Silicon dioxide

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

O2Si

IUPAC Name:

dioxosilane

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Precipitated, CAS-No.: 112926-00-8, Surface area / BET [m2/g]: 160

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

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

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)

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

No death occurred during the study and no adverse clinical symptoms were observed. No effects on food consumption or body weight were seen. The measurements of the spontaneous locomotor activity and the functional observational battery displayed no influence by the treatment. Evaluation of hematological and clinical chemistry parameters did not reveal any treatment related effects. Decreases of the partial thromboplastin time (PTT), white blood cell count (WBC) and lymphocyte count (LYMC) as well as cholinesterase (CHE) and glucose (GLUC) in group 3 (mid dose) after 28 d of exposure were considered not treatment related. Creatinin kinase (CK) and blood urea (UREA) concentration were mildly decreased in group 6 (high dose recovery) after a two week recovery period. All values were within the normal range and the changes were considered not treatment related and to be due to interindividual variability.

During necropsy, no substance-related findings were observed. No effects were seen on organ weights or the organ weight to bodyweight ratio. During histopathological examination, no substance-related findings were observed in the examined organs of males of the control and high dose group.

Applicant's summary and conclusion

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.