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Report No.: 1106G21PHY(03)(c).


Study ID: 1106G21PHY(03).


Number of pages reported: v +21 pages


Printing Serial Number :


Trichoderma R42 (Other) Powder (AP) Trichoderma virens strain R42 (2×108CFU/g) (storage stability and corrosivity - 24 months).


Delegators


Longying Biotechnology Co., Ltd


8th Floor, No. 97, Jingye 1st Road, Zhongshan District, Taipei City, 10462


Test unit


Agricultural Drug Research Institute, Ministry of Agriculture


GLP Laboratory, Agricultural Drug Laboratory


413001 No. 11, Guangming Road, Jiuzhengli, Wufeng District, Taichung City


May 2024

1


Quality Assurance Statement


Report No.: 1106G21PHY(03)(c).


Study ID: 1106G21PHY(03).


Study Name: Trichoderma R42 (Other) Powder (AP) Trichoderma virens strain R42 (2×108CFU/g) (storage stability and corrosivity - 24 months).


This GLP study and summary report have been audited to comply with the OECD GLP specification, and the physical and chemical tests in the study are conducted in accordance with the research plan 1106G21PHY(03)-P1 and the test standard operating procedures specified therein. The content of this research report is true, correct and completely reflects the original data. The following is the type of audit, the stage of the audit, the date of the audit, and the date on which the audit results were notified to the management and the research host.


Check type


Review stage


Date of Audit


Date of notification to management


Inform the study


Host date


What to study


Research proposal and research schedule

2022.04.28

2022.04.28

2022.04.28


What to study


Storage stability test, active ingredient analysis operation and packaging material observation


(18 months).

2023.10.02

2023.10.02

2023.10.02


What to study


Storage stability test, active ingredient analysis operation and packaging material observation (24 months).

2024.03.25

2024.03.25

2024.03.25


What to study


Research reports and raw data

2024.05.23

2024.05.23

2024.05.23


Chen Peiyin quality assurance staff (date).


GLP Laboratory, Agricultural Drug Laboratory


413001 No. 11, Guangming Road, Jiuzhengli, Wufeng District, Taichung City


Good Laboratory Practice (GLP) Declaration of Conformity


Report No.: 1106G21PHY(03)(c).


Study ID: 1106G21PHY(03).


Research Name: Trichoderma R42 (Other) Powder (AP) Trichoderma virens strain R42 (2×108CFU/g) (storage stability and corrosivity - 24 months).


This study complies with the 1997 OECD Good Laboratory Practice (GLP) and related physical and chemical test specifications, and I hereby declare that I am responsible for the correctness and validity of this GLP report. The laboratory confirms the essence of the test substance through the culture certificate (Annex 1) and the content analysis certificate (Annex 2) from the client. Before the start of the study, the active ingredient content was analyzed and the content was confirmed to be in accordance with the nominal level.


Shi Jieyu Research Host (date).


GLP Laboratory, Agricultural Drug Laboratory


413001 No. 11, Guangming Road, Jiuzhengli, Wufeng District, Taichung City


Statement of the management of the physical and chemical laboratory


Report No.: 1106G21PHY(03)(c).


Study ID: 1106G21PHY(03).


Research Name: Trichoderma R42 (Other) Powder (AP) Trichoderma virens strain R42 (2×108CFU/g) (storage stability and corrosivity - 24 months).


The GLP Declaration of Conformity signed by the study host and the Quality Assurance Statement signed by the quality assurance personnel in this study hereby declare that the unit has taken appropriate and necessary measures to ensure compliance with OECD Good Laboratory Practices ( GLP) and the management manual requirements of GLP laboratories of the Agricultural Drug Laboratory.


Xie Fengjia management (date).


GLP Laboratory, Agricultural Drug Laboratory


413001 No. 11, Guangming Road, Jiuzhengli, Wufeng District, Taichung City


Research-related personnel


Representative of the client: Chen Jixin (e-mail: longying056312598@gmail.com).


GLP Lab Management: Xie Fengjia (e-mail: hsiehf@tactri.gov.tw).


Quality assurance staff: Chen Peiyin (e-mail:cpy@tactri.gov.tw).


Study Host: Shi Jieyu (E-mail: scy1214@tactri.gov.tw).


Researcher: Yang Pengwei (performing storage stability and corrosivity tests).


Song Kaizhi (performing storage stability and corrosivity tests).


Physicochemical test research report of GLP laboratory of Agricultural Drug Laboratory

i

TACTRI GLP REPORT NO. 1106G21PHY(03) (c)


directory


Quality Assurance Statement i


Good Laboratory Practice (GLP) Declaration of Conformity II


Physicochemical Laboratory Management Statement III


Research Stakeholders iv


Dir. v


I. Abstract 1


II. Purpose 2


3. Research Schedule 2


4. Test substance and control substance 2


5. The contents of various physical and chemical tests (test guidelines, test methods, test equipment, chemicals, test procedures) 3


(1) Storage stability and corrosivity test: 3


6. Explanation of the revision of the research proposal 6


7. Deviation from the description 6 of the research proposal


Results, discussion and conclusions 6


(1) Storage stability and corrosivity test: 6


9. Research Archive 7


10. Reference document 7


Schedule 8


Table 1: Test results of storage stability and corrosivity of Trichoderma R42 (other) powder (AP) 8


Figure 9


Figure 1: Active ingredient content of Trichoderma R42 (Other) Powder (AP) refrigerated at each storage point9


Appendix I. Copy of the research proposal 11


Appendix II: GLP Laboratory Registration Certificate 15


Note 17 of the supporting annex


Appendix 1: Background information of the strain provided by the client 18


Appendix 2: Certificate of Analysis of Speciate Content provided by the Client 20


Appendix 3: Description of packaging materials provided by the client 21


GLP Laboratory, Agricultural Drug Laboratory


413001 TEL (04) 23302101, No. 11, Guangming Road, Jiuzhengli, Wufeng District, Taichung City


summary


In this study, according to the OPPTS 830.6317 test guideline document and the physicochemical and toxicological test criteria of pesticides, the test substance "Trichoderma R42 (Other) Powder (AP)".Trichoderma virens strain R42 (2×108 CFU/g)".Combined corrosivity tests of packaging materials. The storage stability test of 1106G21PHY (03) in this study is a continuation of the previous study, with a duration of 24 months and a storage temperature of 5±4°C, the packaging container is made of aluminum foil bag (PET/AL/LLDPE), and the test substance (internal number 1106The actual storage date is from the date of receipt 202 2.0331 began to calculate, and the content of the test substance was confirmed to be in accordance with the nominal (the active ingredient content was 1.1×109CFU/g), 24-month storage test results: storage 18After 24 months, there was no obvious change in the appearance of the test substance, no deformation and no leakage in the packaging container (aluminum foil bag), and the content of active ingredientsfor (8.00.)1) × 108 and (3.6 0.4) × 108CFU/g, due to the storage period (2022.). 3.31~2024.3.25), there is no obvious change in the appearance of the test substance, there is no deformation and leakage in the packaging container (aluminum foil bag), and the content of active ingredients is not lower than the nominal levelThe value was 2×108CFU/g, which was evaluated in refrigeration (5°C) shelf life up to 24 months.


Pilot project


Test guidelines or


Test Method


outcome


Storage stability and corrosiveness - 24th month

OPPTS 830.6317


Refrigerated (5°C) for 24 months, the aluminum foil bag (PET/AL/LLDPE) has no deformation and no leakage, not less than the nominal content (2×108CFU/g), assessed for refrigerated storage24 months


objective


This study was commissioned by Longying Biotechnology Co., Ltd. to produce the finished pesticide Trichoderma R42 (other) powderStorage stability of (AP)Trichoderma virens strain R42 (2×108 CFU/g)". Tests for the purpose of pesticide registration information.


Study period


Study start date (date of signing of the research proposal by the study host): May 4, 202


Storage stability and corrosivity test


The test started on September 28, 2022


Date of completion of the test: March 29, 2024


Study Completion Date: Please see the date on which the study host signed the GLP Statement


Test substance and control substance


Test Substance:


1. The following bidders shall provide the test substance and supporting documents for the client


*Name of test substance: Trichoderma R42 (other) powder (AP).


*Name and content of active ingredient: Trichoderma virens strain R42, 2×108 CFU/g


*Name of producer: Longying Biotechnology Co., Ltd


*Producer's address: No. 16, 16th Road, Dapu Meiyuan, Dalin Town, Chiayi County

*批號︰20220301001
*Lot number..20220301001


*Content (Analytical Evidence): 2×108 CFU/g


*Factory manufacturing: Beijing time: 2022.03.01


*Factory expiration date: 2024.02.28


*Factory storage condition: refrigerated


2. Information on the test quality points of the GLP laboratory


Test substance number (internal number): 1106


Date of delivery of test material..2022.03.31


The study host received the date..2022.03.31


The appearance of the test substance at the time of spotting: dark brown powder


Date of content analysis..2022.04.11


Active ingredient content: .1.1×109CFU/g

對照物
Reference Substances:

無。
Not.

各項理化試驗內容(試驗之指引、試驗方法、試驗設備、化學品、試驗步驟)
The contents of various physical and chemical tests (test guidelines, test methods, test equipment, chemicals, test procedures).

貯存安定性及腐蝕性試驗
Storage stability and corrosivity test:

試驗指引:OPPTS 830.6317
Test Guidelines: OPPTS 830.6317

試驗方分別依「貯存安定性(含包裝滲漏)試驗標準操作程序05C-0072-0000(1)」及「單位重量或單位體積中活孢子數測定標準操作程序05C-0015-0000(4)」進行冷藏(5)貯存及各時間點效成分含量分析
Test method: According to the "Standard Operating Procedures for Storage Stability (Including Packaging Leakage) Test 05C-0072-0000(1)" and "Standard Operating Procedures for the Determination of Viable Spore Number per Unit Weight or Unit Volume" respectively05C-0015-0000(4)"Refrigerated (5°C) storage and analysis of active ingredient content at each time point

試驗概述試驗物質送達GLP實驗室試驗物貯存設備日為貯存起始日將試驗物質放置於5±4℃恆溫箱進行持續24個月貯存試驗分別在第1824個月貯存時間點進行含量分析,做為產品有效期間之推估。取樣前先觀察試驗物質及包裝外觀,將測試條件、包裝材質及取樣相關資訊紀錄後取樣進行有效成分含量分析
Test overview: The date on which the test substance is delivered to the test specimen storage equipment of the GLP laboratory is the storage start date, and the test substance is placed in a 5±4°C incubatorThe storage trial lasted for 24 months, at the 18th and 24th months respectivelyThe content analysis is carried out at the storage time point as an estimation of the effective period of the product. Before sampling, observe the appearance of the test substance and packaging, record the test conditions, packaging material and sampling related information, and then take samples for active ingredient content analysis.

試驗設備:
Test Equipment :

設備名稱
The name of the device


use


Label


Model


Device number

最近一次校正日期
The date of the last correction


Electronic balances


Test material sampling weighing

Mettler Toledo

PB3002-S

AB-449-60


2023.05.03 (correction period 12 months)


Electronic balances


Pharmaceutical sampling weighing

SHIMADZU

TX2202L

AA-181-139


2023.05.03 (correction period 12 months)


Sterile workbench


Microbiological manipulation

HIGH TEN

6BH-24

AC-771-44


Exemption from school

低溫箱
Cryogenic chambers

試驗物質冷藏貯存
The test material is stored refrigerated

宏美
Hongmei

NA

AA-340-61

免校
Exemption from school

低溫迴轉式振盪培養箱
Low-temperature rotary vibrating incubator

微生物培養
Microbial culture

FIRSTEK

S300

AC-1120-30

2023.05.18(校正週期12個月)
2023.05.18 (correction period 12 months).

桌上型高壓蒸氣滅菌鍋
Tabletop autoclave

微生物操作
Microbiological manipulation

CHIN KU (光杏)
CHIN KU (LIGHT APRICOT).

AS-1060L

AA-321-23

免校
Exemption from school

藥品與材料
Drugs & Materials:

名稱
name

CAS No.

廠牌
Label

產品編號
Product number

/批號
/lot number

Potassium sorbate

(己二烯酸鉀)
(potassium adipaenoate).

24634-61-5

Alfa Aesar

A12844

/10223676

Rose Bengal

(孟加拉玫瑰紅鈉鹽)
(Bengal rose red sodium salt).

632-69-9

Sigma-Aldrich

198250

/SHBM741

Streptomycin sulfate (鏈黴素)
Streptomycin sulfate

3810-74-0

Bio Bsdiv Inc

SB0494

/P1008080

Difco Potato Dextrose Agar

(馬鈴薯葡萄糖瓊脂)
(Potato dextrose agar).

NA

BD

213400

/9311217

Bacto Agar

(洋菜粉)
(seaweed powder).

NA

BD

214010

/1103620

試驗步驟:
Test Procedure:

以試驗物送達GLP實驗室試驗物儲存室(R402)之貯存設備為貯存起始日,持續為期24個月的貯存安定性試驗,試驗物存放54冷藏箱(設備編號:AA-340-61)分別在第1824個月存時間點時進行觀察分析待試驗物回至室溫,先觀察試驗物及包材外觀是否產生變化,填列貯存測試條件(測試溫度、貯存時間及預訂貯存期間)及包裝容器材質;秤樣分析,記錄試驗物(含瓶)重量、取樣量、取樣後試驗物(含瓶)重量(記錄重量至0.01g)於「貯存安定性(含包裝滲漏)結果紀錄表05C-0072-0001(1)」。
The storage stability test lasts for 24 months on the storage start date of the test substance delivered to the storage equipment of the test object storage room (R402) of GLP laboratory, and the test substance is storedRefrigerated containers at 5 4 °C (equipment number: AA-340-61), respectivelyObservation and analysis were performed at the 18th and 24th month storage time points; When the test object returns to room temperature, observe whether the appearance of the test object and packaging material changes, and fill in the storage test conditions (test temperature, storage time and scheduled storage period) and packaging container material; Scale sample analysis, record the weight of the test object (including bottle), the sample volume, and the weight of the test object (including the bottle) after sampling (record weight to 0.01g) In the "Storage Stability (Including Packaging Leakage) Result Record Form 05C-0072-0001(1)".

有效成分含量分析方法如下依照木黴菌類(Trichoderma spp.)微生物試驗方法,預先配製木黴菌選擇性培養基並記錄於「微生物培養基製備紀錄表05C-0016-0001(3)」,試驗物取三重複各取3 g試驗物配製成30 mL稀釋懸浮母液,將10倍序列稀釋(105~107)試驗物稀釋液,取0.1 mL以滅菌塗抹棒均勻塗佈在木黴菌選擇性培養基(1000 ppm己二烯酸鉀、150 ppm Rose Bengal250 ppm鏈黴素、0.06% PDA1.5% Agar)至無餘液流動,每一稀釋倍數塗抺3個培養皿,置於25℃培養(編號AC-1120-30)培養96小時後,觀察菌落生成數,以下列算式計算單位重量之菌落形成數(CFU/g),並將三次重複結果取平均值:『單位重量之菌落形成數(CFU/g)=平均菌落數(CFU)/0.1 mL×菌液稀釋倍數×30 mL/3 g』,將結果記錄於「單位重量或單位體積中活孢子數測試紀錄表05C-0015-0001(3)」。
The analysis method of active ingredient content is as follows, according to Trichoderma sppMicrobial test method, Trichoderma selective medium was pre-prepared and recorded in the "Microbial Culture Medium Preparation Record Form 05C-0016-0001(3)", and the test object was sampled in three replicates, and 3g of each was takenThe test object was prepared into a 30 mL dilution suspension mother solution, and the 10-fold sequence was diluted (105~107 times) of the test substance dilution, take 0.1 mL and evenly coat it in Trichoderma selective medium (1000 ppm) with a sterilized applicatorpotassium adipaenoate, 150 ppm Rose Bengal, 250 ppm streptomycin, 0.06% PDA, 1.5% Agar) to no residual flow, each dilution was coated3 Petri dishes were placed in a 25 °C incubator (No. AC-1120-30) to incubate 96After hours, the number of colonies formed per unit weight (CFU/g) was calculated using the following equation, and the results of three replicates were averaged: 'Number of colonies formed per unit weight (CFU/g.') ) = Average number of colonies (CFU)/0.1 mL× dilution factor of the bacterial solution ×30 mL/3 g', and the results were recorded in the "Test Record of Viable Spore Count per Unit Weight or Unit Volume". 05C-0015-0001(3)」。


Instructions for the revision of the research proposal


Not.


Deviation from the research proposal description


Due to the transformation of the government, the experimental unit was renamed from the Agricultural Drug and Poison Laboratory of the Agricultural Committee of the Executive Yuan to the Agricultural Drug Laboratory of the Ministry of Agriculture, and the GLP Laboratory of the Agricultural Drug and Poison Laboratory was renamed as the GLP Laboratory of the Agricultural Drug Laboratory.


Due to the adjustment of the position of the original researcher, because the research case is still in progress, after the management has evaluated it in accordance with the relevant operating procedures, Song Kaizhi was assigned as a researcher to continue to implement the unfinished research case.


Results, Discussion and Conclusions


Storage stability and corrosivity test:


After 18 and 24 months of refrigerated storage (5±4°C), the test object appeared to be dark brown powder ( No difference), no leakage (no difference) in the appearance of the packaging container (aluminum foil bag), analysis of the active ingredient content, the unit of three repetitionsThe number of weight colonies formed was (8.00.). 1) × 108 and (3.6 0.4) × 108CFU/g, see Table 1; During storage, the content of active ingredient was greater than 2×108CFU/g and conformed to the nominal content, see Figure 1. Based on the above evaluation, the active ingredient content of the test substance was still higher than the nominal content after 24 months of refrigerated storage, and the refrigerated storage shelf life was estimated to be up to 24 months


Research archiving


After the research report is provided to the client, all records related to the trial (including the research plan, various record forms, and original dataand research reports and other related documents) to be deposited by the archivist in the document archive of the GLP physical and chemical laboratory of the Institute (Room 201, Biopharmaceutical Building) At least 6 years, after the expiration of the period, confirm with the client the destruction or transfer of documents; The remaining test objects are kept in the test object storage room (Room 402, Biopharmaceutical Building).After the expiration date, it will be discarded after confirmation by the research host.


References

United States Environmental Protection Agency OPPTS 830.6317 Storage Stability. EPA 712–C–02–026. June 2002.

Collins, C. H., Patricia M. Lyne, and J. M. Grange. 1989. Microbiological Methods, 6th ed. Butterworth & Co., Oxford, London.

Dhingra, Onkar D., and James B. Sinclair. 1985. Basic Plant Pathology Methods. CRC Press, Boca Raton, Flordia.


schedule


Table 1 Test results of storage stability and corrosivity of Trichoderma R42 (other) powder (AP).


Test conditions


Test temperature: 5°C Test storage time: 18, 24 months


Storage period: March 31, 202 to March 25, 2024day


Packaging container material: aluminum foil bag (PET/AL/LLDPE).


Pack the container after storage


There is no difference in the appearance of the packaging containers


Analyze the project


/ Storage point


(Operation Day).


Appearance of the test specimen after storage


Is there any difference in appearance from the original test subject


Whether there is any difference in the appearance of the packaging material or leakage


Active ingredient content (×108CFU/g).


Repeat 1


Repeat 2


Repeat 3


average

SD


Month 18

(2023.10.2)


Dark brown powder


No difference


No differences, no leakage

8.0

8.1

8.0

8.0

0.1


Month 24

(2024.3.25)


Dark brown powder


No difference


No differences, no leakage

3.0

3.8

3.9

3.6

0.4


Test


outcome


After 18 and 24 months of storage at 5°C, the aluminium foil bag (packaging material) There was no deformation and no leakage, and the average number of colonies formed in the three replicates was (8.00.). 1) × 108 and (3.60.)4)×108CFU/gThe number of colonies formed after storage was above 2×108 CFU/g, which met the nominal standard


Attached drawings


Figure 1: Active ingredient content of Trichoderma R42 (Other) Powder (AP) under refrigeration


Physicochemical test research report of GLP laboratory of Agricultural Drug Laboratory

TACTRI GLP REPORT NO. 1106G21PHY(03) (c) Page 21 of 21


GLP Laboratory, Agricultural Drug Laboratory


413001 TEL (04) 23302101, No. 11, Guangming Road, Jiuzhengli, Wufeng District, Taichung City


appendix


Relevant documents related to the experimental research output of the unit, such as research proposals and supporting materials.


Appendix 1: A copy of the research proposal


Appendix 2: GLP Laboratory Login Certificate


[Blank below this page]


Appendix 1: A copy of the research proposal


Appendix 2: GLP Laboratory Login Certificate


Supporting Attachment Notes


The test substance of this study is provided by the research client, and the laboratory and the client establish a cooperation mechanism to confirm the essence of the test substance, including the source of the strain, the specification of the test substance, and the content of the active ingredient.


Appendix 1: Background information of the strain provided by the client


Appendix 2: Certificate of Analysis of Speciate Content provided by the client


Appendix 3: Description of the packaging materials provided by the client


[Blank below this page]


Appendix 1: Background information of the strain provided by the client


Appendix 2: Certificate of Analysis of Speciate Content provided by the client


Appendix 3: Description of the packaging materials provided by the client