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
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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
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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
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Storage stability and corrosivity test
| The test started on September 28, 2022
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| Date of completion of the test: March 29, 2024
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Study Completion Date: Please see the date on which the study host signed the GLP Statement
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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).
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*Name and content of active ingredient: Trichoderma virens strain R42, 2×108 CFU/g
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*Name of producer: Longying Biotechnology Co., Ltd
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*Producer's address: No. 16, 16th Road, Dapu Meiyuan, Dalin Town, Chiayi County
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*批號︰20220301001 *Lot number..20220301001 |
*Content (Analytical Evidence): ≧2×108 CFU/g
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*Factory manufacturing: Beijing time: 2022.03.01
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*Factory expiration date: 2024.02.28
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*Factory storage condition: refrigerated
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2. Information on the test quality points of the GLP laboratory
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Test substance number (internal number): 1106
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Date of delivery of test material..2022.03.31
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The study host received the date..2022.03.31
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The appearance of the test substance at the time of spotting: dark brown powder
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Date of content analysis..2022.04.11
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Active ingredient content: .1.1×109CFU/g
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對照物質:
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
試驗方法: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℃)貯存及各時間點有效成分含量分析。
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個月之貯存試驗,分別在第18及24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)
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Electronic balances
| Pharmaceutical sampling weighing
| SHIMADZU | TX2202L | AA-181-139 | 2023.05.03 (correction period 12 months)
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Sterile workbench
| Microbiological manipulation
| HIGH TEN | 6BH-24 | AC-771-44 | Exemption from school
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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:
以試驗物質送達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,試驗物質存放於54℃冷藏箱(設備編號:AA-340-61),分別在第18及24個月貯存時間點時進行觀察及分析;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)」,試驗物取樣三重複,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倍)之試驗物稀釋液,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 Bengal、250 ppm鏈黴素、0.06% PDA,1.5% Agar)至無餘液流動,每一稀釋倍數塗抺3個培養皿,置於25℃培養箱(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
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| Packaging container material: aluminum foil bag (PET/AL/LLDPE).
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Pack the container after storage
| There is no difference in the appearance of the packaging containers
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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).
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| | | | 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/g; The number of colonies formed after storage was above 2×108 CFU/g, which met the nominal standard。
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Attached drawings
Figure 1: Active ingredient content of Trichoderma R42 (Other) Powder (AP) under refrigeration