The parent sample consisted of soil from the Apollo 14 mission (sample no. 14003,96). Figure
shows a scanning electron micrograph of the dust containing iron metal inclusions. The material was separated by pneumatic means within a glovebox containing ultrapure nitrogen (0.5 ppm H2
O, 20.6 ppm O2
), using the technique described in Cooper et al.
]. To determine the mechanical irritancy or abrasiveness of the lunar dust, the fraction of material separated by pneumatic means described above with a mean particle size of 50.9
19.8 μm was used for in vivo
Apollo 14 lunar dust image. A scanning electron micrograph image of authentic lunar dust with iron metal inclusions.
To prepare the sample for in vitro
testing, an in-house jet-mill grinding method was used to grind coarse lunar soil grains to produce smaller particles and restore surface reactivity which may have been lost after years of storage. This method allowed us to test for the maximum chemical irritancy potential of the lunar dust, since grinding is expected to lead to a higher degree of surface reactivity due to the generation of silicon- or oxygen-based radicals (“dangling bonds”), which can react with water to produce hydroxyl radicals. It is also possible that grinding of lunar dust exposes reduced iron, which can react with oxygen and water to produce ROS, including hydrogen peroxide or superoxide
]. The material was ground to a median particle diameter of 2.9
1.0 μm. Previous work has shown that surface reactivity generated by grinding is greatly reduced or “passivated” over the course of a few hours by contact with humidity and atmospheric oxygen
]. Consequently, all handling of the lunar soil (both separation and grinding) was performed in an ultrapure nitrogen environment to minimize its exposure to reactive atmospheric species until the tissues were dosed.
In vitro testing
ocular testing was performed by Stillmeadow, Inc. using the commercially available EpiOcularTM
model. This model utilizes human-derived epidermal keratinocytes that have been cultured to form a stratified corneal epithelium
]. Cell viability following exposure to lunar dust was determined by conversion of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) and was expressed as a percentage relative to untreated (negative control) tissues.
Tissue Exposure - MatTek assay medium (MTT-100-ASY) was pre-warmed to 37°C in a 5% CO2 incubator, and 0.9 mL was aliquoted into each well of a sterile 6-well plate. Each insert, containing room-temperature EpiOcularTM tissue, was aseptically placed in one of six wells containing pre-warmed assay media and incubated for one hour at 37°C, under 5% CO2. Following incubation, the media was aspirated and then replaced with an identical volume of pre-warmed media. Approximately 100 mg of ground lunar dust, was applied in duplicate to EpiOcular™ tissues for each exposure time of 3, 30 and 60 minutes, as recommended by the manufacturer (MatTek). Additional tissues were dosed in duplicate each with exposure times of 3, 30 and 60 minutes to serve as dust controls using the following: sodium dodecyl sulfate (SDS) (Mfg: Fisher Bioreagents; Lot: 094466; Exp: Jan 2020), sodium hydroxide (NaOH) (Mfg: MP Biomedicals, LLC; Lot: 7367 J; Exp: Jan 2020) and hydrated amorphous silica (Mfg: Bel-Art Products; CAS 112926-00-8/7631-86-9; Exp: Jan 2020). Two tissues were exposed to 100 μL of deionized (DI) water for 60 minutes to serve as a negative control. The positive control, 0.3% Triton X-100, was used to dose two tissue replicates for each exposure time of 3, 30 and 60 minutes.
After exposure of the tissues was complete, tissues were gently rinsed with calcium- and magnesium-free phosphate buffered saline (PBS) until all test material was removed from each tissue insert, and any liquid remaining on the tissues was aspirated off. Each insert was then submerged in pre-warmed assay media and incubated for 10 minutes at 37°C, under 5% CO2.
Supplied MTT concentrate (MTT-100-CON) was thawed, diluted with the provided diluent (MTT-100-DIL), and mixed well. Three hundred microliters of MTT solution were added to each well of a sterile 24 well plate, and both it and a second empty 24 well plate, to be used in the extraction process, were labelled appropriately for each insert.
Following the 10 minute incubation, tissues were placed in a sterile 24-well plate containing 300 μl MTT solution per well, and returned to a 37°C, 5% CO2 incubator for 3 hours. Following the 3 hour MTT incubation, the tissues were gently rinsed with PBS to remove any remaining MTT solution and blotted with a Kimwipe. Tissue inserts were then added to the pre-labelled 24-well plate, immersed in 2 mL extractant solution (MTT-100-EXT) per well, and sealed inside a zip-top plastic bag to prevent evaporation. The tissues were allowed to extract overnight at room temperature in the dark.
Direct MTT Reduction - A decrease in MTT reduction capacity is used as the indicator of potential irritancy. Therefore, it is important to determine the MTT reduction potential of the test substance itself. To accomplish this, 100 mg of SDS, amorphous silica, NaOH and lunar dust were each added to Eppendorf tubes containing 1 mL MTT solution. One hundred μL of DI water were added to 1 mL MTT solution as a negative control. Tubes were placed in the dark at room temperature for approximately 60 minutes, and then assessed for color change to purple, indicative of auto-reduction. Any direct MTT reduction was measured photometrically and subtracted from the values obtained for the treated tissues.
Determination of ET-50 - After transfer of tissue inserts to MTT plates and extraction were completed, any extraction solution remaining in the inserts was decanted back into the well and mixed thoroughly. Inserts were then discarded, and 200 μL from each well of each replicate were added to a 96 well plate in triplicate. Reduced MTT was then quantified photometrically. The optical density (OD) was measured at 570 nm using a dual wave length MRX Revelation spectrophotometer. The mean of the ODs for the two replicates for each substance and time point were used to calculate the ET-50 of all tissues using the manufacturer-provided spreadsheet. Results were expressed as percent viability in the lunar dust treated tissues relative to the negative control.
Extrapolation of ET-50 to irritation potential
- A proprietary spreadsheet-based model was used to correlate the ET-50 value with the appropriate Draize irritation score. This model was developed by the manufacturer and based on their testing
In vivo testing
ocular testing was performed by Stillmeadow, Inc. according to U.S. Environmental Protection Agency guidelines (OPPTS 870.2400)
]. All procedures were in compliance with Animal Welfare Act Regulations and were approved by the NASA and Stillmeadow, Inc. Institutional Animal Care and Use Committees (IACUC).
Test Substance Administration - Three healthy albino rabbits were released from quarantine five days after receipt. On Day 0, both eyes of each animal were carefully examined prior to treatment using a fluorescein sodium ophthalmic solution and cobalt-filtered light. Tetracaine HCl Ophthalmic Solution (0.5%, Bausch & Lomb, Lot 133541, Exp Sep 2012) was applied immediately prior to the fluorescein staining, with photographs of each eye taken both prior to and following fluorescein staining. Only those animals without extant eye defects or irritation were selected for testing. Each rabbit received 0.1 mg/kg Buprenorphine (Hospira, Lot 77531LL, Exp Sep 2012) parenterally as an analgesic at least 45 minutes prior to dosing.
As recommended by OECD guideline 405, Acute Eye Irritation/Corrosion, a dose equivalent to 0.1 mL volume, or 70
2 mg of lunar dust, was placed into the conjunctival sac of the right eye of each animal by gently pulling the lower lid away from the eyeball to form a cup into which the test substance was poured
]. The lids were gently held together for several seconds to minimize loss of material through the blinking reflex. The untreated left eyes served as comparative controls.
Observations – Gross observation of the treated eyes of all animals were examined without magnification under white room lighting provided by daylight-type fluorescent ceiling fixtures, and (if needed) an additional source of white light affixed to the examination table or using a handheld flashlight. All treated eyes were washed with room temperature DI water for one minute immediately after recording the 1-hour scores. If the test substance was still visible in the rabbit eye after the 1-minute wash, washing was continued for a maximum of 1 additional minute (2 minutes total). Photographs of each eye were taken at the 1-hour observation to document the presence or absence of residual test substance after washing and at each observation period through study termination.
The grades of ocular reaction were recorded at 1, 24, 48 and 72 hours after treatment. The corneas of all treated eyes were examined immediately after the 1- and 24-hour observations with a fluorescein sodium ophthalmic solution. A Finoff ocular transilluminator with cobalt blue filter (Welch Allyn, Skaneateles Falls, NY) was utilized to enhance visualization of fluorescein staining. Slit lamp exams of any fluorescein-stained abrasions were planned, but no abrasions were noted.
Irritation Scoring Method - Individual irritation scores for each animal at each scheduled observation were determined using the standard Draize scale of ocular irritancy (Appendix A). An average irritation score for each scheduled observation was then determined, with a maximum average irritation score derived from the observation yielding the highest average irritation score. The maximum average irritation score was used to rate the test substance according to the definitions presented in Appendix B.