Mill scale (ferrous metal)

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Following deficiencies were observed for the study: non-physiological route of administration (intranasal); number of animals too low; males tested only, one concentration only; justification for selection of concentration, vehicle and route of adminsitration missing; parameters missing (detailed clinical observations, functional tests, gross pathology, body weight and food consumption); incomplete histopathology; individual data missing

Data source

Reference

Reference Type:

publication

Title:

Neurotoxic potential of iron oxide nanoparticles in the rat brain striatum and hippocampus

Author:

Wu J, Ding T, Sun J

Year:

2013

Bibliographic source:

NeuroToxicology 34 (2013) 243–253

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the current study, groups of 6 male Sprague Dawley rats were received 10 µg iron oxide nanoparticles (30 nm; radiolabelled with Iodine-125) in physiological saline via intranasal administration. One group was treated with the test item once (day 1) and animals were sacrifice on day 2. Three groups were given the test item on 7 consecutive days and were sacrificed on days 8, 14 or 21, respectively. A vehicle control group was run concurrently. Clinical signs and mortality were recorded. Furthermore, the brain (olfactory bulb, striatum, hippocampus, frontal cortex, cerebellum and brain stem) was excised and investigated for iron oxide nanoparticle content by radioactivity measurements. Histopathology was conducted on the brain as well as oxidative stress markers (glutathione reductase (GSH-PX), superoxidase dismutase (SOD) activity, hydrogen peroxide (H2O2) and malondiadehyde (MDA)) were measured in the striatum and hippocampus.

GLP compliance:

not specified

Remarks:

not specified in the publication

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Triiron tetraoxide

EC Number:

215-277-5

EC Name:

Triiron tetraoxide

Cas Number:

1317-61-9

Molecular formula:

Fe3O4

IUPAC Name:

Iron oxide

Test material form:

solid: nanoform

Details on test material:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: Sigma Chemical Company (Saint Louis, MO, USA)

The average size of the nanoparticles was confirmed by transmission electron microscopy (TEM, JEM-2010). The sizes and charges of the nanoparticles and agglomerates were also measured by dynamic light scattering (DLS) and zeta potential, respectively. Nanoparticles were suspended in physiological saline or high glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS); the nanoparticle measurements were taken on a ZetaSizer.

Results:
- Average size of nanoparticles (TEM): 30 nm
- Shape of particles: spherical
- Hydrodynamic diameter (physiological saline): 496 ± 86 nm
- Hydrodynamic diameter (DMEM): 570 ± 103 nm
- Zeta potenital (physiological saline): -9.1 ± 2.2 mv
- Zeta potenital (DMEM): -8.5 ± 1.7 mv

RADIOLABELLING INFORMATION
The 125I radiolabelling of the Fe3O4-nanoparticles (Fe3O4-NPs) was described by Xie et al. (2010)*. Amino groups were added to the Fe3O4-NPs via aminopropyltriethoxysilane (APTS) modification. The modified Fe3O4-NPs were then incubated with Bolton–Hunter (BH) reagent, dimethylformamide and Na125I suspended in chloramine. The radiolabelling reaction was terminated by the addition of glycine-supplemented borate buffer. The 125I-Fe3O4-NPs were ultimately separated by column chromatography and characterised by TEM and Zetasizer. Instant thin layer chromatography–silica gel (ITLC-SG) and a γ-counter to identify the 125I-Fe3O4-NPs in solution were used. Prior to the in vivo assay, the stability of 125I-Fe3O4-NPs was analysed in 37 °C physiological saline containing 10% (v%) rat serum. Two microliter samples of the 125IFe3O4-NP suspension were extracted at five time points after instillation (1, 3, 7, 15, and 30 days) and analysed by ITLC-SG paper chromatography. The stability of the 125I-Fe3O4-NPs was expressed as a percentage of radioactivity at the site of instillation compared with the total radioactivity.

Results:
125I-radiolabelled test item:
- Average size of nanoparticles (TEM): 36 nm
- Shape of particles: spherical
- Hydrodynamic diameter (physiological saline): 462 ± 75 nm
- Zeta potenital (physiological saline): -8.4 ± 2.1 mv

Statistical comparisons showed no significant increases in size and zeta potential in the labelled particles (p > 0.05).

125I moved toward the solvent front (Rf = 0.9) faster than 125I-BH (Rf = 0.7), but the 125I-Fe3O4-NPs remained at the point of spotting (Rf = 0) with the labelling yield reaching 95%. Examinations of radiolabelling stability shows that the 125I-Fe3O4-NPs were stable for 30 days in vitro and consistently exceeded 95% in purity.

*References:
- Xie G, Sun J, Zhong G, Shi L, Zhang D. Biodistribution and toxicity of intravenously administered silica nanoparticles in mice. Arch Toxicol 2010;84:10.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SLACCAS Laboratory Animal Co., Ltd. (Shanghai, China)
- Age at study initiation: 4 weeks old
- Housing: 6 rats/cage
- Diet (ad libitum): rodent diet
- Water (ad libitum): water
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 0.5 °C
- Humidity: 50 ± 5 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

other: intranasally

Vehicle:

physiological saline

Remarks on MMAD:

MMAD and GSD not specified

Details on exposure:

TEST SUBSTANCE PREPARATION
125I-Fe3O4-nanoparticles were suspended in physiological saline (1 mg/mL) and sonicated with a Hilscher UP200S; this sonicator generates ultrasonic pulses of 600 W at 20 kHz and was used to sonicate the nanoparticles for 30 minutes to prevent aggregation prior to nasal instillation.

TEST SUBSTANCE ADMINISTRATION
Supine rats were lightly anaesthetised with sodium pentobarbital, and 10 µg (in 10 µL) of 125I-Fe3O4-NPs was intranasally instilled in each rat nostril (20 µg total).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Group 1: administered once
Group 2 to 4: 7 days

Frequency of treatment:

once

Doses / concentrations

Dose / conc.:

10 other: µg

No. of animals per sex per dose:

6 rats/group (total of 24 rats)

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure recovery period (groups 3 and 4): one group (group 3) was treated with the test item for 7 days followed by a recovery period of 7 days (day of sacrifice: 14), whereas the other group (group 4) was treated with the test substance for 7 days followed by a recovery period of 14 days (day of sacrifice: 21).

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: at sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data
OPHTHALMOSCOPIC EXAMINATION: No data

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No data
CHOLINESTERASE ACTIVITY: No data

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: No data
LOCOMOTOR ACTIVITY: No data
AUDITORY STARTLE REFLEX HABITUATION: No data
LEARNING AND MEMORY TESTING: No data

Sacrifice and (histo)pathology:

- Time point of sacrifice (day 1 is day of adminsitration):
- group 1: day 2
- group 2: day 8
- group 3: day 14
- group 4: day 21

- Number of animals sacrificed: all animals of all groups were killed at the relevant time point of sacrifice.

After sacrifice, brain tissues were perfused from every animal. Regions of the sub-brain – including the olfactory bulb, striatum, hippocampus, frontal cortex, cerebellum and brain stem – were separated, gathered, weighed, and counted in a γ-counter for 15 minutes to measure radioactivity (expressed in units of ng/g of tissue).

The following investigations were performed:
1) Oxidative stress-related biomarker assay
The striatum and hippocampus samples of all animals in each group were weighed and suspended in cold protein lysis buffer when the samples were at the ratio of 1:9 (w/v). The protein lysis buffer included 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% 2-mercaptoethanol, 1% NP-40, 0.25% sodium deoxycholate, 5 µg/mL leupeptin, 5 µg/mL asprotinin, 10 µg/mL soybean trypsin inhibitor and 0.2 mM phenylmethyl sulfonylfluoride. Each tissue mixture was homogenised four times with an ultrasonic cell disruptor (Sonics Vibra-Cell, VCX105) for 8 seconds intervals at 4 °C. The solution was centrifuged at 14,000 x g for 5 minutes at 4 °C, and the resulting supernatant was collected for oxidative biomarker analysis (glutathione reductase (GSH-PX), superoxidase dismutase (SOD) activity, hydrogen peroxide (H2O2) and malondiadehyde (MDA)).

2)Histopathological examination
All of the rat brains were excised and immediately fixed in a 10% formalin solution. Histopathological examinations were conducted using standard laboratory procedures. The brains were embedded in paraffin blocks, sectioned into 5-µm-thick slices, mounted on the microscope glass slides and stained with haematoxylin–eosin. The sections were then observed under optical microscopy and photographed.

Positive control:

not specified

Statistics:

The sample radioactivity was determined using the equation C = C0 X e/(0.693159t/T) (with C being the actual radioactivity, C0 is the measured radioactivity, T is the half-life of 125I (59.6 days), and t the time interval between C and C0). The data were expressed as the means ± SD. The statistical analyses were performed using SPSS 12.0, and the statistical comparisons were analysed by the Student’s t-test. The differences were considered to be statistically significant when the p-values were less than 0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

not specified

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 specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BIODISTRIBUTION; ACCUMULATION AND RETENTION
- group 2: sub-brain regions were measured for test item deposition on the seventh day of instillation exposure. The nanoparticles were deposited from highest to lowest concentration in the olfactory bulb, striatum, hippocampus, brain stem, cerebellum, and frontal cortex, respectively.
- there were no significant changes in any of the sub-brain regions (p > 0.05), except in the olfactory bulb.
- a significant increase in nanoparticle deposition from 1 day to 7 days of instillation exposure (p < 0.05) was observed.
- the clearance of the test item from the brain regions was slow, as more than half of the test sustance in the striatum and hippocampus remained 14 days post-instillation.

Please also refer to the section "Overall remarks, attachments".

OXIDATIVE STRESS-RELATED BIOMARKER ASSAY
- striatum: seven days after the nanoparticles were instilled in the striatum, significant increase in the oxidative damage marker hydrogen peroxide (H2O2; p < 0.05) compared with controls was observed. Significant decrease between the control and experimental groups in the striatum levels of glutathione reductase (GSH) were detected 7 days following instillation (p < 0.05).
- hippocampus: the levels of H2O2 in the hippocampal tissue following 7 days of instillation were significantly different from those of the control (p < 0.05). However, there were no significant changes in the other markers (GSH, superoxidease dismutase (SOD), and malondiadehde (MDA)) in the hippocampal tissue at any of the time points.

Please also refer to the section "Overall remarks, attachments".

Details on results:

CLINICAL SIGNS
- none of the animals exhibited any signs of acute toxicity or clinical signs of abnormalities during the experimental period

MORTALITY
- none of the animals died during the test period.

HISTOPATHOLOGICAL FINDINGS. NON-NEOPLASTIC
Histopathological examination showed that no significant exposure-related brain lesions were present in rat brains intranasally instilled with the test item compared with lesions in physiological saline-exposed controls. This finding was observed upon examining rat brains at all experimental time points.

Please also refer to the section "Overall remarks, attachments".

Effect levels

Remarks on result:

other: see "Remarks"

Remarks:

No signs of acute toxicity or clinical signs of abnormalities as well as no mortality were observed after test item administration. Furthermore, histopathological examination of the brain showed no significant exposure-related brain lesions in rats receiving the test item intranasally compared to the control animals that received physiological saline. After intranasal administration of the test item, the highest concentrations of the test item were found in the olfactory bulb, striatum and hippocampus. The clearance of the test substance from the brain regions was slow, as more than half of the test item in the striatum and hippocampus remained in these regions 14 days post instillation (sacrifice on day 21). Furthermore, the striatum exhibited a greater vulnerability to oxidative stress. Seven days after the test substance was instilled significant increase and decrease in the oxidative markers hydrogen peroxide (H2O2) and GSH ( glutathione reductase) was observed compared to the controls, respectively. Apart from H2O2, no significant changes in the remaining markers of oxidative stress (GSH, superoxidase dismutase (SOD), and malondiadehyde (MDA)) were observed in the hippocampus.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Besides the examinations, as described above, in vitro investigations were presented in the publication, which are not relevant for human risk assessment. These investigations are included for the sake of completeness only.

The in vitro studies demonstrated that iron oxide nanoparticles may decrease neuron viability, trigger oxidative stress and activate JNK- and p53-mediated pathways to regulate the cell cyle and apotosis.

Applicant's summary and conclusion

Conclusions:

In the current study, groups of 6 male Sprague Dawley rats were received 10 µg iron oxide nanoparticles (30 nm; radiolabelled with Iodine-125) in physiological saline via intranasal administration. One group was treated with the test item once (day 1) and animals were sacrifice on day 2. Three groups were given the test item on 7 consecutive days and were sacrificed on days 8, 14 or 21, respectively. A vehicle control group was run concurrently.

The results showed no signs of acute toxicity or clinical signs of abnormalities as well as no mortality after test item administration. Furthermore, histopathological examination of the brain showed no significant exposure-related brain lesions in rats receiving the test item intranasally compared to the control animals that received physiological saline. After intranasal administration of the test item, the highest concentrations of the test item were found in the olfactory bulb, striatum and hippocampus. The clearance of the test substance from the brain regions was slow, as more than half of the test item in the striatum and hippocampus remained in these regions 14 days post instillation (sacrifice on day 21). Furthermore, the striatum exhibited a greater vulnerability to oxidative stress. Seven days after the test substance was instilled significant increase and decrease in the oxidative markers hydrogen peroxide (H2O2) and GSH ( glutathione reductase) was observed compared to the controls, respectively. Apart from H2O2, no significant changes in the remaining markers of oxidative stress (GSH, superoxidase dismutase (SOD), and malondiadehyde (MDA)) were observed in the hippocampus.

This reference had several reporting and experimental deficiencies, which do not allow an independent review about the exposure-effect correlation:

Firstly, intranasal administration is a non-physiological route of administration and not relevant for human risk assessment.

The OECD guideline 424 foresees that at least 20 animals (10 females and 10 males) will be tested. At least 5 animals /sex/groups should be used for neurohistopathology However, in the current study groups of 6 male rats were examined. Females were not investigated in this study and, therefore it is impossible to draw any conclusion on the effects of the substance on females. In addition, the overall number of animals used per group is too low, since 6 males only were tested per group. This reduction in number of animals causes a significant reduction of the statistical power.

The OECD guideline 424 foresees that at least three concentrations and a concurrent control should be used. In this study, one concentration of 10 µg was tested only. The usage of one concentration precludes the possibility to demonstrate any concentration-related response and the determination of a No-Observed-Adverse Effect level (NOAEL). Furthermore, the authors did not verify, if the animals received the proposed concentration and they did not state why they chose this concentration as well as the route of administration and vehicle.

Also, some parameters stated by the OECD guideline 424 were not investigated. Detailed clinical observations and functional tests were not conducted. Therefore, it cannot be confirmed that the findings made in the current study do have an adverse effect on the neurobehaviour and survival of the animals. Moreover, gross pathology was missing, and the histopathology conducted was incomplete, which makes it impossible to verify, if the substance had further effects on nervous system. Lastly, food consumption and body weight measurements were not made or presented, which are important parameters for investigating systemic toxicity of a substance.

Lastly, individual data were missing, which precludes the possibility to find outliners. In addition, the results presented in the text and graphics did not correspond with each other, which precludes the possibility to draw any reliable conclusions from this study.