Deuterated form of Iodomethane

Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].

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In vitro GSH conjugation with iodomethane in rat, rabbit, and human blood and tissues [1]
The purpose of this study was to determine the rate constants for the conjugation of iodomethane with GSH in whole blood and cytosols from liver and kidney tissues from female human donors, male Sprague Dawley rats, and pregnant NZW rabbits and fetuses as well as in nasal respiratory and olfactory epithelium from pregnant female NZW rabbits. Iodomethane is metabolized via conjugation with GSH or via a minor CYP450 oxidation to formaldehyde. Potential modes of action for adverse effects due to iodomethane exposure may involve iodide release or GSH depletion. Iodomethane was well metabolized in most of the tissue cytosol samples, but not in blood or fetal rabbit kidney. Limiting velocity Vmax rates were similar in liver and kidney cytosol from rats and human donors (40 and 48 in liver and 15 and 12 nmol/min/mg in kidney for rat and human tissue, respectively), but were lower in rabbit tissues (10 and 0.72 nmol/min/mg in liver and kidney, respectively). The metabolism in olfactory and respiratory epithelial cytosol had Km (Michaelis–Menton) values that were several times higher than for any other tissue (on the order of 25 mM for olfactory and 2.5 mM for respiratory), suggesting an essentially first order rate of metabolism in the nasal area. The results from these in vitro metabolism studies in conjunction with results from the in vivo gas uptake inhalation studies (Thrall et al., Citation2009b) can be used to estimate human in vivo metabolism rate constants using a parallelogram approach.

Iodomethane is a new pre-plant soil fumigant approved in the United States. Human exposure may occur via inhalation due to the high vapor pressure of iodomethane. A quantitative human health risk assessment was conducted for inhalation exposure. The critical effects of acute duration iodomethane exposure are: (1) fetal losses in rabbits, (2) lesions in rat nasal epithelium, and (3) transient neurotoxicity in rats. Chronic exposure of rats resulted in increased thyroid follicular cell tumors from sustained perturbation of thyroid hormone homeostasis. A physiologically based pharmacokinetic (PBPK) model for iodomethane was developed to characterize potential human health effects from iodomethane exposure. The model enabled calculation of human equivalent concentrations (HECs) to the animal no-observed-adverse-effect levels (NOAELs) using chemical-specific parameters to determine the internal dose instead of default assumptions. Iodomethane HECs for workers and bystanders were derived using the PBPK model and NOAELs for acute exposure endpoints of concern. The developmental endpoint NOAEL was 10 ppm and corresponding bystander HEC was 7.4 ppm. The nasal endpoint NOAEL was 21 ppm and the HEC was 4.5 ppm. The transient neurotoxicity endpoint NOAEL was 27 ppm and the HEC was10 ppm. Data demonstrated that humans are less sensitive to the effect that causes developmental toxicity in rabbits and the PBPK model incorporated this information, resulting in a higher HEC for the developmental endpoint than for the nasal endpoint. Nasal olfactory degeneration is the primary endpoint for risk assessment of acute exposure to iodomethane [1].

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Human equivalent concentrations (HECs) corresponding to NOAELs in laboratory animal studies were derived as points of departure for risk assessment of potential iodomethane exposure to humans. HECs for endpoints of concern for acute exposure NOAELs were derived using PBPK models for iodomethane and iodide as described by Sweeney et al. in this issue (Citation2009). PBPK models were not applied to endpoints of concern identified for subchronic or chronic exposure. HECs for intermediate- and long-term exposures to iodomethane were derived using the methodology developed by the EPA Office of Research and Development (ORD) for the derivation of inhalation reference concentrations (RfCs) (EPA, Citation1994). Bystanders potentially exposed to iodomethane were assumed to be exposed for 24 hours in 1 day. Workers exposed to iodomethane were assumed to be exposed for 8 hours per day, 5 days per week.

In this assessment the most sensitive endpoint and the ultimate basis for a given acute exposure assessment scenario is the endpoint represented by the lowest HEC. The lowest HEC may or may not correspond to the lowest animal NOAEL. In both the PBPK modeling and the EPA RfC approaches, different HECs may be identified for the same experimental NOAEL due to: (1) the different algorithms used to derive HECs for systemic versus portal of entry effects; (2) different dose metrics used in the PBPK model; or (3) time adjustments conducted for non-occupational versus occupational exposure scenarios. Differences between systemic versus portal of entry effects may arise from the use of different calculations to estimate the inhalation risk to humans that are dependent on the regional gas dose ratio (RGDR). For non-occupational versus occupational exposure, differences may arise because, while it is presumed that non-occupational exposure may occur 24 hours/day, 7 days/week, occupational exposure occurs only during the course of an average work week (8 hours/day and 5 days/week). An 8-hour work day is not anticipated to result in an 8-hour exposure duration, so this assumption is considered to be conservative. [1]

Acute endpoint studies [1]
The studies listed in Table 1 considered to have endpoints appropriate for risk assessment for acute exposure duration are the acute neurotoxicity study, the developmental toxicity study, and studies that resulted in effects on the rat nasal epithelium. The transient neurotoxicity occurred in a 6-hour exposure, and so the relevance to acute duration assessment is evident. Developmental toxicity is generally considered an effect that could occur due to a single day of exposure; consequently, this effect is subject to a risk assessment for a 24-hour exposure (EPA, Citation1991). The nasal olfactory degeneration observed in a number of subchronic studies is considered a potential endpoint for acute exposure because the effect did not progress in severity with increasing time of exposure, and because studies in the literature report this effect following short exposure to high concentrations of iodomethane (Chamberlain et al., Citation1998b). The studies to be the basis for the acute exposure duration assessments are summarized below.

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Acute neurotoxicity study in rats [1]
In the acute neurotoxicity study, rats were exposed to 0, 27, 93, or 401 ppm iodomethane vapor by inhalation for 6 hours in a whole-body exposure chamber. Rats were evaluated for potential effects on the nervous system using a functional observational battery (FOB). The systemic NOAEL identified was 27 ppm based on FOB findings including decreased motor activity (75 – 78% in males, 81 – 84% in females), repetitive movements of the mouth and jaws (i.e. clonic convulsions) in 1 of 12 females, and decreased body temperature, at the systemic LOAEL (lowest observed adverse effect level) of 93 ppm. These effects were observed on the day of exposure only and did not persist to the day-7 and day-14 evaluations.

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Developmental toxicity studies in rabbits [1]
In a developmental toxicity study, groups of 24 female New Zealand White (NZW) rabbits were exposed to iodomethane vapor (99.6% active ingredient, a.i.) in whole-body inhalation chambers at concentrations of 0, 2, 10, or 20 ppm (0, 0.012, 0.058, or 0.12 mg/L/day) 6 hours per day, gestation days (GDs) 6 through 28. A significant increase in late resorptions per doe was observed following exposure to 20 ppm iodomethane (1.6/doe, compared to 0.1/doe, control), contributing to an increased post-implantation loss of 2.0/doe compared to 0.7/doe in the controls. [1]
The maternal NOAEL identified by the EPA is 20 ppm and no maternal LOAEL was identified (EPA OPP Health Effects Division (HED), Citation2007). The developmental toxicity LOAEL identified by the EPA is 20 ppm based on increased fetal losses, specifically late resorptions, and decreased fetal weights (∼20%). The developmental toxicity NOAEL is 10 ppm. [1]
In a second developmental toxicity study iodomethane (99.7% a.i.) was administered via the inhalation route (whole body) to 24 New Zealand White rabbits/group at concentrations of 0 or 20 ppm during GDs 6 – 28 (control and group 2), GDs 6 – 14 (group 3), GDs 15 – 22 (group 4), GDs 23 – 24 (group 5), GDs 25 – 26 (group 6), or GDs 27 – 28 (group 7) for 6 hours/exposure per day. This study was not intended to fulfill the guideline requirement or establish NOAELs and LOAELs, but rather was conducted to determine if there was a critical period of exposure during gestation that resulted in fetal loss as observed in the standard developmental toxicity study in rabbits. [1]
As in the standard developmental toxicity study, a significant increase in late resorptions/doe was observed in rabbits exposed to 20 ppm iodomethane during GDs 6 – 28 (1.2/doe compared to 0.1/doe control). No other significant increases in late resorptions were observed, but non-significant increases were observed in does exposed during GDs 23 – 24 (0.6/doe), and GDs 25 – 26 (0.7/doe). Thus, a window of sensitivity to iodomethane exposure during GDs 23 – 26 was identified.

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Subchronic inhalation toxicity study in rats (acute effect) [1]
In a subchronic inhalation toxicity study, iodomethane (99.7% a.i.) was administered via whole-body inhalation to Sprague Dawley rats (20/sex/concentration) for 6 hours/day, 5 days/week for 13 weeks at concentrations of 0, 5, 21, or 70 ppm (0, 0.029, 0.12, or 0.41 mg/L/day). Ten rats/sex/concentration were sacrificed after 4 weeks, and the remaining 10 rats/sex/concentration were sacrificed after 13 weeks. [1]
There were no effects of treatment on mortality, ophthalmology, urinalysis, hematology, organ weights, or gross pathology. Irritation of the nasal olfactory epithelium was observed in rats exposed to 70 ppm iodomethane at both interim and terminal sacrifices. The nasal irritation at 4 and 13 weeks was characterized by inflammation and cellular degeneration and regeneration. Body weights and body weight gains were reduced during the first 6 weeks of the study in male and female rats exposed to 70 ppm iodomethane. [1]
The systemic LOAEL identified for this study is 70 ppm based on initial decreases in body weights and body weight gains. The systemic NOAEL is 21 ppm. The port-of-entry LOAEL identified is 70 ppm based on degeneration of the nasal olfactory epithelium. The port-of-entry NOAEL is 21 ppm.

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Subchronic endpoint studies [1]
Two-generation inhalation toxicity study in rats [1]
In a two-generation reproduction toxicity study, iodomethane (99.7% a.i.) was administered via whole-body inhalation to Sprague Dawley rats (30/sex/concentration) for 6 hours/day at concentrations of 0, 5, 21, or 50 ppm. These P generation animals were exposed to the test article for at least 70 days prior to mating to produce the F1 litters. Exposure of the P males continued throughout mating and until the day prior to euthanasia. The P females were exposed throughout mating and through GD 20, at which point exposure was discontinued. Daily exposure of the P females was reinitiated on lactation day (LD) 5 and continued until the day prior to euthanasia. After weaning, F1 animals (30/sex/concentration) were selected, equalized by sex, to become the parents of the F2 generation and, beginning on postnatal day (PND) 28, were exposed to the same concentration test atmosphere as their dam. [1]
The systemic parental NOAEL was 20 ppm and the LOAEL of 50 ppm was based on decreases in body weight, body weight gain, changes in organ weights, and gross pathology and histopathology findings. The developmental NOAEL was 5 ppm based on decreases in body weight gain and delays in vaginal patency observed at 20 ppm.

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Chronic endpoint studies [1]
Combined chronic toxicity/carcinogenicity study in rats [1]
In a combined chronic toxicity/carcinogenicity study in rats, iodomethane (99.7% a.i.) was administered to Sprague Dawley rats via whole body inhalation at concentrations of 0, 5, 20, or 60 ppm for 6 hours/day, 5 days/week for 104 weeks. Sixty animals/sex/concentration were exposed to 0, 5, or 20 ppm iodomethane while 70/sex were exposed at the 60-ppm level. Animals were observed for moribundity and mortality twice daily and clinical signs of toxicity once daily. Once a week a detailed physical examination was conducted, including but not limited to evaluations of changes in appearance, autonomic activity (e.g. lacrimation, piloerection, pupil size, breathing patterns), gait, posture, response to handling, and stereotypic and/or bizarre behavior. In addition, evaluations of clinical chemistry, hematology, urinalysis, gross pathology, and histopathology parameters were conducted. [1]
The systemic NOAEL was 5 ppm based on increased incidence of salivary gland squamous cell metaplasia observed at 20 ppm. The NOAEL for portal of entry effects in the nasal olfactory epithelium was 20 ppm. An increased incidence of thyroid adenomas was observed in male rats exposed to 60 ppm.

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Studies to determine mode of action [1]
Studies were performed to identify the likely MOA of iodomethane in eliciting developmental toxicity in rabbits. It is important to determine the MOA that results in the adverse effect, because that allows determination of the appropriate internal dose metric to be modeled using the PBPK model. The two studies performed to identify the mode of iodomethane action and to support development of the CFD-PBPK model are: (1) a combined baseline inhalation exposure study of iodomethane-related fetotoxicity in rabbits, and (2) a MOA study to evaluate iodomethane-related fetotoxicity in rabbits. The baseline study was performed in rabbits because the ontogeny of rabbit thyroid development had not been described in the literature in detail, and the information available from other iodomethane exposure studies suggested that effects on the developing rabbit thyroid could be important. The goals of each study and the results are summarized briefly below, and presented in full as stand-alone papers in this issue (Sloter et al., Citation2009).

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A baseline study to describe rabbit fetal development was combined with an inhalation exposure study of iodomethane-related fetotoxicity in rabbits [1]
The objective of this study was to identify maternal and/or fetal biomarkers of iodomethane exposure or toxicity in rabbits to be measured in a definitive MOA study. Baseline parameters for each potential biomarker were established using unexposed does and fetuses from GD 21 to GD 27. Potential biomarkers included serum chemistry and hematology parameters, maternal progesterone and estradiol levels, thyroid and pituitary hormones, and glutathione (GSH) concentrations in blood and various tissues. Microscopic examinations of fetal thyroids were conducted to compare the ontogeny of thyroid structure and function in unexposed versus exposed fetuses. Kinetic markers of iodomethane exposure (i.e. hemoglobin adducts and serum iodide levels) were measured in maternal and fetal blood. Iodomethane was administered by whole-body inhalation to two exposure groups (groups 8 and 9) consisting of 10 time-mated female New Zealand White rabbits for 6 hours per day following either a 2-day (GDs 23 – 24, group 8) or 4-day (GDs 23 – 26, group 9) exposure regimen. The gestational day of the laparohysterectomy for each exposure group was GD 24 (group 8) or 26 (group 9) following the 6-hour exposure on each respective day. Seven baseline groups (groups 1 – 7) consisting of 10 time-mated female New Zealand White rabbits were not exposed to the test article; the GD of the laparohysterectomy for the baseline groups ranged from GD 21 (group 1) to 27 (group 7). [1]
Iodide concentrations in maternal and fetal serum following maternal exposure to iodomethane (groups 8 and 9) were 100- to 500-fold higher than baseline concentrations (groups 4 and 6). Serum iodide levels were approximately two-fold higher in does and fetuses following 4 days of maternal exposure (group 9) compared to 2 days of maternal exposure (group 8). Serum iodide concentrations in the fetuses were approximately two-fold higher than maternal concentrations in both exposure groups. Following maternal exposure to iodomethane, iodide accumulation in the developing fetuses was associated with significant effects on fetal serum thyroid and pituitary hormone concentrations. Diminished fetal thyroxine (T4) and increased thyroid stimulating hormone (TSH) serum concentrations correlated with microscopic changes present in the thyroids of 56% (group 8) and 99% (group 9) of fetuses following the 2-day and 4-day exposure regimens, respectively. The changes in the fetal thyroids were characterized by decreased amounts of colloid in the follicular lumen, a hypertrophic follicular epithelium, and vacuolation of the epithelial cytoplasm.

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A follow-on study was performed to characterize the mode of action for iodomethane-related fetotoxicity in rabbits [1]
The objective of the study was to characterize the MOA for iodomethane-related fetotoxicity observed in previous prenatal developmental toxicity studies in rabbits. The previous study suggested the MOA was an increase in fetal iodide level from iodomethane exposure. One group of 40 time-mated female New Zealand White rabbits was exposed by whole body inhalation to 20 ppm iodomethane for 3 or 6 hours daily. A concurrent control group of 40 pregnant females was exposed to filtered air. The females in the control and iodomethane groups were exposed on GDs 23, 24, 25, and/or 26. A comparator group of 40 females received 81.2 μM sodium iodide in sterile water via four, 15-minute intravenous infusions (20.3 μM per infusion) 2 hours apart over a 6-hour period or via two, 15-minute infusions 2 hours apart over a 3-hour period on the same gestation days. Serum iodide levels and tissue GSH concentrations were measured as potential metrics of internal dose and metabolism for the PBPK model, while thyroid hormone levels, thyroid stimulating hormone, 5′-deiodinase inhibition, and thyroid gland histopathology were evaluated to provide insight into the MOA of iodomethane in pregnant rabbits. The hemoglobin adduct S-methylcysteine was examined as a marker of maternal and fetal blood exposure to the parent compound, iodomethane. The pattern of sampling in relation to exposures was designed to characterize the time courses of adsorption, metabolism, distribution, and elimination. [1]
Following maternal exposure to iodomethane on GDs 23 – 26, the mean litter proportion of late fetal resorptions on GD 29 was increased approximately 10-fold (50.4% per litter) compared to the control group (5.0% per litter). In the sodium iodide-exposed group, the mean litter proportions of late fetal resorptions and viable fetuses and mean gravid uterine weight were similar to control group values. Maternal and fetal serum iodide concentrations were increased in the iodomethane (inhalation) and sodium iodide (intravenous infusion) groups and increased with the duration of maternal exposure. Fetal serum iodide concentrations were 2 – 3-fold higher than maternal concentrations in both the iodomethane and sodium iodide exposed groups. Maternal and fetal serum iodide concentrations in the iodomethane group were approximately twice the serum iodide concentrations in the sodium iodide group. Thyroid follicular cell hypertrophy and colloid depletion were apparent in the thyroid glands of fetuses exposed to either iodomethane or sodium iodide. On GDs 23, 24, 25, and 26, the incidence and severity of these fetal thyroid findings were similar between exposure groups and increased with the duration of maternal exposure to iodomethane or sodium iodide. On GD 29 (following 3 days of recovery), the incidence of follicular cell hypertrophy and colloid depletion was similar between the iodomethane and sodium iodide groups; however, the severity remained greater in fetuses exposed to iodomethane. These differences between exposure groups may be due to the fact that the internal dose of iodide in the maternal and fetal serum following sodium iodide infusion was about one-half that achieved by 20 ppm iodomethane inhalation exposure. Microscopic findings in the thyroid glands of does euthanized at GD 26 were generally considered to be within normal limits and could not clearly be distinguished from normal biologic variation. [1]
The time course data show an increase in fetal TSH beginning after 3 days of iodomethane exposure on GD 25, further increasing after 4 days of exposure on GD 26, and decreasing slightly 3 days after exposure was discontinued, on GD 29, though remaining well above the control level. In contrast to the iodomethane-exposed group, fetal TSH levels in the NaI-exposed group returned to near control levels 3 days after exposure was discontinued on GD 29. The mechanism responsible for the increases in TSH appears to be mediated by a prolonged increase in fetal serum iodide level reported in the same experimental subjects.

The pharmacokinetics of sodium iodide in pregnant rabbits [1]
The purpose of this study was to determine the kinetics of radiolabeled sodium iodide (Na131I) in timed-pregnant rabbits and fetuses during the critical period of gestation in which exposures to iodomethane can produce fetal resorptions. Iodomethane releases iodide (I−) via metabolism or reactions with blood and tissue macromolecules. Radioiodide accumulated as expected in the thyroid of maternal animals. Radioiodide also accumulated in fetal blood and tissues, levels of which were consistently higher than maternal levels and, unlike maternal tissues, showed no evidence of clearance over the 24-hr sampling period. In contrast to observations in the maternal animals, fetal stomach contents showed the highest accumulation of radioiodide for both dose groups by 1 – 2 hrs after dosing, followed by the trachea and thyroid tissues, with the lowest concentrations of radioiodide in the amniotic fluid and blood. There was no evidence for preferential accumulation of radioiodide in fetal thyroid tissues on GDs 25 – 26 as there were no significant differences in radioactivity detected between samples of tracheas containing thyroid and samples of trachea alone.

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Studies to support PBPK models [1]
A variety of data were generated to support development of the PBPK models of iodomethane for the rabbit, rat, and human. These PBPK models were developed to address the endpoints of developmental toxicity observed in rabbits and the nasal effects and neurotoxicity observed in rats exposed to iodomethane. The mechanistic studies were intended to either provide compound-specific inputs for the PBPK model or define the dose metric for interspecies extrapolation. The models constitute a sophisticated effort to describe the kinetics of iodomethane following inhalation exposure and the kinetics of iodide as a metabolite. The data developed are critical in the development of the iodomethane models to describe nasal tract dosimetry and GSH depletion in the rat to evaluate nasal toxicity, to describe distribution of iodomethane to tissues including brain, to evaluate transient neurotoxicity, and to capture iodomethane metabolism and iodide kinetics in the pregnant rabbit to address developmental toxicity.

The studies performed to support development of the CFD-PBPK model include: (1) an inhalation mechanistic toxicity study in rats, (2) a pulmonary function study in rabbits, (3) a study to derive iodomethane partition coefficients, (4) computational fluid dynamics simulations to describe rabbit nasal airflows, (5) quantification of nasal absorption of iodomethane in rats and rabbits, (6) quantification of systemic iodomethane absorption in rabbits, (7) characterization of iodide kinetics in pregnant rabbits, and (8) determination of metabolic rate constants. These studies are summarized briefly below, and presented in full as stand-alone papers in this issue.

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Partition coefficients in rat and rabbit tissues and human blood [1]
The objective of this study was to determine tissue:blood partition coefficients in selected tissues from rat and rabbit, and from human blood, to support PBPK modeling. Iodomethane tissue-to-air partition coefficients (PCs) were determined in rat tissues (blood, brain, fat, kidney, liver, muscle, thyroid, nasal tissue), rabbit tissues (fetal blood, maternal blood, brain, fat, kidney, liver, muscle, thyroid, placenta, nasal tissue), human blood (male and female), and saline. The respective PCs for brain, fat, kidney, muscle, and nasal tissue were similar across animal species. The rabbit thyroid PC was three times higher than the rat thyroid PC (rabbit: 39, rat: 11). The rat liver PC was twice as high as the rabbit liver PC (rat: 24, rabbit 13). The rat blood PC was 2.5 times higher than the rabbit blood PC (rat: 39, rabbit: 16). The human blood PC (18) was more similar to the rabbit blood PC than the rat blood PC. The rabbit fetal blood PC (12) was similar to rabbit maternal blood PC (16). The iodomethane data collected for rat, rabbit, and human tissues was used as chemical-specific input into the physiologically based models (Sweeney et al., Citation2009).

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Magnetic resonance imaging and computational fluid dynamics simulations [1]
The purpose of this study was to develop the three-dimensional (3-D) nasal CFD models for the rabbit to determine airflow splits to different regions of the nasal airways, and the corresponding regional surface areas and volumes necessary to construct similar hybrid models in this species. CFD simulations were based upon 3-D computational meshes derived from magnetic resonance images of three adult female NZW rabbits. In the anterior portion of the nose, the maxillary turbinates of rabbits are considerably more complex than comparable regions in rats, mice, monkeys or humans. This leads to a greater surface area to volume ratio in this region, and thus the potential for increased scrubbing of water-soluble or reactive gases and vapors in the anterior portion of the nose, compared to other species such as monkeys and humans. Although there was considerable inter-animal variability in the fine structures of the nasal turbinates and airflows in the anterior portions of the nose, there was remarkable consistency between rabbits in the percentage of total inspired airflows that reached the olfactory epithelium lining the ethmoid turbinate region (∼20%). These latter results (airflows reaching the ethmoid turbinate region) were also consistent with previous published estimates for the male F344 rat.

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Uptake of iodomethane by the rat and rabbit nasal cavities [1]
The purpose of this study was to determine the nasal absorption of iodomethane in the intact nasal cavities of the rat and rabbit to provide experimental data to validate the computational fluid dynamics portion of the models. Uptake of iodomethane in the nasal cavities of the intact rat and rabbit was evaluated by measuring iodomethane concentration in the nasopharynx region of anesthetized animals via a small-diameter air-sampling probe, and comparing that concentration with the concentration in the exposure chamber. The exterior portion of the probe was connected directly to a mass spectrometer to provide a continual real-time analysis of concentrations of iodomethane in the nasal cavity. Rats were placed in a sealed glass chamber and exposed to iodomethane at a chamber concentration of approximately 1 ppm. Studies were conducted on six rats at a single exposure concentration. An average of 63% of iodomethane was absorbed in the rat nasal cavity. Rabbits were placed in a sealed glass chamber and exposed to iodomethane at chamber concentrations ranging from approximately 2 to 50 ppm. The results show that the percent of iodomethane absorbed in the nasal cavity of the rabbit ranged from 57 to 92% (average 72 ± 11) regardless of the initial exposure concentration.

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In vivo gas uptake in rabbits [1]
The purpose of this study was to provide kinetic gas uptake data for rabbits. A series of gas uptake curves were generated for rabbits exposed to varying initial chamber concentrations of iodomethane by inhalation using a closed-chamber gas-uptake system. Studies involved three individual rabbits per dose group exposed at initial chamber concentrations of 50 ppm, 10 ppm, or 2 ppm iodomethane. Animals were unanesthetized and unrestrained throughout the exposures, which were approximately 3.5 – 4 hours in duration.

The general public may be exposed to low levels of iodomethane in air from agricultural uses due to volatilization following application. Specifically, fumigants can off-gas into air and be transported off-site by meteorological processes. Agricultural field workers may be exposed to iodomethane during or after the application process. Bystander exposure to iodomethane following application to agricultural soil is expected to occur over a time period of approximately 24 hours due to the emission patterns of the compound and air dispersion patterns (EPA OPP Health Effects Division (HED), Citation2007). Occupational exposure to iodomethane may occur 8 hours per day, 5 days per week, during the application season. For risk assessment purposes, potential inhalation exposures could be acute (24 hours or less), short-term (1 – 30 days), intermediate-term (1–6 months), or long-term in duration.

The toxicity from iodomethane exposure via the inhalation route has been characterized by generation of a complete set of laboratory studies performed according to US Environmental Protection Agency (EPA) Office of Prevention, Pesticides and Toxic Substances (OPPTS) guidelines to support registration of iodomethane in the USA (EPA OPP Health Effects Division (HED), Citation2007). These studies provide sufficient information to identify critical effects for each relevant exposure scenario and form the bases for a human health risk assessment. A standard risk assessment performed to evaluate the potential risk to bystanders and workers who might be exposed to iodomethane resulting from fumigation activities would apply default methodologies to extrapolate from the no-observed-adverse-effect levels (NOAELs) in the animal studies to estimate human equivalent concentrations to the external animal exposures.

To reduce the uncertainties in the human health risk assessment of iodomethane that result from the use of default assumptions to extrapolate from animal to human exposure, chemical-specific data and physiologically based pharmacokinetic (PBPK) models were developed. A series of studies was conducted to develop and validate hybrid computational fluid dynamics (CFD) models of nasal airflows coupled with PBPK models of the systemic disposition of iodomethane in rats, rabbits, and humans. Studies were performed to identify the appropriate dose metric for each endpoint of potential concern; to characterize the airflow in the nasal passages of rabbits; to describe the kinetic uptake and metabolism of iodomethane in the nasal passages of rats and rabbits; and to determine partition coefficients for iodomethane in a variety of tissues in rats, rabbits, and humans. These studies are summarized briefly in this paper and are published in separate articles in this journal issue. [1] An in vivo 2-day inhalation mechanistic toxicity study—rats (Himmelstein et al., Citation2009). The objective of this study was to evaluate the toxicokinetic behavior of iodomethane in rats exposed by inhalation. Key study endpoints included evaluation of GSH status in selected target tissues, inorganic serum iodide and hemoglobin adducts as measures of internal dose, and clinical chemistry, hematology, thyroid hormone status, and pulmonary function as measures of toxicity. Effects on clinical pathology, thyroid hormone status and uridine diphosphate (UDP)-glucuronyltransferase, and concentrations of S-methylcysteine were determined the morning after 2 days of 6 hour/day whole body inhalation exposure to 0, 25, or 100 ppm iodomethane (n = 10 rats/group). Additional main inhalation exposure groups (n = 3 rats per 0, 25, or 100 ppm) were sampled at 1, 3, 6, 9, 24, 25, 27, 30, 33, and 48 hours for quantification of GSH and inorganic serum iodide. Treatment-related changes from exposure to 25 and 100 ppm iodomethane were minimal to mild increases in total cholesterol concentrations (due to proportional increases in both high density lipoprotein (HDL) and non-HDL cholesterol), and minimal to mild decreases in triglyceride concentrations. Serum TSH concentrations were significantly increased at exposure concentrations of 25 and 100 ppm iodomethane. Serum triiodothyronine (T3) and T4 concentrations were significantly decreased at exposure concentrations of 100 ppm iodomethane, and serum reverse T3 (rT3) concentrations were not altered under the conditions of this study. S-methylcysteine was detected in globin from control rats at an average concentration of 161.2 nmol/g globin, and in rats exposed to 25 and 100 ppm iodomethane, the mean concentrations were increased to 201.6 and 345.3 nmol/g globin, respectively. Iodomethane exposure caused time- and concentration-dependent reductions in tissue GSH concentrations. Depletion was less pronounced in blood, kidney, and liver than in olfactory and respiratory epithelia. Iodomethane exposure of rats resulted in increased inorganic serum iodide concentrations that were concentration and time dependent. Concurrent pulmonary function measurement during exposure of rats to 0, 25, or 100 ppm iodomethane did not lower the breathing frequency, indicating that iodomethane did not induce a respiratory irritant response in rats. A pulmonary function study—rabbits (in Sweeney et al., Citation2009) The objective of this study was to evaluate the toxicokinetic behavior of iodomethane in rabbits exposed by inhalation. To estimate the amount of iodomethane that is actually delivered into the respiratory tract of exposed rabbits, whole-body plethysmography was utilized. Key study endpoints included breathing frequency, tidal volume, serum iodide, and hemoglobin adduct analysis. Rabbits were exposed to average iodomethane concentrations of 0 or 18.46 ppm for 6 hours. None of the rabbits exposed to iodomethane demonstrated clinical signs of respiratory irritation and the overall mean respiratory rate of animals exposed to iodomethane was 4 breaths per minute (bpm) lower than the mean control value 130.6 bpm. Rabbits exposed to iodomethane demonstrated an overall mean minute volume that was 34% higher than the control value of 403.9 mL/min. There was no statistical difference in S-methylcysteine blood values between the control and iodomethane exposed animals. Rabbits exposed to 18.46 ppm iodomethane demonstrated an over 1000-fold increase in inorganic serum iodide levels.

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Endpoints and corresponding modes of action: acute exposure [1]
The critical endpoints identified for acute exposure to iodomethane by the inhalation route include developmental toxicity in rabbits, lesions in the nasal olfactory epithelium, and transient neurotoxicity. Effects on the thyroid have also been reported in a number of the studies of longer duration. Modes of action for these endpoints are summarized briefly below and are described in greater detail in a stand-alone paper in this issue (Kirman et al., Citation2009).

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Developmental toxicity [1]
Developmental and/or offspring toxicity is observed in rabbits and possibly rats. Two developmental toxicity studies in rabbits conducted via the inhalation route have been described. In the guideline study, an increase in fetal losses was noted at the highest exposure concentration. Subsequently, a phased-exposure rabbit developmental toxicity study was conducted in which animals were exposed for different time periods. This second study reproduced the fetal losses seen in the guideline study and defined a narrow dosing window that may elicit this effect. Only exposure on GDs 23 – 24 or GDs 25 – 26 resulted in fetal losses. It is noteworthy that the time of fetal loss coincides with the time of ontogeny of fetal thyroid function in the rabbit (GD 22). Given the essential role of iodine in the proper function of the thyroid gland (both iodine deficiency and excess can have profound effects on thyroid function and thyroid hormone biosynthesis) and the fact that iodomethane exposure may lead to an excess accumulation of iodine in the thyroid, a MOA for the fetal losses involving perturbations of fetal thyroid function as a result of excess iodide has been proposed. In the case of rats, no fetal losses were reported in the developmental toxicity study, yet a decrease in the number of live births was reported in the multigenerational reproduction toxicity study. It is interesting to note that while iodomethane exposure in the developmental study ceased on GD 17 before ontogeny of rat fetal thyroid function, in utero exposure during the multigenerational toxicity continued until GD 20 (i.e. during ontogeny of fetal thyroid function). Thus, the data suggest that fetal losses may have occurred in the rat developmental study had exposure continued beyond GD 17. Similar effects have been reported for another iodine-rich compound, amiodarone (an antiarrhythmic drug), after treatment of pregnant rabbits and rats (FDA, 2003).

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Developmental toxicity—mode of action [1]
The biochemical measure associated with the developmental effects observed in rabbits following iodomethane exposure is fetal iodide accumulation. Data from early studies suggested that there are important species differences in fetal iodide accumulation, such that guinea pigs, sheep, and rabbits possess an active mechanism for transporting iodide from mother to fetus, while other animals such as rats do not concentrate iodide in the fetus (Logothetopoulos & Scott, Citation1956; Roti et al., Citation1983). Recent studies described in this issue confirmed that control fetal rabbits have blood iodide levels that are approximately nine times the blood iodide levels in maternal blood (Sloter et al., Citation2009). The rabbit has been shown to be more sensitive with regard to fetal viability than the rat, hamster, and swine when dosed with potassium iodide or sodium iodide in feed at certain times during gestation (Arrington et al., Citation1965). Iodide accumulation in the fetus is the critical step in the MOA that ultimately leads to fetal loss in rabbits exposed to iodomethane. [1]
The fact that the rabbit fetus concentrates iodide has been known for decades, and in studies performed to support iodomethane registration, the fetuses from unexposed control pregnant rabbits in the GD 23 – 26 timeframe had plasma iodide concentrations that were 9–11-fold higher than their respective mothers (Sloter et al., 2009). Thus, the control rabbit fetal/maternal iodide concentration ratios were 9 to 11. In contrast to rabbits, the human fetus was not believed to concentrate iodide from the maternal circulation in such high ratios. Available data from the literature indicate that normal human fetal iodide concentrations are generally lower than or equal to human maternal concentrations, resulting in a fetal/maternal plasma iodide concentration ratio of approximately 1. A study by Rayburn et al. (Citation2008) was designed to characterize the fetal and maternal plasma iodide concentrations in unexposed fetal maternal pairs of human subjects. These data were used to inform the PBPK modeling and increase the certainty in the human health risk assessment. [1]
The fetal and maternal iodide concentrations and fetal/maternal plasma iodide ratios characterized in the Rayburn study (2008) confirmed that the human conceptus does not highly concentrate iodide relative to the maternal circulation as the rabbit fetus does. Maternal plasma iodide concentrations were 1.6 ± 0.4 μg/dL for premature deliveries and 1.5 ± 0.5 μg/dL for term deliveries. Cord plasma iodide concentrations were 1.4 ± 0.5 μg/dL for premature deliveries and 1.7 ± 0.7 μg/dL for term deliveries. The average fetal:maternal plasma iodide ratio for all subject pairs combined was 1.2 ± 0.7. The average fetal:maternal plasma iodide ratio for premature delivery pairs was 0.9 ± 0.4 (n = 29), and the average fetal:maternal plasma iodide ratio for term deliveries was 1.3 ± 0.8 (n = 92). The human fetal:maternal plasma iodide ratio for premature deliveries is less than one. Thus, the data lead to the conclusion that iodide could not be concentrated in premature infants as it is in the rabbit. This MOA supports the use of the cumulative iodide concentration (area-under-the-curve or AUC) in fetal plasma as an appropriate internal dose measure for PBPK modeling.

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Neurotoxicity [1]
In regards to the potential role of iodomethane as a neurotoxicant, the inhalation acute neurotoxicity study in rats revealed that iodomethane exposure elicited transient behavioral signs of neurotoxicity. These signs of neurotoxicity included: an 80% decrease in motor activity, a 2 – 3°C decrease in body temperature, and repetitive mouth and jaw movement (clonic convulsions), in the absence of neuropathology. All effects were transient, and recovery was complete. Clinical signs of neurotoxicity were not observed in any of the subjects in the subchronic or chronic studies performed on iodomethane.

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Neurotoxicity—mode of action [1]
A wide variety of inhaled solvents are known to produce anesthetic or sedative effects in animals and humans, similar to those reported for iodomethane, by altering nerve cell membrane properties (Snyder & Andrews, Citation1996). Iodomethane may depress spontaneous and evoked activity of neurons in the brain, possibly through non-specific actions with the lipid matrix of the nerve membrane or, as with anesthetic agents, by a more specific action on the g-aminobutyric acid (GABAA) receptor chloride channel, or by inhibition of neurotransmission at excitatory NMDA (N-methyl-d-aspartate) receptors (Balster, Citation1998; Trevor & White, Citation2004). These modes of action have not been linked specifically to iodomethane, but the temporal association, the type of effects, and the transient nature of the effects are similar, and indicate that the effects of iodomethane on the nervous system are due to the concentration of the parent compound in the brain.

[1]. Iodomethane human health risk characterization. Inhal Toxicol. 2009 May;21(6):583-605.

Nasal histopathology [1]
Histopathologic changes caused by iodomethane exposure occurred in the respiratory tract, and the salivary and thyroid glands. The respiratory tract histopathology was characterized by lesions of the nasal cavity described as degeneration of the olfactory epithelium (portal of entry effects). These lesions were identified in the 13-week inhalation toxicity study, the multigenerational reproductive toxicity study, and the combined chronic toxicity/carcinogenicity study in rats and were limited to the extrathoracic region with no involvement of the tracheobronchial or pulmonary regions. Furthermore, they did not appear to progress with time (i.e. nasal lesions of comparable severity were seen after 4, 13, and 52 weeks of exposure at the same concentration), thus suggesting that the nasal lesions were the result of reaching a critical concentration (Cmax) rather than being time-dependent (i.e. C × t; Haber’s law). In contrast, a C × t relationship is assumed for all systemic effects.

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Nasal histopathology—mode of action [1]
The weight of evidence suggests that the MOA responsible for iodomethane effects in the olfactory epithelium of rats is GSH depletion. A variety of potential modes of action of iodomethane on the nasal olfactory epithelium were investigated in a study by Chamberlain et al. (Citation1998b). This in vitro study ruled out two potential modes of action: (1) cellular protein methylation and (2) cytochrome P450 metabolism of iodomethane to formaldehyde. The only parameter studied that correlated with the site-selective lesion of the olfactory epithelium is GSH depletion catalyzed by conjugation of GSH and iodomethane by glutathione S-transferase (GST) theta. [1]
GSH depletion as a key factor in the MOA of iodomethane on the nasal epithelium of rats was confirmed in a second study by Chamberlain et al. (Citation1998a). In the second study, rats pretreated with an isopropyl ester of GSH to increase tissue GSH levels were protected against the nasal effects of iodomethane. Alternatively, rats pretreated with phorone and buthionine sulfoximine to deplete GSH tissue levels exhibited a potentiated response of the nasal effects following iodomethane exposure (Chamberlain et al., Citation1998a). This study concluded that GSH depletion by conjugation with iodomethane is the MOA for the nasal effects and a detoxification pathway for iodomethane.

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Derivation of HECs using PBPK modeling for acute exposures [1]
Iodomethane and iodide PBPK models were used to derive HECs for three potential endpoints for acute iodomethane exposures of 24 hours. The general design and assumptions used in the PBPK models for developmental toxicity, effects on the nasal olfactory epithelium, and acute neurotoxicity, are summarized in the following section and described in detail in the paper presented in this issue by Sweeney et al. (Citation2009).

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PBPK modeling of developmental effects [1]
The endpoint of fetal losses identified in the developmental toxicity studies in rabbits is also considered appropriate for this risk assessment since it is presumed that developmental effects may be the outcome of an acute exposure. In the case of iodomethane, this presumption has been substantiated somewhat by the results of the phased developmental toxicity study in rabbits in which a slight increase in fetal losses was observed after two 6-hr exposures to 20 ppm iodomethane. The MOA identified for this effect is an excess in serum iodide in the rabbit fetus following exposure to 20 ppm iodomethane during GDs 23 through 26. In a MOA study, excess iodide has been shown to lead to fetal thyroid hormone disruptions (possible Wolff–Chaikoff effect) resulting in fetal loss. Consequently, the dose metric used for this assessment is the area under the concentration curve (AUC) for fetal serum inorganic iodide during a single day of exposure. An HEC of 7.4 ppm was calculated for the non-occupational risk assessment, assuming a single 6-hour iodomethane exposure in the rabbit and a corresponding 24-hour bystander iodomethane exposure. This HEC was derived based on the assumption that human fetal serum iodide levels are 120% of the maternal levels at equilibrium (Rayburn et al., Citation2008). [1]
In deriving an HEC that corresponds to the 10-ppm rabbit study NOAEL described in the previous paragraph, the assumption that a single day of iodomethane exposure to the rabbit has the potential to produce an effect was employed. However, given that the window of sensitivity for the effect of fetal loss from iodomethane exposure is GDs 23 through 26, the relevant single day of exposure to produce the critical effect during the study is a single day that was preceded by 17 or more days of iodomethane exposure (Nemec, Citation2002, Citation2003; Nemec et al., Citation2009). In modeling repeated rabbit exposures to 10 ppm iodomethane, rabbit fetal plasma iodide increases with daily iodomethane exposure, approaching steady state around day 5. A repeat exposure modeling scenario was used to derive an AUC corresponding to the fetal plasma iodide level on a single day after four previous days of exposure. This AUC provides an alternate NOAEL-equivalent internal dose for a single-day 10-ppm iodomethane exposure in the rabbit. This 1-day AUC is not the 1-day AUC for the first day of exposure (GD 6), but rather an AUC that reflects the likely iodide profile that occurred during the window of susceptibility. [1]
If 10 ppm is accepted as the rabbit NOAEL and prior exposure of the rabbit to iodomethane is taken into account in the model, the calculated bystander HEC is 26 ppm for this endpoint. This HEC can be compared to the HEC of 7.4 ppm, which results when the impacts of continued daily dosing of the rabbit prior to the window of susceptibility are not taken into account. Similarly, the worker (8 hr/day) HEC is calculated as 67 ppm if prior daily dosing of the rabbit is taken into account, compared to an HEC of 23 ppm when the impact of prior dosing is ignored. The details of these PBPK modeling simulations are described by Sweeney et al. (Citation2009). This repeat dose modeling of the rabbit exposure demonstrates the conservative nature of the proposed HECs of 7.4 ppm (non-occupational) and 23 ppm (occupational), which do not account for the prior days’ dosing.

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Nasal histopathology and PBPK model design [1]
The nasal histopathology was reported in rats after 13 weeks of daily exposure to iodomethane; however, data from the published literature indicate that nasal lesions can occur in rats after acute exposures if the time profile of the exposure concentration leads to an overall iodomethane exposure of greater than or equal to 200 ppm-hr (for example, approximately 2 hours at 100 ppm-hr) (Reed et al., Citation1995). The design of the nasal olfactory epithelium (NOE) compartments of the PBPK model provides for iodomethane exposure to all cell layers in the compartment via diffusion through the cell stacks. Each compartment provides for diffusion of iodomethane from the nasal lumen through a layer of mucus, through four layers of epithelial cells, and the blood exchange layer. This layered or “stacked” structure of the NOE compartment allows the model to approximate the iodomethane concentration gradient across nasal tissue from the mucus layer to the vascularized region under the basal lamina. This corresponds to the degeneration that occurred through the epithelium following exposure to high concentrations of iodomethane. [1]
The proposed MOA for nasal histopathology involves GSH depletion as a key event in the toxicity pathway leading to damage of the nasal olfactory epithelium (Chamberlain et al., Citation1998a, Citation1998b). Consequently, GSH depletion is the dose metric used in the PBPK model for interspecies extrapolation for the nasal lesions and to identify the HEC. Selection of the appropriate degree of GSH depletion to predict nasal olfactory toxicity depends on judgments about the relationship of this measure with toxicity and the time-course of exposure concentrations with the prediction of GSH depletion. The PBPK modeling has been performed assuming that in order for GSH-related toxicity to be produced in the nasal olfactory epithelium, GSH concentrations must drop below 50% of control and remain depleted. A transient 50% depletion of GSH achieved only at the end of a 24-hour iodomethane exposure is an appropriate HEC to the rat NOAEL for the acute exposure endpoint of nasal toxicity because the benchmark GSH depletion would barely be achieved, and not sustained. Average GSH depletion in the four epithelial layers of the human PBPK model is the basis for the HEC.

CD3I

144.96

144.946

865-50-9

2723978

Colorless to light yellow liquid（Density：2.330 g/cm3）

2.2±0.1 g/cm3

40.3±3.0 °C at 760 mmHg

-66.5ºC(lit.)

7.8±10.7 °C

437.1±0.1 mmHg at 25°C

1.528

1.5

0

0

0

2

2

0

C([2H])([2H])([2H])I

INQOMBQAUSQDDS-FIBGUPNXSA-N

InChI=1S/CH3I/c1-2/h1H3/i1D3

trideuterio(iodo)methane

Iodomethane-d3; Iodo(2H3)methane; Methyl-d3 Iodide; Methane-d3, iodo-; EINECS 212-744-5; DTXSID20235599; DTXCID00158090; ...; 865-50-9;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)