FAO SPECIFICATIONS AND EVALUATIONS FOR ...FAO SPECIFICATIONS AND EVALUATIONS FOR FLUAZINAM Page 3 of...

FAO SPECIFICATIONS AND EVALUATIONS

FOR AGRICULTURAL PESTICIDES

FLUAZINAM

3-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)-

α,α,α-trifluoro-2,6-dinitro-p-toluidine

2020

CONTENTS

FLUAZINAM

Page

DISCLAIMER

INTRODUCTION 1 PART ONE SPECIFICATIONS FOR FLUAZINAM 2

FLUAZINAM INFORMATION 3

FLUAZINAM TECHNICAL MATERIAL (SEPTEMBER 2019) 4

FLUAZINAM SUSPENSION CONCENTRATE (SEPTEMBER 2019) 5

PART TWO EVALUATIONS OF FLUAZINAM 8

2019 FAO/WHO EVALUATION REPORT ON FLUAZINAM 9

SUPPORTING INFORMATION 11

ANNEX 1: HAZARD SUMMARY PROVIDED BY PROPOSER 13

ANNEX 2: REFERENCES 15

‘













APPENDIX 1: ANALYTICAL METHOD FOR DETERMINATION

OF IMPURITY 5 IN TC AND SC 52

DISCLAIMER1

FAO specifications are developed with the basic objective of promoting, as far as practicable, the manufacture, distribution and use of pesticides that meet basic quality requirements.

Compliance with the specifications does not constitute an endorsement or warranty of the fitness of a particular pesticide for a particular purpose, including its suitability for the control of any given pest, or its suitability for use in a particular area. Owing to the complexity of the problems involved, the suitability of pesticides for a particular purpose and the content of the labelling instructions must be decided at the national or provincial level.

Furthermore, pesticides which are manufactured to comply with these specifications are not exempted from any safety regulation or other legal or administrative provision applicable to their manufacture, sale, transportation, storage, handling, preparation and/or use.

FAO disclaims any and all liability for any injury, death, loss, damage or other prejudice of any kind that may arise as a result of, or in connection with, the manufacture, sale, trans-portation, storage, handling, preparation and/or use of pesticides which are found, or are claimed, to have been manufactured to comply with these specifications.

Additionally, FAO wishes to alert users to the fact that improper storage, handling, prepara-tion and/or use of pesticides can result in either a lowering or complete loss of safety and/or efficacy. FAO is not responsible, and does not accept any liability, for the testing of pesticides for compliance with the specifications, nor for any methods recommended and/or used for test-ing compliance. As a result, FAO does not in any way warrant or represent that any pesti-cide claimed to comply with a FAO specification actually does so.

1 This disclaimer applies to all specifications published by FAO.


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INTRODUCTION

FAO establishes and publishes specifications* for technical material and related formula-tions of agricultural pesticides, with the objective that these specifications may be used to provide an international point of reference against which products can be judged either for regulatory purposes or in commercial dealings.

From 2002, the development of FAO specifications follows the New Procedure, described in the 1st edition of the “Manual on Development and Use of FAO and WHO Specifications for Pesticides” (2002) - currently available as 3rd revision of the 1st edition (2016) - , which is available only on the internet through the FAO and WHO web sites.

This New Procedure follows a formal and transparent evaluation process. It describes the minimum data package, the procedure and evaluation applied by FAO and the Experts of the FAO/WHO Joint Meeting on Pesticide Specifications (JMPS). [Note: prior to 2002, the Experts were of the FAO Panel of Experts on Pesticide Specifications, Registration Re-quirements, Application Standards and Prior Informed Consent, which now forms part of the JMPM, rather than the JMPS.]

FAO Specifications now only apply to products for which the technical materials have been evaluated. Consequently from the year 2000 onwards the publication of FAO specifications under the New Procedure has changed. Every specification consists now of two parts namely the specifications and the evaluation report(s):

Part One: The Specification of the technical material and the related formulations of the pesticide in accordance with chapters 4 to 9 of the “Manual on development and use of FAO and WHO specifications for pesticides”.

Part Two: The Evaluation Report(s) of the pesticide, reflecting the evaluation of the data package carried out by FAO and the JMPS. The data are provided by the manu-facturer(s) according to the requirements of chapter 3 of the “FAO/WHO Manual on Pesticide Specifications” and supported by other information sources. The Evaluation Report includes the name(s) of the manufacturer(s) whose technical material has been evaluated. Evaluation reports on specifications developed subsequently to the original set of specifications are added in a chronological or-der to this report.

FAO specifications developed under the New Procedure do not necessarily apply to nomi-nally similar products of other manufacturer(s), nor to those where the active ingredient is produced by other routes of manufacture. FAO has the possibility to extend the scope of the specifications to similar products but only when the JMPS has been satisfied that the addi-tional products are equivalent to that which formed the basis of the reference specification.

Specifications bear the date (month and year) of publication of the current version. Evaluations bear the date (year) of the meeting at which the recommendations were made by the JMPS.

* NOTE: PUBLICATIONS ARE AVAILABLE ON THE INTERNET AT

http://www.fao.org/agriculture/crops/thematic-sitemap/theme/pests/jmps/ps-new/en/ OR IN

HARDCOPY FROM THE PLANT PROTECTION INFORMATION OFFICER.

http://www.fao.org/agriculture/crops/thematic-sitemap/theme/pests/jmps/ps-new/en/


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PART ONE

SPECIFICATIONS

SPECIFICATIONS FOR FLUAZINAM 2

FLUAZINAM INFORMATION 3

FLUAZINAM TECHNICAL MATERIAL (SEPTEMBER 2019) 4

FLUAZINAM SUSPENSION CONCENTRATE (SEPTEMBER 2019) 5


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FLUAZINAM

INFORMATION

Identity of the active ingredient

ISO common name Fluazinam (ISO 1750 published)

Chemical name(s)

IUPAC 3-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)- trifluoro-2,6-dinitro-p-to-luidine CA 3-chloro-N-[3-chloro-2,6-dinitro-4-trifluoromethyl) phenyl]-5-(trifluoromethyl)-2-pyridinamine

Synonyms

IKF-1216, B-1216, PP192

Structural formula

Molecular formula

C13H4Cl2F6N4O4

Relative molecular mass 465.1

CAS Registry number 79622-59-6

CIPAC number 521

Identity tests

Retention time in reversed-phase HPLC, UV- and IR-spectra

NH

N

Cl

CF3

O2N

CF3

ClO2N


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FLUAZINAM TECHNICAL MATERIAL

FAO Specification 521 / TC (September 2019*)

This specification, which is PART ONE of this publication, is based on an evaluation of data submitted by the manufacturers whose names are listed in the evaluation reports (521/2008, 521/2017, 521/2018 & 521/2019). It should be applicable to technical materials produced by these manufacturers but it is not an endorsement of those products, nor a guarantee that they comply with the specifications. The specification may not be appropriate for technical materials produced by other manufacturers. The evaluation reports (521/2008, 521/2017, 521/2018 & 521/2019), as PART TWO, form an integral part of this publication.

1. Description

The material shall consist of fluazinam together with related manufacturing impurities, in the form of a yellow solid, having a weak aromatic hydrocarbon-like or strong musty odour, free from visible extraneous matter and added modifying agents.

2. Active Ingredient

2.1 Identity tests (521/TC/M/2, CIPAC Handbook O, p. 69, 2017) The active ingredient shall comply with an identity test and, where the identity remains in doubt with at least one additional test.

2.2 Fluazinam content (521/TC/M/3, CIPAC Handbook O, p. 69, 2017) The fluazinam content shall be declared (not less than 960 g/kg) and, when deter-mined, the average measured content shall not be lower than the declared minimum content.

3. Relevant impurities (Note 1)

3.1 5-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)- α,α,α-trifluoro-4,6-dinitro-o-toluidine (Note 1)

Maximum: 3 g/kg.

________________________

Note 1 This impurity is named «Impurity 5» CAS-Nr.169327-87-1. A peer validated method is provided in Appendix 1.

* Specifications may be revised and/or additional evaluations may be undertaken. Ensure the use of current versions by checking at: http://www.fao.org/agriculture/crops/core-themes/theme/pests/jmps/ps-new/en/

http://www.fao.org/agriculture/crops/core-themes/theme/pests/jmps/ps-new/en/


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FLUAZINAM SUSPENSION CONCENTRATE

FAO Specification 521 / SC (September 2019*)

This specification, which is PART ONE of this publication, is based on an evaluation of data submitted by the manufacturer whose name is listed in the evaluation report (521/2008). It should be applicable to SC produced by this manufacturer but it is not an endorsement of those products, nor a guarantee that they comply with the specifications. The specification may not be appropriate for SC produced by other manufacturers. The evaluation report (521/2008), as PART TWO, forms an integral part of this publication.

1 Description The material shall consist of a suspension of fine particles of technical fluazinam,

complying with the requirements of FAO specification 521/TC (September 2019), in the form of a light-yellow, homogeneous suspension, in an aqueous phase together with suitable formulants. After gentle agitation the material shall be homogeneous (Note 1) and suitable for further dilution in water.

2 Active ingredient

2.1 Identity tests (521/SC/M/2, CIPAC Handbook O, p. 72, 2017)

The active ingredient shall comply with an identity test and, where the identity remains in doubt, shall comply with at least one additional test.

2.2 Fluazinam content (521/SC/M/3, CIPAC Handbook O, p. 72, 2017)

The fluazinam content shall be declared (above 250 up to 500 g/L at 20 2ºC, Note 2) and, when determined, the average content measured shall not differ from that declared by more than ± 5% of the declared content.

3 Relevant impurities 3.1 By-products of manufacture (Note 3) 5-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)- α,α,α-trifluoro-4,6-dinitro-o-toluidine Maximum: 0.3 % of the fluazinam content found under 2.2. 4 Physical properties

4.1 pH range (MT 75.3)

pH range: 7 to 9 (undiluted)

4.2 Pourability (MT 148.1)

Maximum residue: 5%

* Specifications may be revised and/or additional evaluations may be undertaken. Ensure the use of current versions by checking at: http://www.fao.org/agriculture/crops/core-themes/theme/pests/jmps/ps-new/en/

http://www.fao.org/agriculture/crops/core-themes/theme/pests/jmps/ps-new/en/


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4.3 Spontaneity of dispersion (MT 160, CIPAC Handbook F, p. 391, 1995) (Note 4)

A minimum of 90% of the fluazinam content found under 2.2 shall be in suspension after 5 min in CIPAC Standard Water D at 30 ± 2°C.

4.4 Suspensibility (MT 184.1) (Notes 4 & 5)

A minimum of 85 % of the fluazinam content found under 2.2 shall be in suspension after 30 min in CIPAC Standard Water D at 25 ± 5°C.

4.5 Wet sieve test (MT 185, CIPAC Handbook K, p. 149, 2003) (Note 6)

Maximum: 0.1% of the formulation shall be retained on a 75 µm test sieve.

4.6 Persistent foam (MT 47.3, CIPAC Handbook O, p.177, 2017) (Note 7)

Maximum: 25 ml after 1 min. 5 Storage stability

5.1 Stability at 0°C (MT 39.3, Handbook J, p. 126, 2000)

After storage at 0 ± 2°C for 7 days, the formulation shall continue to comply with the clauses for:

- suspensibility (4.4) - wet sieve test (4.5)

5.2 Stability at elevated temperature (MT 46.3, Handbook J, p. 128, 2000)

After storage at 54 ± 2°C for 14 days, the determined average active ingredient content must not be lower than 95 % relative to the determined average content found before storage (Note 8) and the formulation shall continue to comply with the clauses for:

- pH range (4.1) - pourability (4.2) - spontaneity of dispersion (4.3) - suspensibility (4.4) - wet sieve test (4.5)

________________________

Note 1 Before sampling to verify the formulation quality, inspect the commercial container carefully. On standing, suspension concentrates usually develop a concentration gradient from the top to the bottom of the container. This may even result in the appearance of a clear liquid on the top and/or of sediment on the bottom. Therefore, before sampling, homogenize the formulation according to the instructions given by the manufacturer or, in the absence of such instructions, by gentle shaking of the commercial container (for example by inverting the closed container several times). Large containers must be opened and stirred adequately. After this procedure, the container should not contain a sticky layer of non-dispersed matter at the bottom. A suitable and simple method of checking for a non-dispersed sticky layer "cake" is by probing with a glass rod or similar device adapted to the size and shape of the container. All the physical and chemical tests must be carried out on a laboratory sample taken after the recommended homogenization procedure.

Note 2 Unless homogenization is carried out carefully, it is possible for the sample to become aerated. This can lead to errors in the determination of the mass per millilitre and in calculation of the active ingredient content (in g/l) if methods other than MT 3.3 are used. If the buyer requires both g/kg and g/l at 20°C, then in case of dispute the analytical results shall be calculated as g/kg.


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Note 3 This impurity is named «Impurity 5» CAS-Nr.169327-87-1. A peer validated method is provided in Appendix 1.

Note 4 Chemical assay is the only fully reliable method to measure the mass of active ingredient still in suspension. However, simpler methods such as gravimetric and solvent extraction determination may be used on a routine basis provided that these methods have been shown to give equal results to those of the chemical assay method. In case of dispute, the chemical method shall be the referee method.

Note 5 MT 184.1 is the revised version of MT 184 and was adopted at the 2018 CIPAC Meeting in Panama. Prior to its publiction in an next Handbook, copies of the method can be obtained through https://www.cipac.org/index.php/methods-publications/pre-published-methods

Note 6 This test detects coarse particles (e.g. caused by crystal growth) or agglomerates (crust formation) or extraneous materials which could cause blockage of spray nozzles or filters in the spray tank.

Note 7 The mass of sample to be used in the test should be specified at the application rate of use recommended by the supplier.

Note 8 Samples of the formulation taken before and after the storage stability test may be analyzed concurrently after the test in order to reduce the analytical error.

https://www.cipac.org/index.php/methods-publications/pre-published-methods


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PART TWO

EVALUATION REPORTS

FLUAZINAM Page

2019 FAO/WHO evaluation report based on submission of information from

Taizhou Bailly Chemical Co., Ltd. (TC) 9

Supporting information 11

Annex 1: Hazard summary provided by the proposer 13

Annex 2: References 15


Jiangsu Yangnong Chemical Ltd. (TC) 16





Nutrichem (TC) 23





ISK Biosciences (TC, SC) 30




Appendix 1: Analytical method for determination

of impurity 5 in TC and SC 52


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FLUAZINAM

FAO/WHO EVALUATION REPORT 521/2019

Recommendations

The Meeting recommended the following:

(i) The fluazinam TC proposed by Taizhou Bailly Chemical Co., Ltd. should be ac-cepted as equivalent to the fluazinam reference profile.

(ii) The existing TC specification should be extended to the technical material pro-duced by Taizhou Bailly Chemical Co., Ltd.

Appraisal

The Meeting considered data and supporting information submitted in September 2017 by

Taizhou Bailly Chemical Co., Ltd. (Taizhou Bailly) for the determination of the equivalence

for fluazinam TC (FAO Specification 521/TC).

The data submitted were in accordance with the requirements of the Manual on development

and use of FAO and WHO specifications for Pesticides (2016 3rd revision of the 1st Edition)

and supported the draft specifications. The reference specification and supporting data for

fluazinam were provided by ISK Biosciences Europe SA and the FAO specifications had been

provided by ISK Biosciences Europe SA.

Fluazinam is the ISO common name for 3-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)-

α,α,α-trifluoro-2,6-dinitro-p-toluidine (IUPAC). Fluazinam is not under patent.

Taizhou Bailly Chemical Co., Ltd. has not proposed any specification for formulations.

The Meeting was provided with commercially confidential information on the manufacturing

process and five batch analysis data on all impurities present at or above 1 g/kg, as well as

any relevant impurities below 1 g/kg, and their manufacturing limits in the TC. Mass balances

ranged from 99.19% to 99.60% in the 5-batch data. The minimum purity and maximum limits

for the impurities were supported by the 5-batch data and are statistically justified. The com-

pany declared the minimum purity of the fluazinam TC as 980 g/kg and is higher than the

existing FAO specification for fluazinam TC (not less than 960 g/kg).

The manufacturing process, impurity profile and five batch analyses were compared with the

data submitted for the reference profile. Taizhou Bailly utilizes a similar manufacturing pro-

cess as that of the reference product. The Meeting noted that the fluazinam TC manufactured

by Taizhou Bailly has a similar impurity profile to the reference specification and no new im-

purities were present.

A mutagenicity study (Ames test) for fluazinam has been conducted as part of the Tier-1 data

package. Fluazinam TC from Taizhou Bailly does not produce mutagenic effects in an in-vitro


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bacterial assay conducted according to guideline OECD 471. In addition, a mice micronu-

cleus test according to OECD guideline 474 has been conducted as part of Tier-2 and also

provided a negative result.

The analytical method for fluazinam in technical material (including identity tests) is CIPAC

Method 4727 (521/TC/M/3). The content of fluazinam is determined by reversed phase LC,

using UV detection at 240 nm and external standardisation.

Yet, the CIPAC method was not used for the determination of fluazinam in the batch study;

instead an adequately validated in-house HPLC-DAD method was used. Additional bridging

data that demonstrated the comparability of the Taizhou Bailly in-house method with the

CIPAC method were subsequently provided where the same batches were analysed for

fluazinam using both the in-house and CIPAC method. These data demonstrated that the

results from the proposer’s method were comparable to results obtained from analysis by the

CIPAC method.

A fully validated method for analysis using HPLC-DAD was also provided for the relevant

impurity "impurity 5". This method was not the same as the analytical method for the relevant

impurity published in Appendix 1 of the FAO specification for fluazinam. Additional bridging

data where the same batches were analysed using both the in-house and published peer-

validated method were subsequently provided. These data demonstrated that the results

from the proposer’s method were comparable to results obtained from analysis by the method

published in Appendix 1 of the FAO specification for fluazinam.

Other impurities were determined by in-house methods using HPLC-DAD. These methods

were considered properly validated.

Test methods for determination of physico-chemical properties of the technical active ingre-

dient were OECD test methods.

The Meeting was provided with data on melting point and solubility in water. The physical-

chemical properties were in good agreement with the reference material.

The Meeting concluded that fluazinam TC produced by Taizhou Bailly Chemical Co., Ltd. was

equivalent to the fluazinam reference TC based on a Tier-1 evaluation and recommended

extension of the existing fluazinam TC FAO specification to the TC produced by Taizhou

Bailly.

In addition, the Meeting recommended to editorially adjust the numbering of one of the re-

cent fluazinam evaluation reports: the 2018 report was expected to be the first of two, as in

2017 two data packages for fluazinam had been submitted (by Jiangsu Yangnong and by

Taizhou Bailly) and may have led to two 2018 reports. This would have been covered by an

additional number (e.g. 2018.1 Jiangsu Yongnong, published and 2018.2 for Taizhou

Bailly), but as the recommendation for Taizhou Bailly’s TC was in 2019 only, the initial num-

bering of 2018.1 for Yangnong could be adjusted to 2018.


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SUPPORTING INFORMATION

FOR

EVALUATION REPORT 521/2019


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Table 1. Chemical composition and properties of fluazinam technical material (TC)

Manufacturing process, maximum limits

for impurities 1 g/kg, 5 batch analysis data

Confidential information supplied and held on file by FAO. Mass balances were 99.2 - 99.6% and percentages of unknowns were 0.809-0.4%.

Declared minimum fluazinam content 980 g/kg

Relevant impurities 1 g/kg and maxi-mum limits for them

5-chloro-N-(3-chloro-5-trifluoromethyl -2-pyridyl)-α,α,α-trifluoro-4,6-dinitro-o-toluidine

2.0 g/kg max.

Relevant impurities < 1 g/kg and maxi-mum limits for them:

None

Stabilisers or other additives and maxi-mum limits for them:

None.

Parameter Value and conditions Purity % Method reference Study number

Melting temperature range of the TC and/or TK

116.0 °C

[no decomposition or gas evolution occurs]

98.4 OECD 102 B3034

Solubility in water pH 4: 111 μg/L.

pH 7: 253 μg/L

pH 9: 2.2 mg/L

All measured at 20°C.

98.4 OECD 105 B3034


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ANNEX 1

HAZARD SUMMARY PROVIDED BY THE PROPOSER

Note: Taizhou Bailly provided written confirmation that the toxicological and ecotoxicological data included in the following summary were derived from fluazinam having impurity profiles similar to those referred to in Table 1, above.


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Table 2: Mutagenicity profile of fluazinam technical material based on an in vitro test

Species Test Purity % Note2

Guideline, dura-tion, doses and conditions

Result Study number

Mice Micronucleus test

98.1% OECD 474; Dura-tion: 2 years; Dose: 2000 mg/kg;

no micronucleus induction poten-tial

485-1-06-7989

Salmonella typhimurium

TA98, TA100, TA102, TA1535 and TA1537

Bacterial re-verse muta-tion test

98.1% OECD 471

0.10, 0.26, 0.31, 0.63, 1.25, 1.6, 2.5, 4, 5 and 10 µg/plate (with and without S9 mix)

Non-mutagenic 485-1-06-7988

2 Note: Purity is the content of pure active ingredient in the technical material, expressed as a percentage.


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ANNEX 2

REFERENCES

Study number

Author(s) year Study title. Study identification number. Report identifica-tion number. GLP [if GLP]. Company conducting the study.

CPU-009-13

Vincenzo Mori

2013 Fluazinam technical material analytical profile of five batches. GLP. Biotecnologie BT, Italy.

NC-2018-050

Yue Wang 2019 Preliminary Analysis and Validation of Analytical Methods of Fluazinam 98% Tech. Report No. NC-2018-50. GLP. NutriChem Laboratory Co. Ltd., PR China

NCW-2018-054

Yue Wang 2019 Preliminary Analysis of Active Ingredient in Fluazinam TGAI. Report No. NCW-2018-054. GLP. NutriChem Laboratory Co. Ltd., PR China.

NCW-2018-055

Yue Wang 2019 Preliminary Analysis of Isofluazinam Impurity in Fluazinam TGAI. Report No. NCW-2018-055. GLP. NutriChem Laboratory Co. Ltd., PR China.

B3034 Jean-Baptiste C

2013 Physical and chemical properties of one batch of fluazinam TC. Report No. R B3034. GLP. Anadiag Laboratories, France.

485-1-06-7988

2014 Bacterial Reverse Mutation Test of fluazinam 98% min. tech using salmonella typhimurium. Report No. 485-1-06-7988. GLP.,

485-1-06-7989

2014 Micronucleus test of fluazinam 98% min. tech in mice Report No. 485-1-06-7989. GLP.


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FLUAZINAM


Recommendations

The Meeting recommended the following:

(i) The fluazinam TC proposed by Jiangsu Yangnong Chemical Co., Ltd. should be ac-cepted as equivalent to the fluazinam reference profile.

(ii) The existing TC specification should be extended to the technical material produced by Jiangsu Yangnong Chemical Co., Ltd.

Appraisal

The Meeting considered data and supporting information submitted in October 2017 by

Jiangsu Yangnong Chemical Co., Ltd (Jiangsu Yangnong) for the determination of the equiv-

alence for fluazinam TC (FAO specification 521/TC). Fluazinam TC is manufactured by a

subsidiary company of Jiangsu Yangnong, Youjia Crop Protection Co., Ltd, China (Youjia

Crop Protection).

The data submitted were in accordance with the requirements of the Manual on development

and use of FAO and WHO specifications for Pesticides (2016 3rd revision of the 1st Edition)

and supported the draft specifications. The reference specification and supporting data for

fluazinam had been provided by ISK Biosciences Europe SA.

Fluazinam is the ISO common name for 3-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)-

α,α,α-trifluoro-2,6-dinitro-p-toluidine (IUPAC). Fluazinam is not under patent.

Jiangsu Yangnong Chemical Co., Ltd has not proposed any formulation specifications for

fluazinam.

The Meeting was provided with commercially confidential information on the manufacturing

process and five batch analysis data on all impurities present at or above 1g/kg, and their

manufacturing limits in the TC. Mass balances ranged from 99.03% to 99.45% in the 5-batch

data. The maximum limits for the impurities were supported by the 5-batch data and are sta-

tistically justified. The proposer declared the minimum purity of the fluazinam TC as 970 g/kg

which is statistically justified (mean value-3 standard deviation ≈ 984 g/kg) - hence slightly

higher than the existing FAO TC specification (not less than 960 g/kg).


data submitted in the reference profile.

The Youjia Crop Protection manufacturing process utilises a similar synthetic route as that of

the reference product.


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The Meeting noted that Youjia Crop Protection's fluazinam TC contains an additional impurity

that is not present in the reference source. An assessment of publicly available toxicity infor-

mation and data for this impurity confirmed that it is not relevant however, and the Meeting

agreed that sufficient data has been provided to confirm equivalence

A mutagenicity study (Ames test) for fluazinam has been conducted as part of Tier-1 data.

Fluazinam TC from Youjia Crop Protection does not show mutagenicity in an in vitro bacterial

assay conducted according to OECD guideline 471.

The analytical method for the active ingredient (including identity tests) is CIPAC Method

4727 (521/TC/M/3). The content of fluazinam in the technical material is determined by re-

versed phase LC, using UV detection at 240 nm and external standardisation.

The CIPAC method was not used for the determination of fluazinam in the batch study; in-

stead a fully validated in-house HPLC-DAD method was used. Additional bridging data were

provided where the same batches were also analysed for fluazinam using the CIPAC method.

These data demonstrated that the results from the proposer’s method were comparable to

results obtained from analysis by the CIPAC method.

A fully validated method of analysis using reverse phase HPLC was also provided for the

relevant impurity "impurity 5". This method was identical to the analytical method for the rel-

evant impurity published in Appendix 1 of the FAO specification for fluazinam.

Other impurities were determined by in-house methods using GC-FID. These methods were

considered properly validated.

Test methods for determination of physico-chemical properties of the technical active ingre-

dient were OECD test methods.

The Meeting noted, that data on melting point, vapour pressure, octanol/water partition coef-

ficient, solubility in water and organic solvents of the purified fluazinam were submitted alt-

hough not required in an equivalence case, as these intrinsic properties of the pure active

ingredient are not expected to change. The physical-chemical properties were in reasonable

agreement with those of the reference material.

The Meeting therefore concluded that fluazinam TC produced Youjia Crop Protection could

be considered as equivalent to the fluazinam reference TC based on a Tier-1 evaluation and

recommended extension of the existing fluazinam TC FAO specification to the TC proposed

by Yangnong and produced by Youjia Crop Protection.


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FOR



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Table 1. Chemical composition and properties of fluazinam technical material (TC)

Manufacturing process, maximum limits for

impurities 1 g/kg, 5 batch analysis data

Confidential information supplied and held on file by FAO. Mass balances were 99.03 – 99.45 % and per-centages of unknowns were 0.55 – 0.97%.

Declared minimum Fluazinam content 970 g/kg

Relevant impurities 1 g/kg and maximum limits for them

"Impurity 5" (5-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)- α,α,α-trifluoro-4,6-dinitro-o-toluidine): Maxi-mum 2 g/kg

Relevant impurities < 1 g/kg and maximum limits for them:

None

Stabilisers or other additives and maximum limits for them:

None

Melting temperature range of the TC 115.9-117.4 °C


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ANNEX 1


Note: Jiangsu Yangnong provided written confirmation that the toxicological and ecotoxico-logical data included in the following summary were derived from fluazinam having impurity profiles similar to those referred to in Table 1, above.


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Table 2: Mutagenicity profile of fluazinam technical material based on an in vitro test

Species Test Purity % Note3

Guideline, duration, doses and conditions

Result Study num-ber

Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537

Ames Test

98.5

(Batch YJ201706041)

OECD Guideline 471

37±1 °C for 64 hours

0.1,0.4,1.0,4.0 and 12.5 µg/plate mixed in DMSO (with and without S9 mix)

Non-muta-genic

BIO-GT 819



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ANNEX 2

REFERENCES

Study number

Author(s) year Study title. Study identification number. Report identification num-ber. GLP [if GLP]. Company conducting the study.

NC-2012-035 Kong Ling’e 2016 Preliminary Analysis of Fluazinam TGAI. NC-2015-035 + confidential attachment GLP. Nutrichem Laboratory Co., Ltd. China. Unpublished.

YNGF-2017-002

Sun Chun-yan

2017 Determination of active ingredient in Fluazinam TC. YNGF-2017-002. GLP. Jiangsu Yangnong Chemical Co., Ltd.

NC-2015-036 Kong Ling’e 2016 Chemical and Physical Characterization of Fluazinam TGAI: melting Point, Partition Coefficient, Solubility and Vapour Pressure. NC-2015-036. GLP. Nutrichem laboratory Co., Ltd. China. Unpublished

BIO-GT 819

2017 Bacterial Reverse Mutation Test of Fluazinam Using Salmonella Typhi-murium Tester Strains. GLP, unpublished.


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FLUAZINAM


Recommendations

The Meeting recommended that

(i) that the fluazinam TC proposed by Nutrichem Co., Ltd. should be accepted as equivalent to the fluazinam reference profile. (ii) to extend the existing TC specification to the technical material produced by Nutrichem Co., Ltd. Appraisal

The Meeting considered data and supporting information submitted in April 2016 by

Nutrichem Co., Ltd. (Nutrichem) for the determination of the equivalence for fluazinam TC

(FAO specification 521/TC). The data submitted were in accordance with the requirements

of the Manual on Development and Use of FAO and WHO specifications for Pesticides (2016,

3rd revision of the First Edition) and supported the draft specifications. The reference specifi-

cation and supporting data for fluazinam had been provided by ISK Biosciences Europe SA

and the FAO specifications had been published in 2014. Fluazinam has not been evaluated

by the WHO IPCS or by the FAO/WHO JMPR. It was evaluated by US EPA in 2001, and in

the European Union with inclusion into the positive list in 2009.

Nutrichem did not propose specifications for fluazinam formulated products. The Meeting was

provided with commercially confidential information on the manufacturing process and five

batch analysis data on all impurities present at or above 1g/kg, as well as any relevant

impurities below 1g/kg, and their manufacturing limits in the TC. Mass balances ranged from

98.99% to 99.25% in the 5-batch data. The maximum limits for the impurities were supported

by the 5-batch data and they are statistically justified. The proposer declared the minimum

purity of the fluazinam TC as 970 g/kg which is statistically justified and it is somewhat higher

than the existing FAO specification (960 g/kg).


data submitted in the reference profile. Fluazinam TC manufactured by Nutrichem was found

to differ slightly from the impurity profile of the reference: whereas impurity 5 was not detected

above the limit of detection (see Appendix 1), it contains one additional impurity (a positional

isomer of an impurity present in the reference specification). Therefore, a decision on equiv-

alence based on Tier-1 data was not possible and additional toxicological studies were re-

quired.

A mutagenicity study (Ames test) for fluazinam has been conducted as Tier-1 data. Fluazinam

TC does not show mutagenicity in in vitro bacterial assays (OECD 471).


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On request by JMPS, Nutrichem provided a 28-days repeated dose GLP study according to

OECD 407 in rats. The results of the study allow the conclusion that that the Nutrichem fluazi-

nam TC has a no-observed-adverse-effect level (NOAEL) of 5 mg/kg. This value is very sim-

ilar to the NOAEL found in the corresponding study with the reference TC. The Meeting there-

fore concluded that the Nutrichem fluazinam TC is not more hazardous than the reference

TC produced by ISK.

The analytical method for the active ingredient (including identity tests) is CIPAC Method

4727 (521/TC/M/3) now published in Handbook O. The fluazinam content is determined by

reversed phase LC, using UV detection at 240 nm and external standardisation.

A fully validated method for analysis using reverse phase HPLC was also provided for the

relevant impurity.

Test methods for determination of physico-chemical properties of the technical active

ingredient were OECD test methods.

The Meeting concluded that fluazinam TC produced by Nutrichem was equivalent to the

fluazinam reference TC based on Tier-2 evaluation and recommended extension of the ex-

isting TC FAO specification to the technical material produced by Nutrichem Co. Ltd.


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Table 1. Chemical composition and properties of fluazinam technical materials (TC)



Confidential information supplied and held on file by FAO. Mass balances were 98.99 – 99.25 % and per-centages of unknowns were 0.75 – 1.01 %.



None.


none


None.

Parameter Value and conditions Purity %

Method reference Study number

Melting temperature range of the TC

117.5 – 118.2 °C 98.2 EPA Guideline 830.7200

NC-2012-121

Solubility in organic sol-vents

156.07 g/l in methanol at 20 ± 0.5°C

15.18g/l in n-Octanol at 20 ± 0.5°C

6.91g/l in n-hexane at 20 ± 0.5°C

98.2 EPA Guideline 830. 7840

NC-2012-121

FORMULATIONS AND CO-FORMULATED ACTIVE INGREDIENTS

No specifications for formulated products have been proposed by Nutrichem.

METHODS OF ANALYSIS AND TESTING

The analytical method for the active ingredient (including identity tests) is CIPAC Method 4727 (521/TC/M/3). The fluazinam content is determined by reversed phase LC, using UV detection at 240 nm and external standardisation, whereas the method for determination of impurities is a adequately validated reversed-phase HPLC method with UV-DAD detection.

CONTAINERS AND PACKAGING

No special requirements for containers and packaging have been identified.

EXPRESSION OF THE CONTENT OF ACTIVE INGREDIENT

The active ingredient is expressed as fluazinam.


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ANNEX 1


Note: Nutrichem provided written confirmation that the toxicological and ecotoxicological data included in the following summary were derived from fluazinam having impurity profiles similar to those referred to in Table 1, above.


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Table 2. Toxicology profile of the technical material based on repeated administration

Species Test Duration and conditions or guideline adopted

Result Study ref.

Rat, Wistar, females and males

Repeated oral toxicity study

OECD 407, GLP Dose-ranging study 1, 10, 100, 1000 mg/kg bw for 7 days; Main study, 0, 5, 10, 15 mg/kg bw per d for 28 d

NOAEL > 15 mg/kg bw per d.

Polyuria at 100 mg/kg bw and di-arrhoea al de-creased body weight gain at 1000 mg/kg bw

Solanki 2017, confidential re-port

Table 3. Mutagenicity profile of the technical material based on in vitro tests

Species Test Pu-rity % Note4

Guideline, duration, doses and conditions

Result Study number

Salmo-nella typhi-murium TA98, TA100, TA102, TA1535 and TA1537

Ames Test –in vitro

98.1 OECD Guideline 471

65 h 55 min

39.06, 78.13, 156.25, 312.50, 625.00, 1250.00, 2500.00 and 5000.00 μg/plate

Negative

GL00491

The mutagenicity study (Ames test) for fluazinam has been conducted as Tier-1 data. Fluazinam does not show mutagenicity in in vitro bacterial assays (OECD 471).



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ANNEX 2

REFERENCES

(sorted by study number)

Study number

Author(s) year Study title. Study identification number. Report identification number. GLP [if GLP]. Company conducting the study.

FAO/ WHO

2006 Manual on development and use of FAO and WHO specifications for pesticides. February 2006 Revision of First Edition. FAO Plant Produc-tion and Protection Paper. Revised. www.fao.org/ag/AGP/AGPP/Pesticid/Default.htm

FAO 2014 FAO SPECIFICATIONS AND EVALUATIONS FOR AGRICULTURAL PESTICIDES 521

16_150-001

2017 28-day repeated dose study of fluazinam with 14-days recovery period, , Study Nr. 16_150-001, GLP.

NC-2012-137

2012 Vapor pressure of Fluazinam TGAI. Study NC-2012-137. Report NC-2012-137. GLP.

NC-2012-121

2012 Chemical and Physical Characterization of Fluazinam TGAI: Melting Point, Partition Coefficient and Solubility. Study NC-2012-121. Report NC-2012-121. GLP.

GL00491 2016 Bacterial Reverse Mutation Test of Fluazinam TGAI Using Salmonella typhimurium Tester Strain (Ames Test) Report GL00491. GLP.

http://www.fao.org/ag/AGP/AGPP/Pesticid/Default.htm


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FLUAZINAM


Recommendations

The Meeting recommended that

(i) the new specification for fluazinam TC and SC formulations, proposed by ISK Biosciences Europe SA (ISK), and as amended, should be adopted by FAO.

Appraisal

The data for fluazinam were evaluated in support of new FAO specifications.

Fluazinam is not under patent and has not been evaluated previously by the FAO/WHO JMPR and WHO/IPCS. It was evaluated by US EPA in 2001 and is currently under review by the European Commission.

The draft specification and the supporting data were provided by ISK Biosciences Europe SA in 2006, with further clarifications and data provided in 2007. The meeting considered the data on fluazinam, submitted by ISK Biosciences Europe SA (ISK), in support of proposed new FAO specifications for fluazinam TC and SC.

Fluazinam is a yellow solid only slightly soluble in water. It undergoes hydrolysis which is not significantly affected by pH. It is susceptible to fairly rapid photolysis. It has no acid or basic properties.

The meeting were provided with commercially confidential information on the manufacturing process and manufacturing specification for purity and impurities, supported by 5 batch anal-ysis data. Mass balances were >990 g/kg and no unidentified impurities greater than 1 g/Kg were reported. A statement was provided by the UK Pesticides Safety Directorate confirming that the confidential data on the manufacturing process and declaration of composition sub-mitted to the FAO were the same as those submitted to the national regulatory authority. The data provided supported a minimum fluazinam content of 960 g/kg.

The Meeting considered the relevance and limit for the impurity-5 [5-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)-α,α,α-trifluoro-4,6-dinitro-o-toluidine], CAS 169327-87-1) in fluazi-nam

The company had proposed that the impurity-5 be considered relevant and that a limit for it be established at 3 g/kg. Toxicological studies were submitted documenting that impurity-5 causes reversible white matter vacuolation of unknown physiological significance in different parts of the central nervous system in rats, mice, and dogs, and is likely to be the cause of similar findings in animals treated with fluazinam TC.

Based on the information provided by the proposer, the Canadian and European evaluations of the registration by ISK of fluazinam, the Meeting noted that the long-term oral NOAEL of impurity 5 (with reference to white matter vacuolation) is 0.02 mg/kg, the overall NOAEL of fluazinam, 0.38 mg/kg, and the minimum content of the active ingredient is 960 g/kg.


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Based on the rules provided in the Manual (December 2010 - second revision of the First Edition, Appendix J) it can be calculated that the RelHaz is 19.0, and the MTIHaz, 1.79. The latter represents an increase of the toxicity of fluazinam at the maximal theoretical impurity 5 content in fluazinam by 79%. This being in excess of 10%, the impurity is relevant. The max-imum limit for the impurity, Impmax, is 0.50% = 5 g/kg (JMPS, 2010). The limit for the concen-tration of impurity 5 in fluazinam, suggested by the proposer, is 3 g/kg, and thus acceptable to JMPS.

It should be noted that the approach used here is normally limited by the Manual to cases, where the toxicity of the impurity is qualitatively similar to that of the active ingredient, what is not the case here. The justification for nevertheless using this approach is that rather than any specific toxicological end-point, the no effect level from long–term study (life-time studies in rats and mice, and 1-year studies in dogs) is used for both the impurity and active ingredi-ent.

The Meeting is aware that the Canadian authority has established a limit of 1 g/kg, and the European Union, 2 g/kg, for impurity-5 in fluazinam. However, none of the authorities have in their publicly available documents provided the criteria for their established limit concentra-tions.

Methods of analysis

The collaborative validation of a reversed-phase HPLC method for determination of fluazinam and impurity-5 in TC and SC formulations was first presented at the CIPAC Meeting in El Salvador in 2009. Whereas the results presented fulfilled the requirements for a peer valida-tion for the determination of impurity-5 in TC and SC formulation, the collaborative validation for fluazinam itself did not pass the hurdle for a minimum number of participating laboratories providing valid results (8 laboratories) and was again presented with a higher number of la-boratories in 2010 in Slovenia. The method was then adopted as provisional CIPAC method and became a full method in 2011. The methods for impurities are based on reversed-phase HPLC and are adequately validated.

Specification for SC

The test methods proposed for physical properties of the SC were in accordance with the requirements of the Manual (FAO/WHO, 2006) with the following exception:

i) The suspensibility and spontaneity of dispersion properties before and after stor-

age (2 weeks at 54C) were determined used CIPAC standard water C instead of standard water D as the tests were conducted before the use of CIPAC standard water D was required.

The stability study conducted at 0°C used CIPAC standard water D. The proposer has there-fore proposed limits for the clauses based on the data generated using CIPAC Standard water D.


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Uses

Fluazinam is a contact fungicide with a multi-site mode of action. It uncouples oxidative phosphorylation at several metabolic pathways within the fungal cell. Fluazinam is a protect-ant fungicide and when applied to plants, remains primarily on the plant surface. It is used in agriculture on potatoes, vines, apples and onions in Europe against downy mildews such as Phytophtora Infestans.

Identity of the active ingredient

ISO common name Fluazinam (ISO accepted)

Chemical name(s)

IUPAC 3-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)- trifluoro-2, 6-dinitro-p-to-luidine CA 3-chloro-N-[3-chloro-2, 6-dinitro-4-trifluoromethyl) phenyl]-5-(trifluoromethyl)-2-pyridinamine

Synonyms

IKF-1216, B-1216, PP192

Structural formula

Molecular formula

C13H4Cl2F6N4O4

Relative molecular mass 465.1

CAS Registry number 79622-59-6

CIPAC number 521

Identity tests HPLC: retention in reversed-phase HPLC, UV- and IR-spectra

NH

N

Cl

CF3

O2N

CF3

ClO2N


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Table 1. Physico-chemical properties of pure fluazinam

Parameter Value(s) and conditions Purity % Method reference

Vapour pressure (7.5 ± 0.8) x 10-3 Pa at 20 C 99.8 % OECD 104,

Melting point, boil-ing point and/or temperature of de-composition

Melting point: 117 °C

Boiling point: not required as material is solid and does not have a low melt-ing point

Decomposition temperature: not re-quired because the material is solid and does not have a low melting point

99.8 % EEC Base Set Meth-ods (Annex V or dir. 79/831) versions Nov. 1989, Part A.1.

Solubility in water pH 5 – 1.06x10-4 g/L

pH 7 – 1.35x10-4 g/L

pH 9 – 2.72x10-3 g/L

99.8 % OECD 105,

Octanol/water parti-tion coefficient

log POW = 4.03 at 25 C 96.8% 40 CFR 158.190 Pesticide Assess-ment Guidelines Subdivision D: Prod-uct Chemistry Guideline 63-11

Hydrolysis charac-teristics

Half-life = 5 days at 50 °C at pH 4 for fluazinam; 4.5 and 2.7 days at pH 7 for the phenyl and pyridyl labels, re-spectively, and 3.5 and 3.9 days at pH 9 for the phenyl and pyridyl labels, respectively, both at 25 ºC.

[14C-phenyl] fluazi-nam (2.33 GBq mmol-1, 100% ra-diopurity) and [14C-pyridyl] fluazinam (2.37 GBq mmol-1, 97.7% radiopurity)

OECD 111

EPA OPPTS 835.2110,

Photolysis charac-teristics

The half-life in sterile pH 5 buffer was 2.5 days for both labels. The major photolytic product was CO2 after 30 days).

[14C-phenyl] IKF-1216 (57.3 mCi/ mmol, >99%), [14C-pyridyl] IKF-1216 (66.2 mCi/ mmol, >99%), IKF-1216, Lot No. Y911022, 99.6%

United States EPA Guideline 161-2 EC

Dissociation char-acteristics

pKa = 7.34

99.9% 40 CFR 158.190 Pesticide Assess-ment Guidelines, Subdivision D: Prod-uct Chemistry Guideline 63-10


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Table 2. Chemical composition and properties of fluazinam technical materials (TC)



Confidential information supplied and held on file by FAO. Mass balances were 99.19 – 100.92 % and no unknowns were identified.



Impurity-5: 5-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)-α,α,α-trifluoro-4,6-dinitro-o-toluidine Maximum 3 g/kg


None


None

Melting or boiling temperature range of the TC and/or TK

Data available on purified product (see Table 1).

FORMULATIONS AND CO-FORMULATED ACTIVE INGREDIENTS

The main formulation types available are suspension concentrates (SC). These formulations are registered and sold in many countries throughout the world in Europe, USA, Latin Amer-ican Countries, Australia and some Asian countries.

METHODS OF ANALYSIS AND TESTING

The fluazinam content is determined by reversed phase HPLC, using UV detection at 240 nm and external standardisation. The identity of the active ingredient is confirmed by com-parison of retention times in HPLC and IR spectra.

The analytical method for the active ingredient in TC and SC became a full CIPAC method (ISBN 902951661), adopted at the 2011 CIPAC meeting in Beijing. The method is not yet published in a Handbook, but is available as a pre-published method.

Test methods for determination of physical-chemical properties of the technical active ingre-dient were OECD, EPA or EC, respectively.

CONTAINERS AND PACKAGING

No special requirements for containers and packaging have been identified.

EXPRESSION OF THE CONTENT OF ACTIVE INGREDIENT

The active ingredient is expressed as fluazinam.

Fluazinam has not been evaluated by the WHO IPCS or by the FAO/WHO JMPR.

The WHO hazard classification of fluazinam is: There is no hazard classification for fluazinam


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ANNEX 1


Note: ISK provided written confirmation that the toxicological and ecotoxicological data in-cluded in the following summary were derived from fluazinam having impurity profiles simi-lar to those referred to in Table 1, above.


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Table 3. Toxicology profile of fluazinam technical material, based on acute toxicity, irritation and sensitization.

Species Test Duration and conditions or guideline adopted Result

Rat Oral Batch no. 109; purity 95.3 %

Single doses of 2500, 3200, 4000 and 5000 mg/kg bw fluazinam. 14-day ob-servation period.

Japanese MAFF acute oral toxicity study, in accordance with 59 Nohsan no. 4200 (1985), GLP.

LD50: 4500 mg/kg bw (males)

LD50: 4100 mg/kg bw (females)

Rat Dermal Batch no. 8303-2; purity 98.5 %

Single dose of 2000 mg/kg bw fluazinam. 14-day observation period

Japanese MAFF acute oral toxicity study, in accordance with 59 Nohsan no. 4200 (1985), GLP.

LD50 > 2000 mg/kg bw

Rat Inhalation Batch no. A629/1995; purity 97.3 %

Concentrations of 1.0 mg/l, 1.1 mg/L and 4.0 mg/L. 4-hour nose-only expo-sure period.

OECD Guideline 403 and U.S. EPA OPPTS Guideline 870.1300, GLP.

LC50 > 1.1 mg/L

Rabbit Skin irrita-tion

Batch no. 1006; purity 97.9 %

4-hour exposure period, 13 day observation period.

U.S. EPA Pesticide Assessment Guidelines Subdivision F, Series 81-5, GLP.

non-irritant

Rabbit (males and females)

Eye irritation Batch no. 1006; purity 97.9 %

21-day observation period

US EPA Pesticide Assessment Guidelines Subdivision F, 81-4

irritant

Guinea Pig (males and females)

Skin sensiti-sation

Batch no. 8303-2; purity 98.5 %

Dermal responses to primary induction were assessed 24 and 48 hours after administration.

Topical induction (for 48 hours under occlusive dressing at the injection test sites) was carried out on day 8.

On day 22 the challenge phase was performed in the treated group and in the control group by applying 0.2 ml 70 % (w/v) solution of fluazinam in paraffin oil dermally under occlusive dressing for 24 hours on the right flank while the left flank received the vehicle only.

U.S. EPA Pesticide Assessment Guidelines Subdivision F, No. 81-6 (Magnusson and Kligman)

sensitizer

According to the GHS classification system, fluazinam is unclassified.

According to the GHS system, fluazinam would be unclassified. However this classification should be regarded with caution. On the basis of the original interpretation of the results, fluazinam was classified as a sensitizer. Cases of allergic contact dermatitis have been re-ported from workers from manufacturing site and users. Therefore fluazinam should be classified as sensitizing.


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Table 4. Toxicology profile of the technical material based on repeated administration (subacute to chronic)


Result

Mouse subchronic Batch 8203, purity 96.3 %; impurity 5: 0.008%)

Repeated oral dosing, 4-Weeks.

Dose levels: 0, 10, 50, 250 and 3000 ppm fluazinam, equivalent to 0, 1.6, 7.9, 39.5 and 455 mg/kg bw/d.

No specific test guideline, GLP.

Body weight gains of males receiving 3000 ppm and 250 ppm were significantly during the first 10 days of treatment.

Statistically significant higher liver weights in males and females receiving 3000 ppm. Absolute and relative kidney weight of females of the 250 ppm and 3000 ppm dosing groups.

Clinical chemistry findings for both sexes at dose levels of 3000 ppm.

NOAEL: 50 ppm or 7.9 mg/kg bw/d.

Rat subchronic Batch 8203, purity 96.3 %; impurity 5: 0.008%)


Dose levels: 0, 10, 50, 250 and 3000 ppm fluazinam, equivalent to 0, 1.26, 5.21, 26.1 and 305.4 mg/kg bw/d.


Lower body weight and reduced food intake at 3000 and 250 ppm.

Higher relative liver weights in males and fe-males receiving 3000 ppm.

Histopathological changes in the liver at 3000 and 250 ppm.

NOAEL: 50 ppm or 5.21 mg/kg bw/d for males and females.

Dog subchronic Batch 8303-2, purity 98.5 %; impurity 5: <0.005%)


Dose levels: 1, 5, 25 and 150 mg/kg/d bw/d.


Reduced food intake and bodyweight in animals receiving 150 mg/kg bw/d.

Increased liver weights in animals receiving 25 and 150 mg/kg bw/d).

NOAEL: 5 mg/kg bw/d

Rat Short-term Batch 8303-2, purity 98.5 %; impurity 5: <0.005%)

Repeated oral dosing, 90-Days.

Dose levels: 0, 2, 10, 50 and 500 ppm fluazinam, equivalent to 0, 0.16, 0.82, 4.1 and 41 mg/kg bw/d

U.S. EPA Guideline 82-1, GLP.

Increased Liver weights in males at a con-centration of 500 and 50 ppm.

Microscopic liver findings at 500 ppm.

NOAEL: 50 ppm or 4.1 mg/kg bw/d for males and females.


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Result

Dog Short-term Batch 8303-2, purity 98.5 %; impurity 5: <0.005%)

Repeated oral dosing, 90-Days.

Dose levels:1, 10 and 100 mg/kg/d bw/d.


Changes in absolute and relative liver weight and histopathological changes in this organ.

NOAEL: 10 mg/kg bw/d.

Rat Dermal Batch no. 8303-2; purity 98.5 %; impurity 5: <0.005%).

Repeated dermal dosing, 21-days.

Dose levels: 10, 100 and 1000 mg/kg bw/d.


Microscopic liver findings at all dose groups.

A mild toxic effect was also observed in the liver at 1000 mg/kg bw.

Effects to the skin (acanthosis and dermati-tis) observed at all dose groups compared to controls.

NOAEL: <10 mg/kg/day

Dog Long-term Batch 8412-20, purity 95.3 %; impurity 5: 0.20%)

Repeated oral dosing, 1-year.

Dose levels: 1, 10 and 50 mg/kg bw/d


Increase of liver weight and accompanying blood chemistry changes at 50 mg/kg bw/d.

An increased incidence of liquefied contents of the gastro-intestinal tract and mucosal lymphoid hyperplasia of the stomach at 50 mg/kg bw/d and partly in animals at 10 mg/kg bw/d.

Vacuolation of white matter in the brain at 50 mg/kg bw/d. Females of the high dose group showed also vacuolation of white matter in the spinal cord.

NOAEL: 1 mg/kg/day

Rat Long-term/

Carcinogen-icity

Batch 8412-20, purity 95.3 %; impurity 5: 0.20%)

Dose levels: 0, 1, 10, 100 and 1000 ppm equivalent to 0, 0.04, 0.38, 3.82 and 40 mg/kg bw/d for males and 0, 0.05, 0.47, 4.87 and 53 mg/kg bw/d for females.

Repeated oral dosing, 104 weeks

No tumorigenic effects. Non-neoplastic ef-fects in several organs at 100 and 1000 ppm dose levels. NOAEL 0.8 mg/kg per d for males and 0.47 mg/kg per d in females.

No increased vacuolation in the white matter in the brain.


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Result

Rat Long-term/

Carcinogen-icity


Dose levels: 0, 25, 50 and 100 ppm equivalent to 0, 1.0, 1.9 and 3.9 mg/kg bw/d for males and 0, 1.2, 2.4 and 4.9 mg/kg bw/d for females.

Repeated oral dosing, 104 weeks

Japanese MAFF Test Guidelines (1985), U.S. EPA Guidelines and OECD Guideline No. 452, GLP

Slightly increased liver, testes and epididym-ides weights and macroscopically in a higher incidence of small/flaccid testes observed at 100 ppm.

Increased incidence of marked testicular at-rophy in males of the 100 ppm group.

No evidence of any tumorigenic potential in male or female rats.

NOAEL: 50 ppm, equivalent to 1.9 mg/kg bw/d for males and 2.4 mg/kg bw/d for fe-males.

No vacuolation in the white matter of the brain

Mouse Long-term/

Carcinogen-icity

Batch 1030/91; purity 97 %; impurity 5 0.12%.)

Dose levels: 0, 1000, 3000 and 7000 ppm, equivalent to 0, 126, 377, and 964 mg/kg bw/d for males and 0, 162, 453, and 1185 mg/kg bw/d for females.

Repeated oral dosing, 104 weeks.


Incidence of hepatocellular adenomas was elevated in the 3000 ppm group, but not in the 7000 ppm group in the males.

Vacuolation in the brain and cervical spinal cord observed in all treated animal groups in males and females

Mouse Long-term/

Carcinogen-icity

Batch 8412-20; purity 95.3 %; impurity 5: 0.20%)

Dose levels: 0, 1, 10, 100 and 1000 ppm, equivalent to 0, 0.12, 1.12, 10.72 and 107 mg/kg bw/d for males and 0, 0.11, 1.16, 11.72 and 117 mg/kg bw/d for females.

Repeated oral dosing, 104 weeks.


At 1000 ppm, increased liver weight, in-creased macroscopic liver lesions, micro-scopically, higher frequency of basophilic or eosinophilic hepatocytes and higher inci-dence of granulomatous hepatitis and ag-gregates of brown pigmented macrophages in high and mid-dose males and high dosed females.

Increase of vacuolation of white matter in the brain in both sexes of the high dose groups. Increase in liver cell tumours was within historical control data.

NOAEL: 10 ppm, equivalent to 1.12 mg/kg bw/d. Liver cell tumours were observed in the high dose group males, but the inci-dence remained within the historical control range.


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Result

Rat Multigenera-tion /

Reproduc-tion


Dose levels: 0, 20, 100 or 500 ppm


At 500 ppm: reduced body weight and body weight gain of F0 and F1 parental females during maturation and gestation and of F1 and F2 offspring during lactation. Reduced food intake was recorded for F0 females and F1 males and females during maturation. In the F1 generation, conception rate and fertil-ity index were slightly reduced in the 500 ppm group. Gestation length was slightly in-creased in the high and intermediate dose groups. Numbers of implantation sites and mean litter sizes to day 4 post partum were slightly reduced for F1 animals of the high dose group and marginally lower in the inter-mediate group (100 ppm). Relative liver weights were significantly increased in both sexes of the highest dose group and also in females of the intermediate and low dose group of the F0 generation but there was no clear dose response observed. High dose males of the F1 generation showed also an increase of relative liver weight. Histopathologically, a statistically significant increase of periacinar hepatocytic fatty changes were detected in high dose males of F0 and F1 animals and also in F1 males of the 100 ppm group.

NOAEL: for both systemic toxicity and repro-ductive parameters was considered to be 20 ppm, equivalent to approximately 1.5 mg/kg bw/d for males and 1.7 mg/kg bw/d for fe-males.

Rabbit Develop-mental

Batch Lot 8303-2, purity 98.5 %

Dose levels: 0.3, 1 and 3 mg/kg bw /d

Repeated oral dosing from day 6 to 19 of gestation


NOAEL: 3 mg/kg bw/d for maternal toxicity

3 mg/kg bw/d for fetal toxicity.


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Result

Rabbit Develop-mental


Dose levels: 2, 4, 7 and 12 mg/kg bw fluazinam



Slightly reduced maternal body weight gain from day 10 to day 20 of gestation. Reduced food intake in the 7and 12 mg/kg bw/d from day 13 to day 19.

Macroscopic and microscopic lung and liver changes were observed at a dose level of 12 mg/kg bw/d.

Slightly increased incidences of fetal abnor-malities (placental abnormalities, some skel-etal abnormalities including kinked tail tip, fused or incompletely ossified sternebrae and abnormalities of the head bones) were seen at the top dose.

NOAEL: 4 mg/kg/day for maternal toxicity

7 mg/kg bw/day for fetal toxicity

Rat Develop-mental


Dose levels: 10, 50 and 250 mg/kg bw fluazinam


U.S. E.P.A. Guideline No. 83-3

Reduced mean food consumption followed by a reduced rate of weight gain compared to controls at the dose rate of 250 mg/kg bw.

Reduced fetal and placental weights in the 250 and 50 mg/kg bw/d group and indica-tions of fetal immaturity.

Increased incidence of gross morphological fetal abnormalities at the 250 mg/kg bw/d group.

NOAEL for developmental effects: 10 mg/kg bw/d.


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Table 5. Mutagenicity profile of the technical material based on in vitro and in vivo tests

Species Test Conditions Result

Bacteria (E. coli & S. typhi-murium)

Reverse gene muta-tion, in vitro

Batch no. 8412-20, purity 95.3%

Testing Guidelines for Toxicological Studies, 59 NohSan No. 4200, GLP.

Negative

Bacteria (E. coli & S. typhi-murium)

Reverse gene muta-tion, in vitro

Batch no.109, purity 95.3%

Japanese MAFF, NohSan No. 4200 (1985), GLP.

Negative

Mammalian cells (mouse lymphoma)

Gene mutation, in vitro Batch A629/1995, Purity 98.4%

OECD Guideline 476 (1997); Com-mission Directive 2000/32/EC (2000) Annex 4E – B17 (L136, 65); U.S. EPA (1998) Health Effects Guidelines, OPPTS 870.5300, EPA 712-C-98-221.GLP

Negative

Mammalian cells (hamster lymphocyte)

Chromosomal aberra-tion, in vitro

Lot no. 109, Purity 95.3% Testing Guidelines for Toxicological Studies, 59 NohSan No. 4200, GLP.

Negative

Bacteria (B. subtilis) in vi-tro

DNA repair test (Rec-assay), in vitro

Lot no. 109, Purity 95.3% Testing Guidelines for Toxicological Studies, 59 NohSan No. 4200, GLP.

Negative

Mouse Micronucleus, in vivo Batch No. 8412-20, purity of 95.6%

Single oral dose at levels of 500, 1000 and 2000 mg/kg bw OECD Guideline 474 (1997), U.S. EPA Health Effects Guidelines (1991) and Japanese MAFF, 59 NohSan No. 4200 (1985), GLP.

Negative

Fluazinam was tested for genotoxicity in a range of assays, both in vitro and in vivo. There was no evidence of genotoxicity in any of these assays.

Table 6. Supplementary studies (mostly non-guideline) submitted in support of the rele-vance of impurity-5 and effect of vacuolation related to administration of fluazinam technical and impurity 5

1987/1998 B-1216: 52-Week Toxicity Study in Oral Administration to Beagle Dogs. Report No. 86/ISK055/512, Final report; 86/ISK055/512

1994/1998 Technical Fluazinam, Toxicity to Mice by Dietary Administration for Four Weeks. Report No. ISK 49/921049

1988/1998 B-1216 Technical: Potential Carcinogenicity and Chronic Toxicity Study in Dietary Administration to Rats for 104 Weeks. Report No. ISK8/87263

1988/1996 B-1216: Potential Carcinogenicity Study in Dietary Administration to Mice for 104 Weeks. Report No. ISK9/87264

1998 Comparative Study on Susceptibility to Neurotoxicity in Mice, Rats and Dogs. Report No. 98-0020


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1998 Fluazinam Technical; Toxicological Effect on the Brain of Rats and Its Reversibility by Dietary Administration for 14 Days Followed by a 25-Day Recovery Period. Report No. AN-1323

1998 Fluazinam Technical: Toxicological Effect on the Brain of Mice and Its Reversibility by Dietary Administration for Four or Twenty-Eight Days Followed by a 56-Day Re-covery Period. Ltd., Report No. ISK AN-1333

1998 Impurity-5; An Impurity in Fluazinam Technical; Toxicological Effect on Brain and Optic Nerves of Mice Following a Single Oral Administration at Various Stages of Animal Age. Ltd, Report No. AN-1480

1998 Impurity-5; An Impurity in Fluazinam Technical; Sensitivity Comparison on Brain of Mice and Rats Following 14-Day Oral Administration. Ltd., Report No. AN-1481

1998 Impurity-5; An Impurity in Fluazinam Technical; Sensitivity Comparison on the Brain of Rats and Mice in 3- and 10-Week Olds Following 14-Day Oral Administration. Report No. AN-1492

1995 An Acute Neurotoxicity Screening Study in Rats with Technical Fluazinam. Report No. 5603-93-0075-TX-003

Table 7. Ecotoxicology profile of the technical material

Species Test Duration and conditions Result

Oncorhynchus mykiss

Rainbow trout

Acute Toxicity Batch no. 1030/91, purity 96.8 % w/w

Mean measured concentra-tions were 10, 15, 28, 33 and 56 µg/L.

Flow through, 96 hr

U.S. EPA FIFRA Guideline 72-1, GLP.

LC50 = 36 µg ai/L

Daphnia magna

Waterflea

Acute Toxicity Batch no. 1030/91, purity 100% Mean measured concentra-tions were 34, 54, 94, 150 and 260 µg/L.

Flow through, 48 hr

OECD 202, GLP.

EC50 = 220 µg ai/L

Chironomus riparius

Midge

Acute Toxicity Batch no. A629/1995, pu-rity: 97.8 %.

Mean measured concentra-tions were 0, 12, 33, 68, 120, 238 µg/L.

Static, 48 hr

No guideline followed, GLP.

LC50 = 45 µg ai/L

Lemna gibba

Duckweed

7-Day Toxicity Batch no. A626/1995, pu-rity: 98.4 %.

Mean measured concentra-tions were 0.859, 1.73, 4.58, 7.96, 17.5, 35.9 and 69.1 µg/L.

LC50 > 69.1 µg ai/L


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Static renewal, 7-day

ASTM 1991, EPA OPPTS 850.4400, GLP.

Apis mellifera

honey bee

acute oral and contact toxicity

Batch no. 8303-4, purity 98.8 %.

In the contact tests, the concentration range was 0, 20, 50, 100 and 200 µg ai/bee for the technical sub-stance and 0, 1, 2 and 4 µg ai/bee for the formulation.

In the oral tests, the con-centration range for both the technical and formu-lated material was 0, 10, 20, 50 and 100 µg ai/bee.

60 bees per tested dose, 48 hr observation

UK MAFF Pesticides Safety Precautions Scheme, Doc-ument D3 (1979)

Acute LD50: >100 µg a.i./bee – oral Acute LD50: >200 µg a.i./bee - contact

Colinus virginianus

Bobwhite quail

acute toxicity Batch No. 8412-20, purity 95.3 %

Oral intubation at dose levels of 0, 500, 1000 or 2000 mg/kg bw

14-day observation

U.S. EPA FIFRA, No. 71-1, GLP

One mortality occurred at 1000 mg/kg and six at 2000 mg/kg. Birds dosed at 2000 mg/kg exhibited toxic signs including subdued behaviour and ruffled feathers prior to death. Over days 0 to 7, bodyweight gain and female food consumption were slightly reduced at 2000 mg/kg. At post-mortem examination, the intestines of all birds that died during the study were observed to be red in colour.

LD50 = 1782 mg [TGAI]/kg bw

Anas platyrhynchos

Mallard duck

acute toxicity Batch No. 8402-6, purity 95.3 %

Oral intubation at dose levels of 0, 484, 944, 2020, 3050 and 4190 mg/kg bw

14-day observation

U.S. EPA FIFRA, No. 71-1, GLP

No mortalities occurred at any dose level in the study. Female birds in the high-dose group (4190 mg/kg) were subdued on day 2. No other abnormal-ities were noted.

LD50 > 4190 mg [TGAI]/kg bw


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ANNEX 2

REFERENCES

Parameter Author(s) Year

Title Source (where different from company) Company, Report No GLP or GEP status (where relevant), Published or not

Owner

Vapour pres-sure

2002 Determination of the Vapor Pressure of IKF-1216 PAI. Report No. 341134 GLP, unpublished

ISK

Melting point, boiling point and/or temper-ature of decom-position

1992 Determination of the Melting Point/Melting Range of IKF-1216 (PAI)., Report No. 089033 GLP, unpublished

ISK

Solubility in water

2002 IKF-1216 PAI, Determination of the Water Solubility at 3 pH Values. Report No. 341189 GLP, unpublished

ISK

Octanol/Water Partition Coeffi-cient

1992 Fluazinam (IKF-1216) (ASC-66825) - Oc-tanol/Water Partition Coefficient. Report No. 4039-91-0386-AS-001 GLP, unpublished

ISK

Hydrolysis characteris-tics

2003 14C-Fluazinam: Hydrolysis at Three Differ-ent pH Values. Report No. 846211 GLP, unpublished

ISK

Photolysis characteris-tics

1995 A Photolysis Study of IKF-1216 (Fluazinam) in Water at pH 5. Report No. 5312-94-0119-EF-002 GLP, unpublished

ISK

Disociation characteris-tics

1992 Fluazinam (IKF-1216) (ASC-66825) - Disso-ciation Constant. Report No. 4039-91-0387-AS-001 GLP, unpublished

ISK


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Reference list for table 3: Toxicology profile of fluazinam technical material based on acute toxicity, irritation and sensitization

Species/test Author(s) Year


Data Pro-tection

Claimed

Y/N-R/NR

Owner

Rat - oral 1988 Acute Oral Toxicity to Rats of B-1216 Tech-nical (Lot 109). Report No. 881246D/ISK20/AC GLP, unpublished

Y ISK

Rat - dermal 1984 B-1216: Technical: Acute Percutaneous Toxicity in the Rat. Report No. 84/ISK051/0586, Final report; 91/ISK051/0807, Amended report GLP, unpublished

Y ISK

Rat – inhala-tion

2006 Acute inhalation toxicity study of fluazinam technical in albino rats Report no. WIL-282006 GLP, unpublished

Y ISK

Rabbit - skin irritation

. 1992 Primary Dermal Irritation Study in Albino Rabbits with IKF-1216. Report No. 5016-91-0281-TX-001, Final re-port; 5016-91-0281-TX-001-001, Amend-ment 1 GLP, unpublished

Y ISK

Rabbit - eye irritation

1992 Primary Eye Irritation Study in the Albino Rabbits with IKF-1216. Report No. 5016-91-0280-TX-002, Final re-port; 5016-91-0280-TX-002-001, Report amendment GLP, unpublished

Y ISK

Guinea pig – skin sensitiza-tion

. 1984 B-1216: Delayed Contact Hypersensitivity Study in Guinea Pigs Report No. 84/ISK054/686 GLP, unpublished

Y ISK


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Reference list for table 4: Toxicology profile of fluazinam technical material based on repeated ad-ministration

Species/

test

Author(s) Year


Data Pro-tection

Claimed

Y/N-R/NR

Owner

Rat - sub-chronic

1983/1998

B-1216, Four-Week Toxicity Study in Dietary Administration to CD Rats Report no. 82/ISK035/544, Final report; 82/ISK035/544, Addendum 1 GLP, unpublished

Y ISK

Mouse - subchronic

1983/1998

B-1216, Four-Week Toxicity Study in Mice. Report no. 83/ISK036/067, Final report; 83/ISK036/215, Addendum 1; 83/ISK036/067, Addendum 2 GLP, unpublished

Y ISK

Dog - sub-chronic

1984 B-1216, Four week preliminary toxicity study in oral administration to beagle dogs. Report no. 84/ISK038/140, Final Report; 85/ISK038/050, Addendums I; 85/ISK038/248, Addendum II GLP, unpublished

Y ISK

Rat - short-term

Broadmeadow, A.

1984 B-1216: 13-Week Toxicity Study in Dietary Administration to CD Rats. Life Science Research Limited, report no. 84/ISK046/635 Final report, 91/ISK046/0830 Amended final report and addendum 3 (1998). GLP, unpublished

Y ISK

Dog - short-term

1992 B-1216: 13-Week Toxicity Study in Oral Ad-ministration to Beagle Dogs. Report No. 84/ISK048/692 (April 1985), Fi-nal report; 91/ISK048/0832, Amended final report; 84/ISK048/692, Addendum 3 GLP, unpublished

Y ISK

Rat - der-mal

1992 B-1216: 21-Day Percutaneous Toxicity Study in CD Rats Report No. 84/ISK052/690, Final report; 91/ISK052/0824, Amended final report GLP, unpublished

Y ISK

Dog - long-term

1987/1998

B-1216: 52-Week Toxicity Study in Oral Ad-ministration to Beagle Dogs Report No. 86/ISK055/512, Final report; 86/ISK055/512, Addendum 1. GLP, unpublished

Y ISK


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Reference list for table 4: Toxicology profile of fluazinam technical material based on repeated ad-ministration

Species/

test

Author(s) Year


Data Pro-tection

Claimed

Y/N-R/NR

Owner

Rat - long-term

1993 B-1216 Toxicity to Rats by Dietary Admin-istration for Two Years Report No. ISK/43/920649 GLP, unpublished

Y ISK

Mice - long-term

1988 B-1216: Potential Carcinogenicity Study in Dietary Administration to Mice for 104 Weeks Report No. ISK 9/87264

GLP, unpublished

Y ISK

Rat –

reproduction

1987 B-1216: Effects upon reproductive perfor-mance of rats treated continuously through-out two successive generations Report No. 87/ISK068/097 GLP, unpublished

Y ISK

Rabbit –

Develop-mental

1985 Teratology study in the rabbit Report No. 85/ISK049/045 GLP, unpublished

Y ISK

Rabbit –

Develop-mental

1988 Teratology study in the rabbit Report No. 86/ISK069/324 GLP, unpublished

Y ISK

Rat - devel-opmental

1985/

1991

B-1216: Teratology Study in the Rat.

Final Report No. 84/ISK047/606; Amended Final Report No. 91/ISK/047/0820

GLP, unpublished

Y ISK


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Reference list for table 5: Mutagenicity profile of fluazinam technical material based on in vitro and in vivo tests

Species Author(s) Year


Data Pro-tection

Claimed

Y/N-R/NR

Owner

Bacteria 1988 Bacterial Reverse Mutation Test of Fluazi-nam Technical. Report No. T-1674E GLP, unpublished

Y ISK

Bacteria 1989 Bacterial Reverse Mutation Test of Fluazi-nam Technical. Report No. T-1673E GLP, unpublished

Y ISK

Mammalian cells

2000 IKF-1216 Mammalian Cell Mutation Assay Report No. RIA 017/004090 GLP, unpublished

Y ISK

Mammalian cells

1988 Chromosomal Aberration Test of Fluazinam Technical Using Cultured Mammalian Cells. Report No. T-1663E GLP, unpublished

Y ISK

Bacteria 1988 DNA Repair Test of Fluazinam Technical in Bacillus subtilis. Report No. T-1595E GLP, unpublished

Y ISK

mouse 1999 IKF-1216 Technical: Micronucleus Test in Mice. Report No. IET 98-0139 GLP, unpublished

Y ISK


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Reference list for table 6: Ecotoxicology profile of fluazinam technical material

Species Author(s) Year

Title Source (where different from com-pany) Company, Report No GLP or GEP status (where relevant), Published or not

Data Pro-tection

Claimed

Y/N-R/NR

Owner

Acute toxicity Bobwhite quail

1991 B-1216 Technical Acute Oral Toxicity (LD50) to Bobwhite Quail. Report no. ISK 48/91161 GLP, unpublished

Y ISK

Acute toxicity Mallard duck

1991 The Acute Oral Toxicity of B1216 to the Mallard Duck. Report no. ISN 31BT/841207 GLP, unpublished

Y ISK

Acute toxicity Rainbow trout

1992 Technical Fluazinam (IKF-1216) – Acute Toxicity to Rainbow Trout (Oncorhyn-chus mykiss) Under Flow-Through Con-ditions. report no: 5099-91-0422-TX-002 GLP, unpublished

Y ISK

Acute toxicity Daphnia magna

Shults, S. K., Brock, A. W., Laveglia, J.

1992 Acute Toxicity to Daphnids (Daphnia magna) Under Flow-Through Conditions with Technical Fluazinam (IKF-1216). Springborn Laboratories report n o. 5108-91-0418-TX-002 GLP, unpublished

Y ISK

Acute toxicity Chironomus riparius

2003 Acute toxicity ofFluazinam technical to first-instar larvae of Chironomus riparius. RCC Ltd., report no. 851182 GLP, unpublished

Y ISK

Duck weed 2001 IKF-1216: toxicity to the duckweed, Lemna gibba Report no. 2129-SK GLP, unpublished

Y ISK

Acute oral and contact toxicity

1984 B 1216 (PP192): Acute Contact and Oral Toxicity to Honey Bees (Apis mellifera). Report No. RJ0401B GLP Yes, not published

Y ISK


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APPENDIX 1

ANALYTICAL METHOD FOR DETERMINATION OF IMPURITY-5 IN TC AND SC


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3.2 Determination of related impurity 5 by reversed phase HPLC REAGENTS Water HPLC grade Acetonitrile HPLC grade Triethylamine, analytical reagent grade o-phosphoric acid (H3PO4), 85%, analytical reagent grade Impurity 5 reference standard of certified purity Mobile phase eluent A: 0.2 % (v/v) aqueous solution with triethylamine, pH adjusted to 3.0 with o-

phosphoric acid eluent B: acetonitrile

Diluting solvent acetonitrile Calibration solution Weigh in duplicate (to the nearest 0.1 mg) 25 mg of the impurity 5 reference

standard (s mg) into separate volumetric flasks (200 ml). Add acetonitrile (about 180 ml) and place the flasks in an ultrasonic bath until complete dissolution (minimum for 5 minutes). Allow the solu-tions to cool to ambient temperature and fill to the mark with acetonitrile (stock calibration solu-tions C1 and C2). Mix well. Transfer by pipette 10, 5 and 2.5 mL (t mL) of stock calibration solution C1 into separate volumetric flasks (100 mL) and fill to the mark with acetonitrile (calibration solutions C1A, C1B and C1C). Transfer by pipette 15, 7.5 and 4 mL (t' mL) of stock calibration solution C1B into separate volumetric flasks (50 mL) and fill to the mark with acetonitrile (calibration solutions C1D, C1E and C1F). Transfer by pipette 5 mL (t mL) of stock calibration solution C2 into a separate volumetric flask (100 mL) and fill to the mark with acetonitrile (calibration solutions C2B).

APPARATUS High performance liquid chromatograph equipped with a detector suitable for operation at 240 nm

(UV-detection), an oven and an injection system capable of injecting 10 µl Liquid chromatographic column stainless steel, 250 x 4.6 mm (i.d.), packed with Cosmosil C18,

5 µm, Phenomenex or equivalent with the same selectivity Electronic integrator or data system Ultrasonic bath pHmeter PROCEDURE (a) Chromatographic conditions (typical)

Column temperature 50°C Flow rate 0.75 ml/min Detector wavelength 240 nm Injection volume 10 µl Retention time approximately 27 min Gradient programme Time Eluent A Eluent B [ min ] [ % ] [ % ] 0 38 62 29 38 62


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35 25 75 45 25 75 60 38 62 65 38 62

(b) Equilibration of the system. Pump sufficient mobile phase (use gradient programme) through the

column to equilibrate the system. Inject 10 l portions of the calibration solution C1B and repeat the

injections until retention times and peak areas deviate by less than 1 % from the mean for three successive injections.

(c) Sample preparation. Prepare sample solutions in duplicate for each sample. Weigh (to the nearest

0.1 mg) sufficient sample (w mg) to contain about 120 mg fluazinam into a volumetric flask (50 ml). Add acetonitrile (about 40 ml) and place the flasks in an ultrasonic bath until complete dissolution (minimum for 5 minutes). Allow the solutions to cool to ambient temperature and fill to the mark with acetonitrile (sample solutions S1 and S2). Mix thoroughly. Filter a portion of each standard so-lution through a 0.45 µm fitter prior to analysis.

(d) Determination. Inject 10 l portions of the second calibration solution (C2B) for two successive injections. The mean response factor for this solution should deviate by no more than 2% from those for the first calibration solution (C1B) (see paragraph (b) Equilibration of the system), other-wise the calibration solutions should be prepared again.

Inject in duplicate 10 l portions of each sample solution (S1, S2, …,etc.) bracketing them by dupli-cate injections of the six calibration solutions (C1A, C1B, C1C, C1D , C1E and C1F) using the following sequence:

C1A, C1A, S1, S1, S2, S2, C1B, C1B, S3, S3, S4, S4, C1c…

Determine the peak area of impurity 5. (e) Preparation of a calibration curve

Prepare a calibration curve for impurity 5 by plotting peak areas versus the concentration of impu-rity 5 (n µg/ml) in the six calibration solutions. Using the method of least-squares calculate the equation for the straight line that best fits the experimental data. The correlation coefficient should be 0.999 or better. If not, repeat the calibration.

cD

sn

P=

where:

n = concentration of impurity 5 in diluted calibration solution (µg/ml)

P = purity of impurity 5 reference standard (g/kg)

s = mass of impurity 5 reference standard in the stock calibration solution (mg)

Dc = dilution factor of calibration solution.

(f) Calculation Determine the content of impurity 5 of the sample solutions for each sample injection.


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Impurity 5 content 50 x x

)b (

wa

Hw −= [g/kg] (M)

where:

Hw = peak area of impurity 5 in the sample solution

b = intercept of calibration curve

a = slope of calibration curve

w = mass of sample taken (mg)

Repeatability r = 0.017 g/kg at 0.097 g/kg impurity 5 content Reproducibility R = 0.035 g/kg at 0.097 g/kg impurity 5 content

The precision data were determined from an experiment conducted in 2008 involving 8 laboratories. Mean value of precision and concentration level were reported.

Typical HPLC/UV Chromatograms for Impurity 5 determination in technical material 521/TC/M/3.2

m in0 10 20 30 40 50 60

m AU

0

5

10

15

20

25

D AD 1 A , S ig=240,4 R ef=o ff (PE 21550B \F020529 IM P3 2008-02-06 \003-0801.D )

C alibration so lu tion o f IM P U R ITY 5 (about 3 µg /m L)

De

tecto

r R

esp

on

se

at 2

40

nm

26

.90

0 -

im

pu

rity

5 flu

azin

am

m in0 10 20 30 40 50 60

m AU

0

5

10

15

20

25


S am ple so lu tion o f FLU AZIN AM TE C H N IC AL

De

tecto

r R

esp

on

se

at 2

40

nm

26

.90

0 -

im

pu

rity

5 flu

azin

am

m in0 10 20 30 40 50 60

m AU

0

5

10

15

20

25


S olvent (aceton itrile )

De

tecto

r R

esp

on

se

at 2

40

nm


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3.2 Determination of related impurity 5 by reversed phase HPLC

As for fluazinam technical 521/TC/M/3.2 except (c) Sample preparation. Prepare sample solutions in duplicate for each sample. Weigh (to the nearest

0.1 mg) sufficient sample (w mg) to contain about 50 to 70 mg fluazinam into a volumetric flask (25 ml). Add acetonitrile (about 20 ml) and place the flasks in an ultrasonic bath for 5 minutes). Allow the solutions to cool to ambient temperature and fill to the mark with acetonitrile (sample solutions S1 and S2). Mix thoroughly. Filter a portion of each standard solution through a 0.45 µm fitter prior to analysis.

(f) Calculation Determine the content of impurity 5 of the sample solutions for each sample injection.

Impurity 5 content 25 x x

)b (

wa

Hw −= [g/kg] (M)

where:

Hw = peak area of impurity 5 in the sample solution

b = intercept of calibration curve

a = slope of calibration curve

w = mass of sample taken (mg)

Repeatability r = 0.008 g/kg at 0.037 g/kg impurity 5 content Reproducibility R = 0.015 g/kg at 0.037 g/kg impurity 5 content The precision data were determined from an experiment conducted in 2008 involving 8 laboratories. Mean value of precision and concentration level were reported. Typical HPLC/UV Chromatograms for Impurity 5 determination in suspension concentrate 521/SC/M/3.2


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m in0 10 20 30 40 50 60

m AU

0

5

10

15

20

25


C alibration so lu tion o f IM P U R ITY 5 (about 0 .5 µg /m L)

Dete

cto

r R

esp

. :

24

0 n

m

27

.00

5 -

im

pu

rity

5 flu

azin

am

m in0 10 20 30 40 50 60

m AU

0

5

10

15

20

25


S am ple so lu tion o f FLU AZIN AM 500 S C (S H IR LAN )

Dete

cto

r R

esp

. :

24

0 n

m

27

.06

7 -

im

pu

rity

5 flu

azin

am

m in0 10 20 30 40 50 60

m AU

0

5

10

15

20

25


S olvent (aceton itrile )

Dete

cto

r R

esp

. :

24

0 n

m

m in0 10 20 30 40 50 60

m AU

0

5

10

15

20

25


Dete

cto

r R

esp

. :

24

0 n

m

S olution o f FLU AZIN AM 500 S C - B LAN K FO R M U LATIO N (w ithout a .s .)

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