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]

\n\nAcute endpoint studies [1]
\nThe 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.\n

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\n\nAcute neurotoxicity study in rats [1]
\nIn 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.\n

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\n\nDevelopmental toxicity studies in rabbits [1]
\nIn 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.\n [1]
\nThe 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.\n [1]
\nIn 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.\n [1]
\nAs 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.\n

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\n\nSubchronic inhalation toxicity study in rats (acute effect) [1]
\nIn 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.\n [1]
\nThere 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.\n [1]
\nThe 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.\n

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\n\nSubchronic endpoint studies [1]
\nTwo-generation inhalation toxicity study in rats [1]
\nIn 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.\n [1]
\nThe 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.\n

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\n\nChronic endpoint studies [1]
\nCombined chronic toxicity/carcinogenicity study in rats [1]
\nIn 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.\n [1]
\nThe 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.\n

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\n\nStudies to determine mode of action [1]
\nStudies 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).\n

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\n\nA baseline study to describe rabbit fetal development was combined with an inhalation exposure study of iodomethane-related fetotoxicity in rabbits [1]
\nThe 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).\n [1]
\nIodide 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.\n

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\n\nA follow-on study was performed to characterize the mode of action for iodomethane-related fetotoxicity in rabbits [1]
\nThe 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.\n [1]
\nFollowing 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.\n [1]
\nThe 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.

Pharmacokinetics of Sodium Iodide in Pregnant Rabbits [1] This study aimed to determine the in vivo pharmacokinetics of radiolabeled sodium iodide (Na131I) during a critical period of pregnancy (when exposure to iodomethane can lead to fetal uptake). Iodethane releases iodide ions (I−) through metabolism or reaction with macromolecules in blood and tissues. Radioactive iodine accumulated in the thyroid gland of the maternal animals as expected. Radioactive iodine also accumulated in the fetal blood and tissues at levels consistently higher than in the mother, and unlike in the maternal tissues, no clearance was observed during the 24-hour sampling period. Contrary to observations in the maternal animals, the highest accumulation of radioactive iodine was observed in the fetal gastric contents of both dose groups within 1–2 hours after administration, followed by the trachea and thyroid tissues, with the lowest concentrations in the amniotic fluid and blood. No evidence of preferential accumulation of radioactive iodine in the fetal thyroid tissue was found during days 25–26 of gestation, as there was no significant difference in radioactivity detected between tracheal samples containing the thyroid gland and tracheal samples alone.

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Supporting PBPK Models[1]
Various data have been generated to support the development of PBPK models of methyl iodide in rabbits, rats, and humans. These PBPK models were developed to address developmental toxicity endpoints observed in rabbits and nasal effects and neurotoxicity observed in rats exposed to methyl iodide. These mechanistic studies aimed to provide compound-specific inputs for physiological pharmacokinetic (PBPK) models or to determine dose parameters for interspecies extrapolation. These models were designed to comprehensively describe the kinetics of methyl iodide after inhalation exposure and the kinetics of iodides as metabolites. The data obtained are crucial for the development of methyl iodide models that can be used to assess nasal toxicity by describing nasal dosimetry and glutathione (GSH) consumption in rats; to describe the distribution of methyl iodide in tissues including the brain; to assess transient neurotoxicity; and to capture the metabolism and iodide kinetics of methyl iodide in pregnant rabbits for the study of developmental toxicity.
Studies conducted to support the development of CFD-PBPK models include: (1) toxicity studies of rat inhalation mechanisms; (2) rabbit lung function studies; (3) derivation of iodomethane partition coefficients; (4) computational fluid dynamics simulations to describe nasal airflow in rabbits; (5) quantitative analysis of iodomethane absorption in the nasal cavity of rats and rabbits; (6) quantitative analysis of iodomethane absorption in the whole body of rabbits; (7) iodide kinetic characterization in pregnant rabbits; and (8) metabolic rate determination. Constants. These studies are briefly summarized below and will be published in full as independent papers in this issue.

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Partition coefficients in rat and rabbit tissues and human blood[1]
This study aims to determine tissue/blood partition coefficients in specific tissues of rats and rabbits and in human blood to support physiological pharmacokinetics (PBPK) models. The tissue/air partition coefficient (PC) of iodomethane was determined in rat tissues (blood, brain, fat, kidney, liver, muscle, thyroid, and nasal tissues), rabbit tissues (fetal blood, maternal blood, brain, fat, kidney, liver, muscle, thyroid, placenta, and nasal tissues), human blood (male and female), and saline. PC values in brain, fat, kidney, muscle, and nasal tissues were similar across different animal species. The PC value in rabbit thyroid was three times that in rat thyroid (rabbit: 39, rat: 11). The PC value in rat liver was twice that in rabbit liver (rabbit: 24, rabbit: 13). The PC value in rat blood was 2.5 times that in rabbit blood (rabbit: 39, rabbit: 16). The PC value in human blood (18) was closer to that in rabbit blood than in rat blood. The PC value in rabbit fetal blood (12) was similar to that in rabbit maternal blood (16). The collected iodomethane data from rat, rabbit, and human tissues were used as chemically specific inputs for physiological models (Sweeney et al., 2009).

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Magnetic Resonance Imaging and Computational Fluid Dynamics Simulation[1]
The aim of this study was to develop a three-dimensional (3D) CFD model of the rabbit nasal cavity to determine the distribution of airflow to different regions of the nasal airway and the corresponding surface area and volume required to construct a similar hybrid model for this species. The CFD simulation was based on a three-dimensional computational mesh extracted from magnetic resonance images of three adult female New Zealand white rabbits. In the anterior nasal cavity, the maxillary turbinate structure of rabbits is much more complex than the corresponding regions of rats, mice, monkeys or humans. This results in a larger surface area to volume ratio in this region, and therefore a greater clearance capacity of the anterior nasal cavity of rabbits for water-soluble or reactive gases and vapors compared to other species such as monkeys and humans. Although there are significant differences in the fine structure of the turbinates and the airflow in the anterior nasal cavity among different animals, the percentage of inhaled airflow reaching the olfactory epithelium of the ethmoid turbinate region (approximately 20%) showed significant consistency among different rabbits. The latter result (airflow reaching the ethmoid turbinate region) is also consistent with the previously published estimate from male F344 rats.

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Absorption of methyl iodoform in the nasal cavity of rats and rabbits[1]
This study aimed to determine the absorption of methyl iodoform in the intact nasal cavity of rats and rabbits to provide experimental data to validate the computational fluid dynamics part of the model. The absorption of methyl iodoform in the intact nasal cavity of rats and rabbits was assessed by measuring the concentration of methyl iodoform in the nasopharynx of anesthetized animals using a small-diameter air sampling probe and comparing it with the concentration in the exposure chamber. The probe was externally connected to a mass spectrometer for continuous real-time analysis of the concentration of methyl iodoform in the nasal cavity. Rats were placed in a sealed glass chamber and exposed to methyl iodoform at a concentration of approximately 1 ppm. Six rats were studied at a single exposure concentration. The results showed that the rats absorbed an average of 63% of the methyl iodoform in their nasal cavity. Rabbits were placed in a sealed glass chamber and exposed to methyl iodoform at concentrations ranging from approximately 2 to 50 ppm. The results showed that the percentage of methyl iodoform absorbed by the rabbits' nasal cavity ranged from 57% to 92% (mean 72 ± 11%), regardless of the initial exposure concentration.

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Gas Absorption in Rabbits[1]
This study aimed to provide data on gas absorption kinetics in rabbits. A series of gas absorption curves were generated by inhaling rabbits exposed to different initial concentrations of iodomethane using a closed gas absorption system. In this study, three rabbits were selected for each dose group and exposed to iodomethane at initial concentrations of 50 ppm, 10 ppm, or 2 ppm, respectively. The animals were not anesthetized or restricted throughout the exposure, which lasted for approximately 3.5 to 4 hours.

Because methyl iodide volatilizes during agricultural applications, the public may be exposed to low concentrations of methyl iodide in the air. Specifically, the fumigant is released into the air and may spread to other areas through meteorological processes. Agricultural field workers may be exposed to methyl iodide during or after application. Based on methyl iodide emission and airborne dispersion patterns (cited in the Health Impacts Division of the U.S. Environmental Protection Agency, 2007), bystander exposure to methyl iodide is expected within approximately 24 hours after application. During the application season, workers may work 8 hours a day, 5 days a week, increasing their risk of methyl iodide exposure. For risk assessment purposes, potential inhalation exposure can be categorized as acute (24 hours or less), short-term (1–30 days), medium-term (1–6 months), or long-term. The toxicity of methyl iodoform via inhalation has been characterized by a comprehensive series of laboratory studies conducted in accordance with the guidelines of the U.S. Environmental Protection Agency (EPA) Office of Prevention, Pesticides, and Toxic Substances (OPPTS) to support the registration of methyl iodoform in the United States (EPA OPP Health Effects Division (HED), cited 2007). These studies provide sufficient information to identify the key effects of each relevant exposure scenario and form the basis for human health risk assessments. To assess the potential risks to bystanders and workers who may be exposed to methyl iodoform during fumigation operations, a standard risk assessment typically employs a default approach, extrapolating the human equivalent concentration based on the No Observed Adverse Effect Level (NOAEL) in animal studies to estimate the external concentration for animal exposure. To reduce the uncertainty in methyl iodoform human health risk assessments arising from extrapolating human exposure from animal exposure using default assumptions, we developed chemical-specific data and a physiologically based pharmacokinetic (PBPK) model. We conducted a series of studies aimed at developing and validating hybrid computational fluid dynamics (CFD) models that simulate nasal airflow and combine them with PBPK models of the systemic distribution of methyl iodide in rats, rabbits, and humans. These studies aimed to determine appropriate dose parameters for each potential endpoint of interest; and to characterize airflow in the rabbit nasal cavity; this paper aims to describe the kinetics of methyl iodide uptake and metabolism in the nasal cavities of rats and rabbits; and to determine the partition coefficients of methyl iodide in various tissues in rats, rabbits, and humans. These studies are briefly summarized here and have been published in separate articles in this issue of the journal. [1] A 2-day in vivo inhalation mechanism toxicity study in rats (Himmelstein et al., 2009). This study aimed to assess the toxicokinetic behavior of rats after inhalation of methyl iodide. The primary endpoints included assessment of glutathione (GSH) levels in selected target tissues, serum inorganic iodides and hemoglobin adducts as internal dose parameters, and clinical chemistry, hematology, thyroid hormone levels, and lung function as toxicity parameters. After two consecutive days of inhalation of 0, 25, or 100 ppm methyl iodide for 6 hours (n=10 rats per group), clinicopathological findings, thyroid hormone levels, uridine diphosphate glucuronosyltransferase (UDP-glucuronosyltransferase), and S-methylcysteine concentrations were measured the following morning. A separate master inhalation exposure group (0, 25, or 100 ppm, n=3 rats per group) collected samples at 1, 3, 6, 9, 24, 25, 27, 30, 33, and 48 hours for quantitative analysis of glutathione (GSH) and serum inorganic iodides. Treatment-related changes induced by 25 and 100 ppm methyl iodide exposure included a slight to moderate increase in total cholesterol (proportionately due to increases in both high-density lipoprotein (HDL) and non-HDL cholesterol) and a slight to moderate decrease in triglyceride concentrations. Serum thyroid-stimulating hormone (TSH) concentrations were significantly increased at methyl iodide exposure concentrations of 25 ppm and 100 ppm. At a methane iodide exposure concentration of 100 ppm, serum triiodothyronine (T3) and thyroxine (T4) concentrations were significantly decreased, while serum trans-T3 (rT3) concentration remained unchanged under the conditions of this study. S-methylcysteine was detected in globin from control rats, with an average concentration of 161.2 nmol/g globin; in rats exposed to 25 ppm and 100 ppm methane iodide, the average concentrations increased to 201.6 nmol/g globin and 345.3 nmol/g globin, respectively. Methane iodide exposure led to a time- and concentration-dependent decrease in tissue glutathione (GSH) concentrations. GSH consumption in blood, kidneys, and liver was lower than in olfactory and respiratory epithelium. Methane iodide exposure increased serum inorganic iodide concentrations in rats, and the increase was positively correlated with both methane iodide concentration and exposure time. Pulmonary function measurements were performed simultaneously in rats exposed to 0, 25, or 100 ppm methyl iodide. Results showed no decrease in respiratory rate, indicating that methyl iodide did not induce respiratory irritation in rats. A pulmonary function study in rabbits (Sweeney et al., 2009) aimed to assess the toxicokinetics of methyl iodide inhalation in rabbits. Whole-body plethysmography was used to estimate the actual amount of methyl iodide entering the respiratory tract of exposed rabbits. Primary endpoints included respiratory rate, tidal volume, serum iodide, and hemoglobin adduct analysis. Rabbits were exposed to methyl iodide at mean concentrations of 0 or 18.46 ppm for 6 hours. None of the rabbits exposed to methyl iodide showed clinical signs of respiratory irritation, and the mean respiratory rate of the exposed animals was 4 breaths/min lower than the control group's mean of 130.6 bpm. The mean minute ventilation of the exposed rabbits was 34% higher than the control group's 403.9 mL/min. There was no statistically significant difference in S-methylcysteine blood concentrations between the control group and the iodomethane-exposed group. Serum inorganic iodide levels in rabbits exposed to 18.46 ppm iodomethane increased more than 1000-fold.

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Endpoints and corresponding mechanisms of action: Acute exposure[1]
Key endpoints of acute exposure to iodomethane via the inhalation route included developmental toxicity, olfactory epithelial damage, and transient neurotoxicity in rabbits. Effects on the thyroid gland have also been reported in some longer-duration studies. The mechanisms of action of these endpoints are briefly described below, and a more detailed description can be found in an independent paper in this issue (Kirman et al., Citation 2009).

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Developmental toxicity[1]
Developmental and/or progeny toxicity has been observed in rabbits and possibly rats. Two rabbit developmental toxicity studies via the inhalation route have been described. In the guideline study, increased fetal loss was observed at the highest exposure concentration. Subsequently, a phased exposure rabbit developmental toxicity study was conducted in which animals were exposed for different time periods. The second study reproduced the fetal loss observed in the guideline study and identified a narrow dose window that could cause this effect. Fetal loss only occurred on days 23–24 or 25–26 of gestation. Notably, the timing of fetal loss in rabbits coincided with the development of fetal thyroid function (day 22 of gestation). Given the crucial role of iodine in normal thyroid function (both iodine deficiency and excess significantly affect thyroid function and the biosynthesis of thyroid hormones), and the potential for excessive iodine accumulation in the thyroid gland due to iodine methyl iodide exposure, it has been proposed that iodine excess leading to fetal thyroid dysfunction is the mechanism for fetal loss. In rats, developmental toxicity studies did not report fetal loss, but multigenerational reproductive toxicity studies reported a reduction in live births. Notably, while methyl iodide exposure ceased on day 17 of gestation (GD 17) in developmental studies, prior to fetal thyroid function development, intrauterine exposure continued until day 20 of gestation (GD 20) in multigenerational toxicity studies (during fetal thyroid function development). Therefore, the data suggest that fetal death may have occurred in rat developmental studies if methyl iodide exposure continued beyond day 17 of gestation. Another iodine-rich compound, amiodarone (an antiarrhythmic drug), has also been reported to have similar effects after treatment of pregnant rabbits and rats (FDA, 2003).

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Developmental toxicity—Mechanism of action[1]
The biochemical marker related to the developmental effects of iodomethane exposure observed in rabbits is fetal iodide accumulation. Data from earlier studies have shown significant differences in fetal iodide accumulation among different species. Guinea pigs, sheep, and rabbits have active mechanisms for transporting iodides from the mother to the fetus, while other animals (such as rats) do not accumulate iodides in the fetus (Logothetopoulos & Scott, 1956; Roti et al., 1983). The latest study published in this issue confirms that the blood iodide level in the control group of fetal rabbits is about nine times that of the maternal blood iodide level (Sloter et al., 2009). Studies have shown that rabbit fetal survival is more sensitive than that of rats, hamsters and pigs when potassium or sodium iodide is added to the diet during specific periods of pregnancy (Arrington et al., 1965). Iodide accumulation in rabbit fetuses exposed to iodomethane is a key step in its mechanism of action, ultimately leading to fetal death. [1] The fact that rabbit fetuses can accumulate iodide has been known for decades. In studies supporting the iodomethane registry, fetal plasma iodide concentrations in unexposed control pregnant rabbits were 9–11 times higher than in their mothers during days 23–26 of gestation (GD 23–26) (Sloter et al., 2009). Thus, the ratio of fetal to maternal iodide concentrations in control rabbits was 9 to 11. Unlike rabbits, it is thought that human fetuses do not accumulate iodide from the maternal circulation at such a high rate. Existing literature data suggest that the iodine concentration in normal human fetuses is usually lower than or equal to the maternal iodine concentration, resulting in a fetal/maternal plasma iodine concentration ratio of approximately 1. A study conducted by Rayburn et al. (2008) aimed to characterize the concentrations of fetal and maternal plasma iodine in iodine-free human fetal-maternal paired samples. These data were used to guide physiological pharmacokinetic (PBPK) modeling and improve the certainty of human health risk assessment. [1] The fetal and maternal iodine concentrations and the fetal/maternal plasma iodine ratio characterized in the Rayburn study (2008) confirmed that, unlike rabbit fetuses, human embryos do not have a high concentration of iodine relative to the maternal circulation. The maternal plasma iodine concentration in preterm infants was 1.6 ± 0.4 μg/dL, and in term infants it was 1.5 ± 0.5 μg/dL. The umbilical cord plasma iodine concentration in preterm infants was 1.4 ± 0.5 μg/dL, and in term infants it was 1.7 ± 0.7 μg/dL. The mean fetal/maternal plasma iodine ratio for all subjects was 1.2 ± 0.7. The mean fetal/maternal plasma iodine ratio in preterm infants was 0.9 ± 0.4 (n = 29), while the mean fetal/maternal plasma iodine ratio in full-term infants was 1.3 ± 0.8 (n = 92). The fetal/maternal plasma iodine ratio in preterm infants was less than 1. Therefore, the data suggest that the concentration of iodide in preterm infants is not as high as in rabbits. This mechanism of action supports the use of the cumulative concentration of iodide in fetal plasma (area under the curve or AUC) as a suitable internal dose indicator for physiological pharmacokinetics (PBPK) modeling.

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Neurotoxicity [1]
Regarding the potential role of iodomethane as a neurotoxicant, an acute inhalation neurotoxicity study in rats showed that iodomethane exposure caused transient neurotoxic behavioral manifestations. These neurotoxic manifestations included: a decrease in motor activity of 80%, a decrease in body temperature of 2-3°C, and repetitive mouth and jaw movements (clonic jerks) without neuropathological changes. All effects were transient and completely recovered. In subchronic or chronic studies of iodomethane, no clinical symptoms of neurotoxicity were observed in any of the subjects.

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Neurotoxicity—Mechanism of Action[1]
It is known that many inhaled solvents can produce anesthetic or sedative effects similar to iodomethane in animals and humans by altering the properties of nerve cell membranes (Snyder & Andrews, 1996). Iodomethane may inhibit spontaneous and induced activity of brain neurons by acting nonspecifically on the lipid matrix of nerve membranes, or by acting more specifically on chloride channels of γ-aminobutyric acid (GABAA) receptors, or by inhibiting neurotransmission of excitatory NMDA (N-methyl-D-aspartate) receptors (Balster, 1998; Trevor & White, 2004). Although these mechanisms of action are not directly related to iodomethane, their temporal correlation, type of action, and transient nature suggest that the effects of iodomethane on the nervous system are due to the concentration of its parent compound in the brain.

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

Nasal histopathology [1]
The histopathological changes caused by iodomethane exposure occurred in the respiratory tract, salivary glands and thyroid gland. The respiratory tract histopathology features nasal lesions, which manifest as olfactory epithelial degeneration (inlet effect). These lesions were found in a 13-week inhalation toxicity study, a multigenerational reproductive toxicity study and a combined chronic toxicity/carcinogenicity study in rats, and were limited to the extrathoracic region, without involvement of the trachea, bronchi or lung regions. In addition, these lesions did not appear to progress over time (i.e., the severity of nasal lesions was comparable after 4, 13 and 52 weeks of exposure at the same concentration), suggesting that nasal lesions are a result of reaching a critical concentration (Cmax) rather than being time-dependent (i.e. C × t; Hubble's law). In contrast, all systemic effects were assumed to be C × t.

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Nasal histopathology—Mechanism of action [1]
The existing evidence suggests that the mechanism by which iodomethane acts on the rat olfactory epithelium is GSH depletion. A study by Chamberlain et al. (1998b) explored several potential mechanisms of action of iodomethane on the nasal olfactory epithelium. This in vitro study ruled out two potential mechanisms of action: (1) methylation of cellular proteins and (2) cytochrome P450 metabolism of iodomethane to formaldehyde. The only study parameter associated with selective damage to the olfactory epithelium was the depletion of glutathione (GSH), which is caused by the binding of GSH to iodomethane catalyzed by glutathione S-transferase (GST)θ. [1] Another study by Chamberlain et al. (1998a) confirmed that GSH depletion is a key factor in the action of iodomethane on the rat nasal epithelium. In this study, rats pretreated with GSH isopropyl ester to increase tissue GSH levels were protected from iodomethane-induced nasal damage. Conversely, rats pretreated with phorone and sulfoxide imine to decrease tissue GSH levels showed a stronger response to nasal damage after exposure to iodomethane (Chamberlain et al., 1998a). This study concludes that the GSH consumption caused by the binding of methyl iodide to glutathione (GSH) is the mechanism by which it causes nasal effects and is also the detoxification pathway of methyl iodide.

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Using physiological pharmacokinetic (PBPK) models to derive the supercritical values of acute exposure[1]
This study used methyl iodide and iodide PBPK models to derive the supercritical values of three potential endpoints of 24-hour acute methyl iodide exposure. The general design and assumptions of PBPK models for developmental toxicity, nasal olfactory epithelial effects and acute neurotoxicity will be summarized in the following sections and described in detail in the paper published in this issue by Sweeney et al. (Citation 2009).

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PBPK model of developmental effects[1]
Since developmental effects may be the result of acute exposure, the fetal loss endpoint determined in rabbit developmental toxicity studies is also considered applicable to this risk assessment. For methyl iodide, the results of a phased developmental toxicity study in rabbits have to some extent confirmed this hypothesis. The study found that fetal mortality was slightly increased after rabbits were exposed to 20 ppm methyl iodide twice (6 hours each time). The mechanism of action identified in this study is that exposure to 20 ppm methyl iodide during days 23 to 26 of gestation resulted in excessively high serum iodide levels in rabbit fetuses. One mechanistic study indicated that excessive iodide levels could lead to fetal thyroid hormone imbalances (possibly a Wolf-Tchaikov effect), potentially resulting in fetal death. Therefore, the dose-response used in this assessment was the area under the curve (AUC) of fetal serum inorganic iodide concentration over time during a single day of exposure. Assuming a single rabbit exposure to methyl iodide lasting 6 hours and bystander exposure lasting 24 hours, the hazardous exposure concentration (HEC) calculated by the non-occupational risk assessment was 7.4 ppm. This human lethal concentration (HEC) was derived based on the assumption that human fetal serum iodide levels in equilibrium are 120% of maternal levels (Rayburn et al., 2008). [1]
In deriving the HEC corresponding to the No Observed Adverse Effect Level (NOAEL) in the 10 ppm rabbit study described in the previous paragraph, the hypothesis that a single exposure to methyl iodide in rabbits might have an effect was adopted. However, given that the sensitive window for fetal loss due to methyl iodide exposure is days 23 to 26 of gestation, the relevant single exposure day that had a key effect in the study was a single exposure day that had been previously exposed to methyl iodide for 17 days or longer (Nemec, cited in 2002 and 2003; Nemec et al., cited in 2009). In simulating repeated exposure of rabbits to 10 ppm methyl iodide, the concentration of fetal plasma iodide in rabbit fetuses increased with increasing daily exposure to methyl iodide and reached a steady state at approximately day 5. Using a repeated exposure model, the AUC corresponding to the daily fetal plasma iodide concentration 4 days after the previous exposure was derived. This AUC provides an alternative NOAEL equivalent within dose for a single day of exposure to 10 ppm methyl iodide in rabbits. The single-day AUC is not the AUC on the day of first exposure (day 6 of pregnancy), but rather reflects the curve of iodide concentration changes that may occur within the susceptible window. [1]
If 10 ppm is taken as the NOAEL for rabbits and the rabbits' previous exposure to iodomethane is considered in the model, the calculated bystander HEC is 26 ppm. This HEC value can be compared with the HEC value of 7.4 ppm, which is derived without considering the effect of continuous daily administration to rabbits before the susceptible window. Similarly, if the rabbits' previous daily administration is considered, the HEC value for workers (8 hours a day) is calculated to be 67 ppm, while the HEC value is 23 ppm when the effect of previous administration is ignored. Sweeney et al. (Citation 2009) describe the details of these PBPK model simulations. This repeated-dose model of rabbit exposure shows that the proposed HEC values of 7.4 ppm (non-occupational exposure) and 23 ppm (occupational exposure) are conservative because they do not take into account administration over previous days.

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Nasal Histopathology and PBPK Model Design[1]
It has been reported that rats developed nasal histopathological changes after daily exposure to methyl iodide for 13 weeks; however, published data suggest that rats may develop nasal lesions after acute exposure if the exposure concentration-time curve results in a total methyl iodide exposure greater than or equal to 200 ppm·hr (e.g., 100 ppm·hr for about 2 hours) (Reed et al., 1995). The nasal olfactory epithelium (NOE) compartments in the PBPK model are designed to allow methyl iodide to diffuse through cell stacks to all cell layers within the compartments. Each compartment allows methyl iodide to diffuse from the nasal cavity, passing sequentially through a layer of mucus, four layers of epithelial cells, and a blood exchange layer. This layered or “stacked” structure of the NOE compartments allows the model to approximate the concentration gradient of methyl iodide in the nasal tissue from the mucus layer to the vascularized region under the basement membrane. This corresponds to the degenerative changes that occur in the epithelium after exposure to high concentrations of methyl iodide. [1] The mechanism of action proposed in nasal histopathology is glutathione (GSH) depletion, a key event in the toxic pathway leading to nasal olfactory epithelial injury (Chamberlain et al., 1998a, 1998b). Therefore, GSH depletion is a dose-dependent indicator used in physiological pharmacokinetic (PBPK) models for interspecies extrapolation of nasal injury and determination of high lethal concentration (HEC). Choosing an appropriate level of GSH depletion to predict nasal toxicity depends on the assessment of the relationship between this indicator and toxicity, as well as the relationship between changes in exposure concentration over time and the prediction of GSH depletion. The premise of PBPK modeling is that the production of glutathione (GSH)-related toxicity in the nasal olfactory epithelium requires that GSH concentrations be reduced to below 50% of those in the control group and remain in a depleted state. At the end of 24 hours of iodomethane exposure, GSH concentrations only decreased by 50% transiently, which is the appropriate human lethal concentration (HEC) for the no-observed adverse effect level (NOAEL) in rats for the acute exposure endpoint (nasal toxicity), because the baseline GSH depletion level is almost impossible to reach or maintain. The average GSH depletion level of the four layers of epithelial cells in the human PBPK model is the basis of 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.)