Margosa Extract (C0 2 Extl'act from cold pt'essed Neem Seed Oil) Febmary 2006

Transcription

1 Terra stra GmbH Margosa Etract (C0 2 Etl'act from cold pt'essed Neem Seed Oil) Febmary 2006 Anne Point IIA7.1 Acute toicity to fish Reference Data protection Data owner for data protection 1 REFERENCE --(2005) Acute Toicity Testing of Margosa Etract: C0 2 - ~om Cold Pressed Oil from Neem Seed without Shell in Rainbow Trout (Oncorhynchus mykiss) (Teleostei, Salmonidae) GAB Biotechnologie & Analytik GmbH, - 7. vember 2005 Te1rn stra GmbH Data submitted to the MS after 13 May 2000 on eisting [a.s. I b.p.] for the purpose of its [entry into Anne I/IA I authorisation] Official use only 2.1 Guideline study 2.2 GLP 2.3 Deviations 3.1 Test material Lot/Batch number Specification Purity Composition of Product Further relevant properties Method of analysis 2 GUIDELINES AND QUALITY ASSURANCE OECD 203 (1992) EC-Method C. l (92/69/EEC) 3 MATERIALS AND METHODS As given in section As given in section % Margosa Etract 3.2 Preparation of TS see table A solution for poorly soluble or volatile test substances 3.3 Reference substance Method of analysis for reference substance Water samples (1 ml) were transfeited to a HPLC glass vial and mied with 0.1 ml acetonitrile/water/acetic acid (50:50:2 v/v/v) for stabilisation. At the day of HPLC analysis 400 µl acetonitrile was added for desorption of salannin followed by direct injection into the HPLC/MS-MS system. 3.4 Testing procedure n-entry field Dilution water see table A Page 1

2 Terra stra GmbH Margosa Etract (C0 2 Etl'act from cold pt'essed Neem Seed Oil) Febmary 2006 Anne Point IIA7.1 Acute toicity to fish Test organisms Test system Test conditions Duration of the test Test parameter Sampling Monitoring of TS concentration Statistics see table.a see table.a seetable.a h Effects and mortality Observations were ma.de after 3, 6, 24, 48, 72 and 96 h at ea.ch test level, the solvent control and the blank control.a.t 0, 24, 48, 72 and 96 h The LC 50 -values were determined by Speannan-Karber estimator based on the mortality data obse1"ved at different times with the nominal concentrations of the test item solution. 4 RESULTS Limit Test Concentration Number/ percentage of animals showing adverse effects Nature of adverse effects 4.2 Results test substance Initial concentrations of test substance A.ctual concentrations of test substance Effect data (Mortality) Concentration I response cm"ve Other effects 4.3 Results of controls Number/ percentage of Perfo1med 0.1, 1 *, 10* and 100* mg/l * emulsion with oily film and droplets at the sw-fa.ce, at 10 and at 100 mg/l oily agglomerates were adhered to the side wall of the test tank 100 % mortality was obse1"ved at 1 OOmg/L after 48, 72 and 96 h mortality 0, 0.117, 0.187, 0.3 mg/l ( Sa.lannin as analylite) 0, 0.09, 0.14, mg/l ( Sa.lannin as analyte) see table.a Graph of the concentration-mortality cm"ve at test tennina.tion is provided in the study - difficulties with maintenance of equilibrium were obse1"ved at mg/l - fish upside down with loss of equilibrium were observed at 16, 41 and 65.5 mg/l non Page2

3 Terra stra GmbH Margosa Etract (C0 2 Etl'act from cold pt'essed Neem Seed Oil) Febmary 2006 Anne Point IIA7.1 Acute toicity to fish animals showing adverse effects Nature of adverse effects 4.4 T est with reference substance t performed Concentrations Results 5 APPLICANT'S SUMMARY AND CO NCL USION 5.1 Materials and methods A 96 h semi-static test was conducted with Margosa Etract according OECD Test Guideline 203 and EC Method C. l in order to estimate the acute toicity to Rainbow trout (Oncorhynchus mykiss) (Teleostei, Salmonidae). 5.2 R esults and discussion According to the results of the test, the LC50 (96 h) of the test item was detemlined to be 14.6 mg/l (nominal), equivalentto 11.2 mg/lmean measured concentration. The LC 0 (96 h) (=NOEC) was observed at 10 mg/l (nominal), equivalent to 7.64 mg/l mean measured concentration. mortality occwred, neither in the control nor in the solvent group LC0 LC so LC100 Due to the comple composition of the test item he analysis of the test compound was made with Salannin as reference analyte and showed 79.3 % recove1y in fresh test medium 7.64mg/L 11.2 mg/l 19.6 mg/l 5.3 Conclusion see validity criteria summarized in table A 7_4_ l_ Other Conclusions Reliability Deficiencies The chosen analyte Salannin was detected with 79.3 % mean ( fresh) Evaluation by Competent Authorities Use separate "evaluation boes" to provide transparency as to the comments and views submitted EVALUATION BY RAPPORTEUR MEMBE R STATE DE Date Materials and Methods 20 14/08/26 Applicant's version is acceptable considering the following remarks and cot1'ections: 3.2: Contrary to the data given in Table A7 _ 4_ 1_ 1-1, acetone has been used as a solvent/vehicle for both limit and ma.in tests (2.4 ml acetone in 24 L test tank volume, cot1'esponding to a concentration of 0.1 ml/l). In accordance to the guideline, the test also includes solvent controls. Page3

4 Terra stra GmbH Margosa Etract (CO 2 Etract from cold pressed Acute toicity to fish Anne Point IIA7.1 Results and discussion Conclusion Applicant s version is acceptable considering the following remarks: 4.2.1: nominal test concentrations 0, 6.25, 10.0, 16.0, 25.6, 41.0 and 65.5 mg/l. 3 hours after pouring in the water, small droplets of test item were observed at any test concentration, also at the side wall of the test tanks at 25.6 mg/l and at higher concentrations. This did not affect concentrations of salannin, however the test report does not discuss if this conclusion could also be transferred to azadiractin A and B and other components of test substance : Concentrations of salannin were analysed each 24 hours for fresh and aged test media (static-renewal). Based on salannin, mean measured concentration was 76.4 % of nominal. Mean measured initial concentrations were between 77.1 and 81.2 % of nominal. Applicant s version can be accepted considering the following remarks: 5.2.1, 5.2.2, 5.2.3: Results based on mean measured concentration. 3.4: Contrary to the applicants summary, a solvent control / vehicle has been used. 96 h LC 50 = 11.2 mg Margosa Etract/L (mean measured) Reliability 2 Acceptability acceptable Remarks COMMENTS FROM... Date Materials and Methods Results and discussion Conclusion Reliability Acceptability Give date of comments submitted Discuss additional relevant discrepancies referring to the (sub)heading numbers and to applicant's summary and conclusion. Remarks Page 4

5 Terra stra GmbH Margosa Etract (CO 2 Etract from cold pressed Table A7_4_1_1-1: Preparation of TS solution for poorly soluble or volatile test substances Dispersion Vehicle Concentration of vehicle Vehicle control performed Other procedures - Table A7_4_1_1-2: Source Dilution water Dechlorinated drinking water Alkalinity - Hardness mg/l ph Oygen content - Conductance 537 µs/cm Holding water different from dilution water Table A7_4_1_1-3: Species/strain Source Wild caught Age/size Test organisms Oncorhynchus mykiss (Walbaum) Forellenzucht Schneider, D Katzweiler 4 6 cm Kind of food granular rearing food (size 0) Amount of food Feeding frequency Pretreatment Feeding of animals during test 2 % of body weight daily acclimation period > 12 days Page 5

6 Terra stra GmbH Margosa Etract (CO 2 Etract from cold pressed Table A7_4_1_1-4: Test type Renewal of test solution Volume of test vessels Volume/animal Test system Number of animals/vessel 10 Number of vessels/ concentration 1 Test performed in closed vessels due to significant volatility of TS Semistatic Renewal of test medium was made every 24 h 26 L stainless steel ma. loading 1 g fish/l Table A7_4_1_1-5: Test conditions Test temperature C ( mean 16.0 C ) table is provided in the study Dissolved oygen % saturation table is provided in the study ph ( mean 8.19 ) table is provided in the study Adjustment of ph Aeration of dilution water Intensity of irradiation Photoperiod continuous aeration of the test tanks with a membrane pump using a Pasteur pipette t provided h photoperiod daily Table A7_4_1_1-6: Mortality data Test-Substance Mortality Concentration (nominal) Number Percentage [mg/l] 24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h Control Temperature [ C] ph Oygen [mg/l] Page 6

7 Terra stra GmbH Margosa Etract (CO 2 Etract from cold pressed Table A7_4_1_1-7: Effect data 48 h [mg/l] 1 95 % c l. 96 h [mg/l] 1 95 % c l. LC LC LC effect data are based on nominal (n) concentrations Table A7_4_1_1-8: Validity criteria for acute fish test according to OECD Guideline 203 fulfilled t fulfilled Mortality of control animals <10% X Concentration of dissolved oygen in all test vessels > 60% saturation X Concentration of test substance 80% of initial concentration during test X for poorly soluble test substances X Page 7