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Responses collected during the most recent interview were used for these analyses

The 13-items included: social, mellow, creative,top of the world, increased sex drive, energetic, dizzy, nauseous, drowsy, lazy, unable to concentrate, out of control, and guilty. Participants were asked “in the period shortly after you used did it make you feel…”? Responses were scored as present or absent and the item scores summed to make the positive and negative scales.To examine the dimensionality of the 13 subjective experiences examined for each substance, we conducted Mokken Scale Analysis using the statistical software STATA. Mokken scaling analysis extends traditional factor analysis by systematically hierarchically ordering items that are highly correlated. Mokken analysis provides a nonparametric, iterative scale-building technique that identifies the smallest set of internally consistent scales from a given item pool. This model assumes the presence of one or more latent traits that can be measured by subject responses to a set of items. MSA is probabilistic and hierarchical, meaning that the items can be ordered by a degree of “difficulty”;individuals who agree with a more difficultitem will tend to agree with less difficult items. Scales from MSA are formed by taking pairs of items with the highest correlation and including other items until there is no further improvement. Loevinger’s H coefficients, which indicate the fit of an item to the scale, are computed for each item within a scale and for the scale as a whole. H coefficients ranging between 0.3 and 0.4 indicate a weak scale, 0.4–0.5 a medium scale, and 0.5–0.9 a strong scale. In MSA, an item can remain “unscaled” because it could not be added to one of the alternative scales without weakening the scale’s homogeneity. Based on our previous analyses , we used the scaling derived from the CADD sample. Positive and negative scales were standardized by age, sex, vertical outdoor farming and clinical status with two groups. Pairwise correlations between the resulting two scales were then determined for alcohol, tobacco, and marijuana.

Two consistent subjective experience scales were revealed using MSA. The item guilty did not fit into either scale for any substance. The item out of control was dropped due to extremely low endorsement for tobacco and because this item fluctuated between the positive scale and the negative scale. For all three drugs the positive scale included: relaxed, sociable, creative, euphoric, energetic, and increased sex drive. Items included in the negative scale for all three drugs were: lazy, drowsy, unable to concentrate, dizzy, and nauseous.Table 2 provides the H-coefficients for the positive and negative scales for each of the three drugs with the items shown in their hierarchical ordering. For the positive subjective experiences H-coefficients for alcohol,tobacco, and marijuana were 0.45, 0.52, and 0.43, respectively. For the negative scale, the H-coefficients were 0.42, 0.52, and 0.54, respectively. Means and standard deviations for each subjective experience scale across all three drugs are shown in Table 3. Mean scores differed as a function of age, sex, and clinical status. For all but marijuana, younger subjects had significantly lower mean scores on both positive and negative scales. Males scored significantly higher for the positive scales across all drugs than females. Males also had a higher mean score for the alcohol negative scale and a lower mean score for the marijuana negative scale. Mean scores on the positive experiences to tobacco and negative experiences to marijuana scales were higher in our community sample than in our clinical sample. Mean scores were lower in our community sample than in our clinical sample for positive experiences to marijuana and negative experiences to alcohol scales.In the current report, we examined subjective experiences to three commonly used drugs of abuse among young adults from the general community and an area treatment program. In these data, we obtained results that supported previous observations indicating positive and negative subjective experiences for a particular drug were predictive of problem use of that same drug. We then extended this relationship in two ways. First, we obtained results that supported the notion that positive and negative experiences to one drug are similar to those experienced for another drug and second, that subjective experiences to a drug are predictive of the risk for problem use of other drugs.

We interpret these findings to suggest that subjective experiences may be a useful indicator of a common liability towards use and problem use of multiple substances. Following on our previous work on marijuana and subjective experiences , we used Mokken scaling to simultaneously examine whether subjective experiences to three drugs are associated with drug use outcomes. From these analyses we observed that the subjective experience scales for each of the three drugs were comparable to those found in previous studies despite using a different methodology. We observed differences in item means and hierarchical ordering of the items by substance suggesting that subjects are reporting drug specific subjective experiences. This interpretation is consistent with findings from laboratory based studies which have shown that subjects can differentiate between a placebo and a drug or between different drugs based on subjective experiences. As different combinations of alcohol, tobacco, and marijuana use are commonly reported in epidemiological studies, we investigated the relationship between subjective experiences to different drugs in poly-substance users. We observed that subjective experiences to one drug were significantly correlated with experiences to another drug, though the strength of the relationship varied for different drug combinations. The strongest relationships were between alcohol and marijuana, replicating two previous studies , and between alcohol and tobacco. These particular drug combinations target similar neuronal receptor systems and are reported to enhance the overall drug experience when taken together. Further, as subjective experiences are thought to reflect the underlying physiology of a drug’s actions , these cross-substance relationships may provide a closer approximation of a common risk factor suitable for molecular genetic investigation. In this sample of community and clinical subjects, subjective experiences for one drug were associated with outcomes related to a different drug.

Though our results replicate findings that relate positive experiences with greater use of other drugs, we also identified that negative experiences were predictive of abuse and dependence status of a different drug. In particular, negative effects of alcohol and marijuana were associated with misuse of these same drugs as well as tobacco. Although this may appear counter-intuitive, a possible explanation could be that subjects who needed greater amounts of a drug in order to feel its effects drove the observed association. Findings from laboratory-based drug discrimination studies suggest that some subjects are unable to differentiate between drug and placebo at a standard training dose. Differences between the two conditions could, however, be reported as non-discriminators were exposed to greater doses of a drug. Interestingly, those who were able to discriminate between non-exposure and exposure to a drug reported stronger positive and negative subjective experiences, often simultaneously , at greater doses. This underscores the importance of dose in determining individuals’ drug sensitivity as assessed by subjective experiences. The relationship between drug dose, the resulting subjective experiences, and problem drug use has also been examined using self-ratings to the effects of alcohol [SRE; 28]. The SRE primarily assesses negative experiences to alcohol such as dizziness and passing out as related to the dosing levels needed to feel the sedative effects of alcohol. Among adolescent and adult samples of both sexes and family-history positive studies of alcoholics , low levels of response, as measured by the SRE have been implicated as a risk factor for alcohol use disorders. This notion that some drinkers need to ingest greater amounts of alcohol to feel its sedative effects and that this effect is related to greater drinking quantities has been recently supported and extended to include the observation that this relationship is also relevant to those reporting lower levels of stimulant effects during the first five drinks. Our finding that negative alcohol experiences were predictive of problem alcohol use is consistent with this research, despite using a different questionnaire, and extends it to include the potential prediction of other drug use problem behaviors.

Findings from the current study should be considered in light of a number of limitations. First, subjective experiences were collected from participants who ranged between 11 and 30 years of age. Though scaling of the different experiences was consistent between younger and older subjects, the older subjects have typically had a longer use history of alcohol, tobacco, and marijuana. Second, subjective experiences were only collected from those who reported using alcohol and marijuana six or more times and daily use of tobacco for a month. Thus, we were not able to include experiences from those who had used only a few times. Third, we were not able to measure differences in dosage, quality of a drug, depth of inhalation, peer use and drug use setting some of which impact self-reported subjective experiences. Fourth, we were not able to establish the reference point from which people were making their ratings. This is due to the unclear phrasing of the stem questions that asks about experiences “shortly after using.” We cannot know whether subjects were reporting their initial experiences, experiences in the minutes or hours immediately following recent drug intake or the conglomeration of their drug experience, making causal inferences regarding the observed associations not possible. Lastly, the lower endorsement rates for the experiences of tobacco may be due to the dichotomous fashion of responses that limit the sensitivity to detect the milder effects. Further, there more stringent requirements for collecting subjective experiences to tobacco may have made it more difficult to detect significant relationships with subjective experiences to alcohol, tobacco,rolling grow table and their more problematic use behaviors.Despite the high co-morbidity between marijuana and nicotine use, only few studies have directly addressed the mechanisms that lead to their concurrent use. A recent review by Agrawal describes multiple etiologies that influence their comorbidity. This includes route of administration , cross drug adaptation, response to treatments, environmental effects and genetic factors. Others have also alluded to the “gateway drug” hypothesis whereby the use of one drug may potentiate the effects of the other. For example, in a longitudinal study in 14–15 year olds, marijuana use increased the likelihood of initiating nicotine use up to 8 times and developing nicotine dependence up to 3 times suggesting marijuana’s role as a gateway drug. This was further supported by findings showing that women who used marijuana were at 4.4 odds of later developing nicotine use and dependence. The same group also reported in 43,093 adults that nicotine smoking increased the risk for marijuana use and dependence up to 3 times. This latter finding suggests a bi-directional potentiating effect and indicates that more complex factors may drive combined use.

Although the animal literature has characterized the neural mechanisms that may underlie these potentiating effects, it is also possible that personality factors contribute to this phenomenon. Combined marijuana and nicotine use has been associated with differential effects on clinical diagnoses, cognitive and psychosocial problems, and outcomes. For example, Bonn-Miller and colleagues examined associations between negative emotions that discriminate marijuana-only users from co-morbid marijuana and nicotine users. They found that, in general, nicotine-only using individuals had significantly greater negative emotionality than marijuana users, co-morbid marijuana and nicotine users, and non-using controls. Earlier work by Degenhardt showed that while nicotine and marijuana use were both individually associated with increased rates of negative emotion, this relationship appeared to be driven by neuroticism in marijuana users. Taken together, these studies argue for different patterns of co-morbidity in nicotine and marijuana using populations. To date, however, distinctions in trait markers, such as personality factors, have not yet been addressed in this ubiquitous group of co-morbid users. These differences suggest the need for fine-tuning the ability to discriminate risk-profiles between these groups as they also relate to clinical treatment outcomes. Factors that contribute to risk profiles include personality traits that have been examined as putative markers for treatment outcomes. For example, in a prospective four-year study in 112 adults with chronic alcoholism, Krampe et al. determined that the presence of any personality disorder was associated with a decrease in four-year abstinence probability. Similarly, using the NEO Personality Inventory-Revised Betkowska-Korpala found that following treatment, abstinent patients have higher levels of agreeableness and conscientiousness than patients who relapsed within a year following the therapy. This suggests that personality profiles have high predictive values for SUD outcomes and should be considered during treatment programs.

The profile of the changing genes was comparable between tobacco and marijuana exposed cells

Exposure to tobacco smoke can also trigger an inflammatory response and induce oxidative stress through increased levels of reactive oxygen species. Persistent induction of these processes following repeated exposure contributes to loss of normal growth control mechanisms, which is a key step in cancer development. Our study supports many of these findings, with exposure to TSC inducing the expression of genes involved in xenobiotic metabolism , oxidative stress , and DNA damage response as evidenced by changes in the expression of genes involved in cell cycle arrest, protein unfolding,transcription regulation, and inflammation. These same pathways were also significantly affected following MSC exposure, indicating that, as expected, MSC impacts many of the same molecular processes and functions as TSC. Although the effects of the condensates were largely similar, dose–response analysis indicates that the MSC is substantially more potent than TSC, with BMDs that in many instances are an order of magnitude lower than those for TSC. In addition, the results also highlighted some differences in steroid biosynthesis , apoptosis and inflammation, which were more significantly affected following MSC exposure, and cell cycle , which was more affected following TSC exposure.IPA canonical pathways related to the metabolism of xenobiotics were significantly affected in both TSC and MSC exposed cells at both time points. These pathways included Xenobiotic Metabolism Signaling, Metabolism of Xenobiotics by CYP450,microgreen flood table and AHR Signaling. For both TSC and MSC, the number of genes that were significantly affected increased with increasing concentration and the greatest number of genes changing occurred at the 6 + 4 h time point.

Many of the genes that were differentially expressed in TSC exposed cells are among those that have been typically observed to be induced by cigarette smoke [e.g., Nqo1 , Esd , Hmox1 , Cyp1a1 and Cyp1b1. Moreover, the concentration response patterns support the assertion of initial metabolic responses , followed by responses to toxic insult and secondary metabolism. Similar concentration response trends were noted in our previous toxicogenomics analysis of three different TSCs. Although very few studies have been conducted with marijuana smoke, Roth et al. demonstrated the induction of cytochrome P450 genes following exposure of Hepa-1 cells to marijuana tar extracts. Furthermore, the authors showed that tar from marijuana cigarettes tends to be more effective than tar from tobacco at inducing Cyp1a1 gene expression. Since the cannabinoids present in marijuana are capable of acting through the aryl hydrocarbon receptor to induce cytochrome P450 enzymes , and Cyp1a1 is known to bioactivate procarcinogens such as PAHs , questions have been raised about the role of cannabinoids in augmenting the carcinogenic risk posed by marijuana smoke. The question becomes increasingly complex as the cannabinoids THC, CBD and CBN have also been shown to competitively inhibit Cyp1a1, potentially decreasing the production of carcinogens and curtailing negative consequences. In the present study, however, substantial differences in the expression profiles of cytochrome P450 genes between the two smoke types were not observed. The expression of Cyp1a1 following exposure to MSC was comparable to that following TSC exposure, and the micro-array results were supported by RT-PCR. One of the differences in the xenobiotic metabolism responses for the two condensate types is that Hsp90 and Rras2 were only upregulated following MSC exposure. Despite these findings, Hsp90 has been previously observed to be induced following cigarette smoke exposure , and mutations in genes from the Ras family are known to be associated with cigarette-induced cancers.The IPA Canonical Pathway most significantly affected by exposure to TSC was the NRF2-Mediated Oxidative Stress Response Pathway. In this pathway, the transcription factor Nrf2 is phosphorylated following exposure to reactive oxygen, and translocates to the nucleus where it binds to antioxidant response elements. It then activates the expression of detoxification and antioxidant genes that protect the cell against oxidative damage. Of the 192 genes in this pathway, 6–18 genes were perturbed by TSC at the various time points in a concentration dependent manner. The largest expression changes and number of genes were associated with the 6 h time point.

Nrf2-regulated antioxidant genes have been shown to play an important role in protection against the toxic effects of tobacco smoke. Iizuka et al. showed that neutrophilic lung inflammation was significantly enhanced in Nrf2-knockout mice following cigarette smoke exposure. In addition, emphysema was observed 8 and 16 weeks following cigarette smoke exposure in the knockout mice, whereas no pathological abnormalities were observed in wild-type mice. Similarly, Gebel et al. confirmed the protective nature of Nrf2 against the development of emphysema in cigarette smoke exposed wild type mice versus Nrf2 knockout mice, and further investigated the relationships between Nrf2 and inflammation and cell cycle arrest. Comandini et al. conducted a meta-analysis of eight genomic studies on the mechanisms of smoke-induced lung damage in healthy smokers, COPD smokers and non-smokers. They found the Nrf2-mediated oxidative stress response Pathway to be the most significantly altered pathway in healthy smokers compared to non-smokers. In contrast, the Nrf2 pathway was not significantly differentially expressed in COPD smokers, indicating that Nrf2-regulated genes play a key role in protecting against the toxic effects of TSC. The authors suggest that the response of Nrf2- regulated genes may potentially be used as a biomarker for COPD susceptibility. In the present study, we found that the NRF2-Mediated Oxidative Stress Response Pathway is also an important component of the toxicological response to MSC. IPA analyses identified it as one of the top five pathways for both time points and all concentrations of MSC, except for the lowest concentration at the 6 + 4 h time point. A comparison of the Nrf2 pathway at the 6 h time point for the highest exposure concentrations of TSC and MSC shows many similarities. The Nrf2 gene itself was up-regulated along with several basic leucine zipper family transcription factors such as Jun, Atf4, and Maff. In addition, several antioxidant and stress response proteins such as Nqo1, Prdx1, Hmox1, Sod, Txnrd1, Herpud1, Dnajb1/9 were up-regulated. Other studies have also noted that these genes are up-regulated following cigarette smoke exposure. 

However, a notable difference between the two condensates studied here is that Gclc and Gclm, the rate limiting enzymes in glutathione synthesis, were significantly upregulated by TSC , but were not statistically significantly affected in MSC exposed cells. Furthermore Gsta genes were up-regulated in TSC and Gstm genes were down-regulated in MSC exposed cells. These findings were further confirmed by the significant up-regulation of the Glutathione Metabolism Pathway in tobacco exposed cells a tall times and concentrations and the significant down-regulation of this pathway in marijuana exposed cells, particularly at the high concentration at the 6 + 4 h time point. These results suggest that exposure to MSC elicits more severe oxidative stress than exposure to TSC. The relative difference between the two condensates to mount an antioxidant defense may account for the greater cytotoxicity of MSC observed here and in our earlier genotoxicity study, where it appeared that the acute toxicity of MSC prevented the manifestation of micro-nucleus induction. The assertion regarding the relative severity of oxidative stress induced by MSC and TSC is supported by published results from other studies. In a previous study, Sarafian et al. examined reactive oxygen species production and reduced glutathione levels as indicators of oxidative damage following exposure to marijuana smoke. They showed that exposure of human endothelial cells to marijuana smoke resulted in an 80% increase in ROS over control levels, and these levels were as much as three times higher than those resulting from tobacco smoke. Moreover, intracellular glutathione levels following marijuana exposure were lower than for tobacco, and were reduced by 81% relative to controls. The authors argued that the products produced by the pyrolysis of the cannabinoids were likely responsible for the oxidative damage. The same authors also conducted preliminary studies with cultured lung alveolar macrophages from non-smokers and marijuana smokers, and found that marijuana smokers had lower levels of GSH than non-smokers, suggesting a decrease in GSH dependent oxidative defenses in habitual marijuana smokers.

The Biosynthesis of Steroids Pathway was among the most significantly affected IPA Canonical Pathways for MSC exposed cells. This held true both when all of the significantly altered MSC genes were taken into account, and when only the genes unique to MSC were considered. The Biosynthesis of Steroids Pathway is a lipid metabolism pathway that controls the synthesis of cholesterol, which is an essential component of cell membranes and a precursor in the production of bile acids, steroid hormones,seedling grow rack and vitamin D. This pathway was significantly down-regulated for all concentrations of the MSC at both time points, and the number of genes that were significantly affected increased with increasing concentration. The greatest number of genes was affected at the 6 + 4 h time point, and these included Dhcr7, Fdft1, Fdps, Hmgcr, Idi1, Mvd, Mvk, Nqo1, Pmvk, Sc5dl, and Sqle. The majority ofthese genes are involved in the mevalonate and squalene synthesis portions of the pathway. Although no studies have been conducted to specifically investigate the effect of marijuana smoke on lipid metabolism and steroid biosynthesis, early investigations using rodent cells have shown that cannabinoids can affect lipid metabolism, and the effects include an increase in lipolysis in adipose tissue ,the inhibition of corticosteroidogenesis , and the reduced testosterone and progesterone production. The cannabinoid CBD has also been shown to affect cholesterol metabolism in human fibroblasts and aortic medial cells through the inhibition of cholesteryl ester formation. In the present study, HMG-CoA reductase , which is the rate-limiting enzyme for cholesterol synthesis, was notably down-regulated for the medium and high concentrations of MSC at both time points. Previous in vitro investigations with THC have shown that this cannabinoid reduces Hmgcr by 29% , whereas CBD had no effect on Hmgcr levels. When comparing TSC and MSC exposed cells, the Biosynthesis of Steroids Pathway was also significant for TSC, particularly for the 6 + 4 h time point.

However, only one to three genes were perturbed, depending on the concentration. These genes included Fdps, Ggps1, Nqo1, and Hmgcr. The LXR/RXR pathway, which is involved in the regulation of lipid metabolism and cholesterol to bile acid catabolism, was also significantly down-regulated at the 6 + 4 h time point in both MSC and TSC exposed cells. Of note in this pathway is Ldlr, which is the greatest down-regulated gene in MSC exposed cells. This gene was down-regulated 10 fold following the highest MSC exposure concentration but only 1.6 fold following the highest TSC exposure.Exposure to MSC but not TSC appears to have affected apoptosis pathways. Genes in the TWEAK Signaling, TNFR1 and TNFR2 Signaling Pathways were significantly up-regulated following exposure to MSC particularly at the 6 h time point. The up-regulation of these particular genes suggests that MSC inhibits apoptosis and may promote a TNF receptor mediated survival pathway.In a previous study, Sarafian et al. investigated the effects of marijuana smoke and tobacco smoke on apoptosis and necrosis in A549 lung tumor cells. They found that both tobacco and marijuana whole smoke inhibited Fasmediated apoptosis but promoted necrotic cell death. In addition, particulate phase smoke from marijuana was a more potent inhibitor of Fas-induced caspase-3 activity than tobacco. In a later study, the authors also noted the decreased expression of Bax and caspase-8 in human small airway epithelial cells exposed to THC, which they suggest could have accounted for the previously observed suppression in Fas-mediated apoptosis. Although apoptotic pathways were not significantly perturbed following TSC exposure in our present study, Sarafian et al. and other investigators of tobacco smoke effects have found this to be a commonly disrupted pathway. It is suspected that the gene expression fold change cutoff of 2 used in the present study likely prevented a number of apoptotic genes from being included in the analyses. Cursory analyses with a cutoff of 1.5 shows apoptotic pathways as being significant for TSC exposure as well. It is important to note that the marijuana used for this study was obtained from a contracted supplier that provides marijuana for therapeutic use in Canada. It is grown under strictly controlled and documented conditions. Although this study has only examined smoke condensate from a single lot of marijuana, the quality control measures would be expected to minimise differences between marijuana harvests.

Studies in laboratory animals have shown CBD to protect the liver from toxic insults

Assay of the extract was 61% edible fatty acids, 26% phytocannabinoids and 13% other plant chemicals including fatty alkanes, plant sterols, triterpenes, and tocopherols. In the 14-day repeated oral dose-range finding study reported by Marx et al., a No Observed Adverse Effect Level could not be determined, however, the results of a 90-day repeated dose study with a 28-day recovery period in Wistar rats was also reported. In this study, doses of 0 , 100, 360 and 720 mg extract/kg bw per day were used. Significant decreases in body weight, body weight gain, and differences in various organ weights, compared to controls, were reported at the mid and high dose levels, but the authors concluded that many of the findings were reversible as they were trending towards normal at the end of the recovery period. A NOAEL for the hemp extract in Wistar rats in the 90-day study was determined to be 100 mg/ kg bw per day and 360 mg/kg bw per day for males and females, respectively. In the 90-day study being reported here, test article related significant changes in body weights, daily body weight gain and feed efficiency were seen in the males in all treatment groups which was still noted at the end of the recovery period. The magnitude of the significant change in body weights, daily body weight gain and feed efficiency in the low and mid dose groups was less than 10% and showed signs of obvious recovery and were therefore considered to be not toxicologically relevant. The effect in the males receiving 800 mg/kg/day was >10% and was still evident at the end of the recovery period and was considered toxicologically relevant. Reported rodent studies have differing findings on hepatotoxicity when CBD is orally administered in high doses. Hepatocellular hypertrophy with a centrilobular pattern was observed in rat livers in the study being reported.

This pattern of hepatocellular hyperplasia is frequently observed in rats and other animals exposed to agents that induce the CYP family of enzymes and can be associated with activation of peroxisome proliferator-activated receptors. THC has affinity for PPARα,trimming tray and CBD has very low to no affinity for PPARα and high affinity for PPARγ. Interaction with the PPARγ is one of the mechanisms of action for CBD. In our study, we did not show the mechanism of action for the hepatocellular hypertrophy. We did show that the activities of liver enzymes in serum were not significantly changed by treatment with the test article and the hepatocellular hypertrophy was reversed during the 28-day recovery period. In the study reported by Marx et al., no histopathological changes were observed in the livers from the treated and control rats and the liver weights in the male and female rats in the 360 and 720 mg/kg body weight/day were significantly increased at 90 days. The 28-day recovery males and females receiving 720 mg/kg/day retained the significantly increased in hepatic weights. The induction of hepatic drug metabolizing enzymes can be associated with increased liver weights, and hepatocellular hypertrophy and hyperplasia and elevation of hepatic-source enzymes in serum. The evidence in the scientific literature supports a conclusion that the centrilobular pattern of hepatocellular hypertrophy and increased liver weights observed in our study was due to induction of HDMEs and/or peroxisomes. No hepatocellular necrosis and changes in the clinical chemistries occurred which is evidence that liver damage did not occur. This conclusion is further supported by not observing hepatocellular hypertrophy and increased liver weights in the 28-day recovery groups that received the test article.In the study being reported both the treated and control male rats had the same incidence and severity of vacuolization of the adrenal zona fasciculata and the adrenal weights were significantly increased in the Group 4 females. The vacuolization of the adrenal zona fasciculata and increased adrenal weights were not observed in Groups 5 to 8. The histopathological lesions noted in the adrenal glands in the current study was seen in both control and high dose males and is not considered to be due to treatment with test article and not toxicologically relevant. The hemp extract in these studies was shown to be non-mutagenic in a bacterial test system used to evaluate mutagenicity. Marx et al. reported on a GLP-compliant study that concentrations of 5, 000 μg/plate of a CO2 supercritical extract of C. sativa were not mutagenic in a bacterial test system. Our GLP-compliant mutagenicity testing on the diluted extract showed that concentrations of 76,355 μg/plate were not mutagenic with and without the S9 metabolic activation. The extracts produced by isopropanol extraction and supercritical CO2 extraction were not mutagenic with and without S9 metabolic activation at concentrations up to 5000 μg/plate. The bacterial test system with the S9 mix did cause mutagenicity providing evidence that mutagenic metabolites were not produced with any of the extracts. The two additional Ames tests conducted on the undiluted extracts produced by two different extraction methods, were conducted to determine if the method of production or the olive oil diluent impacted the results of the Ames assay. No mutagenicity was noted in any of the tests conducted. Other botanical extracts have been evaluated for mutagenicity. Mutagenic studies on extracts from the plant Euphorbia triaculeata showed that it is not mutagenic and provides protection from the mutagenic effects of cyclophosphamide.A study on a novel taste modulating powder derived from Cordyceps sinensis showed this product was not mutagenic in the Ames test and these results were supported in the micronucleus assay. In a study on the genotoxicity of CBD in Caco-2 cells, 10 μM of CBD did not significantly cause DNA damage after 24 hours of incubation, and CBD was also shown in the comet assay to protect Caco-2 cells from hydrogen peroxide-induced DNA damage. CBD at an oral dose of 1 mg/kg was shown to significantly reduce azoxymethane-induced colonic aberrant crypt foci, colonic polyps and tumors. In summary, the test article, both undiluted and diluted in olive oil, was not mutagenic in a bacterial reverse mutation assay and the NOAEL in the 90-day study was concluded to be 800 mg/kg bw/day and 400 mg/kg bw/day for female and male Sprague Dawley rats, respectively. This assessment adds significant data to the currently available literature as to the safety and toxicology of CBD rich hemp extracts. Given the potential of CBD for a variety of human uses and the limited data currently available, these results support that hemp extracts are likely safe human consumption and additional studies should be conducted to validate this conclusion. Natural lignocellulosic fibers are in past decades gaining increased attention as sustainable materials for polymer composite reinforcement in substitution of energy intensive and non-recyclable synthetic fibers. Indeed, NLF reinforced composites are being considered for applications in civil construction, food packing, automotive components  and ballistic armor. It is worth mentioning that nanocellulose fibrils obtained from NLFs are being investigated as possible reinforcement for novel bio-nanocomposites with special properties for medical applications as well as production of biodegradable plastic films. A well known NLF, the hemp fiber, has been for decades extensively reported as reinforcement of polymer composites in numerous articles and mentioned in most review papers. In fact, hemp fiber/polypropylene has been used as automotive parts. The remarkable mechanical properties of the hemp fiber, tensile strength of 900 MPa and modulus of 70 GPa, offer a possibility of use in ballistic armor reinforcing stronger thermoset polymeric matrices such as epoxy and polyester. This ballistic application has not yet been investigated in hemp fiber composites. Therefore, the objective of the present work is, for the first time, to compare the mechanical properties, evaluated by bend and tensile tests, of both epoxy and polyester composites reinforced with different amounts of hemp fibers. This would allow a definition, in terms of matrix and incorporated volume fraction, of the most suitable composite for application in multilayered armor system.The hemp fibers, Cannabis sativa, investigated in this work were supplied by Designan Fibras Naturais, Brazil, as a bundle illustrated in Fig. 1. Fibers were separated, washed in running water and dried at room temperature for one week. Characterization of the hemp fibers was conducted prior to their processing in composites. One hundred fibers were randomly pick up and measured in a profile projector Nikkon, Japan, to determine the equivalent mean diameter. The average density was obtained by the mass, in a 0.001 g precision scale, divided by the volume considering cylindrical fiber. Both epoxy and polyester resins were purchased from Resinpoxy, Brazil. The epoxy was a diglycidyl ether of the bisphenol A hardened with stoichiometric phr 13 of triethylene tetramine as the catalyst. The polyester was an unsaturated ortoftalic resin hardened with 2 wt% of methyl ethyl ketone for the curing process.Fabrication of the composites for mechanical tests was carried out by laying up continuous and aligned hemp fibers inside a steel mold, Fig. 2, trim tray pollen with internal volume of 150 × 120 × 7mm. Fibers were first cut and weighed according to the volume fraction, calculated by considering their previously evaluated density as well as the densities of epoxy and polyester from. Just before processing, the fibers were dried in a stove at 60 ◦C for 3h. Layers of fibers were placed inside the mold together with either epoxy or polyester resin, already mixed with corresponding hardener. After closing the mold, a pressure of 1 ton was applied and left to cure for 24h at RT. The produced composite plate, Fig. 2, was machined into 150 × 10 × 7mm prismatic specimens for 3 points bend tests as per ASTM D790 standard. Flat tensile specimens were directly fabricated in a specially designed steel mold, Fig. 3, in which hemp fibers were aligned together with the chosen resin already mixed with corresponding hardener. Shape of specimens with 7 × 7mm of gage cross section and 20mm of gage length as per ASTM D 638 standard. A pressure of approximately 1 MPa was applied to the mold’s lid during the cure at RT for 24h. Both flexural and tensile tests  were carried out in a model 5582 Instron machine, USA, operating at RT and a crosshead speed of 0.5mm/min. Fracture analysis of ruptured specimens was performed by scanning electron microscopy in a model SSX-550 Shimadzu microscope, Japan, operating with secondary electron at 20 kV. Fourier transform infrared analysis was performed to detect functional groups in the hemp fibers as well as the effect caused by addition of these fibers in the epoxy functional group. Band spectra were obtained in a model IR-Prestige- 21 Shimadzu spectrometer, Japan, using 2mg pressed ground samples mixed with 110mg of KBr.Frequency histogram for the equivalent diameter of the as-received hemp fibers is shown in Fig. 4. The average length is 76.6mm, while the average diameter, Fig. 4, was found as 65 m. The density of the fibers was obtained as 1.35 ± 0.27 g/cm3 with thinner fibers presenting comparatively higher densities. Fig. 5 shows FTIR spectra of the hemp fiber and three hemp fiber/epoxy composites. The hemp fiber spectrum in Fig. 5 displays intense broad bands centered at 3333 and 2916 cm−1 that could be associated with O H and C–H groups in the fiber cellulose. The sharp band at 1738 cm−1 might be assigned to the C O stretching of carboxylic group of hemicellulose, while the band at 1656 cm−1 was indicated as C C as stretching of unsaturated acids or sterols in tannin. The band at 1510 cm−1 was associated with the aromatic skeletal vibration of lignin. Bands at 1425 and 1377 cm−1 and around were attributed to C–H bending as well as C–H2 rocking vibration of Fig. 4 – Histogram for equivalent diameter of the as-received hemp fibers. groups in lignin and cellulose. The 1160 and 1030 cm−1 bands were assigned to cellulose, respectively, C–O C asymmetric valence vibration and C–O stretching primary alcohol. Finally, the band at 897 cm−1 corresponds to C–H rocking vibrations of cellulose. The FTIR spectra of the hemp fibers/epoxy composites in Fig. 5 revealed bands corresponding to the plain DGEBA/TETA epoxy such as: 2963 and 2866 cm−1 assigned to C–H alkyl group; 1612 and 1510 cm−1 assigned to C C phenyl ring; 1456 and 1257 cm−1 assigned to H C–H groups; 1255 and 1030 cm−1 assigned to C–O C ether; and a small 915 cm−1 band assigned to vibration of epoxide group.