Increasing confidence in new approach methodologies for inhalation risk assessment with multiple end point assays using 5-day repeated exposure to 1,3-dichloropropene.

New Approach Methodologies (NAMs) are being widely used to reduce, refine, and replace, animal use in studying toxicology. For respiratory toxicology, this includes both in silico and in vitro alternatives to replace traditional in vivo inhalation studies. 1,3-Dichloropropene (1,3-DCP) is a volatile organic compound that is widely used in agriculture as a pre-planting fumigant. Short-term exposure of humans to 1,3-DCP can result in mucous membrane irritation, chest pain, headache, and dizziness. In our previous work, we exposed differentiated cells representing different parts of the respiratory epithelium to 1,3-DCP vapor, measured cytotoxicity, and did In Vitro to In Vivo Extrapolation (IVIVE). We have extended our previous study with 1,3-DCP vapors by conducting transcriptomics on acutely exposed nasal cultures and have implemented a separate 5-day repeated exposure with multiple endpoints to gain further molecular insight into our model. MucilAir™ Nasal cell culture models, representing the nasal epithelium, were exposed to six sub-cytotoxic concentrations of 1,3-DCP vapor at the air-liquid interface, and the nasal cultures were analyzed by different methodologies, including histology, transcriptomics, and glutathione (GSH) -depletion assays. We observed the dose-dependent effect of 1,3-DCP in terms of differential gene expression, change in cellular morphology from pseudostratified columnar epithelium to squamous epithelium, and depletion of GSH in MucilAir™ nasal cultures. The MucilAir™ nasal cultures were also exposed to 3 concentrations of 1,3-DCP using repeated exposure 4 h per day for 5 days and the histological analyses indicated changes in cellular morphology and a decrease in ciliated bodies and an increase in apoptotic bodies, with increasing concentrations of 1,3-DCP. Altogether, our results suggest that sub-cytotoxic exposures to 1,3-DCP lead to several molecular and cellular perturbations, providing significant insight into the mode-of-action (MoA) of 1,3-DCP using an innovative NAM model.

Persistence and ingestion characteristics of phytochemical volatiles as bio-fumigants in Sitophilus oryzae adults.

Fumigant toxicity of phytochemical volatiles has been widely reported against stored product insect pests. Such volatiles are considered as natural fumigants and bio-fumigants in post-harvest food protection research. In the present study, persistence and ingestion of diallyl disulfide, citral, eucalyptol, eugenol and menthol were investigated in Sitophilus oryzae adults in comparison with fumigant toxicity and microstructural impact in elytra. The fumigant toxicity bioassay was performed with increasing concentrations of phytochemical volatiles at 25, 125, 250 and 500 µL/L air against S. oryzae adults in 50 mL glass vials. The phytochemical residues were examined from the treated adults by Gas Chromatography coupled with Flame Ionization Detector (GC-FID) and their pathological impacts on the elytral surface was observed under Scanning Electron Microscopy (SEM). After 72 h of fumigation, diallyl disulfide and eucalyptol were identified as potential fumigants with 5.24 and 8.30 µL/L air LC50 values, respectively. GC-FID analyses showed that diallyl disulfide and eucalyptol molecules persistence (1.29 and 2.60 ppb persistence with 0.94 and 0.90 r2 values respectively at 72 h exposure) on the body surface of weevil was positively correlated with the fumigation exposure and toxicity. Whereas, phytochemical molecules ingestion into the body of weevils was not directly linked with the insect mortalities. The SEM observations indicated that diallyl disulfide and eucalyptol molecules caused severe microstructural impacts on the elytra of weevils compared to other molecules. As a result, the present study suggested that phytochemical fumigants are persisted on the body surface and caused insecticidal toxicities in S. oryzae adults. In addition, it was predicted that persisted molecules might be entered into the body of weevils via cuticular penetration.

Histopathological, cytotoxicological, and genotoxic effects of the semi-synthetic compound dillapiole n-butyl ether in Balb/C mice.

Dillapiole n-butyl ether is a substance derived from dillapiole, which exhibits potential insecticidal effects on Aedes aegypti, the principal vector of the Dengue fever, Zika, and Chikungunya viruses, as well as Aedes albopictus, a vector of Dengue fever. As these mosquitoes are resistant to synthetic insecticides, dillapiole n-butyl ether may represent a valuable, plant-based alternative for their control. Dillapiole n-butyl ether has insecticidal and genotoxic effects on A. aegypti and A. albopictus, as shown by the reduction in clutch size and egg viability, and increased mortality rates, as well as a high frequency of micronuclei and chromosomal aberrations. However, the potential cytotoxic and genotoxic effects of this substance in mammals are still unknown. In Balb/C mice, structural changes were detected in hepatic, renal, and cardiac tissues, which were directly proportional to the concentration of the dose applied, in both genders. The induction of genotoxic, mutagenic, and cytotoxic effects was also observed at the highest concentrations (150 and 328 mg/kg). Further research will be necessary to better characterize the potential genotoxicity of this substance at lower concentrations, for the evaluation of the potential health risks related to its presence in environmental features, such as drinking water.

Weight of evidence analysis of the tumorigenic potential of 1,3-dichloropropene supports a threshold-based risk assessment.

1,3-Dichloropropene (1,3-D; CAS #542-75-6) is a fumigant used for preplant treatment of soil to control parasitic nematodes and manage soil borne diseases for numerous fruit, vegetable, field and tree and vine crops across diverse global agricultural areas. In the USA, 1,3-D has historically been classified by the U.S. EPA as likely to be carcinogenic to humans via both oral and inhalation routes. This classification for the oral route was primarily based upon increases in multiple tumor types observed in National Toxicology Program (NTP) cancer bioassays in rats and mice, while the classification for the inhalation route was based upon increased benign bronchioloalveolar adenomas in a mouse study conducted by The Dow Chemical Company. Based on U.S. EPA standard risk assessment methodologies, a low-dose linear extrapolation approach has been used to estimate risks to humans. Furthermore, genotoxicity associated with 1,3-D was historically considered a potential mode of action (MOA) for its tumorigenicity. New information is available and additional studies have been conducted that reveal a different picture of the tumorigenic potential of 1,3-D. These data and information include: (1) initial cancer studies by the NTP were conducted on an antiquated form of 1,3-D which contained a known mutagen/carcinogen, epichlorohydrin, as a stabilizer while current 1,3-D fumigants use epoxidized soybean oil (ESO) as the stabilizer; (2) results from two additional oral rodent cancer bioassays conducted on the modern form of 1,3-D became available and these two studies reveal a lack of carcinogenicity; (3) a newly conducted Big Blue study in F344 rats via the oral route further confirms that 1,3-D is not an in vivo genotoxicant; and (4) a newly conducted repeat dose inhalation toxicokinetic (TK) study shows that linear dose proportionality is observed below 30 ppm, which demonstrates the non-relevance of 60 ppm 1,3-D-induced benign lung tumors in mice for human health assessment. This weight of evidence review is organized as follows: (a) the TK of 1,3-D are presented because of relevant considerations when evaluating test doses/concentrations and reported findings of tumorigenicity; (b) the genotoxicity profile of 1,3-D is presented, including a contemporary study in order to put a possible genotoxicity MOA into perspective; (c) the six available bioassays are reviewed followed by (d) scientifically supported points of departure (PODs) and evaluation of human exposure for use in risk assessment. Through this assessment, all available data support the conclusion that 1,3-D is not a tumorigen at doses below 12.5 mg/kg bw/day via the oral route or at doses below 30 ppm via the inhalation route. These findings and clearly identified PODs show that a linear low dose extrapolation approach is not appropriate and a threshold-based risk assessment for 1,3-D is human health protective. Finally, in 2019, the Cancer Assessment Review Committee (CARC) reevaluated the carcinogenic potential of 1,3-D. In accordance with the EPA's Final Guidelines for Carcinogen Risk Assessment, the CARC classified 1,3-D (Telone) as "Suggestive Evidence of Carcinogenic Potential based on the presence of liver tumors by the oral route in male rats only." Given this finding, EPA stated that "quantification of human cancer risk is not required. The CARC recommends using a non-linear approach (i.e. reference dose (RfD)) that will adequately account for all chronic toxicity including carcinogenicity, that could result from exposure to 1,3-dichloropropene."

In vivo mutagenicity evaluation of the soil fumigant 1,3-dichloropropene.

1,3-Dichloropropene (1,3-D; CAS No. 542-75-6) is a soil fumigant used for the control of nematodes in agriculture. There is an extensive database on the genotoxicity of 1,3-D and many of the published studies are confounded by the presence of mutagenic stabilisers in the test substance. Mixed results were obtained in the in vitro assays, often due to the purity of the 1,3-D sample tested. In order to get further clarity, the mutagenic potential of 1,3-D was investigated in vivo in the transgenic Big Blue rodent models. Inhalation exposure of 150 ppm 1,3-D (×2.5 tumourigenic dose) to transgenic male B6C3F1 mice did not induce lacI mutations in either the lung (tumour target tissue) or liver. Similarly, dietary administration of 1,3-D up to 50 mg/kg/day to transgenic male Fischer 344 rats did not increase the cII mutant frequency in either the liver (tumour target) or kidney. These results, along with other available in vivo data, including the absence of DNA adducts and clastogenic/aneugenic potential, support the conclusion that 1,3-D is efficiently detoxified in vivo and, as such, does not pose a mutagenic hazard or risk.

Peer review of a cancer weight of evidence assessment based on updated toxicokinetics, genotoxicity, and carcinogenicity data for 1,3-dichloropropene using a blinded, virtual panel of experts.

A cancer weight of evidence (WOE) analysis based on updated toxicokinetics, genotoxicity, and carcinogenicity data for 1,3-dichloropropene was peer reviewed by a panel of experts. Historically, 1,3-dichloropropene has been classified in the U.S. as "likely to be carcinogenic to humans" via oral and inhalation exposure routes based upon the results of rodent cancer bioassays conducted in the 1980s. Contemporary studies led the authors of the WOE analysis to conclude that the currently manufactured form of 1,3-dichloropropene is not mutagenic and not carcinogenic below certain doses, pointing to a threshold-based approach for cancer risk assessment. SciPinion conducted a peer review of the WOE analysis using methods for assembling and managing blinded expert panels that maximize expertise while minimizing potential selection/participation bias. The process was implemented through a web-based application that poses a series of questions soliciting the experts' scientific opinions and observations about specific topics. The goal of the peer review was to have experts provide conclusions about the WOE for carcinogenicity classification of 1,3-dichloropropene, identify potential data gaps, and evaluate the validity of a threshold-based risk assessment for 1,3-dichloropropene. Based on a robust peer review of the current scientific information, a cancer WOE classification of "not likely to be carcinogenic to humans" is best supported for 1,3-dichloropropene. This conclusion is reached with a high degree of consensus (consensus score = 0.92) across expert panel members.

The association between 1,3-dichloropropene and asthma emergency department visits in California, USA from 2005 to 2011: a bidirectional-symmetric case crossover study.

Objective: The ever-increasing rate of pesticide use in California farmlands is of great concern due to its potential toxicity on human health. In this study, the association between short term exposure to 1,3-dichloropropene (1,3-D) and asthma emergency department (ED) visits in central and southern California from 2005 to 2011 is investigated.Methods: 3878 ED visits were identified from 2005 to 2011 (1064 days). Conditional logistic regression models were used to obtain the odds ratio (OR) associated with 0.01 ppb increase in 1,3-D. Potential effect modification by sex, race/ethnicity (Non-Hispanic White, Non-Hispanic Black, or Hispanic), and age (2-5, 6-18, 19-40, 41-64 or ≥ 65) are investigated.Results: It was found that a 0.01 ppb increase in 1,3-D at the event day is associated with 13.5% [OR = 1.135, 95% CI: 1.123, 1.149] increase in the odds of having asthma ED visits in central and southern California during October to February of 2005 to 2011. Race had a positive association between 1,3-D and asthma ED visits among Non-Hispanic Black [OR= 1.095 95% CI: 1.035, 1.155] and Hispanic [OR= 1.121 95% CI: 1.064, 1.179]; while Non-Hispanic Whites had no association. Positive association for age was found between 1,3-D and asthma ED visits among patients 2 to 5 [OR= 1.065 95% CI: 1.020, 1.133], 6 to 18 [OR= 1.142 95% CI: 1.086, 1.196], and 19 to 40 [OR= 1.023 95% CI: 1.015, 1.073] years old.Conclusion: These findings suggest a 0.01 ppb increase in 1,3-D concentration increases the odds of having asthma ED visits.

Transcriptomic analysis of the phytotoxic effects of 1-allyl-3-methylimidazolium chloride on the growth and plant hormone metabolic pathways of maize (Zea mays L.) seedlings.

In this study, we investigated the phytotoxicity of an imidazolium-based ionic liquid, 1-allyl-3-methylimidazolium chloride ([Amim]Cl), against maize seedlings. It was found that in response to an increase in [Amim]Cl treatment concentrations, there were significant decreases in growth parameters (fresh weights and lengths) and the photosynthetic pigment contents of maize plants, whereas in contrast, the malondialdehyde content increased. In order to determine the molecular basis of [Amim]Cl-induced plant growth inhibition, an RNA-Seq analysis to examine the gene expression profiles of selected central biological pathways was performed. And a total of 4024 genes that were differentially expressed between control and 400 mg/L [Amim]Cl-treated plants were accordingly identified. Pathway enrichment analysis for the differentially expressed genes revealed that 12 of 15 genes in the porphyrin and chlorophyll metabolic pathways were down-regulated in response to [Amim]Cl treatment. Moreover, all six genes encoding key chlorophyll synthetic enzymes were down-regulated by [Amim]Cl. With regards to plant hormone metabolic pathways, the genes encoding key enzymes involved in ethybilene and abscisic acid (ABA) biosynthesis were up-regulated in response to [Amim]Cl treatment. Genes responsible for gibberellin (GA) inactivation were also stimulated by [Amim]Cl. These observations indicate that [Amim]Cl may promote the biosynthesis of senescence-related hormones (ethylene and ABA) as well as inactivation of growth-promoting hormones (GAs). It might be concluded that the observed [Amim]Cl-induced inhibition of maize seedling growth could be associated with changes in the gene expression profiles of these metabolic pathways.

Metabolic Basis for Nonlinearity in 1,3-Dichloropropene Toxicokinetics and Use in Setting a Kinetically-derived Maximum Inhalation Exposure Concentration in Mice.

1,3-Dichloropropene (1,3-D) showed a statistically increased incidence of bronchioloalveolar adenomas in male B6C3F1 mice at 60 ppm air concentration during previous chronic inhalation testing. No tumors were observed in female mice, nor in either sex of F344 rats up to 60 ppm, the highest dose tested. Therefore, to understand if lung tumors observed in high dose male mice are due to saturation of metabolic clearance, the linearity of 1,3-D concentrations in mouse blood was investigated on day 15 of repeated nose-only inhalation exposure to 0, 10, 20, 40, 60, 90, and 120 ppm (6 h/d, 7 d/week). Additional groups were included at 20, 60, and 120 ppm for blood collection at 1.5 and 3 h of exposure and up to 25 or 40 min post-exposure to determine area-under-the-curve. The data provide multiple lines of evidence that systemic exposures to 1,3-D in the mouse become nonlinear at inhalation exposure levels of 30 ppm or above. A reduction in minute volume occurred at the highest exposure concentration. The glutathione (GSH)-dependent metabolism of 1,3-D results in significant depletion of GSH at repeated exposure levels of 30 ppm and above. This loss of GSH results in decreased metabolic clearance of this test material, with a concomitant increase of the 1,3-D isomers in circulating blood at exposure concentrations ≥30 ppm. Shifts in the ratio of cis- and trans-1,3-D also support nonlinear toxicokinetics well below 60 ppm. Based on this data, a kinetically derived maximum dose for 1,3-D in mice for repeated exposures should be at or below 30 ppm. These results support non-relevance of 1,3-D-induced benign pulmonary tumorigenicity in mice for human health risk assessment.

A fluorescent probe for carbon monoxide based on allyl ether rather than allyl ester: A practical strategy to avoid the interference of esterase in cell imaging.

Pd0-mediated Tsuji-Trost reaction is a practical strategy to design fluorescent probes for carbon monoxide (CO) sensing, and in such reaction CO can reduce Pd2+ to Pd0 in-situ and remove allyl groups on fluorophores. In most of these probes, esters are commonly used to link allyl on fluorophores. We found that the ester groups could be hydrolyzed by esterase activity of fetal bovine serum (FBS), while FBS is a requisite in cell culture, and the hydrolysis could interfere the Pd0-mediated Tsuji-Trost reaction. In this study, we synthesized a fluorescent probe (Cou-CO) using allyl ether as reaction site rather than allyl ester. Cou-CO is non-fluorescence, and could react with CO under the presence of Pd0 to form Cou with strong fluorescence, and the maximum excitation and emission wavelengths of Cou are 464 nm and 495 nm respectively. Cou-CO shows excellent selectivity to CO and could avoid the effect of FBS with the limit of detection for CO is 78 nm. Finally, Cou-CO was successfully applied for imaging of CO in living cells.

Developmental differences in the expression of ABC transporters at rat brain barrier interfaces following chronic exposure to diallyl sulfide.

Many pregnant women and prematurely born infants require medication for clinical conditions including cancer, cardiac defects and psychiatric disorders. In adults drug transfer from blood into brain is mostly restricted by efflux mechanisms (ATP-binding cassette, ABC transporters). These mechanisms have been little studied during brain development. Here expression of eight ABC transporters (abcb1a, abcb1b, abcg2, abcc1, abcc2, abcc3, abcc4, abcc5) and activity of conjugating enzyme glutathione-s-transferase (GST) were measured in livers, brain cortices (blood-brain-barrier) and choroid plexuses (blood-cerebrospinal fluid, CSF, barrier) during postnatal rat development. Controls were compared to animals chronically injected (4 days, 200 mg/kg/day) with known abcb1a inducer diallyl sulfide (DAS). Results reveal both tissue- and age-dependent regulation. In liver abcb1a and abcc3 were up-regulated at all ages. In cortex abcb1a/b, abcg2 and abcc4/abcc5 were up-regulated in adults only, while in choroid plexus abcb1a and abcc2 were up-regulated only at P14. DAS treatment increased GST activity in livers, but not in cortex or choroid plexuses. Immunocytochemistry of ABC transporters at the CSF-brain interface showed that PGP and BCRP predominated in neuroepithelium while MRP2/4/5 were prominent in adult ependyma. These results indicate an age-related capacity of brain barriers to dynamically regulate their defence mechanisms when chronically challenged by xenobiotic compounds.

Effects of fumigation with 1,3-dichloropropene on soil enzyme activities and microbial communities in continuous-cropping soil.

The compound 1,3-D (1,3-dichloropropene) is a potential candidate soil fumigant due to the restrictions on methyl bromide (MB). To date, little is known about the soil microbial community changes induced by 1,3-D fumigation. Therefore, soil properties, related soil enzymes, genes encoding the key enzymes of ammonia oxidation in both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) and bacterial diversity were investigated to assess the effects of 1,3-D fumigation on the soil microbial community. The results exhibited that fumigation with 1,3-D caused accumulation of NH4+-N, but it led to decrease in the rate of NO3--N, and the concentration of NO3--N gradually recovered. At 12 weeks after transplant (WAT) of tomato seedlings, the concentration of NH4+-N and NO3--N were not statistically significant between the 1,3-D treatment groups and the untreated control group. A similar tendency was found for organic matter, soil pH, urease and protease activities. Moreover, quantitative real-time PCR (qPCR) showed that 1,3-D decreased total bacterial abundance, AOA-amoA and AOB-amoA genes. In addition, Illumina MiSeq sequencing analysis revealed that soil bacterial community diversities were significantly reduced at earlier sampling time points, and at later sampling time points, soil bacterial diversity gradually recovered, there was no significant difference compared to the control group. The present study provides useful information to evaluate the environmental safety of 1,3-D.

In vitro evaluation of structural analogs of diallyl sulfide as novel CYP2E1 inhibitors for their protective effect against xenobiotic-induced toxicity and HIV replication.

Diallyl sulfide (DAS) has been shown to prevent xenobiotic (e.g. ethanol, acetaminophen) induced toxicity and disease (e.g. HIV-1) pathogenesis. DAS imparts its beneficial effect by inhibiting CYP2E1-mediated metabolism of xenobiotics, especially at high concentration. However, DAS also causes toxicity at relatively high dosages and with long exposure times. Therefore, the goal of the current study was to investigate the structural analogs of DAS for their improved toxicity profiles and their effectiveness in reducing xenobiotic-induced toxicity and HIV-1 replication. Previously, we identified commercially available analogs that possessed CYP2E1 inhibitory capacity greater than or equal to that of DAS. In this study, we evaluated the toxicity and efficacy of these analogs using hepatocytes, monocytes, and astrocytes where CYP2E1 plays an important role in xenobiotic-mediated toxicity. Our results showed that thiophene, allyl methyl sulfide, diallyl ether, and 2-prop-2-enoxyacetamide are significantly less cytotoxic than DAS in these cells. Moreover, these analogs reduced ethanol- and acetaminophen-induced toxicity in hepatocytes and HIV-1 replication in monocytes more effectively than DAS. Overall, our findings are significant in terms of using these DAS analogs as a tool in vitro and in vivo, especially to examine chronic xenobiotic-induced toxicity and disease pathogenesis that occurs through the CYP2E1 pathway.

Ecotoxicity of 1,3-dichloropropene, metam sodium, and dazomet on the earthworm Eisenia fetida with modified artificial soil test and natural soil test.

1,3-Dichloropropene (1,3-D), metam sodium (MS), and dazomet (DZ) are widely used as preplant soil fumigants to solve soilborne problems. To provide a more scientific and accurate evaluation of 1,3-D, MS, and DZ toxicity to the earthworm Eisenia fetida, modified artificial soil test and natural soil test were studied. The suitable soil moisture to maintain over 90% survival of the earthworms after 4 weeks of treatment in an enclosed system for modified artificial soil test and natural soil test were 26.9 to 86.4% of water-holding capacity (WHC) and 66.2 to 84.3% of WHC, respectively. The optimal soil moisture levels for modified artificial soil test and natural soil test (75 and 55% of WHC, respectively) were finally used to evaluate the toxicity of 1,3-D, MS, and DZ on earthworms. Each desiccator with 10 earthworms and natural or artificial soil was stored at 20 ± 1 °C under constant light of 400 to 800 lx for 2 weeks. The modified artificial soil test showed LC50 values for 1,3-D, MS, and DZ of 3.60, 1.69, and 5.41 mg a.i. kg-1 soil, respectively. The modified natural soil test of the fumigants showed similar LC50 values of 2.77 and 0.65 mg a.i. kg-1 soil, except for DZ at 0.98 mg a.i. kg-1 soil. The present study confirms that both modified artificial soil test and modified natural soil test offer standard methods for acute toxicity test of 1,3-D, MS, and DZ on the earthworms and scientific evidences for assessing the effects of soil fumigants on non-target organisms in the soils. Graphical Abstract Two novel acute toxicity test methods for soil fumigants on the earthworm Eisenia fetida.

Garlic-derived organosulfur compound exerts antitumor efficacy via activation of MAPK pathway and modulation of cytokines in SGC-7901 tumor-bearing mice.

Diallyl trisulfide (DATS), a natural agent derived from garlic, has been tested for its antigastric cancer activities in various preliminary studies. However, more systematic pharmacodymatic (PD) and mechanistic evaluations are clearly needed. The aim of this study was to investigate the antitumor effects of DATS in the treatment of human gastric cancer cell SGC-7901 both in vitro and in vivo using widely recommended study procedures. DATS suppressed cancer cells proliferation and induced cell cycle arrest accompanied by an increase in the expressions of cyclin A2 and cyclin B1 in SGC-7901 cancer cells. DATS also caused an increase in apoptotic cell death, which involved in accumulations of bax, p53, and cytochrome C and reduction of Bcl-2 expressions. Besides, activation of JNK, ERK and p38 phosphorylation in DATS-treated cells suggested that mitogen-activated protein kinase (MAPKs) pathways were involved in DATS-induced apoptosis. Meanwhile, DATS significantly inhibited tumor growth and promoted tumor apoptosis in a xenograft model of gastric cancer cell SGC-7901. DATS inhibited tumor migration and invasion by modulating MMP9 and E-cadherin protein expressions. In addition, DATS treatment evidently increased the cytokine secretions of IL-12, TNF-α and IFN-γ (p<0.05). Biochemical serum analysis and histopathological examination indicated no obvious side effects in major mouse organs. Therefore, our findings provide a framework for further exploration of DATS as a novel chemotherapeutic for human gastric cancer.

Evaluation of potential human health effects associated with the agricultural uses of 1,3-D: Spatial and temporal stochastic risk analysis.

Dow AgroSciences (DAS) markets and sells 1,3-Dichloropropene (1,3-D), the active ingredient in Telone®, which is used as a pre-plant soil fumigant nematicide in economically important crops in California. 1,3-D has been regulated as a "probable human carcinogen" and the California Department of Pesticide Regulation limits use of 1,3-D based on human health risk assessments for bystanders. This paper presents a risk characterization for bystanders based on advances in the assessment of both exposure and hazard. The revised bystander risk assessment incorporates significant advances: 1) new data on residency duration and mobility in communities where 1,3-D is in high demand; 2) new information on spatial and temporal concentrations of 1,3-D in air based on multi-year modeling using a validated model; and 3) a new stochastic spatial and temporal model of long-term exposures. Predicted distributions of long-term, chronic exposures indicate that current, and anticipated uses of 1,3-D would result in lifetime average daily doses lower than 0.002mg/kg/d, a dose associated with theoretical lifetime excess cancer risk of <10(-5) to >95% of the local population based on a non-threshold risk assessment approach. Additionally, examination of 1,3-D toxicity studies including new chronic toxicity data and mechanism of action supports the use of a non-linear, threshold based risk assessment approach. The estimated maximum annual average daily dose of <0.0016mg/kg/d derived from the updated exposure assessment was then compared with a threshold point of departure. The calculated margin of exposure is >1000-fold, a clear indication of acceptable risk for human health. In summary, the best available science supports 1,3-D's threshold nature of hazard and the revised exposure assessment supports that current agricultural uses of 1,3-D are associated with reasonable certainty of no harm, i.e., estimated long-term exposures pose insignificant health risks to bystanders even when the non-threshold approach is assumed.