Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct. 31, 2019 - Oct. 28, 2020; experimental phase: Nov. 19, 2019 - Mar. 17, 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
No validated in vitro method is available for assessing systemic toxicity after repeated exposure.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Jun. 25, 2018
Deviations:
yes
Remarks:
minor ones, not influencing the quality or integrity of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Silicon dioxide
EC Number:
231-545-4
EC Name:
Silicon dioxide
Cas Number:
7631-86-9
Molecular formula:
O2Si
IUPAC Name:
Silicon dioxide
Test material form:
solid: particulate/powder
Remarks:
amorphous
Specific details on test material used for the study:
Name: Sipernat 22 S
CAS No.: 112916-00-8, 7631-86-9
Chemical Name: synthetic amorphous silica
Production route: precipitation
E-Number (Food Permission): E 551
Batch No.: 199021523
Physical State: solid powder
Colour: white
Storage Conditions: at room temperature, protected from moisture
Active Components: >97% (impurities were not taken into account for final formulation)
Date of Manufacture: 15 February 2019
Date of Analysis: 25 March 2019
Expiry Date: 14 February 2021

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex:
male/female
Details on test animals or test system and environmental conditions:
- age at the start of the treatment period: approx. 4-6 weeks old
- body weight at the allocation of the animals to the experimental groups:
males: 148 – 181 g (mean: 162.3 g, ± 20% = 129.9 – 194.8 g)
females: 114 – 151 g (mean: 128.2 g, ± 20% = 102.6 – 153.8 g)

- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats
- Free access to tap water in bottles, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals). The purpose of the acidification was to provide additional protection against microbial contamination. This is a standard practice at BSL Munich and should not be associated with any risk for long-term studies.
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Name: aqua ad injectionem Manufacturer: Delta Medica Batch No.: 906243 Physical State: liquid Storage Conditions: room temperature Expiry Date: May 2022
Details on oral exposure:
The test item formulation or vehicle was administered daily to the animals as a single dose by oral gavage. The application volume for all groups was 7.5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about concentration (±10%) of the test item in the selected vehicle. Investigation of stability was performed in the first study week. Investigation of homogeneity and substance concentration was included on samples from various levels (top, middle and bottom) of prepared formulations study weeks 1, 5, 9 and 13.

Dose Formulation Analysis
Recoveries of reference substance Sipernat 22 S were found between 88 and 95%. Samples of sampling week 5 and 13 fulfilled the requirement of ± 10% nominal test item concentration. Recoveries of study week 1 and 9 reached 88%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrations
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (+ 5 in satellite group for recovery studies)
Control animals:
yes, concurrent vehicle
Details on study design:
additional 28 days of recovery for the satellite group
Positive control:
none

Examinations

Observations and examinations performed and frequency:
Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.
Food consumption was measured weekly during the treatment and recovery period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days. The recovery animals were observed for an additional period of 28 days following the last administration.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed clinical observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and weekly thereafter.

Functional Observations and Ophthalmology
Once before the first exposure and in the last week of the exposure period as well as in the last week of the recovery period multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals.
Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals.

Haematology
Haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.


Sacrifice and pathology:
Blood Coagulation
Coagulation parameters were examined at the end of the treatment and recovery period. After overnight fasting, blood was collected from the abdominal aorta of the animals during terminal anaesthesia in citrate tubes.

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.

Urinalysis
One day after the last administration, urinary parameters from all surviving animals to be subjected to necropsy at the end of the treatment or recovery period were analysed. Urine colour and appearance were recorded. Parameters were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL).

Pathology
One day after the last administration (study day 91) all surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119) were sacrificed using anaesthesia (ketamine/xylazin) and were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Vaginal smears were examined on the day of necropsy to determine the stage of oestrous
cycle.

Organ Weight
The wet weight of the organs of all sacrificed animals was recorded as soon
as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.

Histopathology
The weighted organs were examined histopathologically after preparation of
paraffin sections and haematoxylin-eosin staining for the animals of the control and high dose groups sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a nonparametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or
GraphPad Prism V.6.01 software (p < 0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Few animals showed abnormal breathing. Piloerection was seen in 12 of 30 animals, but this was regarded rather a reaction of discomfort to application and not considered as test item related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
2/30 in total (one male and one female) in treated and recovery groups
No test item related mortality occurred during the treatment or recovery period of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicological relevant effects on mean body weight and body weight development were observed during the treatment or recovery phase of the study in the dose groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicological relevant effects on food consumption were determined during the
treatment or recovery phase of the study in the dose groups.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicological relevant effects on parameters of haematology or blood coagulation were detected at the end of the treatment or recovery period in the dose groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicological relevant effects on hormones or parameters of clinical
biochemistry were detected at the end of the treatment or recovery period in the
dose groups.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicological relevant effects on urinary parameters were detected at the end of the treatment or recovery period in the dose groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No toxicological relevant observations were made during the functional observation
battery during the treatment or recovery phase of the study in dose groups.
Immunological findings:
no effects observed
Description (incidence and severity):
Enhanced immune system evaluation did not reveal indicators for an affection of the immune system by the test substance.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Mean thyroid/parathyroid weights, thyroid/parathyroid to body weight ratios as well as thyroid/parathyroid to brain weight ratios of Sipernat 22 S treated males were slightly increased compared to controls, with differences in thyroid/parathyroid to body weight ratios reaching statistical significance.
Mean weight: 0.045 g vs. 0.033 g in controls
Organ/Body: 0.0116% vs. 0.0075% in controls
Organ/Brain: 2.13% vs. 1.56% in controls
As effects were small and not observed in females, these findings were considered as incidental.

Test item treated males had statistically significantly decreased epididymides to brain weight ratios (77.40%) compared to controls (83.38%). Mean epididymides weights were also decreased, yet not significantly.
Mean weight: Control: 1.74 g, Sipernat 22 S: 1.65 g
As effects were small and not observed in females, these findings were considered as incidental.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Only three of thirty animals treated with Sipernat 22 S showed pathological abnormalities:
- The thymus of male no. 12 had an abnormal brown/red colour, caused by congestion, which was considered to be incidental.
- The lungs of prematurely euthanized male no. 17 had an abnormal spotted colour, failed to collapse at necropsy and were fluid filled and spongy.
Histopathological analyses revealed that in the lungs, there was an intrabronchial foreign material associated with a bronchial/bronchiolar inflammation, a consequence of test item aspiration.
- Prematurely euthanized female no. 120 died of oesophageal perforation by misgavage. Consequently, the thoracic cavity was filled with a white, viscous fluid. Histopathological analysis revealed inflammation in the oesophagus, the
heart and the lung, leading to an adhesion between heart and lung. The thymus also exhibited inflammation and hence was enlarged and had an abnormal brown/red colour. The duodenum, jejunum and ileum were filled with a yellow fluid, a finding which was without a histopathological correlate. The thyroid gland also had an abnormal red colour, which was considered as incidental.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No pathological change was recorded in the nervous system or the intestinal segments, and specifically, no unusual findings were noted in the Peyer’s patches.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related gross lesions or histological findings in any of the organs and tissues examined that could be attributed to treatment with the test item. There were no findings that distinguished controls from test item-treated animals.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related gross lesions or histological findings in any of the organs and tissues examined that could be attributed to treatment with the test item. There were no findings that distinguished controls from test item-treated animals.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
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
immunology
mortality
neuropathology
organ weights and organ / body weight ratios
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

No test item related mortality occurred during the treatment or recovery period of the study. No toxicologically relevant clinical findings were observed in the dose groups during the treatment or recovery period of the study.
Detailed clinical observations and the functional observation battery revealed no noticeable or toxicological relevant findings in the dose groups during the treatment or recovery period of the study.
No toxicological relevant effects on body weight or food consumption were observed during the treatment or recovery phase of the study in the dose groups.
No toxicological relevant effects regarding haematology, clinical biochemistry, hormones, blood coagulation, urine, organ weights or vaginal smears were detected at the end of the treatment or recovery period in the dose groups.
No test item related macroscopic findings were observed during necropsy.
There were no treatment-related gross lesions or histological findings in any of the organs and tissues examined that could be attributed to treatment with the test item. No pathological change was recorded in the nervous system or the intestinal segments, and specifically, no unusual findings were noted in the Peyer’s patches. In addition, enhanced immune system evaluation did not reveal indicators for an affection of the immune system.
Therefore, the oral application of Sipernat 22 S for 90 days at a dose of 1000 mg/kg was deemed to be toxicologically not harmful.

Applicant's summary and conclusion

Conclusions:
The oral application of Sipernat 22 S for 90 days at a dose of 1000 mg/kg was deemed to be toxicologically not harmful.
Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Macrosorb F60, Zeofree 5133 and Sipernat 22 S via oral administration to rats over a period of 90 days. Only the results on the latter are documented here, since Sipernat 22 S (a synthetic amorphous silica) is a structurally related read-across substance to Silicic acid, aluminum sodium salt (CAS 1344-00-9, NAS). No adverse effects were found with the fixed dose of 1000 mg/kg bw/d.