:: obtained from: Charles River Deutschland GmnH, Sulzfeld, Germany
Sex:: male
Details on test animals or test system and environmental conditions:: 6-7 weeks old, 2-3 per cage (but individually exposed), 19-25 °C, 30-70% humidity, feed: SDS RM3 and bottled water ad libitum except during exposure
Route of administration:: inhalation
Type of inhalation exposure:: nose only
Vehicle:: air
Remarks:: humified
Remarks on MMAD:: MMAD / GSD: Mass median aerodynamic diameter of particle size dstribution (MMAD) = 1.71, 1.60, and 1.57 µm. [Note: This particle size distribution is artificial and experimentally produced, but the commercial product has a mean particle size of about 100 µm due to agglomeration of aggregates.]
Details on inhalation exposure:: AEROSOL GENERATION:
Miniature screw conveyor, a dust feeder, (Institute´s design) connected to a low-velocity eductor
in which the test material was aerolised. The eductors were operated with compressed humidified air.

The test material was aerosolised and diluted with a defined amount of humidified air at the entrance of each exposure unit.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Mean actual concentrations: 0.94 (+-0.13), 5.13 (+-0.21), and 25.1 (+-0.5) mg/m3.
Duration of treatment / exposure:: 5 days
Frequency of treatment:: 6 h/d
Dose / conc.:: 1 mg/m³ air (nominal)
Remarks:: actual concentration: 0.94 +/-0.13 mg/m³
Dose / conc.:: 5 mg/m³ air (nominal)
Remarks:: actual concentration: 5.13 +/-0.21 mg/m³
Dose / conc.:: 25 mg/m³ air (nominal)
Remarks:: actual concentration: 25.1 +/-0.5 mg/m³
No. of animals per sex per dose:: 10 males
additionally, satellite groups of 10 males each were exposed correspondingly and kept for a recovery period of one and three months.
Control animals:: yes, concurrent no treatment
Details on study design:: Post-exposure period: 1 or 3 months
Positive control:: Quartz (crystalline silica) was also examined (information included in Part I: 03a_ASASP2003_ZEOSIL 45_inhalation, 5 d, rat)
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
CYTOLOGY on LUNG CELLS in LAVAGE
At necropsy, 5 animals per group and sex were lavaged acc. to standard procedure. The lavage was used for white blood cell count, viability and
cell differentiation (eosinophils, neutrophils, lymphocytes, monocytes/ macrophages, viable cells).
The supernatant of the lavage was used for determination of biochemical parameters (total protein, albumin, ALP, LDH,
N-acetyl glucosaminidase (NAG), SOD, GSH, and TNF-alpha).

SILICON CONTENT
Si content of the lung and tracheobronchial lymph nodes were determined.

HYDROXY OPROLINE CONTENT
The OH-proline of the lung and tracheobronchial lymph nodes were determined.
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, but only kidney, lung and lymphnodes
Other examinations:: see above "Observations and examinations..."
Statistics:: Various procedures acc. to the parameters under test (Report, p. 19)
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: Individual observations in the mornings, i.e. before the start of each day’s exposure, did not reveal any treatment-related findings. Occasional findings were: encrustations, malocclusion of incisors, and a crooked nose in one control animal, malocclusion of incisors in one animal of the low concentration group, haemorrhagic discharge from the penis in one animal of the mid concentration group, and sparsely haired skin which occurred in two males of the high concentration group. No concentration-response relationship was observed. During exposure, the groupwise observation carried out about halfway through each day’s exposure revealed no abnormalities.
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: no effects observed
Water consumption and compound intake (if drinking water study):: not specified
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: Changes in biochemical determinations in lavage fluid were observed in animals of the high concentration group one day after exposure. They consisted of increases in ALP, albumin, LDH, total protein, and NAG; a statistically significant degree was reached in these parameters except for LDH and albumin.
Increases in LDH were observed in one male of the mid concentration group and all males of the high concentration group; however, a statistically significant degree was not reached. Inspection of the individual data revealed that outlying values, which may or may not be related to the exposure, in one animal of the mid concentration group and to a lesser extent in one animal of the low concentration group, highly influenced the statistics. Similarly, although the values in the high concentration group were all higher than all values in the control group, a statistical significant effect of exposure on albumine could not be derived from the ANOVA.
No such changes were observed one month post-exposure. Three months after exposure, a statistically significantly increased NAG value was observed in animals of the mid concentration group but a concentration-response relationship was not observed. ALP was significantly increased in animals of the low concentration group; a concentration-response was also absent.
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: effects observed, treatment-related
Description (incidence and severity):: The day after the last exposure, in males of the high concentration group, a statistically significant increase in the absolute number of neutrophils was observed in bronchoalveolar lavage fluid, which was reflected in a significant increase in total cell number. In these animals, the percentage of neutrophils was also statistically significantly higher with a concomittant, significant, decrease in the percentage of macrophages. A slight, but statistically significant, increase in the percentage of neutrophils and a concomittant, significant, decrease in the percentage of macrophages was also observed in males of the mid concentration group, but this was not reflected in statistically significant changes in absolute numbers. No changes were observed in animals of the low concentration group. Measurements one month post-exposure revealed a slight, though statistically
significant increase in the absolute number of macrophages and as such in the total cell number in animals of the mid concentration group only. Three months after exposure, no changes in absolute or relative cell numbers were observed.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: Absolute and relative weights of lungs and tracheobronchial lymph nodes were significantly increased in animals of the high concentration group one day after exposure. Increases in tracheobronchial lymph node weights were also observed in animals of the low and mid concentration group. However, a statisticially significant degree was only observed in animals of the low concentration group and a concentration-response relationship was not present. No such changes in weights of these organs were observed one month or three months after exposure.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: Gross examination at necropsy did not reveal any treatment-related findings. The changes observed are common findings in rats of this strain and age and occurred only incidentally.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Necropsy one day after exposure
Microscopic examination revealed exposure-related accumulations of alveolar macrophages, occasionally accompanied by a few granulocytes/neutrophils in the lungs of three high concentration group animals and one mid concentration group animal. In addition, very slight reactive epithelial hypertrophy was observed in three high concentration group animals. Two of these animals also showed accumulation of alveolar macrophages, the other one did not. Histopathological changes in the kidneys were observed in one control group animal. No changes were observed in the mediastinal (tracheobronchial) lymph nodes.

Necropsy one month after exposure
The histopathological changes in the kidneys of the control and the high concentration as well as the histopathological findings in the lungs of the control, low, and high concentration groups are common findings in rats of this strain and age and occurred only incidentally. No changes were observed in the mediastinal (tracheobronchial) lymph nodes of the control and high-concentration group.

Necropsy three months after exposure
Microscopic examination after a three months recovery period of the lung lobes, kidneys and tracheobronchial lymph nodes, did not reveal histopathological changes that could be related to exposure to Syloid 74. The histopathological changes observed are common findings in rats of this strain and age and occurred only incidentally or were about equally distributed amongst the groups including controls.
Histopathological findings: neoplastic:: not examined
Other effects:: effects observed, treatment-related
Description (incidence and severity):: One day after the last exposure, slight amounts of silicon were present in the lungs of animals exposed to the high concentration of Syloid 74.
As animals exposed to the mid and low concentration were exposed to 5 or 25- times lower levels of Syloid 74, respectively, it could be expected that silicon levels in animals exposed to the lower concentrations, if present, would be below the detection limit of 15 µg. Silicon levels in tracheobronchial lymph nodes and in controls were below the detection limit (< 15 µg).
One and three months after the last exposure, silicon levels in lungs and tracheobronchial lymph nodes of animals exposed to the high concentration of Syloid 74 were also below the detection limit.
Dose descriptor:: NOAEL
Effect level:: 5.13 mg/m³ air (analytical)
Sex:: male
Basis for effect level:: other: Histopathology: based on the pulmonary response (inflammation reaction) (see details below "Details on results")
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
Dose descriptor:: LOAEL
Effect level:: 25.1 mg/m³ air (analytical)
Sex:: male
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
Dose descriptor:: NOEL
Effect level:: 0.94 mg/m³ air (analytical)
Sex:: male
Basis for effect level:: other: Histopathology: based on the absence of substance-related effects, in particular absence of a pulmonary response (inflammation reaction) (see details below "Details on results")
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 5.13 mg/m³ air (analytical)
System:: immune system
Organ:: lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: From the data of the present study it is concluded that the high concentration of Syloid 74 induced clear treatment-related effects (differential cell count, biochemical parameters, increased weights of lungs and tracheobronchial lymph nodes, and histopathological changes), which seemed to have disappeared one month after exposure. Three months after exposure, however, a slight increase in lung collagen content was observed in animals of this group, which was not confirmed by histopathological changes. In animals of the mid concentration group, the slight but significant increase in the percentage of neutrophils and the concomittant decrease in the percentage of macrophages, although not reflected in changes in absolute numbers, might be the result of exposure to the test material.
No such changes were observed at any time point in animals of the low concentration group exposed to 1 mg/m³, which could, therefore, be considered as a No-Observed-Effect Level (NOEL).
Executive summary:: The short-term inhalation toxicity potential of Syloid 74 was assessed in rats.

Reference 21

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: Aug. 2000 - Feb. 2001
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:: yes
Remarks:: only 5 exposure days; histopathology and organotoxicology limited; no clinical chemistry + haematology, but lung lavage cytology + biochemistry instead
Principles of method if other than guideline:: Method: in accordance with OECD Guide-line 412, 12 May 1981 and directive 92/69/EEC, 29 Dec. 1992, but focus on the respiratory tract (lung and lymph nodes).
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Specific details on test material used for the study:: Zeosil 45
Species:: rat
Strain:: Wistar
Details on species / strain selection:: obtained from: Charles River Deutschland GmnH, Sulzfeld, Germany
The study was conducted with albino rats. This species was used because it is considered the most suitable for this type of study.
Sex:: male/female
Details on test animals or test system and environmental conditions:: 5-6 weeks old, 5 per cage (but individually exposed), 19.5-22.5 °C, 42-70% humidity, feed: SDS RM3 and bottled water ad libitum except during exposure
Route of administration:: inhalation
Type of inhalation exposure:: nose only
Remarks on MMAD:: MMAD / GSD: Mass median aerodynamic diameter of particle size distribution (MMAD) = 2.83, 3.23, 3.27, and for the reference group 2.08 µm.
[Note: This particle size distribution is artificial and experimentally produced, but the commercial product has a mean particle size of about 100 µm due to agglomeration of primary particles.]
Details on inhalation exposure:: AEROSOL GENERATION:
Miniature screw conveyor, a dust feeder, (Institute´s design) connected to a low-velocity eductor
in which the test material was aerolised. The eductors were operated with compressed humidified air.

The test material was aerosolised and diluted with a defined amount of humidified air at the entrance of each exposure unit.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: EXPOSURE LEVELS and PARTICLE SIZE:
Mean actual concentrations: 1.16 (+-0.36), 5.39 (+-0.58), 25.2 (+-1.5)
(and for the control group receiving crystalline silica 24.4 (+-2.9) mg/m3) [Appendix 1.1, Tab. 1.1]
Duration of treatment / exposure:: 5 days
Frequency of treatment:: 6 h/d
Dose / conc.:: 1 mg/m³ air (nominal)
Remarks:: actual concentration: 1.16 +/- 0.36 mg/m³
Dose / conc.:: 5 mg/m³ air (nominal)
Remarks:: actual concentration: 5.39 +/- 0.58 mg/m³
Dose / conc.:: 25 mg/m³ air (nominal)
Remarks:: actual concentration: 25.2 +/- 1.5 mg/m³
No. of animals per sex per dose:: 10 males and females
additionally, satellite groups of 10 each per sex were exposed correspondingly and kept for a recovery period of one and three months.
Control animals:: yes, concurrent no treatment
Details on study design:: Post-exposure period: 1 or 3 months
Positive control:: One extra group was exposed to 25 mg/m3 crystalline silica as a positive control group.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: yes

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
CYTOLOGY on LUNG CELLS in LAVAGE
At necropsy, 5 animals per group and sex were lavaged acc. to standard procedure. The lavage was used for white blood cell count, viability and
cell differentiation (eosinophils, neutrophils, lymphocytes, monocytes/ macrophages, viable cells).
The supernatant of the lavage was used for determination of biochemical parameters (total protein, albumin, ALP, LDH,
N-acetyl glucosaminidase (NAG), SOD, GSH, and TNF-alpha).

SILICON CONTENT
Si content of the lung and tracheobronchial lymph nodes were determined.

HYDROXY OPROLINE CONTENT
The OH-proline of the lung and tracheobronchial lymph nodes were determined.
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, but only kidney, lung and lymphnodes
Other examinations:: see above: "Observation and examinations..."
Statistics:: Various procedures acc. to the parameters under test (Report, p. 19/20)
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: Individual observations in the mornings, i.e. before the start of each day’s exposure, did not reveal any treatment-related findings. The most common finding was sparsely haired skin which occurred in 1-5 males of the control, low, mid and high concentration group, and in 1-10 females of all groups, including the crystalline silica and control group. No concentration-response relationship was observed.
During exposure, the groupwise observation carried out about halfway through each day’s exposure revealed changes in breathing pattern which were generally confined to a slightly visually decreased breathing frequency. This change was seen in all exposure groups during the first days, disappeared somewhat later in animals of the low concentration group and was finally seen in animals of the crystalline silica group only.
Mortality:: mortality observed, non-treatment-related
Description (incidence):: One mid dose animal died.
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: no effects observed
Water consumption and compound intake (if drinking water study):: not specified
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: Changes in biochemicical determinations in lavage fluid were observed in animals of the high concentration group one day after exposure. They consisted of significant increases in ALP, albumin, total protein, LDH and NAG in males, and a significant increase in ALP in females. No such changes were observed one and three months post-exposure.
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: effects observed, treatment-related
Description (incidence and severity):: The day after the last exposure, in males and females of the high concentration group, a statistically significant increase in the absolute number of neutrophils was observed in bronchoalveolar lavage fluid, which was reflected in a slight, though insignificant, increase in total cell number. In these animals, the percentage of neutrophils was also significantly higher with a concomittant significant decrease in the percentage of macrophages. A slight increase in the percentage of neutrophils and a slight decrease in the percentage of macrophages was also observed in males and females of the mid concentration group, but this was not reflected in absolute numbers, and statistical significance was reached in male animals only. No changes were observed in animals of the low concentration group.
Measurements one month post-exposure did not reveal any changes.
Three months after exposure, a slight but significant increase in the percentage of neutrophils and a significant decrease in the percentage of macrophages was observed in males of the high concentration group. However, no significant changes in absolute numbers were observed.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: Weights of lungs and tracheobronchial lymph nodes were (slightly) increased in animals of the high concentration group one day after exposure. Statistically significant differences were observed in absolute lung weight (males), relative lung weight (females) and relative tracheobronchial lymph nodes weight. No such changes in weights of these organs were observed one month or three months after exposure.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: Gross examination at necropsy did not reveal any treatment-related findings. The changes observed are common findings in rats of this strain and age and occurred only incidentally.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Necropsy one day after exposure
Exposure to Zeosil resulted in histopathological changes in the lungs. The changes were mainly characterised by hypertrophy and some hyperplasia of the bronchial/bronchiolar and alveolar duct epithelium (denoted as ‘bronchial/ bronchiolar hypertrophy’). Bronchial/ bronchiolar hypertrophy is unusual for rats of the strain and age used. Therefore, it was considered to be related to the exposure, although it occurred only in one male and two females of the high-concentration group. Very slight to slight polymorpho-nuclear leukocytic inflammatory cell infiltrates were observed around the bronchioli and blood vessels of most high-concentration females and one to several animals in the other exposure groups, including the reference (crystalline silica) group, but not in controls. In males, the incidence and severity of the infiltrates did not demonstrate a concentration-response relationship. In females, the incidence was statistically significantly increased in the high-concentration group when compared to controls, but the severity was not increased. Moreover, polymorphonuclear cell infiltrates are found occasionally in controls, as shown in the controls of subgroup 2. Therefore, this finding was considered to be unrelated to the exposure.
All other histopathological changes in the lungs and the kidneys are common findings in rats of this strain and age and occurred only incidentally or the incidences were about equally distributed amongst the groups including controls.
No changes were observed in the mediastinal (tracheobronchial) lymph nodes.

Necropsy one month after exposure
All the histopathological changes in the lungs and kidneys of the control, the high- concentration as well as the histopathological findings in the lungs of the low-, and mid-concentration groups are common findings in rats of this strain and age and occurred only incidentally. No changes were observed in the mediastinal (tracheobronchial) lymph nodes of the control and high-concentration group.

Necropsy three months after exposure
Microscopic examination of the lung lobes of rats of the low-, mid-, and high- concentration groups, after a three months recovery period, did not reveal histopathological changes that could clearly be related to Zeosil. However, a few observations need to be mentioned. First, a focus of bronchiolar-alveolar hyperplasia (thickened alveolar septa with type-II pneumocyte hyperplasia associated with an increased accumulation of alveolar macrophages) was found in the lung of one male rat of the high-concentration group. Such a focus is an unusual finding in rats of this age. Secondly, a tendency towards increased incidence of alveolar macrophage accumulations was observed in the high- concentration males.Thirdly, the lung lobes of several high-concentration animals exhibited areas with overfilled capillaries in the septa (denoted as ‘hyperemia’)- Although it occurred only in a few animals, it is mentioned here because this hyperemia is unusual. All other histopathological changes in the lungs, the kidneys and lymph nodes are common findings in rats of this strain and age and occurred only incidentally or were about equally distributed amongst the groups including controls.
Histopathological findings: neoplastic:: no effects observed
Other effects:: effects observed, treatment-related
Description (incidence and severity):: One day after the last exposure, slight amounts of silicon (mean ca. 30-40 µg) were present in lungs of animals exposed to the high concentration of Zeosil. Animals exposed to crystalline silica showed 4-5 times higher lung silicon levels (mean ca. 150-160 µg). Silicon levels in tracheobronchial lymph nodes and in controls were below the detection limit (< 25 µg).
One month after the last exposure, silicon levels in animals exposed to the high concentration of Zeosil were below the detection limit. Animals exposed to crystalline silica showed mean levels of about 80 (µg in females, and 140 µg in males. Silicon levels in tracheobronchial lymph nodes were again below the detection limit (< 30 µg).
Three months after the last exposure silicon was detected in males (mean ca. 60 µg) and females (mean ca. 90 µg) exposed to the high concentration of Zeosil. This was a surprising finding since silicon levels were below the detection limit two months earlier. Animals exposed to crystalline silica showed mean levels of about 110 µg in females, and 130 µg in males. Silicon levels in tracheobronchial lymph nodes were again below the detection limit (< 30 µg).
Dose descriptor:: NOAEL
Effect level:: 5.39 mg/m³ air (analytical)
Sex:: male/female
Basis for effect level:: other: Histopathology: based on the pulmonary response (inflammation reaction) (see details below "Details on results")
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
Dose descriptor:: LOAEL
Effect level:: 25.2 mg/m³ air (analytical)
Sex:: male/female
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
Dose descriptor:: NOEL
Effect level:: 1.16 mg/m³ air (analytical)
Sex:: male/female
Basis for effect level:: other: Histopathology: based on the absence of substance-related effects, in particular absence of a pulmonary response (inflammation reaction) (see details below "Details on results")
Remarks on result:: other: Not substance specific, particle-related effect; no systemic effects.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 5.39 mg/m³ air (analytical)
System:: immune system
Organ:: lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Conclusions:: From the data of the present study it is concluded that the high concentration of Zeosil induced treatment-related effects (changes in differential cell count and biochemical parameters, increased weights of lungs and tracheobronchial lymph nodes, and histopathological changes), which seemed to have disappeared one month after exposure, but reappeared to a slight extent three months after exposure in males only. In animals of the mid concentration group changes were confined to a slight increase in relative neutrophil count with a concomittant decrease in relative macrophage count the day after exposure. No such changes were observed at any time point in animals of the low concentration group exposed to 1 mg/m³, which could, therefore, be considered as a No-Observed-Effect Level (NOEL).
Executive summary:: The short-term inhaltion toxicity potential of Zeosil 45 was evaluated in rats.

Reference 22

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Oct 5, 2008 - Jul. 7, 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

only 5-days; for additional minor ones s. below

Principles of method if other than guideline:

Deviations:
- Animal 78 (1.2 mg/m³ group, necropsy day 5) died in the tube just before the second exposure and was therefore replaced with a surplus animal. This animal was exposed for 4 days instead of 5 days.
- Differential cells in the bronchoalveolar lavage were manually counted.
- Accidentally, the bronchoalveolar lavage samples of the animals of the recovery groups necropsied on day 33 were not stored for analysis of TNFalpha and albumin.
The nominal concentration could not be determined for the mid concentration group, since the generation setup was slightly changed during the study.
- Silicon content in lungs and tracheobronchial lymph nodes was not statistically evaluated since the levels of the control group animals were below the detection limit.
These deviations were considered not to have affected the validity of the study.

GLP compliance:

yes

Specific details on test material used for the study:

VA-silica LGS

Species:

rat

Strain:

Wistar

Details on species / strain selection:

obtained from Charles River Deutschland, Sulzfeld, Germany

Sex:

male

Details on test animals or test system and environmental conditions:

ca. 240 g, 5 housed per cage, 12 h light/dark, ca. 20-24 °C, 40-70 % humidity, food: SDS Rat & Mouse No. 3 Breeding Diet RM3, municipal tap water

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

The average particle size (MMAD) was 2.07 μm (gsd of 2.13), 1.85 μm (gsd of 2.34) and 1.57 μm (gsd of 2.10) for the low, mid, and high concentration respectively.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

5 h/d

Frequency of treatment:

for 5 days

Dose / conc.:

1.2 mg/m³ air (analytical)

Remarks:

SD: +/- 0.17 mg/m³

Dose / conc.:

4.97 mg/m³ air (analytical)

Remarks:

SD: +/- 0.78 mg/m³

Dose / conc.:

25.43 mg/m³ air (analytical)

Remarks:

SD: +/- 3.07 mg/m³

No. of animals per sex per dose:

30 (males only)

Control animals:

yes, concurrent vehicle

Details on study design:

up to 3-month observation period

Sacrifice and pathology:

10 the day after final exposure, 10 after one month, 10 after 3 months

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal (1.2 mg/m³ group) died in the tube just before the second exposure.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The slightly lower body weight of the animals of the exposed groups compared to the control animals is not considered treatment related since this is not dose related and it goes together with a slight, but not significant, decrease in food consumption.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A decreased percentage of mononuclear macrophages and an increased percentage and absolute number of neutrophils in bronchoalveolar lavage fluid were seen in animals of the high concentration group necropsied on day 5. These animals also showed increased concentration of ALP, GGT and TNFalpha in their lavage fluid. This indicates an inflammatory response of the lungs. These effects were not found in animals of the low and mid concentration groups necropsied on day 5. Only TNFalpha concentrations were still significantly increased on day 96. No further effects were found in the animals of the recovery groups. Therefore, most of these effects recovered within a month after exposure.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed treatment-related histopathological changes in the lungs of 4/5 high-concentration animals necropsied on day 5, characterised by intraepithelial and peribronchial infiltration of polymorphonuclear inflammatory cells, accompanied by slight hypertrophy and/or hyperplasia of the bronchiolar epithelium. This was not seen in animals of the low and mid concentration group, or in animals of the recovery groups. No other histopathological changes were found.

Details on results:

Exposure of rats for 5 consecutive days to 25.43 mg/m³ VA-Kieselsäure LGS induced inflammation just after exposure, which generally recovered within a month after exposure. Silicon was still detectable 3 months after exposure in the lungs of these animals, and was accompanied by a slight inflammatory response, shown by increased TNFalpha concentrations in bronchoalveolar lavage fluid.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 4.97 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Remarks on result:

other: Not substance specific, particle-related effect; no systemic effects.

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 25.43 mg/m³ air (analytical)

Sex:

male

Basis for effect level:

haematology

histopathology: non-neoplastic

Remarks on result:

other: Not substance specific, particle-related effect; no systemic effects.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25.43 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

It can be concluded that exposure to VA-Kieselsäure LGS at concentrations up to ca. 5 mg/m³ did not induce effects apart from the presence of the test material in the lungs. Hence, the No-Observed-Adverse-Effect-Level of VA-Kieselsäure LGS in rats exposed for 6 hours/day for 5 days and a recovery period up to 90 days is 5 mg/m³.

Executive summary:

The repeated inhalation toxicity potential of VA-silica LGS was evaluated in a 5-day exposure study in rats.

Overall, the effects depicted through this study with a surface-treated synthetic amorphous silica (SAS) show a similar picture of pathology for both non-surface treated SAS and surface treated SAS. Differences in severity of the similar pathological effects might be caused by study design such as exposure conditions, rat strain and test substance differences (particle size, primary structure, surface area, number, density, solubility...). In some studies, at the end of exposure dose-dependent particle related local inflammation in lung and lung associated tissues (lymph nodes) were observed accompanied by corresponding changes of inflammatory marker in the bronchoalveolar fluid. Any clinical effects or morphological changes of other tissues indicating systemic toxicity are not associated with SAS exposure. There are no substantial different pathological findings when comparing different SAS grades. Test item related changes in lungs are dose-dependent and characterized by increased perivascular infiltration, alveolar macrophages and macrophage aggregations, as well as macrophage type II hyperplasia. Accordingly, reactive changes were observed in BALT (bronchus-associated lymphoid tissues) and regional lymph nodes. These effects are not substance-specific, all respirable particles will show the same response directly after exposure1 (day 1). At day 1 the lowest-observed effect concentrations (LOECs) were typically in the range of 0.5 to 50 mg/m³. Based on the biosolubility of SAS reversibility of effects is demonstrated when recovery groups are included in the study with periods of up to one year. Accordingly, in these recovery groups a LOEC and NOAEC are higher. For some SAS grades regional lung lymph nodes show as result of inflammation morphological changes which are not accompanied by any other findings. There are a number of repeated dose studies for SAS available showing a range of LOAEC/NOAEC at the end of recovery. Considering the most conservative values, the LOAEC for SAS is 2.5 mg/m³ at the end of recovery for lung (90-day inhalation toxicity study low specific surface (BET), Fraunhofer ITEM, 2019).

Therefore, in context with "lung overload" described long-term lung effects of low toxic PSPs are not relevant for SAS, confirmed by a Pathology working group (PWG) review of a sub-chronic (13-week) inhalation toxicity study of aerosols of AEROSIL® 200, AEROSIL® R 974, SIPERNAT® 22 S and quartz in rats (Hardisty et al., 2016). Indications for lung fibrosis, which is an irreversible effect, have been published in literature and were based on study materials of one of the applicants' studies according to OECD TG 413 (Sub-chronic inhalation toxicity, Reuzel et al., 1987; 1991). A pathology working group reviewed this finding using the same (original) tissue slides according to the highest current standards and concluded the rapid clearance of the synthetic amorphous silica products will not lead to persistent inflammation and epithelial cell proliferation and therefore will not result in fibrosis/lung tumour induction (Weber et al. 2018, “Aerosols of synthetic amorphous silica do not induce fibrosis in lungs after inhalation: Pathology working group review of histopathological specimens from a sub-chronic 13-week inhalation toxicity study in rats”. Toxicology Research and Application 2: 1–17). Irreversible lung fibrosis has not been associated to any of the eight sub-chronic studies to SAS. These results were confirmed by the recent studies requested by ECHA with high and low surface area pyrogenic SAS, regarded as the worst case of synthetic amorphous silica forms, which showed no lung fibrosis and accordingly no increase of collagen (90-day inhalation toxicity studies, Fraunhofer ITEM, 2019).

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: dermal - local effects

Link to relevant study records

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Reference 1

Endpoint:: short-term repeated dose toxicity: dermal
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
Qualifier:: no guideline available
Principles of method if other than guideline:: 2 dose l, 18 hrs/d, 5 d/wk
21-Day dermal exposure study
GLP compliance:: no
Limit test:: yes
Specific details on test material used for the study:: Cab-O-Sil
Species:: rabbit
Strain:: not specified
Remarks:: albino
Details on species / strain selection:: 1.5 - 1.9 kg
Sex:: male/female
Details on test animals or test system and environmental conditions:: Throughout the study the animals were housed individually in metal cages elevated above the droppings. Food, consisting of Purina Rabbit Pellets and water, was available at all times.
Type of coverage:: not specified
Vehicle:: methylcellulose
Details on exposure:: on intact and abraded skin (2 animals each per dose group) in 0.5 % methyl cellulose
Duration of treatment / exposure:: 3 wks
Frequency of treatment:: 18 h/d, 5 d/wk
Dose / conc.:: 5 000 mg/kg bw/day (nominal)
Dose / conc.:: 10 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:: 2
Control animals:: yes, concurrent vehicle; other: positive control: cosmetic talc
Positive control:: #1625 Cosmetic talc (10 g/kg bw/d)
Clinical signs:: no effects observed
Dermal irritation:: effects observed, non-treatment-related
Description (incidence and severity):: In general the animals of each control and test group exhibited mild erythema and mild to moderate atonia consistently throughout the experimental period. From the beginning of the second week until tne time of sacrifice the exposed skin of each animal showed mild or moderate desquamation. Among the control and test rabbits with abraded skin areas, there appeared to be complete healing of the skin during each week foilowing the periodic abrasions. The skin immediately surrounding the abrasions showed a slightly greater degree of erythema than the remaining exposed skin. Otherwise, the degree of irritation was comparable among the intact and abraded skin animals of each group.
Mortality:: mortality observed, non-treatment-related
Description (incidence):: one death in control group
Body weight and weight changes:: effects observed, non-treatment-related
Description (incidence and severity):: The majority of the animals in test groups receiving CAB-O-SIL gained weignt at a normal rate throughout the experimental period. The initial weight loss among several animals in the control groups was attributed to the initial challenge from infection; however during the latter part of the study these animals showed gains in body weight.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: Examination of the analytical data shows that there was no significant difference in the silicon dioxide content of urine, blood, kidney, liver or spleen between the control and test animals.
Urinalysis findings:: effects observed, non-treatment-related
Description (incidence and severity):: Examination of the analytical data shows that there was no significant difference in the silicon dioxide content of urine, blood, kidney, liver or spleen between the control and test animals.
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: At gross autopsy performed following sacrifice the livers of the majority of the animals contained parasitic areas which resembled coccidiosis. In addition the mesentery of one animal contained parasitic cysts resembling larval tapeworm cysts.Otherwise the organs of each animal appeared grossly within normal limits.
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: effects observed in control and treated groups
: Key result
Dose descriptor:: NOAEL
Effect level:: >= 10 000 mg/kg bw/day (nominal)
Sex:: male/female
: Key result
Critical effects observed:: no
Conclusions:: Under the test conditions employed there was no evidence of systemic toxicity or of gross or microscopic pathology which could be associated witn application of CAB-O-SIL. The silicon dioxide content of blood, urine, spleen, liver, and kidney was comparable for control arid test animals.
Each control material and CAB-O-SIL produced a mild dermal irritation consisting of erythema, atonia, and desquamation. There appeared to be no significant difference in dermal irritative potential between the methyl cellulose solution, #1625 Cosmetic.Talc and CAB-O-SIL.
Executive summary:: The 21d dermal toxicity potential of Cab-O-SIL was examined in rabbits.

Reference 2

Endpoint:: short-term repeated dose toxicity: dermal
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: other:
Critical effects observed:: not specified

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Additional information

The established NOEC and NOAEC levels in the conducted inhalation toxicity studies are valid for rats under the conditions of these studies. In toxicology there are limited models available to translate the obtained results to human (Riediker et al. 2019, Particle and Fiber Toxicology). Anatomy and physiology of the respiratory system differs extremely between both species. This is even not related to gross anatomy but to the inner lung surface and to the histological architecture. The differences are summarized by Miller, Mercer and Crapo (1993). The fundamental differences in pulmonary responses between experimentally-exposed rats, other experimental species and occupationally-exposed humans are reviewed by D.B. Warheit et al. (Toxicology 374, 2016).

The volume of a reactive macrophage may fill the alveolar volume in a rat completely. This is not the case in human, i.e., the volume of macrophage in humane may reach the same size, however, the alveolar volume is higher. The above-mentioned facts result in a fully different translocation of a particulate test item within the lungs. The alveolar lumen is higher. Involved cells are differently distributed. Coughing off material from lower airways is easy for human but not for rats. All these facts making it difficult to translate the effects noted in this study to human, especially, taking into consideration epidemiology studies that did not reveal an impact of SAS in human (EPA, 2017; Merget et al., 2002; OECD, 2017; ECETOC 2012; Bos et al. 2019).

Justification for classification or non-classification

Synthetic amorphous silicas did not induce toxicity or lethality by any route of exposure. For the inhalation route the results confirm biosolubility of SAS over time.
According to Annex I of the Regulation (EC) 1272/2008 and GHS (Globally Harmonized Classification System), the test substance requires no classification and has no mandatory label requirement.

Registration Dossier

Registration Dossier

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Diss Factsheets

Silicon dioxide

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

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Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

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Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Link to relevant study records

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Endpoint conclusion

Additional information

Justification for classification or non-classification

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