Cannabis Science and Technology October 2021

The inter-laboratory variation problem still exists, which means laboratory certification is necessary, but not sufficient to guarantee your samples are analyzed properly. Here, Smith offers suggestions to help improve the situation.

Laboratory Accreditation: Is It Enough to Guarantee Your Samples Are Analyzed Correctly?

The microbial plating of cannabis and hemp derived concentrate has been historically challenging because of issues with consistency. Tween 20, an emulsifying agent, is often added to diluent to aid in extracting the concentrate. In this study, Tween 20 amounts were increased in 1% increments while monitoring two different factors. The first factor was the turbidity of the solution, which determines the saturation point at which no more concentrate can be extracted, given the Tween 20 method. The second factor was the microbial survival rate, measured in colony forming units (CFU) for E. coli and C. albicans. Per the state of Nevada regulations (NCCR effective November 5, 2020), enterobacteria and total yeast and mold are plated for concentrates, thus these two microbes were chosen to be representative. Following analysis, it was determined that a solution of 3% percent Tween 20 was optimal for survival of target microbes and emulsification of concentrate.

The state of Nevada Cannabis Compliance Board (CCB) defines concentrated cannabis as “the extracted or separated resin, whether crude or purified, containing tetrahydrocannabinol (THC) or cannabidiol CBD” (1). Through this extraction process, cannabinoids become highly concentrated while simultaneously making it challenging to extract microorganisms from matrices. Due to this innate difficulty, most manufacturer methods offer a patented emulsifying agent that aids in the breakdown.For plating applications, the use of an emulsifying agent named Tween 20 has been widely adopted. Tween 20 is a non-ionic detergent that aids in emulsification for the preparation of stable oil-in water (2). Ideally, adding the solution in small amounts serves to aid in breaking up the concentrate. However, a caveat of the process is ensuring that a suitable environment for the target microorganisms remains after application of Tween 20.

Though cannabis and cannabis-containing products are illegal on the Federal level in the US, certain states have legalized them to allow for medicinal or recreational products. As a result of this patchwork legalization, the testing (and more specifically the targeted microbiological contaminants) are determined state by state. In Nevada and several other states, concentrated cannabis products need to be tested for enterobacteria and total yeast and mold (1,3). Detection methods are structured via a threshold system wherein microorganism may be detected, but as long as they are below the target threshold then they are considered safe for human consumption or use. In large quantities, it has been found that these categories of microorganisms can lead to human harm if not properly tested for (4). Taking this into account, researchers hypothesized the following: what was the optimal amount of Tween 20 solution that could be added until the environment became unfavorable.(measured in colony forming units [CFU]) for the plated target microorganisms?

 0157:H7 NCTC 12900 was the representative organism. For the study of total yeast and mold, 

 ATCC10231 was the organism used. The procedure used to create viable spikes prior to their dilution into Butterfield’s phosphate buffer and Tween 20 is property of RSR Analytical Laboratories. Incrementally increasing concentration of Tween 20 was diluted appropriately as described in 

. While aseptically creating the dilutions into sterile 150 mL dilution bottles, 100 mL total was created. Of this solution, 9 mL of each respective dilution was added to 1 g of concentrate distillate. This solution was then vortexed for 1 min and then added to a sterile turbidity vial. The vial was allowed to sit, followed by the turbidity procedure. After turbidity was done, either aerobic plate count or yeast and mold method was conducted, dependent on the spike utilized in the preparation process. All remaining milliters of diluent were plated on their respective plates, and resultant CFUs were counted. CFUs were then averaged for each percent of Tween 20 added and analyzed with survival of the target organism as the outcome of interest.

Turbidity of the diluent was measured using a LaMotte TC-3000e turbidity meter. To measure turbidity, the meter was calibrated utilizing known nephelometric turbidity unit (NTU) standards (5).The various concentrations of the Tween 20 solution were then added to the 1 g of cannabis distillate concentrate. The sample was then vortexed for 1 min to completely allow the emulsifying agent, cannabis distillate, and microorganisms to mix completely. The solution was transferred to a sterile turbidity tube and allowed to rest on the bench for 30 s. The turbidity of the sample was taken and recorded. If the turbidity of the liquid was too numerous to calculate, it was subsequently diluted to be in a readable range. Overall the point of this method was to gain a general understanding of how much concentrate was eluted into the buffer.

Rapid aerobic plate count petrifilm by 3M is a self-contained dehydrated media that facilitates the growth of bacteria aerobically for the purpose of enumeration. Following the measurement of turbidity at least 8 replicates, plates of 1 mL of sample were pipetted to the center of the plate and pushed with a 3M flat spreader. These plates were incubated at 35 ±1 °C for 24 ±2 h. This was done in accordance with the instructions given by the manufacturer and AOAC recommendations (6). Following incubation, CFUs were counted as a measure of microbial survival. Though plated on aerobic plate count (APC), this served as a target organism for enterobacteria.

Rapid yeast and mold petrifilm by 3M is a simple ready to use plate system with specialized antibiotics to facilitate the growth of only yeast and mold on the plate in a rapid manner. The plates of 1 mL of sample were pipetted to the center of the plate and pushed with a 3M flat spreader. These plates were incubated at 25 ±1 °C for 60–72 h (7–9).This was done in accordance with the instructions given by the manufacturer and AOAC recommendations. Following incubation, CFUs were counted as a measure of microbial survival.

Data consisted of roughly 90 samples of CFUs from rapid APC and yeast and mold (Y/M) petrifilms taken at 10 levels of Tween 20 (1% to 10%, by increments of 1%), with minimal missing data. To assess whether there was a value of Tween 20 at which the recovery rate of APC and Y/M significantly changed, two threshold regression models with hinge threshold effects were fit with Tween 20 as a continuous predictor, and APC and Y/M as separate outcomes (10).A hinge threshold effect was used; we assumed that the recovery rate would be constant (flat) until the change point value, at which point it would begin decreasing, rather than using a step threshold effect that assumed two constant recovery rates that were different between the threshold value. Alpha for a significant difference in recovery rates above and below the threshold Tween 20 value was set at 0.05. All analysis was conducted using R version 4.0.1.

The results of the turbidity experiment are shown in 

. Following the procedure outlined above, the results were plotted along their respective Tween 20 concentrations. The data appears to follow a logarithmic pattern; no matter how much Tween 20 is added, there is a limit of how much cannabis concentrate can be extracted through this method. Following 7% the growth appears to level off, rendering any additional Tween 20 useless (

Following the proper incubation and CFU counting, statistical analysis was conducted to assess any differences between plates. The results of the threshold regressions with a hinge effect can be seen in Table II; in the APC model, a changepoint was identified at Tween 20 of 6% (95% CI 4, 9), with an increase in a unit of Tween 20 values larger than 6% associated with a decrease in APC recovery rate of 3.98 (95% CI: -14.87, -2.17), while Tween 20 less than or equal to 6% had a constant recovery rate of 63.81 (62.01, 66.41). However, this difference in recovery rate beyond the threshold value of 6% is not significant (p-value=0.22), indicating that there is not sufficient evidence to suggest that 6% is a threshold value associated with a significantly different recovery rate.

In the Y/M model, a changepoint was identified at Tween 20 of 3% (95% CI: 2, 4), with an increase in a unit of Tween 20 values larger than 3% associated with a decrease in Y/M recovery of 5.32 (95% CI: -7.18, -4.23), which was significant (p-value<0.01). Tween 20 values less than or equal to 3% had a constant Y/M recovery rate of 208.69 (95% CI: 203.29, 212.91). So, there was evidence suggesting that Y/M recovery rate significantly decreased at Tween 20 values larger than the 3% threshold, while Tween 20 less than or equal to the threshold value had a constant recovery rate. 

 demonstrate the APC and Y/M log likelihood plots of each threshold value, and frequency plots of each threshold value from 1000 bootstrappings of the analysis, respectively.

 present the fitted lines for the threshold regressions, with a flat pre-threshold value slope, and the post-threshold regression line. While both post-threshold lines have negative slopes, the APC line was not significantly different than the APC pre-threshold slope, whereas the Y/M post-threshold slope was significantly different than the pre-threshold slope.

Innovations in cannabis science allow researchers to push forward in finding new ways of conducting science accurately. Concentrated cannabis has exceptionally difficult matrices to accurately test for microbes because of an oftentimes hard and sticky consistency. While sometimes the amount of extraction, or turbidity, can indicate success, microbiologists must also take into account the environmental conditions of the organisms targeted for analysis. In this study, 

0157:H7 served as the model for enterobacteria and 

 served as the model for total yeast and mold. It was determined that, when utilizing Tween 20 to aid in the emulsification of concentrated cannabis, no more than 3% of Tween 20 to dilution should be added (Figures 3 and 4). The more Tween 20 that is added, the more the turbidity increases, which can contribute to better emulsification of concentrate. However, if it leads to an unfavorable microbial environment, the target organisms might begin to perish and, subsequently, results will be underreported (Figures 3–5).

The goal for cannabis scientists is to keep all consumers, medicinal and recreational, safe. By doing specific research on target matrices, it is possible to incorporate food and environmental testing methods to match cannabis testing needs. Minor changes to the method could be beneficial in testing, and potentially lead to more precision and quality. Many scientists in the cannabis field strive to develop the science; working with state-to-state regulations provides a unique opportunity to aid the entire field. By continuing to publish and grow the grassroots of testing, any increases in testing quality will hopefully permeate across state regulations to the benefit of all cannabis users and producers.

(1) NRS: Chapter 453 - controlled substances. (n.d.). 

https://www.leg.state.nv.us/NRS/NRS-453.html#NRS453Sec042

(2) "TWEEN® 20 for molecular biology, viscous liquid,"Sigma-Aldrich | 9005-64-5. (n.d.). 

https://www.sigmaaldrich.com/US/en/product/sigma/p9416

(3) S. Dailey, "Cannabis microbial testing regulations by state," Medicinal Genomics. 

https://www.medicinalgenomics.com/cannabis-microbial-testing-regulations-by-state/

(4) R. Upton, L. Craker, M. ElSohly, A. Romm, E. Russo, and M. Sexton, "Cannabis inflorescence: Cannabis spp. ; standards of identity, analysis, and quality controlm," 

(5) "LaMotte TC-3000e Manuals," Manuals Library. (n.d.). 

https://www.manualslib.com/products/Lamotte-Tc-3000e-11491053.html

(6) AOAC Official Method 2015.13 Enumeration of Aerobic Bacteria in Food 3M™ Petrifilm™ Rapid Aerobic Count Plate First Action 2015

(7) 3M Petrifilm Rapid Yeast and Mold Count Plate Interpretation Guide. 

https://multimedia.3m.com/mws/media/902046O/3m-petrifilm-rapid-ym-count-plate-interpretation-guide.pdf

(8) P. Bird, J. Flannery, E.Crowley, J. Agin, D. Goins, and R. Jechorek, "Evaluation of the 3M™ Petrifilm™ Rapid Yeast and Mold Count Plate for the Enumeration of Yeast and Mold in Food: Collaborative Study, First Action," 

(10) Y. Fong, Y. Huang, P.B. Gilbert, and S.R. Permar, 

 is the Director of Microbiology at RSR Analytical Laboratories. 

 is a Research Instructor Biostatistician for the Colorado School of Public Health’s Center for Innovative Design and Analysis. Direct correspondence to: 

Articles

In this study, Tween 20 amounts were increased in 1% increments while monitoring two different factors. 

Optimal Dilution of Polysorbate (Tween) 20 for the Determination of Enterobacteria and Total Yeast and Mold on Cannabis Concentrate 

is a Full Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington.

The chemical characterization of different Cannabis sativa spp. cultivars has important regulatory and potential pharmacodynamic implications. While Groups I-III cultivars are distinguishable on the basis of total tetrahydrocannabinol concentrations and the relative ratio to cannabidiol, the expression of other secondary metabolites, such as the terpene family of phytoconstituents, has a more significant bearing on the potential therapeutic outcomes for medicinal end users as a result of the synergies associated with the “entourage effect.” In the work presented here, 131 Group III cannabis cultivars were analyzed for cannabinoid and terpene composition. Principal component and hierarchical cluster analyses were used to assess multidimensional relationships in the overall phytochemical content. Weak correlations were found between individual cannabinoid and terpene species; however, the relative expression of monoterpenoids and terpenoids were the most discriminative variables in terms of differentiation of individual cultivars into two distinct subclasses. The identification of distinct sub-classes within Group III cannabis on the basis of cis-2- pinanol, fenchol, and alpha-terpineol expression, suggests that these classifications can be used to guide more targeted therapeutic outcomes.

With the passage of the 1970 Controlled Substances Act, research into the medicinal properties and phytochemical constituents of cannabis was brought to a halt in the United States. As various states have legalized the use of cannabis within their borders, nascent research is being conducted, but remains fraught with legal peril, as cannabis is still prohibited at the Federal level. The study of cannabidiol (CBD)-rich hemp, now legal to grow in the United States as a result of the 2018 Farm Bill, is not hampered by these restrictions and research on the chemical composition and phytochemical diversity of commercially available cannabis can be performed.

From a regulatory perspective, cannabis is a Schedule 1 controlled substance that differs from hemp on the basis of total ∆

-THC) concentration, with the bifurcation occurring at 0.3% weight/ weight (w/w) total THC. This particular metric is the sum of the amount ∆

-THC plus the amount of its acidic precursor, tetrahydrocannabinolic acid (THCA), after accounting for the loss of mass during decarboxylation (that is, total THC = ∆

-THC + 0.877*THCA). However, and more specifically, cannabis cultivars can be classified into one of three groupings. Group I cannabis cultivars are THC-dominant strains (>20:1 THC/CBD); Group II cannabis cultivars have approximately equal amounts of THC and CBD (that is, ~1:1 THC/ CBD); and Group III cannabis (hemp) cultivars are CBD-dominant (that is, >20:1 CBD/THC). Remarkably, Groups I-III cannabis cultivars generally demonstrate indistinguishable morphological structures, with comparable flavonoid, terpene, and ancillary cannabinoid profiles (1). This lack of discernable phytochemotaxonomy is likely the result of extensive interbreeding and hybridization (2). However, minute differences in phytochemical composition may produce or affect different therapeutic outcomes for patients using medicinal cannabis.

Terpene molecules are ubiquitous hydrocarbons that are naturally abundant in cannabis or hemp and can stimulate a wide array of physiological responses (3). There are currently 20,000+ known quantifiable terpenes, with cannabis producing more than 120 different terpenes species (4). Most terpenes exhibit antimicrobial and anti-inflammatory properties, but other terpenes have been found to modulate blood glucose and attenuate pain (3). Additionally, terpenes such as beta-myrcene, myrcene, and limonene are all perceived as invigorating; whereas linalool, also found in lavender, is perceived to induce a sedative effect. Other terpenes, such as alpha- and beta-pinene produce bronchodilatory effects (5).

In this work, targeted cannabinoid and terpene analyses, in combination with multivariate statistical analysis, were used to identify and select hemp cultivars that produced increased amounts of cannabidiolic acid (CBDA). Using gas chromatography–mass spectrometry (GC–MS), two distinct clusters within Group III cannabis were identified on the basis of their cannabinoid and terpene expression profiles. A parallel analysis by high performance liquid chromatography (HPLC) also resulted in the separation of unique phytochemical clusters, albeit with less discrimination than was afforded by the GC–MS analysis. These results suggest that separate sub-classes within Group III cannabis can be identified on the basis of ancillary phytochemical expression; the elucidation of these discriminatory and bioactive features can possibly be used to guide medicinal end users towards pharmacodynamically more favorable treatment outcomes.

In this study, 131 novel Group III cannabis (hemp) cultivars created by ZED Therapeutics were included. These cultivars were selected on the basis of CBDA expression and the absence of discernible genetic abnormalities or poor performance characteristics. These included hermaphrodism, variegation, extremely long flowering periods, elongated internodal spacing, poor cannabinoid production, and susceptibility to insects, molds, and plant viruses. All plants were grown indoors in an organic blended soil medium (compost, worm casting, perlite, and cocoa coir) under ceramic metal halide (CMH) and high-pressure sodium (HPS) and lighting technologies. During the vegetative cycle, 1000 W CMH lights were used on a 16:8 light:dark regimen, and 1000 W HPS lights were used on a 12:12 light:dark regimen during the flowering cycle.

Samples of final cured flower material were analyzed for cannabinoid and terpene content by GC–MS methods performed by Sunrise Analytical, using the Environmental Protection Agency’s 8270 method. In this work, 10 and 50 different cannabinoid and terpene species were quantified, respectively (please see Supplemental Dataset online). Concentrations of the acidic precursor cannabinoids (that is, THCA and CBDA) were determined via GC–MS after derivatization (6). Calibration was performed with a quadratic calibration curve with 8 reference points and a linearity greater than 0.995. Quantitation was performed within 85–115% of sample matrix reproducibility.

Samples collected two weeks before harvest and of final cured flower material were analyzed by HPLC methods developed by ZED Therapeutics. Briefly, samples were treated with methanol and syringe filtered, prior to separation on a 2.2 µm, 3.0 x 75 mm C18 column and an acetonitrile gradient using Shimadzu’s Cannabis/Hemp Analyzer. Certified reference standards from Cerilliant were used to identify and quantify cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidial (CBD), tetrahydrocannabivarin (THCV), tetrahydrocannabivarinic acid (THCVA), cannabinol (CBN), ∆

-THC), cannaicyclol (CBL), cannabichromene (CBC), tetrahydrocannabinolic acid (THCA), and cannabichromenic acid (CBCA). Calibration was performed using a set of five serial dilutions of each reference standard (2.5, 12.5, 25.0, 125.0, and 250.0 µg/L) with a linearity greater than 0.998. The limits of detection and quantitation were determined experimentally to be 0.5 and 1.0 µg/L, respectively.

Pairwise and correlative analyses were performed using the Prism software suite by GraphPad. Principal component analysis (PCA), hierarchical cluster analysis (HCA), and partial least squares discriminant analysis (PLS-DA) were performed using MetaboAnalyst 4.0 online. Prior to statistical analysis, the data were auto-scaled to CBD and CBDA concentrations to normalize the dataset so that individual constituents could be compared based on their correlation to each other. This is referred to as unit variance scaling where the highest and lowest concentrations are represented by 1 and -1, respectively, with all other concentrations being relative (7). No further data transformation was performed.

The concentrations of CBDA and THCA in Group III cultivars are of particular interest in the hemp industry due to efforts to maximize the former while still maintaining regulatory compliance with the latter. Current United States Department of Agriculture (USDA) regulations specify that in order to fit the classification of hemp, Group III cultivars must exhibit a total THC (∆

-THC + decarboxylated THCA) equal to or less than 0.3 % w/w on a dry weight basis. Therefore, we determined the major acidic cannabinoid precursors (that is, CBDVA, CBDA, CBGA, THCA, and CBCA) in flower material collected two weeks before harvest, as is required by USDA regulations, and in the final cured material (

). All 131 cultivars were devoid of detectable ∆9-THC two weeks prior to harvest, whereas 10 of the 131 cultivars demonstrated total THC levels above 0.3% w/w once THCA decarboxylation was accounted (mean and median values of 0.19% and 0.18%, respectively). The concentrations of all five major acidic precursor cannabinoid species increased significantly (p<0.0001) in the final two weeks of cultivation, while CBDA:THCA ratios remained relatively unchanged (25.0 versus 24.9, p=0.55) between the two collections points. CBDA concentrations rose the most dramatically in the final two weeks of harvest, with a median increase from 5.22% to 13.97% (p<0.0001).

CBDA, THCA, and CBCA concentrations exhibited multicollinearity at both time collection points, whereas CBDA and CBGA exhibited poor correlation two weeks before harvest (R2=0.11), which was accentuated in the final cured flower (R2=0.05) (

). This was expected since CBGA is converted into CBDA, THCA, and CBCA, respectively, via a series of synthase enzymes. Interestingly, the strength of the aforementioned multicollinearity amongst CBDA, THCA, and CBCA decreased over time, and was attributed to the substantial increase in CBDA expression in the final two weeks of cultivation.

An additional two-tailed Pearson correlation analysis was performed to assess the relationships between the major cannabinoids and prominent terpenes in the final cured flower materials (

). This analysis revealed several weak correlations between individual cannabinoid and terpene species (that is, CBDA and fenchol, CBDA and cis-2-pinanol, r ≤ 0.5), compared to stronger correlations between individual terpenes. For example, humulene concentrations strongly correlated with caryophyllene concentrations (r=0.8, p<0.0001). Similarly, fenchol concentrations were correlated strongly with cis-2-pinanol concentrations (r=0.8, p<0.0001) and alpha-terpineol (r=0.7, p<0.0001); and the concentrations of the two pinene isomers were strongly correlated (r=0.8, p<0.0001).

Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to evaluate overall individual phytochemical profiles in relation to each of the Group III cannabis cultivars (see 

). Samples were evaluated based on their terpene content, cannabinoid content, and the combination of the two compound classes. For analysis of the terpene content (see 

), the first four principal components (PCs) had a total variance of 57.8% with PC 1 and 2 explaining 20.8% and 15.0% of the total variance, respectively. Samples were additionally evaluated based on the cannabinoid content. PC1 and PC2 accounted for 58.9% of the total variance, and the first three PCs for 69.2% of the total variance (see 

). Cannabinoids were evaluated using HPLC.

PCA results were similar to those determined by GC–MS. The total variance explained by the first three PCs was 68.7%. PC1 accounted for 30.8% and PC2 accounted for 24.9%. The terpene and cannabis data were combined, which yielded total variance of 52.6% for the first four PCs with PC1 and 2 explaining 22.6% and 12.3%, respectively (

Those compounds with the greatest influence in differentiating the two sub-classes were selected using the following statistical tests: t-test (p-value <0.05), partial least square discriminant analysis (PLS-DA), and random forest (RF). While the t-test is a univariate approach that does not consider correlation between features, PLS-DA and RF are multivariate approaches that can be used with collinear features. The compounds commonly identified by the three approaches for the terpene analysis were cis-2-pinanol, fenchol, α-terpineol, and guaiol—all of which are terpenoids (see 

). The results of the GC–MS analysis of cannabinoids indicated that CBD, THC, ∆

-THC, and THCA were the most influential compounds based on all three statistical tests and that CBD had the greatest influence. The evaluation of the HPLC data revealed that the acid components THCA, CBDA, and CBCA, were the most influential because of the elevated levels in sub-class 1. Based on the RF analysis, CBCA, CBC, and THCA ranked as the top three features because of the low concentration of CBC in sub-class 1; CBDA and THCA were elevated in sub-class 2. When the combined results from the terpenes and cannabinoids quantitation were evaluated, the same sub-class differentiation was evident that was determined as with terpene data alone. The results of the t-test, RF, and PLS-DA statistical tests indicated that cis-2-pinanol and fenchol were the most influential with respect to class differentiation due to lower concentrations in sub-class 1 samples. To a lesser extent, α-terpineol and CBD also affected the differentiation between sub-classes based on the t-test and RF evaluations. However, PLS-DA ranked α-terpineol and THCA third and fourth in significance, respectively.

While the results indicate that THCA concentrations influence the structure of the all-encompassing model space, overall, the variability in terpenoid concentrations, primarily those of cis-2-pinanol, fenchol, and to a lesser degree, alpha-terpineol had the greatest effect on sub-class differentiation. Interestingly, all three of these terpene species are classified as monoterpenoids and contain a hydroxyl (OH) functional group with either a cyclic or bicyclic structural backbone (see Supplemental Table II). Concentrations of terpenoids were 2.44 times higher in sub-class 2 than in sub-class 1. Additionally, the monoterpene concentration for sub-class 1 was 1.32 times higher than that in sub-class 2.

The data presented here document the phytochemical composition of a large dataset (n = 131) of Group III cannabis cultivars and allow for the identification of distinct sub-classes based on the cannabinoid and terpene concentrations expressed in individual cultivars. Weak correlations between individual cannabinoid and terpene species were detected; however, the relative concentrations of monoterpenoids and terpenoids (cis-2- pinanol, fenchol, and alpha-terpineol) were the most discriminative factors in differentiating individual cultivars into two distinct sub-classes. This may be important within the context of pharmacodynamics and potential therapeutic outcomes. For example, fenchol is an antioxidant, anti-inflammatory, and anti-microbial agent (8). Additionally, fenchol and other monoterpenoids have been found to have analgesic activity (9). Therefore, cannabis users looking for pain relief in Group III cannabis may benefit from selecting cultivars with the phytochemical signature of sub-class 2 hemp, which is characterized by higher concentrations of fenchol and other analgesic monoterpenoids compared to subclass 1 hemp. Ultimately, the more resolution that can be provided by cannabis analysis and the resulting classification, the better-informed cannabis users can be when selecting products for the treatment of their individual illnesses. Additional efforts to characterize and quantify different flavonoid species in Group III cannabis may provide additional insight and further sub-class differentiation.

Several tables and figures mentioned in the text as supplementary information are included in the online version only.

(2) E.M. Mudge, S.J. Murch, and P.N. Brown, 

(4) A. Leghissa, Z.L. Hildenbrand, and K.A. Schug, J

(6) A. Leghissa, Z.L. Hildenbrand, F.S. Foss, and K.A. Schug, 

(7) R.A. van dem Berg, H.C.J. Hoefsloot, J.A. Westerhuis, A.K. Smilde, and M.J. van der Werf, 

(9) M. Takaishi, K. Uchida, F. Fujita, and M. Tominaga, 

is with the Department of Chemistry and Biochemistry at The University of Texas at El Paso in El Paso, Texas; the Collaborative Laboratories for Environmental Analysis and Remediation, The University of Texas at Arlington in Arlington, Texas; Curtis Mathes Corporation in Frisco, Texas; and Medusa Analytical, LLC, in Southlake, Texas. 

 is with the Collaborative Laboratories for Environmental Analysis and Remediation, The University of Texas at Arlington; Medusa Analytical, LLC, and the Department of Chemistry and Biochemistry, The University of Texas at Arlington in Arlington Texas. 

MATTHEW SPURLOCK, ADAM M. JACQUES, CHRISTIAN WEST, ORIAH LOVE,

 are with ZED Therapeutics, Inc, in Cheshire, Oregon. 

 is with The Collaborative Laboratories for Environmental Analysis and Remediation, The University of Texas at Arlington; Medusa Analytical, LLC; and the Department of Chemistry and Biochemistry, The University of Texas at Arlington. Direct correspondence to: 

The data presented here document the phytochemical composition of a large dataset of Group III cannabis cultivars and allow for the identification of distinct sub-classes.


Differentiation of Group III Cannabis Cultivars into Novel Sub-Classes

CRATIV, a pioneer in sustainable, child-resistant, cannabis packaging, announced recently a partnership with One Tree Planted to aid in the restoration and reforestation of natural areas impacted by the devasting fires across the US and Canada.

The objectives of this new partnership are:

Empowering consumers to deliver a positive impact

Raising awareness about the importance of ecosystem restoration

Creating a collaborative win for the environment, customers, and cannabis consumers

Enabling the launch of our Plant Based products to enter the market as a force for good

“We have worked hard to provide our customers with a viable alternative to single-use and disposable plastic products, innovation and sustainability are at the core of everything we do at Crativ Packaging,” said CEO and founder Bill Ludlow. “With the introduction of our new environmentally responsible, accelerator and plant-based products, we wanted to go above and beyond, and be a driving force for the betterment of our environment. A partnership with One Tree planted is the perfect fit for our culture, values and mission and enables us to play a role in the vital restoration efforts needed after the destructive fires across the U.S.”

CRATIV Packaging has committed to donating a portion of profits in commemoration of every box of plant-based and accelerator packaging purchased during the sustainable product launch in October. CRATIV Employees are also planning to volunteer their time, and plant trees as part of a community impact event on Earth Day in April 2022.

https://mgretailer.com/press-releases/crativ-announces-collaborative-partnership-with-one-tree-planted-our-planet-our-home-our-responsibility/

CRATIV announced recently a partnership with One Tree Planted to aid in the restoration and reforestation of natural areas impacted by the devasting fires across the US and Canada.

CRATIV Announces Collaborative Partnership with One Tree Planted “Our Planet, Our Home, Our Responsibility”

Pipp Horticulture, a division of Pipp Mobile Storage Systems, Inc., announced that it had acquired the GGS Group of Companies (GGS), which manufactures greenhouses and provides turnkey greenhouse growing solutions including traditional rolling benches, heating, ventilation systems, and curtain systems.

“We are thrilled to welcome the entire GGS team to the Pipp family of companies," said Craig Umans, Pipp president and CEO. "GGS has decades of experience manufacturing greenhouse structures and has become a leading supplier to the horticulture industry. The company has consistently impressed us with its ability to deliver value to its customers, and we look forward to our next phase of growth.”

“We are pleased to partner with PIPP as we build an even more expansive platform to serve our customers.” said Leigh Coulter, president and owner of GGS. “Our expertise in designing, manufacturing and installing commercial greenhouse structures, combined with Pipp's state-of-the-art vertical farming and space optimization solutions, will allow us to offer North American growers a complete and efficient solution.”

“We are delighted to see two such complementary companies come together today. This acquisition is another step in Pipp's strategy to become the leading supplier of turnkey solutions to all segments of horticulture globally," said Marc Paiement, senior partner at Novacap. "We look forward to continuing to work with them throughout this process, providing support and expertise to help them achieve their goal."

Pipp Horticulture’s mission for the past several years has been to assist cultivators all over the world save time and money by creating a more efficient grow facility and help streamline operations. The multi-level mobile cultivation systems company developed a product line to serve the cultivation market and also acquired companies such as, GGS and Vertical Air Solutions, which is a leading provider of air circulation systems for indoor vertical farming.

Following the acquisitions, Coulter will remain president and CEO and operations will remain under the GGS banner.

https://www.greenhousemag.com/article/pipp-horticulture-acquires-ggs/

Pipp Horticulture announced  that it had acquired the GGS Group of Companies (GGS).

Pipp Horticulture Acquires GGS Group of Companies

 is a Princeton, New Jersey-based psychiatrist, writer, speaker, educator, and consultant. He is the founder and board president of Doctors for Cannabis Regulation. Dr. Nathan is a Distinguished Fellow of the American Psychiatric Association and Clinical Associate Professor of Psychiatry at Rutgers Robert Wood Johnson Medical School. He serves on the editorial board of 

As individual US states and the federal government approach legalization of cannabis for medical and adult use, a universal symbol for cannabis products is essential to the protection of public health and safety.

Currently, states that have legalized cannabis for medical or adult use have implemented differing requirements for cannabis labeling. These standards vary widely, and in the absence of federal standardization, the solutions that are best for one jurisdiction may not be suitable for another.

Nonetheless, there is one anchor point to ensure that Americans of all ages and backgrounds can correctly identify and exercise caution with cannabis products: a universal symbol on cannabis packages. Such a symbol is critical for, among other reasons, preventing the accidental ingestion of cannabis by adults and children.

However, no cannabis product symbol has met with approval by more than one or two legalized US states, which is a serious issue of public safety now facing regulators, medical professionals, industry stakeholders, patients, consumers, and the general public.

Working with Doctors for Cannabis Regulation and other stakeholders, David L. Nathan, MD, proposed the Universal Cannabis Product Symbol (UCPS) in 2019. After its publication in 2020 (14), a broad discussion with various stakeholders led to a series of changes to the UCPS. Working with the standards organization ASTM, the authors of this paper co-designed a modified version of the UCPS (8), which was renamed the International Intoxicating Cannabis Product Symbol (IICPS) (

Figure 1: The International Intoxicating Cannabis Product Symbol (IICPS) symbol.

At the time of the publication of this article, the IICPS had already been included in the proposed rules for the state of Montana (15), and other newly legalized states are planning to follow suit. Also at the time of this publication, ASTM was actively engaged in an approval process that would make the IICPS the one and only cannabis product symbol to be designated as an internationally recognized consensus standard.

The word “Intoxicating” was added to the name of the symbol to distinguish hemp products from intoxicating cannabis products. The 

Diagnostic and Statistical Manual of Mental Disorders

, Fifth Edition (DSM-5) defines Cannabis Intoxication as meeting all of four criteria (1):

B. Clinically significant problematic behavioral or psychological changes (for example, impaired motor coordination, euphoria, anxiety, sensation of slowed time, impaired judgment, social withdrawal) that developed during, or shortly after, cannabis use.

C. Two (or more) of the following signs or symptoms developing within 2 hours of cannabis use:

D. The signs or symptoms are not attributable to another medical condition and are not better explained by another mental disorder, including intoxication with another substance.

In 2021, tetrahydrocannabinol (THC) is the main intoxicating compound in all known varieties of the cannabis plant. Thus, any cannabis products surpassing a defined threshold of THC content should bear the IICPS. Definition of this critical threshold of THC content (typically expressed as a weight in milligrams or as a percentage by weight of the product) is a controversial topic beyond the scope of this paper.

Ten US states and the nation of Canada have adopted nine different intoxicating cannabis product symbols. All of them have design problems, generally pertaining to color, shape, symbology, or the prohibited use of text inside a graphical symbol (see 

). The International Intoxicating Cannabis Product symbol (IICPS) is an alternative to the many symbols currently in use, but with a fundamental difference: It is firmly rooted in accepted principles of symbology and consensus standards.

Figure 2: Currently used cannabis product symbols compared to the IICPS.

Table I: Attributes of all symbols in Figure 2. Note that green denotes desirable attributes, while red denotes undesirable attributes, according to existing consensus standards. Consensus standards, as described in the text, dictate that the shape of a safety sign urging caution should be a warning triangle. The ideal number of colors in a graphical symbol is two (and white is considered a color in this context), as more colors unnecessarily increase the cost of packaging. The symbol should have a black and white option for jurisdictions that do not require color printing. This requires that non-white elements of the symbol are separated, or that the symbol uses only two colors. The emphasized color of a symbol should be consistent with existing conventions, in which red implies prohibition, yellow implies warning, and green implies a safe condition (see Table II). Standard safety signs should contain only graphical elements within their borders, not text or punctuation. Only large design elements should be included. Finally, safety signs should be fully compliant with ISO and ANSI consensus standards, as described.

A truly universal symbol must adhere to existing standards of packaging, labeling, and design. And, in order to be accepted by the US federal government, the symbol should be a consensus standard.

Consensus standards are technical specifications issued by standards development organizations (also called voluntary consensus bodies) such as the American National Standards Institute (ANSI), ASTM, and the International Organization for Standardization (ISO) (2–6,10–13). They are developed in an open environment through collaboration by professionals from both the public and private sectors. This is achieved by cooperation of all parties who have an interest in participating in the creation or use of the standards. Consensus standards incentivize universal compliance beyond recommendations, guidelines, or best practices developed by single individuals, organizations, or particular authorities having jurisdiction (AHJs).

Further, the use of consensus standards is mandated by federal law. The National Technology Transfer and Advancement Act of 1995 (NTTAA) requires the federal government to use privately developed consensus standards whenever possible. The NTTAA codifies what was long considered to be best practice within the federal government.

The NTTAA, Section 1115 of Public Law 107-107, Section 12(d) states (7):


(d) UTILIZATION OF CONSENSUS TECHNICAL STANDARDS BY FEDERAL AGENCIES; REPORTS.—


(1) IN GENERAL.—Except as provided in paragraph (3) of this subsection, all Federal agencies and departments shall use technical standards that are developed or adopted by voluntary consensus standards bodies, using such technical standards as a means to carry out policy objectives or activities determined by the agencies and departments.


(2) CONSULTATION; PARTICIPATION.—In carrying out paragraph (1) of this subsection, Federal agencies and departments shall consult with voluntary, private sector, consensus standards bodies and shall, when such participation is in the public interest and is compatible with agency and departmental missions, authorities, priorities, and budget resources, participate with such bodies in the development of technical standards.


(3) EXCEPTION.—If compliance with paragraph (1) of this subsection is inconsistent with applicable law or otherwise impractical, a Federal agency or department may elect to use technical standards that are not developed or adopted by voluntary consensus standards bodies if the head of each such agency or department transmits to the Office of Management and Budget an explanation of the reasons for using such standards. Each year, beginning with fiscal year 1997, the Office of Management and Budget shall transmit to Congress and its committees a report summarizing all explanations received in the preceding year under this paragraph.


(4) DEFINITION OF TECHNICAL STANDARDS.—As used in this subsection, the term ‘‘technical standards’’ means performance-based or design-specific technical specifications and related management systems practices.

Multiple standards development organizations have approved safety signs and caution symbols that are accepted standards in the US and around the world, and those consensus standards may be readily applied to a universal cannabis product symbol.

The international consensus standard for safety symbols is 

(10–12), which corresponds to the similar US consensus standard A

NSI Z535.4: Product Safety Signs and Labels

ISO 3864 standardizes five safety signs as shown in 

ISO 3864-1: Graphical symbols—Safety colours and safety signs—Part 1: Design principles for safety signs and safety marking

For a cannabis product symbol to be compliant with consensus standards, then one of these safety signs must be chosen. Thus, a symbol indicating prohibition, mandatory action, safe condition, or fire equipment would be inappropriate. The only logical option is the standard warning sign, which consists of a black graphical symbol inside a black-bordered yellow equilateral triangle with radiused outer corners.

Letters, numbers, punctuation marks, and mathematical symbols may not be included in standard graphical symbols, with the sole exception of an exclamation point as the graphical symbol in two signs: the General Mandatory Action Sign (ISO 7010-M001) and the General Warning SIgn (ISO 7010-W001) (13). Not one of the hundreds of other registered safety signs in ISO 7010 includes an exclamation point or any other letter, number, or punctuation mark. This strict standard ensures comprehensibility of graphical safety signs by anyone, regardless of age, literacy, and language differences.

People may not recognize “THC” as identifying a cannabis product if they are young, illiterate, do not use the Latin alphabet, or lack specific education about cannabis. Thus, the use of “THC” inside a cannabis product symbol is not just prohibited by consensus standards—it’s also discriminatory.

Creation of the International Intoxicating Cannabis Product Symbol

The International Intoxicating Cannabis Product Symbol (IICPS), intended to symbolize “caution with cannabis,” was the product of a collaboration between cannabis policy experts and designers, utilizing existing standards. This effort is part of a larger project for standardized cannabis labeling initiated by Doctors for Cannabis Regulation (9).

When setting out to design the IICPS, the creators stipulated that it must satisfy a strict set of criteria:

Adhere to existing precedents of standard symbols, so the public will immediately understand the meaning of the symbol.

Use symbology that transcends age, literacy, knowledge of the Latin alphabet, specialized knowledge about cannabis, language, and culture.

Allow for the addition of optional text to meet the needs of authorities having jurisdiction in the US and around the world.

This is not a stop-gap solution until some other design is developed. This is the solution—a symbol designed for permanent use by individual states, the federal government, and other nations.

ISO 3864-2 (11) requires that the standard warning sign include a black graphical symbol within the yellow triangle. ISO 3864-3 (12) specifies that the graphical symbol inside a warning symbol should:

Utilize objects, concepts, and activities, or a combination of these, which are familiar to the target group

Contain only those details that contribute to understanding

Be readily associated with its intended meaning

Be easily distinguishable from other graphical symbols

For cannabis products, the only symbol that satisfies these criteria is a cannabis leaf. It is far and away the most familiar graphical symbol associated with cannabis. A joint, bong, or other paraphernalia, for example, would limit the scope of the symbol and require the consumer to make a logical jump from cannabis to paraphernalia that may be unfamiliar to many. An exclamation point cannot be included in the design, as alphanumeric characters and punctuation marks are forbidden in all consensus standard symbols, except for the generic warning sign with an exclamation point. Also, an exclamation point does not add to recognition of the symbol and would require that the cannabis leaf inside a warning triangle be reduced to an undesirably small size (see California’s cannabis product symbol in Figure 2).

The IICPS creators thus concluded that the cannabis leaf is the only suitable symbol to be placed within the warning triangle (

Figure 3: The cannabis leaf in the IICPS.

The size and shape of the leaf were carefully chosen to include several desirable characteristics:

The leaf has a distinct shape and serrated margins to distinguish it from the leaves of other plant species, thus reinforcing the public’s accurate identification of cannabis plants.

The leaf has well-separated leaflets that remain clear at small sizes, as when printed on vape cartridges or embossed on edible servings.

The leaflets have a silhouette and leaflet count that is intermediate between those of typical mature leaves of the two main varieties of the cannabis plant (

Putting together the standard warning sign and the cannabis leaf, the International Intoxicating Cannabis Product Symbol was born (

). The design of the IICPS is fully compliant with ISO 3864 and ANSI Z535 (

Figure 4: The IICPS on light and dark backgrounds. Note the additional yellow border against the dark background.

Figure 5: Measurements demonstrating the compliance of the IICPS with standards set forth by ISO 3864. The thin red triangle around the cannabis leaf in the first image shows the exclusion zone within which the central cannabis leaf must be contained.

Consistent with ISO 3864, the IICPS is designed to permit the use of text below the symbol, so alphanumeric or special characters may be placed under the symbol for supplemental information (

). This allows for uniformity in the symbol while meeting the varying needs of authorities having jurisdiction in the US and around the world. It also obviates any purported need for the inclusion of letters, numbers, or special characters inside the symbol itself.

Figure 6: ISO compliant versions of the IICPS with text below the symbol.

A black and white version of the IICPS falls outside ISO and ANSI standards for warning signs, which mandate the use of “warning sign yellow” (Pantone 109) as a background color. Thus, it should be used only if color printing is impossible and black and white printing of the IICPS is permitted by the authority having jurisdiction (

We present the IICPS to regulators with a simple message: This is not just the right choice as a universal symbol for cannabis products—it’s also the safe choice.

The IICPS is the only cannabis product symbol in the world that is on track to become a consensus standard (6). It is undergoing approval through the international standards development organization ASTM. The IICPS was unanimously approved by the working group developing a symbol for ASTM’s Committee D37 on Cannabis. The final step is the pending approval of ASTM WK75247—Specification for an International Symbol for Identifying Consumer Products Containing Intoxicating Cannabinoids by the full committee membership of more than 1000 stakeholders.

Typically, once one major standards development organization publishes a consensus standard, the others will support it unless there is a compelling reason to reject it. Regardless, the designers of the IICPS believe that if another standards development organization set out to create a universal symbol for cannabis products, existing precedents for warning signs and the ubiquity of the cannabis leaf as a representative graphical symbol would guide another organization to choose a design that is fundamentally indistinguishable from the IICPS.

When this symbol becomes the ASTM consensus standard, and cannabis is regulated at the national level, US federal regulators will likely be obligated to use the IICPS under the National Technology Transfer and Advancement Act of 1995 (NTTAA).

The authors invite cannabis stakeholders to support the IICPS by formally endorsing its use by states, the federal government, and other authorities having jurisdiction. Please contact 

 to discuss adding your organization’s name to the list of endorsing organizations.

To facilitate immediate adoption by regulators, the designers have published the symbol in multiple digital file formats (for example, PDF, EPS, SVG, and JPG) for distribution and use. The symbol will be available for use by regulators worldwide: on demand, in perpetuity, royalty-free, and at no cost.

Regulators may address any questions or requests for IICPS digital files to 

The authors wish to thank the following individuals for their contributions to the development of the IICPS and/or the preparation of this manuscript: Bryon Adinoff, MD, President, Doctors for Cannabis Regulation; Martha Bajec, Consulting Director, Health & Wellness Research; David Gorelick, MD, PhD, DLFAPA, FASAM, Professor of Psychiatry, University of Maryland School of Medicine; Audra Horridge, VP of Product, Advanced Vapor Devices; Mauricio Mejia, Environmental Administrator, Division of Consumer Services, Florida Department of Agriculture and Consumer Services; Darwin Millard, Chief Science Officer, Canada, Final Bell; Robert J. Morgan, Director, Technical Committee Operations, ASTM International; Karen K. Nathan, EdM; Michelle Niedziela, PhD, VP of Research & Innovation, HCD Research; Jon D. Robinson; Phil Simoes, Principal, Design & Consulting, PS Consulting Services; Christopher Smith, Partner, BGP Advisors; and Leslie Mendoza Temple, Clinical Associate Professor, Family Medicine, University of Chicago Pritzker School of Medicine.

Diagnostic and Statistical Manual of Mental Disorders 

(5th ed.) (American Psychiatric Publishing Co.: Washington, DC, 2013). 

https://doi.org/10.1176/appi.books.9780890425596

American National Standard for Safety Colors.

 American National Standards Institute, Inc. (Secretariat: National Electrical Manufacturers Association): Approved October 20, 2017. 

https://www.nema.org/docs/default-source/standards-document-library/ansi-z535_1-2017-contents-and-scope.pdf?sfvrsn=d7266ce_2

 American National Standards Institute, Inc. (Secretariat: National Electrical Manufacturers Association): Approved July 19, 2011. 

https://webstore.ansi.org/Standards/NEMA/ANSINEMAZ5352011-1373641

Standard Guide for Packaging and Labeling of Consumer Resin Cannabis Products for Sale to Adult Consumers, Legally Authorized Medical Users, and Caregivers in a Business-to-Consumer Retail Environment (Retailers)

Standard Terminology Relating to Cannabis

Specification for an International Symbol for Identifying Consumer Products Containing Intoxicating Cannabinoids

. ASTM: Pending approval, which is expected in early 2022.

https://www.nist.gov/standardsgov/national-technology-transfer-and-advancement-act-1995

(8) K.B. Connelly, “Is a ‘Cannabis Facts Label’ in Our Future?” 

, April 28, 2021. (Note: This article features a later version of the UCPS, which was modified slightly and renamed the IICPS.) 

https://www.packagingdigest.com/cannabis/cannabis-facts-label-our-future

Working with Doctors for Cannabis Regulation and other stakeholders, David L. Nathan, MD, proposed the Universal Cannabis Product Symbol (UCPS) in 2019. After its publication in 2020, a broad discussion with various stakeholders led to a series of changes to the UCPS. Working with the standards organization ASTM, the authors of this paper co-designed a modified version of the UCPS, which was renamed the International Intoxicating Cannabis Product Symbol (IICPS). This article presents the IICPS and next steps for the industry to start implementing its use.

Why All Legalized U.S. Jurisdictions Should (and Probably Will) Adopt the International Intoxicating Cannabis Product Symbol (IICPS)

The Massachusetts Cannabis Control Commission recently announced several appointments so their Cannabis Advisory Board, the members of which will study and make recommendations to the Commission regarding the regulation and taxation of marijuana in the state. The board will reportedly consist of 25 members with five appointees made by the Governor, five appointees by the Treasurer and Receiver General, five appointees by the Attorney General, and 10 ex officio members with expertise and knowledge relevant to the Board’s mission (1).

The recent announcement of five board members came from Treasurer Deborah B. Goldberg (2,3). “I am pleased to appoint these members to the Massachusetts Cannabis Advisory Board,” said Treasurer Goldberg (2,3). “I am confident they will have a meaningful impact on the regulation of the cannabis industry across our state.”

Treasurer-appointed members are required to have expertise in cannabis cultivation, cannabis retailing, cannabis product manufacturing, laboratory sciences and toxicology, and providing legal services to cannabis businesses. Treasurer Goldberg’s new appointees and reappointments to the Cannabis Advisory Board are (1–3): Amanda Rositano, an expert in marijuana product manufacturing; Dr. Alan Balsam, an expert in laboratory sciences and toxicology; Dr. Marion McNabb, an expert in marijuana retailing; Michael Dundas, an expert in marijuana cultivation; and Laury C. Lucien, an expert in providing legal services to marijuana businesses.

Amanda Rositano is the principal of Rositano Strategies, a cannabis business consulting firm focused on building a more inclusive and responsible cannabis industry, and former president of NETA (Parallel, MA), where she served as a key member of the start-up and leadership team that led the launch of the state’s medical marijuana program and successfully transacted the first legal adult-use sale of cannabis east of the Mississippi.


The Massachusetts Cannabis Control Commission recently announced several appointments so their Cannabis Advisory Board, the members of which will study and make recommendations to the Commission regarding the regulation and taxation of marijuana in the state.

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