AOAC CASP SMPRs for Comment

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DRAFT AOAC SMPR 2018.XXX; Version 2, July 23, 2019 1 2 Method Name: 3

Identification and Quantitation of Selected Residual Solvents in Dried

4 5

Cannabis Materials

Intended Use : 6 7 1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics to be used 8 during the evaluation of a method. The evaluation may be an on-site verification, a single-laboratory 9 validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC Stakeholder 10 Panels composed of representatives from the industry, regulatory organizations, contract 11 laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC Expert 12 Review Panels in their evaluation of validation study data for method being considered for 13 Performance Tested Methods or AOAC Official Methods of Analysis , and can be used as acceptance 14 criteria for verification at user laboratories. Consensus-based Reference method.

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

2. Applicability :

Method, or a suite of methods, to identify and quantify selected residual solvents (Table 1) in

cannabis derivatives .

3. Analytical Technique :

Any analytical technique(s) that measures the analytes of interest and meets the following method performance requirements is/are acceptable. More than one analytical technique may be needed.

4. Definitions :

Cannabis Plant Material

Plant material from Cannabis sp. and its hybrids.

Cannabis Derivatives

Products or extracts derived from cannabis plant material.

Limit of Detection (LOD)

The minimum concentration or mass of analyte in a given matrix that can be detected. A minimum

3 to 1 signal to noise ratio (S/N).

Limit of Quantitation (LOQ)

The minimum concentration or mass of analyte in a given matrix that can be reported as a

quantitative result. A minimum 10 to 1 signal to noise ratio (S/N).

Parts Per Million (PPM)

mg of analyte per kg of cannabis derivatives .

Quantitative method

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

Method of analysis where response is the amount of the analyte measured either directly (enumeration in a mass or a volume), or indirectly (color, absorbance, impedance, etc.) in a certain

amount of sample.

Repeatability

Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator and repeating during a short time period. Expressed as the repeatability

standard deviation (SD r

); or % repeatability relative standard deviation (%RSD r ).

Reproducibility

The standard deviation or relative standard deviation calculated from among-laboratory data.

Expressed as the reproducibility standard deviation (SD R

); or % reproducibility relative standard

deviation (% RSD R ).

Recovery

The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed

using the entire method.

5. Method Performance Requirements :

See table 2 and 3.

6. System suitability tests and/or analytical quality control:

Suitable methods will include blank check samples, and check standards at the lowest point and

midrange point of the analytical range.

7. Reference Material(s):

Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements , 19 th Edition of the AOAC INTERNATIONAL Official

Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf

8. Validation Guidance :

USP General Chapter <467><1467>

ICH QC3Regulatory Guidelines

Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method of Analysis; 19 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available

at: http://www.eoma.aoac.org/app_d.pdf

Appendix F: Guidelines for Standard Method Performance Requirements; 19 th Edition of the AOAC

INTERNATIONAL Official Methods of Analysis (2012). Available at:

http://www.eoma.aoac.org/app_f.pdf

Appendix K: Guidelines for Dietary Supplements and Botanicals; 19 th Edition of the AOAC

INTERNATIONAL Official Methods of Analysis (2012). Available on line at:

http://www.eoma.aoac.org/app_k.pdf

100 101

U.S. Food and Drug Administration: Bioanalytical Method Validation Guidance for Industry, May

2018

102 103 104 105 106 107 108

9. Maximum Time-To-Result: None

Table 1: Residual Solvents

Table 1: List of Residual Solvents and Targeted LOQs

Proposed Target LOQ (ppm)

Analytical Range

Concentration Limit (ppm)

Lower

Upper

1 2

30 60

Class 1 Residual Solvents*

Benzene

120

Carbon tetrachloride

150

1,2-Dichloroethane 1,2-Dichloroethane

1870

240

1,1-Dichloroethene

1870

1,1-Dichloroethene 1,1,1-Trichloroethane

1500

45000

Class 2 Residual Solvents* Acetonitrile

410

12300

410

acetonitrile

360

10800

Chlorobenzene

360

60 70

1800 2100

Chloroform

60 70

Cumene

3880 1870

116400

Cyclohexane

3880 1870

56100

1,2-Dichloroethene 1,2-Dimethoxyethane N,N-Dimethylacetamide N,N-Dimethylformamide

100

3000

100

1090

32700 26400 11400

1090

880 380 160 620 220 290

1,4-Dioxane

4800

2-Ethoxyethanol Ethylene glycol

18600

6600 8700

Formamide

Hexane

n-hexane Methanol Methanol

3000

90000

3000

400

50 50

1500 1500

2-Methoxyethanol Methylbutylketone Methylcyclohexane

50 50

1180

35400

1180

600 530

18000 15900

Methylene chloride N-Methylpyrrolidone

600 530

1500 6000 4800

Nitromethane

200 160 720 100 890

Pyridine Sulfolane

200 160 720 100 890

21600

Tetrahydrofuran

3000

Tetralin Toluene Toluene

26700

30 80

2400

Trichloroethylene

2170

65100

Xylene

2170

m,p-xylenes

Class 3 Residual Solvents* Acetic acid

5000 5000 1000 5000 5000 5000 5000 5000 5000 5000 1000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000

5000 5000

150000 150000

Acetone acetone Anisole 1-Butanol 2-Butanol

5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000

150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000

Butyl acetate

tert-Butylmethyl ether

Dimethyl sulfoxide

Ethanol Ethanol

Ethyl acetate Ethyl ether Ethyl formate Formic acid

Heptane

Isobutyl acetate Isopropyl acetate Methyl acetate 3-Methyl-1-butanol Methylethylketone Methylisobutylketone 2-Methyl-1-propanol

Pentane

1-Pentanol 1-Propanol 2-Propanol

isopropanol (2-propanol)

320

5000

150000

Propyl acetate

Additional butane

5000 5000 5000

5000

150000

Butane (sum of n- and iso-)

5000

150000

propane propane

2,2-dimethylbutane 2,3-dimethylbutane

290 290

2-butanone

5000 5000

2-methylbutane 2-methylpentane 3-methylpentane ethylbenzene ethylene oxide isobutane (methyl propane)

290 290

2170

12 12

n-butane n-heptane n-pentane

1000 1000

nitrogen o-xylene

triethylamine

5000

50%

* USP Guideance (467) Residual Solvents

109 110 111

Table 2: Method performance requirements

Parameter

Requirement

LOQ* (ppm)

Specified in Table 1

Analytical Range (ppm)

LOQ to 100*LOQ specified in Table 1

112 113

Table 3: Method performance requirements for pesticides in Table 1.

Analytical Range

100 to 500 ppm 100 - 500 mg/kg

1 to 100 ppm

500 to 1000 ppm

Parameters

1 - 100 mg/kg

500 - 1000 mg/kg > 1000 mg/kg

Recovery (%)

60 – 120

90 - 107

95 - 105

97 - 103

% RSD r

≤ 20

≤ 5

≤ 4

≤ 3

% RSD R

≤ 30

≤ 8

≤ 6

≤ 4

1 DRAFT AOAC SMPR 2019.XXX; Version 5; July 16, 2019

2 3 4 5 6

Method Name:

Quantitation of cannabinoids in plant materials of hemp (low THC varieties

Cannabis spp.)

Intended Use : 7 8 1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics to be 9 used during the evaluation of a method. The evaluation may be an on-site verification, a single- 10 laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC 11 Stakeholder Panels composed of representatives from the industry, regulatory organizations, 12 contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by 13 AOAC Expert Review Panels in their evaluation of validation study data for method being considered 14 for Performance Tested Methods or AOAC Official Methods of Analysis , and can be used as 15 acceptance criteria for verification at user laboratories. Consensus-based Reference method.

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

2. Applicability :

The method will be able to identify, and quantify individual cannabinoids (as listed in Table 1a and Table 1b) in plant materials expressed on a dry weight basis. Method must be able to report total

THC regardless of how it is measured (total THC as defined in this SMPR).

3. Analytical Technique :

Any analytical technique(s) that measures the analytes of interest and meets the following method

performance requirements is/are acceptable.

4. Definitions :

Hemp Plant Materials

Fresh or dried, whole or milled plant material of low THC cultivars of Cannabis spp.

Limit of Quantitation (LOQ)

The minimum concentration or mass of analyte in a given matrix that can be reported as a

quantitative result.

Quantitative method

Method of analysis which response is the amount of the analyte measured either directly (enumeration in a mass or a volume), or indirectly (color, absorbance, impedance, etc.) in a certain

amount of sample.

Repeatability

Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator and repeating during a short time period. Expressed as the repeatability

standard deviation (SD r

); or % repeatability relative standard deviation (%RSD r ).

47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Reproducibility

The standard deviation or relative standard deviation calculated from among-laboratory data.

Expressed as the reproducibility standard deviation (SD R

); or % reproducibility relative standard

deviation (% RSD R ).

Recovery

The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed

using the entire method.

Total THC

the maximum potential percentage w/w delta-9 tetrahydrocannabinol that the test sample could

yield on a dry weight basis including delta 9 THC and delta 9 THCA.

5. Method Performance Requirements :

See table 2 and 3.

6. System suitability tests and/or analytical quality control:

Suitable methods will include blank check samples, and check standards at the lowest point and

midrange point of the analytical range.

A detailed description of your dry weight procedures and calculations must be included.

7. Reference Material(s):

See tables 1A and 1B for sources of reference materials.

Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements , 19 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf

8. Validation Guidance :

Method performance should be demonstrated with homogeneous samples. Inherent variation in the plant may preclude or limit homogeneity for the following reasons: (a) they are resinous, cannabinoids are concentrated in the resin, which can clump during grinding; (b) between flower variation can be high, grinding multiple flowers can impact the homogeneity; (c) grinding can introduce heat, which will cause degradation of cannabidiolic acids into neutral forms, resulting in less accurate results. Grinding would be the best option for homogeneous samples, but in some cases there are issues with clumped resin, highly variable samples and additional grinding would

impact the results and lead to inaccurate data.

Appendix D : Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method of Analysis; 19 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available

at: http://www.eoma.aoac.org/app_d.pdf

Appendix F : Guidelines for Standard Method Performance Requirements; 19 th Edition of the AOAC

INTERNATIONAL Official Methods of Analysis (2012). Available at:

http://www.eoma.aoac.org/app_f.pdf

97 98 99

Appendix K : Guidelines for Dietary Supplements and Botanicals; 19 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available on line at:

100 101 102 103 104 105

http://www.eoma.aoac.org/app_k.pdf

9. Maximum Time-To-Result: None

106

Table 1A: Required Cannabinoids

Common Name

Abbrev -iation

IUPAC Name

CAS Number

Molecular Structure

Reference Material Restek Cerilliant

Cannabidiol

CBD

13956-29-1

2-[(1 R ,6 R )-6-isopropenyl-3- methylcyclohex-2-en-1-yl]-5- pentylbenzene-1,3-diol

Sigma-Aldrich API Standards Echo Pharm Lipomed AG Cerilliant USP Restek Lipomed AG Echo Pharmaceutical

Cannabidiolic Acid

CBDA

1244-58-2

2,4-dihydroxy-3-[(1R,6R)-3- methyl-6-prop-1-en-2- ylcyclohex-2-en-1-yl]-6- pentylbenzoic acid

[SGC: name corrected]

Cannabinol

CBN

521-35-7

Cerilliant Restek

6,6,9-Trimethyl-3-pentyl- benzo[c]chromen-1-ol

Tetrahydro- cannabinol

THC

1972-08-3

Cerilliant USP Echo Pharmaceuticals Cerilliant USP Echo Pharmaceuticals

(−)-(6aR,10aR)-6,6,9-Trimethyl- 3-pentyl-6a,7,8,10a-tetrahydro- 6H-benzo[c]chromen-1-ol

Tetrahydro- cannabinolic acid

THCA

23978-85-0

(6aR,10aR)-1-hydroxy-6,6,9- trimethyl-3-pentyl-6a,7,8,10a- tetrahydro-6h- benzo[c]chromene-2-carboxylic acid

107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127

128

Table 1B: Additional, Desirable Cannabinoids

Name

Abbrev iation

IUPAC Name

CAS Number Molecular Structure Reference Material

Cannabichromene CBC

20675-51-8

Cerilliant Sigma Aldrich Echo Pharmaceuticals

2-Methyl-2-(4-methylpent-3- enyl)-7-pentyl-5-chromenol

Cannabichromenic acid

CBCA

20408-52-0

no reference material

5-Hydroxy-2-methyl-2-(4- methyl-3-penten-1-yl)-7- pentyl-2H-chromene-6- carboxylic acid

Cannabidivarinic acid

CBDVA 2,4-dihydroxy-3-[(1R,6R)- 3-methyl-6-prop-1-en-2- ylcyclohex-2-en-1-yl]-6- propylbenzoic acid

31932-13-5

Cerilliant

Cannabigerol

CBG

25654-31-3

Cerilliant Lipomed AG Echo Pharmaceuticals SPEX Certiprep Tocris (UK)

2-[(2E)-3,7-dimethylocta-2,6- dienyl]-5-pentyl-benzene-1,3- diol

NIST: 2808-33-5

NIST: 1,3-Benzenediol, 2- (3,7-dimethyl-2,6- octadienyl)-5-pentyl-

Cannabigerolic - acid

CBGA

25555-57-1

Cerilliant Echo Pharmaceuticals SPEX Certiprep

3-[(2E)-3,7-dimethylocta- 2,6-dienyl]-2,4-dihydroxy- 6-pentylbenzoic acid

Cannabidivarin

CBDV

24274-48-4

Cerilliant SPEX Certiprep

2-((1 S ,6 S )-3-methyl-6- (prop-1-en-2-yl) cyclohex-2-enyl)-5- propylbenzene-1,3-diol 6,6,9-trimethyl-3-pentyl- 6a,7,10,10a- tetrahydrobenzo[c]chro men-1-ol

Cerilliant SPEX Certiprep

Δ8 Tetrahydro- cannabinol

Δ8 THC

5957-75-5

Tetrahydro- cannabivarin

THCV

28172-17-0

Cerilliant USP

6,6,9-Trimethyl-3-propyl- 6a,7,8,10a-tetrahydro-6 H - benzo[c]chromen-1-ol

Tetrahydrocannab ivarin - acid

THCVA

28172-17-0

No reference material

129 130 131 132

Table 2: Method performance requirements (part 1) for cannabinoids

Parameter

Requirement

Limit of Quantitation (LOQ) (%)

≤ 0.05

Analytical Range (CBD & CBDA) (%)

0. 05 – 35

Analytical Range (others) (%)

0.05 – 5

*All calculated on dry weight basis

133 134 135

Table 3: Method performance requirements (part 2) for cannabinoids

Ranges (%)

Parameters

0.05 – 0.5

> 0.5 - 5

5 - 35

Recovery (%)

85 – 118

90 - 111

95-105

% RSD r

≤ 5 ≤ 8

≤ 3 ≤ 6

≤ 2 ≤ 4

% RSD R

*All calculated on dry weight basis

Only applicable to CBD and CBDA

1 DRAFT AOAC SMPR 2019.XXX; Version 4; July 7, 2019

2 3 4 5 6 7

Method Name:

Standard Method Performance Requirements for Detection of Aspergillus in

Cannabis and Cannabis Products

Intended Use : 8 9 1. Purpose: AOAC Standard Method Performance Requirements SM (SMPRs) describe the minimum 10 recommended performance characteristics to be used during the evaluation of a method. The 11 evaluation may be an on-site verification, a single-laboratory validation, or a multi-site collaborative 12 study. SMPRs are written and adopted by AOAC stakeholder panels composed of representatives from 13 industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic 14 institutions. AOAC SMPRs are used by AOAC expert review panels in their evaluation of validation 15 study data for method being considered for Performance Tested Methods SM or AOAC Official Methods 16 of Analysis SM and can be used as acceptance criteria for verification at user laboratories. [Refer to 17 Appendix F: Guidelines for Standard Method Performance Requirements , Official Methods of Analysis 18 of AOAC INTERNATIONAL (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA.] Consensus-based Reference method.

19 20 21 22 23 24 25 26 27 28 29

2. Applicability :

Candidate methods used to detect Aspergillus ( Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus) in cannabis (plants/flowers) and/or cannabis products (concentrates, infused edibles and infused non-edibles). Candidate methods may be validated for specific matrices, categories or broader claims. See Table 3 for matrix/category claim acceptance criteria.

3. Analytical Technique :

Any analytical technique that meets the method performance requirements is acceptable.

4. Definitions : 30 31 Acceptable minimum detection level (AMDL) . —The predetermined minimum level of an analyte, as 32 specified by an expert committee, which must be detected by the candidate method with an estimated 33 5% lower confidence limit on the probability of detection (POD) of 0.95 or higher. The AMDL is 34 dependent on the intended use. [ISO 16140-1:2016: Microbiology of the food chain—Method 35 validation—Part 1: Vocabulary] 36 37 Candidate Method . — The method submitted for validation [Appendix J: AOAC INTERNATIONAL 38 Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental 39 Surfaces , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, 40 Rockville, MD, USA] 41 42 Candidate Method Presumptive Result . —Preliminary result for a test portion produced by following a 43 candidate method’s instructions for use. 44 45 Candidate Method Confirmed Result . —Final result obtained for a test portion after cultural 46 confirmation of a candidate method.

47 48

Cannabis .—genus of flowering plants within the Cannabinaceae family that commonly contain 9- 49 tetrahydrocannabinol (THC), cannabidiol (CBD), and other cannabinoids and terpenes. Cannabis 50 includes, but is not limited to, high-THC and high-CBD cultivars. 51 52 Cannabis Concentrates . —Extracts (primarily composed of cannabinoids and/or terpenes) 53 manufactured through the extraction and concentration of compounds derived from the cannabis plant 54 or flower. Final products can be many forms including oils, wax, or hash (Category II). 55 56 Cannabis Infused Edibles .—Food and drinks containing extracts of cannabis and/or cannabis materials 57 (Category III). 58 59 Cannabis Infused Non-Edibles .—Products containing extracts of cannabis and/or cannabis materials 60 intended to be applied to the human body or any part thereof. Final products can be many forms 61 including creams, ointments, cosmetics and therapeutic pads (Category IV). 62 63 Cannabis Plant and Flower . —General terms for the structural and flowering unadulterated parts of the 64 cannabis plant (Category I). 65 66 Cannabis Products .—Products (Edible, and non-edible) extracted or infused with compounds derived 67 from the cannabis plant including but not limited to CBD and THC. 68 69 Probability of detection (POD) .—The portion of positive analytical outcomes for a qualitative method 70 for a given matrix at a given analyte level or concentration. This difference in POD values between 71 presumptive and confirmed results is termed dPOD CP . 72 73 Exclusivity .—Study involving pure nontarget strains, which are potentially cross-reactive, that shall be 74 not detected or enumerated by the candidate method. See Table 8 for a list of recommended nontarget 75 strains. [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of 76 Microbiological Methods for Food and Environmental Surfaces , Official Methods of Analysis of AOAC 77 INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] 78 79 Fractional positive .—Validation criterion that is satisfied when an unknown sample yields both positive 80 and negative responses within a set of replicate analyses. The proportion of positive responses should 81 fall within 25 and 75% and should ideally approximate 50% of the total number of replicates in the set. 82 A set of replicate analyses are those replicates analyzed by one method. Only one set of replicates per 83 matrix is required to satisfy this criterion. 84 85 Inclusivity .—Study involving pure target strains that shall be detected or enumerated by the candidate 86 method. See Table 7 for a list of recommended target strains. [Appendix J: AOAC INTERNATIONAL 87 Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental 88 Surfaces , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, 89 Rockville, MD, USA] 90 91 Laboratory probability of detection (LPOD) .—The POD value obtained from combining all valid 92 collaborator data sets for a method for a given matrix at a given analyte level or concentration. 93 [Appendix H: Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative 94

Methods , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, 95 Rockville, MD, USA]

96 97

LCL .—Lower confidence limit. 98 99 Aspergillus .—Filamentous, cosmopolitan and ubiquitous fungus found in nature producing colonies 100 typically of 1-9 cm in size (select species produce 0.5-1 cm colonies). Colonies are powdery in texture 101 and color varies based on species. Reverse color is typically uncolored to pale yellow. Growth is typical 102 at 20-30 o C. Aspergillus fumigatus is thermotolerant and can grow at a temperature range of 20 to 50 103 °C. For all species, hyphae are septate and hyaline. The conidiophores originate from the basal foot cell 104 located on the supporting hyphae and terminate in a vesicle at the apex. Vesicle is the typical formation 105 for the genus Aspergillus . The morphology and color of the conidiophore vary from one species to 106 another. Covering the surface of the vesicle entirely (“radiate” head) or partially only at the upper 107 surface (“columnar” head) are the flask-shaped phialides which are either uniseriate and attached to 108 the vesicle directly or are biseriate and attached to the vesicle via a supporting cell, metula. Over the 109 phialides are the round conidia (2-5 µm in diameter) forming radial chains. Other microscopic structures 110 include sclerotia, cleistothecia, aleuriconidia, and Hulle cells are of key importance in identification of 111 some Aspergillus species. Cleistothecium is a round, closed structure enclosing the asci which carry the 112 ascospores. The asci are spread to the surrounding when the cleistothecium bursts. Cleistothecium is 113 produced during the sexual reproduction stage of some Aspergillus species. Aleuriconidium is a type of 114 conidium produced by lysis of the cell that supports it. The base is usually truncate and carries remnants 115 of the lysed supporting cell. These remnants form annular frills at its base. Hulle cell is a large sterile cell 116 bearing a small lumen. Similar to cleistothecium, it is associated with the sexual stage of some 117 Aspergillus species. See Table 9 & 10 for more macroscopic and microscopic information on Aspergillus 118 species. 119 Chen, S.C.A., Meyer, W., Sorrell, T.C. , Halliday, C. L. (2019) in Manual of Clinical Microbiology , 120 12th Ed, Landry, M.L., McAdam, A.J., Patel, R., & Richter, S.S. (Eds)ASM Press, Washington, D. C., , pp. 121 2103-2131. 122 Anaissie, E.J., McGinnis, M.R., Pfaller, M.A. (2009) in Clinical Mycology , Ed 2, Churchill 123 Livingstone, New York, NY, pp 1-687. 124 Walsh, T.J., Hayden, R.T., Larone, D.H. (2018) in Larones Medically Important Fungi: A Guide to 125 Identification , 6 th Ed, ASM Press, Washington, D.C., pp. 1-500 128 flower/plant and cannabis infused non-edible products a 10 g test portion is used. For cannabis 129 concentrates, a 5 g test portion is used. For cannabis infused edibles, a 25 g test portion is used. A larger 130 test portion can be used in validation studies when appropriate. See Table 2 for minimum test portion 131 requirements. 132 United States Pharmacopeia. Microbiological Examination of Nonsterile Products: Microbial 133 Enumeration Tests (61), USP 40. United States Pharmacopeia. 134 United States Pharmacopeia. Microbiological Examination of Nonsterile Products: Tests for 135 Specified Microorganisms (62), USP 40. United States Pharmacopeia. 136 Feng, P., Weagant, S.D., Grant, M.A., Burkhardt, W. (2017) Bacteriological Analytical Manual: 137 Chapter 4 Enumeration of Escherichia coli and the Coliform 138 Bacteria https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm064948.htm 139 126 127 Test portion . —The test portion is the sample size used in most validation studies. For cannabis

140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182

Andrews, W. H., Wang, H., Jacobson, A., Hammack, T. (2018) Bacteriological Analytical Manual

Chapter 5: Salmonella

https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm

UCL . —Upper confidence limit.

5. System suitability tests and/or analytical quality control:

Positive and negative controls shall be embedded in assays as appropriate. Inhibition controls should be used for method verification for each new matrix. Manufacturer must provide written justification

if controls are not appropriate to an assay.

6. Reference Material(s):

The use of live cultures and/or fungal spores is required for inclusivity and exclusivity testing and for inoculation of test matrices during the matrix studies. Extracted DNA is not suitable for use in validating methods against this SMPR but may be used to develop supplemental information.

7. Validation Guidance :

Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces [ Official Methods of Analysis of AOAC INTERNATIONAL

(2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA]; or ISO 16140-2:2016.

At the time of the publication, no national reference standard exists for the confirmation of Aspergillus from cannabis products. Until one is established the following is recommended for

method developers:

To screen samples for the presence or absence of the target analyte, two methods that employ

different technologies (agar plate, PCR, ELISA) must be used.

To ensure the viability of the inoculating organism (both confirming presumptive results or determining false negative results) an extended primary enrichment (up to at least 48 total hours) followed by plating of the sample to a minimum of two types of agar plates (examples: Dichloran rose bengal chloramphenicol (DRBC), Sabouraud dextrose (SAB-DEX), potato dextrose agar (PDA), Czapek's) is required. Final confirmation can be achieved via matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy, sequencing, or other suitable

confirmatory procedures (microscopic examination, biochemical analysis, etc).

When performing the validation, bulk inoculation of test material is required. In certain instances (ex.

therapeutic patches) individual item inoculation may be required.

183

For the Single Laboratory Validation with artificial contamination, matrix naturally contaminated with

184

non-target organisms (when available) shall be used. For at least one matrix evaluated during the

185

single laboratory validation, competing non-target microflora must be at least 10x the level of the

186

target microorganism. If the concentration of competing microflora does not exceed 10x the target

187

organism for any matrix, artificial contamination of one matrix with non-target organism (s) is

188 189

required.

190

A minimum three level most probably number (MPN) study should be performed to determine the

191

concentration of the target organism used in the validation. See Appendix J guidelines for details on

192 193 194 195 196 197 198 199 200 201 202 203

performing the MPN study.

8. Method Performance Requirements:

See Table 1 for acceptance criteria for validation. See Table 2 for category test portion requirement. See Table 3 for matrix claims acceptance criteria See Table 4 for descriptions of MPN analysis.

See Table 5 condition of inoculating culture and stabilization of matrix for inoculation.

See Table 6 for inclusivity and exclusivity guidance.

Table 1. Validation Acceptance Criteria (Plants/Flowers, Concentrates, Infused Edibles, Infused Non- 204 Edibles) 205

Parameter Requirements

Target Test Concentration a

Parameter

Minimum Acceptable Results

Single Laboratory Validation with artificial contamination

Replicates per matrix: 20 Inoculation procedure: AOAC Appendix J Replicates: 5 Inoculation procedure: AOAC Appendix J

Fractional positive results, 25- 75% positive

Acceptable minimum detection level (low level)

1-10 CFU/Test Portion

95% CI: LCL < 0 < UCL b

dPOD CP

POD of 1.00 c

High Concentration

10-50 CFU/Test Portion

Non-Inoculated (Zero) concentration

POD of 0.00 c

Replicates: 5

0 CFU/Test Portion

Single Laboratory Validation with natural contamination

Fractional positive results, 25- 75% positive for minimum 1 lot

Acceptable minimum detection level (low level)

2 separate lots of 20 replicates

N/A

95% CI: LCL < 0 < UCL b

dPOD CP

Multi Laboratory Validation

0.15 ≥ LPOD ≥ 0.85

1-10 CFU/Test Portion

95% CI: LCL < 0 < UCL b

dPOD CP

LPOD

10-50 CFU/ Test Portion 0 CFU/Test Portion

LPOD ≥ 0.95

LPOD (0)

LPOD ≤ 0.05

a Determined through MPN Procedures (see Table 3) b The range between the lower and upper confidence interval should encompass 0, if not, the results must be investigated, and an explanation provided. c If acceptance criteria is not observed, results must be investigated, and an explanation provided

206 207

Table 2. Category Test Portion Requirements

Minimum Test Portion Size a