Reaction products of sodium glucoheptonate with iron sulfate and ammonium hydroxide

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

A novel platform for pulmonary and cardiovascular toxicological characterization of inhaled engineered nanomaterials

Author:

Sotiriou, G.A., Diaz, E., Long, M.S., Godleski, J., Brain, J., Pratsinis, S.E., Demokritou, P.

Year:

2012

Bibliographic source:

Nanotoxicology. 2012 September ; 6(6): 680–690. doi:10.3109/17435390.2011.604439.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Each day two animals were exposed to Fe2O3 test aerosol and two animals to filtered air (sham) in individual exposure chambers for 5 hours. Four repetitions (days) with a new set of animals each day were carried out. At the end of each five-hour exposure, two animals from each group had in-vivo chemiluminescence IVCL (Boveris et al. 1980). The IVCL technique is a highly sensitive method for identifying cardiopulmonary responses to inhaled ENMs under relatively small doses and acute exposures.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diiron trioxide

EC Number:

215-168-2

EC Name:

Diiron trioxide

Cas Number:

1309-37-1

Molecular formula:

Fe2O3

IUPAC Name:

diiron trioxide

Test material form:

aerosol dispenser: not specified

Details on test material:

nanoparticles: size 4 to 25 nm

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratories (Rensselae, NY)
- Weight at study initiation: 200-250 g
- Housing: housed, and managed according to the NIH guidelines for the care and use of laboratory animals
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
not specified

Male Sprague-Dawley rats (200-250 g) were obtained from Taconic Laboratories (Rensselae, NY), housed, and managed according to the NIH guidelines for the care and use of laboratory animals. Upon arrival, animals were assigned a unique identification number, which determined the exposure date and exposure group (Aerosol or Filtered Air) for the animal. Rats were allowed to acclimate to the animal facility for 4-5 days prior to start of experiments. The Harvard Medical Area’s Animal Use Committee approved the animal protocols used in this study. Each day two animals were exposed to Fe2O3 test aerosol and two animals to filtered air (sham) in individual exposure chambers for 5 hours. Four repetitions (days) with a new set of animals each day were carried out. At the end of each five-hour exposure, two animals from each group had IVCL (Boveris et al. 1980).

Administration / exposure

Route of administration:

inhalation

Details on inhalation exposure:

Please refer to "Any other information on materials and methods"

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Total particle number concentration (CT) of the test ENM aerosol containing Fe2O3 nanoparticles was monitored continuously. It remains constant at ca. 2-3·105 #/cm3. Every 25 minutes there was a 5 minutes break during the system was filled up (gray area).

Duration of exposure:

5 h

Remarks on duration:

Each day two animals were exposed to Fe2O3 test aerosol and two animals to filtered air (sham) in individual exposure chambers for 5 hours. Four repetitions (days) with a new set of animals each day were carried out.

Concentrations:

Total particle number concentration (CT) of the test ENM aerosol containing Fe2O3 nanoparticles remains constant at approximately 2-3·105 #/cm³. Every 25 minutes there was a 5 minutes break during the system was filled up (gray area).

No. of animals per sex per dose:

Each day two animals were exposed to aerosol and two animals to filtered air. Four repetitions (days) with a new set of animals each day were carried out.

Control animals:

yes

Details on study design:

Please refer to "Any other information on materials and methods"

Statistics:

not specified

Results and discussion

Effect levels

Remarks on result:

other: exposures to inhaled nanostructured Fe2O3 can cause both pulmonary and cardiovascular effects.

Remarks:

test concentration: 2-3·10E5 particles/cm³ (this would correspond to approximately to 100-200 μg/m³)

Mortality:

no data

Clinical signs:

other: At the end of each five-hour exposure, two animals from each group showed an in-vivo chemiluminescence (IVCL) difference (counts per second-cps/ cm²) of lungs and hearts, which corresponds to the relative reactive oxygen species (ROS) concentration.

Body weight:

no data

Gross pathology:

no data

Other findings:

no data

Any other information on results incl. tables

Here, a novel technique is presented which is suitable for both ENM in-vivo inhalation and in-vitro toxicological characterization studies. The ability of this technique to generate a variety of industry-relevant, property-controlled exposure atmospheres for inhalation studies was systematically investigated. The suitability of the technique to characterize the pulmonary and cardiovascular effects of inhaled ENM in intact animal models was also demonstrated in an in-vivo study involving Sprague-Dawley rats, using freshly generated nano-iron oxide (Fe₂O₃) as a test aerosol. Both pulmonary and systemic toxicity was demonstrated

using in-vivo chemiluminescence of heart and lung. This novel platform will make it possible for toxicologists to link physico-chemical properties of inhaled ENMs to biological outcomes and help the industry to develop safer ENM.

RESULTS

Performance characterization experiments

Effect of x/y ratio on both the aerosol size (airborne phase) and primary particle size

Figure 2d show the effect of x/y ratio on both the primary particle and the mobility diameter of Fe₂O₃. Figure 2c also shows the XRD patterns of the ex-situ collected and characterized nanoparticles as a function of the x/y ratio. This indicates that - in agreement with literature - larger Fe₂O₃ particles are formed with higher x/y ratio. This is due to their larger residence time in higher temperature zones that result in larger crystals. It is worth pointing out that the XRD-estimated average crystal particle sizes (filled triangles, Figure 2d) are consistent with their average primary particle sizes (open triangles, Figure 2d), with the latter having slightly larger values indicating polycrystalline or aggregated nanoparticles. The modal mobility diameter for the Fe₂O₃ aerosol (Figure 2d, circles, left axis) is not significantly affected by the x/y ratio, as for most ratios it is fairly constant. Furthermore, as it is shown above in the case of SiO₂, there is a difference between mobility diameter (airborne phase) and the primary particle diameter (nanopowder form).

ENM surface modification

The surface of ENMs can be modified in order to add desired attributes such as dispersibility or antibacterial activity. It may also be a useful concept for the formulation of safer ENM. Recently, it was shown that doping ZnO nanoparticles with Fe can reduce the release of Zn ions. This indicates that VENGES has the ability to generate surface modified nanoparticles while maintaining the intended size distribution. This will be useful when studying the comparative toxicity of surface modified nanoparticles in-vivo. By controlling the size, they can influence control the particle deposition in the lungs. In addition, ENMs can also be used as a “vehicle” for delivery of drugs in-vivo.

Pulmonary and cardiovascular effects of inhaled nanostructured Fe₂O₃ using the VENGES platform

The concentration levels remained fairly constant for the whole animal exposure (the 5 minute stopswere necessary in order to replace the syringe with liquid precursor). For this exposure scenario, VENGES was tuned to generate a test aerosol of a total number concentration of 2-3·10⁵ particles/cm³ (this would correspond to approximately to 100-200 μg/m³), approximately 10-20 times higher concentration of indoor conditions (10 μg/m³). The temperature, relative humidity, CO₂, CO and NO₂ concentrations of the test aerosol were identical to the room conditions, ensuring no interference with the in-vivo toxicological results.

Discussion

A promising technological platform suitable for both in-vitro and in-vivo toxicological characterization of engineered nanomaterials, with emphasis on the cardiovascular and pulmonary effects of inhaled ENM, is presented in this study. ENM are produced continuously in the gas phase allowing their continuous transfer to inhalation chambers, with minimal alterations in their state of agglomeration. Defining properties of the generated aerosols (i.e. primary and aerosol particle size, concentration, shape, state of agglomeration, surface chemistry) can be easily modified by adjusting simple process parameters allowing for both in-vitro and in-vivo investigations of toxicity. The ability of the developed technique to generate a variety of industry relevant, property controlled exposure atmospheres for inhalation studies was systematically investigated and documented in the previous section. The suitability of the technique to characterize the pulmonary and cardiovascular effects of inhaled ENM in intact animal models was also demonstrated here using the highly sensitive IVCL assay.

IVCL measures the reactive oxygen species generation (ROS). ROS and free radical generation is considered one of the primary mechanisms of nanoparticle toxicity; it was shown in many ambient particle health effect studies that ROS generation may result in oxidative stress, inflammation, and damage to proteins, membranes and DNA. ROS generation has been also found in many ENMs including carbon based ENMs (fullerenes, carbon nanotubes) and metal oxides. The results indicate that moderate acute exposures to inhaled nanostructured Fe₂O₃ can generate ROS and oxidative stress and cause both pulmonary and cardiovascular effects. The IVCL measurements in the lungs of the exposed animals were about 60 times higher than for the unexposed animals, indicating that the Fe₂O₃ test aerosol increased ROS in the lungs. This oxidative stress was also present in the heart of the animals showing that the inhalation of ENM influences not only the respiratory but also the cardiovascular system with an 11-fold increase in the chemiluminescence of the heart. This substantial effect was found with a moderate mass exposure concentration of 200 μg/m³, a concentration approximately 20 times the fine particle concentration of room air. The substantial IVCL response observed in this study, indicates that these particles reached deep in the lung and evoked a toxicological effect. The increased IVCL response of the heart may indicate a direct effect on the heart (Godleski 2006) but also may be a manifestation of indirect effects via the autonomic nervous system. It should be noted, however, that the proposed platform is not only limited to the evaluation of pulmonary and cardiovascular effects. It can also be used to assess other biological outcomes related to inhaled ENM and it can be a powerful tool to understand the link between certain ENM properties and their bioavailability and toxicity.

Conclusions

In conclusion, this novel approach enables scientists and laboratory personel to generate industry-relevant, property controlled ENM exposure atmospheres suitable for inhalation toxicological studies and assess the link between ENM physico-chemical properties and specific biological outcomes. In addition, the documented in the study ability of the technique to alter in-situ ENM surface properties can be one of the ways to further explore the formulation of safer ENM. Furthermore, this technological platform can be a powerful tool for validation of in-vitro screening assays with adverse biological effects in intact animals, an important element of the strategy recently proposed by the National Nanotechnology Initiative (NNI) on Environmental Health and Safety of Engineered nanomaterials. Its future use will help to assess the cardiovascular, pulmonary and other toxicological effects of inhaled ENM and improve the understanding on the central hypothesis that physical and chemical characteristics of ENM determine their bioavailability, redistribution, and toxicity in the lungs and elsewhere.

Applicant's summary and conclusion

Executive summary:

A novel method is presented which is suitable for assessing in-vivo the link between the physicochemical properties of engineered nanomaterials (ENMs) and their biological outcomes. The ability of the technique to generate a variety of industry-relevant, property-controlled ENM exposure atmospheres for inhalation studies was systematically investigated. The suitability of the technique to characterize the pulmonary and cardiovascular effects of inhaled ENMs in intact animal models is also demonstrated using in-vivo chemiluminescence (IVCL). The IVCL technique is a highly sensitive method for identifying cardiopulmonary responses to inhaled ENMs under relatively small doses and acute exposures.

Each day two animals were exposed to Fe₂O₃test aerosol and two animals to filtered air (sham) in individual exposure chambers for 5 hours. The test concentration was 2-3·10⁵particles/cm³ (this would correspond to approximately to 100-200 μg/m³). The primary particle size for Fe2O3 was controlled from 4 to 25 nm, while the corresponding agglomerate mobility diameter of the aerosol was also controlled and varied from 40 to 120 nm.

Four repetitions (days) with a new set of animals each day were carried out.

It is shown that moderate and acute exposures to inhaled nanostructured Fe₂O₃ can cause both pulmonary and cardiovascular effects.