# Annex B



# Spirotetramat

B.6 Toxicology and metabolism

Reviewed by: US EPA, USA

Peer –reviewed by: AGES, Austria PMRA, Canada– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 2 April 2008

Table of contents

B.6.1 Absorption, distribution, excretion and metabolism (toxicokinetics) (OECD

Annex IIA 5.1)

................................................... ................................................... ....................6 B.6.1.1 Toxicokinetic studies – single dose, oral route in rats (OECD Annex IIA 5.1.1) ......................6 B.6.1.1.1 Metabolism and Pharmacokinetics....................

................................................... ....................6 B.6.1.2 Toxicokinetic studies – second single dose, oral ro ute in rats (OECD Annex IIA 5.1.1

and 5.1.2).........................................

................................................... ....................................19 B.6.1.2.1 Metabolism and Pharmacokinetics (OECD Annex IIA 5.1 .2) ................................................ .19 B.6.1.3 Metabolism (OECD Annex IIA 5.1.4)..................

................................................... .................33 B.6.1.4 Metabolism (PBPK Modeling) (OECD Annex IIA 5.1.5) ..

................................................... ....39 B.6.1.5 Metabolism (PBPK Modeling) (OECD Annex IIA5.1.6) ...

................................................... ....45 B.6.1.6 Metabolism and Pharmacokinetics (OECD Annex IIA 5.1 .6) ................................................ .48 B.6.1.6.1 Metabolism and pharmacokinetics (OECD Annex B.6.1.2 .6)................................................ .59 B.6.1.7 Toxicokinetic studies – repeated dose, oral route i n rats (OECD Annex IIA 5.1.3)................66 B.6.1.8 Summary of ADME studies............................

................................................... ......................75

B.6.2 Acute toxicity including irritancy and skin sensiti zation (OECD Annex IIA 5.2)............77 B.6.2.1 Acute oral toxicity (OECD Annex IIA 5.2.1) .........

................................................... ................77 B.6.2.2 Acute percutaneous toxicity (OECD Annex IIA 5.2.2) .

................................................... ........80 B.6.2.3 Acute inhalation toxicity (OECD Annex IIA 5.2.3)...

................................................... .............83 B.6.2.4 Skin irritation (OECD Annex IIA 5.2.4).............

................................................... ....................89 B.6.2.5 Eye irritation (OECD Annex IIA 5.2.5) ..............

................................................... ...................92 B.6.2.6 Skin sensitisation (OECD Annex IIA 5.2.6) ..........

................................................... ...............96 B.6.2.7 Potentation/interactions of multiple active substan ces or products (OECD Annex IIA

5.2.7).............................................

................................................... .....................................108 B.6.2.8 Summary of acute toxicity..........................

................................................... ........................109

B.6.3 Short term toxicity (OECD Annex IIA 5.3) ...........

................................................... ..........110 B.6.3.1 Oral 28-day toxicity (rodents) (OECD Annex IIA 5.3. 1)................................................. .......110 B.6.3.2 Oral 90-day toxicity (rodents) (OECD Annex IIA 5.3. 2)................................................. .......123 B.6.3.3 Oral 90-day toxicity (dog) (OECD Annex IIA 5.3.3)..

................................................... .........131 B.6.3.4 Oral 1 year toxicity (dog) (OECD Annex IIA 5.3.4)..

................................................... ..........150 B.6.3.5 Repeated dose (28-day/90-day) inhalation toxicity ( rodents) (OECD Annex IIA 5.3.5

and 5.3.6).........................................

................................................... ..................................160 B.6.3.6 Repeated dose (28-day/90-day) percutaneous toxicity (rodents) (OECD Annex IIA

5.3.7 and 5.3.8)...................................

................................................... ...............................160 B.6.3.7 Summary of short-term toxicity studies.............

................................................... .................165

B.6.4 Genotoxicity (OECD Annex IIA 5.4)..................

................................................... ..............166 B.6.4.1 In vitro assays............................................

................................................... ........................166 B.6.4.1.1 Bacterial assay for gene mutation (IIA 5.4.1) ......

................................................... ..............166 B.6.4.1.2 Salmonella Mammalian Activation Gene Mutation................. ..............................................171 B.6.4.1.3 In vitro genotoxicity - Test for clastogenicity in mammalia n cells (OECD Annex IIA 5.4.2.1)...........................................

................................................... ....................................176 B.6.4.1.4 In vitro genotoxicity - Test for gene mutation in mammalian cells (OECD Annex IIA 5.4.3).............................................

................................................... .....................................195 B.6.4.2 In vivo

assays ............................................

................................................... ........................203 B.6.4.2.1 Bone marrow or micronucleus (OECD Annex IIA 5.4.4) .

................................................... ..203– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 3 April 2008

B.6.4.2.2 Metaphase analysis in rodent bone marrow or micronu cleous test in rodents (OECD

Annex IIA 5.4.4) ...................................

................................................... ..............................214 B.6.4.2.3 Unscheduled DNA synthesis (OECD Annex IIA 5.4.5)...

................................................... ...214 B.6.4.2.4 In vivo

studies in germ cells (OECD Annex IIA 5.4.6) ......

................................................... .219 B.6.4.3 Summary and overall conclusions of genotoxicity stu dies ............................................... ....219

B.6.5 Long term toxicity and carcinogenicity (OECD Annex IIA 5.5) ......................................220 B.6.5.1 Long term oral toxicity study in the rat (OECD Anne x IIA 5.5.1 and 5.5.2) ..........................220 B.6.5.2 Carcinogenicity study in the mouse (OECD Annex IIA 5.5.3) ............................................. .242 B.6.5.3 Summary of long term toxicity/carcinogenicity studi es................................................. ........251 B.6.6 Reproductive toxicity (OECD Annex IIA 5.6) .........

................................................... ............251 B.6.6.1 Two generation reproductive toxicity in the rat (OE CD Annex IIA 5.6.1) .............................251 B.6.6.2 REPRODUCTION AND FERTILITY EFFECTS – RANGE-FINDING . .................................268 B.6.6.3 Teratogenicity test by the oral route in the rat (O ECD Annex IIA 5.6.10).............................2 80 B.6.6.4 Teratogenicity test by the oral route in the rabbit (OECD Annex IIA 5.6.11)........................302 B.6.6.5 Summary of reproductive toxicity...................

................................................... ....................315

B.6.7 Neurotoxicity (OECD Annex IIA 5.7) .................

................................................... .............316 B.6.7.1 Acute neurotoxicity – rat (OECD Annex IIA 5.7.1)...

................................................... ..........316 B.6.7.2 Subchronic neurotoxicity – rat 90-day (OECD Annex I IA 5.7.4).......................................... .328 B.6.7.3 Postnatal developmental neurotoxicity (OECD Annex I IA 5.7.5) .........................................3 28 B.6.7.4 Summary on neurotoxicity ...........................

................................................... ......................329

B.6.8 Further toxicological studies......................

................................................... ....................330 B.6.8.1 Mechanistic studies (OECD Annex IIA 5.10)..........

................................................... ...........330 B.6.8.1.1 SPECIAL MALE REPRODUCTIVE TOXICITY STUDY ........... ............................................330 B.6.8.1.2 SPECIAL TESTICULAR/SPERM TOXICITY STUDY ............ ..............................................335 B.6.8.2 Summary of mechanistic studies.....................

................................................... ..................338 B.6.8.3 Toxicity studies on metabolites (OECD Annex IIA 5.8 ) .................................................. ......339 B.6.8.3.1 Acute oral toxicity (OECD Annex IIA 5.8.6) .........

................................................... ..............339 B.6.8.3.2 Salmonella / Escherichia

/Mammalian Activation Gene Mutation Assay .......... ......................349 B.6.8.4 Summary of toxicological studies on metabol ites............................................... ..................380

B.6.9 Medical data and information (OECD Annex IIA 5.9) ..

................................................... .381 B.6.9.1 Report on medical surveillance on manufacturing pla nt personnel (OECD Annex IIA

5.9.1).............................................

................................................... .....................................381 B.6.9.2 Report on clinical cases and poisoning incidents (O ECD Annex IIA 5.9.2) .........................381 B.6.9.3 Observations on exposure of the general population and epidemiological studies

(OECD Annex IIA 5.9.3) .............................

................................................... .......................381 B.6.9.4 Clinical signs and symptoms of poisoning and detail s of clinical tests (OECD Annex

IIA 5.9.4) .........................................

................................................... ...................................381 B.6.9.5 First aid measures (OECD Annex IIA 5.9.5)..........

................................................... ............383 B.6.9.6 Therapeutic regimes (OECD Annex IIA 5.9.6) .........

................................................... .........383 B.6.9.7 Expected effects and duration of poisoning as a fun ction of exposure (OECD Annex

IIA 5.9.7) .........................................

................................................... ...................................383 B.6.9.8 Expected effects and duration of poisoning as a fun ction of time (OECD Annex IIA

5.9.8).............................................

................................................... .....................................383 B.6.9.9 Dermal Penetration (OECD Annex IIA 5.9.9) ..........

................................................... ..........383 B.6.9.9.1 IN VIVO DERMAL PENETRATION STUDY–RAT ....................... ........................................383– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 4 April 2008

B.6.9.9.2 IN VITRO DERMAL PENETRATION STUDY–RAT/HUMAN................. ..............................392 B.6.9.9.3 IN VITRO DERMAL PENETRATION STUDY–RAT/HUMAN................. ..............................399 B.6.9.10 Summary of dermal absorption studies ...............

................................................... ..............407

B.6.10 Summary of mammalian toxicity and overall evaluatio n (OECD Annex IIA 5.11)........408
B.6.10.1 Proposal for ADI...................................

................................................... ..............................418 B.6.10.2 Proposed ArfD ......................................

................................................... .............................418 B.6.10.3 Proposal for drinking water limit..................

................................................... .......................418 B.6.10.4 Proposal for AOEL..................................

................................................... ...........................418

B.6.11 Acute toxicity of the preparations (OECD Annex IIIA 7.1).............................................. 419 B.6.11.1 Acute oral toxicity (OECD Annex IIIA 7.1.1) ........

................................................... ..............419 B.6.11.2 Acute percutaneous (dermal) toxicity (OECD Annex II IA 7.1.2).......................................... .424 B.6.11.3 Acute inhalation toxicity to rats (OECD Annex IIIA 7.1.3)............................................. ........429 B.6.11.4 Skin irritation (OECD Annex IIIA 7.1.4)............

................................................... ..................437 B.6.11.5 Eye irritation (OECD Annex IIIA 7.1.5) .............

................................................... .................443 B.6.11.6 Skin sensitisation (OECD Annex IIIA 7.1.6) .........

................................................... .............448 B.6.11.7 Summary of toxicity studies with the formulation...

................................................... ............461

B.6.12 Dermal absorption (OECD Annex IIIA 7.6) ............

................................................... ........463

B.6.13 Toxicological data on non active substances (OECD A nnex IIIA 7.9)...........................466

B.6.14 Exposure data (OECD Annex IIIA 7.3, 7.4 and 7.5)...

................................................... ....466

B.6.15 References relied on ...............................

................................................... ........................467

Appendix:

B.6.10.1 Proposal for ADI...................................

................................................... ..............................489 B.6.10.2 Proposed ARfD......................................

................................................... ............................489 B.6.10.3 Proposal for drinking water limit..................

................................................... .......................489 B.6.10.4 Proposal for AOEL..................................

................................................... ...........................489

B.6.12 Dermal absorption (annex IIIA 7.6).................

................................................... ................491 B.6.12.1 Dermal absorption, in vivo in the rat ..............

................................................... ....................491 B.6.12.2 Comparative dermal absorption, in vitro using human and rat skin .....................................4 93 B.6.12.3 Summary on dermal absorption.......................

................................................... ..................496

B.6.13 Toxicological data on non active substances (Annex IIIA 7.9) ......................................497 B.6.13.1 Material safety data sheet for each formulant ......

................................................... .............497 B.6.13.2 Available toxicological data for each formulant ....

................................................... .............497

B.6.14 Exposure data (annex IIIA 7.3, 7.4 and 7.5) ........

................................................... ...........498 B.6.14.1 Operator exposure (annex IIIA 7.3) .................

................................................... ..................498 B.6.14.1.1 Estimation of operator exposure assuming personal p rotective equipment is not used ......499 B.6.14.1.2 Estimation of operator exposure assuming personal p rotective equipment is used ............501 B.6.14.1.3 Measurement of operator exposure – (Mixer/Loader/Ap plicator).........................................5 01 B.6.14.1.4 Summary on operator exposure .......................

................................................... .................501– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 5 April 2008

B.6.14.2 Bystander exposure (annex IIIA 7.4) ................

................................................... .................502 B.6.14.2.1 Estimation of bystander exposure ...................

................................................... ..................502 B.6.14.2.2 Measurement of bystander exposure ..................

................................................... ..............503 B.6.14.2.3 Summary on bystander exposure......................

................................................... ................503 B.6.14.3 Worker exposure (annex IIIA 7.5)...................

................................................... ...................504 B.6.14.3.1 Estimation of worker exposure assuming personal pro tective equipment is not used.........504 B.6.14.3.2 Estimation of worker exposure assuming personal pro tective equipment is used...............505 B.6.14.3.3 Estimation of operator exposure assuming personal p rotective equipment is used and

using data generated on dislodgeable residues under the proposed conditions of use ......505 B.6.14.3.4 Measurement of worker exposure .....................

................................................... ................505 B.6.14.3.5 Summary on worker exposure.........................

................................................... ..................505

B.6.15 Dislogeable residues (annex IIIA 7.7) ..............

................................................... ..............506– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 6 April 2008

B.6 Toxicology and metabolism

B.6.1 Absorption, distribution, excretion and metab olism (toxicokinetics) (OECD Annex IIA

5.1)

B.6.1.1 Toxicokinetic studies – single dose, oral r oute in rats (OECD Annex IIA 5.1.1)

B.6.1.1.1 Metabolism and Pharmacokinetics

CITATION : Klempner, A. 2006. [Azaspirodecenyl-3- 14 C]BYI 08330: Absorption, distribution, excretion

and metabolism in the rat. Study No. M01819128. Rep ort No. MEF 048/04, Document No. M-268709-02-2. Bayer CropScience AG Development, M etabolism/Environmental Fate, 40789 Monheim am Rhein, Alfred Nobel Str. 50, Germa ny.

I. MATERIAL AND METHODS:



### A. MATERIAL:



### 1. Test Material:

IUPAC Name: cis-3-(2,5-dimethylphenyl)-8-methoxy-2- oxo-1-azaspiro[4.5]dec-3-en-4-yl ethyl carbonate Code name: BYI 08330 Common name: Spirotetramat (proposed ISO) Empirical formula: C 21 H 27 N O 5 Molar mass: 373.45 g/mol Water solubility: pH 4 = 33.5 mg/L, pH 7 = 29.9 mg /L pH 9 = 19.1 mg/L (unstable) all at 20°C n-Octanol/water partition coefficient: pH 4 = 2.51, pH 7 = 2.51, pH 9 = 2.50

Labelling; Azaspirodecenyl-3- 14 C Specific radioactivity of the original radiolabelled batches: Test 2 – 5: 3.71 MBq/mg (100.2 μCi/mg) Test 8 + 9: 3.67 MBq/mg ( 99.1 μCi/mg)

Specific radioactivity used for administration: Test 2 + 5: 3.71 MBq/mg (100.2 μCi/mg) Test 3 + 4: 0.07 MBq/mg ( 1.9 μCi/mg) Test 8 + 9: 3.67 MBq/mg ( 99.1 μCi/mg) Radiochemical purity: > 98 % or > 99 % (certified, HPLC and TLC with radiodetection) Dose level: Test 2 + 8: 2 mg/kg body weight Test 3 + 4: 100 mg/kg body weight Test 5 + 9: 2 mg/kg body weight, animals pre-tre ated for 14 Days with 2 mg/kg bw non-radiolabelled BYI 08330 Vehicle 0.5 % aqueous tragacanth suspension Stability of the test material The stability of [az aspirodecenyl-3- 14 C]BYI 08330 was demonstrated by radio HPLC analysis of the administration suspensions of each test immediately after dosing– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 7 April 2008



### 2. Test Animals:

Species: Rat ( Rattus norvegicus domesticus )
Strain: Wistar Hsd/Cpb: WU Breeding facility: Harlan & Winkelmann GmbH, Gartenstraße 27, 33178 Borchen, Germany Sex and numbers involved: Males: 12 animals (3 gro ups with 4 animals) Females: 12 animals (3 groups with 4 animals) Age: 9 – 10 weeks (male rats) and 12 – 13 weeks ( female rats) at the time of delivery Body weight: Males: 200 – 216 g at the time of adm inistration 204 – 246 g at the time of sacrifice Females: 197 – 212 g at the time of administration 197 – 212 g at the time of sacrifice
Acclimatization: Makrolon ® cages on wood shavings in the test facility for ab out 7 days prior to the administration. Identification: Cage cards on which the study numbe r, test compound name and individual animal number were displayed. Additional labelling with water-insoluble spots on the tail Housing: After administration of the radiolabelled test item individually in Makrolon ® metabolism cages under conventional hygienic condit ions in air-conditioned

rooms Temperature 22 - 26 °C, relative humidity 37 – 76 %

. 12 / 12 hours light / dark cycle, air change 10 – 1 5 times per hour. Feed and water: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, CH-4303 Kaiseraugst, Switzerland (ca. 16 g per animal and day) last feeding ca. 16 h prior to dosing next feeding ca. 6 h after dosing. tap water from municipal water supply, ad libitum



### B. STUDY DESIGN:

The experimental protocol is summarized in Table B. 6-1.

Table B.6-1: Study designs for assessment of metabo lism and pharmacokinetics of [Azaspriodecenyl-3-14C]BYI 08330 in rats.

Group Dose (mg/kg)

target dose (actual dose) No./sex Sample collection

8 2

(2.02) 4 Plasma: 0.08, 0.17, 0.33, 0.6, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 24, 32,
48 hrs Urine: 4, 8, 12, 24, 48 hrs Feces: 24, 48 hrs Blood, organs, tissues: at 48-hr termination
2 2

(1.84) 4 Plasma: 0.08, 0.17, 0.33, 0.6, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 24, 32,
48 hrs Urine: 4, 8, 24, 48 hrs– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 8 April 2008

Group Dose (mg/kg)

target dose (actual dose) No./sex Sample collection

Feces: 24, 48 hrs Blood, organs, tissues: at 48-hr termination
3 100

(92.7) 4 Same group 2

4 100

(84.9) 4 Same group 2

5 2

(1.65) 4 Same group 2

9 2

(1.87) 4 Same group 2

Dosing Each of the rats (4 animals per group) were adminis tered, by gavage, a 2 or 100 mg/kg bw dose of azaspirodecenyl-3- 14 C labelled BYI 08330 suspended in 0.5% aqueous trag acanth after a 16-hour fasting. The dosing suspensions (0.2 or 10 mg /mL f or the low and high dose level) were prepared one day before dosing and stored at 4 ° C. Each animal received a volume of 2 mL (= 10 mL/k g bw calculated for a nominal body weight of 200 g). Actual adminis tered doses were measured by LSC assay of aliquots of the administration suspensions. The actual mean administered doses of [azaspirodecenyl-3- 14 C]BYI 08330 were in the range of 1.65 - 2.02 mg/kg bw for the low dose tests and 84.9 - 92.7 mg/kg bw for th e high dose tests. The stability of the test compound in the suspensions was assured by radio HPLC analysis of aliquots after administration.

Collection of blood Blood samples were collected separately for each an imal by pressing a capillary coated with heparin in a small cut in the tail vein. The wound was closed wi th adhesive tape. The capillaries were centrifuged at ca. 12000 g for about 10 minutes using a hematocrit centrifuge to separate the plasma from the formed constituents (mainly erythrocytes). After centrifug ation, the capillary was broken at the border betwe en the plasma, and the formed constituents and the pla sma (ca. 30 μL) were each placed onto a small metal dish for weighing. The dish was then placed i nto a scintillation vial for radioactivity measurem ent.

Collection of excreta Urine was collected at various times separately for each animal in a cryogenic trap cooled with dry ic e. The funnels for urine collection were rinsed with d emineralised water at the end of each sampling peri od. The rinsing solutions were drained into the same vi al as the corresponding urine fraction. The radioactivity was determined by LSC. Urine samples were stored in a freezer before analysis.

Faeces were collected at various times separately f or each animal in a cryogenic trap before they were lyophilised (freeze-dried), weighed, and homogenise d. The radioactivity was determined by combustion/LSC. Lyophilised samples were stored at room temperature before extraction/analysis.

Sacrifice and organ/tissue sampling At the respective test end (48 h after administrati on), the animals were anaesthetised using Pentobarbital-Na. They were sacrificed by transecti on of the cervical blood vessels. After transection of the cervical blood vessels, the blood was collected into test tubes coated with heparin and was separated afterwards into plasma and erythrocytes b y centrifugation. The following organs/tissues were collected: erythrocytes, plasma, spleen, gastro-int estinal tract (GIT), liver, kidneys, perirenal fat, adrenal– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 9 April 2008

gland, testis (male only), ovaries (female only), u terus (female only), skeletal muscle, femur bone, h eart, lung, brain, thyroid gland, skin, and carcass.

The organs and tissues prepared at the end of the e xperiments were weighed immediately after the dissection and again after lyophilisation. Finally, they were homogenised and the radioactivity was determined by combustion/LSC. The radioactivity rem aining in the GIT, skin, and residual carcass was determined by combustion/LSC in order to establish the radioactivity balance. For the small organs and tissues (e.g. renal fat, uterus, adrenal glands, th yroid, and ovaries), only the wet weight was weighe d and the samples then solubilised with BTS 450 ® (Beckman Tissue Solubiliser).

Radioactivity measurement The measurement of the radioactivity in liquid samp les was carried out by liquid scintillation countin g (LSC). For this purpose, the volume of the entire s olution was determined and radioactivity of an aliq uot (1 – 3 replicates) of the solution was measured usi ng the following scintillation counters:

Small organs or tissues were solubilised by means o f a tissue solubiliser (e.g. BTS-450 ® ). The solubilised samples or aliquots of them were acidif ied with hydrochloric acid or glacial acetic acid a nd mixed with a suitable scintillation cocktail (e.g. Quickszint 401, Zinsser Analytic GmbH). The radioac tivity was then measured in a scintillation counter. This method was used for the following test materials: r enal fat, adrenal glands, thyroid, ovaries, and uterus.

Solid samples (1 – 5 aliquots) were weighed and com busted in an oxygen atmosphere using the following equipments Oxidizer 307/387 (Packard Inst ruments) for combustion of faeces, erythrocytes, lyophilised organs and tissues like e.g. spleen, li ver, lung, bone muscle, gastrointestinal tract (GIT ), residual carcass and solids (e.g. from faeces after extraction). Carbosorb E (9 mL) was used to trap t he combustion product CO 2 and Permafluor E+ (10 mL; Packard Instruments) as scintillator for LSC.

Metabolite analysis Urine samples were analysed without sample preparat ion. Freeze-dried faeces samples of all animals from 0 - 24 h post administration were combined for each test. The radioactivity in each sample materi al used for analysis was determined by combustion/LSC. Each composite faeces sample was extracted successively 3 times with acetonitrile / water (8: 2, v/v) using a Polytron homogeniser. Each extract was collected by centrifugation and its volume was meas ured. Aliquots of each extract were radioassayed by LSC. The three extracts of each faeces preparation were combined and concentrated to the aqueous remainder and partitioned 3 times against n-hexane. Aliquots of aqueous and organic phases were radioassayed by LSC. The aqueous phase was concentr ated and used for HPLC analysis.

The metabolites in the samples were quantified by H PLC using a reversed phase column (C18) and the eluting solvents water/formic acid = 99: 1 (v/v) an d acetonitrile/water/formic acid = 97: 2: 1 (v/v/v) in the gradient mode. Detection was performed by a UV (254 nm) and a radioisotope detector with a glass scintillator. In order to check the completeness o f the chromatographic elution, several representati ve samples were injected, re-collected, and radioassay ed by LSC. The column recoveries were between >99 and 101% of the injected amount of radioactivit y.

The first method for identification of metabolites in urine and extracts of faeces was HPLC cochromatography with radiolabelled and non-radiolabe lled reference items. One-dimensional TLC on silica 60 F254 (normal phase) plates was used as th e second method for co-chromatographic identification of metabolites. The plates were deve loped over a separation path of ca. 16 cm in a standard TLC-chamber. The following three TLC solv ent systems were used:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 10 April 2008

System 1: Ethyl acetate/propanol-2/water (65/23/12 v/v/v), w ith chamber saturation

System 2 : Dichloromethane/methanol/ammonia solution 25 % (9 0/10/1 v/v/v), with chamber

saturation

System 3 : Trichloromethane/methanol/glacial acetic acid/wat er (40/10/2/1 v/v/v/v), with chamber

saturation

For the TLC confirmation of metabolites, aliquots o f urine and faeces extracts were fractionated with the HPLC profiling method. The collected peaks were the n concentrated to the aqueous remainder. Aliquots of these samples and of the solutions of the refere nce items were applied individually and overlapped with each other on the TLC-plates. Co-chromatograph ic correspondence was assessed either by visual inspection of the plate under UV light at 254 nm an d its associated radioluminogram or by analysis of the radioluminogram only, in cases where radioactive re ference items were used. In addition to cochromatography, the two main urine metabolites were identified by LC-MS and LC-MS/MS of isolated and purified fractions.

II. RESULTS AND DISCUSSION:

A. ABSORPTION:

For all tests, the mean equivalent concentrations ( C) of total radioactivity from four animals were us ed to calculate the basic toxicokinetic parameters from p lasma curve analysis. The pharmacokinetic software TOPFIT (version 2.0) was used with a 3-compartment disposition model for fitting of the data. Correlat ion coefficients were excellent for all tests (0.994-1. 00). BYI 08330 was very rapidly absorbed from the gastro intestinal tract of male and female rats in all tes t groups. The absorption commenced immediately after oral dosing. Values calculated for the absorption half-lives were in the range of 0.6-10 min. No sig nificant differences were observed between the low dose, high dose, and repeated dose tests. BYI 0833 0 was completely absorbed even at the high dose level of 100 mg/kg bw since 89-98% of the total rec overed radioactivity was excreted via the renal rou te. Between 91-100% of the administered doses were reco vered in urine, faeces, and the body of animals. The balance of radioactivity for all tests is prese nted in Table 6-2.

B. DISTRIBUTION:

Radioactivity in plasma of male and female rats fol lowing a single administration of 2 mg/kg bw reache d its calculated maximum at 0.9 and 0.1 hr postdose, respectively. The calculated maximum radioactivity concentrations in plasma were 4.41 and 4.15 mg eq./ kg bw, in males and females, respectively. These peak concentrations amounted to ca. 2 times of the equal distribution concentration of BYI 08330. Afte r reaching the maximum, females showed slightly lower decline of plasma radioactivity concentration as compared with males.

Absorption of the radioactivity was slightly slower for males compared to females. Absorption was followed by a fast initial elimination phase and a moderate terminal elimination phase. The area under the curves (AUC 0- ∞ ) indicated a slightly higher systemic exposure for males than for females. The elimination rate constant was very similar for male s and females. The mean residence time was low in males and slightly higher in females.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 11 April 2008

The results obtained from the repeated low dose exp eriments with pre-treated animals were in the same range as those from the single low dose tests. Abso rption of the radioactivity was similar for males a nd females and was followed by a fast initial eliminat ion phase and a moderate terminal elimination phase

. The mean residence time was low and similar for mal es and females.

The results from the high dose experiments indicate d a sex difference in the maximum plasma concentration. The C max value was significantly higher for males than for females. Compared to all low dose tests and taking the AUC values into account, the plasma concentrations were more or less proportional to the dose ratio. This indicated tha t the absorption process had not been saturated at the high dose level of 100 mg/kg bw. The absorption of the radioactivity was slightly slower than in the low dose experiments and slightly slower for the males compared to females. As in low dose tests, the absorption was followed by a fast initial eliminati on phase and a moderate terminal elimination phase. The areas under the curves (AUC 0- ∞ ) indicated a slightly higher systemic exposure for males than for females. The mean residence time was in the same ra nge as in the low dose test and quite similar for males and females. Comparison of the absorption pha ses of the kinetic curves between the low and high dose groups showed a broader maximum for males of t he high dose test. The maximum concentration (C max ) was reached slightly later at this dose level and the following initial elimination phase was slight ly longer than observed for the low dose level. The cu rves at the end of the terminal elimination phase were comparable. Radioactivity levels in plasma are summarised in Table 6-3. The calculated pharmacokinetic parameters are shown in Table 6-4.

The distribution of radioactive residues in the bod y was analyzed at the time of sacrifice 48 h after dosing by radioactivity measurement of the major organs an d tissues. Less than 0.2% of the administered dose was detected in the body. The residues in all orga ns and tissues at the time of sacrifice were low in all tests and partly below the limit of detection. The highest equivalent concentrations were detected in the liver (0.002-0.18 mg/kg) and kidney (0.001-0.11 mg/ kg), the responsible organs for the degradation and excretion. The distribution of radioactivity levels in organs and tissues of male and female rats 48 h after oral administration of [azaspirodecenyl-3- 14 C]BYI 08330 is shown in Table 6-5.

C. METABOLISM:

No parent compound was detected in urine and faeces of all tests. The main metabolic reaction was cleavage of the ester group which resulted in the f ormation of the primary and most predominant metabolite BYI 08330-enol (53-87% of the administer ed dose). All other identified metabolites could be derived from the enol intermediate. Further metabol ic transformations were oxidative demethylation of the 8-methoxy group to BYI 08330-desmethyl-enol (5- 37 % of the administered dose) and oxidation of the azaspiro moiety to BYI 08330-ketohydroxy (0.5-1 .1 %) and BYI 08330-desmethyl-ketohydroxy (0.1- 0.7 %). Other minor metabolic transformations were conjugation of the enol with glucuronic acid to BYI 08330-enol-GA (0.2-0.8 %) and oxidation of the arom atic methyl group of the enol metabolite to BYI 08330-enol-alcohol (0.4-1.6%). A sex-related diffe rence was observed in metabolism. Male rats exhibited much higher rates of demethylation of BYI 08330-enol to BYI 08330-desmethyl-enol (25-37 %) compared to females rats (5-10 %). Demethylation w as more pronounced in the high dose tests than in the low dose tests.

Identification rates were high in the range of 87-9 5% of the administered dose. Only 0.4-3.5% in total could not be identified. No single unidentified met abolite accounted for >0.7 % of the dose. The proportion of non-identified metabolites was higher for male rats than for female rats.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 12 April 2008

Metabolites excreted with urine and faeces followin g oral administration of [azaspirodecenyl-3- 14 C] BYI 08330 are shown in Table 6-7. The proposed metaboli c pathway of BYI 08330 in the rat is presented in Figure 6-1.

D. EXCRETION:

The excretion of BYI 08330 was fast and almost comp lete 24 h after administration. Significant sex or dose related differences were not observed. Excreti on was mainly renal and quite similar for males and females in low, high and repeated low dose tests. About 88-95% of the administered dose was eliminated via urine and about 2-11% via faeces. I n every dose group, quantitative recovery of dosed radioactivity (91.44-99.78 % of dose) was accomplis hed.

The formation of 14 CO 2 and volatile metabolites was investigated for male and female rats in the autoradiography study (study 5.1.1/01 and Tables 5. 1.1-03 and 5.1.1-04 therein). In this study, no significant exhalation of radioactivity was observe d into the expired air until 48 h postdose, regardl ess of the sex. The excretion of radioactivity following oral administration of [azaspirodecenyl-3- 14 C]BYI 08330 is shown in Table 6-6.

Table B.6-2: Excretion and radiolabelled residues i n organs and tissues of male and female 48 hours after oral administration of [azaspirodecenyl -3-14C]BYI 08330

Radioactivity in % of dose administered

Test 8 Test 2 Test 3 Test 4 Test 5 Test 9 Dose [mg/kg] 2 2 100 100 2 2 (14+1) (14+1)
Pretreatm. Pretreatm.
Sex Male Female Male Female Male Female
Urine 93.34 87.92 89.14 93.78 91.48 94.78
Faeces 5.11 3.34 10.51 2.98 6.59 1.78
Total excreted 98.45 91.26 99.65 96.76 98.07 96.56

Skin 0.004 0.070 0.015 0.014 0.011 0.024 Sum Organs 0.019 0.058 0.112 0.021 0.067 0.036 Body without GIT 0.023 0.129 0.126 0.035 0.078 0.060

GIT 0.014 0.043 0.012 0.012 0.035 0.024 Total Body 0.038 0.171 0.138 0.047 0.113 0.083

Balance 98.49 91.44 99.78 96.81 98.19 96.65
Radioactivity in % of total recovered radioactivity

Urine 94.79 96.06 89.34 96.86 93.23 98.06
Faeces 5.18 3.75 10.52 3.10 6.66 1.85
Total excreted 99.97 99.81 99.86 99.96 99.89 99.91

Skin 0.004 0.078 0.015 0.014 0.012 0.025 Sum Organs 0.019 0.064 0.113 0.022 0.069 0.037 Body without GIT 0.024 0.142 0.127 0.036 0.080 0.062– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 13 April 2008

Radioactivity in % of dose administered

Test 8 Test 2 Test 3 Test 4 Test 5 Test 9

GIT 0.014 0.047 0.012 0.013 0.036 0.025 Total Body 0.038 0.189 0.139 0.049 0.117 0.086

Norm.-Factor 1.015 1.101 1.002 1.034 1.022 1.036

Absorption rate 94.81 96.20 89.47 96.90 93.31 98.12

Absorption rate = radioactivity in urine + radioacti vity in body without GIT

Table B.6-3: Time course of radioactivity measured in the plasma of rats after oral administration of [azaspirodecenyl-3-14C]BYI 08330
Equivalent concentration C [μg/g] in plasma (measu red values)

Test 8 Test 2 Test 3 Test 4 Test 5 Test 9 2 mg/kg 2 mg/kg 100 mg/kg 100 mg/kg 2 mg/kg 2 mg/k g (14+1) (14+1) Time pretreatm. pretreatm. (hours post administration) Male Female Male Female Male Female

0.08 1.435 2.225 14.436 16.071 1.784 1.389
0.17 2.568 3.836 45.424 52.592 3.935 2.301
0.33 3.396 3.239 101.600 89.249 4.891 2.673
0.67 4.139 2.925 157.374 116.044 4.962 2.687
1.00 4.402 2.394 189.735 115.816 4.383 2.179
1.50 4.004 1.598 204.390 100.464 3.401 1.531
2.00 3.390 1.067 201.130 87.011 2.581 0.929
3.00 2.358 0.659 194.017 58.094 1.661 0.395
4.00 1.558 0.515 168.045 40.126 1.114 0.229
6.00 0.676 0.356 122.464 21.094 0.495 0.142
8.00 0.327 0.280 78.375 16.608 0.246 0.132
24.00 0.005 0.035 0.415 0.204 0.009 0.014
32.00 0.003 0.023 0.189 0.150 0.003 0.010
48.00 0.002 0.011 0.154 0.098 0.002 0.009

Table B.6-4: Pharmacokinetic parameters obtained by plasma curve analysis using the software program TOPFIT

Parameter Units Test 8 Test 2 Test 3 Test 4 Test 5 Test 9 Dose mg/kg bw 2 2 100 100 2 (14+1) 2 (14+1) pretreatm. pretreatm.
Sex of rats male female male female male female
No. of animals 4 4 4 4 4 3 Mean rat weights (at administration) g 202 204 205 202 213 208

Renal elimination % of dose
admin. 93.34 87.92 89.14 93.78 91.48 94.78– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 14 April 2008

Parameter Units Test 8 Test 2 Test 3 Test 4 Test 5 Test 9

Correl. coefficient 0.999 0.996 0.996 0.997 1.000 0.994

C max (model) μg/g 4.41 4.15 210 117 5.21 2.98

t max (model) h 0.89 0.09 2.03 0.77 0.45 0.35
C max (experiment) μg/g 4.40 3.84 204 116 4.96 2.69

t max (experiment) h 1.00 0.17 1.50 0.66 0.66 0.66

t 1/2 a h <0.01 <0.01 0.17 0.06 0.10 0.07

t 1/2 e(1) h 0.31 4.79 1.70 0.19 3.62 0.47

t 1/2 e(2) h 20.1 29.7 17.5 27.2 92.7 13.2

t lag a h <0.01 0.08 0.06 0.05 0.03 0.03

AUC (0- ∞ ) μg/g x h 16.4 10.2 1380 451 14.6 7.64

k 1e 1/h 246 258 0.98 3.53 5.61 8.53

CL/f mL/min/kg 2.03 3.27 1.21 3.70 2.28 4.36

CL R mL/min/kg 1.89 2.89 1.08 3.47 2.09 4.13

MRT h 3.39 9.68 4.90 4.26 5.29 4.38

MRT abs h 2.55 1.16 2.53 2.99 1.91 1.94

MRT disp h 0.84 8.52 2.37 1.27 3.38 2.44
Vss L 0.10 1.67 0.17 0.28 0.46 0.64 weighting function* g = 1 g = 1/y g = 1 g = 1/y g = 1 g = 1 compartments number 3 3 3 3 3 3
Remarks:
CL renal = CL total x % urine excretion x 0.01
* for some individual data points weighting factors were used for better curve fitting

Table B.6-5: Distribution of radioactivity in rat t issues/organs at sacrifice, 48 hours after oral administration of [azaspirodecenyl-3-14C]BYI 08330 Equivalent concentration C [μg/g] (mean values)

Test 8 Test 2 Test 3 Test 4 Test 5 Test 9
Dose [mg/kg] 2 2 100 100 2 2 (14+1) (14+1)
pretreatm. pretreatm.
Sex Male Female Male Female Male Female Erythrocytes 0.0010 0.0013 0.0385 0.0250 0.0007 0.00 07
Plasma 0.0011 0.0015 0.0703 0.0267 0.0009 0.0010 Spleen 0.0006 0.0009 0.0626 <LOD 0.0006 0.0006
GIT 0.0024 0.0094 0.0809 0.0999 0.0035 0.0046

Liver 0.0076 0.0035 0.1792 0.0502 0.0094 0.0019 Kidney 0.0009 0.0040 0.1065 0.0609 0.0024 0.0027– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 15 April 2008

Equivalent concentration C [μg/g] (mean values)

Test 8 Test 2 Test 3 Test 4 Test 5 Test 9
Dose [mg/kg] 2 2 100 100 2 2 (14+1) (14+1)
pretreatm. pretreatm.
Sex Male Female Male Female Male Female

Perirenal fat <LOD <LOD <LOD <LOD 0.0047 <LOD Adrenal gland <LOD <LOD <LOD <LOD 0.0062 <LOD

Testis 0.0008 n.a. 0.0622 n.a. 0.0003 n.a.
Ovaries n.a. <LOD n.a. <LOD n.a. <LOD
Uterus n.a. <LOD n.a. <LOD n.a. 0.0015
Skel. muscle <LOD 0.0013 0.0377 <LOD 0.0006 <LOD
Bone femur <LOD 0.0030 0.0855 0.0534 0.0009 <LOD

Heart 0.0006 0.0010 0.0332 0.0189 0.0006 0.0005 Lung 0.0005 0.0011 0.0327 0.0220 0.0006 0.0007
Brain <LOD 0.0005 <LOD <LOD <LOD <LOD Thyroid gland <LOD <LOD <LOD <LOD <LOD <LOD
Skin 0.0008 0.0060 0.0567 0.0529 0.0008 0.0022
Carcass <LOD 0.0015 0.1649 0.0257 0.0009 0.0010 Legend:
<LOD = value below detection limit n.a. = not applicable The given concentrations are mean values from 4 ani mals of each test.

Table B.6-6: Time course of the excretion of radioa ctivity via urine and faeces of male and female rats after oral administration of [azaspirodecenyl- 3-14C]BYI 08330

Cumulative excretion of radioactivity in % of dose administered

Test 8 Test 2 Test 3 Test 4 Test 5 Test 9 Dose [mg/kg] 2 2 100 100 2 2 (14+1) (14+1) pretreatm. pretreatm.
Sex Male Female Male Female Male Female

Urine (time post administration [h])

4 34.48 36.50 17.95 55.19 40.65 56.35

8 77.48 45.10 50.87 78.95 77.77 57.55
12 89.95 * * * * *

24 92.96 85.68 88.32 93.03 90.86 93.16

48 93.34 87.92 89.14 93.78 91.48 94.78

Faeces (time post administration [h])

24 4.89 2.30 9.95 2.79 5.94 1.44

48 5.11 3.34 10.51 2.98 6.59 1.78

Sum excreted 98.45 91.27 99.64 96.76 98.08 96.56
*no sample collected– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 16 April 2008

Table B.6-7: Metabolite distribution in the excreta of male and female rats dosed with [azaspirodecenyl-3-14C]BYI 08330

Percent of administered dose
Test 8 Test 2 Test 3 Test 4 Test 5 Test 9 2 mg/kg 2 mg/kg 100mg/kg 100mg/kg 2 mg/kg 2 mg/kg rep.dose rep.dose (14+1) (14+1) Male Female Male Female Male Female
Excreted with urines 93.34 87.92 89.14 93.78 91.48 94.78
Excreted with faeces 5.11 3.34 10.51 2.98 6.59 1.78
Excreted with urines and faeces 98.45 91.27 99.64 9 6.76 98.08 96.56
Extracted from faeces 4.58 2.30 8.71 1.97 5.43 1.33 Compound (report name):
BYI 08330-
-enol-GA 0.51 0.16 0.79 0.18 0.43 0.15
-enol-alcohol 1.25 0.42 1.58 0.43 1.20 0.81 -desmethyl-enol 26.93 5.01 37.09 10.06 24.73 5.38 -desmethyl-ketohydroxy 0.31 0.21 0.65 0.06 0.36 0. 11
-enol 64.82 80.50 52.92 83.37 66.06 86.66 -ketohydroxy 0.87 1.10 0.49 0.49 0.59 0.80
Total identified 94.69 87.40 93.52 94.59 93.37 93.89 unknown (peak 1) 0.18 - 0.22 - 0.17 - unknown (peak 3) 0.40 0.02 0.51 - 0.42 0.10 unknown (peak 4) 0.54 - 0.72 - 0.59 - unknown (peak 5) 0.21 - 0.31 - 0.26 - unknown (peak 6) 0.39 0.03 0.27 - 0.44 - unknown (peak 8) 0.02 - 0.23 - 0.03 - unknown (peak 9) 0.05 0.02 0.06 0.04 0.07 0.03 unknown (peak 10) 0.06 0.02 0.10 - 0.06 0.03 unknown (peak 12) 0.31 0.09 0.34 0.12 0.28 0.14 unknown (peak 13) 0.55 0.13 0.66 0.18 0.49 0.16 unknown (peak 16) 0.13 0.09 0.05 0.06 0.10 0.10 unknown (peak 18) - - - - - 0.02 unknown (peak 19) - - 0.03 - - 0.01
Total unidentified 2.85 0.40 3.51 0.41 2.92 0.60 Faeces extracts not analysed 0.07 0.07 0.85 0.73 0. 24 0.03
Total uncharacterised = total
unidentified and faeces extracts not analysed 2.92 0.47 4.36 1.14 3.16 0.62

Solids after faeces extraction 0.23 0.11 0.39 0.09 0 .27 0.09 Urines not analysed (24-48 h) 0.38 2.24 0.82 0.75 0 .62 1.62 Faeces not analysed (24-48 h) 0.22 1.05 0.56 0.19 0 .66 0.34 Total excreta samples not analysed 0.84 3.39 1.77 1 .03 1.54 2.05
Total 98.45 91.26 99.65 96.76 98.07 96.56
= Not detected– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 17 April 2008

NH

O O O O

CH 3

CH 3 H 3 C H O CH 3

NH

O O H CH 3

CH 3 H O CH 3 H O

NH

O CH 3

CH 3 CH 3

O O O H

O H

O H O H O

NH

O O CH 3

CH 3 H O

OH CH 3 H OH

NH

O O H CH 3

CH 3 H O

NH

O O H CH 3

CH 2 OH CH 3

NH

O O CH 3

CH 3 H OH

OH BYI08830

BYI08330-enol-alcohol BYI08330-enol BYI08330-enol-GA

BYI08330-desmethyl-enol BYI08330-ketohydroxy

BYI08330-desmethyl-ketohydroxy

Figure B.6-1: Proposed metabolic pathway of BYI 083 30 in rats

III. CONCLUSIONS

A. CONCLUSIONS :

The fate of radiolabelled BYI 08330 in male and fem ale rats following single and repeated oral administration was characterized as follows:

• Absorption : Fast and complete absorption of the radioactive t est compound from the gastrointestinal tract for the low dose, high dose, and repeated dos e groups of male and female rats– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 18 April 2008

• Distribution : Fairly equal distribution of the radioactivity in males and females within the blood and most organs and tissues with preference for the liv er and kidney as the main metabolizing organ also responsible for excretion

• Steady decline of radioactivity concentrations in plasma by several orders of magnitude within 48 hours

• Very low concentrations of radioactivity in tissue s and organs at the time of sacrifice 48 hours postdose which were <LOD for some organs/tissues

• Excretion : Rapid and complete excretion of radioactivity mai nly via the renal route in all dose groups

• Metabolism : Cleavage of the ester group as the major metaboli c transformation resulting in BYI 08330-enol as the major metabolite and key inte rmediate in the metabolic pathway

• Demethylation of the methoxy group in the cyclohex yl ring resulting in BYI 08330-desmethyl-enol as the second prominent metabolite. This metabolic tr ansformation was significantly higher in male rats than in females.

• Further oxidative transformation and conjugation o f BYI 08330-enol were of minor importance

• Very similar metabolic profiles for low dose and r epeated dose tests. Similar profiles for low dose a nd high dose tests but with a somewhat higher proporti on of BYI 08330-desmethyl-enol in the high dose
tests

• Significant difference in metabolic profiles betwe en male and female rats for low dose, high dose and repeated dose groups with male rats exhibiting much higher proportions of BYI 08330-desmethyl-enol than females

• High identification rate of metabolites in all dos e groups (87-95 % of administered dose)

B. REVIEWER COMMENTS :

This disposition study of [Azaspriodecenyl-3- 14 C]BYI 08330 in male and female rats showed excellen t recovery of administered radioactivity (91-99%). T he test article was rapidly absorbed and excreted primarily in the urine (89-98% of dose) as shown by excretion data and by the pharmacokinetic parameters. Total excretion of radioactivity was n early 100% for all test groups indicating little or no potential for bioaccumulation under the conditions of this study. Little potential for sequestration of the test article or its biotransformation products was further demonstrated by extremely low tissue residues. An enol metabolite and the demethylated enol were clearly the major metabolites (53-87% and 5-37% of the dose, respectively), the variabili ty being a notable gender-related difference; males more effective in formation of BYI 08330-enol and B YI 08330-desmethyl-enol.

The investigator’s conclusions are consistent with the data presented in the study. This appeared to be a well-designed, conducted, and reported study.

This study in rats meets current OPPTS 870.7485 Gui delines for a metabolism and pharmacokinetics study. This study in the rat is considered Acceptable / Guideline and meets the guideline requirement for a metabolism and pharmacokinetics study (870.74 85) in rats.

C. STUDY DEFICIENCIES:

There was no indication of randomization of test an imals for experimental group assignments although it was noted that minor body weight variat ions within groups affected actual administered dose; this was not considered a significant flaw. There were no deficiencies identified in the study report that would adversely affect the study result s or their interpretation.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 19 April 2008

B.6.1.2 Toxicokinetic studies – second single dose, oral route in rats (OECD Annex IIA 5.1.1

and 5.1.2)

B.6.1.2.1 Metabolism and Pharmacokinetics (OECD Ann ex IIA 5.1.2)

CITATION : Klempner, A. 2006. [Azaspirodecenyl-3- 14 C]BYI 08330: Depletion of Residues and

Metabolites in Plasma, Urine, Liver, Kidney and Tes tis of the Male Rat. Study No. M41819177. Report No. MEF 06/328, Document No. M-27 5731-01-2. Bayer CropScience AG Development, Metabolism/Environmental Fate, 4078 9 Monheim am Rhein, Alfred Nobel Str. 50, Germany.

I. MATERIAL AND METHODS

A. MATERIAL

1. TEST MATERIAL

IUPAC Name: cis-3-(2,5-dimethylphenyl)-8-methoxy-2- oxo-1-azaspiro[4.5]dec-3- en-4-yl ethyl carbonate Code name: BYI 08330 Common name: Spirotetramat (proposed ISO) Empirical formula: C 21 H 27 N O 5 Molar mass: 373.45 g/mol Water solubility: pH 4 = 33.5 mg/L, pH 7 = 29.9 mg /L pH 9 = 19.1 mg/L (unstable) all at 20°C n-Octanol/water partition coefficient: pH 4 = 2.51, pH 7 = 2.51, pH 9 = 2.50 Labelling: azaspirodecenyl-3- 14 C Specific radioactivity of the original radiolabelled batches: 3.67 MBq/mg (99.1 μCi/mg)

Specific radioactivity used for administration: Low dose tests (test 1 - 3): 3.67 MBq/mg (99.1 μC i/mg) High dose tests (test 4 -6): 0.0073 MBq/mg (0.20 μCi/mg) Radiochemical purity: > 98 % (certified, HPLC and T LC with radiodetection) Dose level: 2 mg/kg and 1000 mg/kg body weight Vehicle: 0.5 % aqueous tragacanth suspension Stability of the test material: The stability of [a zaspirodecenyl-3- 14 C]BYI 08330 was demonstrated by radio HPLC analysis of the administ ration suspensions of each test immediately after dosing– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 20 April 2008

2. Test Animals:

Species: Rat ( Rattus norvegicus domesticus )
Strain: Wistar Hsd/Cpb: WU Breeding facility: Harlan & Winkelmann GmbH, Gartenstraße 27, 33178 Borchen, Germany
Sex and numbers involved: Males: 24 Age: 8 weeks at the time of delivery Body weight: Males: 194 – 205 g at the time of admi nistration 175 – 204 g at the time of sacrifice Acclimatization: Makrolon ® cages on wood shavings in the test facility for ab out 7 days prior to the administration. Identification: Cage cards on which the study numbe r, test compound name and individual animal number were displayed. Additional labelling with waterinsoluble spots on the tail Housing: After administration of the radiolabelled test item individually in Makrolon ® metabolism cages under conventional hygienic condit ions in airconditioned rooms Temperature 22 - 27 °C, relative humidity 43 – 61 %

. 12 / 12 hours light / dark cycle, air change 10 – 1 5 times per hour. Feed and water: rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, CH-4303 Kaiseraugst, Switzerland (ca. 16 g per animal and day) last feeding ca. 16 h prior to dosing next feeding ca. 6 h after dosing. tap water from municipal water supply, ad libitum

B. STUDY DESIGN:

Justification of the dose levels The low dose level was selected at 2 mg/kg bw to ma tch the dose of the low dose tests in the ADME study with BYI 08330 (Report No. MEF-048/04). The h igh dose was selected taking into account the findings from a rat mechanistic study (Report No. S A 04181), where degenerative effects on round / elongating spermatids in the testis of the male rat were found after >10 repeated doses of 1000 mg/kg bw/day. Therefore, 1000 mg/kg bw was selected as th e high dose in this study.

Dosing A summary of the experimental protocol is provided in Table 6-8.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 21 April 2008

Table B.6-8: Experimental protocol summary for met abolism and disposition study of [azaspirodecenyl-3-14C]BYI 08330 in rats

Exp. no. Experiment type/dose Duration (hrs) Comments
1 Single low dose (2 mg/kg) 1 Urine collected at 0-1 hr; blood, testes, liver, kidney, g.i. tract (+contents), skin, carcasss
assessed at termination 2 Single low dose (2 mg/kg) 8 Urine collected at 0-1 and 4-8 hrs; blood, testes, liver, kidney, skin, carcasss assessed at
termination 3 Single low dose (2 mg/kg) 24 Urine collected at 0- 1, 4-8 hrs, 8-24 hrs; blood, testes, liver, kidney, g.i. tract (+contents skin),
carcasss assessed at terminatio 4 Single high dose (1000 mg/kg) 1 Urine collected at 0-1 hr; blood, testes, liver, kidney, skin, carcasss assessed at termination (1 h r post dose) 5 Single high dose (1000 mg/kg) 8 Urine collected at 0-1 and 4-8 hrs; blood, testes, liver, kidney, skin, carcasss assessed at sacrifice (8 hrs post dose) 6 Single high dose (1000 mg/kg) 24 Urine collected a t 0-4, 4-8 hrs, 8-24 hrs; blood, testes, liver, kidney, g.i. tract (+contents skin), carcasss assessed at termination (24 hrs post dose)

Each of the rats in the low dose groups (4 rats per group) of test 1 - 3 orally received 2 mg/kg bw of azaspirodecenyl-3- 14 C BYI 08330 suspended in 0.5 % aqueous tragacanth a fter ca. 16 h of fasting. The dosing suspension (0.2 mg/mL) was prepared one day before dosing and stored at 4 ° C. Each of the rats in the high dose groups (4 rats per group) of test 4 - 6 orally received 1000 mg/kg bw of azaspirodecenyl-3- 14 C BYI 08330 suspended in 0.5 % aqueous tragacanth a fter ca. 16 h of fasting. The dosing suspension (100 mg/mL) was prepared three da ys before dosing and stored at 4 ° C.

The suspensions were administered to the rats by or al gavage using a syringe attached to an animalfeeding knob cannula. Each animal received a volume of 2 mL (= 10 mL/kg bw calculated for a nominal body weight of 200 g). The amounts of the administe red test substance were calculated by dividing the administered radioactivity amounts by the specific radioactivity. The actual mean administered dose of [azaspirodecenyl-3- 14 C]BYI 08330 was 1.83 mg/kg bw for the low dose grou ps and 934 mg/kg bw for the high dose groups. The stability of the test compoun d in the suspensions was assured by radio HPLC analysis of aliquots after administration.

Collection of excreta After administration of the radiolabelled test item , the rats were kept individually in Makrolon® metabolism cages, which allowed for separate and qu antitative collection of urine and faeces. For test 1 and test 4 (sacrifice 1 h after dosing), urine was collected separately for each animal in a cryogenic trap cooled with dry ice for the interval of 1 h, faeces were combined with the gastro-intestinal tract at the time of sacrifice. For test 2 and test 5 (sacrifice 8 h after dosing), urine was collected separately for each animal in a cryogenic trap cooled with dry ice in i ntervals of 4 h and 8 h, faeces were combined with the gastro-intestinal tract at the time of sacrifice. F or test 3 and test 6 (sacrifice 24 h after dosing), Urine was collected separately for each animal in a cryogenic trap cooled with dry ice in intervals of 4 h, 8 h, and 24 h, faeces were collected for the interval 24 h.. Th e funnels for urine collection were rinsed with– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 22 April 2008

demineralised water at the end of each sampling per iod. The rinsing solutions were drained into the same vial as the corresponding urine fraction. The radioactivity was determined by LSC. The faeces samples were lyophilised (freeze-dried), weighed, and homogenised. The radioactivity was determined by combustion/LSC.

Sacrifice and organ/tissue sampling At the respective test end (1 h, 8 h or 24 h after administration), the animals were anaesthetised usi ng Pentobarbital-Na. They were sacrificed by transecti on of the cervical blood vessels. The collected blo od was separated into plasma and erythrocytes by centr ifugation. The following organs/tissues were collected: erythrocytes, plasma, gastro-intestinal tract (GIT), liver, kidneys, testis, skin, and carc ass. Erythrocytes, GIT, carcass, and skin prepared at th e end the experiments were weighed immediately after the dissection and again following lyophilisa tion. Finally, they were homogenised and the radioactivity determined by combustion/LSC. Plasma samples were combined for all animals of eac h test. Liver, kidneys, and testis were weighed and pooled for all animals of each test for the extract ion of radioactive residues.

Radioactivity measurement The measurement of the radioactivity in liquid samp les was carried out by liquid scintillation countin g (LSC) of 1 – 5 replicates. All solid samples and bl ood standards were weighed and combusted in an oxygen atmosphere with an Oxidizer 307/387 (Packard Instruments). For all samples, the limit of detection (LOD) was established at ca. 20 dpm measu red per aliquot after correction for the background radioactivity. The limit of quantification (LOQ) fo r each individual measurement was established as 2 times of the background radioactivity (dpm) of each instrument/method. This background counting rate was in a range between 12 – 30 cpm (approximately e qual to 12 – 30 dpm) and it was automatically subtracted from the measuring results. A quench and counting efficiency correction for transformation of gross counts (cpm) into net counts (dpm) was automa tically performed by the instruments.

Metabolite analysis Urine samples were combined for all animals of a te st group and analysed by HPLC without prior sample preparation. Combined plasma samples (e.g. 2 mL) we re mixed with 5 % formic acid (0.5 mL) and centrifuged. The supernatants were analysed by radi o-HPLC.

The livers, kidneys, and testis of all animals of a test group were combined and cut in small pieces. Then, the composite samples were mixed with acetoni trile/water and defoamer and extracted by maceration with a Polytron or Ultraturrax homogenis er. Extracts and solids were separated by centrifugation or filtration and the solids were fu rther extracted with acetonitrile/water (7/3, v/v) as described above. The volumes of the extracts were m easured and aliquots radioassayed by LSC. The post extraction solids were dried at room temperatu re and aliquots were solubilized with a tissue solubilizer and radioassayed by LSC. All extracts w ere combined, filtered, mixed with 0.1 mL emulsifie r and evaporated to the aqueous remainder which was a nalysed by radio-HPLC for metabolic profiling.

The metabolites in the samples were quantified by H PLC using a reversed phase column (C18) and the eluting solvents water/formic acid = 99: 1 (v/v) an d acetonitrile/water/formic acid = 97: 2: 1 (v/v/v) in the gradient mode. Detection was performed by a UV (254 nm) and a radioisotope detector with a glass scintillator. Metabolites in urine were identified by comparison of the elution pattern of the samples from this study with the urines of male rats (low dose t est no. 8 dosed with 2 mg/kg bw) from the ADME stud y (Report No. MEF-048/04). For plasma and extracts of organs, metabolites were assigned by comparison of profiles with those of urine. Furthermore, the m ost prominent metabolites BYI 08330-enol, BYI 08330

-– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 23 April 2008

desmethyl-enol and BYI 08330-ketohydroxy were ident ified representatively in urine and liver extract b y HPLC co-chromatography with non-radiolabelled refer ence items.

II. RESULTS AND DISCUSSION

A. DISTRIBUTION AND RESIDUES IN ORGANS AND TISSUES :

Recovery of administered radioactivity ranged from 94.8 to 121.8% (Table 6-9). Recovery was slightly lower for the high dose groups all dose groups gene rated acceptable mass balance data.

In low dose test group 1, sacrificed 1 h after dosing, 33.1 % of the dose were detected in the organs and tissues, 32.1 % in the GIT (plus faeces for test 1 and 2) and 12.5 % in urine. About 66 % of the administered dose were already excreted via urine 8 h after administration in test group 2 resulting i n accordingly distinctively lower residues in organs and tissues of only about 12 % of the dose and abou t 9 % in the GIT. The fast excretion and decline of res idues in the body continued, so that 24h after administration only 0.1 % of the dose were found in organs and tissues and 0.2 % in the GIT, but 112 % in urine.

The highest residues in the organs were detected in liver and kidney, which are the primary organs for metabolism and excretion. From 1 h to 24 h, the val ues in liver and kidney decreased from 23 % (liver) and 5 % (kidney) of the dose to <0.1 % of the dose. The corresponding TRR values of liver and kidney were nearly identical 1 h after administration (11. 7 and 11.2 mg/kg) with a rapid decline to 0.02 and 0.01 mg/kg within 24 h (Table 6-10).

The radioactivity in plasma was 3.5 % of the admini stered dose 1h after dosage showing the same fast decline as the other organs to <0.1 % of the dose 2 4 h after dosage. The TRR values of plasma were distinctively lower than in liver and kidney, but s howed a similar fast decline from 4.5 mg/kg after 1 h down to <0.01 mg/kg after 24 h. The TRR values in t estis were much lower than in plasma for all time points (0.7 mg/kg at 1 h declining to <0.01 mg/kg a t 24 h).

In high dose test group 4, sacrificed 1 h after dosing, only 2. 4 % of the dose was detected in the organs and tissues, 85.9 % in the GIT (plus faeces for tes t 4 and 5) and 1.6 % in urine. Only 16 % of the administered dose was excreted via urine 8 h after administration in test group 5. The residues declin ed slowly from 1 h to 8 h from 2.4 % to 1.8 % of the d ose in organs and tissues and from 86 % to 73 % of the dose in the GIT. From 8h to 24h, the decline of radioactive residues was slightly higher resulting in 0.7 % of the dose in organs and tissues, 47 % in th e GIT. In urine, 27 % were found at 24h post dosage

.

In contrast to low dose tests, the highest TRR valu es 1 h and 8 h after dosage were not detected in li ver or kidney, but in plasma. The TRR values in plasma were about 10 - 20 % higher than in liver and kidne y and amounted to 353 mg/kg in plasma after 1h and to 271 mg/kg in plasma after 8h (liver: 315 mg/kg and 246 mg/kg, and kidney: 308 mg/kg and 222 mg/kg) . The decline in all organs was slow from 1 h to 8 h and slightly faster from 8 h to 24 h with 102 mg/ kg for plasma, 109 mg/kg for liver and 85 mg/kg for kidney. The TRR values in testis were much lower th an in plasma for all time points (66 mg/kg at 1 h, 77 mg/kg at 8 h and 28 mg/kg at 24 h).

The total radioactive residues (TRR) detected in pl asma, organs and tissues at sacrifice (expressed as equivalent concentration C, as dose normalised conc entration CN and as percentage of administered dose) are given in Table 6-10.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 24 April 2008

B. METABOLISM:

In the low dose tests, parent compound was not detected in any sam ple or tissue. BYI 08330-enol was the main metabolite in all samples. BYI 08330-desme thyl-enol was the second prominent metabolite in urine, plasma and testis, whereas in liver and kidn ey BYI 08330-ketohydroxy was found as the second prominent metabolite. BYI 08330-ketohydroxy was onl y present at trace levels in plasma, testis and urine. BYI 08330-enol-GA, -enol-alcohol and –desmet hyl-ketohydroxy were detected at low levels. Therefore, they are of minor importance. Residues i n all tissues and plasma 24 h after dosage were too low for quantification of metabolites. Identificati ons rates were high in the range of 89 – 100 %.

The metabolic profiles for urine were in good agree ment with the results of the low dose tests in the ADME study. A summary of the metabolism in urine is presented in Table 6-11; the metabolism in plasma and organs is summarized in Table 6-12.

Similar results were obtained for the high dose tests. Parent compound was not detected in any sam ple or tissue. BYI 08330-enol was the main metabolite i n all samples. BYI 08330-desmethyl-enol was the second prominent metabolite in urine, plasma and or gans. Significant proportions of BYI 08330- ketohydroxy were present in liver and kidney, only. BYI 08330-enol-GA, BYI 08330-enol-alcohol and BYI 08330-desmethyl-ketohydroxy were detected at low le vels and were of minor importance. Identifications rates were high in the range of 95 – 100 %. A summa ry of the metabolism in urine is presented in Table 6-11; the metabolism in plasma and organs for the h igh dose is summarized in Table 6-13.

C. EXCRETION:

The urinary excretion was fast in the low dose test groups 1 – 3 and almost completed within the first day after administration. About 92 % of the recovered r adioactivity (equivalent to 112 % of the administer ed dose) was excreted via urine. About 8 % of the reco vered radioactivity (equivalent to 10 % of the administered dose) were detected in faeces of test 3, 24 h after dosage.

In high dose test groups 4 – 6, excretion was distinctively slo wer than in low dose ones. Only 27 % of the administered dose were excreted via urine 24 h afte r dosage and 18 % via faeces, respectively. The major part of the radioactivity was recovered in th e gastro-intestinal tract of animals. Detailed data for excretion and residues in organs and tissues are pr esented in Table 6-9.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 25 April 2008

Table B.6-9: Balance of radioactivity in excreta, p lasma, organs and tissues of male rats after single oral administration of 2 mg and 1000 mg [aza spirodecenyl-3-14C] BYI 08330/kg w

Radioactivity in % of dose administered (mean value s)

Test no.; Test 1 Test 2 Test 3 Test 4 Test 5 Test 6

No. of animals: 4 4 4 4 4 4

Admin.-route: Oral Oral Oral Oral Oral Oral Dose [mg/kg bw]: 2 2 2 1000 1000 1000 Spec. radioactivity [KBq/mg] 3670 3670 3670 7.34 7.34 7.34

Test duration 1 h 8 h 24 h 1 h 8 h 24 h

Urine

0 - 1 h 12.5 - - 1.6 - -

0 - 4 h - 20.1 37.3 - 4.9 6.0

4 - 8 h - 45.6 39.4 - 10.9 9.4

8 – 24 h - - 35.3 - - 11.4
Sum Urine 12.5 65.7 111.9 1.6 15.8 26.8

Faeces -* -* 9.5 -* -* 17.8

Erythrocytes 1.4 0.3 0.003 0.3 0.2 0.1

Plasma 3.5 1.0 0.003 0.5 0.4 0.1

Liver 22.9 9.2 0.067 1.2 0.9 0.4 Kidney 4.8 1.5 0.005 0.3 0.2 0.1

Testis 0.4 0.2 0.002 0.1 0.1 <0.1 Sum Organs 33.1 12.3 0.080 2.4 1.8 0.7

Skin 12.7 3.2 0.031 3.0 2.4 0.7
Carcass 28.3 10.6 0.097 5.9 6.1 1.8
GIT 32.1 8.6 0.205 85.9 72.9 47.0 Total Body 106.2 34.6 0.413 97.1 83.1 50.2

Balance 118.7 100.3 121.8 98.7 98.9 94.8
* For 1 h and 8 h animals, the faeces were include d in the GIT– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 26 April 2008

Table B.6-10: Total radioactive residues in organs and tissues of male rats sacrificed 1 h, 8 h, and 24 h after single oral administration of 2 mg and 1 000 mg [azaspirodecenyl-3-14C]BYI 08330/kg bw
Test no.; Test 1 Test 2 Test 3 Test 4 Test 5 Test 6

No. of animals: 4 4 4 4 4 4
Admin.-route: oral oral oral oral oral Oral Dose [mg/kg bw]: 2 2 2 1000 1000 1000 Spec. radioactivity [KBq/mg] 3670 3670 3670 7.34 7.34 7.34
Test duration 1 h 8 h 24 h 1 h 8 h 24 h

TRR [mg a.s. equiv./kg] (mean values)

Erythrocytes 1.723 0.412 0.003 199.2 147.9 47.1
Plasma 4.527 1.242 0.004 352.5 271.4 102.0
Liver 11.740 5.073 0.024 315.2 245.9 109.0 Kidney 11.220 3.517 0.011 307.9 221.7 85.3
Testis 0.668 0.342 0.004 66.5 77.4 28.5
Skin 0.978 0.259 0.003 119.2 93.4 31.7
Carcass 0.906 0.355 0.003 97.8 103.3 33.2

Dose normalised concentration CN (mean values)

Erythrocytes 0.951 0.220 0.002 0.212 0.156 0.045
Plasma 2.498 0.663 0.003 0.376 0.287 0.098
Liver 6.487 2.701 0.013 0.336 0.260 0.105 Kidney 6.197 1.873 0.006 0.328 0.235 0.082
Testis 0.369 0.182 0.002 0.071 0.082 0.028
Skin 0.540 0.138 0.001 0.127 0.099 0.031
Carcass 0.500 0.190 0.002 0.104 0.110 0.032

Radioactivity in percent of dose administered (mean values)

Erythrocytes 1.42 0.34 0.003 0.28 0.23 0.06
Plasma 3.52 0.98 0.003 0.52 0.42 0.14
Liver 22.92 9.23 0.067 1.21 0.86 0.38 Kidney 4.83 1.48 0.005 0.26 0.18 0.07
Testis 0.42 0.24 0.002 0.08 0.09 0.04
Skin 12.71 3.22 0.031 2.97 2.37 0.71
Carcass 28.30 10.55 0.097 5.87 6.10 1.81– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 27 April 2008

Table B.6-11: Metabolite distribution in urines of male rats collected 0 – 1 h, 0 – 8 h and 0 – 24 h after single oral administration of 2 mg and 1000 m g [azaspirodecenyl-3-14C]BYI 08330/kg bw

Test Test 1 Test 2 Test 3 Test 4 Test 5 Test 6
Dose [mg/kg bw] 2 2 2 1000 1000 1000 Sacrifice [h] 1 h 8 h 24 h 1 h 8 h 24 h
Sampling interval 0 - 1 h 0 - 8 h 0 - 24 h a 0 - 1 h 0 - 8 h 0 - 24 h a

% of Dose administered: 12.5 65.7 111.9 1.6 15.8 26.8

Peak Report name

no. BYI 08330- % of Administered dose

1 Unknown n.d. n.d. 0.07 n.d. n.d. n.d.

2 -Enol-GA 0.04 0.40 0.55 n.d. 0.12 0.25

3 Unknown n.d. 0.09 0.16 n.d. n.d. 0.02

4 Unknown n.d. 0.12 0.17 n.d. n.d. 0.08

5 Unknown n.d. 0.22 0.36 n.d. n.d. 0.07

6 Unknown n.d. 0.07 0.12 n.d. n.d. 0.04

7 Unknown n.d. n.d. 0.07 n.d. n.d. 0.03

8 Unknown n.d. 0.10 0.13 n.d. n.d. n.d.

9 Unknown n.d. 0.13 0.20 n.d. n.d. n.d.

10 Unknown n.d. n.d. 0.02 n.d. n.d. n.d.

11 -Enol-alcohol 0.26 0.81 1.15 n.d. 0.16 0.33
12 -Desmethyl-enol 1.26 20.50 33.09 0.15 4.18 10.63

13 Unknown 0.10 0.78 1.17 n.d. 0.16 0.29

14 - esmethyl ketohydroxy n.d. 0.13 0.20 n.d. n.d. 0.03

16 -Enol 10.72 42.12 74.07 1.46 11.15 14.99
18 -Ketohydroxy 0.07 0.26 0.37 n.d. n.d. 0.05

Total : 12.45 65.73 111.90 1.61 15.77 26.81

Identified: 12.4 64.2 109.4 1.6 15.6 26.3

Unknown * 0.1 1.5 2.5 n.d. 0.2 0.5

Total: 12.5 65.7 111.9 1.6 15.8 26.8
a Calculated sum of urine pools from 0 – 4 h, 4 – 8 h and 8 – 24 h
* Characterised based on their retention time in HPL C– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 28 April 2008

Table B.6-12: Total radioactive residues of metabol ites in plasma, liver, kidney and testis of male rats sacrificed 1h and 8 h after a single oral admi nistration of 2 mg [azaspirodecenyl-3-14C]BYI 08330/kg bw
Plasma Liver Kidney Testis

Test no. 1 2 1 2 1 2 1 2 Dose [mg/kg bw] 2 2 2 2 2 2 2 2 Sacrifice [h] 1 h 8 h 1 h 8 h 1 h 8 h 1 h 8 h TRR [mg a.s. equiv./kg] 4.527 1.242 11.740 5.073 11.220 3.517 0.668 0.3 42 Report name

BYI 08330- % of TRR

- Enol

- GA n.d. n.d. 0.1 0.2 0.4 0.4 0.1 n.d.

- Enol

- alcohol n.d. n.d. 0.5 0.4 0.2 n.d. 0.2 n.d.

- Desmethyl

- enol 2.3 3.6 7.3 6.8 2.3 3.6 2.5 3.3
Unknown n.d. n.d. 0.7 0.7 0.3 0.3 n.d. n.d.

- Desmethyl

- ketohydrox y n.d. n.d. 1.2 0.7 0.6 0.9 n.d. n.d.
Unknown n.d. n.d. 0.3 0.2 0.5 0.4 n.d. n.d.

- Enol 97.1 96.4 72.1 79.1 73.3 74.4 94.8 83.8
Unknown n.d. n.d. 1.0 0.3 1.7 1.1 n.d. n.d.

- Ketohydroxy 0.6 n.d. 13.2 8.1 20.0 18.7 2.3 2.4
Unknown n.d. n.d. 0.1 n.d. 0.3 n.d. n.d. n.d.
Unknown n.d. n.d. n.d. n.d. n.d. n.d. n.d. 9.4
Unknown n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.7
Subtotal: 100.0 100.0 96.5 96.5 99.5 99.7 99.9 99.4
Identified: 100.0 100.0 94.4 95.3 96.8 97.9 99.9 89.4
Unknown:* n.d. n.d. 2.1 1.2 2.7 1.8 n.d. 10.0 Not analysed / solids

-- -- 3.5 3.5 0.5 0.3 0.1 0.6
Total: 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0

n.d. = not detected
* Characterised based on their retention time in HPL C– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 29 April 2008

Table B.6-13: Total radioactive residues of metabol ites in plasma, liver, kidney and testis of male rats sacrificed 1 h, 8 h, and 24 h after a single o ral administration of 1000 mg [azaspirodecenyl-3- 14C]BYI 08330/kg bw
Plasma Liver Kidney Testis

Test no. 4 5 6 4 5 6 4 5 6 4 5 6 Dose [mg/kg bw] 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1 000 1000 Sacrifice [h] 1 h 8 h 24h 1 h 8 h 24h 1 h 8 h 24h 1 h 8 h 24h TRR [mg a.s. equiv./kg ] 352.5 271.4 102.0 315.2 245.9 109.0 307.9 221.7 85. 3 66.5 77.4 28.5
Report name

BYI 08330- % of TRR

-Enol-GA n.d. n.d. n.d. 0.5 1.2 1.0 0.5 1.0 1.1 n.d . n.d. n.d.

-Enol-alcohol n.d. n.d. n.d. 0.7 0.8 n.d. 0.4 n.d. n.d. n.d. n.d. n.d. -Desmethyl-enol 3.3 7.5 6.8 11.6 14.7 13.0 4.9 13. 1 11.5 2.6 6.4 8.7
-Desmethyl-ketohydroxy n.d. n.d. n.d. 1.0 1.7 1.8 1.1 3.4 3.6 n.d. n.d. n.d.

-Enol 96.7 92.5 93.2 76.4 72.0 74.1 75.4 64.9 65.9 94.9 91.7 89.3

Unknown n.d. n.d. n.d. n.d. n.d. n.d. 1.2 1.3 1.2 n.d. n.d. n.d. -Ketohydroxy n.d. n.d. n.d. 5.5 6.8 6.6 16.3 16.0 16.2 2.2 1.4 1.1

Subtotal : 100.0 100.0 100.0 95.6 97.1 96.4 99.7 99.7 99.5 99. 7 99.5 99.1

Identified: 100.0 100.0 100.0 95.6 97.1 96.4 98.6 9 8.5 98.4 99.7 99.5 99.1

Unknown:* n.d. n.d. n.d. n.d. n.d. n.d. 1.2 1.3 1.2 n.d. n.d. n.d. Not analysed / solids - - - 4.4 2.9 3.6 0.3 0.3 0.5 0.3 0.5 0.9

Total: 100.0 100.0 100.0 100.0 100.0 100.0 100.0 10 0.0 100.0 100.0 100.0 100.0

n.d. = not detected
* Characterised based on their retention time in HPL C

III. CONCLUSIONS

A. CONCLUSIONS :

The first and most important metabolic reaction was the cleavage of the ester bond of the side chain yielding the BYI 08330-enol. The demethylation of t he cyclohexyl-O-methyl group to the respective alcohol (BYI 08330-desmethyl-enol) was a further im portant metabolic reaction as well as the hydroxylation in the azaspiro ring of BYI 08330-eno l resulting in BYI 08330-ketohydroxy. Other metabol ic reactions like conjugation of the BYI 08330-enol wi th glucuronic acid and oxidation of one of the meth yl groups of the phenyl ring forming the BYI 08330-eno l-alcohol were of minor importance.

The results of the low dose tests may be summarized as:

• Absorption, distribution and excretion were rapid. Excretion was mainly renal and nearly completed 24 h after dosage.

• The residues in plasma and organs declined rapidly from the maximum value 1 h after dosage giving low residues in plasma 24 h post administration.

• For all time points, the residues in liver and kid ney were distinctively higher than in plasma. This finding may indicate that metabolites are transferr ed by active transport mechanisms from plasma to the excretory organs.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 30 April 2008

• The residues in testis, carcass and skin were dist inctively lower than in plasma proving the fast excretion of the residues and the non-accumulation of the compound.

• The metabolic profiles in urine were similar to th e results of the ADME study.

• The ratio of the two main metabolites BYI 08330-en ol and BYI 08330-desmethyl-enol in urine was about 2:1.

• BYI 08330-desmethyl-enol was found at lower propor tions in plasma and organs with BYI 08330- enol/BYI 08330-desmethyl-enol ratios of 10:1 to 40: 1. This demonstrates that BYI 08330-desmethylenol was excreted more rapidly than BYI 08330-enol.

• The highest proportions of BYI 08330-desmethyl-eno l were detected in liver, where the compound is formed by metabolic transformation of BYI 08330-eno l. The proportions in plasma, kidney, and testis were comparable and significantly lower than in liv er.

• BYI 08330-ketohydroxy was found as a further promi nent metabolite in liver and kidney, but was only present at trace levels in plasma, testis, and urin e.

For the high dose tests:

• Absorption and excretion were slower and lower tha n in low dose tests. Only 27 % of the dose was renally excreted 24 h after dosage.

• The residues in plasma were slightly higher than i n liver and kidney. This may be caused by the saturation of the active transport mechanisms at hi gh concentrations resulting in a more uniform distribution of the compound in the body. The decli ne of residues was slower than in the low dose
tests.

• As observed in low dose groups, the residues in te stis, carcass and skin were lower than in plasma. Plasma and all organs showed slow depletion of radi oactive residues.

• The metabolism was similar to the metabolism of th e low dose tests, with the exception that BYI 08330-desmethyl-enol was found at higher proportion s in high dose tests.

• As in low dose tests, BYI 08330-desmethyl-enol was found at higher proportions in urine (ratio BYI 08330-enol/BYI 08330-desmethyl-enol = 1.5:1) than i n plasma and organs (ratios of 5:1 to 35:1) due to the rapid excretion of this metabolite.

• The highest percentages of BYI 08330-desmethyl-eno l were detected in liver as well as in kidney. The percentages in plasma, kidney and testis were l ower and comparable.

• Significant percentages of BYI 08330-ketohydroxy w ere present in liver and kidney only.

Based on all results, it can be concluded that due to the saturation of active transport mechanisms in the excretory organs after administration of high doses , the depletion of residues and the excretion via u rine and faeces is slow and with a potential of accumula tion in the body following repeated high doses. A proposed metabolism pathway is shown in Figure 6-2.

B. REVIEWER COMMENTS :

This metabolism study (Report No. MEF 06/328) in ma le rats was well conducted and reported. Mass balance date were excellent. The data clearly indi cate saturation of absorption at the high dose (100 0 mg/kg) versus the low dose (2 mg/kg) resulting in e xpected impact on metabolism, tissue burdens, and excretion. For all test groups, renal excretion wa s prevalent although at the high dose the saturated absorption resulted in greater amounts of radioacti vity appearing in the gastrointestinal tract and fe ces. Clearance of radioactivity from tissues and organs was rapid and nearly complete within 24 hours post dosing. Variability among data was typical for a m etabolism/distribution study and clearly reported. Identification and characterization of metabolites was thorough and clearly reported. The analysis showed the enol metabolite as the primary biotransf ormation product and the demethylated enol as a significant secondary product. Major as well as un known metabolites were quantified and several– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 31 April 2008

quantitatively insignificant metabolites were also identified. A proposed metabolism pathway was consistent with the reported data. There were no ap parent methodological or procedural flaws in the experimental protocol, acqusition/analysis of data or reporting of the data. The investigators express ed variability about the mean as coefficient of variab ility (converted to %) rather than providing standa rd deviation. This is an acceptable approach (C.V. = standard deviation/mean) and has been retained in the summary tables. The reviewer spot checked sever al C.V. values and found them to be accurate. The reviewer is in agreement with the interpretatio n of the study results provided by the investigator

.

This study in rats does meet current OPPTS 870.7485 Guidelines for a metab olism and pharmacokinetics study. This study in the rat is co nsidered Acceptable/Guideline and meets the guideline requirement for a metabolism and pharmaco kinetics study (870.7485) in rats.

C. STUDY DEFICIENCIES:

There were no apparent deficiencies identified in t he study report.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 32 April 2008

NH

O O O O

CH 3

CH 3 H 3 C H O CH 3

NH

O O H CH 3

CH 3 H O CH 3 H O

NH

O CH 3

CH 3 CH 3

O O O H

O H

O H O H O

NH

O O CH 3

CH 3 H O

OH CH 3 H OH

NH

O O H CH 3

CH 3 H O

NH

O O H CH 3

CH 2 OH CH 3

NH

O O CH 3

CH 3 H OH

OH BYI08830

BYI08330-enol-alcohol BYI08330-enol BYI08330-enol-GA

BYI08330-desmethyl-enol BYI08330-ketohydroxy

BYI08330-desmethyl-ketohydroxy

conjugation with glucuronic acid

Figure B.6-2: Proposed metabolic pathway of BYI 083 30 in organs and tissues and excreta– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 33 April 2008

B.6.1.3 Metabolism (OECD Annex IIA 5.1.4)

CITATION : Totis, M. (2006) [Azaspirodecenyl-3- 14 C]-BYI 08330: Comparison of the in vitro metabolism in LiverbeadsTM from male rat, mouse and human. Bay er Crop Science, 355 rue Dostoïevski, BP. 153, 06903 Sophia Antipolis Cedex, France. Report Number SA 05319 Version N°2. July 6, 2006.

I. MATERIAL AND METHODS:

A. MATERIALS:

1. Test compound

Non-Radiolabelled test material:

Lot/batch # mix-batch 08045/0014

Purity 98.1% a.i. (determined by LC/MS)

Empirical formula: C 21 H 27 N O 5

Molar mass: 373.45 g/mol

Water solubility: pH 4 = 33.5 mg/L, pH 7 = 29.9 mg/L pH 9 = 19.1 mg/L (unstable) all at 20°C

n-Octanol/water partition coefficient: pH 4 = 2.51, pH 7 = 2.51, pH 9 = 2.50

Radiolabelled test material:
Description/Labelling: [azaspirodecenyl-3- 14 C]-BYI 08330 Sample ID # BECH 1887
Radiochemical purity: > 98 % (determined by HPLC/MS ; TLC)
Specific radioactivity of the original radiolabelled batches: 3.67 MBq/mg ( 99.1 μCi/mg)

Specific radioactivity used for administration: Low concentration assay: 3.67 MBq/mg ( 99.1 μCi/mg)
High concentration assay: 1.84 MBq/mg ( 46 μCi/mg) radiodilution 1 / 1 with non radiolabelled test compound
Dose level: Low concentration assay: 50 μM
High concentration assay: 520 μM
Stability of the test material: The stability of [a zaspirodecenyl-3- 14 C]BYI 08330 was demonstrated by radio HPLC analysis of the stock solution before in cubation

2. Vehicle: Acetonitrile (ACN)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 34 April 2008

3. Test System:

LiverbeadsTM from male rat, mouse and human. Liverb eadsTM are immobilized hepatocytes entrapped within an alginate matrix. They were purchased fro m Biopredic International, Rennes, France. These hepatocytes were used in the incubation assays to s tudy the in vitro metabolism of BYI 08330:

• Rat: Wistar male rat, batch LVB008001.

• Mouse: CD1 male mice, batch LVB002005

• Human: Male, batch LVB005005

4. Preparation of dosing solutions:

The [azaspirodecenyl-3- 14 C]-BYI 08330 was dissolved in the vehicle ACN, and this stock solution presented an activity of ~ 0.152 μCi/μL (~ 5.62 kBq /μL).

Test substance solutions (non-radiolabel) were prep ared one day before use and stored at -20°C.

For the low concentration assays, 12.5 /μL of this stock solution was added in the test wells, each containing 1 mL (medium + Liverbead). The final co ncentration of the test compound was approximately 50 /μM (~55 kBq), corresponding to ~1 .25% ACN in the incubation medium.

For the high concentration assays, non-radiolabelle d BYI 08330 was first dissolved in ACN to obtain a concentration of 100 mM. Then, a volume of this so lution was mixed to the same volume of the radiolabelled stock solution. Ten (10) μL from the last solution (52 mM) were added in the test wells

, each containing 1 mL (medium + Liverbeads). The fin al concentration of the test compound was approximately 520 μM.

B. STUDY DESIGN AND METHODS:

1. In vitro

metabolism of BYI 08330 by Liverbeads TM from rat, mouse, and human: LiverbeadsTM were thawed according to the procedures described b y the supplier. Then, the cells were pooled in HBSS (Hank’s balanced salt solution) plus penicilli n 50 UI/ml and streptomycin 50 μg/ml plus glucose (25 mM).

The LiverbeadsTM were then seeded in 12 well plates, one plate per concentration. The enzymatic reactions were initiated by the addition of the rad iolabelled product in acetonitrile at the final concentration of approximately 50 μM or 520 μM. Co ntrol samples (Liverbeads + ACN) were also incubated without [ 14 C]BYI 08330. Then, the plates were placed in an in cubator at 37°C under gentle shaking. Each assay (test) was run in duplicate. The time period of incubation was 4 hours.

At the end of the incubation time, the LiverbeadsTM were first dissolved using EDTA Na 2 (100mM), and then the hepatocytes were sonicated during 20 sec. The supernatants including hepatocytes plus incubation medium plus dissolved LiverbeadsTM from e ach well were then transferred into separated vials and immediately stored at -20°C until LC/MS a nalysis.

2. Metabolite analysis :

Hepatocyte incubations were centrifuged and the sup ernatants were directly injected into the HPLC coupled with a UV detector, 14 C detector, and a mass spectrometer. A C18 column with the solvents– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 35 April 2008

0.1% aqueous acetic acid/ acetonitrile and gradient elution was used for quantification of the metabolites by integration of the 14 C signal. The calculated area from each peak was t hen expressed as percentage of the sum of the areas from the diff erent peaks (corresponding to metabolites).

Metabolites were identified by their mass spectra m easured in electrospray positive mode. Identification of some metabolites was supported by comparison of retention times and spectra with those of authentic reference compounds.

II. RESULTS:

At low concentration, analysis of the samples using LC/MS demonstrated the presence of five different metabolites in the mouse, four in the rat and three in the human LiverbeadsTM. The parent compound BYI 08330 was not detected in the LiverbeadsTM from any tested species.

The major metabolites detected in the mouse were BY I 08330-enol and BYI 08330-enol-GA (enol glucuronide) which occurred at 66% and 30% re spectively. The glucurono-conjugation of the main metabolite BYI 08330-enol appeared to be a maj or route in the in vitro degradation (detoxification) of BYI 08330 in the mouse. BYI 08 330-enol-alcohol, BYI 08330-desmethyl-enol and BYI 08330-ketohydroxy were detected at a very low l evel in the mouse (1-2%).

In the rat, the main metabolites were BYI 08330-eno l and BYI 08330-desmethyl-enol which occurred at 87% and 7% respectively. BYI 08330-enol-alcohol an d BYI 08330-ketohydroxy represented 4% and 3% respectively. The BYI 08330-enol-GA (enol glucu ronide) was not detected in vitro in the rat.

The major metabolites detected in human were BYI 08 330-enol and BYI 08330-enol-GA (enol glucuronide), but their relative abundance we re different from those observed in the mouse. They occurred at 92% and 6% respectively. BYI 08330-des methyl-enol was present at a very low level (1%); BYI 08330-enol-alcohol and BYI 08330-ketohydroxy we re not detected in human.

At high concentration, no new metabolite was detect ed in the rat, mouse and human LiverbeadsTM. The low degree of metabolism observed in this exper iment could be indicative of a saturation of the biotransformation enzymatic system.

The results of the individual tests are shown in Ta ble 6-14, and the metabolic pathway for the in vitro test system is presented in Figure 6-3.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 36 April 2008

Table B.6-14: Distribution of the parent compound a nd metabolites after incubation of BYI 08330 to LiverbeadsTM of rat, mouse, and human
Low concentration experiment: distribution of paren t compound and metabolites (sum = 100)

enol-GA (glucuronide)
M=477 enol-alcohol

M=317 desmethylenol
M=287 enol

M=301 keto

hydroxy
M=317 BYI 08330
Rat 0 4 7 87 3 0
Mouse 30 1 1 66 2 0
Human 6 0 1 92 0 0 High concentration experiment: distribution of parent compound and metabolites (su m = 100)
Rat 0 0 0 100 0 0
Mouse 9 1 0 89 1 0
Human 2 0 0 98 0 0

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS :

The enzymatic capabilities of the in vitro system seemed to be saturated at high concentratio n; therefore, no supplementary information was obtaine d.

At low concentrations, BYI 08330 was completely met abolized in the liver cells from all species, and no parent compound was detected at the end of the i ncubation. BYI 08330-enol was the first and most prominent metabolite accounting for 66 – 92% of tot al metabolites. The metabolic profile among the three species at low concentration exhibited marked differences:

• In the rat, the BYI 08330-enol was further metabol ised by oxidation reactions to BYI 08330- desmethyl-enol (oxidative demethylation), BYI 08330 -enol-alcohol (oxidation of aromatic methyl group) and BYI 08330-ketohydroxy (oxidation of the azaspirodecenyl moiety). Oxidation products accounted for ca. 14%. Conjugation was not detecte d as an in vitro metabolic transformation.

• The general picture of the “in vitro” metabolic pa thway in the rat was very similar to the in vivo metabolism in the male rat. The same metabolites w ere detected with BYI 08330-enol and BYI 08330-desmethyl-enol as the two most important degradation products although the proportions of metabolites were different between t he in vivo and in vitro tests.

• In the mouse, oxidative degradation of BYI 08330-e nol was detected as a minor “in vitro” metabolic reaction, only (4% of oxidation products) . Conjugation to BYI 08330-enol-GA was very prominent with the conjugate accounting for ca. 30%

.

• Human liver cells exhibited an “in vitro” metaboli sm more similar to the one in mouse than in the rat. Conjugation to BYI 08330-enol-GA (6%) was mor e prominent than oxidative transformation which was detected as a minor transformation (1%).

B. REVIEWER’S COMMENTS:

The investigator’s conclusions are consistent with the reported data. Metabolism was complete at high and low dose, with no parent compound present at th e end of the incubation period. Although the enol metabolite was the predominant metabolite for all three species at the low dose (87, 92, and 66% for the rat, mouse, and human LiverbeadsTM, respecti vely), species differences were observed in the metabolic profile. The rat LiverbeadsTM also produc ed the keto hydroxyl (3%), enol-alcohol (4%), and– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 37 April 2008

desmethyl-enol (7%) metabolites; the human Liverbea dsTM produced the enol-glucuronide (6%) and desmethyl-enol (1%) metabolites; the mouse Liverbea dsTM produced substantial amounts of the enolglucuronide metabolite (30%) and minor amounts of e nol-alcohol (1%), desmethyl-enol (1%), and keto hydroxyl (2%) metabolites.

C. STUDY DEFICIENCIES:

No study deficiencies were noted.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 38 April 2008

NH

CH 3 CH 3

O O O O C H 3 H O CH 3

NH

CH 3 CH 3

O O H H O CH 3

O

NH

CH 3 CH 3

O O H O CH 3
O H O H

O H O H O

NH

CH 3 CH 3

O O H H OH NH

CH 2 OH CH 3

O O H H O CH 3

NH

CH 3 CH 3

O

H O CH 3

O

OH BYI08330

BYI08330-enol Rat Mouse Human

BYI08330-enol-GA

Mouse Human

Rat

Mouse Human

BYI08330-desmethyl-enol Mouse

BYI08330-enol-alcohol

BYI08330-keto-hydroxy

Mouse Rat Rat

Figure B.6-3: In vitro metabolic pathway of BYI 083 30 in LiverbeadsTM from male rat, mouse, and human (taken from p. 23, Report No. SA05319)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 39 April 2008

B.6.1.4 Metabolism (PBPK Modeling) (OECD Annex IIA 5.1.5)

CITATION : Schmitt, W. (2006) Physiology based pharmacokineti c simulation of BYI 08330 in male rats.

Bayer Technology Services GmbH, PT-AS-Systems Biolo gy, Building E41, 51368 Leverkusen, Germany. Report Number BTS-WSM0602. Ju ly 18, 2006.

I MATERIALS AND METHODS

A. MATERIALS:

No test material was used since this report describ es a computer simulation and not an experimental study.

B. STUDY DESIGN:

Pharmacokinetic simulation

Physiologically based pharmacokinetic (PBPK) simula tions were carried out with the commercially available software PK-Sim (Bayer Technology Service s GmbH). PK-Sim is based on a generic whole body PBPK-model which describes uptake and distribu tion of organic substances after oral or intravenous administration. Results of PBPK-simula tions with PK-Sim consist of concentration time curves for all organs and compartments included in its PBPK-model (Venous Blood, Arterial Blood, Bone, Brain, Fat, Heart, Kidney, Large Intestine, L iver, Lung, Muscle, Pancreas, Portal, Skin, Small Intestine, Spleen, Stomach, Gonads). The concentra tions are given as total tissue concentrations or a s total or unbound concentrations in interstitial or cellular space for the organs. Blood concentration s are available as unbound or total plasma or red blood-c ell concentrations. In order to account for a spec ific fate of the simulated substance, PK-Sim allows incl usion of metabolism in all included organs as well as active transport across all permeation barriers con sidered in the simulation model.

For the physiological parameters of the model (bloo d flow rates, volumes of organs and their subcompartments) default values are provided by PK-Sim for the species mouse, rat, dog and human. These physiological parameters can be set to custom ized values for the simulation of special scenarios such as disease states or particular animal models. Organ/plasma partition coefficients and passive permeabilities are estimated by PK-Sim from physico chemical properties of the compound to be simulated. All other substance-specific model para meters (e.g. clearance, metabolic rates, and rates of active transport) have to be entered directly as in put values. Their values must either be determined experimentally or can be estimated by manually adju sting the simulation for the best fit to experiment al pharmacokinetic data.

A starting parameterization of the generic PBPK-mod el of PK-Sim was achieved as follows:

Physiological parameters: All parameters depending solely on physiology and a natomy of the simulated individual were kept as provided by the b uilt-in database of PK-Sim.

Compound-specific parameters: Parameters depending on the substance to be simul ated were first defined using the PK-Sim functionality to calculate the parameters of passive uptake and distribution processes from physicochemical data. It was stated that because no parent compound was observed in the blood plasma of rats after oral administration of BYI 08330, the first parameterization was made– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 40 April 2008

according to the properties of the major metabolite BYI 08330-enol. The predominant excretion route f or BYI 08330-enol is renal excretion; therefore, the t otal clearance was first ascribed to renal clearanc e. It should be noted that in the disposition study in th e rat (Report No. MEF 048/04), it was not possible to differentiate levels of parent compound from those of the enol metabolite.

The starting parameterization was made by setting v alues of the physicochemical properties of BYI 08330-enol and the clearance to the values give n in Table 6-15.

Table B.6-15: Values of physiochemical and clearanc e values used for starting parameterization

PK-Sim input parameter Value Comment
Lipophilicity 1.76 LogD(pH2) determined by RP-HPLC
Plasma binding -5.3 Logarithmic dissociation consta nt for human serum albumin calculated from chemical structure using in house Q SAR
method.
Molecular Weight [g/mol] 301 g/mol
Solubility [mg/l] 2200 mg/l Water solubility calcul ated for pH6.5 from experimental value of 159 mg/l at pH5
Hepatic Clearance [ml/min/kg] 0
Renal Clearance [ml/min/kg] 1.5 ml/min/kg Obtained by fitting simulation to plasma concentrations of study with dose = 2 mg/kg

The experimental data used within the study for cor relation to simulation results is summarized in Tab le
6-16.

Table B.6-16: Experimental data used in model

Reference
No. Report No.

and Date Title Data used Klempner
2006b MEF-048/04

2006 [Azaspirodecenyl-3- 14 C]BYI 08330: Adsorption,

distribution, excretion and metabolism in the rat – Plasma concentrations (total radioactivity) at doses of 2 mg/kg and 100 mg/kg. – Metabolite spectrum in excreta at 2 mg/kg and 100 mg/kg Klempner
2006a MEF-06/15

2006 [Azaspirodecenyl-3- 14 C]BYI 08330: Distribution

of the total radioactivity in male and female rats determined by Quantitative Whole Body Autoradiography (QWBA) including determination of the total radioactivity
in excreta and exhaled 14 CO 2 – Tissue-concentrations (total radioactivity) in peripheral

organs

Totis 2006 SA 05319

2006 [Azaspirodecenyl-3-14C]-BYI 08330:

Comparison of the in vitro metabolism in LiverbeadsTM from rat, mouse and human. – Fraction of desmethyl-enol
detected after incubation with 50 μM BYI 08330 for 4
h.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 41 April 2008

The following modelling calculations were performed in the study:

• concentration time curves for total radioactivity in plasma after a single oral dose of 2 mg/kg bw in comparison to ADME study; simulation based on passi ve distribution alone

• concentration time curves for total radioactivity in organs and tissues after a single oral dose of 3 mg/kg bw in comparison to quantitative whole body autoradiography study

o simulation based on passive distribution alone

o simulation based on refined model including active uptake into liver and kidney parameters for active uptake optimized for best fit of simulated data to experimental results

• concentration-time curves for total radioactivity in plasma after a single oral dose of 2 mg/kg bw in comparison to ADME study; simulation based on refin ed model including active uptake into liver and kidney using optimized parameters derived from modelling of concentration-time curves in organs and tissues

• concentration time curves for total radioactivity in plasma after a single oral dose of 2 mg/kg bw an d 100 mg/kg bw in comparison to ADME study

o simulation based on refined model including active uptake into liver and kidney plus saturation of renal uptake at high dose level

o simulation based on refined model including active uptake into liver and kidney plus saturation of renal excretion at high dose level

• extrapolation of concentration time curves for tot al radioactivity in plasma after a single oral dose for high dose levels up to 1000 mg/kg bw not studied in the experiment

o simulation based on refined model including active uptake into liver and kidney plus saturation of renal uptake at high dose level

o simulation based on refined model including active uptake into liver and kidney plus saturation of renal excretion at high dose level

o simulation including saturation of hepatic uptake

• dependence of AUC (area under the curve), C max (maximal plasma concentration) and peak/trough ratio (C max /C 24h ) on dose following single dose administration (dos e range 2 – 1000 mg/kg bw).

• dependence of dose normalized area under the curve (AUC norm ) and dose normalized maximal plasma concentration (C max ) on dose following simulated repeated dosing durin g a 28 day period (dose range 2 – 1000 mg/kg bw).

The agreement between calculated and experimental d ata was checked by visual comparison and statistical evaluation.

II. RESULTS:

It was demonstrated that the ADME behaviour of BYI 08330 in male rats can be well described by physiologically based simulations based on availabl e experimental data. It was possible to generate a single PBPK model that adequately described the obs erved plasma concentrations, the non-linearity in dose, organ concentrations, and the metabolism and excretion of BYI 08330 when compared to experimental data.

The increased concentrations in liver and kidney co mpared to plasma and other tissues, as observed by quantitative whole body autoradiography (Report No. MEF-06/15), indicated the presence of active transport processes for the uptake of BYI 08330 met abolites into these tissues.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 42 April 2008

The renal excretion rate of BYI 08330-enol could on ly be explained by active tubular secretion into urine, because the physicochemical properties yield ed a glomerular filtration rate that was much smaller than the observed rate of excretion. It co uld be shown with a high probability that the saturation of the renal transport processes is resp onsible for the experimentally observed non-lineari ty of plasma concentrations between the doses 2 mg/kg and 100 mg/kg (Report No. MEF 048/04). Although the available experimental data did not al low determination if uptake or excretion is the mor e relevant process, it is reasonable to assume that t here is saturation of both processes simultaneously

. This conclusion is based on the fact that the unbou nd concentrations comparable to typical binding constants (in the μmol range) for transport protein s are reached or exceeded in the plasma as well as the kidney cells.

The PBPK-simulations allowed an extrapolation to do ses higher than those investigated experimentally in ADME studies. Calculations were carried out for doses up to 1000 mg/kg, covering the range that was studied in the toxicological experiments. It w as found that saturation of the active transport processes leads to a significant change in the shap e of the plasma concentration curve at the highest doses. This has in turn a significant impact on ph armacokinetic parameters describing the systemic exposure to the compound. While the dose-normalize d maximum concentrations only show a fair increase with the dose, this increase is rather sig nificant for the area under the concentration curve

.

An even more pronounced change was found for the pe ak/trough ratio (C max /C (24h) ). This parameter decreases roughly by a factor of 500 reaching value s as low as 5 – 6 at 1000 mg/kg. Such low peak/trough ratios are indicative of a continuous r ise of systemic concentrations upon repeated daily administration. Such a rise of body burden was fou nd for doses above 500 mg/kg in simulations of daily administration over a period of four weeks. At 1000 mg/kg, the mean daily concentrations increased about twofold with the time until a stead y state was reached after about 15 days. This led to an even higher non linearity of the AUC, with a 7-f old increase in AUC norm between 2 mg/kg and 1000 mg/kg, compared to 5-fold at single administration.

A sensitivity analysis revealed that under certain circumstances, peak/trough ratios below 5 will be obtained. This is the case if at high doses either the fraction of the dose absorbed from the gut is higher or the metabolic rate for the transition of enol to the desmethyl-enol is lower than modeled in the present simulations. The actual values of the resp ective properties might generally differ from those of the simulation because they rely on parameter value s estimated with some uncertainty.

An assessment of the dependence of the disproportio nal increase in systemic exposure on the peak/trough ratio showed that a distinct change in the behaviour occurs between ratios of 5 and 3. Below that range the moderate temporal increase of systemic exposure described above changes into a strong one with concentrations increasing continu ously at least over the whole four weeks investigated here. This leads to predicted systemi c exposures (AUC) at very high doses which are about 20 times higher than those extrapolated linea rly from low dose values.

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS:

An analysis of ADME data of BYI 08330 for male rats (Report No. MEF 048/04) and results of the Quantitative Whole Body Autoradiography study (Repo rt No. MEF-06/15) revealed indications of the– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 43 April 2008

presence of saturable processes that influence the pharmacokinetics of the compound. These indications are a change in shape of the plasma con centration curve with increasing dose and extraordinarily high concentrations in liver and ki dney compared to other tissues. Both organs are kno wn to express various active transport proteins with a broad variety of substrates.

It could be shown that the experimentally determine d ADME behaviour of BYI 08330 in male rats can be well described by PBPK simulations assuming that th e compound enters the systemic circulation as the metabolite BYI 08330-enol that is then further meta bolized to the BYI 08330-desmethyl-enol. The observed plasma concentrations, their non-linearity in dose, organ concentrations and the metabolism and excretion can be described with very good agree ment by simulations based on a common parameterization of the PBPK model.

Based on an analysis of experimental and simulation results the following conclusions about the ADME behaviour of BYI 08330 can be drawn:

• Strongly increased concentrations in liver and kid ney compared to plasma and other tissues, as observed by QWBA, indicate the presence of active t ransport processes for the uptake of BYI 08330 metabolites into these tissues. Inclusion of such processes in the simulation led to a very good agreement between calculated and observed concentra tions in both organs.

• The renal excretion rate of BYI 08330-enol can onl y be explained by active tubular secretion into urine, because the physicochemical properties yield a glomerular filtration rate that is much smaller than the observed rate of excretion.

• It is highly probable that saturation of the renal transport processes is responsible for the change in shape of plasma concentration curves observed at 10 0 mg/kg, although the available experimental data do not allow determination if uptake or secret ion is the more relevant process.

• The extent of metabolism of BYI 08330-enol to BYI 08330-desmethyl-enol, determined by the desmethyl-enol fraction in excreta, is in agreement with the rather low metabolic rate measured in vitro as could be shown by simulations using the in vitro metabolism rate. This is due to the disproportionate hepatic concentration.

Pharmacokinetics simulation were performed for extr apolation of doses up to 1000 mg/kg, which is significantly higher than those investigated experi mentally in the ADME study (maximum 100 mg/kg) and represent dose levels relevant in toxicological studies. The most important results of the extrapolation are:

• The saturation of the active transport processes l eads to a change in the shape of the plasma concentration curve at high doses resulting in a pl ateau-like concentration time curve due to a decreased efficiency of excretion.

• The non-linearity of plasma concentration with dos e leads to a disproportionate increase of systemic exposure with increasing dose. While the dose norm alized maximum concentrations show only a fair increase with dose, this increase is more subs tantial for the area under the concentration curve. The dose normalized area under concentration curve (AUC norm ) is about five times higher at 1000 mg/kg than at 2 mg/kg.

• The peak/trough ratio (C max /C 24h ) changes are even more pronounced with dose. The ratio decreases roughly by a factor of 500 reaching value s as low as 5 – 6 at 1000 mg/kg, thus being indicative of a rise of systemic concentrations upo n repeated daily administration.

In order to assess the impact of this non-linear be haviour on toxicological studies with repeated administrations, the dose dependence of plasma conc entrations during a four-week period with daily– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 44 April 2008

administrations was simulated. In this case a rise in plasma concentrations was found for daily doses above 500 mg/kg. At 1000 mg/kg the mean daily conc entrations increased with time by about a factor of two until a steady state was reached after about 15 days. This caused an even higher non linearity of the AUC, with a 7-fold increase of AUC norm with doses increasing from 2 mg/kg to 1000 mg/kg, compared to 5-fold for a single administration.

The disproportionate increase in plasma concentrati on after repeated administration of BYI 08330 might have an impact on systemic exposure after high dose s in toxicological studies.

B. REVIEWERS COMMENTS:

The PBPK model appears to adequately describe ADME behaviour of BYI 08330 in male rats at doses up to 100 mg/kg bw, when assuming that the compound enters the systemic circulation as the metabolite BYI 08330-enol that is then further metabolized to the BYI 08330-desmethyl-enol. The PBPK model was useful in demonstrating that active transport in th e kidney and liver are involved in the uptake of BY I 08330 metabolites and that active tubular secretion into the urine is likely responsible for the obser ved renal excretion rats. The model also suggests that saturation of the renal transport process is responsible for the nonlinearity of plasma concentr ations evident between doses of 2 to 100 mg/kg bw/day.

Without further supporting data, the simulations at doses greater than 100 mg/kg bw should be viewed with caution, since the PBPK model was developed us ing experimental data only up to a dose of 100 mg/kg bw. However, a subsequent study was conducte d (Report No. MEF 06/328) in which rats were exposed to a dose up to 1000 mg/kg bw, and the PBPK model was adjusted to reflect the new data (Report No. BTS-WSM0603-1). The reader is therefor e referred to this additional PBPK study for conclusions regarding exposure to high doses of BYI 08330.

C. STUDY DEFICIENCIES:

None.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 45 April 2008

B.6.1.5 Metabolism (PBPK Modeling) (OECD Annex IIA5 .1.6)

CITATION : Schmitt, W. (2006) PBPK-Simulation of BYI 08330 in male rats at high doses. Bayer

Technology Services GmbH, PT-AS-Systems Biology, Bu ilding E41, 51368 Leverkusen, Germany. Report Number BTS-WSM0603-1. September 1, 2006.

I. MATERIAL AND METHODS

A. MATERIALS:

No test material was used since this report include s a simulation and not an experimental study.

C. STUDY DESIGN:

Pharmacokinetic simulation

Physiology based pharmacokinetic simulations were c arried out with the commercially available software PK-Sim (Bayer Technology Services GmbH).

The simulation used the physiological parameters op timized in the first PBPK simulation (Report Number BTS-WSM0602) which were refined to match the result s of the plasma and organs metabolism investigations for low and high dose levels (Report No. MEF 06/328).

The following parameters were calculated in the new simulation:

• Time course for plasma and organ concentrations of metabolite BYI 08330-enol after a single dose of 2 mg/kg bw and 1000 mg/kg bw

• Time course for plasma and organ concentrations of metabolite BYI 08330-desmethyl-enol after a single dose of 2 mg/kg bw and 1000 mg/kg bw

• Time course for fractions of renal excretion for m etabolites BYI 08330-enol and BYI 08330- desmethyl-enol after a single dose of 2 mg/kg bw an d 1000 mg/kg bw

• Time course for plasma concentrations of metabolit es BYI 08330-enol and BYI 08330-desmethylenol after repeated daily administration of BYI 083 30 for 4 weeks. The dose range covered was 10 – 1000 mg/kg bw.

The agreement between calculated and experimental d ata was checked by visual comparison and statistical evaluation.

II. RESULTS:

For BYI 08330-enol, the new adjustment of model par ameters had only a minor influence on the simulation results at low doses. Thus at the dose of 2 mg/kg, the correlation between simulation resu lts and observed data remained comparable to the result s of the first PBPK simulation (Report Number BTS-WSM0602). For the high dose (1000 mg/kg), the calculated concentrations were about seven-fold too high compared to the observed values. However, other important results as liver, kidney and testi s tissue/plasma partition coefficients 8 hours after administration, the fractions of administered dose excreted in urine and the peak/trough ratios for bo th dose regimens of the experimental study were as well represented by the simulation of 1000 mg/kg as by that of 2 mg/kg.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 46 April 2008

The model simulation of BYI 08330-desmethyl-enol di d not accurately predict concentrations in plasma, liver, kidney and testis following administration o f 2 mg/kg and 1000 mg/kg BYI 08330. The authors concluded that the observed data points are describ ed satisfactorily considering the sparse experiment al information and the fact that the simulation is bas ed on the output of another simulation.

In addition to the systemic concentrations, the fra ctions of dose which were excreted in urine as BYI 08330-enol and BYI 08330-desmethyl-enol were pr edicted by the simulations and were compared to the respective experimental values. The observed b ehaviour was well reflected by the simulation.

The plasma concentration time curve of the BYI 0833 0-enol after multiple dosing was confirmed by the refined PBPK model. Mean daily plasma concentratio ns were kept constant over time after doses of 10 and 100 mg/kg bw/day. Due to the beginning of the saturation of the uptake process into liver and possibly also kidney cells, the transition from sta ble to rising plasma concentrations is starting at a daily dose of 300 mg/kg bw/day and is fully developed at 500 mg/kg bw/day as is shown in the dose dependence of dose normalized C max and AUC and of the ratio C max(day1) /C max(day28) listed in Table 6-17.

Table B.6-17: Dose-dependency of dose normalized Cm ax, AUC, and ratio Cmax(day1) / Cmax(day28) a
Daily dose [mg/kg] AUC norm [kg.h/l] C max_norm [kg/ml] C max(day 28) /C max(day 1)

10 240 0.0022 1.0

100 662 0.0034 1.0

300 2933 0.0069 1.9

500 7066 0.0172 5.5

1000 6789 0.0168 8.1

a Data taken from text table ; Report No. SA 05319 V ersion No. 2, July 6, 2006.

III. DISCUSSION:

A. CONCLUSIONS:

The parameterization of the PBPK-model for BYI 0833 0 could be significantly improved using the most recent experimental data for liver and kidney conce ntrations at high doses and the concentrations of t he metabolite desmethyl-enol (Report No. MEF 06/328). The results of the PBPK-simulations showed a good to fair agreement with observed data for the t ime course of the plasma and tissue concentrations of BYI 08330-enol and BYI 08330-desmethyl-enol as well as for the excreted concentrations. In most cases, the calculated data show deviations from tho se observed within or below a factor of two. For t he dose of 1000 mg/kg, the calculated enol concentrati ons are 4- to 7-fold higher than those determined experimentally. No explanation could be found for this discrepancy, although several hypotheses were tested. However, other results that depend on the BYI 08330-enol plasma concentration, (e.g. the excreted amounts of BYI 08330-enol and BYI 08330-de smethyl-enol or the BYI 08330-desmethyl-enol plasma and tissue concentrations) are in good agree ment with the respective experimental data.

The model indicated that the saturation of the tran sport processes involved in the excretion is predominantly responsible for the decreases of the peak/trough ratio at high doses; this was experimentally confirmed for 1000 mg/kg. For the a pplication of the model in simulations of scenarios not investigated experimentally, it is important th at despite of the discrepancy in the absolute concentrations found at high doses, the general pha rmacokinetic behaviour with the strong reduction of C max /C (24h) with increasing dose is well represented by the ca lculations. The reduction of the peak/trough– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 47 April 2008

ratio is the main reason for the strong rise of con centrations predicted for repeated administration o f doses larger than 300 mg/kg.

B. REVIEWER’S COMMENTS:

Inclusion of experimental data describing exposure up to 1000 mg/kg bw/day in rats did not significant ly alter the predictions for BYI 08330-enol at low dos es, and improved the BYI 08330-enol PBPK model predictions for exposure to the high dose. The sim ulation adequately predicted liver, kidney and test is tissue/plasma partition coefficients 8 hours after dosing, the fractions of administered dose excreted into urine, and the peak/trough ratios. The deviations f rom the simulated verses experimentally derived tis sue concentrations (plasma, liver, kidney and testis) r ange up to 7-fold, however, indicating that the ADM E process at doses of 1000 mg/kg bw/day is not comple tely described by the PBPK model. It was proposed by the study author that part of the discr epancy is due to a difference between the experimental and simulated fraction absorbed.

Predicted BYI 08330-desmethyl-enol concentrations i n the plasma, liver, kidney, and testes did not correlate satisfactorily with experimental values. As stated by the study author, little experimental data were available to generate this simulation.

Simulations of repeated daily administration of BYI 08330 over four weeks revealed that mean daily plasma concentrations of BYI 08330-enol remained co nstant over time after doses of 10 or 100 mg/kg bw/day, but rising plasma concentrations were seen after dosing with 300 mg/kg bw/day due to the beginning of saturation of the uptake process into liver and kidney cells. It is predicted that mean daily plasma concentrations of BYI 08330-desmethyl-enol a lso remain constant after doses of 10 or 100 mg/kg bw/day, increase linearly with time at daily doses of 300 pr 500 mg/kg bw/day, and increase nearly 100-fold after daily administration of 1000 mg/kg bw/day. As was hypothesized by the authors in the other PBPK modelling study (Report Number SA 05 319 Version N°2. July 6, 2006), the disproportionate increase in plasma concentration o f the metabolites after repeated administration of BYI 08330 might have an impact on systemic exposure aft er high doses in toxicological studies.

C. STUDY DEFICIENCIES:

No study deficiencies were noted.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 48 April 2008

B.6.1.6 Metabolism and Pharmacokinetics (OECD Annex IIA 5.1.6)

CITATION : Klempner, A. 2006. [Azaspirodecenyl-3- 14 C]BYI 08330-ketohydroxy: Absorption,

distribution, excretion and metabolism in the rat. Study No. M01819155. Report No. MEF 06/007, Document No. M-268931-01-2. Bayer CropScien ce AG Development, Metabolism/Environmental Fate, 40789 Monheim am Rhe in, Alfred Nobel Str. 50, Germany.

I. MATERIAL AND METHODS

A. MATERIAL:

1. Test Material:

IUPAC Name: (5s,8s)-3-(2,5-dimethylphenyl)-3-hydrox y-8-methoxy-1- azaspiro[4.5]decane-2,4-dione Code name: BYI 08330-ketohydroxy Common name: Not applicable Empirical formula: C 18 H 23 N O 4 Molar mass: 317.39 g/mol Water solubility: Unknown n-Octanol/water partition coefficient: Unknown

Labelling: Azaspirodecenyl-3- 14 C Specific radioactivity of the original radiolabelled batches: 4.31 MBq/mg = 258,600 dpm/μg

Specific radioactivity used for administration: 4.31 MBq/mg = 258,600 dpm/μg

Radiochemical purity: > 99 % Dose level: 2 mg/kg body weight Vehicle: 0.5 % aqueous tragacanth suspension Stability of the test material: The stability of [a zaspirodecenyl-3- 14 C]BYI 08330-ketohydroxy was demonstrated by radio HPLC analysis of the administ ration suspension immediately after dosing

2. Test Animals:

Species: Rat ( Rattus norvegicus domesticus )
Strain: Wistar Hsd/Cpb: WU Breeding facility: Harlan & Winkelmann GmbH, Gartenstraße 27, 33178 Borchen, Germany
Sex and numbers involved: Males: 4 animals Age: 8 weeks at the time of delivery Body weight: Males: 200 – 212g at the time of admin istration 208 – 220 g at the time of sacrifice Acclimatization: Makrolon ® cages on wood shavings in the test facility for ab out 7 days prior to the administration. Identification: Cage cards on which the study numbe r, test compound name and– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 49 April 2008

individual animal number were displayed. Additional labelling with water-insoluble spots on the tail Housing: After administration of the radiolabelled test item individually in Makrolon ®

metabolism cages under conventional hygienic condi tions in air-conditioned rooms Temperature 22 - 24 °C, relative humidity 50 – 60 %

. 12 / 12 hours light / dark cycle, air change 10 – 1 5 times per hour. Feed and water: rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, CH-4303 Kaiseraugst, Switzerland (ca. 16 g per animal and day) last feeding ca. 16 h prior to dosing next feeding ca. 6 h after dosing. tap water from municipal water supply, ad libitum

B. STUDY DESIGN:

The test item, BYI 08330-ketohydroxy, is a metaboli te of BYI 08330 and was found in the target crop metabolism studies, where it was a main metabolite in cotton. Furthermore, BYI 08330-ketohydroxy was detected in the confined rotational crop study, whe re it was the main metabolite in Swiss chard and turnip roots and a main intermediate in the metabol ic pathway. Based on these results, BYI 08330- ketohydroxy was included in the plant residue metho d and detected in fruiting and leafy crops of European residue trials (unpublished results). BYI 08330-ketohydroxy was detected as a very minor metabolite in urine in the rat ADME study with the active ingredient BYI 08330. Therefore, a supplemental study was conducted to investigate the ADME behaviour of this compound in the rat.

Dosing Each of the rats (4 animals per group) orally recei ved 2 mg/kg bw of azaspirodecenyl-3- 14 C labelled BYI 08330-ketohydroxy suspended in 0.5% aqueous tragaca nth after 16 hr fasting. The dosing suspension (0.2 mg /mL for the low and high dose level) was pr epared one day before dosing and stored at 4 ° C. The suspensions were administered to the rats by oral g avage using a syringe attached to an animal-feeding knob cannula. Each animal received a volume of 2 mL (= 10 mL/kg bw calculated for a nominal body weight of 200 g). Actual administered doses were me asured by LSC assay of aliquots of the administration suspensions. The actual mean adminis tered dose of [azaspirodecenyl-3-[ 14 C]BYI 8330- ketohydroxy was 1.94 mg/kg bw. The stability of the test compound in the suspension was determined by radio HPLC analysis of aliquots after administra tion.

Collection of blood: Blood samples were collected separately for each an imal by pressing a capillary coated with heparin in a small cut in the tail vein. The wound was closed wi th adhesive tape. The capillaries were centrifuged at ca. 12000g for about 10 minutes using a hematocrit centrifuge to separate the plasma from the formed constituents (mainly erythrocytes). After centrifug ation, the capillary was broken at the border betwe en plasma and formed constituents and the plasma (ca. 30 μL) was transferred onto a small metal dish for weighing. The dish was then placed into a scintilla tion vial for radioactivity measurement.

Collection of excreta: Urine was collected at various times separately for each animal in a cryogenic trap cooled with dry ic e. The funnels for urine collection were rinsed with d emineralised water at the end of each sampling peri od. The rinsing solutions were drained into the same vi al as the corresponding urine fraction. The radioactivity was determined by LSC. Urine samples were stored in a freezer before analysis. Faeces– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 50 April 2008

were collected at various times separately for each animal in a cryogenic trap. The individual faeces samples were mixed with a small amount of water and stirred to prepare a homogeneous slurry. The homogenised samples were weighed and the radioactiv ity of aliquots was determined by combustion/ LSC.

Sacrifice and organ/tissue sampling: At the respective test end (48 h after administrati on), the animals were anaesthetised using Pentobarbital-Na. They were sacrificed by transecti on of the cervical blood vessels. After transection of the cervical blood vessels, the oozed out blood was collected into test tubes coated with heparin and was separated afterwards into plasma and erythrocyt es by centrifugation. The following organs/tissues were collected: erythrocytes, plasma, spleen, gastr o-intestinal tract (GIT), liver, kidneys, perirenal fat, adrenal gland, testis, skeleton muscle, femur bone, heart, lung, brain, thyroid gland, skin, and carca ss.

The organs and tissues were weighed immediately aft er dissection, following lyophilisation, and homogenised. Radioactivity was determined by combus tion/LSC. The radioactivity remaining in the GIT, skin, and residual carcass was determined by combus tion/LSC in order to establish the radioactivity balance. For the small organs and tissues (e.g. ren al fat, adrenal glands, and thyroid), only the wet weight was determined and the samples then solubili sed with BTS 450 ® (Beckman Tissue Solubiliser).

Radioactivity measurement: The measurement of the radioactivity in liquid samp les was carried out by liquid scintillation countin g (LSC). For this purpose, the volume of the entire s olution was determined and the radioactivity of an aliquot (1 – 3 replicates) of the solution was meas ured as follows:

Small organs or tissues were solubilised by means o f a tissue solubiliser (e.g. BTS-450 ® ). The solubilised samples or aliquots were acidified with hydrochloric acid or glacial acetic acid and mixed with a suitable scintillation cocktail (e.g. Quickszint 401, Zinsser Analytic GmbH). The radioactivity was then measured in a scintillation counter. This method wa s used for the following test materials: renal fat, adrenal glands, and thyroid.

Solid samples (1 – 5 aliquots) were weighed and com busted in an oxygen atmosphere using an Oxidizer 307/387 (Packard Instruments) for combustion of fae ces, erythrocytes, lyophilised organs and tissues, e.g. spleen, liver, lung, bone muscle, gastrointest inal tract (GIT), residual carcass and solids (e.g. from faeces after extraction). Carbosorb E (9 mL) was u sed to trap the combustion product CO 2 and Permafluor E+ (10 mL; Packard Instruments) was used as scintillator for LSC.

Metabolite analysis Urine samples were analysed without sample preparat ion. Faeces samples of all animals from 0 - 24 h post administration were combined and extracted suc cessively 3 times with acetonitrile / water (8:2, v /v) using a Polytron homogeniser. Each extract was coll ected by centrifugation and its volume measured. Aliquots of each extract were radioassayed by LSC. The three extracts of each faeces preparation were combined and concentrated to the aqueous remainder and partitioned 3 times against n-hexane. Aliquots of aqueous and organic phases were radioas sayed by LSC. The aqueous phase was concentrated and used for HPLC analysis. The radioa ctivity in post extraction solids was determined by combustion analysis.

The metabolites in the samples were quantified by H PLC using a reversed phase column (C18) and the eluting solvents water/formic acid = 99:1 (v/v) and acetonitrile/water/formic acid = 97:2:1 (v/v/v) in the gradient mode. Detection was performed by a UV (254 nm) and a radioisotope detector with a glass– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 51 April 2008

scintillator. Metabolites in urine were identified by direct HPLC-MS-MS analysis of the urine pool 0 – 4 h. One major metabolite was also confirmed by HPLC co- chromatography with the non radiolabelled reference compound. Metabolites in faeces were main ly identified by comparison of the metabolic profil e of faeces with urine. Two major metabolites were al so identified by HPLC co-chromatography with reference compounds.

II. RESULTS AND DISCUSSION

A. ABSORPTION:

BYI 08330-ketohydroxy was rapidly absorbed from the gastrointestinal tract of male rats. The absorptio n commenced immediately after oral dosing. It was co ncluded that at least 55% of the dose was absorbed, based on the rate of renal excretion and the residues in the body without gastro-intestinal tract at the time of sacrifice 48 h after dosing. The abs orption rate was probably much higher since the metabolic profile in faeces was very similar to the one in urine, thus indicating the absorption and systemic availability of this part of the dose too. Approximately 99% of the administered radioactivit y was recovered in urine, faeces, and the body of animals

.

B. DISTRIBUTION:

Radioactivity in plasma of males and female rats fo llowing a single administration of 2 mg/kg bw reach ed its calculated maximum at 0.81 h postdose. Pharmac okinetic parameters were calculated from concentrations of total radioactivity using a 2 com partment disposition model. The calculated maximum radioactivity concentration in plasma was 1.26 mg e q./kg bw. The peak concentration was slightly belo w the equal distribution concentration of 2 mg/kg bw BYI 08330-ketohydroxy. Absorption was followed by a fast elimination phase. The mean residence time w as low at 4.32 h. Radioactivity levels in plasma ar e summarised in Table 6-18.

The distribution of radioactive residues in the bod y was analyzed at the time of sacrifice 48 h after dosing by radioactivity measurement of the major organs an d tissues. Less than 0.2% of the administered dose was detected in the body. Radioactivity concentrati ons were generally low with the highest levels detected in the liver and gastro-intestinal tract. The distribution of radioactivity levels in organs and tissues of male and female rats after oral administ ration of [azaspirodecenyl-3- 14 C]BYI 08330 is shown in Table 6-19.

C. METABOLISM:

The parent compound was not detected in urine. Trac es of BYI 08330-ketohydroxy were found in faeces. BYI 08330-ketohydroxy was completely metabolised fo rming numerous metabolites. The first and most important metabolic reaction was the oxidative deme thylation of the cyclohexyl-O-methyl group to the respective alcohol (BYI 08330-desmethyl-ketohydroxy ). Two isomers of BYI 08330-desmethylketohydroxy were detected as the main components in excreta accounting for 15% of the dose. All other identified metabolites could be derived from BYI 08 330-desmethyl-ketohydroxy as an intermediate. Most of the identified compounds were different mono-, d i- and tri-oxygenated metabolites which are probabl y represented by isomers of mono-hydroxy, di-hydroxy and tri-hydroxy metabolites. A second oxidative transformation was formal loss of two hydrogen atom s of the oxygenated metabolites to “dehydro” derivates. These metabolites probably contain aldeh yde or keto groups or carboxylic acid groups. LCMS/MS clearly proved the metabolic transformations, but did not allow identification of the positions of oxygenation and the functional groups formed.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 52 April 2008

The oxygenated metabolites were classified in three groups to simplify evaluation and quantification: mono-hydroxy, di-hydroxy and tri-hydroxy metabolite s of BYI 08330-desmethyl-ketohydroxy.

About 34% of the dose was accounted for by the vari ous different mono-hydroxy metabolites and 28% by the group of di-hydroxy metabolites of BYI 08330 -desmethyl-ketohydroxy. Tri-hydroxy metabolites were of minor importance. Only one metabolite of th is group was detected accounting for about 3 % of the dose. Conjugation with glucuronic acid or sulfa te was detected for a few metabolites only and at l ow quantities. Cleavage of the azaspirodecane ring of BYI 08330-desmethyl-ketohydroxy was found to a low extent; cleavage was supported by the presence of t wo metabolites: BYI 08330-desmethyl-MA-amide and BYI 08330-desmethyl-glyoxylic amide. Both meta bolites covered ≤ 2.8% of the dose. In total, 85.6% of the administered dose was identified and 8.57% w as characterised. Five minor metabolites (all <1.6 % of the dose) were unidentified but characterised by their HPLC elution behaviour.

Metabolites excreted with urine and faeces followin g oral administration of [azaspirodecenyl-3- 14 C] BYI 08330-ketohydroxy are shown in Table 6-21. The prop osed metabolic pathway is presented in Figure 6-
4.

D. EXCRETION:

The excretion of BYI 08330-ketohydroxy was fast and almost completed 24 h after administration. About 54% of the administered dose was eliminated via uri ne and about 44% via faeces within 48 h after dosage. Quantitative recovery of radioactivity (98. 75% of dose) was accomplished. The excretion of radioactivity following oral administration of [aza spirodecenyl-3- 14 C] BYI 08330-ketohydroxy is shown in Table 6-20.

Table B.6-18: Time course of radioactivity measured in the plasma of male rats after oral administration of 2 mg [azaspirodecenyl-3-14C]BYI 0 8330-ketohydroxy/kg bw Equivalent Concentration Time (hrs) [μg/g]

0.08 0.200

0.16 0.688

0.33 1.113

0.66 1.271

1.00 1.214

1.50 1.010

2.00 0.844

3.00 0.542

4.00 0.345

6.00 0.190

8.00 0.128

24.00 0.008

32.00 0.005

48.00 0.004– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 53 April 2008

Table B.6-19: Distribution of radioactivity in orga ns/tissues of male rats Equivalent Concentration Sample [μg/g]
Erythrocytes 0.0021
Plasma 0.0011 Spleen 0.0012
GIT 0.0103
Liver 0.0182 Kidney 0.0041
Perirenal fat 0.0009 Adrenal gland 0.0029
Testis 0.0036
Skel. Muscle 0.0024
Bone femur 0.0011
Heart 0.0012 Lung 0.0012
Brain <LOD Thyroid gland 0.0066
Skin 0.0020
Carcass 0.0026
<LOD value below detection limit

Table B.6-20: Time course of the cumulative excreti on of radioactivity with urine and faeces of male rats after oral administration of 2 mg [azaspi rodecane-3-14C]BYI 08330-ketohydroxy/kg bw

Cumulative excretion of radioactivity in % of dose administered

Time Males
[hours post admin.] 2 mg/kg bw

Urine

4 23.83

8 43.16

12 48.53

24 53.74

48 54.45

Faeces

24 41.45

48 44.09
Sum (48 h) 98.54– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 54 April 2008

Table B.6-21: Distribution of parent compound and m etabolites in the excreta of rats after oral administration of 2 mg [azaspirodecenyl-3-14C]BYI 0 8330-ketohydroxy/kg bw

Percent of administered dose
urine faeces Sum Time interval [h] 0 - 24 h 0 - 24 h excreta % of dose analysed 53.75 40.46 94.20 Peak Compound (report name) Modification
no. BYI 08330-
1 Unknown 3.71 2.36 6.07 2 -Desmethyl-ketohydroxy * (+ O) 7.30 2.69 9.99 3 -Desmethyl-ketohydroxy * (+ O + Sulfate) 1.01 0.67 1.67
4 Unknown 0.52 0.40 0.92
** 5 a -Desmethyl-ketohydroxy * (+ 2x O - 2x H) b -Desmethyl-ketohydroxy * (+ 2x O) c -Desmethyl-ketohydroxy * (+ 2x O) d -Desmethyl-ketohydroxy * (+ 2x O + conjugate) 10.49 6.17 16.66

6 Unknown 0.34 1.01 1.36 7 -Desmethyl-ketohydroxy * (+ O) 5.55 3.25 8.80 8 -Desmethyl-ketohydroxy * (+ O - 2x H) 3.01 0.08 3.09 9 -Desmethyl-ketohydroxy * (+ 3x O - 2x H) 1.44 1.63 3.07 10 -Desmethyl-ketohydroxy * (+ O) 4.36 0.62 4.98 11 -Desmethyl-ma-amide 0.28 0.23 0.50 12 -Desmethyl-ketohydroxy * (+ O) 0.24 0.35 0.58 13 -Desmethyl-ketohydroxy * (+ 2x O - 2x H) 2.52 9.19 11.71 14 -Desmethyl-ketohydroxy * (+ O - 2x H) 0.60 --- 0.60 15 -Desmethyl-ketohydroxy * (+ O) 0.31 0.29 0.60
16 Unknown 0.14 0.14 17 -Desmethyl-ketohydroxy * (+ O) 0.50 0.19 0.69 18 -Desmethyl-ketohydroxy (Isomer of peak no. 23) 3.72 1.37 5.09
** 19 a -Desmethyl-ketohydroxy (+ glucuronic acid) b -Desmethyl-ketohydroxy * (+ O) 1.26 0.78 2.04

** 20 a -Desmethyl-glyoxylic amide b -Desmethyl-ketohydroxy * (+ 2x O - 2x H) 1.42 0.85 2.27

21 -Desmethyl-ketohydroxy * (+ O) 0.95 1.00 1.95 22 -Desmethyl-ketohydroxy * (+ O) 0.54 0.28 0.82 23 -Desmethyl-ketohydroxy 3.46 6.15 9.60
24 Unknown 0.09 --- 0.09 25 -Ketohydroxy --- 0.90 0.90
Identified: 48.95 36.68 85.63
Characterised 4.80 3.78 8.57 Total analysed: 53.75 40.46 94.20 Not analysed (urine + faeces 24 - 48 h) 0.71 2.64 3.34 Solids (from faeces extraction) N/A 0.96 0.96 Organic phase (from faeces extraction) N/A 0.03 0.03– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 55 April 2008

Total in excreta 54.45 44.09 98.54 * = derivate of BYI 08330-desmethyl-ketohydroxy ** = peak 5 consists of four components, and the pe aks 19 to 20 each of two components
-- not detected– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 56 April 2008

NH

O O CH 3

CH 3 H OH

OH NH

O O CH 3

CH 3 H O

OH CH 3

NH

O O CH 3

CH 3 H OH

OH NH

O O CH 3

CH 3 H OH

OH NH

O O CH 3

CH 3 H OH

OH

NH

O O CH 3

CH 3 H OH

OH NH

O O CH 3

CH 3 H OH

OH NH

O O CH 3

CH 3 H OH

OH NH

O CH 3

CH 3 H OH

HOOC

OH NH

O CH 3

CH 3 H OH

HOOC O

+ 2 O + 3 O + O

+ 2 O - 2 H + 3 O - 2 H + O - 2 H conjugation with glucuronic acid

conjugation with sulfuric acid -desmethyl-MA-amide BYI 08330-ketohydroxy

-desmethyl-ketohydroxy -desmethyl-glyoxylic -amide

Figure B.6-4: Proposed metabolic pathway of BYI 083 30-ketohydroxy in the rat– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 57 April 2008

III. CONCLUSIONS

A. CONCLUSIONS:

The fate of radiolabelled BYI 08330-ketohydroxy in male rats following a single low dose oral administration was characterized as follows:

• Absorption : Fast absorption of the radioactive test compound from the gastrointestinal tract with an absorption rate of at least 54%

• Distribution : Fairly equal distribution of the radioactivity wi thin the blood and most organs and tissues with preference to the liver as the main me tabolizing organ also responsible for excretion

• Steady decline of radioactivity concentrations in plasma by several orders of magnitude within 48 hours

• Concentrations of radioactivity detected in tissue s and organs at the time of sacrifice 48 hours postdose were very low

• Excretion : Rapid and complete excretion of radioactivity via the renal and faecal route

• Metabolism : Demethylation of the cyclohexyl methoxy group as the first metabolic transformation resulting in BYI 08330-desmethyl-ketohydroxy as the key intermediate in the metabolic pathway from which all other identified metabolites could b e derived

• Oxidative conversion of BYI 08330-desmethyl-ketohy droxy to several isomers of mono-, di- and trioxygenated metabolites and corresponding “dehydro” metabolites These metabolites probably contain hydroxy groups, aldehyde or keto groups, and carboxylic acid groups at different positions in the molecule

• Cleavage of the azaspirodecane ring was detected a s a minor metabolic reaction

B. REVIEWER COMMENTS :

This study examined the metabolism and disposition of BYI08330-ketohydroxy (a plant metabolite of Azaspirodecenyl BYI 08330) in male rats following a single oral dose (2 mg/kg). Mass balance showing 99% recovery of administered radioactivity was exce llent. Absorption and excretion were rapid with >98% of the dose being eliminated within 48 hours; 54% in the urine and 44% in the feces. Most excretion occurred within 24 hours. Tissue burdens at 48 hours were negligible suggesting little pote ntial for bioaccumulation at the tested dose. Metabolite profiles for urine and feces were qualitatively si milar but varied quantitatively. The presence of numerou s desmethylhydroxy products affirms the investigator’s contention of demethylation as the i nitial and primary biotransformation process. Unchanged test article was not detected in the urin e and represented <1% of the administered dose in the feces. Minimal radioactivity in tissues and or gans indicated little potential for bioaccumulation in rats at the dose tested. A proposed metabolism pathway is consistent with the data. The reviewer is in agreement with the data interpretation and conclusi ons presented by the investigators.

This study in rats is considered Acceptable/Guideline and meets the guideline requirement for a Tier 1 metabolism and pharmacokinetics study (870.7485) in rats. The study focused on examining the metabolism and disposition of BYI 08330 ketohydroxy , a metabolite of BYI 08330 found in target crop metabolism studies. Although 44% of the administer ed dose was found in the feces, very little parent compound was detected in the feces and the fecal me tabolite profile was well characterized and similar to that found in the urine. Therefore, intravenous administration experiments and biliary excretion t ests would have added little additional information.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 58 April 2008

C. STUDY DEFICIENCIES:

There were no apparent deficiencies identified in t he study report.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 59 April 2008

B.6.1.6.1 Metabolism and pharmacokinetics (OECD Ann ex B.6.1.2.6)

CITATION : Klempner, A. 2006. [Azaspirodecenyl-3-14C]BYI 0833 0-enol-glucoside supplemental Study:

Adsorption, Distribution, Excretion and Metabolism in the rat. Study No. M41819140. Report No. MEF-06/006, Document No. M-268645-01-2. Bayer CropScience AG Development, Metabolism/Environmental Fate, 40789 Monheim am Rhe in, Alfred Nobel Str. 50, Germany.

I. MATERIAL AND METHODS

A. MATERIAL :

1. Test Material:

IUPAC Name: (5s,8s)-3-(2,5-dimethylphenyl)-8-methox y-2-oxo-1-azaspiro[4.5]dec-3- en-4-yl D-glucopyranoside Code name: BYI 08330-enol-glucoside Common name: Not applicable Empirical formula: C 24 H 33 N O 8 Molar mass: 463.53 g/mol Water solubility: Unknown n-Octanol/water partition coefficient: Unknown

Labelling: Azaspirodecenyl-3- 14 C Specific radioactivity of the original radiolabelled batches: 2.96 MBq/mg or 177,600 dpm/μg

Specific radioactivity used for administration: 2.96 MBq/mg or 177,600 dpm/μg

Radiochemical purity: > 99 % (HPLC with radiodetec tion) Dose level: 0.1 mg/kg body weight Vehicle: Aqueous saline (0.9 % NaCl in water adjust ed to pH 5) Stability of the test material: The stability of [a zaspirodecenyl-3- 14 C] BYI 08330-enol-glucoside was demonstrated by radio HPLC analysis of the administ ration solution immediately after dosing

2. Test Animals:

Species: Rat (Rattus norvegicus domesticus) Strain: Wistar Hsd/Cpb: WU Breeding facility: Harlan & Winkelmann GmbH, Garten straße 27, 33178 Borchen, Germany Sex and numbers involved: Males: 1 Age: 8 weeks at the time of delivery Body weight: Males: 199 g at the time of administra tion 202 g at the time of sacrifice

Acclimatization: Makrolon ® cages on wood shavings in the test facility for ab out 7 days prior to the administration. Identification: Cage cards on which the study numbe r, test compound name and– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 60 April 2008

individual animal number were displayed. Additional labelling with water-insoluble spots on the tail Housing: After administration of the radiolabelled test item individually in Makrolon ®

metabolism cages under conventional hygienic condi tions in air-conditioned rooms Temperature 23 - 25 °C, relative humidity 40 – 60 %

. 12 / 12 hours light / dark cycle, air change 10 – 1 5 times per hour. Feed and water: rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, CH-4303 Kaiseraugst, Switzerland (ca. 16 g per animal and day) last feeding ca. 16 h prior to dosing next feeding ca. 6 h after dosing. tap water from municipal water supply, ad libitum

B. STUDY DESIGN:

Dosing: One male rat was given a single oral dose of 0.1 mg azaspirodecenyl-3- 14 C labelled BYI 08330-enolglucoside/kg dissolved in aqueous saline following a 16-hour fasting. The dosing solution was prepared one day before dosing and stored at 4 ° C. The radiolabelled test item administered in this study was isolated from plant material in the BYI 08330 lettu ce metabolism study [Haas and Diederich, 2004, Report MEF-049/04; sample code BD1120D], purified b y HPLC and identified by spectroscopic methods. The solution was administered to the rat by oral ga vage using a syringe attached to an animal-feeding knob cannula. The animal received a volume of 2 mL (equivalent to 10 mL/kg bw calculated for a nominal body weight of 200 g). The concentration of the administration solution was calculated to reac h an administered amount of about 0.1 mg of the paren t compound per kg body weight (bw). The actual administered dose was measured by LSC assay of aliq uots of the administration solution and amounted to 0.063 mg [azaspirodecenyl-3- 14 C]BYI 08330-enol-glucoside / kg bw. The stability of the test compound in the solution was determined by radio HP LC analysis of aliquots after administration.

Collection of blood: Blood samples were collected by pressing a capillar y coated with heparin in a small cut in the tail ve in. The wound was closed with adhesive tape. The capill aries were centrifuged at ca. 12000 g for about 10 minutes using a hematocrit centrifuge to separate t he plasma from the formed constituents (mainly erythrocytes). After centrifugation, the capillary was broken at the border between plasma and formed constituents and the plasma (ca. 30 μL) was pressed onto a small metal dish for weighing. The dish was then placed into a scintillation vial for radioacti vity measurement.

Collection of excreta: Urine was collected at various times in a cryogenic trap cooled with dry ice. The funnel for urine collection was rinsed with demineralised water at t he end of each sampling period. The rinsing solutio ns were drained into the same vial as the correspondin g urine fraction. The radioactivity was determined by LSC. Urine samples were stored in a freezer before analysis.

Faeces were collected at various times separately i n a cryogenic trap before they were lyophilised (freeze-dried), weighed, and homogenised. The radio activity was determined by combustion/LSC. Lyophilised samples were stored at room temperature before extraction/analysis.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 61 April 2008

Sacrifice and organ/tissue sampling: At the respective test end (48 h after administrati on), the animals were anaesthetised using Pentobarbital-Na. They were sacrificed by transecti on of the cervical blood vessels. The following organs/tissues were collected: gastro-intestinal tr act (GIT), skin, and carcass. The organs and tissues prepared at the end of the e xperiments were weighed immediately after the dissection and again following lyophilisation. The radioactivity remaining in the GIT, skin, and resid ual carcass was determined by combustion/LSC in order t o establish the radioactivity balance.

Radioactivity measurement: The measurement of the radioactivity in liquid samp les was carried out by liquid scintillation countin g (LSC). For this purpose, the volume of the entire s olution was determined and radioactivity of an aliq uot (1 – 3 replicates) of the solution was measured: Solid samples (1 – 5 aliquots) were weighed and com busted in an oxygen atmosphere using the following equipments Oxidizer 307/387 (Packard Inst ruments) for combustion of faeces, erythrocytes, lyophilised organs and tissues, e.g. spleen, liver, lung, bone muscle, gastrointestinal tract (GIT), r esidual carcass and solids (e.g. from faeces after extracti on). Carbosorb E (9 mL) was used to trap the combustion product CO 2 and Permafluor E+ (10 mL; Packard Instruments) was used as scintillator for LSC.

Metabolite analysis: Urine samples were analysed without sample preparat ion. Radioactivity in the freeze-dried faeces sample for the time range from 0-24 h post administ ration was determined by combustion/LSC. An aliquot of the sample was extracted 3 times with ac etonitrile / water (8:2; v/v) using a Polytron homogeniser. Each extract was collected by centrifu gation and its volume measured. Aliquots of each extract were radioassayed by LSC. The three extract s were combined, concentrated to the aqueous remainder and partitioned 3 times against n-hexane. Aliquots of aqueous and organic phases were radioassayed by LSC. The aqueous phase was concentr ated and submitted for HPLC analysis.

The metabolites in the samples were quantified by H PLC using a reversed phase column (C18) and the eluting solvents water/formic acid = 99:1 (v/v) and acetonitrile/water/formic acid = 97:2:1 (v/v/v) in the gradient mode. Detection was performed by a UV (254 nm) and a radioisotope detector with a glass scintillator.

Metabolites in urine and extracts of faeces were id entified by comparison of HPLC profiles of samples with those from the BYI 08330 ADME rat study (Repor t No. MEF-048/04) and by co-chromatography with reference compounds.

II. RESULTS AND DISCUSSION

A. ABSORPTION:

The radiolabelled BYI 08330-enol-glucoside was rapi dly absorbed from the gastrointestinal tract of the male rat. The absorption commenced immediately afte r oral dosing as shown by the plasma curve and the calculated absorption half-life of 2.94 hours. It was concluded that about half the dose was absorbed, based on the rate of renal excretion (53. 3%) and the residue in the body without GIT (1.07 % ) at the time of sacrifice 48 h after dosing.

B. DISTRIBUTION:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 62 April 2008

The distribution of the test substance from the cen tral compartment to the different organs and tissue s was followed by measuring the concentration of the total radioactivity in plasma. Plasma radioactivity peaked ca. 4 h after dosing at a level of 0.0232 mg eq./kg bw (values calculated with the pharmacokinetic software TOPFIT). From the maximum value, the levels slowly declined by a factor of ca. 30 at 24 h to levels < 0.0001 at 48 h after tre atment. An AUC of 0.268 ug/g

⋅ hrs was calculated using TOPFIT software.

The radiolabelled residues in skin, GIT and carcass of the animal were determined at sacrifice (48 h after dosing). A negligible amount of radioactivit y was found in skin (0.09%) and GIT (0.11%). The radioactivity in the carcass amounted to 1% of the dose.

C. METABOLISM:

BYI 08330-enol was the main metabolite in excreta a ccounting for about 64% of the dose administered. Minor metabolites were BYI 08330-desmethyl-enol wit h about 5% and BYI 08330-ketohydroxy with about 3% of the dose. The unchanged BYI 08330-enol -glucoside was detected in excreta at about 21% of the administered dose, most of which (20.7%) was found in faeces. In total, ca. 93% of the administered dose could be identified. The distribu tion of metabolites in excreta is presented in Tabl e 6- 23. The proposed metabolic pathway of BYI 08330-eno l-glc in the rat is shown in Figure 6-5.

D. EXCRETION:

Excretion was fast and almost complete 24 h after a dministration. From the total excreted amount of 97.1%, 53.3% and 43.7% of the dose were excreted wi th urine and faeces, respectively. The excretion data are summarized in Table 6-22.

Table B.6-22: Time course of the excretion of radio activity via urine and faeces of the male rat following a single oral administration of 0.1 mg [a zaspirodecenyl-3-14C]BYI 08330-enolglucoside/kg bw
Time (hrs) Animal no.

[h] post admin. 912

Urine

4 0.7
8 21.5
24 52.5
48 53.3

Faeces

24 42.7
48 43.7

Sum 97.1– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 63 April 2008

Table B.6-23: Metabolite distribution in urine and faeces of the male rat dosed with [azaspirodecenyl-3-14C]BYI 08330-enol-glucoside

% of administered dose
Urine Faeces Sum Time intervals (hrs) 0-24 h 0-24h urine + faeces % of dose analysed 52.5 40.9 93.4
Peak ID
HPLC Report name

Reg 2 BYI 08330-enol-glucoside 0.5 20.7 21.2 Reg 3 BYI 08330-desmethyl-enol 4.6 0.6 5.2 Reg 4 BYI 08330-enol 47.4 16.1 63.5 Reg 5 BYI 08330-ketohydroxy - 3.1 3.1
Identified 52.5 40.4 92.9 Reg 1 Unknown - 0.5 0.5 Total analysed 52.5 40.9 93.4 Not analysed (urine + faeces 24-48 h) 0.8 1.1 1.9 Solids (from faeces extraction) N/A 1.2 1.2 Organic phase (from faeces extraction) N/A 0.6 0.6
Total in excreta 53.3 43.8 97.1
--- = not detected– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 64 April 2008

NH

O O H CH 3

CH 3 H O CH 3

H O

NH

O CH 3

CH 3 CH 3

O O O H

O H

O H O H

NH

O O CH 3

CH 3 H O

OH CH 3 H OH

NH

O O H CH 3

CH 3 BYI08330-enol BYI08330-enol-Glc

BYI08330-desmethyl-enol BYI08330-ketohydroxy

Figure B.6-5: Proposed metabolic pathway of BYI 083 30-enol-glucoside in the rat

A. CONCLUSIONS :

BYI 08330-enol-glucoside was a main metabolite of B YI 08330 in the lettuce metabolism study and was therefore included in the plant residue method. BYI 08330-enol-glucoside was detected in fruiting and leafy crops of Europea n residue trials (unpublished results). Because it was not detected in the rat ADME-study, an additional A DME study with this compound was conducted in order to investigate the absorption, distribution, excretion and metabolism of BYI 08330-enol-glucosid e in one male rat.

One male rat was administered by oral gavage a sing le dose of BYI 08330-enol-glucoside in aqueous saline solution at a target dose level of 0.1 mg/kg body weight. The test item was radiolabelled with 14 C in the 3-position of the spiro-ring of the molecule (azaspirodecenyl-3- 14 C label). Microplasma, urine and faeces were collected at various times of post admi nistration. Skin, gastro-intestinal tract and carc ass were sampled at sacrifice.

The fate of radio labelled BYI 08330-enol-glucoside in the male rat following a single oral administra tion was characterized as follows:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 65 April 2008

• Absorption : Fast absorption of the radioactive test compound fr om the gastrointestinal at a rate of ca. 50%

• Distribution : Steady decline of radioactivity concentrations in p lasma by several orders of magnitude within 48 hours

• Excretion : Rapid and complete excretion of radioactivity via t he renal and faecal route in about the same proportion

• Metabolism : Cleavage of the glucoside bond resulting in BYI 083 30-enol as the major metabolite and key intermediate in the metabolic pathway

• Oxidation of the azaspirodecenyl moiety to BYI 083 30-ketohydroxy and demethylation to BYI 08330- desmethyl-enol as minor metabolic reactions

• The metabolism of BYI 08330-enol-glucoside implies the same pathways as the one of the aglycone BYI 08330-enol and the new insecticide BYI 08330.

B. REVIEWER COMMENTS : This special study examined the metabolism and disp osition of a single oral dose of BYI 08330-enolglucoside, a leafy crop metabolite of BYI 08330, in the male rat. Mass balance was excellent. The data clearly indicate rapid absorption and subseque nt excretion via the urine (53.3% of dose) and feces (43.7% of dose) over a 48-hour post dose peri od. Most of the radioactivity in the urine was associated with the enol metabolite resulting from removal of the glucose moiety, while most of the radioactivity in the feces was associated with pare nt compound. A proposed metabolism pathway was provided which is consistent with the study finding s. The reviewer concurs with the interpretations a nd conclusions of the investigators.

This study in rats is considered an Acceptable/NonGuideline special metabolism and pharmacokinetics study in rats. The special study focused on examining the metabolism and disposition of BYI 08330-enol-glucoside, a leafy cr op metabolite of BYI 08330.

C. STUDY DEFICIENCIES: Although the methodology and data appear sound, onl y one male rat was used to assess the metabolism and disposition of the test article. Th ere were no other apparent deficiencies identified in the study report.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 66 April 2008

B.6.1.7 Toxicokinetic studies – repeated dose, oral route in rats (OECD Annex IIA 5.1.3)

CITATION : Klempner, A. 2006. [Azaspirodecenyl-3- 14 C]BYI 08330: Distribution of the total radioactivi ty

in male and female rats determined by quantitative whole body autoradiography (QWBA) including determination of the total radioactivity in excreta and exhaled 14 CO 2 . Study No. M1811287-8. Report No. MEF-06/15, Document No. M-26 9337-01-2. Bayer CropScience AG Development, Metabolism/Environmental Fate, 4078 9 Monheim am Rhein, Alfred Nobel Str. 50, Germany.

I. MATERIAL AND METHODS

A. MATERIAL:

1. Test Material :

IUPAC Name: cis-3-(2,5-dimethylphenyl)-8-methoxy-2- oxo-1-azaspiro[4.5]dec-3- en-4-yl ethyl carbonate Code name: BYI 08330 Common name: Spirotetramat (proposed ISO) Empirical formula: C 21 H 27 N O 5 Molar mass: 373.45 g/mol Water solubility: pH 4 = 33.5 mg/L, pH 7 = 29.9 mg /L pH 9 = 19.1 mg/L (unstable) all at 20°C n-Octanol/water partition coefficient: pH 4 = 2.51, pH 7 = 2.51, pH 9 = 2.50

Labelling: azaspirodecenyl-3- 14 C Specific radioactivity of the original radiolabelled batches: 3.71 MBq/mg (100.2 μCi/mg)

Specific radioactivity used for administration: 3.71 MBq/mg (100.2 μCi/mg)

Radiochemical purity: > 98 % (certified, HPLC and T LC with radiodetection) Dose level: 3 mg/kg body weight Vehicle: 0.5 % aqueous tragacanth suspension Stability of the test material: The stability of [a zaspirodecenyl-3- 14 C]BYI 08330 was demonstrated by radio HPLC analysis of the administ ration suspensions of each test immediately after dosing

2. Test Animals :

Species: Rat ( Rattus norvegicus domesticus )
Strain: Wistar Hsd/Cpb: WU Breeding facility: Harlan & Winkelmann GmbH, Gartenstraße 27, 33178 Borchen, Germany
Sex and numbers involved: Males: 8 + 1 control ani mal Females: 8 + 1 control animal control animals were dosed with non radiolabelled t est item Age: 8 weeks (male rats) and 11 – 12 weeks (female rats)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 67 April 2008

at the time of delivery Body weight: Males: 202 – 217 g at the time of adm inistration 200 – 244 g at the time of sacrifice Females: 194 – 212 g at the time of administration 192 – 208 g at the time of sacrifice Acclimatization: Makrolon ® cages on wood shavings in the test facility for ab out 7 days prior to the administration. Identification: Cage cards on which the study numbe r, test compound name and individual animal number were displayed. Additional labelling with water-insoluble spots on the tail Housing: After administration of the radiolabelled test item individually in Makrolon ®

metabolism cages under conventional hygienic condi tions in air-conditioned rooms Temperature 20 - 24 °C, relative humidity 33 – 63 %

. 12 / 12 hours light / dark cycle, air change 10 – 1 5 times per hour. Feed and water: rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, CH-4303 Kaiseraugst, Switzerland (ca. 16 g per animal and day) last feeding ca. 16 h prior to dosing next feeding ca. 6 h after dosing. tap water from municipal water supply, ad libitum

B. STUDY DESIGN :

Dosing: Each of the rats (8 rats per gender) received a 3 m g/kg bw oral dose of [azaspirodecenyl-3- 14 C]BYI 08330 suspended in 0.5% aqueous tragacanth after ca . 16 h of fasting. A control animal of each gender was treated with 3 mg/kg bw non-radiolabelled BYI 0 8330. The dosing suspensions (0.3 mg/mL) were prepared one day before dosing and stored below 4 ° C. The samples were adjusted to pH 4 to avoid hydrolysis of the test item. The suspensions were administered to the rats by oral gavage using a syringe attached to an animal-feeding knob cannula. Each animal received a volume of 2 mL (= 10 mL/kg bw calculated for a nominal body weight of 20 0 g). The amounts of the administered test substance were calculated by dividing the administe red radioactivity amounts by the specific radioactivity. The actual mean administered dose of [azaspirodecenyl-3- 14 C]BYI 08330 was 2.63 mg/kg bw for male rats and 2.73 mg/kg bw for female rats. The stability of the test compound in the suspensions was assured by radio HPLC analysis of a liquots after administration.

Collection of excreta: After administration of the radiolabelled test item , the rats were kept individually in Makrolon® metabolism cages, which allowed for separate and qu antitative collection of urine, faeces, and the expired air. Urine was collected separately for eac h animal in a cryogenic trap cooled with dry ice at intervals of 4 h, 8 h, 24 h, and every 24 h until 1 68 h. The funnels for urine collection were rinsed with demineralised water at the end of each sampling per iod. The rinsing solutions were drained into the same vial as the corresponding urine fraction. The radioactivity was determined by LSC. The faeces samples were collected every 24 h separately for ea ch animal before they were lyophilised (freezedried), weighed, and homogenised. The radioactivity was determined by combustion/LSC.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 68 April 2008

Trapping of expired air: Carbon dioxide and other volatiles from expired air were collected from four male animals and three female animals for the time ranges 0 – 24 h and 24 – 48 h. The respective metabolism cages were attached to a high velocity air pump and ventilated with ca. 2 L of air per minute and cage. The air w as passed through a trapping system of two gas-washing bottles each containing about 150 – 200 mL of a 1:1-mixture of ethanolamine/ethanol. At sampling, t he exact volume was determined, from which an aliquot was taken for the determination of radioact ivity by LSC.

Sacrifice and preparation of carcass for autoradiog raphy: One animal of each gender was sacrificed using carb on dioxide 1, 4, 8, 24, 48, 72, 120, and 168 h post administration, respectively. One female rat had to be sacrificed prematurely due to bad health condit ion. This animal was excluded from the study. The contro l animals were sacrificed 4 h after dosing.

After sacrifice, the animals were fixed in a stretc hed position using a metal template and immediately frozen at ca. -70°C in a dichloromethane/dry ice ba th. After removal of the template, the animal body together with a series of blood standards ( 14 C labelled compound in bovine blood) was embedded i n a slurry of carboxymethylcellulose (7 to 8 %) on the platform of the cryomicrotome (Leica CM 3600, Leica Instruments GmbH, 69226 Nussloch/ Germany).

Autoradiography: Sagittal sections of 50 μm were cut at ca. -25°C us ing a cryomicrotome, attached to adhesive tape, and lyophilized overnight in the cooling cabinet of the microtome. Four to five sections showing the relev ant organs and tissues were prepared from each animal. Two imaging plates were exposed to the sections at time ranges from 48 to 120 hours. The exposed im aging plates were scanned by laser using the Fuji BAS 5000 ®

image analyser. The sections were stored at about -20°C at all times except during exposure, which was performed at room temperature.

The sections of the control animals were exposed un der identical conditions, using the longest exposur e time which had been chosen for rat sections of anim als treated with the radiolabelled compound. No blackening of typical animal structures could be ob served after an exposure time of 120 hours for male and female animals.

The digital images of the radioluminograms allowed for the assessment of the distribution of radioacti vity concentrations in different organs and tissues. The autoradiograms were quantitatively evaluated using the Tina ®

software (Raytest, version 2.10 g). Defined areas were set and integrated in each organ or tissue or partial structure thereof. After backgro und (Bkd) subtraction, a value of the photostimulat ed luminescence (PSL) per mm 2 was obtained, which is proportional to the equival ent concentration of the radioactivity in that particular tissue.

Two series of 7 calibration standards were prepared by spiking bovine control blood with different concentrations of a 14 C-radiolabelled compound. The concentrations covere d a range from approx. 1,000 to 2,000,000 dpm/g. The radioactivity of each blood calibration standard was determined by combustion/LSC and the mean values of each standard were used to establish a calibration graph for the correlation of (PSL – Bkd)/mm 2 to the activity in dpm/g tissue by linear regressi on analysis. The obtained regression factors were used to calculate the concentration of the radioactivity in dpm/g in the lyophilised rat sections. These values were correct ed for the self-absorption of the various types of tissues using self absorption factors published in the literature. To express the values as equivalent concentrations, the radioactivity concentrations gi ven in dpm/g were divided by the specific radioacti vity of the test substance in dpm/μg.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 69 April 2008

Radioactivity measurement: The measurement of the radioactivity in liquid samp les was carried out by liquid scintillation countin g (LSC) of 1 – 3 replicates. All solid samples and bl ood standards were weighed and combusted in an oxygen atmosphere with an Oxidizer 307/387 (Packard Instruments).

For all samples, the limit of detection (LOD) was e stablished at ca. 20 dpm measured per aliquot after correction for the background radioactivity. The on ly exception was the measurement of radioactivity i n the expired air samples for which all reasonable dp m-values were quantified. The limit of quantificati on (LOQ) for each individual measurement was establish ed as 2 times of the background radioactivity (dpm) of each instrument/method. This background co unting rate was in a range between 12 – 30 cpm (approximately equal to 12 – 30 dpm) and it was aut omatically subtracted from the measuring results. A quench and counting efficiency correction for trans formation of gross counts (cpm) into net counts (dp m) was automatically performed by the instruments.

II. RESULTS AND DISCUSSION

A. DISTRIBUTION:

Blackening by radioactivity that originated from ra diolabelled [azaspirodecenyl-3- 14 C]BYI 08330 was most intense in the gastrointestinal tract, in the urinary bladder, the kidney and in the liver. Amon g the quantitatively analysed organs, tissues, and fluids , the highest equivalent concentrations were observ ed in the liver, kidney and blood. Moderate peak conce ntrations lower than in blood were found in the lun g and myocardium, brown fat, skin, the major glands a nd the reproductive organs. Lower concentrations were reached in all other organs and tissues. The l owest peak concentrations were found in the spinal cord, the brain, and the eye.

Peak concentrations of radioactivity were reached a fter 1 hour of treatment. From the peak values, a continuous decrease of radioactivity concentrations by several orders of magnitude, below the limit of detection was observed for all organs and tissues w ithin 48 hours in male rats and within 24 hours in female rats. Very low residues slightly above the limit of quantification were detected 72 hours afte r dosage in the liver of male rats. All quantitative results are presented in Table 6-24 (male rats) an d Table 6-25 (female rats).

B. EXCRETION

BYI 08330 was excreted rapidly and completely withi n ca. 48 hours. Excretion was mainly renal. Only a minor part of ca. 3-6 % of the dose was excreted vi a faeces. The expiration of 14 C-carbon dioxide and other 14 C-labelled volatiles was tested with male and femal e animals for a period of 48 hours. Less than 0.01% of the administered dose was expired during t he sampling period. This negligible formation of volatile products demonstrates the stability of the azaspirodecenyl-3- 14 C label. The excretion behaviour is summarized in Table 6-26 (male rats) and Table 6 -27 (female rats).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 70 April 2008

Table B.6-24: Distribution of radioactivity in orga ns and tissues of male rats after oral administration of 3 mg [azaspirodecenyl-3-14C]BYI 0 8330/kg bw
Total radioactive residues (TRR) in organs and tiss ues of male rats [μg a.s. equiv./g wet]
Time of sacrifice (hours after administration)

Organ/ Tissue

1 4 8 24 48 72 120 168
Blood 2.711 1.285 0.277 < LOQ - - - -
Liver 7.437 5.439 1.173 0.017 < LOQ 0.006 < LOQ < L OD
Renal cortex 10.635 4.811 1.429 0.008 < LOD - - -
Renal medulla 12.723 7.614 2.391 0.008 < LOD - - -

Brown fat 1.247 0.626 0.177 - - - - -
Perirenal fat --- 0.076 0.043 < LOD - - - -
Skeleton muscle 0.658 0.331 0.064 - - - - - Myocardium 1.611 0.738 0.177 < LOD - - - - Lung 1.097 0.501 0.196 < LOD - - - - Spleen 0.537 0.255 0.065 < LOD - - - -
Pancreas 0.591 0.306 0.068 < LOD - - - -

Bone marrow 0.681 0.298 0.088 - - - - -
Testes 0.509 0.404 0.088 < LOQ - - - -
Brain 0.102 0.056 0.012 < LOD - - - - Spinal cord 0.106 0.058 0.011 - - - - - Pituitary gland 0.966 0.599 0.101 - - - - - Pineal body 1.023 0.479 0.106 - - - - - Adrenal gland 2.005 0.683 0.168 - - - - - Thymus 0.579 0.236 0.053 < LOD - - - - Thyroid gland 1.412 0.639 0.142 - - - - - Salivary gland 1.222 0.506 0.100 - - - - -

Nasal mucosa 0.439 0.319 0.061 - - - - -
Skin 0.955 0.572 0.106 - - - - - Vitreal body (eye) 0.146 0.037 0.024 - - - - - < LOQ = below limit of quantification
< LOD = below limit of detection– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 71 April 2008

Table B.6-25: Distribution of radioactivity in orga ns and tissues of female rats after a single oral administration of 3 mg [azaspirodecenyl-3-14C]BYI 0 8330/kg bw
Total radioactive residues (TRR) in organs and tiss ues of female rats [μg a.s. equiv./g wet]
Time of sacrifice (hours after administration)

Organ/ Tissue

1 4 8 24 48 72 120
Blood 1.195 0.365 0.088 - - - - Liver 4.497 1.318 0.397 < LOD - - - Renal cortex 5.148 1.489 0.438 - - - - Renal medulla 7.306 2.624 0.913 - - - - Brown fat 0.530 0.116 0.037 - - - - Perirenal fat 0.108 0.053 0.010 - - - - Skeleton muscle 0.247 0.070 0.018 - - - - Myocardium 0.749 0.198 0.052 - - - - Lung 0.823 0.116 0.043 - - - - Spleen 0.241 0.068 0.017 - - - - Pancreas 0.281 0.074 0.021 - - - - Bone marrow 0.268 0.089 0.020 - - - - Ovary 0.595 0.111 0.028 - - - - Uterus 0.759 0.170 0.045 - - - - Brain 0.047 0.013 < LOQ - - - - Spinal cord 0.051 0.014 < LOQ - - - - Pituitary gland 0.476 0.134 0.032 - - - - Pineal body 0.504 0.122 0.038 - - - - Adrenal gland 0.877 0.216 0.065 - - - - Thymus 0.214 0.066 0.015 - - - - Thyroid gland 0.567 0.162 0.036 - - - - Salivary gland 0.578 0.147 0.031 - - - - Nasal mucosa 0.172 0.074 0.014 - - - - Skin 0.564 0.140 0.032 - - - - Vitreal body (eye) 0.060 0.040 0.009 - - - - < LOQ = below limit of quantification
< LOD = below limit of detection– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 72 April 2008

Table B.6-26: Excretion of radioactivity in urine, faeces and expired air of male rats after a single oral administration of 3 mg [azaspirodecenyl-3-14C] BYI 08330/kg bw

Radioactivity expressed as percent of dose administ ered

Time of sacrifice
after admin. 1 h 4 h 8 h 24 h 48 h 72 h 120 h 168 h

Time after

admin. Expired air
24 h 0.003 0.002 0.001 0.002
48 h 0.001 0.001 0.001 0.001
Urine
1 h 5.17
4 h 9.07 47.36 51.32 41.79 32.03
8 h 80.63 79.40 19.56 23.91 35.12 36.04
24 h 19.63 30.69 19.44 31.02 38.36
48 h 0.33 0.60 0.77 0.39
72 h 0.14 0.20 0.20
96 h 0.09 0.05
120 h 0.09 0.03
144 h 0.05
168 h 0.02
Faeces
24 h 7.11 4.56 5.44 6.84 5.38
48 h 0.22 0.23 0.34 0.27
72 h 0.04 0.04 0.03
96 h 0.01 0.01
120 h 0.01 0.01
144 h 0.01
168 h 0.01
Urine + faeces + expired air
1 h 5.17
4 h 9.07 47.36 51.32 41.79 32.03
8 h 80.63 79.40 19.56 23.91 35.12 36.04
24 h 26.74 35.25 24.88 37.86 43.74
48 h 0.55 0.83 1.11 0.66
72 h 0.18 0.24 0.23
96 h 0.10 0.06
120 h 0.10 0.04
144 h 0.06
168 h 0.03
Total excreted 5.17 9.07 80.63 106.13 102.73 101.11 116.33 112.90– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 73 April 2008

Table B.6-27: Excretion of radioactivity in urine, faeces and expired air of female rats after a single oral administration of 3 mg [azaspirodecenyl -3-14C]BYI 08330/kg bw

Radioactivity expressed as percent of dose administ ered

Time of sacrifice
after admin. 1 h 4 h 8 h 24 h 48 h 72 h 120 h

Time after

admin. Expired air

24 h 0.001 0.002 0.002
48 h 0.001 0.001 0.001
Urine

1 h 39.55
4 h 33.76 81.32 25.33 74.29
8 h 83.61 73.58 6.72 54.50 19.99
24 h 19.19 5.69 11.92 5.98
48 h 0.39 0.72 0.45
72 h 0.28 0.08
96 h 0.04
120 h 0.05

144 h
168 h
Faeces

24 h 1.80 1.72 2.21 2.84
48 h 0.29 0.09 0.08
72 h 0.02 0.01
96 h 0.01
120 h 0.01

144 h
168 h
Urine + faeces + expired air

1 h 39.55
4 h 33.76 81.32 25.33 74.29
8 h 83.61 73.58 6.72 54.50 19.99
24 h 20.99 7.41 14.13 8.82
48 h 0.68 0.81 0.53
72 h 0.30 0.09
96 h 0.05
120 h 0.06

144 h
168 h
Total excreted 39.55 33.76 83.61 94.57 96.13 95.08 103.83– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 74 April 2008

III. CONCLUSIONS

A. CONCLUSIONS :

The new insecticide BYI 08330 was readily absorbed from the gastrointestinal tract, distributed among almost all organs and tissues, and excreted rapidly mainly via the renal route. Analysis of autoradiograms showed that concentrations of radioa ctivity were most intense in the gastrointestinal tract, in the urinary bladder, the kidney, and in t he liver. The peak value of radioactivity concentra tions was reached 1 hour after dosing followed by a conti nuous decrease by several orders of magnitude, below the limit of detection for all organs and tis sues within 24 hours in female rats and within 48 h ours in male rats. Results of this study indicate that retention or accumulation of BYI 08330-related radioactivity in the animal body is unlikely.

B. REVIEWER COMMENTS :

Results of this supplemental study showed rapid abs orption and excretion of a single 3 mg/kg dose of radiolabelled [azaspirodecen yl-3- 14 C]BYI 08330 in male and female Wistar rats. The primary route of excretion was the urine (nearl y 100% of the dose); feces and expired air accounted for only minute amounts of radioactivity. Administered radioactivity was widely distributed among organs and tissues but occurred in only trace amounts and was rapidly removed (within 24 hrs). Tissues and organs associated with absorptio n, metabolism, and excretion (i.e., liver, kidneys ) contained the greatest amounts of radioactivity. Th e reviewer agrees that at the dose tested, retentio n or accumulation of BYI 08330 or its metabolites is unlikely. The investigator’s conclusions are consistent with the reported data.

This study in the rat is considered an Acceptable/NonGuideline supplement to a metabolism and pharmacokinetics study (870.7485) in rats.

C. STUDY DEFICIENCIES:

There were no deficiencies identified in the study report that would adversely affect the study result s or their interpretation.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 75 April 2008

B.6.1.8 Summary of ADME studies

The disposition of spirotetramat was investigated i n three in vivo and one in vitro studies. Gastroint estinal absorption of spirotetramat was rapid following ora l administration to male and female rats at a singl e dose of 2 or 100 mg/kg bw or repeated doses of 2 mg/kg b w for 14 days. The absorption rate in all tests wa s between 89 and 98% of the total recovered radioacti vity, based upon recovery from the urine and carcas s (without the gastrointestinal tract). No significa nt differences in the absorption rate were observed among any of the dose groups. The absorption rate in the single low dose test was 95% for male rats and 96% for female rats. The maximum of plasma concentration w as reached for all dose groups within 0.09 to 2.03 hours after administration (values calculated by ph armacokinetic modelling). The radioactivity concent rations in plasma declined steadily by several orders of ma gnitude within 48 hours for all dose groups. Radioa ctivity in tissues and organs at the 48-hour termination we re very low (<0.2% and below the limit of detection for some organs/tissues). Urinary excretion was very r apid (essentially complete in 24 hrs) and was the m ajor excretion route at these doses. Faecal excretion ac counted for 2-11% of the dose in male and female ra ts. The excretion behavior was similar for all dose gro ups. BYI 08330 was completely metabolized by the r at in this study; no parent compound was detected in the excreta. Identified metabolites accounted for 87-95 % of the administered dose. Only very minor metabolites (<0.7% of the dose) were not identified. The main metabolic reaction was cleavage of the ester group which resulted in the formation of the primary and most predominant metabolite BYI 08330-enol (53-87% of th e administered dose). All other identified metaboli tes could be derived from the enol intermediate. The se cond prominent metabolic transformation was oxidati ve demethylation of the 8-methoxy group to BYI 08330-d esmethyl-enol (5-37% of the administered dose). Oxidation of the azaspiro moiety to BYI 08330-ketoh ydroxy and BYI 08330-desmethyl-ketohydroxy were detected as minor pathways. Other minor metabolic t ransformations were conjugation of the enol with glucuronic acid to BYI 08330-enol-GA and oxidation of the aromatic methyl group of the enol metabolite to BYI 08330-enol-alcohol. A sex-related difference w as observed in metabolism in this study with male r ats showing much higher rates of demethylation to BYI 0 8330-desmethyl-enol compared to female rats. Simil ar results were obtained when male and female rats wer e administered a single dose of 3 mg/kg bw in anoth er disposition study using quantitative whole body aut oradiography. The highest equivalent concentration s were observed in the liver, kidney, and blood in th at study.

Absorption in male rats administered a single dose of 1000 mg/kg bw spirotetramat was much lower with only 27% of the dose excreted in the urine after 24 hours. Excretion was also distinctively less than for lower-dose animals, and radioactivity in plasma was slightly higher than in liver and kidney. These fi ndings were consistent with saturation of cellular transpo rt mechanisms. In addition, decline of tissue radi oactivity was minimal from 1 h to 8 h post dose and increased only slightly at 24 hours. The metabolism profile was qualitatively similar to that of the lower doses; h owever, BYI 08330-desmethyl-enol occurred at lower levels at 1000 mg/kg bw, and this may be a reflection of t he decreased absorption at this dose. Similar to t he low dose groups, BYI 08330-desmethyl-enol levels were g reater in urine than in plasma and organs. The high est percentage of BYI 08330-desmethyl-enol was detected in liver and kidney. Results of this study indica te that saturation of active transport mechanisms occurs at a dose of 1000 mg/kg but not 2 mg/kg. This result s in decreased excretion via urine and faeces and a pote ntial for accumulation of BYI 08330 related residue s in the body following repeated high doses. The result s of pharmacologically based pharmacokinetic (PBPK) simulations supported this conclusion and suggested that repeated daily doses of ≥ 300 mg spirotetramat/kg bw lead to non-linear elimination kinetics, resulti ng in a high body burden in multiple-dose toxicolog ical studies. This assumes, however, that spirotetramat enters the systemic circulation as the metabolite BYI 08330-enol.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 76 April 2008

In a comparative in vitro metabolism study using he patocytes from male rats, mice, and humans, differe nces in the proportions of several down-stream metabolit es were observed; however, BYI 08330-enol was the f irst and most prominent metabolite and accounted for 66- 100% of all metabolites across species.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 77 April 2008

B.6.2 Acute toxicity including irritancy and skin s ensitization (OECD Annex IIA 5.2)

B.6.2.1 Acute oral toxicity (OECD Annex IIA 5.2.1)

CITATION : Eigenberg, D. (2004) An Acute Oral LD50 Study in th e Rat with BYI08330. Project

Number: 02/A12/NQ. Unpublished study prepared by Ba yer Corp. 19 p. May 6, 2004.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 (Purity: 93.1%); CAS No. 382608-10-8.
Description: Technical grade, beige powder

Lot/Batch no.: NLL 6425-9 Stability of test compound: Stable at room temperature (~ 22 o C)

2. Vehicle and/or positive control: BYI 08330 was formulated, the day of dosing, as a suspension in 0.5% aqueous carboxymethylcellulose
3. Test animals Species: Rat, females

Strain: Wistar Hanover (Crl:WI[Glx/BRL/Han]IGS BR)
Age: 10 weeks
Weight at dosing: Initial body weight range for all five females was 155 g – 159
g Source: Charles River Laboratories, Inc., Raleigh, NC
Acclimatization period: September 23, 2002 to October 4, 2002

Diet: Purina Laboratory Rodent Chow No.5002, ad libitum

Water: Kansas City Water District provides water for our f acility. Water is provided ad libitum using an Edstrom automatic watering system Housing: Individually housed in stainless steel wire mesh ca ges providing a 66.5 square inch area. Minimum space requirement for rats weighing less than 500 grams i s 60 square inches
Environmental conditions: Temperature

and Humidity:

Lighting:

Air changes: Temperature: Set to be maintained at:19° to 25°C

(66° to 77°F); Humidity: Set to be maintained at: 3 0% to 70% RH

12-hour light/dark cycle Airflow in the animal study room, calculated as air changes per hour, averaged at least twelve changes per hour from September 23, 2002, until October 30, 2002. Five in stances of less than ten changes per hour were recorded, bu t the low reading did not cause the daily average to fall bel ow twelve changes per hour– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 78 April 2008

B. Study Design and Methods: 1. Animal assignment and treatment
Dose: The AOT42SStatPgm program, prepared for the US Environmental Protection Agency by Westat, May 2001 ; Updated by USEPA February, 2002 was used for select ing doses. As the oral LD50 was anticipated to be >200 0 mg/kg, the Test Type in the AOT425StatPgm program was chosen as Limit. Application route: Oral by gavage Application volume: 10 ml/kg bw
Fasting time: Fasted overnight Group size: 5 rats Post-treatment observation period: 14 days
Observations: Mortality, clinical signs, body weight, gross necro psy
In life dates: October 22, 2002 – November 6, 2002

II. Results and Discussion: A. Mortality: No mortalities were observed.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of de ath Mortality (%)

Females

2,000 1 st 0 0 5 -- -- 0

LD 50 (females): >2,000 mg/kg bw

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals used; ** according to t he principles of OECD Guideline 423

B. Clinical observations: No treatment-related findings.

C. Body weight: There were no treatment-related effects on body wei ghts or body weight gain.

D. Necropsy: No treatment-related findings.

III. Discussion and Conclusions:
A. Conclusions: BYI 08330 has virtually no acute oral toxicity in rats.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 79 April 2008

B. Reviewer Comments: In an acute oral toxicity study, five young adult f emale Wistar Hanover (Crl:WI[Glx/BRL/Han]IGS BR) rats (age: 10 weeks; weight: 155-159 g; source: Cha rles River Laboratories, Inc., Raleigh, NC) were given a single oral dose of BYI 08330 (Purity: 93. 1%; Batch No. NLL6425-9; beige powder) at 2000 mg/kg body weight. The AOT425StatPgm was used for selecting doses. The test material was formulated as a suspension in 0.5% aqueous carboxym ethylcellulose. The test material was given as a dose volume of 10 ml/kg to fasted rats. Body weights were recorded on the Day(s) 1, 7 and 1 4. On Day 1, the dosed animal was observed for clinical observations: 1) within 30 minutes of dosing, 2) two observations were made between 30 minutes and four hours after dosing, and 3) in the afternoon on the Day 1. Following Day 1, clinical observations were made once each morning and a mort ality check was performed each afternoon until the end of the study. All animals were necro psied and examined for any gross lesions. All animals survived the test and gained body weigh t during the study. No clinical signs or gross pathological findings were observed. Oral LD 50 Females > 2000 mg/kg bw Based on the Oral LD 50 , BYI 08330 is classified as EPA Toxicity Category III.

This acute oral study is classified as Acceptable. It does satisfy the guideline requirement for an acute oral study (OPPTS 870.1100; OECD 425) in the rat

C. Reviewer Conclusions: All countries agree the oral LD 50 > 2000 mg/kg in female rats.

D. Study Deficiencies: 1) The study report should include the report gener ated from the Acute Oral Toxicity Statistical Program (Westat, version 1.0, May 2001). 2) In addition, the registrant should amend the stu dy report to correct an assumed typo under the “Conclusions” where it stated, “According to OECD g uideline 423, the LD 50 can be classified as > 2500 mg/kg.” The LD50 level cannot be placed at a dose level that was not tested. 3) Test material used for the acute oral and acute dermal study is a different isomer of the TGAI than the isomer used for the other acute tests (inh alation, eye and primary irritation and sensitizati on studies).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 80 April 2008

B.6.2.2 Acute percutaneous toxicity (OECD Annex IIA 5.2.2)

CITATION: Eigenberg, D. (2004) An Acute Dermal LD50 Study in the Rat with BYI 08330 Project

Number: 02/A22/NR. Unpublished study prepared by Ba yer Corp. 19 p. April 28,
2004.

I. Materials and Methods

A. Materials

1. Test material: BYI 08330 (Purity: 93.1%) Description: Technical grade, beige powder

Lot/Batch no.: NLL 6425-9 Stability of test compound: Stable at room temperature (~ 22 o C)

2. Vehicle and/or positive control: BYI 08330 was mixed with aqueous 0.5% carboxymethylcellulose in approximately a 1:1 ratio (weight to weight), to form a dry paste
3. Test animals Species: Rat, males and females

Strain: Wistar Hanover (Crl:WI[Glx/BRL/Han]IGS BR)
Age: 10 weeks
Weight at dosing: Males: 271 g – 319 g Females: 182 g – 195 g
Source: Charles River Laboratories, Inc., Raleigh, NC Acclimation period: September 23, 2002 to October 4, 2002

Diet: Purina Laboratory Rodent Chow No.5002, ad libitum

Water: Kansas City Water District provides water for our f acility. Tap water is provided ad libitum using an Edstrom automatic watering system. Housing: Individually housed in stainless steel wire mesh ca ges providing a 66.5 square inch area. Minimum space requirement for rats weighing less than 500 grams i s 60 square inches Environmental conditions: Temperature

and Humidity:

Lighting: Air changes: Temperature: Set to be maintained at:19° to 25°C

(66° to 77°F); Humidity: Set to be maintained at: 3 0% to 70% RH 12-hour light/dark cycle Airflow in the animal study room, calculated as air changes per hour, averaged at least twelve changes per hour from September 23, 2002, until October 30, 200 2. Five instances of less than ten changes per hour we re recorded, but the low reading did not cause the dai ly average to fall below twelve changes per hour.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 81 April 2008

B. Study Design and Methods: 1. Animal assignment and treatment

Dose (mg/kg bw) Surface area (cm2)

males females 2,000

2,000 14.0

14.0 Application route: Dermal, semi-occlusive dressing

Duration: 24 hours Group size: 5 rats/sex/group
Post-treatment observation period: 14 days

Observations: Mortality, clinical signs, skin effects, body weigh t, gross
necropsy
In life dates: 2005-04-21 – 2005-05-05

II. Results and discussion A. Mortality: Mortality was not observed at 2,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological

results* Duration of signs Time of death Mortality [%]

Males
2,000 0 0 5 -- -- 0

Females
2,000 0 0 5 -- -- 0

acute dermal LD 50 > 2,000 mg/kg bw

* 1 st number = number of dead animals:; 2 nd number = number of animals with signs;
3 rd number = number of animals in the group

B. Clinical observations: Clinical signs observed on days 1-3 included the fo llowing: red stain – nose, wetness – urogenital area, yellow stain – urogenital area and red zone – back. No clinical signs were observed after Day 3.

C. Body weight: No treatment-related findings

D. Necropsy: No treatment-related findings– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 82 April 2008

III. Discussion and Conclusions:

A. Conclusions:
The dermal LD50 for BYI 08330 for male and female r ats was >2000 mg/kg.
B. Reviewer Comments: In an acute dermal toxicity study, 5 young Wistar H anover rats of each sex (Crl:WI[Glx/BRL/Han]IGS BR) and (age: 10 weeks; we ight: 271-319 g males; 182-195 g females; source: Charles River Laboratories, Inc., Raleigh, NC) were exposed to a single dermal application of BYI 08330 (Purity: 93.1%; Batch No. NLL6425-9; beige powder) at 2000 mg/kg bw for 24 hours. The test material was mixed with aqueous 0.5% carbo xymethylcellulose in approximately a 1:1 ratio to form a dry paste. The paste was placed on a 2 inch by 2 inch piece of gauze and applied to the shaved area on the back of the animals. The gauze was held in place with medical tape and the torso of the animal was wrapped with porous medical tape. After the 24 hour exposure period, the wrappings were removed and the dose sit e was gently wiped with water-dampened and dry paper towels to remove as much of the test material as possible without damaging the skin. Individual body weights were recorded on the Day(s) 1, 7 and 14. Clinical observations were made once each morning, except on the first two day s of study, when clinical observations were performed in the afternoon. All animals were necro psied and examined for any gross lesions. All animals survived the test and gained body weigh t during the study. Clinical signs observed on days 1-3 included the following: red stain – nose, wetness – urogenital area, yellow stain – urogenital area and red zone – back. No clinical s igns were observed after Day 3. No gross pathological findings were observed. Dermal LD 50 Males > 2000 mg/kg bw Dermal LD 50 Females > 2000 mg/kg bw Dermal LD 50 Combined > 2000 mg/kg bw Based on the Dermal LD 50 , BYI 08330 is classified as EPA Toxicity Category III.

This acute dermal study is classified Acceptable. It does satisfy the guideline requirement for an acute dermal study (OPPTS 870.1200; OECD 402) in th e rat.

C. Reviewer Conclusions: All countries agree the dermal LD 50 for BYI 08330 for male and female rats was > 2000 mg/kg.

D. Study Deficiencies: Test material used for the acute oral and acute der mal study is a different isomer of the TGAI than the isomer used for the other acute tests (inh alation, eye and primary irritation and sensitization studies).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 83 April 2008

B.6.2.3 Acute inhalation toxicity (OECD Annex IIA 5 .2.3)

CITATION: Pauluhn, J. (2002) Acute Inhalation Toxicity in Rat s According to OECD No. 403:

BYI 08330. Project Number: PH-32020. Unpublished st udy prepared by Bayer Ag Inst. of Toxicology. 93 p. May 15, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 (Purity: 96.5%) Description: Technical grade, beige powder

Lot/Batch no.: NLL 6425-9 Stability of test compound: Stable at room temperature (~ 22 o C)

2. Vehicle and/or positive control: Test substance was aerosolized as dust without the use of any additional vehicle 3. Test animals Species: Rat, males and females

Strain: SPF bred Wistar [Hsd Cpb:WU (SPF)] Age: 2 months / males: 183 g – 196 g, females: Weight at dosing: Males: 183 g – 196 g Females: 158 g – 176 g
Source: Harlan-Winkelmann GmbH, Borchen, Germany Acclimation period: Animals were acclimatized to the animal room condit ions for at least 5 days before use
Diet: Ration consisted of a standard fixed-formula diet ( NAFAG No. 9441 W10 pellets maintenance diet for rats and mice), ad libitum
Water: Tap water, ad libitum Housing: During the acclimatization and study periods the an imals were housed singly in conventional Makrolon® Type I I cages (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zsc hr. Versuchstierkunde, 7, 144-153 (1965)). Environmental conditions: Temperature:

Humidity: Lighting:

Ventilation: Light intensity: 22 + 2 o C

approximately 60% 12 h/12 h; artificial light from 6.00 a.m. to 6.00 p.m. Central European Time approximately 10 air changes per hour approximately 14 watt/m2 floor area– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 84 April 2008

B. Study Design and Methods:

1. Animal assignment and treatment

Dose: 0, 1,100 and 4,183 mg BYI 08330/m3 air, the maximum technically attainable gravimetric concentration Application route: Inhalation (nose-only exposure)

Duration: 4 hours Group size: 5 rats/dose/sex
Post-treatment observation period: 14 days

Observations: Mortality, clinical signs, body weights, rectal tem perature, reflex measurements, gross necropsy
In life dates: Not reported

2. Generation of the test atmosphere / chamber desc ription Generation and characterization of chamber atmosphe re Group 1 Group 2 Group 3
Target concentration (mg/m3) Control (air) 1,000 5,000 Actual concentration (mg/m3) -- 1,100.0 4,182.5 Chamber Volume (L) 3.8 3.8 3.8 Total Airflow (L/minute) 15 15 15 Temperature (mean, °C) 22.8 22.9 22.6 Relative humidity (mean, %) <5.4 7.1 <7.3 MMAD (μm) GSD Aerosol mass < 3 μm (%) Mass recovered (mg/m3) --

--

--

-- 3.68*

2.29
41.2 1,126.0 5.13*
2.37
26.8 2,762.5 MMAD = Mass Median Aerodynamic Diameter, GSD = Geom etric Standard Deviation; -- not applicable

.

*With regard to the respirability of the aerosol ge nerated internationally recognized recommendations such as of SOT (1992) were fulfilled at large, i.e. the MMAD w as 3.7-5.1 pm (GSD ~2.3). In order to generate a concentration as high as 4183 mg/m3, which is above (US) or in the range (EU) of the testing guidelin es, the cyclone was omitted. This high concentration could only be attained at the expensive of larger partic le-size. Therefore, this concentration is considered to be t he technically maximum attainable concentration.
Micronization of the test substance was not conside red because at such high concentrations particulate s would agglomerate, i.e., the outcome of study would not b e appreciably different. Moreover, micronization co mmonly increases the electrostatic charges of particulates which promote additional agglomeration. Because o f the
mechanics mentioned above the MMAD value exceeds th e recommended range in the high dose (5.1μm). A cumulative mass percent value of ~47% was demonstra ted for particle sizes up to that of 4.70 μm range therefore, a substantial quantity of test article w as reaching the target tissue. While the cumulativ e mass for the higher dose did not result in at least 50% of the p articulate reaching below 4μm for compound-tissue interaction, all reviewers agrees this it did not i mpact the validity of the study outcome.

Test atmosphere concentration: Nominal concentration: The nominal concentration w as not calculated due to the high weight of the dust gener ator and the– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 85 April 2008

vertical elutriator used to eliminate larger partic les.

Gravimetric concentration: The test-substance conce ntration was determined by gravimetric analysis (filter: Glass-f iber filter, Sartorius, Gottingen, Germany; digital balance). C hamber samples were taken in the vicinity of the breathing zone (see Fig. I). The number of samples taken was sufficient to characterize the test atmosphere and was adjusted so as to accommoda te the sampling duration and/or the need to confirm specif ic concentration values. Optimally, samples were colle cted after the equilibrium concentration had been attained in hour ly intervals. All analytical concentrations reported refer to mg of t est substance/m3 air.
Particle size determination: The samples for the analysis of the particle-size d istribution were also taken in the vicinity of the breathing zone. During each exposure two samples were taken. The particle-size distribution was analyzed using a BERNER-TYPE AERAS low-pressure critical orifice cascade impactor (Hauke, Gmunden, Austria). Specifications and evaluations are provided in the Appendix. The individual impactor stages had been covered by an a luminum foil. An adhesive stage coating (silicone spray) was used to prevent particle bounce and re-entrainment. Gravimetric ana lyses were made using a digital balance.

II. Results and Discussion: A. Mortality: Mortality was not observed at 1,100 or 4,183 mg/m3 air.

Doses, mortality / animals treated

Actual concentration (mg/m3 air) Toxicological results* Duration of signs Time of de ath Mortality [%]

Males

0 0 0 0 0 37.4

1,100 0 4 5 0-5 d 0 35.1**

4,183 0 5 5 0-8 d 0 32.7**

LC 50 (males) > 4,183 mg/m3 air

Females

0 0 0 0 0 37.5

1,100 0 5 5 0-2 d 0 35.6**

4,183 0 5 5 0-8 d 0 33.5**

LC 50 (females) >4,183 mg/m3 air– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 86 April 2008

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals exposed; ** = p ≤ 0.01

B. Clinical observations:

1.1 mg/L dose group: By Day 3, 4/5 males and 5/5 females had either lo st body weight or not gained any weight. By Day 7 all of the males and f emales had surpassed their initial body weights and continued to gain weight till the end of the st udy. Clinical signs observed in this group included the following: ungroomed hair-coat, piloer ection, bradypnea, labored breathing pattern and nasal discharge (serous). No clinical signs we re observed after Day 5. No gross pathological findings were observed.

4.183 mg/L dose group: By Day 3 all the males and females had lost body weight. By Day 7, 4/5 males and 4/5 females had surpassed their initi al body weight and by day 14 all animals exceeded their initial body weights. Clinical sign s observed in this group included the following: ungroomed hair-coat, piloerection, bradypnea, labor ed breathing pattern, breathing sounds, nostrils reddened, nasal discharge, nostrils: red e ncrustations, nose/snout region: red encrustations, stridor, motility reduced, limp and high-legged gain. No clinical signs were noted after Day 8. A battery of reflex measurements was made on the first post exposure day. Rats from this group experienced abnormal reflexes (redu ced grip strength, reduced tonus and impaired righting response). No gross pathological findings were observed.

C. Body weight:

1.1 mg/L dose group: There was no mortality noted. in this dose group. By Day 3, 4/5 males and 5/5 females had either lost body weight or not gained any weight. By Day 7 all of the males and females had surpassed their initial body weight s and continued to gain weight till the end of the study.

4.183 mg/L dose group: There was no mortality noted in this dose group. By Day 3 all the males and females had lost body weight. By Day 7, 4/5 ma les and 4/5 females had surpassed their initial body weight and by day 14 all animals exceeded thei r initial body weights

. D. Necropsy: No gross pathological findings were observed.

III. Discussion and Conclusions:

A. Conclusions: In sumrnary, the aerosolized test substance (solid aerosol) proved to have essentially no acute inhalation toxicity to rats. LC50 > 4183 mg/m3.

B. Reviewer Comments:

In an acute inhalation toxicity study, two groups o f young adult Wistar (Hsd Cpb:WU) rats (5/sex) (age: 2 months; weight: 183-203 g males; 158-181 g females; source: Harlan-Winkelmann GmbH, Borchen, Germany) were exposed via the inhala tion (nose-only exposure route ) to BYI 08330 (Purity: 96.5%; Batch No. NLL 6425-9; beige p owder) for 4 hours at a concentrations of– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 87 April 2008

1.1 mg/L (MMAD = 3.68 μm; GSD = 2.29) and 4.183 mg/ L (MMAD= 5.13 μm; GSD = 2.37)

Body weights were taken before exposure, on Days 3 and 7 and weekly thereafter. On the day of exposure, the test animals were observed several ti mes for clinical signs of toxicity and at the least once daily thereafter. All animals were necr opsied and examined for any gross lesions.

1.1 mg/L dose group:

There was no mortality noted. in this dose group. By Day 3, 4/5 males and 5/5 females had either lost body weight or not gained any weight. By Day 7 all of the males and females had surpassed their initial body weights and continued to gain weight till the end of the study. Clinical signs observed in this group included the following : ungroomed hair-coat, piloerection, bradypnea, labored breathing pattern and nasal disc harge (serous). No clinical signs were observed after Day 5. No gross pathological findin gs were observed.

4.183 mg/L dose group:

There was no mortality noted in this dose group. B y Day 3 all the males and females had lost body weight. By Day 7, 4/5 males and 4/5 females h ad surpassed their initial body weight and by day 14 all animals exceeded their initial body weig hts. Clinical signs observed in this group included the following: ungroomed hair-coat, piloe rection, bradypnea, labored breathing pattern, breathing sounds, nostrils reddened, nasal discharg e , nostrils: red encrustations, nose/snout region: red encrustations, stridor, motility reduce d and limp high-legged gain. No clinical signs were noted after Day 8. A battery of reflex measur ements was made on the first post exposure day. Rats from this group experienced abnormal ref lexes (reduced grip strength, reduced tonus and impaired righting response). No gross patholog ical findings were observed.

LC 50 Males > 4.183 mg/L

LC 50 Females > 4.183 mg/L

LC 50 Combined > 4.183 mg/L

Based on the LC 50 in males and females, BYI 08330 is classified as E PA Toxicity Category IV.

This acute inhalation study is classified as Accept able. It does satisfy the guideline requirement for an acute inhalation study (OPPTS 870.1300; OECD 403) in the rat.

C. Reviewer Conclusions:

All countries agree the inhalation LC 50 for BYI 08330 in male and female rats was > 4.183 mg/L. The study did exhibit some significant clinical sig ns, but those effects resolved by Day 8 and the necropsy findings were unremarkable. In summary, t he aerosolized test substance (solid aerosol) proved to have minimal acute inhalation toxicity to rats.

D. Study Deficiencies:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 88 April 2008

1) The MMAD value exceeds the recommended range in the high dose group (see note under the results and conclusion sections for further details ).

2) Test material used for inhalation, eye and prima ry irritation and sensitization studies is a “cis” isomer of the TGAI test material used for the acute oral and acute dermal studies.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 89 April 2008

B.6.2.4 Skin irritation (OECD Annex IIA 5.2.4)

CITATION: Leuschner, P. (2002) Acute Skin Irritation Test (Pa tch Test) of BYI 08330 in Rabbits.

Project Number: 8147. Unpublished study prepared by Laboratory of Pharmacology & Toxicology. 23 p. April 25, 2002.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 (Purity: 96.5%) Description: Technical grade, beige powder

Lot/Batch no: NLL 6425-9 Stability of test compound: Stable at room temperature (~ 22 o C)

2. Vehicle and/or positive control: The test compound was mixed with 3 ml aqua ad iniec tabilia

3. Test animals Species: Rabbit, males

Strain: Himalayan Age: approx. 6.5 months Weight at dosing: 2.2 kg

Source: LPT Laboratory of Pharmacology and Toxicology KG, Löhndorf/Post Wankendorf, Germany Acclimation period: at least 20 days

Diet: Altromin 2023, Altromin GmbH, D-32791 Lage/Lippe

Water: Tap water, ad libitum Housing: During the exposure period the animals were kept si ngly in special restrainers which allowed free movement of the head but prevented a complete body turn. Before and after the 4-hour exposure period the rabbits were kept se parately in cages with dimensions of 425 mm x 600 mm x 380 m m. Environmental conditions: Temperature:

Humidity: Lighting: Light intensity: 20°C ± 3°C

55% ± 15% darkend for periods of 12 hours 150 lux at approx. 1.5 m room height

B. Study Design and Methods:

1. Animal assignment and treatment
Dose: 500 mg /animal
Application route: Dermal (semi occlusive procedure)

Duration: 4 hours Group size: 3 rabbits

Observations: Clinical signs, skin effects, body weight (at begin ning of– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 90 April 2008

study)
In life dates: February 28, 2002 – March 3, 2002

II. Results and Discussion:

A. Findings: None of the three animals exposed with BYI 08330 sh owed any test substance related lesions. There were no systemic intolerance reactio ns

Summary of irritant effects (Scores)

Animal

no. Before

Dosing 1 hr 24 hrs 48 hrs 72 hrs

E Oe E Oe E Oe E Oe E Oe
1 0 0 0 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0 0 0 0 = no pathological findings; E = erythema/eschar f ormation; h = hour; Oe = oedema formation

III. Discussion and Conclusions:

A. Conclusions:
BYI 08330 has no irritant effect to the skin.

B. Reviewer Comments:

In a primary dermal irritation study, 500 mg of BYI 08330 (Purity: 96.5%; Batch No. NLL 6425-9; beige powder) was applied to the skin of three youn g healthy male Himalayan rabbits (age: 6.5 months; weight: 2.2 kg; source: LPT Laboratory of P harmacology and Toxicology KG, D-24601 Lohndorf/Post Wankendorf).

Approximately 24 hours before exposure, the fur was removed from the dorsal area of the trunk of the animals. A dose of 500 mg was applied to the t est site (approximately 6 cm2). Pulverised solids were moistened sufficiently with water to en sure good contact with skin. The test material was applied to the test site and covered with a gau ze patch. The patch was held in place with semi-occlusive dressing for the exposure period. A fter the 4-hour exposure period the patch was removed and the skin sites were evaluated. Scores were taken at 1, 24, 48 and 72 hours after patch removal.

No dermal irritation was noted at any of the examin ation time points.

Based on no dermal irritation, BYI 08330 is classif ied as EPA Toxicity Category IV.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a primary dermal irritation study (OPPTS 870.2500; OECD 404) in the rabbit.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 91 April 2008

C. Reviewer Conclusions: All countries agree BYI 08330 is not a skin irritan t.

D. Study Deficiencies:

Test material used for inhalation, eye and primary irritation and sensitization studies is a “cis” iso mer of the TGAI test material used for the acute oral a nd acute dermal studies.– –

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B.6.2.5 Eye irritation (OECD Annex IIA 5.2.5)

CITATION: Leuschner, P. (2002) Acute Eye Irritation Study of BYI 08330 by Instillation into the

Conjunctival Sac of Rabbits. Project Number: 8146. Unpublished study prepared by Laboratory of Pharmacology & Toxicology. 26 p. Apr il 25, 2002.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 (Purity: 96.5%) Description: Technical grade, beige powder

Lot/Batch no: NLL 6425-9 Stability of test compound: Stable at room temperature (~ 22 o C)

2. Vehicle and/or positive control: None

3. Test animals Species: Rabbit, males

Strain: Himalayan Age: approx. 6.5 months Weight at dosing: 2.3 kg – 2.5 kg

Source: LPT Laboratory of Pharmacology and Toxicology KG, Löhndorf/Post Wankendorf, Germany Acclimation period: at least 20 days

Diet: Altromin 2023, Altromin GmbH, D-32791 Lage/Lippe

Water: Tap water, ad libitum Housing: During the exposure period the animals were kept si ngly in special restrainers which allowed free movement of the head but prevented a complete body turn. Before and after the 4-hour exposure period the rabbits were kept se parately in cages with dimensions of 425 mm x 600 mm x 380 m m. Environmental conditions: Temperature:

Humidity: Lighting: Light intensity: 20°C ± 3°C

55% ± 15% darkend for periods of 12 hours 150 lux at approx. 1.5 m room height

B. Study Design and Methods:

1. Animal assignment and treatment

Dose: 100 mg /animal
Application route: Instillation into the conjunctival sac of the right eye. The left eye was untreated and served as control. Group size: 3 rabbits

Observations: Clinical signs, eye effects, body weight (at beginn ing of– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 93 April 2008

study)
In life dates: March 5, 2002 – March 14, 2002

II. Results and Discussion: A. Findings Under the present test conditions the following fin dings were noted: Corneal opacity (grade 1) was observed in all animals 24 hours to 6 days after instillation, in animal nos. 1 and 2 until 7 days after instillation.

Irritation of the iris (grade 1) was observed in all animals: - in animal no, 1: 24 hours to 6 days after instill ation, - in animal no. 2: 72 hours to 5 days after instill ation and - in animal no. 3: 24 hours to 5 days after instill ation.

Conjunctival redness (grade 1) was observed in all animals 1 hour to 72 hours after instillation, in animal nos, 2 and 3 until 4 days after instillation

. Conjunctival chemosis (grade 1) was noted in animal no. 3 from 24 to 48 hours after instillation . There were no systemic intolerance reactions,

Summary of Irritant Effects (Scores)

Animal
No. Effects 1 hr 24
hrs 48
hrs 72
hrs 4 days 5 days 6 days 7 days 8 days

Corneal opacity 0 1 1 1 1 1 1 1 0

Iris 0 1 1 1 1 1 1 0 0

Redness conjunctivae* 1 1 1 1 0 0 0 0 0 1

Chemosis conjunctivae* 0 0 0 0 0 0 0 0 0

Corneal opacity 0 1 1 1 1 1 1 1 0

Iris 0 0 0 1 1 1 0 0 0

Redness conjunctivae* 1 1 1 1 1 0 0 0 0 2

Chemosis conjunctivae* 0 0 0 0 0 0 0 0 0

Corneal opacity 0 1 1 1 1 1 1 0 -

Iris 0 1 1 1 1 1 0 0 -

Redness conjunctivae* 1 1 1 1 1 0 0 0 - 3

Chemosis conjunctivae* 0 1 1 0 0 0 0 0 -– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 94 April 2008

- : not examined; cornea: 0=no opacity; 1=scattered or diffuse area s of opacity, details of iris clearly visible iritis: 0=normal; 1=markedly deepened rugae, swel ling, moderate circumcorneal hyperaemia, or injection, iris still react ing to light conjunct. redness: 0=blood vessels normal 1=some bl ood vessels injected conj. chemosis 0= no swelling 1=any swelling abov e normal 2=obvious swelling with partial eversion of li ds *Score of 2 or more required before the observation is considered “positive” for conjunctivae.

III. Discussion and Conclusions:

A. Conclusions: BYI 08330 has an irritant effect to the eye.

B. Reviewer Comments:

In a primary eye irritation study, 100 mg of

BYI 08330 (Purity: 96.5%; Batch No. NLL 6425-9; bei ge powder) was instilled into the conjunctival sac of the right eye in each of the th ree young healthy male Himalayan rabbits (age: 6.5 months; weight: 2.3-2.5 kg; source: LPT Laborat ory of Pharmacology and Toxicology KG, D24601 Lohndorf/Post Wankendorf). The left eye was untreated and served as control.

The eyes were examined ophthalmoscopically with a s lit lamp prior to instillation and also at 1, 24, 48 and 72 hours and on days 4 to 8. The eye re actions were observed and recorded. Twenty-four hours and 7 days after instillation, th e eyes were treated additionally with fluorescein and examined.

Corneal opacity (grade 1) was observed in all eyes from 24 hours to 6 days after instillation and in two eyes until 7 days after instillation. No co rneal opacity was observed on day 8. Iritis (grade 1) was noted in two eyes at 24 and 48 hours, in thr ee eyes at 72 hours and days 4 and 5, and in one eye on day 6 after instillation. No iritis was observed on day 7. Conjunctival redness (grade 1) was noted in all eyes 1 hour to 72 hours after i nstillation and in two eyes until 4 days after instillation. Conjunctival chemosis (grade 1) was noted in one eye at 24 and 48 hours after instillation. All conjunctival irritation was reso lved by day 5.

Based on the corneal opacity observed up to day 7 a fter instillation, BYI 08330 is classified as an eye irritant and placed in EPA Toxicity Category II

.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a primary eye irritation study (OPPTS 870.2400; OECD 405) in the rabbit.

C. Reviewer Conclusions:

All countries agree BYI 08330 should be classified as an eye irritant.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 95 April 2008

D. Study Deficiencies:

Test material used for inhalation, eye and primary irritation and sensitization studies is a “cis” isomer of the TGAI test material used for the acute oral and acute dermal studies.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 96 April 2008

B.6.2.6 Skin sensitisation (OECD Annex IIA 5.2.6)

CITATION: Vohr, H. (2002) Skin Sensitization Effect of BYI 08 330 in Guinea Pigs (Guinea Pig

Maximization Test According to Magnusson and Kligma n). Project Number: PHH32273, T/1071445. Unpublished study prepared by Bay er Ag Inst. of Toxicology. 26 p. July 29, 2002.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 (Purity 96.5%) Description: Technical grade, beige powder

Lot/Batch no: NLL 6425-9 Stability of test compound: Stable at room tempera ture (~ 22 o C) 2. Vehicle and/or positive control: Polyethylene glycol 400 Alpha hexyl cinnamic aldehyde 3. Test animals Species: Guinea pig, females Strain: SPF-bred [Hsd Poc:DH] Age: 5 – 6 weeks Weight at dosing: 292 g – 366 g Source: Harlan Winkelmann GmbH, Borchen, Germany Acclimation period: at least five days before trea tment
Diet: Provimi Kliba 3420; Provimi Kliba AG, Switzerland

Water: Tap water Housing: During the adaptation and study period the animals were conventionally kept in type IV Makrolon® cages, in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. The cag es were exchanged for ones with clean bedding at least two times per week. Low-dust wood shavings supplied by Ssniff Spezialdiäten GmbH, Soest, were used as bedding. Environmental conditions: Temperature:

Humidity: Lighting: Air changes: 22 +/- 3°C (possibly drifting higher at outdoor tem peratures

above 24°C)
40-60 % Twelve hours; artificial lighting from 6 AM to 6 PM

≥ 10 times per hour

B. Study Design and Methods

1. Animal assignment and treatment

Dose:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 97 April 2008

Induction: Intradermal Induction: Starting behind the neck, t hree injections each in a row were made on the left and the right side of the spinal column. The 1st and 2nd injectio ns were made as contiguous as possible and the 3rd injectio ns in a distance of about 2 cm from the 2nd. The volume ap plied per injection site was 0.1 ml. The animals of the three groups were treated as follows: a) Test material g roup - 1. Injection site: cranial/bilateral complete Freund’s adjuvant (DIFCO LAB.) diluted 1:1 with sterile physiological saline solution; 2. Injection site: medial/bilateral 5% BY I 08330 formulated in polyethylene glycol 400; 3. Injection site: caudal/bilateral 5% BYI 08330 formulated at equal p arts in polyethylene glycol 400 and complete Freund’s adjuv ant

Topical Induction: The topical induction was perfor med one week after the intradermal induction. Hypoallergen ic patches (2 x 4 cm) were placed between and on the injection sites, covered with aluminum foil and hel d securely in place on the skin using self-adhesive tape. The patches were treated as follows: a) Test group: 0.5 ml 50% BYI 08330; b) Control group: 0.5 ml polyethylene glycol 400. At the end of the 48-hour exposure period, the remaini ng test material was removed with sterile physiological sal ine solution. Challenge: The challenge was performed three weeks after the intradermal induction. In the meantime, the test m aterial concentrations for the challenge had been determine d in a dose range-finding study using 2 guinea pigs that w ere treated during the inductions in the same manner as the control animals. During the challenge a hypoallerg enic patch loaded with the 25% test material formulation was placed on the right flank of the animals of the tes t group and the control group and held securely in place on the skin with self-adhesive tape for 24 hours. A patch loaded onl y with the vehicle was placed also on the cranial right flank as control. The volume applied in each case was 0.5 ml. At the end of the exposure period, the remaining test material wa s removed with physiological saline solution, and 21 hours later the skin of the animals was shorn in the zone of the challenge area.
Naive Controls: The animals of the control group were treated in th e same manner as the animals of the test group; however, t he formulations for injection site pairs 2 and 3 did n ot contain any test item but in pair 2 undiluted vehicle and i n pair 3 a 1:1 mixture FCA/vehicle. The injection sites were visually assessed 2 and 7 days after the injection. Application route: Dermal (challenge: occlusive application)
Application volume: 0.5 ml/animal Group size: Test item group: 20 females, control group: 10 fema les– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 98 April 2008

Observations: Mortality, clinical signs, skin effects, body weigh t (at beginning and termination of study) Grading of the skin reaction: 0 = no reaction; 1 = slight localized redness; 2 = moderate confluent redness; 3 = severe redness and swelling
In life dates: April 23, 2002 – May 17, 2002

II. Results and Discussion:

A. Findings: One animal of the test item group (animal no. 27) d ied on day 15 of the study. At the end of the study, the mean body weight of the treatment group animals was in the same range than that of the control group animals. The challenge with the 25% test item formulation le d to skin effects (grade 1 - 3) in 18 of 19 animals (95%) in the test item group and no skin ef fects were seen in the control group animals.

Number of animals exhibiting skin effects during ch allenge Test item group (19 animals) Control group (10 ani mals) Test item patch Control patch Test item patch Cont rol patch

Hours 48 72 Total 48 72 48 72 Total 48 72 Challenge
25% 18 18 18 0 0 0 0 0 0 0

Positive Control: The positive control study with alpha-Hexylimtaldeh yd showed that the study

was able to detect sensitizing compounds in the cho sen laboratory conditions.

III. Discussion and Conclusions:

A. Conclusions: BYI 08330 has a skin-sensitization potential under the conditions of the Maximization test.
B. Reviewer Comments:

The Guinea Pig Maximization Test was performed on 30 female Hsd Poc:DH guinea pigs (20 animals for the test group and 10 animals for the c ontrol group) (age: 5-6 weeks; weight: 292-366 g; source: Harlan Winkelmann GmbH Laboratory Animal Breeders, Borchen) to determine whether BYI 08330 (Purity 96.5%; Batch No. NLL6425-9; beige powder) exhibits skin-sensitizing properties. The test material was formulated in po lyethylene glycol 400 to yield a suspension or a paste. An additional two animals were used for dos e-finding for the challenge concentration.

The study was conducted according to OECD Guideline No. 406/ EC Guideline 96/54/EC (22nd Adaptation of Guideline 67/548/EEC)/Health Effects Test Guideline, OPPTS 870.2600 with the following test material concentrations:

Intradermal induction: 5% (= 20 mg test material/an imal)

Topical induction: 50% (= 250 mg test material/anim al)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 99 April 2008

Challenge: 25% (= 125 mg test material/animal)

One animal from the test group died on day 15 of th e study. After the intradermal induction the animal in the control group and in the test group s howed strong effect up to encrustation at the injection sites of the first induction. The challe nge with the 25% test material formulation led to dermal effects (grade 1-3) in 18 of 19 animals (95% ) in the test material group. No dermal effects were observed in the control group animals.

The Guinea Pig Maximization Test methodology was va lidated in a test using alphaHexylimtaldehyd formulated in sterile physicologica l saline solution (Report No. PH 31458; November, 2001).

In this study, BYI 08330 exhibits a dermal sensitization potential

.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a dermal sensitization study (OPPTS 870.2600; OECD 406) in t he Guinea pig.

C. Reviewer Conclusions:

All countries agree that under the conditions of th e Magnusson and Kligman assay, BYI 08330 exhibits dermal sensitization potential.

D. Study Deficiencies:

Test material used for inhalation, eye and primary irritation and sensitization studies is a “cis” isomer of the TGAI test material used for the acute oral and acute dermal studies.

CITATION: Vohr, H. (2004) Study for Skin Sensitization Effect of BYI 08330 in Guinea Pigs

(Buehler Patch Test). Project Number: AT0317. Unpub lished study prepared by Bayer Ag Inst. of Toxicology. 28 p. July 13, 2004.

I. Materials and Methods:

A. Materials
1. Test material: BYI 08330 (Purity: 97.1%) Description: Technical grade, beige powder

Lot/Batch no: mixed batch no. 08045/0014 Stability of test compound: Stable at room temperature (~ 22 o C)

2. Vehicle and/or positive control: Polyethylene glycol 400 Alpha hexyl cinnamic aldehyde 3. Test animals Species: Guinea pig, females– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 100 April 2008

Strain: SPF-bred [Crl:HA] Age: 4-5 weeks Weight at dosing: 309 g – 375 g
Source: Charles River Laboratory, Kißleg, Germany
Acclimation period: at least five days before treatment

Diet: Provimi Kliba 3420; Provimi Kliba AG, Switzerland

Water: Tap water
Housing: During the adaptation and study period the animals were conventionally kept in type IV Makrolon® cages, in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. The cag es were exchanged for ones with clean bedding at least two times per week. Low-dust wood shavings supplied by Ssniff Spezialdiäten GmbH, Soest, were used as bedding. Environmental conditions: Temperature:

Humidity: Lighting: Air changes: 22 +/- 3°C (possibly drifting higher at outdoor tem peratures

above 24°C)
40-70 % Twelve hours; artificial lighting

≥ 10 times per hour

B. Study Design and Methods 1. Animal assignment and treatment

Dose: Range-finding Study: A pilot study was performed using 3 test concentrat ions ( 25%, 50%, 71%) and a vehicle group with 5 guinea pi gs each. The test material was held in place for 6 h ours. The dermal reactions were evaluated 30 and 54 hours aft er application. No dermal reactions were seen in any test group. The 71% concentration was chosen for the ma in
test. Induction (71%): The animals were dermally treated with the test mat erial three times at intervals of seven days. The suitabl e areas of the body were shaved 24 hours before each treatment . The 1st to 3rd inductions were performed with the 71% t est item paste. The animals in the test group were treated with a hypoallergic patch loaded with the test material ap plied to the left flank and held in place on the skin with a n adhesive plaster. The patches were removed after an exposur e period of six hours, and any remaining test item wa s washed off the skin with physiological saline solution. T he volume applied per animal was 500 mg test material mixed w ith 0.2 ml vehicle in the test group (vehicle was polyethyl ene glycol 400). The treatment areas were visually assessed 3 0 hours after initiation of exposure. For this, the treatme nt areas were not shorn or chemically depilated. Challenge (71%): The challenge was performed two weeks after the las t dermal induction. In the meantime, the test materi al– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 101 April 2008

concentrations for the challenge had been determine d in a dose range-finding study using 2 guinea pigs that w ere treated during the inductions in the same manner as the control animals. During the challenge a hypoallerg enic patch loaded with the 71% test material paste was a pplied and fixed to the right flank of each animal in the control and test group. As a control a patch loaded only with t he vehicle was applied and fixed also to the right flank, cran ial to the test material patch. The patches were held securely in place on the skin with self-adhesive tape for 6 hours. T he volume applied for the challenge was 0.5 ml vehicle for co ntrol group, and 500 mg test material mixed with 0.2 ml v ehicle for test item group. At the end of the six-hour ex posure period, the patches were removed and the remaining test item was rinsed away with physiological saline solu tion. Twenty-one hours later the skin of the animals was shorn in the region of the treatment sites.
Naive Controls: In the control group animals, a hypoallergic patch loaded only with the vehicle was applied to the left flank and fixed with a strip of adhesive tape in each of the first to third inductions. The patches were removed after an expos ure period of six hours. The volume applied per animal was 0.5 ml vehicle in the control group. The treatment are as were visually assessed 30 hours after the initiation of exposure. For this, the treatment areas were not shorn or che mically depilated. Application route: Dermal (challenge: occlusive application) Application volume: 0.5 ml/animal Group size: Test item group: 20 females, control group: 10 fema les

Observations: Mortality, clinical signs, skin effects, body weigh t (at beginning and termination of study) Grading of the skin reaction: 0 = no reaction; 1 = slight localized redness; 2 = moderate confluent redness; 3 = severe redness and swelling
In life dates: May 18, 2004 – June 17, 2004

II. Results and Discussion:

A. Findings:

Appearance and behaviour of the test item group wer e not different from the control group. In the control and test item group there were no skin effe cts during the induction treatments. By the end of the study the mean body weight of the treatment group animals was in the same range than that of the control group.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 102 April 2008

Number of animals exhibiting skin effects during ch allenge

Test item group (20 animals) Control group (10 ani mals)
Test item patch Control patch Test item patch Cont rol patch

Hours 30 54 Total 30 54 30 54 Total 30 54
Challenge
71% 0 0 0 0 0 0 0 0 0 0

Positive Control: The study report indicates a positive control study with alpha hexyl cinnamic

aldehyde was performed. It the study author states 95% of the test animals exhibited dermal reactions and therefore demonstrat ed the sensitivity of the study. However, no study individual or tabular res ults were present in the study. Also, EPA guidelines suggest the positive control s tudy be performed within 6 months of the primary test. No dates or references for this positive control study were provided.

III. Discussion and Conclusions

A. Conclusions:

BYI 08330 has no skin-sensitization potential under the conditions of the Buehler test.

B. Reviewer Comments:

The Buehler epicutaneous patch test was performed o n 30 female Crl:HA guinea pigs (20 animals for the test group and 10 animals for the c ontrol group) (age: 4-5 weeks; weight: 309-375 g; source: Charles River Laboratory Animal Breeders . KiBlegg, Germany) to determine whether BYI 08330 (Purity: 97.1%; Batch No. Mix-Batch 08045 /0014; white powder) exhibits skinsensitizing properties. The test material was form ulated in polyethylene glycol 400 to yield a suspension or a paste. An additional two animals w ere used for dose-finding for the challenge concentration.

The study was conducted according to OECD Guideline No. 406/ EC Guideline 96/54/EC (22nd Adaptation of Guideline 67/548/EEC); Health Effects Test Guideline, OPPTS 870.2600 with the following test material concentrations:

1 st to 3 rd induction: 71% Challenge: 71%

In the control and test groups there were no dermal effects during the induction treatments. The challenge with the 71% test material paste produced no dermal effects in the test group and there were no dermal effects in the control group.

The Buehler patch test methodology was validated in a test using alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400, positive con trol. (Report No. PH33315; May, 2004).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 103 April 2008

In this study, BYI 08330 exhibits no dermal sensitization potentia l.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a dermal sensitization study (OPPTS 870.2600; OECD 406) in t he Guinea pig.

C. Reviewer Conclusions:

All countries agree that under the conditions of th e Buehler Patch Test, BYI 08330 exhibits no dermal sensitization potential.

D. Study Deficiencies:

1) The highest soluble concentration tested was 71% This seems to be a deviation from OECD guideline No. 406, where the induction exposure sho uld be the highest concentration to cause mild irritation.

2) Test material was not clearly identified as no CAS No was declared. The test material used for inhalation, eye and primary irritation and othe r sensitization studies is a “cis” isomer of the TGAI test material used for the acute oral and acut e dermal studies.

CITATION : Esdaile, D. (2004) BYI 08330: Evaluation of Potenti al Dermal Sensitization in the

Local Lymph Node Assay. Project Number: SA/04120. U npublished study prepared by Bayer Cropscience. 42 p. September 9, 2004.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 (Purity: 97.2%) Description: Technical grade, beige powder

Lot/Batch no: 08045/0014 Stability of test compound: Stable at room temperature (~ 22 o C)

2. Vehicle and/or positive control: Dimethylformamide Isoeugenol 3. Test animals Species: Mouse, females

Strain: CBA/J Age: 8 weeks Weight at dosing: 22 g – 23 g
Source: R. Janvier, Le Genest St Isle, France Acclimation period: at least five days before study start

Diet: Certified rodent pellet diet: AO4C-10, S.A.F.E. (91 360– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 104 April 2008

Epinay sur Orge, France)
Water: Tap water Housing: The animals were housed in a room within a barrier maintained unit with restricted entry. Mice were ho used individually in suspended, stainless steel, wire-me sh cages. Environmental conditions: Temperature:

Humidity: Lighting: Air changes: 20°C - 24°C

40 - 70% 12-hour light, 12-hour dark cycles (7 am - 7 pm) The Animal Unit ventilation system is constantly mo nitored; regular checks are made in the individual animal ro om for the target specification of 10 to 15 air changes pe r hour.

B. Study Design and Methods

1. Animal assignment and treatment
Dose: Forty five female CBA/J mice were allocated to 9 gr oups of five animals each:

- four groups receiving the test substance at a con centration of 10, 5, 2.5 or 1%,

- four positive control groups receiving the refere nce substance (Isoeugenol) at a concentration of 5, 2.5 , 1 or 0.5%,

- one control group receiving the vehicle, Dimethylformamide (DMF) Treatment preparation and administration: Forty five mice were allocated to 9 groups of five animals each: four groups receiving the test material at a concentration of 10, 5, 2.5 or 1%; four positive co ntrol groups receiving the reference substance (Isoeugeno l) at a concentration of 5, 2.5, 1 or 0.5% and one control group receiving the vehicle, Dimethylformamide (DMF).

The test material, positive control and the vehicle were applied on external surfaces of each ear (i.e. 50 μ l/animal) for three consecutive days (Days 0, 1 and 2) at the appropriate concentration. On Day 5, animals were intravenously injected via the tail vein with 250 μ l of 0.9% sodium chloride containing 20μCi if 3H methyl thymi dine. Five hours after intravenous injection the mice wer e sacrificed and the two auricular lymph nodes were r emoved from each mouse. The nodes from each mouse were placed in an individual tube containing physiologic al saline and were disaggregated by crushing with a plastic p iston. A single cell suspension was obtained, free of connec tive tissue. Cell suspensions were washed with 6 ml of 0.9% physiological saline, centrifuged for 20 minutes at 1800 rpm– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 105 April 2008

and pellets obtained were resuspended in 4 ml of 5% trichloroacetic acid (TCA) and stored overnight at +4°C. After a final centrifugation, the pellets were resu spended in 1 ml of saline, mixed and then placed for 25 minutes in an Ultrasonic Bath to ensure a thoroughly dispersed suspension. Once prepared cell suspensions were add ed to numbered scintillation pots containing 10 ml of sci ntillation fluid and counts were taken in a beta-counter. The results were expressed as disintegrations per minute (DPM) per animal.

Animals were weighed before treatment and at study termination. The site of application was examined for signs of irritation. The animals were checked for clinic al signs and mortality at least once a day during the study. Group size: 9 groups of five animals each

In life dates: June 2, 2004 – June 7, 2004

II. Results and Discussion:

A. Findings:

No mortality and no clinical signs were observed du ring the study. No cutaneous reactions were observed in the vehicle, reference control or treat ed groups. The proliferation index values of the test substance were 5.9, 5.4, 4.3 and 3.4 at treatm ent concentrations of 10, 5, 2.5 and 1% respectively. The proliferation index values of the positive control were 3.4, 1.8, 1.3 and 0.8 at treatment concentrations of 5, 2.5, 1 and 0.5% resp ectively.

Disintegrations per Minute/Mouse (group means):

Dose % Animal
Number Individual Animal
DPM Group Mean DPM Stimulation Index (SI*)

OT1F2434 181
OT1F2435 569
OT1F2436 683
OT1F2437 481 Vehicle Control
DMF

OT1F2438 451 473.0 1.0

OT2F2439 3178
OT2F2440 2632
OT2F2441 2645
OT2F2442 3417 BYI 08330 10% in
DMF

OT2F2443 2116 2797.4 5.9

OT3F2444 1869
OT3F2445 1130
OT3F2446 2911
OT3F2447 2137 BYI 08330 5%
in DMF

OT3F2448 4685 2546.4 5.4– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 106 April 2008

OT4F2449 3141
OT4F2450 1541
OT4F2451 1764

OT4F2452 1377 BYI 08330 2.5% in
DMF

OT4F2453 2367 2038.0 4.3

OT5F2454 727

OT5F2455 838 BYI 08330 1% in
DMF
OT5F2456 2279 1621.7 3.4

OT5F2457 2119

OT5F2458 2145

OT6F2459 1183

OT6F2460 2497

OT6F2461 2104

OT6F2462 577 Isoeugenol 5%
In DMF

OT6F2463 1597 1591.6 3.4

OT7F2464 798

OT7F2465 613

OT7F2466 998

OT7F2467 870 Isoeugenol 2.5%
In DMF

OT7F2468 1031 862.0 1.8

OT8F2469 539

OT8F2470 657

OT8F2471 308

OT8F2472 1095 Isoeugenol 1%
In DMF

OT8F2473 491 618.0 1.3

OT9F2474 539

OT9F2475 509

OT9F2476 352

OT9F2477 268 Isoeugenol 0.5%
In DMF

OT9F2478 166 366.9 0.8– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 107 April 2008

Stimulation Index: Sample Description Test or Control Group Stimulation Index (SI)

Control DMF 1.0

BYI 08330 10% 5.9

BYI 08330 5% 5.4

BYI 08330 2.5% 4.3

BYI 08330 1% 3.4 Isoeugenol 5% 3.4 Isoeugenol 2.5% 1.8 Isoeugenol 1% 1.3 Isoeugenol 0.5% 0.8

III. Discussion and Conclusions:

A. Conclusions: BYI 08330 has a skin-sensitization potential under the conditions of the Local Lymph Node Assay.

B. Reviewer Comments:

In a dermal sensitization study with BYI 08330 (Pur ity: 97.2%; Batch No. 08045/0014), forty five female CBA/J mice (age: 8 weeks; weight: 21.1-24.0 g; source: R. Janvier, Le Genest St Isle, France) were tested using the Local Lymph Node Assa y. The mice were allocated to nine groups of five animals each: four groups receiving the test material each at a concentration of 10, 5, 2.5 or 1%; four positive control groups rece iving the reference substance (Isoeugenol) at a concentration of 5, 2.5, 1 or 0.5% and one contro l group receiving the vehicle, Dimethylformamide (DMF).

The test material, positive control and the vehicle were applied on external surfaces of each ear (i.e. 50 μl/animal) for three consecutive days (Day s 0, 1 and 2) at the appropriate concentration. On Day 5, animals were intravenously injected via t he tail vein with 250 μl of 0.9% sodium chloride containing 20μCi if 3H methyl thymidine. Five hours after intravenous injection the mice were sacrificed and the two auricular lymph nodes w ere removed from each mouse. The lymph nodes from each mouse were placed in an individual tube containing physiological saline and were disaggregated by crushing with a plastic pisto n. A single cell suspension was obtained, free of connective tissue. Cell suspensions were w ashed with 6 ml of 0.9% physiological saline, centrifuged for 20 minutes at 1800 rpm and pellets obtained were resuspended in 4 ml of 5% trichloroacetic acid (TCA) and stored overnight at +4°C. After a final centrifugation, the pellets were resuspended in 1 ml of saline, mixed and then placed for 25 minutes in an Ultrasonic Bath to ensure a thoroughly dispersed suspension. Once p repared cell suspensions were added to numbered scintillation pots containing 10 ml of sci ntillation fluid and counts were taken in a betacounter. The results were expressed as disintegrat ions per minute (DPM) per animal.

Animals were weighed before treatment and at study termination. The site of application was examined for signs of irritation. The animals were checked for clinical signs and mortality at least once a day during the study.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 108 April 2008

The criterion for a positive response was a statist ically significant increase in cell proliferation i n the test concentration groups compared to the vehic le control group and/or SIs greater than or equal to 3.0.

No mortality and no clinical signs were observed du ring the study. Animals in 10, 2.5 and 1% test groups gained body weight while animals in the 5% group lost body weight. No dermal reactions were observed in the vehicle, reference c ontrol or treated groups.

The stimulation index values of the test material w ere 5.9, 5.4, 4.3 and 3.4 at treatment concentrations of 10, 5, 2.5 and 1%, respectively. The stimulation indices of the positive control were 3.4, 1.8, 1.3 and 0.8 at treatment concentrati ons of 5, 2.5, 1 and 0.5%, respectively.

In this study, BYI 08330 exhibited dermal sensitiza tion potential.

In comparison with the positive control substance, BYI 08330 had a skin sensitization potential of approximately five times that of Isoeugenol.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a dermal sensitization study (OPPTS 870.2600; OECD 429) in t he Mouse.

C. Reviewer Conclusions:

All countries agree that under the conditions of th e LLNA Assay, BYI 08330 exhibits dermal sensitization potential.

D. Study Deficiencies:

Test material was not clearly identified as no CAS No was declared. The test material used for inhalation, eye and primary irritation and other se nsitization studies is a “cis” isomer of the TGAI test material used for the acute oral and acute der mal studies.

B.6.2.7 Potentation/interactions of multiple active substances or products (OECD Annex IIA

5.2.7)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 109 April 2008

B.6.2.8 Summary of acute toxicity

BYI 08330 (Spirotetramet Technical) The acute toxicity studies for BYI 08330 were condu cted between 2002 and 2005 at the following testing facilities: Bayer AG Institut für Toxikologie, Baye r AG PH Prof. Toxicology, Bayer CropScience, Bayer CropScience LP, BayerHealthCare AG and LPT Laborato ry of Pharmacology. Four additional acute oral toxicity studies on BYI 08330 metabolites were cond ucted between 2005 and 2006 at BayerHealthCare AG. All studies were fully compliant with Good Laborato ry Practice (GLP) and were conducted in accordance with the relevant prevailing OECD and OPPTS guideline.

The test material for the eight main acute toxicity studies was identified as BYI 08330. However, the purity, batch numbers and description varied between the di fferent testing facilities [(Purity: 93.1%; Batch N o. NLL6425-9; beige powder); (Purity: 96.5%; Batch No. NLL6425-9; beige powder); (Purity: 97.1%; Batch No

. Mix-Batch 08045/0014; white powder) and (Purity: 97 .2%; Batch No. 08045/0014)]. For the metabolite studies the test material was identified as follows : BYI 08330-CIS-Ketohydroxy (Purity: 98.7%; Batch No. NL L 7549-7; light yellow solid), BYI 08330-desmethyl- ketohydroxy (Purity: 94.6%; Batch No. KATH 4726-5-1 ; light beige powder), BYI 08330-di-hydroxy (Purity: 94.5%; Batch No. KATH 4727-3-6; light beige powder) and BYI 08330-mono-hydroxy (Purity: 98.41%; Batch N o. NLL 7635-2D; white powder).

BYI 08330 and its metabolites were found to be of l ow acute toxicity to the rat by the oral route. No treatment-related findings were observed at the lim it dose level of 2000 mg/kg bw. Treatment-related findings in the acute dermal toxicity were limited to minor clinical signs (resolved by day 3) at the dose level of 2000 mg/kg bw. Treatment-related findings in th e acute inhalation toxicity study were limited to m inor bodyweights and clinical signs (resolved by day 8) at the dose levels of 1.1 and 4.183 mg/L.

In the primary eye irritation study, corneal opacit y was observed in all eyes from 24 hours to 6 days after instillation and in two eyes until 7 days after ins tillation. No corneal opacity was observed on day 8. Iritis was noted in two eyes at 24 and 48 hours, in three eyes at 72 hours and days 4 and 5, and in one eye o n day 6 after instillation. No iritis was observed o n day 7. Conjunctival redness was noted in all eye s 1 hour to 72 hours after instillation and in two eyes until 4 days after instillation. Conjunctival chemosis wa s noted in one eye at 24 and 48 hours after instillation. All conjunctival irritation was resolved by day 5.

BYI 08330 was not irritating to rabbit skin. Three dermal sensitization studies were conducted with B YI 08330. In two of the studies, Maximization Test ac cording to Magnusson & Kligman and Local Lymph Node Assay (LLNA), BYI 08330 exhibited a dermal sensitiz ation potential. In the third study, Buehler Patch Test, BYI 08330 exhibited no dermal sensitization potenti al.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 110 April 2008

B.6.3 Short term toxicity (OECD Annex IIA 5.3)

B.6.3.1 Oral 28-day toxicity (rodents) (OECD Annex IIA 5.3.1)

CITATION : Schladt, L., 2001. BYI 08330: Subacute study with mice. Bayer HealthCare AG,

PH-GDD- Toxicology, 42096 Wuppertal, Germany. Laboratory report number M-035927-01- 2, September 13, 2001. Unpublished.

1. MATERIALS AND METHODS:

A. MATERIALS :

1. Test material : BYI 08330 Description: Not reported Batch #: NLL 6425-7 Purity: 97.0% a.i. Compound stability: Stable when stored at room tem perature (Report No. 201363) CAS # if TGAI: Not reported Structure:

2. Vehicle: Basal diet

3. Test animals : Species: Mouse Strain: Crl:CD-1(ICR)BR Age/weight at study initiation: Not reported

Source: Not reported Housing: Not reported Diet: Basal diet (no details given) Water: No data Environmental conditions: Not reported Acclimation period: Not reported

B. STUDY DESIGN :

1. In life dates : Not reported (Year of report: 2001)

2. Animal assignment : The method of animal assignment to the test group s was not reported, but the animals were assigned to various groups as noted be low in Table 6-28.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 111 April 2008

Table B.6-28: Study design

Test group Conc. in diet (ppm) Dose to animal (mg/kg/day) # Male

Control - - 0 5
Low dose 500 136.5 5 High dose 5000 1,415 5

3. Dose selection rationale : No rationale for t he dose selection was provided.

4. Diet preparation and analysis : No details regarding diet preparation were provided . Analyses of test compound in the diet formulations were not performe d. However, homogeneity of BYI 08330 in Purina Mills Certified Rodent Diet 5002 Meal and stability over 7 days at room temperature was confirmed in another Bayer study (Report No. 201363). BYI 08330 was stable in the freezer at 70 ppm for 32 days and at 1200 ppm for 29 days. The mean recovery val ue of fortified ration was 99.8%.

5. Statistics : Not reported

C. METHODS :

1. Observations : Animals were inspected for signs of toxicity and mo rtality daily and more detailed clinical observations were conducted weekly. No details wer e reported.

2. Body weight : Animals were weighed weekly and at the end of the study period.

3. Food consumption and compound intake : Food consumption for each group was reported for th e entire study period and mean daily diet consumption was calculated as g/animal/day and as g food/kg body weight/day. Food efficiency was not calculate d. The method of calculating compound intake (mg/kg bw/day) values was not reported.

4. Ophthalmoscopic examination: Eyes were not examined.

5. Clinical chemistry : The method of blood collection was not reported. However, at study termination the blood was collected from all surviving animals. Th e CHECKED (X) parameters were examined.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 112 April 2008

b. Clinical chemistry :

ELECTROLYTES OTHER

Calcium* Albumin*
Chloride* Creatinine* Magnesium Urea nitrogen* Phosphorus* X Total Cholesterol*
Potassium* Globulins
Sodium* Glucose*

ENZYMES (more than 2 hepatic enzymes eg.,*) Total bilirubin*
X Alkaline phosphatase (ALK)* Total protein (TP)* Cholinesterase (ChE) X Triglycerides Creatine phosphokinase Serum protein electrophore sis Lactic acid dehydrogenase (LDH) X Alanine amino-transferase (also SGPT)* X Aspartate amino-transferase (also SGOT)* Sorbitol dehydrogenase* Gamma glutamyl transferase (GGT)* Glutamate dehydrogenase

6. Urinalysis : Not conducted

.

7. Sacrifice and pathology : All animals were sacrificed by exanguination (hea rt puncture) under deep anesthesia. The liver, adrenals, testes, epidiymide s were weighed and subjected to gross and histolgic al exammination. All collected tissue was fixed in 10 % formalin (except testes which were fixed in Davidson’s fluid), embedded in paraplast, and stain ed with H & E. In addition, cuts from liver samples were stained with Oil Red O.

II. RESULTS:

A. OBSERVATIONS :

1. Clinical signs of toxicity : No treatment-related clinical signs of toxicity we re observed.

2. Mortality: All rats survived to study termination.

B. BODY WEIGHT AND WEIGHT GAIN : There were no treatment-related effects on bo dy weight and body weight gain of treated rats.

C. FOOD CONSUMPTION AND COMPOUND INTAKE :

1. Food consumption : Food consumption was lower than controls by 19% an d 16% at the 500 and 5000 ppm levels, respectively. The lower feed intake in both treatment groups was not considered compound-related, because the changes were not corr elated with the dose.

2. Compound consumption : Refer to Table 6-28 for details.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 113 April 2008

D. CLINICAL CHEMISTRY : Determination of activity of aspartate aminotransfe rase, alanine aminotransferase, alkaline phosphatase, and cholest erol and triglyceride concentrations in peripheral blood did not reveal treatment-related effects.

E. SACRIFICE AND PATHOLOGY :

1. Organ weight : Liver weight was not affected by the treatment.

2. Gross pathology : Organ weights of adrenals, liver, testes and epidi dymides did not differ significantly from the control weights. Necropsy revealed no trea tment-related findings.

3. Microscopic pathology : Histopathological investigations of liver/gall bl adder, adrenal glands, testes and epididymides revealed no evidence of any dose-r elated histopathological changes in animals up to and including 5,000 ppm.

III. CONCLUSIONS:

The subacute toxicity study in male mice establishe d a NOAEL of 5,000 ppm (equal to 1,415 mg/kg/bw/day), based on the lack of treatment-relat ed findings at 5,000 ppm.

REVIEWER COMMENTS: The reviewer agrees with the study author’s overal l conclusions that fourweek dietary treatment of male mice with BYI 08330 at dietary concentrations of 500 and 5000 ppm resulted in no treatment-related effects.

IV. STUDY DEFICIENCIES:

The study did not follow the GLP Standards and many details regarding the methods, material, and parameters measured were not provided. No rational e was provided for using only the male mouse. These deficiencies could affect the interpretation of study results.

CITATION : Krotlinger, F., and F. Mihail. (1998). Cyclic keto enols BSN 3457, BSN 2342,

FHN 7504, FHN 8330 – Subacute exploratory toxicit y studies in rat (Application by feed over 4 weeks). Bayer HealthCare AG, PH-GDD- Toxic ology, 42096 Wuppertal, Germany. Laboratory report number MO-02- 003395, February 13 , 1998. Unpublished.

I. MATERIALS AND METHODS:

A. MATERIALS :

1. Test material : FHN 8330 Description: Technical, white powder (Study Report # 201363)
Batch #: FHN 8330-3 Purity: 98.2% a.i. Compound stability: Stable when stored at room tem perature (Study Report # 201363) CAS # if TGAI: Non-Stereo: 382608-10-8; CIS: 20331 3-25-1– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 114 April 2008

Structure:

2. Vehicle: Basal diet

3. Test animals : Species: Rat
Strain: Hsd/Win:WU Age/weight at study initiation: Not reported

Source: Harlan-Winkelmann, Borchen, FRG Housing: Not reported Diet: Basal diet (no details given)
Water: No data
Environmental conditions: No information provided Acclimation period: No information provided

B. STUDY DESIGN :

1. In life dates : Start: September 1, 1997; End: September 29, 1997

2. Animal assignment : The method of animal assignment to the test group s was not reported; however, the animals were assigned to various groups as note d below in Table 6-29.

Table B.6-29: Study design

Test group Conc. in diet (ppm) Dose to animal (mg/kg/days) # Female

Control - - 0 10
Low dose 500 47.3 5 High dose 5000 501.8 5

3. Dose selection rationale : No rationale for the dose selection was provided

4. Diet preparation and analysis : No details regarding diet preparation were provided . Analyses of test compound in the diet formulations were not performe d. However, homogeneity of BYI 08330 in Purina Mills Certified Rodent Diet 5002 Meal and stability over 7 days at room temperature was confirmed in another Bayer study (Report # 201363, 2005). BYI 0 8330 was stable in the freezer at 70 ppm for 32 days and at 1200 ppm for 29 days. The mean recover y value of fortified ration was 99.8%. In the present study, the test compound was mixed into the diet at the start of the study and two weeks thereafter. One half of each of the mixes was fed immediately and the remaining half after being froz en for a week.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 115 April 2008

Results:

Homogeneity/Stability analysis: Confirmed in Bayer Study Report # 201363.

Concentration analysis: Not provided. Based on the results of homogeneit y analysis (Report # 201363) it was assumed that the animals received the intended concentration of the test compound.

5. Statistics : Not conducted

C. METHODS :

1. Observations : Animals were inspected for signs of toxicity and mo rtality. However, the frequency of observations was not reported.

2. Body weight : Animals were weighed weekly.

3. Food consumption and compound intake : Food consumption for each animal was determined wee kly and mean daily diet consumption was calculated as g food/kg body weight/day. Food efficiency was not calculated. The method of calculating compound int ake (mg/kg bw/day) values was not reported.

4. Ophthalmoscopic examination: Eyes were not examined.

5. Clinical chemistry : The method of blood collection was not reported. However, at study termination the blood was collected from all surviving animals. Th e CHECKED (X) parameters were examined. b. Clinical chemistry :

ELECTROLYTES X OTHER

Calcium* Albumin*

Chloride* Creatinine* Magnesium Urea nitrogen* Phosphorus* X Total Cholesterol*

Potassium* Globulins

Sodium* Glucose*

X ENZYMES (more than 2 hepatic enzymes eg.,*) Total bilirubin*
X Alkaline phosphatase (ALK)* Total protein (TP)* Cholinesterase (ChE) X Triglycerides Creatine phosphokinase Serum protein electrophore sis Lactic acid dehydrogenase (LDH) Alanine amino-transferase (also SGPT)* X Aspartate amino-transferase (also SGOT)* Sorbitol dehydrogenase* X Gamma glutamyl transferase (GGT)* Glutamate dehydrogenase

* Recommended for subchronic non-rodent studies bas ed on Guideline 870.3150

6. Urinalysis : Not conducted

.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 116 April 2008

7. Sacrifice and pathology : All animals were sacrificed by exanguination unde r ether anesthesia. All animals that died and those sacrificed on schedule were subjected to gross pathological examination. The liver was weighed and, in addition to any gross findings, was fixed in 4% formalin and embedded in paraplast. Liver samples were sectioned at 5μm and stained with H & E. In addition, cuts from liver samples were stained with Oil Red O. Tissue sampl es of liver from the subacute rat study (Study # T 0061869) with FHN 8330 were used to determine the l iver cell proliferation index. Quantification of proliferating cell nuclear antigen (PCNA) positive and PCNA negative nuclei was performed for control and high-dose animals. The proliferation index was calculated as the number of PCNA positive nuclei per 1000 nuclei.

II. RESULTS:

A. OBSERVATIONS :

1. Clinical signs of toxicity : No treatment-related clinical signs of toxicity we re observed.

2. Mortality: All rats survived to study termination

B. BODY WEIGHT AND WEIGHT GAIN : There were no treatment-related effects on body wei ght and body weight gain of treated rats.

Table B.6-30: Average body weights and body weight gains during 28 days of treatment a,b
Body weights (g±SD) Total weight gain Dose

rate [ppm] Week -1 Week 1 Week 2 Week 3 Week 4 g % Change
from
Control

0 105±5.1 117±5.71 133±7.28 151±8.15 165±8.46 60 - -

500 102±3.43 113±3.57 126±3.91 144±6.18 155±10.3
1 53 -12

5000 104±6.53 115±8.88 127±10.0
6 145±10.8

9 159±13.0

3 55 -8

a Data obtained from page 20 in the study report.

b Calculated by the reviewer

C. FOOD CONSUMPTION AND COMPOUND INTAKE :

1. Food consumption : Food intake was not affected by the treatment

2. Compound consumption : Refer to Table 6-29 for details.

D. CLINICAL CHEMISTRY : There were no toxicologically relevant changes seen in the parameters measured. The slight decrease in mean triglyceride levels at 500 and 5000 ppm on Day 28 (0.65±0.136 and 0.85±0.209, respectively, vs. 1.01±0.327 in con trol group) occurred in a non-dose related manner.

E. SACRIFICE AND PATHOLOGY :

1. Organ weight : Liver weight was not affected by the treatment– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 117 April 2008

2. Gross pathology : No treatment-related macroscopic changes were seen in treated rats.

3. Microscopic pathology : No treatment-related histopathological findings w ere observed in the liver from treated rats.

4. Cell proliferation assay : The proliferating cell nuclear antigen (PCNA) as say did not reveal a toxicologically significant increase in liver cell proliferation. The proliferation index and nuclear area values for PCNA negative and PCNA positive cells we re similar for both control and treated animals.

III. CONCLUSIONS :

Groups of 5 female rats of strain Hsd/Win:WU were a dministered BYI 08330 daily at dietary dosage levels of 500 and 5000 ppm over a period of 4 weeks . No clinical signs of toxicity were observed and survival was unaffected by the treatment. Body wei ght, body weight gain and food intake of treated ra ts were comparable to the control group. The intake of test compound was proportional to the dietary concentration. Clinical chemistry examination revea led non-dose related decrease in triglyceride level s at 500 and 5000 ppm. The liver weight of treated r ats was comparable to the controls. Gross and histopathology showed no treatment-related effects. The liver cell proliferation assay showed no relev ant increase in cell proliferation or in nuclear area v alues for treated rats. The study author concluded that a four-week treatment with BYI 08330 slightly influen ced the lipid metabolism in female rats.

REVIEWER’S COMMENTS: The reviewer agrees with the study authors’ overall conclusion that fourweek dietary treatment of female rats with BYI 0833 0 at dosages 500 and 5000 ppm resulted in no treatment-related effects. The slight decrease in mean triglyceride level at 500 and 5000 ppm was not dose-dependent or substantiated by any microscopic findings. Therefore, this finding was not considered toxicologically relevant or indicative o f an influence on lipid metabolism. The results of the present study were also supported by the findings i n CD-1 mice in which dietary exposure to BYI 08330 resulted in no treatment-related effects in male mi ce up to a dietary concentration of 5000 ppm (Bayer Study Report # T2070951, 2001).

IV. STUDY DEFICIENCIES:

The study did not follow the GLP Standards and seve ral details regarding the methods and material, concentration analyses for the test diet and indivi dual records of parameters measured were not provided. No rationale was provided for using only the female rat. These deficiencies do not appear to affect the interpretation of study results.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 118 April 2008

CITATION 

### : Eigenberg, D. A. (2004). Technical Grade BYI 08330: A Subacute Toxicity Feeding Study in

the Beagle Dog. Bayer CropScience LP, Toxicology, 1 7745 South Metcalf, Stilwell, Kansas. Study No. 02-S19-JU/Report No. 201012. December 13, 2004. Unpublished.

Animals were assigned to treatment groups as noted in Table 6-31.

Table B.6-31: Study design Conc. in diet (ppm) a Average dose to animal (mg/kg/day) Number of anima ls

Males and Females Males Females Males Females

0 b 0 0 2 2 100 3 3 2 2 400 13 12 2 2 1600 42 70 2 2 6400 104 127 2 2

a Concentration of chemical in diet was confirmed ana lytically during weeks 1, 2, and 4. Homogeneity was verified at the outset of the study.

b Maintained on diet with vehicle only

I. RESULTS AND DISCUSSION:

1. General observations: There was a marked decrease in body weight and body weight gain (Table 6-32), and food consumption (Table 6-33) in the 6,400 ppm dose group of both se xes. Decreases in body weight gain, observed in the other exposure groups, were not dose-related in eit her males or females. Food consumption was slightly depressed in the 1600 ppm males throughout the stud y. Clinical observations were limited to a thin bod y state beginning at day 15 in males and females in t he 6,400 ppm group.

Neurological examinations revealed a mild hind-limb wheelbarrowing deficit at 6,400 ppm in one female and muscular atrophy associated with reduced postural r eactions in the other female and in one male of thi s high-dose group. These alterations might have been secondary effects from the emaciation and thinness observed in these animals. This was supported micro scopically by the lack of any degenerative nervous system changes in these animals.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 119 April 2008

Table B.6-32: Mean Body Weights a (g) Dose Group (ppm) Study Day 0 100 400 1,600 6,400
Males 0 9,952.5 10,681.5 10,777.5 9,125.0 (-8) b 9,355.0 (-6) 7 10,040.0 10,534.5 10,614.0 8,934.0 (-11) 8,42 4.5 (-16) 14 10,268.0 11,319.5 11,317.5 9,265.5 (-10) 8,1 89.0 (-20) 21 10,964.0 11,701.0 11,134.5 9,694.5 (-12) 7,8 43.0 (-28)
28 c 11,230.0 11,421.0 12,398.0 9,830.5 (-12) 7,542. 0 (-33) Gain 1,277.5 739.5 1,620.5 705.5 (-55) -1,813.0
Females 0 7,662.0 8,136.0 7,962.0 7,287.5 (-5) 7,664.5 (0) 7 7,352.0 7,863.5 7,802.0 7,010.0 (-5) 6,766.5 (-8) 14 7,863.5 8,696.5 8,717.5 7,799.0 (-1) 6,788.0 (-14) 21 8,320.0 8,600.0 8,496.0 7,900.0 (-5) 6,603.5 (-21) 28 c 8,596.0 8,991.5 8,928.0 7,871.5 (-8) 6,143.5 (- 29) * Gain 934.0 855.5 966.0 584.0 (-37) -1,521.0 Data obtained from pages 40, 217 and 219 of Report No. 201012

a Values are group means without standard deviations since there were only 2 animals/group

b Values in parentheses are percent differences from control, calculated by the reviewer

c Terminal body weight * = p < 0.05

Table B.6-33: Food consumption (g/animal/day) a Dose Group (ppm) Study Day 0 100 400 1,600 6,400
Males
1 464.5 458.0 374.0 214.0 260.5
2 441.0 368.0 474.5 209.5 147.0
3 440.0 306.0 377.0 218.5 50.0
4 376.5 393.0 359.5 261.0 63.5
5 463.0 410.5 482.5 298.0 105.0
6 460.5 420.0 313.5 360.0 116.5
7 209.0 150.5 167.5 111.0 119.5
14 472.5 447.5 463.5 298.5 192.0
21 487.0 484.5 392.5 345.0 189.0
28 471.0 435.0 536.5 293.5 169.0
29 386.0 200.0 379.0 336.0 176.0 Females 1 324.0 287.0 326.0 199.5 67.0 2 217.5 309.0 197.0 335.5 117.0 3 347.0 313.0 383.5 379.5 82.5 4 307.0 246.0 288.5 391.0 108.5 5 202.5 303.5 227.5 429.0 299.5 6 334.5 367.5 318.5 447.0 309.0 7 116.0 151.5 125.5 232.0 176.0 14 243.5 304.5 308.0 427.5 * 125.5 * 21 376.0 355.5 298.5 504.5 198.5 28 361.0 397.5 217.0 368.0 153.0 29 337.0 245.0 429.0 371.5 108.0 Data obtained from pages 42-45 of Report No. 201012

a Values are group means without standard deviations since there were only 2 animals/group * = p < 0.05– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 120 April 2008

2. Hematology, urinalysis: No treatment-related findings

Clinical chemistry: There were statistically significant decreases in t hyroid hormones (T4 and T3; Tables 6-34 and 6-35) i n the 400, 1,600 and 6,400 ppm dose group males and the 6 ,400 ppm dose group females which were considered compound related. However, these alterations were n ot considered to be biologically significant due to a lack of compensatory increases in TSH (Table 6-36) or ac companying abnormalities in thyroid weight or histopathological appearance of the thyroid gland i n dogs demonstrating hormonal alterations. At 6400 ppm, mean TSH levels were actually decreased in both sex es, and this may reflect normal biological variatio n. In addition, there was no effect on UDP-glucuronyltran sferase activity; increased activity would have pro vided evidence of increased conjugation of T4 and T3 in l iver. Other compound-related changes consisted of decreases in the calcium and albumin levels of the 6,400 ppm dogs, and were considered secondary to emaciation. Calcium and albumin levels are known to be interrelated, for calcium tends to decrease in hypoalbuminemic states (Duncan, J. R. “Endocrine Sy stem” In: Veterinary Laboratory Medicine, Clinical Pathology, 2nd edition, Iowa State University Press , Ames, Iowa, 1986, pp 181-200)

Table B.6-34: Mean thyroxine (T 4) levels a Dose Group (ppm) Parameter 0 100 400 1,600 6,400
Males
T 4 day -11 2.6 2.3 2.1 2.4 2.6
T 4 day 7 1.8 1.7 1.2 0.8 0.3
T 4 day 23 1.5 1.5 0.9 * 0.6 * 0.3 *

Females
T 4 day -11 3.5 2.9 2.3 3.5 2.2
T 4 day 7 1.9 1.6 0.9 1.2 0.4
T 4 day 23 1.9 1.7 1.0 1.4 0.3 * Data obtained from pages 103-118 of Report No. 2010 12

a Values are group means without standard deviations since there were only 2 animals/group

Table B.6-35: Mean triiodothyronine (T 3) levels a Dose Group (ppm) Parameter 0 100 400 1,600 6,400
Males
T 3 day -11 1.0 0.9 0.9 1.1 0.9 T 3 day 7 1.0 1.0 0.8 * 0.7 * 0.4 *
T 3 day 23 0.9 0.9 0.7 0.6 0.4
Females
T 3 day -11 0.8 0.7 0.8 1.1 * 0.8
T 3 day 7 1.1 0.7 0.9 1.0 0.5
T 3 day 23 1.0 0.8 0.9 1.0 0.4 * Data obtained from pages 103-118 of Lab Report No. 201012

a Values are group means without standard deviations since there were only 2 animals/group

Table B.6-36: Mean thyrotropin (TSH) levels a Dose Group (ppm) Parameter 0 100 400 1,600 6,400
Males TSH day -11 0.32 0.21 0.31 0.21 0.16 TSH day 7 0.24 0.16 0.22 0.09 0.07– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 121 April 2008

TSH day 23 0.24 0.10 0.13 0.12 0.06
Females
TSH day -11 0.34 0.09 0.25 0.24 0.12
TSH day 7 0.34 0.17 0.20 0.19 0.03
TSH day 23 0.24 0.13 0.22 0.29 0.02 Data obtained from pages 103-118 of Report No. 2010 12

a Values are group means without standard deviations since there were only 2 animals/group

3. Ophthalmology: No treatment-related effects.

Gross necropsy, organ weights, histopathology:
Emaciation and thinness were noted in one male and in both of the female dogs of the 6,400 ppm group; these were considered secondary to compound adminis tration. Reduced thymus size and/or decreases in absolute and relative (to body weight) thymic weigh ts were observed at 6400 ppm in males and females (Table 6-37), which may possibly be attributed to a non-specific stress response stemming from the dogs’general, overall state of emaciation / thinnes s at the high dose level.

Table B.6-37: Mean thymus weights a Dietary concentration (ppm) Weights 0 100 400 1600 6400 Males Absolute (g) 15.553 17.146 29.299 14.792 7.303 (-53) c Relative (%) b 0.139 0.150 0.232 0.148 0.094 (-32) Females Absolute (g) 22.453 16.053 16.480 10.491 3.866 (-83) Relative (%) b 0.259 0.178 0.184 0.134 0.063 (-76) Data obtained from pages 218, 220, 223 and 225 - of Report No. 20101

a Values are group means without standard deviations since there were only 2 animals/group

b Relative to body weight

c Values in parentheses are percent differences from controls, calculated by the reviewer

Terminal body weight was decreased in male and fema le dogs at 6,400 ppm. The lower terminal body weight mean in 1,600 ppm males was probably related to the lower baseline mean (Table 6-37). At histopathology, sexual immaturity was observed in o ne high-dose group male, which was considered secondary to the animal’s overall state of emaciati on, rather than a specific target-organ effect. Thi s animal weighed approximately 4 kg less than the control ma les and had also a mild diffuse atrophy of the paro tid salivary glands, which may have reflected its decre ased food intake.

II. CONCLUSIONS: The NOAEL of 1,600 ppm (equal to 42 or 70 mg/kg bw/ day in males or females, respectively) in the subacute toxicity feeding study in dogs is based on decreased body weights, food consumption declines, and an overall state of emaciation at 6,400 ppm (eq ual to 104 or 127 mg/kg bw/day in males or females) . No marked toxicity was determined in the present study

.

The NOEL of 400 ppm (equivalent to 13 mg/kg bw/day in males) is based on non-adverse declines in circulating thyroxine (T4) levels at 1,600 ppm, equ ivalent to 42 mg/kg bw/day.

Reviewer’s comments: The reviewer concurs with the investigator’s conclu sion and rationale with regard to the NOAEL in this study. It was evident that neither males nor females toler ated the 6400 ppm dietary level of BYI 08330 since they lost body weight, ate less food and exhibited an em aciated appearance. Apparent secondary effects in s ome– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 122 April 2008

of these animals included decreased albumin and cal cium in both sexes, and atrophy of the parotid sali vary gland and sexual immaturity in one male.

Reduced thymus size and weight (described microscop ically as involution) were seen in both sexes at 64 00 ppm. The effect on the thymus might have been stres s-related since there was no evidence of lymphocyti c changes (degeneration, necrosis, depletion) in any of the other lymphocytic organs. Statistically significant (p<0.05) or non-significa nt decreases in circulating levels of T4 and T3 we re observed at 400, 1600 and 6400 ppm in males and at 6400 ppm in females; non-statistical decreased TSH levels were also seen at 6400 ppm in both sexes. Al though the decreases in T4 and T3, in addition to T SH, appear to be compound-related, they are not conside red toxicologically significant by the USEPA or PMR A, since there was no evidence of correlative effects on the pituitary-thyroid endocrine axis at gross ne cropsy or histopathology. AGES considers the statistically s ignificant decreases in thyroid hormones observed a t ≥ 400 ppm in males (T3, T4) and also in females (T4), alt hough not statistically significant, to be adverse.

The LOAEL (USEPA and PMRA) for BYI 08330 in this st udy is 6400 ppm (104 and 127 mg/kg/day in males and females, respectively) based on decreased thymus size and weight as well as body weight and food consumption, which resulted in emaciation. The NOAEL (USEPA and PMRA) is 1600 ppm (42 and 70 mg/kg/day in males and females, respecti vely).

The LOAEL (AGES) is 400 ppm (13 and 12 mg/kg/day in males and females, respectively) based on decreased thyroid hormone levels. The NOAEL (AGES) is 100 ppm (3 mg/kg/day in males and females).

5. Study/report deficiencies: There were no deficiencies that compromised the acc eptability of the study.

6. Study deficiencies: There were no significant deficiencies in the exper imental design.

7. Report deficiencies:

• Clinical data were summarized for combined sexes.

• Food efficiency was not reported.

• Statistics were referred to but no methods were de scribed on how this was carried out with 2 animals/sex/dose.

• Could not verify reports at 6400 ppm of mild hind- limb wheelbarrowing deficit in one female and muscular atrophy associated with reduced postural r eactions in the other female and one male during neurological examinations.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 123 April 2008

B.6.3.2 Oral 90-day toxicity (rodents) (OECD Annex IIA 5.3.2)

CITATION : Wahle, B.S., “Technical Grade BYI 08330, A Subchro nic Toxicity Testing Study in the Rat”

Bayer Crop Science, Stilwell, KS, Laboratory report number 201136, June 1, 2005, Unpublished

I. Material and methods

A. Materials:

1. Test Material: BYI 08330 Description: Technical grade, beige powder Lot/Batch No.: NLL 6425-9 Purity: 96.5% (12/2001); 93.5% (6/2002); 93.1% (1/2 003) Compound Stability: Stable at room temperature (~ 22 o C)

Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl

carbonate CAS No. of TGAI: trans : 382608-10-8; cis : 203313-25-1

2. Vehicle and/or positive control: None

3. Test animals: Species : Rat, males and females (nulliparous and non-pregnan t) Strain: Wistar Hanover rats (Crl:WI[Glx/BRL/Han]IGS BR) Age/weight at study initiation: Approximately 9 weeks old / 263.4 - 269.6 g mean gr oup weight males;

171.9 - 177.2 g mean group weight females Source: Charles River Laboratories, Inc. (Raleigh, NC) Housing: Individually housed in suspended stainless steel wire-mesh cages, each

containing a feeder, a source of water (pressure-ac tivated water nipples), and deotized (sanitized) cage board in the bedding tray Diet: Purina Mills Certified Rodent Diet 5002 in "m eal" form; ad libitum Water: Municipal water supply of Kansas City, MO; ad libitum . Exception: Control

animals were without pressurized water for approxim ately 1.5 days (between nominal Days 91 and 93). Only the water remaining in the lines would have been available during this time.

Environmental conditions: Temperature:

Humidity: Air changes: Photoperiod: 18 to 26 o C

30 to 70% (relative) 12 air changes per day (daily average) 12 hr of light [7:00 a.m. to 7:00 p.m.] alternating with 12 hr of darkness; lights toggled off during ophthalmi c examinations Acclimatisation: 03/25/02 (receipt) - 04/01/02 (rel ease for study)

B. Study design:

1. In life dates: Start: 04/01/02 (released for study); 04/09/02 (in itiation of exposure)

End: 07/18/02 (terminal sacrifice complete; subchro nic animals)– –

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08/13/02 (terminal sacrifice complete; recovery animals)

2. Animal assignment: 04/02/02; assigned to a control or one of four chem ically-treated groups using a weight stratification -based computer program obtained from INSTEM Computer Syst ems (Stone, Staffordshire, UK). Each animal on study was identified with a microchip (BioMedic Dat a Systems, Inc., Seaford, DE) subcutaneously implan ted on their dorsal surface in the region between the s capulae; the chip was encoded with a unique number, specifying the animal's sex, dose group, cage numbe r, and study affiliation.

Test Group Conc. in Diet (ppm) Dose to Animal (mg/kg; x+ Std) Male Female

Control (A) 0 0 20 20

Low (B) 150 males 8.9 + 1.4 females 11.4 + 1.2 10 10

Mid 1 (C) 600 males 35.9 + 5.7 females 46.1 + 4.5 10 10

Mid 2 (D) 2,500 males 147.9 + 23.2 females 188.3 + 24.2 10 10

High (E) 10,000 males 615.9 + 91.6 females 752.0 + 84.6 20 20

3. Dose selection rationale: Doses were selected based principally upon the toxi cological profile which emerged in the female rat o ver the course of a subacute (4-week) toxicity testing study conducted with the test article at doses of 0 , 500, and 5,000 ppm BYI 08330 (Report No. T0061869). In that study, findings directly attributable to expos ure to the test substance were not observed. Therefore, d oses of 0, 150, 600, 2,500, and 10,000 ppm were chosen at which to conduct the present study. It w as anticipated that the low and high doses chosen o f 150 and 10,000 ppm would constitute NOAEL and maximum t olerated doses, respectively, with the intermediate doses of 600 and 2,500 ppm providing confirmation o f any dose-response relationships that may have emerged.

4. Diet preparation and analysis: The test compound was mixed directly with the feed; control diet consisted of untreated feed. Samples of each batch of feed mixed were taken and retained in the freezer until the study was complete and the analytical data deemed satisfactory. Replacement ad mixtures for each treatment group were prepared weekly and stored under freezer conditions until pr esented to the animals the following week.

BYI 08330 was administered at a constant concentrat ion in the feed for the duration of the study. The concentration of the AI of the test substance in th e various test diets was analytically verified from samples of each dietary level taken during weeks 1, 2, 3, 7, a nd 11. In addition, one recovery sample was analyz ed with each group of samples during the test concentration analyses. The homogeneity and stability of BYI 08 330 when mixed in the dietary carrier was characterized concurrently with the in-life phase of the study. To evaluate the uniformity of distribution/homogene ity of the test substance in the diet, an analysis of feed containing nominal concentrations of 70 and 10,000 ppm BYI 08330 was conducted. Three (3) samples were taken for analysis from each of 3 distinct layers ( top, middle, bottom; 9 samples total) in a Hobart m ixing bowl. A homogeneous distribution of the test subst ance in the feed was defined in terms of a coeffici ent of variation (CV), derived from the 9 samples taken, w hich was < 10%.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 125 April 2008

To evaluate the stability of the test substance in the diet, a time and temperature analysis of feed c ontaining nominal concentrations of 70 and 10,000 ppm BYI 083 30 was conducted. Stability in the feed was assess ed following 1, 3, and 7 days of room temperature stor age (~22 o C) and 7, 14, and 28 days of freezer storage (~
-23 o C). The stability analysis was carried out as foll ows: An initial sample was taken immediately after the test substance was mixed with the ration. Each bat ch of ration, mixed at the 2 concentrations, was th en divided into 2 portions. One portion was placed in a room temperature environment, the other in the f reezer. Feed samples which remained at room temperature wer e sampled for analysis on Days 0, 1, and 7; feed samples which remained in the freezer were sampled for analysis on Days 7, 14, and 28. Chemical stabi lity in the ration was defined in terms of that period o f time that 80% of the test substance, relative to the initial concentration, was recoverable.

The liquid chromatographic methodology used to perf orm the dietary analyses (i.e., homogeneity, stabil ity, and concentration verifications) required for this study has been described previously.

Results:

Homogeneity Analysis: The mean concentrations of BYI 08330 in the feed, s ampled from 3 distinct layers in the mixing bowl an d containing a nominal concentration of either 70 or 10,000 ppm, were determined to be 68.7 ppm (range 6 5.0- 73.1 ppm; CV = 3.2%) and 9,445 ppm (range 8,817-9,9 43 ppm; CV = 3.2%), respectively. Based on a CV < to 10%, BYI 08330 was judged to be homogeneously di stributed in the feed over a concentration range of 70-10,000 ppm.

Stability Analysis: Following 7 days of room temperature storage, the m ean analytically-determined concentration of the AI of the test substance in the 70- or 10,000-ppm admixtu re was determined to be 68.5 ppm (63.3 ppm on Day 0 ) and 9,564 ppm (9,405 ppm on Day 0), respectively. Following 28 days of freezer storage, the mean analytically determined concentration of the AI of the test substance in the 70- and 10,000-ppm admixt ures was determined to be 71.4 ppm and 9,544 ppm, respec tively (63.3 and 9,405 ppm, respectively on Day 0). BYI 08330 mixed in rodent ration was judged to be s table at room temperature for at least 7 days and following freezer storage for a minimum 28 days, ov er a concentration range of 70-10,000 ppm.

Concentration Analysis: Mean analytical concentrations for each dose group were 143, 596, 2,431, and 9,904 ppm, ranging from 9 5- 99% of the corresponding nominal concentrations of 150, 600, 2,500, and 10,000 ppm, respectively. The AI of the test substance was not detected in the contr ol diet. Mean recovery was 99% and ranged from 95-102% for r odent ration spiked with 150, 600, 2,500 or 10,000 ppm BYI 08330.

5. Statistics: Continuous data that were examined statistically we re evaluated initially for equality or homogeneity of variance using Bartlett's test. Group means were f urther analyzed by a one-way analysis of variance (ANOVA) followed by Dunnett's test. In the event o f unequal variances, and at the discretion of the S tudy Director, data were subject to non-parametric proce dures consisting of a Kruskal-Wallis ANOVA followed by the Mann-Whitney-U test for between-group compariso ns. Frequency data were initially examined for trends; data suggestive of a potential effect were then statistically evaluated using the Chi-Square, Fisher Exact, or Chi-Square and Fisher Exact tests. On a c ase-by-case basis, and at the discretion of the stu dy director, data were subject to additional statistic al procedures other than those mentioned above. Fo r the Bartlett test, a probability (p) value < 0.001 was considered significant; for all other statistical t ests,– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 126 April 2008

differences with p values < 0.05 were considered statistically significant. A ll statistical evaluations were performed using software obtained from either INSTE M Computer Systems or SAS Institute Inc.

C. Methods:

1. Observations: 1a. Cageside Observations: For approximately 18 weeks of the study (14 weeks f or subchronic group animals; 18 weeks for recovery animals), a general assessment of all remaining ani mals was carried out at least daily (generally twic e daily during the normal work week and once daily on weeke nds and holidays). These assessments were generally with respect to mo ribundity and mortality, but also included a more general clinical observation for readily apparent c linical signs (in addition to moribundity and morta lity).

1b. Clinical Examinations: Once every week for approximately 18 weeks of the s tudy, detailed physical examinations for clinical s igns of toxicity were performed on all surviving animals , which included evaluation of external surface are as, orifices, posture, general behavior, respiration, a nd excretory products.

1c. Neurological Evaluations: Parameters specifically designed to evaluate possib le neurological effects were not included in this s tudy (i.e., other than those effects that would be ident ified during cage-side or detailed observations). T hese types of investigations were conducted as a part of other toxicology studies, including an acute neurotoxici ty screening study (Report No. 201283) and a 1-year ch ronic rat study (Report No. 201486).

2. Body weight: Individual body weight determinations were performe d weekly throughout the in-life phase of the study on all surviving animals.

3. Food consumption and compound intake: Individual food consumption determinations were per formed weekly throughout the in-life phase of the s tudy on all surviving animals (g consumed/animal/day and g consumed/kg body wt/day). In addition, using specifically defined criteria, food consumption dat a were corrected, as conditions dictated, to accoun t for misleading indications of food intake (i.e., excess ive spillage, clogged feeders, etc.). Test substan ce intake (mg/kg body wt/day) was calculated using weekly mea n body weight and food consumption data. The general relationship used for this calculation was:

[AI in feed (ppm)/1,000] x [feed consumed (g/kg bod y wt/day)] = mg AI/kg body wt/day

4. Ophthalmologic examination: Ophthalmologic exams were conducted on all acclimat ized animals prior to exposure, and then again on a ll surviving animals just prior to their respective te rmination dates (i.e., recovery animals were not ev aluated at the end of the exposure phase, only at the end of t he recovery phase). For listing of the defining co nditions of the 5-level severity grading system in use at th is facility for assigning severity grades to both ophthalmologic and nonneoplastic histopathologic le sions, see the "Histology" section below.

5. Hematology & Clinical Chemistry: Blood was collected from all surviving animals just prior to their respective termination dates (i.e., nonrecovery animals following 13 + 1 weeks of study; recovery animals following 17 + 1 weeks on study). Blood for standard serum chemistry and hematological dete rminations was drawn via the orbital sinus followin g an– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 127 April 2008

overnight fast under light anesthesia (IsoFlo®; Iso flurane). An additional aliquot of blood (non-fast ed) for prothrombin time determinations was taken immediate ly preceding necropsy via cardiac puncture of anima ls under CO 2 anesthesia (diaphragm was cut following blood coll ection).

5a. Hematology: X Hematocrit (Hct)* X Leukocyte differential count* X Hemoglobin (Hgb)* X Mean corpuscular Hgb (MCH)* X Leukocyte count (WBC)* X Mean corpuscular Hgb con centration (MCHC) X Erythrocyte count (RBC)* X Mean corpuscular volum e (MCV)* X Platelet count (PLTS)* X Reticulocyte count (Reti c) X Blood clotting measurements* X Erythrocyte morpho logy (Thromboplastin time) X Heinz bodies (HZ) (Clotting time) X Prothrombin time (PT) * Recommended for 90-day oral rodent studies based on Guideline 870.3100 The CHECKED (X) parameters were examined.

5b. Clinical Chemistry:
ELECTROLYTES OTHER X Calcium (Calc) X Albumin (Alb)* X Chloride (Cl) X Creatinine (Creat)* Magnesium X Blood urea nitrogen (Urea-N)* X Phosphate (Phos) X Total Cholesterol (Chol)* X Potassium (K)* X Globulin (Glob) X Sodium (Na)* X Glucose, fasting (Gluc)* ENZYMES X Total bilirubin (T-Bili) X Alkaline phosphatase (ALP)* X Total protein (T-Pr ot)* Cholinesterase (ChE) X Triglycerides (Trig) X Creatine phosphokinase (CK) Serum protein electr ophoresis X Lactic acid dehydrogenase (LDH) X Uric acid (Uric -A) X Alanine aminotransferase (ALT)* X Thyroxine (T4) X Aspartate aminotransferase (AST)* X Triiodothyron ine (T3) Sorbitol dehydrogenase* X Thyroid Stimulating Horm one (TSH) X Gamma-glutamyltransferase (GGT)* Glutamate dehydrogenase X Cytochrome P-450, hepatic (Cyto P-450) X N-Demethylase, hepatic (N-Demeth) X O-Demethylase, hepatic (O-Demeth) X UDP-Glucuronosyltransferase, hepatic (UDP-GT) * Recommended for 90-day oral rodent studies based on Guideline 870.3100 The CHECKED (X) parameters were examined.

6. Urinalysis:

Urine was collected from all surviving animals just prior to their respective termination dates (i.e., nonrecovery animals following 13 + 1 weeks on study; recovery animals following 17 + 1 weeks of study). The urine was collected on an overnight basis (timed) f rom non-fasted animals.

X Appearance* X Glucose (Glu) X Volume (UVol)* X Ketones (Ket) X Specific gravity (Sp.Gr.)* X Bilirubin (Bil) X pH (pH)* X Leukocytes (U-Leu) X Sediment (microscopic) X Blood (Bld)* X Protein (Pro)* X Nitrite (Nit) X Urobilinogen (UROB) 1 Optional for 90-day oral rodent studies * Recommended for 90-day oral rodent studies The CHECKED (X) parameters were examined.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 128 April 2008

7. Sacrifice and Pathology:

7a. Gross pathology: All animals were sacrificed by CO 2 asphyxiation (or CO 2 anesthesia followed by clipping of the diaphragm) and subject to a postmortem examination, which incl uded (1) documenting and saving all gross lesions, (2) weighing of the designated organs, and (3) collecti ng representative tissue specimens for histopatholo gic evaluation. Tissues were preserved (nominally in 1 0% buffered formalin) and selected organs were weig hed and organ/body weight ratios calculated. Following collection of a liver specimen for histopathologic al analysis, the remaining liver tissue from at least 8 surviving animals (at approximately 14 and 18 wee ks) was snap frozen in liquid nitrogen, pending further bio chemical analyses.

DIGESTIVE SYSTEM CARDIOVASC./HEMAT. NEUROLOGIC X Bile duct (rat) X Aorta* XX Brain*+ X Cecum* X Bone marrow* X Cerebellum X Colon* XX Heart*+ X Cerebrum-Midbrain X Duodenum* X Lymph node, cervical* X Eyes* X Esophagus* X Lymph node, mesenteric* X Medulla/Po ns Gall bladder (not rat)* XX Spleen*+ X Nerve, optic * X Ileum* XX Thymus*+ X Nerve, sciatic* X Jejunum* X Pituitary* XX Liver*+ UROGENITAL X Spinal cord, cervical* X Pancreas* X Cervix X Spinal cord, lumbar* X Rectum* X Clitoral gland^ X Spinal cord, thoracic * X Salivary glands* XX Epididymides*+ X Stomach, glandular* XX Kidneys*+ OTHER X Stomach, non-glandular* X Mammary gland* X Bone, femur X Tongue XX Ovary*+ X Bone marrow X Tooth X Preputial gland^ X Bone, rib/cc jct X Prostate* X Bone, sternum RESPIRATORY X Seminal vesicle* X Gross lesions and masses* X Larynx* XX Testicle*+ X Harderian gland XX Lung* X Urinary bladder* X Joint, fem/tib X Nasal structure XX Uterus*+ X Muscle, protocol X Nasopharynx* X Vagina^ X Physical Identifier (ID chip) X Oral structure X Skin, protocol* X Trachea* GLANDULAR X Zymbal’s gland^ XX Adrenal gland*+ X Exorbital/lacrimal gland^ X Parathyroid* XX Thyroid*

* Recommended for 90-day oral rodent studies based on Guideline 870.3100 + Organ weights required for rodent studies. The CHECKED (X) tissues were collected for histolog ical examination. The organs CHECKED (XX) were als o weighed. The organs CHECKED (^) were preserved for possible micropathologic evaluation only.– –

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7b. Micropathology: With the exception of the physical identifier (micr ochip), the vagina, and the exorbital/lacrimal, cli toral, preputial, and Zymbal glands (of which the latter 5 were taken and preserved for possible micropatholo gic evaluation), representative sections of the tissues shown in Tables on previous page were processed fo r all control (0 ppm) and high-dose (10,000 ppm) animals, embedded in paraffin, sectioned, mounted, and stained with hematoxylin and eosin (H&E) for examin ation under the light microscope. Tissues in which compound-related effects were detected were examine d at lower doses as necessary to establish noobserved-effect levels. Histopathological and opht halmological lesions were assigned a semi-quantitat ive ranking of the severity grade/stage from "Normal (w ithin normal limits)" to the "most severe case (Gra de 5).

II. Results and Discussion:

Findings: General observations: No treatment-related clinical signs or mortalities were observed. Body weight gain was reduced at 10,000 ppm in male rats. The differe nce from controls was 17.6% at the end of the study ; a corresponding decline of 8.4% was noted in absolute body weight at termination of treatment. No releva nt effect on feed intake was determined.

Table B.6-38: Body weight and weight gain (±SEM) a

Dose: Control 150 600 2500 10000
Male Body weight (week -1) 236.0 236.5 238.0 235.7 235.5
Male Body weight (week 1) 269.6±3.5 263.6 267.6 263.7 263.4±4.1
Male Body weight (week 7) 387.2 382.9 375.8 379.2 364.7
Male Body weight (Day 91) 446.7±11.1 432.9 423.3 439.2 409.3±10.5 (-8.4%)
Total weight gain (g) 177.1 169.3 156.0 175.5 145.9*
Weight gain (% of controls) -- 96 88 99 82
Female Body weight (week 1) 172.4±2.1 -- -- 176.6±2.2 173.0±2.1
Female Body weight (day 91) 239.9±4.8 -- -- 246.6±7 231.5±3.1 (-3.5%)
Total weight gain (g) 67.5 -- -- 70 58.5
Weight gain (%) controls

-- -- -- 104% 87%

a Data extracted from pages 49 (males) and 51 (femal es) of the study report * = p < 0.05

Hematology: Not affected. Data are in Tables on pages 132-157 of study report.

Clinical chemistry: Not affected. Data are in Tables on pages 158-171 of study report.

Urinalysis: Not affected. Data are in Tables on pages 178-187 of study report.

Hepatic enzyme profile: Not affected. Data are in Tables on pages 172-177 of study report.

Ophthalmology: No treatment-related effects. Data are in Tables on pages 126-131 of study report.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 130 April 2008

Gross necropsy, organ weights: No treatment-related macroscopic changes were seen. Treatment-related effects in the high dose were decreased kidney (11% ) and testes (6.5%) weights in males. Kidney weigh t for high dose males was similar to controls after recov ery. Kidney weight in females was decreased by 6% (NSS) at termination and equivalent to the control after recovery.

Histopathology:

Epididymis : At the end of the treatment, an increased inciden ce of minimal to severe abnormal spermatozoa and hypospermia were noted in the epididymides of 1 0,000-ppm males. Multi-nucleated giant cells, spherocytes (immature sloughed germ cells), and tis sue debris (recorded as abnormal spermatozoa) were present within the duct of the epididymis at a leve l well above that found in control males. Only one recovery male each showed abnormal spermatozoa or hypospermi a at the end of the 4-week recovery period suggesting reversibility of lesions.

Testes : A minimal to moderate tubular degeneration was no ted in the testes of 10,000-ppm males. Tubular degeneration and vacuolization in the 10,000 ppm do se group was generally multifocal in distribution, and occurred in the germinal epithelial layer of semini ferous tubules with degeneration and loss of epithe lial cells. This finding correlated with the slight decrease in absolute testicular weight. Only one recovery male showed tubular degeneration in the testes at the end of th e 4-week recovery period suggesting reversibility o f lesions.

Table B.6-39: Average incidence and severity (in pa renthesis) of treatment-related microscopic findings Main Dose Group (ppm) Recovery Group (ppm)

0 150 600 2,500 10,000 0 10,000

Males
Epididymis, abnormal spermatozoa 0 - 0 0 9 (1.8) * 0 1 (1.0)

Epididymis, hypospermia 0 * - 0 0 5 (2.6) * 0 1 (5.0)
Testes, tubular degeneration 0 - 0 0 5 (2.0) * 0 1 (2.0)

Testes, vacuolization 0 - 0 0 5 (1.6) * 0 0

Lungs, alveolar macrophages 2

(1.0) 4

(1.3) - 5 (1.4) 9

(1.3) * 3

(1.7) 7

(1.3)

Females
Lungs, alveolar macrophages 1

(1.0) - - 1 (1.0) 7

(1.9) 4

(1.3) 5

(1.4)
* = p < 0.05. Histopathology summary data are on p ages 278-298 of study report.

Lungs : An increased incidence of minimal to slight accum ulation of alveolar macrophages was noted in both sexes at 10,000 ppm. It was stated by the study aut hors that alveolar macrophage accumulation is a common background lesion in the rat. The increased incidences in the 10,000 ppm male and female dose levels, however, were considered to be affected by compound administration. At the end of the recovery period, reversibility was suggested for the affecte d parameter.– –

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III. Conclusions: The subchronic toxicity study in rats established a NOAEL of 2,500 ppm (equal to 148 or 188 mg/kg bw/day) in males or females, respectively , based on the following findings at 10,000 ppm (eq ual to 616 mg/kg bw/day in males and 752 mg/kg bw/day i n females, respectively): reduced body weight, decreased absolute testicular weight, testicular tu bular degeneration, abnormal epididymal spermatozoa and hypospermia, and increased accumulation of alveolar macrophages in both sexes.

CITATION : Wahle, B.S. (2005). Technical grade BYI 08330, A S ubchronic Toxicity Study in the Mouse.

Bayer Crop Science LP, Kansas, Study No.: 201284, J uly 14, 2005. Unpublished.

I. Material and methods

A. Materials:

1. Test Material: BYI 08330 Description: Technical grade, beige powder Lot/Batch No.: NLL 6425-9 Purity: 96.5% (12/2001); 93.5% (6/2002); 93.1% (1/2 003) Compound Stability: Stable at room temperature (~ 22 oC) Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. of TGAI: trans : 382608-10-8; cis : 203313-25-1

2. Vehicle and/or positive control : None

3. Test animals: Species: Mouse; males and females (nulliparous and non-pregnant) Strain: CD-1 [ICR]/BR Age/weight at study initiation (Day 0): Approximately 9 weeks old / 30.1 - 30.9 g mean grou p weight males; 25.3 - 25.9 g mean group weight females Source: Charles River Laboratories, Inc. (Kingston , NY) Housing: Individually housed in suspended stainles s steel wire-mesh cages, each containing a feeder, a source of water (pressure-ac tivated water nipples), and deotized (sanitized) cage board in the bedding tray (animals passing initial shipment exam were co-housed during a porti on of the Acclimatisation) Diet: Purina Mills Certified Rodent Diet 5002 in " meal" form; ad libitum Water: Municipal water supply of Kansas City, MO; ad libitum . Exception: Some animals were without pressurized water for approxim ately 1.5 days (between nominal Days 40 and 42). Only the water re maining in the lines would have been available during this time. Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 19 to 25 o C

30 to 70% (relative) 15 air changes per day (daily average) 12 hr of light [7:00 a.m. to 7:00 p.m.] alternating with 12 hr of darkness Acclimatisation: 05/13/02 (receipt) - 05/20/02 (re lease for study)– –

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B. STUDY DESIGN:

1. In life dates : Start: 05/20/02 (released for study); 05/30/02 (i nitiation of exposure) End: 09/06/02 (terminal sacrifice complete)

2. Animal assignment : 05/22/02; assigned to a control or one of four c hemically-treated groups using a weight stratification-based computer program obtain ed from INSTEM Computer Systems (Stone, Staffordshire, UK). Each animal on study identifie d with a microchip (BioMedic Data Systems, Inc., Se aford, DE) subcutaneously implanted on their dorsal surfac e in the region between the scapulae; the chip was encoded with a unique number, specifying the animal 's sex, dose group, cage number, and study affiliat ion.

Test Group Conc. in Diet (ppm) Dose to Animal (mg/kg; + SD) Male Female

Control (A) 0 0 15 15
Low (B) 70 males 12.8 + 0.9 females 16.0 + 1.3 15 15

Mid 1 (C) 350 males 59.6 + 4.3 females 72.4 + 5.4 15 15

Mid 2 (D) 1,700 males 300 + 28 females 389 + 31 15 15

High (E) 7,000 males 1,305 + 86 females 1,515 + 102 15 15

3. Dose selection rationale: Doses were selected based principally upon the toxi cological profile which was observed in the mouse o ver the course of a subacute (4-week) toxicity testing study conducted with the test article at doses of 0 , 500, and 5,000 ppm BYI 08330 (Report No. T2070951). In that study, findings directly attributable to the exposure to the test substance were not observed. Therefore, doses of 0, 70, 350, 1,700, and 7,000 pp m were chosen at which to conduct the present study. It was anticipated that the low and high doses cho sen of 70 and 7,000 ppm would constitute NOAEL and maximum tolerated doses, respectively, with the intermediate doses of 350 and 1,700 ppm providing c onfirmation of any dose-response relationships that may have emerged.

4. Diet preparation and analysis: The test compound was mixed directly with the feed; control diet consisted of untreated feed. Samples of each batch of feed mixed were taken and retained in the freezer until the study was complete and the analytical data deemed satisfactory. Replacement a dmixtures for each treatment group were prepared every other week and stored under freezer condition s until presented to the animals. BYI 08330 was administered at a constant concentrat ion in the feed for the duration of the study. The concentration of the AI of the test substance i n the various test diets was analytically verified from samples of each dietary level for all batches prepa red. In addition, one recovery sample was analyzed with each group of samples during the test concentration analyses. The homogeneity and stability of BYI 08 330 when mixed in the dietary carrier was characterized prior to the experimental start of the study (10,0 00 ppm; 70 ppm homogeneity). The stability analysis of the 70-ppm ration was determined just following the experimental start of the study. To evaluate the uniformity of distribution/homogene ity of the test substance in the diet, an analysis of feed containing nominal concentrations of 70 and 10,000 ppm BYI 08330 was conducted. Three (3) samples were taken for analysis from each of 3 distinct layers ( top, middle, bottom; 9 samples total) in a Hobart m ixing– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 133 April 2008

bowl. A homogeneous distribution of the test subst ance in the feed was defined in terms of a coeffici ent of variation (CV), derived from the 9 samples taken, w hich was < 10%. To evaluate the stability of the test substance in the diet, a time and temperature analysis of feed c ontaining nominal concentrations of 70 and 10,000 ppm BYI 083 30 was conducted. Stability in the feed was assess ed for up to 7 days of room temperature storage (~22 o C) and up to 28 days of freezer storage (~ -23 oC). The stability analysis was carried out as follows: An initial sample was taken immediately after the test substance was mixed with the ration. Each batch of ration, m ixed at the 2 concentrations, was then divided into 2 portions. One portion was placed in a room tempera ture environment, the other in the freezer. Feed samples which remained at room temperature were sam pled for analysis on Days 0, 1, 3 or 4, and 7; feed samples which remained in the freezer were sampled for analysis on Days 7, 14 or 15, and 28. Chemical stability in the ration was defined in terms of tha t period of time that 80% of the test substance, re lative to the initial concentration, was recoverable. The liquid chromatographic methodology used to perf orm the dietary analyses (i.e., homogeneity, stabil ity, and concentration verifications) required for this study has been described previously.

Results:

Homogeneity Analysis : The mean concentrations of BYI 08330 in the feed, sampled from 3 distinct layers in the mixing bowl and containing a nominal concentrat ion of either 70 or 10,000 ppm, were determined to be 68.7 ppm (range 65.0-73.1 ppm; CV = 3.2%) and 9,445 ppm (range 8,817-9,943 ppm; CV = 3.2%), respectively. Based on a CV < to 10%, BYI 08330 was judged to be homogeneously distributed in the feed over a concentration range of 70-10,000 ppm.

Stability Analysis : Following 7 days of room temperature storage, the mean analytically-determined concentration of the AI of the test substance in th e 70- or 10,000-ppm admixture was determined to be 68.5 ppm (63.3 ppm on Day 0) and 9,564 ppm (9,405 ppm on Day 0), respectively. Following 28 days of freezer storage, the mean anal ytically determined concentration of the AI of the test substance in the 70- and 10,000-ppm admixtures was determined to be 71.4 ppm and 9,544 ppm, respectively (63.3 and 9,405 ppm, respectively on D ay 0). BYI 08330 mixed in rodent ration was judged to be stable at room temperature for at least 7 days a nd following freezer storage for a minimum 28 days, at a concentration range of 70-10,000 ppm.

Concentration Analysis : Mean analytical concentrations for each dose grou p were 70.9, 328, 1,665, and 6,753 ppm, ranging from 94-101% of the correspondin g nominal concentrations of 70, 350, 1,700, and 7,0 00 ppm, respectively. The AI of the test substance wa s not detected in the control diet. Mean recovery was 100% and ranged from 98-102% for rodent ration spiked with 70, 350, 1,700, and 7,000 ppm BYI 08330.

5. Statistics: Continuous data that were examined statistically we re evaluated initially for equality or homogeneity of variance using Bartlett's test (Snedecor and Cochra n, 1967). Group means were further analyzed by a on eway analysis of variance (ANOVA) followed by Dunnet t's test. In the event of unequal variances, and at the discretion of the Study Director, data were subject to non-parametric procedures consisting of a Krusk alWallis ANOVA followed by the Mann-Whitney-U test fo r between-group comparisons. Frequency data were initially examined for trends; data suggestive of a potential effect were then statistically evaluated using the Chi-Square, Fisher Exact, or Chi-Square and Fisher Exact tests. On a case-by-case basis, and at the discretion of the study director, data were subject to additional statistical procedures other than th ose mentioned above. For the Bartlett test, a probabil ity (p) value < 0.001 was considered significant; f or all other statistical tests, differences with p values < 0.05 were considered statistically significant. A ll statistical– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 134 April 2008

evaluations were performed using software obtained from either INSTEM Computer Systems or SAS Institute Inc.

C. Methods:

1. Observations: 1a. Cageside Observations: For approximately 14 weeks of the study, a general assessment of all remaining animals was carried out at least daily (generally twice daily during the norma l work week and once daily on weekends and holidays ). These assessments were generally with respect to mo ribundity and mortality, but also included a more general clinical observation for readily apparent c linical signs (in addition to moribundity and morta lity).

1b. Clinical Examinations: Once each week for approximately 14 weeks of the st udy, detailed physical examinations for clinical si gns of toxicity were performed on all surviving animals, w hich included evaluation of external surface areas, orifices, posture, general behavior, respiration, and excreto ry products.

1c. Neurological Evaluations: Parameters specifically designed to evaluate possib le neurological effects were not included in this s tudy (i.e., other than those effects that would be ident ified during cage-side or detailed observations). These types of investigations were conducted as part of o ther toxicology studies, including an acute neuroto xicity screening study and a 1-year chronic rat study.

2. Body weight: Individual body weight determinations were performe d weekly throughout the in-life phase of the study on all surviving animals (with the exception of 1 animal o n Day 49).

3. Food consumption and compound intake: Individual food consumption determinations (g consu med/animal/day and g consumed/kg body wt/day) were performed weekly throughout the in-life phase of th e study on all surviving animals (with the exceptio n of 1 animal on Day 14). In addition, using specifically defined criteria, food consumption data were correc ted, as conditions dictated, to account for misleading indi cations of food intake (i.e., excessive spillage, c logged feeders, etc.). Test substance intake (mg/kg body w t/day) was calculated using weekly mean body weight and food consumption data. The general relationship used for this calculation was: [AI in feed (ppm)/1,000] x [feed consumed (g/kg bod y wt/day)] = mg AI/kg body wt/day

4. Ophthalmological examination: Ophthalmological examinations were not included in this study. Ophthalmological examinations were included as part of a subchronic investigation with the test substance in rats.

5. Hematology & Clinical Chemistry: Following approximately 13 weeks on study, blood wa s collected from all surviving animals just prior t o termination. Blood for standard serum chemistry an d hematological determinations was drawn via the or bital sinus of non-fasted animals. Due to blood volume c onsiderations, only select serum chemistry evaluati ons were included. A full serum chemistry profile was included as a part of a subchronic investigation wi th the test substance in rats (Report No. 201136).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 135 April 2008

5a. Hematology: X Hematocrit (Hct)* X Leukocyte differential count* X Hemoglobin (Hgb)* X Mean corpuscular Hgb (MCH)* X Leukocyte count (WBC)* X Mean corpuscular Hgb con centration (MCHC) X Erythrocyte count (RBC)* X Mean corpuscular volum e (MCV)* X Platelet count (PLTS)* X Reticulocyte count (Reti c) Blood clotting measurements* X Erythrocyte morphol ogy (Thromboplastin time) X Heinz bodies (HZ) (Clotting time) X Red cell distribution width (RDW) Prothrombin time (PT) X Hemoglobin distribution wi dth (HDW) * Recommended for 90-day oral rodent studies based on Guideline 870.3100 The CHECKED (X) parameters were examined. Data o n pages 77-88 of the study report provide confirmation that these parameters were investigate d.

5b. Clinical Chemistry:
ELECTROLYTES OTHER Calcium (Calc) Albumin (Alb)* Chloride (Cl) X Creatinine (Creat)* Magnesium X Blood urea nitrogen (Urea-N)* Phosphate (Phos) X Total Cholesterol (Chol)* Potassium (K)* Globulin (Glob) Sodium (Na)* Glucose, fasting (Gluc)* ENZYMES Total bilirubin (T-Bili) X Alkaline phosphatase (ALP)* Total protein (T-Pro t)* Cholinesterase (ChE) X Triglycerides (Trig) Creatine phosphokinase (CK) Serum protein electro phoresis Lactic acid dehydrogenase (LDH) Uric acid (Uric-A ) X Alanine aminotransferase (ALT)* Thyroxine (T4) X Aspartate aminotransferase (AST)* Triiodothyroni ne (T3) Sorbitol dehydrogenase* Thyroid Stimulating Hormo ne (TSH) Gamma-glutamyltransferase (GGT)* Glutamate dehydrogenase Cytochrome P-450, hepatic (Cyto P-450) N-Demethylase, hepatic (N-Demeth) O-Demethylase, hepatic (O-Demeth) UDP-Glucuronosyltransferase, hepatic (UDP-GT) * Recommended for 90-day oral rodent studies based on Guideline 870.3100 The CHECKED (X) parameters were examined. Data on pages 90-91 verify that these limited parameters were investigated.

6. Urinalysis: Urinalysis parameters were not included in this stu dy. Urinalysis was included as part of a subchroni c investigation with the test substance in rats (Repo rt No. 201136).

7. Sacrifice and Pathology: 7a. Gross pathology: All animals surviving to scheduled termination were sacrificed by CO2 asphyxiation and subject to a postmortem examination, which included (1) document ing and saving all gross lesions, (2) weighing designated organs, and (3) collecting representativ e tissue specimens for histopathologic evaluation. Tissues were preserved (nominally in 10% buffered f ormalin) and selected organs were weighed and organ/body weight ratios calculated.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 136 April 2008

DIGESTIVE SYSTEM CARDIOVASC./HEMAT. NEUROLOGIC Bile duct (rat) X Aorta* XX Brain*+
X Cecum* X Bone marrow* X Cerebellum
X Colon* XX Heart*+ X Cerebrum-Midbrain X Duodenum* X Lymph node, cervical* X Medulla/Pons X Esophagus* X Lymph node, mesenteric* X Eyes* X Gall bladder (not rat)* XX Spleen*+ X Nerve, opti c* X Ileum* X Thymus*+ X Nerve, sciatic* X Jejunum* X Pituitary*
XX Liver*+ UROGENITAL X Spinal cord, cervical* X Pancreas* X Cervix X Spinal cord, lumbar* X Rectum* X Clitoral gland^ X Spinal cord, thoracic * X Salivary glands* X Epididymides*+ X Stomach, glandular* XX Kidneys*+ OTHER X Stomach, non-glandular* X Mammary gland* X Bone, femur X Tongue XX Ovary*+ X Bone, rib/cc jct X Tooth X Preputial gland^ X Bone, sternum
X Prostate* X Gross lesions and masses* RESPIRATORY X Seminal vesicle* X Harderian gland X Larynx* XX Testicle*+ X Joint, fem/tib XX Lung* X Urinary bladder* X Muscle, protocol X Nasal structure X Uterus*+ X Physical Identifier (ID chip) X Nasopharynx* X Vagina^ X Skin, protocol* X Oral structure X Zymbal’s gland^
X Trachea* GLANDULAR XX Adrenal gland*+ X Exorbital/lacrimal gland^ X Parathyroid* X Thyroid* * Recommended for 90-day oral rodent studies based on Guideline 870.3100 + Organ weights required for rodent studies. The CHECKED (X) tissues were collected for histolog ical examination. The organs CHECKED (XX) were also weighed. The organs CHECKED (^) were preserved for possible micropathologic evaluation only. The summary tables on pages 136-157 of the study report indicate that these organs/tissues were examined. However, no separate entries were made for the cere bellum, cerebrum-midbrain and medulla pons or separate entries for the cervical, lumbar or thorac ic spinal cord although these items are checked as being investigated. Only the brain and spinal cord are li sted in the tables.

7b. Micropathology: With the exception of the physical identifier (micr ochip), the vagina, and the exorbital/lacrimal, cli toral, preputial, and Zymbal glands (of which the latter 5 were taken and preserved for possible micropatholo gic evaluation), representative sections of the tissues were processed for all control (0 ppm) and high-do se (7,000 ppm) animals, embedded in paraffin, sectione d, mounted, and stained with hematoxylin and eosin (H&E) for examination under the light microscope. T issues in which compound-related effects were detec ted were to be examined at lower doses as necessary to establish no-observed-effect levels. Histopathologi cal lesions were assigned a semi-quantitative ranking o f the severity grade/stage from "Normal (within nor mal limits)" to the "most severe case (Grade 5)," as de scribed previously.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 137 April 2008

II. RESULTS AND DISCUSSION:

Findings: General observations : No treatment-related clinical signs or mortalitie s were observed. Body weight was not affected at any dose level in either sex. No releva nt effect on feed intake was determined.

Hematology : Standard hematologic endpoints were not affected.

Clinical chemistry : Selected clinical chemistry endpoints (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, blood urea nitrogen, cholesterol and triglyceride) were not affected.

Gross necropsy, organ weights : No treatment-related macroscopic changes were not ed. Absolute and relative organ weights showed no treatment-related changes.

Histopathology: No treatment-related microscopic findings were noted.

III. DISCUSSION AND CONCLUSIONS

A. CONCLUSIONS :

The subchronic toxicity study in mice established a NOAEL of 7,000 ppm (equal to 1,305 or 1,515 mg/kg bw/day in males or females, respectively), based on the lack of findings at any dietary level tested.

B. REVIEWER COMMENTS :

The reviewer agrees that there were no adverse reac tions to treatment of any kind noted. The LOAEL wa s not established. The NOAEL was 7000 ppm (1305/1515 mg/kg/day [M/F]).

The following table containing body weight and live r weight data was constructed to demonstrate that t here was no effect on either body or liver weight since no actual data were included in the format provided by the registrant.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 138 April 2008

Table B6-40: Body and Liver Weights
Males

Control Low Mid1 Mid2 High Females Control Low Mid1 Mid2 High Body Weight (g±SD)
Initial 30.9±0.6 30.4±0.5 30.9±0.4 30.8±0.5

30.1±0.4 25.9±0.5 25.8±0.4 25.3±0.4 25.3±0.4 25.3±0.4 Week 1 32.5±0.7 31.6±0.5 32.3p0.6 32.2±0.6

31.5±0.5 26.6±0.5 26.7±0.5 26.0±0.4 26.2±0.4 26.3±0.5
Week 2 33.7±0.7 31.5±0.6 32.3±0.9 32.1±0.8

31.9±0.5 27.1±0.6 26.9±0.6 26.6±0.5 26.9±0.4 26.6±0.4 0-2 gain 2.8 1.1 1.4 1.3 1.8 1.2 1.1 1.3 1.6 1.3 Day 91 36.9±0.9 37.2±0.8 36.6±0.5 36.4±1.0

34.9±0.7 30.6±0.5 31.3±0.7 30.4±0.8 30.0±0.4 30.2±0.3 0-91 gain* 6.0 6.8 5.7 5.6 4.8 4.7 5.5 5.1 4. 7 4.9 Terminal Liver Weight (g±SD)
Terminal** 2.075±0.331 1.986 1.970 1.999

2.020±0.126 1.688±0.167 1.679 1.620 1.656 1.712±0.218

Body weight data are from pages 38 (males) and 39 ( females) of the study report. Liver weight data are from pages 130 (males) and 131 (females). *Body weight gain calculated by the EPA reviewer ** Standard deviation (SD) is presented for the con trol and high dose only to conserve space; SD– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 139 April 2008

B.6.3.3 Oral 90-day toxicity (dog) (OECD Annex IIA 5.3.3)

CITATION : Eigenberg, D. A. (2005). Technical Grade BYI 08330: A 90-Day Subchronic Toxicity Feeding

Study in the Beagle Dog. Bayer CropScience LP, Toxi cology, 17745 South Metcalf, Stilwell, Kansas. Study No. 03-S76-PE/Report No. 201223. May 9, 2005 (study completion date). Unpublished.

I. MATERIAL AND METHODS:

A. MATERIALS :

1. Test Material : BYI 08330 Description: Technical grade, white powder, room te mperature storage Lot/Batch No.: 08045/0014 Purity (% ai): 97.8% (5-Dec-03); 97.6% (28-Jun-04) Chemical Name: Ethyl 3-(2,5-dimethylphenyl)-8-metho xy-2-oxo-1-azaspiro[4,5] dec-3-en-4-yl carbonate CAS No. if TGAI: 203313-25-1

2. Vehicle and/or positive control: corn oil and acetone

3. Test animals : Dog, males and females Species: Canis familiaris Strain: Beagle (nulliparous and nonpregnant) Age/weight at study initiation: 7 - 8 months / males: 9.2 - 11.6 kg; females: 6.1 - 8.8 kg

Source: Marshal Farms USA, Inc, North Rose, New Yor k Housing: Individually housed in stainless steel run s Diet: Purina Certified Canine Diet 5006-3 available for ad libitum consumption Water: Tap water provided continuously for ad libitum consumption Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 64 to 84°F

30 to 70% 16 changes/hr average during the dosing period* 12 hrs of light alternating with 12 hrs of darkness Acclimatisation: 7 days prior to release for the st udy *For a 4-week period, there were numerous cases of zero reading for air changes per hour due to an error in the setting of the microprocessor which mo nitored the air flow.

B. STUDY DESIGN :

1. In life dates: Initiation of collection of Data (Day Animals Relea sed for Study): December 17, 2003; Initiation of Dosing: December 22, 2003; Terminal S acrifice: March 23 - 26, 2004

2. Animal assignment: Prior to administering the test substance, male and female animals were randomly assigned, based on weight, to the dose gro ups noted in Table 6-40. The weight variation of the animals was targeted not to exceed + 20% of the mean weight for each sex.– –

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Table B.6-40: Study design Dose Group (ppm) No. Animals/

Dose/Sex Males

(mg/kg/day) Females (mg/kg/day) 0 (control) a 4 0 0 150 4 5 6 300 4 9 10 1,200 4 33 32 2,500* 4 81** 72** * Dogs were dosed for two weeks at 4,000 ppm at which time the dose was lowered to 2,500 ppm due to exce ssive weight loss at 4,000 ppm. ** Dose in mg/kg/day does not include dose received at 4,000 ppm.

a Maintained on diet with vehicle only

3. Dose selection rationale : The nominal concentrations chosen for this study we re 0 (concurrent vehicle control), 150, 300, 1,200, and 2,500 ppm (4/sex/dos e) of technical grade BYI 08330 mixed with dog ration. Dose levels were selected based on the res ults of a 28-day feeding study in the Beagle dog in which BYI 08330 was administered at doses of 0, 100 , 400, 1,600, or 6,400 ppm. A marked reduction in food consumption and body weight was noted in the 6 ,400-ppm animals. Secondary effects on some organ weights were observed in 6,400-ppm animals. C ompound-related decreases in circulating thyroxine (T4) and triiodothyronine (T3) were obser ved at dietary levels of 400, 1600 and 6400ppm in males and at 6,400-ppm females.

4. Diet preparation and analysis : All feed mixtures were prepared weekly by mixing ap propriate amounts of test substance with Purina Certified Canine Diet 5006-3 and then storing the mixture under freezer conditions until administered to the animals. Corn oil, at 1% by weight of the diet, along with aceton e was used as vehicle to suspend the test substance prior to being mixed in the diet. The control diet was prepared similarly to the treated diet, minus the t est substance. A sample of each batch of mixed fee d was taken and retained under freezer conditions unt il the study was complete and the study data deemed satisfactory. Prior to conducting a 4-week pilot study in the dog (Report No. 201012), the homogeneity and stability of BYI 08330 in dog ratio n was confirmed for dietary concentrations ranging from as low as 100 ppm to as high as 6,400 ppm in t he feed. Dietary concentrations of test material us ed in the present study were confirmed and the analyti cal data are included in the study report.

Results: Based on the findings described below, the analytic al data indicated that the mixing procedure was adequate and that the variance between nominal and actual dosage to the animals was acceptable.

Homogeneity analysis: Batches of ration mixed with BYI 08330 at 100 and 6 ,400 ppm were analyzed for homogeneity. Nine samples of ration were taken from each level and analyzed. The mean concentrations were 96.2 ppm (96.2%, CV=4.3%) for t he 100 ppm test level and 6,255 ppm (97.7%, CV= 3.5%) for the 6400 ppm test level. Based on CVs of 4.3 and 3.5%, BYI 08330 mixed in dog ration at concentrations of 100 and 6400 ppm was considere d homogeneously distributed.

Stability analysis: For freezer stability, samples were analyzed on day 0, 7, 14, and 28. After 28 days, there was no decline in concentration for the 100 a nd 6,400 ppm levels. BYI 08330 mixed in dog ration at concentrations of 100 and 6,400 ppm was consider ed stable at freezer temperature for a minimum of 28 days (analytical values were 101 and 114% of act ual concentrations at 100 and 6400 ppm, respectively). For room temperature stability, sam ples were analyzed on day 0, 1, 4, and 7. After 7 days, declines in concentration ranged from 9.8 to 2.1% for 100 and 6,400 ppm levels, respectively. BY I 08330, mixed in dog ration at concentrations of 100 and 6,400 ppm, was considered stable at room temperature for a minimum of 7 days.– –

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Concentration analysis. During the study mixed feed from all dose groups, i ncluding control, was analyzed for stability and concentration during wee ks 1 thru 7 (exceptions during weeks 4 and 5) as we ll as weeks 11 and 14. The mean concentrations for th e study were 99 to 104% of the nominal levels of 150/300 and 4000 ppm, respectively; %RSD values ran ged from 1.38 to 4.59 at 4000 and 1200 ppm, respectively.

5. Statistics: Statistical significance was determined at p< 0.05 for all tests with the exception of Bartlett's test, in which a probability value of p< 0.001 was used. All tests were two-tailed, except for gross and histopathological lesion evaluations, which were on e-tailed. Continuous data were analyzed by Bartlett 's test for homogeneity. If the data were homogeneous an ANOVA was performed, followed by Student's ttest on parameters showing a significant effect by ANOVA. If the data were non-homogeneous a Kruskal-Wallis ANOVA was performed, followed by the Mann-Whitney U-test to identify statistical significance between groups. Frequency data, that were examined statistically, were initially analyze d by a Chi-Square procedure. If there was statistica l significance using the Chi-square test, each treatment group was compared to the control group u sing a Fisher's Exact test.

C. METHODS :

1. Observations :

1a. Cageside observations : All study animals were observed at least twice dail y for clinical signs of toxicity (except once daily on weekends and holiday s).

1b. Clinical examinations : Detailed clinical observations for clinical signs o f toxicity were performed on all animals at study initiation and on a weekly basis thereafter.

2. Body weight: Individual body weights were measured weekly throug hout the study. Body weights were also taken immediately prior to necropsy to allow f or calculation of organ-to-body weight ratios.

3. Food consumption and compound intake : Each animal’s food consumption was measured daily throughout the study. The intake of test compound in mg/kg/day was calculated for males and females using the analytical concentration of BYI 08330 in the feed and the following equation: (average food consumption per week/average body weight per week) x ppm in the feed/1,000.

4. Ophthalmoscopic examination : Following the acclimatisation and prior to initiati on of dosing, ophthalmic examinations were conducted on all anima ls. Ophthalmic examinations were also conducted on all animals just prior to termination of the stu dy.

5. Hematology & clinical chemistry : Clinical chemistry and a complete blood count, incl uding differentials, were performed on all animals (pre-e xposure and during weeks 4, 8, and 13). Animals were fasted overnight prior to the collection of bl ood, which was drawn via jugular venipuncture. The parameters evaluated are marked (x) in the lists be low.

5a. Hematology :

x Hematocrit (HCT)* x Leukocyte differential count* a x Hemoglobin (HGB)* x Mean corpuscular hemoglobin ( MCH)* x Leukocyte count (WBC)* x Mean corpuscular hemoglo bin conc.(MCHC)* x Erythrocyte count (RBC)* x Mean corpuscular volum e (MCV)* x Platelet count* x Reticulocyte count a– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 142 April 2008

Blood clotting measurements* x (Thromboplastin time) (Clotting time) x (Prothrombin time) * Recommended for 90-day oral non-rodent studies ba sed on Guideline 870.3150

a Data were not provided in Study Report

5b. Clinical chemistry:

ELECTROLYTES OTHER x Calcium (calc)* x Albumin (ALB)* x Chloride (Cl)* x Creatinine (Creat)* Magnesium (Mg) x Urea nitrogen (Urea-N)* x Phosphorous (Phos)* x Total Cholesterol (Chol)* x Potassium (K)* x Globulins (Glob) x Sodium (Na)* x Glucose (gluc)* ENZYMES x Total bilirubin (T-Bili)* x Alkaline phosphatase (ALK)* x Total protein (TP)* Cholinesterase (ChE) x Triglycerides (Trig) x Creatine phosphokinase (CK) Serum protein electr ophoresis x Lactic acid dehydrogenase (LD) x Uric Acid (Uric- A) x Alanine aminotransferase (ALT / SGPT)* x Aspartate aminotransferase (AST / SGOT)* x Gamma Glutamyl transferase (GGT)* x Thyroxin (T4) Glutamate dehydrogenase x Triiodothyronine (T3) Sorbitol dehydrogenase* x Thyroid Stimulating Horm one (TSH) *Recommended for 90-day oral non-rodent studies bas ed on Guideline 870.3150

6. Urinalysis: Urine was collected from all animals (pre-exposure and during weeks 4, 8, and 13). The parameters evaluated are marked (x) in the list bel ow.

x Appearance* a x Glucose (Glu)* x Volume (UVol)* x Ketones (Ket) x Specific gravity/osmolality (Sp.Gr.)* x Bilirubi n (Bil) x pH (pH)* x Blood (Bld)*

x Sediment (microscopic) a x Nitrite (Nit) x Protein (Pro) x Urobilinogen (Uro) x Urine Creatinine, 24 hr (Creat) x Leukocytes (U -Leu) *Recommended for 90-day oral non-rodent studies bas ed on Guideline 870.3150

a Data were not provided in Report No. 201223

7. Sacrifice and pathology: Animals were euthanized at the end of the study by intravenous injection of Fatal-Plus® (Vortech Pharmaceuticals, Dearborn, Mic higan). A complete gross examination was performed on all animals. The necropsy consisted of a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. All tissues were processed, embedded in paraffin, sectioned, mounted , and stained with hematoxylin and eosin (H&E) for examination under the light microscope. Histopatho logic evaluation was conducted on all protocolrequired tissues from the control and high-dose ani mals as well as selected tissues (liver, thyroid, thymus, testicles, and epididymides) from the anima ls in the 150, 300 and 1200 ppm groups. Where appropriate, all findings were assigned a severity score where N = tissues within normal histological limits, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked. The mean severity was determined by dividing the sum of the individual animal severity scores by the number of tissues examined in the gro up. A list of the tissues collected (x) and weighed (xx ) at necropsy is presented in the table below.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 143 April 2008

DIGESTIVE SYSTEM CARDIOVASC./HEMAT NEUROLOGIC Tongue x Aorta, thoracic* xx Brain*+ x Salivary glands* xx Heart*+ x Peripheral nerve* x Esophagus* x Bone marrow* x Spinal cord (3 levels )* x Stomach* x Lymph nodes* xx Pituitary* x Duodenum* xx Spleen*+ x Eyes (optic nerve)* x Jejunum* xx Thymus*+ GLANDULAR x Ileum* xx Adrenal gland*+ x Cecum* UROGENITAL Lacrimal gland x Colon* xx Kidney*+ xx Parathyroid*+ x Rectum* x Urinary bladder* xx Thyroid*+ xx Liver*+ xx Testes*+ OTHER xx Gall bladder*+ xx Epididymides*+ x Bone (sternum and/or femur) x Pancreas* x Prostate* x Skeletal muscle

RESPIRATORY xx Ovaries*+ x Skin* x Trachea* xx Uterus*+ x All gross lesions and mass es* x Lung* x Mammary gland* x Nose* x Cervix x Pharynx* x Fallopian tube (oviduct) x Larynx* *Recommended for 90-day oral non-rodent studies bas ed on Guideline 870.3150 + Organ weight required for non-rodent studies

II. RESULTS AND DISCUSSIONS:

A. FINDINGS :

General observations : No mortalities occurred during the study. One fema le in the 2500 ppm group was described as thin during the clinical assessmen t (onset at day 14). The neurotoxicity assessment revealed that this female in this group had abnorma l posture (onset at day 42) and was reluctant to st and (onset at day 63).

Body weight and food consumption: During the first two weeks of the study, there was a treatmentrelated reduction in body weight and body weight ga in for both sexes in the 4,000 ppm dose group (Table 6-41). In males, mean body weight was decrea sed by 11% (NSS), relative to controls, after two weeks of treatment at 4000 ppm. After week two, wh en the 4,000 ppm dose was reduced to 2,500 ppm, these animals gained more body weight than respecti ve controls; however, it was evident that the females did not recover completely since the mean b ody weight at termination was still below the preexposure value and 17% less than controls at termin ation. Mean food consumption was 11% lower in males at the 4000 ppm dietary level during test day s 6-14; after the dietary level of test material wa s decreased to 2500 ppm, decreased food consumption w as no longer observed in these animals. Food consumption in 2500 ppm females was lower than the control throughout the study; the lower values were statistically significant on test days 18, 33, 36, 40, 63, 70, 77, 79 and 84.

The depressed body weight gain and food consumption observed in 2500 ppm females is considered toxicologically significant.– –

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Table B.6-41: Body weights and body weight gains Mean Body Weights (kg ± SD) Weight gain (kg) b Dose (ppm) Day

0 Week

1 Week

2 Week

3 Week

7 Week

13 Total % diff.

Control After

Wk. 2 % diff.

Control

Males

0 10.614

±0.511 10.812

±0.640 11.070

±0.685 11.151

±0.779 11.599

±0.727 11.613

±0.760 1.00 -- 0.462 --

150 10.180

±0.621 10.389

±0.383 10.463

±0.734 10.655

±0.873 10.888

±0.901 11.197

±0.811 1.02 +2 0.542 +17

300 10.296

±0.850 10.534

±0.947 10.516

±0.868 10.608

±0.902 10.931

±1.094 11.054

±1.023 0.76 -24 0.446 -3

1200 10.806

±0.665 10.918

±0.447 11.264

±0.451 11.749

±1.126 11.931

±0.874 12.142

±1.182 1.34 +34 0.393 -15

4000/2500 c 10.733

±0.742 10.104

±0.829 9.816

±0.828 10.196

±0.968 10.895

±0.973 11.023

±0.589 0.29 -71 0.827 +79

Females

0 7.466

±0.941 7.595

±1.019 7.745

±1.055 7.914

±1.083 8.040

±1.073 8.274

±1.076 0.81 -- 0.360 --

150 6.876

±0.767 6.947

±0.719 7.119

±0.672 7.098

±0.712 7.225

±0.507 7.075

±0.568 0.20 -75 -0.023 No gain

300 7.280

±0.933 7.486

±0.897 7.507

±1.039 7.564

±0.924 7.974

±1.292 7.830

±1.528 0.55 -32 0.266 -26

1200 7.048

±0.664 7.190

±0.685 7.267

±0.728 7.266

±0.757 7.625

±0.691 7.990

±0.932 0.94 +16 0.724 +101

4000/2500 c 6.914

±0.650 6.583

±0.790 6.341

±0.879 6.435

±0.756 6.621

±0.915 6.849

±0.656 -0.07 No gain 0.414 +15
Data obtained from page 22 of Report No. AT01512

a Values are group means ± SD b Percent differences from controls and body weight g ains were calculated by the reviewer.

c Dietary concentration of BYI 08339 was decreased fr om 4000 to 2500 ppm on test day 15.

2. Hematology : There were no compound-related findings in males. S light declines in mean values for RBC count, hemoglobin concentration, and hematocrit were seen at the 2500 ppm dietary level in females at 58 days (Table 6-42). The mean values f or these parameters were further decreased at test day 86 (16-17%), and the difference from control wa s statistically significant for each parameter. Oth er findings, such as statistically significant (p<0.05 ), but not dose-related, increases in hemoglobin distribution width (HDW) in 1200 and 2500 ppm femal es at day 58, are considered incidental. The noted compound-related effect on red blood cell parameter s at 2500 ppm in females is considered toxicologically significant.

It should be noted that the study report indicated that differential leukocyte and reticulocyte counts were performed; however, these data were not included in the report.

Table B.6-42: Selected red blood cell parameters in females a

Parameter Dietary concentration (ppm)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 145 April 2008

0 150 300 1200 2500

-3 Days RBC (10 6 /mm 3 ) 6.65 ± 0.37 6.88 ± 0.36 7.25 ± 0.50 7.45 ± 0.47 6.5 9 ± 0.67 HGB (g/dL) 15.6 ± 0.7 16.4 ± 1.0 16.6 ± 1.5 16.9 ± 0.7 15.5 ± 1.4 HCT (%) 45.8 ± 2.0 47.0 ± 2.5 48.2 ± 4.5 49.0 ± 2.1 45.0 ± 3.9 30 Days RBC (10 6 /mm 3 ) 6.78 ± 0.43 7.10 ± 0.53 7.36 ± 052 7.28 ± 0.59 6.6 9 ± 0.84 HGB (g/dL) 16.0 ± 0.9 16.9 ± 1.2 16.8 ± 1.6 16.6 ± 1.0 15.7 ± 1.6 HCT (%) 46.1 ± 2.6 48.8 ± 3.8 48.5 ± 4.4 47.7 ± 3.0 45.1 ± 5.1 58 Days RBC (10 6 /mm 3 ) 7.11 ± 0.67 7.11 ± 0.66 7.26 ± 0.34 7.47 ± 0.33 6.4 1 ± 0.63 HGB (g/dL) 16.8 ± 1.7 16.9 ± 1.6 16.9 ± 0.7 17.1 ± 1.3 15.1 ± 1.2 HCT (%) 47.7 ± 5.0 48.0 ± 4.8 47.5 ± 1.8 48.6 ± 3.3 43.2 ± 3.6 86 Days

RBC (10 6 /mm 3 ) 7.19 ± 0.61 7.12 ± 0.74 7.57 ± 0.55 7.62 ± 0.67 5.95*± 0.79 (17) HGB (g/dL) 17.0 ± 1.5 17.1 ± 1.6 17.5 ± 1.4 17.3 ± 0.9 14.1*± 1.8 (17) HCT (%) 48.5 ± 4.5 48.2 ± 4.8 49.5 ± 4.1 49.7 ± 2.9 40.8*± 4.4 (16) Data obtained from pages 96-102

a Values are group means ± SD * p<0.05

3. Urinalysis : No treatment-related findings

4. Clinical chemistry : There was a treatment-related decrease in T 4 in the 1,200 and 2,500 ppm male and female dose groups (Table 6-43). The findings on st udy day 30 for the males in the 300 ppm dose group were not considered to be treatment-related since o n study days 58 and 86 the T 4 values for males were not statistically significantly below the control g roup. The T 4 values for females in the 150 ppm dose group were statistically significantly lower than t he control group on study days 30, 58 and 86, but t his finding is not considered treatment-related due to the absence of significant declines at 300 ppm. The re was a slight, but statistically significant decreas e in T 3 in the 2,500 ppm male and female dose groups on study days 30 and 58 which is possibly considered t o be treatment-related (Table 6-44). However, on study day 86, T 3 levels were comparable to the control mean. Despit e percentage declines in T 4 at some time points of greater than 50%, no changes in thyr oid weight, thyroid pathology, and no compensating increases in thyrotropin (TSH) (Table 6-45) were se en. Ultimately, based on the total response of the animals to the thyroid profile that emerged over 90 days, the compound-induced changes in circulating thyroid hormones, though significant in magnitude, were judged to be non-adverse. This conclusion was later confirmed when a similar thyroid and toxicolo gical profile emerged in the 1-year chronic dog stu dy.

No other compound-related findings were observed in the assessed clinical chemistry parameters. Statistically significant (p<0.05) changes seen at 2500 ppm in males (increased glucose at day 58 and decreased total bilirubin at day 86) and at 1200 an d 2500 ppm in females at day 86 (increased triglyceride) are considered incidental findings.– –

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Table B.6-43: Mean thyroxine (T 4) levels a Dose Group (ppm) Parameter 0 150 300 1,200 2,500
Males

T 4 day -3 1.70 ± 0.33 2.29

± 0.58 1.76

± 0.43 2.11

± 0.15 1.99

± 0.44

T 4 day 30 1.87 ± 0.23 1.60

± 0.78 1.34

± 0.30 * 0.84

± 0.15 * 0.71

± 0.19 *

T 4 day 58 1.91 ± 0.17 1.67

± 0.94 1.60

± 0.29 1.74

± 0.51 0.80

± 0.23

T 4 day 86 1.85 ± 0.51 1.70

± 0.68 1.42

± 0.30 0.75

± 0.25 * 0.45

± 0.38 *

Females

T 4 day -3 2.21 ± 0.78 1.91

± 0.37 2.99

± 0.58 5.06

± 0.73 * 3.31

± 1.07

T 4 day 30 2.82 ± 0.90 1.49

± 0.09 * 1.94

± 0.95 1.03

± 0.43 * 0.59

± 0.11 *

T 4 day 58 2.82 ± 0.23 1.94

± 0.14 * 2.61

± 0.41 1.24

± 0.44 * 0.94

± 0.37 *

T 4 day 86 2.75 ± 1.08 1.29

± 0.38 * 1.50

± 0.60 1.34

± 0.71 * 1.00

± 0.16 *

Data were obtained from pages 72- 86

a Values are group means ± SD * p < 0.05

Table B.6-44: Mean triiodothyronine (T 3) levels a Dose Group (ppm) Parameter 0 150 300 1,200 2,500
Males

T 3 day -3 0.87 ± 0.11 0.95

± 0.15 1.00

± 0.09 0.98

± 0.15 0.84

± 0.11

T 3 day 30 0.76 ± 0.13 0.77

± 0.19 0.78

± 0.12 0.63

± 0.08 0.49

± 0.06 *

T 3 day 58 0.73 ± 0.10 0.71

± 0.07 0.62

± 0.08 0.64

± 0.13 0.42

± 0.11 *

T 3 day 86 0.97 ± 0.19 0.98

± 0.21 0.97

± 0.11 0.90

± 0.12 0.79

± 0.15 Females

T 3 day -3 0.72 ± 0.03 0.67

± 0.12 0.84

± 0.18 2.76

± 0.39 * 1.99

± 0.17 *

T 3 day 30 0.80 ± 0.09 0.64

± 0.14 0.81

± 0.22 0.73

± 0.20 0.43

± 0.02 *

T 3 day 58 0.65 ± 0.05 0.59

± 0.16 0.69

± 0.13 0.57

± 0.15 0.31

± 0.05 *

T 3 day 86 1.03 ± 0.18 0.75

± 0.13 0.91

± 0.14 0.86

± 0.19 0.72

± 0.04 Data were obtained from pages 72- 86

a Values are group means ± SD * p < 0.05– –

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Table B.6-45: Mean thyrotropin (TSH) levels a Dose Group (ppm) Parameter 0 150 300 1,200 2,500
Males

TSH day -3 0.12 ± 0.06 0.04

± 0.01 * 0.08

± 0.04 0.18

± 0.08 0.10

± 0.07

TSH day 30 0.12 ± 0.05 0.06

± 0.04 0.10

± 0.05 0.11

± 0.07 0.06

± 0.03

TSH day 58 0.09 ± 0.04 0.04

± 0.02 0.06

± 0.03 0.08

± 0.07 0.03

± 0.00

TSH day 86 0.07 ± 0.03 0.06

± 0.02 0.07

± 0.05 0.13

± 0.10 0.06

± 0.04 Females

TSH day -3 0.12 ± 0.09 0.09

± 0.03 0.17

± 0.12 0.16

± 0.04 0.21

± 0.17

TSH day 30 0.14 ± 0.13 0.13

± 0.03 0.19

± 0.07 0.13

± 0.01 0.09

± 0.07

TSH day 58 0.07 ± 0.05 0.11

± 0.06 0.14

± 0.10 0.04

± 0.00 0.10

± 0.15

TSH day 86 0.08 ± 0.06 0.08

± 0.09 0.11

± 0.09 0.05

± 0.01 0.24

± 0.37 Data were obtained from pages 72- 86

a Values are group means ± SD * p < 0.05

5. Ophthalmology : No treatment-related effects.

6. Gross necropsy, organ weights, histopathology : Two females in the 2500 ppm group had rough coat and one female in this group had a thymus that was “reduced in size” (the histopathologist indicated t hat the thymus from this female was atrophied).

There were no treatment-related effects on organ we ights. The statistically significant increase in re lative liver weights for males in the 150, 300, and 2,500 ppm groups is considered to be incidental as there is no dose response, the magnitude of differences were slight, there were no correlative clinical chemist ry or histopathological changes and there was no stati stical difference in the absolute liver weights. Likewise, statistically significantly (p<0.05) lowe r absolute pituitary weights in 1200 and 2500 ppm females are not considered compound-related; corres ponding weights relative to body weights were not significantly different from the control. The only notable compound-related histopathologic change was thymus atrophy in one female at 2500 ppm.

III. CONCLUSION:

The NOAEL of 2,500 ppm (equal to 81 or 72 mg/kg bw/ day in males or females, respectively) in this 90- day feeding study in dogs is based on a body weight decline noted during the first two weeks of the study at 4,000 ppm. No marked toxicity was determin ed in the present study. Despite percentage thyroid hormone declines at some time points of greater tha n 50%, no changes in thyroid weight, thyroid pathology, no compensating increases in thyrotropin (TSH), or no clinical observations (e.g., neurolog ical signs) suggestive of thyroid compromise were detect ed in either sex. Based on the total response of t he animals to the thyroid profile that emerged over 90 days, the compound-induced changes in circulating thyroid hormones, though significant in magnitude, were judged to be non-adverse. This conclusion was later confirmed when a similar thyroid and toxicolo gical profile emerged in the 1-year chronic dog stu dy (see below).– –

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The NOEL of 300 ppm (equivalent to 9 or 10 mg/kg bw/day in m ales or females, respectively) is based on non-adverse declines in circulating thyroxine le vels at 1,200 ppm, equivalent to 33 mg/kg bw/day in males or 32 mg/kg bw/day in females.

IV. REVIEWER’S COMMENTS:

The USEPA and PMRA reviewers concur that the compou nd-related decreases in circulating thyroid levels should not be considered adverse based on mo st of the rationale provided by the investigator. T he reviewers take exception to the conclusion that no clinical signs were seen; abnormal posture and reluctance to stand were seen at 2500 ppm in female s, but not in males.

The USEPA and PMRA reviewers also agree that the NO AEL in males is 2500 ppm (81mg/kg/day), but does not agree that all of the compound-related eff ects observed at 2500 ppm in females should be considered “non-adverse”. Although the females in t his group began to gain body weight after the dieta ry level of test material was reduced from 4000 to 250 0 ppm on day 15, it was evident that they did not recover completely since the mean body weight at te rmination was still below the pre-exposure value. During the clinical assessments, one of these femal es was described as thin. Food consumption values for this group were also depressed throughout the s tudy.

Declines in values for red blood cell parameters (r ed blood cell count, hemoglobin level and hematocri t) were seen in 2500 ppm females at day 58; this decli ne continued throughout the remainder of the study and the difference between the value for each of th ese parameters and the respective control was statistically significant at test day 84.

Gross examinations revealed a thymus in one 2500 pp m female that was described as “reduced in size”; the histopathology report indicated that the thymus in this female was atrophied.

It is the reviewers’ conclusion that the noted comp ound-related effects on body weight gain, food consumption, red blood cell parameters and thymus a t 2500 ppm in females are adverse.

The AGES reviewer considers the statistically signi ficant decrease in thyroid hormone T4 observed at 1200 ppm in both sexes to be adverse.

The LOAEL (USEPA and PMRA) for BYI 08330 in this st udy for female dogs is 2500 ppm (72 mg/kg/day) based on decreased body weight gain and food consumption, depressed RBC parameters (red blood cell count, hemoglobin level and hematocrit) and thymus atrophy; a LOAEL was not established for males. The NOAELs (U SEPA and PMRA) for males and females are 2500 ppm (81 mg/kg/day) and 1200 ppm (32 mg/kg/ day), respectively.

The LOAEL (AGES) is 1200 ppm (33/32 mg/kg/day in ma les/females). The NOAEL (AGES) is 300 ppm (9/10 mg/kg/day in males/females).

A. STUDY/REPORT DEFICIENCIES :

There were no deficiencies that compromised the ac ceptability of the study.

B. STUDY DEFICIENCIES :

The males could have tolerated a higher dosage of B YI 08330.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 149 April 2008

C. REPORT DEFICIENCIES:

• Differential leukocyte and reticulocyte counts, wh ich were reportedly assessed, were not reported.

• Food efficiency was not reported.

• Statistical methods were not described in the stud y report.

• Appearance and presence of any microscopic sedimen t from the assessment of urine were not reported.– –

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B.6.3.4 Oral 1 year toxicity (dog) (OECD Annex IIA 5.3.4)

CITATION : Eigenberg, D. A. (2006). A Chronic Toxicity Feeding Study in the Beagle Dog with Technical

Grade BYI 08330. Bayer CropScience LP, Toxicology, 17745 South Metcalf, Stilwell, Kansas. Study No. 04-C76-VP/Report No. 201486. July 6, 2006. Unpublished.

I. MATERIAL AND METHODS

A. MATERIAL

1. Test material : BYI 08330 Description: Technical grade, white powder Lot/Batch No.: 08045/0014 Purity (% ai)*: 97.8 (12/5/03); 97.6% (6/28/04); 98 .5% (1/27/05); 98.1% (11/17/05) Compound Stability Room temperature storage Chemical Name: Ethyl 3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4 .5]dec-3-en-4-yl carbonate CAS No. if TGAI: Non-Stereo: 382608-10-8; CIS 2033 13-25-1

2. Vehicle and/or positive control : corn oil and acetone

3. Test animals : Dog, males and females Species: Canis familiaris Strain: Beagle (nulliparous and nonpregnant) Age/weight at study initiation: 7 - 8 months / males: 6.6 - 8.5 kg & females: 5.7 - 7.9 kg

Source: Marshal BioResources USA, Inc, North Rose, New York Housing: Individually housed in stainless steel cag es. Diet: Purina Certified Canine Diet Etts 5006-3; presented to the animals for 3-6 hours/day, beginning five days prior to initiation of treatment with the test compound and continuing throughout the study, altho ugh there were two occasions when the length of the timed feeding was not documented. Prior to this time food was available for ad libitum consumption, except for when the animals were fasted overnight for pre-clinical blood analyses. Water: Tap water provided continuously for ad libitum consumption Environmental conditions: Temperature: Humidity:

Air changes:

Photoperiod: Set to be maintained at 18 to 29°C (64 to 84°F).

Set to be maintained at 30 to 70%. There were numerous cases when the relative humidity was below 30% and above 70%. The relative humidity was below 30% during the months when the outside air was cold and dry; the HVAC unit was unable to maintain the relative humidity above 30%. When the relative humidity was above 70%, this was usually caused by flushing; also during the hot humid months the HVAC unit was unable to maintain the relative humidity b elow
70%. Averaged at least 13.00 changes per hour during the dosing period. Approximately 12 hours of light alternating with 12 hours– –

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of darkness. Acclimatisation: 5 days prior to release for the st udy.

B. STUDY DESIGN :

1. In life dates :

Study Initiation (Day protocol was signed): May 28 , 2004 Initiation of collection of Data (Day Animals Relea sed for Study): June 1, 2004 Initiation of Dosing: June 9, 2004 Terminal Sacrifice: June 14, 2005

2. Animal assignment:

The dogs were randomly assigned to dose groups note d in Table 6-46, based on weight, using INSTEM DATATOX ®

. Weight variation of animals used was targeted not to exceed + 20% of the mean weight for each sex. All dogs arrived at the test facility wit h a supplier's identification number tattooed on th e inner part of the ear. This unique identifier was cross-r eferenced with the unique identification number assigned to each animal.

Table B.6-46: Study design Dose Group (ppm) No. Animals/ Dose/Sex Males (mg/kg/day) Females (mg/kg/day) 0 (control) a 4 0 0 200 4 6 5 600 4 20 19 1,800 4 55 48

a Maintained on diet with vehicle only

3. Dose selection rationale:

The nominal concentrations chosen for this study we re 0 (concurrent vehicle control) 200, 600, and 1,800 ppm (4/sex/dose) of technical grade BYI 08330 mixed with dog ration. Selection of these dose levels was based on a 90 day feeding study in the B eagle dog (Report No. 201223), with doses of 0 (concurrent vehicle control) 150, 300, 1,200, and 4 ,000/2,500 ppm in the diet. In this study, there w as a marked reduction in the body weight for both sexes in the 4,000 ppm dose group, which was reduced to 2,500 ppm on study day 15. A dose related decrease in T 4 in the 1,200 and 2,500 ppm male and female dose groups throughout the entire study, and a decr ease in T 3 in the 2,500 ppm male and female dose groups on study days 30 and 58 was observed; these decreases in circulating thyroid hormone levels were not considered toxicologically significant. Co mpound-related inhibition of body weight gain and f ood consumption, depressed RBC parameters (red blood ce ll count, hemoglobin level and hematocrit) and reduced thymus size, observed at 2500 ppm in female s, were considered adverse effects.

4. Diet preparation and analysis :

All feed mixtures were prepared weekly by mixing ap propriate amounts of the test substance with Purina Certified Canine Diet 5006-3 and then storing the m ixture under freezer conditions until given to the animals. Corn oil, at 1% by weight of the diet, alo ng with acetone was used as vehicles to suspend the test substance prior to being mixed in the diet. T he control diet was prepared the same as the treate d diet, excluding only the test substance. A sample of each batch of feed mixed was taken and retained under freezer conditions until the study was comple te and the study data deemed satisfactory. Prior t o– –

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conducting the main study, in a 4-week pilot study in the dog (Report No. 201012), the homogeneity and stability of BYI 08330 in dog ration was confirmed for concentrations of 100 ppm and 6,400 ppm. Dietar y concentrations of test material used in the present study were confirmed and the analytical data are included in the study report.

Results: Based on the findings described below, the analytic al data indicated that the mixing procedure was adequate and that the variance between nominal and actual dosage to the animals was acceptable.

Homogeneity analysis: Batches of ration mixed with BYI 08330 at 100 and 6 ,400 ppm were analyzed for homogeneity. Nine samples of ration were taken from each level and an alyzed. The mean concentrations were 96.2 ppm (96.2%, CV = 4.3%) for the 100 ppm test level and 6 ,255 ppm (97.7%, CV = 3.5%) for the 6,400 ppm test level. Based on CV's of 4.3 and 3.5%, BYI 083 30 mixed in dog ration at concentrations of 100 and 6,400 ppm was considered homogeneously distributed.

Stability analysis: For freezer stability, samples were analyzed on day 0, 7, 14, and 28. After 28 days, there was no decline in concentration for the 100 and 6,400 ppm levels. BYI 08330 at concentrations of 100 and 6,400 ppm was considered stable at freezer temperat ure in dog ration for a minimum of 28 days (analytical values were 101 and 114% of actual conc entrations at 100 and 6400 ppm, respectively). For room temperature stability, samples were analyzed o n day 0, 1, 4, and 7. After 7 days, declines in concentration ranged from 9.8 to 2.1% for 100 and 6 ,400 ppm levels, respectively. BYI 08330, mixed in dog ration at concentrations of 100 and 6,400 ppm, was considered stable at room temperature for a minimum of 7 days .

Concentration analysis: The concentration of the active ingredient in the f eed was verified for the first three weeks of the s tudy and then monthly thereafter. The mean concentratio ns for the study were 94 to 100% of the nominal levels of 200 and 1800 ppm, respectively; %RSD valu es ranged from 4.58 to 6.14 at 600 and 1800 ppm, respectively.

5. Statistics :

Statistical significance was determined at p< 0.05 for all tests with the exception of Bartlett's test, in which a probability value of p< 0.001 was used. All tests were two-tailed, except for gross and histopathological lesion evaluations which were one-tailed. Continuou s data were analyzed by Bartlett's test for homogeneity. If the data were homogeneous an ANOVA was performed, followed by Student's t-test on parameters showing a significant effect by ANOVA. If the data were non-homogeneous a Kruskal-Wallis ANOVA was performed, followed by the Mann-Whitney U -test to identify statistical significance between groups. Frequency data, that were examined statist ically, were initially analyzed by a Chi-Square procedure. If there was statistical significance u sing the Chi-square test, each treatment group was compared to the control group using a Fisher's Exac t test.

A complementary statistical analysis of T 4 and T 3 values was conducted by Dr. Catherine Pallen, PhD, expert in statistics and epidemiology, at Bayer Cro pScience, Sophia Antipolis Cedex, France. The T 4 and
T 3 values were log-transformed and analyzed with a re peated-measures analysis of variance (ANOVA, type III sum of squares) including terms for treatm ent, time and interaction of treatment and time. Repeated measurements analysis was performed assumi ng an unstructured covariance matrix.– –

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When the ANOVA indicated a statistically significan t overall treatment or interaction effect (p< 0.05), multiple t-tests were applied using the ANOVA’s lea st-squares (LS) means and corresponding standard errors. Statistical significances were defined at the 5% and 1% levels of significance.

C. METHODS :

1. Observations :

1a. Cageside observations:

All study animals were observed at least twice dail y for clinical signs of toxicity (except once daily on weekends and holidays).

1b. Clinical examinations:

Detailed clinical observations for clinical signs o f toxicity were performed on all animals at study initiation and on a weekly basis thereafter.

2. Body weight:

Individual body weights were measured weekly throug hout the study. Body weights were also taken immediately prior to necropsy to allow for calculat ion of organ-to-body weight ratios.

3. Food consumption and compound intake :

Each animal’s food consumption was measured daily t hroughout the study. The intake of test compound in mg/kg/day was calculated for males and females u sing the analytical concentration of BYI 08330 in the feed and the following equation: (average food consumption per week/average body weight per week) x ppm in the feed/1,000.

4. Ophthalmoscopic examination:

Following the acclimatisation and prior to initiati on of dosing, ophthalmic examinations were conducte d on all animals. Ophthalmic examinations were also c onducted on all animals just prior to termination of the study.

5. Hematology & clinical chemistry:

Clinical chemistry and a complete blood count, incl uding differentials, were performed on all animals (pre-exposure and during study weeks 13, 25, 38, an d 51). During study week 22, blood was collected from all animals for an additional evaluation of T 4 , T 3 , and TSH. Animals were fasted overnight prior to the collection of blood (except during week 22), wh ich was drawn via jugular venipuncture. The parameters evaluated are marked (x) in the lists be low.

5a. Hematology :

x Hematocrit (HCT)* x Leukocyte differential count* x Hemoglobin (HGB)* x Mean corpuscular hemoglobin ( MCH)* x Leukocyte count (WBC)* x Mean corpuscular hemoglo bin conc.(MCHC)* x Erythrocyte count (RBC)* x Mean corpuscular volum e (MCV)*– –

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x Platelet count* x Reticulocyte count a Blood clotting measurements* x Blood cell morpholo gy x (Thromboplastin time) x Red Blood Cell Distributi on Width (RDW) (Clotting time) x Hemoglobin Distribution Width (H DW) x (Prothrombin time) *Recommended for chronic studies based on Guideline 870.4100

a Data were not provided in Study Report.

5b. Clinical chemistry :

ELECTROLYTES OTHER x Calcium (calc)* x Albumin (ALB)* x Chloride (Cl)* x Creatinine (Creat)* Magnesium (Mg) x Urea nitrogen (Urea-N)* x Phosphorous (Phos)* x Total Cholesterol (Chol)* x Potassium (K)* x Globulins (Glob) x Sodium (Na)* x Glucose (gluc)* ENZYMES (more than 2 hepatic enzymes)* x Total bilirubin (T-Bili) * x Alkaline phosphatase (ALP)* x Total protein (TP)* Cholinesterase (ChE) x Triglycerides (Trig) x Creatine phosphokinase (CK) Serum protein electr ophoresis x Lactic acid dehydrogenase (LD) x Uric Acid (Uric- A) x Alanine aminotransferase (ALT/SGPT)* Bile Acids x Aspartate aminotransferase (AST/SGOT)* x A/G rati o (A\G) x Gamma Glutamyl transferase (GGT)* x Thyroxin (T4) Glutamate dehydrogenase x Triiodothyronine (T3) x Thyroid Stimulating Hormone (TSH) *Recommended for chronic studies based on Guidelin e 870.4100

6. Urinalysis :

Urine was collected from all animals (pre-exposure and during wks 4, 8, and 13). The parameters evaluated are marked with (x) in the list below.

x Appearance* a x Glucose (Glu)* x Volume (UVol)* a x Ketones (Ket) x Specific gravity / osmolality (Sp.Gr.)* x Bilirub in (Bil) x pH (pH)* x Blood (Bld)* x Sediment (microscopic) a x Nitrite (Nit) x Protein (Pro)* x Urobilinogen (Uro) x Urine Creatinine (24 hour) (Creat) x Leucocytes ( U-Leu) *Recommended for chronic studies based on Guidelin e 870.4100

a Data were not provided in the Study Report.

7. Sacrifice and pathology :

Animals were euthanized at the end of the study by intravenous injection of Fatal-Plus ® (Vortech Pharmaceuticals, Dearborn, Michigan). A complete g ross examination was performed on all animals. The necropsy consisted of a systematic gross examin ation of each animal's general physical condition, body orifices, external and internal organs and tis sues. A list of the tissues collected (X) and weig hed (XX) at necropsy is presented in the table below. All tissues were processed, embedded in paraffin, sectioned, mounted, and stained with hematoxylin an d eosin (H&E) for examination under the light microscope. Histopathologic evaluation was conduct ed on all protocol-required tissues from the contro l and high-dose group animals as well as epididymides , lungs, and thyroids from the low- and mid-dose group animals. Where appropriate, all findings wer e assigned a severity score where N = tissues withi n normal histological limits, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked. The mean severity wa s determined by dividing the sum of the individual an imal severity scores by the number of tissues examined in the group.– –

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Tissues Collected at Necropsy, Organs Weighed, and Tissues Examined Microscopically

DIGESTIVE SYSTEM CARDIOVASC./HEMAT. NEUROLOGIC
X Cecum* X Aorta* XX Brain* X Colon* X Bone marrow* X Cerebellum
X Duodenum* XX Heart* X Cerebrum-Midbrain X Esophagus* X Lymph node, mesenteric* X Medulla/Po ns X Gall bladder* X Lymph node, retropharyngeal X E yes* X Ileum* XX Spleen X Nerve, optic* X Jejunum* XX Thymus X Nerve, sciatic XX Liver* XX Pituitary

X Pancreas* UROGENITAL X Spinal cord, cervical* X Rectum* X Cervix X Spinal cord, thoracic* X Salivary glands* Clitoral gland X Spinal cord, l umbar*

X Stomach * XX Epididymides* X Fallopian tube (oviduct) OTHER XX Kidneys* Bone, femur X Mammary gland* X Bone marrow XX Ovary* X Bone, rib/cc jct X Prostate* X Bone, sternum

RESPIRATORY XX Testicle* X Gross lesions*

X Larynx * X Ureter Joint, fem/tib X Lung* X Urinary bladder* X Muscle, protocol
Nasal structure XX Uterus* X Physical Identifier a X Nasopharynx* X Vagina a X Skin, protocol*
Oral structure X Oviduct

X Trachea* GLANDULAR XX Adrenal gland* Exorbital/lacrimal gland X Thyroid (with parathyroid)* *Required for chronic studies based on Guideline 87 0.4100.

a No histopathology performed

I. RESULTS AND DISCUSSIONS:

Findings :

General observations : Notable clinical findings, each observed in a male at 1800 ppm, included dehydration, swelling, decreased activity and react ivity, seizures and ataxia. There were no unscheduled mortalities during the st udy.

There were no compound-related effects on food cons umption and body weights. Body weight gains were not reported, however, it is evident from the body weight data that body weight gains of exposed groups were not depressed. Some lower daily food co nsumption values at 1800 ppm in females were statistically significant (p<0.05) at several inter vals throughout the study, however as previously indicated, no effect on body weight was observed.

Hematology : There were no toxicologically significant findings . The following small, but statistically significant (p<0.05) differences from controls were observed: decreased hemoglobin concentration,– –

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increased white blood cell count, increased count a nd proportion of neutrophils and decreased proportion (but not count) of lymphocytes in 1800 p pm males as well as increased prothrombin time in 600 and 1800 ppm females. These changes are conside red incidental because, in addition to being slight, some were not dose-related, some were trans ient and correlative changes in other parameters were not observed. There were no compound-related e ffects on blood cell morphology.

Urinalysis : No compound-related effects were seen.

Clinical chemistry : Thyroxine (T4) was statistically decreased in male s at 1,800 ppm for all posttreatment time points and at 600 ppm at termination of study (day 357) (Table 6-47). In females, T4 wa s statistically decreased for most time points at 600 and 1,800 ppm. Triiodothyronine (T3) decreases wer e less pronounced and occurred in males at 1,800 ppm for all time points (Table 6-48). There was a tendency for T 4 values to fall below the historical control range a t 1800 ppm in males and females; the same tendency was seen in T 3 values at 1800 ppm in males.

The statistically significant T4 decrease in female s on day 357 at 200 ppm was considered a statistica l aberration due to the high T4 mean in controls. Th e statistically significant T3 decrease in males on day 180 at 200 ppm and 600 ppm was considered a sta tistical aberration due to the relatively high T3 mean in controls. This is also supported by the fac t that T3 levels were comparable to control levels at subsequent time points. Statistical declines in T3 were not noted in females at any dietary level tes ted. Despite declines in thyroid hormones at some time p oints, no changes in thyroid weight or compensating increases in thyrotropin (TSH) Table 6-49) were see n at ≤ 600 ppm. Therefore, decreases in thyroid hormone at ≤ 600 ppm were not considered toxicologically signifi cant.

Other incidental, but statistically significant (p< 0.05), clinical chemistry changes were, in males, decreased albumin at 1800 ppm, increased chloride a t 600 and 1800 ppm, increased phosphorous at 1800 ppm (none of these findings had correlative ch anges) and transient increases of glucose in all dosage groups; in females, transient increases of g lucose (200 and 1800 ppm) and creatinine (1800 ppm) were seen.

Table B.6-47: Mean thyroxine (T 4) levels (μg/dl) Dose group (ppm) Historical range Parameter 0 200 600 1,800 Low High

Males
T 4 day -5 2.14 ± 0.68 2.20 ± 0.23 2.33 ± 0.58 2.74 ± 0.59 1.2 4.1
T 4 day 96 2.35 ± 0.38 0.99 ± 0.21 0.89 ± 0.19 0.53 ± 0.39* 0.9 3.9
T 4 day 155 2.17 ± 0.43 1.30 ± 0.22 1.14 ± 0.40 1.01 ± 0.66 * n.a. n.a. T 4 day 180 2.13 ± 0.66 0.87 ± 0.20 0.70 ± 0.15 0.48 ± 0.44 * 0.7 3.5
T 4 day 271 1.86 ± 0.56 0.94 ± 0.21 0.78 ± 0.14 0.33 ± 0.35 ** 0.9 3.0
T 4 day 357 1.84 ± 0.65 1.16 ± 0.25 1.02 ± 0.12* 0.58 ± 0.14 ** 0.6 3.3 Females T 4 day -5 2.44 ± 0.27 2.57 ± 0.98 2.63 ± 0.62 2.37 ± 0.36 0.6 4.2 T 4 day 96 1.83 ± 0.90 1.19 ± 0.29 0.93 ± 0.46 * 0.69 ± 0.22 * * 0.7 3.6 T 4 day 155 2.56 ± 0.92 1.76 ± 0.17 1.24 ± 0.19 ** 1.12 ± 0.31 ** n.a. n.a. T 4 day 180 2.18 ± 1.02 1.38 ± 0.27 1.32 ± 0.65 0.51 ± 0.18 ** 0.6 4.4 T 4 day 271 1.80 ± 0.57 1.23 ± 0.16 0.90 ± 0.27* 0.58 ± 0.27 ** 0.9 4.0 T 4 day 357 2.88 ± 0.53 1.79 ± 0.52 *  1.11 ± 0.27** 0.85 ± 0.23 ** 0.9 4.6 T 4 values (Group means ± SD) were obtained from pages 364-393 *p < 0.05; ** p < 0.01 (repeated measures ANOVA and t-test using ANOVA ́s least-square means)  statistical aberration due to the high T4 in contr ols; n.a. = not available– –

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Table B.6-48: Mean triiodothyronine (T 3) levels Dose Group (ppm) Historical range Parameter 0 200 600 1,800 Low High

Males

T 3 day -5 0.89 ± 0.07 0.87

± 0.03 0.91

± 0.22 0.95

± 0.25 0.4 1.2

T 3 day 96 0.79 ± 0.12 0.61

± .10 0.68

± 0.14 0.48

± 0.09 ** 0.4 1.3

T 3 day 155 0.71 ± 0.09 0.60

± 0.07 0.62

± 0.08 0.52

± 0.12 * n.a. n.a.

T 3 day 180 0.82 ± 0.05 0.64

± 0.09 **  0.61

± 0.04 **  0.52

± 0.09 ** 0.6 1.4

T 3 day 271 0.64 ± 0.11 0.49

± 0.09 0.50

± 0.06 0.43

± 0.11 * 0.5 1.2

T 3 day 357 0.65 ± 0.09 0.57

± 0.05 0.67

± 0.09 0.46

± 0.02 ** 0.6 2.3

Females

T 3 day -5 0.80 ± 0.16 0.80

± 0.03 0.78

± 0.09 0.87

± 0.11 0.3 1.3

T 3 day 96 0.70 ± 0.30 0.73

± 0.16 0.69

± 0.17 0.62

± 0.10 0.5 1.2

T 3 day 155 0.69 ± 0.16 0.62

± 0.06 0.66

± 0.04 0.59

± 0.10 n.a. n.a.

T 3 day 180 0.85 ± 0.23 0.75

± 0.03 0.82

± 0.16 0.61

± 0.05 0.4 1.4

T 3 day 271 0.61 ± 0.15 0.58

± 0.08 0.63

± 0.11 0.50

± 0.09 0.4 1.3

T 3 day 357 0.73 ± 0.09 0.62

± 0.06 0.63

± 0.07 0.61

± 0.11 0.5 1.1

T 3 values (Group means ± SD) were obtained from pages 364-393 *p < 0.05 ** p < 0.01 (repeated measures ANOVA and t-test usi ng ANOVA ́s least-square means)  statistical aberration due to the relatively high T3 control mean; n.a. = not available

Table B.6-49: Mean thyrotropin (TSH) levels Dose Group (ppm) Historical range Parameter 0 200 600 1,800 Low High

Males TSH day -5 0.18 ± 0.10 0.33 ± 0.31 0.17 ± 0.12 0.22 ± 0.19 0.0 2 0.54 TSH day 96 0.16 ± 0.11 0.17 ± 0.19 0.11 ± 0.11 0.11 ± 0.08 0.0 7 1.10 TSH day 155 0.17 ± 0.10 0.33 ± 0.34 0.15 ± 0.14 0.12 ± 0.11 n.a . n.a. TSH day 180 016 ± 0.14 0.21 ± 0.20 0.13 ± 0.10 0.19 ± 0.13 0.06 0.41 TSH day 271 0.11 ± 0.10 0.16 ± 0.10 0.11 ± 0.11 0.10 ± 0.07 0.0 7 0.39 TSH day 357 0.16 ± 0.13 0.18 ± 0.17 0.11 ± 0.09 0.06 ± 0.05 0.0 4 0.42
Females TSH day -5 0.13 ± 0.09 0.17 ± 0.07 0.11 ± 0.06 0.16 ± 0.04 0.04 0.41 TSH day 96 0.11 ± 0.08 0.12 ± 0.05 0.08 ± 0.07 0.06 ± 0.02 0.03 0.74 TSH day 155 0.12 ± 0.05 0.13 ± 0.09 0.10 ± 0.02 0.0 9 ± 0.03 n.a. n.a. TSH day 180 0.21 ± 0.17 0.21 ± 0.14 0.17 ± 0.12 0.0 7 ± 0.03 0.06 0.49 TSH day 271 0.11 ± 0.05 0.12 ± 0.04 0.09 ± 0.07 0. 09 ± 0.05 0.04 0.39 TSH day 357 0.14 ± 0.08 0.10 ± 0.08 0.08 ± 0.05 0.0 6 ± 0.03 0.07 0.46 TSH values (Group means ± SD) were obtained from pa ges 364-393 * p < 0.05 ** p < 0.01 (repeated measures ANOVA and t-test usi ng ANOVA ́s least-square means) n.a. = not available

Ophthalmology: No treatment-related effects were observed.– –

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Gross necropsy, organ weights: Reduced thymus size and dilated brain were observe d in males at 600 and 1800 ppm; brain dilation was also noted in fema les, but only at 600 ppm. Organ weights were not affected by treatment. Mean thyroid absolute and relative organ weights were not statistically different from controls. Mean abs olute heart weight was statistically low (p<0.05) a t 1800 ppm in males, however, there was no statistical dif ference in mean relative weight and correlative histopathologic changes were not found. Absolute an d relative thymus weights were low (not statistical ly) in males only at 600 ppm.

Histopathology: Two 1,800 ppm males had a slight reduction in the size of the peripheral thyroid follicles. Two additional step-sections from the bl ocks were also evaluated for all control and 1,800 ppm males and females. None of these additional evaluat ions changed the follicular morphology observed in the original slides. Brain dilation was seen at 600 ppm in 1 male (mild) and 1 female (moderate) as we ll as at 1800 ppm in 1 male (moderate); mild axonal de generation was also detected in 1 female at 1800 ppm. Thymus involution was graded mild in 1 male at 600 ppm and moderate in 1 male at 1800 ppm.

III. CONCLUSION: The chronic NOAEL in males was established at 600 p pm (equal to 20 mg/kg bw/day), based on a reduced thyroid follicular size noted in two animal s at 1,800 ppm (equal to 55 mg/kg bw/day). The chro nic NOAEL in females was established at 1,800 ppm (equa l to 48 mg/kg bw/day), based on the absence of adverse compound-induced toxicological responses. D espite thyroid hormone declines at some time points, no changes in thyroid weight, thyroid patho logy, no compensating increases in thyrotropin (TSH ), or no clinical observations (e.g., neurological sig ns) suggestive of thyroid compromise were detected in either sex. Based on the total response of the ani mals to the thyroid profile that emerged over one y ear, the isolated compound-induced changes in circulatin g thyroid hormones observed in this one-year study, though significant in magnitude, were judged to be non-adverse. Based on non-adverse declines in T4 at 600 ppm (equivalent to 20 mg/kg bw/day in males and 19 mg/kg bw/day in females), the overall NOEL for beagle dogs in a chronic one year dog study with te chnical grade BYI 08330 was 200 ppm (equal to 5 mg/kg bw/day).

IV. REVIEWER’S COMMENTS:

USEPA and PMRA reviewers considered the decreases i n thyroid hormones to be non-adverse at ≤ 600 ppm based on the lack of correlative changes in TSH , thyroid weight, or thyroid histopathology. Correlative histopathology was observed in the thyr oid in males at 1800 ppm. AGES reviewers considered the statistically significant decreases in T4 at ≥ 600 ppm to be adverse. PMRA reviewers considered the brain findings to be treatment-relat ed. USEPA reviewers concluded that brain dilation was an equivocal effect and that it should not be u sed as the basis for the LOAEL in the study. Unlik e thymus effects, which were observed in other studie s with spirotetramat in the dog, brain dilation was not observed in any other study in the dog or in an y other species in the database. In addition, brai n dilation has been observed sporadically in several Bayer dog studies of varying durations (from four weeks up to one year). Historically brain dilation , considered a congenital anomaly by veterinary pathologists, has been observed at incidences up to 5% in some dog populations. USEPA concluded that if brain dilation truly were a treatment-relat ed effect, it should have been seen in other dog st udies in the database, but this was not the case. The se izures observed in a single dog in the study were most likely due to the brain dilation in this anima l and not to decreased thyroid hormone. If due to decreased thyroid hormone, the seizures should have been observed in other animals across all of the dog studies, but this was not observed. Based on t he fact that the brain dilation in the one-year dog study is likely not treatment-related, there was no evidence of offspring susceptibility in the developmental toxicity studies or reproduction stud ies with spirotetramat, and the fact that the– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 159 April 2008

structurally related Bayer compounds spirodiclofen and spiromesifen are not neurotoxic in adults or young, USEPA has concluded that there is no concern for neurotoxicity with spirotetramat in the developing animal. PMRA reviewers considered brain dilation to be a treatment-related effect.

The LOAEL (USEPA) for BYI 08330 in this study for m ale dogs is 600 ppm (20 mg/kg/day) based on thymus involution and was not observed in female s. The NOAELs for males and females are 200 ppm (6 mg/kg/day) and 1800 ppm (48 mg/kg/day), respectively.

The LOAEL (PMRA) for male dogs is 600 ppm (20 mg/kg /day) based on thymus involution and brain dilation, and 600 ppm (19 mg/kg/day) in femal es based on a “brain effect” (brain dilation at 600 ppm and axonal degeneration at 1800 ppm). The NOAEL (PMRA) for males and females is 200 ppm (6/5 mg/kg/day in males/females, respective ly).

The LOAEL (AGES) for male dogs is 600 ppm (20 mg/kg /day) based on thymus involution and brain dilation, and 600 ppm (19 mg/kg/day) in femal es based on decreased thyroid hormone levels. The NOAEL (AGES) for males and females is 200 ppm (6/5 mg/kg/day in males/females, respectively).

Study/report deficiencies: There were no deficiencies that compromised the acc eptability of the study.

Study deficiencies: The females could have tolerated a higher dosage of BYI 08330.

Report deficiencies:

• Completed reticulocyte counts were not reported.

• Food efficiency was not reported.

• Appearance, volume and microscopic sediment from t he assessment of urine were not reported.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 160 April 2008

B.6.3.5 Repeated dose (28-day/90-day) inhalation to xicity (rodents) (OECD Annex IIA 5.3.5 and

5.3.6)

No data available.

B.6.3.6 Repeated dose (28-day/90-day) percutaneous toxicity (rodents) (OECD Annex IIA 5.3.7

and 5.3.8)

CITATION : Eigenberg, DA. 2006. A subacute dermal toxicity st udy in rats with BYI 08330. Study No. 05-

S22-YC. Report No. 201505. Bayer CropScience LP, St ilwell, Kansas. Unpublished.

I. MATERIAL AND METHODS

A. MATERIALS :

1. Test material : BYI 08330 Description: Technical grade, white powder, room te mp storage Lot/Batch No.: 08045/0014 Purity (ai%): 97.6% (28-Jun-04); 98.5% (27-Jan-05) ; 98.1% (17-Nov-05) Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. if TGAI: Non-Stereo: 382608-10-8; CIS 2033 13-25-1

2. Vehicle and/or positive control: distilled water

3. Test animals : Species: Rat Strain: Wistar Hanover CRL:WI [GLX/BRL/HAN]IGS BR ( nulliparous and

nonpregnant) Age/weight at study initiation: 7-9 weeks/males: 173- 302 g & females: 149-220 g

Source: Charles River Laboratories, Incorporated, R aleigh, North Carolina Housing: Individually housed in stainless steel wir e mesh cages Diet: Purina Laboratory Certified Rodent Chow 5002 availa ble for ad libitum

consumption Water: Tap water (Kansas City Missouri municipal w ater, dispensed by automatic

watering system) was available for ad libitum consumption. Environmental conditions: Temperature:

Humidity: Air changes: Photoperiod: 18 to 26°C

30 to 70% Averaged at least 11/hr during the dosing period. 12 hrs of light alternating with 12 hrs of darkness

. Acclimatisation: 4 or 10 days prior to release for the study

B. STUDY DESIGN :

1. In-life dates :

Study Initiation : January 6, 2005 Initiation of Dosing: January 10, 2005– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 161 April 2008

Necropsy: February 7 and 8, 2005

2. Animal assignment, test groups and study duratio n : Rats were randomly assigned to dose groups, based on weight, using INSTEM DATATOX®. Four dose groups, three treated and one concurrent control group were dosed dermally with 0 (control g roup), 100, 300, and 1,000 mg/kg of BYI 08330 for 2 8 or 29 days. Each treatment group consisted of ten m ales and ten females. The doses were based on each animal’s body weight on days 0, 7, 14, and 21.

3. Dose selection rationale : A limit dose of 1,000 mg/kg/day was used in the stu dy.

4. Preparation and treatment of animal skin : On study day -3 (i.e., the Friday prior to dosing), the hair was removed from the dorsal and lateral areas of th e trunk of each rat using electric clippers (Note: Rat YC1105 was not shaved until study day 0, due to an oversight on study day -3). During the dosing period, the animals were shaved when necessary due to hair growth. The test material was weighed out for each animal and applied to a large commercially available adhesive bandage (2 in. x 4 in. or 4.5 i n.), and moistened with 1 ml of deionized water. The ad hesive bandage was placed on the shaved skin of the rat and the torso of the animal was then wrappe d with porous medical tape to assure that the bandage remained on the animal during the dosing in terval. The same procedure was performed for control animals with the exception that only the ba ndage, moistened with 1 ml of deionized water, was applied to the dose site. The test substance was h eld in contact with the skin for a minimum of six hrs/day for five consecutive days/week for four wee ks. Each day the bandage and tape were removed and the dose site was gently wiped with water-dampe ned and dry gauze to remove as much test substance residue as feasible without damaging the skin.

5. Statistics : Statistical significance was determined at p < 0.05 for all tests with the exception of Bartlett' s test, in which a probability value of p < 0.001 was used. All tests were two-tailed, except for gross and histopathologic lesion evaluations that were one-ta iled. Continuous data were analyzed by Bartlett's test for homogeneity. If the data were homogeneous, an ANOVA was performed followed by Dunnett's t-test on parameters showing a significant effect by ANOVA . If the data were non-homogeneous, a KruskalWallis ANOVA was performed followed by the Mann-Whi tney U-test to identify statistical significance
among groups.

Frequency data that were examined statistically wer e evaluated using the Chi-Square test. If there wa s statistical significance using the Chi-Square test, each treatment group was compared to the control group using a Fisher's Exact test.

C. METHODS:

1. Observations :

1a. Cageside observations : All animals were observed at least twice daily (AM and PM) during the working week and once on the weekends.

1b. Clinical examinations : During the week, clinical examinations were conduct ed twice a day during the dosing and unwrapping of the animals.

1c. Neurological evaluations : Neurological evaluations are available from other s tudies so they were not performed in this study.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 162 April 2008

2. Body weight : The weight of each animal was determined on study d ays 0, 7, 14, 21, and 28. A terminal body weight was obtained on animals that were found dead or sacrificed in extremis.

3. Food consumption : Food consumption was not measured in the study.

4. Ophthalmoscopic examination : Eyes were examined on all animals pretreatment and during study week (wk) 4. Animals with pretreatment abnormaliti es were excluded from the study.

5. Hematology & clinical chemistry : During the 3rd and 4th week of the study, blood was collected from the orbital sinus of all rats under isoflurane anes thesia. During study wk 3, the blood was analyzed for standard serum chemistry and hematological paramete rs; during wk 4 the blood was analyzed for prothrombin time and thromboplastin time. The anima ls were fasted overnight prior to collecting blood during wk 3. The parameters evaluated are checked w ith (x) in the lists below:

5a. Hematology :

x Hematocrit (HCT) x Leukocyte differential count x Hemoglobin (HGB) x Mean corpuscular hemoglobin (M CH) x Leukocyte count (WBC) x Mean corpuscular hemoglob in conc.(MCHC) x Erythrocyte count (RBC) x Mean corpuscular volume (MCV) x Platelet count x Reticulocyte count Blood clotting measurements: x Erythrocyte morphol ogy x (Activated Partial Thromboplastin time) x Red blo od cell distribution width (RDW) (Clotting time) x Hemoglobin distribution width (HDW) x (Prothrombin time)

5b. Clinical chemistry:

ELECTROLYTES OTHER x Calcium (calc) x Albumin (ALB) x Chloride (Cl) x Cholesterol, total (Chol) Magnesium (Mg) x Creatinine (Creat) x Phosphorous (Phos) x Globulins (Glob) x Potassium (K) x Glucose (Gluc) x Sodium (Na) x Total bilirubin (T-Bili) ENZYMES x Total protein (T-Prot) x Alkaline phosphatase (ALK) x Triglycerides (Trig) Cholinesterase (ChE) x Urin Acid (Uric-A) x Creatine phosphokinase (CK) x A/G ratio (A/G) x Lactic acid dehydrogenase (LD) x Urea nitrogen (U rea-N) x Alanine aminotransferase (ALT / SGPT) Bile Acids x Aspartate aminotransferase (AST / SGOT) Serum pr otein electrophoresis x Gamma Glutamyl transferase (GGT) Glutamate dehydrogenase

6. Urinalysis : Urinalysis was not conducted in the study.

7. Sacrifice and pathology : A complete gross examination was performed on all a nimals. The necropsy consisted of a systematic gross examination of each animal's general physical condition, body orifices

, external and internal organs and tissues. All tiss ues collected were processed, embedded in paraffin, sectioned, mounted, and stained with hematoxylin an d eosin (H&E) for examination (initially control an d high-dose then proceeding to lower doses as necessa ry) under the light microscope. A list of the tiss ues collected (x) and weighed (xx) at necropsy is prese nted in the table below. Only tissues from the cont rol and high dose groups were examined microscopically.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 163 April 2008

DIGESTIVE SYSTEM CARDIOVAS. / HEMAT. NEUROLOGIC Bile Duct (rat) x Aorta xx Brain x Cecum x Bone Marrow x Cerebellum x Colon xx Heart x Cerebrum-Midbrain x Duodenum x Lymph node, cervical* x Medulla/Pons x Esophagus x Lymph node, mesenteric* x Eyes Gall Bladder (not rat) xx Spleen x Nerve, optic x Ileum xx Thymus x Nerve , sciatic x Jejunum UROGENITAL x Pituitary xx Liver x Cervix* x Spinal Cord, cervical x Pancreas x Clitoral Gland* x Spinal Cord, thoraci c x Rectum xx Epididymides x Spinal Cord, lumbar x Salivary Glands xx Kidney OTHER x Stomach, glandular x Mammary Gland Bone, femur x Stomach, non-glandular xx Ovary Bone, rib/cc jct x Tongue* x Preputial Gland* x Bone, sternum x Tooth* x Prostate x Gross lesions

RESPIRATORY x Seminal Vesicle x Harderian Gland* x Larynx xx Testicle x Joint, fem/tib* x Lung x Urinary Bladder x Muscle, protocol* x Nasal structure xx Uterus x Physical identifier*x Nasopharynx x Vagina* x Skin (treated and nontreated) x Oral structure GLANDULAR x Zymbal’s Gland* x Trachea xx Adrenal x Exorbital/Lacrimal Gland* x Thyroid (w/parathyroid)

* No histopathology performed

II. RESULTS:

Findings: General observations : One female at 100 mg/kg bw/day died on study day 22 due to the anesthesia used when collecting blood. This early death was co nsidered to be incidental and not treatment-related

. All other animals survived until the scheduled term ination of the study. No effects on body weight development, food consumption, clinical signs, or l ocal skin reactions were observed.

Ophthalmology : No treatment-related effects.

Hematology:

Statistically significant differences (p ≤ 0.05) were observed in treated groups in haematology, which included increased white blood c ell count, activated partial prothromboblastin time

, prothrombin time, and differential cell counts. Th ese findings were not considered toxicologically or biologically significant since they did not have a clear dose-response pattern, were within historical control ranges, and/or no histological correlations were found.

Clinical chemistry : Statistically significant differences (p ≤ 0.05) were observed in treated groups in clinical chemistry which included decreased creatin e kinase, lactate dehydrogenase, aspartate aminotransferase. These findings were not considere d toxicologically or biologically significant since they did not have a clear dose-response pattern, we re within historical control ranges, and/or no histological correlations were found.

Gross necropsy, organ weights, histopathology : No treatment-related macroscopic changes were seen. Organ weights were not affected by compound a dministration. There were no treatment-related microscopic observations.

III. DISCUSSION AND CONCLUSIONS– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 164 April 2008

A. CONCLUSIONS :

The NOAEL of 1,000 mg/kg in males and females is b ased on the absence of effects at the highest dose tested.

B. REVIEWER COMMENTS :

The reviewer agrees with the study authors’ interpr etation of the data. Dermal treatment of male rats with BYI 08330 at dose levels of 0, 100, 300, and 1 ,000 mg/kg by dermal application for 28 days result ed in no treatment-related adverse effects. Analytical determinations of stability and homogeneity were n ot performed because the test substance was applied un diluted and only moistened with water immediately before application. One mortality of a female at 10 0 mg/kg occurred on Day 22 during blood collection due to isoflurane anesthesia and was not treatment- related. There were no treatment-related effects o n body weight, organ weight, or body weight:organ wei ght ratios. Statistically significant differences observed in treated groups in hematology (increased white blood cell count, activated partial prothromboblastin time, prothrombin time, different ial cell counts) and clinical chemistry (decreased creatine kinase, lactate dehydrogenase, aspartate a minotransferase) measurements were not considered toxicologically or biologically signific ant since they did not have a clear dose-response pattern, were within historical control ranges, and /or no histological correlations were found. Only t issues from the control and high dose groups were examined microscopically, and no treatment-related findings were reported.

A systemic LOAEL for male and female rats was not e stablished. The NOAEL for male and female rats is 1000 mg/kg/day.

A dermal LOAEL for male and female rats was not est ablished. The NOAEL for male and female rats is 1000 mg/kg/day.

This 28-day dermal toxicity study in the rat is Acceptable/Guideline and satisfies the guideline requirement for a 28-day dermal toxicity study (OPP TS 870.3200).

C. STUDY DEFICIENCIES:

No deficiencies were identified

.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 165 April 2008

B.6.3.7 Summary of short-term toxicity studies

The insecticidal mode of action (lipid biosynthesis inhibition) was not reflected in the results of th e short-term toxicological studies in rodents and dogs. Rats, m ice, and dogs did not exhibit changes in plasma lip id parameters such as plasma triglycerides and plasma cholesterol. The thyroid and thymus glands were tar get organs in oral subchronic toxicity studies in the d og. Subchronic (90-day) exposure of dogs to spirot etramat was characterized by statistically significant decl ines in circulating thyroid hormones (T4) at ≥ 1200 ppm (32 mg/kg bw/day); decreased T3 was observed at the hig hest dose tested only (2500 ppm; 72 mg/kg bw/day). However, correlative changes in thyroid weight, thy roid histopathology, or thyroid stimulating hormone (TSH) were not observed in either sex.

In rats, the testes were the target organ following subchronic oral treatment at a high dose. Abnorm al spermatozoa and hypospermia in the epididymis, decr eased testicular weight, and testicular degeneratio n and vacuolation in males were observed in males aft er 90 days of exposure at 10,000 ppm (616 mg/kg bw/day). These effects proved to be reversible in most animals after cessation of treatment. Other e ffects in subchronically treated rats were limited to decline s in terminal body weight in 10,000 ppm male rats a nd an increased incidence of accumulation of alveolar mac rophages in both sexes at 10,000 ppm. It is notewor thy that the thyroid and thymus were unaffected in rats at any dose, while testicular histopathology was unobserved in dogs.

Unlike the rat, no adverse effects of any kind were observed in mice tested orally up to the limit dos e. In vitro results from a comparative metabolism study u sing hepatocytes from male rats, mice, and humans revealed species differences in the metabolism of s pirotetramat. Specifically, mouse hepatocytes were better able than rat or human liver cells to metabo lize BYI 08330-enol via glucuronidation. Potential ly lower levels of the enol metabolite in mice in vivo may a ccount for the lack of testicular toxicity observed in this species.

Subchronic exposure of rats by the dermal route yie lded no evidence of systemic toxicity when spirotet ramat was tested up to 1000 mg/kg bw/day. This result ma y in part be a reflection of the low dermal absorpt ion (approximately 10%) in rats.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 166 April 2008

B.6.4 Genotoxicity (OECD Annex IIA 5.4)

B.6.4.1 In vitro assays

B.6.4.1.1 Bacterial assay for gene mutation (IIA 5. 4.1)

CITATION : Herbold, B. 2006. BYI 08330. Salmonella /Microsome Test Plate Incorporation and Preincubation Method. Study No. T6076454. Report N o.AT03070, Document No. M272000-01-2. Bayer HealthCare AG, PH-GDD Toxicolog y, 42096 Wuppertal, Germany. Unpublished Study.

I. MATERIAL AND METHODS

A. Materials :

1. Test Material: BYI 08330 Description: Fine white powder Lot/Batch No.: SAV5505-085/2 Purity (%ai): 95.2 % (03-29-2006) Compound Stability Room temperature storage Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. of TGAI: 203313-25-1
Solvent Used: DMSO

2. Control Materials: Negative: N/A Solvent (final conc’n): DMSO, (0.1 ml/plate) Positive: Nonactivation: Sodium azide 10 μg/plate TA1535 Nitrofurantoin 0.2 μg/plate TA 100 4-Nitro-1, 2- phenylene diamine 0.5 μg/plate TA 98 Mitomycin C 0.2 μg/plate TA 102 4-Nitro-1, 2- phenylene diamine 10 μg/plate TA1537 Cumene hydroperoxide 50 μg/plate TA 102

Activation: 2-Aminoanthracene 3 μg/plate, all strai ns

3. Activation: S9 derived from x Induced x Aroclor 1254 x Rat x Liver Non-induced Phenobarbitol Mouse Lung None Hamster Other Other Other

Describe S9 mix composition:

Cofactor solution: MgCl 2 x 6 H 2 O : 162.6 mg 70 ml, S9 fraction, 10 ml, KCl 0.15 M, 20 ml KCl: 246.0 mg Glucose-6-phospate, disodium salt: 179.1 mg– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 167 April 2008

NADP, disodium salt: 315.0 mg Phosphate buffer: 100.0 mM

4. Test organisms : S. typhimurium strains

TA97 x TA98 x TA100 x TA102 TA104 x TA1535 x TA1537 TA1538 list any others

Properly maintained? x Yes No Checked for appropriate genetic markers ( rfa mutation, R factor)? x Yes No

5. Test compound concentrations used :

Nonactivated conditions : 0, 16, 50, 158, 500, 1581, 5000 ug/plate, all str ains, 3 replicates/treatment, 2 trials Activated conditions : 0, 16, 50, 158, 500, 1581, 5000 ug/plate, all str ains, 3 replicates/treatment, 2 trials

B. TEST PERFORMANCE

1. Type of Salmonella assay :

x Standard plate test

x Pre-incubation (20 minutes) “Prival” modification (i.e. azo-reduction method) Spot test Other

2. Protocol : In the first trial, a strict plate incorporation me thod was employed (Ames et al. (1973) Proc. Nat.Acad. Sci. 70: 2281-2285, Ames et al. (1975) Mutation Res. 31: 347-364). For the mutant colony count, each treatment level was plated in triplicat e. The negative control consisted of the solvent ab sent the test material. In the plate incorporation proce dure, 0.1 ml of the solvent with or w/o the test ma terial, 0.1 ml of the bacterial inoculum, 0.5 ml of the S9 fraction or buffer, and 2.0 ml of soft agar were wa rmed
to 45 o C in a water bath for 30 seconds, mixed and poured onto a petri dish. The incubation proceeded for 48 hours at 37 °C. The number of mutant colonie s was then recorded.

In the second trial, 0.1 ml of the solvent with or w/o the test material, 0.1 ml of the bacterial inoc ulum, and 0.5 ml of the S9 fraction or buffer were pre-in cubated for 20 minutes at 37 °C, the agar was added and mixed, followed by 48 hours of incubation at 37 °C. Likewise, the number of mutant colonies was recorded. The cytotoxicity of the test material was assessed in three ways. 1). An appraisal of the background growth on the incubation plates was asse ssed. 2). If there was a marked and dosedependent reduction on the mutant count, that was c onsidered to be a measure of toxicity. 3). Total bacterial counts were taken by incubating two plate s per treatment level in the presence of histidine and incubating for 48 hours at 37 °C. A reduction in th e titer in comparison to the negative control was considered to be an indication of toxicity.

3. Statistical Analysis : No statistical analysis was performed on the study data.

4. Evaluation Criteria : Acceptance of an assay required that:

a. The negative controls had to be within the expec ted range as defined by published data or the laboratories’ own historical data. b. The positive controls had to demonstrate a mutag enic effect.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 168 April 2008

c. Titer determinations had to demonstrate sufficie nt bacterial density in the suspension.

In order to achieve a positive result, a reproducib le and dose-related increase in mutant counts for a t least one strain must occur. For TA1535, TA 100 and TA 98, the increase should be at least 2-fold that of the negative controls. For TA1537, there should be a 3-fold increase. For TA102, the increase shoul d be at least 150 colonies over that counted for the negative control.

II. REPORTED RESULTS:

Findings: Doses up to and including 158 ug per plate did not cause any bacteriotoxic effects. Total bacteria cou nts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect. Due to the observed bacteriotoxicity, this range could be used up to 500 ug/plate, and partly also up to and inclu ding 1581 ug per plate for assessment purposes. Evidence of mutagenic activity of BYI 08330 was not seen (Table 6-50). No biologically relevant increase in the mutant count, in comparison with th e negative controls, was observed. The positive controls had a marked mutagenic effect.

Table B.6-50: Summary of mean number of mutant colo nies/plate

Summary of mean values without S9 mix

Strain Table and Group TA 1535 TA 100 TA 1537 TA 98 TA 102

1-5 μg/Plate

0 14 105 7 14 171

16 19 122 6 13 162

50 16 96 6 15 171

158 21 111 8 14 168

500 15 93 6 12 183

1581 15 77 3 9 128

5000 7 54 - 6 -

Na-azide 592

NF 242

4-NPDA 78 147

MMC 268

6-10 μg/Tube

0 17 130 7 20 136

16 18 117 7 23 123

50 17 119 9 23 121

158 11 124 6 18 152

500 9 108 2 17 114

1581 4 85 - 8 65

5000 - 73 - 5 -– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 169 April 2008

Na-azide 790

NF 368

4-NPDA 144 131

Cumene 362

Summary of mean values with S9 mix

1-5 μg/Plate

0 10 126 7 34 183

16 6 103 8 24 177

50 7 92 5 25 175

158 6 121 6 23 169

500 6 104 7 20 166

1581 6 90 5 11 145

5000 4 59 - 8 88

2-AA 59 1500 85 1040 663

6-10 μg/Tube

0 8 129 9 25 136

16 10 132 7 27 146

50 8 116 8 35 141

158 8 141 7 34 126

500 7 124 5 28 136

1581 4 93 3 17 101

5000 - 100 - 9 100

2-AA 67 1367 352 1024 420

III. DISCUSSION AND CONCLUSIONS :

A. CONCLUSIONS :

BYI 08330 was considered to be non-mutagenic withou t and with metabolic activation under the conditions of the Salmonella/microsome test.

B. REVIEWERS COMMENTS:

The reviewer agrees with the investigators’ conclus ions. BYI 08330 was tested to concentrations limit ed by cytotoxicity, proper experimental protocol was f ollowed, and solvent and positive control values we re appropriate. The number of revertants per plate wa s not increased over solvent control values at any experimental point in this study.

This study appears to be a repeat of a study perfor med in 2002 (Report No. AT00056). Results in both studies are similar and support one another.

C. STUDY DEFICIENCIES:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 170 April 2008

No study deficiencies were identified.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 171 April 2008

B.6.4.1.2 Salmonella Mammalian Activation Gene Mutation

CITATION : Herbold, B. 2002. BYI 08330. Salmonella /Microsome Test Plete Incorporation and Preincubation Method. Study No. T5071214. Report N o.AT00056, Document No. M065358-0102. Bayer HealthCare, PH-PD p Health Care Toxicology, Rodents and Genotoxicity, Friedrich-Ebert-Strasse 217-333, D-42 096 Wuppertal, Germany. Unpublished Study.

I. MATERIAL AND METHODS

A. MATERIALS :

1. Test Material: BYI 08330 Description: technical grade, white powder Lot/Batch No.: NLL6425-9 Purity (%ai): 96.5% (11-Dec-01); 93.5% (21-Jun-02) Compound Stability room temperature storage Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. of TGAI: 203313-25-1
Solvent Used: DMSO

2. Control Materials : Negative: N/A Solvent (final conc’n): DMSO, (0.1 ml/plate)

Positive: Nonactivation: Sodium azide Nitrofurantoin 4-Nitro-1, 2- phenylene diamine Mitomycin C 4-Nitro-1, 2- phenylene diamine Cumene hydroperoxide 10 μg/plate TA1535 0.2 μg/plate TA 100 0.5 μg/plate TA 98 0.2 μg/plate TA 102 10 μg/plate TA1537 50 μg/plate TA 102 Activation: 2-Aminoanthracene 3 μg/plate, all str ains

3. Activation: S9 derived from x Induced x Aroclor 1254 x Rat x Liver Non-induced Phenobarbitol Mouse Lung None Hamster Other Other Other

Describe S9 mix composition: Cofactor solution: MgCl 2 x 6 H 2 O : 162.6 mg 70 mls, S9 fraction, 10 ml, KCl 0.15 M, 20 ml KCl: 246.0 mg Glucose-6-phospate, disodium salt: 179.1 mg NADP, disodium salt: 315.0 mg Phosphate buffer: 100.0 Mm– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 172 April 2008

4. Test organisms: S. typhimurium strains TA97 x TA98 x TA100 x TA102 TA104 x TA1535 x TA1537 TA1538 list any others

Properly maintained? x Yes No Checked for appropriate genetic markers ( rfa mutation, R factor)? x Yes No

5. Test compound concentrations used :

Nonactivated conditions : 0, 16, 50, 158, 500, 1581, 5000 ug/plate, all str ains, 3 replicates/treatment, 2 trials Activated conditions : 0, 16, 50, 158, 500, 1581, 5000 ug/plate, all str ains, 3 replicates/treatment, 2 trials

B. TEST PERFORMANCE

1. Type of Salmonella assay:

x standard plate test

x pre-incubation (20 minutes) "Prival" modification (i.e. azo-reduction method) spot test other

2. Protocol : In the first trial, a strict plate incorporation me thod was employed (Ames et al. (1973) Proc. Nat. Acad. Sci. 70: 2281-2285, Ames et al. (1975) Mutation Res. 31: 347-364). For the mutant colony count, each treatment level was plated in triplicat e. The negative control consisted of the solvent ab sent the test material. In the plate incorporation proce dure, 0.1 ml of the solvent with or w/o the test ma terial, 0.1 ml of the bacterial inoculum, 0.5 ml of the S9 fraction or buffer, and 2.0 ml of soft agar were wa rmed
to 45 o C in a water bath for 30 seconds, mixed and poured onto a Petri dish. The incubation proceeded for 48 hours at 37 °C. The number of mutant colonie s was then recorded.

In the second trial, 0.1 ml of the solvent with or w/o the test material, 0.1 ml of the bacterial inoc ulum, and 0.5 ml of the S9 fraction or buffer were pre-in cubated for 20 minutes at 37 °C, the agar was added and mixed, followed by 48 hours of incubation at 37 °C. Likewise, the number of mutant colonies was recorded. The cytotoxicity of the test material was assessed in three ways. 1). An appraisal of the background growth on the incubation plates was asse ssed. 2). If there was a marked and dosedependent reduction on the mutant count, that was c onsidered to be a measure of toxicity. 3). Total bacterial counts were taken by incubating two plate s per treatment level in the presence of histidine and incubating for 48 hours at 37 °C. A reduction in th e titer in comparison to the negative control was considered to be an indication of toxicity.

3. Statistical Analysis : No statistical analysis was performed on the study data.

4. Evaluation Criteria :

Acceptance of an assay required that:

a The negative controls had to be within the expect ed range as defined by published data or the laboratories’ own historical data. b The positive controls had to demonstrate a mutag enic effect. c Titer determinations had to demonstrate sufficien t bacterial density in the suspension.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 173 April 2008

In order to achieve a positive result, a reproducib le and dose-related increase in mutant counts for a t least one strain must occur. For TA1535, TA 100 and TA 98, the increase should be at least 2-fold that of the negative controls. For TA1537, there should be a 3-fold increase. For TA102, the increase shoul d be at least 150 colonies over that counted for the negative control. In the 1st trial, the positive c ontrols for both with and w/o activation in the TA102 assay did not fulfill this requirement. Therefore, the a ssay was performed again using this strain only. The dat a in the results section reflect the outcome of thi s assay.

II. REPORTED RESULTS:

Findings: Doses up to and including 158 μg per plate did not cause any bacteriotoxic effects. Total bacteria co unts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so th at this range could only be used up to and includin g 1,581 μg per plate for assessment purposes. Evidence of m utagenic activity of BYI 08330 was not seen (Table 6-51). No biologically relevant increase in the mu tant count, in comparison with the negative control s, was observed. The positive controls had a marked mu tagenic effect.

Table B.6-51: Summary of mean number of mutant colo nies/plate (Data taken from Study No. T5071214, pp. 26-27)

Summary of mean values without S9 mix

Strain Table and Group TA 1535 TA 100 TA 1537 TA 98 TA 102

1-5 μg/Plate

0 10 122 5 30 213

16 9 143 5 29 225

50 9 136 5 27 245

158 6 146 6 30 237

500 8 120 7 36 189

1581 6 123 4 24 151

5000 4 100 3 19 111

Na-azide 721

NF 330

4-NPDA 105 161

MMC 570

6-10 μg/Tube

0 11 141 7 42 259

16 9 133 6 36 272

50 8 118 7 50 261

158 9 116 6 38 242

500 9 110 5 22 214

1581 6 96 - 20 141– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 174 April 2008

5000 3 91 - 12 104

Na-azide 800

NF 457

4-NPDA 139 191

Cumene 455

Summary of mean values with S9 mix

1-5 μg/Plate

0 10 171 7 39 276

16 10 157 7 43 287

50 11 165 8 44 290

158 14 159 6 32 282

500 7 166 7 33 280

1581 7 147 4 28 234

5000 3 125 4 20 193

2-AA 207 1236 355 1004 540

6-10 μg/Tube

0 10 151 7 55 272

16 11 149 8 49 186

50 12 150 7 54 222

158 11 163 8 45 205

500 8 158 7 46 267

1581 9 104 5 28 252

5000 3 85 2 22 185

2-AA 181 1252 376 999 543– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 175 April 2008

CONCLUSIONS:

BYI 08330 was considered to be non-mutagenic withou t and with metabolic activation under the conditions of the Salmonella/microsome test.

B. REVIEWERS COMMENTS:

The reviewer agrees with the investigators’ conclus ions. BYI 08330 was tested to concentrations limit ed by cytotoxicity, proper experimental protocol was f ollowed, and solvent and positive control values we re appropriate. The number of revertants per plate wa s not increased over solvent control values at any experimental point in this study.

C. STUDY DEFICIENCIES:

No study deficiencies were identified.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 176 April 2008

B.6.4.1.3 In vitro genotoxicity - Test for clastogenicity in mammalia n cells (OECD Annex IIA 5.4.2.1)

In vitro CHROMOSOMAL ABERRATION TEST

CITATION : Herbold, B. (2002) In vitro Chromosome aberration test with Chinese hamster V79 cells.

Rodents and Genotoxicity Unit of Health Care Toxico logy, Bayer HealthCare, Bayer AG,Friedrich-Ebert-Strasse 217-333, D-42096 Wuppert al, F.R.G. Laboratory Report No.: AT00055, Bayer Study No.: T 6071477, Document No.: M-065342-01-02, October 24, 2002. Unpublished

I. MATERIAL AND METHODS

A. MATERIALS :

1. Test Material : BYI 08330 Description: technical grade, white powder Lot/Batch No.: NLL 6425-9 Purity (%ai): 96.5% (11-Dec-01); 93.5% (21-Jun-02) Compound Stability room temperature storage Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. of TGAI: 203313-25-1
Solvent Used: DMSO

2. Control Materials : Negative: Culture Medium Solvent (final conc’n): DMSO (0.2 ml solvent / cul ture) Positive: Nonactivation: Mitomycin C (0.1 μg/ml m edium) Activation: Cyclophosphamide (2 μg/ml medium)

3. Activation: S9 derived from male Sprague-Dawley rats:

x induced x Aroclor 1254 x Rat x Liver non-induced Phenobarbitol Mouse Lung None Hamster Other Other Other

Describe S9 mix composition: For use, frozen aliquots of the S9 fraction (24.9 m g protein/mL) were slowly thawed and mixed with a cofactor solution (4:6). The S9 mix contained 40% S 9 fraction and was kept on ice and used on the same day.

Cofactor solution per 100 ml S9 mix: Sodium phosphate buffer (100 mM; pH 7.4) 60.0 ml MgCl 2 x 6 H 2 O 162.6 mg KCl 246.0 mg Glucose-6-phosphate (disodium salt) 152.0 mg– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 177 April 2008

NADP (disodium salt) 315.2 mg

4. Test cells: mammalian cells in culture

V79 cells were obtained from Dr. Utesch, Merck AG, Darmstadt, Germany
Media: Eagle’s minimal essential media (MEM) supple mented with nonessential amino acids, Lglutamine (2 mM), MEM-vitamins, NaHCO 3 -solution (final concentration 0.225%), penicillin (50 units/mL), streptomycin (50 μg/mL) and heat-inactiv ated fetal calf serum. Properly maintained? x Yes No Periodically checked for Mycoplasma contamination? x Yes No Periodically checked for karyotype stability? x Yes No

5. Test compound concentrations used :

Nonactivated conditions: Cytotoxicity: 0 (solvent control), 1, 5, 10, 50, 10 0, 250, 500, 750 μg/ml Main study: (4 hrs. treatment time) 0 (negative), 0 (solvent control), 10, 20, 30, 50, 70 μg/ml; (18 hrs. treatment time) 0 ( negative), 0 (solvent control), 6, 12, 24, 36, 48 μg/ml Activated conditions: Cytotoxicity: 0 (solvent control), 1, 5, 10, 50, 10 0, 250, 500, 750 μg/ml Main study: (4 hrs. treatment time) 0 (negative), 0 (solvent control), 20, 40, 60, 80, 100 μg/ml

B. TEST PERFORMANCE :

1. Preliminary Cytotoxicity Assay :

One x 10 6 cells were seeded in 20 ml of medium on the day pr ior to treatment. Immediately prior to treatment, this medium was removed from the culture and replaced with 20 mL of MEM plus 2% FCS and 0.2 mL of test material solution (non-activatio n) or 19 mL of MEM plus 2% FCS, 1 ml of S9 mix and 0.2 ml of test material. After 4 hours of incubati on at 37 o C, the medium was removed and the cells washed once with pre-warmed phosphate buffered solu tion. After the addition of MEM plus 10% FCS, the cells were incubated for an additional 20 hours . Cultures were also exposed to the test material for 18 hours without S9-mix and harvested immediately f ollowing treatment. Two hours prior to the cell harvest, 0.2 ml of Colcemid solution (40 μg/ml) was added to each culture. The cells were thereby arrested in the metaphase of mitosis. Duplicate cu ltures of each treatment level were performed. The percentage of mitotic indices and the percentage of cell survival were used as criteria to determine cytotoxicity.

2. Cytogenetic Assay :

a. Cell exposure time : Test Material Solvent Control Positive Control
Non-activated: Activated: 4 h and 18 h
4 h 4 h and 18 h

4 h 4h and 18 h

4h

b. Spindle inhibition Inhibition used/concentration: Colcemid, 40 μg/ml

Administration time: 2 hours (before cell harvest)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 178 April 2008

c. Cell harvest time after termination of
treatment: Test Material Solvent Control Positive Control

Non-activated: Activated: 0, 14 and 26 h 14 and 26 h 0, 14 and 26 h

14 and 26 h 0, 14 and 26 h

14 and 26 h

d. Details of slide preparation :

The medium was removed from each flask and cells we re removed from the bottom of the flask by trypsinization and suspended in medium. This medium was transferred to a centrifuge tube and spun for approximately 5 minutes at 700 rpm. The superna tant was carefully removed. One to 2 mL of a hypotonic solution (0.4% KCl; 37°C) was added to th e tube. Within 4 minutes, the volume was brought to 6 ml with additional hypotonic solution and cells were resuspended. The cells were sedimented in the centrifuge as before and the supe rnatant was removed. A few drops of cold (4°C) fixative [ethanol/acetic acid (3:1)] were added and mixed carefully with the cells. The volume was adjusted to 6 mL with the fixative and mixed again with the cells. The mixture was incubated at room temperature for 20 minutes. Cells were pelleted as before and the supernatant was discarded. Cells were again resuspended in fixative as before and ce ntrifuged. Pelleted cells were resuspended carefully in a small volume of fresh fixative. This suspension was dropped onto clean slides which had been frozen at -20°C previously. The slides we re allowed to dry for at least 2 hours. Thereafter, they were submerged in pure methanol for 3 minutes and stained for 20-30 minutes in 5% Giemsa solution. Slides were rinsed twice in water and on ce in acetone and were then kept in xylene for about 30 minutes. The slides were allowed to dry co mpletely and covered. At least two slides were generated per culture. All solutions used during t his preparation were freshly prepared each time. The Giemsa solution was filtered before usage.

e. Metaphase analysis:

No. of cells examined per dose: Scored for structural? x Yes No Scored for numerical? x Yes, polyploidy metaphases were recorded No

Coded prior to analysis? x Yes No

f. Evaluation criteria: Coded slides were evaluated using a light microscop e at a magnification of about 630. The mitotic index was determined by counting 1000 cells per cul ture. The numbers of mitotic and non-mitotic cells were noted. Duplicate cultures were processed and examined. Coded slides were evaluated using a light microscope at a magnification of abou t 1000. Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from e ach of two parallel cultures, were examined. In most cases at least 100 assessable metaphases we re present on one slide prepared from an individual culture. Therefore, the back-up slide w hich was generated routinely from every culture was normally not utilized for the evaluation. Howev er, in cases when fewer than 100 assessable metaphases were found on the first slide of a cultu re, the back-up slides were evaluated as well until a total of 100 metaphases were reached.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 179 April 2008

Only metaphases containing the modal chromosome num ber (22) were analyzed unless exchanges were detected. In this case, metaphases were evalua ted even if the chromosome number was not equal to 22. The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rieger and Michaelis (1967). Both chromatid and chromosome-type aberrations were assessed. Chromatid-type aberratio ns are clastogenic effects restricted to one of the two corresponding chromatids. Chromosome type a berrations are defined as changes expressed in both corresponding sister chromatids at the same locus. The distinction between chromatid and chromosome type aberrations was not made for exchan ges. The different classes of aberrations were characterized as follows: 1) Gap: A gap is an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) and sma ller than the width of one chromatid. Gaps are found on one chromatid ("gap") or on both chromatid s at apparently identical sites ("isogap"). The biological relevance of gaps of both types is uncle ar. 2) Break: a break is defined as a discontinuit y of one chromatid ("break") or both chromatids, at a pparently the same locus ("isobreak"), with dislocation of the chromatid ends. The dislocated chromatid end(s) has (have) to be present within the respective metaphase. In addition, an achromati c lesion within a chromatid arm without obvious dislocation of the chromatid end(s) but larger than the width of one chromatid is also defined as break or as isobreak, if this occurs in parallel on both chromatids of a chromosome. 3) Fragment: Fragments are parts of chromosomes without centrome r. A fragment is the result of a break. The corresponding defective chromosome is not detectabl e among the chromosomes of the same metaphase. Fragments can be derived from one chroma tid ("fragment") or from both corresponding chromatid regions of a chromosome ("isofragment"). 4) Deletion: A deletion occurs as the result of a break. In case of a deletion, one chromatid ("delet ion") or both corresponding terminal chromatid parts of a chromosome ("isodeletion") are missing w ithin the metaphase under assessment. 5) Exchange: This is an exchange of chromatid-parts b etween different chromosomes (interchange) or within the same chromosome (intrachange). 6) Multi ple aberration: A cell was assessed as to contain "multiple aberrations" when five or more st ructural changes (excluding gaps) occur within one metaphase. In addition to these aberrations, m etaphases showing chromosome disintegration as an indication of a cytotoxic effect were also re corded if they were observed. They were counted separately and were not included among the cells th at were assessed for aberrations. "Chromosome disintegration" was recorded if fewer than half of the chromosomes reveal characteristic structural features within a given metaphase.

g. Statistical analysis :

The statistical analysis was performed by pair-wise comparison of BYI 08330-treated and positive control groups to the respective solvent control gr oup. The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of the corresponding solvent control) using the one-sided chi 2 -test. The numbers of metaphases with aberrations ( including and excluding gaps) and of metaphases with exchanges were compared (pro vided that these data superceded the respective solvent control). Fisher's exact test w as used for the statistical evaluation. A differen ce was considered to be significant, if P<0.05.

II. RESULTS AND DISCUSSIONS:

Findings :

Test material concentrations up to 750 μg/mL did no t change the pH or osmolality of the treatment medium. In the cytogenetic assay without S9 mix, c ytotoxic effects were observed at 50 ug/ml and above after 4 hours treatment and at 24 ug/ml and a bove after 18 hours treatment. With the S9 mix,– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 180 April 2008

cytotoxic effects were observed at 40 ug/ml and abo ve. Therefore, higher concentrations of test mater ial were not evaluated for chromosomal aberration induc tion due to excess cytotoxicity. With one exceptio n the cultures treated with BYI 08330 in the absence and in the presence of S9 mix showed, for the highest concentrations scored [4 hrs: 50 μg/mL (-S9 ), 80 μg/mL (+S9); 18 hrs (-S9): 48 μg/mL], statistically significant but only weakly increased numbers of aberrant metaphases. The vehicle and positive control values were appropriate. Results of the cytogenetic assay using a four-hour exposure are given in Tables 6-52-55 and results using an 18 -hour exposure are shown in Tables 6-56-57.– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism181April 2008Table B.6-52: Chromosomal Aberrations without metabolic Activation 4 Hours TreatmentClasses of aberrationsGapsChromatidtypeChromosometypeotherMetaphases of aberrations(%)ExperimentalGroup andConcentrationin μg/mlHarvestTimeinHoursCultureNumberCellsscoredg i b f d ib if id exmaEmacdIncl.gapsexcl.gapsexchangesDMSO 18 36 100 0 0 0 0 0 0 0 2 0 0 0 0 2.0 2.0 0.00 21 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0200 0 0 0 0 0 0 0 3 0 0 0 0 1.5 1.5 0.0BYI08330 18 25 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.00.010 23 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0200 0 0 0 0 0 0 0 2 0 0 0 0 1.0 1.0 0.0BYI08330 18 32 100 0 0 0 0 0 0 0 5 0 0 0 0 5.0 5.00.030 18 100 0 0 0 1 0 0 0 3 0 0 0 0 3.0 3.0 0.0200 0 0 0 1 0 0 0 8 0 0 0 0 4.0 4.0 0.0BYI08330 18 16 100 0 0 3 0 1 0 0 11 0 0 0 0 12.0 12.0 0.050 42 100 0 0 0 0 0 1 0 4 0 0 0 0 4.0 4.0 0.0200 0 0 3 0 1 1 0 15 0 0 0 0 8.0** 8.0** 0.0Mitomycin C 18 30 100 2 1 6 0 1 10 3 2 18 0 0 0 36.0 33.0 16.00.1 44 100 0 0 10 0 0 16 3 4 15 0 0 0 36.0 36.0 13.0200 2 1 16 0 1 26 6 6 33 0 0 0 36.0** 34.5** 14.5*** p< 0.05** p < 0.01Copied from Lab Report No. AT00055, page 44 (originally called Table 3 in study).g = gap; ig = isogap; b = break; ib = iosbreak; f =fragment; if = isofragment; d = deletion; id = isodeletion; ex = exchange; ma = multiple aberrations;maE = multiple aberrationswith exchange; cd = chromosome disintegration– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism182April 2008Table B.6-53: Chromosomal Aberrations with Metabolic Activation 4 Hours TreatmentClasses of aberrationsGapsChromatidtypeChromosometypeotherMetaphases of aberrations(%)ExperimentalGroup andConcentrationin μg/mlHarvestTimeinHoursCultureNumberCellsscoredg i b f d ib if id exmaEmacdIncl.gapsexcl.gapsexchangesDMSO 18 43 100 0 0 0 0 0 0 0 2 0 0 0 0 2.0 2.0 0.00 37 100 0 0 0 0 0 1 0 2 0 0 0 0 3.0 3.0 0.0200 0 0 0 0 0 1 0 4 0 0 0 0 2.5 2.5 0.0BYI08330 18 39 100 0 0 0 0 0 0 0 2 0 0 0 0 2.0 21.00.020 29 100 1 0 0 0 0 0 1 2 1 0 0 0 4.0 3.0 1.0200 1 0 0 0 0 0 1 4 1 0 0 0 3.0 2.5 0.5BYI08330 18 33 100 0 0 1 0 0 0 0 1 0 0 0 0 2.0 2.00.040 8 100 1 0 0 1 0 3 0 3 2 0 0 0 8.0 8.0 2.0200 1 0 1 1 0 3 0 4 2 0 0 0 5.0 5.0 1.0BYI08330 18 40 100 0 0 0 0 0 1 0 3 1 0 0 0 5.0 5.01.080 12 100 0 0 0 1 0 1 1 8 1 0 0 0 10.0 10.0 1.0200 0 0 0 1 0 2 1 11 2 0 0 0 7.5* 7.5* 1.0Cyclophosphamide18 9 100 1 1 3 1 0 22 4 4 18 0 0 0 38.0 37.0 15.02 26 100 0 2 5 1 0 21 1 2 14 0 0 0 36.0 35.0 13.0200 1 3 8 2 0 43 5 6 32 0 0 0 37.0** 36.0** 14.0*** p< 0.05** p < 0.01Copied from Lab Report No. AT00055, page 44 (originally called Table 4 in study).– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism183April 2008g = gap; ig = isogap; b = break; ib = iosbreak; f =fragment; if = isofragment; d = deletion; id = isodeletion; ex = exchange; ma = multiple aberrations;maE =multiple aberrations with exchange; cd = chromosomedisintegration– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism184April 2008Table B.6-54: Chromosomal Aberrations without and with metabolic Activation 4 Hours TreatmentClasses of aberrationsGapsChromatidtypeChromosometypeotherMetaphases of aberrations(%)ExperimentalGroup andConcentrationin μg/mlHarvestTimeinHoursCultureNumberCellsscoredg i b f d ib if id exmaEmacdIncl.gapsexcl.gapsexchangesWithout metabolic activationDMSO 30 22 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.00 27 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0200 0 0 0 0 0 0 0 2 0 0 0 0 1.0 1.0 0.0BYI08330 30 5 100 0 0 0 0 0 1 0 1 0 0 0 0 2.0 2.0 0.050 28 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0200 0 0 0 0 0 1 0 2 0 0 0 0 1.5 1.5 0.0With metabolic activationDMSO 30 11 100 0 0 0 0 0 0 1 2 0 0 0 0 3.0 3.0 0.00 20 100 0 0 0 0 0 0 0 0 0 0 0 0 0.0 0.0 0.0200 0 0 0 0 0 0 1 2 0 0 0 0 1.5 1.5 0.0BYI08330 30 17 100 0 0 0 0 0 0 0 5 0 0 0 0 5.0 5.00.080 38 100 0 0 0 0 0 1 1 5 0 0 0 0 6.0 6.0 0.0200 0 0 0 0 0 1 1 10 0 0 0 0 5.5* 5.5* 0.0* p< 0.05** p < 0.01Copied from Lab Report No. AT00055, page 45 (originally called Table 5 in study).g = gap; ig = isogap; b = break; ib = iosbreak; f =fragment; if = isofragment; d = deletion; id = isodeletion; ex = exchange; ma = multiple aberrations;maE = multiple aberrationswith exchange; cd = chromosome disintegration– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism185April 2008Table B.6-55: Additionally observed Polyploid Metaphases 4 Hours Treatmentwithout metabolic activationwith metabolic activationExperimental Groupand Concentrationin μg/mlHarvestTime inHoursCultureNumberPolyploidMetaphasesExperimentalGroup andConcentration inμg/mlHarvestTime inHoursCultureNumberPolyploidMetaphasesDMSO 18 36 13 DMSO 18 43 150 21 19 0 37 14BYI 08330 18 25 14 BYI 08330 18 39 1010 23 11 20 29 12BYI 08330 18 32 15 BYI 08330 18 33 1530 18 18 40 8 11BYI 08330 18 16 12 BYI 08330 18 40 550 42 18 80 12 12Mitomycin C 18 30 7 Cyclophosphamide 18 9 100.1 44 3 2 26 4DMSO 30 22 13 DMSO 30 11 90 27 14 0 20 10BYI 08330 30 5 16 BYI 08330 30 17 1350 28 8 80 38 3Copied from Lab Report No. AT00055, page 45 (originally called Table 6 in study).– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism186April 2008Table B.6-56: Chromosomal Aberrations without metabolic Activation 18 Hours TreatmentClasses of aberrationsGapsChromatidtypeChromosometypeotherMetaphases of aberrations(%)ExperimentalGroup andConcentrationin μg/mlHarvestTime inHoursCultureNumberCellsscoredg i b f d ib if id exmaEmacdIncl.gapsexcl.gapsexchangesDMSO 18 46 100 0 0 0 0 0 0 0 2 1 0 0 0 3.0 3.0 1.00 47 100 0 0 0 1 0 0 0 1 0 0 0 0 1.0 1.0 0.0200 0 0 0 1 0 0 0 3 1 0 0 0 2.0 2.0 0.5BYI08330 18 49 100 1 0 2 0 0 1 0 3 3 0 0 0 8.0 7.03.012 58 100 0 0 1 0 0 0 1 2 1 0 0 0 3.0 3.0 1.0200 1 0 3 0 0 1 1 5 4 0 0 0 5.5 5.0 2.0BYI08330 18 55 100 0 0 0 0 0 3 1 3 1 0 0 0 7.0 7.01.024 50 100 0 0 0 2 0 0 1 2 2 0 0 0 4.0 4.0 2.0200 0 0 0 2 0 3 2 5 3 0 0 0 5.5 5.5 1.5BYI08330 18 45 100 0 0 0 0 0 2 0 4 2 0 0 0 8.0 8.02.048 56 100 0 0 0 0 0 4 0 3 0 0 0 0 6.0 6.0 0.0200 0 0 0 0 0 6 0 7 2 0 0 0 7.0* 7.0* 1.0Mitomycin C 18 57 100 1 0 7 0 0 8 1 1 13 1 0 0 25.025.0 12.00.03 53 100 1 1 6 0 0 11 3 4 10 1 0 0 29.0 27.0 10.0200 2 1 13 0 0 19 4 5 23 2 0 0 27.0** 26.0** 11.0*** p< 0.05** p < 0.01Copied from Lab Report No. AT00055, page 47 (originally called Table 9 in study).g = gap; ig = isogap; b = break; ib = iosbreak; f =fragment; if = isofragment; d = deletion; id = isodeletion; ex = exchange; ma = multiple aberrations;maE = multiple aberrationswith exchange; cd = chromosome disintegration– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 187 April 2008

Table B.6-57: Additionally observed Polyploid Metap hases 18 Hours Treatement

without metabolic activation

Experimental Group and Concentration in μg/ml Harvest Time in
Hours Culture

Number Polyploid Metaphases

DMSO 18 46 25

0 47 11

BYI 08330 18 49 16

12 58 13

BYI 08330 18 55 23

24 50 21

BYI 08330 18 45 9

48 56 15
Mitomycin C 18 57 16

0.03 53 14

Copied from Lab Report No. AT00055, page 47 (origin ally called Table 10 in study)

III. CONCLUSION :

BYI 08330 is considered to be weakly positive in th is cytogenetic in vitro study at cytotoxic concentrations only.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 188 April 2008

B.6.4.2 Mammalian Chromosome Aberration Test (In Cu ltured Chinese Hamster Cells) (OECD

Annex IIA 5.4.4.2)

CITATION : Herbold, B. (2003) BYI 08330 cytogenetic screening with Chinese hamster V79 cells. Bayer

HealthCare, PH-PD P Health Care Toxicology, Molecul ar and Genetic Toxicology, FriedrichEbert-Straße 217-333, D-42096 Wuppertal, Germany. Study No. T 5072033. Report No. AT00194. January 13, 2003. Unpublished.

I. MATERIALS AND METHODS:

A. MATERIALS :

1. Test material : BYI 08330 Description: A fine white powder
Lot/Batch #: NLL 6425-14-a Purity: 98.6% a.i.
CAS # of TGAI: 203313-25-1
Structure:

Solvent Used: Dimethyl sulfoxide (DMSO)

2. Control materials : Negative control: None

Solvent control (final conc’n): DMSO [1% (v/v)]

Positive control: Nonactivation: None

Activation: None

3. Activation : S9 derived from

x Induced x Aroclor 1254 x Rat x Liver Non induced Phenobarbitol Mouse Lung
None Hamster Other
Other Other

Describe S9 mix composition: The S9 fraction was isolated in house using male Sp rague Dawley rats. The S9 fraction was derived from the preparation dated May 13, 2002, which had a protein content of 23.1 mg/mL. Frozen aliquots of the S9 fraction were slowly thawed and mixed wit h the cofactor solution at a ratio of 4:6. The S9 m ix thus contained 40% S9 fraction and was kept on ice and used on the same day.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 189 April 2008

Cofactor solution per 100 mL of S9 mix: Sodium phosphate buffer (100 mM; pH 7.4): 60.0 mL MgCl 2 x 6H 2 O: 162.6 mg KCl: 246.0 mg Glucose-6-phosphate (disodium salt): 152.0 mg NADP (disodium salt): 315.2 mg

4. Test cells : mammalian cells in culture

V79 cells (Chinese hamster lung fibroblasts): obta ined from Dr. Utesch, Merck AG, Darmstadt, Germany

Media: Eagle’s minimal essential medium (MEM) with the following supplements: nonessential amino acids, L-glutamine at 2 mM, MEM-vitamens, NaHCO 3 solution at a final concentration of 0.225%, penicillin at 50 units/mL, streptomycin at 50 μg/mL , and heat-inactivated fetal calf serum (FCS). Unless otherwise noted, cells were grown in medium that contained 10% fetal calf serum. The medium used during the treatment contained 2% FCS. Culturing was done in a CO 2 incubator in which the temperature was 37°C and the CO 2 concentration was set at 5%.

Properly maintained? x Yes No Periodically checked for Mycoplasma contamination? x Yes No

Periodically checked for karyotype stability? x Ye s No

5. Test compound concentrations used : Nonactivated conditions: Preliminary cytotoxicity assay: None Main assay: 0, 30, 50, 70, 90, 110 μg/mL, but chrom osomal aberrations were only evaluated at 70 μg/mL Activated conditions: Preliminary cytotoxicity ass ay: None Main assay: 0, 40, 60, 80, 100, 120 μg/ml, but chro mosomal aberrations were only evaluated at 120 μg/mL

B. TEST PERFORMANCE:

1. Preliminary cytotoxicity assay : There was no preliminary cytotoxicity assay.

2. Cytogenetic assay :

a. Cell exposure time : Test material Solvent control Positive control
Non-activated: Activated: 4 h
4 h 4 h

4 h 4 h

4 h

b. Spindle inhibition:
Inhibition Colcemid / 40 μ g/mL– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 190 April 2008

used/concentration: Administration time: 2 hours (before cell harvest )

c. Cell harvest time after
treatment: Test material Solvent control Positive control

Non-activated: Activated: 14 h
14 h 14 h

14 h No positive control

No positive control

d. Details of slide preparation : Cells were removed from each flask by trypsinizati on, suspended in medium, and centrifuged. The resultant pellet was resuspended in a hypotonic solution (0.4% KCl) and recentrifuged. The supernatant was decanted an d a cold ethanol/acetic acid (3:1) fixative was added and mixed with the cells. After remaining at room temperature for 20 minutes, the cells were centrifuged again and the supernatant discarded. A fter an additional treatment with the fixative and another centrifugation, the pelleted cells were res uspended in a small volume of fresh fixative. The suspended cells were then dropped onto slides that had been pre-cooled to -20 o C. The slides were air-dried for at least 2 hours, treated with pure m ethanol for 3 minutes and stained with a 5% Giemsa solution for 20 to 30 minutes. The slides were the n rinsed with water twice, once with acetone, immersed in xylene for about 30 minutes and finally dried and covered. At least two slides were made for each culture.

e. Metaphase analysis:

No. of cells examined per dose: 1000
Scored for structural? X Yes No
Scored for numerical? X Yes Polyploid cells were identified. No Coded prior to analysis? X Yes No

f. Evaluation criteria: Slides were evaluated using a light microscope at m agnifications of approximately 630x and 1000x for the mitotic index and for examination for aberrations, respectively. Duplicate cultures for each treatment level were ev aluated. One hundred metaphases per culture (i.e., 200 metaphases per treatment level) were sco red for structural aberrations. Only metaphases that contained the modal chromosome number of 22 we re analyzed unless exchanges were present. The terminology of Rieger and Michaelis (1967) was used to define the aberrations. Chromatid-type aberrations are clastogenic effects restricted to o ne of the two corresponding chromatids. Chromosome-type aberrations are defined as changes expressed in both corresponding sister chromatids at the same locus. An increased inciden ce of gaps noted for either a single chromatid or on both chromatids at identical sites (i.e., an iso gap) without a concomitant increase in other aberrations was not considered a clastogenic respon se. A test was acceptable if the aberration frequency of the vehicle control fell within the ra nge of the laboratory’s historical control data or within ranges of values found for controls in the l iterature. A test was considered positive if there was a treatment-related and statistically significa nt increase in the aberration rate. A test was considered negative if there was no such increase a t any time interval. A test was also considered negative if there were statistically significant in creases that fell within the range of the historica l vehicle controls. A test was considered equivocal if a statistically significant increase was found t hat was not biologically significant.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 191 April 2008

g. Statistical analysis : Pair-wise comparisons of test material-treated gro ups with the respective control group were made. The mitotic index values were analyzed for significance by means of the one-sided Chi-squared test, providing they showed a reduction. The numbers of metaphases with aberrations and of metaphases with exchanges were c ompared with the respective vehicle control by using Fisher’s exact test. The probability of e rror was established at less than 5%. The reviewer considers the statistical methods appropriate.

II. REPORTED RESULTS:

There was no analytical testing of test solutions i n order to confirm concentrations or to provide information about the stability of the test substan ce in the media and/or solvent.

A. PRELIMINARY CYTOTOXICITY ASSAY :

There was no preliminary cytotoxicity assay. Conce ntrations were chosen on the basis of the results from an earlier study identified by number T 607147 7, and the reviewer has not seen that study.

B. CYTOGENETIC ASSAY :

Data were collected on cytotoxicity as part of the main study, and Tables 6-58 and 6-59 show the resul ts for the mitotic index and the survival index, respe ctively. Mitotix indices (MI) were mainly comparab le to controls at all concentrations, except at 50 ug/mL without S9 mix where the MI was reduced by 38%. Cytotoxic effects were observed at concentrations > 50 μg/ml with and without S9 mix as indicated by th e decrease in relative survival (22-49% and 21-32%, r espectively). Precipitation of BYI 08330 in the medium was not observed. Based on the above cytoto xicity observations, it was decided to examine cells for cytogenetic effects only at 70 μg/mL of B YI 08330 in the absence of metabolic activation, an d only at 120 μg/mL of BYI 08330 in the presence of m etabolic activation. Those results are shown in Table 6-60. None of the treated cultures showed bi ologically relevant or statistically increased numb ers of aberrant metaphases at the one concentration lev el selected for examination with and without S9 mix

. This was true both including and excluding gaps. B ased on earlier work from a study identified as T 6071477, it was said that concentrations of the tes t substance up to 750 μg/mL did not change the pH o r the osmolality of the medium. There were no concur rent positive controls. Numerous tables were presented showing the laboratory’s historical negat ive, vehicle, and positive controls.

Table B.6-58: Results of mitotic index assay of BYI 08330 in cultured Chinese V79 cells with a harvest time of 18 hours 1

Relative mitotic index (%) in the group with Treatment ( μ g/mL) 4-hour exposure without S9-
mix 4-hour exposure with S9-mix

Solvent (DMSO) 100.0 100.0

30 125.3 -

40 - 144.1
50 61.8** -

60 - 161.0

70 85.9 -

80 - 196.3

90 88.2 -

100 - 202.9– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 192 April 2008

Relative mitotic index (%) in the group with

110 100.0 -

120 - 189.7 Data summarized from Report No. AT00194, Table 1 on page 32

1 Results shown are for 4 hours of treatment followed by 14 hours of recovery, which is the same as that for the cells chosen to be evaluated for chromosomal aberrations; results were also shown in the report for a harves t time of 8 hours, which is not considered sufficiently relevan t to include here. *p < 0.05 **p < 00.01

Table B.6-59: Results of survival index assay of BY I 08330 in cultured Chinese V79 cells with a harvest time of 18 hours 1

Relative survival index (%) in the group with Treatment ( μ g/mL) 4-hour exposure without S9-
mix 4-hour exposure with S9-
mix
Solvent (DMSO) 100.0 100.0

30 92.7 -

40 - 92.9
50 78.4* -

60 - 85.9
70 64.7* -

80 - 79.3*

90 50.9* -

100 - 72.7*
110 56.0* -

120 - 68.2*

Data summarized from Report No. AT00194, Table 2 on page 33

1 Results shown are for 4 hours of treatment followed by 14 hours of recovery, which is the same as that for the cells chosen to be evaluated for chromosomal aberrations; results were also shown in the report for a harves t time of 8 hours, which is not considered sufficiently relevan t to include here.
*Said to be a “relevant” reduction from the control , but no statistics were presented.

Table B.6-60: Results of chromosomal aberration ass ay with a 4-hour treatment period with BYI 08330 in Chinese hamster V79 cells

Number of structural aberrations (200 cells/dose, 100 cells per culture) Mean % of cells containing aberration(s) 2 Treatment ( μ g/mL)

ctb cte csb cse Other 1 Gaps (ctg) - gaps + gaps Polyploid cells (%) 2

Without S9-mix, 14-hour recovery period

Solvent (DMSO) 1 0 0 0 1 2 1.0 2.0 9.5

BYI 08330

70 3 0 0 0 1 3 2.0 3.0 16.0

- There was no positive control. With S9-mix, 14-hour recovery period

Solvent 2 0 0 0 2 1 2.0 2.5 12.5– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 193 April 2008

Number of structural aberrations (200 cells/dose, 100 cells per culture) Mean % of cells containing aberration(s) 2 Treatment ( μ g/mL)

ctb cte csb cse Other 1 Gaps (ctg) - gaps + gaps Polyploid cells (%) 2

(DMSO)

BYI 08330

120 1 1 3 0 0 3 0 2.5 2.5 10.0

- There was no positive control.

Data summarized from Report No. AT00194, Table s 3 and 4 on page 34; polyploidy data were not anal yzed. *Statistically significant (p < 0.05)—noting was st atistically significant

1 Includes severely damaged cells

2 Based on 200 cells examined

3 This exchange could be either a chromatid or a chro mosome exchange; it is impossible to tell from the report.
ctb = chromatid break
csb = chromosome break cte = chromatid exchange cse = chromosome exchange ctg = chromatid gap

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS :

Based on the results of this follow-up screening as say, BYI 08330 has no clastogenic potential in mammalian cells in vitro, and that these results suggest that a cytotoxic im purity was present in the initial batch of BYI 08330 that was tested. Further suppor t for his conclusion was said to be reflected in th e negative in vivo

findings summarized in Reports No. AT00048, AR0007 0, and AT00526. The reviewer has seen only the first one of these reports, and t hat one report does support his view.

B. REVIEWER COMMENTS :

The reviewer does not agree with the investigators' conclusions even though the limited data presented show no evidence of induction of chromosomal aberra tions. Because only one concentration was tested in the presence and in the absence of S9 mix, there is too little information to reach a conclusion. The absence of a concurrent positive control in the par t of the assay that used S9 mix means that there wa s no check of whether of S9 mix was effective. The ab sence of concurrent positive controls means that there was no check of the sensitivity of the assay under the conditions that existed for this particul ar experiment, and the extensive historical control da ta that are provided do not overcome this limitatio n— especially in view of the erratic nature of the res ults regarding the mitotic index. There was no QA statement, and the study was not conducted accordin g to Good Laboratory Practices. In view of the many study deficiencies, this is an unacceptable/guideline study

.

C. STUDY DEFICIENCIES:

Minor study deficiency is as follows: (1) The dose formulations were not analyzed for actual concentrations.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 194 April 2008

Major study deficiencies are as follows: (1) no con current positive controls, (2) only one test substa nce concentration was tested in the presence of S9 mix and in the absence of S9 mix, and there should have been at least 3 concentrations tested for each cond ition, (3) there was no QA statement, and (4) the study was not conducted in conformance with GLP sta ndards. It seems unlikely that anything can be done to overcome these deficiencies.

IV. REFERENCES:

Rieger, R. and A. Michaelis. 1967. Die Chromosomenm utationen. VEB Gustav Fisher Verlag, Jena.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 195 April 2008

B.6.4.2.1 In vitro genotoxicity - Test for gene mutation in mammalian cells (OECD Annex IIA 5.4.3)

CITATION : Herbold, B. (2002) Amended report BYI 08330 V79/H PRT-test in vitro for the detection of induced forward mutations. Bayer HealthCare, PH-PD P Health Care Toxicology, Rodents and Genotoxicity, Friedrich-Ebert-Straße 217-333, D -42096 Wuppertal, Germany. Study No. T 7071478. Report No. AT00137A. January 24, 20 02. Unpublished.

I. MATERIALS AND METHODS

A. MATERIALS :

1. Test material : BYI 08330 Description: Light-beige powder
Lot/Batch #: NLL6425-9 Purity: 96.5% a.i. on December 11, 2001; 93.5% a.i. on June 21, 2002
CAS # of TGAI: 203313-25-1
Structure:

Solvent Used: Dimethyl sulfoxide (DMSO)

2. Control Materials : Negative control: Culture medium
Solvent control (final concentration): DMSO [did not exceed 1% (v/v)]

Positive controls: Nonactivation: Ethyl methanesulf onate (EMS) (900 μg/ml; no solvent required) Activation: Dimethybenzanthracene (DMBA) (20 μg/ml DMSO)

3. Activation : S9 derived from

x Induced x Aroclor 1254 x Rat x Liver

Non-induced Phenobarbitol Mouse Lung

None Hamster Other
Other Other

Describe S9 mix composition: Male Sprague Dawley rats were used to make the S9 fraction. The S9 fraction made up 40% (v/v) of the S9 mix; the ot her 60% was sodium phosphate buffer at pH 7.4 that contained the following components: Final concentrations: MgCl 2 x 6 H 2 0 8 mM KCl 33 mM– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 196 April 2008

Glucose-6-phosphate 5 mM
NADP 1 mM Sodium phosphate 150 mM

4. Test cells : mammalian cells in culture Mouse lymphoma L5178Y cells x V79 cells (Chinese h amster lung fibroblasts)Chinese hamster ovary (CHO) cells—CHOK 1 cells list any others

Media: Described below* No Properly maintained? x Yes No Periodically checked for Mycoplasma contamination? x Yes No

Periodically checked for karyotype stability? x Ye s No
*A hypoxanthine-free Eagle’s Minimal Essential Medi um supplemented with non-essential amino acides, Lglutamine (2mM), MEM-vitamins, NaHCO 3 , penicillin (100 units/mL), streptomycin (100 μg/m L) and 10% heatinactivated fetal calf serum (FCS). This was calle d culture media. During treatments with the test c hemical, the
FCS content was reduced to 2%

5

. Locus examined : HGPRT Thymidine kinase (TK) Hypoxanthine-guaninephosphoribosyl transferase (HGPRT) Na + /K + ATPase

Selection agent: Bromodeoxyuridi ne (BrdU) 8-Azaguanine (8-AG) Ouabain

Fluorodeoxyuridi ne (FdU) x 6-Thioguanine (6-TG)
at 10 μ g/mL Trifluorothymidin e (TFT) Other?

6. Test compound concentrations used : Nonactivated conditions: Preliminary cytotoxicity: 0, 0.1, 1, 10, 50, 100, 150, 300, 600, 1000 μ g/mL Main assay, trial 1: 0, 2.5, 5, 10, 20, 40, 80 μ g/mL Main assay, trials 2 and 3: 0, 20, 30, 40, 50, 60, 70 μ g/mL Activated conditions: Preliminary cytotoxicity: 0 , 0.1, 1, 10, 50, 100, 150, 300, 600, 1000 μ g/mL Main assay, trial 1: 0, 20, 40, 60, 80, 100, 120, 1 40 μ g/mL Main assay, trials 2 and 3: 0, 92, 100, 108, 116, 1 24, 132, 140 μ g/mL

B. TEST PERFORMANCE:

1. Cell treatment :

a. Cells were exposed to test compound, negative/solv ent or positive controls for 5 hours (nonactivated) 5 hours (activated).

b. After washing, cells were cultured for 6 days (expression period) before cell selection. Ce lls were subcultured at the midpoint (i.e., after 3 days) of the expression period.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 197 April 2008

c. After expression, 3x10 5 cells/dish ( 8 dishes/culture) were cultured for 6-8 days in selection medium to determine numbers of mutants and 200 cells/dish ( 3 dishes/culture) were cultured for
6-8 days without selective agent to determine cloning efficiency.

2. Statistical methods : The parameters that were analyzed were survival to treatment (i.e., survival to the time when selective agent was applied), population growth, cloning efficiency, and mutant frequency pe r 10 6 clonable cells. The mutant frequency for each trea tment was expressed as the number of TGresistant mutants per 10 6 clonable cells, and it was calculated by dividing the total number of mutant colonies found by the total number of cells seeded into selection dishes and then correcting for the cloning efficiency of the cells. Mutant frequencies were evaluated using weighted analysis of variance as well as weighted recursive regression, both with Po isson derived weights. All acceptable groups were included in the weighted analysis of variance, whic h was followed by pair-wise comparisons to the vehicle control using the Dunnett test and a nomina l significance level of 0.05. Mutant frequencies based on fewer than 5 plates were not included in t he statistical analysis. Also data were not used fr om cultures with any of the following parameters below 10%: relative survival to treatment, relative population growth, and absolute cloning efficiency. The mutant frequencies obtained for both cultures in a group were treated as independent measurements, t hereby increasing the power of the statistical test s applied. Results for the different trials were comp ared to evaluate consistency. The reviewer conside rs the statistical methods to be appropriate.

i. Evaluation criteria : Estimates of cytotoxicity were based on comparisons with the control to obtain relative survival to treatment, relative population growth, and absolute cloning efficiency. In order for a test to be considered acceptable, the following con ditions must be met: (1) the average cloning effici ency of the negative and vehicle controls should be at l east 50%, (2) the average mutant frequency of the vehicle controls should not exceed 25x10 -6 cells, (3) the mutant frequency of the two culture s of the vehicle and/or the negative control should differ o nly to an acceptable extent (with the difference between them generally not exceeding 5x10 -6 ), (4) the positive control should induce an avera ge mutant frequency of at least three times that of the vehic le control, and (5) the test should be limited by cytotoxicity—with the concentration inducing cytoto xicity of about 80 to 90%—or by insolubility, with precipitation being found in the medium at the high est concentration. The survival at the lowest concentration should be in the range of the negativ e control. A trial is considered positive if a concentration-related increase in the mutant freque ncies occurs that is reproducible in other trials. To be considered biologically significant, the increase i n mutant frequencies must be at least two to three times that of the highest negative or vehicle control val ue observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic. In spite of the above criteria, a positive result will only be considered biologically significant if there was no significa nt change in osmolality compared to the vehicle control. Suc h a change could mean that non-physiological cultur e conditions caused the positive result. A result wi ll be considered equivocal if there is no strictly concentration-related increase in mutant frequencie s but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials. An assay will be considered negative if no reproducible and biologic ally signficant increases of mutant frequencies are observed.

II. REPORTED RESULTS:

There was no analytical testing of the concentratio ns of the dosing solutions. However, the batch tha t was used was examined analytically before the study and was approved for use during the test period. The stability of the test chemical in the vehicle w as tested by comparing the amounts of the test– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 198 April 2008

chemical present to the nominal concentrations of 0 .01 and 100 mg/mL both before and after 24 hours of storage at room temperature. At 0.01 mg/mL, the percentages of the nominal concentrations before and after the storage period were both 90%. At 100 mg/mL, the percentages of the nominal concentrations before and after the storage period were 92% and 90%, respectively. It was concluded that the test chemical is stable in the vehicle at room temperature during a period that covers the ti me period from preparation of the formulation to the l ast treatment in the experiments.

A. PRELIMINARY CYTOTOXICITY ASSAY:

V79 cells were exposed for 5 hours to the solvent a lone and to nine concentrations of the test substan ce ranging from 0.1 to 1000 μ g/mL in the absence and presence of S9-mix. Compari sons of relative cloning efficiencies showed cytotoxicity at 50 μ g/mL and above in the absence of S9-mix and at 150 μ g/mL and above in the presence of S9-mix. Results are summa rized in Table 6-61. Cytotoxicity exceeded 90% when concentrations were equal to or greater than 1 00 and 150 μ g/mL in the absence and presence of S9 mix, respectively. Throughout the concentration range tested, the pH and the osmolality were not affected in any meaningful way. Precipitation of t he test substance was seen at concentrations of 600

μ g/mL and above. Based on these findings, in the mu tagenicity test the test substance was tested at concentrations ranging from 2.5 to 80 μ g/mL in the absence of S9-mix and to concentrations ranging from 20 to 140 μ g/mL in the presence of S9-mix.

Table B.6-61: Cytotoxicity assays of BYI 08330 with and without metabolic activation, based on mean colony numbers in three dishes for each group

Treatment ( μ g/mL) With or without S9
activation Absolute cloning efficiency Relative cloning efficiency (%)
Solvent (DMSO) -S9 59.2 100.0

BYI 08330
0.1 -S9 84.7 143.1
1 -S9 114.7 193.8
10 -S9 151.3 255.8
50 -S9 19.2 32.4
100 -S9 1.5 2.5
150 -S9 0.0 0.0
300 -S9 0.0 0.0

600 a -S9 0.0 0.0

1000 a -S9 N b N
Solvent (DMSO) +S9 57.7 100.0

BYI 08330
0.1 +S9 76.3 132.2
1 +S9 120.8 209.5
10 +S9 111.8 193.9
50 +S9 65.2 113.0
100 +S9 69.7 120.8
150 +S9 1.3 2.3
300 +S9 0.0 0.0

600 a +S9 N N

1000 a +S9 N N

Data summarized from Table s 1 and 2, Report No. AT00137A– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 199 April 2008

a There was precipitation of the test substance.

b N means that the cells were not cloned because of e xcessive cytotoxicity.

B. MUTAGENICITY ASSAY :

Cytotoxicity data were also collected in the mutage nicity assay. BYI 08330 induced decreases in survival to treatment (i.e., in survival until the time when the selection procedure was used to revea l mutant colonies) and in relative population growth in both the presence and absence of S9 mix. Relati ve survival and relative population growth were decrea sed by 83-90 % and 95%, respectively, at >40 ug/mL without metabolic activation and 83-97% and 82%, re spectively, at . >116 ug/mL with metabolic activation relative to controls. These results revealed a significant concentration -related cytotoxicity of BYI 08330. Precipitation of BYI 08330 in the cultu re medium was not observed at the concentrations used in the mutagenicity assay. An increase in mut ant frequency (MF) relative to controls was observe d at most concentrations in trial 2 with (3-5X) and w ithout (2-3.8X) metabolic activation. However, in the absence of a clear dose response and reproducibilit y, these results were considered toxicological and biologically insignificant. The positive controls EMS and DMBA had a marked mutagenic effect, as was seen by biologically and statistically significant increases in MFs as compared to the corresponding vehicle controls, and they demonstrated the sensiti vity of the test system and the activity of the S9 mix. Tables 6-62 and 6-63 summarize the results of the m utagenicity assay by averaging the results of both cultures at each test condition. The vehicle, negat ive, and positive control data were well within the range of the laboratory’s historical controls.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 200 April 2008

Table B.6-62: Results of the in vitro CHO/HGPRT mam malian cell gene mutation assay of BYI 08330 without metabolic activation

Treatment a ( μ g/mL) Relative

survival to

treatment (%) Relative

population growth (%) Total mutant

colonies on 10 plates b Absolute

Cloning Efficiency (%) Mutant frequency

(per 10 6 clonable cells)

Negative control trial 1 130.4 80.8 1 + 3 93.6 0.9 Solvent (DMSO) trial 1 100.0 100.0 1 + 1 104.3 0.4

BYI 08330 trial 1

2.5 119.7 49.3 1 + 3 89.8 0.9

5 181.1 69.0 1 + 1 88.5 0.5

10 220.9 99.5 1 + 1 85.3 0.5

20 160.6 62.4 2 + 4 99.1 1.5

40 107.7 21.3 3 + 1 69.5 1.4

80 10.2 - - - - EMS (900) trial 1 80.1 21.5 391 + 303 83.3 173.6*
Negative control trial 2 142.3 94.8 3 + 4 83.5 1.9 Solvent (DMSO) trial 2 100.0 100.0 1 + 3 103.1 0.8

BYI 08330 trial 2

20 119.7 90.3 5 + 4 106.6 1.8

30 87.4 88.9 3 + 17 71.8 5.8

40 47.1 102.4 5 + 5 84.8 2.5

50 25.6 21.3 8 + 21 69.9 9.3

60 16.6 24.4 8 + 12 105.0 5.1

70 14.8 - - - - EMS (900) trial 2 72.6 48.3 551 + 654 39.3 672.6*
Negative control trial 3 110.3 133.4 5 + 4 65.5 3.1 Solvent (DMSO) trial 3 100.0 100.0 12 + 13 59.3 8.9

BYI 08330 trial 3

20 94.3 116.2 12 + 10 59.6 8.3

30 73.0 85.5 21 + 17 65.2 12.1

40 48.3 82.0 5 + 10 63.2 5.2

50 29.6 43.9 11 + 6 71.0 5.7

60 18.8 30.4 9 + 21 68.5 10.3

70 9.8 5.0 8 + 13 49.9 8.7 EMS (900) trial 3 47.5 51.7 859 + 853 52.5 766.0*

Data summarized from Table s 3-5, Report No. AT00137A (data listed are the combined results of two cultu res) *Statistically significant increase in mutant frequ ency over the concurrent solvent control based on t he one-sided
Dunnett Test EMS = Ethyl methanesulfonate

a The trials identified here as trials 1, 2, and 3 we re identified in the report by the dates 5/23/02, 6 /7/02, and 8/27/02, respectively.
b The number before the plus sign is the total number of mutant colonies on 10 plates for the first cult ure, and the other number is the total on 10 plates for the second cul ture.

Table B.6-63: Results of the in vitro CHO/HGPRT mam malian cell gene mutation assay of BYI– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 201 April 2008

08330 with metabolic activation

Treatment a ( μ g/mL) Relative

survival to

treatment (%) Relative

population growth (%) Total mutant

colonies on 10 plates b Absolute

Cloning Efficiency (%) Mutant frequency

(per 10 6 clonable cells)

Negative control trial 1 114.8 62.5 4 + 8 102.8 2.5 Solvent (DMSO) trial 1 100.0 100.0 6 + 5 99.8 2.3

BYI 08330 trial 1

20 103.3 53.3 1 + 9 118.6 1.8

40 87.3 31.1 5 + 3 125.8 1.3

60 153.0 37.6 1 + 8 102.0 1.8

80 109.9 57.5 3 + 2 93.3 1.1

100 70.5 63.9 7 + 3 101.3 2.3

120 54.5 40.6 4 + 9 92.7 2.8
140 8.7 17.9 27 + 73 106.7 19.3*
DMBA (20) trial 1 70.0 36.2 328 + 202 114.3 98.3*
Negative control trial 2 90.8 93.0 2 + 4 82.7 1.6 Solvent (DMSO) trial 2 100.0 100.0 8 + 7 107.8 3.0

BYI 08330 trial 2

92 87.0 52.5 34 + 8 84.6 11.4

100 59.0 33.3 2 + 4 84.9 1.4

108 100.8 26.4 5 + 18 103.6 4.6

116 89.5 21.5 3 + 14 123.6 3.4

124 91.3 35.0 21 + 15 111.5 6.8

132 97.3 29.0 26 + 12 127.8 6.0

140 0.0 - - - -
DMBA (20) trial 2 70.4 50.1 189 + 153 76.4 94.9*
Negative control trial 3 121.4 107.8 6 + 8 76.0 3.8 Solvent (DMSO) trial 3 100.0 100.0 4 + 1 82.6 1.3

BYI 08330 trial 3

92 41.5 91.8 2 + 1 65.3 1.5

100 28.3 65.7 2 + 2 60.0 2.3

108 31.3 83.9 10 + 4 59.2 4.7

116 17.2 74.0 1 + 3 68.6 1.3

124 13.8 50.6 6 + 1 57.5 2.4

132 10.6 33.1 2 + 1 53.5 1.1

140 3.5 18.3 3 + 5 51.7 3.3
DMBA (20) trial 3 80.5 123.7 100 + 90 56.7 69.8*

Data summarized from Table s 6-8, Report No. AT00137A (data listed are the combined results of two cultu res) *Statistically significant increase in mutant frequ ency over the concurrent solvent control based on t he one-sided
Dunnett Test DMBA = Dimethybenzanthracene

a The trials identified here as trials 1, 2, and 3 we re identified in the report by the dates 5/22/02, 6 /4/02, and 8/16/02, respectively.

b The number before the plus sign is the total number of mutant colonies on 10 plates for the first cult ure, and the other number is the total on 10 plates for the second cul ture.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 202 April 2008

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS :

The investigator concluded that BYI 08330 is non-mu tagenic in the V79/HGPRT forward mutation assay, both with and without S9 mix. He noted that it cau sed concentration-related cytotoxicity.

B. REVIEWER COMMENTS :

The reviewer agrees with these conclusions. Cytoto xicity was observed as decreases in relative surviv al and relative population growth at concentrations > 40 ug/mL with and without metabolic activation . An increase in MF was observed relative to controls in trial 2 with and without metabolic activation but was considered insignificant toxicologically and biolog ically in the absence of a dose response and reproducibility. Solvent and positive controls gav e appropriate responses and agreed well with the laboratory’s extensive historical control. The stud y followed proper experimental protocol and met the criteria for a valid test and a negative result. There was no evidence of induced mutant colonies ov er background. The study is classified as Acceptable/ Guideline.

C. STUDY DEFICIENCIES:

None– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 203 April 2008

B.6.4.3 In vivo assays

B.6.4.3.1 Bone marrow or micronucleus (OECD Annex I IA 5.4.4)

B.6.4.3.1.1 In vivo mammalian cytogenetics – erythrocyte micronucleus assay (OECD annex IIA 5.4.4.1)

STUDY TYPE : In Vivo

Mammalian Cytogenetics - Erythrocyte Micronucleus assay in mouse ; OPPTS 870.5395 [ § 84-2]; OECD 474.

CITATION : Herbold, B. (2002) BYI 08330 micronucleus-test on the male mouse. Bayer HealthCare,

PH-PD P Health Care Toxicology, Rodents and Genotox icity, Friedrich-Ebert-Straße 217- 333, D-42096 Wuppertal, Germany. Study No. T 80714 79. Report No. AT00048. October 24, 2002.

I. MATERIALS AND METHODS:

A. MATERIALS :

1. Test material : BYI 08330 Description: Fine brownish powder
Lot/Batch #: NLL6425-9 Purity: 96.5% a.i. on December 11, 2001; 93.5% a.i. on June 21, 2002
CAS # of TGAI: 203313-25-1
Structure:

Solvent Used: 0.5% aqueous Cremophor emulsion

2. Control materials : Negative control (if not vehicle) : Final volume: Route:

Vehicle: 0.5% aqueous Cremophor

emulsion Final volume: 10 mL/kg bw Route: i.p. injection Positive control : Cyclophosphamide (CP) Final dose (s): 20 mg/kg bw Route: i.p. injection

3. Test animals : Species: Mouse
Strain: Hsd/Win: NMRI Age/weight at study initiation: Approximately 6 to 12 weeks of age based on weight / weight was 38-44 g Source: Harlan Winkelmann GmbH, Borchen, Germany No. animals used per 5 Males 0 Females– –

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dose Properly Maintained? x Yes - No
Five additional males were treated at the highest d ose; they were to be used as replacements in case s ome of the others died from the treatment.

4. Test compound administration :

Dose levels (mg/kg bw) Final volume Route Preliminary (2 x 200), (2 x 500), (2 x 1,000), with 24 hours between fractions 10 ml/kg bw i.p. injections

Main study (2 x 0), (2 x 125), (2 x 250),

(2 x 500), with 24 hours between fractions) 10 mL/kg bw i.p. injections

B. TEST PERFORMANCE:

1. Treatment and sampling times :

a. Test compound : Dosing: once x twice (24 hrs apart) Other ) Sampling (after dose): 6 hr 12 hr x 24 hr 48 hr 72 hr
Other:

b . Negative and/or vehicle control: 0.5% aqueous Cre mophor emulsion, same schedule as for test chemical

c. Positive control: Dosing: x once twice (24 hrs apart) Other Sampling (after dose): 6 hr 12 hr x 24 hr 48 hr 72 hr
Other:

2. Tissues and cells examined : Were erythrocytes from bone marrow examined?: Yes No. of polychromatic erythrocytes (PCEs) examined per animal: 2000 No. of normochromatic erythrocytes (NCE; more matu re RBCs) examined per animal: Number of NCEs found up until 2000 PCEs had been found
Other

3. Details of slide preparation : Bone marrow from at least one intact femur per ani mal was flushed out using a syringe into a tube that contained fetal ca lf serum. The tube containing the serum and bone marrow was centrifuged at approximately 1,000 rpm f or five minutes. The supernatant was removed with a suitable pipette, leaving only a small remainder. The sediment was mixed to produce a homogeneous suspension. One drop of the viscous suspension was placed on a well-cleaned slide and spread with a suitable object to allow proper evaluation of the s mear. The labeled slides were dried overnight. If fresh smears needed to be stained, they were dried with h eat for a short period. The smears were stained– –

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automatically with an Ames Hema-Tek Slide Stainer f rom the Miles Company. The slides were then "destained" with methanol, rinsed with deionized wa ter, and left to dry before being covered with cove r slips. The slides were not evaluated until the cove ring agent had dried. Slides were coded to avoid b ias.

4. Evaluation criteria : Slides were evaluated using a light microscope at a magnification of about 1,000. Micronuclei appear as stained chromatin particles i n the enucleated PCEs. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. The number of NCEs per 2,000 PCEs was determined for each animal. If the NCE:PCE ratio for a single animal amounts to distinctly more than 6,000:2,000, or if such a rati o seems likely without other animals in the group showing similar effects, then that particular anima l may be regarded as pathological and possibly one that would show effects unrelated to treatment. Su ch an animal may be omitted from the evaluation. I t can only be concluded that there is a relevant trea tment-related alteration of the PCE:NCE ratio if th at ratio is clearly lower for a majority of the animal s in the treated group than in the vehicle control. The number of NCEs showing micronuclei was also determi ned; however, it was realized by the time the report was written that an increase in the number o f micronucleated NCEs, without a preceding increase in micronucleated PCEs, would be meaningless, and t hat such data are of almost no use when bone marrow is collected so soon after treatment. An ass ay is considered acceptable if the frequencies of micronucleated PCEs of the vehicle and positive con trols are within the expected ranges, based on laboratory’s experience and the literature. A test is considered positive if there is a biologically s ignificant (termed “relevant and significant”) increase in the frequency of micronucleated PCEs in comparison to the vehicle control. A test is concluded to show a negative response if there is no relevant or sign ificant increase in the frequency of micronucleated PCEs. A test is also considered negative if there is a statistically significant increase in micronucleate d PCEs that is within the range of the laboratory’s historical negative (or vehicle) controls. A test is considered equivocal if there is an increase of micronucleated PCEs above the range of the laborato ry’s historical negative (or vehicle) controls but that increase is not statistically significant in compar ison to the concurrent control. A repeat test will then be performed.

5. Statistical methods : The parameters analyzed were the MNPCE (i.e., mic ronucleated PCE) frequencies, the MNNCE frequencies (unreported here since almost useless), and the PCE:NCE ratios. The BYI 08330 group(s) with the highest mean freque ncy of MNPCEs (provided this was higher than the mean of the negative control mean), and the positiv e control, were compared with the vehicle control using Wilcoxon's nonparametric rank sum test. This was also to be done for NCEs if the treatment was shown to induce MNPCEs. A variation was considered statistically significant if its error probability was below 5%. For NCEs they would use a one-sided Chi- square test, again at the 5% level. Standard deviations were calculated for all means. The revi ewer considers the analyses used to be appropriate.

II. REPORTED RESULTS:

There was no analytical testing of the concentratio ns of the dosing solutions. However, the stability of the test chemical in the vehicle was tested by comparin g the amounts of the test chemical present to the nominal concentrations of 10 and 100 mg/mL both bef ore and after 4 hours of storage at room temperature. At 10 mg/mL, the percentages of the n ominal concentrations before and after the 4-hour storage period were 97% and 98%, respectively. At 1 00 mg/mL, these percentages were 96% and 94%, respectively. It was concluded that the test chemic al is stable during an interval that spans the time period from preparation of the formulation to the last tre atment in the experiments.– –

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A. PRELIMINARY TOXICITY ASSAY :

The preliminary toxicity assay was done using anima ls of the same source, strain, and age. Groups of three males and three females were given two intrap eritoneal (i.p.) injections of the test chemical separated by 24 hours for each dose. At the 200 mg/ kg bw dose, for at least 96 hours the males experienced apathy, a semi-anaesthetized state, rou ghened fur, loss of weight, a staggering gait, late ral and sternal recumbency, spasms, palmospasms, leapin g spasms, twitching, rolling over, periodical stretching of the body, breathing difficulties, eye lids stuck together, closed eyes, and diarrhea. In addition, 1 of 3 males in the 500 mg/kg bw group di ed, and all males in the 1000 mg/kg group died. At the 200 mg/kg bw dose, females experienced all of t he same symptoms as males except for a staggering gait, palmospasms, and closed eyes. Als o, all females in the 1000 mg/kg bw group died. It was concluded that toxicity was so similar in the t wo sexes that the micronucleus assay could be done on males only. Based on these results, 500 mg/kg w as chosen as the maximum dose for the micronucleus assay.

B. MICRONUCLEUS ASSAY :

After two i.p. injections of BYI 08330, treated mal es show the following test substance related sympto ms until they were killed to harvest bone marrow: apat hy, a semi-anaesthetized state, roughened fur, loss of weight, lateral recumbency, spasms, extension spasm s, leaping spasms, twitching, rolling over, periodi cal stretching of the body, breathing difficulties, eye lids stuck together resulting in slitted eyes, and diarrhea. Two of 10 and 1/5 males receiving 500 and 250 mg/kg bw, respectively, died from its toxic effects befo re the harvesting of bone marrow. No symptoms (includ ing no deaths) were observed in the vehicle and postive control groups. Results of this assay are s ummarized in Table 6-64. No biologically or statis tically significant differences were found between test sub stance treated and vehicle control mice for the frequencies of micronucleated PCEs. The PCE:NCE ra tio showed a significant difference at the highest dose, with there being significantly fewer PCEs at that dose. Vehicle and positive control values for all parameters were appropriate, and the vehicle contro l and positive control frequencies of micronucleate d PCEs and of PCE/NCE ratios were well within the ran ges of the laboratory’s historical controls. The do se levels and the route of exposure were also appropri ate. It was pointed out that micronucleated PCEs normally occur in the vehicle control up to a frequ ency of approximately 6.0 per 2000 cells.– –

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Table B.6-64: Summary of micronucleus assay with BY I 08330 at 24 hours after treatment

Treatment Number of
PCEs scored per mouse for
micronuclei MNPCEs/2000
PCEs (Mean ± SD) No. of NCEs per 2000
PCEs (Mean ± SD %)

MALES

Vehicle control (0.5% aqueous Cremophor emulsion) 2000 2.0 ± 1.6 1264 ± 353

BYI 08330 (mg/kg bw) 2 x 125 (24-hours between doses) 2 x 250 (24-hours between doses) 2 x 500 (24-hours between doses) 2000
2000
2000 1.6 ± 2.1

3.5 ± 1.7
5.4 ± 3.2 1722 ± 720
4202 ± 1098
4948 ± 1943*

Cyclophosphamide (20 mg/kg bw) 2000 31.0 ± 6.2** 1071 ± 169

Data obtained from Report No. AT00048. October 24, 2002, Table 6, PCEs = polychromatic (immature) erythrocytes NCEs = normochromatic (mature) erythrocytes
MNPCEs = micronucleated PCEs *Statistically significantly higher than vehicle co ntrol, p < 0.05 **Statistically significantly higher than vehicle c ontrol, p < 0.01

III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS :

The investigator concluded that intraperitoneally-a dministered BYI 08330 was not clastogenic in the micronucleus test in male mice. He also concluded that the treatment caused a shift in the PCE:NCE ratio, which shows that the chemical was affecting the bone marrow.

B. REVIEWER COMMENTS :

The reviewer agrees with the investigator’s conclus ions. The dose was clearly limited by toxicity, an d it was adequately demonstrated that the toxicity is si milar enough in the two sexes to make it acceptable to do this assay only using males. The positive and n egative controls induced the appropriate response a nd were within the ranges of the laboratory’s historic al controls. There was not a significant increase in the frequency of micronucleated polychromatic eryth rocytes in bone marrow after any treatment time.

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.– –

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C. STUDY DEFICIENCIES: Minor study deficiency is as follows:

• The dose formulations were not analyzed for actual concentrations.– –

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STUDY TYPE : In Vivo Mammalian Cytogenetics - Erythrocyte chromosomal a berration assay in mice OPPTS 870.5385 [§84-2]; OECD 475.

CITATION : Herbold, B. (2003) Chromosome aberration assay in bone marrow cells of the mouse with

BYI 08330. RCC Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, D-64380 Rossdorf, Germany. Laboratory Report No.: AR00070, Bayer Study No.: T 4072032, RCCCCR Study No.: 765100, March 24, 2003.

I. MATERIAL AND METHODS

A. MATERIALS :

1. Test Material : BYI 08330 Description: technical grade, white powder, room te mperature storage Lot/Batch No.: NLL 6425-9 Purity (a.i.%): 93.5% (June 21, 2002); 92.7% (Dec ember 19, 2002) Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. of TGAI: 203313-25-1

2. Control Materials : Negative control (if not vehicle): Final Volume: Route:

Vehicle: 0.5% aqueous Cremophor Final Volume: 10 mL/kg bw Route: ip Positive control: Cyclophosphamide Final Dose(s): 40 mg/kg Route: ip

3. Test animals : Species: Mouse Strain: NMRI Age/weight at study initiation: 8 - 10 weeks old / 38.2 + 3.2 g Source: RCC Ltd., Biotechnology & Animal Breeding D ivision CH-4414 Füllinsdorf No. animals used per dose 6 males (5/dose evaluated for cytogenetic effects); 0 females Properly Maintained? Yes

4. Test compound administration : Dose Levels Final Volume Route Toxicity tests: First Second Third Main Study: 1000 mg/kg bw

500 mg/kg bw 750 mg/kg bw 0, 125, 250, 500 mg/kg bw 10 mL/kg bw 10 mL/kg bw 10 mL/kg bw 10 mL/kg bw ip

ip ip ip– –

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B. TEST PERFORMANCE :

1. Treatment and Sampling Times :

a. Test compound : Dosing: x once twice (24 hrs apart) Other (high dose only) Sampling (after last dose): x 24 hr 12 hr 24 hr x 48 hr 72 hr
Other:

b. Negative and/or vehicle control : Dosing: x once twice (24 hrs apart) Other Sampling (after last dose): x 24 hr 12 hr 24 hr 48 hr 72 hr
Other:

c. Positive control : Dosing: x once twice (24 hrs apart) Other Sampling (after last dose): x 24 hr 12 hr 24 hr 48 hr 72 hr
Other:

2. Preparation of the Bone Marrow :

The animals were sacrificed by cervical dislocation . The femora were removed, the epiphyses were cut off and the marrow was flushed out with approximate ly 5 ml hypotonic potassium chloride solution (0.56 % w/v, prewarmed to 37 °C). The hypotonic cell susp ension was then incubated for 20 min at 37 °C. The cells were sedimented by a brief centrifugation (1, 000 rpm), the hypotonic supernatant was discarded and the cell pellet was fixed with 3+1 absolute met hanol+glacial acetic acid fixative for 60 min. Then the cell pellet was gently resuspended with fixative an d stored overnight at 4°C. Prior to making slides t he fixative was changed and enough fixative was added to make a relatively thin cell suspension. The fixative-cell suspension was spread by flame-drying and stained with Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). One or mor e slides were made from each bone marrow sample.

3. ANALYSIS OF METAPHASE CELLS :

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Gaps, breaks, fragments, deletions, exchanges and c hromosomal disintegrations were recorded as structural chromosome aberrations. At least 100 wel l spread metaphases per animal were scored for cytogenetic damage on coded slides. The number of c hromosome aberrations per metaphase was determined. Only metaphases with the characteristic chromosome number of 40+ 2 were included in the analysis. To describe a cytotoxic effect, the mitot ic index (% cells in mitosis; 1,000 cells are score d) was determined.

4. EVALUATION / ACCEPTANCE CRITERIA :

The aberration types were defined as follows:– –

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1) Gap : A gap is an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) and smaller than the width of one chromatid. Gaps are found on one chromatid ("gap") or on both chromatids at apparently identic al sites ("isogap"). The biological relevance of gaps of both types is unclear;

2) Break:

A break is defined as a discontinuity of one chrom atid ("break") or both chromatids, at apparently the same locus ("isobreak"), with disloc ation of the chromatid ends. The dislocated chromatid end(s) has (have) to be present within th e respective metaphase. In addition, an achromatic lesion within a chromatid arm without ob vious dislocation of the chromatid end(s) but larger than the width of one chromatid is also defi ned as break or as isobreak, if this occurs in parallel on both chromatids of a chromosome.

3) Fragment: Fragments are parts of chromosomes without centrome r. A fragment is the result of a break. The corresponding defective chromosome is no t detectable among the chromosomes of the same metaphase. Fragments can be derived from one c hromatid ("fragment") or from both corresponding chromatid regions of a chromosome ("i sofragment").

4) Deletion : A deletion occurs as the result of a break. In ca se of a deletion, one chromatid ("deletion") or both corresponding terminal chromatid parts of a chromosome ("isodeletion") are missing within the metaphase under assessment. 5) Exchange: This i s an exchange of chromatid-parts between different chromosomes (interchange) or within the s ame chromosome (intrachange). 6) Multiple aberration: A cell was assessed as to contain "mult iple aberrations" when five or more structural changes (excluding gaps) occur within one metaphase . 7) Chromosome disintegration: A chromosome disintegration is recorded if fewer than half of the chromosomes reveal characteristic structural features within a given metaphase. Five animals per group were evaluated as described. The test is acceptable if the positive control show s a statistically significant response and if the aberration rate of the vehicle control (excl. gaps) is below 2 %.

5. Evaluation of Results / Statistical Methods :

A test item is classified as mutagenic if it induce s either a dose-related increase or a clear increas e in the number of structural chromosomal aberrations in a s ingle dose group. Statistical evaluation (nonparametric Mann-Whitney test) was used as an ai d in evaluating the results. However, the primary point of consideration is the biological relevance of the results. A test item that fails to produce a biologically relevant increase in the number of str uctural chromosomal aberrations is considered nonmutagenic in this system.

II. RESULTS:

Findings : No substantial reduction of the mitotic indices c ould be observed after treatment with the test item, indicating that the test item at the doses ad ministered was not cytotoxic in the bone marrow. No biologically relevant or statistically significant increase in the frequency of aberrant cells occurre d after treatment with the test item as compared to the veh icle control. The appropriate reference mutagen (cyclophosphamide) showed a distinct and statistica lly significant increase of induced aberration frequency and the vehicle control value was appropr iate. Historical control values are included as an appendix to this review. Results of the cytogeneti c assay are summarized in Table 6-65.– –

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Table B.6-65: Summary of the Cytogenetic Assay with BYI 08330

Number of cells with aberrations 1 % Aberrant
cells Treatment (mg/kg bw) Gaps Break

s Frag. MultAbb Exch Pulver + gaps - gaps Mitotic Index (%)

Vehicle 1 2 1 0 0 0 0.8 0.6 6.92
BYI 08330
125 2 1 0 0 1 0 0.8 0.4 7.38
250 1 0 0 0 0 0 0.2 0 8.38 500 (24 hour) 1 0 0 0 0 0 0.2 0 5.50

500 (48 hour) 1 4 0 0 0 1 1.0 0.8 7.54

CP (40) 11 26 34 43 16 6 36.4 36.0* 3.92
Table 1 summarized from Laboratory Report No.: AR00070, (originally called Table 7 in study).

1 Columns for isochromatid gaps, breaks and fragment s are not included because none were found with the exception of one iso-fragment in the positive control. 500 cells/dose (100/mouse) were scored for aberrati ons in the vehicle and test material treated groups . 250 cells (50/mouse) were scored for the positive control. Frag. = fragments. MultAbb = multiple aberrations (more than 5 in one cell). Exch = exchanges. Pulver = chromosome pulverization. CP = Cyclophosphamide. * Statistically significant at p < 0.01– –

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APPENDIX. HISTORICAL CONTROL VALUES

Preparation time (h) Negative

control % abbarant

cells excl. gaps Positive control

Cyclophophamide Dose mg/kg
b.w. % abbarant

cells excl. gaps

24 A. deion. 1.4 CPA 40 24.6

24 PEG 400 0.8 CPA 40 42.4

24 CMC 1.0 CPA 40 44.0
24 Cremophor 0.6 CPA 30 29.4

24 CMC 0.4 CPA 40 37.0
24 Cremophor 0.48 CPA 20 12.7
24 Cremophor 0.59 CPA 20 30.8

24 A. deion. 0.48 CPA 20 30.8

24 CMC 0 CPA 20 27.8

12 A. deion. 0.37 CPA 20 7.5

24 DMSO 0.5 CPA 20 24.7

18 A. deion. 0.2 CPA 20 10.3

24 Corn oil 0.26 CPA 40 27.6

24 PEG 400 0.6 CPA 40 22.6– –

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B.6.4.3.2 Metaphase analysis in rodent bone marrow or micronucleous test in rodents (OECD

Annex IIA 5.4.4)

B.6.4.3.3 Unscheduled DNA synthesis (OECD Annex IIA 5.4.5)

In Vivo/in vitro

Mammalian UDS test OPPTS 870.5550 [ § 84-2]; OECD 486.

CITATION : Brendler-Schwaab, S. (2003) Unscheduled DNA synthe sis test with rat liver cells in vivo.

Department of Molecular and Genetic Toxicology, Tox icology, Bayer HealthCare, FriedrichEbert-Strasse 217-333, D-42096 Wuppertal, F.R.G.. L aboratory Report No.: AT00526, Bayer Study No.: T 3072031, July 10, 2003. Unpublis hed.

I. MATERIAL AND METHODS

A. MATERIALS :

1. Test material : BYI 08330 Description: technical grade, white powder, room te mperature storage Lot/Batch No.: NLL6425-9 Purity (%ai): 93.5% (6/2002); 92.7% (12/2002) Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. of TGAI: 203313-25-1 Solvent Used: 0.5% Cremophor

2. Control materials : Negative: Not used Vehicle: 0.5% Cremophor Positive: 2-Acetylaminofluorene (2-AAF) / 100 mg/kg bw; sacrifice time of 16 hours N,N'-Dimethylhydrazine (DMH) / 40 mg/kg bw; sacrif ice time of 4 hours

3. Test animals : Species: Rat Strain: Wistar rats Crl:(WI)BR Age/weight at study initiation: 5-7 wks of age and 101-150 g at arrival

Source: Charles River Deutschland, Sulzfeld, German y No. animals used per dose 4 males; 0 females

Properly Maintained? Yes

4. Test compound administration : Dose Levels (nominal) Final Volume Route

0, 1,000, 2,000 mg/kg 20 ml/kg p.o.– –

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B. TEST PERFORMANCE :

1. Treatment and sampling times :

a. Test compound :
Dosing: once x once Other
Sampling (after last dose) 24 hr x 4 hr x 16 hr 48 hr 72 hr

Other:

b. Negative and/or vehicle control :
Dosing: once x once Other
Sampling (after last dose) 24 hr x 4 hr x 16 hr 48 hr 72 hr

Other:

c. Positive control :
Dosing: x once twice (24 hrs apart) Other
Sampling (DMH): x 4 hr
Sampling (2-AAF): x 16 hr

Other:

2. Preparation of hepatocyte cultures:

For each animal, cells were seeded in two 60 mm-Pet ri dishes (7.5 x10 5 viable cells per dish) precoated with collagen to determine cell viability, attachme nt rate and morphology about 1.5 hours after establishment of the cultures. For the determinati on of the genotoxicity of the test substance in the UDS assay a Thermanox 25-mm round plastic coverslip pre coated with collagen was placed into each well of 6-well culture dishes. Approximately 3.75 x 10 5 viable cells were seeded per well (in 2.5 ml cultu re medium 1), whereby 4 wells per animal including the control groups were established. All cultures wer e incubated for 90 min in a 37°C incubator in a humid ified atmosphere containing approximately 5% CO 2
.

3. Culture labelling:

After the attachment period, cell number and viabil ity of the cultures were determined by the method o f trypan blue exclusion employing the two additional 60 mm-Petri dishes. Also, following the attachment period, cells in the 6-well dishes were washed with culture medium 2 (culture medium 1 without dexamethasone, containing 1% FCS) and finally incub ated in the same medium. To each culture 10 uCi/mL 3 H-thymidine (specific activity: 15.8 Ci/mmole and 6 .2 Ci/mmole, respectively) was added. The cultures were then placed in the incubator.

After 4 h incubation, hepatocytes were washed twice with culture medium 3 (culture medium 2 without gentamycin sulfate) containing unlabelled thymidine and subsequently cultivated in this medium overnight (cold chase).– –

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Thereafter, the cultures were washed twice with PBS in the 6-well dishes. Subsequently, a 1% sodium citrate solution was added to swell the nuclei for 5-10 min. The cells on the coverslips were then fix ed by two changes of a 1:4 acetic acid:absolute ethanol s olution for a total fixing time of at least 30 min. Wells were then washed 2-6 times each with deionized dist illed water and air dried.

4. Autoradiography and grain counting:

The entire autoradiography procedure is performed i n the dark. Air-dried coverslips were mounted cell

side-up on microscope slides. In a darkroom, these were dipped in a NTB-2 photographic emulsion (Kodak), diluted 1:1 with distilled water, and left to dry in the air overnight. The next day, the coa ted slides were stored in light-tight boxes in the pres ence of a drying agent (Drierite) for 14 days at -2 0°C in order to reduce the CG background and therefore to increase the sensitivity of the assay. The photographic emulsion was then developed for 3-4 mi n in Kodak D-19 developer at temperatures below 15°C. The slides were rinsed afterwards with distil led water, fixed in Kodak Fixer for 7-8 min, and ai r dried. Slides were then stained with hematoxylin an d eosin. Grain counting was done by eye using a Zeiss microscope interfaced to a TV color screen (S ony) with a Sony high resolution TV color camera. UDS was determined as follows: Evaluation was perf ormed with coded slides. Each slide was examined by counting 50 cells per slide. Three slides per an imal were evaluated (total of 150 nuclei for each r at). Only cells viable at the time of fixation were scor ed; isolated nuclei and cells with abnormal morphol ogy were excluded. A starting point was randomly select ed on the lower third of the slide, and cells were scored in a regular fashion by bringing new cells i nto the field of view, moving along the X-axis. If the total number of 50 cells had not been reached befor e coming to the edge of the slide, the stage was moved on the Y-axis, and counting resumed in the op posite X-direction, parallel to the first line. UDS was measured by counting nuclear grains and subtrac ting the average number of grains in 3 cytoplasmic areas of the same size as the corresponding nucleus . This value was referred to as the nuclear net gra in (NNG) count of the cell. The mean NNG count per ani mal was routinely determined from triplicate coverslips. The mean NNG value per dose group was c alculated from the mean NNG value of each animal of the respective dose group. The number of cells in repair (nuclei with 5 or more net grains) was also determined. Grains were counted by eye. Data w ere transferred to a PC and processed with the released and Windows NT 4.0 based software "UDS-Tes t" (V02.02) of Bayer AG.

5. Assay evaluation criteria:

1. For each of the evaluated 50 cells per slide, th e number of nuclear grains is scored, as well as numbers of three cytoplasmic grain counts from nucl ear-sized areas adjacent to each nucleus. 2. Only cells viable at the time of fixation and wi th nuclei evenly coated with emulsion will be score d. Cells with abnormal morphology, such as those with pyknotic or lysed nuclei, will not be counted. Isolated nuclei not surrounded by cytoplasm will no t be counted. 3. For the conditions described here, a number of 5 NNG or more is chosen as a conservative estimate as to whether a particular cell is responding (cell "in repair"). 4. Cells with heavily labelled nuclei (= S-phase ce lls) will be excluded from scoring.

6. Statistical methods:

Descriptive statistical methods were used to calcul ate means and standard deviations. The means and standard deviations in the tables were calculated f rom the means calculated individually for each of t he three coverslips per animal. Statistical evaluation of % cells in repair was performed using the Chi 2 test. The following indices were calculated:– –

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Mean Net Grains per Nucleus (NNG) = average number of the mean nuclear net grain counts of each evaluable coverslip; 50 cells per coverslip Mean Grains per Nucleus (NG) = average number of th e mean nuclear grain counts of each evaluable coverslip; 50 cells per coverslip Mean Cytoplasmic Grain Count (CG) = average number of the mean cytoplasmic grain counts (3 areas per cell) of each evaluable coverslip; 50 cells per coverslip. % Nuclei with 5 or more grains = (number of cells w ith 5 or more net nuclear grain counts per dose / N o. of evaluated cells per dose) x 100 Absolute Survival (%) = % of viable cells after iso lation

II. RESULTS:

Findings: Rats treated with BYI 08330 showed symptoms of toxi city after administration of both doses. Signs included roughened fur and pallor at both dos es and rapid breathing at 2,000 mg/kg only. No relevant cytotoxicity was observed in hepatocytes a fter isolation. After single oral treatment with 1, 000 mg/kg bw or 2,000 mg/kg bw BYI 08330, no indication of UDS-induction was found following either a four-hour or 16-hour exposure time. The positive c ontrols 2-Acetylaminofluorene and N,N'- Dimethylhydrazine had clear genotoxic effects.

Table B.6-66: Summary of the UDS assay (16-hour exp osure)

Dose Group Mean NNG +/- S.D. Mean NG +/- S.D. Mean CG +/- S.D.

Vehicle Control -2.01 +/- 0.30 2.67 +/- 1.11 4.68 + /- 1.32

Test Substance

1000 mg/kg -1.28 +/- 0.26 3.60 +/- 1.07 4.88 +/- 1. 30

2000 mg/kg -1.38 +/- 0.55 4.20 +/- 1.42 5.58 +/- 1. 59

Positive Control 5.81 +/- 0.40 9.90 +/- 1.25 4.09 + /- 0.86

copied from Laboratory Report No.: AT00526, (origin ally called Table 5 in study)

Table B.6-67: Summary of the UDS assay (4- hour exp osure)

Dose Group Mean NNG +/- S.D. Mean NG +/- S.D. Mean CG +/- S.D.

Vehicle Control -1.57 +/- 0.15 3.46 +/- 0.90 5.04 + /- 0.89

Test Substance

1000 mg/kg -0.86 +/- 0.20 4.57 +/- 1.80 5.43 +/- 1. 94

2000 mg/kg -0.84 +/- 0.19 4.13 +/- 0.78 4.97 +/- 0. 78– –

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Positive Control 4.63 +/- 0.38 10.12 +/- 2.02 5.49 +/- 1.87

copied from Laboratory Report No.: AT00526, (origin ally called Table 6 in study)

III. CONCLUSION :

BYI 08330 was negative in the in vivo/in vitro UDS assay with rat liver cells.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 219 April 2008

B.6.4.3.4 In vivo studies in germ cells (OECD Annex IIA 5.4.6)

B.6.4.4 Summary and overall conclusions of genotoxi city studies

Overall, assays for point mutations and chromosomal aberrations (both in vivo and in vitro) were negat ive for spirotetramat. A weak positive finding was noted in a single in vitro chromosomal aberration test, but at cytotoxic concentrations only. Negative findings in two in vivo chromosomal aberration studies and one in vivo/in vitro unscheduled DNA synthesis assay using rat hepatocytes do not suggest a genotoxic concern for spirotetramat.– –

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B.6.5 Long term toxicity and carcinogenicity (OECD Annex IIA 5.5)

B.6.5.1 Long term oral toxicity study in the rat (O ECD Annex IIA 5.5.1 and 5.5.2)

CITATION : Whale BS. (2005). Technical Grade BYI08330 (Common Name Spirotetramat): A Chronic

Toxicity Study in the Rat. Bayer Crop Science LP, S tilwell, Kansas, November 15, 2005, Unpublished.

I. Material and methods

A. Materials:

1. Test Material: BYI 08330 Description: Technical grade, white powder Lot/Batch No.: 08045/0014 Purity (% a.i.): 97.5% (7/2003); 97.8% (12/2003); 97.6% (6/2004); 98.5% (1/2005) Compound Stability: Stable at room temperature (~ 22 o C) Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. for TGAI: trans : 382608-10-8; cis : 203313-25-1

2. Vehicle and/or positive control: None

3. Test animals: Species: Rat; male and female (nulliparous and non -pregnant) Strain: Wistar Hanover rats (Crl:WI[Glx/BRL/Han]IG S BR) Age/weight at study initiation: Approximately 8 weeks old / 241.8 - 243.9g mean gro up weight males; 154.4 - 157.2g mean group weight females Source: Charles River Laboratories, Inc. (Raleigh, NC) Housing: Individually housed in suspended stainles s steel wire-mesh cages, each containing a feeder, a source of water (pressure-ac tivated water nipples), and deotized (sanitized) cage board in the bedding tray. One nylabone® was placed in the cage of each animal. Cage racks were rotated monthly. Diet: Purina Mills Certified Rodent Diet 5002 in " meal" form; ad libitum Water: Municipal water supply of Kansas City, MO; ad libitum

. Environmental conditions: Temperature: Humidity:

Air changes: Photoperiod: 18 to 26 o C

30 to 70% (relative); high range relative humidity was 72% on days 24 and 27, and 71% on day 66. 10.27 changes/hr (minimum daily average) 12 hr of light [7:00 a.m. to 7:00 p.m., or 8:00 a.m . to 8:00 p.m.] alternating with 12 hr of darkness. Deviations in the light/dark cycle occurred 9 times (5 planned; 4 unplanned) over the course of the study. None of the deviations was of a duration as to impa ct study outcome. Acclimatisation: 08/25/03 (receipt) - 09/03/03 (re lease for study)– –

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B. Study design:

1. In life dates: Start: 09/03/03 (released for study); 09/09/03 (in itiation of exposure) End: 09/16/04 (terminal sacrifice complete; chro nic animals)

2. Animal assignment: 09/04/03; assigned to a control or one of three che mically-treated groups (as noted in Table 6-68) using a weight stratification-based computer program obtained from INSTEM Computer Systems (Stone, Staffordshire, UK). Each animal on study identified with a microchip (BioMedic Data Systems, Inc., Seaford, DE) subcutaneously implante d on their dorsal surface in the region between the scapulae; the chip was encoded with a unique number , specifying the animal's sex, dose group, cage number, and study affiliation.

Table B.6-68: Study design Main Study 12 months Test Group Conc. in Diet (ppm) Dose to animal (mg/kg; x+ Std) Male Female

Control 0 0 25 25

Low (LDT) 250 males 13.2 + 3.4 females 18.0 + 3.4 25 25

Mid (MDT) 3,500 males 189 + 49 females 255 + 39 25 25

High (HDT) males 7,500 females 12,000 males 414 + 106

females 890 + 156 25 25

3. Dose selection rationale: Doses were based principally upon the toxicological profile which emerged in the rat over the course o f a subchronic toxicity testing study (Report No. 20113 6). In that study, the toxicological response of the ma le rat was principally characterized by decreased r ate of body weight gain, as well as structural and/or func tional alterations in testes- and lung-related endp oints. A decline in rate of body weight gain of 17.6% was noted in 10,000-ppm males; a corresponding decline of 8.4% was noted in absolute body weight. Extrapolat ing these subchronic changes out over a prolonged chronic exposure (nominally 1 year), which may have increased the severity of the body weight effect, a high-dose of 7,500 ppm was chosen for males in the present study. In the female, the toxicological response was chara cterized by a structural and/or functional alterati on in the lung (the same lesion noted in 10,000-ppm males ). The lung lesion was considered a sign of genera l toxicity, and progression over the 1-year period co uld not be excluded. However, unlike the male, a c lear body weight effect (a classic predictor of a maxima lly-tolerated dose; MTD) was not established. Thes e results suggested a different sensitivity in the tw o sexes, indicating that females could be treated w ith concentrations somewhat higher than males to achiev e roughly the same effect (i.e., males are more sensitive than females). Thus, a high-dose of 12,0 00 ppm was chosen for females in the present study.

It was anticipated that the low and high doses chos en of 250 ppm and 7,500 / 12,000 ppm would constitute a no-observed-adverse-effect level and a maximum tolerated dose , respectively, with the intermediate dosage of 3,500 ppm serving to confirm any dose response relationships that may have emerged. The probable routes of human exposure to BYI 08330 are via ingestion of foodstuffs that might contain low residues of the test substance, accidental ingestio n, or dermal contact during manufacture or use. Th us,– –

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formulation with the feed was an appropriate route of administration with which to develop the chronic toxicological profile of the test substance in a gi ven test species.

4. Diet preparation and analysis: The test compound was mixed directly with the feed; control diet consisted of untreated feed. Samples of each batch of feed mixed were taken and retained in the freezer until the study was complete and the analytical data deemed satisfactory. Replacement a dmixtures for each treatment group were prepared weekly and stored under freezer conditions until pr esented to the animals the following week. BYI 08330 was administered at a constant concentrat ion in the feed for the duration of the study. The concentration of the AI of the test substance in th e various test diets was analytically verified from samples of each dietary level taken during Weeks 1, 2, 3, 7 , 11, 16, 20, 24, 28, 32, 36, 40, 45, and 49. In a ddition, one recovery sample was analyzed with each group of samples during the test concentration analyses. The homogeneity and stability of BYI 08330 when mix ed in the dietary carrier was characterized prior t o the in-life phase of the study.

To evaluate the uniformity of distribution/homogene ity of the test substance in the diet, an analysis of feed containing nominal concentrations of 70 and 12,000 ppm BYI 08330 was conducted. Three (3) samples were taken for analysis from each of 3 distinct lay ers (top, middle, bottom; 9 samples total) in a Hob art mixing bowl. A homogeneous distribution of the tes t substance in the feed was defined in terms of a percent relative standard deviation (%RSD, same as coefficient of variation; CV), derived from the 9 samples taken, which was < 10%. Additional homogeneity analyses were perform ed for two other BYI 08330 rodent studies utilizing identical rodent rat ion mixing procedures in March of 2005. Those stud ies included separate oncogenicity studies in both rats and mice, and were initiated at about the same tim e as the present study. To evaluate the stability of the test substance in the diet, a time and temperature analysis of feed containing nominal concentrations of 70 and 12,000 ppm BYI 08330 was conducted. Stability in the feed was assessed following 1 and 7 days of room tempera ture storage (~22 o C) and 7, 14, and 29, and 32 days of freezer storage (~ -23 o C; 12,000 ppm evaluated at 29 days; 70 ppm evaluate d at 32 days). The stability analysis was carried out as follows: An initial sample was taken immediately after the test substance was mixed with the ration. Each batch of ration, mixed at the 2 concentrations, was then divided into 2 portions. One portion was placed in a room temperature environment, the other in the freezer. Feed samples which remained at room tempe rature were sampled for analysis on Days 0, 1, and 7; feed samples which remained in the freezer were sampled for analysis on Days 7, 14, 29 (12,000 ppm only), and 32 (70 ppm only). Chemical stability in the ration was defined in terms of that period of time that 80% of the test substance, relative to the initial concentration, was recoverable. The liquid chromatographic methodology used to perf orm the dietary analyses required for this study (i .e., homogeneity, stability, and concentration verificat ions) has been described previously.

Results:

Homogeneity Analysis: The mean concentrations of BYI 08330 in the feed, sampled from 3 distinct layers in the mixing bowl and containing a nominal concentration of either 70 or 12,000 ppm, were determined to be 68.9 ppm (range 64.9-73.2 ppm; %RS D = 3.8%) and 12,157 ppm (range 11,844-12,567 ppm; %RSD = 2.1%), respectively. Based on a %RSD < to 10%, BYI 08330 was judged to be homogeneously distributed in the feed over a concen tration range of 70-12,000 ppm. Similarly, additio nal homogeneity analyses performed for two other rodent studies with BYI 08330 (utilizing identical rodent ration mixing procedures) confirmed the homogeneity of the test substance in the rodent ration.– –

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Stability Analysis: Following 7 days of room temperature storage, the analytically-determined concentration of the AI of the test substance in th e 70- or 12,000-ppm admixture was determined to be 69.8 ppm (68.9 ppm on day 0) and 11,948 ppm (12,157 ppm on day 0), respectively. Following 29 and 32 days of freezer storage, the analytically determine d concentration of the AI of the test substances in the 12,000- and 70-ppm admixtures was determined to be 12,474 and 68.0 ppm, respectively (12,157 and 68.9 ppm, respectively on day 0). BYI 08330 mixed in rodent ration was judged to be stable at room temperature for at least 7 days and following freez er storage for a minimum 29 days, over a concentrat ion range of 70-12,000 ppm.

Concentration Analysis: Mean analytical concentrations for each dose grou p were 241, 3,373, 7,366, and 11,549 ppm, ranging from 96-98% of the correspo nding nominal concentrations of 250, 3,500, 7,500, and 12,000, respectively. The AI of the test subst ance was not detected in the control diet. Mean recovery was 97% and ranged from 87-104% for rodent ration spiked with 250, 3,500, 7,500, or 12,000 ppm BYI 08330.

5. Statistics: Continuous data that were examined statistically we re evaluated for equality or homogeneity of varianc e using Bartlett's test. Group means were further ana lyzed by a one-way variance analysis (ANOVA) followed by Dunnett's test. Frequency data that we re examined statistically were evaluated using the Chi-square and/or Fisher exact tests. Continuous d ata collected in the FOB that were examined statistically were evaluated using an ANOVA with p ost-hoc comparisons using Dunnett's test. Categorical data collected in the FOB that were exa mined statistically were evaluated using General Linear Modeling and Categorical Modeling (CATMOD) P rocedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respec tively. For the Bartlett test, a probability (p) l evel < 0.001 was considered significant; for all other sta tistical tests, differences with p values < 0.05 were considered statistically significant. Additional s tatistical tests may have been used to evaluate dat a generated from this study when deemed appropriate b y the study director. All statistical evaluations were performed using software obtained from either INSTE M Computer Systems or SAS Institute Inc.

C. Methods:

1. Observations: 1a. Cageside Observations: For 52 weeks of the study, a general assessment of all remaining animals was carried out at least dail y (generally twice daily during the normal work week and once daily on weekends and holidays). These assessments were generally with respect to moribund ity and mortality, but also included a more general clinical observation for readily apparent clinical signs (in addition to moribundity and mortality).

1b. Clinical Examinations: Once each week for 52 weeks of the study, detailed physical examinations for clinical signs of toxicit y were performed on all surviving animals. These exa minations included evaluation of external surface areas, orifices, posture, general behavior, respira tion, and excretory products, and a general open fi eld assessment (qualitative; utilizing a standard arena ). General observations in the standard arena were not required during the week in which the animals a re subject to a more detailed open field observatio n as part of the full functional observational batter y.

1c. Neurological Evaluations: During month 12 of the study, the last 10 surviving rats/sex/dose were subject to a functional observational battery to assess motor activity, gri p strength, and sensory reactivity to stimuli of di fferent– –

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types (e.g., visual, auditory, and proprioceptive s timuli). Parameters evaluated included home cage observations

- posture, piloerection, involuntary motor movemen ts (clonic or tonic), gait abnormalities, vocalizations, decreased activity, repetitive head bobbing (nutation), increased reactivity; observations during handling - ease of removing the rat from its cage, reaction to being handled, muscle tone, palpebral closure, lacrimation, salivation, nasal d ischarge, stains, alopecia, emaciation, bite marks, exophthalmia, broken teeth/malocclusion, missing to e nails, dehydration, cool-to-touch; open field observations

- piloerection, respiratory abnormalities, posture , involuntary motor movements-clonic or tonic, stereotypy, bizarre behavior, gait abnormali ties, vocalizations, rearing, defecation, urination ; and reflex/physiologic observations and measurements - approach response, touch response, auditory response, tail pinch response, grip strength, body weight, body temperature). Pupil size and pupil response were evaluated during routine ophthalmic e xaminations.

2. Body weight: Individual body weight determinations were carried out weekly for the first 13 weeks of the study and every 4 + 1 weeks thereafter on all surviving animals. Body weights were also measured immediately prior to all necropsies to allow for calculation of organ to body weight ratios.

3. Food consumption and compound intake: Individual food consumption determinations were car ried out weekly for the first 13 weeks of the study and every 4 + 1 weeks thereafter on all surviving animals. In a ddition, using specifically defined criteria, food consumption data were corrected, as conditions dictated, to account for misleading indications of food intake (i.e., excessive spillage, clogged feed ers, etc.). Test substance intake (mg/kg body wt/d ay) was calculated using weekly mean body weight and fo od consumption data. The general relationship used for this calculation was:

[AI in feed (ppm)/1,000] x [feed consumed (g/kg bod y wt/day)] = mg AI/kg body wt/day

4. Ophthalmologic examination: Ophthalmologic exams were conducted on all acclimat ized animals prior to exposure, and then again on all surviving animals just prior to termination. O phthalmological lesions were assigned a semiquantitative ranking of the severity grade/stage fr om "Normal (within normal limits)" to the "most sev ere case (Grade 5)," as described previously.

5. Hematology and Clinical Chemistry: Blood was collected at approximately 3, 6, and 12 m onths (+ 0.5 month/interval) into the study. Blood was drawn via the orbital sinus following an overni ght fast from all surviving animals while under lig ht anesthesia (IsoFlo®; Isoflurane). To the extent po ssible, 15 of the blood samples were used for hematologic (excluding prothrombin time) and clinic al determinations; the remaining blood samples (nominally 10) were used for prothrombin time deter minations. Differential blood counts were determin ed on all animals sacrificed due to moribundity.

5a. Hematology:
X Hematocrit (Hct)* X Leukocyte differential count* X Hemoglobin (Hgb)* X Mean corpuscular Hgb (MCH)* X Leukocyte count (WBC)* X Mean corpuscular Hgb con centration (MCHC) X Erythrocyte count (RBC)* X Mean corpuscular volum e (MCV)* X Platelet count (PLTS)* X Reticulocyte count (Reti c) X Blood clotting measurements* X Erythrocyte morpho logy– –

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Thromboplastin time Activated partial thromboplastin time- (a) X Heinz bodies (HZ)

Clotting time X Prothrombin time (PT)

* Recommended for chronic studies based on Guidelin e 870.4100. (a) Added by the HED reviewer. Also data were provi ded for “RDW” and “HDW” but these acronyms were not defined. The CHECKED (X) parameters were examined.

5b. Clinical Chemistry:

ELECTROLYTES OTHER X Calcium (Calc) X Albumin (Alb)* X Chloride (Cl) X Creatinine (Creat)* Magnesium X Blood urea nitrogen (Urea-N)* X Phosphate (Phos) X Total Cholesterol (Chol)* X Potassium (K)* X Globulin (Glob) X Sodium (Na)* X Glucose, fasting (Gluc)*
ENZYMES (more than 2 hepatic enzymes)* X Total bilirubin ( T-Bili) X Alkaline phosphatase (ALP)* X Total protein (T-Pr ot)* Cholinesterase (ChE) X Triglycerides (Trig) X Creatine phosphokinase (CK) Serum protein electr ophoresis X Lactic acid dehydrogenase (LDH) X Uric acid (Uric -A) X Alanine aminotransferase (ALT)* X Thyroxine (T4) X Aspartate aminotransferase (AST)* X Triiodothyron ine (T3) Sorbitol dehydrogenase* X Thyroid Stimulating Horm one (TSH) X Gamma-glutamyltransferase (GGT)* Glutamate dehydrogenase Cytochrome P-450, hepatic (Cyto P-450) N-Demethylase, hepatic (N-Demeth) O-Demethylase, hepatic (O-Demeth) UDP-Glucuronosyltransferase, hepatic (UDP-GT)

* Recommended for chronic studies based on Guidelin e 870.4100. The CHECKED (X) parameters were examined.

6. Urinalysis: Urine was collected at approximately 3, 6, and 12 m onths (+ 0.5 month/interval) into the study. Non-f asted urine was collected on an overnight basis (timed) f rom the first 10 surviving rats/sex/dose the week p rior to the week of bleeding.

X Appearance* X Glucose (Glu)* X Volume (UVol)* X Ketones (Ket) X Specific gravity (Sp.Gr.)* X Bilirubin (Bil) X pH (pH)* X Leukocytes (U-Leu) X Sediment (microscopic) X Blood (Bld)* X Protein (Pro)* X Nitrite (Nit) X Urobilinogen (UROB) * Recommended for chronic studies based on Guidelin e 870.4100. The CHECKED (X) parameters were examined.– –

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7. Sacrifice and Pathology:

7a. Gross pathology: A complete postmortem examination was conducted on all animals (surviving, found dead, sacrificed in extremis ) which included (1) documenting and saving all gro ss lesions, (2) weighing designated organs, and (3) collecting representative tissue specimens for histopathologic evaluation. Animals surviving at termination were sacrificed by CO 2 asphyxiation. Tissues were preserved and organ/bo dy weight ratios were calculated for weighed organs.

DIGESTIVE SYSTEM CARDIOVASC./HEMAT. NEUROLOGIC Bile duct (rat) X Aorta* XX Brain*+
X Cecum* X Bone marrow* X Cerebellum*
X Colon* XX Heart*+ X Cerebrum-Midbrain* X Duodenum* X Lymph node, cervical* X Medulla/Pons* X Esophagus* X Lymph node, mesenteric* X Eyes* Gall bladder (not rat)* XX Spleen*+ X Nerve, optic * X Ileum* XX Thymus+ X Nerve, sciatic* X Jejunum* X Pituitary*

XX Liver*+ UROGENITAL X Spinal cord, cervical* X Pancreas* X Cervix X Spinal cord, lumbar* X Rectum* X Clitoral gland^ X Spinal cord, thoracic * X Salivary glands* XX Epididymides*+ X Stomach, glandular* XX Kidneys*+ OTHER X Stomach, non-glandular* X Mammary gland* X Bone, femur X Tongue XX Ovary*+ X Bone marrow X Tooth X Preputial gland^ X Bone, rib/cc jct
XX Prostate* X Bone, sternum
RESPIRATORY X Seminal vesicle* X Gross lesions and masses* X Larynx* XX Testicle*+ X Harderian gland XX Lung* X Urinary bladder* X Joint, fem/tib X Nasal structure XX Uterus*+ X Muscle, protocol X Nasopharynx* X Vagina^ X Physical Identifier (ID chip) X Oral structure X Skin, protocol*

X Trachea* GLANDULAR X Zymbal’s gland^ XX Adrenal gland*+ X Exorbital/lacrimal gland^ X Parathyroid* XX Thyroid* * Required for chronic studies based on Guideline 8 70.4100. + Organ weights required for rodent studies. The CHECKED (X) tissues were collected for histolog ical examination. The organs CHECKED (XX) were als o weighed. The organs CHECKED (^) were preserved for possible micropathologic evaluation only.

7b. Micropathology: With the exception of the physical identifier (micr ochip), the vagina, and the exorbital/lacrimal, cli toral, preputial, and Zymbal glands (of which the latter 5 were taken and preserved for possible micropatholo gic– –

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evaluation), representative sections of the tissues shown in Table 6-71 were processed for all control (0 ppm) and high-dose (7,500 / 12,000 ppm) animals, em bedded in paraffin, sectioned, mounted, and stained with hematoxylin and eosin (H&E) for examin ation under the light microscope. Tissues in which compound-related effects were detected were examine d at lower doses as necessary to establish noobserved-effect levels. Target organs and gross le sions were examined for all animals. Histopathological lesions were assigned a semi-quan titative ranking of the severity grade/stage from "Normal (within normal limits)" to the "most severe case (Grade 5)," as described previously.

II. Results

General observations: Clinical observations were limited to an increased incidence of yellow and brown staining of the perigenital area and tail in 12,000 -ppm females. [Note added by HED reviewer: There were 11 incidents of yellow staining with a mean on set time of 194 days and also 11 incidents of brown staining with a mean onset time of 226 days; these conditions were therefore late-developing]. No ot her treatment-related clinical signs and no substance-r elated mortality were observed. Neurobehavioral parameters (e.g. weekly open field assessments; FOB conducted during Month 12) provided no indication of a neurological effect attributable to exposure to the test substance. Feed intake was no t toxicologically relevantly affected. Body weights w ere slightly reduced (-6.6%) in 12,000 ppm females at end of treatment. Overall mean body weight gain in this group was 86% of controls. Male body weights were not affected.

Table B.6-69: Mean body weights (g) and cumulative body weight gain (g) Dose Group (ppm) Parameter 0 250 3,500 7,500/12,000

Males
Initial body weight 244 243 242 243
Final body weight (week 51) 544±9.7 550±14 541±6 53 6±10
Body weight gain (week 1) 31 34 31 30
Body weight (week 13) 429±6.4 427±8 423±6 420±6
Body weight gain (week 13) 185 184 182 177
Body weight gain (week 27) 239 243 238 235
Body weight gain (week 51) 300 306 299 293

Females
Initial body weight 157 154 157 157
Final body weight (week 51) 295±8 306±8 287±6 276±6
Body weight gain (week 1) 16 17 15 15
Body weight (week 13) 231±2.9 231±3.1 227±3.2 232±3 .1
Body weight gain (week 13) 74 76 69 67
Body weight gain (week 27) 101 107 91 87*
Body weight gain (week 51) 138 152 130 118
* = p < 0.05; Mean body weight data are on pages 33 -42 of the study report. Mean body weight gain dat a are on pages 43-50.

Hematology: Not affected. Mean haematology data are on pages 341 to 373 of the study report.– –

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Clinical chemistry: Not affected. Mean clinical chemistry data are on pages 322 to 341.

Urinalysis: Not affected. Mean urinalysis data are on pages 3 74 to 386.

Ophthalmology: No treatment-related effects. Mean ophthalmology d ata are on pages 319 to 321.

Gross necropsy, organ weights: Gross necropsy showed an increased incidence of dis coloration of the lung in 12,000-ppm females (0, 1, 0, 5). No other i ndication of a BYI 08330-induced change was noted i n either sex at any dose tested. An increase in relat ive liver weights was noted in 7,500-ppm males and in 12,000-ppm females which was considered treatment-r elated. Relative kidney weights were also slightly increased in females at 12,000 ppm.

Table B.6-70: Mean absolute (g±SD) and relative (%) organ weights Dose Group (ppm) Parameter 0 250 3,500 7,500/12,000

Males
Final body weight 548.7 553.5 544.0 542.6
Liver weight, absolute 17.792 17.945 18.599 18.913
Liver weight, relative (a) 3.240±0.277 3.249 3.435 3.530±0.324 *

Females
Final body weight 296.0 306.3 289.0 275.2
Liver weight, absolute 9.790 10.147 9.820 10.476
Liver weight, relative (a) 3.321±0.328 3.328 3.415 3.820±0.375*
Kidney weight, absolute 2.037 2.057 2.030 2.048
Kidney weight, relative (a) 0.694±0.068 0.678 0.706 0.750±0.099*
* = p < 0.05; There were 23-25 animals/sex in each group available for organ weight determinations. Me an organ weight data are on page 429 to 438 of the study rep ort.

Histopathology: Testis/epididymis: An increased incidence of exfoliated germ cells/de bris was observed in the epididymis of males at 7500 ppm (3/25 vs. 0/24 in controls). Abno rmal spermatozoa were also observed in high dose males (2/25 vs. 0/25 in controls). Although the inc reases were not statitistically significant, they w ere considered toxicologically relevant, since testicul ar/epididymal histopathology was observed at higher incidences following treatment with spirotetramat f or 24 months. Lung : An increased incidence of minimal to slight accum ulation of alveolar macrophages was noted in the lungs of 3,500- and 7,500-ppm males, and 12,000-ppm females. This finding correlated with the discoloration of the lung in the 12,000-ppm females (see above). The increased incidence noted in this study was attributed to exposure to the test substance. N o other indication of microscopic changes attributa ble to exposure to the test substance was observed.

Table B.6-71: Incidence and average severity (figur es in parenthesis) of microscopic findings Dose Group (ppm) Parameter 0 250 3,500 7,500/12,000
Males Lungs, macrophages, alveolar 1 (1.0) 2 (1.0) 6 (1.0 )* 11 (1.2) * Testes, abnormal spermatozoa 0 0 0 2 (1.5)– –

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Epididymides, exfoliated germ
cells/debris 0 0 0 3 (2.0)

Females Lungs, macrophages, alveolar 5 (1.0) 3 (1.0) 2 (1.0 ) 21 (1.3) * * = p < 0.05. There were 25 animals/sex in each gr oup examined. The pathology summary table is on page 439 to 471 of the study report.

Conclusions: The chronic NOAEL was established at 250 ppm (equa l to 13.2 mg/kg bw/day) in male rats, based on an increased incidence of accumulation of alveolar macrophages at 3,500 ppm (equal to 189 mg/kg bw/day). Some evidence of testicular toxicit y was observed in a total number of 25 male rats at 7,500 ppm (equal to 414 mg/kg bw/day) examined histopatho logically after one year of treatment. The chronic NOAEL in females was established at 3,5 00 ppm (equal to 255 mg/kg bw/day), based on decreased body weight and body weight gain, yellow and brown staining in the perigenital area and tail

, discoloration of the lung and increased incidence o f accumulation of alveolar macrophages at 12,000 pp m (equal to 890 mg/kg bw/day).

CITATION: Whale B. (2006). Technical Grade BYI 08330 (Common Name Spirotetramat): An

Oncogenicity Testing Study in the Rat. Bayer Crop Science LP, Stilwell, KS, May 31, 2006, Unpublished.

I. Material and methods

A. Materials:

1. Test Material: BYI 08330 Description: Technical grade, white powder Lot/Batch No.: 08045/0014

Purity: 97.5% (7/2003); 97.8% (12/2003); 97.6% (6/2004); 98 .5% (1/2005); 97.4% (5/2005); 98.1% (11/2005) Compound Stability Stable at room temperature (~ 22 o C) Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate CAS No. of TGAI: trans : 382608-10-8; cis : 203313-25-1

2. Vehicle and/or positive control: None

3. Test animals: Species: Rat; males and females (nulliparous and n on-pregnant) Strain: Wistar Hanover rats (Crl:WI[Glx/BRL/Han]IG S BR) Age/weight at treatment initiation: Approximately 7 weeks old / 204.7 - 206.5 g mean gr oup weight males; 151.1 - 152.2 g mean group weight females Source: Charles River Laboratories, Inc. (Raleigh, NC) Housing: Individually housed in suspended stainles s steel wire-mesh cages, each containing a feeder, a source of water (pressure-ac tivated water nipples), and deotized (sanitized) cage board in the bedding tray. One nylabone® was placed in the cage of each animal. Cage racks were rotated monthly. Diet: Purina Mills Certified Rodent Diet 5002 in " meal" form; ad libitum Water: Municipal water supply of Kansas City, MO; ad libitum

.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 230 April 2008

Environmental conditions: Temperature: Humidity:

Air changes: Photoperiod: 18 to 26 o C

30 to 70% (relative); high range for relative humid ity was 72% on Days 232, 295, 436, and 540; 76% on Day 541; 71% on Days 548 and 562; and 75% on Day 569. 10.08 changes/hr (minimum daily average) 12 hr of light [7:00 a.m. to 7:00 p.m., or 8:00 a.m . to 8:00 p.m.] alternating with 12 hr of darkness. Deviations in the light/dark cycle occurred 8 times (3 planned; 5 unplanned) over the course of the study. The light cycle is unknown for Days 343 (Edstrom version 5 installed), 378 (from 6 p.m. through 12:0 0 a.m.), 379 (for approx. 1.5 hours; Edstrom system backup and reconfiguration), and 405 (power outage, smart box damaged by lightning strike). None of th e deviations was of a duration as to impact study
outcome. Acclimatisation: 07/07/03 (receipt) - 07/14/03 (an imals release for study)

B. Study design:

1. In-life dates: Start: 07/11/03 (study initiation); 07/14/03 (rele ased for study); 07/16/03 (initiation of exposure; males); 07/1 7/03 (initiation of exposure; females) End: 07/21/05 (terminal sacrifice complete)

2. Animal Assignment/Dose Levels: 07/14/03 (weighed) and 07/15/03 (randomized); assig ned to a control or one of three chemically-treated groups (as noted in Table 6-72) using a weight stratification-based computer program obtained from INSTEM Computer Syst ems (Stone, Staffordshire, UK). Each animal on study was identified with a microchip (IPTT-200; Bi oMedic Data Systems, Inc., Seaford, DE) subcutaneou sly implanted on their dorsal surface in the region bet ween the scapulae; the chip was encoded with a uniq ue number, specifying the animal's sex, dose group, ca ge number, and study affiliation.

Table B.6-72: Study design Main Study
24 months Test Group Conc. in Diet (ppm) Dose to animal (mg/kg; x+ Std)

Male Female

Control 0 0 55 55

Low (LDT) 250 males 12.5 + 4.4
females 16.8 + 3.9 55 55

Mid (MDT) 3,500 males 169 + 60
females 229 + 53 55 55

High (HDT) males 7,500 females 12,000 males 373 + 134
females 823 + 157 55 55

3. Dose Selection: Doses were based principally upon the toxicological profile which emerged in the rat over the course o f a subchronic toxicity testing study.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 231 April 2008

In that study, the toxicological response of the ma le rat was principally characterized by decreased r ate of body weight gain, as well as structural and/or func tional alterations in testes- and lung-related endp oints. A decline in rate of body weight gain of 17.6% was noted in 10,000-ppm males; a corresponding decline of 8.4% was noted in absolute body weight. Extrapolat ing these subchronic changes out over a prolonged lifetime exposure (nominally 2 years), which may ha ve increased the severity of the body weight effect , a high-dose of 7,500 ppm was chosen for males in the present study. In the female, the toxicological response was chara cterized by a structural and/or functional alterati on in the lung (the same lesion noted in 10,000-ppm males ). The lung lesion was considered a sign of genera l toxicity, and progression over the 2-year period co uld not be excluded. However, unlike the male, a c lear body weight effect (a classic predictor of a maxima lly-tolerated dose; MTD) was not established. Thes e results suggested a different sensitivity in the tw o sexes, indicating that females could be treated w ith concentrations somewhat higher than males to achiev e roughly the same effect (i.e., males are more sensitive than females). Thus, a high-dose of 12,0 00 ppm was chosen for females in the present study.

It was anticipated that the low and high doses of 2 50 ppm and 7,500 / 12,000 ppm would constitute a noobserved-adverse-effect level and a maximum tolerated dose , respectively, with the intermediate dosage of 3,500 ppm serving to confirm any dose response r elationships that may have emerged. The probable routes of human exposure to BYI 08330 are via inges tion of foodstuffs that might contain low residues of the test substance, accidental ingestion, or dermal contact during manufacture or use. Thus, formulat ion with the feed was an appropriate route of administr ation with which to develop the chronic toxicologic al profile of the test substance in a given test speci es.

4. Diet preparation and analysis: The test substance was mixed directly with the feed ; control diet consisted of untreated feed. Sample s of each batch of feed mixed were taken and retained in the freezer until the study was complete and the analytical data deemed satisfactory. Replacement a dmixtures for each treatment group were prepared weekly and stored under freezer conditions until pr esented to the animals the following week. BYI 083 30 was administered at a constant concentration in the feed for the duration of the study. The concentra tion of the AI of the test substance in the control, 250 -, 3,500-, 7,500-, and 12,000-ppm test diets was analytically verified from samples taken during Wee ks 1, 2, 3, 6, 11, 15, 19, 24, 28, 32, 36, 40, 44, 48, 53, 57, 62, 66, 70, 74, 79, 83, 87, 92, 96, 100, and 10 5. In addition, one recovery sample was analyzed w ith each group of samples during the test concentration analyses. The homogeneity and stability of BYI 08330 when mixed in the dietary carrier was charact erized prior to the in-life phase of the study and again just prior to termination of the in-life phase of t he study. To evaluate the uniformity of distribution/homogene ity of the test substance in the diet, an analysis of feed containing nominal concentrations of 70 and 12,000 ppm BYI 08330 was conducted. Three (3) samples were taken for analysis from each of three distinct layers (top, middle, bottom; 9 samples total) in a Hobart mixing bowl. A homogeneous distribution of the tes t substance in the feed was defined in terms of a percent relative standard deviation (%RSD, same as coefficient of variation; CV), derived from the 9 samples taken, which was < 10%. Homogeneity was again analyzed, just prior t o termination, using one sample from the top, middle, and bottom mixing bowl s containing nominal concentrations of 250 and 12,000 ppm BYI 08330. To evaluate the stability of the test substance in the diet, a time and temperature analysis of feed containing nominal concentrations of 70 and 12,000 ppm BYI 08330 was conducted. Stability in the feed was assessed following 1 and 7 days of room tempera ture storage (~22 o C) and 7, 14, and 29, and 32 days of freezer storage (~ -23 o C; 12,000 ppm evaluated at 29 days; 70 ppm evaluate d at 32 days).

The stability analysis was carried out as follows: An initial sample was taken immediately after the test substance was mixed with the ration. Each batch of ration, mixed at the 2 concentrations, was then– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 232 April 2008

divided into 2 portions. One portion was placed in a room temperature environment, the other in the freezer. Feed samples which remained at room tempe rature, were sampled for analysis on Days 0, 1, and 7; feed samples which remained in the freezer, were sampled for analysis on Days 7, 14, 29 (12,000 ppm only), and 32 (70 ppm only). Chemical stability in the ration was defined in terms of that period of time that 80% of the test substance, relative to the initial concentration, was recoverable. The liquid chromatographic methodology used to perf orm the dietary analyses required for this study (i .e., homogeneity, stability, and concentration verificat ions) has been described previously.

Results:

Homogeneity Analysis: The mean concentrations of BYI 08330 in the feed, sampled from three distinct layers in the mixing bowl and containing a nominal concentration of either 70 or 12,000 ppm, were determined to be 68.9 ppm (range 64.9-73.2 ppm; %RS D = 3.8%) and 12,157 ppm (range 11,844-12,567 ppm; %RSD = 2.1%), respectively. Based on a %RSD < to 10%, BYI 08330 was judged to be homogeneously distributed in the feed over a concen tration range of 70-12,000 ppm. Homogeneity analysis conducted just prior to study termination again confirmed homogeneity of the test substance i n rodent ration. The mean concentrations of BYI 0833 0 in the feed, sampled from three distinct layers i n the mixing bowl and containing a nominal concentration of either 250 or 12,000 ppm, were determined to be 250 ppm (range 248-254 ppm; %RSD = 1.3%) and 11,288 ppm (range 11,245-11,374 ppm; %RSD = 0.7%), respectively.

Stability Analysis: Following 7 days of room temperature storage, the analytically-determined concentration of the AI of the test substance in th e 70- or 12,000-ppm admixture was determined to be 69.8 ppm (68.9 ppm on Day 0) and 11,948 ppm (12,157 ppm on Day 0), respectively. Following 29 and 32 days of freezer storage, the analytically-determ ined concentration of the AI of the test substance in the 12,000- and 70-ppm admixtures was determined to be 12,474 and 68.0 ppm, respectively (12,157 and 68.9 ppm, respectively on Day 0). BYI 08330 mixed in rodent ration was judged to be stable at room temperature for at least 7 days and following freez er storage for a minimum 29 days, over a concentrat ion range of 70-12,000 ppm.

Concentration Analysis: Mean analytical concentrations for each dose grou p were 249, 3,407, 7,342, and 11,722 ppm, ranging from 97-100% of the correspondi ng nominal concentrations of 250, 3,500, 7,500, and 12,000 ppm, respectively. The AI of the test subst ance was not detected in the control diet. Mean recovery was 98% and ranged from 88-105% for rodent ration spiked with 250, 3,500, 7,500, and 12,000 ppm BYI 08330.

5. Statistics: Continuous data that were examined statistically we re evaluated initially for equality or homogeneity of variance using Bartlett's test. Group means were f urther analyzed by a one-way analysis of variance (ANOVA) followed by Dunnett's test for pair-wise co mparisons. Frequency data were initially examined for trends; data suggestive of a potential effect were then statistically evaluated using the chi-square, Fisher exact, or chi-square and Fisher exact tests. On a case-by-case basis, and at the discretion of the st udy director, data were subject to additional statistic al procedures other than those mentioned above (e.g

., Thakur "TREND" program, Ajit, K. Thakur). For the Bartlett test, a probability (p) value < 0.001 was considered significant; for all other statistical t ests, differences with p values < 0.05 were considered statistically significant. All statistical evaluat ions were performed using software obtained from ei ther INSTEM Computer Systems or SAS Institute Inc.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 233 April 2008

C. Methods:

1. Observations
2. 1a. Cageside Observations: For approximately 105 weeks of the study, a general assessment of all remaining animals was carried ou t at least daily (generally twice daily during the no rmal work week and once daily on weekends and holidays). These assessments were generally with r espect to moribundity and mortality, but also inclu ded a more general clinical observation for readily app arent clinical signs (in addition to moribundity an d mortality).

1b. Clinical Examinations: Once each week for approximately 104 weeks of the s tudy, detailed physical examinations for clinical signs of toxicity were performed on all surviving a nimals. These examinations included evaluation of external surface areas (visual and palpation), orif ices, posture, general behavior, respiration, and excretory products.

2. Body weight: Individual body weight determinations were carried out weekly for the first 13 weeks of the study and every 4 + 1 weeks thereafter on all surviving animals. Body weights were also measured immediately prior to all necropsies to allow for calculation of organ/body weight ratios with the following except ions. Terminal body weights for animals RK3010 (term sac) and RK3151 (sacrificed in extremis ) were not measured. Final live body weights were used to cal culate organ/body weight ratios as appropriate.

3. Food consumption and compound intake: Individual food consumption determinations were car ried out weekly for the first 13 weeks of the study and every 4 + 1 weeks thereafter on all surviving animals. In a ddition, using specifically defined criteria, food consumption data were corrected, as conditions dict ated, to account for misleading indications of food intake (i.e., excessive spillage, clogged feeders, etc.). Test substance intake (mg/kg body wt/day) w as calculated using weekly mean body weight and food c onsumption data. The general relationship used for this calculation was: [AI in feed (ppm)/1,000] x [feed consumed (g/kg bod y wt/day)] = mg AI/kg body wt/day

4. Ophthalmologic examination: Ophthalmologic examinations were not conducted.

5. Hematology & Clinical Chemistry: Blood was collected at approximately 12, 18, and 24 months into the study from all surviving animals f or differential determinations. In all cases, blood w as sampled via the orbital sinus from non-fasted an imals. Differential blood counts were determined on all an imals sacrificed due to moribundity with the except ion of RK1022 (had been bled for this purpose earlier i n the week; scheduled blood collection). Clinical Chemistry determinations were not performe d.

5a. Hematology:
Hematocrit (Hct) X Leukocyte differential count* Hemoglobin (Hgb) Mean corpuscular Hgb (MCH) Leukocyte count (WBC) Mean corpuscular Hgb concen tration (MCHC) Erythrocyte count (RBC) Mean corpuscular volume ( MCV) Platelet count (PLTS) Reticulocyte count (Retic)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 234 April 2008

Blood clotting measurements X Erythrocyte morpholo gy Thromboplastin time Heinz bodies (HZ) Clotting time Prothrombin time (PT) * Minimum required for carcinogenicity studies (Con t. and HDT unless effects are observed) based on Gu ideline
870.4200 & OECD 451. The CHECKED (X) parameters were examined.

6. Urinalysis: Urinalysis determinations were not performed.

7. Sacrifice and Pathology:

7a. Gross pathology: A complete postmortem examination was conducted on all animals (surviving, found dead, sacrificed in extremis ) which included (1) documenting and saving all gro ss lesions, (2) weighing designated organs, and (3) collecting representative tissue specimens for histopathologic evaluation.

Animals surviving at termination were sacrificed by CO 2 asphyxiation. Tissues were preserved and organ/body weight ratios were calculated for weighe d organs.

DIGESTIVE SYSTEM CARDIOVASC./HEMAT. NEUROLOGIC Bile duct* (rat) X Aorta* XX Brain*+
X Cecum* X Bone marrow* X Cerebellum
X Colon* XX Heart*+ X Cerebrum-Midbrain X Duodenum* X Lymph node, cervical* X Medulla/Pons X Esophagus* X Lymph node, mesenteric* X Eyes* X Gall bladder* (not rat) XX Spleen*+ X Nerve, opti c* X Ileum* X Thymus X Nerve, sciatic* X Jejunum* X Pituitary*

XX Liver*+ UROGENITAL X Spinal cord, cervical* X Pancreas* X Cervix X Spinal cord, lumbar* X Rectum* X Clitoral gland^ X Spinal cord, thoracic * X Salivary glands* X Epididymides*+ X Stomach, glandular* XX Kidneys*+ OTHER X Stomach, non-glandular* X Mammary gland* X Bone, femur X Tongue XX Ovary*+ X Bone, rib/cc jct X Tooth X Preputial gland^ X Bone, sternum
X Prostate* X Gross lesions and masses*
RESPIRATORY X Seminal vesicle* X Harderian gland X Larynx* XX Testicle*+ X Joint, fem/tib XX Lung*++ X Urinary bladder* X Muscle, protocol X Nasal structure* X Uterus*+ X Physical Identifier (ID chip) (1) X Nasopharynx* X Vagina^ X Skin, protocol* X Oral structure X Zymbal’s gland^

X Trachea* GLANDULAR– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 235 April 2008

XX Adrenal gland*+ X Exorbital/lacrimal gland^ X Parathyroid* X Thyroid* * Required for carcinogenicity studies based on Gui deline 870.4200. + Organ weight required in carcinogenicity studies. ++ Organ weight required if inhalation route. The CHECKED (X) tissues were collected for histolog ical examination. The organs CHECKED (XX) were als o weighed. The organs CHECKED (^) were preserved for possible micropathologic evaluation only. (1) The ID chip for animal RK1001 was cut during th e necropsy process. The tail was marked with an in delible marker and retained as the physical identifier.

7b. Micropathology: With the exception of the physical identifier (micr ochip or tail), the vagina, and the exorbital/lacri mal, clitoral, preputial, and Zymbal glands (of which th e latter 5 were taken and preserved for possible micropathologic evaluation), representative section s of the tissues shown in Table 6-76 were processed for all control (0 ppm) and high-dose (7,500 / 12,0 00 ppm) animals, embedded in paraffin, sectioned, mounted, and stained with hematoxylin and eosin (H& E) for examination under the light microscope. Tissues in which compound-related effects were dete cted were examined at lower doses as necessary to establish no-observed-effect levels. Target organs and gross lesions were examined for all animals. Histopathological lesions were assigned a semi-quan titative ranking of the severity grade/stage from "Normal (within normal limits)" to the "most severe case (Grade 5)," as described previously. The "gr ade number" was used to calculate an average grade for a particular observation within a dose group. Neoplastic lesions were generally not assigned a gr ade.Tissues from approximately 101 animals were processed as described above by Charles River Labor atories, Pathology Associates (Kansas City, MO). These animals represented all early deaths through day 682 (05/28/05). All other histology was perfor med by in-house (Bayer CropScience LP - Toxicology). Th e initial micropathologic evaluation of this study was performed by Dr. Barry Stuart (Bayer CropScience LP - Toxicology). An internal peer review was later conducted by Dr. Stuart and Dr. Steve Lake, and inc luded examination of various selected and randomlyselected slides.

II. Results

Findings: General observations: Survival was unaffected by treatment, as the incid ence of mortality was comparable between groups. Early deaths/survival nu mbers in males were 15/40, 21/34, 11/44, 20/35 and 16/39, 16/39, 18/37, 13/42 in females in ascending order of dose levels. Clinical observations reveale d an increased incidence of “body, scaly” and yellow and brown staining on the perigenital, perianal area a nd tail in 7500 ppm males and 12,000-ppm females. Feed intake was not toxicologically relevantly affected

. Significant decreases in mean body weight and body weight gain were noted in both sexes at the highest doses tested. Based on final body weight measureme nts (week 103), body weight declines of 10% and 14% were seen for 7,500-ppm males and 12,000-ppm fe males, respectively.

Table B.6-73: Incidence of treatment-related clinic al observations DOSE GROUP (PPM) PARAMETER 0 250 3,500 7,500/12,000
MALES STAINS, YELLOW, PGA 6 9 5 16* STAINS, BROWN # 1 5 2 9*– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 236 April 2008

BODY, SCALY 6 5 7 13
FEMALES STAINS, YELLOW, PGA 9 9 13 31* STAINS, BROWN # 0 1 6* 23* BODY, SCALY 1 0 3 15* * = p < 0.05; # includes tail, perigenital area (PGA) and perianal locations Note added by the HED reviewer: The clinical obser vations are reported on Pages 89 to 127 of the stud y report.– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism237April 2008Table B.6-74: Mean body weights (g) and cumulativebody weight gain (g)DOSE GROUP (PPM)PARAMETER0 250 3,500 7,500/12,000MALESINITIAL BODY WEIGHT 206.5 204.8 205.3 204.7FINAL BODY WEIGHT WEEK 103 629.4 596.7 619.5 564.9 *BODY WEIGHT GAIN WEEK 2 43.0 42.9 43.1 38.7 *MEAN BODY WEIGHT WEEK 13 433.1±4.4 432.7±5.7 428.6±5.5 408.9±4.3BODY WEIGHT GAIN WEEK 13 226.6 228.0 224.3 204.2*BODY WEIGHT GAIN WEEK 26 286.4 289.3 286.7 263.3*BODY WEIGHT GAIN WEEK 54 367.0 371.6 363.1 334.0*BODY WEIGHT GAIN WEEK 78 426.9 419.3 424.4 380.8 *BODY WEIGHT GAIN WEEK 103 422.0 399.9 414.9 361.2 *FEMALESINITIAL BODY WEIGHT 152.0 151.1 152.2 151.6FINAL BODY WEIGHT WEEK 103 386.6 393.3 398.0 331.8 *BODY WEIGHT GAIN WEEK 2 18.8 17.9 17.5 15.6 *MEAN BODY WEIGHT WEEK 13 243.3±2.5 237.5±2.3 239.3±3.1 230.5±2.8*BODY WEIGHT GAIN WEEK 13 91.2 86.4 87.1 78.9 *BODY WEIGHT GAIN WEEK 26 116.0 109.6 111.2 99.0*BODY WEIGHT GAIN WEEK 54 176.9 156.1* 163.0 134.2 *BODY WEIGHT GAIN WEEK 78 231.3 218.5 231.1 171.9*BODY WEIGHT GAIN WEEK 103 233.9 241.1 246.8 180.5 ** = p < 0.05Note added by HED reviewer. The mean body weight ±the standard deviation for week 13 were added by the HED reviewer to illustrate body weight variability. Mean bodyweight data are on pages 37 to 49 and mean body weight gain data are on pages 50 to 60 of the study report.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 238 April 2008

Hematology: Leukocyte differential counts and erythrocyte morp hology determined at approximately 12, 18, and 24 months into the study provided no indication of a BYI 08330-induced change in either sex at any dose tested.

Gross necropsy, organ weights: Gross necropsy showed an increased incidence of foc al to multifocal (1-4 mm) discoloured zones (sometimes raised) of the lun g in 12,000-ppm females (1, 1, 3, 32*) which were attributed to compound administration. Relative lun g weight was slightly increased in the 7,500 ppm ma le and the 12,000 ppm female group. The lung weight ch anges in 12,000 ppm females correlated with increased focal to multifocal discolorations observ ed in the lungs at necropsy. Absolute kidney weigh ts were decreased in both sexes at 3,500 and 7,500/12,000 p pm and were associated with microscopic findings noted in the same groups. Any other apparent change in absolute kidney weight (e.g., 250 ppm males) wa s considered to be incidental, due to the highly vari able kidney weight data (particularly in males) and the lack of microscopic evidence of a direct toxicological i nsult by the test substance on the kidney that woul d account for a decrease in kidney weight. All other apparent organ weight changes were attributable to changes in the body weight profile.

Table B.6-75: Mean absolute (g) and relative (%) or gan weights (for survivors – added by HED reviewer). DOSE GROUP (PPM) PARAMETER 0 250 3,500 7,500/12,000
MALES
FINAL BODY WEIGHT 620.6 600.6 614.8 566.6* LIVER WEIGHT,
ABSOLUTE 19.396±3.39 18.551 19.219 18.437±2.282

LIVER WEIGHT,
RELATIVE 3.119 3.104 3.133 3.274

LUNG WEIGHT,
ABSOLUTE 2.245±0.309 2.362 2.290 2.420±0.503

LUNG WEIGHT,
RELATIVE 0.366 0.402 0.377 0.435*

KIDNEY WEIGHT,
ABSOLUTE 4.329±0.926 3.910* 3.886* 3.697±0.533*

KIDNEY WEIGHT,
RELATIVE 0.709 0.659 0.635 0.662

FEMALES
FINAL BODY WEIGHT 377.0 395.4 396.5 328.2* LIVER WEIGHT,
ABSOLUTE 12.621±2.87 12.907 12.777 12.360±2.89

LIVER WEIGHT,
RELATIVE 3.379 3.295 3.245 3.779*

LUNG WEIGHT,
ABSOLUTE 1.949±0.478 1.899 1.852 1.934±0.303

LUNG WEIGHT,
RELATIVE 0.527 0.492 0.479 0.600*

KIDNEY WEIGHT,
ABSOLUTE 2.979±0.669 2.851 2.658* 2.572±0.313*

KIDNEY WEIGHT,
RELATIVE 0.820 0.741 0.691* 0.796– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 239 April 2008

* = p < 0.05. Standard deviation data added by the US EPA Reviewer. Mean summary data are on pages 833 to 836 in the study report. The number of animals examine d was the number of survivors.

Histopathology: Non-neoplastic changes : Statistically significant changes in the incidenc e of non-neoplastic lesions were detected in the lung, kidneys, testis/epididymis an d liver.

Lung : An increased incidence of alveolar macrophage acc umulation (M: 7, 17*, 17*, 3; F: 20, 14, 18, 3), a common background lesion in the r at, and a complex of changes described as interstit ial pneumonia (M: 22,15,19,44*; F: 4,13*,14*,52*) was n oted in the lungs of 7,500-ppm males, and 12,000-pp m females. Both findings were attributable to exposu re to the test substance and were interpreted as a continuum of morphologic changes (correlating with the gross lung observations noted in 12,000 ppm females) and a such were evaluated together (M: 29, 32, 36, 47*; F: 24, 27, 32, 55*). Macrophage accumulation without additional change was coded as such. Interstitial pneumonia was diagnosed with th e presence of one or more of the following changes: p resence of lymphocytes, cholesterol clefts, interst itial thickening of the alveolar septa by connective tiss ue, or increased alveolar pneumocytes and presence of occasional extravasation of erythrocytes (micro hem orrhage). The lung effects described above were foc al to multifocal in distribution and involved a small ove rall portion of the lung tissue triggering no respi ratory symptoms. It was noted by the HED reviewer that th e alveolar macrophages and interstitial pneumonia d id not occur in the same animal in female control, low and mid dose groups.

Kidney : An increased incidence of tubular dilatation was observed in the outer medulla (presumed to be in the loop of Henle) in both sexes of the 3,500 ppm g roups and in the 7,500/12,000 ppm groups. The affec ted renal tubules were dilated and were either empty or contained pale eosinophilic proteinacious material

, usually granular. Negligible cellular death, regene ration, or inflammatory responses to the tubular di latation was present. In addition, there were no associated tubular casts in more distal tubular locations such as the collecting ducts. This lesion in the 3,500 and 7,50 0/12,000 ppm animals was considered treatment-relat ed and was presumed to be reflected in the decreased k idney weights although there was no associated loss of tissue. It was also stated that the high backgroun d incidence of chronic nephropathy greatly influenc ed the variability of the kidney weights measured in this study which is commonly noted in 2-year rat studies

.

Testis : In males, there was a background incidence of typ ical aging changes including variably severe testicular degeneration, oftentimes noted grossly a s soft, brown, or reduced in size (12,8,10, and 6 f or the control, low, mid and high dose groups, respectivel y – added by HED reviewer). In addition to this cha nge in the 7,500-ppm males, there were a few animals with generally slight morphologic testicular change, mor e subtle than distinct tubular degeneration, characte rized by a depletion, asynchrony, and degeneration of the latter stage spermatids (0,0,0, and 9* for the cont rol, low, mid and high dose groups, respectively – added by HED reviewer).

Epididymis : An increased incidence of immature/exfoliated ger m cells/debris was observed in the lumen contents of the head, body, and tail of the epididy mides at 7,500 ppm (6,10,6, and 31* for the control , low, mid and high dose groups, respectively – added by t he HED reviewer), which correlates with the testicu lar change. There was no morphologic change noted in th e anatomical structure of the epididymis tissue in conjunction with the luminal content of exfoliated immature / abnormal testicular stages.

Liver : In the bile duct, a nonstatistical increased inci dence of hyperplasia/fibrosis with associated minim al periportal mononuclear cell infiltrate was noted in 12,000-ppm females.

Neoplastic changes:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 240 April 2008

Type, incidence and organ distribution of all neopl astic lesions was not significantly different betwe en treated and control rats.

Table B.6-76: Average incidence and severity (figur es in parenthesis) of microscopic findings DOSE GROUP (PPM) PARAMETER 0 250 3,500 7,500/12,000
MALES LUNG, MACROPHAGES,
ALVEOLAR 7(1.3) 17(1.2)
* 17(1.4)* 3(1.0)

LUNG, PNEUMONIA,
INTERSTITIAL 22 (1.6) 15(1.7) 19(1.7)(A)* 44(2.3)*

COMBINED 29 32 36 47* KIDNEY, DILATATION,
TUBULAR A
(N = 55 FOR ALL GROUPS) 0 0 8(1.8)* 19(1.9)*

KIDNEY, NEPHROPATHY,
CHRONIC 44(2.3) 47 (1.7) 43 (2.0) 42(1.8)

TESTIS, DEGENERATION 12(3.4) 8(3.5) 10(2.9 6(3.2) TESTIS, SPERMATID DEGNERATION, DEPLETION,
ASYNCHRONY 0 0 0 9(1.2)*

EPIDIDYMIS, N(B) GERM CELL, EXFOLIATED, DEBRIS 55 6 (2.2) 55

10(1.9) 54

6(1.8) 54

31(2.5)*

LIVER, BILIARY
HYPERPLASIA
/FIBROSIS 14(1.3) 1(1.0) 0 9(1.3)

FEMALES LUNG, MACROPHAGES,
ALVEOLAR 20(1.3) 14(1.1) 18(1.1) 3(1.7)

LUNG, PNEUMONIA,
INTERSTITIAL 4(1.5) 13(1.4)* 14(2.1)* 52(3.4)*

COMBINED 24 27 32 55* KIDNEY, DILATATION,
TUBULAR A
(N = 55 FOR ALL GROUPS) 0 0 16 (1.8)* 42(2.5)*

KIDNEY, NEPHROPATHY,
CHRONIC 31(1.9) 29(1.4) 22 (1.3) 22(1.6)

LIVER, BILIARY
HYPERPLASIA
/FIBROSIS 18(1.9) 24(1.7) 24(1.8) 32(1.7)

* = p < 0.05; a thin (descending) limb of the loop of Henle Notes added by the US EPA reviewer. The pathology summary reports are on pages 837 to 9 999. The individual animal sheets are on pages 100 0 to
2395. (a) asterisk removed from the original summary repo rt by the HED reviewer. (b) the number of animals examined was added by the reviewer.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 241 April 2008

Conclusions: The chronic (24 months) NOAEL was established at 2 50 ppm (equal to 12.5 mg/kg bw/day) in male rats, based on decreased absolute kidney weigh ts and an increased incidence of renal tubular dilatation at 3,500 ppm (equal to 169 mg/kg bw/day) . There was a slightly increased incidence of testi cular tubular degeneration and immature/exfoliated germ c ells in the epididymal lumen in 7,500 ppm male rats after two years of treatment (equal to 373 mg/kg bw /day). The chronic (24 months) NOAEL in females was establ ished at 250 ppm (equal to 16.8 mg/kg bw/day), based on decreased absolute kidney weights and an i ncreased incidence of renal tubular dilatation at 3 ,500 ppm (equal to 229 mg/kg bw/day). Based on type, in cidence and organ distribution of neoplastic lesion s in treated and control rats, there was no indication f or an oncogenic effect of BYI 08330.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 242 April 2008

B.6.5.2 Carcinogenicity study in the mouse (OECD An nex IIA 5.5.3)

CITATION : Whale, B. 2006 Technical grade BYI08830 (Common Na me Spirotetramat): An Oncogenicity

Testing Study in Mouse. Bayer Crop Science, Stilwel l, KS. March 13 (original) 17 (amended), 2006, Unpublished.

Reviewer’s Discussion

US EPA has prepared the following table to illustra te that there were no adverse effects on key parame ters:

Parameter Males

Control 70 1700 7000 Females
Control 70 1700 6000 Body Weight (g±SEM) b

Initial 30.7±0.3 30.6 30.7 30.8±0.3 25.1±0.3 24.6 24. 4 24.7±0.2
Week 1 32.4±0.3 32.3 32.6 32.5±0.3 25.8±0.2 25.6 2 5.5 25.4±0.2
Week 2 33.1±0.3 33.4 33.5 33.4±0.3 26.9±0.2 26.7 26.7 26.7±0.3
Terminal 43.5±0.7 43.0 43.0 42.3±0.7 36.5±0.8 36.8 35 .7 36.0±0.7 Liver weight (g±SEM) terminal b

Terminal 2.381±0.458 2.322 2.314 2 .307±0.395 1.922±0.305 1.925 2.052 2.059±0.437 Liver Histopathology (All animals) a

N Hyperplasia
Adenoma
Carcinoma 55 19 21 54

1 0 0 0
3 4 4 3
2 2 1 2 55 21 16 55
0 0 0 1
1 0 0 0
1 0 0 0

a All liver lesions are “hepatocellular”. Data are f rom page 936 (males) and 955 (females) of the study report.

b The SEM for the control and high dose only are pres ented. The SEM for the low and mid dose groups wer e similar. SD, not presented, is considered a more a ppropriate measure of variability around the mean t han SEM. Body weight data are from pages 33 (males) an d 34 (females) of the study report. Liver weight d ata are from pages 195 (males) and 196 (females).

Comments:

a. The subchronic study (2005; ) in mice suggest ed a possible slight body weight effect in the high dose during the first weeks of the study. No similar ef fect was noted in this definitive study in mice.

b. The mice are being dosed with the limit dose of spirotetramat. However, there was no indication of increased liver weight or hypertrophy/hyperplasia t hat would be associated with increased capacity to metabolize and facilitate the excretion of spirotet ramat.

c. The histopathology report indicated that the b rain had more incidents of mineralization in the hi gh dose group (13 in males and 12 in females) than in the c ontrol (6 in males and 8 in females). However, ther e was no increase in severity, with most cases being mini mal. Thus, no effect of spirotetramat on brain mineralization was concluded.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 243 April 2008

d. The rat subchronic (2005, Lab Report No. 20113 6), chronic (2005) and carcinogenicity (2006) studi es all indicated the presence of “alveolar macrophages” an d/or interstitial pneumonia (refer to the evaluatio for the rat carcinogenicity study for a discussion). There was no similar effect in the lung in this mouse st udy.

e. The rat feeding studies (see above) also suggest ed possible effects on the male reproductive system such as testicular degeneration, vacuolation, abnormal s permatozoa and hypospermia (refer to the DER for th e rat carcinogenicity study for a discussion). No si milar effects were noted in this mouse study.

Material and methods

A. Materials:

1. Test Material: 3-(2,5-Dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro [4.5]dec-3-en-4-yl ethyl carbonate (BYI 08330; comm on name spirotetramat) Description: Technical grade, white powder Lot/Batch #: 08045/0014

Purity: 97.5% (7/2003); 97.8% (12/2003); 97.6% (6/2004); 98 .5% (1/2005); 97.4% (5/2005) Compound Stability: Stable at room temperature (~ 22 o C) CAS # of TGAI: trans : 382608-10-8; cis : 203313-25-1

2. Vehicle and/or positive control: None

3. Test animals: Species: Mouse; male and female (nulliparous and n on-pregnant) Strain: CD-1; (CD-1 [ICR]/BR) Age/weight at study initiation: Approximately 8 weeks old / 30.6 - 30.8g mean group weight males; 24.4 - 25.1g mean group weight females Source: Charles River Laboratories, Inc. (Kingston , NY) Housing: Housed in suspended stainless steel wire- mesh cages, each containing a feeder, a source of water (pressure-activated water nipples), and deotized (sanitized) cage board in the bedding tray. Animal s passing the initial shipment exam were co-housed during a portion of th e Acclimatization and later housed individually (for the remainder of the Acclimatization and throughout the exposure period). Cage racks were r otated monthly. Diet: Purina Mills Certified Rodent Diet 5002 in " meal" form; ad libitum Water: Municipal water supply of Kansas City, MO; ad libitum

.– –

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Environmental conditions: Temperature: Humidity:

Air changes: Photoperiod 18 to 26 o C

30 to 70% (relative); high range relative humidity was 71% on Days -10 (pre-exposure) and 39, and 73% on Day 1 1. 13.58 changes/hr (minimum daily average) 12 hr of light [7:00 a.m. to 7:00 p.m., or 8:00 a.m . to 8:00 p.m.] alternating with 12 hr of darkness. Deviatio ns in the light/dark cycle occurred 3 times (2 planned; 1 unp lanned) over the course of the study. The light cycle is u nknown for Days 202 (Edstrom version 5 installed) and 234 (pow er outage occurred, smart box replaced due to lightnin g strike). None of the deviations was of a duration as to impa ct study
outcome. Acclimatisation: 10/20/03 (receipt) - 10/27/03 (re lease for study)

B. Study design:

1. In-life dates : Start: 10/27/03 (released for study); 10/28/03 (st udy initiation); 11/03/03 (initiation of exposure) End: 05/11/05 (terminal sacrifice complete)

2. Animal Assignment/Dose Levels: 10/28/03; assigned to a control or one of three che mically-treated groups (as noted in the table below) using a weight stratification-based computer program obtained fro m INSTEM Computer Systems (Stone, Staffordshire, UK). Each animal on study identified with a microchip (BioMedic Data Systems, Inc., Seaford, DE) subcutan eously implanted on their dorsal surface in the reg ion between the scapulae; the chip was encoded with a u nique number, specifying the animal's sex, dose gro up, cage number, and study affiliation.

Main Study
18 months Test Group Conc. in Diet (ppm) Dose to animal (mg/kg; + SD)
Male Female

Control 0 0 55 55

Low (LDT) 70 m: 10.9+ 1.4
f: 13.7+ 2.2 55 55

Mid (MDT) 1,700 m: 263+ 35
f: 331+ 49 55 55

High (HDT) 7,000 (1) m: 1022+ 196
f: 1319+ 290 55 55

(1) The high-dose level for this study was initiate d with a dietary concentration of 7,000 ppm. Starting with Week 12 of the study, this level was reduced to 6,000 ppm in order to achieve an average active ingredient intake of approximately 1,000 mg/kg/day through 18 months of
exposure.

3. Dose selection rationale: Doses were based principally upon the lack of a tox icological response in the mouse over the course of a subchronic toxicity testing study at doses up to an d including 7,000 ppm (Report No. 201284). It was– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 245 April 2008

anticipated that the low and high doses chosen of 7 0 ppm and 7,000 would constitute a no-observedadverse-effect level

and a limit dose , respectively, with the intermediate dosage of 1,7 00 ppm serving to confirm any dose response relationships that may ha ve emerged. The probable routes of human exposure to BYI 08330 are via ingestion of foodstuf fs that might contain low residues of the test substance, accidental ingestion, or dermal contact during manufacture or use. Thus, formulation with the feed was an appropriate route of administration wit h which to develop the toxicological profile of the test substance in a given test species.

4. Diet preparation and analysis: The test compound was mixed directly with the feed; control diet consisted of untreated feed. Samples of each batch of feed mixed were taken and retained in the freezer until the study was complete and the analytical data deemed satisfactory. Replacement a dmixtures for each treatment group were prepared weekly and stored under freezer conditions until pr esented to the animals the following week. BYI 08330 was administered at a constant concentrat ion in the feed for the duration of the study. The concentration of the AI of the test substance in th e control, 70-ppm, and 1,700-ppm test diets was analytically verified from samples taken during Wee ks 1, 2, 3, 8, 12, 16, 20, 24, 28, 32, 37, 41, 46, 50, 54, 58, 63, 67, 71, and 76. The concentration of the A I of the test substance was analytically verified f rom samples of the 7,000-ppm test diet taken during Wee ks 1, 2, 3, and 8, and from samples of the 6,000- ppm test diet taken during Weeks 12, 16, 20, 24, 28 , 32, 37, 41, 46, 50, 54, 58, 63, 67, 71, and 76. In addition, one recovery sample was analyzed with eac h group of samples during the test concentration analyses. The homogeneity and stability of BYI 083 30 when mixed in the dietary carrier was characterized prior to the in-life phase of the stu dy and again just prior to termination of the in-li fe phase of the study. To evaluate the uniformity of distribution/homogene ity of the test substance in the diet, an analysis of feed containing nominal concentrations of 70 and 12 ,000 ppm BYI 08330 was conducted. Three (3) samples ere taken for analysis from each of 3 disti nct layers (top, middle, bottom; 9 samples total) i n a Hobart mixing bowl. A homogeneous distribution of the test substance in the feed was defined in terms of a percent relative standard deviation (%RSD, sam e as coefficient of variation; CV), derived from th e 9 samples taken, which was < 10%. Homogeneity was again analyzed, just prior t o termination, using 1 sample from the top, middle, and bottom mixing bowl s containing nominal concentrations of 70 and 6,000 ppm BYI 08330. To evaluate the stability of the test substance in the diet, a time and temperature analysis of feed containing nominal concentrations of 70 and 12,000 ppm BYI 08330 was conducted. Stability in the feed was assessed following 1 and 7 days of room tempera ture storage (~22 o C) and 7, 14, and 29, and 32 days of freezer storage (~ -23 o C; 12,000 ppm evaluated at 29 days; 70 ppm evaluate d at 32 days). The stability analysis was carried out as follows: An initial sample was taken immediately after the test substance was mixed with the ration. Each batch of ration, mixed at the 2 concentrations, was then divided into 2 portions. One portion was placed in a room temperature environment, the other in the freezer. Feed samples which remained at room tempe rature were sampled for analysis on Days 0, 1, and 7; feed samples which remained in the freezer w ere sampled for analysis on Days 7, 14, 29 (12,000 ppm only), and 32 (70 ppm only). Chemical stabilit y in the ration was defined in terms of that period of time that 80% of the test substance, relative to th e initial concentration, was recoverable. The liqu id chromatographic methodology used to perform the die tary analyses required for this study (i.e., homogeneity, stability, and concentration verificat ions) has been described previously.

Results:

Homogeneity Analysis: The mean concentrations of BYI 08330 in the feed, sampled from 3 distinct layers in the mixing bowl and containing a nominal concentration of either 70 or 12,000 ppm, were– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 246 April 2008

determined to be 68.9 ppm (range 64.9-73.2 ppm; %RS D = 3.8%) and 12,157 ppm (range 11,844-12,567 ppm; %RSD = 2.1%), respectively. Based on a %RSD < to 10%, BYI 08330 was judged to be homogeneously distributed in the feed over a concen tration range of 70-12,000 ppm. Homogeneity analysis conducted just prior to study termination again confirmed homogeneity of the test substance i n rodent ration. The mean concentrations of BYI 0833 0 in the feed, sampled from 3 distinct layers in th e mixing bowl and containing a nominal concentration of either 70 or 6,000 ppm, were determined to be 65.8 ppm (range 61.9-70.1 ppm; %RSD = 6.3%) and 5,9 51 ppm (range 5,923-5,993 ppm; %RSD = 0.6%), respectively.

Stability Analysis: Following 7 days of room temperature storage, the analytically-determined concentration of the AI of the test substance in th e 70- or 12,000-ppm admixture was determined to be 69.8 ppm (68.9 ppm on Day 0) and 11,948 ppm (12,157 ppm on Day 0), respectively. Following 29 and 32 days of freezer storage, the analytically determ ined concentration of the AI of the test substances in the 12,000- and 70-ppm admixtures was determined to be 12,474 and 68.0 ppm, respectively (12,157 and 68.9 ppm, respectively on Day 0). BYI 08330 mi xed in rodent ration was judged to be stable at roo m temperature for at least 7 days and following freez er storage for a minimum 29 days, over a concentration range of 70-12,000 ppm.

Concentration Analysis: Mean analytical concentrations for each dose grou p were 67.7, 1,655, 5,986, and 6,873 ppm, ranging from 97-100% of the correspo nding nominal concentrations of 70, 1,700, 6,000, and 7,000 ppm, respectively. The AI of the test su bstance was not detected in the control diet. Mean recovery was 97% and ranged from 89-103% for rodent ration spiked with 70, 1,700, 6,000, and 7,000 ppm BYI 08330.

5. Statistics: Continuous data that were examined statistically we re evaluated initially for equality or homogeneity of variance using Bartlett's test (Snedecor and Cochra n, 1967). Group means were further analyzed by a one-way variance analysis (ANOVA) (Snedecor and Coc hran, 1967) followed by Dunnett's test (Dunnett, 1955, 1964). Frequency data were initially examine d for trends; data suggestive of a potential effect were then statistically evaluated using the chi-squ are, Fisher exact, or chi-square and Fisher exact t ests. On a case by case basis, and at the discretion of t he study director, data were subject to additional statistical procedures other than those mentioned a bove. For the Bartlett test, a probability (p) val ue < 0.001 was considered significant; for all other sta tistical tests, differences with p values < 0.05 were considered statistically significant. All statisti cal evaluations were performed using software obtai ned from either INSTEM Computer Systems or SAS Institut e Inc. (Cary, NC).

C. Methods:

1. Observations:

1a. Cageside Observations: For approximately 79 weeks of the study, a general assessment of all remaining animals was carried out at least daily (generally twice daily during the no rmal work week and once daily on weekends and holidays). These assessments were generally with r espect to moribundity and mortality, but also included a more general clinical observation for re adily apparent clinical signs (in addition to morib undity and mortality).

1b. Clinical Examinations:– –

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Once each week for approximately 79 weeks of the st udy, detailed physical examinations for clinical signs of toxicity were performed on all surviving a nimals. These examinations included evaluation of external surface areas, orifices, posture, general behavior, respiration, and excretory products.

2. Body weight: Individual body weight determinations were carried out weekly for the first 13 weeks of the study and every 4 + 1 weeks thereafter on all surviving animals. Body weights were also measured immediately prior to all necropsies to allow for calculation of organ to body weight ratios.

3. Food consumption and compound intake: Individual food consumption determinations were car ried out weekly for the first 13 weeks of the study and every 4 + 1 weeks thereafter on all surviving animals. In a ddition, using specifically defined criteria, food consumption data were corrected, as conditions dictated, to account for misleading indications of food intake (i.e., excessive spillage, clogged feed ers, etc.). Test substance intake (mg/kg body wt/d ay) was calculated using weekly mean body weight and fo od consumption data. The general relationship used for this calculation was:

[AI in feed (ppm)/1,000] x [feed consumed (g/kg bod y wt/day)] = mg AI/kg body wt/day

4. Ophthalmologic examination: Ophthalmologic examinations were not conducted.

5. Hematology & Clinical Chemistry: Blood was collected at approximately 12 and 18 mont hs into the study from all surviving animals for differential determinations with the exception of a nimal RM3155 at 12 months. In all cases, blood was sampled via the orbital sinus from non-fasted anima ls. Differential blood counts were determined on a ll animals sacrificed due to moribundity with the foll owing exceptions: RM1105, RM1131, RM2006, and RM2051. Clinical Chemistry determinations were not performed.

5a. Hematology
Hematocrit (Hct) X Leukocyte differential count* Hemoglobin (Hgb) Mean corpuscular Hgb (MCH) Leukocyte count (WBC) Mean corpuscular Hgb concen tration (MCHC) Erythrocyte count (RBC) Mean corpuscular volume ( MCV) Platelet count (PLTS) Reticulocyte count (Retic) Blood clotting measurements X Erythrocyte morpholo gy (Thromboplastin time) Heinz bodies (HZ) (Clotting time) Prothrombin time (PT) * Minimum required for carcinogenicity studies (Con t. and HDT unless effects are observed) based on
Guideline 870.4200 & OECD 451. The CHECKED (X) parameters were examined.

6. Urinalysis: Urinalysis determinations were not performed.

7. Sacrifice and Pathology:– –

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7a. Gross pathology: A complete postmortem examination was conducted on all animals (surviving, found dead, sacrificed in extremis ) which included (1) documenting and saving all gro ss lesions, (2) weighing designated organs, and (3) collecting representative tissue specimens for histopathologic evaluation. Animals surviving at termination were sacrificed by CO 2 asphyxiation. Tissues were preserved and organ/bo dy weight ratios were calculated for weighed organs.

DIGESTIVE SYSTEM CARDIOVASC./HEMAT NEUROLOGIC Bile duct* (rat) X Aorta* XX Brain*+
X Cecum* X Bone marrow* X Cerebellum
X Colon* XX Heart*+ X Cerebrum-Midbrain X Duodenum* X Lymph node, cervical* X Medulla/Pons X Esophagus* X Lymph node, mesenteric* X Eyes* X Gall bladder* (not rat) XX Spleen*+ X Nerve, opti c* X Ileum* X Thymus X Nerve, sciatic* X Jejunum* X Pituitary*
XX Liver*+ UROGENITAL X Spinal cord, cervical* X Pancreas* X Cervix X Spinal cord, lumbar* X Rectum* X Clitoral gland^ X Spinal cord, thoracic * X Salivary glands* X Epididymides*+ X Stomach, glandular* XX Kidneys*+ OTHER X Stomach, non-glandular* X Mammary gland* X Bone, femur X Tongue XX Ovary*+ X Bone, rib/cc jct X Tooth X Preputial gland^ X Bone, sternum
X Prostate* X Gross lesions and masses*
RESPIRATORY X Seminal vesicle* X Harderian gland X Larynx* XX Testicle*+ X Joint, fem/tib XX Lung*++ X Urinary bladder* X Muscle, protocol X Nasal structure* X Uterus*+ X Physical Identifier (ID chip) (1) X Nasopharynx* X Vagina^ X Skin, protocol* X Oral structure X Zymbal’s gland^

X Trachea* GLANDULAR XX Adrenal gland*+ X Exorbital/lacrimal gland^ X Parathyroid* X Thyroid*

* Required for carcinogenicity studies based on Gui deline 870.4200. +Organ weight required in carcinogenicity studies. ++Organ weight required if inhalation route. The CHECKED (X) tissues were collected for histolog ical examination. The organs CHECKED (XX) were also weighed. The organs CHECKED (^) were preserve d for possible micropathologic evaluation only. (1) Starting on 04/15/05 (nominal Day 529), indeli ble tail marks (animal number) were used in lieu of replacing defective or "non-reading" ID chips. In such cases, the tail was collected.

7b. Micropathology: With the exception of the physical identifier (micr ochip and/or tail), the vagina, and the exorbital/l acrimal,– –

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clitoral, preputial, and Zymbal glands (of which th e latter 5 were taken and preserved for possible micropathologic evaluation), representative section s of the tissues shown in the table above were processed for all control (0 ppm) and high-dose (7, 000 ppm) animals, embedded in paraffin, sectioned, mounted, and stained with hematoxylin and eosin (H& E) for examination under the light microscope. Tissues in which compound-related effects were dete cted were examined at lower doses as necessary to establish no-observed-effect levels. Target organs and gross lesions were examined for all animals. Histopathological lesions were assigned a semi-quan titative ranking of the severity grade/stage from "Normal (within normal limits)" to the "most severe case (Grade 5)," as described previously (Squire, 1989; Hardisty, 1990). The "grade number" was used to calculate an average grade for a particular observation within a dose group. Neoplastic lesion s were generally not assigned a grade. The initia l micropathologic evaluation of this study was perfor med by Dr. Laura Elcock (Charles River Laboratories

, Pathology Associates, Kansas City, MO). This evalu ation was later reviewed by pathologists associated with Bayer CropScience LP, and included examination of various selected and randomly-selected slides.

II. Results

Findings: General observations: Survival was unaffected by treatment, as the incid ence of mortality was comparable between groups. Early deaths/survival nu mbers in males were 12/43, 13/42, 14/41, 11/44 in males and 19/36, 20/35, 14/41, 18/37 in females in ascending order of dose levels. Clinical observati ons attributable to exposure to the test substance were not observed in either sex at any dose tested. Th e general observation profile was consistent with tha t of the aging mouse.

Body Weight: (Note: See also the body weight table prepared by t he HED reviewer at the beginning of this document). There were no treatment-related eff ects on body weight and food consumption in either sex at any dose tested.

Hematology: Evaluation of the data from blood collected at appr oximately 12 and 18 months into the study provided no indication of a BYI 08330-induced change in either sex at any dose tested. Determinations included leukocyte differential coun ts and erythrocyte morphology only.

Gross necropsy, organ weights: Evaluation of the general profile of absolute and r elative organ weights as well as of gross lesions observed in thi s study did not indicate a BYI 08330-induced change in either sex at any dose tested.

Histopathology: Non-neoplastic changes: Microscopic findings (non-neoplastic) attributable to exposure to the test substance were not observed in this study.

Neoplastic changes: Microscopic findings (neoplastic) attributable to e xposure to the test substance were not observed in this study. Two incidences of tumors arising from brown fat were noted in high-dose males. Microscopicall y, the tumors were determined to be benign, and were c omposed of well-differentiated cells that resembled brown fat. However, there were occasional cells th at appeared to originate from white fat. As such, the term “lipomatous neoplasm” was assigned to the lesi on. In both cases, the tumors were noted grossly (1.5 to 2.0 cm in size) and were located in the int erscapular region. Both animals lived until study termination. Similar tumors were not observed in fe males. The report of this type of tumor is rare. One consideration that was made concerned the use of im plantable identification transponders (ID chips),– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 250 April 2008

which were placed between the scapula (subcutaneous ly) prior to study. No direct evidence of involvement was documented in this study; however, implantable transponders (microchips) have been associated with the formation of various other tumo r types in both mice and rats (Elcock et al ., 2001: Tumors in long-term rat studies associated with mic rochip animal identification devices. Exp Toxic Pat hol 52 , 483-491; Ghanta and Edmondson, 1990: Tissue react ion to an implantable identification device in mice. Toxicol Pathol 18 (3), 412-416.; Palmer et al ., 1998: Fibrosarcomas associated with passive integrated transponder implants. Toxicol Pathol (meeting abstract) 26 (1), 170). Any possible influence of the transponder (i.e., foreign-body tumorigenesis) under the circumstances described for this specific case is unclear. In this study, the data suggest t hat these tumors were spontaneous in nature. The incidence in the high dose was not significantly di fferent from controls and no similar tumors were observed in any female animal. Both tumors were be nign in nature and did not result in premature mortality in either case.

III. Conclusion

The NOAEL of the chronic feeding study in mice was established at 7,000/6,000 ppm (equal to 1022 or 1319 mg/kg bw/day in males or females, respectively ), based on the absence of compound-induced toxicological responses.

Based on type, incidence and organ distribution of neoplastic lesions in treated and control mice, the re was no indication for an oncogenic effect of BYI 08 330.– –

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B.6.5.3 Summary of long term toxicity/carcinogenici ty studies

The thymus and thyroid were also target organs foll owing one-year oral exposure of dogs to spirotetram at. In addition, the brain was also considered to be a tar get organ by PMRA. Thymus involution and brain dil ation with dose-related severity were observed in one mal e each at 600 ppm (20 mg/kg bw/day) and 1800 ppm (55 mg/kg bw/day). Brain dilation was observed in one female at the mid dose (600 ppm) only, and axon al degeneration in the hypothalamus was observed in on e female at 1800 ppm (48 mg/kg bw/day). PMRA considered these brain effects to be treatment-rela ted. Brain dilation has been observed in up to 5% o f Beagle dog colonies and is considered a congenital anomaly by Bayer CropScience. Brain dilation was n ot observed in any other study in the dog or in any ot her species tested with spirotetramat. For these r easons, and since brain dilation was also observed sporadic ally in several other Bayer dog studies of varying durations (from four weeks up to one year), USEPA c onsidered the observation to be equivocal at best a nd did not consider its use appropriate for risk asses sment. PMRA, however, considered brain dilation to be treatment-related. Clinical signs of neurotoxicity (dehydration, swelling, decreased activity and reac tivity, seizures and ataxia) were also observed in one male at the highest dose tested, a finding that was considered consistent by USEPA with the congenital brain dilation in this animal. Clinical signs of neurotoxicity were not observed in any other animal in the study. Declines in circulating thyroid hor mone (T4) were observed in males and females at ≥ 600 ppm (20 mg/kg bw/day) Reduction in follicle s ize, a possible indication of a reduced amount of colloid in the organ, and decreased T3 levels were observed in 1800-ppm males (55 mg/kg bw/day) only. Correlative changes in thyroid weight or thyroid stimulating hormone (TSH) were not observed in either sex. No morphological changes in the testes were observed i n dogs at any dose.

In rats, abnormal sperm or spermatid degeneration i n the testes and germ cell exfoliated debris in the epididymis were observed in male rats at the high d ose only (7500 ppm; 414 or 373 mg/kg bw/day) follow ing 12 (not statistically significant) or 24 months of oral exposure. Other target organs in rats followi ng exposure for 2 years were the kidney (both sexes) at mid and high doses and the liver (females) at the highest dose only. These results are consistent with the excret ory and/or detoxification roles of the kidney and l iver. Similar kidney toxicity was also observed at higher doses (419.3 or 484.7 mg/kg bw/day) in parental animals of the F1 generation in the 2-generation reproducti ve toxicity study in rats. No adverse findings were observed in mice up to the limit dose following long-term treatment with spirotetramat. Treatment-related increases in tumor incidence were not observed in either sex in rats or mice. Spirotetramat was also negative for mutagenicity an d clastogenicity in several standard in vivo and in vitro
assays.

B.6.6 Reproductive toxicity (OECD Annex IIA 5.6)

B.6.6.1 Two generation reproductive toxicity in the rat (OECD Annex IIA 5.6.1)

CITATION : Young, A.D. (2006). Technical grade BYI 08330: A two generation reproductive toxicity

study in the Wistar rat. Bayer CropScience LP, Tox icology, 17745 South Metcalf, Stilwell, KS 66085-9104. Report Number 201426-1, July 13, 20 06. Unpublished.

I. MATERIAL AND METHODS

A. Materials :– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 252 April 2008

1. Test material : BYI 08330 Description: White Powder Lot/Batch No.: 08045/0014 Purity: 97.8% (12/03), 97.6% (6/04), 98.5% (1/05) , 97.4% (5/05) Compound Stability: stable at room temperature (~ 22 o ) Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate; Spirotetramat CAS No. of TGAI: Non-stereo: 382608-10-8; cis: 20 3313-25-1

2. Vehicle and/or positive control: None. Compound was dry mixed and administered thr ough the diet.

3. Test animals : Species: Rat, males and females Strain: Number of animals: Wistar Hanover rats Crl:WI(Glx/BRL/Han)IGS BR Total animals ordered: 280; Number used on study: 130 male and 130 female 10 animals/sex were used for vendor surveillance, w hich included a gross necropsy and serological evaluation. 5 animals/sex were sacrificed prior to initiation of exposure and 5 animals/sex were sa crificed approximately four weeks after initiation of exposure. Age at study initiation: (P) 8-9 wks

Wt. at study initiation: (P) Males: 240.9 - 307.7 g; Females: 175.4 - 213.5 g

Source: Animal Receipt: Charles River Laboratories, Raleigh, NC Upon receipt, animals were examined by a veterinari an prior to release for study. Housing: Animals passing the initial shipment exam were housed individually (except during the mating phase and as noted below for the F 1 pups) in suspended stainless steel cages and deotized cage b oard in the bedding trays. During gestation and lactation, individual dams (and litters) were housed in polycarbonate cages with corn-cob bedding Diet: ad libitum - Purina Mills Rodent Lab Chow 5002 meal Water: ad libitum - pressure-activated water nipples Temperature: Humidity: Room temperature 18 to 26 o C

Relative humidity 30 to 70%, exceptions stated in t he Protocol Amendments and Deviations section Environmental conditions:

Air changes:

Photoperiod: Minimum of 10 air changes per hour, based on averag e

number of changes per hour per day Daily photoperiod of 12 hrs of light [6:00 a.m. to 6:00 p.m.] alternating with 12 hrs of darkness , excepti ons stated in the Protocol Amendments and Deviations section Acclimatisation: 8 days

All procedures associated with the animals were app roved by the Institution’s Animal Care and Use Committee (IACUC); and during the course of the stu dy, room conditions were monitored and recorded in a consistent and timely manner. Feed and water samples were collected periodically and analyzed fo r potential contaminants; the concentrations outlined in the “Certification Profile” for Purina Mills Ce rtified Lab Chows were used as a standard of comparison for judging the acceptability of any contaminants detected in the analyzed feed samples.

B. PROCEDURES AND STUDY DESIGN:– –

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1. Mating procedure : Males and females were exposed to the test materi al for ten weeks prior to mating. Mating was accomplished by co-housing one female wi th one male for up to 14 consecutive days with exceptions stated in the Protocol Amendments and De viations section. Approximately four to six animals from each dose group were co-housed daily b eginning on the first day of the mating phase and continuing until all animals had been cohoused. Du ring the mating phase vaginal smears were taken each morning and examined for the presence of sperm and/or internal vaginal plug. Females found to be inseminated were placed in a polycarbonate nesti ng cage. The day on which insemination was observed in the vaginal smear was designated day 0 of gestation for that female. In order to evaluate those females which may have been inseminated witho ut exhibiting sperm in the vaginal smear or an internal vaginal plug, all remaining females were p laced in polycarbonate nesting cages following the 14- day mating period.

2. Study schedule : One hundred and twenty female and one hundred and twenty male rats were assigned to one of four treatment groups (30 animals/group): nominal doses of 0, 250, 1,000, and 6,000 ppm technical grade BYI 08330 in the diet. Animals wer e exposed to the treated feed throughout the entire study. The parental animals (P and F 1 generations) were given test diets for 10 weeks pr ior to mating. Selection of F 1 -pups to evaluate preputial separation and vaginal patency was made when the pups were 21 days of age. The F 2 -generation pups were maintained until 21 days old.

3. Animal assignment : Following a minimum of 6 days of acclimation and prior to initiation of treatment, study animals were examined by a veterinarian and r eleased for study use. The animals were then assigned to either a control or one of three chemic ally-treated groups using a weight stratification-b ased computer program (INSTEM Computer Systems, Stone, S taffordshire, UK) and those animals falling within +/- 20% of the mean for all animals were put on study (Table 6-77). Once animals were distributed into their dose groups, each rat on stu dy had a microchip (BioMedic Data Systems, Inc., Seaford, Delaware) subcutaneously implanted on thei r dorsal surface in the region between their scapulae. At the very least, the chip was encoded with a unique number, specifying the animal’s sex, dose group, cage number, and study affiliation. In addition, a laminated cage card, essentially duplicating the information present on the chip, wa s attached to the outside of each animal’s cage. P ups born alive were identified by tattoo and pups found dead were identified with a marking pen. All anim als not placed on study (for whatever reason) were ulti mately sacrificed; however, prior to that time the animals were available, subject to the approval of the IACUC, for alternative uses which may have been unrelated to this study.

Table B.6-77: Animal assignment and study design Animals/group Test Group Dose in Diet a

(ppm) P Males P Females F 1 Males F 1 Females
Control 0 ppm 30 30 30 30
Low (LDT) 250 ppm 30 30 30 30
Mid (MDT) 1,000 ppm 30 30 30 30
High (HDT) 6,000 ppm 30 30 30 30

Data taken from Text Table 1, p. 18, Report No. 201426-1

a Diets were administered from beginning of the stud y until sacrifice.

4. Dose selection rationale: Doses were selected based upon the preliminary re sults which emerged in the rat over the course of a pilot reproductive tox icity testing study conducted with the test chemica l at doses of 0, 200, 500, 6,000 and 10,000 ppm BYI 0833 0/kg body weight/day (Report No. 201300-1). In that study, the P-generation produced no births in the 10,000 ppm dose group in the absence of effects– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 254 April 2008

on body weight, food consumption or clinical observ ations. In this same dose group, declined epididymal weights were observed. Sperm analysis r evealed declined sperm motility (67%) and % progressive sperm (79%), as well as, abnormal sperm in the epididymis and cauda epididymis. The F 1 - generation 8-9 week old males of the 6,000 ppm dose group also exhibited, to a much lesser extent, declined sperm motility (15%) and % progressive spe rm (15%), as well as, abnormal sperm in the epididymis and cauda epididymis. Based on these in terim results, the doses selected for the twogeneration reproduction toxicity study were 0, 250, 1,000 and 6,000 ppm BYI 08330.

5. Dosage preparation and analysis : The test compound was mixed directly with the fee d. Treated diet was mixed at room temperature; aliquots of the chem ical were taken from the original test batch and transferred to the mixing area. The control test d iet was taken directly from the bag without mixing. A sample of each batch of feed mixed was taken and re tained in the freezer until the study was complete and the analytical data deemed satisfactory. Repla cement admixtures for each treatment group, for each week, were stored under freezer conditions unt il presented to the animals the following week. Additionally, the entire batch of replacement admixture for each treatment g roup was used for subsequent weekly feedings if within freezer stabil ity limits.

Feed samples were taken from each dietary level and analyzed for concentration for study weeks 1, 2, and 3 and at monthly intervals thereafter.

Results Homogeneity Analysis: The homogeneity and stability of the test substan ce in rodent feed was verified. The mean concentrations of BYI 08330 in the feed, sampled from three distinct layers in the mixing bowl and containing a nominal concentration of either 70, 150, or 10,000 ppm, were determined to be 68.7 ppm (range 65.0-73.1 ppm; CV = 3.2%), 14 4 ppm (range 127-153 ppm; CV = 5.5%), and 9,445 ppm (range 8,817-9,943; CV = 3.2%), respectiv ely. Based on a %RSD< 10%, BYI 08330 was judged to be homogeneously distributed in the feed over a concentration range of 70-10,000 ppm.

Stability Analysis: Following 7 days of room temperature storage, the analytically-determined concentration of the AI of the test substance in th e 70-, 150-, or 10,000-ppm admixture was determined to be 68.5 ppm (63.3 ppm on day 0); 131 ppm (151 pp m on day 0); and 9,564 ppm (9,405 ppm on day 0), respectively. Following 28 days of freezer sto rage, the analytically-determined concentration of the AI of the test substances in the 70-, 150-, and 10, 000-ppm admixtures was determined to be 71.4 ppm (63.3 ppm on day 0); 146 ppm (151 ppm on day 0); an d 9,544 ppm (9,405 ppm on day 0), respectively.

BYI 08330 mixed in rodent ration was judged to be s table at room temperature for at least seven days and following freezer storage for a minimum of 28 d ays, over a concentration range of 70-10,000 ppm.

Concentration Analysis: The concentration of BYI 08330 in the various tes t diets was determined using liquid chromatography. Mean analytical conce ntrations for each dose group were 244, 1,006 and 5,898 ppm, ranging from 98-101% of the correspondin g nominal concentrations of 250, 1,000 and 6,000 ppm, respectively. The AI of the test substance wa s not detected in the control diet. Mean recovery was 99% and ranged from 94-103% for rodent ration s piked with 250, 1,000 or 6,000 ppm BYI 08330.

C. OBSERVATIONS

1. Parental animals :– –

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Mortality and clinical observations : Mortality checks (cageside observations) were perf ormed twice daily (AM and PM), during the work week and once da ily on weekends and holidays. Cageside observations characterized mortality, moribundity, behavioral changes, signs of difficult or prolonged delivery, and overt toxicity by viewing the animal in the cage. In the event a possible clinical sign was observed during the cageside evaluation, the animal was removed from the cage and a detailed assessment conducted. A detailed evaluation of cli nical signs, which included both observing the anim al in the cage and removing the animal to perform a ph ysical examination, was conducted once per week throughout the entire in-life phase of the study.

Body weights and food consumption: Body weights and food consumption were measured a nd fresh feed provided once/week for both males and females during the 10-week premating period with exceptions stated in the Protocol Amendments and De viations section. During the mating period and until sacrifice, body weights for the males were ta ken once per week. Body weights were also taken during the mating period for unmated females. Also during the mating period, fresh feed was provided for both males and unmated females once/week but fo od consumption was not measured. During gestation, dam body weights were measured on days 0 , 6, 13 and 20, and fresh feed was provided and food consumption measured once/week. During lactat ion, dam body weights were measured on days 0, 4, 7, 14 and 21. Fresh feed was provided and food consumption measured once/week, with the exception of week one when food consumption was mea sured twice (days 0-4 and 4-7).

Estrous cyclicity : The estrous cycle (determined by examining daily vaginal smears) was characterized for all P- and F 1 -generation females, over a three-week period prior to mating. Additionally, the estrous cycle stage was determine d for all females just prior to termination.

Sperm parameters:

For all P and F 1 -generation males at termination, sperm was collect ed from one testis and one epididymis for enumeration of homoge nization-resistant spermatids and cauda epididymal sperm reserves, respectively. In addition, an eval uation of the morphology and motility was performed on sperm sampled from the distal portion (closest t o the urethra) of the vas deferens. Sperm motility and counts were conducted using the IVOS (Integrated Vi sual Operating Systems) sperm analyzer. Morphology and testis counts were conducted on the control and highest dose group and in all dose groups for epididymal counts for the first generati on. Morphology and counts were conducted on all dose groups for the second generation.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 256 April 2008

2. Litter observations : The following litter observations (X) were made.

Time of observation (lactation day) Observation
Days (0-21) Day 0 Day 4 a Day 4 b Day 7 Day

14 Day 21

Number of live pups X
Pup weight X X X X X

External alterations X X X X X
Number of dead pups X
Sex of each pup (M/F) X
Preputial Separation Performed postweaning for F 1 pups Vaginal Patency Performed postweaning for F 1 pups

a Before standardization (culling) b After standardization (culling) Exceptions stated in the Protocol Amendments and De viations section

The size of each litter was adjusted on lactation D ay 4 to yield, as close as possible, four males and females per litter. If the number of male or femal e pups was less than four each, a partial adjustmen t was made (e.g., three males and five females). No adjustment was made for litters of fewer than eight pups. Adjustments were made by random selection of the pups using software provided by SAS. Culled pups were sacrificed by intracranial injection of 0 .01-0.05cc Fatal Plus (Vortech Pharmaceuticals, Dearborn, MI). Grossly abnormal pups underwent a g ross internal and external examination and all culled pups were discarded. The pups not culled on lactation day 4 were maintained with the dam until weaning (lactation day 21). At 21-days of age a su fficient number of F 1 - pups/sex/litter was maintained to produce the next generation. F 1 - pups not selected to become parents of the next gen eration and all F 2 - pups were sacrificed and examined macroscopically f or any structural abnormalities or pathological changes, particularly as they may relate to the org ans of the reproductive system. Dead pups were examined grossly for external and internal abnormal ities and a possible cause of death was determined for pups stillborn or found dead.

3. Postmortem observations :

1) Parental animals : All surviving parental males were sacrificed as s oon as possible after the last litters were produced. Maternal animals were sacri ficed following the weaning of their respective litters (lactation day 21). These animals were sub ject to examinations as follows.

P

- generation males and females (10 animals/sex/level) were bled from the orbital sinus utilizing a glass capillary tube under brief isofluorane anesth esia for possible evaluation of clinical chemistry parameters. Liver samples were also collected for the same animals and immediately placed in liquid nitrogen prior to placing in -70 o C freezer for possible evaluation of liver enzymes. Analysis of clinical chemistries and liver enzymes was deemed u nnecessary.

Male rats were sacrificed by carbon dioxide asphyxi ation, terminal body weights were taken, and a gross external examination was performed. For all males at termination, sperm was collected from one testes and one epididymis for enumeration of ho mogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. In additi on, an evaluation of the morphology and motility– –

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was performed on sperm sampled from the distal port ion (closest to the urethra) of the vas deferens. Sperm motility and counts were conducted using IVOS (Integrated Visual Operating Systems).

Each dam was terminated by carbon dioxide asphyxiat ion and a gross external examination was performed. Terminal body weights were taken and th e abdomen and thoracic cavities were opened and a gross internal examination was performed. Th e uterus was excised and the implantation sites, if present, were counted.

Females which were found sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed after gestation Day 24. Females wh ich were never observed as being inseminated and/or with an internal vaginal plug and did not de liver at least 24 days after the completion of the mating phase, were sacrificed and necropsied. A gr oss necropsy was performed on these animals as described above. In addition, patency of the ce rvical/uterine os in these females was examined via flushing of the uterine horns with deionized wa ter.

The following tissues were collected (X) and weighe d (XX). Micropathology (in addition to collection and weights) was performed on those tissues designa ted with (XXX):

XX Brain XX Thyroid XXX Prostate X Cervix XXX Pituitary XX Thymus XXX Seminal Vesicle X Vagin a
XXX Liver XXX Adrenal XXX Testis X Oviduct XXX Kidney XXX Epididymis XXX Uterus X Coagulating Gland XXX Spleen XXX Ovary X Gross Lesions

Animals found moribund while on study were sacrific ed if their status dictated and a gross necropsy was performed. Animals found dead were necropsied as soon as possible. Necropsy examinations included those parameters previously described.

2) Offspring : The F 1 -offspring not selected as parental animals and all F 2 -offspring were sacrificed at 21-days of age. These animals were subject to post mortem examinations (macroscopic and/or microscopic examination) as follows. The following tissues from 21-day weanlings were collected and weighed: brain, thymus, spleen, and uterus. An y gross lesion was documented and collected. Micropathology was performed on the following tissu es: From 1 female/litter; uterus, ovary, vagina, cervix, oviduct (fallopian tube) and from 1 male/li tter; testis, epididymis, prostate, coagulating gla nd, seminal vesicles. Exceptions are stated in the Pro tocol Amendments and Deviations section.

Pups found dead or terminated in a moribund conditi on underwent a gross necropsy for possible defects and/or cause of death.

D. DATA ANALYSIS

1. Statistical analyses : The data were analyzed using applications provid ed by DATATOX (Instem Computer Systems), SAS (SAS Institute, Inc.), or TA SC (Toxicology Analysis Systems Customized). Parametric data (including body weight gain and foo d consumption) were analyzed using a univariate Analysis of Variance (ANOVA) and if significant dif ferences were observed, a Dunnett's Test was performed. Nonparametric data (e.g., number of est rous cycles, litter size, and number of implantatio n sites) were first analyzed by the Kruskal-Wallis te st and then by the Dunn's Test if significant diffe rences were identified. Nonparametric dichotomous data (e .g. fertility and gestation indices) were initially analyzed by the Chi-Square Test and if significance was observed between groups then by the Fisher's– –

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Exact Test with the Bonferroni adjustment. To the extent possible, the frequency of gross lesions was first examined visually, then, in the event of ques tionable distribution, by statistical analysis usin g the Chi-square and Fisher's exact tests. Differences b etween the control and test compound-treated groups was considered statistically significant when p < 0 .05 or p < 0.01.

2. Indices : Reproductive indices: The following reproductive i ndices were calculated from breeding and parturition records of animals in the study:

100 used ales co-ho No. of fem es

ated femal min of inse No. ex (%) Mating Ind a ×

=

100

females d inseminate of No. nt females of pregna No. Index (%) Fertility b
×

=

100 females pregnant of No. pups

live with females of No. Index (%) Gestation ×

=

a Includes pregnant females not observed sperm positi ve or with an internal vaginal plug. b Includes females which did not deliver, but had im plantation sites.

Offspring viability indices : The following viability indices were calculated f rom lactation records of litters in the study:

100 s/litter ation site of implant Total No. orn/litter

of pups b Total No. x (%) Birth Inde ×

=

100 tter of pups/li Total No. tter

ps born/li of live pu No. Index (%) Livebirth ×

=

100 n/litter e pups bor No. of liv ing)

(pre-cull 4 on day ps/litter of live pu No. Index (%) Viability ×

=

100 ing) (post-cul 4 ter on day e pups/lit No. of liv 21 on day ups/litter of live p No. Index (%) Lactation ×

=

3. Historical control data : Historical control data were provided in Attachmen t IV of the study report. This data were obtained from reproduction studies perfor med in this laboratory (1998-2004) in the Wistar ra t.

II RESULTS

Findings:

a) Parental toxicity:

General observations : There were no treatment-related mortalities or cli nical signs observed during the course of the study. Toxicologically significant ef fects on feed intake were determined at 6,000 ppm i n females during lactation. Body weight and/or body weight gain was reduced at 6,000 ppm in males and females. Selected body weight and food consumption data are given in Tables 6-78 and 6-79 respectively; compound intake is given in Table 6-8 0. Details are as follows:– –

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Premating : A decline in body weight gain (Wk 1-10) was noted in P-generation males (13%; NSS), but not females, of the 6,000 ppm group. In the F 1 -generation, the males of the 6,000 ppm group exhib ited statistically significant declines in body weight ( mean of 6%) from days 14-70 and declines in overall body weight gain (8%). In F 1 -generation females of the 6,000 ppm group, statist ically significant declines in body weight (6%, week 10 of premating) and decli nes in overall body weight gain (11%) were observed at the end of the premating phase.

Gestation : A decline in body weight gain (8%) was noted in F 1 -generation females of the 6,000 ppm group which was accompanied by statistically signif icantly lower body weight means (7%) from day 0-20 of gestation.

Lactation : P-generation females of the 6,000 ppm group showe d statistically significant declines in food consumption on both a g/kg/day (9-14%) and g/animal /day (13.4%) basis. In the F 1 -generation, females of the 6,000 ppm group exhibited statistically sign ificant body weight declines (6-8%) throughout lactation, as well as significantly declined food c onsumption (lactation days 7-21) on both a g/kg/day (8- 9%) and g/animal/day (13.9%) basis. Statistically s ignificant declines in food consumption were also observed in F1-generation females of the 1,000 ppm group (lactation days 14–21) and are not considered toxicologically significant in the absen ce of changes in body weight or body weight gain.

Table B.6-78: Mean body weight (mean g ± S.E.) and weight gain (g)a of parental animals Dose Group (ppm) En Endpoint 0 250 1,000 6,000 P generation males (pre-mating) Body weight – Wk 0 270.8 ± 2.43 273.5 ± 2.26 275.6 ± 2.59 277.2 ± 2.10 Body weight – Wk 10 430.3 ± 4.67 438.5 ± 5.38 425.6 ± 6.00 415.7 ± 6.41 Weight gain – Wk 1-10 159.5 165.0 150.0 138.5
F 1 generation males (pre-mating) Body weight – Wk 0 198.5 ± 3.94 198.9 ± 5.48 197.1 ± 5.24 189.4 ± 4.25 Body weight – Wk 10 401.0 ± 6.42 416.0 ± 7.60 404.8 ± 4.96 375.9* ± 7.13 (-6) b Weight gain – Wk 1-10 202.5 217.1 207.7 186.5 (-8) P generation females (pre-mating) Body weight – Wk 0 191.7 ± 1.44 192.2 ± 1.82 194.6 ± 1.82 193.2 ± 1.71 Body weight – Wk 10 241.4 ± 2.74 244.0 ± 2.37 241.7 ± 2.92 240.2 ± 2.45 Weight gain – Wk 1-10 49.7 51.8 47.1 47.0 P generation females (gestation) Body weight – GD 20 346.6 ± 5.09 344.3 ± 4.69 341.9 ± 4.35 335.0 ± 3.86 Weight gain – GD 0-20 102.8 ± 3.59 98.3 ± 3.38 93.9 ± 2.28 93.7 ± 3.15
F 1 generation females (pre-mating) Body weight – Wk 0 147.2 ± 2.73 146.2 ± 2.77 148.8 ± 2.57 143.2 ± 2.22 Body weight – Wk 10 226.7 ± 3.03 226.2 ± 3.03 226.1 ± 2.64 213.7* ± 3.09 (-6) Weight gain – Wk 1-10 79.5 80.0 77.3 70.5 (-11)
F 1 generation females (gestation) Body weight – GD 20 318.0 ± 3.70 324.5 ± 5.50 310.8 ± 4.54 296.7** ± 3.17 (-7) Weight gain – GD 0-20 88.9 ± 2.50 95.3 ± 3.09 83.8 ± 2.74 82.2 ± 1.86 (-8)
F 1 generation females (lactation) Body weight – LD 0 247.5 ± 3.78 254.7 ± 4.45 245.9 ± 3.53 232.0** ± 3.37 (-6) Body weight – LD 4 258.9 ± 3.42 259.2 ± 3.69 255.0 ± 3.71 238.6** ± 3.90 (-8) Body weight – LD 7 267.0 ± 3.54 268.9 ± 3.45 263.1 ± 3.61 248.6** ± 3.62 (-7) Body weight – LD 14 277.4 ± 3.92 282.8 ± 3.82 277.5 ± 3.73 260.4** ± 4.67 (-6) Body weight – LD 21 270.2 ± 3.63 275.7 ± 3.65 264.9 ± 3.12 254.4** ± 3.51 (-6)

Data taken from Table s 2, 8, and 13, pp. 54-61, 84-85, and 99-100, respe ctively, Report No. 201426-1– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 260 April 2008

a Premating weight gain calculated from group mean va lues; not analyzed statistically. b Number in parentheses is percent difference from co ntrol. * = p < 0.05,** = p < 0.01

Table B.6-79: Mean food consumption by females duri ng lactation (g/kg/day ± S.E.) Dose group (ppm) Interval 0 250 1,000 6,000 P generation females (lactation) LD 0-4 124.1 ± 4.43 111.4 ± 5.49 108.3* ± 3.21 (-13 ) a 106.5** ± 3.35 (-14)
LD 4-7 164.4 ± 4.25 152.4 ± 4.53 151.8 ± 3.77 144.4 ** ± 3.49 (-12)
LD 7-14 181.6 ± 4.21 175.0 ± 4.14 179.7 ± 2.39 165. 5** ± 3.02 (-9)
LD 14-21 214.0 ± 5.69 207.5 ± 5.59 208.0 ± 3.75 191 .1** ± 3.83 (-11)
F 1 generation females (lactation)
LD 0-4 117.7 ± 5.07 107.3 ± 4.58 104.3 ± 4.88 109.0 ± 3.01
LD 4-7 156.9 ± 3.09 147.1 ± 2.99 153.2 ± 4.14 158.2 ± 5.66
LD 7-14 185.4 ± 2.60 182.1 ± 2.47 184.9 ± 4.44 169. 8** ± 2.81 (-8) LD 14-21 212.8 ± 2.54 208.5 ± 2.62 197.8* ± 4.79 (- 7) 194.1** ± 2.14 (-9)

Data taken from Table 14, pp. 102-103, Report No. 201426-1

a Number in parentheses is percent difference from co ntrol. *=p < 0.05, ** = p < 0.01

Table B.6-80: Average compound intake (mg/kg/day) Phase of Study 250 ppm a 1,000 ppm a 6,000 ppm a

Premating (P-gen) - Male 17.2 70.7 419.3
Premating (P-gen) - Female 20.0 82.5 484.7
Gestation (P-gen) - Female 19.6 76.7 467.4
Lactation (P-gen) - Female 39.4 162.9 895.7
Premating (F 1 -gen) - Male 19.3 79.5 486.7
Premating (F 1 -gen) - Female 21.7 90.3 539.5
Gestation (F 1 -gen) - Female 17.8 69.8 434.7
Lactation (F 1 -gen) - Female 39.4 161.0 930.6

a Individual values were based on the means for each particular phase for each generation.

Gross pathology, organ weig hts: No treatment-related macroscopic changes were obse rved at necropsy in P- and F 1 -generation rats. Significant declines in terminal body weight were observed in both F 1 generation males and females of the 6,000 ppm grou p. There were various organ weight changes which were statistically significantly diff erent from controls (testes, epididymis, spleen, th yroid). Those findings were considered to be unrelated to t reatment as most of those statistically different o rgan weights fell within their respective weight ranges from each of the study ́s concurrent control groups, or were not correlated with dose, and/or were correlat ed with a lower terminal body weight with no effect s on relative organ weight (Table 6-81). Decreases i n absolute and/or relative kidney weight at 6000 pp m in F 1 generation males and females were considered toxic ologically significant in the presence of histopathology of the kidney.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 261 April 2008

Table B.6-81: Mean absolute (g) and relative (%) or gan weights Dose Group (ppm) Parameter 0 250 1,000 6,000

F 1 generation males Final body weight 424.0 ± 36.9 442.0 ± 44.8 428.9 ± 33.7 397.3* ± 41.2 Kidney weight, absolute -left 1.470 ± 0.129 1.410 ± 0.172 1.322* ± 0.209 1.313* ± 0.142 Kidney weight, absolute -right 1.519 ± 0.092 1.480 ± 0.145 1.419* k ± 0.226 1.347* k

± 0.132 Kidney weight, relative -left 0.347 ± 0.023 0.320* k ± 0.026 0.310* k

± 0.050 0.332* k

± 0.028 Kidney weight, relative -right 0.360 ± 0.027 0.336* ± 0.023 0.331* ± 0.043 0.340 ± 0.029 P generation females Final body weight 285.4 ± 25.2 284.5 ± 22.1 284.7 ± 21.9 287.9 ± 19.1 Kidney weight, absolute -left 1.088 ± 0.116 1.053 ± 0.104 1.044 ± 0.087 1.038 ± 0.099 Kidney weight, absolute -right 1.141 ± 0.125 1.091 ± 0.109 1.110 ± 0.107 1.076 ± 0.089 Kidney weight, relative -left 0.382 ± 0.027 0.371 ± 0.026 0.369 ± 0.032 0.361* ± 0.023 Kidney weight, relative -right 0.400 ± 0.027 0.384 ± 0.030 0.392 ± 0.032 0.374* ± 0.025 F 1 generation females Final body weight 268.4 ± 20.6 269.7 ± 24.5 261.8 ± 17.5 252.8* ± 17.9 Kidney weight, absolute -left 1.081 ± 0.114 1.023 ± 0.118 1.008* ± 0.101 0.949* ± 0.132 Kidney weight, absolute -right 1.136 ± 0.108 1.092 ± 0.119 1.051* ± 0.112 1.011* ± 0.131 Kidney weight, relative -left 0.404 ± 0.038 0.379* ± 0.027 0.385 ± 0.028 0.374* ± 0.032 Kidney weight, relative -right 0.424 ± 0.040 0.405 ± 0.029 0.401* ± 0.030 0.399* ± 0.034 Data taken from Table s OW1K-SUM and OW2K-SUM, pp. 491-501 and 503-513, r espectively, Pathology Report, Report No. 201426-1

* = p < 0.05 (ANOVA+Dunnett`s test); * k = p < 0.05 (Kruskal-Wallis ANOVA+Mann-Whitney U-te st)

Histopathology : Histopathology findings considered to be compound- related were confined to the kidneys of the F 1 -generation male and female rats of the 6,000-ppm g roup (Table 6-82) and the epididymis of a single F 1 male from the 6,000-ppm dose group. In F 1 -generation male and female rats of the 6,000 ppm group, a minimal to moderate multifoc al tubular dilatation was observed in the outer portion of the medulla, containing occasional prote inaceous material. In general, both kidneys were affected with the severity and incidence being grea ter in males.

The epididymis of one single F 1 -generation male rat (animal number: SJ3514) had a moderate number of abnormal sperm within the tubules. Although mic roscopically there was only one animal with abnormal sperm, the sperm were similar in appearanc e (amorphous sperm heads) to abnormal sperm observed microscopically in the males of the 6,000- ppm F 1 eight-to-nine week old interim animals and the 10,000 ppm P males in the Dose Range-Finding St udy (Report No. 201300-1).

No treatment-related findings were observed in the reproductive tract of females of either generation (ovaries, oviducts, uterus, cervix, vagina). Ovaria n follicles were counted from control and 6,000-ppm dose F 1 -generation females. There was no difference in the number of primordial follicles, antral follicles, or corpora lutea in the 6,000-ppm females, when com pared to controls.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 262 April 2008

Table B.6-82: Incidence of treatment-related change s in the kidneys from F1 animals Dose group (ppm) 0 250 1,000 6,000
F 1 generation males
Number of animals examined 30 30 30 30 Kidney, tubular dilatation Minimal Slight Moderate Total Affected 0 0 0 0 0

0 0 0 0

0 0 0 13

5 5 23 F 1 generation females
Number of animals examined 30 30 30 30 Kidney, tubular dilatation Minimal Slight Moderate Total Affected 0 0 0 0 0

0 0 0 0

0 0 0 7

8 0 15

b) Reproductive function : Estrus cycle staging did not indicate treatment-rel ated effects on mean cycle length and number of normally cycling females in both P- and F 1 -generation females at any dietary level tested.

In P-generation males, sperm motility (percent moti le and percent progression) and epididymal sperm count were evaluated for the controls and all treatment levels (Table 6-83). Morphology and testicular counts were evaluated for the controls a nd 6,000 ppm dose group. There were no compound-related effects on any sperm parameter at any dietary level. Statistical differences observed on epididymal counts for the 1,000- and 6, 000-ppm males are not considered to be compound-related, since there were no effects on te sticular weight or testicular sperm count and similar findings were not observed in the second ge neration.

Table B.6-83: Mean sperm measures Dose Group (ppm) Sperm Analysis 0 250 1,000 6,000 P generation males Motility: % motile 83.7 82.8 83.9 85.0 Motility: % progressive 58.8 59.0 60.6 61.7 Sperm counts: sperm/g testis 89.95 N/A N/A 87.43 Sperm counts: sperm/g epididymis 622.95 562.85 481.82* 472.27* Morphol.: number of normal sperm cells 197.3 N/A N/A 196.0 Morphol.: number of abnormal sperm cells 2.0 N/A N/A 2.8 Morphol: number with detached heads 0.8 N/A N/A 1.2 F 1 generation males Motility: % motile 81.1 82.8 83.9 79.9 Motility: % progressive 56.6 59.7 59.3 55.7 Sperm counts: sperm/g testis 87.8 90.3 89.9 79.3 Sperm counts: sperm/g epididymis 517.2 490.3 520.1 496.5 Morphol.: number of normal sperm cells 196.3 196.8 197.3 189.9 Morphol.: number of abnormal sperm cells 2.9 2.6 1. 9 8.9 Morphol: number with detached heads 0.8 0.6 0.9 1.3 Data taken from Table 22, pp. 137-138, Report No. 201426-1 * = p < 0.05– –

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In F 1 -generation males, sperm motility (percent motile a nd percent progression), total sperm count (epididymal and testicular), and morphology were ev aluated at all dietary levels (Table 6-83). A morphologic effect on sperm, presenting as amorphou s sperm heads, was noted and is consistent with what was seen in the one-generation study with BYI 08330 (Report No. 201300-1). Variation in susceptibility to this finding was observed as 9/30 males exhibited a minimal effect (one to four amorphous sperm heads were noted out of 200 viewed) but only one outlying male (No. 3514) was affected to the extent that it compromised fertiliz ing capabilities for this one animal (i.e. the male presented 82% total abnormal sperm with 77% of thes e sperm presenting as amorphous heads, and the female found sperm positive from this male did not become pregnant). This same male showed abnormal sperm in the epididymis (see histopatholog y). There were no compound-related effects on sperm motility or total sperm count (epididymal or testicular) observed at any dietary level tested.

c) Reproductive performance : Female reproductive performance was not affected f or any parameter measured (e.g., mating, fertility or gestation indi ces, days to insemination, gestation length, or the median number of implants) in either generation at any dietary level tested. However, as stated above, the female found sperm positive from the hig h-dose male with abnormal sperm did not become pregnant. Comparison of the number of uteri ne implantation sites at necropsy with the number of delivered pups revealed the absence of an y treatment-related effect on post-implantation loss in both generations.

The litter parameters determined at birth (pup weig ht at birth, total number of pups born, stillborn pups, live birth index, percentages of male pups bo rn, mean litter size at birth) were not affected in both generations.

d) Neonatal toxicity:

General observations: There were no treatment-related effects on the via bility parameters of the pups (viability index, lactation index), and no cli nical signs and no malformations were observed in both F 1 and F 2 pups.

Offspring body weight and body weight gain data are given in Table 6-84. Pup weights at birth were not affected (F 1 and F 2 ). On lactation day 21, the F 1 pup body weights for both sexes in the 6,000 ppm group was significantly lower than control (8.2 %) with females also significantly lower on day 14 (6%). The pup body weight gains for 6,000-ppm males and females were statistically decreased (9%) during days 4-21 of lactation.

On lactation days 14 and 21, the F 2 pup body weights for both sexes in the 6,000-ppm g roup were significantly less than control (7-8% and 9-11%, re spectively). The pup body weight gains for 6,000- ppm males and females were statistically decreased (12-14%) during days 4-21 of lactation.– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism264April 2008Table B.6-84: Mean body weight and body weight gain(g ± S.E.) of the F1 and F2 pups during lactationLactationDay0 ppm 250 ppm 1000 ppm 6000 ppm 0 ppm250 ppm 1000 ppm 6000 ppmF1Pups – maleF1Pups – female06.0 ± 0.09 6.0 ± 0.09 5.8 ± 0.08 5.9 ± 0.06 5.6 ± 0.08 5.7 ± 0.08 5.6 ± 0.07 5.5 ± 0.054(postcull)9.3 ± 0.25 9.4 ± 0.21 9.4 ± 0.23 8.9 ± 0.18 9.0 ± 0.23 9.2 ± 0.18 9.0 ± 0.22 8.5 ± 0.16714.8 ± 0.41 15.0 ± 0.36 15.1 ± 0.32 14.3 ± 0.26 14.3 ± 0.40 14.7 ± 0.26 14.6 ± 0.30 13.6 ± 0.251430.4 ± 0.65 30.9 ± 0.57 30.9 ± 0.50 28.9 ± 0.46 29.6 ± 0.61 30.5 ± 0.46 30.0 ± 0.5027.7* ± 0.41 (-6)a2146.1 ± 0.96 47.5 ± 0.90 47.1 ± 0.8942.5** ± 0.55 (-8)44.3 ± 0.93 46.4 ± 0.75 44.7 ± 0.9140.6** ± 0.55 (-8)Wt gain days 0-43.3 ± 0.19 3.5 ± 0.16 3.6 ± 0.16 3.1 ± 0.15 3.3 ± 0.19 3.5 ± 0.15 3.4 ± 0.17 3.0 ± 0.13Wt gain days 4-75.5 ± 0.19 5.6 ± 0.22 5.7 ± 0.12 5.4 ± 0.12 5.4 ± 0.20 5.5 ± 0.16 5.6 ± 0.12 5.1 ± 0.13Wt gain days 7-1415.6 ± 0.32 15.9 ± 0.36 15.8 ± 0.2714.5* ± 0.29 (-7)15.3 ± 0.29 15.8 ± 0.35 15.4 ± 0.28 14.1* ± 0.31 (-8)Wt gain days 14-2115.7 ± 0.51 16.6 ± 0.44 16.3 ± 0.4913.6** ± 0.25 (-13)14.7 ± 0.47 15.9 ± 0.39 14.7 ± 0.4812.9** ± 0.23 (-12)Wt gain days 4-2136.8 ± 0.78 38.2 ± 0.82 37.7 ± 0.7233.5** ± 0.46 (-9)35.4 ± 0.74 37.2 ± 0.71 35.6 ± 0.7632.1** ± 0.48 (-9)F2Pups – maleF2Pups – female06.0 ± 0.08 5.8 ± 0.09 6.0 ± 0.11 6.0 ± 0.11 5.6 ± 0.08 5.5 ± 0.08 5.8 ± 0.12 5.6 ± 0.114(postcull)10.0 ± 0.18 9.5 ± 0.22 9.6 ± 0.25 9.9 ± 0.26 9.5 ±0.18 9.2 ± 0.20 9.5 ± 0.29 9.4 ± 0.26715.8 ± 0.24 15.2 ± 0.36 15.4 ± 0.38 15.1 ± 0.39 15.2 ± 0.25 14.7 ± 0.34 15.0 ± 0.41 14.4 ± 0.381431.4 ± 0.41 30.9 ± 0.63 31.3 ± 0.7029.0** ± 0.63 (-8)30.2 ± 0.37 30.2 ± 0.60 30.8 ± 0.6928.0** ± 0.59 (-7)2147.4 ± 0.60 46.5 ± 0.91 46.0 ± 0.8842.4** ± 0.86 (-11)45.3 ± 0.58 45.2 ± 0.92 45.0 ± 0.9441.0** ± 0.78 (-9)Wt gain days 0-44.0 ± 0.11 3.7 ± 0.13 3.7 ± 0.21 3.9 ± 0.18 3.9 ± 0.11 3.7 ± 0.13 3.7 ± 0.19 3.8 ± 0.17Wt gain days 4-75.9 ± 0.12 5.8 ± 0.16 5.8 ± 0.16 5.3** ± 0.15 (- 5.7± 0.11 5.5 ± 0.17 5.6 ± 0.18 5.0** ± 0.15 (-– –Spirotetramat – Volume 3, Annex B.6 Toxicology andmetabolism265April 200810)12)Wt gain days 7-1415.5 ± 0.26 15.7 ± 0.33 15.9 ± 0.3813.9** ± 0.35 (-10)15.0 ± 0.23 15.5 ± 0.31 15.8 ± 0.3513.6** ± 0.35 (-9)Wt gain days 14-2116.4 ± 0.35 15.6 ± 0.3314.7** ± 0.36 (-10)13.4** ± 0.37 (-18)15.1 ± 0.31 15.0 ± 0.41 14.2 ± 0.3613.0** ± 0.37 (-14)Wt gain days 4-2137.8 ± 0.48 37.0 ± 0.73 36.4 ± 0.7132.5** ± 0.65 (-14)35.8 ± 0.46 36.0 ± 0.76 35.5 ± 0.7431.6** ± 0.58 (-12)Data taken fromTables 18 and 19, pp. 116-121 and 123-128, respectively,Report No. 201426-1aNumber in parentheses is percent difference from control.*=p<0.05, **=p<0.01.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 266 April 2008

Gross pathology, organ weights : In F 1 and F 2 weanlings no treatment-related macroscopic alterat ions were observed at any dietary level tested. Absolute brain weights were statistically significantly decreased (F 2 males, F 2 combined sexes) and relative brain weights were in creased (F 1 and F 2 males and females; F 1 and F 2 combined sexes) at 6,000 ppm (Table 6-85). At 6,0 00 ppm, absolute spleen weights were decreased in combined F 1 weanlings and in F 2 male and female weanlings. F 2 males and females also had a decreased absolute thymus weight at 6,000 ppm (Table 6-85). The decreases in brain, spleen, and thymus weight were considered se condary to the lower mean body weights of the 6,000 ppm pup dose group.

Table B.6-85: Mean absolute (g) and relative (%) or gan weights of weanlings Dose Group (ppm) Parameter 0 250 1,000 6,000

F 1 male+female weanlings (combined) Brain weight, absolute 1.454 ± 0.0132 1.449 ± 0.012 3 1.465 ± 0.0108 1.421 ± 0.0114 Brain weight, relative 3.252 ± 0.0524 3.096 ± 0.048 7 3.231 ± 0.0565 3.444** ± 0.0332 Spleen weight, absolute 0.205 ± 0.0057 0.229 ± 0.00 96 0.219 ± 0.0081 0.185* ± 0.0059 Spleen weight, relative 0.454 ± 0.0075 0.483 ± 0.01 50 0.476 ± 0.0112 0.445 ± 0.0114
F 2 male+female weanlings (combined) Brain weight, absolute 1.479 ± 0.0084 1.467 ± 0.009 8 1.474 ± 0.0099 1.444* ± 0.0105 Brain weight, relative 3.194 ± 0.0380 3.233 ± 0.051 6 3.274 ± 0.0622 3.491** ± 0.0508 Spleen weight, absolute 0.200 ± 0.0049 0.204 ± 0.00 66 0.203 ± 0.0087 0.160** ± 0.0056 Spleen weight, relative 0.430 ± 0.0084 0.445 ± 0.00 93 0.443 ± 0.0114 0.382** ± 0.0078 Thymus weight, absolute 0.214 ± 0.0050 0.207 ± 0.00 62 0.202 ± 0.0056 0.187** ± 0.0055 Thymus weight, relative 0.459 ± 0.0084 0.453 ± 0.00 96 0.445 ± 0.0092 0.450 ± 0.0101
Data taken from Table s OW3K-SUM, pp. 515-519, Pathology Report, Report N o. 201426-1

* = p < 0.05; ** = p < 0.01

Histopathology : There were no compound-related microscopic finding s observed in F 1 and F 2 weanlings of either sex at any dietary level tested

.

Developmental milestones : Maturation of external sexual organs (balanopreput ial separation, vaginal opening) was not affected in F 1 weanlings. Anogenital distance was not triggered t o be measured in this study.

III. CONCLUSION:

The NOAEL for male parental toxicity was establishe d at 1,000 ppm (equal to premating doses of 70.7 mg/kg bw/day or 79.5 mg/kg bw/day in P- or F 1 -generation males, respectively), based on body wei ght gain declines in P-generation males at 6,000 ppm (e qual to 419.3 mg/kg bw/day) and body weight gain declines, decreased terminal body weights and incre ased renal multifocal tubular dilatation in F 1 - generation males at 6,000 ppm (equal to 486.7 mg/kg bw/day).

The NOAEL for female parental toxicity was establis hed at 1,000 ppm (equal to premating doses of 82.5 mg/kg bw/day or 90.3 mg/kg bw/day in P- or F 1 -generation females, respectively), based on reduce d food consumption during lactation in P-generation f emales at 6,000 ppm(equal to 484.7 mg/kg bw/day) and reduced food consumption during lactation, redu ced body weights (end premating), body weight gain declines (premating), decreased terminal body weights and increased renal multifocal tubular dilatation in F 1 -generation females at 6,000 ppm (equal to 539.5 mg /kg bw/day).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 267 April 2008

The NOAEL for reproductive toxicity was established at 1,000 ppm in males (equal to premating doses of 79.5 mg/kg bw/day in F 1 generation males), based on abnormal sperm cell mo rphology in F 1 - generation males at 6,000 ppm (equal to 486.7 mg/kg bw/day), which included one animal with abnormal epididymal sperm cells and pronounced abno rmal spermatozoa leading to compromised fertilizing capabilities and other animals (9/30) s howing one to four amorphous sperm heads out of 200 sperm cells viewed. Reproductive performance was no t impaired at 6,000 ppm in either generation.

The NOAEL for reproductive toxicity was established at 6,000 ppm in females (equal to premating doses of 484.7 mg/kg bw/day or 539.5 mg/kg bw/day in P- o r F 1 -generation females, respectively), based on the absence of effects at 6,000 ppm on mean estrus cycle length, number of normally cycling females, mating-, fertility- and gestation indices, mean ges tation duration, mating performance, vaginal openin g, litter parameters determined at birth (pup weight, total number of pups born, stillborn pups, viabilit y index on lactation day 0, sex ratio, mean litter si ze).

The NOAEL for offspring toxicity was established at 1,000 ppm, based on decreased body weights on lactation day 21 and decreased body weight gain in F 1 pups and decreased body weights on lactation days 14 and 21 and decreased body weight gain in F 2 pups at 6,000 ppm. Pup weight declines corresponded with maternal body weight and food con sumption declines.

Reviewer’s Conclusions : Systemic toxicity in parental animals was eviden t at 6000 ppm as decreases in body weight and body weight gain in P-generation males and F 1 -generation males and females, kidney histopathology, and decreased kidney weight in the F 1 males and females. Reproductive performance was affected in one F 1 male that had an increase in abnormal sperm and re duced fertility at 6000 ppm. Offspring survival was not affected b y treatment. However, F 1 and F 2 pup growth was decreased during lactation at 6000 ppm.

The parental systemic LOAEL for BYI 08330 in male a nd female Wistar rats is 6000 ppm (419.3 and 484.7 mg/kg bw/day in males and females, respec tively) based on decreases in body weight (F 1 males and females), weight gain (P males, F 1 males and females), and kidney histopathology and decreased kidney weights (F 1 males and females). The parental systemic NOAEL i s 1000 ppm (70.7 and 82.5 mg/kg bw/day in males and female s, respectively).

The reproductive toxicity LOAEL for BYI 08330 in ma le Wistar rats is 6000 ppm (419.3 mg/kg bw/day in males) based on abnormal sperm cells and decreased fertility in the F 1 males. The reproductive toxicity NOAEL in males is 1000 ppm (7 0.7 mg/kg bw/day in males). The reproductive toxicity LOAEL was not observed in fem ales; the reproductive NOAEL in females was 6000 ppm (484.7 mg/kg bw/day).

The offspring toxicity LOAEL for BYI 08330 in male and female Wistar rats is 6000 ppm (419.3 and 484.7 mg/kg bw/day in males and females, respec tively) based on decreased body weight and body weight gain during lactation in both F 1 and F 2 generations. The offspring NOAEL is 1000 ppm (70.7 and 82.5 mg/kg/ bw/day in males and females, respectively).

IV. STUDY DEFICIENCIES:

No major deficiencies were identified in the conduc t of this study.– –

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B.6.6.2 Reproduction and fertility effects – range- finding

CITATION : Young, A.D. (2006). Technical grade BYI 08330: A dose range-finding reproductive toxicity

study in the Wistar rat. Bayer CropScience LP, Tox icology, 17745 South Metcalf, Stilwell, KS 66085-9104. Report Number 201300-1, July 12, 20 06. Unpublished.

I. MATERIAL AND METHODS

A. MATERIALS :

1. Test Material: BYI 08330 Description: White Powder Lot/Batch No.: 08045/0014 Purity: 97.5% (7/2003), 97.8% (12/2003), 97.6% (6 /2004) Compound Stability: stable at room temperature (~ 22 o ) Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-o xo-1-azaspiro[4.5]dec-3-en-4-yl carbonate; Spirotetramat CAS No. of TGAI: Non-stereo: 382608-10-8; cis: 20 3313-25-1

2. Vehicle and/or positive control : None. Compound was dry mixed and administered thr ough the diet.

3. Test animals : Species: Rat, males and females Strain: Number of animals: Wistar Hanover rats Crl:WI(Glx/BRL/Han)IGS BR Total animals ordered: 120; Number used on study: 50 male and 50 female. Age at study initiation: (P) 14 wks

Wt. at study initiation: (P) Males: 296.8 – 353.0 g; Females: 180.1 – 217.6 g

Source: Animal Receipt: Charles River Laboratories, Raleigh, NC Upon receipt, animals were examined by a veterinari an prior to release for study. Housing: Animals passing the initial shipment exam were housed individually (except during the mating phase and as noted below for the F 1 pups) in suspended stainless steel cages and deotized cage b oard in the bedding trays. During gestation and lactation, individual dams (and litters) were housed in polycarbonate cages with corn-cob bedding Diet: ad libitum - Purina Mills Rodent Lab Chow 5002 meal Water: ad libitum - pressure-activated water nipples Temperature: Humidity: Room temperature 19 to 25 o C

Relative humidity 30 to 70% Environmental conditions: Air changes:

Photoperiod: Minimum of 10 air changes per hour, based on averag e

number of changes per hour per day Daily photoperiod of 12 hrs of light [6:00 a.m. to 6:00 p.m.] alternating with 12 hrs of darkness Acclimatisation: 7 days

All procedures associated with the animals were app roved by the Institution’s Animal Care and Use Committee (IACUC); and during the course of the stu dy, room conditions were monitored and recorded in a consistent and timely manner. Feed and water samples were collected periodically and analyzed for potential contaminants; the concentrations outl ined in the “Certification Profile” for Purina Mill s– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 269 April 2008

Certified Lab Chows (PMI Nutrition International, 1 998) were used as a standard of comparison for judging the acceptability of any contaminants detec ted in the analyzed feed samples.

B. PROCEDURES AND STUDY DESIGN

1. Mating procedure : Males and females were exposed to the test material for ten weeks prior to mating. Mating was accomplished by co-housing one female with one male for up to seven consecutive days. During the mating phase vaginal smears were taken each morning and examined for the presence of sperm and/or internal vaginal plug. Females found to be insemin ated were placed in a polycarbonate nesting cage. The day on which insemination was observed in the v aginal smear was designated day 0 of gestation for that female. In order to evaluate those females which may have b een inseminated without exhibiting sperm in the vaginal smear or an internal vaginal plug, all rema ining females were placed in polycarbonate nesting cages following the 7-day mating period.

2. Study schedule : Fifty female and fifty male rats were assigned to o ne of five treatment groups (10 animals/group): nominal doses of 0, 200, 500, 6,000, and 10,000 ppm technical grade BYI 08330 in the diet. Animals were exposed to the treated feed throughout the ent ire in-life phase of the study. The parental anima ls (P-generation) were given test diets for 10 weeks p rior to mating. Selection of F 1 -pups to evaluate preputial separation and vaginal patency, as well a s sperm analysis, was made when the pups were 21 days of age. The F 1 -generation male pups were maintained until 8-9 wee ks old and the females until 5- 6 weeks old prior to sacrifice.

3. Animal assignment : Following a minimum of six days of acclimation and prior to initiation of treatment, study animals wer e examined by a veterinarian and released for study u se. The animals were then assigned to either a control or one of four chemically-treated groups us ing a weight stratification-based computer program (INSTEM Computer Systems, Stone, Staffordshire, UK) . Only those animals falling within +/- 20% of the mean for all animals were put on study (Table 6-86) . Once animals were distributed into their dose groups, each rat on study had a microchip (BioMedic Data Systems, Inc., Seaford, Delaware) subcutaneously implanted on their dorsal surface in the region between their scapulae. At the very least, the chip was encoded with a unique number, s pecifying the animal’s sex, dose group, cage number, and study affiliation. In addition, a lami nated cage card, essentially duplicating the inform ation present on the chip, was attached to the outside of each animal’s cage. Pups born alive were identifi ed by tattoo and pups found dead were identified with a marking pen. All animals not placed on study (fo r whatever reason) were ultimately sacrificed; howeve r, prior to that time the animals were available, subject to the approval of the IACUC, for alternati ve uses which may have been unrelated to this study

.– –

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Table B.6-86: Animal assignment and study design Animals/group Test Group Dose in Diet a

(ppm) P Males P Females F 1 Males Day 21/Days 59-64 F 1 Females Day 21/Days 37-42
Control 0 ppm 10 10 14/17 11/15
Low (LDT) 250 ppm 10 10 12/13 13/13
Mid 1 (MDT 1) 1,000 ppm 10 10 16/16 15/16
Mid 2 (MDT 2) 6,000 ppm 10 10 12/15 16/18
High (HDT) 10,000 ppm 10 10 0/0 0/0 Data taken from Text Table 1, p. 19, Report No. 201 300-1

a Diets were administered from beginning of the stud y until sacrifice.

4. Dose selection rationale : Doses were based principally upon the toxicologic al profile which emerged in the rat over the course of a subchronic toxicity testing study (Report No. 201136) and two developmental studies (Report Nos. AT01413 and AT01 512). It was anticipated that by using such a broad range of doses that a more definitive toxicol ogical profile would emerge in the animal that woul d allow the selection of appropriate doses at which t o conduct a subsequent two-generation reproductive toxicity testing study in the rat.

5. Dosage preparation and analysis : The test compound was mixed directly with the fee d. Treated diet was mixed at room temperature; aliquots of the chem ical were taken from the original test batch and transferred to the mixing area. The control test d iet was taken directly from the bag without mixing. A sample of each batch of feed mixed was taken and re tained in the freezer until the study was complete and the analytical data deemed satisfactory. Repla cement admixtures for each treatment group were prepared every two weeks during the 10-week premati ng period and weekly (or at greater intervals depending on freezer stability) thereafter and stor ed under freezer conditions until presented to the animals the following week (or weeks). Additionall y, the entire batch of replacement admixture for each treatment group was used for subsequent weekly feed ings.

The concentration of BYI 08330 in the various test diets was analytically verified for batches intende d for weeks 1, 2/3, 4/5, 6/9, and 10/14/16/20 (Bayer Crop science LP, Environmental Research, Plant and Animal Residue Group, 17745 S. Metcalf, Stilwell, K S). The homogeneity and stability of BYI 08330 when mixed in the rodent feed was characterized pri or to study initiation.

Results Homogeneity Analysis: The mean concentrations of BYI 08330 in the feed, s ampled from three distinct layers in the mixing bow l and containing a nominal concentration of either 70 , 150, or 10,000 ppm, were determined to be 68.7 ppm (range 65.0-73.1 ppm; CV = 3.2%), 144 ppm (rang e 127-153 ppm; CV = 5.5%), and 9,445 ppm (range 8,817-9,943; CV = 3.2%), respectively. Based on a %RSD< 10%, BYI 08330 was judged to be homogeneously distr ibuted in the feed over a concentration range of 70-10,000 ppm.

Stability Analysis: Following 7 days of room temperature storage, the a nalytically-determined concentration of the AI of t he test substance in the 70-, 150-, or 10,000-ppm admi xture was determined to be 68.5 ppm (63.3 ppm on day 0); 131 ppm (151 ppm on Day 0); and 9,564 ppm ( 9,405 ppm on day 0), respectively. Following 28 days of freezer storage, the analytically-determine d concentration of the AI of the test substances in the– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 271 April 2008

70-, 150-, and 10,000-ppm admixtures was determined to be 71.4 ppm (63.3 ppm on day 0); 146 ppm (151 ppm on day 0); and 9,544 ppm (9,405 ppm on day 0), respectively. BYI 08330 mixed in rodent ration was judged to be stable at room temperature for at least seven days and following freezer stora ge for a minimum of 28 days, over a concentration rang e of 70-10,000 ppm.

Concentration Analysis: Mean analytical concentrations for each dose group were 197, 498, 5,997, and 10,051 ppm, ranging from 98-101% of the corresponding nominal concentrations of 200, 500, 6,000 and 10,000 ppm, respectively. The AI of the test substance was not detected in th e control diet. Mean recovery was 101% and ranged from 97-103% for rodent ration spiked with 200, 500 , 6,000, or 10,000 ppm BYI 08330.

C. OBSERVATIONS

1. Parental animals :

Mortality and clinical observations: Mortality checks (cageside observations) were perf ormed twice daily (AM and PM), during the work week and once da ily on weekends and holidays with exceptions stated in the Protocol Amendments and Deviations se ction. Cageside observations characterized mortality, moribundity, behavioral changes, signs o f difficult or prolonged delivery, and overt toxici ty by viewing the animal in the cage. In the event a pos sible clinical sign was observed during the cagesid e evaluation, the animal was removed from the cage an d a detailed assessment conducted. A detailed evaluation of clinical signs includes both observin g the animal in the cage and removing the animal to perform a physical examination and was conducted on ce per week throughout the entire in-life phase of the study.

Body weights and food consumption: Body weights and food consumption were measured an d fresh feed provided once/week for both males and females during the 10-week premating period. During the mating period and until sacrifice, body weights for the males were taken once per week. Also during t he mating period, fresh feed was provided for both mal es and unmated females once/week but food consumption was not measured. During gestation, da m body weights were measured on GD 0, 6, 13, and 20, and fresh feed was provided and food consum ption measured once/week. During lactation, dam body weights were measured on LD 0, 4, 7, 14, and 2 1. Fresh feed was provided and food consumption measured once/week, with the exception of week one when food consumption was measured twice (LD 0-4 and 4-7).

Estrous cyclicity: Estrous cycling was not performed in this one-gener ation study.

Sperm parameters: For all males at termination, sperm was collected f rom one testis and one epididymis for enumeration of homogenization-resist ant spermatids and cauda epididymal sperm reserves, respectively. In addition, an evaluation of the morphology and motility was performed on sperm sampled from the distal portion (closest to t he urethra) of the vas deferens. Sperm motility an d counts were conducted using IVOS (Integrated Visual Operating Systems). Morphology and counts were conducted for all groups.

2. Litter observations: The following litter observations (X) were made.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 272 April 2008

Time of observation (lactation day) Observation Day 0 Day 4 a Day 4 b Day 7 Day 14 Day 21 Days 0- 21
Number of live pups X
Pup weight X X X X X

External alterations X X X X X
Number of dead pups X
Sex of each pup (M/F) X
Preputial Separation Performed postweaning
Vaginal Patency Performed postweaning

a Before standardization (culling) b After standardization (culling) Exceptions stated in the Protocol Amendments and De viations section

The size of each litter was adjusted on lactation D ay 4 to yield, as closely as possible, four males a nd four females per litter. If the number of male or female pups was less than four, a partial adjustmen t was made (e.g., three females and five males). No adju stment was made for litters of fewer than eight pups. Adjustments were made by random selection of the pu ps using software provided by SAS. Culled pups were sacrificed by intracranial injection of 0.01-0.05cc Fatal Plus (V ortech Pharmaceuticals, Dearborn, MI), grossly abnormal pups underwent a gross intern al and external examination, and all culled pups were discarded. The pups not culled on lactation D ay 4 were maintained with the dam until weaning (lactation day 21). At 21-days of age, a sufficien t number of F 1 pups/sex/litter were maintained and observed for vaginal opening and preputial separati on.

The F 1 -males were maintained for an additional 5 to 6 wee ks prior to performing sperm analysis (motility, counts, and morphology). F 1 -pups not selected for further evaluations were sac rificed and examined macroscopically for any structural abnormalities or pathological changes, particularly as they may rel ate to the organs of the reproductive system.

Dead pups were examined grossly for external and in ternal abnormalities, and a possible cause of death was determined for pups stillborn or found dead.

3. Postmortem observations :

1) Parental animals : All surviving parental males were sacrificed as s oon as possible after the last litters were produced. Maternal animals were sacrificed fo llowing the weaning of their respective litters (lactation day 21). These animals were subject to examinations as follows.

Male rats were sacrificed by carbon dioxide asphyxi ation, terminal body weights were taken, and a gros s external examination was performed. For all males at termination, sperm was collected from one testis and one epididymis for enumeration of homogenizatio n-resistant spermatids and cauda epididymal sperm reserves, respectively. In addition, an eval uation of the morphology and motility was performed on sperm sampled from the distal portion (closest t o the urethra) of the vas deferens. Sperm motility and counts were conducted using IVOS (Integrated Visual Operating Systems).

Each dam was terminated by carbon dioxide asphyxiat ion and a gross external examination was performed. Terminal body weights were taken and th e abdomen and thoracic cavities were opened and– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 273 April 2008

a gross internal examination was performed. The ut erus was excised and the implantation sites, if present, were counted.

Females which were found sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed after gestation Day 24. Females which w ere never observed as being inseminated and/or with an internal vaginal plug and did not deliver at lea st 24 days after the completion of the mating phase

, were sacrificed and necropsied. A gross necropsy w as performed on these animals as described above. In addition, patency of the cervical/uterine os in these females was examined via flushing of the uter ine horns with deionized water.

The following tissues were collected and weighed (X X). Micropathology (in addition to collection and weights) was performed on those tissues designated with (XXX):

XX Ovaries XXX Testes XX Uterus XXX Epididymides

XX Brain XX Prostate XX Pituitary XX Seminal vesicles

XX Liver XX Kidney XX Spleen XX Thyroid XX Thymus XX Adrenal

Animals found moribund while on study were sacrific ed if their status dictated, and a gross necropsy w as performed. Animals found dead were necropsied as s oon as possible. Necropsy examinations included those parameters previously described.

2) Offspring : Approximately half of the F 1 offspring were sacrificed at 21 days of age. Thes e animals were subjected to postmortem examinations (macrosco pic and/or microscopic examination) as follows. The following tissues from 21-day weanlings were co llected and weighed: Brain, thymus, spleen, and uterus. Any gross lesion was documented and collec ted. Exceptions are stated in the Protocol Amendments and Deviations section.

At 21-days of age, a sufficient number of F 1 male pups/litter was maintained for an additional 5 to 6 weeks prior to performing sperm analysis (motility, counts, and morphology).

Pups found dead or terminated in a moribund conditi on underwent a gross necropsy for possible defects and/or cause of death.

D. DATA ANALYSIS:

1. Statistical analyses : The data were analyzed using applications provid ed by DATATOX (Instem Computer Systems), SAS (SAS Institute, Inc.), or TA SC (Toxicology Analysis Systems Customized). Parametric data (including body weight gain and foo d consumption) were analyzed using a univariate Analysis of Variance (ANOVA) and if significant dif ferences were observed, a Dunnett's Test was performed. Nonparametric data (e.g., number of est rous cycles, litter size, and number of implantatio n sites) were first analyzed by the Kruskal-Wallis te st and then by the Dunn's Test if significant diffe rences were identified. Nonparametric dichotomous data (e .g. fertility and gestation indices) were initially analyzed by the Chi-Square Test and if significance was observed between groups then by the Fisher's Exact Test with the Bonferroni adjustment. To the extent possible, the frequency of gross lesions was first examined visually, then, in the event of ques tionable distribution, by statistical analysis usin g the– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 274 April 2008

Chi-square and Fisher's exact tests. Differences b etween the control and test compound-treated groups were considered statistically significant when p < 0.05 or p < 0.01.

2. Indices : Reproductive indices: The following reproductive indices were calculate d from breeding and parturition records of animals in the study:

100 housed

- co females of No. es

ated femal min inse No. (%) Index Mating a ×

=



### 100 females ated min inse No. of b

s nt female No. pregna ) Index (% Fertility ×

=

100

females pregnant of No pups

live with females of No. (%) Index Gestation ×

=

a Includes pregnant females not observed sperm positi ve or with an internal vaginal plug. b Includes females which did not deliver, but had im plantation sites.

Offspring viability indices : The following viability indices were calculated f rom lactation records of litters in the study:



### 100 er sites/litt on implantati of No. Total r

born/litte pups No. Total (%) Index Birth ×

=

100 tter of pups/li Total No. n/litter e pups bor No. of liv Index (%) Livebirth ×

=

100 n/litter e pups bor No. of liv ing )

(pre-cull 4 er on day e pups/lit No. of liv Index (%) Viability ×

=

100 ling) (post-cul 4 ter on day e pups/lit No. of liv 21 n day s/litter o No. of pup Index (%) Lactation ×

=

3. Historical control data : Historical control data were provided in Attachmen t IV of the study report. This data were obtained from reproduction studies perfor med in the laboratory (1998-2004) in the Wistar rat

.

II RESULTS

1. Findings :

a. Parental toxicity :

General observations: There were no treatment-related mortalities or cli nical signs observed during the course of the study. Hair thinning was noted on three males at 10,000 ppm. During premating, no effects were observed on body weight, body weigh t gain, or food consumption in parental animals up to 500 ppm (Table 6-87). At 10,000 ppm, decreas ed mean premating body weight gain (NSS) was observed in male (10%) and female (12%) parenta l animals. Females showed nonsignificant body weight gain declines at 6,000 ppm during prema ting (11%) and lactation (12%). Females of the 10,000 ppm group were sacrificed after the gestatio n phase due to non-pregnancy (see below). The– –

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mean daily intake of the test substance (mg BYI 083 30/kg bw/day) throughout this one-generation reproduction study at nominal dietary concentration s of 0, 200, 500, 6,000 or 10,000 ppm, respectively, is summarized in Table 6-88.

Table B.6-87: Mean (± s.e.) body weight (g) and bod y weight gain (g) Dose Group (ppm) Body weight 0 200 500 6,000 10,000 a P generation males (pre-mating)
BW – week 0 325.4 ± 4.41 329.3 ± 5.17 325.6 ± 5.21 325.8 ± 5.66 326.7 ± 4.38
BW – week 10 441.3 ± 8.98 444.4 ± 10.47 439.4 ± 8.2 6 438.3 ± 7.76 431.4 ± 7.99 BW gain b – week 1- 10 115.9 115.1 113.8 112.5 104.7 (10)

P generation females (pre-mating)
BW – week 0 202.1 ± 3.67 199.6 ± 3.70 202.8 ± 2.96 200.3 ± 3.32 202.1 ± 4.24
BW – week 10 246.1 ± 4.75 245.1 ± 5.27 247.2 ± 5.04 239.4 ± 5.54 240.7 ± 3.94 BW gain – week 1-10 44.0 45.5 44.4 39.1 (11) c 38.6 (12) P generation females (lactation)
BW LD 0 273.2 ± 5.41 270.6 ± 7.26 262.1 ± 7.86 263. 6 ± 6.49 -
BW LD 4 282.2 ± 6.09 280.4 ± 7.75 276.1 ± 8.13 273. 3 ± 5.48 -
BW LD 7 289.4 ± 5.10 288.6 ± 9.02 282.8 ± 7.40 279. 8 ± 5.15 -
BW LD 14 306.8 ± 6.51 304.6 ± 7.32 301.5 ± 8.36 291 .5 ± 4.41 -
BW LD 21 293.4 ± 5.70 290.1 ± 7.04 286.3 ± 8.66 281 .4 ± 6.12 - BW gain b – days 1- 21 20.2 19.5 24.2 17.8 (12) -
Data taken from Tables 2 and 13, pp. 45-48 and 73, respectively, Report No. 201300-1

a = no pups at this dietary level b Weight gain values were calculated from group means

. c Number in parenthesis is percent difference from co ntrol.

Table B.6-88: Mean test article intake

Phase of Study 200 ppm in mg/kg/day a 500 ppm in

mg/kg/day a 6,000 ppm in

mg/kg/day a 10,000 ppm in

mg/kg/day a

Premating ( P

-gen) - Male 10.5 27.8 320.1 537.9

Premating ( P

-gen) - Female 12.8 31.4 384.1 645.7

Gestation ( P

-gen) - Female 12.9 31.9 393.9 NA b

Lactation ( P

-gen) - Female 26.7 74.4 831.0 NA b
Data taken from Text Table 7, Report No. 201300-1

a Individual values were based on the means for each particular phase for each generation. b No pregnancies occurred in the 10,000 ppm dose gro up.

b. Reproductive function : In P generation males a significant decline in both motility and progression of sperm was noted in the 10,000 ppm dose group as wel l as a slight decline in epididymal counts and a significant increase in abnormal sperm (Table 6-89) . The abnormal sperm observed in the morphological exam presented as amorphous sperm heads. These find ings resulted in no pregnancies at this dietary level. Sperm analysis revealed no effects in P gene ration males at 6,000 ppm and below.

In F 1 eight-to-nine week old interim males, slight declin es in motility and progression of sperm was observed in the 6,000 ppm dose group (Table 6-89). Morphology showed an increase in abnormal sperm presenting as amorphous sperm heads. There we re no sperm effects observed in F1 eight-tonine week old interim animals at 500 ppm and below.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 276 April 2008

Table B.6-89: Mean sperm measures Dose Group (ppm) Sperm Analysis 0 200 500 6,000 10,000 a P generation males (n ≈ 10/group) Motility: % motile 74.1 74.0 79.0 75.2 30.6 Motility: % progressive 57.4 53.7 58.3 52.4 14.1 Sperm counts: sperm/g testis 98.8 128.4 109.7 111.0 97.7 Sperm counts: sperm/g epididymis 985.6 732.8 798.1 944.2 464.0 Morphology: number of normal cells 199.2 199.3 199.5 199.0 105.0 Morphol.: number of amorphous heads 0.7 0.7 0.5 0.8 95.5 Morphology: number of small heads 0.1 0.0 0.0 0.2 0.0 F1 eight-to-nine week old interim animals (n ≈ 15/group) Motility: % motile 89.5 89.7 88.3 75.5 - Motility: % progressive 69.2 63.5 62.5 55.7 - Sperm counts: sperm/g testis 81.8 75.1 73.1 73.0 - Sperm counts: sperm/g epididymis 503.6 485.7 507.1 463.4 - Morphol.: number of normal cells 197.2 198.5 198.7 179.6 - Morphol.: number of amorphous heads 2.8 1.5 1.3 20. 2 - Morphol.: number of small heads 0.1 0.0 0.0 0.2 - Data taken from Table 22, pp. 98-99, Report No. 201 300-1 a = no pups at this dietary level

c Reproductive performance : The 10,000 ppm dietary level was associated with n o fertility in the P generation animals. There were no implantation site s noted in the females at this same level. At the 2 00- , 500-, and 6,000-ppm dietary levels, there were no compound-related effects on any parameter (e.g., mating, fertility, or gestation indices, days to in semination, gestation length, or the median number of implants). The litter parameters determined at birt h (litter size, percentage of males born, birth ind ex, live birth index, viability index, lactation index), wer e also not affected.

Gross pathology, organ weights: No treatment-related macroscopic changes were obse rved at necropsy in P generation rats. Absolute and relati ve weights of the cauda epididymis were decreased b y 29% and 27%, respectively, in the 10,000 ppm males (Table 6-90). In the females of the 10,000 ppm dose group, terminal body weight declines (NSS), as well as declines in absolute and relative kidney a nd liver weights were observed.

Table B.6-90: Affected organ weight

Dose Group (ppm) Weight (g) ±

s.d. 0 200 500 6,000 10,000 P generation males Cauda epid. abs. 0.337 ± 0.038 0.321 ± 0.022 0.346 ± 0.036 0.311 ± 0.071 0.239* ± 0.030 Cauda epid. rel. 0.074 ± 0.009 0.071 ± 0.005 0.076 ± 0.009 0.069 ± 0.017 0.054* ± 0.005 P generation females a Body weight 283.4 ± 26.0 279.1 ± 24.7 280.4 ± 25.2 272.8 ± 22.7 254.5 ± 16.2 (10) Liver, abs. 11.318±2.271 11.544±2.415 12.131±2.283 12.039±2.355 8.531*±0.886 Liver, rel. 3.963±0.535 4.103±0.624 4.313±0.650 4.3 94±0.682 3.351±0.248 Kidney, abs. 2.092±0.251 2.044±0.264 2.044±0.261 1. 956±0.211 1.732*±0.240 Kidney, rel. 0.738±0.048 0.731±0.064 0.729±0.079 0. 717±0.055 0.681±0.082 Data taken from Tables OW1K-SUM and OW2K-SUM, pp. 3 09-313 and 315-319, Pathology Report, Report No. 201300-1

a The 10,000 ppm group females were never pregnant. s.d. = standard deviation; epid. = epididymis; abs. = absolute; rel. = relative; * = p < 0.05– –

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Histopathology: P generation males in the 10,000 ppm group showed abnormal sperm in the epididymis (10/10) and cauda epididymis (9/9). The abnormal sperm consisted of what appears to be the retention of the residual body to the tail of t he spermatozoa. Severity grade consisted of minima l to moderate change. There was no evidence of change i n the epithelium lining the seminiferous tubules of the testes in 10,000 ppm generation males. There we re no abnormal sperm cells observed in P generation males at 6,000 ppm and below.

D NEONATAL TOXICITY:

1. General observations : No treatment-related clinical signs and no malform ations were observed in F1 pups. Pup weights at birth were not affected. At 6, 000 ppm, significant declines in offspring body wei ght (14%) were noted at lactation day 21 with a total d ecline in body weight gain of 22% and 15% for lactation days 14-21 and 4-21, respectively (Table 6-91).

Table B.6-91: Mean (s.e.) Pup Body Weights (g) and weight gains – male and female combined Dose Group (ppm) Lactation Day 0 200 500 6,000 10,000 c F1 pups (21 day old) BW day 0 6.3 ± 0.25 6.1 ± 0.21 5.8 ± 0.09 5.9 ± 0.19 - BW day 4 a 10.9 ± 0.59 10.5 ± 0.68 9.9 ± 0.1 10.1 ± 0.40 - BW day 4 b 10.9 ± 0.57 10.5 ± 0.69 9.9 ± 0.1 10.0 ± 0.41 - BW day 7 17.4 ± 0.87 16.7 ± 1.01 15.9 ± 0.24 15.8 ± 0.53 - BW day 14 34.9 ± 1.84 33.4 ± 1.21 31.9 ± 0.68 31.5 ± 0.80 - BW day 21 53.1 ± 2.38 50.1 ± 1.57 48.4 ± 0.89 45.7* ± 1.17 (14) d -
BW Gain days 0-4 4.6 ± 0.40 4.4 ± 0.50 4.2 ± 0.10 4.2 ± 0.27 - BW Gain days 4-7 6.5 ± 0.37 6.2 ± 0.32 5.9 ± 0.21 5.8 ± 0.20 - BW Gain days 7-14 17.5 ± 1.07 16.7 ± 0.56 16.0 ± 0.52 15.7 ± 0.45 - BW Gain days 14-21 18.2 ± 0.060 16.7 ± 0.49 16.5 ± 0.35 14.2** ± 0.50 (22) -
BW Gain days 4-21 42.2 ± 1.94 39.6 ± 0.96 38.5 ± 0.83 35.7* ± 0.89 (15) - Data taken from Tables 18 and 19, pp. 85-87 and 89- 91, respectively, Report No. 201300-1

a = before standardization (culling); b =after standardization; c = no pups at this dietary level d Number in parentheses is percent difference from co ntrol. * = p < 0.05; s.d. = standard deviation

Gross pathology, organ weights: In F1 pups of 21 days of age, no treatment-related macroscopic alterations were observed and no significant differ ences in terminal body weights or absolute and rela tive organ weights were noted at any dietary level teste d. In F1 eight-to-nine week old males, a significa nt decline (8%) in terminal body weight was observed a t 6,000 ppm (Table 6-92). There were no statistically significant effects observed on the o rgans weighed (testis, epididymis, cauda epididymis ) at any dietary level tested.

Table B.6-92: Mean (s.d.) terminal body weights (g) Dose Group (ppm) Body weight 0 200 500 6,000 10,000 a F1 eight-to-nine week old interim animals
Terminal BW 278.4 ± 26.7 289.6 ± 21.5 277.6 ± 8.8 2 57.0* ± 17.1 (8) -
Data taken from Table OW1K-SUM, p. 311, Pathology R eport, Report No. 201300-1.

a = no pups at this dietary level; * = p < 0.05; s.d . = standard deviation– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 278 April 2008

Histopathology: In F1 weanlings the testis and epididymis of the c ontrol and 6,000 ppm animals did not contain sperm due to the immaturity of the animals. The epithelium appeared normal.

F1 eight-to-nine week old interim animals of the 6, 000 ppm dose group showed abnormal sperm in the epididymis (4/15) and cauda epididymis (4/14). Sev erity grade consisted of minimal or moderate change. A single control animal had a moderate num ber of abnormal sperm within the epididymis and cauda epididymis. There was no evidence of change i n the epithelium lining the seminiferous tubules of the testes in F1 eight-to-nine week old interim ani mals in the 6,000 ppm group. There were no abnormal sperm cells observed in F1 eight-to-nine week old i nterim animals at 500 ppm and below.

Sexual maturation: There were no treatment-related findings observed i n the F1 generation on either preputial separation or vaginal patency at any diet ary level tested.

III. CONCLUSION:

The NOAEL for male parental toxicity in this pilot study was established at 6,000 ppm (equal to premating doses of 320 mg/kg bw/day), based on redu ced cauda epididymal weights and effects on sperm at 10,000 ppm (equal to 538 mg/kg bw/day). Th e highest dietary exposure level (10,000 ppm) caused treatment-related effects on sperm cells of P generation males and included decreased sperm motility and progression, decline in epididymal spe rm count and increase in abnormal sperm in the epididymis and the cauda epididymis. The NOAEL for female parental toxicity in this pilot was established at 500 ppm (equal to lactating doses of 74.4 mg/kg bw/day), based on slight body weight gain declines during lactation in the P-generation females at 6000 ppm (equal to 831 mg/kg bw/day).

The NOAEL for male reproductive toxicity in this pi lot study was established at 6,000 ppm in Pgeneration males (equal to premating doses of 320 m g/kg bw/day), based on decreased sperm motility and progression, decreased epididymal counts, and i ncrease in abnormal sperm in the epididymis and the cauda epididymis in P-generation males at 10,00 0 ppm (equal to 538 mg/kg bw/day). Other than the lack of pregnancies in the 10,000 ppm dose grou p, considered to be attributed to the effects on th e sperm of the males, there were no reproductive effe cts observed in the females during the course of th is pilot study.

The NOAEL for offspring toxicity in this pilot stud y was established at 500 ppm, based on decreased body weights on lactation day 21 at 6,000 ppm. Pup weight declines corresponded with maternal body weight declines.

The NOAEL for male parental toxicity in F 1 eight-to-nine week old interim males in this pilot study was established at 500 ppm based on decreased terminal body weights at 6,000 ppm.

The NOAEL for male reproductive toxicity in F 1 eight-to-nine week old interim males in this pilot study was established at 500 ppm based on decreased sperm motility and progression and an increase in abnormal sperm in the epididymis and the cauda epid idymis at 6,000 ppm.

Reviewers’ Conclusion : Systemic toxicity was limited to body weight gain decreases in the F 1 - generation animals. The target organ is the testes with progressive effects over successive generations: the F 1 males were affected at a lower dose than the P-gen eration males. Adverse effects on the testes (sperm) resulted in decreased reprodu ctive performance (fertility).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 279 April 2008

The parental systemic toxicity LOAEL for BYI 08330 in male and female Wistar rats is 6,000 ppm (320.1 and 384.1 mg/kg bw/day in males and females, respec tively) based on decreased body weight gain (P females) and terminal body weight (F 1 males). The parental systemic NOAEL is 500 ppm (2 7.8 and 31.4 mg/kg bw/day in males and females, respectivel y).

The reproductive toxicity LOAEL for BYI 08330 in ma le and female Wistar rats is 6000 ppm (320.1 and 384.1 mg/kg bw/day in males and females, respective ly) based on decreased sperm motility and progression and increased abnormal sperm cells in t he F 1 males. The reproductive toxicity NOAEL is 500 ppm (27.8 and 31.4 mg/kg bw/day in males and fe males, respectively).

The offspring toxicity LOAEL for BYI 08330 in male and female Wistar rats is 6000 ppm (320.1 and 384.1 mg/kg bw/day in males and females, respective ly) based on decreased body weight and body weight gain during lactation in F 1 pups. The offspring NOAEL is 500 ppm (27.8 and 31 .4 mg/kg/ bw/day in males and females, respectively).

IV. STUDY DEFICIENCIES:

No major deficiencies were identified in the conduc t of this study. In the Pathology Report, the histopathology summary table for the P-generation m ales is incorrect. Results were verified from individual animal data.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 280 April 2008

B.6.6.3 Teratogenicity test by the oral route in th e rat (OECD Annex IIA 5.6.10)

CITATION : Klaus, A.M. (2004). BYI 08330: Developmental toxic ity study in rats after oral administration.

Bayer HealthCare AG (Wuppertal, Germany). Laborato ry report number AT01413, August 23, 2004. Unpublished.

Klaus, A.M. (2001). BYI 08330: Pilot study on deve lopmental toxicity in rats after oral

administration. Bayer HealthCare AG (Wuppertal, Ger many). Laboratory report number T3068559, May 8, 2001. Unpublished.

Klaus, A.M. (2004). BYI 08330: Supplementary devel opmental toxicity study in rats after oral

administration. Bayer HealthCare AG (Wuppertal, Ger many). Laboratory report number AT01512, October 7, 2004. Unpublished.

I. MATERIALS AND METHODS

A. MATERIALS :

1. Test material : BYI 08330 Description: Technical, whitish powder
Lot/batch #: NLL 6425-14-a Purity: 99.0% (cis-isomer) Compound stability: Room temperature storage
CAS #of TGAI: 203313-25-1
Structure: Structure available on p17, Lab Report No. AT01413

2. Vehicle and/or positive control : 0.5% carboxymethylcellulose (CMC high viscosity, by Fluka BioChemica, 9471-Buchs, Switzerland) in demineraliz ed water as a suspension, (Lot/Batch # and purity were not reported).

3. Test animals : Species: Rat (nulliparous, non-pregnant females only)
Strain: Wistar, Hsd Cpb:WU Age/weight at study initiation: 13 to 15 weeks old; 202 to 247 g (GD 0)

Source: Harlan-Winkelmann GmbH, Borchen, Germany Housing: Individually housed in Type III Makrolon cages
Diet: Provimi Kliba Mouse and Rat Maintenance Diet, Art-N o.: 3883.0.15 ad libitum
Water: Tap water ad libitum
Environmental conditions: Temperatur

e: Humidity:
Air changes: Photoperiod 21± 2 o C

approx. 50% at least 10/hr 12 hrs dark/12 hrs light– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 281 April 2008

: Acclimation period: At least 7 days

B. PROCEDURES AND STUDY DESIGN

1. In life dates : Start: May 14, 2002; End: June 25, 2002

2. Mating : At the time of mating, males were mature to breed w eighing more than 300 grams. Males were the same strain and source as females. The an imals were paired by placing two females overnight into a Type III cage together with one male rat. If sperm was detected i n the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation (GD 0).

3. Animal assignment : After insemination was ascertained, the animals wer e assigned to their respective study group according to a computer-generated rando mization procedure as in Table 6-93.

Table B.6-93: Animal assignment
Dose (mg/kg bw/day) 0 20 140 1000

Number of Females 25 25 25 25

4. Dose selection rationale : The dose levels used were selected according to a dose-range finding-study in rats with dose levels of 0, 50, 200, 800 and 100 0 mg/kg bw/day, administered daily by oral gavage from GD 6-19 (Lab Report T3068559; see Appendix 1). Maternal effects were seen at the 800 mg/kg bw/day dose level (body weight loss, impaired body weight development) and at the 1000 mg/kg bw/day dose level (respiratory findings, piloerection, bod y weight loss, impaired body weight gain, in- or decreased water intake, increased urination, light colored feces). Effects on intrauterine developmen t could not be completely excluded at the 200 mg/kg b w/day level (possibly marginally reduced fetal weight), were evident at a dose level of 800 mg/kg bw/day (reduced placental and possibly fetal weight

, retarded ossification, wavy ribs) and were clearly observed at the 1000 mg/kg bw/day dose level (distinctly reduced fetal and placental weight, nec rotic placental borders, retarded ossification, wav y and 14th ribs and possibly marginally increased inciden ce of common malformations).

5. Dosage preparation and analysis : Test material-vehicle suspensions for each concentr ation were prepared after 2 to 7 days of use by mixing appropr iate amounts of test substance with 0.5% carboxymethyl-cellulose in demineralised water with storage at room temperature. Prior to the start of the study, stability of the test substance in aqueo us 0.5% carboxymethyl-cellulose was evaluated for a period of 8 days at room temperature. Concentratio n and homogeneity (top, middle, and bottom) of the test mixture were evaluated once prior to the study and once during the study. Investigations on the stability and homogeneity and a content check of th e active ingredient in samples of 1 mg/ml and 100 mg/ml were performed before the start of this study and covered the concentrations used in this study. Two content checks of the formulations of all conce ntrations as well as checks on homogeneity of the low and high concentrations were carried out during the in-life period of the study (formulations prep ared May 22, and June 20, 2002, respectively).

The first content check of the formulations of all concentrations as well as checks on homogeneity of the low and high concentrations revealed no significant deviation of the active ingredient content from th e nominal value in the formulations of any of the tre atment groups and homogeneity of the low and high concentrations also complied. Homogeneity of the 2 mg/ml concentration was marginally out of the accepted range (RSD 11.82% versus limit value of 10 %) during the second content check. However, this deviation lay only marginally above the accept ed range, the content of this formulation was– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 282 April 2008

confirmed and only 7 females were treated with this formulation for 3 or 4 days at the end of gestatio n. Thus, an impact of this finding on the outcome of t he study was excluded.

Results :

Homogeneity Analysis: The analytical data verify that the test substance was homogeneously distributed, with stirring, in the 1 and 100 mg/ml formulation; content was 95% and 106% of nominal, respectively.

Nominal Value (mg/ml) Content (mg/ml) Content as % of nominal value %RSD
1 0.952 95 0.31
100 106.249 106 4.76

Stability Analysis: The analytical data verify that the 1 and 100 mg/ml formulations were stable at room temperature for at least 8 days; after 8 days of st orage at room temperature content was within 3% of initial values.

Content in % Content as % of Nominal Value Nominal Value (mg/ml) Start After 8 Days Start After 8 days
1 0.952 0.927 95 93 100 106.249 107.642 106 108

Concentration Analysis: The analytical data verify that the test substance was homogeneously distributed in the 2 and 100 mg/ml formulations and that the contents were within defined limits.

Date of formulation preparation – May 22, 2002
Nominal Value (mg/ml) Content (mg/ml) Content as % of nominal value %RSD

2 1.756 88 3.14 14 14.094 101 Content only
100 102.277 102 1.52

Date of formulation preparation – June 20, 2002
Nominal Value (mg/ml) Content (mg/ml) Content as % of nominal value %RSD

2 1.822 91 11.82* 14 13.659 98 Content only
100 109.999 110 4.43 *Value not within defined limits

The analytical data indicate that the mixing proced ure was adequate and the differences between the nominal and actual dosage to the study animals was acceptable

6. Dosage administration : The females were treated once daily by oral gavage between 06:00 and 12:30 a.m. from GD 6-19, in a volume of 10 ml/kg body wei ght. The females of the control group received vehicle only (0.5% aqueous CMC) at the same volume. Dosing was based on the body weight from the most recent body weight determination.– –

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C. OBSERVATIONS :

1. Maternal observations and evaluations : The animals were checked for mortality or clinical signs at least once daily. Body weight was recorded on gest ation day 0 and daily between days 6 and 20. Food consumption was measured on GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, and 18-20, based on the differences in weight of feed provided and feed whi ch remained unconsumed. Dams were sacrificed on GD 20 using cardiotomy under deep carbon dioxide an esthesia. Examinations at sacrifice consisted of a gross pathological evaluation. Each uterus was wei ghed and dissected; weights and appearance of placentas were recorded. The number of corpora lute a and implantations were determined along with early resorptions, late resorptions, and live and d ead fetuses. In females without visible implantati on sites, the number of implantations was determined a fter staining of the uterus with a solution of 10% ammonium sulfide.

2. Fetal evaluations: The number of live fetuses was noted and their sex and body weight were recorded. All of the fetuses were evaluated for external abno rmalities. Every other fetus was assigned to eithe r the visceral or skeletal examinations. In the visceral examination, the fetus was sectioned according to the modified Wilson technique and the organs in the tho racic, abdominal and pelvic regions were evaluated for abnormalities. The fetuses included in the ske letal examination were eviscerated, treated with th e cartilage staining technique of Inouye, using alcia n blue GX, cleared with a dilute potassium hydroxid e solution and stained with Alizarin red S. Fetuses were sacrificed by carbon dioxide asphyxia.

D. DATA ANALYSIS :

1. Statistical analyses : The following parameters were analyzed using ANOVA with pairwise comparison for statistical significance by the Dunnett’s test: food consumption, body weight (including body weig ht gain and corrected body weight gain), uterine weigh t, number of corpora lutea per female, number of implantations per female, number of live fetuses pe r female and as a percentage of implantations per female, placental weights per female and fetal weig hts per female. The Chi square test was used to evaluate fertility and gestation rates, number of i mplantations per group, number of preimplantation losses per group, number of postimplantation losses , early resorptions, late resorptions or dead fetus es per group, the number of live fetuses per group in percent of implantations, number of male or female fetuses or fetuses with undeterminable sex per grou p, number of placentas with findings or litters wit h placental findings per group, number of fetuses or litters with external, visceral or skeletal finding s, and number of fetuses or litters with malformations. I f a significant difference existed, Fisher’s exact test with the Bonferroni correction was used for pairwise com parison. The Chi squared test also was used to evaluate the number of fetuses or litters with visc eral or cartilaginous tissue observations. The Krus kalWallis test was used to evaluate the number of prei mplantation losses per female, the number of postimplantation losses, early resorptions, late re sorptions, or dead fetuses per female, number of ma le or female fetuses or fetuses of undeterminable sex per female, and the proportion of placental, fetal external or visceral findings and fetal malformatio ns and external deviations per female. If a signif icant difference existed, Dunn’s test was used for pairwi se comparisons. Females without implantation sites or females which died or were sacrificed in moribun d conditions were excluded from any statistical evaluation. Differences were considered significan t when p<0.05.

2. Indices : The following indices were calculated from cesarea n section data of study animals:

Corrected body weight gain = Body weight gain GD0-20 - uterus weight GD20– –

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100 pora lutea No. of cor lantations

No. of imp a lutea - of corpor No. ion loss eimplantat Pr ×

=

100 lantations No. of imp ad fetuses

No. of de esorptions of late r No. ss ntation lo Post impla × +

=

100

of fetuses Total No. etuses of male f No. ratio Fetal sex ×

=

3. Historical control data : Historical control data from studies conducted be tween 1987 and 2003 were cited for comparison with concurrent study data, bu t were not provided for review.

II. RESULTS:

A. MATERNAL TOXICITY :

1. Mortality and clinical observations : Mortality, appearance and behavior were not affect ed by treatment with BYI 08330. Light colored feces were observed as an intermittent finding or at the end of gestation in few females of the 1000 mg/kg bw/day g roup, although the discoloration may be related to the high amount of white colored test substance giv en. A single female of the 140 mg/kg bw/day group (no.78) showed intermittently (GD 9 to 15) sunken f lanks, piloerection, severely reduced food intake a nd no or reduced amount of feces. Due to the lack a d ose relationship and single transient appearance, treatment relationship was not assumed for these fi ndings.

2. Body weight : Body weight data are summarized in Table 6-94. Transient body weight loss occurred on GD 7 in dams treated at 1000 mg/kg bw/day, and fina l body weight and weight gain was significantly reduced during gestation by approximately 7% and 20 %, respectively at 1000 mg/kg bw/day. Corrected body weight gain at 1000 mg/kg bw/day also was redu ced significantly by 29 % relative to controls.

At the 140 mg/kg bw/day dose level body weight loss was not observed, but body weight gain during the treatment (GD 6-19, 14% decrease relative to contro ls, p<0.05) was reduced which resulted in a statistically significantly reduced final body weig hts (5% decrease relative to of control, p<0.05). T hese findings were possibly both related to incidentally lower litter size in the 140 mg/kg bw/day dose gro up and to incidental morbidity of female no.78 (unrela ted to treatment), as effects on carcass weight and corrected body weight gain were not evident in the 140 mg/kg bw/day dose group and statistical significance was no longer evident for final body w eights when this female was excluded from calculations.

A supplementary oral developmental toxicity study w ith BYI 08330 in Wistar rats (Lab Report No. AT01512; see Appendix 2) at dose levels of 0, 10, 3 5 and 140 mg/kg bw/day revealed no effects on body weight development at dosages up to and includ ing 140 mg/kg bw/day. Body weight gain during treatment (GD 6-19) (corrected body weight gain fro m GD 0-20 in parentheses), in grams, in that study was 82.5 (50.8), 85.0 (53.8), 82.5 (49.4), and 81.4 (52.7) in the 0, 10, 35, and 140 mg/kg bw/day dose groups, respectively. Maternal body weight was als o unaffected in the rat developmental toxicity rang efinding study at a similar dose. Thus a treatment- related effect on body weight development was not assumed at the 140 mg/kg bw/day dose level in the m ain study. Necropsy revealed no treatment related findings at a dose level up to and including 1000 m g/kg bw/day.

Body weight and body weight gain were not affected at the 20 mg/kg bw/day dose level.– –

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Table B.6-94: Mean maternal body weight and body we ight gain (g ±SD) a
Dose in mg/kg bw/day (# of Dams) Interval 0 (20) 20 (24) 140 (23) 1000 (22)

Final body weight on day 20 343.9 ± 18.86 337.3 ± 16.23 327.7 ± 26.38* (-5%) b 319.9 ± 23.49**

(-7%)
Pretreatment: days 0-6 26.0 ± 4.11 25.8 ± 5.18 26.1 ± 4.36 22.8 ± 5.19

Treatment: days 6-19 86.8 ± 10.05 85.2 ± 10.94 74.8 ± 18.79* (-14%) 69.5 ± 13.25**

(-20%) Throughout gestation: days 0-20 123.4 ± 13.62 121.0 ± 13.40 111.1 ± 21.56 100.3 ± 18.81** (-19%) Corrected BW gain from day 0-
20 50.3 ± 9.73 46.9 ± 11.27 47.3 ± 12.34 35.6 ± 11.83**
(-29%)

a Data obtained from pages 64-68 in the study report (Lab Report AT01413) b Numbers in parentheses are percent change relative to control. * Statistically different (p <0.05) from the contr ol. ** Statistically different (p <0.01) from the contr ol.

3. Food consumption : Food consumption data are summarized in Table 6-95 . Food consumption at 1000 mg/kg bw/day was significantly reduced by 19%, 10%, 9% and 11% relative to controls on gestation days 6–9, GD 9–12, GD 15–18 and GD 18–20, respectiv ely. No treatment-related effect occurred at lower levels. Findings from a supplementary study ( Lab Report No. AT01512; see Appendix 2) confirmed no treatment-related effects on food consumption at doses of 10, 35, and 140 mg/kg bw/day. In that study, one female at the 140 mg/kg bw/day dose exhi bited distinctly to severely reduced food consumption on GD 9-15. This is the same animal th at exhibited a decrease in body weight on GD 11- 15. The morbidity of this animal was considered in cidental to treatment.

Table B.6-95: Mean (±S.D.) maternal food consumptio n (g/animal/day) a

Dose in mg/kg bw/day (# of Dams) Food consumption 0 (20) 20 (24) 140 (23) 1000 (22)
Day 0

- 3 20.35 ± 1.91 20.51 ± 2.34 20.41 ± 1.74 19.56 ± 1.34 Day 3 - 6 21.00 ± 2.28 21.21 ± 2.25 20.81 ± 1.67 19.94 ± 2.14 Day 6 - 9 21.37 ± 1.82 21.06 ± 1.90 20.62 ± 1.82 17.32 ± 2.74 ** (-19%) b
Day 9 -12 22.02 ± 1.79 21.81 ± 2.11 21.36 ± 3.31 19.80 ± 2.82* (-10%) Day 12 - 15 22.48 ± 1.97 22.61 ± 1.79 21.43 ± 4.20 20.64 ± 2.37 Day 15 – 18 25.85 ± 2.24 26.04 ± 1.93 24.78 ± 2.81 23.53 ± 2.87** (-9%) Day 18 – 20 26.42 ± 3.15 25.73 ± 2.24 25.83 ± 2.55 23.59 ± 2.89** (-11%)

a Data obtained from page 63 in the study report (La b Report AT01413).

b Numbers in parentheses are percent change relative to control. * Statistically different (p <0.05) from the contr ol. ** Statistically different (p <0.01) from the contr ol.

4. Gross pathology : No test-substance related gross pathological find ings were ascertained in the females of the dosed groups at dose levels up to and includ ing 1000 mg/kg bw/day.

5. Cesarean section data : Data are summarized in Table 6-96 and as follows: No effects occurred on gestation rate, postimplantation loss, number of fe tuses, sex distribution, and placental weight at a dose– –

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up to and including 1000 mg/kg bw/day. A marginal r eduction of placental weight (not statistically significant) together with a more distinct reductio n of fetal weight (86% of control, p<0.01) was obse rved in the 1000 mg/kg bw/day dose group. The marginally lower fetal weight of the 140 mg/kg bw/day group was considered incidental and not related to treatm ent. Effects on fetal weight were not evident in a supplementary oral developmental toxicity study wit h BYI 08330 (Lab Report No. AT01512; see Appendix 2). Here mean fetal weights in the 0, 10, 35 and 140 mg/kg bw/day dose groups were 3.60, 3.61, 3.57 and 3.60 grams, respectively, which rend ers a treatment related effect at the 140 mg/kg bw/day dose level in the present study unlikely. E ffects on fetal weight were not evident at the 20 m g/kg bw/day dose level.

Preimplantation loss was slightly increased in the 140 mg/kg bw/day group, and thus mean number of implantations was marginally lower. However, preim plantation loss and mean number of fetuses available for evaluation lay in the range of histor ical control data. Furthermore, a treatment relate d effect was not evident in the supplementary study (Lab Rep ort No. AT01512; see Appendix 2) at dose levels of 10, 35, and 140 mg/kg bw/day. Thus, an impact on t he outcome of the study due to unequal distribution of females in the different groups with respect to preimplantation loss and number of implantation sit es was excluded.– –

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Table B.6-96: Cesarean section observations a [mean ±SD values, as appropriate]
Dose (mg/kg bw/day) Observation 0 20 140 1000

# Animals assigned (mated) 25 25 25 25 # Animals pregnant 20 24 23 22 Pregnancy rate (%) 80 96 92 88 # Nonpregnant 5 1 2 3 Maternal wastage 0 0 0 0

No. died 0 0 0 0 No. died pregnant 0 0 0 0 No. died nonpregnant 0 0 0 0

No. aborted 0 0 0 0 No. Premature delivery 0 0 0 0 Total No. corpora lutea (Corpora lutea/dam) 289 (14.4 ± 1.88) 372

(15.5 ± 2.65) 347

(15.1 ± 2.78) 321

(14.6 ± 2.54) Total No. implantations (Implantations/dam) 260 (13.0 ± 1.75) 314

(13.1 ± 1.53) 277**

(12.0 ± 2.85) 285

(13.0 ± 1.91)

Total No. litters 20 24 23 22

Total No. live fetuses (Live fetuses/dam) 247 (12.4 ± 1.84) 301

(12.5 ± 1.69) 253

(11.0 ± 3.19) 270

(12.3 ± 2.12)

Total No. dead fetuses (Dead fetuses/dam) 0 (0) 0

(0) 0

(0) 0

(0) Total No. resorptions 13 13 24 15 Early 0 0 0 0

Late 13 13 24 15 Resorptions/dam 0.6 0.5 1.0 0.7 Early 0 0 0 0

Late 0.6 ± 0.81 0.5 ± 0.83 1.0 ± 1.46 0.7 ± 0.99 Litters with total resorptions 0 0 0 0 Mean fetal weight (g) 3.63 ± 0.157 3.58 ± 0.276 3.50 ± 0.341 3.12 ± 0.233**

Males 3.72 ± 0.178 3.69 ± 0.289 3.62 ± 0.302 3.20 ± 0.239**

Females 3.53 ± 0.192 3.47 ± 0.276 3.35 ± 0.328 3.04 ± 0.229** Sex ratio (% male/litter) 54.2 ± 13.64 47.7 ± 15.76 47.9 ± 20.13 48.0 ± 11 .20 Preimplantation loss (%) (Preimplantation loss/ female) 10.0 (1.5 ± 1.61) 15.6

(2.4 ± 2.06) 20.2

(3.0 ± 2.12)* 11.2

(1.6 ± 1.53) Postimplantation loss (%) (Postimplantation loss/ female) 5.0 (0.6 ± 0.81) 4.1

(0.5 ± 0.83) 8.7

(1.0 ± 1.46) 5.3

(0.7 ± 0.99) Placental weight (g) 0.62 ± 0.050 0.64 ± 0.106 0.65 ± 0.175 0.57 ± 0.061

a Data obtained from pages 70-76 in the study repo rt (Lab Report AT01413). * Statistically different (p <0.05) from the contr ol. ** Statistically different (p <0.01) from the contr ol.– –

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B. DEVELOPMENTAL TOXICITY :

Table 6-97 summarizes fetal malformations, categori zed as external, visceral, or skeletal. Both the fe tal and litter incidences of malformations were within or at the upper limit of the historical control inc idence range (up to 6.9% of fetuses and 40.0% of litters). Although two of the findings are rare observation s (cleft palate, co-arctation of aortic arch), a spec ific potential of BYI 08330 to induce these types o f malformations was not assumed since these findings occurred only once in the 1000 mg/kg bw/day group, were observed spontaneously in control or lo w dose groups of recent developmental toxicity studies and were different in type.

1. External examination : Fetal external malformations included: cleft pala te (1000 mg/kg bw/day: 1 case), microphthalmia (1 case each in the 0, 20, and 1000 mg/kg bw/day groups), anophthalmia (20 mg/kg bw/day: 1 case), and a combined finding of shortene d upper jaw, macroglossia, domed head, dyplasia of limbs, skull and vertebral column (140 mg/kg bw/day : 1 case). Fetal external deviations were not observed in this study, at dose levels up to and in cluding 1000 mg/kg bw/day.

2. Visceral examination: Fetal visceral malformations included: co-arctati on of aortic arch (1000 mg/kg bw/day: 1 case), atrial septal defect of the heart (1 case each in the 0, 20, and 1000 mg/kg bw/day groups), and absent lobe of the thyroid gland (0 mg /kg bw/day: 3 fetuses in 2 litters; 140 mg/kg bw/da y: 1 case). The incidence and type of visceral deviat ions of fetuses was not affected up to and includin g 1000 mg/kg bw/day. Common visceral deviations repr esenting the normal range of scattering for the rat strain used included findings at the eyes (slightly folded retina), brain (slight dilation of ventricl es; dorsal brain slightly misshapen), cochlea (blood in cochle a), thyroid (reduction in size), arteries (left com mon carotid artery septated), liver (small additional l iver lobe/s), kidneys and ureters (slightly increas ed renal pelvis cavitation, absence of renal papilla, hydrou reter), testes (slightly displaced), bladder (sligh tly distended), genital tubercle (slightly elongated) a nd hemorrhages in different structures.

T– –

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able B.6-97: Malformations a
Dose (mg/kg bw/day) Observations b

0 20 140 1000 No. Fetuses(litters) examined 247 (20) 301 (24) 253 (23) 270 (22) No. Fetuses(litters) affected with malformations 7 (4) 5 (4) 2 (2) 12 (9) % Malformed fetuses (litters) per group 2.83 (20.0) 1.66

(16.7) 0.79 (8.7) 4.44 (40.9) External malformations Cleft palate 0 0 0 1 (1) Microphthalmia, unilateral 1 (1) 1 (1) 0 1 (1) Anophthalmia, unilateral 1 (1) 1 (1) 0 0 Upper jaw shortened, macroglossia, domed head, dysplasia fore- and hindlimbs, skull and vertebral column 0 0 1 (1) 0

Visceral malformations Lobe of thyroid gland absent 3(2) 0 1 (1) 0 Atrial septal defect of the heart 1 (1) 1 (1) 0 1 (1) Co-arctation of aortic arch between left carotid an d left subclavian arteries, ascending aorta reduced i n size, left subclavian artery arises from descending aorta 0 0 0 1 (1)

Skeletal malformations Dysplasia of forelimb bones 1 (1) 2 (2) 0 (0) 4 (4) Altered appearance of sacral vertebral arch; pelvic shift 0 0 0 3 (3)

Supernumerary lumbar vertebra 0 0 0 1 (1)

a Data obtained from pages 78-79 and 132-136 (visceral data excluded) in the s tudy report (Lab Report AT01413).

b Some observations may be grouped together.

c Fetal (litter) incidence– –

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3. Skeletal examination : Table 6-98 includes fetal skeletal malformations. Skeletal evaluation revealed a slightly increased number of fetuses and litters wi th generally common unspecific malformations (4.4% and 40.9%, respectively) at the maternally toxic 10 00 mg/kg bw/day dose level. Skeletal malformations included dysplastic forelimb bones (0 mg/kg bw/day: 1 case; 20 mg/kg bw/day: 2 fetuses in 2 litters; 1000 mg/kg bw/day: 4 fetuses in 4 litters), a super numerary lumbar vertebra (1000 mg/kg bw/day: 1 case) and altered appearance of sacral vertebral ar ches and pelvic shift (1000 mg/kg bw/day: 3 fetuses in 3 litters).

Table 6-98 below lists the skeletal deviations tha t were statistically significantly different on a f etal and/or litter basis. Statistically significantly retarded ossification in various locations (phalanges, stern ebrae, vertebrae, skull bones) were noted at the 1000 mg/k g bw/day dose level in the osseus and/or cartilagineous parts. Increased incidences of skele tal variations (wavy ribs, 14 th ribs, combined osseous and cartilagineous findings) were seen as well. All these findings can be ascribed to the observed gro wth retardation at this dose level and are considered t o be an indirect effect of treatment.

Retarded ossification was noted in the 140 mg/kg bw /day dose group and in the 20 mg/kg bw/day dose group on a fetal basis at single bones of the fore- and hindlimbs without clear dose relationship, tog ether with incomplete ossification of the 6 th sternal segment and the supraoccipital bone (at 20 mg/kg bw/day only). None of these findings were statistically si gnificantly different on a litter basis and all fin dings were within the range of historical control values. Furt hermore, the changes of the low- and mid-dose group s were poorly correlated with dose. In addition, a s upplemental study (Lab Report No. AT01512; see Appendix 2) revealed no relevant effects on the deg ree of ossification at dose levels of 10, 35 and 14 0 mg/kg bw/day. Moreover, no statistically significan t changes were noted in ossification means of all b ut two of the respective bones up to and including 140 mg/kg bw/day and this significance was only seen on a fetal basis. Based on the clarifying results o f the supplementary study, a treatment-related effe ct for retarded ossification in the present study at doses of 20 and 140 mg/kg bw/day is considered unlikely.

The number of individual ribs with wavy appearance and sum of wavy ribs was statistically significantl y increased on a fetal basis in the 140 and 20 mg/kg bw/day dose group, but not on a litter basis. The f etal incidences at 20 and 140 mg/kg bw/day for this skel etal variant (17% and 18%, respectively) were slightly outside the historical control incidence r anges of 2.7 – 15.1% (historical control range deri ved from 22 studies performed between 1996 and 2001). H owever, the mean fetal control incidence was rather low and a dose-response relationship was not clearly evident for the mid- and low-dose group, assuming that the statistical differences noted for wavy ribs were apparently due to chance variation. Based on the clarifying results of the supplementar y study, which revealed no indications for a treatm ent related increase of wavy ribs at dose levels up to 140 mg/kg bw/day [13 (8), 12 (5), 19 (6), and 11 (4 ) fetuses (litters) in the 0, 10, 35, and 140 mg/kg b w/day dose groups, respectively], a treatment-relat ed effect for the occurrence of wavy ribs in the prese nt study at doses of 20 and 140 mg/kg bw/day is excluded.– –

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Table B.6-98: Skeletal examinations a
Dose (mg/kg bw/day)

Observations b 0 20 140 1000
#Fetuses (litters) examined 127 (20) 159 (24) 135 (23) 146 (22) #Fetuses (litters) affected N/A c N/A N/A N/A Incomplete ossification Distal phalanx digits, right

-1 st

-2 nd

-3 rd

-4 th

-5 th
Distal phalanx digits, left

-1 st

-2 nd

-3 rd

-4 th

-5 th 65 (19) d
64 (19) 20 (12) 39 (16) 108 (20)

77 (19) 74 (19) 25 (13) 50 (18) 109 (20) 112** (24)

103* (24) 45* (15) 77** (23) 149 (24)

121* (24) 112 (24) 59** (18) 85 (23) 149 (24) 89 (20)

81 (20) 29 (10) 61 (18) 124 (23)

100 (23) 90 (22) 37 (12) 76* (20) 123 (23) 133** (22)

120** (22) 78** (18) 111** (22) 142** (22)

134** (22) 126** (22) 87** (20) 118** (22) 142** (22) Distal phalanx toes, right
-1 st

-2 nd

-3 rd

-4 th
Distal phalanx toes, left

-1 st

-2 nd

-3 rd

-4 th 57 (19) 34 (14) 21 (9) 45 (17)

74 (19) 41 (16) 26 (12) 45 (16) 101** (23)

67* (22) 44 (18) 75 (22)

123** (24) 75* (22) 53 (18) 84* (22) 87** (23)

52 (16) 39 (12) 75** (20)

99* (23) 62* (19) 48* (16) 75** (20) 111** (22)

95** (22*) 84** (22**) 101** (22)

110** (22) 93** (22) 82** (22**) 101** (22)
Sternebra
-2 nd

-3 rd

-4 th

-5 th

-6 th 28 (16) 2 (2) 2 (2) 28 (14) 4 (4) 35 (16)

2 (2) 5 (4) 33 (17) 21** (12) 29 (16)

3 (2) 4 (3) 32 (17) 21** (13) 97** (21)

14* (10*) 21** (12**) 85** (22*) 56** (16**) Thoracic vertebral body, 1 st 7 (7) 5 (5) 8 (6) 30** (14) Sacral vertebral arch(es), right
-3 rd

-4 th
Sacral vertebral arch(es), left
-3 rd

-4 th 1 (1) 20 (11)

1 (1) 20 (11) 7 (3)

35 (14)

7 (3) 32 (12) 4 (2)

37 (18)

4 (2) 27 (11) 12* (7)

77** (20*)

13** (8) 80** (21**) Premaxillary bone, bilateral 0 (0) 1 (1) 0 (0) 8* (4) Mandible, bilateral 1 (1) 0 (0) 0 (0) 11* (4) Nasal bone, bilateral 4 (3) 9 (7) 2 (1) 44** (14**) Frontal bone, bilateral 1 (1) 2 (2) 0 (0) 10* (7) Parietal bone, bilateral 24 (11) 44 (18) 20 (10) 48* (18) Interparietal bone 30 (13) 54 (20) 32 (16) 70** (20) Supraoccipital bone 8 (5) 29** (10) 8 (6) 32** (13)– –

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Dose (mg/kg bw/day) Observations b 0 20 140 1000 Temporal bone 5 (5) 7 (6) 8 (5) 41** (18**) Zygomatic bone, bilateral 0 (0) 2 (2) 3 (2) 20** (13**)

No ossification Proximal phalanx digits, right
-3 rd

-4 th
Proximal phalanx digits, left
-3 rd

-4 th 82 (19) 87 (19)

92 (20) 98 (20) 120 (23)

132* (24)

129 (24) 138 (23) 98 (21)

107 (22)

107 (23) 110 (23) 139** (22)

144** (22)

143** (22) 144** (22)

5 th Metacarpal, right
5 th Metacarpal, left 7 (6) 10 (8) 24* (11)

27 (12) 18 (9)

21 (8) 53** (15)

61** (15) Distal phalanx toes, right
-1 st

-2 nd

-3 rd

-4 th

-5 th
Distal phalanx toes, left
-1 st

-2 nd

-3 rd

-4 th

-5 th 5 (5) 5 (5) 5 (5) 5 (5) 7 (6)

3 (3) 4 (4) 3 (3) 5 (5) 5 (5) 7 (4)

11 (7) 11 (7) 12 (7) 11 (7)

8 (4) 11 (7) 11 (7) 12 (7) 11 (7) 2 (2)

5 (2) 4 (2) 4 (2) 4 (2)

4 (3) 3 (2) 3 (2) 4 (2) 7 (4) 19* (7)

22** (8) 23** (8) 28** (9) 28** (10)

16* (6) 23** (9) 24** (10) 29** (10) 29** (10) Supraoccipital bone, round area 12 (11) 15 (10) 19 (12) 32* (19)

Wavy ribs, sum of fetal incidence 7 (6) 27** (10) 24** (12) 84** (21**)

14 th rib, sum of fetal incidence 19 (10) 17 (12) 17 (11) 67** (19) Thoracic vertebral body, dumbbell shaped

-11 th

-12 th

-13 th 28 (14) 32 (14) 15 (12) 21 (12)

27 (12) 5* (5*) 19 (12)

21 (12) 11 (9) 69** (20)

75** (21) 49** (19)

Fontanelle, slightly enlarged 11 (7) 30 (12) 8 (6) 34** (13)

a Data obtained from pages 80-129 in the study report (Lab Report AT01413).

b Some observations may be grouped together.

c N/A, total fetal (litter) incidence was not availa ble.

d Fetal (litter) incidence; values that are statisti cally significant on a fetal and litter basis are i n bold type. * Statistically different (p <0.05) from the contr ol. ** Statistically different (p <0.01) from the contr ol.

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS :

The LOAEL for maternal toxicity was 1000 mg/kg bw/d ay, based on reduced food consumption, transient body weight loss, reduced terminal body weight, and reduced corrected body weight gain at 1000 mg/kg– –

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bw/day, and the maternal toxicity NOAEL was 140 mg/ kg bw/day. The LOAEL for developmental toxicity was established at 1000 mg/kg bw/day, based on a ma rginal reduction of placental weight, distinctly decreased fetal weights, slightly increased inciden ce of common unspecific malformations, increased incidence of skeletal variations (wavy ribs, 14 th ribs, combined osseous and cartilaginous findings) , and increased incidence of retarded ossification at 100 0 mg/kg bw/day, and the developmental toxicity NOAEL was 140 mg/kg bw/day. No evidence for a prima ry embryotoxic or teratogenic potential of BYI 08330 was determined.

B. REVIEWER COMMENTS :

1. Maternal toxicity : Mortality, appearance, and behavior were not affect ed by treatment with BYI 08330 at dose levels up to and including 1000 mg/kg bw/day. At 1000 mg/kg bw/day, body weight development was reduced compared to controls. Final body weigh t was decreased 7% relative to control; weight gain during treatment and throughout gestation was decre ased by 20% and 19% relative to control, respectively; and corrected body weight gain was de creased 29% relative to control. At 140 mg/kg bw/day, final body weight was decreased 5% relative to control and weight gain during treatment was decreased 14% relative to control. However, the co rrected body weight gain was not statistically significantly affected and effects were not reprodu ced in the supplementary study (Lab Report AT01512; see Appendix 2) at dose levels of 10, 35, and 140 m g/kg bw/day, so toxicological relevance at 140 mg/kg bw/day was not assumed.

In the range-finding study (Lab Report T3068559), i mpaired body weight gain was evident at 800 mg/kg bw/day. At 1000 mg/kg bw/day, food consumption was decreased during intervals GD 6-9 (19% decrease relative to control), GD 9-12 (10% decreas e relative to control), GD15-18 (9% decrease relative to control), and GD 18-20 (11% decrease re lative to control). No gross pathological findings were found at doses up to and including 1000 mg/kg bw/day.

The oral maternal toxicity LOAEL for BYI 08330 in r ats is 1000 mg/kg bw/day, based on impaired food consumption, transient body weight loss, impai red body weight gain, and reduced final body weight. The maternal NOAEL is 140 mg/kg bw/da y.

2. Developmental toxicity:

a. Deaths/resorptions : There were no fetal deaths. Preimplantation loss (d ue to increased late resorptions) was higher in dams of the 140 mg/kg bw /day dose group compared to controls, resulting in marginally lower mean number of total implantati ons. However, an effect was not evident in the 1000 mg/kg bw/day dose group or in dams from the 14 0 mg/kg bw/day group in the supplementary study with BYI 08330 (Lab Report AT01512; see Appen dix 2). Therefore, a toxicologically relevant effect is not assumed.

b. Altered growth : Reduced fetal weight (86% of control) was observed in the 1000 mg/kg bw/day dose group. No effect on fetal weight was evident at doses of 140 mg/kg bw/day and below (10, 20, and 35 mg/kg bw/day) in either the main study or th e supplementary study. In the range-finding study (Lab Report T3068559), a marginal reduction o f fetal weight could not be excluded at doses of 200 and 800 mg/kg bw/day; final evaluation at these levels could not be determined in the pilot study.

c. Developmental variations : At 1000 mg/kg bw/day, developmental variations incl uded incomplete ossification of the distal phalanx digits and toes, 2 nd through 5 th sternebra, 1 st thoracic vertebral body,– –

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3 rd and 4 th sacral vertebral arches, and several skull bones ( e.g., mandible, and premaxillary, nasal, frontal, parietal, interparietal, supraoccipital, t emporal, and zygomatic bones). No ossification was evident at the 3 rd and 4 th proximal phalanx digits, 5 th metacarpals, distal phalanx toes, and the round area of the supraoccipital bone. Further, the incid ence of the sum of 14 th rib deviations, dumbbell shaped 11 th , 12 th , and 13 th thoracic vertebral bodies, and slightly enlarged f ontanelle was greater in the 1000 mg/kg bw/day dose group. The total incide nce of wavy ribs was increased compared to control values at all dose levels in the main study (20, 140, and 1000 mg/kg bw/day), but was not reproduced in the supplementary study at doses of 1 0, 35, and 140 mg/kg bw/day. Thus, toxicological relevance was assumed at the 1000 mg/ kg bw/day dose level only. In the range-finding study, the incidence of wavy ribs was slightly incr eased (data not available for review) at dose level s of 800 and 1000 mg/kg bw/day.

Statistically significant effects on ossification a lso were evident at the 20 and 140 mg/kg bw/day dos e levels, but there was no dose relationship and, wit h the exception of incomplete ossification of the left 4 th distal phalanx digit and the left 2 nd distal phalanx toe at 140 mg/kg bw/day, the effect s were not reproduced in the supplementary study with BYI 08330 at doses up to and including 140 mg/kg bw/day. For the two exceptions, toxicological rele vance is not assumed since no dose relationship was evident in either the main study or the supplem entary study.

d. Malformations : The total incidence of fetal (litter) malformation s was higher (not statistically significant) in the 1000 mg/kg bw/day dose group, i ncluding two single observations considered rare, cleft palate and co-arctation of the aortic arch. A specific potential of BYI 08330 to induce these types of malformations was not assumed.

The oral developmental toxicity LOAEL for BYI 08330 in rats is 1000 mg/kg bw/day, based on reduced fetal weight, increased incidence of malfor mations, and increased incidence of skeletal deviations (retarded ossification of skull bones, fore- and hindlimbs, sternum and vertebral column; wavy ribs, 14 th ribs, and combined osseous and cartilaginous findi ngs). The developmental NOAEL is 140 mg/kg bw/day.

C. STUDY DEFICIENCIES :

A minor deficiency was that, the total number of fe tuses (litters) affected by skeletal deviations cou ld not be determined from the study report (also the case for all data except the visceral evaluation data in the supplementary study report (Lab Report No. AT01512)

.

Homogeneity of the 2 mg/ml concentration was margin ally out of the accepted range (RSD 11.82% versus limit value of 10%) during the 2 nd content check. However, this deviation lay only ma rginally above the accepted range, the content of this formu lation was confirmed and only 7 females were treated with this formulation for 3 or 4 days at th e end of gestation. Thus, an impact of this finding on the outcome of the study was excluded.

APPENDIX 1 : Oral Rat Developmental Toxicity Dose Ranging Stud y

CITATION : Klaus, A.M. (2001). BYI 08330: Pilot Study on Deve lopmental Toxicity in Rats After Oral

Administration. Bayer HealthCare AG (Wuppertal, Ger many). Laboratory report number T3068559, May 8, 2001. Unpublished.– –

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In a range finding oral developmental toxicity stud y (Lab Report No. T3068559), BYI 08330 (98,8% a.i., batch # NLL 6425-5) was administered to five groups of 7 inseminated Wistar (Hsd Cpb:WU) rats/dose in 0.5% aqueous carboxymethylcellulose by oral gava ge at dose levels of 0, 50, 200, 800 and 1000 mg/kg bw/day from GD 6-19. For better clarification of toxic effects, 7 additional females were added later on to the 1000 mg/kg bw/day dose group. The f etuses were delivered by cesarean section on GD 20 and dams were killed and subjected to necropsy. Investigations were performed on general tolerance of the test compound by the females as we ll as on its effect on intrauterine development (including external, skeletal and visceral evaluati on of fetuses).

Maternal toxicity: Treatment related effects on respiration (transien tly gasping or labored breathing, respiratory sounds), water intake (increased or dec reased water intake at the end of treatment), excretion (increased urination and light colored fe ces) and appearance (piloerection) were restricted to the 1000 mg/kg bw/day group. Slight body weight los s for one day (GD 6 to 7) occurred at a dose level of 800 mg/kg bw/day and above; the 1000 mg/kg bw/da y group additionally exhibited slightly reduced food consumption during treatment and a more pronou nced reduction at the end of treatment. Reduced carcass weight and corrected body weight gain were observed at the 800 mg/kg bw/day level (marginal effects) and to a more distinct degree in the 1000 mg/kg bw/day group. Final body weight and body weight gain during gestation were reduced distinctl y in the 1000 mg/kg bw/day group. Necropsy revealed no treatment related findings at a dose le vel up to 800 mg/kg bw/day and toxicological relevance was not assumed for a slight renal pelvis dilation seen in one female of the 1000 mg/kg bw/day group due to comparable findings in historic al controls. Therefore, the maternal toxicity LOAEL was 800 mg/kg bw/day based on body weight los s and impaired body weight gain. The maternal toxicity NOAEL was 200 mg/kg bw/day.

Developmental toxicity: Reproductive parameters (i.e. gestation rate, post implantation loss, litter size and fetal sex distribution) were not affected to a toxicologically relevant degree by treatment at a d ose level up to and including 1000 mg/kg bw/day. A marg inal reduction of placental weight was seen at a dose level of 800 mg/kg bw/day and above; in the 10 00 mg/kg bw/day group together with increased incidence of necrotic placental borders and possibl y engorged placentae. A marginal effect on fetal weight (reduction) could not be completely excluded at dose levels of 200 and 800 mg/kg bw/day and was clearly evident at the 1000 mg/kg bw/day dose l evel. Final evaluation of this finding at the 200 a nd 800 mg/kg bw/day dose levels was not possible in th is pilot study.

Overall incidence of common unspecific malformation s (dysplastic tubular bones, interatrial septal defect of the heart) was marginally increased at th e distinctly maternal toxic 1000 mg/kg bw/day dose level (i.e. 5.5% of fetuses and 35.7% of litters af fected) though within the upper range of historical control data. One case each of dysplastic tubular b ones was seen in the other dose groups; at the 800 mg/kg bw/day dose level together with one case of i nteratrial septal defect. Since these malformations are common findings in the rat strain used and thei r incidence lay within the range of historical cont rol data, toxicological relevance was not assumed for t hese findings up to and including the 800 mg/kg bw/day dose level. Final evaluation was not possibl e at the 1000 mg/kg bw/day level in this pilot stud y. External and visceral fetal evaluation gave no furt her indication for toxicologically relevant effects up to and including 1000 mg/kg bw/day. Retarded fetal oss ification together with marginally to slightly increased incidence of wavy ribs (variation) occurr ed at a dose level of 800 mg/kg bw/day and above. Incidence of additional 14th ribs (variation) was a s well slightly increased at the 1000 mg/kg bw/day level. The developmental toxicity LOAEL was 200 mg/kg bw/d ay based on marginally reduced fetal weight. The developmental toxicity NOAEL was 50 mg/kg bw/day.– –

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Based on these results, it is concluded that the do se level of 1000 mg/kg bw/day is suitable for the h igh dose level in the main developmental toxicity study of BYI 08330 in rats. It is also considered that the dose levels of approximately 140 mg/kg bw/day and 2 0 mg/kg bw/day are suitable for the mid and low dose levels, respectively.– –

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APPENDIX 2 : Oral Rat Developmental Toxicity Supplementary Stu dy

CITATION : Klaus, A.M. (2004). BYI 08330: Supplementary devel opmental toxicity study in rats after oral

administration. Bayer HealthCare AG (Wuppertal, Ger many). Laboratory report number AT01512, October 7, 2004. Unpublished.

A supplementary developmental toxicity study was pe rformed for clarification of results of the main developmental toxicity study with BYI 08330 in rats (Lab Report AT01413). In the main study, equivoca l retarded ossification of single localisations and e quivocal increased incidence of wavy ribs were note d in the 140 and in the 20 mg/kg bw/day dose group witho ut clear dose relation.

Groups of 25 inseminated Wistar (Hsd Cpb:WU) rats ( 12-17 weeks old; 194-235 g on GD 0) were treated daily, orally by gavage, with BYI 08330 (99 .1% a.i., batch # NLL 6425-14-a), suspended in 0.5 % aqueous carboxymethylcellulose, from gestation day (GD) 6 to day 19 at doses of 0, 10, 35, and 140 mg/kg bw/day (in-life dates: February 5 to March 13 , 2003). The fetuses were delivered by cesarean section on GD 20. The stability and homogeneity of 1.0 and 100 mg/mL formulations, and the achieved concentrations of all formulations, were confirmed by analysis during the study. The study methodology was identical to the former study with the followin g exceptions: the liver of the dams were weighed an d those of the control and high dose groups were subj ected to histopathology. Since no treatment related effects on the liver were evident in the high dose group following histopathology, low and medium dose group livers were not subjected to histopathology. Blood samples were collected from maternal animals at necropsy for measurement of red blood cell param eters (erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular haemoglobin, a nd mean corpuscular hemoglobin concentration) and clinical chemistry parameters (aspartate aminot ransferase, alanine aminotransferase, alkaline phosphatase,  -glutamyltransferase, cholesterol, triglycerides, c reatine, urea, calcium, and phosphorus).

The total numbers of fetuses examined (number of li tters) was 266 (22), 263 (22), 275 (22), and 227 (19) for the 0, 10, 35, and 140 mg/kg bw/day groups , respectively. All live fetuses were weighed, sex ed, and examined for external abnormalities. Every oth er fetus within a litter was prepared for either skeletal or visceral evaluation. During fetal eval uation, it was observed that offspring of one male revealed a specific type of malformation (missing h ead of 1 st rib or microphthalmia or both) irrespective of dose group, indicating a paternally mediated eff ect. The litters (fetuses) sired by this male were excluded from calculation of the mean values for fe tal external, visceral and skeletal findings as wel l as from calculation of mean values for incidence of ma lformations and external and visceral deviations. Although only 19 litters were available at the 140 mg/kg bw/day dose level after exclusion of the 4 females mentioned above, the remaining numbers of f etuses (litters) available for evaluation were stil l regarded adequate to fulfil the guideline requireme nts of approximately 20 litters per group.

Maternal toxicity: No treatment related maternal effects were observe d at any dose level with respect to mortality, clinical signs, food consumption, bod y weight development, hematology and clinical chemistry parameters, liver weight, necropsy findin gs and histopathology of the liver at levels up to and including 140 mg/kg bw/day. The oral maternal toxicity LOAEL for BYI 08330 in r ats was not established at dose levels up to and including 140 mg/kg bw/day. The maternal toxicity NOAEL was 140 mg/kg bw/day, based on the absence of effec ts at 140 mg/kg bw/day.

Developmental toxicity: There were no treatment related effects at any dose level on reproductive parameters, (i.e. gestation rate, postimplantation loss, litter size, placental weight and appearance, fetal weight and fetal sex distribution). The incidence a nd type of fetal malformations were unaffected by– –

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treatment at levels up to and including 140 mg/kg b w/day. Meaningful fetal external or visceral deviations were not evident at a dose level up to a nd including 140 mg/kg bw/day in this study. Fetal skeletal ossification and incidence of skeletal var iations including evaluation of cartilaginous struc tures revealed no evidence for treatment related effects at a dose level up to and including 140 mg/kg bw/da y. The incidence of wavy ribs was not affected at a do se level up to and including 140 mg/kg bw/day. No evidence for a primary embryotoxic or teratogenic p otential of BYI 08330 was observed. The oral developmental toxicity LOAEL for BYI 08330 in rats not established at dose levels up to and including 140 mg/kg bw/day. The developmental toxi city NOAEL was 140 mg/kg bw/day, based on the absence of treatment-related deviations at 1 40 mg/kg bw/day.

More detailed results of developmental toxicity are included below:

Malformations : Table 6-99 summarizes fetal malformations, categori zed as external, visceral, or skeletal. External, visceral and skeletal evaluatio n of fetuses revealed no treatment related malformations at a dose level up to and including 1 40 mg/kg bw/day when excluding offspring of male no. 16 from evaluation of this study. The remaining malformations were generally of a common type and/or showed no dose response relationship on a fe tal and/or litter basis either in comparison to the concurrent control or to the results of the precedi ng study.

Isolated malformations of the eyes (uni- and/or bil ateral microphthalmia) was the most common malformation in this study (35 mg/kg bw/day: 5 case s; 140 mg/kg bw/day: 1 case), but displayed no dose relationship in the actual and in comparison t o the preceding study (Lab Report AT01413). Thus the incidence of microphthalmia in this study was r egarded as incidental and unrelated to treatment. One lobe of the thyroid gland was absent in a fetus of the 35 mg/kg bw/day dose group and in 2 fetuses (in 2 litters) of the 140 mg/kg bw/day dose group. Treatment relationship was excluded for these findi ngs due to lack of dose relationship in comparison to t he preceding study with dose levels up to 1000 mg/k g bw/day (control: 3 fetuses in 2 litters; 140 mg/kg bw/day: 1 case; 20 mg/kg bw/day and 1000 mg/kg bw/day: 0 cases). The remaining malformations in t he treated groups, i.e. atrial septal defect of the heart, displaced azygos vein, right-sided aortic sp ur and dysplastic forelimb bones were single findin gs without dose response relationship and were compara ble to the actual or historical controls so that treatment relationship was not evident for these fi ndings.– –

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Table B.6-99: Malformations a
Dose (mg/kg bw/day) Observations b

0 10 35 140 No. Fetuses(litters) examined 266 (22) 263 (22) 275 (22) 227 (19) No. Fetuses(litters) affected with malformations 5 (5) 2 (2) 9 (9) 3 (3) % Malformed fetuses (litters) per group 1.9 (22.7) 0.8 (9.1) 3.3 (40.9) 1.3

(15.8) External malformations Microphthalmia, unilateral and/or bilateral 0 0 5(5) 1 Visceral malformations Lobe of thyroid gland absent 0 0 1 2(2) Right sided aortic spur 0 0 1 0 Right sided azygos vein 0 0 1 0 Atrial septal defect of the heart 1 1 0 0 Skeletal malformations
4 th thoracic vertebral body unilateral one ossificatio n center missing and cartilage bipartite; bone and ca rtilage of 3 rd and 5 th thoracic vertebral bodies asymmetric 1 0 0 0

4 th and 5 th thoracic vertebral bodies fused 1 0 0 0 Dysplasia of forelimb bones (scapula, humerus, radi us and/or ulna) 2(2) 1 1 0

Supernumerary lumbar vertebra 0 0 0 1

a Data obtained from pages 134-138 in the study report (Lab Report No. AT01512 )

b Some observations may be grouped together.

c Fetal (litter) incidence

Developmental variations : Fetal external and visceral examinations revealed n o treatment-related deviations at a dose level up to and including 140 mg/kg bw/day. Table 6-100 summarizes fetal skeletal deviations. Evaluation of fetal skeletal deviation s showed considerably lower incidences of wavy ribs at all dose levels tested, when compared to the main s tudy. Furthermore all incidences were comparable to the control group [13 (8), 12 (5), 19 (6), and 1 1 (4) fetuses (litters) in the 0, 10, 35, and 140 m g/kg bw/day dose groups, respectively] and thus revealed no indication for a treatment related effect. This also renders a treatment related effect for occurre nce of wavy ribs at doses up to and including 140 mg/kg bw/day in the former study unlikely.

Fetal skeletal examinations revealed statistically significantly retarded ossification of isolated bon es on a fetal basis, scattered across all dose groups teste d. However, a relationship to treatment was not assumed, based on the lack of reproducibility in al most all cases when compared with the preceding study. Moreover, none of the fetal means of the dos e groups exceeded the limits of historical controls and no statistically significant changes have been observed on a litter basis. Furthermore, the doseresponse correlation was generally poor. Thus a tre atment related effect on ossification of forelimbs, hindlimbs, sacral vertebral arches and skull bones or on the incidence of supernumerary ribs was not assumed at a dose level up to and including 140 mg/ kg bw/day in the actual study.

The findings are described in detail as follows. S tatistically significantly retarded ossification of digits of the forelimbs was noted on a fetal basis. The stati stically significant findings at the 5 th right digit showed no statistical significance in the preceding study at a dose level up to and including 140 mg/kg bw/da y and the other localizations affected in the 140 mg/ kg bw/day dose group in the actual study were not affected in the preceding study (except for distal phalanx of 4 th digit left). Retarded ossification of distal phalanges of toes was seen with statistical signifi cance on a fetal basis for distal phalanx of 3 rd toe right and 2 nd and 5 th toe left. In the preceding study, only the 2 nd toe left was affected at 140 mg/kg bw/day,– –

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but the percentage in the preceding study at 140 mg /kg bw/day (47.3%) was nearly identical to the control value of the actual study (45.3%). Incompl ete ossification of 4 th sacral vertebral arch left occurred in the 35 and 140 mg/kg bw/day groups with statistical significance on a fetal basis but with out dose relation. Further, incomplete ossification of the sacral vertebral arch was not evident in the preceding study at doses up to and including 140 mg /kg bw/day.

One bone of the skull (sphenoid bone) showed statis tically significantly retarded ossification at the 140 mg/kg bw/day dose level on a fetal basis while ossi fication of the supraoccipital bones was more progressed (reduced number of incompletely ossified bones) at the 140 mg/kg bw/day dose level on a fetal and litter basis. On a litter basis neither s tatistical significance nor dose relationship were evident for retarded ossification of the sphenoid bone. In the preceding study effects on ossification of supraoccipital and sphenoid bone were not observed at a dose level up to and including 140 mg/kg bw/day.

Statistically significantly increased numbers of pu nctiform and comma-shaped supernumerary ribs (14 th ribs) were observed in the 140 mg/kg bw/day dose gr oup. However, statistical significance was not observed for percentage of overall number of 14 th ribs on a fetal or litter basis. The highest overa ll percentage of 14 th ribs on litter basis occurred in the control group (54.5% of litters affected versus 47.4% in the 140 mg/kg bw/day group). Furthermore, a treatment related effect on incidence of 14 th ribs was not evident in the preceding study with BYI 083 30 at a dose level up to and including 140 mg/kg bw/day.– –

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Table B.6-100: Skeletal examinations a
Dose (mg/kg bw/day)

Observations b 0 10 35 140
#Fetuses(litters) examined 140 (22) 136 (22) 144 (22) 118 (19) #Fetuses(litters) affected N/A c N/A N/A N/A Incomplete ossification Distal phalanx digits, right
-2 nd

-4 th

-5 th
Distal phalanx digits, left
-2 nd

-3 rd

-4 th 72 (18) d
53 (16) 117 (22)

85 (20) 34 (13) 64 (16) 83 (21)

63 (20) 128* (22)

96 (21) 38 (18) 81 (21) 76 (21)

66 (21) 134* (22)

87 (22) 45 (17) 70 (21) 82* (19)

70** (19) 113** (19)

89* (19) 50** (18) 75* (19) Distal phalanx toes, right, 3 rd
Distal phalanx toes, left, 2 nd 35 (14) 63 (18) 55* (19)

81 (20) 44 (14)

75 (20) 52** (16)

73* (18) Sternebra, 5 th 18 (9) 37* (15) 25 (12) 23 (12) Sacral vertebral arch(es), left, 4 th 18 (10) 33 (14) 38* (14) 30* (12) Sphenoid bone 1 (1) 8 (7) 4 (4) 9* (6)

No ossification Proximal phalanx digits, right, 4 th
Proximal phalanx digits, left, 3 rd 104 (21) 105 (22) 114 (22)

116 (22) 119 (22)

126* (22) 103* (19)

100 (19) Distal phalanx toes, left, 5 th 10 (22) 13 (22) 17 (22) 22* (19)

Wavy ribs, sum of fetal incidence 13 (8) 12 (5) 19 (6) 11 (4)

14 th rib, sum of fetal incidence 20 (12) 18 (9) 21 (8) 31 (9)

14 th rib, punctiform, bilateral 0 3 (2) 1 (1) 8** (3)

14 th rib, comma-shaped 2 (2) 5 (4) 2 (2) 10* (5)

a Data obtained from pages 94-132 in the study report (Lab Report No. AT01512) b Some observations may be grouped together.

c N/A, total fetal (litter) incidence was not availa ble.

d Fetal (litter) incidence. * Statistically different (p <0.05) from the contr ol. ** Statistically different (p <0.01) from the contr ol– –

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B.6.6.4 Teratogenicity test by the oral route in th e rabbit (OECD Annex IIA 5.6.11)

CITATION : Klaus, A.M. (2004). BYI 08330: Developmental toxic ity study in rabbits after oral

administration. Bayer HealthCare AG (Wuppertal, Ger many). Laboratory report number AT01003, February 17, 2004. Unpublished.

Holzum, B. (2001). BYI 08330: Pilot developmental t oxicity study in rabbits after oral administration. Bayer HealthCare AG (Wuppertal, Ger many). Laboratory report number T3062735, November 14, 2001. Unpublished.

I. MATERIALS AND METHODS

A. MATERIALS :

1. Test material : BYI 08330 Description: Technical, light

- beige powder Lot/batch #: 08045/0004 Purity: 98.9 % Compound stability : Room temperature storage CAS #of TGAI: 203313

- 25

- 1 Structure: Structure available on p16, Lab Report No. AT01003

2. Vehicle and/or positive control : 0.5% carboxymethylcellulose (CMC high viscosity, by Fluka BioChemica, 9471-Buchs, Switzerland) in demineraliz ed water as a suspension, (Lot/Batch # and purity were not reported).

3. Test animals : Species: Rabbit, Himalayan (nulliparous, non

- pregnant females only) Strain: CHBB.HM Age/weight at study initiation: 106 to 192 days old 1929 to 2805g (GD 0)

Source: Charles River Deutschland GmbH, 88535

- KiBlegg, Germany Housing: Individually housed in Makrolon cages Diet: Ssniff rabbit diet K- Z ad libitum (Ssniff Spezialdiaten GmbH, 59494-Soest; Germany)
Water: Tap water ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 20 ± 2 o C 50 + 20% at least 10/hr 12 hrs dark/12 hrs light

Acclimation period: At least 7 days

B. PROCEDURES AND STUDY DESIGN

1. In life dates : Start: March 25, 2003; End: June 25, 2003

2. Mating : The mating was performed between 05:00 and 10:00 CE T. One male rabbit was mated with one female rabbit under observation. About one hou r after the first mating had occurred the same animals were mated again. It was recorded which fe male was mated with which male. The day on which the copulation was observed was considered as day 0 of gestation (GD 0).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 303 April 2008

3. Animal assignment : After copulation, 88 females were sequentially al located to 4 experimental groups according to a computer-generated randomization pro cedure (see Table 6-101). During the conduct of the study, 10 additional females were allocated to the study without randomization: 2 females to the 4 0 mg/kg bw/day dose group and 8 females to the 160 mg /kg bw/day dose group. The 2 dams were added to the 40 mg/kg bw/day group because low fertility was suspected. The 8 dams were added to the 160 mg/kg bw/day group because 5 dams in this group wer e sacrificed in moribund condition, 1 dam was found dead, and 3 dams had no implantation sites at caesarean section.

Table B.6-101: Animal assignment
Dose (mg/kg bw/day) 0 10 40 160

Number of Females 22 22 24 30

4. Dose selection rationale : The dose levels used were selected according to a dose-range finding-study in rabbits with dose levels of 0, 5, 25, 100, 160, 250 or 500 mg/kg bw/day, administered daily by oral gavage from gestation GD 6-28 (Report No. T3062735) . Maternal effects were noted at the 160 mg/kg dose level (body weight loss, impaired body weight development, decreased food consumption) and were severe (hypoactivity, labored breathing, convu lsion, severe body weight loss, severely decreased food consumption) at levels of 250 mg/kg bw/day and above resulting in death or premature sacrifice in moribund conditions in all animals treated at 500 m g/kg bw/day and in one animal of the 250 mg/kg bw/day dose level. Necropsy revealed in single anim als hardened abdominal fatty tissue, round depressions in the gastric mucosa, enlarged gall bl adder, or gaseous content in the intestine and hemorrhages in the renal capsule. The gestation rat e in the 250 mg/kg group was decreased by one abortion and by one total resorption. Due to these findings and due to early sacrifice or death of the remaining females in the 250 and 500 mg/kg bw/day g roups the evaluation of the remaining reproduction parameters was limited to dose levels up to and inc luding 160 mg/kg. Effects on intrauterine development could not be completely excluded at the 160 mg/kg bw/day level (possibly marginally reduced fetal and placental weights). Appendix 1 ha s more detail on the pilot range-finding study. Bas ed on these results, it is concluded that the dose lev el of 160 mg/kg bw/day is suitable for the high dos e level in the main developmental study of BYI 08330 in rabbits. The dose levels of 40 mg/kg bw/day and 10 mg/kg bw/day are considered suitable for the mid dle and low dose levels, respectively.

6. Dosage preparation and analysis : Test material-vehicle suspensions for each concentr ation were prepared by mixing appropriate amounts of test subs tance with 0.5% carboxymethyl-cellulose in demineralised water with storage at room temperature. Investigations on the stability and ho mogeneity and a content check of the active ingredient in sam ples of 1.0 and 100 mg/ml revealed no significant deviations from the content determined on the day o f preparation after 8 days of storage. Two content checks of the dose preparations (2, 8 and 32 mg/ml) as well as checks on homogeneity of the low and high concentrations revealed no significant deviati on of the active ingredient content from the nomina l values. Test substance was not detectable in the 0 mg/ml preparation.

Results :

Homogeneity Analysis: The analytical data verify that the test substance was homogeneously distributed, with stirring, in the 1 and 100 mg/ml formulation; content was 95% and 106% of nominal, respectively.

Nominal Value (mg/ml) Content (mg/ml) Content as % of nominal value %RSD– –

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1 0.952 95 0.31 100 106.249 106 4.76

Stability Analysis: The analytical data verify that the 1 and 100 mg/ml formulations were stable at room temperature for at least 8 days; after 8 days of st orage at room temperature content was within 3% of initial values.

Content in % Content as % of Nominal Value Nominal Value (mg/ml) Start After 8 Days Start After 8 days 1 0.952 0.927 95 93
100 106.249 107.642 106 108

Concentration Analysis: The analytical data verify that the test substance was homogeneously distributed in the 2 and 32 mg/ml formulations and that the contents were within defined limits.

Date of formulation preparation – March 31, 2003
Nominal Value (mg/ml) Content (mg/ml) Content as % of nominal value %RSD

0 Not detec table - - 2 1.856 93 2.74
8 8.086 101 Content only 32 32.011 100 0.41

Date of formulation preparation –May 19, 2003 Nominal Value (mg/ml) Content (mg/ml) Content as % of nominal value %RSD

0 Not detec table - - 2 1.869 93 0.92 8 7.775 97 Content only

32 32.011 100 0.41
*Value not within defined limits

The analytical data indicate that the mixing proced ure was adequate and the differences between the nominal and actual dosage to the study animals was acceptable. Test substance was not identified in the 0 mg/ml samples.

6. Dosage administration : The females were treated once daily by oral gavage between 06:00 and 12:00 CET from GD 6-28, in a volume of 5 ml/kg body weigh t. The females of the control group received vehicle only (0.5% aqueous CMC) at the same volume. Dosing was based on the body weight from the most recent body weight determination, prior to eac h administration.

C. OBSERVATIONS :

1. Maternal observations and evaluations : The animals were checked for mortality or clinical signs at least once daily. Body weight was recorded on gest ation day 0 and daily from day 6 to 29. Food consumption was measured on GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20, 20-21, 21-24, 24-27, and 27-29, based on the differences in weight of feed p rovided and feed which remained unconsumed. Dams were sacrificed on GD 29 by intravenous inject ion of 2 ml T61 ® ad us.vet. (Hoechst Roussel Vet.) and subjected to cesarean section and necropsy. Ex aminations at sacrifice consisted of a gross pathological evaluation. Females which aborted were killed and necropsied. For females that survived– –

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until scheduled sacrifice, each uterus was weighed and dissected; weights and appearance of placentas were recorded. The number of corpora lutea and impl antations were determined along with early resorptions, late resorptions, and live and dead fe tuses. In females without visible implantation sit es, the number of implantations was determined after staini ng of the uterus with 10% ammonium sulphide solution.

2. Fetal evaluations: The number of live fetuses was noted and their sex and body weight were recorded. All of the fetuses were evaluated for external abno rmalities, and visceral and skeletal varitions and malformations. Fetuses were killed on dry ice. In t he visceral examination, the fetuses were sectioned according to the modified Staples technique and the organs in the thoracic, abdominal and pelvic regio ns and in the brain were evaluated for abnormalities. For skeletal examination, fetuses were eviscerated

, treated with the cartilage staining technique of Da wson/Inouye, using alcian blue, cleared with a dilu te potassium hydroxide solution and stained with Aliza rin red S. Processing and evaluation were performed after cesarean section without knowledge of treatment groups.

D. DATA ANALYSIS :

1. Statistical analyses : The following parameters were analyzed using ANOVA with pairwise comparison for statistical significance by the Dunnett’s test: food consumption, body weight (including body weig ht gain and corrected body weight gain), uterine weigh t, number of corpora lutea per female, number of implantations per female, number of live fetuses pe r female and as a percentage of implantations per female, placental weights per female and fetal weig hts per female. The Chi square test was used to evaluate fertility and gestation rates, number of i mplantations per group, number of preimplantation losses per group, number of postimplantation losses , early resorptions, late resorptions or dead fetus es per group, the number of live fetuses per group in percent of implantations, number of male or female fetuses or fetuses with undeterminable sex per grou p, number of placentas with findings or litters wit h placental findings per group, number of fetuses or litters with external, visceral or skeletal finding s, and number of fetuses or litters with malformations. I f a significant difference existed, Fisher’s exact test with the Bonferroni correction was used for pairwise com parison. The Chi square test also was used to evaluate the number of fetuses or litters with visc eral or cartilaginous tissue observations. The Krus kalWallis test was used to evaluate the number of prei mplantation losses per female, the number of postimplantation losses, early resorptions, late re sorptions, or dead fetuses per female, number of ma le or female fetuses or fetuses of undeterminable sex per female, and the proportion of placental, fetal external or visceral findings and fetal malformatio ns and external deviations per female. If a signif icant difference existed, Dunn’s test was used for pairwi se comparisons. Females without implantation sites or females which aborted, died or were sacrificed i n moribund condition were excluded from any statistical evaluation. Cases of skeletal localizat ions with mechanical damage were excluded from the calculation of percentages of affected localization s. Differences were considered significant when p<0.05.

2. Indices : The following indices were calculated from cesarea n section data of study animals:

Corrected body weight gain = Body weight gain GD0-29 - uterus weight GD29

100 pora lutea No. of cor ons

implantati of No. - lutea corpora of No. loss ation Preimplant ×

=

100 ons implantati of No. fetuses

dead of No. s resorption late of No. loss on implantati Post × +

=– –

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100 of fetuses Total No. etuses of male f No. ratio Fetal sex ×

=

3. Historical control data : Historical control data from studies conducted be tween 1987 and 2003 were cited for comparison with concurrent study data.

II. RESULTS:

A. MATERNAL TOXICITY :

1. Mortality and clinical observations : Appearance, behavior and mortality were unaffected at the 10 mg/kg level, while abortion in relation to severe m aternal toxicity was seen at the 40 mg/kg and 160 mg/kg dose levels. Death, sacrifice in moribund con dition, cold ears, alopecia, altered fecal amount/appearance and reddish excretions occurred a t the 160 mg/kg level. One female of the 160 mg/kg group was found dead after having shown r eddish excretion and soft feces and 5 females had to be sacrificed in moribund condition after ha ving shown severely reduced to no food consumption, severe body weight loss, cold ears, alopecia, reduc ed amount or no feces, diarrhea (in one female) and soft and light colored feces, reddish excretion, de creased water intake and urination and discolored urination most probably indicating concentration of urine. Abortion was observed most likely as a consequence of maternal toxicity in two other femal es of the 160 mg/kg group and in one female of the 40 mg/kg group showing clinical signs, impaired foo d consumption and body weight loss before abortion. The remaining females of the 160 mg/kg dose group r evealed cold ears, alopecia and soft, mucoid and light colored feces. A summary of maternal clinica l signs is presented in Table 6-102.

Table B.6-102: Maternal clinical observations durin g treatment (GD6-29) a
Dose in mg/kg bw/day (# of Dams) Observation 0 (22) 10 (22) 40 (24) 160 (30)

Normal 361/1 b 345/1 324/0 286/0
Sacrificed moribund 0/0 0/0 0/0 5/5
Found dead 0/0 0/0 0/0 1/1
Abortion 0/0 0/0 1/1 2/2
Cold ears 22/9 21/9 38/10 124/23 Alopecia 48/4 71/7 73/7 136/13
Reduced amount of feces 5/3 11/7 27/9 78/15
Soft feces 20/4 32/12 50/14 88/21 Light colored feces 0/0 3/1 3/1 9/3
Decreased urination 3/2 11/5 11/4 46/9 Discolored urination (light yellow) 0/0 2/2 5/3 32/6

Reddish excretion 0/0 1/1 0/0 12/4
Mucoid feces 0/0 1/1 0/0 3/1 Decreased water consumption 3/2 8/4 19/8 64/13

a Data obtained from page 60 in the study report ( Lab Report No. AT01003). b Frequency/ animals

2. Body weight : Body weight data are summarized in Table 6-103. Marked to severe body weight loss was observed in individual females of the 40 and 16 0 mg/kg bw/day dose groups which aborted or had– –

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to be sacrificed in moribund condition (body weight loss of -257 to -639 grams), while body weight changes in the remaining females did not show meani ngful treatment related effects. Statistically significantly reduced body weight gain during treat ment (GD 6-29) and the overall gestation period (GD 0-29) was noted in the 10 mg/kg bw/day dose group w hich resulted in a marginal body weight loss when corrected body weight gain was taken into considera tion. Impaired body weight development at the 10 mg/kg bw/day dose level was regarded as an incid ental finding and without relation to treatment due to lack of dose dependency. Mortality or early sacr ifice at the 40 and 160 mg/kg bw/day dose levels di d not contradict the assumed lack of dose dependency since only one female was sacrificed due to abortion in the 40 mg/kg bw/day dose group and a ne gative impact of mortality on the evaluation of bod y weight development could thus be excluded at a dose level up to and including 40 mg/kg bw/day.

Table B.6-103: Mean maternal body weight and body w eight gain (g ± SD) a
Dose in mg/kg bw/day (# of Dams) Interval 0 (22) 10 (22) 40 (21) 160 (19)

Final body weight on day 29 2735.5 ±
173.09 2751.1 ± 184.81 2815.8 ± 237.16 2738.1 ± 160.98

Pretreatment: days 0-6 111.5 ± 49.62 89.0 ± 33.80 121.8 ± 60.59 90.8 ± 60. 02

Treatment: days 6-29 362.0 ± 84.20 287.9 ± 90.74* 355.3 ± 107.63 327.3 ± 76.03

Throughout gestation: days 0-29 473.5 ± 96.67 376.9 ±
100.57** 477.0 ± 89.68 418.2 ± 106.21

Corrected BW gain from day 0-
29 65.2 ± 92.61 -0.8 ± 93.27 60.8 ± 82.88 46.5 ± 96.43

a Data obtained from pages 64-68 in the study repo rt (Lab Report No. AT01003). * Statistically different (p <0.05) from the cont rol. ** Statistically different (p <0.01) from the contr ol.

3. Food consumption : A statistically significant reduction in food con sumption was noted in the 10 mg/kg bw/day dose group (GD 27-29). This finding was rega rded as incidental because it was not dose related and was within the range of historical control data so that the finding was regarded as unrelated to treatment. Severely decreased or lack of food consu mption was observed after start of treatment and/or later on in females of the 160 mg/kg bw/day group w hich aborted or had to be sacrificed in moribund condition. Decreased food consumption also was seen in one female of the 40 mg/kg bw/day group before abortion on GD 24 (food consumption decrease d 19% from GD 18-20 to GD 20-21). The females which aborted or were sacrificed in moribund condit ion were excluded from statistical evaluation and data were not provided for review.

4. Gross pathology : Females of the 160 mg/kg group which had to be s acrificed in moribund condition revealed gross pathological findings of the intesti ne (fluid and/or gaseous contents in caecum), gall bladder (black or whitish mottled), and the liver ( green discolored in the area of the gall bladder/li ght discoloration). Treatment relationship was assumed for these findings. Incidental findings among all other females included cysts at the oviduct (0 mg/k g: 3 females; 10 mg/kg: 2 females; 160 mg/kg: 3 females); whitish punctiform areas in the caecum wa ll (10 mg/kg: 1 female) and gaseous contents in the rectum (40 mg/kg: 1 female).

5. Cesarean section data : Data are summarized in Table 6-104 and as follows: The gestation rate was decreased by one abortion in the 40 mg/kg dose grou p and two abortions in the 160 mg/kg group. No– –

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effects of treatment were noted in postimplantation loss in the remaining females, the number of fetus es as well as placental and fetal weight and appearanc e, and fetal sex distribution.

Table B.6-104: Cesarean section observations a [mea n±SD values, as appropriate]
Dose (mg/kg bw/day) Observation 0 10 40 160

# Animals assigned (mated) 22 22 24 30 # Animals pregnant 22 22 22 27 Pregnancy rate (%) 100 100 91.7 90 # Nonpregnant 0 0 0 3 Maternal wastage 0 0 1 8 No. died (pregnant) 0 0 0 1

No. aborted 0 0 1 2 No. premature delivery 0 0 0 0

No. sacrificed moribund 0 0 0 5 Total No. corpora lutea (Corpora lutea/dam) 186

8.5 ± 2.09 176

8.0 ± 1.57 186

8.9 ± 2.41 151

7.9 ± 2.46 Total No. implantations (Implantations/dam) 180

8.2 ± 1.84 165

7.5 ± 1.74 180

8.6 ± 2.44 138

7.3 ± 2.23

Total No. litters b 22 22 21 19

Total No. live fetuses (Live fetuses/dam) 170

7.7 ± 1.64 152

6.9 ± 1.51 172

8.2 ± 2.23 132

6.9 ± 2.09

Total No. dead fetuses (Dead fetuses/dam) 0 0 0 0

Total No. resorptions 10 13 8 6 Early 0 0 0 0

Late 10 13 8 6 Resorptions/dam Early 0 0 0 0

Late 0.5 ± 0.80 0.6 ± 0.73 0.4 ± 0.74 0.3 ± 0.48 Litters with total resorptions 0 0 0 0 Mean fetal weight (g) 37.78 ± 3.369 38.11 ± 3.137 36.25 ± 3.432 38.48 ± 4 .588

Males 37.69 ± 4.169 38.56 ± 3.002 37.15 ± 3.862 38.14 ± 4 .384

Females 38.17 ± 3.036 37.46 ± 3.608 35.60 ± 3.772 37.55 ± 5 .769 Sex ratio (% male/litter) 49.5 ± 19.05 47.2 ± 23.89 46.0 ± 14.85 51.4 ± 23.08 Preimplantation loss (%) (Preimplantation loss/ dam) 3.2

0.3 ± 0.77 6.3

0.5 ± 1.22 3.2

0.3 ± 0.72 8.6

0.7 ± 1.25 Postimplantation loss (%) (Postimplantation loss/ dam) 5.6

0.5 ± 0.80 7.9

0.6 ± 0.73 4.4

0.4 ± 0.74 4.3

0.3 ± 0.48 Placental weight (g) 4.46 ± 0.528 4.57 ± 0.717 4.27 ± 0.765 4.48 ± 0.925

a Data obtained from pages 70-77 in the study repor t (Lab Report No. AT01003). b Including only females with viable litters were con sidered when calculating “per dam” or “per litter” values.

B. DEVELOPMENTAL TOXICITY :– –

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Table 6-105 summarizes fetal malformations, categor ized as external, visceral or skeletal. Malformatio ns were not dose related and/or lay well within the ra nge of normal scattering of the rabbit strain used and/or were also seen in the current control. Indee d, the highest total incidence of malformations was in the 10 mg/kg group (5.3% of fetuses, 31.8% of litte rs). Single malformations which appeared only once in the 160 mg/kg dose group were different in type and comparable with historical control data so that a treatment related effect was not assumed for these malformations. Thus, a teratogenic effect of BYI 08330 was excluded at a dose level up to and includ ing 160 mg/kg bw/day. As some fetuses of the 10 mg/kg and 160 mg/kg group revealed more than one ma lformation, the number of malformations is higher than the number of affected fetuses in these groups.

1. External examination : External malformations included malpositioned for elimbs (one of the most common malformations in the rabbit strain used and most likely due to restriction of fetal movement in the uterus) in all dose groups and single cases of domed head and cleft palate in the 160 mg/kg dose group. No external deviations were evident at dose s up to and including 160 mg/kg.

2. Visceral examination: Visceral malformations included ventricular septal defects of the heart (3 fetuses in 3 litters in the 10 mg/kg dose group) and single cases of microphthalmia and encephalomeningocele in the 160 mg/kg dose group. Table 6-106 summarize s visceral deviations, including a possibly increased incidence of fetuses with distinct liver lobulation in the 160 mg/kg group. The toxicologica l significance of this finding was questioned, since the liver is normally lobed and abnormal liver lobu lation was not reported. Other visceral deviations noted a mong dose groups included a slight dilation of late ral brain ventricles and whitish discoloration of the l iver. The findings were comparable either to the cu rrent controls (slight dilation of brain ventricles) or t o historical controls (up to 2.8% of fetuses and 14 .3% of litters with whitish liver discoloration) and revea led no dose dependency.

3. Skeletal examination : Skeletal malformations were observed in the cartila ginous parts of ribs/cervical ribs/supernumerary ossification centers, scattered among all dose groups. Fetal skeletal evaluations (retardations, variations) including cartilagineous tissues revealed no treatment related effects at a dose level up to and including 160 mg/kg bw/day. Single statistically significant differences were observed at the 10 mg/kg and 40 mg/kg levels, on a fetal basis (retarded ossification of the 5 th sternebra in the 10 mg/kg dose group and of the 8 th caudal vertebral arches in the 10 mg/kg and 40 mg/ kg group). All values regarding retarded ossification at the 10 mg/kg and 40 mg/kg levels lay well within the normal range o f scattering for these findings in developmental toxi city studies in the strain of rabbits used. A dose dependency was not evident for these findings and s tatistical significance was not seen when calculati on was done on a litter basis.– –

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Table B.6-105: Malformations a,b

Dose (mg/kg bw/day) Observations c

0 10 40 160
No. Fetuses(litters) examined 170 (22) 152 (22) 172 (21) 132 (19)
No. Fetuses(litters) affected with malformations 5 (5) 8 (7) 4 (4) 7 (4)
% Malformed fetuses (litters) per group 2.9 (22.7) 5.3 (31.8) 2.3 (19.0) 5.3 (21.1)
External malformations
Domed head 0 0 0 1 Cleft palate 0 0 0 1 Malpositioned forelimbs 1 3(2) 1 5(2) Visceral malformations Eyeball reduced in size (microphthalmia) 0 0 0 1 Encephalomeningocele 0 0 0 1 Ventricular septal defect of the heart 1 3(3) 0 0 Skeletal malformations Fusion, bifurcation in the cartilaginous part, or s light thickening of ribs with/without supernumerary ossification center above the 1 st 1 3(3) 1 1

Sternal segment or at 8 th rib cervical rib, supernumerary ossification center (with cartilage) above the 1 st sternal segment fused with it 0 0 1 0

Missing presacral vertebra 2(2) 0 0 0 Supernumerary presacral vertebra 0 0 1 0

a Data obtained from pages 103-108 in the study rep ort (Lab Report No. AT01003).

b Fetal (litter) incidence.

c Some observations may be grouped together

Table B.6-106: Visceral deviations a, b

Dose (mg/kg bw/day) Observations c

0 10 40 160
No. Fetuses(litters) examined 170 (22) 152 (22) 172 (21) 132 (19)
No. Fetuses(litters) affected 5 (2) 3 (3) 8 (5) 10* (3)
% Affected fetuses (litters) per group 2.9 (9.1) 2.0 (13.6) 4.7 (23.8) 7.6 (15.8) Slight dilation of lateral brain ventricles 5(2) 2(2) 3(2) 0(0) Whitish discoloration of the liver 0 1 3(2) 2(1) Distinct liver lobulation 0(0) 0(0) 2(1) 8**(2)

a Data obtained from pages 79-80 in the study re port (Lab Report No. AT01003).

b Fetal (litter) incidence.

c Some observations may be grouped together ** Statistically different (p <0.01) from the contr ol.

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS :

The NOAEL for maternal toxicity was 10 mg/kg bw/day , based on abortion in one animal at 40 mg/kg bw/day with clinical signs, impaired food consumpti on and body weight loss before abortion. The NOAEL for abortion was 10 mg/kg bw/day, based on abortion in one animal at 40 mg/kg bw/day, most likely secondary to severe systemic maternal toxicity. The NOAEL for embryo/fetal development was 40 mg/kg– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 311 April 2008

bw/day, based on a possibly increased incidence of fetuses with distinct liver lobulation at 160 mg/kg bw/day. There was no evidence for an embryotoxic or a teratogenic potential.

B. REVIEWER COMMENTS :

1. Maternal toxicity : Appearance, behavior and mortality were unaffected at the 10 mg/kg level. Abortion was observed in the 40 mg/kg bw/day (1 dam) and 160 mg/kg bw/day (2 dams) groups; clinical signs, impaired food consumption and body weight loss were evident before abortion. Death (1 dam), sacrifice in moribund condition (5 dams), cold ears (23 dams) , alopecia (13 dams), reddish excretions (4 dams) and altered feces occurred at the 160 mg/kg bw/day level. Females in moribund condition were reported to have severely reduced food consumption, severe b ody weight loss, cold ears, alopecia, reduced amount/altered appearance of feces, reddish excreti on, decreased water intake and decreased/discolored urination, although the body w eight and food consumption data for these animals were not included in the study report for review. G ross pathology of these females revealed treatmentrelated findings of the intestine (fluid and/or gas eous contents in caecum), gall bladder (black or wh itish mottled), and the liver (green discolored in the ar ea of the gall bladder/light discoloration). Body weight changes in the remaining females did not show meani ngful treatment related effects.

The oral maternal toxicity LOAEL for BYI 08330 in r abbits is 40 mg/kg bw/day, based on abortion, clinical signs, impaired food and water consumption and body weight loss. The maternal NOAEL is 10 mg/kg bw/day.

2. Developmental toxicity:

a. Resorptions : . There were no fetal deaths among litters with via ble fetuses. The number of resorptions ranged from 6 to 13 (0.3 to 0.6 per dam ) and were scattered without dose dependency among all dose groups.

b. Altered growth : Fetal weight was not affected among litters with v iable fetuses.

c. Developmental variations : Visceral deviations included an increased incidence of fetuses (8 fetuses in 2 litters) with distinct liver lobulatio n in the 160 mg/kg group; statistical significance was evident on a fetal, but not litter, basis. The tox icological significance of this finding was questio ned, since the liver is normally lobed and abnormal live r lobulation was not reported. Other visceral deviations noted (slight dilation of lateral brain ventricles and whitish discoloration of the liver) revealed no dose dependency. Fetal skeletal evaluat ions revealed no treatment related effects at a dose level up to and including 160 mg/kg bw/day. St atistically significant retarded ossification of th e 5 th sternebra was observed on a fetal basis in the 10 mg/kg dose group, but since a dose dependency was not evident, this finding was consid ered incidental to treatment.

d. Malformations : The total number of fetuses(litters) affected with malformations was 5(5), 8(7), 4(4) and 7(4) in the 0, 10, 40 and 160 mg/kg dose groups, respectively. Thus, malformations were not dose related and/or also were seen in the current control. External malformations included malpositioned forelimbs in 1(1), 3(2), 1(1) and 5(2 ) fetuses(litters) in the 0, 10, 40 and 160 mg/kg dose groups, respectively, and single cases of dome d head and cleft palate in the 160 mg/kg dose group. Visceral malformations included ventricular septal defects of the heart in 3(3) fetuses(litters ) in the 10 mg/kg group and single cases of microphthalm ia and encephalomeningocele in the 160 mg/kg dose group. A treatment related effect was no t assumed for the malformations which appeared only once in the 160 mg/kg dose group (dom ed head/ encephalomeningocele, cleft palate,– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 312 April 2008

and microphthalmia). Skeletal malformations were ob served in the cartilaginous parts of ribs/cervical ribs/supernumerary ossification centers, scattered among all dose groups. A teratogenic effect of BYI 08330 was excluded at a dose level up to and includ ing 160 mg/kg bw/day.

The oral developmental toxicity LOAEL for BYI 08330 in rabbits was not established. The developmental NOAEL is 160 mg/kg bw/day.

C. STUDY DEFICIENCIES : A minor deficiency was that data from moribund cond ition was not provided for review.– –

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APPENDIX 1 : Oral Rabbit Developmental Toxicity Dose Ranging S tudy

CITATION : Holzum, B. (2001). BYI 08330: Pilot developmental toxicity study in rabbits after oral

administration. Bayer HealthCare AG (Wuppertal, Ger many). Laboratory report number T3062735, November 14, 2001. Unpublished.

In a range finding oral developmental toxicity stud y (Lab Report No. T3062735), BYI 08330 (97.0% a.i., batch # NLL 6425-7) was administered to groups of 3 inseminated female Himalayan rabbits/dose in 0.5% aqueous carboxymethylcellulose by oral gavage at dose levels of 0, 5, 25, 100, 160, 250, or 500 mg/kg bw/day from days 6 through 28 of gestation (G D 6-28). The fetuses were delivered by cesarean section on GD 29 and dams were killed and subjected to necropsy. Investigations were performed on general tolerance of the test compound by the femal es as well as on its effect on intrauterine development (including external, visceral and skele tal evaluation of fetuses).

Maternal toxicity: Maternal effects were noted at the 160 mg/kg bw/day dose level (body weight loss, impaired body weight development, decreased food co nsumption, cold ears) and were severe (hypoactivity, labored breathing, convulsion, sever e body weight loss, severely decreased food consumption) at levels of 250 mg/kg bw/day and abov e resulting in death or premature sacrifice in moribund conditions in all animals treated at 500 m g/kg bw/day and in one animal of the 250 mg/kg bw/day dose level.

One female of the 500 mg/kg bw/day group died on GD 10. The remaining two females of this group and one female of the 250 mg/kg bw/day group were kille d for humane reasons on GD 10 or 18 (500 mg/kg bw/day) or GD 23 (250 mg/kg bw/day). One additional female of the 250 mg/kg bw/day group was killed after abortion on GD 24. Two females of the 500 mg/ kg bw/day group and one female of the 250 mg/kg bw/day group which was killed in moribund condition exhibited wounds in the region of the head, throat or forelimbs. Hypoactivity (lying on side) on a sin gle day occurred in one female each of the 500 and 250 mg/kg bw/day groups as well as in the female in the 250 mg/kg bw/day group that aborted which also showed labored breathing and convulsion. Cold ears, severely decreased or no food consumption and distinct to severe body weight loss (up to - 34 3 g) also occurred in the females of the 500 and 25 0 mg/kg bw/day groups. Necropsy revealed hardened fat ty tissue in the abdominal cavity and at the border of the pancreas and round depressions in the gastric mucosa in one female of the 500 mg/kg bw/day group, an enlarged gall bladder in one femal e and gaseous contents in the stomach and intestine as well as hemorrhages in the renal capsu le in a further female of the 250 mg/kg bw/day grou p.

Cold ears occurred for several days in all females of the 160 mg/kg bw/day group. Food consumption was transiently distinctly to severely decreased in two females at the 160 mg/kg bw/day level, which a lso showed moderate to distinct body weight loss (-191 or -273 g). Gross necropsy did not reveal treatment related findings at levels up to and including 160 mg/kg bw/day. The maternal toxicity LOAEL was 160 mg/kg bw/day based on body weight loss, impaired bo dy weight development, decreased food consumption and cold ears. The maternal toxicity N OAEL was 100 mg/kg bw/day.

Developmental toxicity: The gestation rate in the 250 mg/kg bw/day group w as decreased by one abortion and by one total resorption. Due to these findings and due to early sacrifice or death of the remaining females in the 250 and 500 mg/kg bw/day g roups the evaluation of the remaining reproduction parameters was limited to dose levels up to and including 160 mg/kg bw/day. The resorption rate and correspondingly the number of f etuses as well as fetal sex distribution were unaffected at levels up to and including 160 mg/kg bw/day. Placental and fetal weights in the 160 mg/k g bw/day group were slightly decreased when compared to the control group which was, however, most– –

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likely due to the incidentally higher litter sizes in the 160 mg/kg group rather than a treatment-rela ted effect. A final assessment is, however, not possibl e due to the low number of females in a pilot study

. The isolated malformations seen in one fetus each o f the 100 mg/kg bw/day group (malposition of forelimb) and in the 25 and 160 mg/kg bw/day groups (cardiac ventricular septal defect with/without truncus arteriosus) are considered incidental as th ey are known as common findings in the strain of rabbits used. Thus, effects on intrauterine develop ment could not be completely excluded at the 160 mg/kg bw/day level (possibly marginally reduced fet al and placental weights). The developmental toxicity LOAEL was 160 mg/kg bw/day based on margin ally reduced fetal and placental weights. The developmental toxicity NOAEL was 100 mg/kg bw/d ay.

Based on these results, it is concluded that the do se level of 160 mg/kg bw/day is suitable for the hi gh dose level in the main developmental study of BYI 0 8330 in rabbits. The dose levels of approximately 40 mg/kg bw/day and 10 mg/kg bw/day are considered suitable for the middle and low dose levels, respectively.– –

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B.6.6.5 Summary of reproductive toxicity

The reproductive and developmental toxicity potenti als of spirotetramat were tested in a multigenerati on study in rats and in developmental toxicity studies in rats and rabbits. The observed effects on male reproductive function and performance triggered the conduct of several mechanistic studies and pharmacokinetic investigations. The developmental toxicity study in rats was supplemented by a second study to further elucidate the findings of the firs t (main) study.

Reproductive toxicity In addition to testicular histopathology observed f ollowing subchronic and chronic exposure of male ra ts to spirotetramat, evidence of male reproductive toxici ty was provided in the 2-generation reproductive to xicity study. Abnormal sperm cells were reported in F1-ge neration male rats treated with 6000 ppm (419 mg/kg bw/day) spirotetramat in the diet, and decreased re productive performance was also observed in one of these males. Similar results were obtained in the 1-generation reproductive toxicity range-finding st udy, in which decreased sperm motility and progression and increased abnormal sperm cells in the epididymides were observed in F1 males at ≥ 6000 ppm (320 mg/kg bw/day). The difference in dos e between the two studies was attributed to increased food consumptio n of parental animals in the 2-generation study. R enal toxicity was also observed in F1 adults in the 2-, but not 1-generation reproductive toxicity study. Offspring toxicity was limited to decreased body we ights in F 1 and F 2 pups of both sexes during lactation at 6000 ppm ( ≥ 419.3 mg/kg bw/day). Decreased body weights were a lso observed in parental animals at the same dose. Development of the sexual organs of off spring (balano-preputial separation, vaginal openin g) was unaffected in either study.

In an investigative study designed to explore the t ime of onset of testicular toxicity in rats, decrea sed epididymal sperm counts were recorded ≥ 10 days of treatment with 1000 mg/kg bw/day by gava ge. Repeated dosing, therefore, is necessary to produce male reproductive toxicity in rats. In a second investigative study, male rats were treated by gava ge with the enol metabolite of spirotetramat for 21 days at a dose of 800 mg/kg bw/day. Spermatotoxicity, abno rmal sperm, and Sertoli cell vacuolation were obser ved in the testes-epididymides of treated animals. The refore, male reproductive toxicity in rats is likel y due to the enol metabolite of spirotetramat.

Developmental toxicity Developmental toxicity in the absence of maternal t oxicity was not observed in either the rat or rabbi t. In the developmental toxicity study in rats, reduced fetal weight and increased incidences of malformations a nd skeletal deviations were observed at 1000 mg/kg bw/ day. Malformations at the high dose included singl e cases of cleft palate, co-arctation of aortic arch, atrial septal defect of the heart, microphthalmia, and supernumerary lumbar vertebra, four cases of dyspla stic forelimb bones, and three cases of malformed sacral vertebral arches with pelvic shift. Skeleta l findings at this dose were primarily incomplete o r delayed ossification and skeletal variations. Toxicity to the offspring was observed in the presence of mater nal toxicity, including decreased food consumption and body weight/gain, at 1000 mg/kg bw/day. Increased incidences of wavy ribs and retarded ossification a t 20 and 140 mg/kg bw/day in the main study were considered equivocal, and when irreproducible at th e same or lower doses in the supplementary rat developmental toxicity study, they were not conside red treatment-related.

In the developmental toxicity study in rabbits, off spring toxicity was not observed at any dose up to 160 mg/kg bw/day. However, maternal toxicity was obser ved at ≥ 40 mg/kg bw/day, including a dose-dependent increase in abortion and other clinical signs of to xicity in affected animals. The maternal LOAEL of 40 mg/kg bw/day in the study was considered conservative, si nce late abortion, clinical signs, impaired food an d water consumption, and body weight loss were observed in one animal only.– –

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B.6.7 Neurotoxicity (OECD Annex IIA 5.7)

B.6.7.1 Acute neurotoxicity – rat (OECD Annex IIA 5 .7.1)

CITATION : Gilmore, R.G. (2005) An acute oral neurotoxicity s creening study with technical grade BYI

08330 in Wistar rats. Bayer CropScience LP Toxicolo gy, Stilwell, KS. Study number 04-N12- UU, April 13, 2005. Unpublished.

I. MATERIAL AND METHODS:

A. MATERIALS :

1. Test material : BYI 08330 Description: Technical grade, white powder, room te mp storage Lot/Batch No.: Mix batch 08045/0014 Purity (a.i. %): 97.8 % (12-2003); 98.5% (12-2005 ) Chemical Name: Ethyl 3-(2,5-dimethylphenyl)-8-metho xy-2-oxo-1-azaspiro[4.5]dec-3-en-4- yl carbonate (cis and trans isomers) CAS No. of TGAI: trans : 382608-10-8; cis : 203313-25-1 Structure: Not available

2. Vehicle and/or positive control : The vehicle used was 0.5% methylcellulose/0.4% Twee n 80 in deionized water

3. Test animals : Species: Rat; male and female [nulliparous and non- pregnant] Strain: Wistar Hanover (Crl:WI[Glx/BRL/Han]IGS BR) Age/weight at dosing: ~9 weeks / males: 238-304 g; females: 155-164 g Source: Charles River Laboratories, Inc., Raleigh, NC Housing: Animals were individually housed in suspen ded stainless steel siremesh cages Diet: Purina Mills Rodent Lab Chow 5002 was provide d ad libitum Water: Tap water was provided ad libitum Environmental conditions: Temperature

: Humidity: Air changes: Photoperiod: 18-26 o C

30-70% 10 air changes/hr minimum 12 hrs light / 12 hrs dark

Acclimatisation: At least 6 days

B. STUDY DESIGN :

1. In life dates : Start: 4/5/2004; End: 11/25/2004

2. Animal assignment and treatment : The animals were weighed and those with body weight s that were more or less than 20% of the mean weight for each s ex were rejected. Using software, the remaining animals were randomly assigned to the control or a treatment group so that, for each sex, groups had– –

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comparable body weights when treatment was started. Nonfasted rats were administered a single dose by gavage in 0.5% methylcellulose/0.4% Tween 80 in deionized water at a dosing volume of 10 ml/kg. Animals were observed at least once daily for morta lity or clinical signs of moribundity and were weig hed (as part of the FOB) one week prior to treatment, a pproximately 4 hours after treatment, and 7 and 14 days after treatment.

The rationale for dose selection was based on the r esults of an acute oral toxicity (LD 50 ) study in young adult female Wistar rats. In that study, fasted fe male Wistar rats were administered an acute oral (gavage) dose of 2,000 mg/kg as an aqueous suspensi on in 0.5% carboxymethylcellulose, at a dosing volume 10 ml/kg. Animals were observed for mortali ty and clinical signs for 14 days after treatment. The test substance produced no clinical signs or mortal ity at the limit dose. These results support the u se of a limit dose (2,000 mg/kg) in the neurotoxicity stu dy but provided no information to establish the tim e of peak effect.

The results from a study using radio-labeled BYI 08 330 were examined to estimate the time of peak effect. In that study, adult Wistar rats received a single oral (gavage) dose of 100 mg/kg (both sexe s) or 1,000 mg/kg (males only) BYI 08330 in 0.5% (w/v) tr agacanth in water adjusted to pH 4 with acetic acid

, at a dosing volume of 10 ml/kg. In this study, the group mean peak blood concentrations occurred 1.0 t o 3.0 hours (males) or 0.67 to 1.0 hour (females) aft er treatment at 100 mg/kg and 1.0 to 8.0 hours (mal es only) following administration at 1,000 mg/kg. Pla sma concentrations in males at a dose of 1,000 mg/k g (2% cremophor in water) changed very little at 1.0, 2.0, 4.0 and 8.0 hours following treatment (570, 6 00, 770 and 700 μM, respectively). Based on these coll ective results, the doses selected for the initial study were 0, 200, 500 and 2,000 mg/kg for both sexes. Th e 2,000 mg/kg dose was selected as a limit dose that may produce slight evidence of toxicity, the m iddle dose was selected to produce minimal or no effect and the low dose was expected to be an overa ll NOEL in both sexes.

Based on the initial findings, it was necessary to conduct a follow-up study to establish a NOAEL for clinical observations, FOB and measures of activity . In that study, doses selected were 0, 50, 100 an d 500 mg/kg. The 500 mg/kg dose was selected to repro duce the findings in the initial study. The remaining treated doses were selected to establish an overall NOAEL. Based on the estimated time of peak blood concentrations at these dose ranges, the FOB began approximately 90 minutes (minimum) following dose administration, with the automated t est of activity concluding at about 4 hours after treatment. Animals were assigned to the test group s noted in Table 6-107.

Table B.6-107: Study design Dose group (mg/kg bw) Experimental parameter 0 50 100 200 500 2,000 Total no. animals/sex/group 12/sex 12/sex 12/sex 12 /sex 12/sex 12/sex
Behavioral testing 12/sex 12/sex 12/sex 12/sex 12/s ex 12/sex Neuropathology 6/sex – – 6/sex 6/sex 6/sex

n =12 animals/dose group for each study; a total of 2 4 animals/sex were tested at 500 mg/kg. – = not examined at this dose level in the follow-u p study.

3. Test substance preparation and analysis : The test substance was suspended in 0.5% methylcellulose/0.4% Tween 80 in deionized water. No information on how the test substance was stored was provided. Results of stability, concent ration, and homogeneity analyses follow below.

Results :– –

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Homogeneity Analysis: Homogeneity of the test substance in the vehicle wa s accepted for the range of concentrations used here, as the 4, 15 and 200 m g/ml concentrations (equivalent to doses of 40, 150 and 2,000 mg/kg, respectively) had percent relative standard deviations (%RSD) of 1.04%, 2.1% and 2.5%, respectively.

Stability Analysis: After eight days of storage, the nominal concentrat ions of 4, 15 or 200 mg/ml levels were 100%, 103% and 97% of the initial concentratio ns, respectively.

Concentration Analysis: For the initial study, doses of 0, 200, 500 and 2,000 mg/kg for mal es and females ranged from 91% to 103% of the nominal conc entrations. Based on these results, the analytically-confirmed doses for males and females were 0, 182, 515 and 1920 mg/kg. For the followup study, doses of 0, 50, 100 and 500 mg/kg for males and females ranged from 95% to 105% of the nominal concentrations. Based on these results, th e analytically-confirmed doses for males and females were 0, 47.4, 99.8, and 523 mg/kg.

The analytical data indicated that the mixing proce dure was adequate and that the variance between nominal and actual dosage to the animals was accept able.

4. Statistics : Generally, continuous data were analyzed using ANOV A, followed by Dunnett’s test. For the FOB, continuous data were first analyzed using Repe ated-Measures ANOVA followed by a one-way ANOVA followed by Dunnett’s test. Categorical data were analyzed using General Linear Modeling and Categorical Modeling with post-hoc comparisons usin g Dunnett’s test and an Analysis of Contrasts, respectively. Motor and locomotor activity were an alyzed using ANOVA procedures followed by Dunnett’s test. Continuous homogeneous neuropathol ogy data were analyzed using ANOVA, followed by Dunnett’s test while continuous non-homogeneous data were analyzed using the non-parametric Kruskall-Wallis test followed by a Mann-Whitney U T est for pair-wise comparisons. Micropathology frequency data were analyzed using a Chi-Square Tes t followed by a one-tailed Fisher’s Exact Test.

C. Methods / Observations :

1. Mortality and clinical observations : Animals were observed at least once daily for morta lity and morbidity. Detailed clinical observations were rec orded daily.

2. Body weight : Animals were weighed weekly as a component of the FOB.

3. Food consumption : Food consumption was not recorded. The test substa nce was administered by gavage in a single dose.

4. Cholinesterase Determination : Cholinesterase activities were not determined.

5. Neurobehavioral Assessment :

a. Functional Observational Battery (FOB) : Observations for all animals were performed by the same observer throughout the study. Technicians wer e blind with respect to group assignment of an individual animal. The test room was a standard an imal room. The animals were allowed to acclimate in this room for at least 30 minutes. Thi s room was maintained on the same light/dark cycle and settings for temperature and relative hum idity as the animal room with testing conducted during the light phase. In the initial study, the FOB was conducted one week prior to treatment, 90 minutes after dosing and again seven and 14 days fo llowing treatment. For the follow-up study, FOB– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 319 April 2008

testing was conducted one week prior to treatment a nd approximately 90 minutes after dosing. Scoring criteria were given for the measured parame ters. No information on the equipment used in measuring grip strength was presented in the test r eport.

The checked (X) parameters were examined.

HOME CAGE OBSERVATIONS HANDLING OBSERVATIONS OPEN FIELD OBSERVATIONS
X Posture* X Reactivity* X Mobility

Biting X Lacrimation* / chromodacryorrhea X Rearing+

X Convulsions* X Salivation* X Arousal/ general act ivity level*

X Tremors* Piloerection* X Convulsions*

X Abnormal Movements* Fur appearance X Tremors*

Palpebral closure* X Palpebral closure* X Abnormal movements*

X Piloerection Respiratory rate+ X Urination / def ecation*

X Gait Abnormalities X Red/crusty deposits* Groomi ng

SENSORY OBSERVATIONS Mucous membranes /eye /skin colour X Gait abnormalities / posture*

X Approach response+ X Eye prominence* Gait score*

X Touch response+ X Muscle tone* X Bizarre / stereo typic behaviour*

X Startle response* X Backing

X Pain response* Time to first step

X Pupil response*

Eyeblink response PHYSIOLOGICAL OBSERVATIONS NEUROMUSCULAR OBSERVATIONS Forelimb extension X Body weight* Hindlimb extens or strength

Hindlimb extension X Body temperature+ X Forelimb grip strength*

X Air righting reflex+ X Hindlimb grip strength*

Olfactory orientation X Landing foot splay*

X Pupil size OTHER OBSERVATIONS Rotarod performance

*Required parameters; +Recommended parameters

b. Motor activity : For the initial study, motor activity testing was c onducted one week prior to treatment, on the day of dosing, and again 7 and 14 days following treatment. For the follow-up study, motor activity testing was conducted one wee k prior to treatment and approximately 90 minutes after dosing. Testing was staggered over a 2-day interval for each sex on days 7 and 14. Sets of 8 animals (maximum) were evaluated approxim ately 30 minutes after the last animal was tested in the FOB. All 8 rats were placed into fig ure-eight mazes for motor activity evaluations. Motor and locomotor activities were evaluated by te sting the animals individually in a figure-eight maze that had 8 infrared emitter/detector pairs. E valuations were conducted for 10-minute intervals for a total of 60 minutes. Motor activity was defi ned as the number of beam interruptions that occurred during the test session. Locomotor activi ty was measured by eliminating consecutive counts for a given beam. Habituation was defined a s a decrement in activity during the test session.– –

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6. Sacrifice and Pathology : For the initial study, a complete gross necropsy was performed on all animals. In the follow-up study, a necropsy was no t conducted since a NOAEL was established for postmortem pathology in the initial study. The necrops y involved an examination of all organs, body cavit ies, cut surfaces, external orifices, and surfaces. On day 14, the first 6 males and 6 females at each dos e level were selected for perfusion and collection of tissues. If perfusion was inadequate, one of the remaining animals was used. Animals selected for p erfusion were deeply anesthetized using an intraperitoneal dose of 50 mg/kg of pentobarbital a nd then perfused via the left ventricle with a sodi um nitrite (in phosphate buffer) flush followed by uni versal fixative (4% EM-grade formalin and 1% glutaraldehyde) in phosphate buffer. The entire br ain and spinal cord, both eyes with optic nerves, a nd selected (bilateral) peripheral nerves (sciatic, ti bial, and sural), the gasserian ganglion, gastrocne mius muscle, and physical identifier were dissected from each animal and post-fixed in 10% buffered formali n. The brain was weighed upon removal from the skull, prior to placement into formalin, and the brain:bod y weight ratio was calculated. Surviving animals not perfused were sacrificed by CO 2 asphyxiation. Tissues from the control and high-dose group animal s and any gross lesions collected at necropsy were processed for microscopic examination. Eight coron al sections of the brain and sections from 3 levels of the spinal cord (cervical, thoracic, and lumbar) an d the cauda equina were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Dorsal root ganglia (including dorsal and ventral r oot fibers) from the cervical and lumbar swellings, gas serian ganglion, eyes, optic nerves, and gastrocnemius muscle were embedded in glycol methac rylate. Peripheral nerve tissues (sciatic, tibial, and sural nerves) were embedded in glycol methacryl ate and cut in cross/transverse section as well as longitudinal section. Glycol methacrylate-embedded tissues were sectioned at 2-3 μm and stained using hematoxylin and eosin.

7. Positive controls : The positive control data cited were collected in 1 993 and 2002; references are provided in the study.

II. RESULTS:

A. Observations :

1. Clinical signs : Selected clinical observations from the initial stu dy are presented in Table 6-108. Compound-related clinical signs were evident in the initial study at all dose levels. Urine stain was observed at all treated dose levels and was conside red to be related to treatment (1/12 males and 1/12 females in the 200 mg/kg dose group). Perianal sta in was observed in males (one each) at the two highest dose levels but not at the lowest dose or i n females at any dose level. These signs were evid ent on day 0 and generally resolved within 1 - 4 days a fter treatment. For the follow-up study, urine stain was evident in six females treated with 500 mg/kg, but not at lower levels or in males at any dose lev el. This sign was first observed on day 0 and resolved within 1-4 days after treatment. Based on these results, the NOAEL for clinical signs is 100 mg/kg for males and females.

Table B.6-108: Clinical observations a
Dose (mg/kg) Observation Control 50 100 200 500 2000

Males

Urine stain (initial study) (follow-up study) 0
0 0 0 1 (0-1, 7) b 3 (0)

0 5 (0-2)– –

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Dose (mg/kg) Observation Control 50 100 200 500 2000

Perianal stain (initial study) 0 1 (0) 1 (0)

Females

Urine stain (initial study) (follow-up study) 0
0 0 0 1 (0) 6 (0-1) 6 (0-3) 6 (0-3)

a Data obtained from pages 32 and 34, Report No. 201 283.

b Incidence for n=12 (range of days observed).

2. Mortality : There were no compound-related deaths at any dose level.

B. BODY WEIGHT AND BODY WEIGHT GAIN :

Body weight was not affected by treatment in males or females at any dose.

C. FOOD CONSUMPTION :

Food consumption was not measured.

D. CHOLINESTERASE ACTIVITIES :

Cholinesterase activities were not measured.

E. NEUROBEHAVIORAL RESULTS :

Body weight was not affected by treatment in males or females at any dose.

1. FOB findings : In the initial study, urine stain was observed on day 0 in males ( one each) at the lowand mid-dose levels. There were no findings relate d to treatment observed in high-dose males; therefore, the effect is not considered treatment-r elated. Females at any dose level were not affecte d. In the follow-up study , there were no treatment-related findings at any dos e level in either sex. Based on these results, the NOAEL for functional observat ions is 2,000 mg/kg for males and females.

2. Motor activity : Total motor and locomotor activity in the initial study were significantly reduced at 2,000 mg/kg in males (48% and 64%, respectively) an d females (40% and 65%, respectively) on day 0. At 500 mg/kg, motor activity was reduced (32%) and locomotor activity was significantly reduced (44%) in males only on day 0. Locomotor activity was sli ghtly reduced (29%; NSS) in males of the low dose group (200 mg/kg). In addition, interval motor and locomotor activity on day 0 was significantly redu ced in males at several intervals at 500 and 2,000 mg/k g; subsession locomotor activity (interval 3 only) was also reduced in males at 200 mg/kg on day 0. A sim ilar trend was evident in females at 500 and 2,000 mg/kg for motor activity and at 2000 mg/kg for loco motor activity, but the difference from control was not statistically significant. In the follow-up study, total motor and locomotor activity was sligh tly reduced (21% and 30%, respectively; NSS) on day 0 in males dosed at 500 mg/kg, but not at lower levels (50 and 100 mg/kg) or in females at any dose. Interval motor and locomotor activity was reduced (NSS) at several intervals in both sexes at 500 mg/kg on day 0. Based on dose-dependent decreases in these measures of activity, the NOAEL for motor/locomotor activity is 100 mg/kg for males and 200 mg/kg for females. There were no compound-related effects on days 7 or 14 after treatment and habituation was not affected by treatment on any day, at any dose.– –

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Table B.6-109: Mean (±S.D.) motor and locomotor act ivity data (total activity counts for session) a
Dose (mg/kg) Test Day 0 200 500 2000

Males
Motor Activity

Pretreatment 416 ± 219 318 ± 121 385 ± 138 383± 135

Post-treatment

1 516 ± 182 546 ± 420 350 ± 141 ( ↓ 32) 270* ± 83 ( ↓ 48) b

Post-treatment

2 493 ± 170 573 ± 370 409 ± 79 459 ± 128

Post-treatment
3 410 ± 118 429 ± 125 346 ± 103 374 ± 96

Locomotor Activity

Pretreatment 265 ± 172 202 ± 93 239 ± 91 238 ± 93

Post-treatment

1 312 ± 140 222 ± 102 ( ↓ 29) 174* ± 80 ( ↓ 44) 113* ± 39 ( ↓ 64)

Post-treatment

2 307 ± 126 270 ± 65 233 ± 45 271 ± 75

Post-treatment
3 236 ± 82 227 ± 83 175 ± 43 221 ± 80

Females
Motor Activity

Pretreatment 508 ± 169 399 ± 166 567 ± 136 539 ± 11 6
Post-treatment

1 491 ± 142 443 ± 162 444 ± 110 294* ± 135 ( ↓ 40)

Post-treatment

2 520 ± 246 450 ± 294 530 ± 130 477 ± 160

Post-treatment
3 409 ± 160 494 ± 190 428 ± 209 469 ± 177

Locomotor Activity

Pretreatment 329 ± 108 220* ± 122 363 ± 117 345 ± 8 1
Post-treatment

1 292 ± 90 262 ± 112 251 ± 104 102* ± 47 ( ↓ 65)

Post-treatment

2 294 ± 105 256 ± 170 315 ± 76 261 ± 88

Post-treatment
3 239 ± 108 303 ± 163 263 ± 153 240 ± 95

a Data obtained from pages 160-161 and 166-167, Repo rt No. 201283.

b Percentage decrease from control value calculated by the reviewer. N = 12/sex/group. * Significantly different from control, p ≤ 0.05. Pre-treatment = one week prior to treatment. Post-treatment 1 = day of dosing. Post-treatment 2 = 7 days following treatment. Post-treatment 3 = 14 days following treatment.– –

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Table B.6-110: Mean (±SD) sub-session motor activit y (#movements/10 minute interval) a
Dose (mg/kg)

0 200 500 2000 0 200 500 2000 Interval

Males Females

Pretreatment

1 96 ± 27 87 ± 15 93 ± 27 90 ± 15 104 ± 19 92 ± 29 116 ± 13 106 ± 20

2 80 ± 48 76 ± 31 80 ± 20 78 ± 37 89 ± 29 78 ± 50 1 07 ± 30 93 ± 19

3 77 ± 49 68 ± 39 80 ± 30 85 ± 25 98 ± 46 73 ± 42 1 04 ± 31 100 ± 24

4 65 ± 44 65 ± 44 58 ± 29 65 ± 26 87 ± 48 65 ± 35 1 07 ± 35 94 ± 25

5 58 ± 49 19 ± 21 52 ± 47 43 ± 38 70 ± 37 46 ± 50 8 0 ± 41 88 ± 35

6 39 ± 28 4* ± 97 22 ± 31 22 ± 31 61 ± 49 45 ± 43 5 3 ± 44 59 ± 43

Post-treatment 1

1 115 ±

31 118 ± 48 91 ± 29 80* ± 13 118 ± 36 114 ± 21 105 ± 2 4 76 ± 30

2 105 ±

34 109 ± 88 73 ± 22 64 ± 55 95 ± 49 86 ± 27 78 ± 30 50 ± 26

3 113 ±

48 97 ± 67 55* ± 22 34* ± 17 93 ± 36 73 ± 40 76 ± 24 5 0 ± 31

4 82 ± 36 97 ± 86 54 ± 29 28* ± 19 79 ± 35 70 ± 38 74 ± 30 33 ± 29

5 65 ± 47 75 ± 103 41 ± 27 37 ± 24 63 ± 28 52 ± 42 52 ± 22 49 ± 40

6 36 ± 36 50 ± 75 35 ± 34 26 ± 18 43 ± 38 49 ± 39 6 0 ± 29 35 ± 28

Post-treatment 2

1 120 ±

38 124 ± 32 117 ± 22 121 ± 21 126 ± 37 132 ± 51 143 ± 34 138 ± 23

2 98 ± 29 87 ± 27 80 ± 16 86 ± 17 105 ± 29 98 ± 57 115 ± 31 108 ± 39

3 87 ± 36 104 ± 82 77 ± 15 76 ± 29 81 ± 34 73 ± 52 93 ± 19 85 ± 37

4 68 ± 40 146 ± 275 67 ± 26 72 ± 35 63 ± 27 53 ± 60 70 ± 33 66 ± 36

5 63 ± 31 56 ± 24 40 ± 29 61 ± 43 50 ± 33 48 ± 52 6 7 ± 62 46 ± 40

6 57 ± 39 56 ± 30 28 ± 23 44 ± 36 96 ± 223 47 ± 50 43 ± 37 34 ± 29

Post-treatment 3

1 115 ±

36 122 ± 27 115 ± 29 107 ± 24 121 ± 46 136 ± 44 124 ± 60 128 ± 42

2 83 ± 30 90 ± 24 76 ± 17 81 ± 10 88 ± 37 108 ± 45 92 ± 51 101 ± 39

3 78 ± 24 77 ± 21 65 ± 34 73 ± 20 70 ± 33 86 ± 46 6 9 ± 38 77 ± 48

4 59 ± 34 61 ± 30 41 ± 18 45 ± 17 52 ± 34 69 ± 53 5 8 ± 43 59 ± 40

5 42 ± 27 38 ± 33 27 ± 32 30 ± 20 43 ± 26 47 ± 30 4 9 ± 37 57 ± 44

6 33 ± 21 41 ± 35 21 ± 24 39 ± 34 36 ± 27 49 ± 18 3 6 ± 45 48 ± 32

a Data obtained from pages 172-179, Report No. 201 283. N = 12/sex/group. * Significantly different from control, p ≤ 0.05. Pre-treatment = one week prior to treatment.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 324 April 2008

Post-treatment 1 = day of dosing. Post-treatment 2 = 7 days following treatment. Post-treatment 3 = 14 days following treatment.

Table B.6-111: Mean (±SD) sub-session locomotor act ivity (#movements/10 minute interval) a
Dose (mg/kg)
0 200 500 2000 0 200 500 2000 Interval

Males Females

Pretreatment

1 62 ± 24 57 ± 14 63 ± 23 60 ± 12 70 ± 17 55 ± 18 7 7 ± 15 70 ± 13

2 51 ± 39 46 ± 24 46 ± 14 48 ± 25 55 ± 21 44 ± 36 6 9 ± 28 54 ± 14

3 52 ± 39 44 ± 27 52 ± 22 52 ± 21 64 ± 32 43 ± 34 6 4 ± 26 60 ± 23

4 42 ± 34 41 ± 29 34 ± 19 42 ± 19 59 ± 35 36 ± 25 7 1 ± 31 66 ± 24

5 38 ± 36 11 ± 14 31 ± 29 25 ± 24 45 ± 24 25 ± 26 5 1 ± 27 55 ± 23

6 19 ± 18 3 ± 6 13 ± 20 11 ± 16 36 ± 27 18 ± 21 31 ± 29 40 ± 29
Post-treatment 1

1 76 ± 21 65 ± 23 54* ± 15 45* ± 14 78 ± 19 70 ± 12 62 ± 20 35 ± 16
2 64 ± 30 44 ± 24 34* ± 14 19* ± 9 53 ± 22 51 ± 20 47 ± 27 18 ± 11
3 68 ± 38 44* ± 24 28* ± 16 14* ± 10 51 ± 21 43 ± 2 4 44 ± 22 14 ± 8
4 50 ± 29 39 ± 26 29 ± 22 13* ± 11 45 ± 21 39 ± 26 41 ± 23 11 ± 8
5 34 ± 30 18 ± 17 15* ± 14 12* ± 14 39 ± 20 32 ± 30 26 ± 15 15 ± 12

6 20 ± 25 13 ± 19 14 ± 16 9 ± 7 26 ± 27 28 ± 27 30 ± 18 9 ± 6
Post-treatment 2

1 84 ± 29 86 ± 25 74 ± 16 78 ± 10 86 ± 27 85 ± 32 9 9 ± 25 84 ± 19

2 61 ± 24 50 ± 22 45 ± 13 50 ± 9 72 ± 23 57 ± 38 74 ± 26 63 ± 28

3 54 ± 30 43 ± 12 46 ± 12 45 ± 18 56 ± 26 39 ± 27 5 8 ± 20 45 ± 21

4 41 ± 27 39 ± 14 36 ± 16 43 ± 25 36 ± 18 29 ± 34 3 8 ± 27 34 ± 20

5 38 ± 21 29 ± 14 20 ± 16 35 ± 28 26 ± 23 23 ± 25 2 6 ± 22 21 ± 19

6 30 ± 27 24 ± 15 13 ± 11 21 ± 21 18 ± 20 23 ± 30 2 0 ± 17 15 ± 15
Post-treatment 3

1 76 ± 21 72 ± 18 66 ± 14 71 ± 21 82 ± 35 92 ± 41 8 9 ± 47 82 ± 29

2 48 ± 22 48 ± 13 38 ± 11 46 ± 7 49 ± 24 67 ± 37 60 ± 40 57 ± 22

3 43 ± 18 40 ± 16 32 ± 16 41 ± 19 41 ± 20 52 ± 41 4 1 ± 26 40 ± 21

4 31 ± 22 31 ± 19 18 ± 9 27 ± 14 25 ± 22 35 ± 39 32 ± 28 26 ± 19

5 21 ± 15 18 ± 21 12 ± 14 16 ± 13 24 ± 16 29 ± 23 2 5 ± 21 14 ± 14

6 17 ± 15 19 ± 22 9 ± 11 20 ± 21 18 ± 19 28 ± 12 17 ± 23 21 ± 22

a Data obtained from pages 186-193, Report No. 2012 83. N = 12/sex/group. * Significantly different from control, p ≤ 0.05. Pre-treatment = one week prior to treatment. Post-treatment 1 = day of dosing. Post-treatment 2 = 7 days following treatment. Post-treatment 3 = 14 days following treatment.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 325 April 2008

Table B.6-112: Mean (±S.D.) motor and locomotor act ivity data (total activity counts for session) a
Dose (mg/kg/day) Test Day 0 50 100 500

Males Motor Activity

Pretreatment 404 ± 160 460 ± 100 383 ± 100 382 ± 16 0
Post-treatment

1 373 ± 124 387 ± 111 349 ± 97 296 ± 74 ( ↓ 21)

Locomotor Activity

Pretreatment 276 ± 128 323 ± 82 242 ± 76 240 ± 104
Post-treatment

1 257 ± 100 274 ± 84 228 ± 73 179 ± 50 ( ↓ 30)

Females
Motor Activity

Pretreatment 474 ± 222 483 ± 109 539 ± 162 443 ± 15 1
Post-treatment

1 440 ± 178 485 ± 192 470 ± 189 375 ± 175

Locomotor Activity

Pretreatment 319 ± 171 306 ± 97 350 ± 112 312 ± 122

Post-treatment

1 293 ± 131 323 ± 142 321 ± 152 236 ± 131

a Data obtained from pages 163-164 and 169-170, Repor t No. 201283. N = 12/sex/group. Pre-treatment = one week prior to treatment. Post-treatment 1 = day of dosing.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 326 April 2008

Table B.6-113: Mean (±SD) sub-session motor activit y (#movements/10 minute interval) a
Dose (mg/kg)

0 50 100 500 0 50 100 500 Interval

Males Females

Pretreatment

1 96 ± 32 105 ± 18 101 ± 16 104 ± 37 98 ± 23 97 ± 1 6 115 ± 18 103 ± 19

2 85 ± 30 107 ± 27 77 ± 17 87 ± 32 82 ± 32 89 ± 24 92 ± 30 80 ± 21

3 75 ± 24 98 ± 21 67 ± 25 73 ± 36 84 ± 45 94 ± 31 8 8 ± 35 82 ± 39

4 65 ± 43 68 ± 39 60 ± 32 49 ± 31 76 ± 48 94 ± 28 9 3 ± 37 75 ± 41

5 47 ± 40 48 ± 34 45 ± 30 40 ± 34 73 ± 47 58 ± 29 7 8 ± 28 61 ± 38

6 36 ± 30 35 ± 36 34 ± 24 29 ± 31 60 ± 50 52 ± 34 7 2 ± 38 42 ± 35

Post-treatment 1

1 92 ± 28 95 ± 23 81 ± 15 84 ± 22 129 ± 50 119 ± 29 118 ± 22 87* ± 32

2 82 ± 26 78 ± 20 74 ± 16 58 ± 19 99 ± 36 119 ± 27 93 ± 21 77 ± 25

3 75 ± 20 72 ± 32 58 ± 30 47 ± 21 77 ± 37 87 ± 41 8 8 ± 35 63 ± 38

4 55 ± 21 60 ± 20 55 ± 25 38 ± 22 59 ± 43 71 ± 46 6 2 ± 48 58 ± 38

5 41 ± 25 49 ± 22 51 ± 24 34 ± 18 45 ± 35 48 ± 40 6 3 ± 54 40 ± 34

6 29 ± 24 32 ± 30 31 ± 24 35 ± 29 32 ± 29 41 ± 48 4 6 ± 37 50 ± 43

a Data obtained from pages 181-184, Report No. 2012 83. N = 12/sex/group. * Significantly different from control, p ≤ 0.05. Pre-treatment = one week prior to treatment. Post-treatment 1 = day of dosing.

Table B.6-114: Mean (±SD) sub-session locomotor act ivity (#movements/10 minute interval) a
Dose (mg/kg)
0 50 100 500 0 50 100 500 Interval

Males Females

Pretreatment

1 73 ± 28 81 ± 17 73 ± 15 70 ± 17 68 ± 19 65 ± 13 7 6 ± 13 77 ± 16

2 56 ± 28 78 ± 27 49 ± 15 60 ± 27 57 ± 25 55 ± 21 5 7 ± 22 53 ± 17

3 51 ± 22 72 ± 22 46 ± 19 48 ± 24 56 ± 37 64 ± 23 5 5 ± 24 57 ± 34

4 44 ± 33 45 ± 26 36 ± 21 31 ± 22 54 ± 38 60 ± 25 6 4 ± 28 52 ± 30

5 30 ± 30 28 ± 24 26 ± 20 20 ± 24 48 ± 31 34 ± 23 5 4 ± 22 45 ± 32

6 21 ± 21 20 ± 26 13 ± 13 10 ± 17 37 ± 36 28 ± 24 4 3 ± 21 29 ± 26
Post-treatment 1

1 73 ± 23 76 ± 18 62 ± 12 61 ± 14 99 ± 39 89 ± 27 8 8 ± 20 64* ± 25

2 57 ± 23 57 ± 16 50 ± 15 37 ± 15 71 ± 28 79 ± 21 64 ± 20 48 ± 20

3 51 ± 19 53 ± 25 37 ± 22 30 ± 17 53 ± 29 58 ± 33 6 1 ± 29 39 ± 28

4 38 ± 19 42 ± 15 32 ± 18 23 ± 14 33 ± 30 45 ± 37 4 2 ± 39 33 ± 27

5 22 ± 18 31 ± 17 28 ± 16 16 ± 10 23 ± 27 28 ± 28 4 4 ± 40 22 ± 22

6 16 ± 15 17 ± 19 17 ± 16 12 ± 10 15 ± 15 23 ± 29 2 2 ± 22 30 ± 28

a Data obtained from pages 195-198, Report No.20128 3.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 327 April 2008

N = 12/sex/group. * Significantly different from control, p ≤ 0.05. Pre-treatment = one week prior to treatment. Post-treatment 1 = day of dosing.

F. SACRIFICE AND PATHOLOGY :

1. Gross pathology : There were no compound-related gross lesions in mal es or females at any dose.

2. Brain weight : Brain weight was not affected by treatment in eithe r sex at any dose.

3. Neuropathology : Compound-related microscopic lesions were not evide nt in high-dose males or females administered 2,000 mg/kg bw.

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS:

The study author concluded that evidence of toxicit y was limited to clinical signs and decreased activ ity on the day of treatment, with complete recovery by day 7. There were no gross or microscopic lesions or evidence of neurotoxicity at any dose. Based on these results, the NOAEL for BYI 08330 by acute oral administration is 100 mg/kg for males and fema les.

B. REVIEWER COMMENTS :

In the initial study, evidence of acute oral toxici ty was observed in both sexes at 200, 500 and 2,000 mg/kg bw. Evidence of toxicity was limited to clin ical signs (urine stain at all doses, perianal stai n in males at 500 and 2,000 mg/kg) and decreased activit y in the figure-eight maze (in females at 500 and 2,000 mg/kg and in males at 200, 500 and 2,000 mg/k g) beginning on the day of treatment and with complete recovery by day 7. A follow-up study conf irmed findings (clinical signs in females and decreased motor activity in both sexes) in the main study at 500 mg/kg bw, with no compound-related effects at 50 and 100 mg/kg. Although only 1/12 an imals of each sex treated with 200 mg/kg showed the clinical sign of urine stain, evidence of a dose-r esponse at the higher doses combined with reduced motor activity in males (29% decrease) confirms cho ice of 200 mg/kg as the LOAEL.

The LOAEL for acute neurotoxicity of spirotetramat technical in rats was 200 mg/kg based on clinical signs in males and females and decreased m otor activity in males. The NOAEL was 100 mg/kg.

C. STUDY DEFICIENCIES :

The methods and equipment used for FOB testing were not described. Food consumption was not measured.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 328 April 2008

B.6.7.2 Subchronic neurotoxicity – rat 90-day (OECD Annex IIA 5.7.4)

No data available

B.6.7.3 Postnatal developmental neurotoxicity (OECD Annex IIA 5.7.5)

No data available– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 329 April 2008

B.6.7.4 Summary on neurotoxicity

Clinical signs of toxicity and/or decreased motor a ctivity were observed following a single dose of 20 0 mg/kg bw spirotetramat to rats. These effects were obser ved with greater severity at doses higher than 200 mg/kg bw in this study, as well as in a follow-up study i n rats. In the larger database, following one-year administration of spirotetramat to dogs, brain dila tion with dose-related severity was observed in mal es at
≥ 600 ppm (20 mg/kg bw/day), while axonal degeneratio n in the hypothalamus was observed in one female at 1800 ppm (48 mg/kg bw/day). Clinical signs of neur otoxicity (dehydration, swelling, decreased activit y and reactivity, seizures and ataxia) were also observed in one male with brain dilation at the highest dos e tested. Similar effects were not observed in either rats or mice.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 330 April 2008

B.6.8 Further toxicological studies

B.6.8.1 Mechanistic studies (OECD Annex IIA 5.10)

B.6.8.1.1 Special male reproductive toxicity study

CITATION : Kennel, P. 2005. BYI 08330: Evaluation of the po tential reproductive toxicity in the male rat

following daily oral administration by gavage. Bay er CropScience, 355, rue Dostoievski, BP 153, 06903 Sophia Antipolis Cedex, France. Laborat ory Report No. SA 041181, May 23, 2005. Unpublished

METHODS AND MATERIALS:

Groups of 8 Wistar rats (Crl:WI[Glx/BRL/Han]IGS BR strain) were administered BYI 08330 (97.2% purity, Batch No. MIX-batch08045/0014) by gavage (0 .5% methylcellulose vehicle) at a dose of 0 or 1000 mg/kg bw/day for 3, 10, 21, or 41 days. The d osing volume was 5 mL/kg bw based on the most recent body weight. The dosing suspension was prepa red three times during the study by suspending the test material in aqueous 0.5% methyl cellulose.

Clinical observations were recorded daily; body wei ght was measured on the first day of treatment, weekly thereafter, and at necropsy. Food consumpti on was measured weekly. A detailed physical examination was performed at weekly intervals throu ghout the study. The animals were anesthetized and sacrificed by exsanguination 4 hours after admi nistering the last dose. All sacrificed animals wer e necropsied; the testes, epididymis, and prostate we re weighed. The ventral prostate, left testis, and left epididymis were preserved in fixative and processed for microscopic examination. The right epididymis was collected for sperm count and assessment of spe rm morphology. The cauda epididymis was removed and weighed. Sperm count was expressed as absolute count and relative count based on cauda weight. The right testis was stored frozen.

RESULTS AND DISCUSSION:

GENERAL OBSERVATIONS:

One animal died on day 31. This animal showed incre ased salivation on days 17 and 21 and a reduced body weight gain between days 21 and 28 (0.7 g vs 2 .1 g for controls). This animal was not necropsied ; therefore, the cause of death could not be determin ed. Most clinical observations were seen in only o ne or two animals each, but localized soiled fur was o bserved in eight animals and generalized soiled fur

, increased salivation, and wasted appearance were ob served in three animals each.

Body weight and weight gain data are presented in T able 6-115. Mean body weight was reduced from day 3 to study termination; treated animals weighed 7-12% less than controls from day 10 to 41. Treated animals also had body weight loss between d ays 1 and 10 and showed a continued decrease in weight gain for the remainder of the study resultin g in an overall 60% decrease in weight gain. Mean food consumption was statistically decreased by 22% from day 1 to 8 (first week), and was slightly reduced by 6-10 % (N.S.) from day 8 to 22.– –

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Table B.6-115: Body weight (g) and body weight gain (g) Dose group (mg/kg) Parameter 0 1,000

Body weight day 1 391 ± 12 392 ±13

Body weight day 3 399 ± 13 391 ± 15* (98)

Body weight day 10 417 ± 13 388 ± 32** (93)

Body weight day 15 428 ± 17 395 ± 29** (92)

Body weight day 21 442 ± 19 406 ± 32** (92)

Body weight day 28 453 ± 22 404 ± 31** (89)

Body weight day 35 466 ± 23 411 ± 30** (88)

Body weight day 41 478 ± 23 421 ± 27** (88)

Weight gain day 1-10 26 -4

Weight gain day 10-28 36 16 (44) a

Weight gain day 28-41 25 17 (68) a

Overall body weight gain (day 1-41) 90 ± 12 36 ± 23 ** (40)

Data taken from pages 37 and 41, Lab Report No. SA 041181

a Numbers in parentheses are percent of control calcu lated by the reviewer.
* p<0.05; **p<0.01, statistically significant compa red with the control group.

ORGAN WEIGHTS AND GROSS FINDINGS: Weight changes and gross findings in the testis and epididymis of control and treated rats are presented in Table 6-116. In treated animals, the absolute weight of the left testis and left epididy mis were significantly decreased by 10-11% on day 3; th e relative weight (% bw) of the left testis was significantly increased by 12% and the absolute wei ght of the left epididymis was significantly decreased by 11% on day 10. No significant organ w eight changes were observed on day 21, but absolute right and left testes and epididymides wer e significantly decreased by 22-26% and relative weights were decreased by 11-15% on day 41. No tre atment-related effect was observed on prostate weight. The decreased organ weights were due in pa rt to the 12% decrease in terminal body weight in treated animals.

A small testis was observed in one rat treated for 3 days; otherwise, no treatment-related gross findi ngs were observed until day 41. Gross findings on day 41 included small prostate in 2/7 animals, small and/or soft testes in 6/7 animals, and small epidid ymis in 7/7 animals compared with none in the control group. The small testis observed in one rat after treatment for three days is not considered treatment related.

Table B.6-116: Weight and gross findings in the tes tes and epididymis

Day 3 Day 10 Day 21 Day 41 Control 1000 mg/kg Control 1000 mg/kg Control 1000 mg/kg Control 1000 mg/kg
Absolute (g) and relative (% bw) left testes weigh t

1.81 ± 0.14 1.61 ± 0.18* 1.78 ± 0.10 1.78 ± 0.14 1.83 ± 0.18 1.79 ± 0.12 1.91 ± 0.16 1.49 ± 0.21**– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 332 April 2008

Day 3 Day 10 Day 21 Day 41 Control 1000 mg/kg Control 1000 mg/kg Control 1000 mg/kg Control 1000 mg/kg

0.450 ± 0.05 0.409 ± 0.04 0.428 ± 0.02 0.479 ± 0.04* 0.413 ± 0.04 0.431 ± 0.03 0.400 ± 0.04 0.355 ± 0.05

Absolute (g) and relative (% bw) right testes weig ht

1.78 ± 0.18 0.441 ± 0.06 1.72 ± 0.13

0.437 ± 0.03 1.77 ± 0.13

0.424 ± 0.02 1.78 ± 0.14

0.480 ± 0.04** 1.83 ± 0.18

0.413 ± 0.04 1.77 ± 0.12

0.428 ± 0.04 1.94 ± 0.13

0.407 ± 0.04 1.50 ± 0.20**

0.357 ± 0.05*

Absolute (g) and relative (% bw) left epididymis w eight

0.68 ± 0.05 0.168 ± 0.02 0.61 ± 0.05*

0.155 ± 0.01 0.73 ± 0.06

0.175 ± 0.01 0.65 ± 0.09*

0.173 ± 0.02 0.68 ± 0.04

0.152 ± 0.01 0.65 ± 0.06

0.156 ± 0.02 0.78 ± 0.05

0.163 ± 0.02 0.58 ± 0.04**

0.138 ± 0.01** Absolute (g) and relative (% bw) right epididymis weight

0.69 ± 0.08 0.170 ± 0.02 0.65 ± 0.06

0.166 ± 0.01 0.73 ± 0.06

0.175 ± 0.01 0.66 ± 0.09

0.177 ± 0.01 0.70 ± 0.05

0.157 ± 0.01 0.66 ± 0.05

0.160 ± 0.01 0.78 ± 0.04

0.164 ± 0.01 0.61 ± 0.04**

0.144 ± 0.01** Gross findings: testes - small and/or soft

0/8 1/8 0/8 0/8 0/8 0/8 0/8 6/7 Gross findings: epididymides: small

0/8 0/8 0/8 0/8 0/8 0/8 0/8 7/7 Gross findings: prostate: small

0/8 0/8 0/8 0/8 0/8 0/8 0/8 2/7
Data taken from pages 47-50 and 42-55, Lab Report N o. SA 041181 *p<0.05; **p< 0.01, statistically significant compa red with the control group.

HISTOPATHOLOGIC FINDINGS: Microscopic findings are presented in Table 6-117. Two of eight rats had increased intraluminal aberrant cells in the epididymis after treatment fo r 3 but not after 10 days compared with 0/8 control s. No other treatment-related microscopic lesions were observed in the testis or epididymis on day 3 or day 10 or in the prostate at any sacrifice time. Tr eatment-related changes were observed in the testes (spermatid and Sertoli cell changes) and epididymid es (aberrant spermatocytes) on days 21 and 41 days of treatment.

After treatment for 21 days, marked degenerating el ongating spermatids (steps 9 to 14 of the maturation cycle) were observed in 8/8 treated anim als, together with multinucleated giant spermatids in 2/8 rats and moderate to marked degenerating rou nd spermatids in 4/8 rats. The investigators noted that these testicular findings are consistent with a treatment-related effect on round spermatids or l ate stage spermatocytes. At final sacrifice on day 41, slight to severe degenerating elongating spermatid s (steps 9 to 14) were observed in 7/7 treated animal s, resulting in the loss of elongating spermatids (steps 9 to 19) in 5/7 treated rats. Multinucleate d giant spermatids and marked degenerating round spermatids (around steps 7-8) were observed in 4/7 and 5/7 rats, respectively. The investigators note d that the microscopic findings in the testes at 41 d ays also are consistent with a treatment-related ef fect on round spermatids. Slight to moderate Sertoli ce ll vacuolation was observed in the testes of 6/7 ra ts on day 41. There was an increase of intraluminal a bnormal aberrant cells in the epididymides of all treated animals after treatment for 21 and 41 days. This finding was slight to moderate on day 21 and had progressed to marked severity by 41 days. Olig ospermia was not observed on day 21 but was observed in all treated rats on day 41. The investi gators noted that the findings in the epididymides were a consequence of degenerating spermatids in th e testis.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 333 April 2008

Table B.6-117: Incidence of treatment-related micro scopic findings in the testes and epididymides

Parameter Day 21 Day 41
Dose Group (mg/kg) 0 mg/kg 1,000 mg/kg 0 mg/kg 1,00 0 mg/kg

Number of animals examined 8 8 8 7

Testes
Degenerating round spermatids (steps 7-8) 0 4 (2.50 ) a 0 5 (3.00)
Multinucleated giant spermatids 0 2 (2.50) 0 4 (1.2 5)
Degenerating elongating spermatids (steps 9- 14) 0 8 (3.0) 0 7 (2.86)

Loss of elongating spermatids (steps 9-14) 0 0 0 5 (3.00)
Sertoli cell vacuolation 0 0 0 6 (1.17)
Epididymis
Increase of intraluminal aberrant cell types 0 8 (1 .75) 0 7 (3.00)
Oligospermia 0 0 0 7 (2.86)
Data taken from pages 24-26,

a Numbers in parentheses are average severity grade i n affected animals: 1 = slight, 2 = moderate, 3 = m arked, 4

= severe.

SPERM ANALYSIS: Sperm count and morphology data are presented in Ta ble 6-118. Small statistically significant decreases (-13% to -15%) were observed in the absol ute and relative (to cauda epididymis weight) sperm count on day 10 and the absolute sperm count on day 21. The absolute and relative sperm counts on 41 days of treatment were markedly decrea sed (P<0.01) by 77% and 68%, respectively, compared with that of controls.

Analysis of sperm morphology showed that the percen t abnormal sperm was significantly increased in the treated group on day 21. The greater-than-twofo ld increase in abnormal sperm on day 21 is considered toxicologically significant and is likel y related to the increased incidences in degenerati ng spermatid in the testis and aberrant cell types in the epididymis on day 21. The percent abnormal sperm in treated rats was markedly increased on day 41; 72% of the sperm were abnormal compared with only 3.8% in the control group.

Table B.6-118: Sperm count and morphology

Day 3 Day 10 Day 21 Day 41 Control 1000 mg/kg Control 1000 mg/kg Control 1000 mg/kg Control 1000 mg/kg
Epididymal sperm count (absolute) x 10 6

184.4 ± 25.5 160.6 ± 33.9

(-13%) a 218.4 ±

31.5 188.5 ± 19.5*

(-14%) 199.1 ± 26.9 169.6 ± 24.1* (-15%) 214.3 ±

11.6 50.3 ± 9.5**

(-77%) Relative sperm count to caudal epididymal weight x 10 6

849.6 ± 81.0 801.7 ± 121.3

(-6%) 940.6 ±

84.3 818.0 ±

102.4* (-13%) 871.5 ± 82.5 808.0 ± 95.4 (-7%) 878.1 ±

63.7 281.4 ±

37.6** (-68%) Sperm morphology (% abnormal cells)

4.1 ± 0.8 2.8 ± 1.7 3.2 ± 1.3 3.7 ± 0.5 2.5 ± 1.5 6 .4 ± 2.6** 3.8 ± 1.4 72.0 ± 19.4**– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 334 April 2008

Data taken from pages 21 and 45, Lab Report No. SA 04118

a Percent different from control *p<0.05; **p< 0.01, statistically significant compa red with the control group.

CONCLUSION: The investigator concluded that treatment with BYI 05330 at 1000 mg/kg induced treatment-related effects on round spermatids or late stage spermatoc ytes in male rats. This conclusion was based on decreased weight of the testes and epididymides, sm all testes and epididymides observed after treatment for 41 days, increased incidence of rats with degenerating round spermatids, degenerating elongating spermatids, and multinucleated giant spe rmatids on day 21 of treatment and a loss of elongating spermatids on day 41. In addition, an i ncreased incidence of intraluminal aberrant cells w as observed in the epididymis of treated rats on day 2 1 and oligospermia was observed on day 41.

REVIEWER’S CONCLUSION: In male rats treated with BYI 05330 at 1000 mg/kg f or up to 41 days, weight loss was observed early in the study followed by decreased body weight gain fo r the remainder of the study resulting in a 12% decrease in body weight at the end of treatment. F ood consumption was significantly reduced during th e first week of treatment. The most prevalent clinic al observations included soiling of fur along with increased salivation and wasting appearance in seve ral rats. One treated rat died, but this animal wa s not necropsied to determine the cause of death; the refore, the death cannot be attributed to treatment with the test material.

Postmortem examination showed decreased testis and epididymis weight in animals sacrificed on day 41; these animals also had small testes and epididy mides. Microscopic findings confirming an effect o f BYI 05330 treatment on the testes included degenera ting round spermatids (Steps 7-8), degenerating elongating spermatid (Steps 9-14), and multinucleat ed giant spermatids in the testes and increase of intraluminal aberrant cells in the epididymis after treatment for 21 days. The effect on the testes progressed to loss of elongating spermatids in the testes and an increase in severity of intraluminal aberrant cells and the presence of oligospermia in the epididymides after treatment for 10 days. Continued treatment with the test material for 41 d ays also had an effect on Sertoli cells.– –

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B.6.8.1.2 Special testicular/sperm toxicity study

CITATION : Tinwell, H. 2006. BYI 08330-ENOL: Investigation of the testicular/sperm toxicity in the rat

following 21 days of exposure by gavage. Bayer Cro pScience, 355, rue Dostoievski, BP 153, 06903 Sophia Antipolis Cedex, France. Laborat ory Report No. SA 06011, June 30, 2006. Unpublished.

METHODS AND MATERIALS:

Groups of five Wistar rats Rj:WI (IOPS HAN) were ad ministered BYI 08330-ENOL (96.8% purity, Batch No. 692-101-09-0005, beige powder) by gavage (0.5% methylcellulose vehicle) at a dose of 0 or 800 mg/kg bw/day for 21 days. The dosing volume was 5 mL/kg bw based on the body weight measured each day. The dosing suspension was prepared three times during the study by suspending the test material in aqueous 0.5% methyl cellulose.

All animals were checked twice a day for moribundit y and mortality except on weekends or holidays when they were checked once a day. A detailed physical examination was performed at weekly intervals throughout the study. The body weight of each anim al was measured once during acclimation, daily from day -1 to day 21, and at necropsy. Food consumptio n for each animal was measured weekly. Plasma was prepared from heparinized blood samples collect ed from the abdominal aorta at the time of sacrific e and stored frozen, but was not analyzed for metabol ites. The animals were anesthetized with isofluran e and sacrificed by exsanguination 4 hours after admi nistering the last dose. All sacrificed animals wer e necropsied; the testes, epididymis, and liver were weighed. Both testes and left epididymis were preserved in fixative and processed for microscopic examination. The right epididymis was weighed; th e cauda epididymis was dissected and weighed. The ep ididymal sperm were counted and sperm morphology was assessed.

RESULTS AND DISCUSSION:

GENERAL OBSERVATIONS:

No mortalities were recorded during the course of t his study. Treatment-related clinical observations included localized soiled fur on the anogenital reg ion of 4/5 rats (day 7-21) and on the mouth of 5/5 rats (day 19-21), increased salivation in 5/5 rats (day 12-20) and reduced motor activity and wasted appearance in 2/5 rats each (day 20-21). Mean body weight of treated rats was slightly below (up to - 8%) that of controls throughout treatment, but weig ht gain was markedly reduced by 78%. Treated rats had a net weight gain of only 1 g during the last 1 0 days of the study compared with 25 g for controls

. Mean food consumption was statistically reduced by 14% (p<0.05) during study days 1-8 and by 8% (N.S.) during study days 8-15. The mean food consum ption was similar to control levels during the last week of exposure.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 336 April 2008

Table B.6-119: Body weight (g) and body weight gain (g)

Parameter 0 mg/kg 800 mg/kg

Body weight day 1 454 ± 7 455 ± 6

Body weight day 3 456 ± 8 448 ± 4

Body weight day 10 471 ± 16 462 ± 14

Body weight day 15 490 ± 17 465 ± 25

Body weight day 21 500 ± 19 465 ± 38

Weight gain, day 11-21 25 1

Overall body weight gain 46 10 (22%) a

Data taken from pages 27-28, Study No. SA 06011.

a Number in parenthesis is the percent of the control value calculated by the reviewer.

ORGAN WEIGHTS AND GROSS FINDINGS: The mean absolute weight of the right and left test is of treated rats was increased by 8% (p<0.05 for left testis only) and the relative weight was incre ased by 17% (p<0.05) compared with that of controls

. The increase in relative weight was due in part to decreased terminal body weight of treated rats. No significant difference was observed in the weight o f the epididymides in treated rats compared with controls. Soiled fur was observed on four treated rats during necropsy compared with none of the controls; this finding was consistent with the clin ical observations. No other treatment-related gros s changes were observed.

HISTOPATHOLOGY: The incidences of treatment-related findings in the testes and epididymides are presented in Table 6- 120. Degenerating round spermatids (step 7-8) was observed in 1/5 treated rats, and degenerating elongating spermatids (Step 9 to 14) along with dif fuse sloughing of germ cells were found in all trea ted animals. Multinucleated giant spermatids were obser ved in 2/5 rats, and Sertoli cell vacuolation of minimal degree was noted in 1/5 rats. In the left epididymis, minimal to slight exfoliation of germ c ells was found in all treated animals. This finding is considered a consequence of degenerating spermatids observed in the testes.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 337 April 2008

Table B.6-120: Incidence of treatment-related chang es in the testis and epididymis

Parameter 0 mg/kg 800 mg/kg

Number of animals examined 5 5

Testes

Degenerating round spermatids (step 7-8) 0 1 (2.00 ) a

Degenerating elongating spermatids (step 9-14) 0 5 (2.80)

Multinucleated giant spermatids 0 2 (1.50)

Sloughing of germ cells 0 5 (1.60)

Sertoli cell vacuolation 0 1 (1.00)

Epididymis

Exfoliated germ cells: focal/multifocal 0 5 (1.8)

Data taken from pages 18 and 41, Study No. SA 06011

.

a Numbers in parentheses are average severity grade i n affected animals: 1 = slight, 2 = moderate, 3 = m arked, 4

= severe.

SPERM ANALYSIS: The data for sperm count and morphology are summari zed in Table 6-121. No statistically significant change in absolute or the relative (based on epidid ymal weight) numbers of epididymal spermatozoa was observed after 21 days of treatment. The data were highly variable for the control and treated groups; the relative sperm count ranged from 756-11 09 x 10 6 spermatozoa/g tissue for controls and
690-929 x 10 6 spermatozoa/g tissue for treated animals.

The percentage of abnormal spermatozoa was signific antly increased after 21 days of treatment with BYI 08330-ENOL when compared with control values (1 4.9% vs 3.2% in the control group). The majority of the abnormalities consisted of isolated heads wi th normal morphology (60%) and spermatozoa with a normal head and an abnormal mid-piece (33.3%).

Table B.6-121: Sperm count and morphology (mean ± S D)
Control group 800 mg/kg

Epididymal sperm count (absolute)

189.8 ± 19.0 199.7 ± 24.3

Epididymal sperm count per gram cauda

918.7 ± 136.5 846.1 ± 93.8

Sperm morphology (% abnormal cells)

3.2 ± 0.4 14.9 ± 6.3*

Data taken from page 41, Study No. SA 06011.
*p<0.05, statistically significant compared with th e control group.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 338 April 2008

CONCLUSION : The investigators noted that treatment of male rats with BYI 08330-ENOL caused an overall decrease in body weight gain, decrease in relative testes weigh t, and effects in the testes and epididymides. Testicular and epididymal effects consisted primari ly of degeneration and sloughing of spermatids (round and elongating) in the testes, exfoliation o f germ cells in the epididymis, and an increase in the percentage of abnormal epididymal spermatozoa. The se effects are similar to those observed in male rats administered the parent compound BYI 08330 for up to 41 days. The investigator concluded that testicular/sperm toxicity caused by BYI 08330 is un likely due to the acyl chain but to the enol.

REVIEWER’S CONCLUSION: This study was conducted to determine whether the a cyl or enol metabolite of BYI 08330 caused the testicular/sperm toxicity in male rats administered the parent compound. It was stated that the acyl chain was highly unstable; therefore, this study wa s conducted with the enol. Male rats were administered BYI 08330-ENOL at 800 mg/kg bw by gava ge for 21 days; this dose is equivalent to 1000 mg/kg of the parent compound. Male rats administer ed the enol showed effects similar to those of male rats administered the parent compound, BYI 08330 fo r 21 or 41 days. Effects in rats administered the enol consisted primarily of soiled fur and salivati on (clinical observations), a marked decrease in bo dy weight gain, a slight decrease in testes weight, an d microscopic changes in the testes (degeneration o f round and elongating spermatids, sloughing of germ cells, the presence of multinucleated giant spermatids, and Sertoli cell vacuolation) and epidi dymis (exfoliation of germ cells). There was an increase in the percentage in abnormal spermatozoa consisting primarily of isolated normal heads and spermatozoa with an abnormal mid-piece but no treat ment-related change in sperm count.

In conclusion, testicular/sperm toxicity in male ra ts administered BYI 08330 is due to the enol and is unlikely due to the acyl chain alone. This conclus ion in agreement with that of the investigators.

B.6.8.2 Summary of mechanistic studies

In an investigative study designed to explore the t ime of onset of testicular toxicity in rats, decrea sed epididymal sperm counts were recorded =10 days of t reatment with 1000 mg/kg bw/day spirotetramat by gavage. Repeated dosing, therefore, is necessary t o produce male reproductive toxicity in rats. In a second investigative study, male rats were treated by gava ge with the enol metabolite of spirotetramat for 21 days at a dose of 800 mg/kg bw/day. Spermatotoxicity, abno rmal sperm, and Sertoli cell vacuolation were obser ved in the testes-epididymides of treated animals. Bas ed on these and similar results with the parent com pound, it was concluded that male reproductive toxicity in rats is caused either by the parent compound alone or by its enol metabolite following enzymatic cleavage.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 339 April 2008

B.6.8.3 Toxicity studies on metabolites (OECD Annex IIA 5.8)

B.6.8.3.1 Acute oral toxicity (OECD Annex IIA 5.8.6 )

CITATION: Schungel, M. (2005) Acute Toxicity in the Rat after Oral Administration of BYI 08330.

Project Number: T1075865. Unpublished study prepare d by Bayer Ag Inst. of Toxicology. 24 p. October 14, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330-CIS-Ketohydroxy (Purity: 98.7
Description: light yellow solid

Lot/Batch no.: NLL 7549-7 Stability of test compound: Not reported

2. Vehicle and/or positive control: Tap water with the aid of 2% Cremophor EL

3. Test animals Species: Rat, females

Strain: Wistar [HsdCpb:Wu] Age: 10-12 weeks Weight at dosing: 141 g – 165 g
Source: Harlan Winkelmann GmbH, Horst, NL Acclimatization period: At least 5 days

Diet: Provimi Kliba 3883.0.15

Water: Tap water ad libitum Housing: The animals were group caged conventionally in polycarbonate cages.
Environmental conditions: Temperature and

Humidity: Lighting: Air changes: 22 ± 2°C

55 ± 5% 12 hours rhythm Approximately 10 changes per hour

B. Study Design and Methods:

1. Animal assignment and treatment
Dose: 2,000 mg/kg bw
Application route: Oral by gavage
Application volume: 10 ml/kg bw– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 340 April 2008

Fasting time: For administration, food was withheld from the anim als for approximately 16 - 24 h before administration of th e test compound, and they were fed again approximately 2 - 4 h after administration. Group size: 3 rats/group
Post-treatment observation period: 14 days

Observations: Mortality, clinical signs, body weight, gross necro psy
In life dates: 2005-08-17 – 2005-09-14

II. Results and Discussion: A. Mortality: Mortality was not observed at 2,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of de ath Mortality (%)

Females

2,000 1 st 0 0 3 -- -- 0

2,000 2 nd 0 0 3 -- -- 0

LD 50 >2,000 mg/kg bw

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals used

B. Clinical observations: No clinical signs were observed.

C. Body weight: Body weight and body-weight gain was not affected b y the treatment

D. Necropsy: No particular gross pathological changes were obser ved in animals sacrificed at the end of the study period.

III. Discussion and Conclusions:

A. Conclusions: According to OECD guideline 423 the L050 cut-off o f BYI 08330-CIS-Ketohydroxy is ≥ 5000 mg/kg bw (Category 5 / unclassified of the Gl obally Harmonized Classification System).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 341 April 2008

B. Reviewer Comments: In an acute oral toxicity study, two groups of thre e female Wistar HsdCpb:Wu rats (age: 10-12 weeks; weight: 141-165 g ; source: Harlan/Winklemann GmbH, 5960 AD Horst, Netherlands) were given a single oral dose o f BYI 08330-CIS-Ketohydroxy (Purity: 98.7%; Batch No. NL L 7549-7; light yellow solid) at 2000 mg/kg body weight. The test material was formulated in tap water with the aid of 2% Cremopho r EL. The administration volume was 10 ml/kg body weight. Food was withheld from the animals fo r approximately 16-24 hours before administration of the test material. Individual body weights were recorded on Day(s) 1, 8 and 15. Animals were observed for clinical signs of toxicity and mortality several times on th e Day 1 and at least once daily for 14 days thereafter. All animals were necropsied and examine d for any gross lesions. All animals survived the test and gained weight dur ing the study. No clinical signs or gross pathological findings were observed. Oral LD 50 Females > 2000 mg/kg bw Based on no mortality at 2000 mg/kg, BYI 08330-CIS- Ketohydroxy is classified as EPA Toxicity Category III.

This acute oral study is classified as Acceptable. It does satisfy the guideline requirement for an acute oral study (OPPTS 870.1100; OECD 423) in the rat



### .

C. Reviewer Conclusions: All countries agree the oral LD 50 > 2000 mg/kg in female rats for BYI 08330-CIS-Ketohydroxy.

D. Study Deficiencies: None.

CITATION: Schungel, M. (2006) Acute Toxicity in the Rat after Oral Administration of BYI 08330.

Project Number: T9076402. Unpublished study prepare d by Bayer Ag Inst. of Toxicology. 27 p. April 6, 2006.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330-desmethyl-ketohydroxy (Purity: 94.6%) Synonym: FHN 15271 Description: light beige powder

Lot/Batch no.: KATH 4726-5-1 Stability of test compound: Not reported

2. Vehicle and/or positive control: Tap water with the aid of 2% Cremophor EL

3. Test animals Species: Rat, females

Strain: Wistar [HsdCpb:Wu] Age: 10-12 weeks Weight at dosing: 180 g – 194 g– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 342 April 2008

Source: Harlan Winkelmann GmbH, Borchen, Germany Acclimatization period: At least 5 days

Diet: Provimi Kliba 3883.0.15

Water: Tap water ad libitum Housing: The animals were group caged conventionally in polycarbonate cages. Environmental conditions: Temperature

and Humidity: Lighting: Air changes: 22 ± 2°C

55 ± 5% 12 hours rhythm Approx. 10 changes per hour

B. Study Design and Methods:

1. Animal assignment and treatment

Dose: 2,000 mg/kg bw
Application route: Oral by gavage
Application volume: 10 ml/kg bw
Fasting time: For administration, food was withheld from the anim als for approximately 16 - 24 hr before administration of t he test compound, and they were fed again approximately 2 - 4 hr after administration, Group size: 3 rats/group
Post-treatment observation period: 14 days

Observations: Mortality, clinical signs, body weight, gross necro psy
In life dates: 2006-03-02 – 2006-03-22

II. Results and Discussion:

A. Mortality: Mortality was not observed at 2,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of de ath Mortality (%)

Females

2,000 1 st 0 0 3 -- -- 0

2,000 2 nd 0 0 3 -- -- 0

LD 50 >2,000 mg/kg bw

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals used;

B. Clinical observations: No clinical signs were observed.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 343 April 2008

C. Body weight: Body weight and body-weight gain was not affected b y the treatment

D. Necropsy: No particular gross pathological changes were obser ved in animals sacrificed at the end of the study period.

III. Discussion and Conclusions:

A. Conclusions: According to OECD guideline 423 the LD5O cut-off of BYT 08330-desmethyl-ketohydroxy is ≥ 5000 mg/kg bw. (Category 5 / unclassified of the Globally Harmonized Classification System).

B. Reviewer Comments: In an acute oral toxicity study, two groups of thre e female Wistar HsdCpb:Wu rats (age: 10-12 weeks; weight: 180-194 g ; source: Harlan/Winklemann GmbH, 33178 Borchen, Germany) were given a single oral dose of BYI 08330-desmethyl-ketohydroxy (Purity: 94.6%; Batch No. KATH 4726-5-1; light beige powder) at 2000 mg/kg body weight. The test material was formulated in tap water with the aid of 2% Crem ophor EL. The administration volume was 10 ml/kg body weight. Food was withheld from the anim als for approximately 16-24 hours before administration of the test material. Individual body weights were recorded on Days 1, 8 and 15. Animals were observed for clinical signs of toxicity and mortality several times on the Day 1 and at least once daily for 14 days thereafter. All animals were necropsied and examined for any gross lesions. All animals survived the test and gained weight dur ing the study. No clinical signs or gross pathological findings were observed. Oral LD 50 Females > 2000 mg/kg bw Based on the lack of mortality at 2000 mg/kg, BYI 0 8330-desmethyl-ketohydroxy is classified as EPA Toxicity Category III.

This acute oral study is classified as Acceptable. It does satisfy the guideline requirement for an acute oral study (OPPTS 870.1100; OECD 423) in the rat.

C. Reviewer Conclusions: All countries agree the oral LD 50 > 2000 mg/kg in female rats for BYI 08330-desmethyl-ketohydroxy.

D. Study Deficiencies: None.

CITATION: Schungel, M. (2006) Acute Toxicity in the Rat after Oral Administration of BYI 08330.

Project Number: T3076767. Unpublished study prepare d by Bayer Ag Inst. of Toxicology. 26 p. May 5, 2006.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330-di-hydroxy (Purity: 94.5%) Synonym: FHN15104– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 344 April 2008

Description: light beige powder

Lot/Batch no.: KATH 4727-3-6 Stability of test compound: Not reported

2. Vehicle and/or positive control: 0.5% aqueous tylose

3. Test animals Species: Rat, females

Strain: Wistar [HsdCpb:Wu] Age: 10-12 weeks Weight at dosing: 159 g – 191 g
Source: Harlan Winkelmann GmbH, Borchen, Germany Acclimatization period: At least 5 days

Diet: Provimi Kliba 3883.0.15

Water: Tap water ad libitum Housing: The animals were group caged conventionally in polycarbonate cages.
Environmental conditions: Temperature and

Humidity: Lighting: Air changes: 22 ± 2°C

55 ± 5% 12 hours rhythm Approx. 10 changes per hour

B. Study Design and Methods:

1. Animal assignment and treatment

Dose: 2,000 mg/kg bw
Application route: Oral by gavage
Application volume: 10 ml/kg bw
Fasting time: Food was withheld from the animals for approximatel y 16- 24 hours before administration of the test material

. Group size: 3 rats/ group
Post-treatment observation period: 14 days

Observations: Mortality, clinical signs, body weight, gross necro psy
In life dates: 2006-04-05 – 2006-04-26

II. Results and Discussion:

A. Mortality: Mortality was not observed at 2,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of de ath Mortality (%)

Females

2,000 1 st 0 0 3 -- -- 0– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 345 April 2008

2,000 2 nd 0 0 3 -- -- 0

LD 50 >2,000 mg/kg bw

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals used

B. Clinical observations: No clinical signs were observed.

C. Body weight: Body weight and body-weight gain was not affected b y the treatment

D. Necropsy: No particular gross pathological changes were obser ved in animals sacrificed at the end of the study period.

III. Discussion and Conclusions:

A. Conclusions: According to OECD guideline 423 the LD5O cut-off of IIYI 08330-di-hydroxy is ≥ 5000 mg/kg bw. (Category 5 / unclassified of the Globally Harmonized Classific ation System).

B. Reviewer Comments: In an acute oral toxicity study, two groups of thre e female Wistar HsdCpb:Wu rats (age: 10-12 weeks; weight: 159-191 g ; source: Harlan/Winklemann GmbH, 33178 Borchen, Germany) were given a single oral dose of BYI 08330-di-hydroxy (Purity: 94.5%; Batch No. KATH 4727-3-6; light beige powder) at 2000 mg/kg bo dy weight of. The test material was formulated in 0.5% aqueous tylose. The administration volume was 10 ml/kg body weight. Food was withheld from the animals for approximately 16-24 hours befo re administration of the test material. Individual body weights were recorded on Days 1, 8 and 15. Animals were observed for clinical signs of toxicity and mortality several times on the Day 1 and at least once daily for 14 days thereafter. All animals were necropsied and examined for any gross lesions. All animals survived the test and gained weight dur ing the study. No clinical signs or gross pathological findings were observed. Oral LD 50 Females > 2000 mg/kg bw Based on the lack of mortality at 2000 mg/kg, BYI 0 8330-di-hydroxy is classified as EPA Toxicity Category III.

This acute oral study is classified as Acceptable. It does satisfy the guideline requirement for an acute oral study (OPPTS 870.1100; OECD 423) in the rat.

C. Reviewer Conclusions: All countries agree the oral LD 50 > 2000 mg/kg in female rats for BYI 08330-di-hydroxy.

D. Study Deficiencies: None.

CITATION: Schungel, M. (2005) Acute Toxicity in the Rat After Oral Administration: BYI 08330-

mono-hydroxy. Project Number: T/2076045, AT02687. U npublished study prepared by Bayer Ag Inst. of Toxicology. 24 p. December 8, 2005.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 346 April 2008

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330-mono-hydroxy (Purity: 98.41%) Synonym: FHN 15436, NLL 7635, AE 1796847 Description: white powder

Lot/Batch no.: NLL 7635-2D Stability of test compound: Not reported

2. Vehicle and/or positive control: Tap water with the aid of 2% Cremophor EL

3. Test animals Species: Rat, females

Strain: Wistar [HsdCpb:Wu] Age: 10-12 weeks Weight at dosing: 171 g – 182 g
Source: Harlan Winkelmann GmbH, Horst, Netherlands Acclimatization period: At least 5 days

Diet: Provimi Kliba 3883.0.15

Water: Tap water ad libitum Housing: The animals were group caged conventionally in polycarbonate cages. Environmental conditions: Temperature

and Humidity: Lighting: Air changes: 22 ± 2°C

55 ± 5% 12 hours rhythm Approx. 10 changes per hour

B. Study Design and Methods:

1. Animal assignment and treatment

Dose: 2,000 mg/kg bw
Application route: Oral by gavage
Application volume: 10 ml/kg bw
Fasting time: For administration, food was withheld from the anim als for approximately 16 - 24 h before administration of th e test compound, and they were fed again approximately 2 - 4 h after administration. Group size: 3 rats/group
Post-treatment observation period: 14 days

Observations: Mortality, clinical signs, body weight, gross necro psy
In life dates: 2005-10-05 – 2005-10-26– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 347 April 2008

II. Results and Discussion:

A. Mortality: Mortality was not observed at 2,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of de ath Mortality (%)

Females

2,000 1 st 0 0 3 -- -- 0

2,000 2 nd 0 0 3 -- -- 0

LD 50 >2,000 mg/kg bw

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals used

B. Clinical observations: No clinical signs were observed.

C. Body weight: Body weight and body-weight gain was not affected b y the treatment

D. Necropsy: No particular gross pathological changes were obser ved in animals sacrificed at the end of the study period.

III. Discussion and Conclusions:

A. Conclusions: According to OECD guideline 423 the LD5O cut-off of BYI 08330-mono-hydroxy is

≥ 5000 mg/kg bw. (Category 5 / unclassified of the G lobally Harmonized Classification System).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 348 April 2008

B. Reviewer Comments: In an acute oral toxicity study, two groups of thre e female Wistar HsdCpb:Wu rats (age: 10-12 weeks; weight: 171-182 g ; source: Harlan/Winklemann GmbH, 5960 AD Horst, Netherlands) were given a single oral dose o f 2000 mg/kg body weight of BYI 08330-monohydroxy (Purity: 98.41%; Batch No. NLL 7635-2D; whi te powder). The test material was formulated in tap water with the aid of 2% Cremophor EL. The administration volume was 10 ml/kg body weight. . Food was withheld from the animals for approxima tely 16-24 hours before administration of the test material. Individual body weights were recorded on Days 1, 8 and 15. Animals were observed for clinical signs of toxicity and mortality several times on the Day 1 and at least once daily for 14 days thereafter. All animals were necropsied and examined for any gross lesions. All animals survived the test and gained weight dur ing the study. No clinical signs or gross pathological findings were observed. Oral LD 50 Females > 2000 mg/kg bw Based on the lack of mortality at 2000 mg/kg, BYI 0 8330-mono-hydroxy is classified as EPA Toxicity Category III.

This acute oral study is classified as Acceptable. It does satisfy the guideline requirement for an acute oral study (OPPTS 870.1100; OECD 423) in the rat.

C. Reviewer Conclusions: All countries agree the oral LD 50 > 2000 mg/kg in female rats for BYI 08330-mono-hydroxy.

D. Study Deficiencies: None.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 349 April 2008

B.6.8.3.2 Salmonella / Escherichia

/Mammalian Activation Gene Mutation Assay

CITATION : Herbold, B. (2005). BYI 08330-CIS-Ketohydroxy (P roject BYI 08330):

Salmonella/microsome test – plate incorporation and preincubation method. Bayer HealthCare AG, PH-R&D Toxicology, 42096 Wuppertal, Germany. Report No. AT02735, December 15, 2005. Unpublished.

I. MATERIALS AND METHODS:

A. MATERIALS :

1. Test Material: BYI 08330-cis-ketohydroxy (metabolite of BYI 08330) Description: white powder, room temp storage Lot/Batch No.: NLL7549-7 Purity (ai%): 98.7% (27-Jun-05) CAS No. of TGAI: unavailable Structure:

2. Control materials : Negative: N/A Solvent (final conc’n): DMSO, 0.1 ml/plate Positive: Nonactivation: Sodium azide 10 μg/plate TA1535 Nitrofurantoin 0.2 μg/plate TA 100 4-Nitro-1, 2- phenylene diamine 0.5 μg/plate TA 98 4-Nitro-1, 2- phenylene diamine 10 μg/plate TA1537 Mitomycin C 0.2 μg/plate TA 102 (plate incorporat ion) Cumene hydroperoxide 50 μg/plate TA 102 (preincub ation) Activation : 2-Aminoanthracene 3 μg/plate, all strains

3. Activation: S9 derived from male Sprague-Dawley rats

x Induced x Aroclor 1254 x Rat x Liver Non-induced Phenobarbitol Mouse Lung
None Hamster Other
Other Other

Describe S9 mix composition : Cofactor solution: 70 mL MgCl 2 x 6 H 2 O 162.6 mg KCl 246.0 mg Glucose-6-phospate, disodium salt 179.1 mg NADP, disodium salt 315.0 mg Phosphate buffer (pH not stated) 100.0 mM– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 350 April 2008

S9-fraction:cofactor mix 1:9 (v/v)

4. Test organisms: S. typhimurium strains
TA97 x TA98 x TA100 x TA102 TA104

x TA1535 x TA1537 TA1538 list any others

Properly maintained? x Yes No Checked for appropriate genetic markers ( rfa mutation, R factor)? x Yes No

5. Test compound concentrations used :

Standard plate test and preincubation test: (all st rains, triplicate plating) Nonactivated conditions : 0, 16, 50, 158, 500, 1,581, 5,000 μ g/plate Activated conditions : 0, 16, 50, 158, 500, 1,581, 5,000 μ g/plate

B. TEST PERFORMANCE:

1. Type of Salmonella assay :

X Standard plate test

X Pre-incubation ( 20 minutes)

"Prival" modification (i.e. azo-reduction method) Spot test
Other

2. Protocol: In the first trial, a standard plate incorporati on method was employed (Ames et al. (1973) Proc. Nat. Acad. Sci. 70: 2281-2285, Ames et al. (1975) Mutation Res. 31: 347-364). Three plates were used for each strain and dose, both with and withou t S9 mix. Based on solubility, 5,000 μg or 5 μL pe r plate was used as the highest dose. The test was co nducted by adding 0.1 mL of test material solution or vehicle, 0.1 mL bacterial culture, and 0.5 mL of either S9-mix for tests with activation or phospha te buffer for tests without activation to 2 mL of molt en soft agar (composition not provided) at 45 ° C in a test tube. After mixing, the contents were poured onto the surface of petri dishes containing solid agar (composition not provided) and the plates incubated at 37 ° C for 48 hours. The number of revertant colonies per plate were then counted using an autom atic counter. The results of the first experiment were considered as a pre-test for toxicity, but if there was a positive response or if at least three doses could be used for assessment, the first trial was i ncluded in the mutagenicity assessment. If the sec ond test confirmed the results of the first, no additio nal repeat was performed.

The preincubation test was conducted by incubating 0.1 mL of test material solution or vehicle, 0.1 mL of a bacterial suspension and 0.5 mL of either S9-mix or phosphate buffer at 37 ° C for about 20 minutes. Two mL of soft agar was then added, the contents mi xed and poured onto agar plates and the test then continued as described for the standard plate test. To distinguish between the standard plate incorporation test and the preincubation test, dose s were given as μg/plate and μg/tube, respectively.

All plating for both protocols was in triplicate.

The toxicity of the substance was assessed by deter mining (1) background growth on the plates of the mutation assay, (2) mutant counts per plate, compar ed to the negative controls, and looking for a dose

dependent reduction, and (3) the bacterial titer, u sing a 1:1,000,000 dilution of the bacterial suspen sions, except that the histidine concentration in the soft agar was increased five-fold to permit the complet e– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 351 April 2008

growth of bacteria. The tests were performed both w ith and without S9 mix. The count was made after the plates had been incubated for 48 hours at 37°C.

3. Statistical analysis : No statistical analysis was conducted.

4. Evaluation criteria : The following criteria determined the acceptance of an assay: a) the negative controls had to be within the expected range, as de fined by published data and/ or the laboratories' o wn historical data; b) the positive controls had to sh ow sufficient effects, as defined by the laboratori es' experience; and c) titer determinations had to demo nstrate sufficient bacterial density in the suspens ion. Only trials which complied with all three of the ab ove criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a tri al was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

II. REPORTED RESULTS:

No analytical report was included for the batch of BYI 08330-CIS-ketohydroxy (NLL7549-7) used in this study. The test material (0.01 mg/mL – 50 mg/mL) w as shown to be stable in solvent over a period of 4 hours (p. 46 of Report No. AT02735).

A. PRELIMINARY CYTOTOXICITY ASSAY:

No preliminary cytotoxicity assay was conducted per se , rather, cytotoxicity was assessed during the course of the mutagenicity experiments.

B. MUTAGENICITY ASSAYS :

BYI 08330-CIS-ketohydroxy was tested at six concen trations ranging from 16 to 5000 μ g/plate using all five tester strains with and without S9-mix, in bot h a standard plate test and a 20-minute preincubati on assay. Marked cytotoxicity was observed at 5,000 μ g/plate in the preincubation assay for all strains with and without activation. No substance precipitation occurred. No evidence of a mutagenic effect was obtained in either assay. The solvent and positive controls induced the appropriate responses in the respective strains. The bacterial counts for the D MSO control with strain TA98 were somewhat higher than the range of medians for 1996-2004 (Table 6-12 4) however, there was no difference in results between DMSO and any treatment dose. Results of th e standard plate test and preincubation assay without S9 activation are summarized in Table 6-122 , and with S9 activation are summarized in Table 6- 123. Historical solvent control data are summarize d in Table 6-124.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 352 April 2008

Table B.6-122: Summary of the mutation assays with BYI 08330-CIS-Ketohydroxy without activation
Mean number of revertants/plate or tube ± standard deviation BYI 08330-CIS- Ketohydroxy ( μ g/plate or tube) S9 TA1535 TA100 TA1537 TA98 TA102 Plate incorporation method 0 (DMSO) - 8 ± 1 118 ± 16 6 ± 1 60 ± 3 233 ± 19

16 - 10 ± 2 103 ± 2 4 ± 1 58 ± 5 207 ± 7

50 - 9 ± 1 131 ± 18 6 ± 1 59 ± 7 245 ± 6

158 - 8 ± 1 131 ± 28 5 ± 1 54 ± 3 210 ± 11

500 - 8 ± 2 136 ± 15 6 ± 2 56 ± 8 215 ± 7

1581 - 10 ± 3 124 ± 5 5 ± 2 54 ± 7 212 ± 16

5000 - 6 ± 2 104 ± 20 4 ± 1 60 ± 5 161 ± 28
Sodium azide - 666 ± 30

Nitrofurantoin - 331 ± 45

4-NPDA - 90 ± 23 189 ± 4 Mitomycin C - 644 ± 57

Preincubation method 0 (DMSO) - 17 ± 1 143 ± 13 8 ± 2 26 ± 3 238 ± 6

16 - 14 ± 3 149 ± 11 8 ± 3 27 ± 3 285 ± 22

50 - 20 ± 2 142 ± 14 5 ± 1 32 ± 6 250 ± 15

158 - 18 ± 2 135 ± 3 9 ± 2 35 ± 5 254 ± 27

500 - 18 ± 6 138 ± 12 6 ± 1 41 ± 9 235 ± 13

1581 - 15 ± 4 127 ± 18 3 ± 1 46 ± 7 194 ± 3

5000 - 40 ± 8 5 ± 3 136 ± 10
Sodium azide - 573 ± 67

Nitrofurantoin - 442 ± 26

4-NPDA - 112 ± 19 167 ± 31
Cumene HP - 468 ± 45
Data summarized from Report No. AT02735, Tables 1 - 10, pages 48-57. 4-NPDA = 4-Nitro-1,2-phenylene diamine Cumene HP = Cumene hydroperoxide– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 353 April 2008

Table B.6-123: Summary of the mutation assays with BYI 08330-CIS-Ketohydroxy with activation
Mean number of revertants/plate or tube ± standard deviation BYI 08330-CIS- Ketohydroxy ( μ g/plate or tube) S9 TA1535 TA100 TA1537 TA98 TA102 Plate incorporation method 0 (DMSO) + 11 ± 3 148 ± 14 6 ± 1 63 ± 8 257 ± 21

16 + 11 ± 3 152 ± 12 5 ± 1 62 ± 10 266 ± 23

50 + 8 ± 3 181 ± 21 5 ± 1 62 ± 5 273 ± 11

158 + 8 ± 4 162 ± 33 6 ± 3 58 ± 10 287 ± 23

500 + 9 ± 3 137 ± 3 8 ± 2 69 ± 9 261 ± 15

1581 + 12 ± 4 162 ± 25 4 ± 0 70 ± 1 251 ± 38

5000 + 7 ± 1 123 ± 19 6 ± 3 69 ± 9 252 ± 21
2-Aminoanthracene + 109 ± 9 1631 ± 37 284 ± 30 1454 ± 77 647 ± 53

Preincubation method 0 (DMSO) + 13 ± 3 140 ± 3 7 ± 1 45 ± 8 289 ± 15

16 + 14 ± 3 131 ± 3 6 ± 3 49 ± 8 301 ± 28

50 + 9 ± 3 164 ± 3 7 ± 1 53 ± 7 299 ± 63

158 + 12 ± 3 171 ± 3 7 ± 1 48 ± 3 309 ± 27

500 + 12 ± 3 163 ± 3 8 ± 3 51 ± 1 325 ± 45

1581 + 11 ± 3 149 ± 3 8 ± 3 53 ± 9 325 ± 32

5000 + 3 ± 3 91 ± 3 -- 22 ± 3 175 ± 24
2-Aminoanthracene + 142 ± 3 1739 ± 3 314 ± 40 1304 ± 46 646 ± 8
Data summarized from Report No. AT02735, pp. 26-27 and 48-57.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 354 April 2008

Table B.6-124: Historical control data, presented a s range of medians for years 1996-2004 Compound S9 TA1535 TA100 TA1537 TA98 TA102 Plate incorporation method
DMSO - 8-16 70-144 6-8 14-34 174-227
Sodium azide - 629-780

Nitrofurantoin - 217-358

4-NPDA - 91-126 137-173
Cumene HP - 331-556

DMSO + 9-12 89-169 8-10 23-41 248-294

2-Aminoanthracene + 150-235 1128-1526 148-286 1164 -1524 477-721

Preincubation method

DMSO - 7-15 72-145 6-8 15-32 209-271
Sodium azide - 609-747

Nitrofurantoin - 256-463

4-NPDA - 98-143 139-183
Cumene HP - 429-602

DMSO + 8-12 88-159 8-10 23-40 251-337

2-Aminoanthracene + 141-196 1190-1627 63-345 1117-1 442 466-613 Data summarized from Report No. AT02735, pages 32-4 5. 4-NPDA = 4-Nitro-1,2-phenylene diamine Cumene HP = Cumene hydroperoxide

III. DISCUSSION and CONCLUSIONS:

A. CONCLUSIONS :

The investigators concluded that BYI 08330-CIS-keto hydroxy was non-mutagenic without or with metabolic activation under the conditions of the Sa lmonella/microsome test.

B. REVIEWER COMMENTS :

The reviewer agrees with the investigator’s conclu sion. BYI 08330-CIS-ketohydroxy was tested to the limit dose for the assay, proper experimental proto col appears to have been followed, and the solvent and positive control values were appropriate. The bacterial counts for the DMSO control with strain TA98 were somewhat higher than the range of medians for 1996-2004, however, there was no difference in results between DMSO and any treatmen t dose. The study left out some methods details, such as the composition of the overlay and underlay agar, but followed the standard Ames protocol, so this is not considered a study deficiency. This is an Acceptable study.

C. STUDY DEFICIENCIES:

No study deficiencies were identified.

D. REFERENCES:

Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee. 1973. Carcinogens are mutagens: A simple test system combining liver homogenates for activation a nd bacteria for detection. Proc. Nat. Acad. Sci. (USA) 70, 2281-2285.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 355 April 2008

Ames, B.N., J. McCann and E. Yamasaki. 1975. Meth ods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity tes t. Mutation Res. 31, 347-364.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 356 April 2008

Salmonella/escherichia/mammalian activation gene mu tation assay

CITATION : Wirnitzer, U. 2006. BYI 08330-desmethyl-ketohydr oxy (Project BYI 08330):

Salmonella/microsome test – plate incorporation and preincubation method. Bayer HealthCare AG, PH-GDD Toxicology, 42096 Wuppertal, Germany. Study T 2076126, May 9, 2006. Report No. AT03027. Unpublished.

I. MATERIALS AND METHODS:

A. MATERIALS :

1. Test Material: BYI 08330- desmethyl-ketohydroxy (metabolite of BYI 08330) Description: white powder, room temp storage Lot/Batch No.: KATH 4726-5-1 Purity (ai%): 94.6% (05-Jan-06) CAS No. of TGAI: 203313-25-1 Structure:

2. Control Materials: Negative: N/A Solvent (final conc’n): DMSO, 0.1 ml/plate Positive: Nonactivation: Sodium azide 10 μg/plate TA1535 Nitrofurantoin 0.2 μg/plate TA 100 4-Nitro-1, 2- phenylene diamine 0.5 μg/plate TA 98 4-Nitro-1, 2- phenylene diamine 10 μg/plate TA1537 Mitomycin C 0.2 μg/plate TA 102 (plate incorporat ion) Cumene hydroperoxide 50 μg/plate TA 102 (preincub ation) Activation : 2-Aminoanthracene 3 μg/plate, all strains

3. Activation: S9 derived from male Sprague-Dawley rats

x Induced x Aroclor 1254 x Rat x Liver Non-induced Phenobarbitol Mouse Lung
None Hamster Other
Other Other

Describe S9 mix composition : Cofactor solution: 70 mL MgCl 2 x 6 H 2 O 162.6 mg KCl 246.0 mg Glucose-6-phospate, disodium salt 179.1 mg NADP, disodium salt 315.0 mg Phosphate buffer (pH not stated) 100.0 mM S9-fraction:cofactor mix 1:9 (v/v)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 357 April 2008

4. Test organisms: S. typhimurium strains
TA97 x TA98 x TA100 x TA102 TA104 x TA1535 x TA1537 TA1538 list any others

Properly maintained? x Yes No Checked for appropriate genetic markers ( rfa mutation, R factor)? x Yes No

5. Test compound concentrations used :

Standard plate test and preincubation test: (all st rains, triplicate plating) Nonactivated conditions : 0, 16, 50, 158, 500, 1,581, 5,000 μ g/plate Activated conditions : 0, 16, 50, 158, 500, 1,581, 5,000 μ g/plate

B. TEST PERFORMANCE:

1. Type of Salmonella assay :

X Standard plate test

X Pre-incubation ( 20 minutes)

"Prival" modification (i.e. azo-reduction method) Spot test
Other

2. Protocol: In the first trial, a standard plate incorporati on method was employed (Ames et al. (1973) Proc. Nat. Acad. Sci. 70: 2281-2285, Ames et al. (1975) Mutation Res. 31: 347-364). Based on solubility, 5,000 μg or 5 μL per plate was used as the highest dose. The test was conducted by adding 0.1 mL of test material solution or vehicle, 0.1 mL bacterial culture, and 0.5 mL of either S9-mix for tests with activation or phosphate buffer for tests witho ut activation to 2 mL of molten soft agar (composit ion not provided) at 45 ° C in a test tube. After mixing, the contents were poured onto the surface of petri dishes containing solid agar (composition not provi ded) and the plates incubated at 37 ° C for 48 hours. The number of revertant colonies per plate were the n counted using an automatic counter. The results o f the first experiment were considered as a pre-test for toxicity, but if there was a positive response or if at least three doses could be used for assessment, the first trial was included in the mutagenicity assessment. If the second test confirmed the resul ts of the first, no additional repeat was performed

.

The preincubation test was conducted by incubating 0.1 mL of test material solution or vehicle, 0.1 mL of a bacterial suspension and 0.5 mL of either S9-mix or phosphate buffer at 37 ° C for about 20 minutes. Two mL of soft agar was then added, the contents mi xed and poured onto agar plates and the test then continued as described for the standard plate test. To distinguish between the standard plate incorporation test and the preincubation test, dose s were given as (μg/plate) and μg/tube, respectivel y, although the two terms could actually be used synon ymously.

All plating for both protocols was in triplicate.

The toxicity of the substance was assessed by deter mining (1) background growth on the plates of the mutation assay, (2) mutant counts per plate, compar ed to the negative controls, and looking for a dose

dependent reduction, and (3) the bacterial titer, u sing a 1:1,000,000 dilution of the bacterial suspen sions, except that the histidine concentration in the soft agar was increased five-fold to permit the complet e growth of bacteria. The tests were performed both w ith and without S9 mix. The count was made after the plates had been incubated for 48 hours at 37°C.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 358 April 2008

3. Statistical analysis : No statistical analysis was conducted.

4. Evaluation criteria : The following criteria determined the acceptance of an assay: a) the negative controls had to be within the expected range, as de fined by published data and/ or the laboratories' o wn historical data; b) the positive controls had to sh ow sufficient effects, as defined by the laboratori es' experience; and c) titer determinations had to demo nstrate sufficient bacterial density in the suspens ion. Only trials which complied with all three of the ab ove criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a tri al was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

II. REPORTED RESULTS:

No analytical report was included for the batch of BYI 08330-desmethyl-ketohydroxy (KATH 4726-5-1) used in this study. The test material (0.01 mg/mL – 50 mg/mL) was shown to be stable in solvent over a period of 4 hours at room temperature (p. 47 of Rep ort No. AT03027).

A. PRELIMINARY CYTOTOXICITY ASSAY:

No preliminary cytotoxicity assay was conducted per se , rather, cytotoxicity was assessed during the course of the mutagenicity experiments.

B. MUTAGENICITY ASSAYS :

BYI 08330-desmethyl-ketohydroxy was tested at six concentrations ranging from 16 to 5000 μ g/plate using all five tester strains with and without S9-m ix, in both a standard plate test and a 20-minute preincubation assay. Slight to moderate cytotoxici ty ( ≤ 60% growth inhibition) was observed at 1,581 and 5,000 μg/plate for TA102 and TA1535 (preincubation) and at 5000 μg/plate for TA1535 (plate incorporation). No substance precipitation occurre d. No evidence of a mutagenic effect was obtained in either assay. The solvent and positive controls in duced the appropriate responses (essentially within the laboratory’s historical control ranges) in the resp ective strains. Results of the standard plate test and preincubation assay without S9 activation are summa rized in Table 6-125, and with S9 activation are summarized in Table 6-126. Historical solvent cont rol data are summarized in Table 6-127.

Table B.6-125: Summary of the mutation assays with BYI 08330-desmethyl-ketohydroxy without activation
Mean number of revertants/plate or tube ∀ standard deviation BYI 08330-desmethyl- ketohydroxy
( μ g/plate or tube) S9 TA1535 TA100 TA1537 TA98 TA102

Plate incorporation method
0 (DMSO) - 16 ± 5 118 ± 14 4 ± 1 35 ± 2 201 ± 32

16 - 15 ± 3 104 ± 21 5 ± 2 35 ± 4 181 ± 10

50 - 16 ± 3 114 ± 3 6 ± 1 36 ± 13 179 ± 8

158 - 12 ± 1 126 ± 26 5 ± 1 34 ± 6 208 ± 21

500 - 14 ± 5 108 ± 12 4 ± 3 29 ± 6 180 ± 5

1581 - 14 ± 2 112 ± 19 4 ± 1 36 ± 6 171 ± 12– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 359 April 2008

5000 - 11 ± 5 107 ± 6 3 ± 2 32 ± 11 183 ± 9

Sodium azide - 653 ± 43

Nitrofurantoin - 275 ± 16

4-NPDA - 78 ± 11 156 ± 18
Mitomycin C - 646 ± 36

Preincubation method
0 (DMSO) - 15 ± 4 160 ± 11 9 ± 1 31 ± 2 216 ± 17

16 - 17 ± 3 149 ± 14 8 ± 2 33 ± 4 189 ± 17

50 - 22 ± 4 138 ± 22 5 ± 3 29 ± 8 197 ± 15

158 - 16 ± 1 155 ± 8 5 ± 1 30 ± 10 260 ± 32

500 - 13 ± 2 177 ± 31 8 ± 1 26 ± 6 246 ± 20

1581 - 18 ± 5 139 ± 9 9 ± 4 34 ± 3 201 ± 19

5000 - 14 ± 5 145 ± 23 7 ± 3 27 ± 4 174 ± 17

Sodium azide - 435 ± 76

Nitrofurantoin - 426 ± 12

4-NPDA - 122 ± 16 183 ± 28

Cumene HP - 402 ± 5

Data summarized from Report AT03027, pp. 25-26 and 49-58. 4-NPDA = 4-Nitro-1,2-phenylene diamine Cumene HP = Cumene hydroperoxide– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 360 April 2008

Table B.6-126: Summary of the mutation assays with BYI 08330-desmethyl-ketohydroxy with activation
Mean number of revertants/plate or tube ∀ standard deviation BYI 08330-CIS- Ketohydroxy ( μ g/plate or tube) S9 TA1535 TA100 TA1537 TA98 TA102

Plate incorporation method
0 (DMSO) + 9 ± 2 154 ± 9 8 ± 4 47 ± 9 237 ± 31

16 + 9 ± 2 133 ± 8 4 ± 1 41 ± 5 243 ± 26

50 + 12 ± 1 145 ± 5 5 ± 2 40 ± 8 237 ± 20

158 + 8 ± 3 150 ± 16 6 ± 3 41 ± 7 237 ± 76

500 + 9 ± 3 125 ± 5 7 ± 3 43 ± 5 242 ± 44

1581 + 8 ± 1 126 ± 12 6 ± 5 46 ± 8 213 ± 22

5000 + 5 ± 2 107 ± 16 4 ± 2 40 ± 4 224 ± 21

2-Aminoanthracene + 129 ± 8 1435 ± 103 112 ± 30 904 ± 163 582 ± 29

Preincubation method
0 (DMSO) + 10 ± 2 155 ± 8 8 ± 1 45 ± 11 279 ± 6

16 + 12 ± 3 148 ± 20 7 ± 2 32 ± 5 266 ± 17

50 + 10 ± 3 132 ± 8 6 ± 2 34 ± 4 265 ± 25

158 + 11 ± 3 140 ± 14 7 ± 1 48 ± 9 283 ± 34

500 + 10 ± 2 131 ± 15 10 ± 1 39 ± 5 315 ± 19

1581 + 11 ± 3 135 ± 20 9 ± 3 40 ± 11 288 ± 20

5000 + 8 ± 3 106 ± 8 5 ± 2 34 ± 6 242 ± 24

2-Aminoanthracene + 206 ± 21 1532 ± 83 191 ± 13 944 ± 46 576 ± 111

Data summarized from Report No. AT03027, pp. 25-26 and 49-58. 4-NPDA = 4-Nitro-o-phenylendiamine– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 361 April 2008

Table B.6-127: Historical control data, presented a s range of medians for years 1996-2004 Compound S9 TA1535 TA100 TA1537 TA98 TA102
Plate incorporation method

DMSO - 8-16 70-144 6-8 14-34 174-227

Sodium azide - 629-780

Nitrofurantoin - 217-358

4-NPDA - 91-126 137-173

Cumene HP - 331-556

DMSO + 9-12 89-169 8-10 23-41 248-294

2-Aminoanthracene + 150-235 1128-1526 148-286 1164 -1524 477-721

Preincubation method

DMSO - 7-15 72-145 6-8 15-32 209-271

Sodium azide - 609-747

Nitrofurantoin - 256-463

4-NPDA - 98-143 139-183

Cumene HP - 429-602

DMSO + 8-12 88-159 8-10 23-40 251-337

2-Aminoanthracene + 141-196 1190-1627 63-345 1117-1 442 466-613
Data summarized from Report No. AT02735, pages 32-4 5. 4-NPDA = 4-Nitro-1,2-phenylene diamine Cumene HP = Cumene hydroperoxide

III. DISCUSSION and CONCLUSIONS:

A. CONCLUSIONS :

The investigators concluded that BYI 08330-desmeth yl-ketohydroxy was non-mutagenic without or with metabolic activation under the conditions of the Sa lmonella/microsome test.

B. REVIEWER COMMENTS :

The reviewer agrees with the investigators’ conclu sion. BYI 08330-desmethyl-ketohydroxy was tested t o the limit dose for the assay, proper experimental p rotocol appears to have been followed, and the solvent and positive control values were appropriat e. The study left out some methods details, such a s the composition of the overlay and underlay agar, b ut followed the standard Ames protocol, so this is not considered a study deficiency. This is an Acceptab le study.

STUDY DEFICIENCIES: Minor study deficiency is as follows:

(1) The dose formulations were not analyzed for ac tual concentrations.

D. REFERENCES:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 362 April 2008

Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee. 1973. Carcinogens are mutagens: A simple test system combining liver homogenates for activation a nd bacteria for detection. Proc. Nat. Acad. Sci. (USA) 70, 2281-2285.

Ames, B.N., J. McCann and E. Yamasaki. 1975. Meth ods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity tes t. Mutation Res. 31, 347-364.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 363 April 2008

Salmonella/escherichia/mammalian activation gene mu tation assay

CITATION : Herbold, B. (2005). BYI 08330-di-hydroxy (Projec t BYI 08330): Salmonella /microsome test – plate incorporation and preincubation method. Ba yer HealthCare AG, PH-R&D Toxicology, 42096 Wuppertal, Germany. Report No. A T03069, May 24, 2005. Unpublished.

I. MATERIAL AND METHODS

A. MATERIALS :

1. Test material : BYI 08330-di-hydroxy Description: Fine white powder, room temp storage Lot/Batch No.: KATH 4727-3-6 Purity (ai%): 94.5% (March 7, 2006) CAS No. of TGAI: Unavailable Name / Structure: (5s,8s)-3-(2,5-dimethylphenyl)-3 ,4-dihydroxy-8-methoxy-1- azaspiro[4.5]decan-2-one

Solvent Used: DMSO

2. Control materials : Negative: N/A Solvent (final conc’n): DMSO, (0.1 ml/plate)

Positive: Nonactivation: Sodium azide 10 μg/plate TA1535 Nitrofurantoin 0.2 μg/plate TA 100 4-Nitro-1, 2- phenylene diamine 0.5 μg/plate TA 98 4-Nitro-1, 2- phenylene diamine 10 μg/plate TA1537 Mitomycin C 0.2 μg/plate TA 102 Cumene hydroperoxide 50 μg/plate TA 102 Activation: 2-Aminoanthracene 3 μg/plate, all strains

3. Activation: S9 derived from male Sprague-Dawl ey rats

x induced x Aroclor 1254 x Rat x Liver non-induced Phenobarbitol Mouse Lung
None Hamster Other
Other Other

Describe S9 mix composition: Cofactor solution: (per 70 mL) MgCl 2 x 6 H2O 162.6 mg– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 364 April 2008

KCl 246.0 mg Glucose-6-phospate, disodium salt 179.1 mg NADP, disodium salt 315.0 mg Phosphate buffer 100.0 mM S9 fraction 10 %

4. Test organisms : S. typhimurium strains

TA97 x TA98 x TA100 x TA102 TA104

x TA1535 x TA1537 TA1538 list any others

Properly maintained? x Yes No Checked for appropriate genetic markers (rfa mutati on, R factor)? x Yes No

5. Test compound concentrations used :

Nonactivated conditions: 0, 16, 50, 158, 500, 1,581 , 5,000 μg/plate, all strains, triplicate plating Activated conditions: 0, 16, 50, 158, 500, 1,581, 5 ,000 μg/plate, all strains, triplicate plating

B. TEST PERFORMANCE :

1. Type of Salmonella assay: x Standard plate test x Pre-incubation (20 minutes) “Prival” modification (i.e. azo-reduction method) spot test

2. Protocol :

In the first trial, a plate incorporation method wa s employed (Ames et al. (1973) Proc. Nat. Acad. Sci . 70: 2281-2285, Ames et al. (1975) Mutation Res. 31: 347 -364). For the mutant count, three plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with t he solvent minus the test substance, comprised the neg ative control. Each positive control also contained three plates per strain. The amount of solvent for the test substance and for the controls was 0.1 ml/plate. The doses for the first trial were routi nely determined on the basis of a standard protocol : if not limited by solubility 5,000 μg or 5 μl per plate we re used as the highest dose. At least five addition al doses were routinely used. If less than three dose s were used for assessment, at least two repeats were performed. The results of the first experimen t were then considered as a pre-test for toxicity. However, in case of a positive response or if at le ast three doses could be used for assessment, the f irst trial was included in the assessment. If the second test confirmed the results of the first, no additi onal repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment. The independent repeat was performed a s preincubation in a water bath at 37°C for 20 minutes. At the end of the preincubation period 2 m l of molten soft agar were added to the tubes, the content mixed and plated. For the mutant count, th ree plates were used for each strain and dose. An equal number of plates, filled with the solvent min us the test substance, comprised the negative contr ol. Each positive control also contained three plates p er strain. The doses of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as μg/tube for better separ ation of plate incorporation and preincubation trials, de spite the fact that μg/plate and μg/tube could be u sed synonymously. Following incubation for 48 hours a t 37°C, revertants colonies were counted using an– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 365 April 2008

Artek automatic colony counter. If precipitation i nterfered with automatic counting, colonies were counted manually.

Cytotoxicity was assessed by evaluating the backgro und lawn of bacteria, the bacterial titer and by checking for a reduction in the background number o f revertants per plate. A toxic effect of the substance was assumed when there was a marked and d ose-dependent reduction in the mutant count per plate, compared to the vehicle controls

3. Statistical analysis : No statistical analysis was performed on the stud y data.

4. Evaluation criteria :

The following criteria determined the acceptance of an assay:

a. The negative controls had to be within the expec ted range, as defined by published data and/ or the laboratories' own historical data; b) the positive controls had to show sufficient effects, as defined by the laboratories' experience; and c) titer determin ations had to demonstrate sufficient bacterial density in the suspension. Only trials which compl ied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the tes t compound. Furthermore, an unacceptable trial would have been repeated. Criteria for a positive response were, in general, a reproducible and dose-related increase in the number of revertants p er plate in at least one tester strain. The increase should be at least two-fold over the respe ctive vehicle control value for strains TA98, TA100 and TA1535, at least a three-fold over the respecti ve vehicle control value for strain TA1537 and an increase in the number of revertants per plate of a t least 100 over the respective vehicle control for TA102.

II. REPORTED RESULTS:

A. PRELIMARY CYTOTOXICITY ASSAY :

The results of the first experiment in the mutageni city assay were considered as a pre-test for toxici ty.

B. MUTAGENICITY ASSAY:

Doses up to and including 5,000 μg/plate did not ca use any bacteriotoxic effects. Total bacteria count s remained unchanged and no inhibition of growth was observed. Precipitation did not occur at any concentration in any strain with or without metabol ic activation. There was no evidence of mutagenic activity of BYI 08330-di-hydroxy in any tester stra in with or without S9-mix using either the standard plate assay or the preincubation protocol. No biological ly relevant increase in the mutant count, in compar ison with the vehicle controls, was observed. Results of the mutation assays without and with S9-mix are summarized in Tables 6-128 and 6-129, respectively. The vehicle and positive control values were appropriate for the respective strains and were wit hin the historical control ranges. Historical cont rol values are summarized in Table 6-130. This is an Ac ceptable/Guideline study.

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 366 April 2008

B. REVIEWER’S COMMENTS :

BYI 08330-di-hydroxy was tested up to the limit dos e, 5000 ug/mL. It was not bacteriotoxic nor was there precipitation. There was no increase in the number of revertants per plate relative to the vehi cle control values observed at any test material concen tration in any tester strain with or without S9-mix in either assay. BYI 08330-di-hydroxy was considered to be non-mutagenic without and with metabolic activation under the conditions of the Salmonella/m icrosome test.

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for Test Guideline OPPTS 870.5100 1 ; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) dat a.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 367 April 2008

Table B.6-128: Summary of the mutation assays with BYI 08330-di-hydroxy without activation
Mean number of revertants/plate + standard deviation BYI 08330-mono- hydroxy ( μ g/plate or tube) S9 TA1535 TA100 TA1537 TA98 TA102

Plate incorporation method
0 (DMSO) - 17 ± 3 129 ± 5 6 ± 1 36 ± 4 195 ± 16

16 - 20 ± 1 137 ± 13 5 ± 2 33 ± 4 185 ± 7

50 - 24 ± 2 131 ± 23 6 ± 3 52 ± 4 193 ± 8

158 - 21 ± 3 135 ± 33 6 ± 1 38 ± 8 213 ± 20

500 - 24 ± 2 124 ± 20 6 ± 3 32 ± 6 153 ± 14

1581 - 22 ± 2 129 ± 10 7 ± 1 40 ± 9 169 ± 10

5000 - 25 ± 2 131 ± 8 3 ± 1 30 ± 10 171 ± 25
Sodium azide (10) - 689 ± 6 *
Nitrofurantoin (0.2) - 281 ± 14 *

4-NPDA - 65 ± 14 1 * 157 ± 11 2 *

Mitomycin C (0.2) - 526 ± 3 *

Preincubation method
0 (DMSO) - 18 ± 2 120 ± 24 8 ± 1 26 ± 2 164 ± 10

16 - 15 ± 3 107 ± 5 7 ± 1 29 ± 7 168 ± 4

50 - 19 ± 6 127 ± 6 7 ± 2 26 ± 4 176 ± 11

158 - 16 ± 6 132 ± 15 5 ± 0 30 ± 5 168 ± 9

500 - 23 ± 4 136 ± 19 6 ± 2 25 ± 4 170 ± 12

1581 - 25 ± 5 119 ± 8 6 ± 2 23 ± 2 167 ± 6

5000 - 19 ± 4 119 ± 8 6 ± 4 26 ± 6 171 ± 11
Sodium azide (10) - 678 ± 18 *
Nitrofurantoin (0.2) - 460 ± 66 *

4-NPDA - 107 ± 12 1 * 174 ± 35 2 *

Cumene HP (50) - 392 ± 9 *

Data summarized from Report No. AT02716, Tables 1 - 10, pages 48-57. 4-NPDA = 4-Nitro-1,2-phenylene diamine Cumene HP = Cumene hydroperoxide

1 10 μg/plate

2 0.5 μg/plate * Positive response– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 368 April 2008

Table B.6-129: Summary of the mutation assays with BYI 08330-di-hydroxy with activation
Mean number of revertants/plate + standard deviation BYI 08330-CIS- Ketohydroxy ( μ g/plate or tube) S9 TA1535 TA100 TA1537 TA98 TA102

Plate incorporation method
0 (DMSO) + 10 ± 2 146 ± 17 7 ± 1 43 ± 4 233 ± 32

16 + 6 ± 1 121 ± 1 6 ± 2 49 ± 5 254 ± 18

50 + 6 ± 2 116 ± 20 6 ± 2 53 ± 10 230 ± 17

158 + 6 ± 2 150 ± 22 6 ± 1 49 ± 7 251 ± 26

500 + 8 ± 1 140 ± 18 6 ± 2 44 ± 9 221 ± 14

1581 + 7 ± 3 132 ± 22 5 ± 1 39 ± 5 241 ± 33

5000 + 3 ± 2 122 ± 8 5 ± 2 44 ± 3 259 ± 6

2-Aminoanthracene (3) + 81 ± 6 * 1344 ± 122 * 47 ± 3 * 960 ± 93 * 590 ± 42 *

Preincubation method
0 (DMSO) + 10 ± 3 158 ± 20 8 ± 1 34 ± 4 245 ± 4

16 + 8 ± 1 139 ± 6 6 ± 1 41 ± 4 253 ± 5

50 + 6 ± 1 154 ± 22 6 ± 1 35 ± 8 254 ± 6

158 + 8 ± 1 169 ± 21 8 ± 1 42 ± 13 240 ± 4

500 + 7 ± 1 166 ± 19 6 ± 2 39 ± 3 243 ± 3

1581 + 9 ± 1 152 ± 16 7 ± 1 40 ± 6 253 ± 7

5000 + 6 ± 1 145 ± 6 5 ± 2 28 ± 2 258 ± 4

2-Aminoanthracene (3) + 90 ± 32 * 1190 ± 218 * 178 ± 45 * 1013 ± 121 * 41 0 ± 13 *

Data summarized from Report No. AT02735, Tables 1 - 10, pages 48-57. 4-NPDA = 4-Nitro-o-phenylendiamine * Positive response

STUDY DEFICIENCY: The following minor deficiency was noted, but does not change the conclusions of this DER:

- The dose formulations were not analyzed for actua l concentrations.

REFERENCES :

Ames, B.N., W.E. Durston, E. Yamasaki et al. (1973) Carcinogens are mutagens: Asinple test system combining liver homogenates for activation and bact eria for detection. Proc. Nat. Acad. Sci. (USA) 70:
2281-2285.

Ames, B.N., J. McCann and E. Yamasaki (1975) Method s for detecting carcinogens and mutagens with the Salmonella

/mammalian-microsome mutagenicity test. Mutation Re s. 31: 347-364.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 369 April 2008

Table B.6-130: Historical control data, presented a s range of medians for years 1996-2004 Compound S9 TA1535 TA100 TA1537 TA98 TA102
Plate incorporation method

DMSO - 8-16 70-144 6-8 14-34 174-227

Sodium azide - 629-780

Nitrofurantoin - 217-358

4-NPDA - 91-126 137-173

Cumene HP - 331-556

DMSO + 9-12 89-169 8-10 23-41 248-294

2-Aminoanthracene + 150-235 1128-1526 148-286 1164 -1524 477-721

Preincubation method

DMSO - 7-15 72-145 6-8 15-32 209-271

Sodium azide - 609-747

Nitrofurantoin - 256-463

4-NPDA - 98-143 139-183

Cumene HP - 429-602

DMSO + 8-12 88-159 8-10 23-40 251-337

2-Aminoanthracene + 141-196 1190-1627 63-345 1117-1 442 466-613
Data summarized from Report No. AT02735, pages 32-4 5. 4-NPDA = 4-Nitro-1,2-phenylene diamine Cumene HP = Cumene hydroperoxide– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 370 April 2008

Ten-day range finding

CITATION : Tinwell, H. (2006). BYI 08330-ENOL: Exploratory 10-day toxicity study in the rat by

gavage. Bayer CropScience, 355, rue Dostolevski, BP 153, 06903 Sophia Antipolis Cedex, France. Laboratory report number SA 05323, June 30, 2006. U npublished.

I. MATERIALS AND METHODS:

A. MATERIALS :

1. Test material : BYI 08330-enol; principal metabolite of BYI08330 Description: Technical, beige powder
Batch #: 692-101-09-0005 Purity: 96.8% a.i. Compound stability: Stable when stored at room te mperature CAS # if TGAI: Not available
Structure: Not available

2. Vehicle: 0.5% Methylcellulose 3. Test animals : Species: Rat Strain: Rj: WI (IOPS HAN) Age/weight at study initiation: 11 weeks/445 g-457 g

Source: R. Janvier, Le Genest St Isle, France Housing: Stainless steel wire-mesh cages Diet: Certified rodent pelleted and irradiated diet A04C-10
Water: No data Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 20-24 ° C

40-70% 10-15/hr 12 hrs dark/ 12 hrs light Acclimation period: 2 weeks

B. STUDY DESIGN :

1. In life dates : Start: January 24, 2006; End: March 6, 2006

2. Animal assignment : Out of five animals ordered three were randomly selected based on body weight. The test group consisted of three males. No explana tion was offered for the choice of males and no control group was maintained.

3. Dose selection rationale : Rats received the test substance daily in 0.5% meth ylcellulose in distilled water by gavage (5 mL/kg) based on body weight. The dose level of 800 mg/kg/day chosen for this study is equivalent to 1000 mg/kg/day of the parent compound (BYI 08330). This dose of parent compound was shown to cause clinical signs of toxic ity and a significant reduction in mean body weight as well as one death in rats (Bayer Study Report # SA 04181).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 371 April 2008

4. Dose preparation and analysis : The test compound was mixed with 0.5% methylcellulo se in distilled water once and stored at ±3oC when not in use. The solution was mixed continuously before and during dosing using an electromagnetic stirrer. No analys es of test compound in the dose formulations were performed.

Results:

Homogeneity/Stability/Concentration analysis: Not available

5. Statistics : No statistical analyses were performed. Only mean s and standard deviations were calculated for body weight and food consumption usi ng computer software.

C. METHODS :

1. Observations : A nimals were inspected twice daily for signs of toxi city and mortality. Observed signs (nature, onset, severity, reversibility and duratio n of clinical signs) were recorded at least once da ily. Cageside inspections for signs of ill-health were c onducted.

2. Body weight : Animals were weighed daily.

3. Food consumption and compound intake : The weight of the food supplied was recorded on Stu dy Days 1 and 7. The remaining food was recorded on D ays 7 and 10.

4. Sacrifice : All animals were sacrificed under deep anesthesia by isoflurane inhalation. All animals were weighed prior to sacrifice

II. RESULTS:

A. OBSERVATIONS :

1. Clinical signs of toxicity : No treatment-related clinical signs of toxicity we re observed.

2. Mortality: All rats survived to study termination.

B. BODY WEIGHT AND WEIGHT GAIN : No control group was available for comparison and therefore, no statistical analyses were performed on body weight parameters. However, a co mparison of mean body weight for Days 2 through Day 10 was made with the mean body weight on Day 1 of treatment. This comparison showed a slight reduction in body weight on Day 2, 3 and 8 (0.9%, 0 .4% and 0.9% compared to that on Day 1) and a marginal increase on Day 4 (0.9%) and Day 7 (1.1%). The mean terminal body weight was the same as that recorded on Day 1 of treatment. For the remain der of the days, the mean body weight was the same as that recorded for Day 1. The mean overall gain f or treated group was only 1g. For individual rats the body weight change varied from –3 g to +4 g.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 372 April 2008

Table B.6-131: Average body weights and body weight gains of rats fed 800 mg/kg/day BYI 08330 during 10 days of treatment a
Days of

treatment (n=3) 1 2 3 4 5 6 7 8 9 10

Mean body weight (g±SD)

%
Decrease

or increase of Day 1 451±6

- - 447±8

-0.9 449±7

-0.4 455±8

+0.9 453±6

- - 452±8

- - 456±8

+1.1 447±8

-0.9 451±11

- - 452±7

- -

Mean body weight gain (g) - - -4±2 2±1 6±2 -2±2 -1±3 4±2 -9±2 5±5 1±5

a Data obtained from page 20 in the study report

C. FOOD CONSUMPTION : The mean food consumption at Day 10 (21±2g) was red uced by 19% compared to that recorded on Study Day 7 (26±g). T he overall mean food consumption over the 10-day period was not provided.

D. SACRIFICE : No abnormal necropsy findings were recorded.

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS : A group of 3 male Rj: WI (IOPS HAN) Wistar rats were administe red BYI 08330-enol daily by gavage at a dose level of 800 mg/kg/day for 10 days. The stu dy was intended to determine the toxic potential of BYI 08330-enol so as to ensure that no unscheduled deaths would occur in a future investigative study. No clinical signs of toxicity were observed and sur vival was unaffected by the treatment. Mean food consumption on Day 10 was reduced (19%) compared to that on Day 7. Mean body weight fluctuated on a day-to-day basis but did not differ significan tly from the mean body weight on Day 1. The mean overall gain for treated group was only 1 g compare d to 26 g for control rats of the similar age durin g a similar duration. The study author concluded that 8 00 mg/kg/day of BYI 08330-enol induced general signs of toxicity (lack of body weight gain) during the treatment period, but was not overtly toxic to male Wistar rats when administered by oral gavage for 10 days.

B. REVIEWER COMMENTS :

The reviewer disagrees with the study author’s conc lusion that 10-day oral gavage administration of BY I 08330-enol was not overtly toxic to male rats. The test compound caused a lack of body weight gain– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 373 April 2008

over a 10-day period. Although the study examined limited parameters the available finding is suggestive of systemic toxicity.

C. STUDY DEFICIENCIES:

The study did not maintain a control group, employe d only one dose group consisting of only 3 animals, and measured limited parameters. Concentration ana lysis of the dosing solution was not available. No rationale was provided for using only male rat. T hese deficiencies may affect interpretation of the study results.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 374 April 2008

Salmonella/escherichia/mammalian activation gene mu tation assay

CITATION : Herbold, B. (2005). BYI 08330-mono-hydroxy (Proj ect BYI 08330): Salmonella /microsome test – plate incorporation and preincubation method . Bayer HealthCare AG, PH-R&D Toxicology, 42096 Wuppertal, Germany. Report No. A T02716, December 13, 2005. Unpublished.

I. MATERIAL AND METHODS

A. MATERIALS :

1. Test material : BYI 08330-mono-hydroxy Description: Fine white powder, room temp storage Lot/Batch No.: NLL 7635-2D Purity (ai%): 98.41% (August 26, 2005) CAS No. of TGAI: Unavailable Name / Structure: 3-(2,5-dimethylphenyl)-4-hydroxy -8-methoxy-1-azaspiro[4.5]decan-2-one

Solvent Used: DMSO

2. Control materials : Negative: N/A Solvent (final conc’n): DMSO, (0.1 ml/plate)

Positive: Nonactivation: Sodium azide 10 μg/plate TA1535 Nitrofurantoin 0.2 μg/plate TA 100 4-Nitro-1, 2- phenylene diamine 0.5 μg/plate TA 98 4-Nitro-1, 2- phenylene diamine 10 μg/plate TA1537 Mitomycin C 0.2 μg/plate TA 102 Cumene hydroperoxide 50 μg/plate TA 102 Activation: 2-Aminoanthracene 3 μg/plate, all strains

3. Activation: S9 derived from male Sprague-Dawley rats:

x Induced x Aroclor 1254 x Rat x Liver Non-induced Phenobarbitol Mouse Lung
None Hamster Other
Other Other

Describe S9 mix composition: Cofactor solution: (per 70 mL) MgCl 2 x 6 H2O 162.6 mg KCl 246.0 mg Glucose-6-phospate, disodium salt 179.1 mg– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 375 April 2008

NADP, disodium salt 315.0 mg Phosphate buffer 100.0 mM S9 fraction 10 %

4. Test organisms: S. typhimurium strains:

TA97 x TA98 x TA100 x TA102 TA104 x TA1535 x TA1537 TA1538 list any others

Properly maintained? x Yes No Checked for appropriate genetic markers (rfa mutati on, R factor)? x Yes No

5. Test compound concentrations used :

Nonactivated conditions: 0, 16, 50, 158, 500, 1,581 , 5,000 μg/plate, all strains, triplicate plating Activated conditions: 0, 16, 50, 158, 500, 1,581, 5 ,000 μg/plate, all strains, triplicate plating

B. TEST PERFORMANCE :

1. Type of Salmonella assay : x Standard plate test x Pre-incubation (20 minutes) “Prival” modification (i.e. azo-reduction method) Spot test

2. Protocol:

In the first trial, a plate incorporation method wa s employed (Ames et al. (1973) Proc. Nat. Acad. Sci . 70: 2281-2285, Ames et al. (1975) Mutation Res. 31: 347 -364). For the mutant count, three plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with t he solvent minus the test substance, comprised the neg ative control. Each positive control also contained three plates per strain. The amount of solvent for the test substance and for the controls was 0.1 ml/plate. The doses for the first trial were routi nely determined on the basis of a standard protocol : if not limited by solubility 5,000 μg or 5 μl per plate we re used as the highest dose. At least five addition al doses were routinely used. If less than three dose s were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at le ast three doses could be used for assessment, the f irst trial was included in the assessment. If the second test confirmed the results of the first, no additi onal repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment. The independent repeat was performed a s preincubation in a water bath at 37°C for 20 minutes. At the end of the preincubation period 2 m l of molten soft agar were added to the tubes, the content mixed and plated. For the mutant count, th ree plates were used for each strain and dose. An equal number of plates, filled with the solvent min us the test substance, comprised the negative contr ol. Each positive control also contained three plates p er strain. The doses of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as μg/tube for better separ ation of plate incorporation and preincubation trials, de spite the fact that μg/plate and μg/tube could be u sed synonymously. Following incubation for 48 hours a t 37°C, revertants colonies were counted using an Artek automatic colony counter. If precipitation i nterfered with automatic counting, colonies were counted manually.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 376 April 2008

Cytotoxicity was assessed by evaluating the backgro und lawn of bacteria, the bacterial titer and by checking for a reduction in the background number o f revertants per plate. A toxic effect of the substance was assumed when there was a marked and d ose-dependent reduction in the mutant count per plate, compared to the vehicle controls

3. Statistical analysis : No statistical analysis was performed on the stu dy data.

4

. Evaluation criteria :

The following criteria determined the acceptance of an assay:

a. The negative controls had to be within the expec ted range, as defined by published data and/ or the laboratories' own historical data; b) the positive controls had to show sufficient effects, as defined by the laboratories' experience; and c) titer determin ations had to demonstrate sufficient bacterial density in the suspension. Only trials which compl ied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the tes t compound. Furthermore, an unacceptable trial would have been repeated. Criteria for a positive response were, in general, a reproducible and dose-related increase in the number of revertants p er plate in at least one tester strain. The increase should be at least two-fold over the respe ctive vehicle control value for strains TA98, TA100 and TA1535, at least a three-fold over the respecti ve vehicle control value for strain TA1537 and an increase in the number of revertants per plate of a t least 100 over the respective vehicle control for TA102.

II. REPORTED RESULTS:

A. PRELIMARY CYTOTOXICITY ASSAY :

The results of the first experiment in the mutageni city assay were considered as a pre-test for toxici ty.

B. MUTAGENICITY ASSAY:

Doses up to 5,000 μg/plate did not cause any bacter iotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation did not occur. There was no evidence of mutagenic activity of BYI 08330-mono -hydroxy in any tester strain with or without S9-mi x using either the standard plate assay or the preinc ubation protocol. No biologically relevant increas e in the mutant count, in comparison with the vehicle co ntrols, was observed. Results of the mutation assay s without and with S9-mix are summarized in Tables 6- 132 and 6-133 respectively. The vehicle and positive control values were appropriate for the re spective strains and were within the historical con trol ranges. Historical control values are summarized i n Tables 6-134. This is an Acceptable/Guideline study.

III. CONCLUSIONS:

BYI 08330-mono-hydroxy was tested up to the limit dose, 5000 ug/mL. It was not bacteriotoxic nor was there precipitation. There was no increase in the number of revertants per plate relative to the vehi cle control values observed at any test material concen tration in any tester strain with or without S9-mix in either assay. BYI 08330-mono-hydroxy was considere d to be non-mutagenic without and with metabolic activation under the conditions of the Salmonella/m icrosome test.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 377 April 2008

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for Test Guideline OPPTS 870.5100 1 ; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) dat a.

C. STUDY DEFCIENCIES : The following minor deficiency was noted, but do es not change the conclusions of this DER:

- The dose formulations were not analyzed for actua l concentrations.

Table B.6-132: Summary of the mutation assays with BYI 08330-mono-hydroxy without activation
Mean number of revertants/plate ∀ standard deviation BYI 08330-mono- hydroxy ( μ g/plate or tube) S9 TA1535 TA100 TA1537 TA98 TA102

Plate incorporation method
0 (DMSO) - 16 ± 4 149 ± 13 6 ± 1 46 ± 2 249 ± 30

16 - 19 ± 1 156 ± 7 7 ± 2 51 ± 4 212 ± 23

50 - 14 ± 2 171 ± 3 6 ± 1 52 ± 6 238 ± 17

158 - 17 ± 2 147 ± 14 6 ± 2 47 ± 4 264 ± 13

500 - 15 ± 4 141 ± 9 5 ± 1 59 ± 10 245 ± 12

1581 - 17 ± 5 133 ± 18 7 ± 2 51 ± 6 219 ± 12

5000 - 16 ± 3 164 ± 5 7 ± 2 44 ± 4 235 ± 16
Sodium azide (10) - 610 ± 88 *
Nitrofurantoin (0.2) - 340 ± 33 *

4-NPDA - 94 ± 6 1 * 150 ± 13 2 *

Mitomycin C (0.2) - 704 ± 20 *

Preincubation method
0 (DMSO) - 11 ± 3 110 ± 8 9 ± 1 53 ± 2 244 ± 36

16 - 8 ± 1 107 ± 12 7 ± 2 59 ± 2 210 ± 23

50 - 9 ± 3 120 ± 5 7 ± 1 50 ± 1 229 ± 10

158 - 13 ± 1 143 ± 18 7 ± 1 57 ± 4 278 ± 23

500 - 10 ± 2 143 ± 14 9 ± 3 59 ± 12 247 ± 32

1581 - 10 ± 2 112 ± 11 6 ± 2 59 ± 10 252 ± 13

5000 - 8 ± 1 99 ± 5 6 ± 1 54 ± 11 240 ± 20
Sodium azide (10) - 570 ± 57 *
Nitrofurantoin (0.2) - 379 ± 3 *

4-NPDA - 121 ±28 1 * 178 ± 25 2 *

Cumene HP (50) - 479 ± 65 *

Data summarized from Report No. AT02716, Tables 1 - 10, pages 48-57. 4-NPDA = 4-Nitro-1,2-phenylene diamine Cumene HP = Cumene hydroperoxide

1 10 μg/plate

2 0.5 μg/plate– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 378 April 2008

* Positive response

Table B.6-133: Summary of the mutation assays with BYI 08330-CIS-Ketohydroxy with activation
Mean number of revertants/plate ∀ standard deviation BYI 08330-CIS- Ketohydroxy ( μ g/plate or tube) S9 TA1535 TA100 TA1537 TA98 TA102

Plate incorporation method
0 (DMSO) + 12 ± 2 162 ± 10 7 ± 2 61 ± 7 242 ± 30

16 + 10 ± 2 169 ± 18 4 ± 1 56 ± 11 251 ± 45

50 + 10 ± 3 188 ± 13 7 ± 2 55 ± 3 244 ± 19

158 + 9 ± 2 171 ± 19 6 ± 2 68 ± 4 228 ± 24

500 + 11 ± 2 179 ± 8 5 ± 1 48 ± 6 250 ± 11

1581 + 10 ± 1 167 ± 18 7 ± 3 46 ± 8 223 ± 14

5000 + 9 ± 1 166 ± 17 5 ± 1 54 ± 12 236 ± 16

2-Aminoanthracene (3) + 122 ± 4 * 1690 ± 46 * 318 ± 39 * 1321 ± 251 * 880 ± 24 *

Preincubation method
0 (DMSO) + 12 ± 3 191 ± 15 8 ± 1 59 ± 8 278 ± 18

16 + 12 ± 3 188 ± 24 8 ± 4 60 ± 19 275 ± 21

50 + 10 ± 5 175 ± 15 8 ± 4 47 ± 5 285 ± 12

158 + 8 ± 3 191 ± 22 9 ± 1 56 ± 9 258 ± 41

500 + 10 ± 4 180 ± 6 8 ± 3 58 ± 5 265 ± 27

1581 + 8 ± 1 180 ± 14 6 ± 2 53 ± 8 266 ± 29

5000 + 7 ± 1 155 ± 15 6 ± 1 50 ± 12 290 ± 12

2-Aminoanthracene (3) + 145 ± 5 * 1631 ± 74 * 360 ± 59 * 1297 ± 21 * 662 ± 38 *

Data summarized from Report No. AT02735, Tables 1 - 10, pages 48-57. 4-NPDA = 4-Nitro-o-phenylendiamine * Positive response

REFERENCES :

Ames, B.N., W.E. Durston, E. Yamasaki et al. (1973) Carcinogens are mutagens: Asinple test system combining liver homogenates for activation and bact eria for detection. Proc. Nat. Acad. Sci. (USA) 70:
2281-2285.

Ames, B.N., J. McCann and E. Yamasaki (1975) Method s for detecting carcinogens and mutagens with the Salmonella

/mammalian-microsome mutagenicity test. Mutation Re s. 31: 347-364.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 379 April 2008

Table B.6-134: Historical control data, presented a s range of medians for years 1996-2004 Compound S9 TA1535 TA100 TA1537 TA98 TA102
Plate incorporation method

DMSO - 8-16 70-144 6-8 14-34 174-227

Sodium azide - 629-780

Nitrofurantoin - 217-358

4-NPDA - 91-126 137-173

Cumene HP - 331-556

DMSO + 9-12 89-169 8-10 23-41 248-294

2-Aminoanthracene + 150-235 1128-1526 148-286 1164 -1524 477-721

Preincubation method

DMSO - 7-15 72-145 6-8 15-32 209-271

Sodium azide - 609-747

Nitrofurantoin - 256-463

4-NPDA - 98-143 139-183

Cumene HP - 429-602

DMSO + 8-12 88-159 8-10 23-40 251-337

2-Aminoanthracene + 141-196 1190-1627 63-345 1117-1 442 466-613
Data summarized from Report No. AT02735, pages 32-4 5. 4-NPDA = 4-Nitro-1,2-phenylene diamine Cumene HP = Cumene hydroperoxide– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 380 April 2008

B.6.8.4 Summary of toxicological studies on metabol ites

Decreased body weight was observed following a 10-d ay exposure of rats by gavage to the enol metabolit e of spirotetramat (800 mg/kg bw/day). Following a 2 1-day exposure of rats to the enol metabolite at th e same dose by gavage, clinical signs of toxicity, decreas ed body weight gain, and testicular/spermatotoxicit y were observed. The ketohydroxy, desmethyl-ketohydroxy, dihydroxy, and monohydroxy metabolites of spirotetramat were non-mutagenic when tested in the Ames test.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 381 April 2008

B.6.9 Medical data and information (OECD Annex IIA 5.9)

B.6.9.1 Report on medical surveillance on manufactu ring plant personnel (OECD Annex IIA

5.9.1)

Two cases of Type IV hypersensitivity (facial) were reported and confirmed by patch testing in spirote tramat manufacturing plant personnel in 2004. Due to the two cases of type 4 sensitization, a survey was conducted among all potentially exposed persons. I t did not reveal any additional cases.

B.6.9.2 Report on clinical cases and poisoning inci dents (OECD Annex IIA 5.9.2)

B.6.9.3 Observations on exposure of the general pop ulation and epidemiological studies

(OECD Annex IIA 5.9.3)

B.6.9.4 Clinical signs and symptoms of poisoning an d details of clinical tests (OECD Annex

IIA 5.9.4)

The databases listed below were investigated via li terature research to check whether incidences were reported globally regarding poisoni ng to cover following points in the EU Annex II dossier for spirotetramat:

5.9.2 Report on clinical cases and poisoning incide nts 5.9.3 Epidemiological studies 5.9.4 Clinical signs and symptoms of poisoning

Databases checked: Data base name or abbreviation Agricola CAB-Abstr. CAplus CA-Registry CCRIS Chapman&HallChemical Chemikalienkontakt Chemlist Crossfile/Biosis CSNB Derwent CropProt Derwent Drug File DOSE Embase HSDB JICST Marketletter Medline MSDS Nioshtic Oshline– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 382 April 2008

Pesticide Fact File RTECS Scriplus Ulidat

Results: None of the checked data bases gave hints to human poisoning concerning spirotetramat. No negative effects were reported.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 383 April 2008

B.6.9.5 First aid measures (OECD Annex IIA 5.9.5)

B.6.9.6 Therapeutic regimes (OECD Annex IIA 5.9.6)

B.6.9.7 Expected effects and duration of poisoning as a function of exposure (OECD Annex

IIA 5.9.7)

B.6.9.8 Expected effects and duration of poisoning as a function of time (OECD Annex IIA

5.9.8)

B.6.9.9 Dermal Penetration (OECD Annex IIA 5.9.9)

B.6.9.9.1 In vivo dermal penetration study–rat

CITATION : Odin-Feurted, M. (2006) [ 14 C]-BYI 08330 OD150 formulation: In vivo dermal absorption study in the male rat. Bayer CropScience, 355 rue Dostoievski, BP. 153, 06903 Sophia Antipolis, France. Study No. SA 06009. July 27, 20 06. Unpublished.

I. MATERIALS AND METHODS:

A. MATERIALS :

1. Test material : [ 14 C]-BYI 08330 Description: Solid in distilled water
Lot/batch #: Reference synthesis KML 2978-2, BECH 1925 Purity: 99% a.i. Compound stability: Not available
CAS # for TGAI: Not available
Structure :

Vehicle/Solvent used: Distilled water

Radiolabelling: Indicated in structure by asterisk Specific Activity: 3.67 MBq/mg (99.1 μCi/mg)
Radiochemical Purity: >99%

Source: Bayer CropScience AG, Product Technology, Isotope C hemistry, Wuppertal, Germany
Other comments: None

2. Relevance of test material to proposed formulati on(s ): Not relevant

3. Test animals :– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 384 April 2008

Species: Rat
Strain: Wistar RJ: WI (IOPS HAN) Age/weight at study initiation: Age not specified/182-302g
Source: R. Janvier, France Housing: Individually in suspended, ss wire-mesh cages durin g acclimation period and in metabolism cages (Jencon’ s Metabowls or Radleys) during treatment phase.
Diet: Certified rodent diet A04C-10, Scientific Animal Fo od and Engineering, Route de Saint Bris, Augy, France; ad libitum
Water: Softened municipal tap; ad libitum
Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 20-24 ° C

40-70% 10-15/hr 12 hrs dark/12 hrs light Acclimation period: ≥ 14 days

B. STUDY DESIGN:

1. Dose :

Rationale: Dose selection was based on anticipated dermal dep osition of recommended field dilution.

Nominal doses : 100.0, 15.0 and 5.0 μg a.i./cm 2 skin.

Actual doses : 1.2, 0.2, and 0.06 mg a.i./12 cm 2 skin (see Table 6-135 for details). Actual doses were calculated as the mean dose applied to each of 16 animals/dose rate as determined by radiochemical analysis of the dosing solutions befo re, during, and after dosing. The homogeneity of each dose was also determined by assay of 5 aliquot s of each dosing solution before administration of doses.

Dose volume: 10 μ L/cm 2 skin

Duration of exposures (time from dose to skin wash) : 1, 10, or 24 hrs for determination of absorption. A fourth group per dose level was expo sed for 10 hrs and excretion was followed up to termination at 168 hrs.

Termination periods (time from dose to sacrifice): For each dose (high, medium, or low), 16 animals were exposed (groups of 4 animals/dose/exposure per iod). Groups of 4 male rats/dose were exposed for 1, 10, or 24 hrs and sacrificed at termination of the exposure period. One group of 4 animals per dose was exposed for 10 hrs, then given a skin wash and left in individual metabolism cages until sacrifice at 168 hrs to quantify excretion.

Number of animals/group: 4 male rats/dose/exposure period

2. Animal preparation : Prior to dosing, an area of dorsal skin was shaved and swabbed with acetone. Care was taken not to abrade the skin or non-abrade d areas were selected for application. The site of application (approximately 6 cm 2 /site; 2 sites/animal) was defined and protected by gluing plastic saddles to the unabraded skin using a cyanoacrylate-based g lue. To protect the application site, after dosing , a– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 385 April 2008

strip of perforated plastic was placed over the sad dle and held in place with surgical tape. The cove r prevented loss of material but allowed ventilation of the underlying skin.

3. Dose preparation, administration and quantificat ion :

Preparation: Dosing suspensions of the three formulations of r adiolabeled BYI 08330 were prepared by the Research and Development Centre of Bayer CropSc ience, Germany and stored at approximately -20 C prior to dosing.

Application: The dosing solutions were thawed and stirred contin uously during dosing. The required volume (120 μ L/rat) of the dose formulation was applied and spre ad evenly across the surface of the skin site using a calibrated positive displacement pipette. The dosing site was covered by a perforated plastic cover held in place with surgical tape. Th e perforated plastic cover was not in direct contac t with the test material applied to the skin. Sixteen rat s were dosed per exposure concentration. The pipet te tip used to apply the dose was retained for subsequ ent extraction of residual activity for calculation of the actual dose applied. Dosed animals were placed in glass metabolism cages equipped for collection of urine and feces. Provision was not made for CO 2 collection.

Quantification: Radioactivity (dpm) and radiochemical purity were determined before dosing. Homogeneity of the doses was determined from radioc hemical analysis of five dose-sized aliquots of the dosing solutions taken before, during and after dos ing. Pre-dose mid-dose and post-dose aliquots were analyzed to confirm radioactivity levels and homoge neity. Data are summarized in Table 6-135.

Table 6-135: Dosing

Amount of formulation applied/animal

(120 μL) Dose
level ( μ g/cm 2 ) Radio-label (kBq) Actual (mg a.i.) Nominal activity of dosing soln. (MBq/mL) Actual activity

of dosing soln. (MBq/mL) Active

ingredient (g/L)

100 608.4 1.2 4.98 5.07 10.1

15.0 669.6 0.2 4.57 5.58 1.52

5.0 211.2 0.06 1.52 1.76 0.48 Data taken from pp. 21, 46-48; Study No. SA06009

4. Skin wash and removal of stratum corneum : For each dosage group (high, medium, or low) four animals were sacrificed after 1, 10, and 24 hrs. F our additional rats were exposed at each dose rate for 10 hrs, skin washed and observed to termination at 168 hrs. At termination, the skin was swabbed with mild liquid soap solution, using natural sponge swa bs, to remove non-absorbed radioactivity. Swabbing was continued until no radioactivity was detected i n swabs using a Geiger counter. The skin was again shaved and tape-stripped to remove the stratum corn eum. Adhesive tape was applied for 5 seconds, then removed and the process was repeated until the skin appeared shiny, indicating the stratum corneum removal was complete.

5. Sample collection : Urine and feces were collected from each group of f our animals for each time point (1, 10, 24, and 168 hr) and each dosing level (100. 0, 15.0 and 5.0 μg a.i./cm 2 skin). At each termination period, 4 rats per dose level were exsanguinated un der halothane anaesthesia and a blood sample was taken by cardiac puncture. Dressings (saddle, gauz e, cover, and tape) were removed and saved for analysis. The treated area of skin was removed and saved for analysis. An area of skin immediately surrounding the treated area (approximately 1 cm wi de) was also taken for analysis as well as an isolated control portion of untreated skin. The re sidual carcass was also saved for analysis of– –

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radioactivity. Washings, bandages, sponges, blood, urine, feces, skin, and carcasses were stored at - 20 C until analyzed. Individual organs and other tis sues were not analyzed separately.

6. Sample preparation and analysis : Details of sample preparation are provided in Table 6-136. Liquid scintillation counting results (cpm) were converted to dpm using external standardization and appropriate quench correction data entered into the instrument computer of a Packard Tricarb 1900 TR Liquid Scintillation Counter (LSC). Efficiency cor relation curves were prepared for each scintillatio n cocktail and checked using [ 14 C]-n-hexadecane standards. The limit of detection (LOD) was taken to be two times the background count for blank samples pr epared using the appropriate scintillation cocktail

. Total amounts of radioactivity in samples were repo rted as percentage of the applied dose

Table 6-136: Sample preparation details
Sample media Preparation details
Skin wash swabs Sponges used for skin washings were solubilized in Soluene tissue digestant. Duplicate aliquots were mixed with scintillant and counted directly by liquid scintillation counting. Feces Mixed with distilled water and homogenized to a paste. Triplicat samples were oxidized by combustion mixed with cellulose powder and the 14 CO 2 generated was trapped in CarboSorb and mixed with scintillant pri or to analysis by liquid scintillation counting. Urine Duplicate aliquots of urine were added direct ly to scintillant and analyzed by liquid scintillation counting. Cardiac blood Triplicate weighed aliquots were dri ed on Combusto-pads, combusted in Combustocones and absorbed samples were added directly to s cintillant and analyzed by liquid scintillation counting. Cage wash Duplicate aliquots of cage washings were weighed, added directly to scintillant, and analyzed by liquid scintillation counting. Saddle, tapes, gauze and cover (dressing) Extracted with acetonitrile. Duplicate weighed ali quots of the extractant were added to scintillant and analyzed by liquid scintillation counting. Carcass Solubilized in alcoholic potassium hydroxid e and duplicate weighed samples analyzed by liquid scintillation counting. Fur samples Fur samples from treated skin (taken pr ior to tape stripping) were extracted with acetonitrile and duplicate aliquots of the extracta nt were added to scintillant and analyzed by liquid scintillation counting. Skin samples Skin was solubilized in Soluene tissue digestant and analyzed by liquid scintillation counting. Tape strips with attached

stratum corneum Solubilized in tetrahydrofuran, samples added direc tly to scintillant, and analyzed by liquid scintillation counting.

II. RESULTS

A. SIGNS AND SYMPTOMS OF TOXICITY :

All animals were examined for mortality and signs o f toxicity throughout the study period. The result s of these observations were not reported.

B. SUMMARY TABLES :– –

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Table 6-137: Amount of radiolabel recovered in each matrix at specified hours post-application of a 100 μg/cm 2 dose of [ 14 C]-BYI 08330
Residues in matrix (% of applied dose) Matrix analyzed 1 hr (Group 1) 10 hr (Group 2) 24 hr (Group 3) 10/1 68 (Group 4) a
Swabs, initial 84.88±8.22 76.07±6.68 77.60±4.92 79.59±2.67 Swabs, final 0.12±0.04 0.16±0.03 0.07±0.02 0.12±0.04 Swabs, area surrounding
site 0.04±0.05 0.08±0.08 0.02±0.02 0.03±0.02

Total swabs 85.04±8.09 76.31±6.73 77.70±4.96 79.74± 2.64 Fur (dose site) N.S N.S. N.S. 2.13±0.82 Dressing 1.56±2.31 0.33±0.19 1.27±0.39 1.85±0.51
Surface dose 2.46±1.90 4.66±3.23 4.24±2.05 0.58±0.31
Total non-absorbed
radiolabel 89.07±5.59 81.30±4.26 83.21±4.96 84.30±1.57

Stratum corneum 5.06±4.18 14.26±5.26 9.66±2.74 2.60±1.58
Treated skin 1.57±0.71 1.63±0.34 1.73±1.04 0.28±0.17
Total dose site 6.62±4.58 15.89±4.97 11.39±3.72 2.8 7±1.75
Urine 0.00±0.00 0.26±0.18 0.79±0.19 2.06±0.90
Feces 0.00±0.00 0.00±0.00 0.04±0.04 0.18±0.05 Cage wash 0.00±0.00 0.10±0.04 0.34±0.17 4.01±2.68
Total excreted 0.00±0.00 0.36±0.21 1.17±0.27 6.25±3 .39
Blood 0.01±0.01 0.02±0.00 0.02±0.01 0.00±0.00
Carcass 0.42±0.20 0.31±0.06 0.40±0.04 0.13±0.03
Non-treated skin 0.53±0.50 0.38±0.31 0.18±0.07 0.24±0.12 Surrounding skin 1.24±0.80 0.65±0.32 2.62±1.58 1.07±0.24 Total directly absorbed 2.20±1.45 1.73±0.55 4.39±1. 83 7.69±3.41 Total potentially absorbable 8.83±5.06 17.62±4.93 1 5.78±5.33 10.57±4.68
Total recovered 97.89±1.27 98.92±7.49 98.99±0.66 94 .87±4.22

Data taken from Table s 2 and 8, pp. 23 and 32; Report No. MEF-06/15.

a 10 hours of exposure with observation period extend ed to termination at 168 hrs.

Table 6-138: Amount of radiolabel recovered in eac h matrix at specified hours post-application of a 15 μg/cm 2 dose of [ 14 C]-BYI 08330
Residues in matrix (% of applied dose) Matrix analyzed 1 hr (Group 5) 10 hr (Group 6) 24 hr (Group 7) 10/1 68 (Group 8) a
Swabs, initial 83.35±6.04 80.73±7.62 70.22±5.33 83.89±4.93 Swabs, final 0.13±0.08 0.47±0.16 0.65±0.14 0.30±0.13 Swabs, area surrounding
site 0.06±0.03 0.13±0.16 0.04±0.04 0.02±0.04

Total swabs 83.55±5.82 81.33±7.28 70.90±5.16 84.21± 4.76 Fur (dose site) N.S. N.S. N.S. 1.64±0.84 Dressing 0.48±0.31 1.10±1.28 0.22±0.08 1.25±0.82
Surface dose 1.83±1.48 2.72±2.56 4.16±1.54 0.41±0.15
Total non-absorbed
radiolabel 85.86±4.04 85.15±6.23 75.28±5.43 87.10±3.16

Stratum corneum 4.68±4.53 7.32±6.46 14.29±4.96 1.53±0.80– –

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Treated skin 0.73±0.34 1.11±1.13 1.36±0.50 0.19±0.13
Total dose site 5.41±4.76 8.43±7.57 15.65±5.37 1.72 ±0.88
Urine N.S. 0.24±0.15 0.80±0.30 1.83±0.45
Feces 0.01±0.01 0.04±N.A. 0.09±0.03 0.16±0.03 Cage wash 0.02±0.01 0.20±0.23 0.23±0.07 0.44±0.10
Total excreted 0.02±0.01 0.45±0.39 1.12±0.30 2.42±0 .35
Blood 0.00±0.01 0.02±0.00 0.02±0.01 0.00±0.00
Carcass 0.17±0.06 0.23±0.08 0.24±0.05 0.14±0.01
Non-treated skin 0.18±0.07 0.11±0.01 0.13±0.04 0.18±0.07 Surrounding skin 0.69±0.42 1.12±1.79 0.43±0.27 0.39±0.28 Total directly absorbed 1.07±0.50 1.92±2.25 1.94±0. 49 2.99±0.41 Total potentially absorbable 6.48±5.17 10.36±6.52 1 7.59±5.79 4.71±0.96
Total recovered 92.34±2.19 95.50±0.46 92.87±0.42 91 .81±2.33 Data taken from Tables 4 and 8, pp. 26 and 32; Repo rt No. MEF-06/15.

a 10 hours of exposure with observation period extend ed to termination at 168 hrs.

Table 6-139: Amount of radiolabel recovered in eac h matrix at specified hours post-application of a 5 μg/cm 2 dose of [ 14 C]-BYI 08330
Residues in matrix (% of applied dose) Matrix analyzed 1 hr (Group 9) 10 hr (Group 10) 24 hr (Group 11) 10 /168 (Group 12) a
Swabs, initial 91.53±1.57 85.30±2.79 85.58±2.33 0.31±0.08 Swabs, final 0.07±0.03 0.67±0.04 0.07±0.02 82.33±2.35 Swabs, area surrounding
site 0.04±0.04 0.10±0.07 0.00±0.00 0.06±0.05

Total swabs 91.64±1.55 86.08±2.69 85.65±2.36 82.71± 2.37 Fur (dose site) N.S. N.S. N.S. 1.99±0.99 Dressing 0.13±0.06 0.14±0.11 0.79±0.30 2.22±1.17
Surface dose 0.80±0.62 2.20±0.78 1.52±0.60 0.71±0.43
Total non-absorbed
radiolabel 92.57±1.25 88.42±3.05 87.96±2.19 87.62±1.54

Stratum corneum 1.63±0.48 10.02±3.86 7.11±1.96 1.53±0.45
Treated skin 0.51±0.23 0.58±0.2 1.99±0.59 0.30±0.30
Total dose site 2.14±0.49 10.60±3.69 9.10±1.67 1.82 ±0.59
Urine 0.01±0.01 0.34±0.24 0.93±0.78 1.49±0.56
Feces 0.00±0.00 0.06±0.09 0.19±0.12 0.20±0.08 Cage wash 0.02±0.02 0.21±0.14 0.56±0.23 0.78±0.21
Total excreted 0.03±0.02 0.61±0.32 1.68±1.07 2.47±0 .81
Blood 0.01±0.01 0.02±0.02 0.02±0.01 0.00±0.00
Carcass 0.57±0.08 0.45±0.12 0.85±0.11 0.51±0.11
Non-treated skin 0.15±0.03 0.16±0.03 0.35±0.08 0.35±0.12 Surrounding skin 0.13±0.06 0.19±0.09 0.64±0.43 0.66±0.36 Total directly absorbed 0.88±0.15 1.43±0.47 3.54±1. 58 3.99±1.36 Total potentially absorbable 3.02±0.41 12.03±3.51 1 2.64±1.17 5.81±1.92
Total recovered 95.59±0.97 100.45±1.28 100.61±2.49 93.43±2.68

Data taken from Table s 6 and 8, pp. 29 and 32; Report No. MEF-06/15.

a 10 hours of exposure with observation period extend ed to termination at 168 hrs.– –

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C. TOTAL ABSORBED DOSE :

Results of analysis are summarized in Tables 6-137, 6-138, and 6-139

. Recovery of the applied dose (material balance) was acceptable. Group means ranged from 94.9-99.0, 91. 8-95.5, and 93.4-100.6% for the 100.0, 15.0, and 5.0 μg/cm 2 dose groups, respectively. The majority of the ad ministered dose was recovered from skin washings, and as non-absorb ed radiolabel (81.3-89.1, 75.3-87.1, and 87.6- 92.6% for the high, medium, and low dose groups, re spectively). Of the applied dose, 2.87-11.39, 1.76- 15.6, and 1.82-10.6% was retained at the applicatio n site for the high, medium, and low dose groups, respectively. Estimates of dermal absorption (total absorbed dose ) were based on the sum of residues recovered in urine, cage wash, feces, carcass, non- treated skin, and surrounding skin and range from 1.73-7.69, 1.07-2.99, and 0.88-3.99% for the high, medium, and low dose groups, respectively. The study authors excluded residues retained at the ski n site in estimates of dermal absorption because th e values declined over time (168 hrs) and could mask any increase of residues in the rest of the body ov er the observation period.

The mean potential absorption level was defined by the study authors to be the total directly absorbed label + the total radioactivity recovered from the dose site and is used interchangeably with total indirectly absorbed radioactivity. This value appe ared to increase between 1 and 10 hrs exposure in t he three dose groups, due to retention in the stratum corneum, but only significantly for the high and lo w dose groups and remained stable between 10 and 24 h rs exposure for all three dose groups. The mean potential dermal absorption values for the high dos e group ranged from 8.83% of applied dose at 1 hr t o 15.78% after 24 hrs exposure. For the medium dose groups, the mean levels ranged between 6.48% after 1 hr exposure to 17.59% after 24 hrs exposure , and for the low dose group between 3.02% after 1 hr to 12.64% after 24 hrs exposure. The dose level did not appear to significantly affect the degree of potential/indirect dermal absorption.

Based on the likely worker exposure time frame, it is considered most appropriate to adopt the dermal absorption value calculated for the groups of anima ls for which the skin site was washed after 10 hour s, and which were sacrificed after 168 hrs. Mean derm al absorption for these groups of animals was 10.57, 4.71, and 5.81% for the high, medium, and lo w dose groups respectively. This value includes th e potentially absorbable radioactivity present in the stratum corneum and the skin at the application si te (2.87, 1.72, and 1.82% for the high, medium, and lo w dose groups, respectively). These values were included, because at every dose the amount of radio activity at the dose site (including stratum corneu m) decreased during the 168-hr time period while the a mount of radioactivity in excreta increased, thereb y implying that the compound continued to be absorbed after dosing was stopped.

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS :

Following a 1, 10 or 24 hr in vivo dermal exposure of male rats to [ 14 C]-BYI 08330 at dose levels of 100, 15, or 5 μg a.i./cm 2 , the patterns of absorption, distribution (skin) a nd excretion were investigated. Male rats were sacrificed after each exposure period. A dditional groups were exposed to the high, medium, and low doses for 10 hrs and sacrificed 168 hrs pos t-treatment.

The majority of the applied radioactivity was found to be non-absorbed which accounted for means of 81.30-89.07, 75.28-85.86, and 87.96-92.57% of the a pplied radiolabel in the high, medium, and low dose groups, respectively.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 390 April 2008

For the high dose group there was no significant di fference between the mean levels of direct dermal absorption following 1, 10 or 24 hours of exposure. The observed mean levels were 2.20, 1.73, and 4.39% of the applied dose after 1, 10, and 24 hrs e xposure, respectively. For the medium dose animals

, the mean levels of direct dermal absorption were 1. 07, 1.92, and 1.94% after 1, 10, and 24hrs exposure

, respectively and for the low dose the mean levels w ere 0.88, 1.43, and 3.54% after 1, 10, and 24 hrs exposure, respectively. Generally there appeared t o be a slight trend towards higher mean dermal absorption values with increasing duration of expos ure. These increases were significant between 1 and 24 hours exposure for the medium and low dose g roups but not for the high dose group. There was a significant increase observed in the mean absorpt ion levels at 10 and 24 hours for the high dose animals. Total direct dermal absorption did not app ear to be significantly affected by the level of ap plied dose.

The data from the 168-hour depuration groups indica te that only a portion of the radioactivity remaini ng at the dose site (treated skin plus stratum corneum ) after 10 hours exposure (46, 16, and 29% for the high, medium and low dose groups, respectively) is likely to be absorbed during the depuration period. The remainder is expected to be sloughed off with t he renewal of the stratum corneum.

B. REVIEWER COMMENTS :

The total recovered radioactivity for the 12 groups of male Wistar rats exposed dermally to [ 14 C]-BYI 08330 in this in vivo

dermal penetration study was excellent and ranged from 91.81 to 100.61%. The total absorbed values for the 1, 10, 24, and 168 ho ur post-treatment sacrifice periods for the high, medium and low dose levels are given in Tables 6-13 7, 6-138, and 6-139.

The mean potential absorption level was defined by the study authors to be the total directly absorbed label + the total radioactivity recovered from the dose site and was used interchangeably with total indirectly absorbed radioactivity. Indirect recover y values are also normally considered part of the estimated value for dermal absorption. The estimate d value is the sum of the total radioactivity recov ered from the skin at the application site, non-treated skin, surrounding skin, urine, feces, cage washes, blood, and carcass. This value is considered conser vative since a portion of the active ingredient was retained at the skin site and it is unlikely that a ll of the skin residues will become systemically av ailable. This value appeared to increase between 1 and 10 hr s exposure in the three dose groups, due to retention in the stratum corneum, but only signific antly for the high and low dose groups and remained stable between 10 and 24 hrs exposure for the three dose groups. The mean potential dermal absorption values for the high dose group ranged fr om 8.83% of applied dose at 1 hr to 15.78% after 24 hrs exposure. For the medium dose groups, the mean levels ranged between 6.48%after 1 hr exposure to 17.59 after 24 hrs exposure, and for the low dos e group between 3.02% after 1 hr to 12.64 after 24 hrs of exposure. The dose level did not appear to significantly affect the degree of potential/indire ct dermal absorption.

As stated previously, based on the likely worker ex posure time frame and the more accurate measurement of absorbed dose, it is considered most appropriate to adopt the dermal absorption value calculated for the groups of animals for which the skin site was washed after 10 hours and which were sacrificed after 168 hrs.

This study in the rat is acceptable/guideline and satisfies the guideline requirement for an in vivo dermal penetration study (870.7600) in rats.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 391 April 2008

C. STUDY DEFICIENCIES:

There were no study deficiencies that would impact the integrity of the study results. Unlabeled pare nt material, if used, was not characterized. The resu lts of examinations for mortality and clinical sign s of toxicity were not reported.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 392 April 2008

B.6.9.9.2 In vitro dermal penetration study - rat/human

CITATION : Capt A. (2006) [ 14 C]-BYI 08330 in OD150 formulation: Comparative in vitro dermal absorption study using human and rat skin. Bayer Cr opScience, 355 rue Dostoievski, BP. 153, 06903 Sophia Antipolis, France. Study No. SA 0 5254. April 05, 2006. Unpublished.

I. MATERIALS AND METHODS:

A. MATERIALS :
A. MATERIALS : 1. Test material : [ 14 C]-BYI 08330 Description: White powder dissolved in distilled water
Lot/batch #: Reference synthesis KML 2978-2, BECH1855 Purity: 99% a.i. Compound stability: Not available
CAS # for TGAI: Not available
Structure:

Vehicle/Solvent used: Distilled water

Radiolabelling: Indicated in structure by asterisk Specific Activity: 3.67 MBq/mg (99.1 μCi/mg)
Radiochemical Purity: Chemical Purity >99%
>99%

Source: Bayer CropScience AG, Product Technology, Isotope C hemistry, Wuppertal, Germany
Other comments: None

2. Relevance of test material to proposed formulati on(s ): Not relevant

3. Skin membrane source : Species: Rat/Human
Strain: Rat: Wistar Rj: EWI (IOPS HAN) Human: NA Age/weight at study initiation: Rat: 6-8 wks;Human: 39-66 yrs
Source: Housing (rat):

Diet (rat): Water (rat): Environmental conditions (rat): Rat: R. Janvier (France) Human: Tissue Bank (Bio predic, Rennes, France) Together (10) in wire-mesh bottomed, stainless stee l cage during the acclimation period Pelleted; A04 C-10 . SAFE. Rte de St Bros. Aigu, Fr ance; ad libitum– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 393 April 2008

Acclimation period (rat): Filtered municipal tap; ad libitum Temperature: 20-24 C Humidity: 40-70% Air changes: 10-15/hr Photoperiod: 12 hrs dark/12hrs light ≥ 5 days

B. STUDY DESIGN:

1. Dose :

Rationale : Dose selection was based on direct exposure to for mulation concentrate 150 mg/mL) or anticipated dermal deposition of field spray diluti ons (1.5 or 0.05 mg/mL).

Nominal doses : 150, 1.5 or 0.05 mg a.i./mL dosing solution.

Actual doses :

The actual concentration of [ 14 C]-BYI 08330 in the dosing solutions were 1.58, 0.0 13, or 0.0004 mg/10 μL dose (see Table 6-140 for detail s). Actual doses were calculated as the mean dose applied to each of 6 dermal membranes/dose rate/exp osure period (8 hours) for both rat and human skin by weight as verified by radiochemical analysis of the three dosing solutions (high, medium, and low) as close to the time of application as practicable.

Dose volume : 10 μ L/cm 2 skin (2.54 cm 2 /membrane)

Duration of exposures (time from dose to terminatio n): 24 hours with skin wash after 8 hrs exposure.

Termination periods (24 hrs): A reservoir was placed atop each membrane for recep tor fluid which was heated to 32 C using a water-heated manifold. The lower reservoir of the diffusion chambers were connected to a fraction collector via effluent tubi ng. Fractions were collected hourly following dosi ng and collected until termination at 24 hrs. The recepto r fluid chamber contents were stirred continuously and pumped at a rate of 1.5 mL/hr. At termination the diffusion sells were dismantled and subjected to ma ss balance procedures.

Number of membranes /group : Six membranes (target number before exclusion of da maged membranes) were tested in individual diffusion cell s/dose group/exposure period. The actual numbers used are given in the data tables (Tables 6-141, 6- 142, and 6-143).

2. Membrane preparation : Following acclimation each animal was sacrificed by cervical dislocation and a dorsal area of skin was clipped and excised for mem brane preparation. The skin was dermatomed using a mini-dermatome (Decadermatone microsystem motor, Thackray Surgery, Leeds, UK)). Dermal membranes 420-510 μM thick were prepared using this apparatus. Human skin membranes from 13 donors were prepared similarly using the mini-derma tome. Prior to use, the integrity of the selected membranes was assessed by measuring the trans-epide rmal water loss (TEWL) from the stratum corneum. The membranes were assembled in diffusion cells (Franz cell modified, Gallas, France) and an evaporimeter probe (Dermalab, Cortex Technology, Hadsund, Denmark) was placed atop the receptor cell and water diffusion through the skin was measured (g/m 2 /hr). Human and rat skin with a TEWL of >40g/m2/hr were considered potentially dama ged and were not used.

3. Dose preparation, application to membranes and q uantification :– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 394 April 2008

Preparation : Dosing suspensions (three formulations) were prep ared in the Monheim Research and Development Centre of Bayer CropScience, Germany, o n the day of dosing or as close as practicable. Details of preparation were not given in the study report. The high, medium, and low dose formulation s were tested for solubility in the receptor fluid an d found to be soluble for 24 hrs at 2.4mg in 3 mL o f receptor fluid (Eagle’s medium supplemented with 5% bovine serum albumin and 50 mg/L gentamicin) which exceeded the maximum volume of the diffusion cells. The amount tested for solubility exceeded the total quantity of [ 14 C]-BYI 08330 applied to the membranes of any cell. Doses were checked for radioactivity before during and after dosing. Homo geneity was checked by taking five aliquots of each dose formulation before application. The formulati ons were considered to be homogeneous.

Assembly of diffusion cells and application of dosi ng solutions : Glass flow-through diffusion cells (Franz cell modified, Gallas, France) were used wit h an exposed membrane area of 2.54cm 2 . A cell consisted of a donor and a receptor chamber between which the skin was positioned. The upper chamber with receptor fluid (Eagle’s medium with 5% bovine serum albumin and gentamicin at 50 mg/L, pH 7.4-7.5), in contact with the skin was warmed to 32 C. The lower chamber contents were stirred continuously with a magnetic stirring bar and pumpe d to a fraction collector at a flow rate of 1.5 mL/ hr. The dosing solutions (high, medium, or low dose) we re applied to the split-thickness skin samples with a pipette at 10 μL/cm 2 skin surface. Each dosing solution was assayed fo r radioactivity by LSC using dose checks before, during, and after dosing.

Quantification : Radioactivity (dpm) and radiochemical purity were determined before application of the dosing solutions to membranes. Radiochemical purit y was determined by TLC and HPLC analysis of the dosing preparations. Homogeneity of the doses was d etermined from radiochemical analysis of dosesize aliquots of the dosing solutions taken before, during and after dosing. Data are summarized in Table 6-140.

Table 6-140: Dose levels achieved

Dose Application rate

(μL/cm 2 ) Actual [ 14 C]-BYI
08330
concentration (mg/mL) Applied dose (μg

[ 14 C]-BYI 08330 /cell) Applied dose

(kBq/cell)

High 10 150 1580 52.1
Medium 10 15.0 13.3 48.6
Low 10 0.05 0.4 1.4 Data taken from Appendix Tables C4-C6, pp. 48-51; S tudy No. SA 05254. Means in columns 4 and 5 calcul ated by study reviewer.

4. Sample collection and preparation : Diffusion cell contents were sampled by collection of cell eluants hourly using a fraction collector set to cycle each hour at a flow rate of 1.5 mL/hr. Cells were expos ed to the high, medium, or low doses of [ 14 C]-BYI 08330 for 8 hrs and washed by repeated swabb ing after exposure to remove residual radioactivity. Tween 6 0 (1%) in phosphate buffered saline was used as the swabbing agent. Swabbing was continued until no ra dioactivity could be detected on the sponges using a Geiger counter. Sampling of receptor fluid conti nued hourly to termination at 24 hrs. Membranes we re swabbed again at termination and all swabs were sav ed for radiochemical analysis by LSC. At termination membranes were tape-stripped to remove the stratum corneum. Tape stripping was repeated until the epidermis was shiny, indicating stratum corneum removal was complete. The tape strips were saved for analysis and the epidermal me mbranes were removed and saved for analysis by LSC. Receptor fluid at termination was removed fro m cells for analysis, the diffusion cell components– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 395 April 2008

were washed, and the washings saved for analysis fo r mass balance. Samples were stored at -20 C until analyzed.

5. Sample analysis : Material balance samples, receptor fluid samples, a nd aliquots of dosing solutions were analyzed using Liquid Scintillation Counting ( LSC) techniques. Receptor fluid samples were analyzed directly by addition to scintillant for co unting. Skin swabs were solubilized in Soluene and scintillation fluid was added for LSC analysis. Ta pe strips were solubilized using tetrahydrofuran an d scintillation fluid was added for direct LSC analys is. Skin samples were solubilized in soluene, scintillation fluid was added and samples were coun ted by LSC. Diffusion cell components, including stirrers and tubing, were soaked in acetonitrile/di stilled water (50:50 v/v) for 12 hrs and the washin gs were analyzed by LSC. All samples were counted usi ng a Packard 1900 TR Liquid Scintillation Spectrometer with on-line computing. Quenching eff ects were determined using external standardization and spectral quench parameter (tSIE ) methods. Efficiency correlation curves were prepared for each scintillation cocktail using radi olabeled n-hexadecane internal standards. The limi t of detection was considered to be twice the background values for blank samples in scintillation cocktail

. The calculation of % recovered dose, means and stan dard deviations were accomplished with a personal computer using Debra 5.2a software and Mic rosoft Excel.

II. RESULTS:

A. MATERIAL BALANCE AND SPIROTETRAMAT DISTRIBUTION :

The material balance for dermal penetration of [ 14 C]-BYI 08330 through rat and human epidermis at the high, medium, and low dose rates are shown in Table s 6-141, 6-142, and 6-143, respectively. Recovery of radioactive material was excellent with means ra nging from 96.0-100.1% of the applied dose for rats and 96.0-99.0% for humans. For the formulation con centrate, skin washing removed the majority of the applied dose from the skin surface after 8 hrs expo sure and at termination (24 hrs). For rat skin, th e washings removed 92.0, 65.1, and 70.1% at the high, medium, and low dose levels, respectively. Washing removed 95.5, 93.0, and 84.7% of the high, medium, and low doses, respectively, applied to human skin. The residual radioactivity remaining w ith the epidermis for rat skin after 24 hrs. was 0. 75, 4.34, and 1.26% for the high, medium, and low doses , respectively; for human skin, 1.14, 15.6, and 7.1% of the high, medium, and low doses remained wi th the epidermis after 24 hrs. In addition, a portion of the applied doses remained in the stratu m corneum of both species. For the rat, 0.12, 1.7, and 1.9% of the respective high, medium and low doses r emained with the stratum corneum. For human skin, 0.1, 0.7, and 3.24% of the added radioactivit y remained within the stratum corneum for the high, medium, and low doses, respectively. At terminatio n, <0.005% of the three applied doses remained in the diffusion chambers for the rat and <0.005, 0.00 5, and 1.55% of the respective high, medium, and low doses remained for humans.

The portion of the applied dose which was absorbed by rat and human skin (i.e., penetrated into receptor fluid) is discussed in the following secti on.

Table 6-141: Material balance and in vitro absorpti on of [ 14 C]-BYI 08330 through human and rat epidermis expressed as percentage of the applied do se (high dose = 1500 μg/membrane)

Parameter Human skin (n=5) Rat skin (n=5) Surface dose (non-absorbed) 029±0.48 1.32±0.77 Skin swabs (8 hr) 95.01±3.60 91.07±2.32 Skin swabs (24 hr) 0.53±0.74 0.95±0.56 Skin swabs (8 + 24 hr) 95.54±3.07 92.02±2.08– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 396 April 2008

Residual dose in chamber 0.14±0.10 0.51±0.61
Total % non-absorbed 95.97±2.80 93.84±1.49
Skin 0.11±0.13 0.75±0.53
Stratum corneum 0.12±0.15 1.14±0.28
Total % dose site 0.23±0.28 1.88±0.79 Receptor fluid (0-24hr) 0.01±0.01 0.24±0.10 Receptor fluid terminal <0.005±<0.005 0.02±0.01 Receptor chamber <0.005±<0.005 <0.005±<0.005 Total % directly absorbed 0.01±0.01 0.26±0.10 Total % potentially absorbable 0.24±0.28 2.14±0.82 Total % recovery (material balance) 96.21±2.65 95.98±0.82

Data taken from Table 1, page 24; Study No. SA05254.

Table 6-142: Material balance and in vitro absorpti on of [ 14 C]-BYI 08330 through human and rat epidermis expressed as percentage of the applied do se (medium dose = 15 μg/membrane)

Parameter Human skin (n=4) Rat skin (n=5) Surface dose (non-absorbed) 0.68±0.46 8.52±6.08 Skin swabs (8 hr) 91.56±4.69 61.94±11.48 Skin swabs (24 hr) 1.45±1.14 3.21±3.55 Skin swabs (8 + 24 hr) 93.01±4.64 65.15±8.83
Residual dose in chamber 2.55±3.16 0.56±0.24
Total % non-absorbed 96.24±4.27 74.23±13.99
Skin 0.67±0.25 4.34±4.70
Stratum corneum 1.66±1.91 15.62±7.11
Total % dose site 2.32±2.13 19.96±8.40 Receptor fluid (0-24hr) 0.36±0.29 2.94±0.82 Receptor fluid terminal 0.05±0.09 0.31±0.17 Receptor chamber <0.005±<0.005 <0.005±<0.005 Total % directly absorbed 0.41±0.36 3.25±0.96 Total % potentially absorbable 2.74±1.87 23.21±8.31 Total % recovery (material balance) 98.98±2.54 97.44±6.60

Data taken from Table 2, page 26; Study No. SA 05254.

Table 6-143: Material balance and in vitro absorpti on of [ 14 C]-BYI 08330 through human and rat epidermis expressed as percentage of the applied do se (low dose = 0.5 μg/membrane)

Parameter Human skin (n=5) Rat skin (n=7) Surface dose (non-absorbed) 2.46±1.47 5.57±5.05 Skin swabs (8 hr) 83.83±8.63 68.59±9.70 Skin swabs (24 hr) 0.89±1.34 1.53±1.01 Skin swabs (8 + 24 hr) 84.72±7.64 70.12±9.46
Residual dose in chamber 0.41±0.58 1.66±1.82
Total % non-absorbed 87.59±6.09 77.35±4.84
Skin 3.24±3.12 1.26±1.22
Stratum corneum 4.65±3.29 9.22±4.22
Total % dose site 7.89±5.68 10.49±4.62– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 397 April 2008

Receptor fluid (0-24hr) 0.56±0.67 10.45±5.07 Receptor fluid terminal <0.005±<0.005 0.22±90.12 Receptor chamber <0.005±<0.005 1.55±2.66 Total % directly absorbed 0.56±0.67 12.22±6.04 Total % potentially absorbable 8.45±6.05 22.71±6.25 Total % recovery (material balance) 96.04±3.79 100.06±4.95

Data taken from Table 3, page 28 Study No. SA 05254

B. TOTAL ABSORBED DOSE :

Absorbed dose data for the high, medium, and low do ses, applied to rat and human skin membranes, are given in Tables 6-141, 6-142, and 6-143, respec tively. The absorbed dose is expressed as the percentage of [C 14 ]-BYI 08330 recovered in the receptor fluid during the 24 hour flow-through period, the amount in the receptor fluid at termination and the residual radioactivity retained in the receptor ch amber at termination (24 hrs). This amounted to 0.01% an d 0.26% of the applied high dose in the human and rat skin, respectively. For the medium dose, the m ean total amount of radioactivity directly absorbed was 0.41 and 3.25% for human and rat skin, respecti vely. For the low dose, the mean total radioactivi ty directly absorbed was 0.56 and 12.22% for the human and rat skin, respectively.

The study authors considered the potentially absorb able portion of the administered dose to be that found in the skin compartment excluding surface res idual (mean percent directly absorbed + mean percent at the dose site). For the high dose, 0.24 and 2.14% of the dose in the human and rat skin wa s considered potentially absorbable. For the medium dosage, 2.73 and 23.21% of the dose to human and rat skin, respectively, was considered potentially absorbable. For the low dose, 8.45 and 22.71% percent of the administered dose was considered pot entially absorbable in the human and rat skin, respectively.

Given the uncertainty regarding actual deposition u nder actual field conditions, it is considered appropriate to derive an estimate of dermal absorpt ion based on the results from the low dose group (0.05 mg a.i./mL dosing solution), since percent de rmal absorption was greatest at this dose level. T his value was not reported by the study authors as the amount by weight per unit area of skin but was presented as the percentage of absorbed radiolabel /dosed area of membrane (1 cm 2 ). The radioactivity present in the stratum corneum of the human skin at termination was considered not to be available for further absorption but to be labile t o loss by desquamation.

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS :

This study (No. SA 05254) was carried out to compar e the in vitro dermal penetration of BYI 08330 following single dermal application of [ 14 C]-BYI 08330 to excised human skin and rat dermatom ed skin. Dermal absorption was determined at three dose conc entrations of an OD150 formulation, i.e., neat (150 mg/mL) and two spray dilutions at 1.5 and 0.05 mg/mL referred to as the high, medium, and low doses. The three formulations were applied to epid ermal membranes at a rate of 10μL/cm 2 . The membranes were held between chambers of flow throug h diffusion cells and receptor fluid was passed through the membranes at 1.5 mL/hr flow rate and co llected from the effluent tubing using a fraction collector. After 8 hours exposure, the residual doses were was hed off the skin and hourly sampling– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 398 April 2008

was continued to termination at 24 hours. At termi nation the skin membranes were tape-stripped to remove residual surface dose and the stratum corneu m.

The percentage of [ 14 C]-BYI 08330 directly absorbed over the 24 hr obser vation period was 0.01 and 0.26% of the high dose, 0.41 and 3.25% of the mediu m dose and 0.56 and 12.22% of the low dose for human and rat skin, respectively.

The percentage of [ 14 C]-BYI 08330 considered potentially absorbable over the 24 hr observation period was 0.24 and 2.14% for the high dose, 2.74 a nd 23.21% for the medium dose and 8.45 and 22.71% for the low dose for human and rat skin, res pectively. The absorption factor differences between the two species were 8.9 for the high dose, 8.5 for the medium dose and 2.7 for the low dose.

B. REVIEWER COMMENTS :

This study was carried out to determine the compara tive in vitro percutaneous absorption of BYI 08330 through rat and human epidermis following exposure to a [ 14 C]-BYI 08330 formulation concentrate or two simulated aqueous spray dilutions of the OD150 formulation. The former (neat formulation) was included to assess exposure to mixer/loaders and th e latter dilutions to simulate exposure of spray applicators in the field. The exposure period was 8 hours with observation to termination at 24 hrs. The experiments were carried out using diffusion chambe rs with rat or human epidermal membranes prepared from skin obtained by excising clipped ski n from rats or from human skin obtained from a tissue bank. The doses of formulation concentrate or spray dilutions were applied to the intact rat o r human epidermal membranes and allowed to diffuse in to receptor fluid contained in the diffusion cells. Effluents were pumped to a fraction collector at a flow rate of 1.5 mL/hr. Collection tubes in the fr action collector were cycled every hour. Total radioactiv ity recovered from the chambers, skin wash, epiderm al membranes, and receptor fluids was excellent, rangi ng between 96.0-96.2% for the formulation concentrate (high dose) and 97.4-99.0% and 96.0-100 .1% for the aqueous dilutions of the formulation concentrate (medium and low doses, respectively). The amount of radioactivity directly absorbed ranged between 0.01-0.26%, 0.41-3.25%, and 0.56-12. 22% of the applied dose for the high, medium, and low doses, respectively. The potentially absor bable radioactivity ranged between 0.24-2.14%, 2.74

- 23.21%, and 8.45-22.71% of the applied radioactivit y for the high, medium, and low doses, respectively

. The absorption data for the formulation concentrate and simulated spray dilutions indicated that BYI 08330 was poorly absorbed through rat and human ski n in vitro. Rat skin appeared to be more permeable than human skin. The potential absorptio n differences for rat and human skin differed by factors of 8.9, 8.5, and 2.7 for the high, medium, and low doses, respectively.

All values reported in this study were expressed as percentage of applied dose (radioactivity) and not as actual amounts recovered as expressed by weight of active ingredient per unit area of exposed skin (μg/cm 2 ). Although this omission does not affect the outc ome of the experiments, data interpretation would have been more meaningful had the actual abso rbed or potentially absorbable amounts of BYI 08330 been calculated and reported.

C. STUDY DEFICIENCIES:

Data were reported as percent of applied radioactiv ity recovered and not as actual amounts of active ingredient by weight. Details of dose solution pre paration were not given in the report. These omissi ons did not appear to affect the results of this in vitro dermal penetration study using membranes prepared from rat and human skin.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 399 April 2008

B.6.9.9.3 In vitro dermal penetration study–rat/human

CITATION : Capt A. (2006) [ 14 C]-BYI 08330 in SC240 formulation: Comparative in vitro dermal absorption study using human and rat skin. Bayer Cr opScience, 355 rue Dostoievski, BP. 153, 06903 Sophia Antipolis, France. Study No. SA 05255. April 05, 2006. Unpublished.

I. MATERIALS AND METHODS:

A. MATERIALS:
A. MATERIALS : 1. Test material : [ 14 C]-BYI 08330 Description: White powder dissolved in distilled water
Lot/batch #: Reference synthesis KML 2978-2, BECH1855 Purity: 99% a.i. Compound stability: Not available
CAS # for TGAI: Not available
Structure:

Vehicle/Solvent used: Distilled water

Radiolabelling: Indicated in structure by asterisk Specific Activity: 3.67 MBq/mg (99.1 μCi/mg)
Radiochemical Purity: Chemical Purity >99%
>99%

Source: Bayer CropScience AG, Product Technology, Isotope C hemistry, Wuppertal, Germany
Other comments: None

2. Relevance of test material to proposed formulati on(s ): Not relevant

3. Skin membrane source : Species: Rat/Human
Strain: Rat: Wistar Rj: EWI (IOPS HAN) Human: NA Age/weight at study initiation: Rat: 6-8 wks Human: 38-66 yrs
Source: Housing (rat):

Diet (rat): Water (rat): Rat: R. Janvier (France) Human: Tissue Bank (Bio predic, Rennes, France) Together (11 in wire-mesh bottomed, stainless stee l cage during the acclimation period) Pelleted; A04 C-10 . SAFE. Rte de St Bros. Aigu, Fr ance; ad– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 400 April 2008

Environmental conditions (rat):

Acclimation period (rat): libitum Filtered municipal tap; ad libitum Temperature: 20-24 C Humidity: 40-70% Air changes: 10-15/hr Photoperiod: 12 hrs dark/12hrs light ≥ 5 days

B. STUDY DESIGN:

1. Dose :

Rationale : Dose selection was based on direct exposure to for mulation concentrate (240 mg/mL) or anticipated dermal deposition of field spray diluti ons (1.5 or 0.05 mg/mL).

Nominal doses : 240, 1.5 or 0.05 mg a.i./mL dosing solution.

Actual doses : The actual concentration of [ 14 C]-BYI 08330 in the dosing solutions were 2.68, 0.0 088, or 0.0005 mg/10 μL dose (see Table 6-144 for detail s). Actual doses were calculated as the mean dose applied to each of 6 dermal membranes/dose rate/exp osure period (8 hours) for both rat and human skin by weight as verified by radiochemical analysis of the three dosing solutions (high, medium, and low) as close to the time of application as practicable.

Dose volume : 10 μ L/cm 2 skin (2.54 cm 2 /membrane)

Duration of exposures (time from dose to terminatio n ): 24 hours with skin wash after 8 hrs exposure.

Termination periods (24 hrs): A reservoir was placed atop each membrane for rece ptor fluid which was heated to 32 C using a water heated manifold. The lower reservoir of the diffusion chambers were connected to a fraction collector via effluent tubi ng. Fractions were collected hourly following dosi ng and collected until termination at 24 hrs. The recepto r fluid chamber contents were stirred continuously and pumped at a rate of 1.5 mL/hr. At termination the diffusion sells were dismantled and subjected to ma ss balance procedures.

Number of membranes /group : Six membranes (target number before exclusion of da maged membranes) were tested in individual diffusion cell s/dose group/exposure period. The actual numbers used are given in the data tables (Tables 6-145, 6- 146, and 6-147).

2. Membrane preparation : Following acclimation each animal was sacrificed by cervical dislocation and a dorsal area of skin was clipped and excised for mem brane preparation. The skin was dermatomed using a mini-dermatome (Decadermatone microsystem motor, Thackray Surgery, Leeds, UK). Dermal membranes 420-510 μM thick were prepared using this apparatus. Human skin membranes from 13 donors were prepared similarly using the mini-derma tome. Prior to use, the integrity of the selected membranes was assessed by measuring the trans-epide rmal water loss (TEWL) from the stratum corneum. The membranes were assembled in diffusion cells (Franz cell modified, Gallas, France) and an evaporimeter probe (Dermalab, Cortex Technology, Hadsund, Denmark) was placed atop the receptor cell and water diffusion through the skin was measured (g/m 2 /hr). Human and rat skin with a TEWL of >40g/m 2 /hr were considered potentially damaged and were no t used.

3. Dose preparation, application to membranes and q uantification :– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 401 April 2008

Preparation : Dosing suspensions (three formulations) were prep ared in the Monheim Research and Development Centre of Bayer CropScience, Germany, a nd details of preparation were not given in the study report. On the day of dosing or as close as practicable, the high, medium, and low dose formulations were tested for solubility in the rece ptor fluid and found to be soluble for 24 hrs at an amount in 3 mL of receptor fluid (Eagle’s medium su pplemented with 5% bovine serum albumin and 50 mg/L gentamicin) which exceeded the maximum volume of the diffusion cells. The amount tested exceeded the total quantity of { 14 C]-BYI 08330 applied to the membranes of any cell. Doses were checked for radioactivity before during and after d osing. Homogeneity was checked by taking five aliquots of each dose formulation before applicatio n. The formulations were considered to be homogeneous.

Assembly of diffusion cells and application of dosi ng solutions : Glass flow-through diffusion cells (Franz cell modified, Gallas, France) were used wit h an exposed membrane area of 2.54 cm 2 . A cell consisted of a donor and a receptor chamber between which the skin was positioned. The upper chamber with receptor fluid (Eagle’s medium with 5% bovine serum albumin and gentamicin at 50 mg/L, pH 7.4-7.5), in contact with the skin, was warmed t o 32 C. The lower chamber contents were stirred continuously with a magnetic stirring bar and pumpe d to a fraction collector at a flow rate of 1.5 mL/ hr. The dosing solutions (high, medium, or low dose) we re applied to the split-thickness skin samples with a pipette at 10 μL/cm 2 skin surface. Each dosing solution was assayed fo r radioactivity by LSC using dose checks before, during, and after dosing.

Quantification : Radioactivity (dpm) and radiochemical purity were determined before application of the dosing solutions to membranes. Radiochemical purit y was determined by TLC and HPLC analysis of the dosing preparations. Homogeneity of the doses was d etermined from radiochemical analysis of dosesize aliquots of the dosing solutions taken before, during, and after dosing. Data are summarized in Table 6-144.

Table 6-144: Dose levels achieved

Dose Application rate

(μL/cm 2 ) Actual [ 14 C]-BYI
08330
concentration (mg/mL) Applied dose (μg

[ 14 C]-BYI 08330 /cell) Applied dose

(kBq/cell)

High 10 240 2682 55.8
Medium 10 15.0 8.75 32.3
Low 10 0.05 0.5 1.75 Data taken from Appendix Table s C4-C6, pp. 48-51; Study No. SA 05255. Means in c olumns 4 and 5 calculated by study reviewer.

4. Sample collection and preparation : Diffusion cell contents were sampled by collection of cell eluants hourly using a fraction collector set to cycle each hour at a flow rate of 1.5 mL/hr. Cells were expos ed to the high, medium, or low doses of [ 14 C]-BYI 08330 for 8 hrs and washed by repeated swabb ing after exposure to remove residual radioactivity. Tween 6 0 (1%) in phosphate buffered saline was used as the swabbing agent. Swabbing was continued until no ra dioactivity could be detected on the sponges using a Geiger counter. Sampling of receptor fluid conti nued hourly until termination at 24 hrs. Membranes were swabbed again at termination and all swabs wer e saved for radiochemical analysis by LSC. At termination, membranes were tape-stripped to remove the stratum corneum. Tape stripping was repeated until the epidermis was shiny, indicating stratum corneum removal was complete. The tape strips were saved for analysis and the epidermal me mbranes were removed and saved for analysis by– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 402 April 2008

LSC. Receptor fluid at termination was removed fro m cells for analysis and the diffusion cell components were washed and the washings saved for a nalysis for mass balance. Samples were stored at -20 C until analyzed.

5. Sample analysis : Material balance samples, receptor fluid samples, a nd aliquots of dosing solutions were analyzed using Liquid Scintillation Counting ( LSC) techniques. Receptor fluid samples were analyzed directly by addition to scintillant for co unting. Skin swabs were solubilized in Soluene and scintillation fluid added for LSC analysis. Tape-s trips were solubilized using tetrahydrofuran and scintillation fluid was added for direct LSC analys is. Skin samples were solubilized in soluene, scintillation fluid was added, and samples were cou nted by LSC. Diffusion cell components, including stirrers and tubing, were soaked in acetonitrile/di stilled water (50:50 v/v) for 12 hrs and the washin gs analyzed by LSC. All samples were counted using a Packard 1900 TR Liquid Scintillation Spectrometer with on-line computing. Quenching effects were det ermined using external standardization and spectral quench parameter (tSIE) methods. Efficiency correl ation curves were prepared for each scintillation cocktail using radiolabeled n-hexadecane internal s tandards. The limit of detection was considered to be twice the background values for blank samples in sc intillation cocktail. The calculation of percent recovered dose, means and standard deviations were accomplished with a personal computer using Debra 5.2a software and Microsoft Excel.

II. RESULTS:

C. MATERIAL BALANCE AND RADIOLABEL DISTRIBUTION :

The material balance for dermal penetration of [ 14 C]-BYI 08330 through rat and human epidermis at the high, medium, and low dose rates are shown in Table s 6-145, 6-146, and 6-147, respectively. Recovery of radioactive material was excellent with means ra nging from 92.88-99.46% of the applied dose for rat s and 92.08-99.46% for humans. For the formulation c oncentrate, skin washing removed the majority of the applied dose from the skin surface after 8 hrs exposure and at termination (24 hrs). For rat skin , the washings removed 87.97, 78.49, and 74.33% at the hi gh, medium, and low dose levels, respectively. Washing removed 91.37, 91.62, and 79.59% of the hig h, medium, and low doses, respectively, applied to human skin. The residual radioactivity remainin g with the epidermis for rat skin after 24 hrs. was 0.07, 0.95, and 2.60% for the high, medium, and low dose, respectively; for human skin, 0.20, 0.47, an d 3.41% of the high, medium, and low doses remained w ith the epidermis after 24 hrs. In addition, a portion of the applied doses remained in the stratu m corneum of both species. For the rat, 2.54, 9.33

, and 6.73% of the respective high, medium and low do ses remained with the stratum corneum. For human skin, 0.11, 0.87, and 7.82% of the added radi oactivity remained in the stratum corneum for the high, medium, and low doses, respectively. At term ination, <0.005% of the three applied doses remained in the diffusion chambers for the human an d <0.005, 0.16, and <0.005% of the respective high, medium, and low doses remained for rats.

The portion of the applied dose which was absorbed by (penetrated into receptor fluid) rat and human skin is discussed in the following section.– –

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Table 6-145: Material balance and in vitro absorpti on of [ 14 C]-BYI 08330 through human and rat epidermis expressed as percentage of the applied do se (high dose = 2400 μg/membrane)

Parameter Human skin (n=5) Rat skin (n=4) Surface dose (non-absorbed) 0.25±0.22 1.84±0.58 Skin swabs (8 hr) 91.28±1.30 87.69±1.99 Skin swabs (24 hr) 0.09±0.05 0.28±0.19 Skin swabs (8 + 24 hr) 91.37±1.33 87.97±2.13
Residual dose in chamber 0.05±0.04 0.18±0.24
Total % non-absorbed 91.67±1.13 89.98±1.75
Skin 0.20±0.28 0.07±0.07
Stratum corneum 0.11±0.13 2.54±1.09
Total % dose site 0.31±0.41 2.61±1.16 Receptor fluid (0-24hr) 0.08±0.13 0.29±0.45 Receptor fluid terminal 0.02±0.05 <0.005±<0.005 Receptor chamber <0.005±<0.005 <0.005±<0.005 Total % directly absorbed 0.10±0.14 0.29±0.45 Total % potentially absorbable 0.41±0.39 2.90±1.60 Total % recovery (material balance) 92.08±0.80 92.88±1.12

Data taken from Table 1, page 24; Study No. SA 05255

Table 6-146: Material balance and in vitro absorpti on of [ 14 C]-BYI 08330 through human and rat epidermis expressed as percentage of the applied do se (medium dose = 15 μg/membrane)

Parameter Human skin (n=5) Rat skin (n=6) Surface dose (non-absorbed) 1.60±3.39 9.06±12.25 Skin swabs (8 hr) 90.46±8.06 71.75±26.92 Skin swabs (24 hr) 1.16±2.41 6.74±8.82 Skin swabs (8 + 24 hr) 91.62±6.28 78.49±19.44
Residual dose in chamber 0.11±0.17 0.15±0.14
Total % non-absorbed 93.33±4.77 87.70±17.65
Skin 0.47±0.42 0.95±0.84
Stratum corneum 0.87±1.43 9.33±18.54
Total % dose site 1.34±1.79 10.27±18.24 Receptor fluid (0-24hr) 0.07±0.04 1.07±0.99 Receptor fluid terminal <0.005±<0.005 0.26±0.51 Receptor chamber <0.005±<0.005 0.16±0.31 Total % directly absorbed 0.07±0.04 1.49±0.84 Total % potentially absorbable 1.41±1.77 11.76±17.9 6 Total % recovery (material balance) 94.73±4.70 99.46±7.34

Data taken from Table 2, page 26; Study No. SA 05255– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 404 April 2008

Table 6-147: Material balance and in vitro absorpti on of [14C]-BYI 08330 through human and rat epidermis expressed as percentage of the applied do se (low dose = 0.5 μg/membrane)

Parameter Human skin (n=6) Rat skin (n=4) Surface dose (non-absorbed) 3.6±1.64 3.22±1.66 Skin swabs (8 hr) 78.67±2.00 73.57±4.08 Skin swabs (24 hr) 0.92±0.53 0.76±0.25 Skin swabs (8 + 24 hr) 79.59±2.27 74.33±4.24
Residual dose in chamber 0.20±0.27 0.29±0.58
Total % non-absorbed 83.43±1.44 77.83±3.45
Skin 3.41±1.30 2.60±1.05
Stratum corneum 7.82±2.19 6.73±2.18
Total % dose site 11.23±1.89 9.33±1.47 Receptor fluid (0-24hr) 0.24±0.17 6.70±3.27 Receptor fluid terminal <0.005±<0.005 0.29±0.05 0.05Receptor chamber <0.005±<0.005 <0.005±<0.005 Total % directly absorbed 0.24±0.17 6.99±3.29 Total % potentially absorbable 11.47±1.93 16.32±3.2 9 Total % recovery (material balance) 94.90±1.68 94.15±2.18

Data taken from Table 3, page 28 Study No. SA 05255

D. TOTAL ABSORBED DOSE :

Absorbed dose data for the high, medium, and low do ses, applied to rat and human skin membranes, are given in Tables 6-145, 6-146, and 6-147, respec tively. The absorbed dose is expressed as the percentage of [C 14 ]-BYI 08330 recovered in the receptor fluid during the 24 hour flow-through period, the amount in the receptor fluid at termination and the residual radioactivity retained in the receptor ch amber at termination (24 hrs). This amounted to 0.01% an d 0.29% of the applied high dose in the human and rat skin, respectively. For the medium dose, the m ean total amount of radioactivity directly absorbed was 0.07% and 1.49% for human and rat skin, respect ively. For the low dose, the mean total radioactivity directly absorbed was 0.24% and 6.99% for the human and rat skin, respectively.

The study authors considered the potentially absorb able portion of the administered dose to be that found in the skin compartment excluding surface res idual (mean percent directly absorbed + mean percent at the dose site). For the high dose 0.41% and 2.80% of the dose in the human and rat skin was considered potentially absorbable. For the med ium dosage, 1.41% and 11.76% of the dose to human and rat skin, respectively, was considered po tentially absorbable. For the low dose, 11.47% and 16.32% percent of the administered dose was conside red potentially absorbable in the human and rat skin, respectively.

Given the uncertainty regarding actual deposition u nder actual field conditions, it is considered appropriate to derive an estimate of dermal absorpt ion based on the results from the low dose group (0.05 mg a.i./mL dosing solution) since percent der mal absorption was greatest at this dose level. Th is value was not reported by the study authors as the amount by weight per unit area of skin, but was presented as the percentage of absorbed radiolabel/ dosed area of membrane (1 cm 2 ). The radioactivity present in the stratum corneum of the human skin at termination was considered by the study authors not to be available for further absorption but to b e labile to loss by desquamation.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 405 April 2008

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS :

This study (No. SA 05255) was carried out to compar e the in vitro dermal penetration of BYI 08330 following single dermal application of [ 14 C]-BYI 08330 to excised human skin and rat dermatom ed skin. Dermal absorption was determined at three dose conc entrations of an OD240 formulation, i.e., neat (240 mg/mL) and two spray dilutions at 1.5 and 0.05 mg/mL referred to as the high, medium, and low doses. The three formulations were applied to epid ermal membranes at a rate of 10μL/cm 2 . The membranes were held between chambers of flow throug h diffusion cells, and receptor fluid was passed through the membranes at 1.5 mL/hr flow rate and co llected from the effluent tubing using a fraction collector. After 8 hours exposure, the residual doses were was hed off the skin and hourly sampling was continued to termination at 24 hours. At termi nation, the skin membranes were tape-stripped to remove residual surface dose and the stratum corneu m.

The percentage of [ 14 C]-BYI 08330 directly absorbed over the 24 hr obse rvation period was 0.1 and 0.29% of the high dose, 0.07, and 1.49% of the medi um dose and 0.24 and 6.99% of the low dose for human and rat skin, respectively.

The percentage of [ 14 C]-BYI 08330 considered potentially absorbable over the 24 hr observation period was 0.41 and 2.90% for the high dose, 1.41 a nd 11.76% for the medium dose and 11.47 and 16.32% for the low dose for human and rat skin, res pectively. The absorption factor differences between the two species were 7.1 for the high dose, 8.3 for the medium dose and 1.4 for the low dose.

B. REVIEWER COMMENTS :

This study was carried out to determine the compara tive in vitro percutaneous absorption of BYI 08330 through rat and human epidermis following exposure to a [ 14 C]-BYI 08330 formulation concentrate or two simulated aqueous spray dilutions of the OD240 formulation. The neat formulation was included to assess exposure to mixer/loaders and the latter dil utions to simulate exposure of spray applicators in the field. The exposure period was 8 hours with ob servation to termination at 24 hrs. The experiment s were carried out using diffusion chambers with rat or human epidermal membranes prepared from skin obtained by excising clipped skin from rats or from human skin obtained from a tissue bank. The doses of formulation concentrate or spray dilutions were applied to the intact rat or human epidermal membranes and allowed to diffuse into receptor flui d contained in the diffusion cells. Effluents were pumped to a fraction collector at a flow rate of 1. 5 mL/hr. Collection tubes in the fraction collecto r were cycled every hour. Total radioactivity recovered f rom the chambers, skin wash, epidermal membranes, and receptor fluids was acceptable, ranging between 92.08-92.88% for the formulation concentrate (high dose) and 94.73-99.46% and 94.15-94.90% for t he aqueous dilutions of the formulation concentrate (medium and low doses, respectively). The amount of radioactivity directly absorbed ranged between 0.10-0.29%, 0.07-1.49%, and 0.24-6.9 9% of the applied dose for the high, medium, and low doses, respectively. The potentially absor bable radioactivity ranged between 0.41-2.90%, 1.41-11.76%, and 11.47-16.32% of the applied radioa ctivity for the high, medium, and low doses, respectively. The absorption data for the formula tion concentrate and simulated spray dilutions indicated that BYI 08330 was poorly absorbed throug h rat and human skin in vitro. Rat skin appeared to be more permeable than human skin. The potentia l absorption differences for rat and human skin differed by factors of 7.1, 8.3, and 1.4 for the hi gh, medium and low doses, respectively.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 406 April 2008

All values reported in this study were expressed as percentage of applied dose (radioactivity) and not as actual amounts recovered as expressed by weight of active ingredient per unit area of exposed skin (μg/cm 2 ). Although this omission does not affect the outc ome of the experiments, data interpretation would have been more meaningful had the actual abso rbed or potentially absorbable amounts of BYI 08330 been calculated and reported.

C. STUDY DEFICIENCIES:

Data were reported as percent of applied radioactiv ity recovered and not as actual amounts of active ingredient by weight. Details of dose solution pre paration were not given in the report. These omissions did not appear to affect the results of t his in vitro dermal penetration study using membranes prepared from rat and human skin.– –

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B.6.9.10 Summary of dermal absorption studies

The extent of dermal absorption of spirotetramat wa s investigated in vivo in rats and in vitro on huma n and rat skin. In the in vivo study, male Wistar rats w ere administered nominal doses of 100, 15, or 5 μg a.i./cm 2 of radiolabeled spirotetramat (OD150 formulation) t o approximately 12 cm 2 of body surface for periods of 1, 10, or 24 hours and monitored up to 168 hr post-dos ing. Based on the likely worker exposure time fram e and the increased absorption of spirotetramat from the site of application following exposure, it was considered most appropriate to adopt the dermal abs orption value calculated for the groups of animals for which the skin site was washed after 10 hours, and which were sacrificed after 168 hrs. Mean dermal absorption for these groups of animals was 10.57, 4 .71, and 5.81% for the high, medium, and low dose groups respectively. This value includes the poten tially absorbable radioactivity present in the stra tum corneum and the skin at the application site (2.87, 1.72, and 1.82% for the high, medium, and low dose groups, respectively). These values were included, because at every dose the amount of radioactivity at the dose site (including stratum corneum) decreased dur ing the 168-hr time period while the amount of radioactivity in excreta increased, thereby implyin g that the compound continued to be absorbed after dosing was stopped. Dermal absorption values calculated f rom the 10hr exposure/168 hr post-exposure scenario

, rather than from the 10 hr exposure scenario alone, were chosen for risk assessment, because the forme r scenario contained measures from excreta, which pro vided a more accurate estimate of actual and potent ial dermal absorption following a 10-hr worker exposure

.

In vitro dermal absorption data are available from two studies performed with the OD 150 and SC 240 formulations. In the in vitro dermal absorption st udy with the OD 150 formulation, diffusion cells wi th epidermal membranes derived from rat or human skin were administered doses of 1.58, 0.013 or 0.0004 mg of [ 14 C]-BYI 08330 to 2.54cm 2 of epidermal surface and exposed for 8 hours, wash ed and monitored to termination at 24 hours post-dosing. The chosen do ses were those from formulation concentrate and two simulated aqueous spray dilutions of the OD150 form ulation. Rat skin was more permeable to the treatments, and the potential absorption factor dif ferences between the two species were 8.9 for the h igh dose, 8.5 for the medium dose, and 2.7 for the low dose.

In the in vitro dermal absorption study with the SC 240 formulation, diffusion cells with epidermal me mbranes derived from rat or human skin were administered do ses of 2.68, 0.088 or 0.0005 mg of [ 14 C]-BYI 08330 to
2.54cm 2 of epidermal surface and exposed for 8 hours, wash ed, and monitored to termination at 24 hours post-dosing. Rat skin was more permeable to the tr eatments, and the potential absorption factor diffe rences between the two species were 7.1 for the high dose, 8.3 for the medium dose, and 1.4 for the low dose.

In the US and Canada, in vitro data are not conside red currently in the calculation of in vivo human d ermal absorption from in vivo rat dermal absorption data. Therefore, the proposed dermal absorption for the OD 150 formulation in the US is based exclusively on t he in vivo study. The proposed dermal absorption v alue for spirotetramat is 10%, and this is based on the actual and potential absorption measured 168 hrs fo llowing a high-dose 10-hour exposure.

The results of the in vivo study performed with the OD 150 formulation (using an exposure time of 10 h ours and a period of 168 hours post application) togethe r with the results of the in vitro study performed with the OD 150 formulation are used to determine the dermal absorption to be used for risk assessment for the OD 150 formulation for the EU. The in vitro studies d emonstrate that no significant difference in dermal absorption is detected between the OD 150 and SC 24 0 formulations. The same might be expected for the in vivo results. Therefore, the results of the in viv o study performed with the OD 150 formulation toget her with the results of the in vitro study performed with th e SC 240 formulation are used to develop a proposal for dermal absorption of BYI 08330 in the SC 240 formul ation for the EU.– –

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B.6.10 Summary of mammalian toxicity and overall ev aluation (OECD Annex IIA 5.11)

The acute, short-term, and long-term toxicity of sp irotetramat is well understood. Spirotetramat tech nical demonstrated moderate to low acute toxicity (Toxici ty Category III or IV) via the oral, dermal, and in halation routes. Spirotetramat is non-irritating to the ski n (Toxicity Category IV), although it is an irritan t to the eyes (Toxicity Category II) and exhibits a skin-sensitiz ation potential in animals and humans. The thyroid and thymus glands were target organs in oral subchronic toxicity studies in the dog; whereas, the testesepididymides were the target organs following subch ronic oral treatment of rats. Long-term toxicity s tudies reflected the short-term toxicological profile of s pirotetramat with the thymus, thyroid, and brain as target organs following one-year oral exposure of dogs. C hronic exposure of rats to spirotetramat also refle cted the subchronic pattern of testicular toxicity. No evidence of tumor formation was found following lon g-term studies of rodents, and spirotetramat was also nega tive for mutagenicity and clastogenicity in several standard in vivo

and in vitro

assays.

The reproductive and developmental toxicity potenti al of spirotetramat was tested in rats and rabbits. In addition to testicular histopathology observed foll owing subchronic and chronic exposure of rats to spirotetramat, male reproductive toxicity was recor ded in the 2-generation reproductive toxicity study

. However, development of the sexual organs of offspr ing (balano-preputial separation, vaginal opening) was unaffected. In an investigative study designed to explore the time of onset of testicular toxicity in rats, decreased epididymal sperm counts were noted after 10 days of exposure. Therefore, repeated dosing wi th spirotetramat is necessary to produce male reproduc tive toxicity in rats. Similar effects were observ ed after repeated dosing with the enol metabolite of spirote tramat. Developmental toxicity was not observed wi th spirotetramat in the absence of maternal toxicity i n either the rat or rabbit.

Summary of mammalian toxicity and overall evaluatio n by AGES

ADME-behaviour The disposition of spirotetramat was investigated i n three in vivo and one in vitro studies. Gastroint estinal absorption of spirotetramat was rapid following ora l administration to male and female rats at a singl e dose of 2 or 100 mg/kg bw or repeated doses of 2 mg/kg b w for 14 days. The absorption rate in all tests wa s between 89 and 98% of the total recovered radioacti vity, based upon recovery from the urine and carcas s (without the gastrointestinal tract). No significa nt differences in the absorption rate were observed among any of the dose groups. The absorption rate in the single low dose test was 95% for male rats and 96% for female rats. The maximum of plasma concentration w as reached for all dose groups within 0.09 to 2.03 hours after administration (values calculated by ph armacokinetic modelling). The radioactivity concent rations in plasma declined steadily by several orders of ma gnitude within 48 hours for all dose groups. Radioa ctivity in tissues and organs at the 48-hour termination we re very low (<0.2% and below the limit of detection for some organs/tissues). Urinary excretion was very r apid (essentially complete in 24 hrs) and was the m ajor excretion route at these doses. Faecal excretion ac counted for 2-11% of the dose in male and female ra ts. The excretion behavior was similar for all dose gro ups.

Spirotetramat (BYI 08330) was completely metabolize d by the rat in this study; no parent compound was detected in the excreta. Identified metabolites acc ounted for 87-95% of the administered dose. Only v ery minor metabolites (<0.7% of the dose) were not iden tified. The main metabolic reaction was cleavage o f the ester group which resulted in the formation of the primary and most predominant metabolite BYI 08330-e nol (53-87% of the administered dose). All other identi fied metabolites could be derived from the enol intermediate. The second prominent metabolic transf ormation was oxidative demethylation of the 8-metho xy group to BYI 08330-desmethyl-enol (5-37% of the adm inistered dose). Oxidation of the azaspiro moiety t o BYI 08330-ketohydroxy and BYI 08330-desmethyl-ketoh ydroxy were detected as minor pathways. Other– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 409 April 2008

minor metabolic transformations were conjugation of the enol with glucuronic acid to BYI 08330-enol-GA and oxidation of the aromatic methyl group of the enol metabolite to BYI 08330-enol-alcohol. A sex-relate d difference was observed in metabolism in this study with male rats showing much higher rates of demethylation to BYI 08330-desmethyl-enol compared to female rats. Similar results were obtained when male and female rats were administered a single dos e of 3 mg/kg bw in another disposition study using quantitative whole body autoradiography. The highe st equivalent concentrations were observed in the l iver, kidney, and blood in that study.

Absorption in male rats administered a single dose of 1000 mg/kg bw spirotetramat was much lower with only 27% of the dose excreted in the urine after 24 hours. Excretion was also distinctively less than for lower-dose animals, and radioactivity in plasma was slightly higher than in liver and kidney. These fi ndings were consistent with saturation of cellular transpo rt mechanisms. In addition, decline of tissue radi oactivity was minimal from 1 h to 8 h post dose and increased only slightly at 24 hours. The metabolism profile was qualitatively similar to that of the lower doses; h owever, BYI 08330-desmethyl-enol occurred at lower levels at 1000 mg/kg bw, and this may be a reflection of t he decreased absorption at this dose. Similar to t he low dose groups, BYI 08330-desmethyl-enol levels were g reater in urine than in plasma and organs. The high est percentage of BYI 08330-desmethyl-enol was detected in liver and kidney. Results of this study indica te that saturation of active transport mechanisms occurs at a dose of 1000 mg/kg but not 2 mg/kg. This result s in decreased excretion via urine and faeces and a pote ntial for accumulation of BYI 08330 related residue s in the body following repeated high doses. The result s of pharmacologically based pharmacokinetic (PBPK) simulations supported this conclusion and suggested that repeated daily doses of ≥ 300 mg spirotetramat/kg bw lead to non-linear elimination kinetics, resulti ng in a high body burden in multiple-dose toxicolog ical studies. This assumes, however, that spirotetramat enters the systemic circulation as the metabolite BYI 08330-enol.

In a comparative in vitro metabolism study using he patocytes from male rats, mice, and humans, differe nces in the proportions of several down-stream metabolit es were observed; however, BYI 08330-enol was the f irst and most prominent metabolite and accounted for 66- 100% of all metabolites across species.

Acute toxicity Spirotetramat technical demonstrated moderate to lo w acute toxicity via the oral (LD50 > 2,000 mg/kg b w), dermal (LD50 > 2,000 mg/kg bw), and inhalation (LC5 0 > 4.183 mg/L air) routes. Spirotetramat is nonirritating to the skin, although it is an irritant to the eyes and exhibits a skin sensitization poten tial under the conditions of the guinea pig maximization test and the local lymph node assay, but not the Buehler pat ch
test.

Table B.6-135: Summarised results of acute toxicity studies with Spirotetramat Type of Study Species Vehicle Results Reference Acute Oral rat Carboxymethylcellulose sodium salt solution LD 50 > 2000mg/kg bw Eigenberg D.A. (2004) Acute Dermal rat Carboxymethylcellulose sodium salt solution LD 50 > 2000mg/kg bw Eigenberg D.A. (2004)
Acute Inhalation (solid aerosol) rat None LC 50 > 4,183 mg/l/4h Pauluhn J. (2002)
Skin Irritation rabbit Water Not irritant Leuschner J. (2002) Eye Irritation rabbit test substance was used as supplied Irritant Leuschner J. (2002)
Skin sensitisation (maximization test) Guinea pig Propylene glycol 400 Skin sensitizing Vo hr H.W. (2002) Skin sensitisation Guinea pig Propylene glycol 400 Not a sensitiser Vohr H.W.– –

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Type of Study Species Vehicle Results Reference (Buehler test) (2004) Skin sensitisation (LLNA) Mouse dimethylformamide (DMF) Skin sensitizing Esdaile D. (2004)

Short term toxicity The insecticidal mode of action (lipid biosynthesis inhibition) was not reflected in the results of th e short-term toxicological studies in rodents and dogs. Rats, mi ce, and dogs did not exhibit changes in plasma lipi d parameters such as plasma triglycerides and plasma cholesterol.

The thyroid and thymus glands were target organs in a 90-day feeding study with spirotetramat in dogs. Statistically significant declines in circulating t hyroid hormones (T4 and/or T3) at ≥ 1200 ppm (32 mg/kg bw/day) were observed. However, correlative changes in thyroid weight, thyroid histopathology, or thyr oid stimulating hormone (TSH) were not observed in eith er sex. The NOAEL was considered to be 300 ppm, equivalent to 9 mg/kg bw/d in males and 10 mg/kg bw /d in females.

The thymus, thyroid, and brain were target organs f ollowing one-year oral exposure of dogs to spirotet ramat. Thymus involution and brain dilation with dose-rela ted severity were observed in males at ≥ 600 ppm (20 mg/kg bw/day), while axonal degeneration in the hyp othalamus was observed in one female at 1800 ppm (48 mg/kg bw/day). Brain dilation was observed in o ne female at the mid dose (600 ppm) also, however, it was not observed at the high dose (1800 ppm). Both sexes showed statistically significant compound rel ated decreases in circulating thyroid hormone T4 at 600 and 1800 ppm and males also of T3 at 1800 ppm. Reduction in thyroid follicle size, a possible indi cation of a reduced amount of colloid in the organ, was observed in 1800-ppm males (55 mg/kg bw/day) only. Correlative changes in thyroid weight or thyroid stimulating hormone (TSH) were not observed in eith er sex. Clinical signs of neurotoxicity (dehydratio n, swelling, decreased activity and reactivity, seizur es and ataxia) were observed in one male at the hig hest dose tested, a finding that was consistent with the multi-organ toxicity observed at this dose. No morphological changes in the testes were observed i n dogs at any dose. The NOAEL was considered to be 200 ppm, equivalent to 6 mg/kg bw/d in males and 5 mg/kg bw/d in females.

In rats, the testes were the target organ following subchronic oral treatment (14 weeks) at a high dos e. Abnormal spermatozoa and hypospermia in the epididy mis, decreased testicular weight, and testicular degeneration and vacuolation in males were observed after 90 days of exposure at 10000 ppm (616 mg/kg bw/d). These effects proved to be reversible in mos t animals after cessation of treatment. Other effec ts in subchronically treated rats were limited to decline s in terminal body weight in 10000 ppm male rats an d an increased incidence of accumulation of alveolar mac rophages in both sexes at 10000 ppm. Thyroid and thymus were unaffected in rats at any dose. The NOA EL was considered to be 2500 ppm, equivalent to 148 mg/kg bw/d in male and 188 mg/kg bw/d in female rat s.

Unlike the rat, no adverse effects of any kind were observed in mice tested orally up to the limit dos e. In vitro results from a comparative metabolism study using h epatocytes from male rats, mice, and humans reveale d species differences in the metabolism of spirotetra mat. Specifically, mouse hepatocytes were better ab le than rat or human liver cells to metabolize BYI 083 30-enol via glucuronidation. Potentially lower leve ls of the enol metabolite in mice in vivo may account for the lack of testicular toxicity observed in this speci es. The NOAEL was considered to be 7000 ppm, equivalent to 1305 mg/kg bw/d in males and 1515 mg/kg bw/d in females.

Subchronic exposure of rats by the dermal route yie lded no evidence of systemic toxicity when spirotet ramat was tested up to 1000 mg/kg bw/day.– –

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Table B.6-136: Summarised results of subacute/subch ronic toxicity studies with Spirotetramat Study; Reference Dose levels NOAEL Relevant effects a t LOAEL Wistar rats, 90 days oral Wahle B.S.; 2005 0, 150, 600, 2500, 10000 ppm/diet (equivalent to 0, 9, 36, 148 and 616 mg/kg bw ( ♂ ); 0, 11, 46, 188 and 752 mg/kg bw ( ♀ ) 2500 ppm (148 mg/kg bw ♂ ; 188 mg/kg bw ♀ ) -bodyweight ↓ ( ♂

, ♀ ) -bodyweight gain ↓ ( ♂

, ♀ ) -kidney weight ↓ ( ♂

, ♀ ) -testicular weight ↓ -histopathological changes in epididymidis and testis -alveolar macrophages in lungs ( ♂

, ♀ ) CD-1 mice, 90 days oral Wahle B.S.; 2005 0, 70, 350, 1700, 7000 ppm/diet (equivalent to 0, 13, 60, 300 and 1305 mg/kg bw ( ♂ ); 0, 16, 72, 389 and 1515 mg/kg bw ( ♀ ) 7000 ppm (1305 mg/kg bw ♂ ; 1515 mg/kg bw

♀ ) No toxicologically

significant changes at the highest dose tested

Beagle dogs, 90 days oral Eigenberg D. A; 2005 0, 150, 300, 1200, 2500 ppm/diet (equivalent to 0, 5, 9, 33, 81 mg/kg bw ( ♂ ); 0, 6, 10, 32, 72 mg/kg bw ( ♀ ) 300 ppm (9 mg/kg bw/d ♂ ; 10 mg/kg bw ♀ ) -T3, T4 ↓

Beagle dogs, 1 year oral Eigenberg D. A; 2006 0, 200, 600, 1800 ppm/diet (equivalent to 0, 6, 20, 55 mg/kg bw ( ♂ ); 0, 5, 19, 48 mg/kg bw ( ♀ ) 200 ppm (6 mg/kg bw/d ♂ ; 5 mg/kg bw ♀ ) -T3, T4 ↓ -brain dilation -thymus involution

Wistar rats, 28 days dermal Eigenberg D. A; 2006 0, 100, 300, 1000 mg/kg bw/d 1000 mg/kg bw/d No tox icologically significant changes at the highest dose tested

Genotoxicity Overall, assays for point mutations and chromosomal aberrations (both in vivo and in vitro) were negat ive for spirotetramat. A weak positive finding was noted in a single in vitro chromosomal aberration test, but at cytotoxic concentrations only. Negative findings in two in vivo chromosomal aberration studies and one in vivo/in vitro unscheduled DNA synthesis assay using rat hepatocytes do not suggest a genotoxic concern for spirotetramat.

Table B.6-150: Summary of genotoxicity studies Type of Study Test system Dose levels Results Refere nce In vitro studies
Bacterial reverse mutation Salmonella

typhimurium

(TA 100, TA1535, TA1537, TA98 and TA102) +/-S9 mix: 0, 16, 50,

158, 500, 1581, 5000 μg/plate Negative Herbold B. (2002)

Bacterial reverse mutation Salmonella

typhimurium

(TA 100, TA1535, TA1537, TA98 and TA102) +/-S9 mix: 0, 16, 50,

158, 500, 1581, 5000 μg/plate Negative Herbold B. (2006)

Chromosome aberration test Chinese Hamster V79 cells +S9 mix: 0, 20, 40, 80 μg/ml -S9 mix: 0, 10, 30, 50 μg/ml Weakly positive Herbold B. (2002)

Chromosome aberration test Chinese Hamster V79 cells +S9 mix: 0, 40, 60, 80,

100, 120 μg/ml -S9 mix: 0, 30, 50, 70, 90, 110 μg/ml Negative Herbold B. (2006)– –

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Type of Study Test system Dose levels Results Refere nce V79/HPRT Chinese Hamster V79 cells +S9 mix: 0, 20, 40, 60,

80, 100, 120, 140 μg/ml -S9 mix: 0, 2.5, 5, 10, 20, 40, 80 μg/ml Negative Herbold B. (2002)

In vivo studies
Mouse bone marrow micronucleus bone marrow of male mice 0, 125, 250, 500 mg/kg bw Negative Herbold B. (2002)

Mouse bone marrow chromosome aberration test bone marrow of male mice 0, 125, 250, 500 mg/kg bw Negative Honarvar N. (2003)

Unscheduled DNA synthesis – rat liver liver of male rats 0, 1000, 2000 mg/kg bw Negative Herbold B. (2003)

Long term toxicity/carcinogenicity: Chronic toxicity/carcinogenicity was tested in rats and mice following application of spirotetramat fo r one and two years, respectively. Target organs in rats were the kidney (both sexes) at mid and high doses and the liver (females) at the highest dose only. These res ults are consistent with the excretory and/or detox ification roles of the kidney and liver. Consistent with the subchronic study the lung demonstrated treatment-re lated presence of alveolar macrophages in mid and high do se males and high dose females. Abnormal sperm or spermatid degeneration in the testes and germ cell exfoliated debris in the epididymis were observed i n male rats at the high dose (7500 ppm; 414 or 373 mg/kg b w/day) following 12 (not statistically significant) or 24 months of oral exposure. In the last month of the one year chronic toxicity study, 10 rats/sex/dose were evaluated in an FOB assessment that included evaluation of motor activi ty and responses to sensory stimuli. Treatment-rela ted effects in the FOB assessments were not observed.

No adverse findings were observed in mice up to the limit dose following long-term treatment with spirotetramat.

Treatment-related increases in tumor incidence were not observed in either sex in rats or mice.

Table B.6-151: Summarised results of chronic toxici ty/cancerogenicity studies with Spirotetramat Study; Reference Dose levels NOAEL Relevant effects a t LOAEL Wistar rats,1 year oral Whale B; 2005 0, 250, 3500, 7500/12000 ( ♂ / ♀ )

ppm/diet, equivalent to 0, 13.2, 189, 414 mg/kg bw ( ♂ ) and 0, 18, 255, 890 mg/kg bw ( ♀ ) ♂ : 250 ppm (13.2 mg/kg bw) ♀ : 3500 ppm (255 mg/kg

bw) ♂ : -alveolar macrophages

in lungs ♀ :-bodyweight ↓ -bodyweight gain ↓ -relative liver weight ↑ -relative kidney weight ↑ -discoloration of lungs -alveolar macrophages in lungs -yellow/brown staining of perigenital area and tail Wistar rats, 2 year oral cancerogenicity study Whale B; 2006 0, 250, 3500, 7500/12000 ( ♂ / ♀ )

ppm/diet, equivalent to 0, 12.5, 169, 373 mg/kg bw ( ♂ ) and 0, 16.8, 229, 823 mg/kg bw ( ♀ ) ♂ / ♀ : 250 ppm (12.5/16.8 mg/kg bw) ♂ / ♀ : absolute kidney

weight ↓ , tubular dilation ♂ / ♀ : interstitial pneumonia ♂ : alveolar macrophages in lungs ♀ : discoloration of lungs ♀ :-yellow/brown staining of perigenital area and tail CD-1 mice, 18 0, 70, 1700, 7000/6000 ( ♂ / ♀ ) ♂ / ♀ : 7000/6000 ppm No toxicologically– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 413 April 2008

Study; Reference Dose levels NOAEL Relevant effects a t LOAEL months oral Whale B; 2006 ppm/diet (equivalent to 0, 10.9,

263 and 1022 mg/kg bw ( ♂ ) and 0, 13.7, 331 and 1319 mg/kg bw

( ♀ )) (1022/1319 mg/kg bw) significant changes at the highest dose tested

Reproductive/Developmental toxicity In addition to testicular histopathology observed f ollowing subchronic and chronic exposure of male ra ts to spirotetramat, evidence of male reproductive toxici ty was provided in the 2-generation reproductive to xicity study. Abnormal sperm cells were reported in F1-gen eration male rats treated with 6000 ppm (419 mg/kg bw/day) spirotetramat in the diet, and decreased re productive performance was also observed in one of these males. Similar results were obtained in the 1 -generation reproductive toxicity range-finding stu dy, in which decreased sperm motility and progression and increased abnormal sperm cells in the epididymides were observed in F1 males at ≥ 6000 ppm (320 mg/kg bw/day). The highest dose level of 10000 ppm, equivalent to 538 mg/kg bw/d, was associated with n o fertility in parental generation animals. There w ere no implantation sites noted in the females due to trea tment-related effects on sperm cells of males at th is dose level (increased numbers of abnormal sperms, reduce d epididymal sperm counts, decline in both motility and progression of epididymal sperm cells). Absolute an d relative weight of the cauda epididymis was decre ased in parental males. Histopathology showed abnormal s perm cells of minimal to moderate severity in the epididymis and the cauda epididymis.

Renal toxicity was also observed in F1 adults in th e 2-, but not 1-generation reproductive toxicity st udy. Offspring toxicity was limited to decreased body we ights in both studies, observed in F 1 and F 2 pups respectively of both sexes during lactation at 6000 ppm (320 and 419.3 mg/kg bw/day respectively). Decreased body weights were also observed in parent al animals at the same dose. The difference in dose between the two studies was attributed to increased food consumption of parental animals in the 2- generation study.

Development of the sexual organs of offspring (bala no-preputial separation, vaginal opening) was unaff ected in both studies. Developmental toxicity in the abse nce of maternal toxicity was not observed in either the rat or rabbit.

In February 2008 the notifier submitted a position paper ( High dose reproductive effects in male rats and their relevance to humans; Temerowski M., 2008 ).The main statements and conclusions from this pap er are:

• Only in rats and only at very high doses the male r eproductive system was affected (reproductive performance, sperm count, morphology and motility, weight of cauda epididymis, testicular and/or epididymal morphology). This is a high-dose phenome non with a steep dose-response curve and reversibility after cessation of exposure. The over all NOAEL for these effects in the reproductive, subchronic and chronic studies is 320 mg/kg bw/day.

• Mice are insensitive to effects on the male reprodu ctive system in the subchronic and chronic studies even at doses exceeding the limit dose of 1000 mg/k g bw/day.

• From multiple special mechanistic studies, the path omechanism and toxicokinetics in rats and the relevance for humans are well understood:
1. More than 10 days of treatment with repeated dai ly doses of 1000 mg BYI 08330 per kg bw were needed to cause spermatid degeneration in rats.
2. There was no evidence of a hormonal dysregulatio n.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 414 April 2008

3. These effects are obviously caused by disturbed metabolic support of germ cells by Sertoli cells.

4. As mentioned above, these effects are reversible after cessation of exposure.
5. These effects were caused by BYI 08330-enol, the main rat metabolite which predominates in blood ( ~ 90%;) at physiological pH in the anionic form (pKa of enol = 5.2).
6. The low toxic potency (high-dose phenomenon) and the delayed onset of effects are related to specia l toxicokinetic features: Due to the anionic state of the enol, hepatic and renal elimination of BYI 083 30- enol occur via very efficient active transport proc esses (against a concentration gradient) which can only be overwhelmed (saturated) by very high (appro x. 500 mg/kg bw/day) and repeated doses. Lower-dose (<320 mg/kg bw day) and short-term (<10 days) exposure can be efficiently handled by the active elimination.
7. Results from a comparative in vitro metabolism s tudy using hepatocytes from male rats, mice and humans revealed species differences in the proporti on of spirotetramat metabolites formed. Specifically, mouse hepatocytes were better able th an rat liver cells to metabolize BYI 08330-enol via glucuronidation. The metabolic profile generated fr om the human hepatocytes was more comparable to that of the mouse than of the rat. In both mouse and human tissue, conjugation was prevalent compared to the unexceptional oxidative transformat ion noted in rats. Results obtained from rat hepatocytes corresponded to in vivo metabolism find ings in the rat.

8. This efficient metabolic elimination in mice is the obvious reason for the absence of testicular to xicity even at doses >1000 mg/kg bw. Based on these metabo lic features, it can be expected that humans are, similar to mice, less sensitive than rats to e ffects of BYI 08330 on the male reproductive system

. 9. The rat is therefore not the relevant species fo r human risk assessment. But even if humans would b e as sensitive as rats, humans will not develop testi cular toxicity under practical conditions (>6000-fo ld margin of safety between rat testicular NOAEL (320 mg/kg bw/day) and AOEL / ADI (0.05 mg/kg bw/day)). And even high-dose accidental exposure or self-poisoning will not cause testicular toxicity in humans due to the high-dose phenomenon, the repeate d-dose prerequisite, and the recovery of testicular toxicity: Additional Note: To reach the experimental NOAEL for testicular toxicity in rats, a 70 kg person would have to ingest 22,000 mg BYI 08330 for more than 10 consecutive days.

10. It is therefore not justified to classify BYI 08330 to any category of reproductive effects.

Comment: The statement of the notifier can not be agreed to. In the 1-generation reproductive toxicity range-fin ding study, decreased sperm motility and progressio n and increased abnormal sperm cells in the epididymides were observed in F1 males at ≥ 320 – 400 mg/kg bw/day and the highest dose level of 538 mg/kg bw/d was associated with no fertility in parental gener ation animals. The NOAEL for reproductive toxicity was 70 mg/kg bw /day. So the effects on the male reproductive syste m

are no “high dose phenomenon”. The effects on testicular spermatogenesis were attr ibuted to the BYI 08330-enol, because it is the mai n metabolite in the rat. But it can not be excluded t hat other metabolites can cause the same effects. BYI 08330-enol is further metabolised by oxidation reactions to BYI 08330-desmethylenol, BYI 08330-eno lalcohol and BYI 08330-ketohydroxy. Oxidation produc ts accounted for approximately 14%. Conjugation was not detected. In the mouse, conjugation of BYI 0833 0-enol with glucuronic acid accounted for approxima tely 30%. In human hepatozytes, conjugation to BYI 08330 -enol-glucuronic acid was 6%. The in vitro conjugat ion rate is dependent on the concentration used and dec lined in mice from 30% to 9% and in humans from 6% to 2% at liver concentrations of 19 μ g/g and 190 μ g/g BYI 08330, respectively. Glucuronidation of the BYI 08330-enol in mice leads to much lower systemic lev els of free BYI 08330-enol when compared to the rat

.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 415 April 2008

The conjugation enables the mouse to utilize separa te active transport systems in the kidneys, thus av oiding a saturation of the elimination process. Thus, the utilization of different transport systems renders the mouse less sensitive to BYI 08330-mediated testicular tox icity when compared to the rat. In contrast to mice , for humans the ability to conjugate BYI 08330-enol with glucuronic acid is fivefold lower than for the mou se (dependent on the concentration, in humans 6% respe ctively 2%, in mice 30% respectively 9%). Therefore a similarity in the metabolic pathway can not be foll owed. As for humans conjugation is only 2% at high doses, it can not be assumed that humans are less sensitiv e to Spirotetramat than rats.

Due to abnormal sperm cells, decreased sperm motili ty and progression and decreased reproductive performance, spirotetramat should be classified to “category 3 of reproductive substances “ and labelled with the risk phrase “R 62 – Possible risk of impaired f ertility“ according to Annex VI of the EC Council D irective 67/548/EEC.

In the developmental toxicity study in rats, toxici ty to the offspring was observed in the presence of maternal toxicity, including decreased food consumption and body weight/gain, at 1000 mg/kg bw/day. Reduced fet al weight and increased incidences of skeletal malform ations and skeletal deviations were observed at 100 0 mg/kg bw/day. Malformations at the high dose includ ed one case of supernumerary lumbar vertebra, one case of cleft palate and one case of co-arctation o f aortic arch. One case of atrial septal defect of the heart and microphthalmia were observed in the control, lo w and high dose each, but not at the mid dose. Four cases of dysplastic forelimb bones (1.5 %) and three cases of malformed sacral vertebral arches w ith pelvic shift (1.1 %) were observed in the high dose . Historical control data of the performing laborat ory (Bayer HealthCare AG) for dysplastic forelimb bones in studies conducted in the years 1999 – 2004 show ed 26 affected animals out of 1975 animals, a percenta ge of 1.3 % [range 0.4 – 4.3% due to one study conducted in the year 2000, were 10 animals out of 232 were affected (4.3%)]. An incidence of 1.5 % in the study with spirotetramat is therefore outside the c oncurrent control (0.4%) and the historical control data (1.3%).

Statistically significantly increased incidences of sacral vertebral alterations (1.1 %) were observed at a dose level of 1000 mg/kg bw spirotetramat in comparison to the concurrent controls (0.0 %). The incidence i n historical controls in studies conducted 1999 – 200 6 showed 2 affected animals out of 6554 animals, a percentage of 0.03 % (range 0 – 0.4%).

Table B.6-137: Percentage of main skeletal observat ions in the rat developmental toxicity study with Spirotetramat (%) Dose (mg/kg/d) Observations 0 20 140 1000 % of Historical control data

Dysplasia of forelimb bones (%) 0.4 0.7 0.0 1.5** 1.3 (26 out of 1975 animals) [1999 – 2004] Altered appearance of sacral vertebral arch; pelvic shift (%) 0.0 0.0 0.0 1.1** 0.03 (2 out of 6554 animals) [1999 – 2006]

* p<0.05; **P<0.01 (Fisher’s exact test)

Statistically significantly increased incidences of wavy ribs were observed at all dose levels compare d to concurrent and historical control values . Statisti cally significantly increased numbers of fetuses wi th 14 th ribs were observed at a dose level of 1000 mg/kg/d spiro tetramat.

Table B.6-153: Percentage of observations at the ri bs Dose (mg/kg/d) Observations 0 20 140 1000 Historical control data (range)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 416 April 2008

Wavy ribs 5.5 17** 17.8** 57.5** (2.7 – 15.1) [1996 – 2001]
14 th rib 15 10.7 12.6 45.9** (0.0 – 13) [1996 – 2000]

The maternal and the developmental NOAEL for this s tudy was set at 140 mg/kg bw/d. Due to increased incidences of skeletal malformatio ns and skeletal deviations and according to Annex V I of the EC Council Directive 67/548/EEC, spirotetramat should be classified to “category 3 of reproductive substances “ and labelled with the risk phrase “R 63 – Possibl e risk of harm to the unborn child“.

In the developmental toxicity study in rabbits, off spring toxicity was not observed at any dose up to 160 mg/kg bw/day. However, maternal toxicity was observ ed at ≥ 40 mg/kg bw/day, including a dose-dependent increase in abortion and clinical signs of toxicity in affected animals (severely reduced food consump tion, body weight loss, alopecia, altered appearance of f eces, discoloured urination). Gross pathology revea led treatment-related fluid/gaseous contents in caecum, mottled gall bladder, discolouration of the liver. The maternal NOAEL was set at 10 mg/kg bw/d.

Table B.6-154: Summarised results of reproductive t oxicity/teratogenicity studies with Spirotetramat Study; Reference Dose levels NOAEL Relevant effects Multigeneration, rats Young A. D.; 2006 0, 250, 1000, 6000 ppm/diet (equivalent to 0, 17, 70 and 419 mg/kg bw ( ♂ ) and 0, 20, 82, 485 mg/kg bw ( ♀ )) Parental/reproductive/ developmental: 1000 ppm (70 mg/kg bw ♂

, 82 mg/kg bw ♀ ) parental: -body weight ↓ -body weight gain ↓ -food consumption ↓ -kidney weight ↓ -tubular dilation reproductive: -abnormal sperm cells -reproductive performance ↓ developmental: -body weight ↓ -body weight gain ↓ Developmental toxicity, rats Klaus A. M.; 2004 0, 20, 140, 1000 mg/kg bw maternal/ developmental: 140 mg/kg bw maternal: -food consumption ↓ -body weight loss developmental: -fetal weight ↓ -skeletal variations ↑ -retarded ossification ↑
-skeletal malformations ↑ Supplementary developmental toxicity, rats Klaus A. M.; 2004 0, 10, 35, 140 mg/kg bw maternal/ developmental: 140 mg/kg bw No toxicologically significant changes at the highest dose tested

Developmental toxicity, rabbits Klaus A. M.; 2004 0, 10, 40, 160 mg/kg bw maternal: 10 mg/kg bw developmental: 160 mg/kg bw maternal: -food consumption ↓ -body weight loss -abortion ↑ -clinical signs developmental: no effects

Neurotoxicity: Spirotetramat has been assessed for potential neuro toxicity in two acute neurotoxicity studies in the rat ( Gilmore, 2005

) and was shown to have no neurotoxic potential in these studies.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 417 April 2008

In the first acute neurotoxicity study, rats receiv ed single oral doses of 0, 200, 500 or 2000 mg/kg b w spirotetramat via gavage. Evidence of acute oral to xicity was observed in both sexes at all dose level s but was limited to clinical signs (urine and perianal s tain) and decreased activity in the figure-eight ma ze beginning on the day of treatment and with complete recovery by day 7.

In a follow-up study, rats received single oral dos es of 0, 50, 100 or 500 mg/kg bw spirotetramat via gavage. The findings from the first study were confirmed at a dose level of 500 mg/kg bw. No compound-related effects were observed at 50 and 100 mg/kg bw. The NOAEL for systemic effects in this study was es tablished at 100 mg/kg bw spirotetramat. Neurotoxic effects were not observed up to a limit dose of 200 0 mg/kg bw.

Further toxicological studies

Toxicity studies on metabolites Acute oral (rat) and genotoxicity (bacterial revers e mutation) testing was conducted on the following four plant metabolites of Spirotetramat (BYI 08330) exce eding 0.01 mg/kg raw foodstuff for human consumptio n or exceeding 0.05 mg/kg raw animal fodder: 1. BYI 08330-cis-ketohydroxy 2. BYI 08330-desmethyl-ketohydroxy 3. BYI 08330-mono-hydroxy 4. BYI 08330-di-hydroxy

In the ADME study with Spirotetramat ( Klempner A., 2006 ; Rep.Nr. MEF 048/04 ) these plant metabolites were not identified in the excreta of the rat or we re identified in the excreta to a minor degree only . However, in a later supplemental metabolism study in the rat ( Klempner A., 2006 ; Rep.Nr. MEF 06/328 ), BYI 08330- cis-ketohydroxy

was identified as the second prominent metabolite (beside the enol) in liver and kidneys after a single oral administration of 2 mg/kg bw an d BYI 08330-desmethyl-ketohydroxy was also detected in liver and kidneys in this study.

A supportive low dose ADME study with radiolabelled BYI 08330-cisketohydroxy in male rats ( Klempner A.,
2006 ; Rep.Nr. MEF 06/007 ) revealed a complete metabolization mainly to BYI 08330-desmethylketohydroxy followed by single and multiple oxidation steps res ulting in different mono-, di- and trioxygenated me tabolites which are probably represented by isomers of mono-h ydroxy, di-hydroxy and tri-hydroxy metabolites. Thu s, there is no toxicological concern for the BYI 08330-cis-ketohydroxy residues and BYI 08330-desmethylketohydroxy

residues found in plants because these plant metab olites were formed in relevant amounts in rats and have therefore been co-tested in the toxic ity studies with BYI 08330. With all four metabolites, the results of the acute oral study (LD50 > 2,000 mg/kg) and the bacterial reverse mutation test (negative = no evidence of mutagenici ty) were equivalent to those observed with the pare nt compound and uncritical.

Toxicity tests were not performed with the plant me tabolite BYI 08330-enol-glucoside . An additional single low dose toxicokinetic study ( Klempner A., 2006 ; Rep.Nr. MEF 06/006 ) implies that the metabolism of BYI 08330-enol-glucoside

follows the same metabolic pathways as the one of the aglycone BYI 08330-enol and the parent compound (BYI 08330).

Mechanistic studies In a mechanistic study designed to explore the time of onset of testicular toxicity of spirotetramat i n rats ( Kennel P., 2005; Rep.Nr. SA 04181 ), decreased epididymal sperm counts were recorded ≥ 10 days of– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 418 April 2008

treatment with 1000 mg/kg bw/day by gavage. Repeate d dosing, therefore, is necessary to produce male reproductive toxicity in rats.

In a second mechanistic study, male rats were treat ed by gavage with the enol metabolite of spirotetra mat for 21 days at a dose of 800 mg/kg bw/day ( Tinwell H., 2006; Rep.Nr. SA 06011 ). Spermatotoxicity, abnormal sperm, and Sertoli cell vacuolation were o bserved in the testes-epididymides of treated anima ls. Therefore, male reproductive toxicity in rats is li kely due to the enol metabolite of spirotetramat.

For the Toxicological Risk Assessment for the EU pl ease see Appendix.

B.6.10.1 Proposal for ADI

EPA and AGES: The acceptable daily intake (ADI) was determined t o be 0.05 mg/kg bw/day, based on the NOAEL of 5 mg/kg bw/day obtained in the one-year or al toxicity study in the dog and an uncertainty fac tor of 100 (10 for intra-species variability and 10 for in terspecies extrapolation).
PMRA: In addition, it was considered appropriate to reta in the PCPA factor, but that it could be reduced fr om 10-fold to 3-fold. The PCPA 3-fold factor was added to the standard 100-fold, resulting in a composite assessment factor of 300 in the calculation of the ADI. So the ADI was determined to be 0.02 mg/kg bw/ day.

B.6.10.2 Proposed ArfD

The acute reference dose (ARfD) was determined to b e 1 mg/kg bw, based on the NOAEL of 100 mg/kg bw obtained in the acute neurotoxicity study in the ra t and an uncertainty factor of 100 (10 for intra-sp ecies variability and 10 for interspecies extrapolation).

B.6.10.3 Proposal for drinking water limit

The determination of a maximum allowable concentrat ion (MAC) value in drinking water is not necessary, because according to Directive 91/414/EC only the A DI, ARfD and AOEL values have to be determined. According to Directive 98/83/EC a drinking water li mit of 0.1 μg spirotetramat/l is established.

B.6.10.4 Proposal for AOEL

The acceptable operator exposure level (AOEL) was d etermined to be 0.05 mg/kg bw/day, based on the NOAEL of 5 mg/kg bw/day obtained in the one-year or al toxicity study in the dog and an uncertainty fac tor of 100 (10 for intra-species variability and 10 for in terspecies extrapolation).– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 419 April 2008

B.6.11 Acute toxicity of the preparations (OECD Ann ex IIIA 7.1)

B.6.11.1 Acute oral toxicity (OECD Annex IIIA 7.1.1 )

CITATION: Schungel, M. (2005) BYI 08330 150 OD: Acute Toxicit y in the Rat After Oral

Administration. Project Number: T0075503, AT02161. Unpublished study prepared by Bayer Ag Inst. of Toxicology. 24 p. July 1, 200 5.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 150 OD
Article no.: 00-06424376 Description: Light brown suspension
Lot/Batch no.: 08030/0189(0152)
Content: 148.89 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -10

2. Vehicle and/or positive control: Tap water

3. Test animals Species: Rat, females
Strain: Wistar (Hsd CpPb:WU) Age: 10-12 weeks approximately Weight at dosing: 169 g – 189 g
Source: Harlan/Winkelmann GmbH, Borchen, Germany Acclimatization period: At least 5 days
Diet: Provimi Kliba 3883.0.15; Kaiseraugst, Switzerland
Water: Tap water ad libitum Housing: The animals were group caged conventionally in polycarbonate cages on low dust wood granulate bedd ing (J. Rettenmaier & Söhne, 73479 Ellwangen-Holzmühle, Germany). The cages of the animals were placed on racks, in ascending group number order. The wood granulate was randomly checked for contaminants at regular intervals and the results have been stored at the Department for Laboratory Animal Services, Bayer HealthCare AG, Wuppertal, Germany. Room temperature: 22 ± 2°C; Air humidity: 55 ± 5%; Ventilation: approx. 10 changes per hour; Light/ Dark cycle : 12 hour rhythm B. Study Design and Methods:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 420 April 2008

1. Animal assignment and treatment
Dose: 2,000 mg/kg bw Application route: Oral by gavage Application volume: 10 ml/kg bw Fasting time: For administration, food was withheld from the anim als for approximately 16 - 24 h before administration of th e test compound, and they were fed again approximately 2-4 h after administration Group size: 3 rats/dose group Post-treatment observation period: 14 days
Observations: Mortality, clinical signs, body weight, gross necro psy

In life dates: 2005-04-21 – 2005-05-11

II. Results and Discussion:

A. Mortality: Mortality was not observed at 2,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of de ath Mortality (%)

Females
2,000 1 st 0 0 3 -- -- 0

2,000 2 nd 0 0 3 -- -- 0

acute oral LD 50 **: > 5,000 mg/kg bw

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals used; ** according to t he principles of OECD Guideline 423

B. Clinical observations: No clinical signs were observed.

C. Body weight: Body weight and body-weight gain was not affected b y the treatment

D. Necropsy: No particular gross pathological changes were obser ved in animals sacrificed at the end of the study period.

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 150 OD is non-toxic to rats after acute oral administration.

B. Reviewer Comments: In an acute oral toxicity study, two groups of thre e female Wistar HsdCpb:Wu rats (age: 10-12 weeks; weight: 169-189 g ; source: Harlan/Winklemann GmbH, Borchen, Germany) were given a single oral dose of 2000 mg/kg body weight of BYI 08330 150 OD [Batch No. 08030/0189(0152); light-brown suspension ]. The test material was formulated in tap– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 421 April 2008

water and the administration volume was 10 ml/kg bo dy weight. For dosing, food was withheld from the animals for approximately 16-24 hours before do sing of the test material.

Individual body weights were recorded Days 1, 8 and 15. Animals were observed for clinical signs of toxicity and mortality several times on the day of dosing and at least once daily thereafter for 14 da ys after dosing. A gross necropsy examination was perf ormed on all animals at scheduled euthanasia.

All animals survived and gained weight during the s tudy. No clinical signs or gross pathological findings were observed.

Oral LD 50 Females > 2000 mg/kg bw

Based on no mortality at 2000 mg/kg, BYI 08330 150 OD is classified as EPA Toxicity Category III.

This acute oral study is classified as Acceptable. It does satisfy the guideline requirement for an acute oral study (OPPTS 870.1100; OECD 423) in the rat.

C. Reviewer Conclusions: All countries agree the oral LD 50 for BYI 08330 150 OD for female rats was > 2000 mg/kg.

D. Study Deficiencies: None.

CITATION: Schungel, M. (2005) BYI 08330 240 SC: Acute Toxicit y in the Rat after Oral

Administration. Project Number: AT02162, T2075523. Unpublished study prepared by Bayer Ag Inst. of Toxicology. 24 p. July 1, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 240 SC Development no.: 30-00360972 Description: White suspension
Lot/Batch no.: 07218/0073(0020)
Content: 244.48 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -08

2. Vehicle and/or positive control: Tap water

3. Test animals Species: Rat, females
Strain: Wistar (Hsd CPb:WU) Age: 10 - 12 weeks approximately Weight at dosing: 161 g - 177 g– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 422 April 2008

Source: Harlan Winkelmann GmbH, Borchen, Germany Acclimatization period: At least 5 days
Diet: Provimi Kliba 3883.0.15; Kaiseraugst, Switzerland
Water: Tap water ad libitum Housing: The animals were group caged conventionally in polycarbonate cages on low dust wood granulate bedd ing (J. Rettenmaier & Söhne, 73479 Ellwangen-Holzmühle, Germany). The cages of the animals were placed on r acks, in ascending group number order. The wood granulate wa s randomly checked for contaminants at regular interv als and the results have been stored at the Department for Laboratory Animal Services, Bayer HealthCare AG, Wuppertal, Germany. Room temperature : 22 ± 2°C; Air humidity : 55 ± 5% ; Ventilation : approx. 10 changes per hour; Light/ D ark cycle : 12 hour rhythm B. Study Design and Methods

1. Animal assignment and treatment
Dose: 2,000 mg/kg bw Application route: Oral by gavage Application volume: 10 ml/kg bw Fasting time: For administration, food was withheld from the anim als for approximately 16 - 24 h before administration of th e test compound, and they were fed again approximately 2 - 4 h after administration Group size: 3 rats/dose group Post-treatment observation period: 14 days
Observations: Mortality, clinical signs, body weight, gross necro psy

In life dates: 2005-04-27 – 2005-05-18

II. Results and Discussion:

A. Mortality: Mortality was not observed at 2,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of death Mortality (%)

Females
2,000 1 st 0 0 3 -- -- 0

2,000 2 nd 0 0 3 -- -- 0

acute oral LD 50 **: > 5,000 mg/kg bw– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 423 April 2008

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals used; ** according to t he principles of OECD Guideline 423

B. Clinical observations: No clinical signs were observed.

C. Body weight: Body weight and body-weight gain was not affected b y treatment.

D. Necropsy: No particular gross pathological changes were obser ved in animals sacrificed at the end of the study period.

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 240 SC is non-toxic to rats after acute oral administration.

B. Reviewer Comments: In an acute oral toxicity study, two groups of thre e female Wistar HsdCpb:Wu rats (age: 10-12 weeks; weight: 161-177 g; source: Harlan/Winklemann GmbH, Borchen, Germany) were given a single oral dose of 2000 mg/kg body weight of BYI 08330 240 SC [Batch No. 07218/0073(0020)]. The test material was formulated in tap water and t he administration volume was 10 ml/kg body weight.

Individual body weights were recorded Days 1, 8 and 15. Animals were observed for clinical signs of toxicity and mortality several times on the day of dosing and at least once daily thereafter for 14 da ys after dosing. A gross necropsy examination was performed on all animals at scheduled euthanasia. All animal s survived and gained weight during the study. No cl inical signs or gross pathological findings were observed.

Oral LD 50 Females > 2000 mg/kg bw

Based on the lack of mortality at 2000 mg/kg, BYI 0 8330 240 SC is classified as EPA Toxicity Category III.

This acute oral study is classified as Acceptable. It does satisfy the guideline requirement for an a cute oral study (OPPTS 870.1100; OECD 425) in the rat.

C. Reviewer Conclusions: All countries agree the oral LD 50 for BYI 08330 240 SC for female rats was > 2000 mg/kg.

D. Study Deficiencies: None.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 424 April 2008

B.6.11.2 Acute percutaneous (dermal) toxicity (OECD Annex IIIA 7.1.2)

CITATION: Schungel, M. (2005) BYI 08330 150 OD: Acute Toxicit y in the Rat After Dermal

Application. Project Number: T 1075504. Unpublished study prepared by Bayer Ag Inst. of Toxicology. 26 p. July 1, 2005.

I. Materials and Methods

A. Materials

1. Test material: BYI 08330 150 OD
Article no.: 00-06424376 Description: Light brown suspension
Lot/Batch no.: 08030/0189(0152)
Content: 148.89 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -10

2. Vehicle and/or positive control: 

### None

3. Test animals Species: Rat, males and females
Strain: Wistar (Hsd Cpb:WU) Age: 9 – 13 weeks approximately Weight at dosing: Males: 213 g – 232 g Females: 211 g – 219 g Source: Harlan/Winkelmann GmbH, Borchen, Germany Acclimation period: At least 5 days
Diet: Provimi Kliba 3883.0.15; Kaiseraugst, Switzerland
Water: Tap water ad libitum Housing: The animals were caged individually in polycarbonat e cages on low dust wood granulate bedding (J. Rettenmaier & Söhne, 73479 Ellwangen-Holzmühle, Germany). The cages of the animals were placed on racks, in ascending group number order. The wood granulate was randomly checked for contaminants at regular intervals and the results have been stored at the Department for Laboratory Animal Services, Bayer HealthCare AG, Wuppertal, Germany. Room temperature: 22 ± 2°C; Air humidity: 55 ± 5%; Ventilation: approx. 10 changes per hour; Light/Dark cycle: 12 hour rhythm. B. Study Design and Methods: 1. Animal assignment and treatment
Dose (mg/kg bw) Surface area Range of doses– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 425 April 2008

(cm2) (mg/cm2)

males females 4,000

4,000 14.0

14.0 60.9-66.3

60.3-62.6 Application route: Dermal, semi-occlusive dressing
Duration: 24 hours Group size: 5 rats/sex/group Post-treatment observation period: 14 days
Observations: Mortality, clinical signs, skin effects, body weigh t, gross
necropsy In life dates: 2005-04-21 – 2005-05-05

II. Results and discussion

A. Mortality: Mortality was not observed at 4,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of death Mortality [%]

Males
4,000 0 0 5 -- -- 0
Females
4,000 0 0 5 -- -- 0

acute dermal LD 50 : > 4,000 mg/kg bw

* 1 st number = number of dead animals:; 2 nd number = number of animals with signs;
3 rd number = number of animals in the group

B. Clinical observations: No clinical signs were observed.

C. Body weight: Body weight and body weight gain was not affected b y treatment in males. A slight decrease in body weight was observed on day 8 in tw o females and on day 15 in two other females. This effect is assumed to be caused by the stress o f the occlusive dressing, and is not considered test compound related.

D. Necropsy: No particular gross pathological changes were obser ved in animals sacrificed at the end of the study period.

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 150 OD is non-toxic a fter acute dermal administration.

B. Reviewer Comments: In an acute dermal toxicity study, five young Wista r (HsdCpb:Wu) rats/sex (age: 9-13 weeks; weight: 213-232 g; 211-219 g fema les; source: Harlan/Winklemann GmbH, Borchen, Germany) were exposed to a single dermal a pplication of BYI 08330 150 OD [Batch No. 08030/0189(0152); light-brown suspension] for 24 ho urs.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 426 April 2008

Individual body weights were recorded Days 1, 8 and 15. Animals were observed for clinical signs of toxicity and mortality several times on the day of dosing and at least once daily thereafter for 14 da ys after dosing. A gross necropsy examination was perf ormed on all animals at scheduled euthanasia.

All animals survived the study. All male animals g ained body weight during the study. A slight decreased in body weight was observed on Day 8 of t he study in two females and on Day 15 in two other female animals. No clinical signs or gross p athological findings were observed.

Dermal LD 50 Males > 4000 mg/kg bw Dermal LD 50 Females > 4000 mg/kg bw Dermal LD 50 Combined > 4000 mg/kg bw

Based on no mortality at 4000 mg/kg bw, BYI 08330 150 OD is classified as EPA Toxicity Category
III.

This acute dermal study is classified Acceptable. It does satisfy the guideline requirement for an acute dermal study (OPPTS 870.1200; OECD 402) in th e rat.

C. Reviewer Conclusions: All countries agree the dermal LD 50 for BYI 08330 150 OD for male and female rats was > 4000 mg/kg.

D. Study Deficiencies: None.

CITATION: Schungel, M. (2005) BYI 08330 240 SC: Acute Toxicit y in the Rat after Dermal

Application. Project Number: AT02165, T3075524. Unp ublished study prepared by Bayer Ag Inst. of Toxicology. 25 p. July 1, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 240 SC Development no.: 30-00360972 Description: White suspension
Lot/Batch no.: 07218/0073(0020)
Content: 244.48 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -08

2. Vehicle and/or positive control: None

3. Test animals Species: Rat, males and females
Strain: Wistar (Hsd CPb:WU) Age: 9 - 13 weeks approximately Weight at dosing: Males: 206 g - 222 g– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 427 April 2008

Females: 208 g - 225 g Source: Harlan Winkelmann GmbH, Borchen, Germany Acclimation period: At least 5 days
Diet: Provimi Kliba 3883.0.15; Kaiseraugst, Switzerland
Water: Tap water ad libitum Housing: The animals were caged individually in polycarbonat e cages on low dust wood granulate bedding (J. Rettenmaier & Söhne, 73479 Ellwangen-Holzmühle, Germany). The cages of the animals were placed on racks, in ascending group number order. The wood granulate was randomly checked for contaminants at regular intervals and the results have been stored at the Department for Laboratory Animal Services, Bayer HealthCare AG, Wuppertal, Germany. Room temperature : 22 ± 2°C; Air humidity : 55 ± 5%;Ventilation : approx. 10 changes per hour; Light/Dark cycle : 12 hour rhythm. B. Study Design and Methods:

1. Animal assignment and treatment

Dose (mg/kg bw) Surface area (cm2) Range of doses

(mg/cm2)

males females 4,000

4,000 16.0

14.0 51.5 – 55.5 59.4

– 64.3

Application route: Dermal, semi-occlusive dressing
Duration: 24 hours Group size: 5 rats/sex/group Post-treatment observation period: 14 days
Observations: Mortality, clinical signs, skin effects, body weigh t, gross
necropsy In life dates: 2005-04-27 – 2005-05-11

II. Results and Discussion:

A. Mortality: Mortality was not observed at 4,000 mg/kg bw.

Doses, mortality / animals treated

Dose (mg/kg bw) Toxicological results* Duration of signs Time of death Mortality [%]

Males
4,000 0 0 5 -- -- 0

Females
4,000 0 0 5 -- -- 0

acute dermal LD 50 : > 4,000 mg/kg bw– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 428 April 2008

* 1 st number = number of dead animals:; 2 nd number = number of animals with signs;
3 rd number = number of animals in the group

B. Clinical observations: The only local sign is a yellow discoloration of th e treatment area. This is not regarded as a toxicologically relevant sign.

C. Body weight: There were no toxicological effects on body weight or body weight development in males and females. The slight decrease in body weig ht gain of the females in the first week is assumed to be caused by the stress of the applicati on procedure, and is not considered test compound related.

D. Necropsy: No particular gross pathological changes were obser ved in animals sacrificed at the end of the study period.

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 240 SC is non-toxic after acute dermal administration.

B. Reviewer Comments: In an acute dermal toxicity study, groups of five y oung Wistar (HsdCpb:Wu) rats/sex (age: 9-13 weeks; weight: 206-222 g for m ales; 208-225 g for females; source: Harlan/Winklemann GmbH, Borchen, Germany) were expo sed to a single dermal application of BYI 08330 240 SC [Batch No. 07218/0073(0020); white sus pension] for 24 hours.

Individual body weights were recorded Days 1, 8 and 15. Animals were observed for clinical signs of toxicity and mortality several times on the day of dosing and at least once daily thereafter for 14 da ys after dosing. A gross necropsy examination was perf ormed on all animals at scheduled euthanasia.

All animals survived the study. All male animals g ained body weight during the study. A slight, transient decrease in body weight was observed at D ay 8 of the study in two females. By Day 15, four females exceeded their initial body weight and one female was back to its initial body weight at Day 1

. The only local sign was a yellow discoloration of t he treatment area. No gross pathological findings were observed.

Dermal LD 50 Males > 4000 mg/kg bw Dermal LD 50 Females > 4000 mg/kg bw Dermal LD 50 Combined > 4000 mg/kg bw

Based on no mortality at 4000 mg/kg bw, BYI 08330 2 40 SC is classified as EPA Toxicity Category III.

This acute dermal study is classified Acceptable. It does satisfy the guideline requirement for an ac ute dermal study (OPPTS 870.1200; OECD 402) in the rat.

C. Reviewer Conclusions: All countries agree the dermal LD 50 for BYI 08330 240 SC for male and female rats was > 4000 mg/kg.

D. Study Deficiencies: None.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 429 April 2008

B.6.11.3 Acute inhalation toxicity to rats (OECD An nex IIIA 7.1.3)

CITATION: Pauluhn, J. (2005) BYI 08330 150 OD: Acute Inhalati on Toxicity in Rats. Project

Number: T6075301, AT02396. Unpublished study prepar ed by Bayer Ag Inst. of Toxicology. 74 p. September 14, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 150 OD
Article no.: 00-06424376 Description: Light brown suspension
Lot/Batch no.: 08030/0189(0152)
Content: 148.89 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -10

2. Vehicle and/or positive control: None

3. Test animals Species: Rat, males and females
Strain: SPF Wistar (Hsd Cpb:WU) Age: Approximately two months Weight at dosing: Males: 168.0 g – 199.0 g Females: 168.0 g – 175.0 g Source: Harlan/Winkelmann GmbH, Borchen, Germany Acclimation period: At least 5 days
Diet: Provimi Kliba 3883 9441 pellets, Kaiseraugst, Switz erland
Water: tap water ad libitum Housing: During the acclimation and study periods, the anima ls were housed singly in conventional Makrolon® Type I II cages (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zsc hr. Versuchstierkunde, 7, 144-153 (1965)). Cages were changed twice a week while unconsumed feed and wate r bottles were changed once per week. The legal requirements for housing experimental animals (Dire ctive 86/609 EEC) were followed. Bedding consisted of typ e BK8/15 low-dust wood granulate from Ssniff, Soest/Westfalen, Germany. The wood granulate was randomly checked for harmful constituents at the re quest of the Laboratory Animal Services, Bayer Healthcare AG. B. Study Design and Methods: 1. Animal assignment and treatment– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 430 April 2008

Dose: 0 – 1,760 mg/m3 air (maximum technically attainable concentration) Application route: Inhalation (nose-only exposure)
Duration: 4 hours Group size: 5 rats/dose/sex
Post-treatment observation period: 14 days

Observations: Mortality, clinical signs, body weights, rectal tem perature, reflex measurements, gross necropsy In life dates: 2005-05-03 – 1005-05-18

2. Generation of the test atmosphere / chamber desc ription Generation and characterization of chamber atmosphe re Group 1 Group 2

Target concentration (mg/m3) Control 5,000
Actual concentration (mg/m3) -- 1,760
Chamber Volume (L) 3.8 3.8
Total Airflow (L/minute) 15 15
Temperature (mean, °C) 22.8 22.5
Relative humidity (mean, %) < 5 < 6.8*
MMAD (μm) GSD Aerosol mass < 3 μm (%) Mass recovered (mg/m3) --

--

--

-- 2.21

2.21 65.2 1,831
MMAD = Mass Median Aerodynamic Diameter, GSD = Geom etric Standard Deviation; -- not applicable. * Humidity was lower due to the use of dry air for the dispersion of the test substance. This deviati on from the guideline had no apparent negative impact on the ou tcome of the study



### .

Test atmosphere concentration: The nominal concentration was calculated from the r atio of the quantity of test article nebulized, i.e. the weight loss of nebulizer before and after each use, and the total throughput of air through the inhalation chamber. Specific information concer ning air flows and test atmosphere concentrations are provided in the table above. The lower analytical concentrations compared with the nominal concentrations are attributed to the evapor ation of constituents contained in the formulation which als o contributes to the overall mass loss. Particle size determination: The samples for the analysis of the particle-size distribution were also taken in the vicinity of the breathing zone. D uring each exposure two samples were taken. The particle-size distribution was analyzed using a BERNER-TYPE AERAS low- pressur e critical orifice cascade impactor (Hauke, Gmunden, Austria). Specifications and evaluations are provided in the Appendix. The individual impactor stages had been covered by an a luminum foil and glass fiber filter which were subjected to grav imetric analysis– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 431 April 2008

as described in section 7.7. An adhesive stage coat ing (silicone spray) was not used to prevent particle bounce and re-entrainment due to the adsorptive properties of the glass fiber filter. Gravimetric analyses were made using a digital balance.

II. Results and Discussion: A. Mortality: Mortality was not observed at 1,760 mg/m3 air.

Doses, mortality / animals treated


### Actual
concentration (mg/m3 air) Toxicological results* Duration of signs Time of death Mortalit y [%]

Males

0 0 0 5 -- -- 0
1,760 0 5 5 0 d – 3 d -- 0

Females

0 0 0 5 -- -- 0
1,760 0 5 5 0 d – 2 d -- 0

acute inhalative LC 50 

### : > 1,760 mg/m3 air

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals exposed

B. Clinical observations: The following clinical signs were observed in the t est substance groups: Males : labored breathing patterns, bradypnea, motility r educed, piloerection, hair-coat ungroomed, high-legged gait, nose: red encrustations, nasal di scharge (serous). Females : labored breathing patterns, bradypnea, piloerecti on, hair-coat ungroomed, high-legged gait, nose: red encrustations and reddened.

C. Body weight: Comparisons between the control and exposure group revealed mild, although statistically significant decreased body weights.

D. Necropsy: Animals sacrificed at the end of the observation pe riod: Macroscopic findings amongst the groups were indistinguishable.

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 150 OD is of very low toxicity after acute inhalation
to rats.

B. Reviewer Comments: In an acute inhalation toxicity study, two groups of young adult Wistar (Hsd Cpb:WU) rats (5/sex) (age: not reported; weigh t: 168-199 g males; 168-175 g females; source: Harlan-Winkelmann GmbH, Borchen, Germany) were expo sed via the inhalation (nose-only exposure) route to BYI 08330 150 OD [Batch No. 08030/0189(0152); light-bro wn suspension] for 4 hours at a concentration of 1.76 mg/L (MMAD = 2.2 μ m; GSD = 2.2). The liquid aerosol generated (undiluted test material) was respirable to rats.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 432 April 2008

Body weights were measured before the exposure peri od, on days 3 and 7, and weekly thereafter. The period of observation was for two weeks. On th e day of exposure, the test animals were observed several times for clinical signs of toxici ty and at the least once daily thereafter. A gross necropsy examination was performed on all animals a t the time of scheduled euthanasia.

All animals survived the study. One male and two f emale animals had decreased body weights on Day 3 of study. By Day 7, these animals had surpas sed their initial body weights and continued to gain weight till the end of the study. The remaini ng animals showed increased body weights at each weighing interval. Clinical signs observed in male s included the following: labored breathing patterns, bradypnea, motility reduced, piloerection , hair-coat ungroomed, high-legged gait, nose: red encrustations, nasal discharge (serous). Clinical signs for female included the following: labored breathing patterns, bradypnea, piloerection, hair-c oat ungroomed, high-legged gait, nose: red encrustations, nose: reddened. All animals were wi thout clinical signs on the fourth post-exposure day. No gross pathological findings were observed.

LC 50 Males > 1.760 mg/L LC 50 Females > 1.760 mg/L LC 50 Combined > 1.760 mg/L

Based on the lack of mortality at the LC 50 , BYI 08330 150 OD is classified as EPA Toxicity Category
III.

This acute inhalation study is classified as Accept able. It does satisfy the guideline requirement for an acute inhalation study (OPPTS 870.1300; OECD 403 ) in the rat.

C. Reviewer Conclusions: All countries agree the inhalation LC 50 for BYI 08330 150 OD for male and female rats was > 1.760 mg/L.

D. Study Deficiencies: None.

CITATION: Pauluhn, J. (2005) BYI 08330 240 SC: Acute Inhalati on Toxicity in Rats. Project

Number: AT02374, T5075300. Unpublished study prepar ed by Bayer Ag Inst. of Toxicology. 73 p. September 8, 2005.

I. Materials and Methods:

1. Test material: BYI 08330 240 SC Development no.: 30-00360972 Description: White suspension
Lot/Batch no.: 07218/0073(0020)
Content: 244.48 /l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -08

2. Vehicle and/or positive control: 

### None

3. Test animals– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 433 April 2008

Species: Rat, males and females
Strain: Wistar (Hsd Cpb:WU) Age: Approximately 2 months Weight at dosing: Males: 174.0 g - 199.0 g Females: 169.0 g - 175.0 g Source: Harlan Winkelmann GmbH, Borchen, Germany Acclimation period: At least 5 days
Diet: Provimi Kliba 3883 9441 pellets, Kaiseraugst, Switz erland
Water: Tap water ad libitum Housing: During the acclimation and study periods, the anima ls were housed singly in conventional Makrolon® Type I II cages (based on A. Spiegel and R. Gönnert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zsc hr. Versuchstierkunde, 7, 144-153 (1965)). Cages were changed twice a week while unconsumed feed and wate r bottles were changed once per week. The legal requirements for housing experimental animals (Dire ctive 86/609 EEC) were followed. Bedding consisted of typ e BK8/15 low-dust wood granulate from Ssniff, Soest/Westfalen, Germany. The wood granulate was randomly checked for harmful constituents at the re quest of the Laboratory Animal Services, Bayer Healthcare AG. B. Study Design and Methods: 1. Animal assignment and treatment
Dose: 0 – 3,013 mg/m3 air (maximum technically attainable concentration) Application route: Inhalation (nose-only exposure)
Duration: 4 hours Group size: 5 rats/dose/sex
Post-treatment observation period: 14 days

Observations: Mortality, clinical signs, body weights, rectal tem perature, reflex measurements, gross necropsy

In life dates: 2005-05-10 – 2005-05-25– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 434 April 2008

2. Generation of the test atmosphere / chamber desc ription Generation and characterization of chamber atmosphe re Group 1 Group 2

Target concentration (mg/m3) Control (water) 5,000
Actual concentration (mg/m3) -- 3,013
Chamber Volume (L) 3.8 3.8
Total Airflow (L/minute) 15 15
Temperature (mean, °C) 22.8 22.3
Relative humidity (mean, %) <5 88.7
MMAD (μm) GSD Aerosol mass < 3 μm (%) Mass recovered (mg/m3) --

--

--

-- 3.59

2.07 40.5 1,073.0
MMAD = Mass Median Aerodynamic Diameter, GSD = Geom etric Standard Deviation; -- not applicable



### .

Test atmosphere concentration: The nominal concentration was calculated from the r atio of the quantity of test article nebulized, i.e. the weight loss of nebulizer before and after each use, and the total throughput of air through the inhalation chamber. Specific information concer ning air flows and test atmosphere concentrations are provided in the table above. The lower analytical concentrations compared with the nominal concentrations are attributed to the evapor ation of constituents contained in the formulation which als o contributes to the overall mass loss. Particle size determination: The samples for the an alysis of the particle-size distribution were also taken in the vicinity of the breathing zone. D uring each exposure two samples were taken. The particle-size distribution was analyzed using a BERNER-TYPE AERAS low- pressur e critical orifice cascade impactor (Hauke, Gmunden, Austria). Specifications and evaluations are provided in the Appendix. The individual impactor stages had been covered by an a luminum foil and glass fiber filter which were subjected to grav imetric analysis as described in section 7.7. An adhesive stage coat ing (silicone spray) was not used to prevent particle bounce and re-entrainment due to the adsorptive properties of the glass fiber filter. Gravimetric analyses were made using a digital balance.

II. Results and Discussion:

A. Mortality: Mortality was not observed at 3,013 mg/m3 air.

Doses, mortality / animals treated

Actual concentration (mg/m3 air) Toxicological results* Duration of signs Time of de ath Mortality [%]

Males– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 435 April 2008

0 0 0 5 -- -- 0
3,013 0 5 5 0 d – 2 d -- 0

Females

0 0 0 5 -- -- 0
3,013 0 5 5 0 d – 3 d -- 0

acute inhalative LC 50 : > 3,013 mg/m3 air

* 1 st number = number of dead animals; 2 nd number = number of animals with signs;
3 rd number = number of animals exposed

B. Clinical observations: Group 1: All rats tolerated the exposure without sp ecific signs. Group 2/males: Piloerection, hair-coat ungroomed, b radypnea, labored breathing pattern, motility reduced, high-legged gait. Group 2/females: Piloerection, hair-coat ungroomed, labored breathing pattern, irregular breathing pattern, high-legged gait.

C. Body weight: Comparisons between the control and the exposure gr oup did not reveal toxicologically significant decreases in body weigh ts.

D. Necropsy: Animals sacrificed at the end of the observation pe riod: Macroscopic findings amongst the groups were indistinguishable.

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 240 SC is of very lo w toxicity after acute inhalation
to rats.
B. Reviewer Comments: : In an acute inhalation toxicity study, two groups of young adult Wistar (Hsd Cpb:WU) rats (5/sex) (age: not reported; weigh t: 174-199 g males; 171-175 g females; source: Harlan-Winkelmann GmbH, Borchen, Germany) were expo sed via the inhalation (nose-only exposure) route to BYI 08330 240 SC [Batch No. 0721 8/0073(0020); white suspension] for 4 hours at a concentration of 3.013 mg/L (MMAD = 3.59 μm; GSD = 2.07). The liquid aerosol generated (undiluted test material) was respirable to rats.

Body weights were measured before the exposure peri od, on days 3 and 7, and weekly thereafter. The period of observation was for two weeks. On th e day of exposure, the test animals were observed several times for clinical signs of toxici ty and at the least once daily thereafter. A gross necropsy examination was performed on all animals a t the time of scheduled euthanasia.

All animals survived and gained body weight during the study. Clinical signs observed during the study included the following: piloerection, hair-co at ungroomed, bradypnea, labored breathing pattern, irregular breathing pattern, motility redu ced and high-legged gait. All clinical signs resol ved by fourth post-exposure day. No gross pathological findings were observed.

LC 50 Males > 3.013 mg/L LC 50 Females > 3.013 mg/L LC 50 Combined > 3.013 mg/L

Based on no mortality at the limit dose, BYI 08330 240 SC is classified as EPA Toxicity Category IV.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 436 April 2008

This acute inhalation study is classified as Accept able. It does satisfy the guideline requirement for an acute inhalation study (OPPTS 870.1300; OECD 403 ) in the rat.

C. Reviewer Conclusions: All countries agree the inhalation LC 50 for BYI 08330 240 SC for male and female rats was > 3.013 mg/L.

D. Study Deficiencies: None.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 437 April 2008

B.6.11.4 Skin irritation (OECD Annex IIIA 7.1.4)

CITATION : Schungel, M. (2005) BYI 08330 150 OD: Acute Skin Ir ritation/Corrosion on Rabbits.

Project Number: T6075365, AT02359. Unpublished stud y prepared by Bayer Ag Inst. of Toxicology. 20 p. August 31, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 150 OD
Article no.: 00-06424376 Description: Light brown suspension
Lot/Batch no: 08030/0189(0152)
Content: 148.89 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -10

2. Vehicle and/or positive control: None

3. Test animals Species: Rabbit, females
Strain: Albino (Crl:KBL(NZW)BR) Age: Young adult animals Weight at dosing: 2.3 kg – 2.8 kg
Source: Charles River, Kißlegg, Germany Acclimation period: At least 5 days
Diet: Ssniff K-Z, Soest, Germany
Water: Tap water ad libitum Housing: The animals were housed individually in cage units Metall/Noryl by EBECO. Excrement trays below the ca ges contained low dust wood granulate bedding (J. Rette nmaier & Söhne, 73479 Ellwangen-Holzmühle, Germany). The wood granulate was changed at least twice weekl y. The water bottles were changed weekly. The animals were regularly transferred to clean cag es. The animal room had a standardized climate: Room temperature: 23 ± 3°C; Air humidity: 50 ± 25%; Light/ Dark cycle: 12 hour rhythm. B. Study Design and Methods: 1. Animal assignment and treatment
Dose: 0.5 ml pure liquid test substance/animal Application route: Dermal (semi occlusive procedure)
Duration: 4 hours– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 438 April 2008

Group size: 3 rabbits
Observations: Clinical signs, skin effects, body weight (at begin ning of study) In life dates: 2005-05-10 – 2005-05-17

II. Results and Discussion:

A. Findings: Under the present test conditions the following fin dings were noted: Animal no. one: erythema (grade 2) up to 72 hours e xamination time point. Animal no. two: erythema (grade 1) up to 72 hours e xamination time point. Animal no. three: erythema (grade 1) at 24 and 72 h ours examination time points and grade 2 after 48 hours. There were no systemic intolerance reactions.

Summary of irritant effects (Scores)

Animal

no. 24h 48h 72h Mean

scores Response Reversible (days)

E O E O E O E O E O E 0

1 2 0 2 0 2 0 2.0 0.0 + - 7 na

2 1 0 1 0 1 0 1.0 0.0 - - 7 na

3 1 0 2 0 1 0 1.3 0.0 - - 7 na

Abbreviation: 0 = no pathological findings, E = Ery thema and eschar formation, O = Oedema formation; n a = not applicable

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 150 OD is non-irrita nt to the skin.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 439 April 2008

B. Reviewer Comments: In a primary dermal irritation study, 0.5 ml of BY I 08330 150 OD [Batch No. 08030/0189(0152); light-brown suspension] was appli ed to the skin of three healthy female young adult Crl:KBL(NZW)BR rabbits (age: not reported; weight: 2.3-2.8 kg; source: Charles River, KiBlegg, Germany). On the day before the test, the fur was shorn on the right and left side form the dorso-lat eral area of the trunk of each of the rabbits. The test material, 0.5 ml, was applied to the skin of the a nimal under a gauze patch. The treated skin area was app roximately 2.5 cm by 2.5 cm in size. The patch was held in place with non-irritating tape for the duration of the exposure period. After the exposur e period the dressing and patch were removed and the exposed skin area carefully washed with water.

For animal welfare reasons one animal had three tes t patches applied to determine the extent of dermal irritation. The first patch was removed after thre e minutes. As no serious dermal reactions were observed, the second patch was removed after one ho ur. At this stage the dermal observations indicated that the exposure could be allowed to ext end to 4 hours, therefore the third patch was removed after four hours and the responses were gra ded one hour later. The study was completed using two additional animals, exposed for four hour s.

Dermal irritation was scored at 1, 24, 48 and 72 ho urs after patch removal. If no irritation was obse rved after 72 hours the study was completed. If dermal irritation was observed, animals were monitored usually on day 7 and 14 after patch removal until t he changes had completely subsided. The degree of dermal irritation was recorded as specified by Drai ze and any serious lesions or toxic effects other t han dermal irritation were also recorded.

At one hour, all three animals were observed with v ery slight erythema (score 1). At 24 hours, two animals were observed with very slight erythema (sc ore 1) and one animal with well defined erythema (score 2). At 48 hours, two animals were observed with well defined erythema and one animal with very slight erythema. At 72 hours, two animals were obs erved with very slight erythema and one animal with well defined erythema. By day 7, all dermal irrita tion was resolved. Primary Dermal Irritation Inde x (PDII) = 1.33.

Based on the observation of very slight to well def ined erythema observed at 72 hours, BYI 08330150 OD is classified as EPA Toxicity Category IV.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a primary derma l irritation study (OPPTS 870.2500; OECD 404) in the rabbit.

C. Reviewer Conclusions: All countries agree BYI 08330 150 OD causes sligh t dermal irritation.

D. Study Deficiencies: None.

CITATION: Schungel, M. (2005) BYI 08330 240 SC: Acute Skin Ir ritation/Corrosion on Rabbits.

Project Number: AT02291, T7075366. Unpublished stud y prepared by Bayer Ag Inst. of Toxicology. 20 p. August 19, 2005.

I. Materials and Methods:

A. Materials– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 440 April 2008

1. Test material: BYI 08330 240 SC Development no.: 30-00360972 Description: White suspension
Lot/Batch no: 07218/0073(0020)
Content: 244.48 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -08

2. Vehicle and/or positive control: None

3. Test animals Species: Rabbit, females
Strain: Albino (Crl:KBL(NZW)BR) Age: Young adult animals Weight at dosing: 2.3 kg – 2.7 kg
Source: Charles River Animal Breeder, Kißlegg, Germany Acclimation period: At least 5 days
Diet: Ssniff K-Z, Soest, Germany
Water: Tap water ad libitum Housing: The animals were housed individually in cage units Metall/Noryl by EBECO. Excrement trays below the ca ges contained low dust wood granulate bedding (J. Rette nmaier & Söhne, 73479 Ellwangen-Holzmühle, Germany). The wood granulate was changed at least twice weekl y. The water bottles were changed weekly. The animals were regularly transferred to clean cag es. The animal room had a standardized climate: Room temperature: 23 ± 3°C; Air humidity: 50 ± 25%; Light/ Dark cycle: 12 hour rhythm.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 441 April 2008

B. Study Design and Methods: 1. Animal assignment and treatment
Dose: 0.5 ml pure liquid test substance/animal Application route: Dermal (semi occlusive procedure)
Duration: 4 hours Group size: 3 rabbits
Observations: Clinical signs, skin effects, body weight (at begin ning of study)

II. Results and Discussion:

A. Findings: None of the three animals showed skin irritating ef fects. There were no systemic intolerance reactions.

Summary of irritant effects (Scores)

Animal no. 24h 48h 72h Mean

scores Response Reversible (days)

E O E O E O E O E O E O

1 0 0 0 0 0 0 0.0 0.0 -- -- na na

2 0 0 0 0 0 0 0.0 0.0 -- -- na na

3 0 0 0 0 0 0 0.0 0.0 -- -- na na

Abbreviation: 0 = no pathological findings, E = Ery thema and eschar formation, O = Oedema formation; n a = not applicable

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 240 SC is non-irrita nt to the skin.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 442 April 2008

B. Reviewer Comments: In a primary dermal irritation study, 0.5 ml of BYI 08330 240 SC [Batch No. 07218/0073(0020); white suspension] was applied to the skin of three healthy female young adult Crl:KBL (NZW) BR rabbits (age: young adult; weight: 2.3-2.7 kg; source: Charles River, KiBlegg, Germany). On the day before the test, the fur was shorn on the right and left side form the dorsolateral area of the trunk of each of the rabbits. The test material, 0.5 ml, was applied to the skin of the animal under a gauze patch. The treated skin area was approximately 2.5 cm by 2.5 cm in size. The patch was held in place with non-irritating tape fo r the duration of the exposure period. After the exposure period the dressing and patch were removed and the exposed skin area carefully washed with water.

For animal welfare reasons one animal had three tes t patches applied to determine the extent of dermal irritation. The first patch was removed aft er three minutes. As no serious dermal reactions were observed, the second patch was removed after o ne hour. At this stage the dermal observations indicated that the exposure could be allowed to ext end to 4 hours, therefore the third patch was removed after four hours and the responses were gra ded one hour later. The study was completed using two additional animals, exposed for four hour s.

Dermal irritation was scored at 1, 24, 48 and 72 ho urs after patch removal. If no irritation was observed after 72 hours the study was completed. T he degree of dermal irritation was recorded as specified by Draize and any serious lesions or toxi c effects other than dermal irritation were also recorded. No dermal irritation was noted in any of the treated animals.

Based on the lack of any observed dermal irritation , BYI 08330 240 SC is classified as EPA Toxicity Category IV. PDII is 0.0.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a primary derma l irritation study (OPPTS 870.2500; OECD 404) in the rabbit.

C. Reviewer Conclusions: All countries agree BYI 08330 240 SC is not an ir ritant to the skin.

D. Study Deficiencies: None.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 443 April 2008

B.6.11.5 Eye irritation (OECD Annex IIIA 7.1.5)

CITATION: Schungel, M. (2005) BYI 08330 150 OD: Acute Eye Irr itation on Rabbits. Project

Number: T2075370, AT02358. Unpublished study prepar ed by Bayer Ag Inst. of Toxicology. 21 p. August 31, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 150 OD
Article no.: 00-06424376 Description: Light brown suspension
Lot/Batch no: 08030/0189(0152)
Content: 148.89 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -10

2. Vehicle and/or positive control: None

3. Test animals Species: Rabbit, females
Strain: Albino (Crl:KBL(NZW)BR) Age: Young adult animals Weight at dosing: 2.4 kg – 2.8 kg
Source: Charles River, Kißlegg, Germany Acclimation period: At least 5 days
Diet: Ssniff K-Z, Soest, Germany
Water: Tap water ad libitum Housing: The animals were housed individually in cage units Metall/Noryl by EBECO. Excrement trays below the ca ges contained low dust wood granulate bedding (J. Rette nmaier & Söhne, 73479 Ellwangen-Holzmühle, Germany). The wood granulate was changed at least twice weekly. T he animals were regularly transferred to clean cages. The animal room had a standardized climate: Room temperature: 20 ± 3°C; Air humidity: 50 ± 25%; Ligh t/Dark cycle: 12 hour rhythm.

B. Study Design and Methods:

1. Animal assignment and treatment
Dose: 0.1 ml pure liquid test substance/animal Application route: Instillation into the conjunctival sac of one eye. The eye was not rinsed for at least 24 hours following instilla tion.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 444 April 2008

Group size: 3 rabbits
Observations: Clinical signs, eye effects, body weight (at beginn ing of study) In life dates: 2005-05-24 – 2005-06-14

II. Results and Discussion:

A. Findings Under the present test conditions the following fin dings were noted: Animal no. one: Corneal opacity (grade 2) after 24 hours and grade 1 after 48 and 72 hours.

Conjunctival redness (grade 3) after 24 hours, grad e 2 after 48 hours and grade 1 after 72 hours. Conjunctival chemosis (grade 2) a fter 24 hours and grade 1 after 48 hours. Animal no. two: Corneal opacity (grade 1) after 2 4 hours and grade 1 up to 72 hours.

Conjunctival redness (grade 2) after 24 hours and u p to 72 hours. Conjunctival chemosis (grade 2) after 24 hours and up to 72 hour s. Animal no. three: Corneal opacity (grade 2) after 24 hours and grade 1 after 48 and 72 hours.

Conjunctival redness (grade 3) after 24 hours and u p to 72 hours. Conjunctival chemosis (grade 2) after 24 hours and grade 1 after 48 and 72 hours.

Summary of Irritant Effects (Scores)

Animal
No. 24 h 48 h 72 h mean

scores response reversible [days]

1 corneal opacity

iritis conjunctivae*
- redness

- chemosis 2
0

3

2 1

0

2

1 1

0

1
0 1.3

0.0

2.0
1.0 -

-

-

- 21

na

14
3

2 corneal opacity

iritis conjunctivae*
- redness

- chemosis 1
0

2

2 1

0

2

2 1

0

2

2 1.0

0.0

2.0
2.0 -

-

-

+ 21

na

14

14

3 corneal opacity

iritis conjunctivae*
- redness

- chemosis 2
0

3

2 1

0

3

1 1

0

3

1 1.3

0.0

3.0
1.3 -

-

+

- 21

na

21

21
*Score of 2 or more required to be considered “posi tive” for conjunctivae. response: corneal opacity: mean scores: <2 = --; > 2<3 = +; > 3 = ++ iritis: mean scores: <1 = --; > 1<2 = +; =2 = ++ conj. redness: mean scores: <2.5 = --; > 2.5 = + conj. oedema: mean scores: <2 = --; > 2 = + na: not applicable

III. Discussion and Conclusions:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 445 April 2008

A. Conclusions: The formulation Spirotetramat 150 OD is non-irritan t to the eyes.

B. Reviewer Comments: In a primary eye irritation study, 0.1 ml of 08330 150 OD [Batch No. 08030/0189(0152); light-brown suspension] was insti lled into the conjunctival sac of one eye in each of the three young healthy female Crl:KBL(NZW)BR ra bbits (age: not reported; weight: 2.4-2.8 kg; source: Charles River, KiBlegg, Germany). The othe r untreated eye served as control. The eye was not rinsed for at least 24 hours following instilla tion. If one hour after instillation a severe irri tation was not observed, two further rabbits were treated.

Eye irritations were scored and recorded at 1, 24, 48 and 72 hours after instillation. If no irritati on indices were observed after 72 hours, the study was completed. If eye irritations were observed, animals were monitored usually on day 7, 14 and 21 after instillation until changes had completely subsided. The degree of ocular irritation was reco rded as specified by Draize.

Corneal opacity was observed at 24, 48, and 72 hour s and on days 7 and 14. Scores for corneal opacity ranged from 1 to 2 through day 14 and the o pacity cleared by day 21. No iritis was observed at any scoring interval. Conjunctival redness and chemosis was observed at 1, 24, 48 and 72 hours and on days 7 and 14. Scores for the conjunctival irritation ranged from 1 to 3. All conjunctival irritation resolved by day 21.

Based on the corneal opacity observed up to day 14, BYI 08330150 OD is classified as EPA Toxicity Category II.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a primary eye irritation study (OPPTS 870.2400; OECD 405) in the rabbit.

C. Reviewer Conclusions: All countries agree BYI 08330 150 OD is an eye ir ritant.

D. Study Deficiencies: None.

CITATION: Schungel, M. (2005) BYI 08330 240 SC: Acute Eye Irr itation on Rabbits. Project

Number: AT02290, T0075369. Unpublished study prepa red by Bayer Ag Inst. of Toxicology. 20 p. August 19, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 240 SC Development no.: 30-00360972 Description: White suspension
Lot/Batch no: 07218/0037(0020)
Content: 240 g/l Stability of test compound: guaranteed for study duration, expiry date: 2006-03 -08– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 446 April 2008

2. Vehicle and/or positive control: None

3. Test animals Species: Rabbit, females
Strain: Albino (Crl:KBL(NZW)BR) Age: Young adult animals Weight at dosing: 2.8 kg - 3.0 kg
Source: Charles River, Kißlegg, Germany Acclimation period: At least 5 days
Diet: Ssniff K-Z, Soest, Germany
Water: Tap water ad libitum Housing: The animals were housed individually in cage units Metall/Noryl by EBECO. Excrement trays below the ca ges contained low dust wood granulate bedding (J. Rette nmaier & Söhne, 73479 Ellwangen-Holzmühle, Germany). The wood granulate was changed at least twice weekly. T he animals were regularly transferred to clean cages. The animal room had a standardized climate: Room temperature: 20 ± 3°C; Air humidity: 50 ± 25%; Ligh t/Dark cycle: 12 hour rhythm.

B. Study design and Methods:

1. Animal assignment and treatment
Dose: 0.1 ml pure liquid test substance/animal Application route: Instillation into the conjunctival sac of one eye Group size: 3 rabbits
Observations: Clinical signs, eye effects, body weight (at beginn ing of study)
In life dates: 2005-05-24 – 2005-05-27

II. Results and Discussion: A. Findings: Animal no. one: Conjunctival redness (grade 1) at 2 4 hours examination time point. Animal no. three: Conjunctival redness (grade 2) at 24 hours examination time point. There were no systemic intolerance reactions.

Summary of Irritant Effects (Scores)

Animal
No. 24 h 48 h 72 h mean

scores response reversible [days]
1 corneal opacity

iritis conjunctivae*
- redness

- chemosis 0
0

1
0 0

0

0

0 0

0

0
0 0.0

0.0

0.3
0.0 -

-

-

- na

na

2

na– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 447 April 2008

2 corneal opacity

iritis conjunctivae*
- redness

- chemosis 0
0

0
0 0

0

0
0 0

0

0
0 0.0

0.0

0.0
0.0 -

-

-

- na

na

1*

na
3 corneal opacity

iritis conjunctivae*
- redness

- chemosis 0
0

2
0 0

0

0
0 0

0

0
0 0.0

0.0

0.7
0.0 -

-

-

- na

na

2

na *Score of 2 or more required to be considered “posi tive” for conjunctivae. response: corneal opacity: mean scores: <2 = --; > 2<3 = +; > 3 = ++ iritis: mean scores: <1 = --; > 1<2 = +; =2 = ++ conj. redness: mean scores: <2.5 = --; > 2.5 = + conj. chemosis: mean scores: <2 = --; > 2 = + na: not applicable; *: in respect of the result 1 h post application

III. Discussion and Conclusions:

A. Conclusions: The formulation Spirotetramat 240 SC is non-irritan t to eyes.

B. Reviewer Comments: In a primary eye irritation study, 0.1 ml of BYI 08 330 240 SC [Batch No. 07218/0073(0020); white suspension] was instilled i nto the conjunctival sac of one eye in each of the three young healthy female Crl:KBL (NZW) BR rabbits (age: not reported; weight: 2.8-3.0 kg; source: Charles River, KiBlegg, Germany). The other untreated eye served as control. The eye was not rinsed for at l east 24 hours following instillation.

Eye irritations were scored and recorded at 1, 24, 48 and 72 hours after instillation. If no irritati on indices were observed after 72 hours, the study was complet ed. The degree of ocular irritation was recorded a s specified by Draize.

No corneal opacity or iritis was observed during th e study. Conjunctival redness was observed in all eyes at 1 hour (score 1) and two eyes at 24 hours (score 1 and 2). All irritation was resolved by 48 hours

.

BYI 08330 240 SC is classified as EPA Toxicity Cate gory III based on the conjunctival redness observed at 24 hours.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a primary eye i rritation study (OPPTS 870.2400; OECD 405) in the rabbit.

C. Reviewer Conclusions: All countries agree BYI 08330 240 SC is slightly irritating to the eyes based on the conjunctival redness (score of 2) observed a t 24 hours

D. Study Deficiencies: None.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 448 April 2008

B.6.11.6 Skin sensitisation (OECD Annex IIIA 7.1.6)

CITATION: Vohr, H. (2005) BYI 08330 150 OD: Study for the Ski n Sensitization Effect in Guinea Pigs

[Buehler Patch Test]. Project Number: T6075581, AT0 2570. Unpublished study prepared by Bayer Ag Inst. of Toxicology. 25 p. November 23 , 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 150 OD Development no.: 30-00364846 Description: Light brown liquid
Lot/Batch no: 08030/0189(0152)
Content: 148.89 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -10

2. Vehicle and/or positive control: Lewatit water. A 0.48 % concentration was formulate d (ready to use concentration) 3. Test animals Species: Guinea pig, females
Strain: SPF-bred (Crl:HA) Age: 5 - 6 weeks Weight at dosing: 290 g – 388 g
Source: Charles River Laboratory Breeder, Kißlegg, Germany Acclimation period: At least 5 days
Diet: Provimi Kliba 3420, Kaiseraugst, Switzerland
Water: Tap water ad libitum Housing: During the adaptation and study period the animals were conventionally kept in type IV Makrolon® cages, in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. The cages wer e exchanged for ones with clean bedding two times per week. Low-dust wood shavings supplied by Rettenmaier & Sö hne, GmbH & Co, 73494 Rosenberg, Germany were used as bedding. The wood shavings were spot checked for contaminant levels. Records of these test results a re filed at Bayer HealthCare AG. Room temperature: 22 +/- 3°C ( possibly drifting higher at outdoor temperatures above 24°C) ; Relative humidity: 40 - 70 %; Light/dark cycle: Twelve hours rhythm; artificial lighting: Air exchange rate: >= 10 times per hour.

B. Study Design and Methods 1. Animal assignment and treatment– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 449 April 2008

Dose:

1 st to 3 rd induction: The animals were dermally treated with the test mat erial 3 times at intervals of 7 days. The 1 st to 3 rd induction were performed with the 0.48% test item concentration. T he animals in the test item group were treated with a hypoalle rgic patch loaded with the test item applied to the left flank and held in place on the skin with adhesive plaster. The volum e applied per animal was 0.5 ml. The patches were removed af ter an exposure period of six hours, and any remaining tes t item was removed with physiological saline solution. The tr eatment areas were visually assessed 30 hours after initiat ion of exposure. For this, the treatment areas were not sh orn or chemically depilated. Challenge: The challenge was performed 2 weeks after the last dermal induction. For the challenge the backs and right f lanks of the animals were shorn one day prior to the challenge. During the challenge a hypoallergenic patch loaded with the 0. 48% test item concentration was applied and fixed to the rig ht flank of each animal in the control and test item group. As a control a patch loaded only with the vehicle was applied and fixed also to the right flank, cranial to the test item patch. Th e patches were held securely in place on the skin with self-adhesi ve tape for 6 hours. The volume applied per animal was 0.5 ml. At the end of the six-hour exposure period, the patches were r emoved and the remaining test item was rinsed away with steril e physiological saline solution. Twenty-one hours lat er the skin of the animals was shorn in the region of the treatmen t sites.
Naive Controls: In the control group animals, a hypoallergic patch loaded only with the vehicle was applied to the left flank and fixed with a strip of adhesive tape in each of the first to thir d inductions. The patches were removed after an exposure period of si x hours, and any remaining test item was removed with physio logical saline solution. The volume applied per animal was 0.5 ml. The treatment areas were visually assessed 30 hours aft er initiation of exposure. For this, the treatment areas were not shorn or chemically depilated. Application route: Dermal (challenge: occlusive application) Application volume: 0.5 ml/animal Group size: Test item group: 20 females, control group: 10 fema les

Observations: Mortality, clinical signs, skin effects, body weigh t (at beginning and termination of study) Grading of the skin reaction: 0 = no reaction; 1 = slight localized redness; 2 = moderate confluent redness; 3 = severe redness and swelling
In life dates: 2005-09-13 - 2005-10-13

II. Results and Discussion:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 450 April 2008

A. Findings: There were no skin effects in the animals of the te st item group and the control group during the first to third induction treatment. The challenge with the 0.48 % test item concentration l ed to skin effect (grade 1) in 1 of 20 animals of the tes t item group (5 %) and to no skin effects in the co ntrol group. Single animals showing slight reaction (grad e 1) can be detected from time to time not dependin g on whether they were treated with vehicle or test i tem. Thus, such skin effects of single animals migh t appear by chance.

Number of animals exhibiting skin effects during ch allenge

Test item group (20 animals) Control group (10 anim als)
Test item patch Control patch Test item patch Contr ol patch

Hours 30 54 Total 30 54 30 54 Total 30 54
Challenge
0.48 % 1 1 1 0 0 0 0 0 0 0

Positive Control: The positive control study with a lpha hexyl cinnamic aldehyde showed that the study

was able to detect sensitizing compounds in the cho sen laboratory conditions.

III. Discussion and Conclusions:

A. Conclusions: Under the conditions of the Buehler Patch Test and with respect to the evaluation criteria, the “ready to use” test formulation exhib its no skin sensitization potential– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 451 April 2008

B. Reviewer Comments: The Buehler epicutaneous patch test was performed o n 30 female Crl:HA guinea pigs (20 animals for the test Item group and 10 control animals) (age: 5-6 weeks; weight: 290-3 88 g; source: Charles River Laboratory Animal Breeders , KiBlegg, Germany) to determine whether the test item “BYJ 08330 150 OD ready to use dilution” (cont ent of the active ingredient: BYI 08330: 148.89 g/l ) exhibits skin-sensitizing properties.

The study was conducted with the following test ite m concentrations:

1 st to 3 rd induction: 0.48% Challenge: 0.48%

The test item was diluted with Lewatit® water to th e 0.48% ready to use concentration (suspension).

There were no skin effects in the animals of the te st item group and the control group during the firs t to third induction treatment. The challenge with the 0 .48% test item concentration led to a skin effect ( grade 1) in 1 of 20 animals of the test item group (5%) a nd to no skin effects in the control group.

The Buehler patch test methodology was validated in a test using alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400. (Report No. PH034010, August, 2005.)

In summary, by comparing the effects in the test it em group and in the control group it can be conclud ed that under the conditions of the Buehler Patch Test and with respect to the evaluation criteria the BY I 08330 150 OD ready to use dilution exhibits no skin -sensitization potential.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a dermal sensit ization study (OPPTS 870.2600; OECD 406) in the Guinea pig.

C. Reviewer Conclusions: All countries agree that under the conditions of the Buehler Patch Test, BYI 08330 150 OD ready to use dilution exhibits no derm al sensitization potential.

D. Study Deficiencies: None.

CITATION: Vohr, H. (2005) BYI 08330 150 OD: Study for the Ski n Sensitization Effect in Guinea

Pigs. Project Number: T9074945, AT01873. Unpublishe d study prepared by Bayer Ag Inst. of Toxicology. 27 p. February 18, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 150 OD Development no.: 30-00364846 Description: Brown suspension
Lot/Batch no: 08030/0180(0152)
Content: 144.65 g/l– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 452 April 2008

Stability of test compound: Guaranteed for study duration; expiry date: 2005-11 -29

2. Vehicle and/or positive control: Sterile physiological saline solution
3. Test animals Species: Guinea pig, females
Strain: SPF-bred (Crl:HA) Age: 4 -5 weeks Weight at dosing: 255 g – 351 g
Source: Charles River Animal Breeder, Kißlegg, Germany Acclimation period: At least 5 days
Diet: Provimi Kliba 3420, Kaiseraugst, Switzerland
Water: Tap water ad libitum
Housing: During the adaptation and study period the animals were conventionally kept in type IV Makrolon® cages, in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. The cages wer e exchanged for ones with clean bedding two times per week. Low-dust wood shavings supplied by Rettenmaier & Sö hne, GmbH & Co, 73494 Rosenberg, Germany were used as bedding. The wood shavings were spot checked for contaminant levels. Records of these test results a re filed at Bayer HealthCare AG. Room temperature: 22 +/- 3°C ( possibly drifting higher at outdoor temperatures above 24°C) ; Relative humidity: 40 - 70 %; Light/dark cycle: Twelve hours rhythm; artificial lighting: Air exchange rate: >= 10 times per hour.

B. Study Design and Methods:

1. Animal assignment and treatment
Dose:

1 st to 3 rd induction: The animals were dermally treated with the test ite m 3 times at intervals of 7 days. The 1 st to 3 rd induction were performed with the 100% test item concentration. The animals in t he test item group were treated with a hypo allergic patch loade d with the test material applied to the left flank and held in place on the skin with adhesive plaster. The patches were remov ed after an exposure period of six hours, and any remaining tes t item was removed under running water. The volume applied pe r animal was 0.5 ml. The treatment areas were visually asse ssed 30 hours after initiation of exposure. For this, the t reatment areas were not shorn or chemically depilated.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 453 April 2008

Challenge: The challenge was performed 2 weeks after the last dermal induction. For the challenge the backs and right f lanks of the animals were shorn one day prior to the challenge. During the challenge a hypoallergenic patch loaded with the 10 0% test material concentration was applied and fixed to the right flank of each animal in the control and test item group. As a control a dry patch was applied and fixed also to the right f lank, cranial to the test item patch. The patches were held securely in place on the skin with self-adhesive tape for 6 hours. The volume applied per animal was 0.5 ml. At the end of the s ix-hour exposure period, the patches were removed and the r emaining test item was rinsed away with sterile physiologica l saline solution. Twenty-one hours later the skin of the an imals was shorn in the region of the treatment sites. Naive Controls: In the case of the control group animals, a hypo al lergic dry patch was applied to the left flank and fixed with a strip of adhesive tape in each of the first to third inducti ons. The patches were removed after an exposure period of si x hours, and any remaining test item was removed under runni ng water. The volume applied per animal was 0.5 ml. The trea tment areas were visually assessed 30 hours after initiat ion of exposure. For this, the treatment areas were not sh orn or chemically depilated. Application route: Dermal (challenge: occlusive application) Application volume: 0.5 ml/animal Group size: Test item group: 20 females, control group: 10 fema les
Observations: Mortality, clinical signs, skin effects, body weigh t (at beginning and termination of study) Grading of the skin reaction: 0 = no reaction; 1 = slight localized redness; 2 = moderate confluent redness; 3 = severe redness and swelling

In life dates: 2005-01-11 – 2005-02-10

II. Results and Discussion:

A. Findings: There were no skin effects in the animals of the te st item group and the control group during the first induction treatment. After the sec ond induction there were skin effects (grade 1) in 11 of 20 animals and after the third induction (grade 1-3) i n 20 of 20 animals of the test item group and no sk in effects in the animals of the control group. The ch allenge with the 100 % test item concentration led to skin effects (grade 1-2) in 20 of 20 animals of the test item group (100 %) and no skin effects in the control group.

Number of animals exhibiting skin effects during ch allenge Test item group (20 animals) Control group (10 ani mals) Test item patch Control patch Test item patch Cont rol patch

Hours 30 54 Total 30 54 30 54 Total 30 54
Challenge
100 % 20 20 20 0 0 0 0 0 0 0– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 454 April 2008

Positive Controls: The positive control study with alpha hexyl cinnami c aldehyde showed that the study was able to detect sensitizing compounds in t he chosen laboratory conditions.

III. Discussion and Conclusions:

A. Conclusions: Under the conditions of the Buehler Patch Test and with respect to the evaluation criteria, the test item therefore exhibits a skin-s ensitization potential.

B. Reviewer Comments: The Buehler epicutaneous patch test was performed i n 2005 on 30 female Crl:HA guinea pigs (20 animals for the test item gr oup and 10 control animals) (age: 4-5 weeks; weight : 255-351 g; source: Charles River Laboratory Animal Breeders, KiBlegg, Germany) to determine whether the test item BYI 08330 150 OD” (content of the act ive ingredient: BYI 08330: 144.65 g/l) exhibits ski nsensitizing properties. Additional two animals were used for dose-finding for the challenge concentrat ion. For the dose range-finding study the test item was formulated in sterile physiological saline solution to yield a suspension.

The study was conducted with the following test ite m concentrations:

1 st to 3 rd induction: 100% Challenge: 100%

There were no skin effects in the animals of the te st item group and the control group during the firs t induction treatment. After the second induction the re were skin effects (grade 1) in 11 of 20 animals and after the third induction (grade 1-3) in 20 of 20 a nimals of the test item group and no skin effects i n the animals of the control group.

The challenge with the 100% test item concentration led to skin effects (grade 1-2) in 20 of 20 animal s of the test item group (100%). No skin effects were n oted in the control group.

The Buehler patch test methodology was validated in a test using alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400. (Report No. PH33529, October, 2004.)

In summary, by comparing the effects in the test it em group and in the control group it can be conclud ed that under the conditions of the Buehler Patch Test , BYI 08330 150 OD exhibits skin-sensitization potential.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a dermal sensit ization study (OPPTS 870.2600; OECD 406) in the Guinea pig.

C. Reviewer Conclusions: All countries agree that under the conditions of the Buehler Patch Test, BYI 08330 150 OD exhibits dermal sensitization potentia l.

D. Study Deficiencies: None.

CITATION: Vohr, H. (2005) BYI 08330 240 SC: Study for the Ski n Sensitization Effect in Guinea Pigs– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 455 April 2008

(Buehler Patch Test). Project Number: AT01876, T807 4944. Unpublished study prepared by Bayer Ag Inst. of Toxicology. 27 p. February 25 , 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 240 SC Development no.: 30-00360972 Description: White suspension
Lot/Batch no: 07218/0019(0019)
Content: 244.48 g/l Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -08

2. Vehicle and/or positive control: Sterile physiological saline solution

3. Test animals Species: Guinea pig, females
Strain: SPF-bred (Crl:HA) Age: 4 -5 weeks Weight at dosing: 265 g - 348 g
Source: Charles River Animal Breeder, Kißlegg, Germany Acclimation period: At least 5 days
Diet: Provimi Kliba 3420, Kaiseraugst, Switzerland
Water: Tap water ad libitum
Housing: During the adaptation and study period the animals were conventionally kept in type IV Makrolon® cages, in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. The cages wer e exchanged for ones with clean bedding two times per week. Low-dust wood shavings supplied by Rettenmaier & Sö hne, GmbH & Co, 73494 Rosenberg, Germany were used as bedding. The wood shavings were spot checked for contaminant levels. Records of these test results a re filed at Bayer HealthCare AG. Room temperature: 22 +/- 3°C ( possibly drifting higher at outdoor temperatures above 24°C) ; Relative humidity: 40 - 70 %; Light/dark cycle: Twelve hours rhythm; artificial lighting: Air exchange rate: >= 10 times per hour.

B. Study Design and Methods:

1. Animal assignment and treatment
Dose:– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 456 April 2008

1 st to 3 rd induction: The animals were dermal treated with the test item three times at intervals of seven days. The 1 st to 3 rd induction was performed with the 100% test item concentration. T he volume applied per animal was 0.5 ml. Challenge: The challenge was performed two weeks after the las t dermal induction. The volume applied per animal was 0.5 m l. Naive Controls: In the case of the control animals, a hypo allergic dry patch was applied to the left flank in each of the first to t hird inductions. The volume applied per animal was 0.5 ml per animal

. Application route: Dermal (Challenge: occlusive application) Application volume: 0.5 ml/animal Group size: Test item group: 20 females, control group: 10 fema les
Observations: Mortality, clinical signs, skin effects, body weigh t (at beginning and termination of study) Grading of the skin reaction: 0 = no reaction; 1 = slight localized redness; 2 = moderate confluent redness; 3 = severe redness and swelling In life dates: 2005-01-11 – 2005-02-10

II. Results and Discussion:

A. Findings: Appearance and behaviour of the test item group wer e not different from the control group. There were no skin effects in the animals of the te st item group and the control group during the firs t and second induction treatments. After the third induct ion there were skin effects (grade 1) in 3 of 20 an imals of the test item group and no skin effects in the a nimals of the control group.

The challenge with the 100% test item concentration led to skin effects (grade 1) in 7 of 20 animals o f the test item group (35%) and no skin effects in the co ntrol group.

Number of animals exhibiting skin effects during ch allenge Test item group (20 animals) Control group (10 ani mals) Test item patch Control patch Test item patch Cont rol patch

Hours 30 54 Total 30 54 30 54 Total 30 54 Challenge
100 % 7 6 7 0 0 0 0 0 0 0

Positive Control: The positive control study with alpha hexyl cinna mic aldehyde showed that the study was able to detect sensitizing compounds in the cho sen laboratory conditions.

III. Discussion and Conclusions:

A. Conclusions: Under the conditions of the Buehler Patch Test an d with respect to the evaluation criteria, the test item therefore exhibits a skin-s ensitization potential.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 457 April 2008

B. Reviewer Comments: The Buehler epicutaneous patch test was performed i n 2005 on 30 female Crl:HA guinea pigs (20 animals for the test item gr oup and 10 control animals) (age: 4-5 weeks; weight : 265-348 g; source: Charles River Laboratory Animal Breeders. KiBlegg, Germany) to determine whether the test item “BYI 08330 240 SC” (content of the ac tive ingredient: BYI 08330: 236.50 g/L) exhibits sk insensitizing properties. Additional two animals were used for dose-finding for the challenge concentrat ion.

The study was conducted with the following test ite m concentrations:

1 st to 3 rd induction: 100% Challenge: 100%

The doses for the induction and challenge groups we re based on the results of a range-finding study formulated into a suspension with sterile physiolog ical saline solution.

There were no skin effects in the animals of the te st item group and the control group during the firs t and second induction treatments. After the third induct ion there were skin effects (grade 1) in 3 of 20 an imals of the test item group and no skin effects in the a nimals of the control group.

The challenge with the 100% test item concentration led to skin effects (grade 1) in 7 of 20 animals o f the test item group (35%) and no skin effects in the co ntrol group.

In summary, by comparing the effects in the test it em group and in the control group it can be conclud ed that under the conditions of the Buehler Patch Test and with respect to the evaluation criteria the te st item exhibits a skin-sensitization potential.

The Buehler patch test methodology was validated in a test using alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400, positive con trol. (Report No. PH33529, October, 2004).

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a dermal sensit ization study (OPPTS 870.2600; OECD 406) in the Guinea pig.

C. Reviewer Conclusions: All countries agree that under the conditions of the Buehler Patch Test, BYI 08330 240 SC exhibits dermal sensitization potentia l.

D. Study Deficiencies: None.

CITATION: Vohr, H. (2005) BYI 08330 240 SC: Study for the Ski n Sensitization Effect in Guinea Pigs. Project Number: AT02598, T5075616. Unp ublished study prepared by Bayer Ag Inst. of Toxicology. 25 p. November 8, 2005.

I. Materials and Methods:

A. Materials

1. Test material: BYI 08330 240 SC Development no.: 30-00360972– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 458 April 2008

Description: White suspension
Lot/Batch no: 07218/0019(0019)
Content: 244.48 g/L Stability of test compound: Guaranteed for study duration; expiry date: 2006-03 -10

2. Vehicle and/or positive control: The test item was diluted with Lewatit water to the 0.3% ready to use concentration. An adjuvant was added to the formulation with a concentration of 0.15% v/v. 3. Test animals Species: Guinea pig, females

Strain: SPF-bred (Crl:HA) Age: 5 - 6 weeks Weight at dosing: 313 g - 385 g

Source: Charles River Laboratory Breeder, Kißlegg, Germany Acclimation period: At least 5 days

Diet: Provimi Kliba 3420, Kaiseraugst, Switzerland

Water: Tap water ad libitum Housing: During the adaptation and study period the animals were conventionally kept in type IV Makrolon® cages, in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. The cages wer e exchanged for ones with clean bedding two times per week. Low-dust wood shavings supplied by Rettenmaier & Sö hne, GmbH & Co, 73494 Rosenberg, Germany were used as bedding. The wood shavings were spot checked for contaminant levels. Records of these test results a re filed at Bayer HealthCare AG. Room temperature: 22 +/- 3°C ( possibly drifting higher at outdoor temperatures above 24°C) ; Relative humidity: 40 - 70 %; Light/dark cycle: Twelve hours rhythm; artificial lighting: Air exchange rate: >= 10 times per hour.

B. Study Design and Methods:

1. Animal assignment and treatment
Dose:

1 st to 3 rd induction: The animals were dermally treated with the test ite m three times at intervals of seven days. The 1 st to 3 rd inductions were performed with the 0.3% test item concentration. T he volume per animal was 0.5 ml. Challenge: The challenge was performed two weeks after the las t dermal induction. The volume per animal was 0.5 ml Naïve Controls: For the control animals, a hypo allergic patch load ed only with the vehicle was applied to the left flank in the fi rst to third inductions. The volume per animal was 0.5 ml. Application route: Dermal (challenge: occlusive application) Application volume: 0.5 ml/animal– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 459 April 2008

Group size: Test item group: 20 females, control group: 10 fema les
Observations: Mortality, clinical signs, skin effects, body weigh t (at beginning and termination of study) Grading of the skin reaction: 0 = no reaction; 1 = slight localized redness; 2 = moderate confluent redness; 3 = severe redness and swelling In life dates: 2005-09-13 – 2005-10-13

II. Results and Discussion: A. Findings: Three of the twenty animals of the test item group showed clinical signs and two of these animals died at day 12 and day 13 after treatment. There were no skin effects in the animals of the te st item group and the control group during the first t o third induction treatment. The challenge with the 0.3% test item concentration led to no skin effects in t he animals of the test item group and no skin effec ts in the control group.

Number of animals exhibiting skin effects during ch allenge Test item group (18 animals) Control group (10 ani mals) Test item patch Control patch Test item patch Cont rol patch

Hours 30 54 Total 30 54 30 54 Total 30 54 Challenge
0.3 % 0 0 0 0 0 0 0 0 0 0

Positive Control: The positive control study with alpha hexyl cinnamic aldehyde showed that the study was able to detect sensitizing compounds in the cho sen laboratory conditions.

III. Discussion and Conclusions:
A Conclusions: Under the conditions of the Buehler Patch Test and with respect to the evaluation criteria, the “ready to use” test formulation exhib its no skin sensitisation potential.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 460 April 2008

B. Reviewer Comments: The Buehler epicutaneous patch test was performed i n 2005 on 30 female Crl:HA guinea pigs (20 animals for the test item gr oup and 10 control animals) (age: 5-6 weeks; weight : 313-385 grams; source: Charles River Laboratory Ani mal Breeders, KiBlegg, Germany) to determine whether the test item “BYI 08330 240 SC ready to us e dilution” (content of the active ingredient: BYI 08330: 244.48 g/l) exhibits skin-sensitizing proper ties.

The study was conducted with the following test ite m concentrations:

1 st to 3 rd induction: 0.3% Challenge: 0.3%

The test item was diluted with Lewatit® water to th e 0.3% ready to use concentration. An adjuvant was added to the formulation with a concentration of 0. 15% v/v. The formulation was visually described as suspension.

The Buehler patch test methodology was validated in a test using alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400, positive con trol. (Report No. PH034010, August 2005.)

There were no skin effects in the animals of the te st item group and the control group during the firs t to third induction treatment.

The challenge with the 0.3% test item concentration led to no skin effects in the animals of the test item group and no skin effects in the control group.

In summary, by comparing the effects in the test it em group and in the control group it can be conclud ed that under the conditions of the Buehler Patch Test , BYI 08330 240 SC ready to use dilution exhibits n o skin-sensitization potential.

This study is classified as Acceptable. It does sa tisfy the guideline requirement for a dermal sensit ization study (OPPTS 870.2600; OECD 406) in the Guinea pig.

C. Reviewer Conclusions: All countries agree that under the conditions of the Buehler Patch Test, BYI 08330 240 SC ready to use dilution exhibits no derm al sensitization potential.

D. Study Deficiencies: None.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 461 April 2008

B.6.11.7 Summary of toxicity studies with the formu lation

BYI 08330 150 OD (MOVENTO® 150 OD Insecticide) All of the BYI 08330 150 OD (MOVENTO® 150 OD Insect icide) acute toxicity studies were conducted in 2005 at the Bayer HealthCare AG. All studies were fully compliant with Good Laboratory Practice (GLP) and were conducted in accordance with the relevant prev ailing OECD and OPPTS guideline. Six of the studie s were conducted using BYI 08330 150 OD [Batch No. 08030/0189 (0152); light-br own suspension] as the test material. The test material for the dermal sensiti zation study was identified as BYI 08330 150 OD ready to use dilution. An additional dermal sensitization s tudy was conducted using BYI 08330 150 OD [Batch No

. 08030/180(0152); liquid brown suspension] as the te st material.

BYI 08330 150 OD was found to be of low acute toxic ity to the rat by the oral, dermal and inhalation r outes. No treatment-related findings were observed in the acute oral and dermal toxicity studies at the limit dose levels of 2000 and 4000 mg/kg bw, respectively. Tr eatment-related findings in the acute inhalation to xicity study were limited to minor bodyweights and clinica l signs (resolved by day 4) at the dose level of 1. 760 mg/L.

In the primary eye irritation study, corneal opacit y was observed at 24, 48, and 72 hours and on days 7 and 14. Scores for corneal opacity ranged from 1 to 2 through day 14 and the opacity cleared by day 21. Conjunctival redness and chemosis was observed at 1 , 24, 48 and 72 hours and on days 7 and 14. Scores for the conjunctival irritation ranged from 1 to 3. All conjunctival irritation resolved by day 21. BYI 08330 150 OD is considered an eye irritant.

In the primary dermal irritation study, all three a nimals were observed with very slight erythema at o ne hour. At 24 hours, two animals were observed with very sl ight erythema and one animal with well defined erythema. At 48 hours, two animals were observed w ith well defined erythema and one animal with very slight erythema. At 72 hours, two animals were obs erved with very slight erythema and one animal with well defined erythema. By day 7, all dermal irritation was resolved. BYI 08330 150 OD causes slight dermal irritation.

Two dermal sensitization studies were submitted for BYI 08330 150 OD. The test material for the first study was identified as BYI 08330 150 OD ready to use dil ution. By comparing the effects in the test item gr oup and in the control group it can be concluded that u nder the conditions of the Buehler Patch Test and w ith respect to the evaluation criteria the BYI 08330 15 0 OD ready to use dilution exhibits no skin-sensiti zation potential. The test material for the second materi al was identified as BYI 08330 150 OD but with a di fferent batch number than the other dermal sensitization an d acute toxicity studies. By comparing the effects in the test item group and in the control group it can be concluded that under the conditions of the Buehler Patch Test, BYI 08330 150 OD exhibits skin-sensitization potential.

BYI 08330 240 SC ((MOVENTO® 240 SC Insecticide) All of the BYI 08330 240 SC (MOVENTO® 240 SC Insect icide) acute toxicity studies were conducted in 2005 at the Bayer HealthCare AG. All studies were fully compliant with Good Laboratory Practice (GLP) and were conducted in accordance with the relevant prev ailing OECD and OPPTS guideline. Six of the studie s were conducted using BYI 08330 240 SC [Batch No. 07 218/0073(0020); white suspension] as the test material. An additional dermal sensitization study was conducted using BYI 08330 240 SC [Batch No. 07218/0019(0019); liquid white suspension] as the t est material.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 462 April 2008

BYI 08330 240 SC was found to be of low acute toxic ity to the rat by the oral, dermal and inhalation r outes. No treatment-related findings were observed in the acute oral and dermal toxicity studies at the limit dose levels of 2000 and 4000 mg/kg bw, respectively. Tr eatment-related findings in the acute inhalation to xicity study were limited to minor clinical signs (resolve d by day 4) at the dose level of 3.013 mg/L.

BYI 08330 240 SC was not irritating to rabbit skin and caused minimal, reversible ocular irritation. Two dermal sensitization studies were submitted. The t est material of the first study was identified as B YI 08330 240 SC but with a different batch number than the o ther acute toxicity studies. By comparing the effe cts in the test item group and in the control group it can be concluded that under the conditions of the Bueh ler Patch Test and with respect to the evaluation crite ria the test item exhibits a skin-sensitization pot ential. The test material in the second study was identified as BYI 08330 240 SC ready to use. By comparing the effects in the test item group and in the control g roup it can be concluded that under the conditions of the Buehler Patch Test, BYI 08330 240 SC ready to use d ilution exhibits no skin-sensitization potential.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 463 April 2008

B.6.12 Dermal absorption (OECD Annex IIIA 7.6)

See B.6.9.9– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 464 April 2008

B.6.12.1 Summary on Dermal absorption (AGES)

The results showed that the rate of absorption of [ 14 C]-BYI 08330 through human and rat epidermis from t he concentrate formulation and its v/v aqueous dilutio ns was slow. The majority of the applied dose (huma n: 85 – 95%; rat: 65 – 92%) was removed by mild skin wash ing after an exposure period of 8 hours. These data predict that the dermal absorption of BYI 08330 thr ough human or rat skin from potential exposure to t he OD formulation, either as the concentrate formulation or as the v/v aqueous spray strength dilutions, wou ld be low. In these experiments, rat skin was shown to be more permeable than human skin. The estimated dermal absorption for humans is calculated by adjusting the dermal absorption f igures for rats obtained in the in vivo

studies by the correction factors derived from the in vitro studies in rat and human skin. In order to assess the risk to workers potent ially exposed to BYI 08330, the formulation concent rate was included to assess exposure to mixer/loaders an d the spray strength dilutions are most relevant to spray operators.

Following a 10 hour exposure period, the in vivo dermal absorption of [ 14 C]-BYI 08330 in 150 OD formulation at the 3 concentrations accounted for means of appr oximately 17.6%, 10.4% and 12% of the dose respectively.

Over 168 hours following a 10 hour exposure period, the in vivo dermal absorption of [ 14 C]-BYI 08330 in 150 OD formulation accounted for means of approximately 10.6%, 4.8% and 5.8% for the high, intermediate an d low dose, respectively.

In the in-vitro

skin penetration study with human skin, an absorpt ion rate of 0.24 % for the concentrate and 2.74 % and 8.45 % for the dilutions, respectively, was obtained after the 8 hour exposure. For rat skin, an absorption rate of 2.15% for the c oncentrate and 23.21% and 22.70% for the dilutions, respectively, was obtained after the 8 hour exposur e.

So for the concentrate, the mean factor for species difference would be 8.9. For the aqueous dilutions , mean factors for species difference would be 8.5 and 2.7 , respectively.

Taking into account for the rat in vivo study the mean factor for species difference of 8. 9, an absorption rate for humans of 1.19 % can be proposed for the concen trate. For the field dilutions, absorption rates of 0.56 % and 2.15 % will be assumed.

Table 6-155: Calculation of species differences in dermal absorption Concentrate ( 150 mg/ml formulation) Dilution ( 1.5 mg/ml formulation) Dilution ( 0.05 mg/ml formulation) Human epidermis in vitro (8h) 0.24 2.74 8.45 Rat epidermis in vitro (8h) 2.15 23.21 22.70 Species difference correction factor 8.9 8.5 2.7

Table 6-156: Calculation of dermal absorption of B YI 08330 in humans Concentrate ( 150 mg/ml formulation) Dilution

( 1.5 mg/ml formulation) Dilution ( 0.05 mg/ml formulation) mean proportion of dose absorbed (%) rat epidermis in vivo (10 hour exposure, 168 hour observation) 10.6 4.8 5.8– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 465 April 2008

Species difference correction factor 8.9 8.5 2.7 Dermal absorption: Human 1.19 0.56 2.15– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 466 April 2008

B.6.13 Toxicological data on non active substances (OECD Annex IIIA 7.9)

Toxicological data on non active substances please see Appendix.

B.6.14 Exposure data (OECD Annex IIIA 7.3, 7.4 and 7.5)

Toxicological Risk Assessment for the EU please see Appendix.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 467 April 2008

B.6.15 References relied on

Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

Annex II Data and Information KIIA 5.1.1 /01 Klempner, A. 2006 [Azaspirodecenyl-3 -14C]BYI 08330: Distribution of the total radioactivity in male and female rats determined by quantitative whole body autoradiography (QWBA) including determination of the total radioactivity in excreta and exhaled 14CO2 Bayer CropScience AG, Report No.: MEF-06/15, Edition Number: M-269337-01-2 Date: 21.02.2006 GLP, unpublished Yes BCS

KIIA 5.1.1 /02 Klempner, A. 2006 [Azaspirodecenyl-3 -14C]BYI 08330: Adsorption, distribution, excretion and metabolism in the rat Bayer CropScience AG, Report No.: MEF-048/04, Edition Number: M-268709-02-2 Date: 15.02.2006, Amended: 15.09.2006 GLP, unpublished Yes BCS

KIIA 5.1.1 /03 Schmitt, W. 2006 Physiology based ph armacokinetic simulation of BYI 08330 in male rats Bayer Technology Services GmbH, Leverkusen, Germany Bayer CropScience AG, Report No.: BTS-WSM0602, Edition Number: M-274844-01-2 Date: 21.07.2006 Non GLP, unpublished Yes BCS

KIIA 5.1.1 /04 Klempner, A. 2006 [Azaspirodecenyl-3 -14C]BYI 08330: Depletion of residues and metabolites in plasma, urine, liver, kidney and testis of the male rat Bayer CropScience AG, Report No.: MEF-06/328, Edition Number: M-275731-01-2 Date: 11.08.2006 GLP, unpublished Yes BCS

KIIA 5.1.1 /05 Schmitt, W. 2006 PBPK-Simulation of BYI 08330 in male rats at high doses Bayer Technology Services GmbH, Leverkusen, Germany Bayer CropScience AG, Report No.: BTS-WSM0603-1, Edition Number: M-274847-02-2 Date: 21.07.2006, Amended: 01.09.2006 Non GLP, unpublished Yes BCS– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 468 April 2008

Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIA 5.1.1 /06 Totis, M. 2006 [Azaspirodecenyl-3-14 C]-BYI 08330: Comparison of the in vitro metabolism in liverbeads from male rat, mouse and human Bayer CropScience SA, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA05319, Edition Number: M-274118-02-2 Date: 06.07.2006, Amended: 09.08.2006 GLP, unpublished Yes BCS

KIIA 5.1.1 /07 Klempner, A. 2006 [Azaspirodecenyl-3 -14C]BYI 08330-enol-glucoside supplemental study: adsorption, distribution, excretion and metabolism in the rat Bayer CropScience AG, Report No.: MEF-06/006, Edition Number: M-268645-01-2 Date: 09.02.2006 GLP, unpublished Yes BCS

KIIA 5.1.1 /08 Klempner, A. 2006 [Azaspirodecane-3- 14C]BYI 08330-ketohydroxy: Adsorption, distribution, excretion and metabolism in the rat Bayer CropScience AG, Report No.: MEF-06/007, Edition Number: M-268931-01-2 Date: 20.02.2006 GLP, unpublished Yes BCS

KIIA 5.2.1 /01 Eigenberg, D. A. 2004 An acute oral LD50 study in the rat with BYI 08330 Bayer CropScience LP, Research Triangle Park, NC, USA Bayer CropScience AG, Report No.: 200398, Edition Number: M-069299-01-1 Date: 06.05.2004 GLP, unpublished Yes BCS

KIIA 5.2.2 /01 Eigenberg, D. A. 2004 An acute derma l LD50 study in the rat with BYI 08330 Bayer CropScience LP, Stilwell, KS, USA Bayer CropScience AG, Report No.: 200399, Edition Number: M-066937-01-2 Date: 28.04.2004 GLP, unpublished Yes BCS

KIIA 5.2.3 /01 Pauluhn, J. 2002 BYI 08330 (c.n.: -- ) - Study on acute inhalation toxicity in rats according to OECD no. 403 Bayer AG, Wuppertal, Germany Bayer CropScience AG, Report No.: 32020, Edition Number: M-064654-01-2 Date: 15.05.2002 GLP, unpublished Yes BCS– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 469 April 2008

Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIA 5.2.4 /01 Leuschner, J. 2002 Acute skin irrita tion test (patch test) of BYI 8330 in rabbits LPT Laboratory of Pharmacology and Toxicology KG, Hamburg, Germany Bayer CropScience AG, Report No.: R8147, Edition Number: M-062870-01-2 Date: 25.04.2002 GLP, unpublished Yes BCS

KIIA 5.2.5 /01 Leuschner, J. 2002 Acute eye irritat ion study of BYI 08330 by instillation into the conjunctival sac of rabbits LPT Laboratory of Pharmacology and Toxicology KG, Hamburg, Germany Bayer CropScience AG, Report No.: R8146, Edition Number: M-062864-01-3 Date: 25.04.2002 GLP, unpublished Yes BCS

KIIA 5.2.6 /01 Vohr, H.-W. 2002 BYI 08330 - Study f or the skin sensitization effect in guinea pigs (guinea pig maximization test according to Magnusson and Kligman) Bayer AG, Wuppertal, Germany Bayer CropScience AG, Report No.: 32273, Edition Number: M-076253-01-2 Date: 29.07.2002 GLP, unpublished Yes BCS

KIIA 5.2.6 /02 Vohr, H. W. 2004 BYI 08330 - Study f or the skin sensitization effect in guinea pigs (Buehler Patch test) Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT01317, Edition Number: M-078494-01-2 Date: 13.07.2004 GLP, unpublished Yes BCS

KIIA 5.2.6 /03 Esdaile, D. 2004 BYI 08330 - Evaluat ion of potential dermal sensitization in the local lymph node assay Bayer CropScience SA, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA 04120, Edition Number: M-090707-01-2 Date: 09.09.2004 GLP, unpublished Yes BCS

KIIA 5.3.1 /01 Mihail, F.;

Kroetlinger, F. 1998 Cyclic ketoenols BSN 3457, BSN 2342, FHN 7504, FHN 8330 - Subacute exploratory toxicity studies in rats (application by feed over 4 weeks) Bayer AG, Wuppertal, Germany Bayer CropScience AG, Report No.: T0061869, Edition Number: M-040236-01-2 Date: 13.02.1998 Non GLP, unpublished Yes BCS– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 470 April 2008

Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIA 5.3.1 /02 Schladt, L. 2001 BYI 08330 - Subacut e study with mice (keto-enol design) Bayer AG, Wuppertal, Germany Bayer CropScience AG, Report No.: T2070951, Edition Number: M-035927-01-2 Date: 13.09.2001 Non GLP, unpublished Yes BCS

KIIA 5.3.1 /03 Eigenberg, D. A. 2004 Technical grad e BYI 08330: A subacute toxicity feeding study in the Beagle dog Bayer CropScience LP, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201012, Edition Number: M-182239-01-1 Date: 13.12.2004 GLP, unpublished Yes BCS

KIIA 5.3.2 /01 Wahle, B. S. 2005 Technical grade BY I 08330: A subchronic toxicity testing study in the rat Bayer CropScience LP, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201136, Edition Number: M-252787-01-1 Date: 01.06.2005 GLP, unpublished Yes BCS

KIIA 5.3.2 /02 Wahle, M. S. 2005 Technical grade BY I 08330: A subchronic toxicity testing study in the mouse Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201284, Edition Number: M-255359-01-1 Date: 14.07.2005 GLP, unpublished Yes BCS

KIIA 5.3.3 /01 Eigenberg, D. A. 2005 Technical grad e BYI 08330 - A 90-day subchronic toxicity feeding study in the Beagle dog Bayer CropScience LP, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201223, Edition Number: M-254183-01-1 Date: 09.05.2005 GLP, unpublished Yes BCS

KIIA 5.3.4 /01 Eigenberg, D. A. 2006 A chronic toxi city feeding study in the beagle dog with technical grade BYI 08330 Bayer CropScience, Kansas City, MO, USA Bayer CropScience AG, Report No.: 201486, Edition Number: M-274969-01-1 Date: 06.07.2006 GLP, unpublished Yes BCS– –

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KIIA 5.3.7 /01 Eigenberg A. D. 2006 A subacute derm al toxicity study in rats with BYI 08330 Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201505, Edition Number: M-275227-01-1 Date: 20.06.2006 GLP, unpublished Yes BCS

KIIA 5.4.1 /01 Herbold, B. 2002 BYI 08330 - Salmone lla/microsome test plate incorporation and preincubation method Bayer HealthCare, Wuppertal, Germany Bayer CropScience AG, Report No.: AT00056, Edition Number: M-065358-01-2 Date: 24.10.2002 GLP, unpublished Yes BCS

KIIA 5.4.1 /02 Herbold, B. 2006 BYI 08330 - Salmone lla/microsome test - Plate incorporation and preincubation method Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT03070, Edition Number: M-272000-01-2 Date: 24.05.2006 GLP, unpublished Yes BCS

KIIA 5.4.2 /01 Herbold, B. 2002 BYI 08330 - In vitr o chromosome aberration test with chinese hamster V79 cells Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT00055, Edition Number: M-065342-01-2 Date: 24.10.2002 GLP, unpublished Yes BCS

KIIA 5.4.2 /02 Herbold, B. 2003 BYI 08330 - Cytogen etic screening with chinese hamster V79 cells Bayer HealthCare, Wuppertal, Germany Bayer CropScience AG, Report No.: AT00194, Edition Number: M-075136-01-2 Date: 13.01.2003 Non GLP, unpublished Yes BCS

KIIA 5.4.3 /01 Herbold, B. 2002 BYI 08330 - V79/HPR T-test in vitro for the detection of induced forward mutations Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT00137, Edition Number: M-072857-02-2 Date: 03.12.2002, Amended: 04.05.2006 GLP, unpublished Yes BCS

KIIA 5.4.4 /01 Herbold, B. 2002 BYI 08330 - Micronu cleus-test on the male mouse Bayer HealthCare, Wuppertal, Germany Bayer CropScience AG, Report No.: AT00048, Edition Number: M-065314-01-2 Date: 24.10.2002 GLP, unpublished Yes BCS– –

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Y/N-R/NR Owner

KIIA 5.4.4 /02 Honarvar, N. 2003 Chromosome aberrat ion assay in bone marrow cells of the mouse with BYI 08330 RCC Cytotest Cell Research, Rossdorf, Germany Bayer CropScience AG, Report No.: AR00070, Edition Number: M-084116-01-2 Date: 24.03.2003 GLP, unpublished Yes BCS

KIIA 5.4.5 /01 BrendlerSchwaab, S. 2003 BYI 08330 - Unscheduled DNA synthesis test wit h rat liver cells in vivo Bayer HealthCare, Wuppertal, Germany Bayer CropScience AG, Report No.: AT00526, Edition Number: M-116087-01-2 Date: 10.07.2003 GLP, unpublished Yes BCS

KIIA 5.5.1 /01 Wahle, B. S. 2005 Technical grade BY I 08330 (common name Spirotetramat): a chronic toxicity testing study in the rat Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201285, Edition Number: M-260765-01-1 Date: 15.11.2005 GLP, unpublished Yes BCS

KIIA 5.5.2 /01 Wahle, B. S. 2006 Technical grade BY I 08330 (common name Spirotetramat): An oncogenicity testing study in the rat Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201358, Edition Number: M-273643-01-1 Date: 08.03.2006 Non GLP, unpublished Yes BCS

KIIA 5.5.3 /01 Wahle, B. S. 2006 Revised report : T echnical grade BYI 08330 (common name Spirotetramat): An oncogenicity testing study in the mouse Bayer CropScience LP, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201359-1, Edition Number: M-275506-02-1 Date: 13.03.2006, Amended: 17.03.2006 Non GLP, unpublished Yes BCS

KIIA 5.5.3 /02 Jensen, T. L. 2005 A revised homogen eity and stability study of BYI 08330 technical in rodent ration Bayer CropScience, Kansas City, MO, USA Bayer CropScience AG, Report No.: 201363, Edition Number: M-258710-01-1 Date: 04.10.2005 GLP, unpublished Yes BCS– –

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Y/N-R/NR Owner

KIIA 5.5.4 /01 Kennel, P. 2005 BYI 08330 - Evaluati on of the potential reproductive toxicity in the male rat following dai ly oral administration by gavage Bayer CropScience SA, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA 04181, Edition Number: M-252001-01-2 Date: 23.05.2005 GLP, unpublished Yes BCS

KIIA 5.5.4 /02 Tinwell, H. 2006 BYI 08330-Enol - In vestigation of the testicular/sperm toxicity in the rat following 21 d ays of exposure by gavage Bayer CropScience SA, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA06011, Edition Number: M-273959-01-1 Date: 30.06.2006 Non GLP, unpublished Yes BCS

KIIA 5.6.1 /01 Young, A. D. 2006 Technical grade BY 1 08330 - A dose range-finding reproductive toxicity study in the Wistar rat (revised report) Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201300-1, Edition Number: M-273578-02-1 Date: 30.05.2006, Amended: 12.07.2006 GLP, unpublished Yes BCS

KIIA 5.6.1 /02 Young, A. D. 2006 Technical grade BY I 08330 (common name Spirotetramat): A two generation reproductive toxicity study in the Wistar rat Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201426-1, Edition Number: M-274619-02-1 Date: 30.05.2006, Amended: 13.07.2006 GLP, unpublished Yes BCS

KIIA 5.6.10 /01 Klaus, A. M. 2001 BYI 08330 - Pilot study on develo pmental toxicity in rats after oral administration Bayer AG, Wuppertal, Germany Bayer CropScience AG, Report No.: T3068559, Edition Number: M-021476-01-2 Date: 08.05.2001 Non GLP, unpublished Yes BCS

KIIA 5.6.10 /02 Klaus, A.M. 2004 BYI 08330, Synonym: FHN 08330 - Developmental toxicity study in rats after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT01413, Edition Number: M-086404-01-2 Date: 23.08.2004 GLP, unpublished Yes BCS– –

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KIIA 5.6.10 /03 Klaus, A. M. 2004 BYI 08330 - Synonym: FHN 08330 - Supplementary developmental toxicity study in rats after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT01512, Edition Number: M-091750-01-2 Date: 07.10.2004 GLP, unpublished Yes BCS

KIIA 5.6.10 /04 Temerowski, M. 2008 Spirotetramat (syn. BYI 08330) - Request for historical control data Bayer CropScience AG, Report No.: M-296860-01-1, Edition Number: M-296860-01-1 Date: 30.01.2008 Non GLP, unpublished Yes BCS

KIIA 5.6.11 /01 Holzum, B. 2001 BYI 08330 - Pilot developmental tox icity study in rabbits after oral administration Bayer AG, Wuppertal, Germany Bayer CropScience AG, Report No.: T3062735, Edition Number: M-084392-01-2 Date: 14.11.2001 Non GLP, unpublished Yes BCS

KIIA 5.6.11 /02 Klaus, A.-M. 2004 BYI 08330 - Developmental toxicit y study in rabbits after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT01003, Edition Number: M-122324-01-2 Date: 17.02.2004 GLP, unpublished Yes BCS

KIIA 5.7.1 /01 Gilmore, R. G.;

Fickbohm, B. L. 2005 An acute oral neurotoxicity screening study wi th technical grade BYI 08330 in Wistar Rats Bayer CropScience LP, Stilwell, KS, USA Bayer CropScience AG, Report No.: 201283, Edition Number: M-254187-01-1 Date: 13.04.2005 GLP, unpublished Yes BCS

KIIA 5.8 /01 Schuengel, M. 2005 BYI 08330-CIS-Ketoh ydroxy - Acute toxicity in the rat after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02506, Edition Number: M-258306-01-2 Date: 14.10.2005 GLP, unpublished Yes BCS– –

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Y/N-R/NR Owner

KIIA 5.8 /02 Herbold, B. 2005 BYI 08330-CIS-Ketohyd roxy (Project: BYI 08330) - Salmonella/microsome test - Plate incorporation and preincubation method Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02735, Edition Number: M-262850-01-3 Date: 15.12.2005 GLP, unpublished Yes BCS

KIIA 5.8 /03 Schuengel, M. 2006 BYI 08330-desmethyl -ketohydroxy - Acute toxicity in the rat after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02927, Edition Number: M-269279-01-2 Date: 06.04.2006 GLP, unpublished Yes BCS

KIIA 5.8 /04 Wirnitzer, U. 2006 BYI 08330-desmethyl -ketohydroxy (Project: BYI 08330) - Salmonella/microsome test - Plate incorporation and preincubation method Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT03027, Edition Number: M-271090-01-2 Date: 09.05.2006 GLP, unpublished Yes BCS

KIIA 5.8 /05 Schuengel, M. 2005 BYI 08330-mono-hydr oxy - Acute toxicity in the rat after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02687, Edition Number: M-262070-01-2 Date: 08.12.2005 GLP, unpublished Yes BCS

KIIA 5.8 /06 Herbold, B. 2005 BYI 08330-mono-hydrox y (Project: BYI 08330) - Salmonella/microsome test - Plate incorporation and preincubation method Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02716, Edition Number: M-262976-01-2 Date: 13.12.2005 GLP, unpublished Yes BCS

KIIA 5.8 /07 Schuengel, M. 2006 BYI 08330-di-hydrox y - Acute toxicity in the rat after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02995, Edition Number: M-270700-01-2 Date: 05.05.2006 GLP, unpublished Yes BCS– –

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Y/N-R/NR Owner

KIIA 5.8 /08 Herbold, B. 2006 BYI 08330-di-hydroxy (Project: BYI 08330) - Salmonella/microsome test - Plate incorporation and preincubation method Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT03069, Edition Number: M-271980-01-2 Date: 24.05.2006 GLP, unpublished Yes BCS

KIIA 5.9.1 /01 Steffens, W. 2005 Assessment of pote ntial skin sensitization incidence in workers in handling BYI 08330 Bayer CropScience AG, Report No.: M-257035-01-1, Edition Number: M-257035-01-1 Date: 05.09.2005 Non GLP, unpublished Yes BCS

KIIA 5.9.1 /02 Kehrig, B.;

Steffens, W. 2006 Occupational medical experiences with Spirotetramat/BYI 08330 Bayer Industry Services, Dormagen, Germany Bayer CropScience AG, Report No.: M-277039-01-1, Edition Number: M-277039-01-1 Date: 30.08.2006 Non GLP, unpublished Yes BCS

KIIA 5.9.2 /01 Temerowski, M. 2006 BYI 08330 (Spiro tetramat) - Assessment of literature research in various databases Bayer CropScience AG, Report No.: M-275046-01-1, Edition Number: M-275046-01-1 Date: 27.07.2006 Non GLP, unpublished Yes BCS

KIIA 5.9.3 /01 Temerowski, M. 2006 BYI 08330 (Spiro tetramat) - Assessment of literature research in various databases Bayer CropScience AG, Report No.: M-275046-01-1, Edition Number: M-275046-01-1 Date: 27.07.2006 Non GLP, unpublished Yes BCS

KIIA 5.9.4 /01 Temerowski, M. 2006 BYI 08330 (Spiro tetramat) - Assessment of literature research in various databases Bayer CropScience AG, Report No.: M-275046-01-1, Edition Number: M-275046-01-1 Date: 27.07.2006 Non GLP, unpublished Yes BCS

KIIA 5.10 /01 Temerowski, M. 2008 Spirotetramat (sy n. BYI 08330) - High dose reproductive effects in male rats and their relevance to humans Bayer CropScience AG, Report No.: M-297775-01-1, Edition Number: M-297775-01-1 Date: 20.02.2008 Non GLP, unpublished Yes BCS– –

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Y/N-R/NR Owner

KIIA 5.10 /02 Temerowski, M. 2008 Spirotetramat (sy n. BYI 08330) - Setting of an ARfD for Spirotetramat Bayer CropScience AG, Report No.: M-296931-01-1, Edition Number: M-296931-01-1 Date: 31.01.2008 Non GLP, unpublished Yes BCS

KIIA 5.10 /03 Cappon, G. D.;

Fleeman, T. L.; Chapin, R. E.; Hurtt, M. E. 2005 Effects of feed restriction during organogenes is on embryo-fetal development in rabbit Publisher:2005 Wiley-Liss, Inc., Journal:Birth Defects Research (Part B), Volume:74, Pages:424-430, Year:2005, Report No.: Lit. 8639, Edition Number: M-274544-01-1 Non GLP, published also filed: KIIIA1 10.3.1.3 /01 No

KIIA 5.10 /04 Clark, R. L.;

Robertson, R. T.; Peter, C. P.; Bland, J. A.; Nolan, T. E.; Oppenheimer, L.; Bokelman, D. L. 1986 Association between adverse maternal and embryo-fetal effects in Norfloxacin-treated and food-deprived rabbits Publisher:Society of Toxicology, Journal:Fundamental and Applied Toxicology, Volume:7, Pages:272-286, Year:1986, Report No.: M-296863-01-1, Edition Number: M-296863-01-1 Non GLP, published No

KIIA 5.10 /05 Matsuzawa, T.;

Nakata, , M.; Goto, I.; Tsushima, M. 1981 Dietary deprivation induces fetal loss and abo rtion in rabbits Publisher:Elsevier/North-Holland Scientific Publishers Ltd., Location:Netherlands, Journal:Toxicology, Volume:22, Issue:--, Pages:255-259, Year:1981, Report No.: M-296871-01-1, Edition Number: M-296871-01-1 Non GLP, published No

KIIA 5.10 /06 Petrere, J. A.;

Rohn, W. R.; Grantham, L. E.; Anderson, J. A. 1993 Food restriction during organogenesis in rabbi ts: Effects ob reproduction and the offspring Publisher:Society of Toxicology, Journal:Fundamental and Applied Toxicology, Volume:21, Issue:--, Pages:517-522, Year:1993, Report No.: M-296872-01-1, Edition Number: M-296872-01-1 Non GLP, published No– –

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KIIA 5.10 /07 Matsuoka, T.;

Mizoguchi, Y.; Serizawa, K.; Ishikura, T.; Mizuguchi, H.; Asano, Y. 2006 Effects of stage and degree of restricted feed ing on pregnancy outcome in rabbits Journal:The Journal of Toxicological Science, Volume:31, Issue:2, Pages:169-175, Year:2006, Report No.: M-298488-01-1, Edition Number: M-298488-01-1 Non GLP, published No

KIIA 5.10 /08 Christenson, W.

R. 2008 Technical grade spirotetramat (BYI 08330): Further examination of the finding of brain dilatation in the on year dog study Bayer CropScience LP, RTP, NC, USA Bayer CropScience AG, Report No.: G201862, Edition Number: M-298505-01-1 Date: 03.03.2008 Non GLP, unpublished Yes BCS

KIIA 5.10 /09 Christenson, W.

R. 2008 Comparison of neurotoxic potential, declines i n thyroid hormone and data on sensitivity of the young in studies conducted with Spiromesifen, Spirodiclofen, and Spirotetramat Bayer CropScience, Research Triangle Park, NC, USA Bayer CropScience AG, Report No.: M-298729-01-1, Edition Number: M-298729-01-1 Date: 10.03.2008 Non GLP, unpublished Yes BCS

Annex III information, tests and studies Movento 150 OD
KIIIA1 7.1.1 /01 Schuengel, M. 2005 BYI 08330 150 OD - Acute toxicit y in the rat after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02161, Edition Number: M-254331-01-2 Date: 01.07.2005 GLP, unpublished Yes BCS

KIIIA1 7.1.2 /01 Schuengel, M. 2005 BYI 08330 150 OD - Acute toxicit y in the rat after dermal application Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02164, Edition Number: M-254642-01-2 Date: 01.07.2005 GLP, unpublished Yes BCS– –

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Y/N-R/NR Owner

KIIIA1 7.1.3 /01 Pauluhn, J. 2005 BYI 08330 150 OD - Acute inhalatio n toxicity in rats Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02396, Edition Number: M-257809-01-2 Date: 14.09.2005 GLP, unpublished also filed: KIIIA2 7.1.3 /01 Yes BCS

KIIIA1 7.1.4 /01 Schuengel, M. 2005 BYI 08330 150 OD - Acute skin ir ritation/corrosion on rabbits Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02359, Edition Number: M-256984-01-2 Date: 31.08.2005 GLP, unpublished Yes BCS

KIIIA1 7.1.5 /01 Schuengel, M. 2005 BYI 08330 150 OD - Acute eye irr itation on rabbits Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02358, Edition Number: M-256988-01-3 Date: 31.08.2005 GLP, unpublished Yes BCS

KIIIA1 7.1.6 /01 Vohr, H.-W. 2005 BYI 08330 150 OD (Project: BYI 083 30) - Study for the skin sensitization effect in guinea pigs (Buehler patch test) Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT01873, Edition Number: M-246231-02-2 Date: 18.02.2005 GLP, unpublished Yes BCS

KIIIA1 7.1.6 /02 Vohr, H.-W. 2005 BYI 08330 150 OD ready to use dilu tion (Project: BYI 08330) - Study for the skin sensitization effec t in guinea pigs (Buehler patch test) Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02570, Edition Number: M-260111-01-2 Date: 03.11.2005 GLP, unpublished Yes BCS

KIIIA1 7.3.1 /01 Lundehn, J. R.;

Westphal, D.; Kieczka,, H.; Krebs, B.; Loecher-Bolz, S.; Maasfeld, W.; Pick, E. D. 1992 Uniform principles for safeguarding the health of applicators of plant protection products (Uniform principles for operator protection) Publisher:Kommissionsverlag Paul Parey, Location:Berlin, Germany, Journal:Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft, Volume:277, Pages:1-112, Year:1992, Report No.: MO-99-018858, Edition Number: M-001230-02-1 Non GLP, published No– –

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Y/N-R/NR Owner

KIIIA1 7.3.1 /02 Anon. 1992 UK Predictive Operator Exposure Model (P OEM): A users guide Publisher:PSD, Journal:Pesticides Safety Directorate, Year:1992, Report No.: MO-01-012223, Edition Number: M-054618-01-1 Non GLP, published No

KIIIA1 7.3.1 /03 Szilvasi, S. 2001 Guide to the agronomic parameters used in reaching a provisional estimate of the risks associated with the use and application of agricultural antiparasitic products Journal:Ministere de l'agriculture, France, Pages:1-19, Year:2001, Report No.: MO-04-005969, Edition Number: M-103300-01-2 Non GLP, published No

KIIIA1 7.3.1 /04 Mich, G. 1996 Operator exposure in greenhouses duri ng practical use of plant protection products - Final report ECON, Ingelheim, Germany Bayer CropScience AG, Report No.: EF 94-02-03, Edition Number: M-024096-01-1 Date: 06.06.1996 GLP, unpublished Yes BCS

KIIIA1 7.4.1 /01 Ganzelmeier, H.;

Rautmann, D.; Spangenberg, R.; Streloke. M.; Herrmann, M.; Wenzelburger, H. J.; Walter, H. F. 1995 Studies on the spray drift of plant protection products Publisher:Blackwell Wissenschaftsverlag GmbH, Location:Berlin/Wien, Journal:Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft, Volume:305, Pages:1-113, Year:1995, Report No.: MO-98-000356, Edition Number: M-001224-01-1 Non GLP, published No

KIIIA1 7.4.1 /02 Lundehn, J. R.;

Westphal, D.; Kieczka,, H.; Krebs, B.; Loecher-Bolz, S.; Maasfeld, W.; Pick, E. D. 1992 Uniform principles for safeguarding the health of applicators of plant protection products (Uniform principles for operator protection) Publisher:Kommissionsverlag Paul Parey, Location:Berlin, Germany, Journal:Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft, Volume:277, Pages:1-112, Year:1992, Report No.: MO-99-018858, Edition Number: M-001230-02-1 Non GLP, published No– –

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Y/N-R/NR Owner

KIIIA1 7.5.1 /01 Hoernicke, E.;

Nolting, H.G.; Westphal, D. 1998 Label instructions for the protection of worke rs reentering crop growing areas after application of plant protection products Publisher:Eugen Ulmer GmbH & Co., Location:Stuttgart, Journal:Nachrichtenbl. Deut. Pflanzenschutzd., Volume:50, Issue:10, Pages:267-269, Year:1998, Report No.: MO-04-000845, Edition Number: M-107544-01-1 Non GLP, published No

KIIIA1 7.5.1 /02 Brouwer, D. H.;

Haan, M. de; Hemmen, J. J.

van 2001 Modeling re-entry exposure estimates: techniqu es and application rates Publisher:CRC Press, Journal:Worker exposure to agrochemicals, Volume:119, Pages:119-138, Year:2001, Report No.: MO-03-011549, Edition Number: M-110514-01-1 Non GLP, published No

KIIIA1 7.5.1 /03 Krieger, R. I.;

Ross, J. H.; Thongsinthusak, T. 1992 Assessing human exposure to pesticides Publisher:Springer-Verlag, Location:New York, USA, Journal:Reviews of Environmental Contamination and Toxicology, Volume:128, Issue:--, Pages:1-15, Year:1992, Report No.: MO-02-004605, Edition Number: M-049968-01-1 Non GLP, published No

KIIIA1 7.5.1 /04 Nigg, H.N.;

Stamper, J.H.; Queen, R.M. 1984 The devleopment and use of a univeral model to predict tree crop harvester pesticide exposure Publisher:Springer-Verlag, Location:New York, Journal:Florida Agricultural Experiment Stations Journal Series No. 4586, Pages:5, Year:1984, Report No.: PR 182-186, Edition Number: M-245489-01-1 Non GLP, published No– –

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Y/N-R/NR Owner

KIIIA1 7.5.1 /05 Stamper, J.H.;

Nigg, H.N.; Queen, M. 1986 Predicted of pesticide dermal exposure and urinary metabolite level of tree crop harvesters from field residues Publisher:Springer-Verlag, Location:New York, Journal:Florida Agricultural Experiment Station Journal No. 6267, Pages:8, Year:1986, Report No.: 36-693-700, Edition Number: M-245482-01-1 Non GLP, published No

KIIIA1 7.6.1 /01 Odin-Feurtet, M. 2003 [14C]-BYI 08330 in OD150 form ulation: In vivo dermal absorption study in the male rat Bayer CropScience SA, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA06009, Edition Number: M-276579-01-2 Date: 02.10.2003 GLP, unpublished Yes BCS

KIIIA1 7.6.2 /01 Capt, A. 2006 [14C]-BYI 08330 in OD150 formulation: Comparative in vitro dermal absorption study using human and rat skin Bayer CropScience SA, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA05254, Edition Number: M-269449-01-2 Date: 05.04.2006 GLP, unpublished Yes BCS

KIIIA1 7.6.2 /02 Capt, A. 2006 [14C]-BYI 08330 in SC240 formulation: Comparative in vitro dermal absorption study using human and rat skin Bayer CropScience SA, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA05255, Edition Number: M-269195-01-2 Date: 05.04.2006 GLP, unpublished Yes BCS

Movento 240 SC
KIIIA1 7.1.1 /01 Schuengel, M. 2005 BYI 08330 240 SC - Acute toxicit y in the rat after oral administration Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02162, Edition Number: M-254639-01-1 Date: 01.07.2005 GLP, unpublished Yes Bayer CropScie

nce

KIIIA1 7.1.2 /01 Schuengel, M. 2005 BYI 08330 240 SC - Acute toxicit y in the rat after dermal application Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02165, Edition Number: M-254643-01-1 Date: 01.07.2005 GLP, unpublished Yes Bayer CropScie

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Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIIA1 7.1.3 /01 Pauluhn, J. 2005 BYI 08330 240 SC - Acute inhalatio n toxicity in rats Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02374, Edition Number: M-257562-01-1 Date: 08.09.2005 GLP, unpublished Yes Bayer CropScie

nce

KIIIA1 7.1.4 /01 Schuengel, M. 2005 BYI 08330 240 SC - Acute skin ir ritation/corrosion on rabbits Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02291, Edition Number: M-256010-01-1 Date: 19.08.2005 GLP, unpublished Yes Bayer CropScie

nce

KIIIA1 7.1.5 /01 Schuengel, M. 2005 BYI 08330 240 SC - Acute eye irr itation on rabbits Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02290, Edition Number: M-255999-01-1 Date: 19.08.2005 GLP, unpublished Yes Bayer CropScie

nce

KIIIA1 7.1.6 /01 Vohr, H.-W. 2005 BYI 08330 240 SC (Project: BYI 083 30) - Study for the skin sensitization effect in guinea pigs (Buehl er patch test) Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT01876, Edition Number: M-246259-01-1 Date: 25.02.2005 GLP, unpublished Yes Bayer CropScie

nce

KIIIA1 7.1.6 /02 Vohr, H.-W. 2005 BYI 08330 240 SC ready to use dilu tion (Project: BYI 08330) - Study for the skin sensitization effec t in guinea pigs (Buehler patch test) Bayer HealthCare AG, Wuppertal, Germany Bayer CropScience AG, Report No.: AT02598, Edition Number: M-260366-01-1 Date: 08.11.2005 GLP, unpublished Yes Bayer CropScie

nce

KIIIA1 7.3.1 /01 Lundehn, J. R.;

Westphal, D.; Kieczka,, H.; Krebs, B.; Loecher-Bolz, S.; Maasfeld, W.; Pick, E. D. 1992 Uniform principles for safeguarding the health of applicators of plant protection products (Uniform principles for operator protection) Publisher:Kommissionsverlag Paul Parey, Location:Berlin, Germany, Journal:Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft, Volume:277, Pages:1-112, Year:1992, Report No.: MO-99-018858, Edition Number: M-001230-02-1 Non GLP, published No– –

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Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIIA1 7.4.1 /01 Ganzelmeier, H.;

Rautmann, D.; Spangenberg, R.; Streloke. M.; Herrmann, M.; Wenzelburger, H. J.; Walter, H. F. 1995 Studies on the spray drift of plant protection products Publisher:Blackwell Wissenschaftsverlag GmbH, Location:Berlin/Wien, Journal:Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft, Volume:305, Pages:1-113, Year:1995, Report No.: MO-98-000356, Edition Number: M-001224-01-1 Non GLP, published No

KIIIA1 7.4.1 /02 Lundehn, J. R.;

Westphal, D.; Kieczka,, H.; Krebs, B.; Loecher-Bolz, S.; Maasfeld, W.; Pick, E. D. 1992 Uniform principles for safeguarding the health of applicators of plant protection products (Uniform principles for operator protection) Publisher:Kommissionsverlag Paul Parey, Location:Berlin, Germany, Journal:Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft, Volume:277, Pages:1-112, Year:1992, Report No.: MO-99-018858, Edition Number: M-001230-02-1 Non GLP, published No

KIIIA1 7.5.1 /01 Hoernicke, E.;

Nolting, H.G.; Westphal, D. 1998 Label instructions for the protection of worke rs reentering crop growing areas after application of plant protection products Publisher:Eugen Ulmer GmbH & Co., Location:Stuttgart, Journal:Nachrichtenbl. Deut. Pflanzenschutzd., Volume:50, Issue:10, Pages:267-269, Year:1998, Report No.: MO-04-000845, Edition Number: M-107544-01-1 Non GLP, published No

KIIIA1 7.5.1 /02 Brouwer, D. H.;

Haan, M. de; Hemmen, J. J.

van 2001 Modeling re-entry exposure estimates: techniqu es and application rates Publisher:CRC Press, Journal:Worker exposure to agrochemicals, Volume:119, Pages:119-138, Year:2001, Report No.: MO-03-011549, Edition Number: M-110514-01-1 Non GLP, published No– –

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Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIIA1 7.5.1 /03 Krieger, R. I.;

Ross, J. H.; Thongsinthusak, T. 1992 Assessing human exposure to pesticides Publisher:Springer-Verlag, Location:New York, USA, Journal:Reviews of Environmental Contamination and Toxicology, Volume:128, Issue:--, Pages:1-15, Year:1992, Report No.: MO-02-004605, Edition Number: M-049968-01-1 Non GLP, published No

KIIIA1 7.5.1 /04 Spencer, J., R.;

Sanborn, J., R.; Hernandez, B.,Z., Schneider, F., A.; Margetich, S., S. 1991 Long and short intervals of dermal exposure of peach harvesters to foliar Azinphos-methyl residues Publisher:California Department of Food and Agriculture, Location:Sacramento, California, Pages:1 - 15, Year:1991, Report No.: HS-1578, Edition Number: M-243413-01-1 Non GLP, published No

KIIIA1 7.5.1 /05 Spencer, J., R.;

Hernandez, B.,Z.; Schneider, F., A.; Sanborn, J., R.; Margetich, S.,S.; Begum, S.; Wilson, B., W. 1993 Dermal and urinary monitoring of peach and app le harvesters exposed to organophosphate resideus in sutter, stanislaus and madera countries, 1989 and 1990 Publisher:California Environmental Protection Agency, Location:Sacramento, California, Pages:1 - 31, Year:1993, Report No.: HS-1577, Edition Number: M-243429-01-1 Non GLP, published No

KIIIA1 7.5.1 /06 Schneider, F.;

Spencer , J.; Sanborn J.; Alcoser,m D.; Garza, R.; Margetich, S.; Valle, M. 1990 Dermal and urinary monitoring of nectarine harvesters exposed to azinphos-methyl residues in fresno country California 1988 Publisher:California Department of Food and Agriculture Division of, Location:Fresno, Journal:Protection, and worker safety Worker Health and Safety Branch 1220 N Street, Sacramento, California, Pages:19, Year:1990, Report No.: HS-1532, Edition Number: M-245496-01-1 Non GLP, published No– –

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Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIIA1 7.5.1 /07 Urtizberea, M. 2002 Post-application worker exposur e study and determination of transfer coefficient during harvesting of peaches treated by Rovral Aqua Flo Aventis CropScience, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA 98151, Edition Number: M-075639-01-1 Date: 03.04.2002 GLP, unpublished Yes BCS

KIIIA1 7.6.2 /01 Capt, A. 2006 [14C]-BYI 08330 in SC240 formulation: Comparative in vtiro dermal absorption study using human and rat skin Bayer CropScience SA, Sophia Antipolis, France Bayer CropScience AG, Report No.: SA05255, Edition Number: M-269195-01-2 Date: 05.04.2006 GLP, unpublished Yes Bayer CropScie

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# Appendix



# Spirotetramat

Toxicological Risk Assessment for the EU (Annex III)– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 488 April 2008

Table of contents

B.6.10.1 Proposal for ADI...................................

................................................... ..............................489 B.6.10.2 Proposed ARfD......................................

................................................... ............................489 B.6.10.3 Proposal for drinking water limit..................

................................................... .......................489 B.6.10.4 Proposal for AOEL..................................

................................................... ...........................489

B.6.12 Dermal absorption (annex IIIA 7.6).................

................................................... ................491 B.6.12.1 Dermal absorption, in vivo in the rat ..............

................................................... ....................491 B.6.12.2 Comparative dermal absorption, in vitro using human and rat skin .....................................4 93 B.6.12.3 Summary on dermal absorption.......................

................................................... ..................496

B.6.13 Toxicological data on non active substances (Annex IIIA 7.9) ......................................497 B.6.13.1 Material safety data sheet for each formulant ......

................................................... .............497 B.6.13.2 Available toxicological data for each formulant ....

................................................... .............497

B.6.14 Exposure data (annex IIIA 7.3, 7.4 and 7.5) ........

................................................... ...........498 B.6.14.1 Operator exposure (annex IIIA 7.3) .................

................................................... ..................498 B.6.14.1.1 Estimation of operator exposure assuming personal p rotective equipment is not used ......499 B.6.14.1.2 Estimation of operator exposure assuming personal p rotective equipment is used ............501 B.6.14.1.3 Measurement of operator exposure – (Mixer/Loader/Ap plicator).........................................5 01 B.6.14.1.4 Summary on operator exposure .......................

................................................... .................501 B.6.14.2 Bystander exposure (annex IIIA 7.4) ................

................................................... .................502 B.6.14.2.1 Estimation of bystander exposure ...................

................................................... ..................502 B.6.14.2.2 Measurement of bystander exposure ..................

................................................... ..............503 B.6.14.2.3 Summary on bystander exposure......................

................................................... ................503 B.6.14.3 Worker exposure (annex IIIA 7.5)...................

................................................... ...................504 B.6.14.3.1 Estimation of worker exposure assuming personal pro tective equipment is not used.........504 B.6.14.3.2 Estimation of worker exposure assuming personal pro tective equipment is used...............505 B.6.14.3.3 Estimation of operator exposure assuming personal p rotective equipment is used and

using data generated on dislodgeable residues under the proposed conditions of use ......505 B.6.14.3.4 Measurement of worker exposure .....................

................................................... ................505 B.6.14.3.5 Summary on worker exposure.........................

................................................... ..................505

B.6.15 Dislogeable residues (annex IIIA 7.7) ..............

................................................... ..............506
Appendix I: Detailed Exposure Calculations (UK POEM ).................................................. .......................508
Appendix II: Detailed Exposure Calculations (BBA Mo del) ............................................... ......................510– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 489 April 2008

B.6.10.1 Proposal for ADI

The estimation of the Acceptable Daily Intake (ADI) is based on the lowest no-observed adverse effect level (NOAEL) observed in subchronic and chronic toxicity , carcinogenicity, reproduction and neurotoxicity s tudies with spirotetramat. Given the results from all rele vant studies (see table 1.1.1-1), the lowest NOAEL of 5.0 mg/kg bw/d was found in the 1 year toxicity study i n dogs. Applying the standard uncertainty factor of 100 to account for intraspecies and interspecies variabili ty to the NOAEL of 5.0 mg/kg bw/d from the dog stud y mentioned, results in an ADI of 0.05 mg/kg bw/d

.

Remark : The notifier proposed an ADI of 0.132 mg/kg bw/d based upon the NOAEL of 13.2 mg/kg bw/d established in the 1 year toxicity study in rats.

B.6.10.2 Proposed ARfD

For the determination of the Acute Reference Dose ( ARfD), results from oral studies that used acute or short term exposure are considered to be the most relevan t. Spirotetramat is of low acute oral toxicity. The lowest relevant NOAEL of 10 mg/kg bw/d was established in the developmental toxicity study in rabbits. Aborti on due to systemic maternal toxicity occurred in one f emale at the next higher dose level of 40 mg/kg bw/ day on gestation day 24. The selection of this endpoint is overestimating the actual risk upon acute exposure and the NOAEL for this endpoint is therefore not the mo st appropriate one to derive the ARfD. For this pur pose, the acute neurotoxicity study in rats is considered appropriate. In this study, a NOAEL of 100 mg/kg b w/d was established, based on urine and perianal stain and decreased motor activity at 200 mg/kg bw/d. So taking into account the standard uncertainty fac tor of 100, an ARfD of 1 mg/kg bw/d is proposed.

B.6.10.3 Proposal for drinking water limit

The determination of a maximum allowable concentrat ion (MAC) value in drinking water is not necessary, because according to Directive 91/414/EC only the A DI, ARfD and AOEL values have to be determined. According to Directive 98/83/EC a drinking water li mit of 0.1 μg spirotetramat/l is established.

B.6.10.4 Proposal for AOEL

According to the principles of Annex VI to Directiv e 91/414 EEC, the proposed acceptable operator expo sure level should be established on the basis of the hig hest dose at which no adverse effect is observed in relevant studies in the most sensitive species. The setting of an AOEL is usually based on mid-term st udies (i.e. subacute/ subchronic and reproduction or deve lopmental toxicity studies) since these studies in most cases can be considered a more appropriate model fo r the actual operator exposure to be expected. The lowest NOAEL of all relevant studies were found in the 1 year dog study, which is considered a mid -term study. If a 100 fold safety factor (for intraspecie s and interspecies variability) is applied to the s ystemic NOAEL, a systemic AOEL of 0.05 mg/kg bw/d is proposed.

Remark : The notifier proposed a systemic AOEL of 0.1 mg/k g bw/d based on the NOAEL of 10 mg/kg bw/d derived in the developmental toxicity study in rabb its.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 490 April 2008

Table 6.10-1: Summary of repeat oral dose studies w ith spirotetramat for setting the ADI/AOEL/
ARfD Study; Reference Dose levels NOAEL Relevant effects Beagle dogs, 1 year oral Eigenberg D; 2006 0, 6, 20, 55 mg/kg bw/d ( ♂ ), 0, 5, 19, 48 mg/kg bw/d ( ♀ ) 6 mg/kg bw/d ( ♂ ) 5 mg/kg bw/d ( ♀ ) -thymus involution

-brain dilation -T3, T4 ↓ Wistar rats, 1 year oral Wahle B; 2005 0, 13.2, 189, 414 mg/kg bw/d ( ♂ ), 0, 18, 255, 890 mg/kg bw/d ( ♀ ) 13.2 mg/kg bw/d ( ♂ ) 255 mg/kg bw/d ( ♀ ) -bodyweight ↓

-bodyweight gain ↓ -alveolar macrophages ↑ -discoloration of the lung ( ♀ ) -relative liver weight ↑ -relative kidney weight ↑ ( ♀ ) Wistar rats, 2 year oral Wahle B; 2006 0, 12.5, 169 and 373 mg/kg bw ( ♂ ), 0, 16.8, 229 and 823 mg/kg bw ( ♀ ) 12.5 mg/kg bw ( ♂ ); 16.8 mg/kg bw ( ♀ ) -bodyweight ↓

-bodyweight gain ↓ -alveolar macrophages ↑ ( ♂ ) -interstitial pneumonia - relative lung weight ↑ ( ♀ ) -relative liver weight ↑ ( ♀ ) -biliary hyperplasia/fibrosis ( ♀ ) -absolute kidney weight ↓ -tubular dilation -spermatid degeneration -immature/exfoliated germ cells CD-1 mice, 18 months oral Wahle B; 2006 0, 10.9, 263 and 1022 mg/kg bw ( ♂ ), 0, 13.7, 331 and 1319 mg/kg bw ( ♀ ) 1022 mg/kg bw ♂ ; 1319 mg/kg bw ♀ )

Multigeneration, rats Young A.D., 2006 0, 17.2, 70.7, 419.3 mg/kg bw/d ( ♂ ), 0, 20, 82.5 and 484.7 mg/kg bw/d ( ♀ ) Parental, reproductive and : developmental: 70 mg/kg bw Parental:

-bodyweight ↓ -bodyweight gain ↓ -kidney weight ↓ -histopath. changes in kidney Reproductive: -abnormal sperm cells -fertility ↓ -food consumption ↓ Offspring: -bodyweight ↓ -bodyweight gain ↓ Developmental toxicity, rats Klaus A.M.; 2004 0, 20, 140, 1000 mg/kg bw/d Maternal and developmental: 140 mg/kg bw maternal:

-food consumption ↓ -body weight loss developmental: fetal weight ↓ skeletal deviations Developmental toxicity, rabbits Klaus A.M.; 2004 0, 10, 40, 160 mg/kg bw maternal: 10 mg/kg bw developmental: 160 mg/kg bw maternal:

-food and water consumption ↓ -body weight loss -clinical signs -abortion developmental: no effects Acute neurotoxicity Gilmore R.G.; 2005 0, 50, 100, 500 mg/kg bw systemic: 100 mg/kg bw neurotox.: 500 mg/kg bw systemic:

-clinical signs motor activity ↓ ( ♂ ) no evidence of neurotoxicity– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 491 April 2008

B.6.12 Dermal absorption (annex IIIA 7.6)

B.6.12.1 Dermal absorption, in vivo in the rat

Reference: [ 14 C]-BYI 08330 in OD150 formulation: In vivo dermal a bsorption study in the

male rat Study No.: SA 06009 Author(s), year: Odin-Feurtet, M.; 2006 Guideline(s): OPPTS Guideline Nr. 870.7600 (1998) GLP: Yes Deviations: None Validity: Yes

Material and methods: The in vivo

dermal absorption of BYI 08330 was determined by u sing [ 14 C]-BYI 08330 in 150 OD formulation (Batch no. KML 2978-2; radiochemical purity > 99%) following single dermal application to male rats (s train: Wistar Rj: WI (IOPS HAN) derived, source: R. Janvie r, France) at 3 concentrations: high dose (nominal value 100 μg a.i./cm 2 ), intermediate dose (nominal value 15 μg a.i./cm 2 ) and low dose (nominal value 5 μg a.i./cm 2 ). For each dose, 4 groups consisting of 4 animals each, were used:

Table 6.12.1-1: Experimental design

Study section Group number Number of animals Time of exposure & sacrifice

1 4 1
2 4 10
3 4 24 High dose (100 μg a.i./cm 2 )
4 4 10 (168)*
5 4 1
6 4 10
7 4 24 Intermediate dose (15 μg a.i./cm 2 )
8 4 10 (168)*
9 4 1
10 4 10
11 4 24 Low dose (5 μg a.i./cm 2 )
12 4 10 (168)*

*: time of exposure 10 hours, time of sacrifice 168 hours after application

Measured amounts (120 μl) of each dose formulation were applied to 12 cm 2 of skin, corresponding to nominal doses of 1.2 mg/rat, 0.2 mg/rat and 0.06 mg /rat respectively. The application site was semioccluded with a perforated plastic cover (to allow ventilation), held in place over the plastic saddle with surgical tape. The exposure time of the formulation on the skin wa s identical with the time of sacrifice except for g roups 4, 8 and 12. In these groups the animals were exposed for 10 hours, then the dose site was washed and the animals sacrificed 168 hours post dose application. The application sites of all rats were washed with mild liquid soap solution using natural sponge swab s to remove non-absorbed radioactivity. Swabbing was con tinued until no radioactivity was detected in swabs using a Geiger counter. The skin was shaved again a nd tape-stripped to remove the stratum corneum. Adhesive tape was applied for 5 seconds, then remov ed and this was repeated until the stratum corneum removal was complete.– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 492 April 2008

Urine, faeces and cage wash were collected at 1, 10 , 24 and 168 hours post dose. Prior to sacrifice, b lood samples were taken. The treated area of skin and a surrounding annular ring of untreated skin (approximately 1cm) were exc ised and taken for analysis as well as an isolated contr ol portion of untreated skin. The residual carcass was retained for analysis also. The amounts detected in urine, faeces, cage wash, c arcass, blood, stratum corneum (tape strips 3 to th e last tape strip), treated skin (skin after tape-strippin g), non-treated skin and surrounding skin were cons idered as absorbed [the amount located in the skin should be considered as being absorbed in case the experiment is terminated before serial non-detects in excreta are observed and/or no clear decline in excreta is mea sured (Sanco/222/2000 rev.7)]. Radioactivity recovered from the swabs, dressing (s addle, surgical tape, gauze and cover), fur and sur face dose (tape strips 1 & 2) was considered to be unabs orbed. Radioactivity in samples was analysed by liquid sci ntillation counting (LSC).

Findings: Recovery of radiolabelled test material of the appl ied dose was acceptable, with recovery rates of 94. 9 – 99.0% for the high dose (100 μg a.i./cm 2 ), 92.3 – 95.5% for the intermediate dose (15 μg a. i./cm 2 ) and 93.4 – 100.6% for the low dose (5 μg a.i./cm 2 ). Based on the likely operator/worker exposure time f rame, the most appropriate exposure period would be 10 hours. So the dermal absorption values calculated f or the groups of animals for which the skin was was hed after 10 hours (groups 2, 6, and 10) and for those for which the skin was washed after 10 hours and wh ich were sacrificed after 168 hours (groups 4, 8 and 12 ) were taken into account for determination of derm al absorption of BYI 08330 in vivo. After 10 hours exposure, the in vivo dermal absorption of [ 14 C]-BYI 08330 in 150 OD formulation at the 3 concentrations accounted for means of approximately 17.6%, 10.4% and 12% of the dose respectively. Over 168 hours following a 10 hour exposure period, the in vivo dermal absorption of [ 14 C]-BYI 08330 in 150 OD formulation at the 3 concentrations accounted fo r means of approximately 10.6%, 4.8% and 5.8% of th e dose respectively. At least more than 80% of the ap plied radioactivity was readily washed from the ski n surface after the 10 hour exposure interval. Skin stripping showed that most radioactivity at th e application site was in the stratum corneum. The amount of radioactivity in the stratum corneum, taken afte r the 10 hour application period and after skin was h, was 14.26% for the high dose, 7.32% for the intermediat e and 10.02% for the low dose, respectively. Termination after 168 hours showed that mean radioactivity in t he stratum corneum decreased (2.6% for the high and 1.53% for the intermediate and low dose, respective ly), indicating further resorption from this depots

. The absorption and excretion pattern and the mean p ercentage of the total dose found in the different compartments are presented in table 1.5.1-2.

Table 6.12.1-2 Absorption and excretion after percu taneous administration of [ 14 C]-BYI 08330 (mean values expressed as % of the dose applied)

High dose 100 μg a.i./cm 2 Intermediate dose 15 μg a.i./cm 2 Low dose 5 μg a.i./cm 2

Time group (h) 1 10 24 10/16 8 1 10 24 10/16 8 1 10 24 10/16 8

Urine 0.0 0.26 0.79 2.06 N.S. 0.24 0.8 1.83 0.01 0. 34 0.93 1.49

Faeces 0.0 0.0 0.04 0.18 0.01 0.04 0.09 0.16 0.00 0 .06 0.19 0.2

Cage wash 0.0 0.1 0.34 4.01 0.02 0.2 0.23 0.44 0.02 0.21 0.56 0.78

Blood 0.01 0.02 0.02 0.0 0.0 0.02 0.02 0.0 0.01 0.0 2 0.02 0.0– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 493 April 2008

High dose 100 μg a.i./cm 2 Intermediate dose 15 μg a.i./cm 2 Low dose 5 μg a.i./cm 2

Carcass 0.42 0.31 0.4 0.13 0.17 0.23 0.24 0.14 0.57 0.45 0.85 0.51

Stratum

corneum 5.06 14.26 9.66 2.6 4.68 7.32 14.29 1.53 1.63 10.02 7.11 1.53

Treated skin 1.57 1.63 1.73 0.28 0.73 1.11 1.36 0.19 0.51 0.58 1 .99 0.3

Untreated skin 0.53 0.38 0.18 0.24 0.18 0.11 0.13 0.18 0.15 0.16 0 .35 0.35

Surroun. skin 1.24 0.65 2.62 1.07 0.69 1.12 0.43 0.39 0.13 0.19 0 .64 0.66

Total absorbed * 8.83 17.61 15.78 10.57 6.48 10.39 17.59 4.86 3.03 1 2.03 12.64 5.82

Swabs 85.04 76.3 77.7 79.74 83.55 81.33 70.9 84.21 91.64 86.08 85.65 82.71

Fur N.S. N.S. N.S. 2.13 N.S. N.S. N.S. 1.64 N.S. N. S. N.S. 1.99

Dressing 1.56 0.33 1.27 1.85 0.48 1.1 0.22 1.25 0.1 3 0.14 0.79 2.22

Surface dose 2.46 4.66 4.24 0.58 1.83 2.72 4.16 0.41 0.8 2.2 1.5 2 0.71

Total recovery 97.89 98.92 98.99 94.87 92.34 95.54 92.87 92.37 95. 59 100.4 5 100.6

1 93.43

N.S.: No Sample; * urine, faeces, cage wash, carcass, blood, stratum c orneum, treated, untreated and surrounding skin

Conclusion: After topical application of [ 14 C]-BYI 08330 in 150 OD formulation to male rats at 3 dose levels (100, 15 and 5 μg a.i./cm 2 ), the dermal absorption was considered to be low. Means of 80% of all doses were readily washed from the skin surface using mild soap soluti on and water washes. Following a 10 hour exposure period, the in vivo dermal absorption of [ 14 C]-BYI 08330 in 150 OD formulation at the 3 concentrations accounted for means of appr oximately 17.6%, 10.4% and 12% of the dose respectively. Over 168 hours following a 10 hour exposure period, the in vivo dermal absorption of [ 14 C]-BYI 08330 in 150 OD formulation accounted for means of approximately 10.6%, 4.8% and 5.8% for the high, intermediate an d low dose, respectively.

B.6.12.2 Comparative dermal absorption, in vitro us ing human and rat skin

Reference: [ 14 C]-BYI 08330 in OD150 formulation: Comparative In v itro dermal absorption

study using human and rat skin Study No.: SA 05254 Author(s), year: Capt, A.; 2006 Guideline(s): OECD Guideline 428 (2004a,b) ECETOC, Monograph No. 20 (1993) Sanco/222/2000 rev. 7 (2004) GLP: Yes Deviations: None Validity: Yes

Material and methods: The absorption and distribution of BYI 08330 in vit ro through human and rat epidermis was determined b y using [ 14 C]-BYI 08330 (Batch no. KML 2978-2; radiochemical p urity > 99%) in 150 OD formulation concentrate and two field spray dilutions. The neat formulation was included to assess exposure to– –

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mixer/loaders and the latter dilutions to simulate exposure of spray applicators in the field. The nom inal doses were 150, 1.5 and 0.05 mg a.i./ml formulation ) The dose volume was 10 l/cm 2 skin.

Skin preparation: From 10 male rats (strain: Wistar Rj: WI; source: R. Janvier, France) a dorsal area of skin was clipped and excised for membrane preparation. T he skin was dermatomed by using a mini-dermatome. Dermal membranes 420 – 510 μM thick were prepared. Human skin membranes from 13 donors were prepared s imilarly using the mini-dermatome. Prior to use, the integrity of the selected membran es was assessed by measuring the trans-epidermal wa ter loss (TEWL) from the stratum corneum. The membranes were assembled in diffusion cells and an evaporimeter probe was placed atop the receptor cel l and water diffusion through the skin was measured (g/m 2 /hr). Human and rat skin with a TEWL > 40 g/m 2 /hr were considered potentially damaged and were no t used.

Dose preparation: The doses were applied as a 150 mg/ml OD formulati on and two spray strength dilutions of the formulation in water (1.5 mg/ml and 0.05 mg/ ml). The dose preparations were applied to the spli tthickness skin samples with a pipette at the rate o f approximately 10μl/cm 2 exposed skin.

Sampling of receptor fluid: The receptor fluid passing through the receptor ch amber was collected in glass vials held in a fraction collector which was starte d after dose application for each group was complet e. Samples were then collected hourly for the duration of the experiment (24 hours). At 8 hours post-application, the skin was swabbed w ith 1% v/v Tween 80 in PBS (phosphate buffer saline ) using sponge swabs, in order to remove and retain t he non-absorbed dose, until no radioactivity was detected with a Geiger counter. 24 hours after application, the treated skin cell w as swabbed again prior to the tape-stripping. The t apestripping procedure was continued until a “shiny” a ppearance of the epidermis was evident, which indic ated that the stratum corneum had been removed. The tape -strips were collected into scintillation vials. Th e remaining skin was removed and taken for analysis.

Analysis: Receptor fluid, skin wash, donor chamber, tape str ips (stratum corneum) and epidermis were analysed by LSC.

Findings: Mean recovery of radiolabelled test material for th e concentrate and the dilutions was between 96 – 99 % of the applied dose for human skin and 96 – 100% for r at skin. The majority (65 - 95%) of the applied dose was rem oved from the surface of human and rat skin by skin washing. For the formulation concentrate ( 150 mg/ml formulation), the remaining proportion re covered from stratum

corneum for human skin was 0.12% after the 24 hour exposur e, leaving a mean of 0.11% of the applied dose in the remaining epidermal tissue . For rat skin, recovery from stratum corneum was 1.14%, for the epidermal tissue 0.75%. For the 1.5 mg/ml formulation dilution, the remaini ng proportion recovered from stratum corneum was 1.66% (human) and 15.62% (rat) leaving a mean of 0.67% an d 4.34% respectively of the applied dose in the remaining epidermal tissue. For the 0.05 mg/ml formulation dilution, the remain ing proportion recovered from stratum corneum was 4.65% (human) and 9.22% (rat) leaving a mean of 3.2 4% and 1.26% respectively of the applied dose in th e remaining epidermal tissue. The recovery of [ 14 C]-BYI 08330 (expressed as % of dose applied) found in the different compartments after application of the concentrate and the dilutions ar e given in table1.5.2-1.– –

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Table 6.12.2-1 Mean % of applied dose detected in d ifferent compartments after application of the concentrate and the dilutions to human and rat skin

Concentrate (150 mg/ml formulation) Dilution (1.5 mg/ml formulation) Dilution (0.05 mg/ml formulation)

Human Rat Human rat human rat

Surface dose (tape strips 1 & 2) 0.29 1.32 0.68 8.52 2.46 5.57

Skin swabs (8 + 24 h) 95.54 92.02 93.01 65.15 84.72 70.12

Donor chamber 0.14 0.51 2.55 0.56 0.41 1.66

Total % nonabsorbed 95.97 93.85 96.24 74.23 87.59 77.35

Stratum corneum (tape strips excluding 1 & 2) 0.12 1.14 1.66 15.62 4.65 9.22

Epidermis (skin after tape-stripping) 0.11 0.75 0.67 4.34 3.24 1.26

Receptor fluid 0.01 0.26 0.41 3.25 0.56 12.22

Total absorbed* 0.24 2.15 2.74 23.21 8.45 22.70

total recovered 96.21 96.0 98.98 97.44 96.04 100.05

*stratum corneum, epidermis, receptor fluid

Conclusion: Mean recovery of radioactivity was in a range of 96 .0 – 100.05% of the applied dose for the formulatio n concentrate and the dilutions for human and rat ski n. The majority of the applied dose was removed by skin washing (65 - 95%). Regarding the amounts of [ 14 C]-BYI 08330 recovered in the receptor fluid, strat um corneum and epidermis as systemically available, the total penetration ra te after 8 hour exposure was 0.24% (human) and 2.15 % (rat) for the concentrate. For the dilutions, penet ration rates were 2.74% and 8.45% (human) and 23.21 % and 22.70% (rat), respectively. Rat skin appeared to be more permeable than human s kin. The potential absorption differences for human and rat skin differed by factors of 8.9, 8.5 and 2. 7 for the high, medium and low doses, respectively.– –

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B.6.12.3 Summary on dermal absorption

The results showed that the rate of absorption of [ 14 C]-BYI 08330 through human and rat epidermis from t he concentrate formulation and its v/v aqueous dilutio ns was slow. The majority of the applied dose (huma n: 85 – 95%; rat: 65 – 92%) was removed by mild skin wash ing after an exposure period of 8 hours. These data predict that the dermal absorption of BYI 08330 thr ough human or rat skin from potential exposure to t he OD formulation, either as the concentrate formulation or as the v/v aqueous spray strength dilutions, wou ld be low. In these experiments, rat skin was shown to be more permeable than human skin. The estimated dermal absorption for humans is calculated by adjusting the dermal absorption f igures for rats obtained in the in vivo

studies by the correction factors derived from the in vitro studies in rat and human skin. In order to assess the risk to workers potent ially exposed to BYI 08330, the formulation concent rate was included to assess exposure to mixer/loaders an d the spray strength dilutions are most relevant to spray operators. Following a 10 hour exposure period, the in vivo dermal absorption of [ 14 C]-BYI 08330 in 150 OD formulation at the 3 concentrations accounted for means of appr oximately 17.6%, 10.4% and 12% of the dose respectively. Over 168 hours following a 10 hour exposure period, the in vivo dermal absorption of [ 14 C]-BYI 08330 in 150 OD formulation accounted for means of approximately 10.6%, 4.8% and 5.8% for the high, intermediate an d low dose, respectively. In the in-vitro

skin penetration study with human skin, an absorpt ion rate of 0.24 % for the concentrate and 2.74 % and 8.45 % for the dilutions, respectively, was obtained after the 8 hour exposure. For rat skin, an absorption rate of 2.15% for the c oncentrate and 23.21% and 22.70% for the dilutions, respectively, was obtained after the 8 hour exposur e. So for the concentrate, the mean factor for species difference would be 8.9. For the aqueous dilutions , mean factors for species difference would be 8.5 and 2.7 , respectively. Taking into account for the rat in vivo study the mean factor for species difference of 8. 9, an absorption rate for humans of 1.19 % can be proposed for the concen trate. For the field dilutions, absorption rates of 0.56 % and 2.15 % will be assumed.

Table 6.12.3-1: Calculation of species differences in dermal absorption
Concentrate (150 mg/ml formulation) Dilution (1.5 mg/ml formulation) Dilution (0.05 mg/ml formulation) Human epidermis in vitro (8h) 0.24 2.74 8.45 Rat epidermis in vitro (8h) 2.15 23.21 22.70 Species difference correction factor 8.9 8.5 2.7

Table 6.12.3-2: Calculation of dermal absorption of BYI 08330 in humans
Concentrate (150 mg/ml formulation) Dilution (1.5 mg/ml formulation) Dilution (0.05 mg/ml formulation) mean proportion of dose absorbed (%) rat epidermis in vivo (10 hour exposure, 168 hour observation) 10.6 4.8 5.8

Species difference correction factor 8.9 8.5 2.7 Dermal absorption: Human 1.19 0.56 2.15– –

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B.6.13 Toxicological data on non active substances (Annex IIIA 7.9)

The formulation “BYI 08330 150 OD” contains the act ive ingredient Spirotetramat (150 g/l spirotetramat technical) as well as an emulsifier, a surfactant, dispersing agents, an antifoam,an antioxidant, a bu ffer and a co-solvent. The notifier provided Material Safety D ata Sheets (MSDS) for all these auxiliaries; only l imited information is available concerning the toxicity of these substances.

B.6.13.1 Material safety data sheet for each formul ant

The acute toxicity profile of all co-formulants in the representative formulation is sufficiently cove red by studies performed with the preparation. On the basi s of all data available and according to Commission Directives 67/548/EEC (as amended) and 1999/45/EEC, “BYI 08330 150 OD” has to be classified as sensitizing (hazard symbol Xi, risk phrase R 43).

B.6.13.2 Available toxicological data for each form ulant

Table 6.13.2-1: Toxicological data relating to the co-formulants
Coformulants CAS Number Function MSDS date Labellin g/Comments with respect to toxicology Genapol X-080 (poly(oxy-1,2- ethanediyl) α

isotridecyl- ω -hydroxy9043-30-5 Emulsifier; dispersant 08/05 Xn, R 22, R 41

Arlatone T (V)(sorbitan, poly(oxy1,2-ethanediyl)derives, hepta-9- octadecenoate 54846-79-6 Surfactant 10/97 -

ZephrymPD 2206 ( 70142-34-6 Dispersing agent 01/03 -

Borresperse NA (lignosulfonic acid, sodium salt) 8061-51-6 Dispersing agent 02/02 -

Silfoam SC 1132 (polysiloxanes, di-Me) 63148-62-9 Antifoaming agent 01/05 -

Vulkanox BHT crystalline (phenol, 2,6-bis(1,1- dimethylethyl)-4-methyl128-37-0 Antioxidant 11/04 -

Citric acid, anhydrous(1,2,3-propanetricarboxylic acid, 2-hydroxy77-92-9 Buffer 03/03 Xi, R 36

Sunflower oil 8001-21-6 Co-solvent 09/01 -– –

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B.6.14 Exposure data (annex IIIA 7.3, 7.4 and 7.5)

B.6.14.1 Operator exposure (annex IIIA 7.3)

The representative formulation “Spirotetramat OD 15 0” (BYI 08330 150 OD) is an oil dispersion formulat ion containing 150 g/l spirotetramat. “Spirotetramat OD 150” is an insecticide intended for the control of scales, aphids, mealy bugs and mites on citrus and aphids o n lettuce, for the latter both in the field as well as in the greenhouse. Applications of “Spirotetramat OD 150” will be achieved via field crop sprayers, broadcast air assisted sprayers and hand-held sprayers. Hand-held sprayers are used in citrus and in the greenhouse. Water will be the diluent/carrier in all situations

. The recommended use conditions are summarised in th e following table:

Table 6.14.1-1: Crops and use pattern proposed for “Spirotetramat OD 150”

Application rate per treatment Interval betw. appl. PHI

(days) Crop F/G kg

as/hl water l/ha kg as/ha Product

L/ha Application techniques

Citrus F 0.0096 1000 l/ha and m CH*, max. 3000 l/ha 0.096

g/ha and m CH*,

max. 0.288 kg/ha (3 metres) 0.64 –

1.92 21 14 Broadcast air assisted sprayer and Hand held sprayer

Lettuce F 0.0072-

0.0144 500-1000 0.072 0.48 14 7 Field crop sprayer

Lettuce G 0.0072-

0.0144 500-1000 0.072 0.48 14 7 Hand held sprayer

CH*: Canopy height

“Spirotetramat OD 150” is of low acute toxicity by the oral, dermal and inhalation route. It is not an irritant to skin and eyes but has a potential for skin sensitis ation. Recommended protective clothing are gloves, a coverall and solid shoes. Operator exposure estimates for outdoor cultures ar e calculated using both the German model (Uniform Principles for Safeguarding the Health of Applicato rs of Plant Protection Products [Uniform Principles for Operator Protections]; Mitteilungen aus der Biologi schen Bundesanstalt für Land- und Forstwirschaft, B erlinDahlem, no. 277, 1992) and the UK POEM (Scientific Subcommittee on Pesticides and British Agrochemicals Joint Medical Panel., Estimation of E xposure and Absorption of Pesticides by Spray Operators [UK MAFF] 1986 and the Predictive Operato r Exposure Model [POEM], UK MAFF 1992). Exposure is calculated for each application techniq ue with the maximum dose rate (German model and UK POEM) and minimum spray volume (UK POEM). Lower dos es and higher spray volumes will be covered by this calculation. Hand applications in citrus are calculated using th e German model as exposure calculation for the high level hand held scenario cannot be made with the UK POEM.– –

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Table 6.14.1-2: Data used for the calculations Area treated per day: Citrus, Broadcast air assiste d sprayer: 8 ha for the calculations using the German Model

, 15 ha for the calculations using the UK POEM Citrus, hand held sprayer: 1 ha for the calculations using the German Model Lettuce, field crop sprayer 20 ha for the calculations using the German Model 50 ha for the calculations using the UK POEM Application rates (maximum); 0.288 kg as/ha citrus 0.072 kg as/ha lettuce Spray volume (minimum): (for UK POEM only) 3000 l/ha for citrus 500 l/ha for lettuce Package size and type: (for UK POEM only) 5 l for tractor and for hand held applications

Standard operator body weight: 70 kg for German mod el 60 kg for UK POEM

Dermal absorption has been determined for “Spirotet ramat OD 150” formulated as the 150 mg/ml OD formulation in vivo and in vitro. For details see B.1.5. For the active ingredient Spirotetramat an acceptab le operator exposure level (AOEL) of 0.05 mg/kg bw/ d was established from the 1 year toxicity study in d ogs with a safety factor of 100 (NOAEL 5 mg/kg bw/d ). No correction of oral absorption was made (96 % oral a bsorption).

B.6.14.1.1 Estimation of operator exposure assuming personal protective equipment is not used

Operator exposure values have been calculated accor ding to the exposure models and model parameters described above. The results of the operator exposure estimates and their percent account of the proposed AOEL are summarized in the following tables. For details of the calculations refer to Appendices I and II.

Table 6.14.1.1-1: POEM model: Estimated operator ex posure values and % of the AOEL – without
PPE Total systemic exposure (mg/kg bw/day) % of AOEL (0.05 mg/kg bw/day) Tractor boom applications – low crop
Lettuce 0.0038 7.6 Tractor broadcast air-assist applications – high cr op Citrus 0.0064 12.8

Table 6.14.1.1-2: German model: Estimated operator exposure values and % of the AOEL – without PPE Total systemic exposure (mg/kg bw/day) % of AOEL (0.05 mg/kg bw/day) Tractor boom applications – low crop
Lettuce 0.0015 3 Tractor broadcast air-assist applications – high cr op Citrus 0.0097 19.4 Hand-held applications – high crop
Citrus 0.0151 30.2

Glasshouse application:– –

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In the surrogate glasshouse exposure study (Final R eport: Operator Exposure in Greenhouse During Practical Use of Plant Protection Products, (Mich, G., June 6, 1996 Projekt No. EF-94-02-03, ECON Forschungs- und Bewertungskonzepte für die Umwelt u nd Gesundheitssicherheit GmbH)) the exposure of 12 operators during mixing and loading and applicat ion were investigated for the two scenarios “low cr ops on tables” and “high crops on ground”. Dermal exposure of the body was determined via cell ulose patches located on the outside of the operato r’s clothing (potential dermal exposure), as well as by analysing whole body underwear (actual dermal exposure). Hand exposure was measured via glove rinsing (poten tial dermal exposure) and hand rinsing (actual derm al exposure). For determination of head exposure a pat ch was fixed to a cap. Inhalation exposure was measured via adsorbent tubes fixed to the garments at the height of the operators mouth and connected to a personal battery powered pump. During application the inhalation exposure was 0.4 mg/person x kg a.s. handled when low cultures grown on tables were sprayed. The dermal exposure resulted i n 0.7 mg/person x kg a.s. handled and 7.5 mg/person x kg a.s. for the actual and potential exposure, resp ectively. The summary results of the geometric mean exposures are presented in the following table:

Table 6.14.1.1-3: Surrogate glasshouse exposure stu dy: Summary results of the geometric mean
exposures Dermal (hands) 0.735 0.009

Dermal (head) 0.439 0.439 Dermal (body) 6.350 0.223 Dermal (total) 7.524 0.671 application low culture

inhalation 0.398 0.398

This greenhouse study provides appropriate data for the hand held scenario in greenhouses. Application data may be used for all types of hand held applica tion. Mixing/loading data are only to be used if a WP formulation and a Knapsack sprayer is used. In the absence of available data, the mixing/loading data for formulations other than WP and for other equipment used (e.g. stationary tank) may be taken from the available exposure models. It is considered that th e process of mixing/loading for both indoor and out door are similar. The exposure estimates for mixing/loading of an oil dispersion formulation such as “Spirotetramat OD 1 50” are based on data of the German model. The low crop scenario is chosen to calculate the exposure in lettuce. The use of a large tank will be the usual practice when large areas are treated. The risk ass essment presented assumes a work rate of 1 ha/d in the gree nhouse. The spray gun/lance connected to a tank wil l be used for this scenario, especially if high water vo lumes are used. A use rate of 0.072 kg a.s./ha, a dermal absorption rate of 1.19 % and an operator body weight of 60 k g were estimated.

Table 6.14.1.1-4: Glasshouse application - low crop s: Estimated operator exposure values and % of the AOEL – without PPE

Task Route of exposure Calculation [potential exp. (unprotected operator) X dose rate x work

rate] Exposure estimate [mg a.s./person/d]

Mixing/loading Inhalation 0.0006 x 0.072 x 1 0.0000 4 Dermal (hands) 2.4 x 0.072 x 1 0.1728 Application Inhalation 0.398 x 0.072 x 1 0.0287 Dermal (hands) 0.735 x 0.072 x 1 0.0529 Dermal (head) 0.439 x 0.072 x 1 0.0316– –

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Task Route of exposure Calculation [potential exp. (unprotected operator) X dose rate x work

rate] Exposure estimate [mg a.s./person/d]

Dermal (body) 6.350 x 0.072 x 1 0.4572

Systemic exposure* 0.0425 Mg/kg bw/d Systemic exposure** 0.0007 (1.4 % of AOEL) *: Dermal absorption: 1.19% concentrate, 2.15% spra y dilution, 100% inhalation **: body weight 60 kg

According to the applied models the estimated opera tor exposure account for much less than 100% of the proposed AOEL (0.05 mg/kg bw/d) for “Spirotetramat OD 150” even if no PPE is used.

B.6.14.1.2 Estimation of operator exposure assuming personal protective equipment is used

Model calculations of the estimated operator exposu re assuming PPE is used are not required since mode l calculations without PPE predict the systemic expos ure to be well within the acceptable exposure level

. B.6.14.1.3 Measurement of operator exposure – (Mixe r/Loader/Applicator)

Measurement of operator exposure is not required si nce model calculations predict the systemic exposur e to be well within the acceptable exposure level.

B.6.14.1.4 Summary on operator exposure

The proposed systemic AOEL of “Spirotetramat OD 150 ” is 0.05 mg/kg bw/d (based on the NOAEL of 5 mg/kg bw/d in the 1 year toxicity study in dogs wit h a safety factor of 100). No correction of oral ab sorption was made (96 % oral absorption). For the scenarios calculated, the results of the exposure estimations according to the POEM and the German model and to application in glasshouses do not exceed the proposed systemic AOEL of 0.05 mg/kg bw/d even if n o personal protective equipment is used.– –

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B.6.14.2 Bystander exposure (annex IIIA 7.4)

B.6.14.2.1 Estimation of bystander exposure

A calculation of exposure and risk is shown for a u se scenario that is considered to result in the hig hest level of spray drift which may contaminate the bystander – tractor-mounted air-assisted application in citru s.
The following scenario is assessed:
Tractor-mounted airblast sprayers: 0.288 kg a.i./ha , 3000 l/ha. The potential routes of exposure for bystanders are via dermal and inhalation exposure. Bystander expo sure will result primarily from drift. Such exposure is likely to be brief and not occurring repeatedly to the same individual. The AOEL is therefore considered to be a very conservative toxicological reference value. For the following assessment it is assumed that the person is located at the edge of the field, at a distance of 10 m from the application. The person is assumed to wear some clothing (T-shirt and trousers) which provide s some protection to the covered body parts. Exposure is calculated for the non-covered body areas (face

, neck front and back, lower arms, hands, feet). Expo sure to an incidental bystander is calculated based on the following parameters:

Maximum application rate [mg/m 2 ] AR 0.288 kg a.i./ha = 28.8 mg/m 2

Spray concentration [mg/ml] C 0.096 mg a.i./ml Dermal absorption [%] DA 2.15 % Drift at a distance of 7.5 m [%] (90%-ile) 1 D Tractor air-assist sprayer: 2.62% (late season ap plication)

Exposed body surface [m 2 ] BS 0.4250 m 2 (corresponds to the unprotected surface of an adul t wearing a T-shirt and trousers) 2 Body weight [kg] BW 50 kg Inhalation exposure [ml/hr] (90%-ile) 3 IE Tractor air-assist sprayer: 0.06 ml spray/hr (based on 0.06 ml spray/m 3 and a breathing rate of 1 m 3 /hr) Duration of exposure [min] T 5 min

Dermal exposure calculates as follows:



# [ ] BW BS D AR day / bw kg / mg Exposure Dermal × ×

=

Inhalation exposure calculates as follows:



# [ ] 60

/ / × ×

×

= BW T IE C day bw kg mg Exposure Inhalation

Total systemic exposure calculates as follows:



# [ ] Exposure Inhalation DA Exposure Dermal day / bw kg / mg Exposure Systemic Total + × =

1 Ganzelmeier et al

.: Studies on the spray drift of plant protection p roducts. Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft, Berlin -Dahlem, Heft 305, 1995. Updated: BBA (2000:1). Bekanntmachung über Abdriftw erte, die bei der Prüfung und Zulassung von Pflanzenschutzmitteln herangezogen werden. Bundesan zeiger Nr. 100, 26 Mai 2000, 9879-9881. 2 US EPA (1996). OPPTS Occupational and Residential Exposure Test Guidelines. Series 875 3 Lloyd G.A. & Bell G.J. (1983). Hydraulic nozzles: Comparative spray drift study. AHU report no. 122– –

Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 503 April 2008

Based on this scenario, total systemic exposure for an incidental bystander calculates as follows:

Table 6.14.2.1-1: Estimated bystander exposure valu e for “Spirotetramat OD 150” and % of the AOEL Spirotetramat OD 150 (AOEL = 0.05 mg/kg bw/d) Dermal exposure (mg/kg bw/day) 0.006413 Inhalation exposure (mg/kg bw/day) 0.0000096 Total systemic exposure (mg/kg bw/day) 0.00015 % of AOEL 0.3

The applied approach provides a conservative assess ment of the exposure risk for incidental bystanders

. The calculated exposure to “Spirotetramat OD 150” i s well below AOEL.

B.6.14.2.2 Measurement of bystander exposure

Measurement of bystander exposure is not required s ince model calculations predict the systemic exposu re to be well within the acceptable exposure level.

B.6.14.2.3 Summary on bystander exposure

A calculation of exposure and risk is shown for a u se scenario that is considered to result in the hig hest level of spray drift which may contaminate the bystander – tractor-mounted air-assisted application in citru s. These results indicate that short exposure of bysta nders outside the treatment areas would be 0.3 % of AOEL and should therefore not give rise to concern.– –

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B.6.14.3 Worker exposure (annex IIIA 7.5)

The type of crop, product type and the time point o f application of “Spirotetramat OD 150” may require reentry activities such as crop inspection, pruning a nd harvesting which may lead to worker exposure. An 8 hour period of exposure is considered appropriate f or a harvesting task and an example calculation of exposure and risk will be shown for this scenario.

There are no foliar dislodgeable residue (FDR) data available for “Spirotetramat OD 150” on the crops described above, nor specific transfer coefficients for the work activities that may be performed in t hese crops. A foliar dislodgeable residue study was perf ormed on grapes and hops, with a max. application r ate of 110g ai/ha ( Fischer, 2006, see B.1.6 ).The worker re-entry exposure model published by H oernicke et al (1998) has been used to provide a conservative esti mate of exposure during the conduct of re-entry wor k activities 4

.

For the foliar dislodgeable residues (FDR) the Germ an re-entry model proposes the overly conservative transfer coefficient (TF) of 30,000 cm 2 /hour, which reflects tasks with the most intensive contact with the treated crop, such as pruning or harvesting. As a w orst case Tier 1 approach this value is applied in the following calculations.

B.6.14.3.1 Estimation of worker exposure assuming p ersonal protective equipment is not used

Worker exposure is calculated by the following appr oach:

Dermal exposure (D) [mg/person/day] = FDR x TF x A x R

Dermally absorbed dose (D) [mg/person/day] = D x AF

Absorbed dose [mg/kg bw/day] = D/bw

Parameters used for calculation and the resulting e xposure levels are summarised in the following tabl es:

Table 6.14.3.1-1: Re-entry worker calculations of e xposure to “Spirotetramat OD 150” (no PPE) Symbol Definition Quantity Units DFR Foliar dislodgeable residues 0.943 μ g/cm 2 /kg a.i.
TF Transferfactor 30000 cm 2 /person x h A Work rate/day 8 h/day P penetration through clothing 5 % R application rate 2 x 0.288 kg a.i./ha D= DFR x TF x A x R (dermal exposure unprotected) 1 30,36 mg/person AF dermal absorption 2.15 % Bw Body weight 60 Kg Syst.exp.unprot.= D x AF/60 kg 0,047 mg/kg bw
% Account of AOEL 93,4 %

4 Hoernicke E. et al ., (1998) Label instructions for the protection of workers re-entering crop growing areas after application of plant protection products. Nachricht enbl. Deut. Pflanzenschutzd. 50 (10), 267-269 (1998 ) (doc. No.: M128767-01-1).– –

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B.6.14.3.2 Estimation of worker exposure assuming p ersonal protective equipment is used

Estimation of worker exposure assuming PPE is used is not required since model calculations without PP E predict the systemic exposure to be well within the acceptable exposure level.

B.6.14.3.3 Estimation of operator exposure assuming personal protective equipment is used and using data generated on dislodgeable residues under the proposed conditions of use

Estimation of worker exposure using PPE and actual dislodgeable residues data were not performed.

B.6.14.3.4 Measurement of worker exposure

Measurement of worker exposure is not required sinc e model calculations predict the systemic exposure to be well within the acceptable exposure level.

B.6.14.3.5 Summary on worker exposure

The exposure of re-entry workers during a longer te rm activity such as pruning or harvesting has been calculated according to the German re-entry model t o provide a conservative assessment of risk. The calculated exposure levels for “Spirotetramat OD 15 0” are below the respective AOEL (0.05 mg/kg bw/d) even without specific protective equipment (93,4 % of AOEL).– –

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B.6.15 Dislogeable residues (annex IIIA 7.7)

Reference: Determination of Dislodgeable Foliar Residue on Gra pes and Hops treated with

BYI 08330 150 OD Report No.: RAFNY040 Author(s), year: Fischer, D.; 2006 Guideline(s): DACO No’s.: 5.9 & USEPA Series 875: G roup B GLP: Yes Deviations: -Cartridge samples from trial FN344-05D were not shipped according to shipment

schedule outlined in the protocol. However, all sam ples were received safely at the analytical laboratory and field recovery samples we re stored and transported with the samples to verify analyte stability. No adverse eff ect on the study. -Cation exchange capacity data for soil from plot f rom trial FN343-05D were not collected and are not available. Since these data a re not critical for this type of study, there was no adverse effect. -Sample shipment chain of custody form for the cart ridge samples from trial FN339- 05D shipped on October 5, 2005 was not available. T hese samples were reserve samples held at the field site, then shipped follow ing safe receipt of the primary samples to the analytical lab. Since all primary s amples were received safely at the laboratory, these samples were not needed for analy sis, and therefore this deviation had no effect on the study. Validity: Yes

Materials and methods: Four field trials were conducted on grapes and two field trials were conducted on hops to determine th e amount of spirotetramat residue that can be dislodg ed from grape and hops foliage, respectively, follo wing foliar application of BYI 08330 150 OD (150g ai/l). Trials on grapes were conducted in Pennsylvania, California (2x) and Washington. Trials on hops were conducted in Washington and Oregon. Applications t o both crops were made at a target rate of 110g ai/h/application . The intervals between applications to grapes and hops were 30 days and 14 days, respectively. Al l applications were made using airblast equipment.

Leaf punch sampling (400 cm 2 double-sided surface area) were collected prior to the first application (control samples) and at various time intervals after the fi rst and second applications up to 35 days following the second application. Three treated samples were coll ected at each interval. Sample residue was dislodge d from each sample with 200 ml of detergent solution. Aliquots of the dislodging solution were applied t o solid phase extraction cartridges, and the cartridges wer e washed, dried, and frozen. Residues were eluted f rom the cartridges and quantitated by liquid chromatogr aphy/mass spectrometry-mass spectrometry (lc/ms-ms)

. The limit of quantitation was 0.01 μg/cm 2 . The measured residues were corrected for any loss es that occurred from sample collection through sample anal ysis.

Findings: The following tables summarize the dislodgeable fol iar residue values found in grapes and hops:

Table 6.15.-1: Dislodgeable foliar residue values after one and two applications of spirotetramat to
grapes

Trial location A μg/cm 2 B

μg/cm 2 C

μg/cm 2 D

μg/cm 2 Half life (d)

Pennsylvania 0.13 < LOQ 0.12 0.03 18– –

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Trial location A μg/cm 2 B

μg/cm 2 C

μg/cm 2 D

μg/cm 2 Half life (d)

California 1 0.08 0.02 0.12 0.04 22
California 2 0.19 0.14 0.30 0.12 33 Washington 0.29 0.05 0.36 0.07 16 A: Sampling interval – immediately after first treatm ent B: Sampling interval – 30 days after first applicatio n; prior to 2 nd treatment C: Sampling interval – immediately after 2 nd treatment D: Sampling interval – 35 days after 2 nd treatment

Table 6.15.-2: Dislodgeable foliar residue values a fter one and two applications of spirotetramat to hops

Trial location A μg/cm 2 B

μg/cm 2 C

μg/cm 2 D

μg/cm 2 Half life (d)

Washington 0.13 0.08 0.20 0.10 47 Oregon 0.10 0.01 0.09 < LOQ 5 A: Sampling interval – immediately after first treatm ent B: Sampling interval – 14 days after first applicatio n; prior to 2 nd treatment C: Sampling interval – immediately after 2 nd treatment D: Sampling interval – 35 days after 2 nd treatment

Conclusion: For the 4 trials conducted on grapes, the average d islodgeable foliar residue (DFR) on grape leaves immediately after the first and second application of BYI 08330 150 OD ranged from 0.08 to 0.29 μg/cm 2 and from 0.12 to 0.36 μg/cm 2 , respectively. 35 days after the second applicatio n, the average DFR from the 4 grape trials had declined to values ranging from 0. 03 to 0.12 μg/cm 2 . The half life of BYI 08330 150 OD on grape leaves at the 4 trial locations following the second application was 18, 22, 33 and 16 days, respectively.

For the 2 trials conducted on hops, the average DFR on hop leaves immediately after the first and seco nd application of BYI 08330 150 OD ranged from 0.10 to 0.13 μg/cm 2 and from 0.09 to 0.20 μg/cm 2

, respectively. 35 days after the second application, the average DFR from the 2 hop trials had declined to values ranging from < LOQ to 0.10 μg/cm 2 . The half life of BYI 08330 150 OD on hop leaves a t the 2 trial locations following the second application was 47 a nd 5 days, respectively.

The recommended crops and use conditions of BYI 083 30 150 OD are citrus and salad, with a max. application rate per treatment of 288 g/ha and 72 g /ha respectively.

So for the estimation of dislodgeable foliar residu es for spirotetramat, application to citrus, 288 g ai/h/application , based on data for grapes (0.36 μg/cm 2 immediately after the second application of BYI 08330 150 OD) were taken into account (worst case s cenario). So for the estimation of worker exposure, dislodgeable foliar residues of 0.943 μg/cm 2 were assumed.– –

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## Appendix I: Detailed Exposure Calculations (UK POE M)

Table I-1: Tractor-mounted/trailed boom sprayer - l ow crops (lettuce), Unprotected Operator

EXPOSURE DURING MIXING AND LOADING
Container size 5 litres Hand contamination/operation 0.01 Ml Application dose 0.48 litres product/ha
Work rate 50 Ha/day Number of operations 5 /day
Hand contamination 0.05 ml/day Protective clothing None
Transmission to skin 100 % Dermal exposure to formulation 0.05 ml/day
DERMAL EXPOSURE DURING SPRAY APPLICATION Application technique Tractor-mounted/trailed boom sprayer: hydraulic noz zles Application volume 500 spray/ha
Volume of surface contamination 10 ml/h
Distribution Hands Trunk Legs
65% 10% 25% Clothing None Permeable Permeable
Penetration 100% 5% 15% Dermal exposure 6.5 ml/h 0.05 ml/h 0.375 ml/h Duration of exposure 6 H Total dermal exposure to spray 41.55 ml/day
ABSORBED DERMAL DOSE Mix/load Application Dermal exposure 0.05 ml/day 41.55 ml/day Concen. of a.s. product or spray 150 mg/ml 0.144 mg /ml Dermal exposure to a.s. 7.5 mg/day 5.9832 mg/day
Percent absorbed 1.19% 2.15%
Absorbed dose 0.08925 mg/day 0.1286388 mg/day

INHALATION EXPOSURE DURING SPRAYING Inhalation exposure 0.01 ml/h Duration of exposure 6 H Concentration of a.s. in spray 0.144 Mg/ml Inhalation exposure to a.s. 0.00864 Mg/day
Percent absorbed 100 %
Absorbed dose 0.00864 Mg/day
PREDICTED EXPOSURE
Total absorbed dose 0.2265288 Mg/day Operator body weight 60 Kg Operator exposure 0.00377548 Mg/kg bw/day
PERCENT AOEL 7.6– –

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Table I-2: Tractor Air-assisted Sprayer - high crop s (citrus), Unprotected Operator

EXPOSURE DURING MIXING AND LOADING
Container size 5 litres Hand contamination/operation 0.01 Ml Application dose 1.92 litres product/ha
Work rate 15 Ha/day Number of operations 6 /day
Hand contamination 0.06 ml/day Protective clothing None
Transmission to skin 100 % Dermal exposure to formulation 0.06 ml/day
DERMAL EXPOSURE DURING SPRAY APPLICATION Application technique Tractor-mounted/trailed broadcast air-assisted spra yer Application volume 3000 spray/ha
Volume of surface contamination 400 ml/h
Distribution Hands Trunk Legs
10% 65% 25% Clothing None Permeable Permeable
Penetration 100% 2% 5% Dermal exposure 10 ml/h 5.2 ml/h 5 ml/h Duration of exposure 6 H Total dermal exposure to spray 121.2 ml/day
ABSORBED DERMAL DOSE Mix/load Application Dermal exposure 0.06 ml/day 121.2 ml/day Concen. Of a.s. product or spray 150 mg/ml 0.096 mg /ml Dermal exposure to a.s. 9 mg/day 11.6352 mg/day
Percent absorbed 1.19% 2.15%
Absorbed dose 0.1071 mg/day 0.2501568 mg/day

INHALATION EXPOSURE DURING SPRAYING Inhalation exposure 0.05 ml/h Duration of exposure 6 H Concentration of a.s. in spray 0.096 Mg/ml Inhalation exposure to a.s. 0.0288 Mg/day
Percent absorbed 100 %
Absorbed dose 0.0288 Mg/day
PREDICTED EXPOSURE
Total absorbed dose 0.3860568 Mg/day Operator body weight 60 Kg Operator exposure 0.00643428 Mg/kg bw/day
PERCENT AOEL 12.8– –

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## Appendix II: Detailed Exposure Calculations (BBA Mo del)

Table II-1: Tractor mounted boom sprayer - low crop s (lettuce) Use rate (kg a.i./ha) 0.072 kg a.i./ha Treated surface per working day (ha) 20 ha
A. Model Data Exposure during mixing/loading per kg a.i. handled: 2.4 mg on hands 0.0006 mg by inhalation Exposure during spray operation per kg a.i. handled : 0.06 mg on head 0.38 mg on hands 1.6 mg/on body surface 0.001 mg/by inhalation B. Potential Field Operator Exposure per Working Da y (mg) Dermal Exposure (mg) Inhalation (mg) Head Hands Body Activity: Mixing/loading Exposure/kg a.i. handled: 2.4 0.0006 Exposure/person and working day: 3.456 0.000864 Spraying Exposure/kg a.i. handled: 0.06 0.38 1.6 0.001 Exposure/person and working day: 0.0864 0.5472 2.30 4 0.00144 C. Dermal Exposure Without Protective Clothing Exposed Body Parts No PPE

Mixing/loading:
Hands 3.456 mg/person Spraying:
Hands: 0.5472 mg/person
Head: 0.0864 mg/person Body: 2.304 mg/person D.Systemic Exposure Related to Activity
No PPE Mixing/loading Dermal exposure 3.456 mg/person Dermal absorption 1.19 % Absorbed dose 0.0411264 mg/person
Inhalation 0.000864 mg/person Absorption 100 % Absorbed dose 0.000864 mg/person Total absorbed mix/load/person 0.0419904 mg/person Spraying Dermal exposure 2.94 mg/person Dermal absorption 2.15 % Absorbed dose 0.06 mg/person
Inhalation 0.001 mg/person Absorption 100 % Absorbed dose 0.001 mg/person Total absorbed spraying/person 0.06 mg/person E. Total Systemic Exposure Systemic exposure/person 0.11 mg/person Systemic exposure /kg b.w. (70 kg) 0.0015 mg/kg b.w

. % of AOEL (0.0035 mg/kg bw/d) 3 %– –

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Table II-2: Tractor mounted airblast sprayer - high crops (citrus) Use rate (kg a.i./ha) 0.288 kg a.i./ha Treated surface per working day (ha) 8 ha
A. Model Data Exposure during mixing/loading per kg a.i. handled: 2.4 mg on hands 0.0006 mg by inhalation Exposure during spray operation per kg a.i. handled : 1.2 mg on head 0.7 mg on hands 9.6 mg/on body surface 0.018 mg/by inhalation B. Potential Field Operator Exposure per Working Da y (mg) Dermal Exposure (mg) Inhalation (mg) Head Hands Body Activity: Mixing/loading Exposure/kg a.i. handled: 2.4 0.0006 Exposure/person and working day: 5.5296 0.0013824 Spraying Exposure/kg a.i. handled: 1.2 0.7 9.6 0.018 Exposure/person and working day: 2.7648 1.6128 22.1 184 0.041472 C. Dermal Exposure Without Protective Clothing Exposed Body Parts No PPE Mixing/loading:
Hands 5.5296 mg/person Spraying:
Hands: 1.6128 mg/person
Head: 2.7648 mg/person Body: 22.1184 mg/person D.Systemic Exposure Related to Activity
No PPE Mixing/loading Dermal exposure 5.5296 mg/person Dermal absorption 1.19 % Absorbed dose 0.0658 mg/person
Inhalation 0.0013824 mg/person Absorption 100 % Absorbed dose 0.0013824 mg/person Total absorbed mix/load/person 0.06718464 mg/person Spraying Dermal exposure 26.496 mg/person Dermal absorption 2.15 % Absorbed dose 0.569664 mg/person
Inhalation 0.041472 mg/person Absorption 100 % Absorbed dose 0.0415 mg/person Total absorbed spraying/person 0.6111 mg/person E. Total Systemic Exposure Systemic exposure/person 0.6783mg/person Systemic exposure /kg b.w. (70 kg) 0.0097 mg/kg b.w

. % of AOEL (0.0035 mg/kg bw/d) 19.4 %– –

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Table II-3: Hand held application - high crops (cit rus) Use rate (kg a.i./ha) 0.288 kg a.i./ha Treated surface per working day (ha) 1 ha
A. Model Data Exposure during mixing/loading per kg a.i. handled: 205 mg on hands 0.05 mg by inhalation Exposure during spray operation per kg a.i. handled : 4.8 mg on head 10.6 mg on hands 25 mg/on body surface 0.3 mg/by inhalation B. Potential Field Operator Exposure per Working Da y (mg) Dermal Exposure (mg) Inhalation (mg) Head Hands Body Activity: Mixing/loading Exposure/kg a.i. handled: 205 0.05 Exposure/person and working day: 59.04 0.0144 Spraying Exposure/kg a.i. handled: 4.8 10.6 25 0.3 Exposure/person and working day: 1.3824 3.0528 7.2 0.0864 C. Dermal Exposure Without Protective Clothing Exposed Body Parts No PPE Mixing/loading:
Hands 59.04 mg/person Spraying:
Hands: 3.0528 mg/person
Head: 1.3824 mg/person Body: 7.2 mg/person D.Systemic Exposure Related to Activity
No PPE Mixing/loading Dermal exposure 59.04 mg/person Dermal absorption 1.19 % Absorbed dose 0.7026 mg/person
Inhalation 0.0144 mg/person Absorption 100 % Absorbed dose 0.0144 mg/person Total absorbed mix/load/person 0.7170 mg/person Spraying Dermal exposure 11.6352 mg/person Dermal absorption 2.15 % Absorbed dose 0.2502 mg/person
Inhalation 0.0864 mg/person Absorption 100 % Absorbed dose 0.0864 mg/person Total absorbed spraying/person 0.3366 mg/person E. Total Systemic Exposure Systemic exposure/person 1.0535 mg/person Systemic exposure /kg b.w. (70 kg) 0.0151 mg/kg b.w

. % of AOEL (0.0035 mg/kg bw/d) 30.2 %