Annex B 

Spirotetramat 

B.6 Toxicology and metabolism 

Reviewed by: 

US EPA, USA 

Peer –reviewed by: 

AGES, Austria 

PMRA, Canada 
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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 route 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 in 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 sensitization (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 substances 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 mammalian 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
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B.6.4.2.2 Metaphase analysis in rodent bone marrow or micronucleous 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 studies ...................................................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 Annex 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 studies.........................................................251

B.6.6 Reproductive toxicity (OECD Annex IIA 5.6) ........................................................................251

B.6.6.1 Two generation reproductive toxicity in the rat (OECD 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 (OECD Annex IIA 5.6.10).............................280

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 IIA 5.7.4)...........................................328

B.6.7.3 Postnatal developmental neurotoxicity (OECD Annex IIA 5.7.5) .........................................328

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 metabolites.................................................................380

B.6.9 Medical data and information (OECD Annex IIA 5.9) ......................................................381

B.6.9.1 Report on medical surveillance on manufacturing plant personnel (OECD Annex IIA 

5.9.1).....................................................................................................................................381

B.6.9.2 Report on clinical cases and poisoning incidents (OECD 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 details 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 function of exposure (OECD Annex 

IIA 5.9.7) ...............................................................................................................................383

B.6.9.8 Expected effects and duration of poisoning as a function 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
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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 evaluation (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 IIIA 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 Annex 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 .....................................493

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 protective equipment is not used ......499

B.6.14.1.2 Estimation of operator exposure assuming personal protective equipment is used ............501

B.6.14.1.3 Measurement of operator exposure – (Mixer/Loader/Applicator).........................................501

B.6.14.1.4 Summary on operator exposure ...........................................................................................501
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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 protective equipment is not used.........504

B.6.14.3.2 Estimation of worker exposure assuming personal protective equipment is used...............505

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 ......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
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B.6 Toxicology and metabolism 

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

5.1) 

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

B.6.1.1.1 Metabolism and Pharmacokinetics 

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

and metabolism in the rat. Study No. M01819128. Report No. MEF 048/04, Document No. 

M-268709-02-2. Bayer CropScience AG Development, Metabolism/Environmental Fate, 

40789 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: C21 H27 N O5

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-3-14C 

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-treated 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 [azaspirodecenyl-3-14C]BYI 08330 was demonstrated by radio 

HPLC analysis of the administration suspensions of each test immediately 

after dosing 
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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 groups 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 administration 

 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 about 7 days prior to 

the administration. 

Identification: Cage cards on which the study number, 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 conditions in air-conditioned 

rooms 

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

12 / 12 hours light / dark cycle, air change 10 – 15 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 metabolism 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 
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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 administered, by gavage, a 2 or 100 mg/kg bw dose of 

azaspirodecenyl-3-14C labelled BYI 08330 suspended in 0.5% aqueous tragacanth after a 16-hour 

fasting. The dosing suspensions (0.2 or 10 mg /mL for 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/kg bw calculated 

for a nominal body weight of 200 g). Actual administered doses were measured by LSC assay of aliquots 

of the administration suspensions. The actual mean administered doses of [azaspirodecenyl-3-14C]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 the 

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 animal by pressing a capillary coated with heparin in a 

small cut in the tail vein. The wound was closed with 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 centrifugation, the capillary was broken at the border between 

the plasma, and the formed constituents and the plasma (ca. 30 μL) were each placed onto a small 

metal dish for weighing. The dish was then placed into a scintillation vial for radioactivity measurement. 

Collection of excreta

Urine was collected at various times separately for each animal in a cryogenic trap cooled with dry ice. 

The funnels for urine collection were rinsed with demineralised water at the end of each sampling period. 

The rinsing solutions were drained into the same vial 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 for each animal in a cryogenic trap before they were

lyophilised (freeze-dried), weighed, and homogenised. 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 administration), the animals were anaesthetised using 

Pentobarbital-Na. They were sacrificed by transection 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 by centrifugation. The following organs/tissues were

collected: erythrocytes, plasma, spleen, gastro-intestinal tract (GIT), liver, kidneys, perirenal fat, adrenal 
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gland, testis (male only), ovaries (female only), uterus (female only), skeletal muscle, femur bone, heart, 

lung, brain, thyroid gland, skin, and carcass. 

The organs and tissues prepared at the end of the experiments were weighed immediately after the 

dissection and again after lyophilisation. Finally, they were homogenised and the radioactivity was 

determined by combustion/LSC. The radioactivity remaining 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, thyroid, and ovaries), only the wet weight was weighed 

and the samples then solubilised with BTS 450®

 (Beckman Tissue Solubiliser). 

Radioactivity measurement

The measurement of the radioactivity in liquid samples was carried out by liquid scintillation counting 

(LSC). For this purpose, the volume of the entire solution was determined and radioactivity of an aliquot 

(1 – 3 replicates) of the solution was measured using the following scintillation counters: 

Small organs or tissues were solubilised by means of a tissue solubiliser (e.g. BTS-450®

). The 

solubilised samples or aliquots of them 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 was used for the following test materials: renal 

fat, adrenal glands, thyroid, ovaries, and uterus. 

Solid samples (1 – 5 aliquots) were weighed and combusted in an oxygen atmosphere using the 

following equipments Oxidizer 307/387 (Packard Instruments) for combustion of faeces, erythrocytes, 

lyophilised organs and tissues like e.g. spleen, liver, lung, bone muscle, gastrointestinal tract (GIT), 

residual carcass and solids (e.g. from faeces after extraction). Carbosorb E (9 mL) was used to trap the 

combustion product CO2 and Permafluor E+ (10 mL; Packard Instruments) as scintillator for LSC. 

Metabolite analysis

Urine samples were analysed without sample preparation. Freeze-dried faeces samples of all animals 

from 0 - 24 h post administration were combined for each test. The radioactivity in each sample material 

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 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 

radioassayed by LSC. The aqueous phase was concentrated and used for HPLC analysis. 

The metabolites in the samples were quantified by HPLC 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. In order to check the completeness of the chromatographic elution, several representative 

samples were injected, re-collected, and radioassayed by LSC. The column recoveries were between 

>99 and 101% of the injected amount of radioactivity. 

The first method for identification of metabolites in urine and extracts of faeces was HPLC cochromatography with radiolabelled and non-radiolabelled reference items. One-dimensional TLC on 

silica 60 F254 (normal phase) plates was used as the second method for co-chromatographic 

identification of metabolites. The plates were developed over a separation path of ca. 16 cm in a 

standard TLC-chamber. The following three TLC solvent systems were used: 
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System 1: Ethyl acetate/propanol-2/water (65/23/12 v/v/v), with chamber saturation 

System 2: Dichloromethane/methanol/ammonia solution 25 % (90/10/1 v/v/v), with chamber 

saturation 

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

saturation 

For the TLC confirmation of metabolites, aliquots of urine and faeces extracts were fractionated with the 

HPLC profiling method. The collected peaks were then concentrated to the aqueous remainder. Aliquots 

of these samples and of the solutions of the reference items were applied individually and overlapped 

with each other on the TLC-plates. Co-chromatographic correspondence was assessed either by visual 

inspection of the plate under UV light at 254 nm and its associated radioluminogram or by analysis of the 

radioluminogram only, in cases where radioactive reference 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 used to 

calculate the basic toxicokinetic parameters from plasma curve analysis. The pharmacokinetic software 

TOPFIT (version 2.0) was used with a 3-compartment disposition model for fitting of the data. Correlation 

coefficients were excellent for all tests (0.994-1.00). 

BYI 08330 was very rapidly absorbed from the gastrointestinal tract of male and female rats in all test 

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 significant differences were observed between the low 

dose, high dose, and repeated dose tests. BYI 08330 was completely absorbed even at the high dose 

level of 100 mg/kg bw since 89-98% of the total recovered radioactivity was excreted via the renal route. 

Between 91-100% of the administered doses were recovered in urine, faeces, and the body of animals. 

The balance of radioactivity for all tests is presented in Table 6-2. 

B. DISTRIBUTION:

Radioactivity in plasma of male and female rats following a single administration of 2 mg/kg bw reached 

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. After 

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 (AUC0-∞) indicated a slightly higher systemic exposure for males than for females. The 

elimination rate constant was very similar for males and females. The mean residence time was low in 

males and slightly higher in females. 
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The results obtained from the repeated low dose experiments with pre-treated animals were in the same 

range as those from the single low dose tests. Absorption of the radioactivity was similar for males and 

females and was followed by a fast initial elimination phase and a moderate terminal elimination phase. 

The mean residence time was low and similar for males and females. 

The results from the high dose experiments indicated a sex difference in the maximum plasma 

concentration. The Cmax 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 that 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 elimination phase and a moderate terminal elimination phase. 

The areas under the curves (AUC0-∞) indicated a slightly higher systemic exposure for males than for 

females. The mean residence time was in the same range as in the low dose test and quite similar for 

males and females. Comparison of the absorption phases of the kinetic curves between the low and high 

dose groups showed a broader maximum for males of the high dose test. The maximum concentration 

(Cmax) was reached slightly later at this dose level and the following initial elimination phase was slightly 

longer than observed for the low dose level. The curves 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 body was analyzed at the time of sacrifice 48 h after dosing 

by radioactivity measurement of the major organs and tissues. Less than 0.2% of the administered dose 

was detected in the body. The residues in all organs 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-3-14C]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 formation of the primary and most predominant 

metabolite BYI 08330-enol (53-87% of the administered dose). All other identified metabolites could be

derived from the enol intermediate. Further metabolic 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 aromatic methyl group of the enol metabolite to BYI 

08330-enol-alcohol (0.4-1.6%). A sex-related difference 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 was more pronounced in the high dose tests than in 

the low dose tests. 

Identification rates were high in the range of 87-95% of the administered dose. Only 0.4-3.5% in total

could not be identified. No single unidentified metabolite accounted for >0.7 % of the dose. The 

proportion of non-identified metabolites was higher for male rats than for female rats. 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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Metabolites excreted with urine and faeces following oral administration of [azaspirodecenyl-3-14C] BYI 

08330 are shown in Table 6-7. The proposed metabolic pathway of BYI 08330 in the rat is presented in 

Figure 6-1. 

D. EXCRETION:

The excretion of BYI 08330 was fast and almost complete 24 h after administration. Significant sex or 

dose related differences were not observed. Excretion 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. In every dose group, quantitative recovery of dosed 

radioactivity (91.44-99.78 % of dose) was accomplished. 

The formation of 14CO2 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 observed into the expired air until 48 h postdose, regardless of 

the sex. The excretion of radioactivity following oral administration of [azaspirodecenyl-3-14C]BYI 08330 

is shown in Table 6-6. 

Table B.6-2: Excretion and radiolabelled residues in 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

 
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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 + radioactivity 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 (measured 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/kg 

 (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 
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 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 

 

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

 tmax (model) h 0.89 0.09 2.03 0.77 0.45 0.35 

 Cmax (experiment) μg/g 4.40 3.84 204 116 4.96 2.69 

 tmax (experiment) h 1.00 0.17 1.50 0.66 0.66 0.66 

 

 t1/2 a h <0.01 <0.01 0.17 0.06 0.10 0.07 

 t1/2 e(1) h 0.31 4.79 1.70 0.19 3.62 0.47 

 t1/2 e(2) h 20.1 29.7 17.5 27.2 92.7 13.2 

 

 tlag 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 

 

 k1e 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 

 CLR 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 

 MRTabs h 2.55 1.16 2.53 2.99 1.91 1.94 

 MRTdisp 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:

 CLrenal = CLtotal 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 tissues/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 0.0007 

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 
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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 animals of each test. 

Table B.6-6: Time course of the excretion of radioactivity 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 
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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 96.76 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 
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NH

O

O

O O

CH3

CH3

H3C

H

O CH3

NH

O

HO

CH3

CH3

H

O

CH3

H

O

NH

O

CH3

CH3

CH3

O

O

HO

HO

HO

HO

O

NH

O

O

CH3

CH3

H

O

OH

CH3

H

OH

NH

O

HO

CH3

CH3

H

O

NH

O

HO

CH3

CH2OH

CH3

NH

O

O

CH3

CH3

H

OH

OH

BYI08830

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

BYI08330-ketohydroxy BYI08330-desmethyl-enol

BYI08330-desmethyl-ketohydroxy

Figure B.6-1: Proposed metabolic pathway of BYI 08330 in rats

III. CONCLUSIONS

A. CONCLUSIONS: 

The fate of radiolabelled BYI 08330 in male and female rats following single and repeated oral 

administration was characterized as follows: 

• Absorption: Fast and complete absorption of the radioactive test compound from the gastrointestinal 

tract for the low dose, high dose, and repeated dose groups of male and female rats 
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• Distribution: Fairly equal distribution of the radioactivity in males and females within the blood and 

most organs and tissues with preference for the liver 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 tissues and organs at the time of sacrifice 48 hours 

postdose which were <LOD for some organs/tissues 

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

• Metabolism: Cleavage of the ester group as the major metabolic transformation resulting in 

BYI 08330-enol as the major metabolite and key intermediate in the metabolic pathway 

• Demethylation of the methoxy group in the cyclohexyl ring resulting in BYI 08330-desmethyl-enol as 

the second prominent metabolite. This metabolic transformation was significantly higher in male rats 

than in females. 

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

• Very similar metabolic profiles for low dose and repeated dose tests. Similar profiles for low dose and 

high dose tests but with a somewhat higher proportion of BYI 08330-desmethyl-enol in the high dose 

tests 

• Significant difference in metabolic profiles between 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 dose groups (87-95 % of administered dose) 

B. REVIEWER COMMENTS: 

This disposition study of [Azaspriodecenyl-3-14C]BYI 08330 in male and female rats showed excellent 

recovery of administered radioactivity (91-99%). The 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 nearly 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 variability being a notable gender-related difference; males

more effective in formation of BYI 08330-enol and BYI 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 Guidelines 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 (870.7485) in rats.

C. STUDY DEFICIENCIES:

There was no indication of randomization of test animals for experimental group assignments 

although it was noted that minor body weight variations 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 results or their interpretation. 
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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 Annex IIA 5.1.2) 

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

Metabolites in Plasma, Urine, Liver, Kidney and Testis of the Male Rat. Study No. 

M41819177. Report No. MEF 06/328, Document No. M-275731-01-2. Bayer CropScience 

AG Development, Metabolism/Environmental Fate, 40789 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: C21 H27 N O5

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-3-14C 

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 μCi/mg) 

High dose tests (test 4 -6): 0.0073 MBq/mg (0.20 μCi/mg) 

Radiochemical purity: > 98 % (certified, HPLC and TLC 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 [azaspirodecenyl-3-14C]BYI 08330 was 

demonstrated by radio HPLC analysis of the administration 

suspensions of each test immediately after dosing 
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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 administration 

 175 – 204 g at the time of sacrifice 

Acclimatization: Makrolon® cages on wood shavings in the test facility for about 7 days 

prior to the administration. 

Identification: Cage cards on which the study number, 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 conditions in airconditioned rooms 

Temperature 22 - 27 °C, relative humidity 43 – 61 %. 

12 / 12 hours light / dark cycle, air change 10 – 15 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 match the dose of the low dose tests in the ADME 

study with BYI 08330 (Report No. MEF-048/04). The high dose was selected taking into account the 

findings from a rat mechanistic study (Report No. SA 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 the high dose in this study. 

Dosing 

A summary of the experimental protocol is provided in Table 6-8. 
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Table B.6-8: Experimental protocol summary for metabolism 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 hr 

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 at 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-3-14C BYI 08330 suspended in 0.5 % aqueous tragacanth after 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-3-14C BYI 08330 suspended in 0.5 % aqueous tragacanth after ca. 16 h of fasting. The 

dosing suspension (100 mg/mL) was prepared three days before dosing and stored at 4°C. 

The suspensions were administered to the rats by oral 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 administered test substance were calculated by dividing the 

administered radioactivity amounts by the specific radioactivity. The actual mean administered dose of

[azaspirodecenyl-3-14C]BYI 08330 was 1.83 mg/kg bw for the low dose groups and 934 mg/kg bw for the 

high dose groups. The stability of the test compound 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 quantitative 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 intervals of 4 h and 8 h, faeces were combined with the 

gastro-intestinal tract at the time of sacrifice. For 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.. The funnels for urine collection were rinsed with 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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demineralised water at the end of each sampling period. 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 using 

Pentobarbital-Na. They were sacrificed by transection of the cervical blood vessels. The collected blood 

was separated into plasma and erythrocytes by centrifugation. The following organs/tissues were 

collected: erythrocytes, plasma, gastro-intestinal tract (GIT), liver, kidneys, testis, skin, and carcass. 

Erythrocytes, GIT, carcass, and skin prepared at the end the experiments were weighed immediately 

after the dissection and again following lyophilisation. Finally, they were homogenised and the 

radioactivity determined by combustion/LSC. 

Plasma samples were combined for all animals of each test. Liver, kidneys, and testis were weighed and

pooled for all animals of each test for the extraction of radioactive residues. 

Radioactivity measurement 

The measurement of the radioactivity in liquid samples was carried out by liquid scintillation counting 

(LSC) of 1 – 5 replicates. All solid samples and blood 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 measured per aliquot after correction for the background

radioactivity. The limit of quantification (LOQ) for 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 equal 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 automatically performed by the instruments. 

Metabolite analysis

Urine samples were combined for all animals of a test group and analysed by HPLC without prior sample 

preparation. Combined plasma samples (e.g. 2 mL) were mixed with 5 % formic acid (0.5 mL) and 

centrifuged. The supernatants were analysed by radio-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 acetonitrile/water and defoamer and extracted by 

maceration with a Polytron or Ultraturrax homogeniser. Extracts and solids were separated by 

centrifugation or filtration and the solids were further extracted with acetonitrile/water (7/3, v/v) as 

described above. The volumes of the extracts were measured and aliquots radioassayed by LSC. The 

post extraction solids were dried at room temperature and aliquots were solubilized with a tissue 

solubilizer and radioassayed by LSC. All extracts were combined, filtered, mixed with 0.1 mL emulsifier 

and evaporated to the aqueous remainder which was analysed by radio-HPLC for metabolic profiling. 

The metabolites in the samples were quantified by HPLC 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 were identified by comparison of the elution pattern of the samples from 

this study with the urines of male rats (low dose test no. 8 dosed with 2 mg/kg bw) from the ADME study 

(Report No. MEF-048/04). For plasma and extracts of organs, metabolites were assigned by comparison 

of profiles with those of urine. Furthermore, the most prominent metabolites BYI 08330-enol, BYI 08330-
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desmethyl-enol and BYI 08330-ketohydroxy were identified representatively in urine and liver extract by 

HPLC co-chromatography with non-radiolabelled reference 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 generated 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 in 

accordingly distinctively lower residues in organs and tissues of only about 12 % of the dose and about 9 

% in the GIT. The fast excretion and decline of residues 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 values 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 administered dose 1h after dosage showing the same fast 

decline as the other organs to <0.1 % of the dose 24 h after dosage. The TRR values of plasma were 

distinctively lower than in liver and kidney, but showed 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 testis were much lower than in plasma for all time 

points (0.7 mg/kg at 1 h declining to <0.01 mg/kg at 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 test 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 declined 

slowly from 1 h to 8 h from 2.4 % to 1.8 % of the dose 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 the GIT. In urine, 27 % were found at 24h post dosage. 

In contrast to low dose tests, the highest TRR values 1 h and 8 h after dosage were not detected in liver 

or kidney, but in plasma. The TRR values in plasma were about 10 - 20 % higher than in liver and kidney 

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 than 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 plasma, organs and tissues at sacrifice (expressed as

equivalent concentration C, as dose normalised concentration CN and as percentage of administered 

dose) are given in Table 6-10. 
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B. METABOLISM:

In the low dose tests, parent compound was not detected in any sample or tissue. BYI 08330-enol was 

the main metabolite in all samples. BYI 08330-desmethyl-enol was the second prominent metabolite in 

urine, plasma and testis, whereas in liver and kidney BYI 08330-ketohydroxy was found as the second 

prominent metabolite. BYI 08330-ketohydroxy was only present at trace levels in plasma, testis and 

urine. BYI 08330-enol-GA, -enol-alcohol and –desmethyl-ketohydroxy were detected at low levels. 

Therefore, they are of minor importance. Residues in all tissues and plasma 24 h after dosage were too

low for quantification of metabolites. Identifications rates were high in the range of 89 – 100 %. 

The metabolic profiles for urine were in good agreement 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 sample 

or tissue. BYI 08330-enol was the main metabolite in all samples. BYI 08330-desmethyl-enol was the 

second prominent metabolite in urine, plasma and organs. 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 levels and were of minor importance. Identifications 

rates were high in the range of 95 – 100 %. A summary of the metabolism in urine is presented in Table

6-11; the metabolism in plasma and organs for the high 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 radioactivity (equivalent to 112 % of the administered 

dose) was excreted via urine. About 8 % of the recovered 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 slower than in low dose ones. Only 27 % of the 

administered dose were excreted via urine 24 h after dosage and 18 % via faeces, respectively. The 

major part of the radioactivity was recovered in the gastro-intestinal tract of animals. Detailed data for 

excretion and residues in organs and tissues are presented in Table 6-9. 
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Table B.6-9: Balance of radioactivity in excreta, plasma, organs and tissues of male rats after 

single oral administration of 2 mg and 1000 mg [azaspirodecenyl-3-14C] BYI 08330/kg w 

Radioactivity in % of dose administered (mean values) 

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 included in the GIT 
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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 1000 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 

 
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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 mg [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 ha

0 - 1 h 0 - 8 h 0 - 24 ha 

% 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 

-

esmethylketohydroxy 

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 HPLC 
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Table B.6-12: Total radioactive residues of metabolites in plasma, liver, kidney and testis of male 

rats sacrificed 1h and 8 h after a single oral administration 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 0.342

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-ketohydroxy 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 HPLC 
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Table B.6-13: Total radioactive residues of metabolites in plasma, liver, kidney and testis of male 

rats sacrificed 1 h, 8 h, and 24 h after a single oral 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 1000 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 98.5 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: 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 

n.d. = not detected 

* Characterised based on their retention time in HPLC 

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 the cyclohexyl-O-methyl group to the respective 

alcohol (BYI 08330-desmethyl-enol) was a further important metabolic reaction as well as the 

hydroxylation in the azaspiro ring of BYI 08330-enol resulting in BYI 08330-ketohydroxy. Other metabolic 

reactions like conjugation of the BYI 08330-enol with glucuronic acid and oxidation of one of the methyl 

groups of the phenyl ring forming the BYI 08330-enol-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 kidney were distinctively higher than in plasma. This 

finding may indicate that metabolites are transferred by active transport mechanisms from plasma to 

the excretory organs. 
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• The residues in testis, carcass and skin were distinctively 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 the results of the ADME study. 

• The ratio of the two main metabolites BYI 08330-enol and BYI 08330-desmethyl-enol in urine was 

about 2:1. 

• BYI 08330-desmethyl-enol was found at lower proportions 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-enol were detected in liver, where the compound is 

formed by metabolic transformation of BYI 08330-enol. The proportions in plasma, kidney, and testis 

were comparable and significantly lower than in liver. 

• BYI 08330-ketohydroxy was found as a further prominent metabolite in liver and kidney, but was only 

present at trace levels in plasma, testis, and urine. 

For the high dose tests: 

• Absorption and excretion were slower and lower than in low dose tests. Only 27 % of the dose was 

renally excreted 24 h after dosage. 

• The residues in plasma were slightly higher than in liver and kidney. This may be caused by the 

saturation of the active transport mechanisms at high concentrations resulting in a more uniform 

distribution of the compound in the body. The decline of residues was slower than in the low dose 

tests. 

• As observed in low dose groups, the residues in testis, carcass and skin were lower than in plasma. 

Plasma and all organs showed slow depletion of radioactive residues.

• The metabolism was similar to the metabolism of the low dose tests, with the exception that BYI 

08330-desmethyl-enol was found at higher proportions 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 in 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-enol were detected in liver as well as in kidney. 

The percentages in plasma, kidney and testis were lower and comparable. 

• Significant percentages of BYI 08330-ketohydroxy were 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 urine 

and faeces is slow and with a potential of accumulation 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 male rats was well conducted and reported. Mass 

balance date were excellent. The data clearly indicate saturation of absorption at the high dose (1000 

mg/kg) versus the low dose (2 mg/kg) resulting in expected impact on metabolism, tissue burdens, and 

excretion. For all test groups, renal excretion was prevalent although at the high dose the saturated

absorption resulted in greater amounts of radioactivity appearing in the gastrointestinal tract and feces. 

Clearance of radioactivity from tissues and organs was rapid and nearly complete within 24 hours post 

dosing. Variability among data was typical for a metabolism/distribution study and clearly reported. 

Identification and characterization of metabolites was thorough and clearly reported. The analysis 

showed the enol metabolite as the primary biotransformation product and the demethylated enol as a 

significant secondary product. Major as well as unknown metabolites were quantified and several 
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quantitatively insignificant metabolites were also identified. A proposed metabolism pathway was 

consistent with the reported data. There were no apparent methodological or procedural flaws in the 

experimental protocol, acqusition/analysis of data or reporting of the data. The investigators expressed 

variability about the mean as coefficient of variability (converted to %) rather than providing standard 

deviation. This is an acceptable approach (C.V. = standard deviation/mean) and has been retained in 

the summary tables. The reviewer spot checked several C.V. values and found them to be accurate. 

The reviewer is in agreement with the interpretation of the study results provided by the investigator. 

This study in rats does meet current OPPTS 870.7485 Guidelines 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 (870.7485) in rats. 

C. STUDY DEFICIENCIES: 

There were no apparent deficiencies identified in the study report. 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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NH

O

O

O O

CH3

CH3

H3C

H

O

CH3

NH

O

HO

CH3

CH3

H

O CH3

H

O

NH

O

CH3

CH3

CH3

O

O

HO

HO

HO

HO

O

NH

O

O

CH3

CH3

H

O

OH

CH3

H

OH

NH

O

HO

CH3

CH3

H

O

NH

O

HO

CH3

CH2OH

CH3

NH

O

O

CH3

CH3

H

OH

OH

BYI08830

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

BYI08330-ketohydroxy BYI08330-desmethyl-enol

BYI08330-desmethyl-ketohydroxy

conjugation with

glucuronic acid

Figure B.6-2: Proposed metabolic pathway of BYI 08330 in organs and tissues and excreta 
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B.6.1.3 Metabolism (OECD Annex IIA 5.1.4) 

CITATION: Totis, M. (2006) [Azaspirodecenyl-3-14C]-BYI 08330: Comparison of the in vitro metabolism 

in LiverbeadsTM from male rat, mouse and human. Bayer 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: C21 H27 N O5

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-3-14C]-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 [azaspirodecenyl-3-14C]BYI 08330 was demonstrated by 

radio HPLC analysis of the stock solution before incubation 

2. Vehicle: Acetonitrile (ACN) 
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3. Test System:

LiverbeadsTM from male rat, mouse and human. LiverbeadsTM are immobilized hepatocytes entrapped 

within an alginate matrix. They were purchased from Biopredic International, Rennes, France. These 

hepatocytes were used in the incubation assays to study 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-3-14C]-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 prepared 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 concentration 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-radiolabelled BYI 08330 was first dissolved in ACN to obtain a 

concentration of 100 mM. Then, a volume of this solution 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 final concentration of the test compound was 

approximately 520 μM. 

B. STUDY DESIGN AND METHODS: 

1. In vitro metabolism of BYI 08330 by LiverbeadsTM from rat, mouse, and human: LiverbeadsTM 

were thawed according to the procedures described by the supplier. Then, the cells were pooled in 

HBSS (Hank’s balanced salt solution) plus penicillin 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 radiolabelled product in acetonitrile at the final 

concentration of approximately 50 μM or 520 μM. Control samples (Liverbeads + ACN) were also 

incubated without [14C]BYI 08330. Then, the plates were placed in an incubator 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 Na2 (100mM), and 

then the hepatocytes were sonicated during 20 sec. The supernatants including hepatocytes plus 

incubation medium plus dissolved LiverbeadsTM from each well were then transferred into separated 

vials and immediately stored at -20°C until LC/MS analysis. 

2. Metabolite analysis: 

Hepatocyte incubations were centrifuged and the supernatants were directly injected into the HPLC 

coupled with a UV detector, 14C detector, and a mass spectrometer. A C18 column with the solvents 
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0.1% aqueous acetic acid/ acetonitrile and gradient elution was used for quantification of the 

metabolites by integration of the 14C signal. The calculated area from each peak was then expressed 

as percentage of the sum of the areas from the different peaks (corresponding to metabolites). 

Metabolites were identified by their mass spectra measured 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 BYI 08330-enol and BYI 08330-enol-GA 

(enol glucuronide) which occurred at 66% and 30% respectively. The glucurono-conjugation of the 

main metabolite BYI 08330-enol appeared to be a major route in the in vitro degradation 

(detoxification) of BYI 08330 in the mouse. BYI 08330-enol-alcohol, BYI 08330-desmethyl-enol and 

BYI 08330-ketohydroxy were detected at a very low level in the mouse (1-2%). 

In the rat, the main metabolites were BYI 08330-enol and BYI 08330-desmethyl-enol which occurred at 

87% and 7% respectively. BYI 08330-enol-alcohol and BYI 08330-ketohydroxy represented 4% and 

3% respectively. The BYI 08330-enol-GA (enol glucuronide) was not detected in vitro in the rat. 

The major metabolites detected in human were BYI 08330-enol and BYI 08330-enol-GA 

(enol glucuronide), but their relative abundance were different from those observed in the mouse. They

occurred at 92% and 6% respectively. BYI 08330-desmethyl-enol was present at a very low level (1%); 

BYI 08330-enol-alcohol and BYI 08330-ketohydroxy were not detected in human. 

At high concentration, no new metabolite was detected in the rat, mouse and human LiverbeadsTM. 

The low degree of metabolism observed in this experiment could be indicative of a saturation of the 

biotransformation enzymatic system. 

The results of the individual tests are shown in Table 6-14, and the metabolic pathway for the in vitro 

test system is presented in Figure 6-3. 
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Table B.6-14: Distribution of the parent compound and metabolites after incubation of BYI 08330 

to LiverbeadsTM of rat, mouse, and human 

Low concentration experiment: distribution of parent 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 (sum = 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 concentration; 

therefore, no supplementary information was obtained. 

At low concentrations, BYI 08330 was completely metabolized in the liver cells from all species, and 

no parent compound was detected at the end of the incubation. BYI 08330-enol was the first and most 

prominent metabolite accounting for 66 – 92% of total metabolites. The metabolic profile among the 

three species at low concentration exhibited marked differences: 

• In the rat, the BYI 08330-enol was further metabolised 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 detected as an in vitro metabolic transformation. 

• The general picture of the “in vitro” metabolic pathway in the rat was very similar to the in vivo 

metabolism in the male rat. The same metabolites were 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 the in vivo and in vitro tests. 

• In the mouse, oxidative degradation of BYI 08330-enol 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” metabolism more similar to the one in mouse than in the 

rat. Conjugation to BYI 08330-enol-GA (6%) was more 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 the 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, respectively), species differences were observed in the 

metabolic profile. The rat LiverbeadsTM also produced the keto hydroxyl (3%), enol-alcohol (4%), and 
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desmethyl-enol (7%) metabolites; the human LiverbeadsTM produced the enol-glucuronide (6%) and 

desmethyl-enol (1%) metabolites; the mouse LiverbeadsTM produced substantial amounts of the enolglucuronide metabolite (30%) and minor amounts of enol-alcohol (1%), desmethyl-enol (1%), and keto 

hydroxyl (2%) metabolites. 

C. STUDY DEFICIENCIES:

No study deficiencies were noted. 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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NH

CH3

CH3

O

O

H3C O O

H

O

CH3

NH

CH3

CH3

O

HO

H

O

CH3

O

NH

CH3

CH3

O

O

H

O

CH3

HO

HO

HO

HO

O

NH

CH3

CH3

O

HO

H

OH

NH

CH2OH

CH3

O

HO

H

O

CH3

NH

CH3

CH3

O

H

O

CH3

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 08330 in LiverbeadsTM from male rat, mouse, and 

human (taken from p. 23, Report No. SA05319)
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B.6.1.4 Metabolism (PBPK Modeling) (OECD Annex IIA 5.1.5) 

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

Bayer Technology Services GmbH, PT-AS-Systems Biology, Building E41, 51368 

Leverkusen, Germany. Report Number BTS-WSM0602. July 18, 2006. 

I MATERIALS AND METHODS

A. MATERIALS:

No test material was used since this report describes a computer simulation and not an experimental 

study. 

B. STUDY DESIGN:

Pharmacokinetic simulation

Physiologically based pharmacokinetic (PBPK) simulations were carried out with the commercially 

available software PK-Sim (Bayer Technology Services GmbH). PK-Sim is based on a generic whole 

body PBPK-model which describes uptake and distribution of organic substances after oral or 

intravenous administration. Results of PBPK-simulations 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, Liver, Lung, Muscle, Pancreas, Portal, Skin, Small 

Intestine, Spleen, Stomach, Gonads). The concentrations are given as total tissue concentrations or as 

total or unbound concentrations in interstitial or cellular space for the organs. Blood concentrations are 

available as unbound or total plasma or red blood-cell concentrations. In order to account for a specific 

fate of the simulated substance, PK-Sim allows inclusion of metabolism in all included organs as well as 

active transport across all permeation barriers considered in the simulation model. 

For the physiological parameters of the model (blood 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 customized 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 physicochemical properties of the compound to be 

simulated. All other substance-specific model parameters (e.g. clearance, metabolic rates, and rates of 

active transport) have to be entered directly as input values. Their values must either be determined 

experimentally or can be estimated by manually adjusting the simulation for the best fit to experimental 

pharmacokinetic data. 

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

Physiological parameters: All parameters depending solely on physiology and anatomy of the 

simulated individual were kept as provided by the built-in database of PK-Sim. 

Compound-specific parameters: Parameters depending on the substance to be simulated 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 
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according to the properties of the major metabolite BYI 08330-enol. The predominant excretion route for 

BYI 08330-enol is renal excretion; therefore, the total clearance was first ascribed to renal clearance. It 

should be noted that in the disposition study in the 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 values of the physicochemical properties of 

BYI 08330-enol and the clearance to the values given in Table 6-15. 

Table B.6-15: Values of physiochemical and clearance 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 constant for human serum albumin 

calculated from chemical structure using in house QSAR 

method. 

Molecular Weight [g/mol] 301 g/mol 

Solubility [mg/l] 2200 mg/l Water solubility calculated 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 correlation to simulation results is summarized in Table 

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-3-14C]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-3-14C]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

– 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 

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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 passive 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 refined 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 and 

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 total 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), Cmax (maximal plasma concentration) and peak/trough 

ratio (Cmax/C24h) on dose following single dose administration (dose range 2 – 1000 mg/kg bw). 

• dependence of dose normalized area under the curve (AUCnorm) and dose normalized maximal 

plasma concentration (Cmax) on dose following simulated repeated dosing during a 28 day period 

(dose range 2 – 1000 mg/kg bw). 

The agreement between calculated and experimental data 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 available experimental data. It was possible to generate a

single PBPK model that adequately described the observed 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 compared 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 metabolites into these tissues. 
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The renal excretion rate of BYI 08330-enol could only be explained by active tubular secretion into 

urine, because the physicochemical properties yielded a glomerular filtration rate that was much 

smaller than the observed rate of excretion. It could be shown with a high probability that the 

saturation of the renal transport processes is responsible for the experimentally observed non-linearity 

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 allow determination if uptake or excretion is the more 

relevant process, it is reasonable to assume that there is saturation of both processes simultaneously. 

This conclusion is based on the fact that the unbound concentrations comparable to typical binding 

constants (in the μmol range) for transport proteins are reached or exceeded in the plasma as well as 

the kidney cells. 

The PBPK-simulations allowed an extrapolation to doses 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 was found that saturation of the active transport 

processes leads to a significant change in the shape of the plasma concentration curve at the highest 

doses. This has in turn a significant impact on pharmacokinetic parameters describing the systemic 

exposure to the compound. While the dose-normalized maximum concentrations only show a fair 

increase with the dose, this increase is rather significant for the area under the concentration curve. 

An even more pronounced change was found for the peak/trough ratio (Cmax/C(24h)). This parameter 

decreases roughly by a factor of 500 reaching values as low as 5 – 6 at 1000 mg/kg. Such low 

peak/trough ratios are indicative of a continuous rise of systemic concentrations upon repeated daily 

administration. Such a rise of body burden was found 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 steady state was reached after about 15 days. This led to 

an even higher non linearity of the AUC, with a 7-fold increase in AUCnorm 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 respective properties might generally differ from those of 

the simulation because they rely on parameter values estimated with some uncertainty. 

An assessment of the dependence of the disproportional 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 continuously at least over the whole four weeks 

investigated here. This leads to predicted systemic exposures (AUC) at very high doses which are 

about 20 times higher than those extrapolated linearly 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 (Report No. MEF-06/15) revealed indications of the 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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presence of saturable processes that influence the pharmacokinetics of the compound. These 

indications are a change in shape of the plasma concentration curve with increasing dose and 

extraordinarily high concentrations in liver and kidney compared to other tissues. Both organs are known 

to express various active transport proteins with a broad variety of substrates. 

It could be shown that the experimentally determined ADME behaviour of BYI 08330 in male rats can be 

well described by PBPK simulations 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 

observed plasma concentrations, their non-linearity in dose, organ concentrations and the metabolism 

and excretion can be described with very good agreement 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 kidney compared to plasma and other tissues, as 

observed by QWBA, indicate the presence of active transport 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 concentrations in both organs. 

• The renal excretion rate of BYI 08330-enol can only 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 100 mg/kg, although the available experimental 

data do not allow determination if uptake or secretion 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 extrapolation of doses up to 1000 mg/kg, which is 

significantly higher than those investigated experimentally 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 leads to a change in the shape of the plasma 

concentration curve at high doses resulting in a plateau-like concentration time curve due to a 

decreased efficiency of excretion. 

• The non-linearity of plasma concentration with dose leads to a disproportionate increase of systemic 

exposure with increasing dose. While the dose normalized maximum concentrations show only a 

fair increase with dose, this increase is more substantial for the area under the concentration curve. 

The dose normalized area under concentration curve (AUCnorm) is about five times higher at 1000 

mg/kg than at 2 mg/kg. 

• The peak/trough ratio (Cmax/C24h) changes are even more pronounced with dose. The ratio 

decreases roughly by a factor of 500 reaching values as low as 5 – 6 at 1000 mg/kg, thus being 

indicative of a rise of systemic concentrations upon repeated daily administration. 

In order to assess the impact of this non-linear behaviour on toxicological studies with repeated 

administrations, the dose dependence of plasma concentrations during a four-week period with daily 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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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 concentrations 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 AUCnorm with doses increasing from 2 mg/kg to 1000 mg/kg, compared 

to 5-fold for a single administration. 

The disproportionate increase in plasma concentration after repeated administration of BYI 08330 might

have an impact on systemic exposure after high doses 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 the kidney and liver are involved in the uptake of BYI 

08330 metabolites and that active tubular secretion into the urine is likely responsible for the observed 

renal excretion rats. The model also suggests that saturation of the renal transport process is 

responsible for the nonlinearity of plasma concentrations 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 using experimental data only up to a dose of 100 

mg/kg bw. However, a subsequent study was conducted (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 therefore referred to this additional PBPK study for 

conclusions regarding exposure to high doses of BYI 08330.

C. STUDY DEFICIENCIES:

None. 
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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, Building 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 includes a simulation and not an experimental study. 

C. STUDY DESIGN: 

Pharmacokinetic simulation

Physiology based pharmacokinetic simulations were carried out with the commercially available software

PK-Sim (Bayer Technology Services GmbH). 

The simulation used the physiological parameters optimized in the first PBPK simulation (Report Number

BTS-WSM0602) which were refined to match the results 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 metabolites BYI 08330-enol and BYI 08330-

desmethyl-enol after a single dose of 2 mg/kg bw and 1000 mg/kg bw 

• Time course for plasma concentrations of metabolites BYI 08330-enol and BYI 08330-desmethylenol after repeated daily administration of BYI 08330 for 4 weeks. The dose range covered was 10 

– 1000 mg/kg bw. 

The agreement between calculated and experimental data was checked by visual comparison and 

statistical evaluation. 

II. RESULTS: 

For BYI 08330-enol, the new adjustment of model parameters had only a minor influence on the 

simulation results at low doses. Thus at the dose of 2 mg/kg, the correlation between simulation results 

and observed data remained comparable to the results 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 testis 

tissue/plasma partition coefficients 8 hours after administration, the fractions of administered dose 

excreted in urine and the peak/trough ratios for both dose regimens of the experimental study were as 

well represented by the simulation of 1000 mg/kg as by that of 2 mg/kg. 
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The model simulation of BYI 08330-desmethyl-enol did not accurately predict concentrations in plasma, 

liver, kidney and testis following administration of 2 mg/kg and 1000 mg/kg BYI 08330. The authors 

concluded that the observed data points are described satisfactorily considering the sparse experimental 

information and the fact that the simulation is based on the output of another simulation. 

In addition to the systemic concentrations, the fractions of dose which were excreted in urine as 

BYI 08330-enol and BYI 08330-desmethyl-enol were predicted by the simulations and were compared to 

the respective experimental values. The observed behaviour was well reflected by the simulation. 

The plasma concentration time curve of the BYI 08330-enol after multiple dosing was confirmed by the 

refined PBPK model. Mean daily plasma concentrations 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 stable 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 Cmax and AUC and of the ratio Cmax(day1)/Cmax(day28) listed in Table 6-17. 

Table B.6-17: Dose-dependency of dose normalized Cmax, AUC, and 

ratio Cmax(day1) / Cmax(day28)a

Daily dose 

[mg/kg] 

AUCnorm 

[kg.h/l] 

Cmax_norm 

[kg/ml] 

Cmax(day 28)/Cmax(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 Version No. 2, July 6, 2006. 

III. DISCUSSION: 

A. CONCLUSIONS:

The parameterization of the PBPK-model for BYI 08330 could be significantly improved using the most 

recent experimental data for liver and kidney concentrations at high doses and the concentrations of the 

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 time 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 those observed within or below a factor of two. For the 

dose of 1000 mg/kg, the calculated enol concentrations 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-desmethyl-enol or the BYI 08330-desmethyl-enol 

plasma and tissue concentrations) are in good agreement with the respective experimental data. 

The model indicated that the saturation of the transport 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 application of the model in simulations of scenarios

not investigated experimentally, it is important that despite of the discrepancy in the absolute 

concentrations found at high doses, the general pharmacokinetic behaviour with the strong reduction of

Cmax/C(24h) with increasing dose is well represented by the calculations. The reduction of the peak/trough 
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ratio is the main reason for the strong rise of concentrations predicted for repeated administration of 

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 significantly 

alter the predictions for BYI 08330-enol at low doses, and improved the BYI 08330-enol PBPK model 

predictions for exposure to the high dose. The simulation adequately predicted liver, kidney and testis 

tissue/plasma partition coefficients 8 hours after dosing, the fractions of administered dose excreted into 

urine, and the peak/trough ratios. The deviations from the simulated verses experimentally derived tissue 

concentrations (plasma, liver, kidney and testis) range up to 7-fold, however, indicating that the ADME 

process at doses of 1000 mg/kg bw/day is not completely described by the PBPK model. It was 

proposed by the study author that part of the discrepancy is due to a difference between the 

experimental and simulated fraction absorbed. 

Predicted BYI 08330-desmethyl-enol concentrations in 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 constant 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 also 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 05319 Version N°2. July 6, 2006), the 

disproportionate increase in plasma concentration of the metabolites after repeated administration of BYI 

08330 might have an impact on systemic exposure after high doses in toxicological studies. 

C. STUDY DEFICIENCIES:

No study deficiencies were noted. 
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B.6.1.6 Metabolism and Pharmacokinetics (OECD Annex IIA 5.1.6) 

CITATION: Klempner, A. 2006. [Azaspirodecenyl-3-14C]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 CropScience AG Development, 

Metabolism/Environmental Fate, 40789 Monheim am Rhein, Alfred Nobel Str. 50, Germany. 

I. MATERIAL AND METHODS 

A. MATERIAL:

1. Test Material:

IUPAC Name: (5s,8s)-3-(2,5-dimethylphenyl)-3-hydroxy-8-methoxy-1-

azaspiro[4.5]decane-2,4-dione 

Code name: BYI 08330-ketohydroxy 

Common name: Not applicable 

Empirical formula: C18 H23 N O4

Molar mass: 317.39 g/mol 

Water solubility: Unknown 

n-Octanol/water partition 

coefficient: 

Unknown 

Labelling: Azaspirodecenyl-3-14C 

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 [azaspirodecenyl-3-14C]BYI 08330-ketohydroxy was 

demonstrated by radio HPLC analysis of the administration 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 administration 

 208 – 220 g at the time of sacrifice 

Acclimatization: Makrolon® cages on wood shavings in the test facility for about 7 days 

prior to the administration. 

Identification: Cage cards on which the study number, test compound name and 
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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 conditions in 

air-conditioned rooms 

Temperature 22 - 24 °C, relative humidity 50 – 60 %. 

12 / 12 hours light / dark cycle, air change 10 – 15 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 metabolite 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, where it was the main metabolite in Swiss chard and 

turnip roots and a main intermediate in the metabolic pathway. Based on these results, BYI 08330-

ketohydroxy was included in the plant residue method 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 received 2 mg/kg bw of azaspirodecenyl-3-14C labelled BYI 

08330-ketohydroxy suspended in 0.5% aqueous tragacanth after 16 hr fasting. The dosing suspension 

(0.2 mg /mL for the low and high dose level) was prepared one day before dosing and stored at 4°C. 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 200 g). Actual administered doses were measured by LSC assay of aliquots of the 

administration suspensions. The actual mean administered dose of [azaspirodecenyl-3-[14C]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 administration. 

Collection of blood:

Blood samples were collected separately for each animal by pressing a capillary coated with heparin in a 

small cut in the tail vein. The wound was closed with 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 centrifugation, the capillary was broken at the border between 

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 scintillation vial for radioactivity measurement. 

Collection of excreta:

Urine was collected at various times separately for each animal in a cryogenic trap cooled with dry ice. 

The funnels for urine collection were rinsed with demineralised water at the end of each sampling period. 

The rinsing solutions were drained into the same vial as the corresponding urine fraction. The 

radioactivity was determined by LSC. Urine samples were stored in a freezer before analysis. Faeces 
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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 radioactivity of aliquots was determined by combustion/ 

LSC. 

Sacrifice and organ/tissue sampling: 

At the respective test end (48 h after administration), the animals were anaesthetised using 

Pentobarbital-Na. They were sacrificed by transection 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 erythrocytes by centrifugation. The following organs/tissues 

were collected: erythrocytes, plasma, spleen, gastro-intestinal tract (GIT), liver, kidneys, perirenal fat, 

adrenal gland, testis, skeleton muscle, femur bone, heart, lung, brain, thyroid gland, skin, and carcass. 

The organs and tissues were weighed immediately after dissection, following lyophilisation, and 

homogenised. Radioactivity was determined by combustion/LSC. The radioactivity remaining 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, adrenal glands, and thyroid), only the wet 

weight was determined and the samples then solubilised with BTS 450®

 (Beckman Tissue Solubiliser). 

Radioactivity measurement:

The measurement of the radioactivity in liquid samples was carried out by liquid scintillation counting 

(LSC). For this purpose, the volume of the entire solution was determined and the radioactivity of an 

aliquot (1 – 3 replicates) of the solution was measured as follows: 

Small organs or tissues were solubilised by means of 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 was used for the following test materials: renal fat,

adrenal glands, and thyroid. 

Solid samples (1 – 5 aliquots) were weighed and combusted in an oxygen atmosphere using an Oxidizer 

307/387 (Packard Instruments) for combustion of faeces, erythrocytes, lyophilised organs and tissues, 

e.g. spleen, liver, lung, bone muscle, gastrointestinal tract (GIT), residual carcass and solids (e.g. from 

faeces after extraction). Carbosorb E (9 mL) was used to trap the combustion product CO2 and 

Permafluor E+ (10 mL; Packard Instruments) was used as scintillator for LSC. 

Metabolite analysis

Urine samples were analysed without sample preparation. Faeces samples of all animals from 0 - 24 h 

post administration were combined and extracted successively 3 times with acetonitrile / water (8:2, v/v) 

using a Polytron homogeniser. Each extract was collected 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 radioassayed by LSC. The aqueous phase was 

concentrated and used for HPLC analysis. The radioactivity in post extraction solids was determined by

combustion analysis. 

The metabolites in the samples were quantified by HPLC 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 
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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 mainly identified by comparison of the metabolic profile 

of faeces with urine. Two major metabolites were also 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 absorption 

commenced immediately after oral dosing. It was concluded 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 absorption 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 radioactivity was 

recovered in urine, faeces, and the body of animals. 

B. DISTRIBUTION:

Radioactivity in plasma of males and female rats following a single administration of 2 mg/kg bw reached 

its calculated maximum at 0.81 h postdose. Pharmacokinetic parameters were calculated from 

concentrations of total radioactivity using a 2 compartment disposition model. The calculated maximum 

radioactivity concentration in plasma was 1.26 mg eq./kg bw. The peak concentration was slightly below 

the equal distribution concentration of 2 mg/kg bw BYI 08330-ketohydroxy. Absorption was followed by 

a fast elimination phase. The mean residence time was low at 4.32 h. Radioactivity levels in plasma are 

summarised in Table 6-18. 

The distribution of radioactive residues in the body was analyzed at the time of sacrifice 48 h after dosing 

by radioactivity measurement of the major organs and tissues. Less than 0.2% of the administered dose 

was detected in the body. Radioactivity concentrations 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 administration of [azaspirodecenyl-3-14C]BYI 08330 is shown in 

Table 6-19. 

C. METABOLISM:

The parent compound was not detected in urine. Traces of BYI 08330-ketohydroxy were found in faeces. 

BYI 08330-ketohydroxy was completely metabolised forming numerous metabolites. The first and most 

important metabolic reaction was the oxidative demethylation 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 08330-desmethyl-ketohydroxy as an intermediate. Most 

of the identified compounds were different mono-, di- and tri-oxygenated metabolites which are probably 

represented by isomers of mono-hydroxy, di-hydroxy and tri-hydroxy metabolites. A second oxidative 

transformation was formal loss of two hydrogen atoms of the oxygenated metabolites to “dehydro” 

derivates. These metabolites probably contain aldehyde 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. 
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The oxygenated metabolites were classified in three groups to simplify evaluation and quantification: 

mono-hydroxy, di-hydroxy and tri-hydroxy metabolites of BYI 08330-desmethyl-ketohydroxy. 

About 34% of the dose was accounted for by the various 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 this group was detected accounting for about 3 % of 

the dose. Conjugation with glucuronic acid or sulfate was detected for a few metabolites only and at low 

quantities. Cleavage of the azaspirodecane ring of BYI 08330-desmethyl-ketohydroxy was found to a low 

extent; cleavage was supported by the presence of two metabolites: BYI 08330-desmethyl-MA-amide 

and BYI 08330-desmethyl-glyoxylic amide. Both metabolites covered ≤2.8% of the dose. In total, 85.6% 

of the administered dose was identified and 8.57% was 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 following oral administration of [azaspirodecenyl-3-14C] BYI 

08330-ketohydroxy are shown in Table 6-21. The proposed 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 urine 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 [azaspirodecenyl-3-14C] 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 08330-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 
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Table B.6-19: Distribution of radioactivity in organs/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 excretion of radioactivity with urine and faeces of 

male rats after oral administration of 2 mg [azaspirodecane-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 
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Table B.6-21: Distribution of parent compound and metabolites in the excreta of rats after oral 

administration of 2 mg [azaspirodecenyl-3-14C]BYI 08330-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
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Total in excreta 54.45 44.09 98.54

* = derivate of BYI 08330-desmethyl-ketohydroxy 

** = peak 5 consists of four components, and the peaks 19 to 20 each of two components 

-- not detected 
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NH

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O

CH3

CH3

H

OH

HOOC

O

+ O + 2 O + 3 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 08330-ketohydroxy in the rat 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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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 within the blood and most organs and 

tissues with preference to the liver as the main metabolizing 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 tissues 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 be derived 

• Oxidative conversion of BYI 08330-desmethyl-ketohydroxy 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 as 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 excellent. 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 potential 

for bioaccumulation at the tested dose. Metabolite profiles for urine and feces were qualitatively similar 

but varied quantitatively. The presence of numerous desmethylhydroxy products affirms the 

investigator’s contention of demethylation as the initial and primary biotransformation process. 

Unchanged test article was not detected in the urine and represented <1% of the administered dose in 

the feces. Minimal radioactivity in tissues and organs 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 conclusions 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 administered dose was found in the feces, very little parent 

compound was detected in the feces and the fecal metabolite profile was well characterized and similar

to that found in the urine. Therefore, intravenous administration experiments and biliary excretion tests 

would have added little additional information. 
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C. STUDY DEFICIENCIES:

There were no apparent deficiencies identified in the study report. 
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B.6.1.6.1 Metabolism and pharmacokinetics (OECD Annex B.6.1.2.6) 

CITATION: Klempner, A. 2006. [Azaspirodecenyl-3-14C]BYI 08330-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 Rhein, Alfred Nobel Str. 50, Germany. 

I. MATERIAL AND METHODS 

A. MATERIAL: 

1. Test Material:

IUPAC Name: (5s,8s)-3-(2,5-dimethylphenyl)-8-methoxy-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: C24 H33 N O8

Molar mass: 463.53 g/mol 

Water solubility: Unknown 

n-Octanol/water partition 

coefficient: 

Unknown 

Labelling: Azaspirodecenyl-3-14C 

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 radiodetection) 

Dose level: 0.1 mg/kg body weight 

Vehicle: Aqueous saline (0.9 % NaCl in water adjusted to pH 5) 

Stability of the test material: The stability of [azaspirodecenyl-3-14C] BYI 08330-enol-glucoside was 

demonstrated by radio HPLC analysis of the administration solution 

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: 1 

Age: 8 weeks at the time of delivery 

Body weight: Males: 199 g at the time of administration 

 202 g at the time of sacrifice 

Acclimatization: Makrolon®

 cages on wood shavings in the test facility for about 7 days 

prior to the administration. 

Identification: Cage cards on which the study number, test compound name and 
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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 conditions 

in air-conditioned rooms 

Temperature 23 - 25 °C, relative humidity 40 – 60 %. 

12 / 12 hours light / dark cycle, air change 10 – 15 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-3-14C 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 lettuce metabolism study [Haas and Diederich, 2004, 

Report MEF-049/04; sample code BD1120D], purified by HPLC and identified by spectroscopic methods. 

The solution was administered to the rat by oral gavage 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 reach 

an administered amount of about 0.1 mg of the parent compound per kg body weight (bw). The actual 

administered dose was measured by LSC assay of aliquots of the administration solution and amounted 

to 0.063 mg [azaspirodecenyl-3-14C]BYI 08330-enol-glucoside / kg bw. The stability of the test 

compound in the solution was determined by radio HPLC analysis of aliquots after administration. 

Collection of blood:

Blood samples were collected by pressing a capillary coated with heparin in a small cut in the tail vein. 

The wound was closed with 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 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 radioactivity 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 the end of each sampling period. The rinsing solutions 

were drained into the same vial 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 in a cryogenic trap before they were lyophilised 

(freeze-dried), weighed, and homogenised. The radioactivity was determined by combustion/LSC. 

Lyophilised samples were stored at room temperature before extraction/analysis. 
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Sacrifice and organ/tissue sampling:

At the respective test end (48 h after administration), the animals were anaesthetised using 

Pentobarbital-Na. They were sacrificed by transection of the cervical blood vessels. The following 

organs/tissues were collected: gastro-intestinal tract (GIT), skin, and carcass. 

The organs and tissues prepared at the end of the experiments were weighed immediately after the 

dissection and again following lyophilisation. The radioactivity remaining in the GIT, skin, and residual 

carcass was determined by combustion/LSC in order to establish the radioactivity balance. 

Radioactivity measurement:

The measurement of the radioactivity in liquid samples was carried out by liquid scintillation counting 

(LSC). For this purpose, the volume of the entire solution was determined and radioactivity of an aliquot 

(1 – 3 replicates) of the solution was measured: 

Solid samples (1 – 5 aliquots) were weighed and combusted in an oxygen atmosphere using the 

following equipments Oxidizer 307/387 (Packard Instruments) for combustion of faeces, erythrocytes, 

lyophilised organs and tissues, e.g. spleen, liver, lung, bone muscle, gastrointestinal tract (GIT), residual 

carcass and solids (e.g. from faeces after extraction). Carbosorb E (9 mL) was used to trap the 

combustion product CO2 and Permafluor E+ (10 mL; Packard Instruments) was used as scintillator for 

LSC. 

Metabolite analysis:

Urine samples were analysed without sample preparation. Radioactivity in the freeze-dried faeces 

sample for the time range from 0-24 h post administration was determined by combustion/LSC. An 

aliquot of the sample was extracted 3 times with acetonitrile / water (8:2; v/v) using a Polytron 

homogeniser. Each extract was collected by centrifugation and its volume measured. Aliquots of each 

extract were radioassayed by LSC. The three extracts 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 concentrated and submitted for HPLC analysis. 

The metabolites in the samples were quantified by HPLC 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 identified by comparison of HPLC profiles of samples 

with those from the BYI 08330 ADME rat study (Report No. MEF-048/04) and by co-chromatography 

with reference compounds. 

II. RESULTS AND DISCUSSION 

A. ABSORPTION:

The radiolabelled BYI 08330-enol-glucoside was rapidly absorbed from the gastrointestinal tract of the

male rat. The absorption commenced immediately after 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:
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The distribution of the test substance from the central compartment to the different organs and tissues 

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 treatment. 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 radioactivity 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 accounting for about 64% of the dose administered. 

Minor metabolites were BYI 08330-desmethyl-enol with 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 distribution of metabolites in excreta is presented in Table 6-

23. The proposed metabolic pathway of BYI 08330-enol-glc in the rat is shown in Figure 6-5. 

D. EXCRETION:

Excretion was fast and almost complete 24 h after administration. From the total excreted amount of 

97.1%, 53.3% and 43.7% of the dose were excreted with urine and faeces, respectively. The excretion 

data are summarized in Table 6-22. 

Table B.6-22: Time course of the excretion of radioactivity via urine and faeces of the male rat 

following a single oral administration of 0.1 mg [azaspirodecenyl-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 
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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 
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NH

O

HO

CH3

CH3

H

O

CH3

H

O

NH

O

CH3

CH3

CH3

O

O

HO

HO

HO

HO

NH

O

O

CH3

CH3

H

O

OH

CH3

H

OH

NH

O

HO

CH3

CH3

BYI08330-enol

BYI08330-enol-Glc

BYI08330-ketohydroxy BYI08330-desmethyl-enol

Figure B.6-5: Proposed metabolic pathway of BYI 08330-enol-glucoside in the rat 

A. CONCLUSIONS: 

 

BYI 08330-enol-glucoside was a main metabolite of BYI 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 European residue trials (unpublished results). Because it was 

not detected in the rat ADME-study, an additional ADME study with this compound was conducted in 

order to investigate the absorption, distribution, excretion and metabolism of BYI 08330-enol-glucoside 

in one male rat. 

One male rat was administered by oral gavage a single 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 14C 

in the 3-position of the spiro-ring of the molecule (azaspirodecenyl-3-14C label). Microplasma, urine and 

faeces were collected at various times of post administration. Skin, gastro-intestinal tract and carcass 

were sampled at sacrifice. 

The fate of radio labelled BYI 08330-enol-glucoside in the male rat following a single oral administration 

was characterized as follows: 
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• Absorption: 

Fast absorption of the radioactive test compound from the gastrointestinal at a rate of ca. 50% 

• Distribution: 

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

48 hours 

• Excretion: 

Rapid and complete excretion of radioactivity via the renal and faecal route in about the same 

proportion 

• Metabolism: 

Cleavage of the glucoside bond resulting in BYI 08330-enol as the major metabolite and key 

intermediate in the metabolic pathway 

• Oxidation of the azaspirodecenyl moiety to BYI 08330-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 disposition 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 subsequent excretion via the urine (53.3% of dose) and 

feces (43.7% of dose) over a 48-hour post dose period. 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 parent compound. A proposed metabolism pathway was 

provided which is consistent with the study findings. The reviewer concurs with the interpretations and 

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 crop metabolite of BYI 08330. 

C. STUDY DEFICIENCIES:

Although the methodology and data appear sound, only one male rat was used to assess the 

metabolism and disposition of the test article. There were no other apparent deficiencies identified in 

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

CITATION: 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. Study No. 

M1811287-8. Report No. MEF-06/15, Document No. M-269337-01-2. Bayer CropScience 

AG Development, Metabolism/Environmental Fate, 40789 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: C21 H27 N O5

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-3-14C 

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 TLC with radiodetection) 

Dose level: 3 mg/kg body weight 

Vehicle: 0.5 % aqueous tragacanth suspension 

Stability of the test material: The stability of [azaspirodecenyl-3-14C]BYI 08330 was 

demonstrated by radio HPLC analysis of the administration 

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 animal 

Females: 8 + 1 control animal 

control animals were dosed with non radiolabelled test item 

Age: 8 weeks (male rats) and 11 – 12 weeks (female rats) 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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at the time of delivery 

Body weight: Males: 202 – 217 g at the time of administration 

 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 about 7 days 

prior to the administration. 

Identification: Cage cards on which the study number, 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 conditions in 

air-conditioned rooms 

Temperature 20 - 24 °C, relative humidity 33 – 63 %. 

12 / 12 hours light / dark cycle, air change 10 – 15 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 mg/kg bw oral dose of [azaspirodecenyl-3-14C]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 08330. 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 200 g). The amounts of the administered test 

substance were calculated by dividing the administered radioactivity amounts by the specific 

radioactivity. The actual mean administered dose of [azaspirodecenyl-3-14C]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 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 quantitative collection of urine, faeces, and the 

expired air. Urine was collected separately for each animal in a cryogenic trap cooled with dry ice at

intervals of 4 h, 8 h, 24 h, and every 24 h until 168 h. The funnels for urine collection were rinsed with 

demineralised water at the end of each sampling period. 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 each animal before they were lyophilised (freezedried), weighed, and homogenised. The radioactivity was determined by combustion/LSC. 
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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 was 

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, the exact volume was determined, from which an 

aliquot was taken for the determination of radioactivity by LSC. 

Sacrifice and preparation of carcass for autoradiography: 

One animal of each gender was sacrificed using carbon 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 condition. 

This animal was excluded from the study. The control animals were sacrificed 4 h after dosing. 

After sacrifice, the animals were fixed in a stretched position using a metal template and immediately

frozen at ca. -70°C in a dichloromethane/dry ice bath. After removal of the template, the animal body 

together with a series of blood standards (14C labelled compound in bovine blood) was embedded in 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 using a cryomicrotome, attached to adhesive tape, and

lyophilized overnight in the cooling cabinet of the microtome. Four to five sections showing the relevant 

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 imaging 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 under identical conditions, using the longest exposure 

time which had been chosen for rat sections of animals treated with the radiolabelled compound. No 

blackening of typical animal structures could be observed 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 radioactivity 

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 background (Bkd) subtraction, a value of the photostimulated 

luminescence (PSL) per mm2

 was obtained, which is proportional to the equivalent 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 14C-radiolabelled compound. The concentrations covered 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)/mm2 to the activity in dpm/g tissue by linear regression 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 corrected 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 given in dpm/g were divided by the specific radioactivity 

of the test substance in dpm/μg. 
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Radioactivity measurement: 

The measurement of the radioactivity in liquid samples was carried out by liquid scintillation counting 

(LSC) of 1 – 3 replicates. All solid samples and blood 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 measured per aliquot after

correction for the background radioactivity. The only exception was the measurement of radioactivity in 

the expired air samples for which all reasonable dpm-values were quantified. The limit of quantification 

(LOQ) for 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 equal 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 automatically performed by the instruments. 

II. RESULTS AND DISCUSSION 

A. DISTRIBUTION:

Blackening by radioactivity that originated from radiolabelled [azaspirodecenyl-3-14C]BYI 08330 was 

most intense in the gastrointestinal tract, in the urinary bladder, the kidney and in the liver. Among the 

quantitatively analysed organs, tissues, and fluids, the highest equivalent concentrations were observed 

in the liver, kidney and blood. Moderate peak concentrations lower than in blood were found in the lung 

and myocardium, brown fat, skin, the major glands and the reproductive organs. Lower concentrations 

were reached in all other organs and tissues. The lowest peak concentrations were found in the spinal 

cord, the brain, and the eye. 

Peak concentrations of radioactivity were reached after 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 within 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 after 

dosage in the liver of male rats. All quantitative results are presented in Table 6-24 (male rats) and 

Table 6-25 (female rats). 

B. EXCRETION

BYI 08330 was excreted rapidly and completely within ca. 48 hours. Excretion was mainly renal. Only a 

minor part of ca. 3-6 % of the dose was excreted via faeces. The expiration of 14C-carbon dioxide and 

other 14C-labelled volatiles was tested with male and female animals for a period of 48 hours. Less than 

0.01% of the administered dose was expired during the sampling period. This negligible formation of 

volatile products demonstrates the stability of the azaspirodecenyl-3-14C label. The excretion behaviour 

is summarized in Table 6-26 (male rats) and Table 6-27 (female rats).
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Table B.6-24: Distribution of radioactivity in organs and tissues of male rats after oral 

administration of 3 mg [azaspirodecenyl-3-14C]BYI 08330/kg bw 

Total radioactive residues (TRR) in organs and tissues 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 < LOD 

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 
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Table B.6-25: Distribution of radioactivity in organs and tissues of female rats after a single oral 

administration of 3 mg [azaspirodecenyl-3-14C]BYI 08330/kg bw 

Total radioactive residues (TRR) in organs and tissues 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
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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 administered 

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 
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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 administered 

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
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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 radioactivity were most intense in the gastrointestinal 

tract, in the urinary bladder, the kidney, and in the liver. The peak value of radioactivity concentrations 

was reached 1 hour after dosing followed by a continuous decrease by several orders of magnitude, 

below the limit of detection for all organs and tissues within 24 hours in female rats and within 48 hours 

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 absorption and excretion of a 

single 3 mg/kg dose of radiolabelled [azaspirodecenyl-3-14C]BYI 08330 in male and female Wistar rats. 

The primary route of excretion was the urine (nearly 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 absorption, metabolism, and excretion (i.e., liver, kidneys) 

contained the greatest amounts of radioactivity. The reviewer agrees that at the dose tested, retention 

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 results or 

their interpretation. 
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B.6.1.8 Summary of ADME studies 

The disposition of spirotetramat was investigated in three in vivo and one in vitro studies. Gastrointestinal 

absorption of spirotetramat was rapid following oral administration to male and female rats at a single dose 

of 2 or 100 mg/kg bw or repeated doses of 2 mg/kg bw for 14 days. The absorption rate in all tests was 

between 89 and 98% of the total recovered radioactivity, based upon recovery from the urine and carcass 

(without the gastrointestinal tract). No significant 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 was reached for all dose groups within 0.09 to 2.03 

hours after administration (values calculated by pharmacokinetic modelling). The radioactivity concentrations 

in plasma declined steadily by several orders of magnitude within 48 hours for all dose groups. Radioactivity 

in tissues and organs at the 48-hour termination were very low (<0.2% and below the limit of detection for 

some organs/tissues). Urinary excretion was very rapid (essentially complete in 24 hrs) and was the major 

excretion route at these doses. Faecal excretion accounted for 2-11% of the dose in male and female rats. 

The excretion behavior was similar for all dose groups. BYI 08330 was completely metabolized by the rat 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 the administered dose). All other identified metabolites 

could be derived from the enol intermediate. The second prominent metabolic transformation was oxidative 

demethylation of the 8-methoxy group to BYI 08330-desmethyl-enol (5-37% of the administered dose). 

Oxidation of the azaspiro moiety to BYI 08330-ketohydroxy and BYI 08330-desmethyl-ketohydroxy were 

detected as minor pathways. Other 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-related 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 dose of 3 mg/kg bw in another 

disposition study using quantitative whole body autoradiography. The highest equivalent concentrations 

were observed in the liver, 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 findings 

were consistent with saturation of cellular transport mechanisms. In addition, decline of tissue radioactivity 

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; however, BYI 08330-desmethyl-enol occurred at lower levels 

at 1000 mg/kg bw, and this may be a reflection of the decreased absorption at this dose. Similar to the low 

dose groups, BYI 08330-desmethyl-enol levels were greater in urine than in plasma and organs. The highest 

percentage of BYI 08330-desmethyl-enol was detected in liver and kidney. Results of this study indicate that 

saturation of active transport mechanisms occurs at a dose of 1000 mg/kg but not 2 mg/kg. This results in 

decreased excretion via urine and faeces and a potential for accumulation of BYI 08330 related residues in 

the body following repeated high doses. The results 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, resulting in a high body burden in multiple-dose toxicological 

studies. This assumes, however, that spirotetramat enters the systemic circulation as the metabolite BYI 

08330-enol. 
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In a comparative in vitro metabolism study using hepatocytes from male rats, mice, and humans, differences 

in the proportions of several down-stream metabolites were observed; however, BYI 08330-enol was the first 

and most prominent metabolite and accounted for 66-100% of all metabolites across species. 
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B.6.2 Acute toxicity including irritancy and skin sensitization (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 the Rat with BYI08330. Project 

Number: 02/A12/NQ. Unpublished study prepared by Bayer 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 oC) 

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 facility. 

Water is provided ad libitum using an Edstrom automatic 

watering system 

 Housing: Individually housed in stainless steel wire mesh cages 

providing a 66.5 square inch area. Minimum space 

requirement for rats weighing less than 500 grams is 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: 30% 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 instances 

of less than ten changes per hour were recorded, but the low 

reading did not cause the daily average to fall below twelve 

changes per hour 
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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 selecting 

doses. As the oral LD50 was anticipated to be >2000 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 necropsy 

 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 death Mortality 

(%) 

Females 

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

LD50 (females): >2,000 mg/kg bw 

* 1st number = number of dead animals; 2nd number = number of animals with signs; 

3

rd number = number of animals used; ** according to the principles of OECD Guideline 423 

B. Clinical observations: No treatment-related findings. 

C. Body weight: There were no treatment-related effects on body weights 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. 
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B. Reviewer Comments: 

In an acute oral toxicity study, five young adult female Wistar Hanover (Crl:WI[Glx/BRL/Han]IGS BR) 

rats (age: 10 weeks; weight: 155-159 g; source: Charles 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 carboxymethylcellulose. 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 14. 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 mortality check was performed each afternoon 

until the end of the study. All animals were necropsied and examined for any gross lesions. 

All animals survived the test and gained body weight during the study. No clinical signs or gross 

pathological findings were observed. 

Oral LD50 Females > 2000 mg/kg bw 

Based on the Oral LD50, 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 LD50 > 2000 mg/kg in female rats. 

D. Study Deficiencies: 

1) The study report should include the report generated from the Acute Oral Toxicity Statistical 

Program (Westat, version 1.0, May 2001). 

2) In addition, the registrant should amend the study report to correct an assumed typo under the 

“Conclusions” where it stated, “According to OECD guideline 423, the LD50 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 (inhalation, eye and primary irritation and sensitization 

studies).
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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 Bayer 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 oC) 

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 facility. 

Tap water is provided ad libitum using an Edstrom 

automatic watering system. 

 Housing: Individually housed in stainless steel wire mesh cages 

providing a 66.5 square inch area. Minimum space 

requirement for rats weighing less than 500 grams is 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: 30% 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 instances of less than ten changes per hour were 

recorded, but the low reading did not cause the daily 

average to fall below twelve changes per hour. 
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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 weight, 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 LD50 > 2,000 mg/kg bw 

* 1st number = number of dead animals:; 2nd 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 following: 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 
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III. Discussion and Conclusions: 

A. Conclusions: 

The dermal LD50 for BYI 08330 for male and female rats was >2000 mg/kg. 

B. Reviewer Comments: 

In an acute dermal toxicity study, 5 young Wistar Hanover rats of each sex 

(Crl:WI[Glx/BRL/Han]IGS BR) and (age: 10 weeks; weight: 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% carboxymethylcellulose 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 site 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 days of study, when clinical observations were 

performed in the afternoon. All animals were necropsied and examined for any gross lesions. 

All animals survived the test and gained body weight 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 signs were observed after Day 3. No gross 

pathological findings were observed. 

Dermal LD50 Males > 2000 mg/kg bw 

Dermal LD50 Females > 2000 mg/kg bw 

Dermal LD50 Combined > 2000 mg/kg bw 

Based on the Dermal LD50, 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 the rat. 

C. Reviewer Conclusions: 

All countries agree the dermal LD50 for BYI 08330 for male and female rats was > 2000 mg/kg. 

D. Study Deficiencies: 

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 (inhalation, eye and primary irritation and 

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

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

BYI 08330. Project Number: PH-32020. Unpublished study 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 oC) 

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 conditions 

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 animals 

were housed singly in conventional Makrolon® Type II 

cages (based on A. Spiegel and R. Gönnert, Zschr. 

Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. 

Versuchstierkunde, 7, 144-153 (1965)). 

Environmental 

conditions: 

Temperature: 

Humidity: 

Lighting: 

Ventilation: 

Light intensity: 

22 + 2 oC 

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 
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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 temperature, 

reflex measurements, gross necropsy 

 In life dates: Not reported 

2. Generation of the test atmosphere / chamber description 

Generation and characterization of chamber atmosphere 

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 = Geometric Standard Deviation; 

-- not applicable. 

*With regard to the respirability of the aerosol generated internationally recognized recommendations such as of 

SOT (1992) were fulfilled at large, i.e. the MMAD was 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 guidelines, the 

cyclone was omitted. This high concentration could only be attained at the expensive of larger particle-size. 

Therefore, this concentration is considered to be the technically maximum attainable concentration. 

Micronization of the test substance was not considered because at such high concentrations particulates would 

agglomerate, i.e., the outcome of study would not be appreciably different. Moreover, micronization commonly 

increases the electrostatic charges of particulates which promote additional agglomeration. Because of the 

mechanics mentioned above the MMAD value exceeds the recommended range in the high dose (5.1μm). A 

cumulative mass percent value of ~47% was demonstrated for particle sizes up to that of 4.70 μm range 

therefore, a substantial quantity of test article was reaching the target tissue. While the cumulative mass for the 

higher dose did not result in at least 50% of the particulate reaching below 4μm for compound-tissue 

interaction, all reviewers agrees this it did not impact the validity of the study outcome. 

Test atmosphere 

concentration: 

Nominal concentration: The nominal concentration was not 

calculated due to the high weight of the dust generator and the 
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vertical elutriator used to eliminate larger particles. 

Gravimetric concentration: The test-substance concentration was 

determined by gravimetric analysis (filter: Glass-fiber filter, 

Sartorius, Gottingen, Germany; digital balance). Chamber 

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 accommodate the 

sampling duration and/or the need to confirm specific 

concentration values. Optimally, samples were collected after the 

equilibrium concentration had been attained in hourly intervals. All 

analytical concentrations reported refer to mg of test 

substance/m3 air. 

Particle size determination: The samples for the analysis of the particle-size distribution 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 aluminum foil. 

An adhesive stage coating (silicone spray) was used to prevent 

particle bounce and re-entrainment. Gravimetric analyses 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 death 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** 

LC50 (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** 

LC50 (females) >4,183 mg/m3 air 
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* 1st number = number of dead animals; 2nd number = number of animals with signs; 

 3rd 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 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 discharge (serous). No clinical signs were 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 initial body weight and by day 14 all animals 

exceeded their initial body weights. Clinical signs observed in this group included the following: 

ungroomed hair-coat, piloerection, bradypnea, labored breathing pattern, breathing sounds, 

nostrils reddened, nasal discharge, nostrils: red encrustations, 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 (reduced 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 weights 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 males and 4/5 females had surpassed their initial 

body weight and by day 14 all animals exceeded their 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 of 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 inhalation (nose-only exposure route ) to BYI 

08330 (Purity: 96.5%; Batch No. NLL 6425-9; beige powder) for 4 hours at a concentrations of 
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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 times for clinical signs of toxicity and at the 

least once daily thereafter. All animals were necropsied 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 discharge (serous). No clinical signs were 

observed after Day 5. No gross pathological findings were observed. 

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 males and 4/5 females had surpassed their initial body weight and by 

day 14 all animals exceeded their initial body weights. Clinical signs observed in this group 

included the following: ungroomed hair-coat, piloerection, bradypnea, labored breathing pattern, 

breathing sounds, nostrils reddened, nasal discharge , nostrils: red encrustations, nose/snout 

region: red encrustations, stridor, motility reduced and limp 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 (reduced grip strength, reduced tonus 

and impaired righting response). No gross pathological findings were observed. 

LC50 Males > 4.183 mg/L 

LC50 Females > 4.183 mg/L 

LC50 Combined > 4.183 mg/L 

Based on the LC50 in males and females, BYI 08330 is classified as EPA Toxicity Category IV. 

This acute inhalation study is classified as Acceptable. 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 LC50 for BYI 08330 in male and female rats was > 4.183 mg/L. 

The study did exhibit some significant clinical signs, but those effects resolved by Day 8 and the 

necropsy findings were unremarkable. In summary, the aerosolized test substance (solid aerosol) 

proved to have minimal acute inhalation toxicity to rats. 

D. Study Deficiencies: 
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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 primary irritation and sensitization studies is a “cis” 

isomer of the TGAI test material used for the acute oral and acute dermal studies. 
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B.6.2.4 Skin irritation (OECD Annex IIA 5.2.4) 

CITATION: Leuschner, P. (2002) Acute Skin Irritation Test (Patch 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 oC) 

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

 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 singly 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 separately 

in cages with dimensions of 425 mm x 600 mm x 380 mm. 

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 beginning of 
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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 showed any test substance related 

lesions. There were no systemic intolerance reactions

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 formation; 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 young healthy male Himalayan rabbits (age: 6.5 

months; weight: 2.2 kg; source: LPT Laboratory of Pharmacology 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 test site (approximately 6 cm2). Pulverised 

solids were moistened sufficiently with water to ensure good contact with skin. The test material 

was applied to the test site and covered with a gauze patch. The patch was held in place with 

semi-occlusive dressing for the exposure period. After 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 examination time points. 

Based on no dermal irritation, BYI 08330 is classified as EPA Toxicity Category IV. 

This study is classified as Acceptable. It does satisfy the guideline requirement for a primary 

dermal irritation study (OPPTS 870.2500; OECD 404) in the rabbit. 
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C. Reviewer Conclusions: 

All countries agree BYI 08330 is not a skin irritant. 

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. 
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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. 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 oC) 

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 singly 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 separately 

in cages with dimensions of 425 mm x 600 mm x 380 mm. 

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 beginning of 
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study) 

 In life dates: March 5, 2002 – March 14, 2002 

II. Results and Discussion: 

A. Findings 

Under the present test conditions the following findings 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 instillation, 

- in animal no. 2: 72 hours to 5 days after instillation and 

- in animal no. 3: 24 hours to 5 days after instillation. 

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 - 
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- : not examined; 

cornea: 0=no opacity; 1=scattered or diffuse areas of opacity, details of iris clearly visible 

iritis: 0=normal; 1=markedly deepened rugae, swelling, moderate circumcorneal 

 hyperaemia, or injection, iris still reacting to light 

conjunct. redness: 0=blood vessels normal 1=some blood vessels injected 

conj. chemosis 0= no swelling 1=any swelling above normal 

 2=obvious swelling with partial eversion of lids 

*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; beige powder) was instilled into the 

conjunctival sac of the right eye in each of the three young healthy male Himalayan rabbits (age: 

6.5 months; weight: 2.3-2.5 kg; source: LPT Laboratory 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 slit lamp prior to instillation and also at 1, 

24, 48 and 72 hours and on days 4 to 8. The eye reactions were observed and recorded. 

Twenty-four hours and 7 days after instillation, the 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 corneal opacity was observed on day 8. Iritis (grade

1) 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 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 instillation 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 resolved by day 5. 

Based on the corneal opacity observed up to day 7 after instillation, BYI 08330 is classified as an 

eye irritant and placed in EPA Toxicity Category II. 

This study is classified as Acceptable. It does satisfy 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. 
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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. 
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B.6.2.6 Skin sensitisation (OECD Annex IIA 5.2.6) 

CITATION: Vohr, H. (2002) Skin Sensitization Effect of BYI 08330 in Guinea Pigs (Guinea Pig 

Maximization Test According to Magnusson and Kligman). Project Number: PHH32273, T/1071445. Unpublished study prepared by Bayer 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 temperature (~ 22 oC) 

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 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 cages 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 temperatures 

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:
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 Induction: Intradermal Induction: Starting behind the neck, three 

injections each in a row were made on the left and the right 

side of the spinal column. The 1st and 2nd injections were 

made as contiguous as possible and the 3rd injections in a 

distance of about 2 cm from the 2nd. The volume applied 

per injection site was 0.1 ml. The animals of the three 

groups were treated as follows: a) Test material group - 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% BYI 08330 

formulated in polyethylene glycol 400; 3. Injection site: 

caudal/bilateral 5% BYI 08330 formulated at equal parts in 

polyethylene glycol 400 and complete Freund’s adjuvant 

Topical Induction: The topical induction was performed one 

week after the intradermal induction. Hypoallergenic 

patches (2 x 4 cm) were placed between and on the 

injection sites, covered with aluminum foil and held 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 remaining test 

material was removed with sterile physiological saline 

solution. 

 Challenge: The challenge was performed three weeks after the 

intradermal induction. In the meantime, the test material 

concentrations for the challenge had been determined in a 

dose range-finding study using 2 guinea pigs that were 

treated during the inductions in the same manner as the 

control animals. During the challenge a hypoallergenic 

patch loaded with the 25% test material formulation was 

placed on the right flank of the animals of the test group and 

the control group and held securely in place on the skin with 

self-adhesive tape for 24 hours. A patch loaded only 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 was 

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 the same 

manner as the animals of the test group; however, the 

formulations for injection site pairs 2 and 3 did not contain 

any test item but in pair 2 undiluted vehicle and in 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 females 
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 Observations: Mortality, clinical signs, skin effects, body weight 

(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) died 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 led to skin effects (grade 1 - 3) in 18 of 19 

animals (95%) in the test item group and no skin effects were seen in the control group animals. 

Number of animals exhibiting skin effects during challenge

 Test item group (19 animals) Control group (10 animals) 

 Test item patch Control patch Test item patch Control 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-Hexylimtaldehyd showed that the study 

was able to detect sensitizing compounds in the chosen 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 control 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 polyethylene glycol 400 to yield a suspension or a 

paste. An additional two animals were 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: 

Intradermal induction: 5% (= 20 mg test material/animal) 

Topical induction: 50% (= 250 mg test material/animal) 
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Challenge: 25% (= 125 mg test material/animal) 

One animal from the test group died on day 15 of the study. After the intradermal induction the 

animal in the control group and in the test group showed strong effect up to encrustation at the 

injection sites of the first induction. The challenge 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 validated in a test using alphaHexylimtaldehyd formulated in sterile physicological 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 satisfy the guideline requirement for a dermal 

sensitization study (OPPTS 870.2600; OECD 406) in the Guinea pig. 

C. Reviewer Conclusions: 

All countries agree that under the conditions of the 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. Unpublished 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 oC) 

2. Vehicle and/or positive control: Polyethylene glycol 400 

Alpha hexyl cinnamic aldehyde 

 3. Test animals 

 Species: Guinea pig, females 
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 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 cages 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 temperatures 

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 concentrations ( 

25%, 50%, 71%) and a vehicle group with 5 guinea pigs 

each. The test material was held in place for 6 hours. The 

dermal reactions were evaluated 30 and 54 hours after 

application. No dermal reactions were seen in any test 

group. The 71% concentration was chosen for the main 

test. 

 Induction (71%): The animals were dermally treated with the test material 

three times at intervals of seven days. The suitable areas of 

the body were shaved 24 hours before each treatment. The 

1st to 3rd inductions were performed with the 71% test item 

paste. The animals in the test group were treated with a 

hypoallergic patch loaded with the test material applied to 

the left flank and held in place on the skin with an adhesive 

plaster. The patches were removed after an exposure 

period of six hours, and any remaining test item was washed 

off the skin with physiological saline solution. The volume 

applied per animal was 500 mg test material mixed with 0.2 

ml vehicle in the test group (vehicle was polyethylene glycol 

400). The treatment areas were visually assessed 30 hours 

after initiation of exposure. For this, the treatment areas 

were not shorn or chemically depilated. 

 Challenge (71%): The challenge was performed two weeks after the last 

dermal induction. In the meantime, the test material 
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concentrations for the challenge had been determined in a 

dose range-finding study using 2 guinea pigs that were 

treated during the inductions in the same manner as the 

control animals. During the challenge a hypoallergenic 

patch loaded with the 71% test material paste was applied 

and fixed to the right flank of each animal in the control and 

test group. As a control a patch loaded only with the vehicle 

was applied and fixed also to the right flank, cranial to the 

test material patch. The patches were held securely in place 

on the skin with self-adhesive tape for 6 hours. The volume 

applied for the challenge was 0.5 ml vehicle for control 

group, and 500 mg test material mixed with 0.2 ml vehicle 

for test item group. At the end of the six-hour exposure 

period, the patches were removed and the remaining test 

item was rinsed away with physiological saline solution. 

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 exposure 

period of six hours. The volume applied per animal was 0.5 

ml vehicle in the control group. The treatment areas were 

visually assessed 30 hours after the 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 females 

 Observations: Mortality, clinical signs, skin effects, body weight 

(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 were not different from the control group. In the 

control and test item group there were no skin effects 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. 
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Number of animals exhibiting skin effects during challenge

 Test item group (20 animals) Control group (10 animals) 

 Test item patch Control patch Test item patch Control 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 demonstrated the sensitivity of the 

study. However, no study individual or tabular results were present in the study. 

Also, EPA guidelines suggest the positive control study 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 on 30 female Crl:HA guinea pigs (20 

animals for the test group and 10 animals for the control 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 formulated in polyethylene glycol 400 to yield a 

suspension or a paste. An additional two animals were 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 3rd 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 control. (Report No. PH33315; May, 2004). 
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In this study, BYI 08330 exhibits no dermal sensitization potential. 

This study is classified as Acceptable. It does satisfy the guideline requirement for a dermal 

sensitization 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 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 should 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 other sensitization studies is a “cis” isomer of the 

TGAI test material used for the acute oral and acute dermal studies. 

CITATION: Esdaile, D. (2004) BYI 08330: Evaluation of Potential Dermal Sensitization in the 

Local Lymph Node Assay. Project Number: SA/04120. Unpublished 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 oC) 

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. (91360 
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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 housed 

individually in suspended, stainless steel, wire-mesh 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 monitored; 

regular checks are made in the individual animal room for 

the target specification of 10 to 15 air changes per hour. 

B. Study Design and Methods 

1. Animal assignment and treatment 

 Dose: Forty five female CBA/J mice were allocated to 9 groups of 

five animals each: 

- four groups receiving the test substance at a concentration 

of 10, 5, 2.5 or 1%, 

- four positive control groups receiving the reference 

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 control 

groups receiving the reference substance (Isoeugenol) 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 μl/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 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 were removed 

from each mouse. The nodes from each mouse were 

placed in an individual tube containing physiological saline 

and were disaggregated by crushing with a plastic piston. A 

single cell suspension was obtained, free of connective 

tissue. Cell suspensions were washed with 6 ml of 0.9% 

physiological saline, centrifuged for 20 minutes at 1800 rpm 
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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 prepared cell suspensions were added to 

numbered scintillation pots containing 10 ml of scintillation 

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 clinical 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 during the study. No cutaneous reactions were 

observed in the vehicle, reference control or treated groups. The proliferation index values of the 

test substance were 5.9, 5.4, 4.3 and 3.4 at treatment 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% respectively. 

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 
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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 
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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 (Purity: 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 Assay. 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 receiving the reference substance (Isoeugenol) 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 μl/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 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 were 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 piston. A single cell suspension was obtained, 

free of connective tissue. Cell suspensions were washed 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 prepared cell suspensions were added to 

numbered scintillation pots containing 10 ml of scintillation fluid and counts were taken in a betacounter. 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 clinical signs and mortality at 

least once a day during the study. 
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The criterion for a positive response was a statistically significant increase in cell proliferation in 

the test concentration groups compared to the vehicle control group and/or SIs greater than or 

equal to 3.0. 

No mortality and no clinical signs were observed during 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 control or treated groups. 

The stimulation index values of the test material were 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 concentrations of 5, 2.5, 1 and 0.5%, respectively. 

In this study, BYI 08330 exhibited dermal sensitization 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 satisfy the guideline requirement for a dermal 

sensitization study (OPPTS 870.2600; OECD 429) in the Mouse. 

C. Reviewer Conclusions: 

All countries agree that under the conditions of the 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 sensitization studies is a “cis” isomer of the TGAI 

test material used for the acute oral and acute dermal studies.

B.6.2.7 Potentation/interactions of multiple active substances or products (OECD Annex IIA 

5.2.7) 
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B.6.2.8 Summary of acute toxicity 

BYI 08330 (Spirotetramet Technical) 

The acute toxicity studies for BYI 08330 were conducted between 2002 and 2005 at the following testing

facilities: Bayer AG Institut für Toxikologie, Bayer AG PH Prof. Toxicology, Bayer CropScience, Bayer 

CropScience LP, BayerHealthCare AG and LPT Laboratory of Pharmacology. Four additional acute oral 

toxicity studies on BYI 08330 metabolites were conducted between 2005 and 2006 at BayerHealthCare AG. 

All studies were fully compliant with Good Laboratory 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 different testing facilities [(Purity: 93.1%; Batch No. 

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 No. NLL 7635-2D; white powder). 

BYI 08330 and its metabolites were found to be of low acute toxicity to the rat by the oral route. No

treatment-related findings were observed at the limit 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 the acute inhalation toxicity study were limited to minor 

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 opacity was observed in all eyes from 24 hours to 6 days after 

instillation and in two eyes until 7 days after instillation. 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 on 

day 6 after instillation. No iritis was observed on day 7. Conjunctival redness was noted in all eyes 1 hour to 

72 hours after instillation and in two eyes until 4 days after instillation. Conjunctival chemosis was 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 BYI 

08330. In two of the studies, Maximization Test according to Magnusson & Kligman and Local Lymph Node

Assay (LLNA), BYI 08330 exhibited a dermal sensitization potential. In the third study, Buehler Patch Test, 

BYI 08330 exhibited no dermal sensitization potential. 
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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 temperature (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 groups was not reported, but the 

animals were assigned to various groups as noted below in Table 6-28. 
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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 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 performed. 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 value of fortified ration was 99.8%. 

5. Statistics: Not reported 

C. METHODS:

1. Observations: Animals were inspected for signs of toxicity and mortality daily and more detailed clinical 

observations were conducted weekly. No details were 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 the 

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 calculated. 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. The CHECKED (X) parameters were examined. 
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 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 electrophoresis 

 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 (heart puncture) under deep 

anesthesia. The liver, adrenals, testes, epidiymides were weighed and subjected to gross and histolgical 

exammination. All collected tissue was fixed in 10% formalin (except testes which were fixed in 

Davidson’s fluid), embedded in paraplast, and stained 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 were observed. 

2. Mortality: All rats survived to study termination. 

B. BODY WEIGHT AND WEIGHT GAIN: 

 There were no treatment-related effects on body 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% and 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 correlated with the dose. 

2. Compound consumption: Refer to Table 6-28 for details. 
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D. CLINICAL CHEMISTRY: Determination of activity of aspartate aminotransferase, alanine 

aminotransferase, alkaline phosphatase, and cholesterol 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 epididymides did not differ significantly 

from the control weights. Necropsy revealed no treatment-related findings. 

3. Microscopic pathology: Histopathological investigations of liver/gall bladder, adrenal glands, testes 

and epididymides revealed no evidence of any dose-related histopathological changes in animals up to 

and including 5,000 ppm. 

III. CONCLUSIONS:

The subacute toxicity study in male mice established a NOAEL of 5,000 ppm (equal to 1,415 

mg/kg/bw/day), based on the lack of treatment-related findings at 5,000 ppm. 

REVIEWER COMMENTS: The reviewer agrees with the study author’s overall 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 rationale 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 ketoenols BSN 3457, BSN 2342, 

 FHN 7504, FHN 8330 – Subacute exploratory toxicity studies in rat (Application by feed 

over 4 weeks). Bayer HealthCare AG, PH-GDD- Toxicology, 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 temperature (Study Report # 201363) 

CAS # if TGAI: Non-Stereo: 382608-10-8; CIS: 203313-25-1 
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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 groups was not reported; however, 

the animals were assigned to various groups as noted 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 performed. 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 08330 was stable in the freezer at 70 ppm for 32 

days and at 1200 ppm for 29 days. The mean recovery 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 frozen 

for a week. 
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Results:

Homogeneity/Stability analysis: Confirmed in Bayer Study Report # 201363. 

Concentration analysis: Not provided. Based on the results of homogeneity 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 mortality. 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 weekly 

and mean daily diet consumption was calculated as g food/kg body weight/day. Food efficiency was not 

calculated. 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. The 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 electrophoresis 

 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 based on Guideline 870.3150

6. Urinalysis: Not conducted.
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7. Sacrifice and pathology: All animals were sacrificed by exanguination under 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 samples of liver from the subacute rat study (Study # T 

0061869) with FHN 8330 were used to determine the liver 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 were observed. 

2. Mortality: All rats survived to study termination 

B. BODY WEIGHT AND WEIGHT GAIN: 

There were no treatment-related effects on body weight 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 control group) occurred in a non-dose related manner. 

E. SACRIFICE AND PATHOLOGY: 

1. Organ weight: Liver weight was not affected by the treatment 
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2. Gross pathology: No treatment-related macroscopic changes were seen in treated rats. 

3. Microscopic pathology: No treatment-related histopathological findings were observed in 

the liver from treated rats. 

4. Cell proliferation assay: The proliferating cell nuclear antigen (PCNA) assay 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 were similar for both control and treated animals. 

III. CONCLUSIONS: 

Groups of 5 female rats of strain Hsd/Win:WU were administered 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 weight, body weight gain and food intake of treated rats 

were comparable to the control group. The intake of test compound was proportional to the dietary 

concentration. Clinical chemistry examination revealed non-dose related decrease in triglyceride levels 

at 500 and 5000 ppm. The liver weight of treated rats was comparable to the controls. Gross and 

histopathology showed no treatment-related effects. The liver cell proliferation assay showed no relevant 

increase in cell proliferation or in nuclear area values for treated rats. The study author concluded that a 

four-week treatment with BYI 08330 slightly influenced 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 08330 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 of an influence on lipid metabolism. The results of the 

present study were also supported by the findings in CD-1 mice in which dietary exposure to BYI 08330 

resulted in no treatment-related effects in male mice 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 several details regarding the methods and material, 

concentration analyses for the test diet and individual 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. 
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CITATION: Eigenberg, D. A. (2004). Technical Grade BYI 08330: A Subacute Toxicity Feeding Study in 

the Beagle Dog. Bayer CropScience LP, Toxicology, 17745 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 animals 

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 analytically 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 sexes. Decreases in body weight gain, observed in the

other exposure groups, were not dose-related in either males or females. Food consumption was slightly

depressed in the 1600 ppm males throughout the study. Clinical observations were limited to a thin body 

state beginning at day 15 in males and females in the 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 reactions in the other female and in one male of this 

high-dose group. These alterations might have been secondary effects from the emaciation and thinness 

observed in these animals. This was supported microscopically by the lack of any degenerative nervous 

system changes in these animals. 
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Table B.6-32: Mean Body Weightsa (g) 

Study Day Dose Group (ppm) 

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) 8,424.5 (-16) 

14 10,268.0 11,319.5 11,317.5 9,265.5 (-10) 8,189.0 (-20) 

21 10,964.0 11,701.0 11,134.5 9,694.5 (-12) 7,843.0 (-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) 

28c 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

Study Day Dose Group (ppm) 

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 
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2. Hematology, urinalysis: No treatment-related findings 

Clinical chemistry:

There were statistically significant decreases in thyroid hormones (T4 and T3; Tables 6-34 and 6-35) in 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 not considered to be biologically significant due to a lack 

of compensatory increases in TSH (Table 6-36) or accompanying abnormalities in thyroid weight or 

histopathological appearance of the thyroid gland in dogs demonstrating hormonal alterations. At 6400 ppm, 

mean TSH levels were actually decreased in both sexes, and this may reflect normal biological variation. In 

addition, there was no effect on UDP-glucuronyltransferase activity; increased activity would have provided 

evidence of increased conjugation of T4 and T3 in liver. 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 System” 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) levelsa

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. 201012

 a Values are group means without standard deviations since there were only 2 animals/group 

Table B.6-35: Mean triiodothyronine (T 3) levelsa

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) levelsa

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
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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. 201012

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 administration. Reduced thymus size and/or decreases in 

absolute and relative (to body weight) thymic weights 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 / thinness at the high dose level. 

Table B.6-37: Mean thymus weightsa

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 female 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 one high-dose group male, which was considered 

secondary to the animal’s overall state of emaciation, rather than a specific target-organ effect. This animal 

weighed approximately 4 kg less than the control males and had also a mild diffuse atrophy of the parotid 

salivary glands, which may have reflected its decreased 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 (equal 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, equivalent to 42 mg/kg bw/day. 

Reviewer’s comments:

The reviewer concurs with the investigator’s conclusion and rationale with regard to the NOAEL in this study. 

It was evident that neither males nor females tolerated the 6400 ppm dietary level of BYI 08330 since they 

lost body weight, ate less food and exhibited an emaciated appearance. Apparent secondary effects in some 
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of these animals included decreased albumin and calcium in both sexes, and atrophy of the parotid salivary 

gland and sexual immaturity in one male. 

Reduced thymus size and weight (described microscopically as involution) were seen in both sexes at 6400 

ppm. The effect on the thymus might have been stress-related since there was no evidence of lymphocytic 

changes (degeneration, necrosis, depletion) in any of the other lymphocytic organs. 

Statistically significant (p<0.05) or non-significant decreases in circulating levels of T4 and T3 were 

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. Although the decreases in T4 and T3, in addition to TSH, 

appear to be compound-related, they are not considered toxicologically significant by the USEPA or PMRA, 

since there was no evidence of correlative effects on the pituitary-thyroid endocrine axis at gross necropsy or 

histopathology. AGES considers the statistically significant decreases in thyroid hormones observed at ≥400 

ppm in males (T3, T4) and also in females (T4), although not statistically significant, to be adverse.

The LOAEL (USEPA and PMRA) for BYI 08330 in this study 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, respectively). 

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 acceptability of the study. 

6. Study deficiencies: There were no significant deficiencies in the experimental design. 

7. Report deficiencies: 

• Clinical data were summarized for combined sexes. 

• Food efficiency was not reported. 

• Statistics were referred to but no methods were described 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 reactions in the other female and one male 

during neurological examinations. 
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B.6.3.2 Oral 90-day toxicity (rodents) (OECD Annex IIA 5.3.2) 

CITATION: Wahle, B.S., “Technical Grade BYI 08330, A Subchronic 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% (1/2003) 

Compound 

Stability: 

Stable at room temperature (~ 22 oC) 

Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-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-pregnant) 

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 group 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-activated water nipples), 

and deotized (sanitized) cage board in the bedding tray 

Diet: Purina Mills Certified Rodent Diet 5002 in "meal" form; ad libitum

Water: Municipal water supply of Kansas City, MO; ad libitum. Exception: Control 

animals were without pressurized water for approximately 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 oC 

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 ophthalmic 

examinations 

Acclimatisation: 03/25/02 (receipt) - 04/01/02 (release for study) 

B. Study design:

1. In life dates: Start: 04/01/02 (released for study); 04/09/02 (initiation of exposure) 

End: 07/18/02 (terminal sacrifice complete; subchronic 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 chemically-treated groups using a weight stratification-based 

computer program obtained from INSTEM Computer Systems (Stone, Staffordshire, UK). Each animal on 

study was identified with a microchip (BioMedic Data Systems, Inc., Seaford, DE) subcutaneously implanted 

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. 

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 toxicological profile which emerged in the female rat over 

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 exposure to 

the test substance were not observed. Therefore, doses of 0, 150, 600, 2,500, and 10,000 ppm were 

chosen at which to conduct the present study. It was anticipated that the low and high doses chosen of 150 

and 10,000 ppm would constitute NOAEL and maximum tolerated doses, respectively, with the intermediate

doses of 600 and 2,500 ppm providing confirmation 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 admixtures for each treatment group were prepared 

weekly and stored under freezer conditions until presented to the animals the following week. 

BYI 08330 was administered at a constant concentration in the feed for the duration of the study. The 

concentration of the AI of the test substance in the various test diets was analytically verified from samples of 

each dietary level taken during weeks 1, 2, 3, 7, and 11. In addition, one recovery sample was analyzed with 

each group of samples during the test concentration analyses. The homogeneity and stability of BYI 08330 

when mixed in the dietary carrier was characterized concurrently with the in-life phase of the study. 

To evaluate the uniformity of distribution/homogeneity 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 mixing 

bowl. A homogeneous distribution of the test substance in the feed was defined in terms of a coefficient of 

variation (CV), derived from the 9 samples taken, which was < 10%. 
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To evaluate the stability of the test substance in the diet, a time and temperature analysis of feed containing 

nominal concentrations of 70 and 10,000 ppm BYI 08330 was conducted. Stability in the feed was assessed 

following 1, 3, and 7 days of room temperature storage (~22 oC) and 7, 14, and 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, 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 temperature 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, and 28. 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 perform the dietary analyses (i.e., homogeneity, stability, 

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 concentration 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 the 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 

analytically 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 Day 0). 

BYI 08330 mixed in rodent ration was judged to be stable at room temperature for at least 7 days and 

following freezer storage for a minimum 28 days, over 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 95-

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 control diet. 

Mean recovery was 99% and ranged from 95-102% for rodent ration spiked with 150, 600, 2,500 or 

10,000 ppm BYI 08330. 

5. Statistics:

Continuous data that were examined statistically were evaluated initially for equality or homogeneity of 

variance using Bartlett's test. Group means were further analyzed by a one-way analysis of variance 

(ANOVA) followed by Dunnett'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 Kruskal-Wallis ANOVA followed by 

the Mann-Whitney-U test for 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 those mentioned above. For the 

Bartlett test, a probability (p) value < 0.001 was considered significant; for all other statistical tests, 
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differences with p values < 0.05 were considered statistically significant. All statistical evaluations were 

performed using software obtained from either INSTEM Computer Systems or SAS Institute Inc. 

C. Methods: 

1. Observations: 

1a. Cageside Observations: 

For approximately 18 weeks of the study (14 weeks for subchronic group animals; 18 weeks for recovery 

animals), a general assessment of all remaining animals was carried out at least daily (generally twice daily 

during the normal work week and once daily on weekends and holidays). 

These assessments were generally with respect to moribundity 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 every week for approximately 18 weeks of the study, detailed physical examinations for clinical signs 

of toxicity were performed on all surviving animals, which included evaluation of external surface areas, 

orifices, posture, general behavior, respiration, and excretory products. 

1c. Neurological Evaluations: 

Parameters specifically designed to evaluate possible neurological effects were not included in this study 

(i.e., other than those effects that would be identified during cage-side or detailed observations). These types 

of investigations were conducted as a part of other toxicology studies, including an acute neurotoxicity 

screening study (Report No. 201283) and a 1-year chronic rat study (Report No. 201486). 

2. Body weight:

Individual body weight determinations were performed weekly throughout the in-life phase of the study on all 

surviving animals. 

3. Food consumption and compound intake:

Individual food consumption determinations were performed weekly throughout the in-life phase of the study 

on all surviving animals (g consumed/animal/day and g consumed/kg body wt/day). In addition, 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 feeders, etc.). Test substance intake 

(mg/kg body wt/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 body wt/day)] = mg AI/kg body wt/day 

4. Ophthalmologic examination:

Ophthalmologic exams were conducted on all acclimatized animals prior to exposure, and then again on all 

surviving animals just prior to their respective termination dates (i.e., recovery animals were not evaluated at 

the end of the exposure phase, only at the end of the recovery phase). For listing of the defining conditions 

of the 5-level severity grading system in use at this facility for assigning severity grades to both 

ophthalmologic and nonneoplastic histopathologic lesions, 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 determinations was drawn via the orbital sinus following an 
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overnight fast under light anesthesia (IsoFlo®; Isoflurane). An additional aliquot of blood (non-fasted) for 

prothrombin time determinations was taken immediately preceding necropsy via cardiac puncture of animals 

under CO2 anesthesia (diaphragm was cut following blood collection). 

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 concentration (MCHC) 

X Erythrocyte count (RBC)* X Mean corpuscular volume (MCV)* 

X Platelet count (PLTS)* X Reticulocyte count (Retic) 

X Blood clotting measurements* X Erythrocyte morphology 

 (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-Prot)* 

 Cholinesterase (ChE) X Triglycerides (Trig) 

X Creatine phosphokinase (CK) Serum protein electrophoresis 

X Lactic acid dehydrogenase (LDH) X Uric acid (Uric-A) 

X Alanine aminotransferase (ALT)* X Thyroxine (T4) 

X Aspartate aminotransferase (AST)* X Triiodothyronine (T3) 

 Sorbitol dehydrogenase* X Thyroid Stimulating Hormone (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) from 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. 
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7. Sacrifice and Pathology:

7a. Gross pathology: 

All animals were sacrificed by CO2 asphyxiation (or CO2 anesthesia followed by clipping of the diaphragm) 

and subject to a postmortem examination, which included (1) documenting and saving all gross lesions, (2) 

weighing of the designated organs, and (3) collecting representative tissue specimens for histopathologic 

evaluation. Tissues were preserved (nominally in 10% buffered formalin) and selected organs were weighed 

and organ/body weight ratios calculated. Following collection of a liver specimen for histopathological 

analysis, the remaining liver tissue from at least 8 surviving animals (at approximately 14 and 18 weeks) was 

snap frozen in liquid nitrogen, pending further biochemical 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/Pons 

 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 histological examination. The organs CHECKED (XX) were also weighed. 

The organs CHECKED (^) were preserved for possible micropathologic evaluation only. 
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7b. Micropathology: 

With the exception of the physical identifier (microchip), the vagina, and the exorbital/lacrimal, clitoral, 

preputial, and Zymbal glands (of which the latter 5 were taken and preserved for possible micropathologic 

evaluation), representative sections of the tissues shown in Tables on previous page were processed for 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 examination under the light microscope. Tissues in which

compound-related effects were detected were examined at lower doses as necessary to establish noobserved-effect levels. Histopathological and ophthalmological lesions were assigned a semi-quantitative 

ranking of the severity grade/stage from "Normal (within normal limits)" to the "most severe case (Grade 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 difference 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 relevant 

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 (females) 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. 
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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 weight for 

high dose males was similar to controls after recovery. 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 incidence of minimal to severe abnormal spermatozoa 

and hypospermia were noted in the epididymides of 10,000-ppm males. Multi-nucleated giant cells, 

spherocytes (immature sloughed germ cells), and tissue debris (recorded as abnormal spermatozoa) were 

present within the duct of the epididymis at a level well above that found in control males. Only one recovery 

male each showed abnormal spermatozoa or hypospermia at the end of the 4-week recovery period 

suggesting reversibility of lesions. 

Testes: A minimal to moderate tubular degeneration was noted in the testes of 10,000-ppm males. Tubular 

degeneration and vacuolization in the 10,000 ppm dose group was generally multifocal in distribution, and 

occurred in the germinal epithelial layer of seminiferous tubules with degeneration and loss of epithelial 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 the 4-week recovery period suggesting reversibility of 

lesions. 

Table B.6-39: Average incidence and severity (in parenthesis) 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 pages 278-298 of study report. 

Lungs: An increased incidence of minimal to slight accumulation of alveolar macrophages was noted in both 

sexes at 10,000 ppm. It was stated by the study authors 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 affected 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 (equal 

to 616 mg/kg bw/day in males and 752 mg/kg bw/day in females, respectively): reduced body weight, 

decreased absolute testicular weight, testicular tubular 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 Subchronic Toxicity Study in the Mouse. 

Bayer Crop Science LP, Kansas, Study No.: 201284, July 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% (1/2003) 

Compound Stability: Stable at room temperature (~ 22 oC) 

Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-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 group weight males; 

25.3 - 25.9 g mean group weight females 

 Source: Charles River Laboratories, Inc. (Kingston, NY) 

 Housing: Individually 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 (animals passing 

initial shipment exam were co-housed during a portion 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 approximately 1.5 days 

(between nominal Days 40 and 42). Only the water remaining in the lines 

would have been available during this time. 

 Environmental 

conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

19 to 25 oC 

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 (release for study) 
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B. STUDY DESIGN: 

1. In life dates: Start: 05/20/02 (released for study); 05/30/02 (initiation of exposure) 

 End: 09/06/02 (terminal sacrifice complete) 

2. Animal assignment: 05/22/02; assigned to a control or one of four chemically-treated groups 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 implanted 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. 

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 toxicological profile which was observed in the mouse over 

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 ppm 

were chosen at which to conduct the present study. It was anticipated that the low and high doses chosen 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 confirmation 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 admixtures for each treatment group were prepared 

every other week and stored under freezer conditions until presented to the animals. 

BYI 08330 was administered at a constant concentration in the feed for the duration of the study. 

The concentration of the AI of the test substance in the various test diets was analytically verified from 

samples of each dietary level for all batches prepared. In addition, one recovery sample was analyzed with 

each group of samples during the test concentration analyses. The homogeneity and stability of BYI 08330 

when mixed in the dietary carrier was characterized prior to the experimental start of the study (10,000 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/homogeneity 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 mixing 
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bowl. A homogeneous distribution of the test substance in the feed was defined in terms of a coefficient of 

variation (CV), derived from the 9 samples taken, which was < 10%. 

To evaluate the stability of the test substance in the diet, a time and temperature analysis of feed containing 

nominal concentrations of 70 and 10,000 ppm BYI 08330 was conducted. Stability in the feed was assessed 

for up to 7 days of room temperature storage (~22 oC) 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, 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 temperature were sampled 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 that period of time that 80% of the test substance, relative to the 

initial concentration, was recoverable. 

The liquid chromatographic methodology used to perform the dietary analyses (i.e., homogeneity, stability, 

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 concentration 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 the 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 analytically 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 Day 0). BYI 08330 mixed in rodent ration was judged to 

be stable at room temperature for at least 7 days and 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 group were 70.9, 328, 1,665, and 

6,753 ppm, ranging from 94-101% of the corresponding nominal concentrations of 70, 350, 1,700, and 7,000 

ppm, respectively. The AI of the test substance was 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 were evaluated initially for equality or homogeneity of 

variance using Bartlett's test (Snedecor and Cochran, 1967). Group means were further analyzed by a oneway analysis of variance (ANOVA) followed by Dunnett'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 KruskalWallis ANOVA followed by the Mann-Whitney-U test for 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 those 

mentioned above. For the Bartlett test, a probability (p) value < 0.001 was considered significant; for all 

other statistical tests, differences with p values < 0.05 were considered statistically significant. All statistical 
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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 normal work week and once daily on weekends and holidays). 

These assessments were generally with respect to moribundity 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 approximately 14 weeks of the study, detailed physical examinations for clinical signs of 

toxicity were performed on all surviving animals, which included evaluation of external surface areas, orifices, 

posture, general behavior, respiration, and excretory products. 

1c. Neurological Evaluations: 

Parameters specifically designed to evaluate possible neurological effects were not included in this study 

(i.e., other than those effects that would be identified during cage-side or detailed observations). These 

types of investigations were conducted as part of other toxicology studies, including an acute neurotoxicity 

screening study and a 1-year chronic rat study. 

2. Body weight:

Individual body weight determinations were performed weekly throughout the in-life phase of the study on all 

surviving animals (with the exception of 1 animal on Day 49). 

3. Food consumption and compound intake:

Individual food consumption determinations (g consumed/animal/day and g consumed/kg body wt/day) were 

performed weekly throughout the in-life phase of the study on all surviving animals (with the exception of 1 

animal on Day 14). In addition, 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 

feeders, etc.). Test substance intake (mg/kg body wt/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 body 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 was collected from all surviving animals just prior to

termination. Blood for standard serum chemistry and hematological determinations was drawn via the orbital 

sinus of non-fasted animals. Due to blood volume considerations, only select serum chemistry evaluations 

were included. A full serum chemistry profile was included as a part of a subchronic investigation with the 

test substance in rats (Report No. 201136). 
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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 concentration (MCHC) 

X Erythrocyte count (RBC)* X Mean corpuscular volume (MCV)* 

X Platelet count (PLTS)* X Reticulocyte count (Retic) 

 Blood clotting measurements* X Erythrocyte morphology 

 (Thromboplastin time) X Heinz bodies (HZ) 

 (Clotting time) X Red cell distribution width (RDW) 

 Prothrombin time (PT) X Hemoglobin distribution width (HDW) 

 * Recommended for 90-day oral rodent studies based on Guideline 870.3100 

 The CHECKED (X) parameters were examined. Data on pages 77-88 of the study report provide 

confirmation that these parameters were investigated. 

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-Prot)* 

 Cholinesterase (ChE) X Triglycerides (Trig) 

 Creatine phosphokinase (CK) Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) Uric acid (Uric-A) 

X Alanine aminotransferase (ALT)* Thyroxine (T4) 

X Aspartate aminotransferase (AST)* Triiodothyronine (T3) 

 Sorbitol dehydrogenase* Thyroid Stimulating Hormone (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 study. Urinalysis was included as part of a subchronic 

investigation with the test substance in rats (Report 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) documenting and saving all gross lesions, (2) weighing 

designated organs, and (3) collecting representative tissue specimens for histopathologic evaluation. 

Tissues were preserved (nominally in 10% buffered formalin) and selected organs were weighed and 

organ/body weight ratios calculated. 
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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, optic* 

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 histological 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 cerebellum, cerebrum-midbrain and medulla pons or 

separate entries for the cervical, lumbar or thoracic spinal cord although these items are checked as being 

investigated. Only the brain and spinal cord are listed in the tables. 

7b. Micropathology: 

With the exception of the physical identifier (microchip), the vagina, and the exorbital/lacrimal, clitoral, 

preputial, and Zymbal glands (of which the latter 5 were taken and preserved for possible micropathologic 

evaluation), representative sections of the tissues 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 detected 

were to be examined at lower doses as necessary to establish no-observed-effect levels. Histopathological 

lesions were assigned a semi-quantitative ranking of the severity grade/stage from "Normal (within normal 

limits)" to the "most severe case (Grade 5)," as described previously. 
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II. RESULTS AND DISCUSSION:

Findings:

General observations: No treatment-related clinical signs or mortalities were observed. Body weight was not 

affected at any dose level in either sex. No relevant 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 noted. 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 reactions to treatment of any kind noted. The LOAEL was 

not established. The NOAEL was 7000 ppm (1305/1515 mg/kg/day [M/F]).

The following table containing body weight and liver weight data was constructed to demonstrate that there 

was no effect on either body or liver weight since no actual data were included in the format provided by the 

registrant. 
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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 control and high dose only to conserve space; SD 
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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, Toxicology, 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 temperature 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-methoxy-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 York 

Housing: Individually housed in stainless steel runs 

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 study 

*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 monitored the air flow.

B. STUDY DESIGN: 

1. In life dates: Initiation of collection of Data (Day Animals Released for Study): December 17, 2003; 

Initiation of Dosing: December 22, 2003; Terminal Sacrifice: 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 groups 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 excessive 

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 were 0 (concurrent vehicle 

control), 150, 300, 1,200, and 2,500 ppm (4/sex/dose) of technical grade BYI 08330 mixed with dog 

ration. Dose levels were selected based on the results 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. Compound-related decreases in circulating 

thyroxine (T4) and triiodothyronine (T3) were observed 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 appropriate 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 acetone 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 test substance. A sample of each batch of mixed feed 

was taken and retained under freezer conditions until 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 ration was confirmed for dietary concentrations ranging 

from as low as 100 ppm to as high as 6,400 ppm in the feed. Dietary 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 analytical 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 the 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 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 mixed in dog ration 

at concentrations of 100 and 6,400 ppm was considered stable at freezer temperature for a minimum of 

28 days (analytical values were 101 and 114% of actual concentrations at 100 and 6400 ppm, 

respectively). For room temperature stability, samples 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. 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. 
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Concentration analysis. During the study mixed feed from all dose groups, including control, was 

analyzed for stability and concentration during weeks 1 thru 7 (exceptions during weeks 4 and 5) as well 

as weeks 11 and 14. The mean concentrations for the study were 99 to 104% of the nominal levels of 

150/300 and 4000 ppm, respectively; %RSD values ranged 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 one-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 analyzed 

by a Chi-Square procedure. If there was 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 study animals were observed at least twice daily for clinical signs of 

toxicity (except once daily on weekends and holidays). 

1b. Clinical examinations: Detailed clinical observations for clinical signs of toxicity were performed 

on all animals at study initiation and on a weekly basis thereafter.

2. Body weight: Individual body weights were measured weekly throughout the study. Body weights were 

also taken immediately prior to necropsy to allow for 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 initiation of dosing, 

ophthalmic examinations were conducted on all animals. Ophthalmic examinations were also conducted 

on all animals just prior to termination of the study. 

5. Hematology & clinical chemistry: Clinical chemistry and a complete blood count, including 

differentials, were performed on all animals (pre-exposure and during weeks 4, 8, and 13). Animals 

were fasted overnight prior to the collection of blood, which was drawn via jugular venipuncture. The 

parameters evaluated are marked (x) in the lists below. 

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 hemoglobin conc.(MCHC)* 

x Erythrocyte count (RBC)* x Mean corpuscular volume (MCV)* 

x Platelet count* x Reticulocyte counta
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 Blood clotting measurements* 

x (Thromboplastin time) 

 (Clotting time) 

x (Prothrombin time) 

* Recommended for 90-day oral non-rodent studies based 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 electrophoresis 

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 Hormone (TSH) 

*Recommended for 90-day oral non-rodent studies based 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 below. 

x Appearance*ax Glucose (Glu)* 

x Volume (UVol)* x Ketones (Ket) 

x Specific gravity/osmolality (Sp.Gr.)* x Bilirubin (Bil) 

x pH (pH)* x Blood (Bld)* 

x Sediment (microscopic)ax 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 based 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, Michigan). 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. Histopathologic evaluation was conducted on all protocolrequired tissues from the control and high-dose animals as well as selected tissues (liver, thyroid, 

thymus, testicles, and epididymides) from the animals 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 group. 

A list of the tissues collected (x) and weighed (xx) at necropsy is presented in the table below. 
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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 masses* 

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 based 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 female in the 2500 ppm group 

was described as thin during the clinical assessment (onset at day 14). The neurotoxicity assessment 

revealed that this female in this group had abnormal posture (onset at day 42) and was reluctant to stand 

(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 gain for both sexes in the 4,000 ppm dose group 

(Table 6-41). In males, mean body weight was decreased by 11% (NSS), relative to controls, after two 

weeks of treatment at 4000 ppm. After week two, when the 4,000 ppm dose was reduced to 2,500 ppm, 

these animals gained more body weight than respective controls; however, it was evident that the 

females did not recover completely since the mean body weight at termination was still below the preexposure value and 17% less than controls at termination. Mean food consumption was 11% lower in 

males at the 4000 ppm dietary level during test days 6-14; after the dietary level of test material was 

decreased to 2500 ppm, decreased food consumption was 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 

4000/2500c 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 

4000/2500c 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 gains were calculated by the reviewer. 

c Dietary concentration of BYI 08339 was decreased from 4000 to 2500 ppm on test day 15. 

2. Hematology: There were no compound-related findings in males. Slight 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 for these parameters were further decreased at test 

day 86 (16-17%), and the difference from control was statistically significant for each parameter. Other 

findings, such as statistically significant (p<0.05), but not dose-related, increases in hemoglobin 

distribution width (HDW) in 1200 and 2500 ppm females at day 58, are considered incidental. The noted

compound-related effect on red blood cell parameters 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 femalesa 

Parameter 

Dietary concentration (ppm) 
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0 150 300 1200 2500 

-3 Days 

RBC (106/mm3) 6.65 ± 0.37 6.88 ± 0.36 7.25 ± 0.50 7.45 ± 0.47 6.59 ± 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 (106/mm3) 6.78 ± 0.43 7.10 ± 0.53 7.36 ± 052 7.28 ± 0.59 6.69 ± 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 (106/mm3) 7.11 ± 0.67 7.11 ± 0.66 7.26 ± 0.34 7.47 ± 0.33 6.41 ± 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 (106/mm3) 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 T4 in the 1,200 and 2,500 ppm male and 

female dose groups (Table 6-43). The findings on study day 30 for the males in the 300 ppm dose group 

were not considered to be treatment-related since on study days 58 and 86 the T4 values for males were 

not statistically significantly below the control group. The T4 values for females in the 150 ppm dose 

group were statistically significantly lower than the control group on study days 30, 58 and 86, but this 

finding is not considered treatment-related due to the absence of significant declines at 300 ppm. There 

was a slight, but statistically significant decrease in T3 in the 2,500 ppm male and female dose groups on 

study days 30 and 58 which is possibly considered to be treatment-related (Table 6-44). However, on 

study day 86, T3 levels were comparable to the control mean. Despite percentage declines in T4 at some 

time points of greater than 50%, no changes in thyroid weight, thyroid pathology, and no compensating 

increases in thyrotropin (TSH) (Table 6-45) were seen. 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 toxicological profile emerged in the 1-year chronic dog study. 

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 and 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) levelsa

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) levelsa

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) levelsa

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 that 

the thymus from this female was atrophied). 

There were no treatment-related effects on organ weights. The statistically significant increase in relative 

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 chemistry 

or histopathological changes and there was no statistical difference in the absolute liver weights. 

Likewise, statistically significantly (p<0.05) lower absolute pituitary weights in 1200 and 2500 ppm 

females are not considered compound-related; corresponding 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 determined in the present study. Despite percentage thyroid

hormone declines at some time points of greater than 50%, no changes in thyroid weight, thyroid 

pathology, no compensating increases in thyrotropin (TSH), or no clinical observations (e.g., neurological 

signs) suggestive of thyroid compromise were detected in either sex. 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 toxicological profile emerged in the 1-year chronic dog study 

(see below). 
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The NOEL of 300 ppm (equivalent to 9 or 10 mg/kg bw/day in males or females, respectively) is based 

on non-adverse declines in circulating thyroxine levels 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 compound-related decreases in circulating thyroid 

levels should not be considered adverse based on most of the rationale provided by the investigator. The 

reviewers take exception to the conclusion that no clinical signs were seen; abnormal posture and 

reluctance to stand were seen at 2500 ppm in females, but not in males. 

The USEPA and PMRA reviewers also agree that the NOAEL in males is 2500 ppm (81mg/kg/day), but 

does not agree that all of the compound-related effects observed at 2500 ppm in females should be 

considered “non-adverse”. Although the females in this group began to gain body weight after the dietary 

level of test material was reduced from 4000 to 2500 ppm on day 15, it was evident that they did not 

recover completely since the mean body weight at termination was still below the pre-exposure value. 

During the clinical assessments, one of these females was described as thin. Food consumption values 

for this group were also depressed throughout the study. 

Declines in values for red blood cell parameters (red blood cell count, hemoglobin level and hematocrit) 

were seen in 2500 ppm females at day 58; this decline continued throughout the remainder of the study 

and the difference between the value for each of these parameters and the respective control was 

statistically significant at test day 84. 

Gross examinations revealed a thymus in one 2500 ppm 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 compound-related effects on body weight gain, food 

consumption, red blood cell parameters and thymus at 2500 ppm in females are adverse. 

The AGES reviewer considers the statistically significant 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 study 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 (USEPA 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 males/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 acceptability of the study. 

B. STUDY DEFICIENCIES: 

The males could have tolerated a higher dosage of BYI 08330. 
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C. REPORT DEFICIENCIES: 

• Differential leukocyte and reticulocyte counts, which were reportedly assessed, were not reported. 

• Food efficiency was not reported. 

• Statistical methods were not described in the study report. 

• Appearance and presence of any microscopic sediment 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 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: 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 cages. 

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, although 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 below 

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 study. 

B. STUDY DESIGN: 

1. In life dates: 

Study Initiation (Day protocol was signed): May 28, 2004 

Initiation of collection of Data (Day Animals Released 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 noted 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 with a supplier's identification number tattooed on the inner 

part of the ear. This unique identifier was cross-referenced 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 were 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 Beagle 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 was 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 T4 in the 1,200 and 2,500 ppm male and female 

dose groups throughout the entire study, and a decrease in T3 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. Compound-related inhibition of body weight gain and food 

consumption, depressed RBC parameters (red blood cell count, hemoglobin level and hematocrit) and 

reduced thymus size, observed at 2500 ppm in females, were considered adverse effects. 

4. Diet preparation and analysis: 

All feed mixtures were prepared weekly by mixing appropriate amounts of the test substance with Purina

Certified Canine Diet 5006-3 and then storing the mixture under freezer conditions until given to the 

animals. Corn oil, at 1% by weight of the diet, along with acetone was used as vehicles to suspend the

test substance prior to being mixed in the diet. The control diet was prepared the same as the treated 

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 complete and the study data deemed satisfactory. Prior to 
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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. Dietary 

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 analytical 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 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 08330 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 temperature in dog ration for a minimum of 28 days 

(analytical values were 101 and 114% of actual concentrations at 100 and 6400 ppm, respectively). For 

room temperature stability, samples 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. 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 feed was verified for the first three weeks of the study 

and then monthly thereafter. The mean concentrations for the study were 94 to 100% of the nominal 

levels of 200 and 1800 ppm, respectively; %RSD values 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. Continuous 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 statistically, were initially analyzed by a Chi-Square 

procedure. If there was statistical significance using the Chi-square test, each treatment group was 

compared to the control group using a Fisher's Exact test. 

A complementary statistical analysis of T4 and T3 values was conducted by Dr. Catherine Pallen, PhD,

expert in statistics and epidemiology, at Bayer CropScience, Sophia Antipolis Cedex, France. The T4 and 

T3 values were log-transformed and analyzed with a repeated-measures analysis of variance (ANOVA, 

type III sum of squares) including terms for treatment, time and interaction of treatment and time. 

Repeated measurements analysis was performed assuming an unstructured covariance matrix. 
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When the ANOVA indicated a statistically significant overall treatment or interaction effect (p<0.05), 

multiple t-tests were applied using the ANOVA’s least-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 daily for clinical signs of toxicity (except once daily on 

weekends and holidays).

1b. Clinical examinations:

Detailed clinical observations for clinical signs of toxicity were performed on all animals at study 

initiation and on a weekly basis thereafter.

2. Body weight:

Individual body weights were measured weekly throughout the study. Body weights were also taken 

immediately prior to necropsy to allow for 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 initiation of dosing, ophthalmic examinations were conducted 

on all animals. Ophthalmic examinations were also conducted on all animals just 

prior to termination of the study. 

5. Hematology & clinical chemistry: 

Clinical chemistry and a complete blood count, including differentials, were performed on all animals 

(pre-exposure and during study weeks 13, 25, 38, and 51). During study week 22, blood was collected 

from all animals for an additional evaluation of T4, T3, and TSH. Animals were fasted overnight prior to 

the collection of blood (except during week 22), which was drawn via jugular venipuncture. The 

parameters evaluated are marked (x) in the lists below. 

5a. Hematology: 

x Hematocrit (HCT)* x Leukocyte differential count*

x Hemoglobin (HGB)* x Mean corpuscular hemoglobin (MCH)* 

x Leukocyte count (WBC)* x Mean corpuscular hemoglobin conc.(MCHC)* 

x Erythrocyte count (RBC)* x Mean corpuscular volume (MCV)* 
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x Platelet count* x Reticulocyte counta 

 Blood clotting measurements* x Blood cell morphology 

x (Thromboplastin time) x Red Blood Cell Distribution Width (RDW) 

 (Clotting time) x Hemoglobin Distribution Width (HDW) 

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 electrophoresis 

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 ratio (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 Guideline 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*ax Glucose (Glu)* 

x Volume (UVol)*a x Ketones (Ket) 

x Specific gravity / osmolality (Sp.Gr.)* x Bilirubin (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 Guideline 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 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. A list of the tissues collected (X) and weighed 

(XX) at necropsy is presented in the table below. All tissues were processed, embedded in paraffin, 

sectioned, mounted, and stained with hematoxylin and eosin (H&E) for examination under the light 

microscope. Histopathologic evaluation was conducted on all protocol-required tissues from the control 

and high-dose group animals as well as epididymides, lungs, and thyroids from the low- and mid-dose 

group animals. 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 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/Pons 

X Gall bladder* X Lymph node, retropharyngeal X Eyes* 

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, lumbar* 

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 Identifiera

X Nasopharynx* X Vaginaa

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 870.4100. 

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 reactivity, seizures and ataxia. 

There were no unscheduled mortalities during the study. 

There were no compound-related effects on food consumption 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 consumption values at 1800 ppm in females were 

statistically significant (p<0.05) at several intervals 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 and proportion of neutrophils and decreased 

proportion (but not count) of lymphocytes in 1800 ppm males as well as increased prothrombin time in 

600 and 1800 ppm females. These changes are considered incidental because, in addition to being 

slight, some were not dose-related, some were transient and correlative changes in other parameters 

were not observed. There were no compound-related effects on blood cell morphology. 

Urinalysis: No compound-related effects were seen. 

Clinical chemistry: Thyroxine (T4) was statistically decreased in males 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 was 

statistically decreased for most time points at 600 and 1,800 ppm. Triiodothyronine (T3) decreases were 

less pronounced and occurred in males at 1,800 ppm for all time points (Table 6-48). There was a 

tendency for T4 values to fall below the historical control range at 1800 ppm in males and females; the 

same tendency was seen in T3 values at 1800 ppm in males. 

 

The statistically significant T4 decrease in females on day 357 at 200 ppm was considered a statistical 

aberration due to the high T4 mean in controls. The statistically significant T3 decrease in males on

day 180 at 200 ppm and 600 ppm was considered a statistical aberration due to the relatively high T3 

mean in controls. This is also supported by the fact 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 tested. 

Despite declines in thyroid hormones at some time points, no changes in thyroid weight or compensating

increases in thyrotropin (TSH) Table 6-49) were seen at ≤600 ppm. Therefore, decreases in thyroid 

hormone at ≤600 ppm were not considered toxicologically significant. 

Other incidental, but statistically significant (p<0.05), clinical chemistry changes were, in males, 

decreased albumin at 1800 ppm, increased chloride at 600 and 1800 ppm, increased phosphorous at 

1800 ppm (none of these findings had correlative changes) and transient increases of glucose in all 

dosage groups; in females, transient increases of glucose (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 

T4 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 controls; 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 

T3 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 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 0.02 0.54 

TSH day 96 0.16 ± 0.11 0.17 ± 0.19 0.11 ± 0.11 0.11 ± 0.08 0.07 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 0.07 0.39 

TSH day 357 0.16 ± 0.13 0.18 ± 0.17 0.11 ± 0.09 0.06 ± 0.05 0.04 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 0.09 ± 0.03 n.a. n.a. 

TSH day 180 0.21 ± 0.17 0.21 ± 0.14 0.17 ± 0.12 0.07 ± 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 0.06 ± 0.03 0.07 0.46 

TSH 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) 

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 observed in males at 600 

and 1800 ppm; brain dilation was also noted in females, 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 absolute heart weight was statistically low (p<0.05) at 1800 

ppm in males, however, there was no statistical difference in mean relative weight and correlative 

histopathologic changes were not found. Absolute and relative thymus weights were low (not statistically) 

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 blocks were also evaluated for all control and 1,800 ppm 

males and females. None of these additional evaluations 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 well 

as at 1800 ppm in 1 male (moderate); mild axonal degeneration 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 ppm (equal to 20 mg/kg bw/day), based on a 

reduced thyroid follicular size noted in two animals at 1,800 ppm (equal to 55 mg/kg bw/day). The chronic 

NOAEL in females was established at 1,800 ppm (equal to 48 mg/kg bw/day), based on the absence of 

adverse compound-induced toxicological responses. Despite thyroid hormone declines at some time 

points, no changes in thyroid weight, thyroid pathology, no compensating increases in thyrotropin (TSH), 

or no clinical observations (e.g., neurological signs) suggestive of thyroid compromise were detected in 

either sex. Based on the total response of the animals to the thyroid profile that emerged over one year, 

the isolated compound-induced changes in circulating 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 technical grade BYI 08330 was 200 ppm (equal to 5 

mg/kg bw/day). 

IV. REVIEWER’S COMMENTS: 

USEPA and PMRA reviewers considered the decreases in 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 thyroid 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-related. USEPA reviewers concluded that brain dilation 

was an equivocal effect and that it should not be used as the basis for the LOAEL in the study. Unlike 

thymus effects, which were observed in other studies with spirotetramat in the dog, brain dilation was

not observed in any other study in the dog or in any other species in the database. In addition, brain 

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-related effect, it should have been seen in other dog studies 

in the database, but this was not the case. The seizures observed in a single dog in the study were 

most likely due to the brain dilation in this animal 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 the 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 studies with spirotetramat, and the fact that the 
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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 male dogs is 600 ppm (20 mg/kg/day) based 

on thymus involution and was not observed in females. 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 females 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, respectively). 

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 females 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 acceptability 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 the assessment of urine were not reported. 
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B.6.3.5 Repeated dose (28-day/90-day) inhalation toxicity (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 study in rats with BYI 08330. Study No. 05-

S22-YC. Report No. 201505. Bayer CropScience LP, Stilwell, Kansas. Unpublished. 

I. MATERIAL AND METHODS 

A. MATERIALS: 

1. Test material: BYI 08330

Description: Technical grade, white powder, room temp 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-oxo-1-azaspiro[4.5]dec-3-en-4-yl 

carbonate 

CAS No. if TGAI: Non-Stereo: 382608-10-8; CIS 203313-25-1

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, Raleigh, North Carolina 

Housing: Individually housed in stainless steel wire mesh cages 

Diet: Purina Laboratory Certified Rodent Chow 5002 available for ad libitum

consumption 

Water: Tap water (Kansas City Missouri municipal water, 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 
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Necropsy: February 7 and 8, 2005 

2. Animal assignment, test groups and study duration: 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 group), 100, 300, and 1,000 mg/kg of BYI 08330 for 28 

or 29 days. Each treatment group consisted of ten males 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 study. 

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 the 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 in.), 

and moistened with 1 ml of deionized water. The adhesive bandage was placed on the shaved skin of 

the rat and the torso of the animal was then wrapped with porous medical tape to assure that the 

bandage remained on the animal during the dosing interval. The same procedure was performed for 

control animals with the exception that only the bandage, moistened with 1 ml of deionized water, was 

applied to the dose site. The test substance was held in contact with the skin for a minimum of six 

hrs/day for five consecutive days/week for four weeks. Each day the bandage and tape were removed 

and the dose site was gently wiped with water-dampened 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-tailed. 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-Whitney U-test to identify statistical significance 

among groups. 

Frequency data that were examined statistically were evaluated using the Chi-Square test. If there was 

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 conducted twice a day 

during the dosing and unwrapping of the animals. 

1c. Neurological evaluations: Neurological evaluations are available from other studies so they 

were not performed in this study. 
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2. Body weight: The weight of each animal was determined on study days 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 abnormalities 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 anesthesia. During study wk 3, the blood was analyzed for 

standard serum chemistry and hematological parameters; during wk 4 the blood was analyzed for 

prothrombin time and thromboplastin time. The animals were fasted overnight prior to collecting blood 

during wk 3. The parameters evaluated are checked with (x) in the lists below: 

5a. Hematology: 

x Hematocrit (HCT) x Leukocyte differential count 

x Hemoglobin (HGB) x Mean corpuscular hemoglobin (MCH) 

x Leukocyte count (WBC) x Mean corpuscular hemoglobin conc.(MCHC) 

x Erythrocyte count (RBC) x Mean corpuscular volume (MCV) 

x Platelet count x Reticulocyte count 

 Blood clotting measurements: x Erythrocyte morphology 

x (Activated Partial Thromboplastin time) x Red blood 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 (Urea-N) 

x Alanine aminotransferase (ALT / SGPT) Bile Acids

x Aspartate aminotransferase (AST / SGOT) Serum protein 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 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 collected were processed, embedded in paraffin,

sectioned, mounted, and stained with hematoxylin and eosin (H&E) for examination (initially control and 

high-dose then proceeding to lower doses as necessary) under the light microscope. A list of the tissues 

collected (x) and weighed (xx) at necropsy is presented in the table below. Only tissues from the control 

and high dose groups were examined microscopically.
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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, thoracic 

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 considered to be incidental and not treatment-related. 

All other animals survived until the scheduled termination of the study. No effects on body weight 

development, food consumption, clinical signs, or local 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 cell count, activated partial prothromboblastin time, 

prothrombin time, and differential cell counts. These 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 creatine kinase, lactate dehydrogenase, aspartate 

aminotransferase. These 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. 

Gross necropsy, organ weights, histopathology: No treatment-related macroscopic changes were 

seen. Organ weights were not affected by compound administration. There were no treatment-related 

microscopic observations. 

III. DISCUSSION AND CONCLUSIONS 
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A. CONCLUSIONS: 

 The NOAEL of 1,000 mg/kg in males and females is based on the absence of effects at the highest dose 

tested. 

B. REVIEWER COMMENTS: 

The reviewer agrees with the study authors’ interpretation 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 resulted 

in no treatment-related adverse effects. Analytical determinations of stability and homogeneity were not 

performed because the test substance was applied undiluted and only moistened with water immediately 

before application. One mortality of a female at 100 mg/kg occurred on Day 22 during blood collection 

due to isoflurane anesthesia and was not treatment-related. There were no treatment-related effects on 

body weight, organ weight, or body weight:organ weight ratios. Statistically significant differences 

observed in treated groups in hematology (increased white blood cell count, activated partial 

prothromboblastin time, prothrombin time, differential cell counts) and clinical chemistry (decreased 

creatine kinase, lactate dehydrogenase, aspartate aminotransferase) measurements 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. Only tissues 

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 established. The NOAEL for male and female 

rats is 1000 mg/kg/day. 

A dermal LOAEL for male and female rats was not established. 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 (OPPTS 870.3200). 

C. STUDY DEFICIENCIES: 

No deficiencies were identified.
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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 the short-term 

toxicological studies in rodents and dogs. Rats, mice, and dogs did not exhibit changes in plasma lipid 

parameters such as plasma triglycerides and plasma cholesterol. The thyroid and thymus glands were target 

organs in oral subchronic toxicity studies in the dog. Subchronic (90-day) exposure of dogs to spirotetramat 

was characterized by statistically significant declines in circulating thyroid hormones (T4) at ≥1200 ppm (32 

mg/kg bw/day); decreased T3 was observed at the highest dose tested only (2500 ppm; 72 mg/kg bw/day). 

However, correlative changes in thyroid weight, thyroid 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. Abnormal 

spermatozoa and hypospermia in the epididymis, decreased testicular weight, and testicular degeneration 

and vacuolation in males were observed in males after 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 effects in 

subchronically treated rats were limited to declines in terminal body weight in 10,000 ppm male rats and an 

increased incidence of accumulation of alveolar macrophages in both sexes at 10,000 ppm. It is noteworthy 

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 dose. In 

vitro results from a comparative metabolism study using hepatocytes from male rats, mice, and humans 

revealed species differences in the metabolism of spirotetramat. Specifically, mouse hepatocytes were

better able than rat or human liver cells to metabolize BYI 08330-enol via glucuronidation. Potentially lower 

levels of the enol metabolite in mice in vivo may account for the lack of testicular toxicity observed in this 

species. 

Subchronic exposure of rats by the dermal route yielded no evidence of systemic toxicity when spirotetramat 

was tested up to 1000 mg/kg bw/day. This result may in part be a reflection of the low dermal absorption 

(approximately 10%) in rats. 
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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 No.AT03070, Document No. M272000-01-2. Bayer HealthCare AG, PH-GDD Toxicology, 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-oxo-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 strains 

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: MgCl2 x 6 H2O: 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 
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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 strains, 3 replicates/treatment, 2 

trials 

Activated conditions: 0, 16, 50, 158, 500, 1581, 5000 ug/plate, all strains, 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 method 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 triplicate. The negative control consisted of the solvent absent 

the test material. In the plate incorporation procedure, 0.1 ml of the solvent with or w/o the test material, 

0.1 ml of the bacterial inoculum, 0.5 ml of the S9 fraction or buffer, and 2.0 ml of soft agar were warmed 

to 45oC 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 colonies 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 inoculum, 

and 0.5 ml of the S9 fraction or buffer were pre-incubated 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 assessed. 2). If there was a marked and dosedependent reduction on the mutant count, that was considered to be a measure of toxicity. 3). Total 

bacterial counts were taken by incubating two plates per treatment level in the presence of histidine and 

incubating for 48 hours at 37 °C. A reduction in the 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 expected range as defined by published data or the 

laboratories’ own historical data. 

b. The positive controls had to demonstrate a mutagenic effect. 
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c. Titer determinations had to demonstrate sufficient bacterial density in the suspension. 

In order to achieve a positive result, a reproducible and dose-related increase in mutant counts for at 

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 should 

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 counts 

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 including 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 the negative controls, was observed. The positive 

controls had a marked mutagenic effect. 

Table B.6-50: Summary of mean number of mutant colonies/plate 

Summary of mean values without S9 mix 

Table and Strain 

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 - 
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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 without and with metabolic activation under the 

conditions of the Salmonella/microsome test. 

B. REVIEWERS COMMENTS:

The reviewer agrees with the investigators’ conclusions. BYI 08330 was tested to concentrations limited 

by cytotoxicity, proper experimental protocol was followed, and solvent and positive control values were 

appropriate. The number of revertants per plate was not increased over solvent control values at any 

experimental point in this study. 

This study appears to be a repeat of a study performed in 2002 (Report No. AT00056). Results in both 

studies are similar and support one another. 

C. STUDY DEFICIENCIES:
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No study deficiencies were identified. 
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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 No.AT00056, Document No. M065358-0102. Bayer HealthCare, PH-PD p Health Care Toxicology, Rodents and 

Genotoxicity, Friedrich-Ebert-Strasse 217-333, D-42096 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-oxo-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 strains 

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: MgCl2 x 6 H2O: 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 
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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 strains, 3 replicates/treatment, 2 

trials 

Activated conditions: 0, 16, 50, 158, 500, 1581, 5000 ug/plate, all strains, 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 method 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 triplicate. The negative control consisted of the solvent absent 

the test material. In the plate incorporation procedure, 0.1 ml of the solvent with or w/o the test material, 

0.1 ml of the bacterial inoculum, 0.5 ml of the S9 fraction or buffer, and 2.0 ml of soft agar were warmed 

to 45oC 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 colonies 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 inoculum, 

and 0.5 ml of the S9 fraction or buffer were pre-incubated 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 assessed. 2). If there was a marked and dosedependent reduction on the mutant count, that was considered to be a measure of toxicity. 3). Total 

bacterial counts were taken by incubating two plates per treatment level in the presence of histidine and 

incubating for 48 hours at 37 °C. A reduction in the 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 expected range as defined by published data or the 

laboratories’ own historical data. 

b The positive controls had to demonstrate a mutagenic effect. 

c Titer determinations had to demonstrate sufficient bacterial density in the suspension. 
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In order to achieve a positive result, a reproducible and dose-related increase in mutant counts for at 

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 should 

be at least 150 colonies over that counted for the negative control. In the 1st trial, the positive controls 

for both with and w/o activation in the TA102 assay did not fulfill this requirement. Therefore, the assay 

was performed again using this strain only. The data in the results section reflect the outcome of this 

assay. 

II. REPORTED RESULTS: 

Findings: 

Doses up to and including 158 μg per plate did not cause any bacteriotoxic effects. Total bacteria counts 

remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a 

strong, strain-specific bacteriotoxic effect, so that this range could only be used up to and including 1,581 

μg per plate for assessment purposes. Evidence of mutagenic activity of BYI 08330 was not seen (Table 

6-51). No biologically relevant increase in the mutant count, in comparison with the negative controls, 

was observed. The positive controls had a marked mutagenic effect. 

Table B.6-51: Summary of mean number of mutant colonies/plate (Data taken from Study No. 

T5071214, pp. 26-27) 

Summary of mean values without S9 mix 

Table and Strain 

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 
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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 
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CONCLUSIONS: 

BYI 08330 was considered to be non-mutagenic without and with metabolic activation under the 

conditions of the Salmonella/microsome test. 

B. REVIEWERS COMMENTS:

The reviewer agrees with the investigators’ conclusions. BYI 08330 was tested to concentrations limited 

by cytotoxicity, proper experimental protocol was followed, and solvent and positive control values were 

appropriate. The number of revertants per plate was not increased over solvent control values at any 

experimental point in this study. 

C. STUDY DEFICIENCIES:

No study deficiencies were identified. 
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B.6.4.1.3 In vitro genotoxicity - Test for clastogenicity in mammalian 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 Toxicology, Bayer HealthCare, Bayer 

AG,Friedrich-Ebert-Strasse 217-333, D-42096 Wuppertal, 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-oxo-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 / culture) 

 Positive: Nonactivation: Mitomycin C (0.1 μg/ml medium) 

 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 mg protein/mL) were slowly thawed and mixed with a 

cofactor solution (4:6). The S9 mix contained 40% S9 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 

MgCl2 x 6 H2O 162.6 mg 

KCl 246.0 mg 

Glucose-6-phosphate (disodium salt) 152.0 mg 
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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) supplemented with nonessential amino acids, Lglutamine (2 mM), MEM-vitamins, NaHCO3-solution (final concentration 0.225%), penicillin (50 

units/mL), streptomycin (50 μg/mL) and heat-inactivated 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, 100, 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, 100, 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 106 cells were seeded in 20 ml of medium on the day prior 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-activation) or 19 mL of MEM plus 2% FCS, 1 ml of S9 mix and 

0.2 ml of test material. After 4 hours of incubation at 37oC, the medium was removed and the cells 

washed once with pre-warmed phosphate buffered solution. 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 following 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 cultures 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) 
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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 were 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 supernatant was carefully removed. One to 2 mL of a 

hypotonic solution (0.4% KCl; 37°C) was added to the 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 supernatant 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 centrifuged. 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 were 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 once in acetone and were then kept in xylene for 

about 30 minutes. The slides were allowed to dry completely and covered. At least two slides were 

generated per culture. All solutions used during this 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 microscope at a magnification of about 630. The mitotic 

index was determined by counting 1000 cells per culture. 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 about 1000. Chromosomes of approximately 200 

metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined. 

In most cases at least 100 assessable metaphases were present on one slide prepared from an 

individual culture. Therefore, the back-up slide which was generated routinely from every culture 

was normally not utilized for the evaluation. However, in cases when fewer than 100 assessable 

metaphases were found on the first slide of a culture, the back-up slides were evaluated as well until

a total of 100 metaphases were reached. 
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Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges 

were detected. In this case, metaphases were evaluated 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 aberrations are clastogenic effects restricted to one of 

the two corresponding chromatids. Chromosome type aberrations 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 exchanges. 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 smaller than the width of one chromatid. Gaps are 

found on one chromatid ("gap") or on both chromatids at apparently identical sites ("isogap"). The 

biological relevance of gaps of both types is unclear. 2) Break: a break is defined as a discontinuity 

of one chromatid ("break") or both chromatids, at apparently 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 achromatic 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 centromer. A fragment is the result of a break. The 

corresponding defective chromosome is not detectable among the chromosomes of the same 

metaphase. Fragments can be derived from one chromatid ("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 ("deletion") or both corresponding terminal chromatid 

parts of a chromosome ("isodeletion") are missing within the metaphase under assessment. 5) 

Exchange: This is an exchange of chromatid-parts between different chromosomes (interchange) or 

within the same chromosome (intrachange). 6) Multiple aberration: A cell was assessed as to 

contain "multiple aberrations" when five or more structural changes (excluding gaps) occur within 

one metaphase. In addition to these aberrations, metaphases showing chromosome disintegration 

as an indication of a cytotoxic effect were also recorded if they were observed. They were counted 

separately and were not included among the cells that 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 group. The mitotic index was statistically analyzed 

(provided that it was reduced compared to the mean of the corresponding solvent control) using the 

one-sided chi2

-test. The numbers of metaphases with aberrations (including and excluding gaps) 

and of metaphases with exchanges were compared (provided that these data superceded the 

respective solvent control). Fisher's exact test was used for the statistical evaluation. A difference 

was considered to be significant, if P<0.05. 

II. RESULTS AND DISCUSSIONS: 

Findings: 

Test material concentrations up to 750 μg/mL did not change the pH or osmolality of the treatment 

medium. In the cytogenetic assay without S9 mix, cytotoxic effects were observed at 50 ug/ml and 

above after 4 hours treatment and at 24 ug/ml and above after 18 hours treatment. With the S9 mix, 
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cytotoxic effects were observed at 40 ug/ml and above. Therefore, higher concentrations of test material 

were not evaluated for chromosomal aberration induction due to excess cytotoxicity. With one exception 

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. 

 
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Table B.6-52: Chromosomal Aberrations without metabolic Activation 4 Hours Treatment 

Classes of aberrations 

Gaps Chromatid 

type 

Chromosome 

type 

other 

Metaphases of aberrations 

(%) 

Experimental 

Group and 

Concentration 

in μg/ml 

Harve

st 

Time 

in 

Hours 

Culture 

Number 

Cells 

scored 

g i b f d ib if id ex 

ma

E 

m

a 

c

d 

Incl. 

gaps 

excl. 

gaps 

excha

nges 

DMSO 18 36 100 0 0 0 0 0 0 0 2 0 0 0 0 2.0 2.0 0.0 

0 21 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0 

 200 0 0 0 0 0 0 0 3 0 0 0 0 1.5 1.5 0.0 

BYI08330 18 25 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0 

10 23 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0 

 200 0 0 0 0 0 0 0 2 0 0 0 0 1.0 1.0 0.0 

BYI08330 18 32 100 0 0 0 0 0 0 0 5 0 0 0 0 5.0 5.0 0.0 

30 18 100 0 0 0 1 0 0 0 3 0 0 0 0 3.0 3.0 0.0 

 200 0 0 0 1 0 0 0 8 0 0 0 0 4.0 4.0 0.0 

BYI08330 18 16 100 0 0 3 0 1 0 0 11 0 0 0 0 12.0 12.0 0.0 

50 42 100 0 0 0 0 0 1 0 4 0 0 0 0 4.0 4.0 0.0 

 200 0 0 3 0 1 1 0 15 0 0 0 0 8.0** 8.0** 0.0 

Mitomycin C 18 30 100 2 1 6 0 1 10 3 2 18 0 0 0 36.0 33.0 16.0 

0.1 44 100 0 0 10 0 0 16 3 4 15 0 0 0 36.0 36.0 13.0 

 200 2 1 16 0 1 26 6 6 33 0 0 0 36.0** 34.5** 14.5** 

* p< 0.05 ** p < 0.01 

Copied 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 aberrations 

with exchange; cd = chromosome disintegration 
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Table B.6-53: Chromosomal Aberrations with Metabolic Activation 4 Hours Treatment 

Classes of aberrations 

Gaps Chromatid 

type 

Chromosome 

type 

other 

Metaphases of aberrations 

(%) 

Experimental 

Group and 

Concentration 

in μg/ml 

Harve

st 

Time 

in 

Hours 

Culture 

Number 

Cells 

scored 

g i b f d ib if id ex 

ma

E 

m

a 

c

d 

Incl. 

gaps 

excl. 

gaps 

excha

nges 

DMSO 18 43 100 0 0 0 0 0 0 0 2 0 0 0 0 2.0 2.0 0.0 

0 37 100 0 0 0 0 0 1 0 2 0 0 0 0 3.0 3.0 0.0 

 200 0 0 0 0 0 1 0 4 0 0 0 0 2.5 2.5 0.0 

BYI08330 18 39 100 0 0 0 0 0 0 0 2 0 0 0 0 2.0 21.0 0.0 

20 29 100 1 0 0 0 0 0 1 2 1 0 0 0 4.0 3.0 1.0 

 200 1 0 0 0 0 0 1 4 1 0 0 0 3.0 2.5 0.5 

BYI08330 18 33 100 0 0 1 0 0 0 0 1 0 0 0 0 2.0 2.0 0.0 

40 8 100 1 0 0 1 0 3 0 3 2 0 0 0 8.0 8.0 2.0 

 200 1 0 1 1 0 3 0 4 2 0 0 0 5.0 5.0 1.0 

BYI08330 18 40 100 0 0 0 0 0 1 0 3 1 0 0 0 5.0 5.0 1.0 

80 12 100 0 0 0 1 0 1 1 8 1 0 0 0 10.0 10.0 1.0 

 200 0 0 0 1 0 2 1 11 2 0 0 0 7.5* 7.5* 1.0 

Cyclophosphamide 18 9 100 1 1 3 1 0 22 4 4 18 0 0 0 38.0 37.0 15.0 

2 26 100 0 2 5 1 0 21 1 2 14 0 0 0 36.0 35.0 13.0 

 200 1 3 8 2 0 43 5 6 32 0 0 0 37.0** 36.0** 14.0** 

* p< 0.05 ** p < 0.01 

Copied from Lab Report No. AT00055, page 44 (originally called Table 4 in study). 
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g = 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 = chromosome disintegration 
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Table B.6-54: Chromosomal Aberrations without and with metabolic Activation 4 Hours Treatment 

Classes of aberrations 

Gaps Chromatid 

type 

Chromosome 

type 

other 

Metaphases of aberrations 

(%) 

Experimental 

Group and 

Concentration 

in μg/ml 

Harve

st 

Time 

in 

Hours 

Culture 

Number 

Cells 

scored 

g i b f d ib if id ex 

ma

E 

m

a 

c

d 

Incl. 

gaps 

excl. 

gaps 

excha

nges 

Without metabolic activation 

DMSO 30 22 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0 

0 27 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0 

 200 0 0 0 0 0 0 0 2 0 0 0 0 1.0 1.0 0.0 

BYI08330 30 5 100 0 0 0 0 0 1 0 1 0 0 0 0 2.0 2.0 0.0 

50 28 100 0 0 0 0 0 0 0 1 0 0 0 0 1.0 1.0 0.0 

 200 0 0 0 0 0 1 0 2 0 0 0 0 1.5 1.5 0.0 

With metabolic activation 

DMSO 30 11 100 0 0 0 0 0 0 1 2 0 0 0 0 3.0 3.0 0.0 

0 20 100 0 0 0 0 0 0 0 0 0 0 0 0 0.0 0.0 0.0 

 200 0 0 0 0 0 0 1 2 0 0 0 0 1.5 1.5 0.0 

BYI08330 30 17 100 0 0 0 0 0 0 0 5 0 0 0 0 5.0 5.0 0.0 

80 38 100 0 0 0 0 0 1 1 5 0 0 0 0 6.0 6.0 0.0 

 200 0 0 0 0 0 1 1 10 0 0 0 0 5.5* 5.5* 0.0 

* p< 0.05 ** p < 0.01 

Copied 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 aberrations 

with exchange; cd = chromosome disintegration
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Table B.6-55: Additionally observed Polyploid Metaphases 4 Hours Treatment 

without metabolic activation with metabolic activation 

Experimental Group 

and Concentration 

in μg/ml 

Harvest 

Time in 

Hours 

Culture 

Number 

Polyploid 

Metaphases 

Experimental 

Group and 

Concentration in 

μg/ml 

Harvest 

Time in 

Hours 

Culture 

Number 

Polyploid 

Metaphases 

DMSO 18 36 13 DMSO 18 43 15 

0 21 19 0 37 14 

BYI 08330 18 25 14 BYI 08330 18 39 10 

10 23 11 20 29 12 

BYI 08330 18 32 15 BYI 08330 18 33 15 

30 18 18 40 8 11 

BYI 08330 18 16 12 BYI 08330 18 40 5 

50 42 18 80 12 12 

Mitomycin C 18 30 7 Cyclophosphamide 18 9 10 

0.1 44 3 2 26 4 

DMSO 30 22 13 DMSO 30 11 9 

0 27 14 0 20 10 

BYI 08330 30 5 16 BYI 08330 30 17 13 

50 28 8 80 38 3 

Copied from Lab Report No. AT00055, page 45 (originally called Table 6 in study). 
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Table B.6-56: Chromosomal Aberrations without metabolic Activation 18 Hours Treatment 

Classes of aberrations 

Gaps Chromatid 

type 

Chromosome 

type 

other 

Metaphases of aberrations 

(%) 

Experimental 

Group and 

Concentration 

in μg/ml 

Harvest 

Time in 

Hours 

Culture 

Number 

Cells 

scored 

g i b f d ib if id ex 

ma

E 

m

a 

c

d 

Incl. 

gaps 

excl. 

gaps 

excha

nges 

DMSO 18 46 100 0 0 0 0 0 0 0 2 1 0 0 0 3.0 3.0 1.0 

0 47 100 0 0 0 1 0 0 0 1 0 0 0 0 1.0 1.0 0.0 

 200 0 0 0 1 0 0 0 3 1 0 0 0 2.0 2.0 0.5 

BYI08330 18 49 100 1 0 2 0 0 1 0 3 3 0 0 0 8.0 7.0 3.0 

12 58 100 0 0 1 0 0 0 1 2 1 0 0 0 3.0 3.0 1.0 

 200 1 0 3 0 0 1 1 5 4 0 0 0 5.5 5.0 2.0 

BYI08330 18 55 100 0 0 0 0 0 3 1 3 1 0 0 0 7.0 7.0 1.0 

24 50 100 0 0 0 2 0 0 1 2 2 0 0 0 4.0 4.0 2.0 

 200 0 0 0 2 0 3 2 5 3 0 0 0 5.5 5.5 1.5 

BYI08330 18 45 100 0 0 0 0 0 2 0 4 2 0 0 0 8.0 8.0 2.0 

48 56 100 0 0 0 0 0 4 0 3 0 0 0 0 6.0 6.0 0.0 

 200 0 0 0 0 0 6 0 7 2 0 0 0 7.0* 7.0* 1.0 

Mitomycin C 18 57 100 1 0 7 0 0 8 1 1 13 1 0 0 25.0 25.0 12.0 

0.03 53 100 1 1 6 0 0 11 3 4 10 1 0 0 29.0 27.0 10.0 

 200 2 1 13 0 0 19 4 5 23 2 0 0 27.0** 26.0** 11.0** 

* p< 0.05 ** p < 0.01 

Copied 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 aberrations 

with exchange; cd = chromosome disintegration
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Table B.6-57: Additionally observed Polyploid Metaphases 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 (originally called Table 10 in study) 

III. CONCLUSION: 

BYI 08330 is considered to be weakly positive in this cytogenetic in vitro study at cytotoxic 

concentrations only. 
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B.6.4.2 Mammalian Chromosome Aberration Test (In Cultured 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, Molecular 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 Sprague 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 with the cofactor solution at a ratio of 4:6. The S9 mix 

thus contained 40% S9 fraction and was kept on ice and used on the same day. 
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Cofactor solution per 100 mL of S9 mix: 

Sodium phosphate buffer (100 mM; pH 7.4): 60.0 mL 

MgCl2 x 6H2O: 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): obtained 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, NaHCO3 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 CO2 incubator in 

which the temperature was 37°C and the CO2 concentration was set at 5%. 

 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: 

Preliminary cytotoxicity assay: None 

Main assay: 0, 30, 50, 70, 90, 110 μg/mL, but chromosomal aberrations 

were only evaluated at 70 μg/mL 

 Activated conditions: Preliminary cytotoxicity assay: None 

Main assay: 0, 40, 60, 80, 100, 120 μg/ml, but chromosomal 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 μg/mL 
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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 trypsinization, suspended in 

medium, and centrifuged. The resultant pellet was resuspended in a hypotonic solution (0.4% KCl) 

and recentrifuged. The supernatant was decanted and 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. After an additional treatment with the fixative and 

another centrifugation, the pelleted cells were resuspended in a small volume of fresh fixative. The 

suspended cells were then dropped onto slides that had been pre-cooled to -20o C. The slides were 

air-dried for at least 2 hours, treated with pure methanol for 3 minutes and stained with a 5% Giemsa 

solution for 20 to 30 minutes. The slides were then 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 magnifications of 

approximately 630x and 1000x for the mitotic index and for examination for aberrations, respectively. 

Duplicate cultures for each treatment level were evaluated. One hundred metaphases per culture 

(i.e., 200 metaphases per treatment level) were scored for structural aberrations. Only metaphases 

that contained the modal chromosome number of 22 were 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 one of the two corresponding chromatids. 

Chromosome-type aberrations are defined as changes expressed in both corresponding sister 

chromatids at the same locus. An increased incidence of gaps noted for either a single chromatid or 

on both chromatids at identical sites (i.e., an isogap) without a concomitant increase in other 

aberrations was not considered a clastogenic response. A test was acceptable if the aberration 

frequency of the vehicle control fell within the range of the laboratory’s historical control data or 

within ranges of values found for controls in the literature. A test was considered positive if there

was a treatment-related and statistically significant increase in the aberration rate. A test was 

considered negative if there was no such increase at any time interval. A test was also considered 

negative if there were statistically significant increases that fell within the range of the historical 

vehicle controls. A test was considered equivocal if a statistically significant increase was found that 

was not biologically significant. 
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g. Statistical analysis: Pair-wise comparisons of test material-treated groups 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 compared with the respective vehicle control 

by using Fisher’s exact test. The probability of error was established at less than 5%. The reviewer

considers the statistical methods appropriate. 

II. REPORTED RESULTS: 

There was no analytical testing of test solutions in order to confirm concentrations or to provide 

information about the stability of the test substance in the media and/or solvent. 

A. PRELIMINARY CYTOTOXICITY ASSAY: 

There was no preliminary cytotoxicity assay. Concentrations were chosen on the basis of the results 

from an earlier study identified by number T 6071477, 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 results 

for the mitotic index and the survival index, respectively. Mitotix indices (MI) were mainly comparable 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 the 

decrease in relative survival (22-49% and 21-32%, respectively). Precipitation of BYI 08330 in the 

medium was not observed. Based on the above cytotoxicity observations, it was decided to examine 

cells for cytogenetic effects only at 70 μg/mL of BYI 08330 in the absence of metabolic activation, and 

only at 120 μg/mL of BYI 08330 in the presence of metabolic activation. Those results are shown in 

Table 6-60. None of the treated cultures showed biologically relevant or statistically increased numbers 

of aberrant metaphases at the one concentration level selected for examination with and without S9 mix. 

This was true both including and excluding gaps. Based on earlier work from a study identified as T 

6071477, it was said that concentrations of the test substance up to 750 μg/mL did not change the pH or 

the osmolality of the medium. There were no concurrent positive controls. Numerous tables were 

presented showing the laboratory’s historical negative, 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 hours1

Relative mitotic index (%) in the group with 

Treatment (μg/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 
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Relative mitotic index (%) in the group with 

110 100.0 - 

120 - 189.7 

Data summarized from Report No. AT00194, Table 1 on page 32 

1Results 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 harvest time of 8 

hours, which is not considered sufficiently relevant to include here. 

*p < 0.05 

**p < 00.01 

Table B.6-59: Results of survival index assay of BYI 08330 in cultured Chinese V79 cells with a 

harvest time of 18 hours1

Relative survival index (%) in the group with 

Treatment (μg/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 

1Results 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 harvest time of 8 

hours, which is not considered sufficiently relevant 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 assay 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 

(μg/mL) 

ctb cte csb cse Other1 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 
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Number of structural aberrations 

(200 cells/dose, 100 cells per culture) 

Mean % of cells 

containing 

aberration(s)2

Treatment 

(μg/mL) 

ctb cte csb cse Other1 Gaps 

(ctg) 

- gaps + gaps 

Polyploid 

cells (%)2

(DMSO) 

BYI 08330 

120 1 13 0 0 3 0 2.5 2.5 10.0 

- There was no positive control. 

Data summarized from Report No. AT00194, Tables 3 and 4 on page 34; polyploidy data were not analyzed. 

*Statistically significant (p < 0.05)—noting was statistically significant 

1

Includes severely damaged cells 

2

Based on 200 cells examined 

3

This exchange could be either a chromatid or a chromosome 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 assay, BYI 08330 has no clastogenic potential in 

mammalian cells in vitro, and that these results suggest that a cytotoxic impurity was present in the initial 

batch of BYI 08330 that was tested. Further support for his conclusion was said to be reflected in the 

negative in vivo findings summarized in Reports No. AT00048, AR00070, and AT00526. The reviewer 

has seen only the first one of these reports, and that 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 aberrations. 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 part of the assay that used S9 mix means that there was 

no check of whether of S9 mix was effective. The absence of concurrent positive controls means that 

there was no check of the sensitivity of the assay under the conditions that existed for this particular 

experiment, and the extensive historical control data that are provided do not overcome this limitation—

especially in view of the erratic nature of the results regarding the mitotic index. There was no QA 

statement, and the study was not conducted according 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.
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Major study deficiencies are as follows: (1) no concurrent positive controls, (2) only one test substance 

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 condition, (3) there was no QA statement, and (4) the 

study was not conducted in conformance with GLP standards. It seems unlikely that anything can be 

done to overcome these deficiencies. 

IV. REFERENCES: 

Rieger, R. and A. Michaelis. 1967. Die Chromosomenmutationen. VEB Gustav Fisher Verlag, Jena. 
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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/HPRT-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, 2002. 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 methanesulfonate (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 other 60% was sodium phosphate buffer at pH 7.4 that

contained the following components: 

Final concentrations: 

MgCl2 x 6 H20 8 mM 

KCl 33 mM 
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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 hamster lung 

fibroblasts) 

Chinese hamster ovary (CHO) cells—CHOK1 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 Yes No 

*A hypoxanthine-free Eagle’s Minimal Essential Medium supplemented with non-essential amino acides, Lglutamine (2mM), MEM-vitamins, NaHCO3, penicillin (100 units/mL), streptomycin (100 μg/mL) and 10% heatinactivated fetal calf serum (FCS). This was called culture media. During treatments with the test chemical, 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 μg/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 μg/mL 

Main assay, trial 1: 0, 2.5, 5, 10, 20, 40, 80 μg/mL 

Main assay, trials 2 and 3: 0, 20, 30, 40, 50, 60, 70 μg/mL 

Activated conditions: Preliminary cytotoxicity: 0, 0.1, 1, 10, 50, 100, 150, 300, 600, 1000 μg/mL 

Main assay, trial 1: 0, 20, 40, 60, 80, 100, 120, 140 μg/mL 

Main assay, trials 2 and 3: 0, 92, 100, 108, 116, 124, 132, 140 μg/mL 

B. TEST PERFORMANCE:

1. Cell treatment: 

a. Cells were exposed to test compound, negative/solvent or positive controls for 5 hours 

(nonactivated) 5 hours (activated). 

b. After washing, cells were cultured for 6 days (expression period) before cell selection. Cells were 

subcultured at the midpoint (i.e., after 3 days) of the expression period. 
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c. After expression, 3x105

 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 per 

106 clonable cells. The mutant frequency for each treatment was expressed as the number of TGresistant mutants per 106

 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 Poisson derived weights. All acceptable groups were 

included in the weighted analysis of variance, which was followed by pair-wise comparisons to the 

vehicle control using the Dunnett test and a nominal significance level of 0.05. Mutant frequencies 

based on fewer than 5 plates were not included in the statistical analysis. Also data were not used from 

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, thereby increasing the power of the statistical tests 

applied. Results for the different trials were compared to evaluate consistency. The reviewer considers 

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 conditions must be met: (1) the average cloning efficiency 

of the negative and vehicle controls should be at least 50%, (2) the average mutant frequency of the 

vehicle controls should not exceed 25x10-6 cells, (3) the mutant frequency of the two cultures of the 

vehicle and/or the negative control should differ only to an acceptable extent (with the difference 

between them generally not exceeding 5x10-6), (4) the positive control should induce an average mutant 

frequency of at least three times that of the vehicle control, and (5) the test should be limited by 

cytotoxicity—with the concentration inducing cytotoxicity of about 80 to 90%—or by insolubility, with 

precipitation being found in the medium at the highest concentration. The survival at the lowest 

concentration should be in the range of the negative control. A trial is considered positive if a 

concentration-related increase in the mutant frequencies occurs that is reproducible in other trials. To be 

considered biologically significant, the increase in mutant frequencies must be at least two to three times 

that of the highest negative or vehicle control value 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 significant change 

in osmolality compared to the vehicle control. Such a change could mean that non-physiological culture 

conditions caused the positive result. A result will be considered equivocal if there is no strictly 

concentration-related increase in mutant frequencies 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 biologically signficant increases of mutant frequencies are

observed. 

II. REPORTED RESULTS: 

There was no analytical testing of the concentrations of the dosing solutions. However, the batch that 

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 was tested by comparing the amounts of the test 
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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 time 

period from preparation of the formulation to the last treatment in the experiments. 

A. PRELIMINARY CYTOTOXICITY ASSAY:

V79 cells were exposed for 5 hours to the solvent alone and to nine concentrations of the test substance 

ranging from 0.1 to 1000 μg/mL in the absence and presence of S9-mix. Comparisons of relative cloning 

efficiencies showed cytotoxicity at 50 μg/mL and above in the absence of S9-mix and at 150 μg/mL and 

above in the presence of S9-mix. Results are summarized in Table 6-61. Cytotoxicity exceeded 90% 

when concentrations were equal to or greater than 100 and 150 μg/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 the test substance was seen at concentrations of 600

μg/mL and above. Based on these findings, in the mutagenicity test the test substance was tested at 

concentrations ranging from 2.5 to 80 μg/mL in the absence of S9-mix and to concentrations ranging 

from 20 to 140 μg/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 

(μg/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 

600a -S9 0.0 0.0 

1000a -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 

600a +S9 N N 

1000a +S9 N N 

Data summarized from Tables 1 and 2, Report No. AT00137A 
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a

There was precipitation of the test substance. 

bN means that the cells were not cloned because of excessive cytotoxicity. 

B. MUTAGENICITY ASSAY: 

Cytotoxicity data were also collected in the mutagenicity assay. BYI 08330 induced decreases in 

survival to treatment (i.e., in survival until the time when the selection procedure was used to reveal 

mutant colonies) and in relative population growth in both the presence and absence of S9 mix. Relative 

survival and relative population growth were decreased by 83-90 % and 95%, respectively, at >40 ug/mL 

without metabolic activation and 83-97% and 82%, respectively, 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 culture medium was not observed at the concentrations 

used in the mutagenicity assay. An increase in mutant frequency (MF) relative to controls was observed 

at most concentrations in trial 2 with (3-5X) and without (2-3.8X) metabolic activation. However, in the 

absence of a clear dose response and reproducibility, 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 sensitivity of the test system and the activity of the S9 mix. 

Tables 6-62 and 6-63 summarize the results of the mutagenicity assay by averaging the results of both 

cultures at each test condition. The vehicle, negative, and positive control data were well within the

range of the laboratory’s historical controls. 
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Table B.6-62: Results of the in vitro CHO/HGPRT mammalian cell gene mutation assay of BYI 

08330 without metabolic activation 

Treatmenta

(μg/mL) 

Relative 

survival to 

treatment 

(%) 

Relative 

population 

growth (%) 

Total mutant 

colonies on 

10 platesb

Absolute 

Cloning 

Efficiency 

(%) 

Mutant frequency 

(per 106 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 Tables 3-5, Report No. AT00137A (data listed are the combined results of two cultures) 

*Statistically significant increase in mutant frequency over the concurrent solvent control based on the one-sided 

Dunnett Test 

EMS = Ethyl methanesulfonate 

a

The trials identified here as trials 1, 2, and 3 were 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 culture, and the other 

number is the total on 10 plates for the second culture.

Table B.6-63: Results of the in vitro CHO/HGPRT mammalian cell gene mutation assay of BYI 
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08330 with metabolic activation 

Treatmenta

(μg/mL) 

Relative 

survival to 

treatment 

(%) 

Relative 

population 

growth (%) 

Total mutant 

colonies on 

10 platesb 

Absolute 

Cloning 

Efficiency 

(%) 

Mutant frequency 

(per 106 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 Tables 6-8, Report No. AT00137A (data listed are the combined results of two cultures) 

*Statistically significant increase in mutant frequency over the concurrent solvent control based on the one-sided 

Dunnett Test 

DMBA = Dimethybenzanthracene 

a

The trials identified here as trials 1, 2, and 3 were 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 culture, and the other 

number is the total on 10 plates for the second culture.
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III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS:

The investigator concluded that BYI 08330 is non-mutagenic in the V79/HGPRT forward mutation assay, 

both with and without S9 mix. He noted that it caused concentration-related cytotoxicity. 

B. REVIEWER COMMENTS: 

The reviewer agrees with these conclusions. Cytotoxicity was observed as decreases in relative survival 

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 biologically in the absence of a dose response and 

reproducibility. Solvent and positive controls gave appropriate responses and agreed well with the 

laboratory’s extensive historical control. The study followed proper experimental protocol and met the

criteria for a valid test and a negative result. There was no evidence of induced mutant colonies over 

background. The study is classified as Acceptable/Guideline.

C. STUDY DEFICIENCIES: 

None 
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B.6.4.3 In vivo assays 

B.6.4.3.1 Bone marrow or micronucleus (OECD Annex IIA 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 Genotoxicity, Friedrich-Ebert-Straße 217-

333, D-42096 Wuppertal, Germany. Study No. T 8071479. 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 dose; they were to be used as replacements in case some 

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 Cremophor 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 mature 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 animal was flushed out 

using a syringe into a tube that contained fetal calf serum. The tube containing the serum and bone 

marrow was centrifuged at approximately 1,000 rpm for 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 smear. The labeled slides were dried overnight. If fresh 

smears needed to be stained, they were dried with heat for a short period. The smears were stained 
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automatically with an Ames Hema-Tek Slide Stainer from the Miles Company. The slides were then 

"destained" with methanol, rinsed with deionized water, and left to dry before being covered with cover 

slips. The slides were not evaluated until the covering agent had dried. Slides were coded to avoid bias. 

4. Evaluation criteria: Slides were evaluated using a light microscope at a magnification of about 1,000. 

Micronuclei appear as stained chromatin particles in 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 ratio seems likely without other animals in the group 

showing similar effects, then that particular animal may be regarded as pathological and possibly one 

that would show effects unrelated to treatment. Such an animal may be omitted from the evaluation. It 

can only be concluded that there is a relevant treatment-related alteration of the PCE:NCE ratio if that 

ratio is clearly lower for a majority of the animals in the treated group than in the vehicle control. The 

number of NCEs showing micronuclei was also determined; however, it was realized by the time the 

report was written that an increase in the number of micronucleated NCEs, without a preceding increase

in micronucleated PCEs, would be meaningless, and that such data are of almost no use when bone 

marrow is collected so soon after treatment. An assay is considered acceptable if the frequencies of 

micronucleated PCEs of the vehicle and positive controls are within the expected ranges, based on 

laboratory’s experience and the literature. A test is considered positive if there is a biologically significant 

(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 significant 

increase in the frequency of micronucleated PCEs. A test is also considered negative if there is a 

statistically significant increase in micronucleated 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 laboratory’s historical negative (or vehicle) controls but that 

increase is not statistically significant in comparison to the concurrent control. A repeat test will then be 

performed. 

5. Statistical methods: The parameters analyzed were the MNPCE (i.e., micronucleated PCE) 

frequencies, the MNNCE frequencies (unreported here since almost useless), and the PCE:NCE ratios. 

The BYI 08330 group(s) with the highest mean frequency of MNPCEs (provided this was higher than the 

mean of the negative control mean), and the positive 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 reviewer considers the analyses used to be appropriate.

II. REPORTED RESULTS: 

There was no analytical testing of the concentrations of the dosing solutions. However, the stability of the 

test chemical in the vehicle was tested by comparing the amounts of the test chemical present to the 

nominal concentrations of 10 and 100 mg/mL both before and after 4 hours of storage at room 

temperature. At 10 mg/mL, the percentages of the nominal concentrations before and after the 4-hour 

storage period were 97% and 98%, respectively. At 100 mg/mL, these percentages were 96% and 94%, 

respectively. It was concluded that the test chemical is stable during an interval that spans the time period 

from preparation of the formulation to the last treatment in the experiments. 
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A. PRELIMINARY TOXICITY ASSAY: 

The preliminary toxicity assay was done using animals of the same source, strain, and age. Groups of 

three males and three females were given two intraperitoneal (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, roughened fur, loss of weight, a staggering gait, lateral 

and sternal recumbency, spasms, palmospasms, leaping spasms, twitching, rolling over, periodical 

stretching of the body, breathing difficulties, eyelids stuck together, closed eyes, and diarrhea. In

addition, 1 of 3 males in the 500 mg/kg bw group died, and all males in the 1000 mg/kg group died. At 

the 200 mg/kg bw dose, females experienced all of the same symptoms as males except for a 

staggering gait, palmospasms, and closed eyes. Also, all females in the 1000 mg/kg bw group died. It

was concluded that toxicity was so similar in the two sexes that the micronucleus assay could be done 

on males only. Based on these results, 500 mg/kg was chosen as the maximum dose for the 

micronucleus assay. 

B. MICRONUCLEUS ASSAY: 

After two i.p. injections of BYI 08330, treated males show the following test substance related symptoms 

until they were killed to harvest bone marrow: apathy, a semi-anaesthetized state, roughened fur, loss of 

weight, lateral recumbency, spasms, extension spasms, leaping spasms, twitching, rolling over, periodical 

stretching of the body, breathing difficulties, eyelids 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 before 

the harvesting of bone marrow. No symptoms (including no deaths) were observed in the vehicle and 

postive control groups. Results of this assay are summarized in Table 6-64. No biologically or statistically 

significant differences were found between test substance treated and vehicle control mice for the 

frequencies of micronucleated PCEs. The PCE:NCE ratio 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 control and positive control frequencies of micronucleated 

PCEs and of PCE/NCE ratios were well within the ranges of the laboratory’s historical controls. The dose 

levels and the route of exposure were also appropriate. It was pointed out that micronucleated PCEs 

normally occur in the vehicle control up to a frequency of approximately 6.0 per 2000 cells. 
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Table B.6-64: Summary of micronucleus assay with BYI 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 control, p < 0.05 

**Statistically significantly higher than vehicle control, p < 0.01 

III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS:

The investigator concluded that intraperitoneally-administered 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 conclusions. The dose was clearly limited by toxicity, and it 

was adequately demonstrated that the toxicity is similar enough in the two sexes to make it acceptable to 

do this assay only using males. The positive and negative controls induced the appropriate response and 

were within the ranges of the laboratory’s historical controls. There was not a significant increase in 

the frequency of micronucleated polychromatic erythrocytes 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 aberration 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 temperature storage 

Lot/Batch No.: NLL 6425-9 

Purity (a.i.%): 93.5% (June 21, 2002); 92.7% (December 19, 2002) 

Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-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 Division 

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 approximately 5 ml hypotonic potassium chloride solution (0.56

% w/v, prewarmed to 37 °C). The hypotonic cell suspension 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 methanol+glacial acetic acid fixative for 60 min. Then the 

cell pellet was gently resuspended with fixative and stored overnight at 4°C. Prior to making slides the 

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 more 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 chromosomal disintegrations were recorded as 

structural chromosome aberrations. At least 100 well spread metaphases per animal were scored for 

cytogenetic damage on coded slides. The number of chromosome 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 mitotic index (% cells in mitosis; 1,000 cells are scored) 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 identical sites ("isogap"). The biological relevance of 

gaps of both types is unclear; 

2) Break: A break is defined as a discontinuity of one chromatid ("break") or both chromatids, at 

apparently 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 

achromatic 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 centromer. A fragment is the result of a 

break. The corresponding defective chromosome is not detectable among the chromosomes of the 

same metaphase. Fragments can be derived from one chromatid ("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 ("deletion") 

or both corresponding terminal chromatid parts of a chromosome ("isodeletion") are missing within 

the metaphase under assessment. 5) Exchange: This is an exchange of chromatid-parts between 

different chromosomes (interchange) or within the same chromosome (intrachange). 6) Multiple 

aberration: A cell was assessed as to contain "multiple 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 shows 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 induces either a dose-related increase or a clear increase in the 

number of structural chromosomal aberrations in a single dose group. Statistical evaluation 

(nonparametric Mann-Whitney test) was used as an aid 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 structural chromosomal aberrations is considered nonmutagenic in this system. 

II. RESULTS: 

Findings: No substantial reduction of the mitotic indices could be observed after treatment with the test 

item, indicating that the test item at the doses administered was not cytotoxic in the bone marrow. No

biologically relevant or statistically significant increase in the frequency of aberrant cells occurred after 

treatment with the test item as compared to the vehicle control. The appropriate reference mutagen 

(cyclophosphamide) showed a distinct and statistically significant increase of induced aberration 

frequency and the vehicle control value was appropriate. Historical control values are included as an

appendix to this review. Results of the cytogenetic 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 

Treatment cells 

(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 fragments 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 aberrations 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 synthesis test with rat liver cells in vivo. 

Department of Molecular and Genetic Toxicology, Toxicology, Bayer HealthCare, FriedrichEbert-Strasse 217-333, D-42096 Wuppertal, F.R.G.. Laboratory Report No.: AT00526, 

Bayer Study No.: T 3072031, July 10, 2003. Unpublished. 

I. MATERIAL AND METHODS 

A. MATERIALS: 

1. Test material: BYI 08330 

Description: technical grade, white powder, room temperature 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-oxo-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; sacrifice 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, Germany 

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-Petri dishes (7.5 x105 viable cells per dish) precoated 

with collagen to determine cell viability, attachment rate and morphology about 1.5 hours after 

establishment of the cultures. For the determination of the genotoxicity of the test substance in the UDS 

assay a Thermanox 25-mm round plastic coverslip precoated with collagen was placed into each well of 

6-well culture dishes. Approximately 3.75 x 105

 viable cells were seeded per well (in 2.5 ml culture 

medium 1), whereby 4 wells per animal including the control groups were established. All cultures were 

incubated for 90 min in a 37°C incubator in a humidified atmosphere containing approximately 5% CO2. 

3. Culture labelling:

After the attachment period, cell number and viability of the cultures were determined by the method of 

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 incubated in the same medium. To each culture 10 

uCi/mL 3H-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 fixed by 

two changes of a 1:4 acetic acid:absolute ethanol solution for a total fixing time of at least 30 min. Wells 

were then washed 2-6 times each with deionized distilled water and air dried. 

4. Autoradiography and grain counting:

The entire autoradiography procedure is performed in the dark. Air-dried coverslips were mounted cellside-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 coated 

slides were stored in light-tight boxes in the presence of a drying agent (Drierite) for 14 days at -20°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 min in Kodak D-19 developer at temperatures below 

15°C. The slides were rinsed afterwards with distilled water, fixed in Kodak Fixer for 7-8 min, and air 

dried. Slides were then stained with hematoxylin and eosin. Grain counting was done by eye using a 

Zeiss microscope interfaced to a TV color screen (Sony) with a Sony high resolution TV color camera. 

UDS was determined as follows: Evaluation was performed with coded slides. Each slide was examined 

by counting 50 cells per slide. Three slides per animal were evaluated (total of 150 nuclei for each rat). 

Only cells viable at the time of fixation were scored; isolated nuclei and cells with abnormal morphology 

were excluded. A starting point was randomly selected on the lower third of the slide, and cells were 

scored in a regular fashion by bringing new cells into the field of view, moving along the X-axis. If the 

total number of 50 cells had not been reached before coming to the edge of the slide, the stage was 

moved on the Y-axis, and counting resumed in the opposite X-direction, parallel to the first line. UDS

was measured by counting nuclear grains and subtracting 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 grain 

(NNG) count of the cell. The mean NNG count per animal was routinely determined from triplicate 

coverslips. The mean NNG value per dose group was calculated 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 were transferred to a PC and processed with the 

released and Windows NT 4.0 based software "UDS-Test" (V02.02) of Bayer AG. 

5. Assay evaluation criteria:

1. For each of the evaluated 50 cells per slide, the number of nuclear grains is scored, as well as 

numbers of three cytoplasmic grain counts from nuclear-sized areas adjacent to each nucleus. 

2. Only cells viable at the time of fixation and with nuclei evenly coated with emulsion will be scored. 

Cells with abnormal morphology, such as those with pyknotic or lysed nuclei, will not be counted. 

Isolated nuclei not surrounded by cytoplasm will not 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 cells) will be excluded from scoring. 

6. Statistical methods:

Descriptive statistical methods were used to calculate means and standard deviations. The means and 

standard deviations in the tables were calculated from the means calculated individually for each of the 

three coverslips per animal. Statistical evaluation of % cells in repair was performed using the Chi2

 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 the 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 with 5 or more net nuclear grain counts per dose / No. 

of evaluated cells per dose) x 100 

Absolute Survival (%) = % of viable cells after isolation 

II. RESULTS: 

Findings: Rats treated with BYI 08330 showed symptoms of toxicity after administration of both doses. 

Signs included roughened fur and pallor at both doses and rapid breathing at 2,000 mg/kg only. No 

relevant cytotoxicity was observed in hepatocytes after 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 controls 2-Acetylaminofluorene and N,N'-

Dimethylhydrazine had clear genotoxic effects. 

Table B.6-66: Summary of the UDS assay (16-hour exposure) 

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, (originally called Table 5 in study) 

Table B.6-67: Summary of the UDS assay (4- hour exposure) 

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, (originally called Table 6 in study) 

III. CONCLUSION: 

BYI 08330 was negative in the in vivo/in vitro UDS assay with rat liver cells. 
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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 genotoxicity studies 

Overall, assays for point mutations and chromosomal aberrations (both in vivo and in vitro) were negative 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 (OECD 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, Stilwell, 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 oC) 

Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-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]IGS BR) 

 Age/weight at study 

initiation: 

Approximately 8 weeks old / 241.8 - 243.9g mean group weight males; 

154.4 - 157.2g 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-activated 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 oC 

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 impact 

study outcome. 

 Acclimatisation: 08/25/03 (receipt) - 09/03/03 (release for study) 
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B. Study design: 

1. In life dates: Start: 09/03/03 (released for study); 09/09/03 (initiation of exposure) 

 End: 09/16/04 (terminal sacrifice complete; chronic animals) 

2. Animal assignment: 09/04/03; assigned to a control or one of three chemically-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 implanted 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 of a 

subchronic toxicity testing study (Report No. 201136). 

In that study, the toxicological response of the male rat was principally characterized by decreased rate of 

body weight gain, as well as structural and/or functional alterations in testes- and lung-related endpoints. 

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. Extrapolating 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 characterized by a structural and/or functional alteration in 

the lung (the same lesion noted in 10,000-ppm males). The lung lesion was considered a sign of general 

toxicity, and progression over the 1-year period could not be excluded. However, unlike the male, a clear 

body weight effect (a classic predictor of a maximally-tolerated dose; MTD) was not established. These 

results suggested a different sensitivity in the two sexes, indicating that females could be treated with 

concentrations somewhat higher than males to achieve roughly the same effect (i.e., males are more 

sensitive than females). Thus, a high-dose of 12,000 ppm was chosen for females in the present study.

It was anticipated that the low and high doses chosen 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 ingestion, or dermal contact during manufacture or use. Thus, 
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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 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 admixtures for each treatment group were prepared 

weekly and stored under freezer conditions until presented to the animals the following week. 

BYI 08330 was administered at a constant concentration in the feed for the duration of the study. The

concentration of the AI of the test substance in the 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 addition, 

one recovery sample was analyzed with each group of samples during the test concentration analyses. 

The homogeneity and stability of BYI 08330 when mixed in the dietary carrier was characterized prior to 

the in-life phase of the study. 

To evaluate the uniformity of distribution/homogeneity 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 layers (top, middle, bottom; 9 samples total) in 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, same as coefficient of variation; CV), derived from the 9 

samples taken, which was < 10%. Additional homogeneity analyses were performed for two other BYI 

08330 rodent studies utilizing identical rodent ration mixing procedures in March of 2005. Those studies 

included separate oncogenicity studies in both rats and mice, and were initiated at about the same time 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 temperature storage (~22 oC) and 7, 14, and 29, and 32 

days of freezer storage (~ -23 oC; 12,000 ppm evaluated at 29 days; 70 ppm evaluated 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 temperature 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 perform the dietary analyses required for this study (i.e., 

homogeneity, stability, and concentration verifications) 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; %RSD = 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 concentration range of 70-12,000 ppm. Similarly, additional 

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 the 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 determined 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 freezer storage for a minimum 29 days, over a concentration 

range of 70-12,000 ppm. 

Concentration Analysis: Mean analytical concentrations for each dose group were 241, 3,373, 7,366, 

and 11,549 ppm, ranging from 96-98% of the corresponding nominal concentrations of 250, 3,500, 7,500, 

and 12,000, respectively. The AI of the test substance 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 were evaluated for equality or homogeneity of variance 

using Bartlett's test. Group means were further analyzed by a one-way variance analysis (ANOVA) 

followed by Dunnett's test. Frequency data that were examined statistically were evaluated using the 

Chi-square and/or Fisher exact tests. Continuous data collected in the FOB that were examined 

statistically were evaluated using an ANOVA with post-hoc comparisons using Dunnett's test. 

Categorical data collected in the FOB that were examined statistically were evaluated using General 

Linear Modeling and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using 

Dunnett's test and an Analysis of Contrasts, respectively. For the Bartlett test, a probability (p) level <

0.001 was considered significant; for all other statistical tests, differences with p values < 0.05 were 

considered statistically significant. Additional statistical tests may have been used to evaluate data 

generated from this study when deemed appropriate by the study director. All statistical evaluations were 

performed using software obtained from either INSTEM 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 daily 

(generally twice daily during the normal work week and once daily on weekends and holidays). These 

assessments were generally with respect to moribundity 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 toxicity 

were performed on all surviving animals. These examinations included evaluation of external surface 

areas, orifices, posture, general behavior, respiration, and excretory products, and a general open field 

assessment (qualitative; utilizing a standard arena). General observations in the standard arena were

not required during the week in which the animals are subject to a more detailed open field observation 

as part of the full functional observational battery. 

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, grip strength, and sensory reactivity to stimuli of different 
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types (e.g., visual, auditory, and proprioceptive stimuli). Parameters evaluated included home cage 

observations - posture, piloerection, involuntary motor movements (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 discharge, stains, alopecia, emaciation, bite marks,

exophthalmia, broken teeth/malocclusion, missing toe nails, dehydration, cool-to-touch; open field 

observations - piloerection, respiratory abnormalities, posture, involuntary motor movements-clonic or 

tonic, stereotypy, bizarre behavior, gait abnormalities, 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 examinations. 

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 carried out weekly for the first 13 weeks of the study

and every 4 + 1 weeks thereafter on all surviving animals. In addition, 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 feeders, etc.). Test substance intake (mg/kg body wt/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 body wt/day)] = mg AI/kg body wt/day 

4. Ophthalmologic examination: 

Ophthalmologic exams were conducted on all acclimatized animals prior to exposure, and then again on 

all surviving animals just prior to termination. Ophthalmological lesions were assigned a semiquantitative ranking of the severity grade/stage from "Normal (within normal limits)" to the "most severe 

case (Grade 5)," as described previously. 

5. Hematology and Clinical Chemistry: 

Blood was collected at approximately 3, 6, and 12 months (+ 0.5 month/interval) into the study. Blood 

was drawn via the orbital sinus following an overnight fast from all surviving animals while under light 

anesthesia (IsoFlo®; Isoflurane). To the extent possible, 15 of the blood samples were used for 

hematologic (excluding prothrombin time) and clinical determinations; the remaining blood samples 

(nominally 10) were used for prothrombin time determinations. Differential blood counts were determined 

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 concentration (MCHC) 

X Erythrocyte count (RBC)* X Mean corpuscular volume (MCV)* 

X Platelet count (PLTS)* X Reticulocyte count (Retic) 

X Blood clotting measurements* X Erythrocyte morphology 
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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 Guideline 870.4100. 

(a) Added by the HED reviewer. Also data were provided 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-Prot)* 

 Cholinesterase (ChE) X Triglycerides (Trig) 

X Creatine phosphokinase (CK) Serum protein electrophoresis 

X Lactic acid dehydrogenase (LDH) X Uric acid (Uric-A) 

X Alanine aminotransferase (ALT)* X Thyroxine (T4) 

X Aspartate aminotransferase (AST)* X Triiodothyronine (T3) 

 Sorbitol dehydrogenase* X Thyroid Stimulating Hormone (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 Guideline 870.4100. 

The CHECKED (X) parameters were examined. 

6. Urinalysis: 

Urine was collected at approximately 3, 6, and 12 months (+ 0.5 month/interval) into the study. Non-fasted 

urine was collected on an overnight basis (timed) from the first 10 surviving rats/sex/dose the week prior 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 Guideline 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 gross lesions, (2) weighing designated organs, 

and (3) collecting representative tissue specimens for histopathologic evaluation. Animals surviving at 

termination were sacrificed by CO2 asphyxiation. Tissues were preserved and organ/body 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 870.4100. 

+ Organ weights required for rodent studies. 

The CHECKED (X) tissues were collected for histological examination. The organs CHECKED (XX) were also weighed. 

The organs CHECKED (^) were preserved for possible micropathologic evaluation only. 

7b. Micropathology: 

With the exception of the physical identifier (microchip), the vagina, and the exorbital/lacrimal, clitoral, 

preputial, and Zymbal glands (of which the latter 5 were taken and preserved for possible micropathologic 
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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, 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 detected were examined at lower doses as necessary to establish noobserved-effect levels. Target organs and gross lesions were examined for all animals. 

Histopathological lesions were assigned a semi-quantitative 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 onset 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 other 

treatment-related clinical signs and no substance-related 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 not 

toxicologically relevantly affected. Body weights were 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±6 536±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 data 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 374 to 386. 

Ophthalmology: No treatment-related effects. Mean ophthalmology data are on pages 319 to 321. 

Gross necropsy, organ weights: Gross necropsy showed an increased incidence of discoloration of the 

lung in 12,000-ppm females (0, 1, 0, 5). No other indication of a BYI 08330-induced change was noted in 

either sex at any dose tested. An increase in relative liver weights was noted in 7,500-ppm males and in 

12,000-ppm females which was considered treatment-related. 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. Mean organ 

weight data are on page 429 to 438 of the study report. 

Histopathology: 

Testis/epididymis: An increased incidence of exfoliated germ cells/debris was observed in the epididymis of 

males at 7500 ppm (3/25 vs. 0/24 in controls). Abnormal spermatozoa were also observed in high dose 

males (2/25 vs. 0/25 in controls). Although the increases were not statitistically significant, they were 

considered toxicologically relevant, since testicular/epididymal histopathology was observed at higher

incidences following treatment with spirotetramat for 24 months. 

Lung: An increased incidence of minimal to slight accumulation 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. No other indication of microscopic changes attributable to 

exposure to the test substance was observed. 

Table B.6-71: Incidence and average severity (figures 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 group examined. The pathology summary table is on page 439 to 

471 of the study report. 

Conclusions: The chronic NOAEL was established at 250 ppm (equal 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 toxicity was observed in a total number of 25 male rats at 7,500 

ppm (equal to 414 mg/kg bw/day) examined histopathologically after one year of treatment. 

The chronic NOAEL in females was established at 3,500 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 of accumulation of alveolar macrophages at 12,000 ppm 

(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 oC) 

Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-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-pregnant) 

 Strain: Wistar Hanover rats (Crl:WI[Glx/BRL/Han]IGS BR) 

 Age/weight at 

treatment initiation: 

Approximately 7 weeks old / 204.7 - 206.5 g mean group weight males; 

151.1 - 152.2 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-activated 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. 
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 Environmental 

conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

18 to 26 oC 

30 to 70% (relative); high range for relative humidity 

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 12:00 

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 the 

deviations was of a duration as to impact study 

outcome. 

 Acclimatisation: 07/07/03 (receipt) - 07/14/03 (animals release for study) 

B. Study design:

1. In-life dates: Start: 07/11/03 (study initiation); 07/14/03 (released for study); 

 07/16/03 (initiation of exposure; males); 07/17/03 (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); assigned 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 Systems (Stone, Staffordshire, UK). Each animal on 

study was identified with a microchip (IPTT-200; BioMedic Data Systems, Inc., Seaford, DE) subcutaneously 

implanted 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.6-72: 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 of a 

subchronic toxicity testing study. 
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In that study, the toxicological response of the male rat was principally characterized by decreased rate of 

body weight gain, as well as structural and/or functional alterations in testes- and lung-related endpoints. 

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. Extrapolating these subchronic changes out over a prolonged 

lifetime exposure (nominally 2 years), 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 characterized by a structural and/or functional alteration in 

the lung (the same lesion noted in 10,000-ppm males). The lung lesion was considered a sign of general 

toxicity, and progression over the 2-year period could not be excluded. However, unlike the male, a clear 

body weight effect (a classic predictor of a maximally-tolerated dose; MTD) was not established. These 

results suggested a different sensitivity in the two sexes, indicating that females could be treated with 

concentrations somewhat higher than males to achieve roughly the same effect (i.e., males are more 

sensitive than females). Thus, a high-dose of 12,000 ppm was chosen for females in the present study.

It was anticipated that the low and high doses of 250 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 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 ingestion, or dermal contact during manufacture or use. Thus, formulation 

with the feed was an appropriate route of administration with which to develop the chronic toxicological 

profile of the test substance in a given test species. 

4. Diet preparation and analysis: 

The test substance 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 admixtures for each treatment group were prepared 

weekly and stored under freezer conditions until presented to the animals the following week. BYI 08330 

was administered at a constant concentration in the feed for the duration of the study. The concentration 

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 Weeks 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 105. In addition, one recovery sample was analyzed with 

each group of samples during the test concentration analyses. The homogeneity and stability of BYI 

08330 when mixed in the dietary carrier was characterized prior to the in-life phase of the study and again 

just prior to termination of the in-life phase of the study. 

To evaluate the uniformity of distribution/homogeneity 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 test 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 to termination, using one 

sample from the top, middle, and bottom mixing bowls 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 temperature storage (~22 oC) and 7, 14, and 29, and 32 

days of freezer storage (~ -23 oC; 12,000 ppm evaluated at 29 days; 70 ppm evaluated 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 
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divided into 2 portions. One portion was placed in a room temperature environment, the other in the 

freezer. Feed samples which remained at room temperature, 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 perform the dietary analyses required for this study (i.e., 

homogeneity, stability, and concentration verifications) 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; %RSD = 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 concentration range of 70-12,000 ppm. Homogeneity 

analysis conducted just prior to study termination again confirmed homogeneity of the test substance in 

rodent ration. 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 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 the 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-determined 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 freezer storage for a minimum 29 days, over a concentration 

range of 70-12,000 ppm. 

Concentration Analysis: Mean analytical concentrations for each dose group were 249, 3,407, 7,342, and 

11,722 ppm, ranging from 97-100% of the corresponding nominal concentrations of 250, 3,500, 7,500, and

12,000 ppm, respectively. The AI of the test substance 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 were evaluated initially for equality or homogeneity of 

variance using Bartlett's test. Group means were further analyzed by a one-way analysis of variance 

(ANOVA) followed by Dunnett's test for pair-wise 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 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 tests, differences with p values < 0.05 were considered 

statistically significant. All statistical evaluations were performed using software obtained from either 

INSTEM Computer Systems or SAS Institute Inc. 
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C. Methods:

1. Observations 

2. 

1a. Cageside Observations: 

For approximately 105 weeks of the study, a general assessment of all remaining animals was carried out 

at least daily (generally twice daily during the normal work week and once daily on weekends and 

holidays). These assessments were generally with respect to moribundity 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 approximately 104 weeks of the study, detailed physical examinations for clinical 

signs of toxicity were performed on all surviving animals. These examinations included evaluation of 

external surface areas (visual and palpation), 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/body weight ratios with the following exceptions. 

Terminal body weights for animals RK3010 (term sac) and RK3151 (sacrificed in extremis) were not 

measured. Final live body weights were used to calculate organ/body weight ratios as appropriate. 

3. Food consumption and compound intake: 

Individual food consumption determinations were carried out weekly for the first 13 weeks of the study and 

every 4 + 1 weeks thereafter on all surviving animals. In addition, 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 feeders, etc.). Test substance intake (mg/kg body wt/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 body 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 for 

differential determinations. In all cases, blood was sampled via the orbital sinus from non-fasted animals. 

Differential blood counts were determined on all animals sacrificed due to moribundity with the exception 

of RK1022 (had been bled for this purpose earlier in the week; scheduled blood collection). 

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 concentration (MCHC) 

 Erythrocyte count (RBC) Mean corpuscular volume (MCV) 

 Platelet count (PLTS) Reticulocyte count (Retic) 
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 Blood clotting measurements X Erythrocyte morphology 

 Thromboplastin time Heinz bodies (HZ) 

 Clotting time 

 Prothrombin time (PT) 

* Minimum required for carcinogenicity studies (Cont. 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: 

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 gross lesions, (2) weighing designated organs, 

and (3) collecting representative tissue specimens for histopathologic evaluation. 

Animals surviving at termination were sacrificed by CO2 asphyxiation. Tissues were preserved and 

organ/body 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, optic* 

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 
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 XX Adrenal gland*+ 

 X Exorbital/lacrimal gland^ 

 X Parathyroid* 

 X Thyroid* 

* Required for carcinogenicity studies based on Guideline 870.4200. 

+ Organ weight required in carcinogenicity studies.

++ Organ weight required if inhalation route. 

The CHECKED (X) tissues were collected for histological examination. The organs CHECKED (XX) were also weighed. 

The organs CHECKED (^) were preserved for possible micropathologic evaluation only. 

(1) The ID chip for animal RK1001 was cut during the necropsy process. The tail was marked with an indelible marker 

and retained as the physical identifier. 

7b. Micropathology: 

With the exception of the physical identifier (microchip or tail), the vagina, and the exorbital/lacrimal, 

clitoral, preputial, and Zymbal glands (of which the latter 5 were taken and preserved for possible 

micropathologic evaluation), representative sections of the tissues shown in Table 6-76 were processed

for all control (0 ppm) and high-dose (7,500 / 12,000 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 detected 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-quantitative ranking of the severity grade/stage from 

"Normal (within normal limits)" to the "most severe case (Grade 5)," as described previously. The "grade 

number" was used to calculate an average grade for a particular observation within a dose group. 

Neoplastic lesions were generally not assigned a grade.Tissues from approximately 101 animals were 

processed as described above by Charles River Laboratories, Pathology Associates (Kansas City, MO). 

These animals represented all early deaths through day 682 (05/28/05). All other histology was performed 

by in-house (Bayer CropScience LP - Toxicology). The 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 included examination of various selected and randomlyselected slides. 

II. Results 

Findings: 

General observations: Survival was unaffected by treatment, as the incidence of mortality was 

comparable between groups. Early deaths/survival numbers 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 revealed an 

increased incidence of “body, scaly” and yellow and brown staining on the perigenital, perianal area and 

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 measurements (week 103), body weight declines of 10% and 

14% were seen for 7,500-ppm males and 12,000-ppm females, respectively. 

Table B.6-73: Incidence of treatment-related clinical 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*
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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 observations are reported on Pages 89 to 127 of the study report. 
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Table B.6-74: 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 206.5 204.8 205.3 204.7 FINAL 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±4.3 BODY 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 * FEMALES INITIAL BODY WEIGHT 152.0 151.1 152.2 151.6 FINAL 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 < 0.05 Note 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 body weight data are on pages 37 to 49 and mean body weight gain data are on pages 50 to 60 of the study report.
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Hematology: Leukocyte differential counts and erythrocyte morphology 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 focal to multifocal (1-4 

mm) discoloured zones (sometimes raised) of the lung in 12,000-ppm females (1, 1, 3, 32*) which were 

attributed to compound administration. Relative lung weight was slightly increased in the 7,500 ppm male 

and the 12,000 ppm female group. The lung weight changes in 12,000 ppm females correlated with 

increased focal to multifocal discolorations observed in the lungs at necropsy. Absolute kidney weights were 

decreased in both sexes at 3,500 and 7,500/12,000 ppm and were associated with microscopic findings 

noted in the same groups. Any other apparent change in absolute kidney weight (e.g., 250 ppm males) was 

considered to be incidental, due to the highly variable kidney weight data (particularly in males) and the lack 

of microscopic evidence of a direct toxicological insult by the test substance on the kidney that would 

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 (%) organ 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
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* = 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 examined was the number of survivors. 

Histopathology: 

Non-neoplastic changes: Statistically significant changes in the incidence of non-neoplastic lesions were 

detected in the lung, kidneys, testis/epididymis and liver. 

Lung: An increased incidence of alveolar macrophage accumulation (M: 7, 17*, 17*, 3; F: 

20, 14, 18, 3), a common background lesion in the rat, and a complex of changes described as interstitial 

pneumonia (M: 22,15,19,44*; F: 4,13*,14*,52*) was noted in the lungs of 7,500-ppm males, and 12,000-ppm 

females. Both findings were attributable to exposure 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 the 

presence of one or more of the following changes: presence of lymphocytes, cholesterol clefts, interstitial 

thickening of the alveolar septa by connective tissue, or increased alveolar pneumocytes and presence of 

occasional extravasation of erythrocytes (micro hemorrhage). The lung effects described above were focal to 

multifocal in distribution and involved a small overall portion of the lung tissue triggering no respiratory 

symptoms. It was noted by the HED reviewer that the alveolar macrophages and interstitial pneumonia did 

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 groups and in the 7,500/12,000 ppm groups. The affected 

renal tubules were dilated and were either empty or contained pale eosinophilic proteinacious material, 

usually granular. Negligible cellular death, regeneration, or inflammatory responses to the tubular dilatation 

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 7,500/12,000 ppm animals was considered treatment-related 

and was presumed to be reflected in the decreased kidney weights although there was no associated loss of 

tissue. It was also stated that the high background incidence of chronic nephropathy greatly influenced 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 typical aging changes including variably severe 

testicular degeneration, oftentimes noted grossly as soft, brown, or reduced in size (12,8,10, and 6 for the 

control, low, mid and high dose groups, respectively – added by HED reviewer). In addition to this change in 

the 7,500-ppm males, there were a few animals with generally slight morphologic testicular change, more 

subtle than distinct tubular degeneration, characterized by a depletion, asynchrony, and degeneration of the 

latter stage spermatids (0,0,0, and 9* for the control, low, mid and high dose groups, respectively – added by 

HED reviewer). 

Epididymis: An increased incidence of immature/exfoliated germ cells/debris was observed in the lumen 

contents of the head, body, and tail of the epididymides at 7,500 ppm (6,10,6, and 31* for the control, low, 

mid and high dose groups, respectively – added by the HED reviewer), which correlates with the testicular 

change. There was no morphologic change noted in the 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 incidence of hyperplasia/fibrosis with associated minimal 

periportal mononuclear cell infiltrate was noted in 12,000-ppm females. 

Neoplastic changes: 
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Type, incidence and organ distribution of all neoplastic lesions was not significantly different between treated 

and control rats. 

Table B.6-76: Average incidence and severity (figures 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 9999. The individual animal sheets are on pages 1000 to 

2395. 

(a) asterisk removed from the original summary report by the HED reviewer. 

(b) the number of animals examined was added by the reviewer. 
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Conclusions: The chronic (24 months) NOAEL was established at 250 ppm (equal to 12.5 mg/kg bw/day) in 

male rats, based on decreased absolute kidney weights 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 testicular 

tubular degeneration and immature/exfoliated germ cells 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 established at 250 ppm (equal to 16.8 mg/kg bw/day), 

based on decreased absolute kidney weights and an increased incidence of renal tubular dilatation at 3,500 

ppm (equal to 229 mg/kg bw/day). Based on type, incidence and organ distribution of neoplastic lesions in 

treated and control rats, there was no indication for an oncogenic effect of BYI 08330. 
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B.6.5.2 Carcinogenicity study in the mouse (OECD Annex IIA 5.5.3) 

CITATION: Whale, B. 2006 Technical grade BYI08830 (Common Name Spirotetramat): An Oncogenicity 

Testing Study in Mouse. Bayer Crop Science, Stilwell, KS. March 13 (original) 17 

(amended), 2006, Unpublished. 

Reviewer’s Discussion 

 

US EPA has prepared the following table to illustrate that there were no adverse effects on key parameters: 

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 25.5 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) terminalb

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 from page 936 (males) and 955 (females) of the study report. 

b

The SEM for the control and high dose only are presented. The SEM for the low and mid dose groups were 

similar. SD, not presented, is considered a more appropriate measure of variability around the mean than 

SEM. Body weight data are from pages 33 (males) and 34 (females) of the study report. Liver weight data 

are from pages 195 (males) and 196 (females). 

Comments:

a. The subchronic study (2005; ) in mice suggested a possible slight body weight effect in the high dose 

during the first weeks of the study. No similar effect 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 that would be associated with increased capacity to 

metabolize and facilitate the excretion of spirotetramat. 

c. The histopathology report indicated that the brain had more incidents of mineralization in the high dose 

group (13 in males and 12 in females) than in the control (6 in males and 8 in females). However, there was 

no increase in severity, with most cases being minimal. Thus, no effect of spirotetramat on brain 

mineralization was concluded. 
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d. The rat subchronic (2005, Lab Report No. 201136), chronic (2005) and carcinogenicity (2006) studies all 

indicated the presence of “alveolar macrophages” and/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 study. 

e. The rat feeding studies (see above) also suggested possible effects on the male reproductive system such 

as testicular degeneration, vacuolation, abnormal spermatozoa and hypospermia (refer to the DER for the 

rat carcinogenicity study for a discussion). No similar 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; common 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 oC) 

 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 non-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. Animals passing the initial 

shipment exam were co-housed during a portion of the Acclimatization and 

later housed individually (for the remainder of the Acclimatization and 

throughout the exposure period). 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. 
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 Environmental 

conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod 

18 to 26 oC 

30 to 70% (relative); high range relative humidity was 71% 

on Days -10 (pre-exposure) and 39, and 73% on Day 11. 

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. Deviations in the 

light/dark cycle occurred 3 times (2 planned; 1 unplanned) 

over the course of the study. The light cycle is unknown for 

Days 202 (Edstrom version 5 installed) and 234 (power 

outage occurred, smart box replaced due to lightning strike). 

None of the deviations was of a duration as to impact study 

outcome. 

 Acclimatisation: 10/20/03 (receipt) - 10/27/03 (release for study) 

B. Study design:

1. In-life dates: Start: 10/27/03 (released for study); 10/28/03 (study 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 chemically-treated 

groups (as noted in the table below) 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 implanted 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. 

Main Study 

Test Group Conc. in Diet (ppm) 18 months

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 initiated 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 toxicological response in the mouse over the course of a 

subchronic toxicity testing study at doses up to and including 7,000 ppm (Report No. 201284). It was 
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anticipated that the low and high doses chosen of 70 ppm and 7,000 would constitute a no-observedadverse-effect level and a limit dose, respectively, with the intermediate dosage of 1,700 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 ingestion, or dermal contact during manufacture or use. Thus, formulation with the 

feed was an appropriate route of administration with 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 admixtures for each treatment group were prepared 

weekly and stored under freezer conditions until presented to the animals the following week. 

BYI 08330 was administered at a constant concentration in the feed for the duration of the study. The

concentration of the AI of the test substance in the control, 70-ppm, and 1,700-ppm test diets was 

analytically verified from samples taken during Weeks 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 AI of the test substance was analytically verified from 

samples of the 7,000-ppm test diet taken during Weeks 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 each group of samples during the test concentration 

analyses. The homogeneity and stability of BYI 08330 when mixed in the dietary carrier was 

characterized prior to the in-life phase of the study and again just prior to termination of the in-life phase 

of the study. 

To evaluate the uniformity of distribution/homogeneity 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 distinct layers (top, middle, bottom; 9 samples total) in 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, same as coefficient of variation; CV), derived from the 9 

samples taken, which was < 10%. Homogeneity was again analyzed, just prior to termination, using 1 

sample from the top, middle, and bottom mixing bowls 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 temperature storage (~22 oC) and 7, 14, and 29, and 32 

days of freezer storage (~ -23 oC; 12,000 ppm evaluated at 29 days; 70 ppm evaluated 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 temperature 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 perform the dietary analyses required for this study (i.e., 

homogeneity, stability, and concentration verifications) 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 
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determined to be 68.9 ppm (range 64.9-73.2 ppm; %RSD = 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 concentration range of 70-12,000 ppm. Homogeneity 

analysis conducted just prior to study termination again confirmed homogeneity of the test substance in 

rodent ration. 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 6,000 ppm, were determined to be 

65.8 ppm (range 61.9-70.1 ppm; %RSD = 6.3%) and 5,951 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 the 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 determined 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 freezer storage for a minimum 29 days, over a 

concentration range of 70-12,000 ppm. 

Concentration Analysis: Mean analytical concentrations for each dose group were 67.7, 1,655, 5,986, 

and 6,873 ppm, ranging from 97-100% of the corresponding nominal concentrations of 70, 1,700, 6,000, 

and 7,000 ppm, respectively. The AI of the test substance 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 were evaluated initially for equality or homogeneity of 

variance using Bartlett's test (Snedecor and Cochran, 1967). Group means were further analyzed by a 

one-way variance analysis (ANOVA) (Snedecor and Cochran, 1967) followed by Dunnett's test (Dunnett, 

1955, 1964). 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 those mentioned above. For the Bartlett test, a probability (p) value <

0.001 was considered significant; for all other statistical tests, differences with p values < 0.05 were 

considered statistically significant. All statistical evaluations were performed using software obtained 

from either INSTEM Computer Systems or SAS Institute 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 normal work week and once daily on weekends and 

holidays). These assessments were generally with respect to moribundity and mortality, but also 

included a more general clinical observation for readily apparent clinical signs (in addition to moribundity 

and mortality). 

1b. Clinical Examinations: 
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Once each week for approximately 79 weeks of the study, detailed physical examinations for clinical 

signs of toxicity were performed on all surviving animals. 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 carried out weekly for the first 13 weeks of the study

and every 4 + 1 weeks thereafter on all surviving animals. In addition, 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 feeders, etc.). Test substance intake (mg/kg body wt/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 body 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 months into the study from all surviving animals for 

differential determinations with the exception of animal RM3155 at 12 months. In all cases, blood was

sampled via the orbital sinus from non-fasted animals. Differential blood counts were determined on all 

animals sacrificed due to moribundity with the following 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 concentration (MCHC) 

 Erythrocyte count (RBC) Mean corpuscular volume (MCV) 

 Platelet count (PLTS) Reticulocyte count (Retic) 

 Blood clotting measurements X Erythrocyte morphology 

 (Thromboplastin time) Heinz bodies (HZ) 

 (Clotting time) 

 Prothrombin time (PT) 

* Minimum required for carcinogenicity studies (Cont. 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 gross lesions, (2) weighing designated organs, 

and (3) collecting representative tissue specimens for histopathologic evaluation. Animals surviving at 

termination were sacrificed by CO2 asphyxiation. Tissues were preserved and organ/body 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, optic* 

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 Guideline 870.4200. 

+Organ weight required in carcinogenicity studies. 

++Organ weight required if inhalation route. 

The CHECKED (X) tissues were collected for histological examination. The organs CHECKED (XX) were 

also weighed. The organs CHECKED (^) were preserved for possible micropathologic evaluation only. 

(1) Starting on 04/15/05 (nominal Day 529), indelible 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 (microchip and/or tail), the vagina, and the exorbital/lacrimal, 
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clitoral, preputial, and Zymbal glands (of which the latter 5 were taken and preserved for possible 

micropathologic evaluation), representative sections 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 detected 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-quantitative 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 lesions were generally not assigned a grade. The initial 

micropathologic evaluation of this study was performed by Dr. Laura Elcock (Charles River Laboratories, 

Pathology Associates, Kansas City, MO). This evaluation 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 incidence of mortality was 

comparable between groups. Early deaths/survival numbers 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 observations 

attributable to exposure to the test substance were not observed in either sex at any dose tested. The 

general observation profile was consistent with that of the aging mouse. 

Body Weight: (Note: See also the body weight table prepared by the HED reviewer at the beginning of 

this document). There were no treatment-related effects on body weight and food consumption in either 

sex at any dose tested. 

Hematology: Evaluation of the data from blood collected at approximately 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 counts and erythrocyte morphology only. 

Gross necropsy, organ weights: Evaluation of the general profile of absolute and relative organ 

weights as well as of gross lesions observed in this 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 exposure to the test substance were not observed in this 

study. Two incidences of tumors arising from brown fat were noted in high-dose males. Microscopically, 

the tumors were determined to be benign, and were composed of well-differentiated cells that resembled

brown fat. However, there were occasional cells that appeared to originate from white fat. As such, the 

term “lipomatous neoplasm” was assigned to the lesion. In both cases, the tumors were noted grossly 

(1.5 to 2.0 cm in size) and were located in the interscapular region. Both animals lived until study 

termination. Similar tumors were not observed in females. The report of this type of tumor is rare. One 

consideration that was made concerned the use of implantable identification transponders (ID chips), 
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which were placed between the scapula (subcutaneously) 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 tumor types in both mice and rats (Elcock et al., 2001: 

Tumors in long-term rat studies associated with microchip animal identification devices. Exp Toxic Pathol 

52, 483-491; Ghanta and Edmondson, 1990: Tissue reaction 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 that these tumors were spontaneous in nature. The 

incidence in the high dose was not significantly different from controls and no similar tumors were 

observed in any female animal. Both tumors were benign 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, there 

was no indication for an oncogenic effect of BYI 08330. 
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B.6.5.3 Summary of long term toxicity/carcinogenicity studies 

The thymus and thyroid were also target organs following one-year oral exposure of dogs to spirotetramat. In 

addition, the brain was also considered to be a target organ by PMRA. Thymus involution and brain dilation 

with dose-related severity were observed in one male 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 axonal 

degeneration in the hypothalamus was observed in one female at 1800 ppm (48 mg/kg bw/day). PMRA 

considered these brain effects to be treatment-related. Brain dilation has been observed in up to 5% of 

Beagle dog colonies and is considered a congenital anomaly by Bayer CropScience. Brain dilation was not 

observed in any other study in the dog or in any other species tested with spirotetramat. For these reasons, 

and since brain dilation was also observed sporadically in several other Bayer dog studies of varying 

durations (from four weeks up to one year), USEPA considered the observation to be equivocal at best and 

did not consider its use appropriate for risk assessment. PMRA, however, considered brain dilation to be 

treatment-related. Clinical signs of neurotoxicity (dehydration, swelling, decreased activity and reactivity, 

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 hormone 

(T4) were observed in males and females at ≥600 ppm (20 mg/kg bw/day) Reduction in follicle size, 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 in 

dogs at any dose. 

In rats, abnormal sperm or spermatid degeneration in the testes and germ cell exfoliated debris in the

epididymis were observed in male rats at the high dose only (7500 ppm; 414 or 373 mg/kg bw/day) following 

12 (not statistically significant) or 24 months of oral exposure. Other target organs in rats following 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 excretory and/or detoxification roles of the kidney and liver. 

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 reproductive 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 and 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, Toxicology, 17745 South Metcalf, Stilwell, 

KS 66085-9104. Report Number 201426-1, July 13, 2006. Unpublished. 

I. MATERIAL AND METHODS

A. Materials:
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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 (~22o) 

Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-yl 

carbonate; Spirotetramat 

 CAS No. of TGAI: Non-stereo: 382608-10-8; cis: 203313-25-1 

2. Vehicle and/or positive control: None. Compound was dry mixed and administered through 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, which included a gross 

necropsy and serological evaluation. 5 animals/sex were sacrificed prior 

to initiation of exposure and 5 animals/sex were sacrificed 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 veterinarian 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 F1 pups) in 

suspended stainless steel cages and deotized cage board 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 26oC 

Relative humidity 30 to 70%, exceptions stated in the 

Protocol Amendments and Deviations section 

 Environmental 

conditions: 

Air changes: 

Photoperiod: 

Minimum of 10 air changes per hour, based on average 

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 , exceptions 

stated in the Protocol Amendments and Deviations 

section 

 Acclimatisation: 8 days 

All procedures associated with the animals were approved by the Institution’s Animal Care and Use 

Committee (IACUC); and during the course of the study, 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 outlined in the “Certification Profile” for Purina Mills Certified 

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 material for ten weeks prior to mating. 

Mating was accomplished by co-housing one female with one male for up to 14 consecutive days with 

exceptions stated in the Protocol Amendments and Deviations section. Approximately four to six 

animals from each dose group were co-housed daily beginning on the first day of the mating phase and 

continuing until all animals had been cohoused. 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 inseminated were placed in a polycarbonate nesting 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 without exhibiting sperm in the vaginal smear or an 

internal vaginal plug, all remaining females were placed 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 were exposed to the treated feed throughout the entire

study. The parental animals (P and F1 generations) were given test diets for 10 weeks prior to mating. 

Selection of F1-pups to evaluate preputial separation and vaginal patency was made when the pups 

were 21 days of age. The F2-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 released for study use. The animals were then 

assigned to either a control or one of three chemically-treated groups using a weight stratification-based 

computer program (INSTEM Computer Systems, Stone, Staffordshire, 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 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, 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, was attached to the outside of each animal’s cage. Pups 

born alive were identified by tattoo and pups found dead were identified with a marking pen. All animals 

not placed on study (for whatever reason) were ultimately 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 Dieta

(ppm) P Males P Females F1 Males F1 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 study until sacrifice. 

4. Dose selection rationale: Doses were selected based upon the preliminary results which emerged in 

the rat over the course of a pilot reproductive toxicity testing study conducted with the test chemical at 

doses of 0, 200, 500, 6,000 and 10,000 ppm BYI 08330/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
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on body weight, food consumption or clinical observations. In this same dose group, declined 

epididymal weights were observed. Sperm analysis revealed declined sperm motility (67%) and % 

progressive sperm (79%), as well as, abnormal sperm in the epididymis and cauda epididymis. The F1-

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 sperm (15%), as well as, abnormal sperm in the 

epididymis and cauda epididymis. Based on these interim 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 feed. Treated diet 

was mixed at room temperature; aliquots of the chemical were taken from the original test batch and 

transferred to the mixing area. The control test diet was taken directly from the bag without mixing. A 

sample of each batch of feed mixed was taken and retained in the freezer until the study was complete 

and the analytical data deemed satisfactory. Replacement admixtures for each treatment group, for 

each week, were stored under freezer conditions until presented to the animals the following week. 

Additionally, the entire batch of replacement admixture for each treatment group was used for 

subsequent weekly feedings if within freezer stability 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 substance 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%), 144 ppm (range 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 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 the 70-, 150-, or 10,000-ppm admixture 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-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); 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 storage for a minimum of 28 days, over a concentration range of 70-10,000 ppm. 

Concentration Analysis: The concentration of BYI 08330 in the various test diets was determined 

using liquid chromatography. Mean analytical concentrations for each dose group were 244, 1,006 and 

5,898 ppm, ranging from 98-101% of the corresponding nominal concentrations of 250, 1,000 and 6,000 

ppm, respectively. The AI of the test substance was not detected in the control diet. Mean recovery 

was 99% and ranged from 94-103% for rodent ration spiked 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 performed twice 

daily (AM and PM), during the work week and once daily 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 clinical signs, which included both observing the animal 

in the cage and removing the animal to perform a physical 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 and fresh 

feed provided once/week for both males and females during the 10-week premating period with 

exceptions stated in the Protocol Amendments and Deviations section. During the mating period and 

until sacrifice, body weights for the males were taken 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 food 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 lactation, 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 measured twice (days 0-4 and 4-7). 

Estrous cyclicity: The estrous cycle (determined by examining daily vaginal smears) was 

characterized for all P- and F1-generation females, over a three-week period prior to mating. 

Additionally, the estrous cycle stage was determined for all females just prior to termination. 

Sperm parameters: For all P and F1 -generation males at termination, sperm was collected from one 

testis and one epididymis for enumeration of homogenization-resistant 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 the urethra) of the vas deferens. Sperm motility and 

counts were conducted using the IVOS (Integrated Visual 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 generation. Morphology and counts were conducted on all 

dose groups for the second generation. 
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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 F1 pups 

Vaginal Patency Performed postweaning for F1 pups 

a

 Before standardization (culling) 

b

 After standardization (culling) 

Exceptions stated in the Protocol Amendments and Deviations section 

The size of each litter was adjusted on lactation Day 4 to yield, as close as possible, four males and

females per litter. If the number of male or female pups was less than four each, a partial adjustment 

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 gross 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 sufficient number of F1-pups/sex/litter was maintained to 

produce the next generation. F1-pups not selected to become parents of the next generation and all F2-

pups were sacrificed and examined macroscopically for any structural abnormalities or pathological 

changes, particularly as they may relate to the organs of the reproductive system. Dead pups were 

examined grossly for external and internal 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 soon as possible after the last 

litters were produced. Maternal animals were sacrificed following the weaning of their respective 

litters (lactation day 21). These animals were subject 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 anesthesia 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 -70oC freezer for possible evaluation of liver enzymes. Analysis of 

clinical chemistries and liver enzymes was deemed unnecessary. 

Male rats were sacrificed by carbon dioxide asphyxiation, 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 homogenization-resistant spermatids and cauda 

epididymal sperm reserves, respectively. In addition, an evaluation of the morphology and motility 
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was performed on sperm sampled from the distal portion (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 asphyxiation and a gross external examination was 

performed. Terminal body weights were taken and the abdomen and thoracic cavities were opened 

and a gross internal examination was performed. The 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 which were never observed as being inseminated 

and/or with an internal vaginal plug and did not deliver at least 24 days after the completion of the 

mating phase, were sacrificed and necropsied. A gross necropsy was performed on these animals 

as described above. In addition, patency of the cervical/uterine os in these females was examined 

via flushing of the uterine horns with deionized water. 

The following tissues were collected (X) and weighed (XX). Micropathology (in addition to collection 

and weights) was performed on those tissues designated with (XXX): 

XX Brain XX Thyroid XXX Prostate X Cervix 

XXX Pituitary XX Thymus XXX Seminal Vesicle X Vagina 

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 sacrificed 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 F1-offspring not selected as parental animals and all F2-offspring were sacrificed at 

21-days of age. These animals were subject to postmortem examinations (macroscopic and/or 

microscopic examination) as follows. The following tissues from 21-day weanlings were collected 

and weighed: brain, thymus, spleen, and uterus. Any gross lesion was documented and collected. 

Micropathology was performed on the following tissues: From 1 female/litter; uterus, ovary, vagina, 

cervix, oviduct (fallopian tube) and from 1 male/litter; testis, epididymis, prostate, coagulating gland, 

seminal vesicles. Exceptions are stated in the Protocol Amendments and Deviations section. 

Pups found dead or terminated in a moribund condition underwent a gross necropsy for possible 

defects and/or cause of death. 

D. DATA ANALYSIS

1. Statistical analyses: The data were analyzed using applications provided by DATATOX (Instem 

Computer Systems), SAS (SAS Institute, Inc.), or TASC (Toxicology Analysis Systems Customized). 

Parametric data (including body weight gain and food consumption) were analyzed using a univariate 

Analysis of Variance (ANOVA) and if significant differences were observed, a Dunnett's Test was 

performed. Nonparametric data (e.g., number of estrous cycles, litter size, and number of implantation 

sites) were first analyzed by the Kruskal-Wallis test and then by the Dunn's Test if significant differences 

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 questionable distribution, by statistical analysis using the 

Chi-square and Fisher's exact tests. Differences between 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 indices were calculated from breeding and 

parturition records of animals in the study: 

100

No. of females co-housed

No.of inseminated females Mating Index (%) 

a

= ×

100

No.of inseminate dfemales

No. of pregnant females FertilityIndex (%)

b

= ×

100

No. of pregnant females

No. of females with live pups Gestation Index (%) = ×

a

Includes pregnant females not observed sperm positive or with an internal vaginal plug. 

b

 Includes females which did not deliver, but had implantation sites. 

Offspring viability indices: The following viability indices were calculated from lactation records of litters in the 

study: 

100

Total No. of implantation sites/litter

Total No. of pups born/litter Birth Index (%) = ×

100

Total No. of pups/litter

No.of live pups born/litter Livebirth Index (%) = ×

100

No. of live pups born/litter

No.of live pups/litter on day 4 (pre-culling) Viability Index (%) = ×

100

No. of live pups/litter on day4 (post-culing)

No. of live pups/litter on day 21 Lactation Index (%) = ×

3. Historical control data: Historical control data were provided in Attachment IV of the study report. This 

data were obtained from reproduction studies performed in this laboratory (1998-2004) in the Wistar rat. 

II RESULTS 

Findings: 

a) Parental toxicity:

General observations: There were no treatment-related mortalities or clinical signs observed during the 

course of the study. Toxicologically significant effects on feed intake were determined at 6,000 ppm in 

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 6-80. 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 F1-generation, the males of the 6,000 ppm group exhibited 

statistically significant declines in body weight (mean of 6%) from days 14-70 and declines in overall

body weight gain (8%). In F1-generation females of the 6,000 ppm group, statistically significant declines 

in body weight (6%, week 10 of premating) and declines 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 F1-generation females of the 6,000 ppm 

group which was accompanied by statistically significantly lower body weight means (7%) from day 0-20 

of gestation. 

Lactation: P-generation females of the 6,000 ppm group showed statistically significant declines in food 

consumption on both a g/kg/day (9-14%) and g/animal/day (13.4%) basis. In the F1-generation, females 

of the 6,000 ppm group exhibited statistically significant body weight declines (6-8%) throughout 

lactation, as well as significantly declined food consumption (lactation days 7-21) on both a g/kg/day (8-

9%) and g/animal/day (13.9%) basis. Statistically significant 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 absence 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) EnEndpoint 

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 

F1 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 

F1 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) 

F1 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) 

F1 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 Tables 2, 8, and 13, pp. 54-61, 84-85, and 99-100, respectively, Report No. 201426-1 
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a

Premating weight gain calculated from group mean values; not analyzed statistically. 

bNumber in parentheses is percent difference from control. 

* = p < 0.05,** = p < 0.01 

Table B.6-79: Mean food consumption by females during 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) 

F1 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 

aNumber in parentheses is percent difference from control. 

*=p < 0.05, ** = p < 0.01 

Table B.6-80: Average compound intake (mg/kg/day) 

Phase of Study 250 ppma 1,000 ppma 6,000 ppma

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 (F1 -gen) - Male 19.3 79.5 486.7 

Premating (F1 -gen) - Female 21.7 90.3 539.5 

Gestation (F1 -gen) - Female 17.8 69.8 434.7 

Lactation (F1 -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 weights: No treatment-related macroscopic changes were observed at 

necropsy in P- and F1-generation rats. Significant declines in terminal body weight were observed in 

both F1 generation males and females of the 6,000 ppm group. There were various organ weight 

changes which were statistically significantly different from controls (testes, epididymis, spleen, thyroid). 

Those findings were considered to be unrelated to treatment as most of those statistically different organ 

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 correlated with a lower terminal body weight with no effects 

on relative organ weight (Table 6-81). Decreases in absolute and/or relative kidney weight at 6000 ppm 

in F1 generation males and females were considered toxicologically significant in the presence of 

histopathology of the kidney. 
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Table B.6-81: Mean absolute (g) and relative (%) organ weights 

Dose Group (ppm) Parameter 0 250 1,000 6,000 

F1 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 

F1 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 Tables OW1K-SUM and OW2K-SUM, pp. 491-501 and 503-513, respectively, Pathology Report, 

Report No. 201426-1 

* = p < 0.05 (ANOVA+Dunnett`s test); *

k

 = p < 0.05 (Kruskal-Wallis ANOVA+Mann-Whitney U-test) 

Histopathology: Histopathology findings considered to be compound-related were confined to the 

kidneys of the F1-generation male and female rats of the 6,000-ppm group (Table 6-82) and the 

epididymis of a single F1 male from the 6,000-ppm dose group. In F1-generation male and female rats of 

the 6,000 ppm group, a minimal to moderate multifocal tubular dilatation was observed in the outer 

portion of the medulla, containing occasional proteinaceous material. In general, both kidneys were 

affected with the severity and incidence being greater in males. 

The epididymis of one single F1-generation male rat (animal number: SJ3514) had a moderate number 

of abnormal sperm within the tubules. Although microscopically there was only one animal with 

abnormal sperm, the sperm were similar in appearance (amorphous sperm heads) to abnormal sperm 

observed microscopically in the males of the 6,000-ppm F1 eight-to-nine week old interim animals and 

the 10,000 ppm P males in the Dose Range-Finding Study (Report No. 201300-1). 

No treatment-related findings were observed in the reproductive tract of females of either generation 

(ovaries, oviducts, uterus, cervix, vagina). Ovarian follicles were counted from control and 6,000-ppm

dose F1-generation females. There was no difference in the number of primordial follicles, antral follicles, 

or corpora lutea in the 6,000-ppm females, when compared to controls. 
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Table B.6-82: Incidence of treatment-related changes in the kidneys from F1 animals 

Dose group (ppm) 

0 250 1,000 6,000 

F1 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 

F1 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-related effects on mean 

cycle length and number of normally cycling females in both P- and F1-generation females at any 

dietary level tested. 

In P-generation males, sperm motility (percent motile 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 and 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 testicular weight or testicular sperm count and 

similar findings were not observed in the second generation. 

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 

F1 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 F1-generation males, sperm motility (percent motile and percent progression), total sperm count 

(epididymal and testicular), and morphology were evaluated at all dietary levels (Table 6-83). A 

morphologic effect on sperm, presenting as amorphous 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 fertilizing capabilities for this one animal (i.e. the male

presented 82% total abnormal sperm with 77% of these 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 histopathology). 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 for any parameter 

measured (e.g., mating, fertility or gestation indices, 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 high-dose male with abnormal sperm did not 

become pregnant. Comparison of the number of uterine implantation sites at necropsy with the 

number of delivered pups revealed the absence of any treatment-related effect on post-implantation 

loss in both generations. 

The litter parameters determined at birth (pup weight at birth, total number of pups born, stillborn 

pups, live birth index, percentages of male pups born, mean litter size at birth) were not affected in

both generations. 

d) Neonatal toxicity:

General observations: There were no treatment-related effects on the viability parameters of the 

pups (viability index, lactation index), and no clinical signs and no malformations were observed in 

both F1 and F2 pups. 

Offspring body weight and body weight gain data are given in Table 6-84. Pup weights at birth were 

not affected (F1 and F2). On lactation day 21, the F1 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 F2 pup body weights for both sexes in the 6,000-ppm group were 

significantly less than control (7-8% and 9-11%, respectively). The pup body weight gains for 6,000-

ppm males and females were statistically decreased (12-14%) during days 4-21 of lactation. 
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Table B.6-84: Mean body weight and body weight gain (g ± S.E.) of the F1 and F2 pups during lactation 

Lactation 

Day 0 ppm 250 ppm 1000 ppm 6000 ppm 0 ppm 250 ppm 1000 ppm 6000 ppm 

F1 Pups – male F1 Pups – female 

0 6.0 ± 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 ± 0.05 4 

(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 ± 0.16 

7 14.8 ± 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 ± 0.25 

14 

30.4 ± 0.65 30.9 ± 0.57 30.9 ± 0.50 28.9 ± 0.46 29.6 ± 0.61 30.5 ± 0.46 30.0 ± 0.50 27.7* ± 0.41 (-

6)a

21 

46.1 ± 0.96 47.5 ± 0.90 47.1 ± 0.89 42.5** ± 0.55 (-

8) 

44.3 ± 0.93 46.4 ± 0.75 44.7 ± 0.91 40.6** ± 0.55 (-

8) 

Wt gain days 0-4 3.3 ± 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 ± 0.13 Wt gain days 4-7 5.5 ± 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 ± 0.13 Wt gain days 7-14 15.6 ± 0.32 15.9 ± 0.36 15.8 ± 0.27 14.5* ± 0.29 (-

7) 15.3 ± 0.29 15.8 ± 0.35 15.4 ± 0.28 14.1* ± 0.31 (-8) 

Wt gain days 14-21 15.7 ± 0.51 16.6 ± 0.44 16.3 ± 0.49 13.6** ± 0.25 (-

13) 

14.7 ± 0.47 15.9 ± 0.39 14.7 ± 0.48 12.9** ± 0.23 (-

12) 

Wt gain days 4-21 36.8 ± 0.78 38.2 ± 0.82 37.7 ± 0.72 33.5** ± 0.46 (-

9) 

35.4 ± 0.74 37.2 ± 0.71 35.6 ± 0.76 32.1** ± 0.48 (-

9) 

F2 Pups – male F2 Pups – female 

0 6.0 ± 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 ± 0.11 4 

(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 ± 0.26 

7 15.8 ± 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 ± 0.38 

14 

31.4 ± 0.41 30.9 ± 0.63 31.3 ± 0.70 29.0** ± 0.63 (-

8) 

30.2 ± 0.37 30.2 ± 0.60 30.8 ± 0.69 28.0** ± 0.59 (-

7) 

21 

47.4 ± 0.60 46.5 ± 0.91 46.0 ± 0.88 42.4** ± 0.86 (-

11) 

45.3 ± 0.58 45.2 ± 0.92 45.0 ± 0.94 41.0** ± 0.78 (-

9) 

Wt gain days 0-4 4.0 ± 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 ± 0.17 Wt gain days 4-7 5.9 ± 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 (-
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10) 12) 

Wt gain days 7-14 15.5 ± 0.26 15.7 ± 0.33 15.9 ± 0.38 13.9** ± 0.35 (-

10) 

15.0 ± 0.23 15.5 ± 0.31 15.8 ± 0.35 13.6** ± 0.35 (-

9) 

Wt gain days 14-21 16.4 ± 0.35 15.6 ± 0.33 14.7** ± 0.36 (-

10) 

13.4** ± 0.37 (-

18) 

15.1 ± 0.31 15.0 ± 0.41 14.2 ± 0.36 13.0** ± 0.37 (-

14) 

Wt gain days 4-21 37.8 ± 0.48 37.0 ± 0.73 36.4 ± 0.71 32.5** ± 0.65 (-

14) 

35.8 ± 0.46 36.0 ± 0.76 35.5 ± 0.74 31.6** ± 0.58 (-

12) 

Data taken from Tables 18 and 19, pp. 116-121 and 123-128, respectively, Report No. 201426-1 a Number in parentheses is percent difference from control. 

 *=p<0.05, **=p<0.01.
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Gross pathology, organ weights: In F1 and F2 weanlings no treatment-related macroscopic alterations 

were observed at any dietary level tested. Absolute brain weights were statistically significantly 

decreased (F2 males, F2 combined sexes) and relative brain weights were increased (F1 and F2 males 

and females; F1 and F2 combined sexes) at 6,000 ppm (Table 6-85). At 6,000 ppm, absolute spleen 

weights were decreased in combined F1 weanlings and in F2 male and female weanlings. F2 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 secondary to the lower mean body weights of the 

6,000 ppm pup dose group. 

Table B.6-85: Mean absolute (g) and relative (%) organ weights of weanlings 

Dose Group (ppm) Parameter 0 250 1,000 6,000 

F1 male+female weanlings (combined) 

Brain weight, absolute 1.454 ± 0.0132 1.449 ± 0.0123 1.465 ± 0.0108 1.421 ± 0.0114 

Brain weight, relative 3.252 ± 0.0524 3.096 ± 0.0487 3.231 ± 0.0565 3.444** ± 0.0332 

Spleen weight, absolute 0.205 ± 0.0057 0.229 ± 0.0096 0.219 ± 0.0081 0.185* ± 0.0059 

Spleen weight, relative 0.454 ± 0.0075 0.483 ± 0.0150 0.476 ± 0.0112 0.445 ± 0.0114 

F2 male+female weanlings (combined) 

Brain weight, absolute 1.479 ± 0.0084 1.467 ± 0.0098 1.474 ± 0.0099 1.444* ± 0.0105 

Brain weight, relative 3.194 ± 0.0380 3.233 ± 0.0516 3.274 ± 0.0622 3.491** ± 0.0508 

Spleen weight, absolute 0.200 ± 0.0049 0.204 ± 0.0066 0.203 ± 0.0087 0.160** ± 0.0056 

Spleen weight, relative 0.430 ± 0.0084 0.445 ± 0.0093 0.443 ± 0.0114 0.382** ± 0.0078 

Thymus weight, absolute 0.214 ± 0.0050 0.207 ± 0.0062 0.202 ± 0.0056 0.187** ± 0.0055 

Thymus weight, relative 0.459 ± 0.0084 0.453 ± 0.0096 0.445 ± 0.0092 0.450 ± 0.0101 

Data taken from Tables OW3K-SUM, pp. 515-519, Pathology Report, Report No. 201426-1 

* = p < 0.05; ** = p < 0.01 

Histopathology: There were no compound-related microscopic findings observed in F1 and F2

weanlings of either sex at any dietary level tested. 

Developmental milestones: Maturation of external sexual organs (balanopreputial separation, vaginal 

opening) was not affected in F1 weanlings. Anogenital distance was not triggered to be measured in this 

study. 

III. CONCLUSION: 

The NOAEL for male parental toxicity was established 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 F1-generation males, respectively), based on body weight 

gain declines in P-generation males at 6,000 ppm (equal to 419.3 mg/kg bw/day) and body weight gain 

declines, decreased terminal body weights and increased renal multifocal tubular dilatation in F1-

generation males at 6,000 ppm (equal to 486.7 mg/kg bw/day). 

The NOAEL for female parental toxicity was established 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 F1-generation females, respectively), based on reduced 

food consumption during lactation in P-generation females at 6,000 ppm(equal to 484.7 mg/kg bw/day) 

and reduced food consumption during lactation, reduced body weights (end premating), body weight 

gain declines (premating), decreased terminal body weights and increased renal multifocal tubular 

dilatation in F1-generation females at 6,000 ppm (equal to 539.5 mg/kg bw/day). 
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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 F1 generation males), based on abnormal sperm cell morphology in F1-

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 abnormal spermatozoa leading to compromised 

fertilizing capabilities and other animals (9/30) showing one to four amorphous sperm heads out of 200

sperm cells viewed. Reproductive performance was not 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- or F1-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 gestation duration, mating performance, vaginal opening, 

litter parameters determined at birth (pup weight, total number of pups born, stillborn pups, viability 

index on lactation day 0, sex ratio, mean litter size). 

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 F1 pups and decreased body weights on lactation 

days 14 and 21 and decreased body weight gain in F2 pups at 6,000 ppm. Pup weight declines 

corresponded with maternal body weight and food consumption declines. 

Reviewer’s Conclusions: Systemic toxicity in parental animals was evident at 6000 ppm as decreases 

in body weight and body weight gain in P-generation males and F1-generation males and females, 

kidney histopathology, and decreased kidney weight in the F1 males and females. Reproductive 

performance was affected in one F1 male that had an increase in abnormal sperm and reduced fertility 

at 6000 ppm. Offspring survival was not affected by treatment. However, F1 and F2 pup growth was 

decreased during lactation at 6000 ppm. 

The parental systemic 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, respectively) based on decreases in body weight 

(F1 males and females), weight gain (P males, F1 males and females), and kidney histopathology 

and decreased kidney weights (F1 males and females). The parental systemic NOAEL is 1000 

ppm (70.7 and 82.5 mg/kg bw/day in males and females, respectively).

The reproductive toxicity LOAEL for BYI 08330 in male Wistar rats is 6000 ppm (419.3 mg/kg 

bw/day in males) based on abnormal sperm cells and decreased fertility in the F1 males. The 

reproductive toxicity NOAEL in males is 1000 ppm (70.7 mg/kg bw/day in males). The 

reproductive toxicity LOAEL was not observed in females; 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, respectively) based on decreased body weight 

and body weight gain during lactation in both F1 and F2 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 conduct 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, Toxicology, 17745 South Metcalf, Stilwell, 

KS 66085-9104. Report Number 201300-1, July 12, 2006. 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 (~22o

) 

Chemical Name: 3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-yl 

carbonate; Spirotetramat 

 CAS No. of TGAI: Non-stereo: 382608-10-8; cis: 203313-25-1 

2. Vehicle and/or positive control: None. Compound was dry mixed and administered through 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 veterinarian 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 F1 pups) in 

suspended stainless steel cages and deotized cage board 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 25oC 

Relative humidity 30 to 70% 

 Environmental 

conditions: 

Air changes: 

Photoperiod:

Minimum of 10 air changes per hour, based on average 

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 approved by the Institution’s Animal Care and Use 

Committee (IACUC); and during the course of the study, 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 outlined in the “Certification Profile” for Purina Mills 
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Certified Lab Chows (PMI Nutrition International, 1998) 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

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 inseminated were placed in a polycarbonate nesting 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 without exhibiting sperm in the 

vaginal smear or an internal vaginal plug, all remaining 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 one 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 entire in-life phase of the study. The parental animals 

(P-generation) were given test diets for 10 weeks prior to mating. Selection of F1-pups to evaluate 

preputial separation and vaginal patency, as well as sperm analysis, was made when the pups were 21 

days of age. The F1-generation male pups were maintained until 8-9 weeks 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 were 

examined by a veterinarian and released for study use. The animals were then assigned to either a 

control or one of four chemically-treated groups using 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, 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, was attached to the outside of each animal’s cage. Pups born alive were identified 

by tattoo and pups found dead were identified with a marking pen. All animals not placed on study (for 

whatever reason) were ultimately 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. 
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Table B.6-86: Animal assignment and study design 

Animals/group 

Test Group Dose in Dieta

(ppm) P Males P Females F1 Males 

Day 21/Days 59-64 

F1 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. 201300-1 

a

 Diets were administered from beginning of the study until sacrifice. 

4. Dose selection rationale: Doses were based principally upon the toxicological 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 AT01512). It was anticipated that by using such a 

broad range of doses that a more definitive toxicological profile would emerge in the animal that would 

allow the selection of appropriate doses at which to 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 feed. Treated diet 

was mixed at room temperature; aliquots of the chemical were taken from the original test batch and 

transferred to the mixing area. The control test diet was taken directly from the bag without mixing. A 

sample of each batch of feed mixed was taken and retained in the freezer until the study was complete 

and the analytical data deemed satisfactory. Replacement admixtures for each treatment group were 

prepared every two weeks during the 10-week premating period and weekly (or at greater intervals 

depending on freezer stability) thereafter and stored under freezer conditions until presented to the 

animals the following week (or weeks). Additionally, the entire batch of replacement admixture for each 

treatment group was used for subsequent weekly feedings. 

The concentration of BYI 08330 in the various test diets was analytically verified for batches intended for 

weeks 1, 2/3, 4/5, 6/9, and 10/14/16/20 (Bayer Cropscience LP, Environmental Research, Plant and 

Animal Residue Group, 17745 S. Metcalf, Stilwell, KS). The homogeneity and stability of BYI 08330 

when mixed in the rodent feed was characterized prior to study initiation. 

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, 150, or 10,000 ppm, were determined to be 68.7 

ppm (range 65.0-73.1 ppm; CV = 3.2%), 144 ppm (range 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 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 the 70-, 150-, or 10,000-ppm admixture 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-determined concentration of the AI of the test substances in the 
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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 storage 

for a minimum of 28 days, over a concentration range 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 the 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 performed twice 

daily (AM and PM), during the work week and once daily on weekends and holidays with exceptions 

stated in the Protocol Amendments and Deviations section. 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 clinical signs includes both observing the animal in the cage and removing the animal to

perform a physical examination and 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 and 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 the 

mating period, fresh feed was provided for both males and unmated females once/week but food 

consumption was not measured. During gestation, dam body weights were measured on GD 0, 6, 13, 

and 20, and fresh feed was provided and food consumption measured once/week. During lactation, dam 

body weights were measured on LD 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 measured twice (LD 

0-4 and 4-7). 

Estrous cyclicity: Estrous cycling was not performed in this one-generation study. 

Sperm parameters: For all males at termination, sperm was collected from one testis and one 

epididymis for enumeration of homogenization-resistant 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 the urethra) of the vas deferens. Sperm motility and 

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. 
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Time of observation (lactation day) 

Observation 

Day 0 Day 4a Day 4b

 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 Deviations section 

The size of each litter was adjusted on lactation Day 4 to yield, as closely as possible, four males and 

four females per litter. If the number of male or female pups was less than four, a partial adjustment was 

made (e.g., three females and five males). 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 gross 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 sufficient number of F1 pups/sex/litter were maintained and 

observed for vaginal opening and preputial separation. 

The F1-males were maintained for an additional 5 to 6 weeks prior to performing sperm analysis (motility, 

counts, and morphology). F1-pups not selected for further evaluations were sacrificed and examined 

macroscopically for any structural abnormalities or pathological changes, particularly as they may relate 

to the organs of the reproductive system. 

Dead pups were examined grossly for external and internal 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 soon as possible after the last litters 

were produced. Maternal animals were sacrificed following the weaning of their respective litters 

(lactation day 21). These animals were subject to examinations as follows. 

Male rats were sacrificed by carbon dioxide asphyxiation, terminal body weights were taken, and a gross 

external examination was performed. For all males at termination, sperm was collected from one testis

and one epididymis for enumeration of homogenization-resistant 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 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 asphyxiation and a gross external examination was 

performed. Terminal body weights were taken and the abdomen and thoracic cavities were opened and 
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a gross internal examination was performed. The 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 which were never observed as being inseminated and/or with

an internal vaginal plug and did not deliver at least 24 days after the completion of the mating phase, 

were sacrificed and necropsied. A gross necropsy was performed on these animals as described above. 

In addition, patency of the cervical/uterine os in these females was examined via flushing of the uterine 

horns with deionized water. 

The following tissues were collected and weighed (XX). 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 sacrificed 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: Approximately half of the F1 offspring were sacrificed at 21 days of age. These animals 

were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows. 

The following tissues from 21-day weanlings were collected and weighed: Brain, thymus, spleen, and 

uterus. Any gross lesion was documented and collected. Exceptions are stated in the Protocol 

Amendments and Deviations section. 

At 21-days of age, a sufficient number of F1 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 condition underwent a gross necropsy for possible defects 

and/or cause of death. 

D. DATA ANALYSIS:

1. Statistical analyses: The data were analyzed using applications provided by DATATOX (Instem 

Computer Systems), SAS (SAS Institute, Inc.), or TASC (Toxicology Analysis Systems Customized). 

Parametric data (including body weight gain and food consumption) were analyzed using a univariate 

Analysis of Variance (ANOVA) and if significant differences were observed, a Dunnett's Test was 

performed. Nonparametric data (e.g., number of estrous cycles, litter size, and number of implantation 

sites) were first analyzed by the Kruskal-Wallis test and then by the Dunn's Test if significant differences 

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 questionable distribution, by statistical analysis using the 
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Chi-square and Fisher's exact tests. Differences between 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 calculated from breeding and parturition 

records of animals in the study: 

100

No.of femalesco -housed

No. inseminated females Mating Index (%)

a

= ×

100

No. of inseminated females

 b No. pregnant females

Fertility Index (%) = ×

 100

No of pregnant females

No. of females with live pups Gestation Index (%) = ×

a

Includes pregnant females not observed sperm positive or with an internal vaginal plug. 

b

 Includes females which did not deliver, but had implantation sites. 

Offspring viability indices: The following viability indices were calculated from lactation records of litters in 

the study: 

 100

Total No. of implantati on sites/litter

Total No. pups born/litte r

Birth Index (%) = ×

100

Total No. of pups/litter

No. of live pups born/litter Livebirth Index (%) = ×

100

No. of live pups born/litter

No. of live pups/liter on day4 (pre-culling ) Viability Index (%) = ×

100

No. of live pups/litter on day 4 (post-culling)

No. of pups/litter on day 21 Lactation Index (%) = ×

3. Historical control data: Historical control data were provided in Attachment IV of the study report. This 

data were obtained from reproduction studies performed 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 clinical 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 weight gain, or food consumption in parental animals 

up to 500 ppm (Table 6-87). At 10,000 ppm, decreased mean premating body weight gain (NSS) 

was observed in male (10%) and female (12%) parental animals. Females showed nonsignificant 

body weight gain declines at 6,000 ppm during premating (11%) and lactation (12%). Females of the 

10,000 ppm group were sacrificed after the gestation phase due to non-pregnancy (see below). The 
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mean daily intake of the test substance (mg BYI 08330/kg bw/day) throughout this one-generation 

reproduction study at nominal dietary concentrations 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 body 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 ± 8.26 438.3 ± 7.76 431.4 ± 7.99 

BW gainb – 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 gainb – 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 

bWeight gain values were calculated from group means. 

cNumber in parenthesis is percent difference from control. 

Table B.6-88: Mean test article intake 

Phase of Study 200 ppm in 

mg/kg/daya

500 ppm in 

mg/kg/daya

6,000 ppm in 

mg/kg/daya

10,000 ppm in 

mg/kg/daya

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 NAb

Lactation (P-gen) - Female 26.7 74.4 831.0 NAb

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 group. 

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 well 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 findings resulted in no pregnancies at this dietary 

level. Sperm analysis revealed no effects in P generation males at 6,000 ppm and below. 

In F1 eight-to-nine week old interim males, slight declines 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 were no sperm effects observed in F1 eight-tonine week old interim animals at 500 ppm and below.
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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. 201300-1 

a = no pups at this dietary level 

c Reproductive performance: The 10,000 ppm dietary level was associated with no fertility in the P 

generation animals. There were no implantation sites noted in the females at this same level. At the 200-

, 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 insemination, gestation length, or the median number of 

implants). The litter parameters determined at birth (litter size, percentage of males born, birth index, live 

birth index, viability index, lactation index), were also not affected. 

Gross pathology, organ weights: No treatment-related macroscopic changes were observed at 

necropsy in P generation rats. Absolute and relative weights of the cauda epididymis were decreased by 

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 and 

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 femalesa 

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 4.394±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. 309-313 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 the spermatozoa. Severity grade consisted of minimal to 

moderate change. There was no evidence of change in the epithelium lining the seminiferous tubules of

the testes in 10,000 ppm generation males. There were 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 malformations were observed in F1 

pups. Pup weights at birth were not affected. At 6,000 ppm, significant declines in offspring body weight 

(14%) were noted at lactation day 21 with a total decline 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 

dNumber in parentheses is percent difference from control. 

* = 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 differences in terminal body weights or absolute and relative 

organ weights were noted at any dietary level tested. In F1 eight-to-nine week old males, a significant 

decline (8%) in terminal body weight was observed at 6,000 ppm (Table 6-92). There were no 

statistically significant effects observed on the organs 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 ± 8.8 257.0* ± 17.1 

(8) 

- 

Data taken from Table OW1K-SUM, p. 311, Pathology Report, Report No. 201300-1. 

a

 = no pups at this dietary level; * = p < 0.05; s.d. = standard deviation 
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Histopathology: In F1 weanlings the testis and epididymis of the control 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). Severity grade consisted of minimal or moderate 

change. A single control animal had a moderate number of abnormal sperm within the epididymis and 

cauda epididymis. There was no evidence of change in the epithelium lining the seminiferous tubules of

the testes in F1 eight-to-nine week old interim animals in the 6,000 ppm group. There were no abnormal

sperm cells observed in F1 eight-to-nine week old interim animals at 500 ppm and below. 

Sexual maturation: There were no treatment-related findings observed in the F1 generation on either 

preputial separation or vaginal patency at any dietary 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 reduced cauda epididymal weights and effects on 

sperm at 10,000 ppm (equal to 538 mg/kg bw/day). The 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 sperm 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 pilot study was established at 6,000 ppm in Pgeneration males (equal to premating doses of 320 mg/kg bw/day), based on decreased sperm motility 

and progression, decreased epididymal counts, and increase in abnormal sperm in the epididymis and 

the cauda epididymis in P-generation males at 10,000 ppm (equal to 538 mg/kg bw/day). Other than 

the lack of pregnancies in the 10,000 ppm dose group, considered to be attributed to the effects on the 

sperm of the males, there were no reproductive effects observed in the females during the course of this 

pilot study. 

The NOAEL for offspring toxicity in this pilot study 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 F1 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 F1 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 epididymis at 6,000 ppm. 

Reviewers’ Conclusion: Systemic toxicity was limited to body weight gain decreases in the F1-

generation animals. The target organ is the testes with progressive effects over successive 

generations: the F1 males were affected at a lower dose than the P-generation males. Adverse effects 

on the testes (sperm) resulted in decreased reproductive performance (fertility). 
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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, respectively) based on decreased body weight gain (P 

females) and terminal body weight (F1 males). The parental systemic NOAEL is 500 ppm (27.8 and 

31.4 mg/kg bw/day in males and females, respectively). 

The reproductive 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, respectively) based on decreased sperm motility and 

progression and increased abnormal sperm cells in the F1 males. The reproductive toxicity NOAEL is 

500 ppm (27.8 and 31.4 mg/kg bw/day in males and females, 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, respectively) based on decreased body weight and body 

weight gain during lactation in F1 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 conduct of this study. In the Pathology Report, the 

histopathology summary table for the P-generation males is incorrect. Results were verified from 

individual animal data. 
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B.6.6.3 Teratogenicity test by the oral route in the rat (OECD Annex IIA 5.6.10) 

CITATION: Klaus, A.M. (2004). BYI 08330: Developmental toxicity study in rats after oral administration. 

Bayer HealthCare AG (Wuppertal, Germany). Laboratory report number AT01413, August 

23, 2004. Unpublished. 

 Klaus, A.M. (2001). BYI 08330: Pilot study on developmental toxicity in rats after oral 

administration. Bayer HealthCare AG (Wuppertal, Germany). Laboratory report number 

T3068559, May 8, 2001. Unpublished. 

 Klaus, A.M. (2004). BYI 08330: Supplementary developmental toxicity study in rats after oral 

administration. Bayer HealthCare AG (Wuppertal, Germany). 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 demineralized 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-No.: 3883.0.15 

ad libitum 

Water: Tap water ad libitum 

Environmental conditions: Temperatur

e: 

Humidity: 

Air 

changes: 

Photoperiod

21± 2oC 

approx. 50% 

at least 10/hr 

12 hrs dark/12 hrs light 
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: 

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 weighing more than 300 grams. Males 

were the same strain and source as females. The animals were paired by placing two females 

overnight into a Type III cage together with one male rat. If sperm was detected in 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 were assigned to their respective 

study group according to a computer-generated randomization 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 1000 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, body weight loss, impaired body weight gain, in- or 

decreased water intake, increased urination, light colored feces). Effects on intrauterine development 

could not be completely excluded at the 200 mg/kg bw/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, necrotic placental borders, retarded ossification, wavy and 

14th ribs and possibly marginally increased incidence of common malformations). 

5. Dosage preparation and analysis: Test material-vehicle suspensions for each concentration were 

prepared after 2 to 7 days of use by mixing appropriate 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 aqueous 0.5% carboxymethyl-cellulose was evaluated for a

period of 8 days at room temperature. Concentration 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 the 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 concentrations as well as checks on homogeneity of the 

low and high concentrations were carried out during the in-life period of the study (formulations prepared 

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 the 

nominal value in the formulations of any of the treatment 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 accepted range, the content of this formulation was 
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confirmed and only 7 females were treated with this formulation for 3 or 4 days at the end of gestation. 

Thus, an impact of this finding on the outcome of the 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 storage 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 procedure 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 weight. 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 gestation 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 which remained unconsumed. Dams were sacrificed on 

GD 20 using cardiotomy under deep carbon dioxide anesthesia. Examinations at sacrifice consisted of a

gross pathological evaluation. Each uterus was weighed and dissected; weights and appearance of 

placentas were recorded. The number of corpora lutea and implantations were determined along with 

early resorptions, late resorptions, and live and dead fetuses. In females without visible implantation 

sites, the number of implantations was determined after 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 abnormalities. Every other fetus was assigned to either the 

visceral or skeletal examinations. In the visceral examination, the fetus was sectioned according to the 

modified Wilson technique and the organs in the thoracic, abdominal and pelvic regions were evaluated 

for abnormalities. The fetuses included in the skeletal examination were eviscerated, treated with the 

cartilage staining technique of Inouye, using alcian blue GX, cleared with a dilute potassium hydroxide 

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 weight 

gain and corrected body weight gain), uterine weight, number of corpora lutea per female, number of 

implantations per female, number of live fetuses per female and as a percentage of implantations per 

female, placental weights per female and fetal weights per female. The Chi square test was used to 

evaluate fertility and gestation rates, number of implantations per group, number of preimplantation 

losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses 

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 group, number of placentas with findings or litters with 

placental findings per group, number of fetuses or litters with external, visceral or skeletal findings, and 

number of fetuses or litters with malformations. If a significant difference existed, Fisher’s exact test with 

the Bonferroni correction was used for pairwise comparison. The Chi squared test also was used to 

evaluate the number of fetuses or litters with visceral or cartilaginous tissue observations. The KruskalWallis test was used to evaluate the number of preimplantation losses per female, the number of 

postimplantation losses, early resorptions, late resorptions, or dead fetuses per female, number of male 

or female fetuses or fetuses of undeterminable sex per female, and the proportion of placental, fetal 

external or visceral findings and fetal malformations and external deviations per female. If a significant 

difference existed, Dunn’s test was used for pairwise comparisons. Females without implantation sites

or females which died or were sacrificed in moribund conditions were excluded from any statistical 

evaluation. Differences were considered significant when p<0.05. 

2. Indices: The following indices were calculated from cesarean section data of study animals: 

Corrected body weight gain = Body weight gain GD0-20 - uterus weightGD20 
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100

No. of corpora lutea

No. of corpora lutea - No. of implantations Pr eimplantation loss = ×

100

No. of implantations

No. of late resorptions No. of dead fetuses Post implantation loss ×+

=

100

Total No. of fetuses

No. of male fetuses Fetal sex ratio = ×

3. Historical control data: Historical control data from studies conducted between 1987 and 2003 were 

cited for comparison with concurrent study data, but were not provided for review. 

II. RESULTS:

A. MATERNAL TOXICITY:

1. Mortality and clinical observations: Mortality, appearance and behavior were not affected 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 group, although the discoloration may be related to 

the high amount of white colored test substance given. A single female of the 140 mg/kg bw/day group 

(no.78) showed intermittently (GD 9 to 15) sunken flanks, piloerection, severely reduced food intake and 

no or reduced amount of feces. Due to the lack a dose relationship and single transient appearance, 

treatment relationship was not assumed for these findings. 

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 final 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 reduced 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 controls, p<0.05) was reduced which resulted in a 

statistically significantly reduced final body weights (5% decrease relative to of control, p<0.05). These 

findings were possibly both related to incidentally lower litter size in the 140 mg/kg bw/day dose group 

and to incidental morbidity of female no.78 (unrelated 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 weights when this female was excluded from 

calculations. 

A supplementary oral developmental toxicity study with BYI 08330 in Wistar rats (Lab Report No. 

AT01512; see Appendix 2) at dose levels of 0, 10, 35 and 140 mg/kg bw/day revealed no effects on 

body weight development at dosages up to and including 140 mg/kg bw/day. Body weight gain during 

treatment (GD 6-19) (corrected body weight gain from 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 also unaffected in the rat developmental toxicity rangefinding 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 main study. Necropsy revealed no treatment related 

findings at a dose level up to and including 1000 mg/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 weight 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 control. 

** Statistically different (p <0.01) from the control. 

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, respectively. 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 exhibited distinctly to severely reduced food 

consumption on GD 9-15. This is the same animal that exhibited a decrease in body weight on GD 11-

15. The morbidity of this animal was considered incidental to treatment. 

Table B.6-95: Mean (±S.D.) maternal food consumption (g/animal/day) a

Food Dose in mg/kg bw/day (# of Dams) 

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 (Lab Report AT01413). 

b Numbers in parentheses are percent change relative to control. 

* Statistically different (p <0.05) from the control. 

** Statistically different (p <0.01) from the control. 

4. Gross pathology: No test-substance related gross pathological findings were ascertained in the females 

of the dosed groups at dose levels up to and including 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 fetuses, sex distribution, and placental weight at a dose 
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up to and including 1000 mg/kg bw/day. A marginal reduction of placental weight (not statistically 

significant) together with a more distinct reduction of fetal weight (86% of control, p<0.01) was observed 

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 treatment. Effects on fetal weight were not evident in a 

supplementary oral developmental toxicity study with 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 renders a treatment related effect at the 140 mg/kg 

bw/day dose level in the present study unlikely. Effects on fetal weight were not evident at the 20 mg/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, preimplantation loss and mean number of fetuses 

available for evaluation lay in the range of historical control data. Furthermore, a treatment related effect 

was not evident in the supplementary study (Lab Report No. AT01512; see Appendix 2) at dose levels of 

10, 35, and 140 mg/kg bw/day. Thus, an impact on the outcome of the study due to unequal distribution

of females in the different groups with respect to preimplantation loss and number of implantation sites 

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 report (Lab Report AT01413). 

* Statistically different (p <0.05) from the control. 

** Statistically different (p <0.01) from the control. 
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B. DEVELOPMENTAL TOXICITY: 

Table 6-97 summarizes fetal malformations, categorized as external, visceral, or skeletal. Both the fetal 

and litter incidences of malformations were within or at the upper limit of the historical control incidence 

range (up to 6.9% of fetuses and 40.0% of litters). Although two of the findings are rare observations 

(cleft palate, co-arctation of aortic arch), a specific potential of BYI 08330 to induce these types of 

malformations was not assumed since these findings occurred only once in the 1000 mg/kg bw/day 

group, were observed spontaneously in control or low dose groups of recent developmental toxicity 

studies and were different in type. 

1. External examination: Fetal external malformations included: cleft palate (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 shortened 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 including 1000 mg/kg bw/day. 

2. Visceral examination: Fetal visceral malformations included: co-arctation 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/day: 

1 case). The incidence and type of visceral deviations of fetuses was not affected up to and including 

1000 mg/kg bw/day. Common visceral deviations representing the normal range of scattering for the rat

strain used included findings at the eyes (slightly folded retina), brain (slight dilation of ventricles; dorsal 

brain slightly misshapen), cochlea (blood in cochlea), thyroid (reduction in size), arteries (left common 

carotid artery septated), liver (small additional liver lobe/s), kidneys and ureters (slightly increased renal 

pelvis cavitation, absence of renal papilla, hydroureter), testes (slightly displaced), bladder (slightly 

distended), genital tubercle (slightly elongated) and hemorrhages in different structures. 

T 
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able B.6-97: Malformations a

Observations Dose (mg/kg bw/day) 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 

External malformations (40.9)

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 and 

left subclavian arteries, ascending aorta reduced in 

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 study 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 with generally common unspecific malformations (4.4% 

and 40.9%, respectively) at the maternally toxic 1000 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 supernumerary lumbar vertebra (1000 mg/kg bw/day: 1 

case) and altered appearance of sacral vertebral arches and pelvic shift (1000 mg/kg bw/day: 3 fetuses

in 3 litters). 

 Table 6-98 below lists the skeletal deviations that were statistically significantly different on a fetal and/or 

litter basis. Statistically significantly retarded ossification in various locations (phalanges, sternebrae, 

vertebrae, skull bones) were noted at the 1000 mg/kg bw/day dose level in the osseus and/or 

cartilagineous parts. Increased incidences of skeletal variations (wavy ribs, 14th ribs, combined osseous 

and cartilagineous findings) were seen as well. All these findings can be ascribed to the observed growth 

retardation at this dose level and are considered to 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, together 

with incomplete ossification of the 6th sternal segment and the supraoccipital bone (at 20 mg/kg bw/day 

only). None of these findings were statistically significantly different on a litter basis and all findings were 

within the range of historical control values. Furthermore, the changes of the low- and mid-dose groups 

were poorly correlated with dose. In addition, a supplemental study (Lab Report No. AT01512; see 

Appendix 2) revealed no relevant effects on the degree of ossification at dose levels of 10, 35 and 140 

mg/kg bw/day. Moreover, no statistically significant changes were noted in ossification means of all but 

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 of the supplementary study, a treatment-related effect 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 significantly 

increased on a fetal basis in the 140 and 20 mg/kg bw/day dose group, but not on a litter basis. The fetal 

incidences at 20 and 140 mg/kg bw/day for this skeletal variant (17% and 18%, respectively) were 

slightly outside the historical control incidence ranges of 2.7 – 15.1% (historical control range derived 

from 22 studies performed between 1996 and 2001). However, 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 supplementary study, which revealed no indications for a treatment 

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 bw/day dose groups, respectively], a treatment-related 

effect for the occurrence of wavy ribs in the present 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/Ac N/A N/A N/A 

Incomplete ossification

Distal phalanx digits, right 

 -1st

 

 -2nd

 -3rd

 -4th

 -5th 

Distal phalanx digits, left

 -1st 

 -2nd

 -3rd

 -4th

 -5th 

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 

 -1st 

 -2nd

 -3rd

 -4th

Distal phalanx toes, left 

 -1st 

 -2nd

 -3rd

 -4th

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 

 -2nd

 -3rd

 -4th

 -5th 

 -6th

 

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, 1st  7 (7) 5 (5) 8 (6) 30** (14) 

Sacral vertebral arch(es), right 

 -3rd

 -4th

Sacral vertebral arch(es), left 

 -3rd

 -4th

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 

 -3rd 

 -4th 

Proximal phalanx digits, left 

 -3rd 

 -4th

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 

 -1st 

 -2nd

 -3rd

 -4th

 -5th 

Distal phalanx toes, left 

 -1st

 

 -2nd

 -3rd

 -4th

 -5th

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**) 

14th rib, sum of fetal incidence 19 (10) 17 (12) 17 (11) 67** (19) 

Thoracic vertebral body, dumbbell 

shaped 

 -11th

 

 -12th

 

 -13th 

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 available.

d

 Fetal (litter) incidence; values that are statistically significant on a fetal and litter basis are in bold type. 

* Statistically different (p <0.05) from the control. 

** Statistically different (p <0.01) from the control. 

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS: 

The LOAEL for maternal toxicity was 1000 mg/kg bw/day, 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 marginal reduction of placental weight, distinctly 

decreased fetal weights, slightly increased incidence of common unspecific malformations, increased 

incidence of skeletal variations (wavy ribs, 14th ribs, combined osseous and cartilaginous findings), and 

increased incidence of retarded ossification at 1000 mg/kg bw/day, and the developmental toxicity 

NOAEL was 140 mg/kg bw/day. No evidence for a primary embryotoxic or teratogenic potential of BYI 

08330 was determined. 

B. REVIEWER COMMENTS: 

1. Maternal toxicity: Mortality, appearance, and behavior were not affected 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 weight was decreased 7% relative to control; weight gain

during treatment and throughout gestation was decreased by 20% and 19% relative to control, 

respectively; and corrected body weight gain was decreased 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 corrected body weight gain was not statistically 

significantly affected and effects were not reproduced in the supplementary study (Lab Report AT01512;

see Appendix 2) at dose levels of 10, 35, and 140 mg/kg bw/day, so toxicological relevance at 140 

mg/kg bw/day was not assumed. 

In the range-finding study (Lab Report T3068559), impaired 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% decrease relative to control), GD15-18 (9% decrease 

relative to control), and GD 18-20 (11% decrease relative 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 rats is 1000 mg/kg bw/day, based on impaired 

food consumption, transient body weight loss, impaired body weight gain, and reduced final 

body weight. The maternal NOAEL is 140 mg/kg bw/day. 

2. Developmental toxicity:

a. Deaths/resorptions: There were no fetal deaths. Preimplantation loss (due 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 implantations. However, an effect was not evident in the 

1000 mg/kg bw/day dose group or in dams from the 140 mg/kg bw/day group in the supplementary 

study with BYI 08330 (Lab Report AT01512; see Appendix 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 the supplementary study. In the range-finding 

study (Lab Report T3068559), a marginal reduction of 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 included incomplete 

ossification of the distal phalanx digits and toes, 2nd through 5th sternebra, 1st thoracic vertebral body, 
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3

rd and 4th sacral vertebral arches, and several skull bones (e.g., mandible, and premaxillary, nasal, 

frontal, parietal, interparietal, supraoccipital, temporal, and zygomatic bones). No ossification was 

evident at the 3rd and 4th proximal phalanx digits, 5th metacarpals, distal phalanx toes, and the round 

area of the supraoccipital bone. Further, the incidence of the sum of 14th rib deviations, dumbbell 

shaped 11th, 12th, and 13th thoracic vertebral bodies, and slightly enlarged fontanelle was greater in 

the 1000 mg/kg bw/day dose group. The total incidence 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 10, 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 increased (data not available for review) at dose levels 

of 800 and 1000 mg/kg bw/day. 

Statistically significant effects on ossification also were evident at the 20 and 140 mg/kg bw/day dose 

levels, but there was no dose relationship and, with the exception of incomplete ossification of the 

left 4th distal phalanx digit and the left 2nd distal phalanx toe at 140 mg/kg bw/day, the effects 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 relevance is not assumed since no dose relationship 

was evident in either the main study or the supplementary study. 

d. Malformations: The total incidence of fetal (litter) malformations was higher (not statistically 

significant) in the 1000 mg/kg bw/day dose group, including 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 malformations, and increased incidence of 

skeletal deviations (retarded ossification of skull bones, fore- and hindlimbs, sternum and 

vertebral column; wavy ribs, 14th ribs, and combined osseous and cartilaginous findings). 

The developmental NOAEL is 140 mg/kg bw/day.

C. STUDY DEFICIENCIES: 

A minor deficiency was that, the total number of fetuses (litters) affected by skeletal deviations could 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 marginally out of the accepted range (RSD 11.82% 

versus limit value of 10%) during the 2nd content check. However, this deviation lay only marginally 

above the accepted range, the content of this formulation was confirmed and only 7 females were 

treated with this formulation for 3 or 4 days at the 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 Study 

CITATION: Klaus, A.M. (2001). BYI 08330: Pilot Study on Developmental Toxicity in Rats After Oral 

Administration. Bayer HealthCare AG (Wuppertal, Germany). Laboratory report number 

T3068559, May 8, 2001. Unpublished. 
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In a range finding oral developmental toxicity study (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 gavage 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 fetuses 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 well as on its effect on intrauterine development 

(including external, skeletal and visceral evaluation of fetuses). 

Maternal toxicity: Treatment related effects on respiration (transiently gasping or labored breathing, 

respiratory sounds), water intake (increased or decreased water intake at the end of treatment), 

excretion (increased urination and light colored feces) and appearance (piloerection) were restricted to 

the 1000 mg/kg bw/day group. Slight body weight loss 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/day group additionally exhibited slightly reduced 

food consumption during treatment and a more pronounced 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 distinctly in the 1000 mg/kg bw/day group. Necropsy 

revealed no treatment related findings at a dose level 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 historical controls. Therefore, the maternal toxicity 

LOAEL was 800 mg/kg bw/day based on body weight loss and impaired body weight gain. The 

maternal toxicity NOAEL was 200 mg/kg bw/day.

Developmental toxicity: Reproductive parameters (i.e. gestation rate, postimplantation loss, litter size 

and fetal sex distribution) were not affected to a toxicologically relevant degree by treatment at a dose 

level up to and including 1000 mg/kg bw/day. A marginal reduction of placental weight was seen at a 

dose level of 800 mg/kg bw/day and above; in the 1000 mg/kg bw/day group together with increased 

incidence of necrotic placental borders and possibly 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 level. Final evaluation of this finding at the 200 and 

800 mg/kg bw/day dose levels was not possible in this pilot study. 

Overall incidence of common unspecific malformations (dysplastic tubular bones, interatrial septal 

defect of the heart) was marginally increased at the distinctly maternal toxic 1000 mg/kg bw/day dose 

level (i.e. 5.5% of fetuses and 35.7% of litters affected) though within the upper range of historical

control data. One case each of dysplastic tubular bones was seen in the other dose groups; at the 800 

mg/kg bw/day dose level together with one case of interatrial septal defect. Since these malformations

are common findings in the rat strain used and their incidence lay within the range of historical control 

data, toxicological relevance was not assumed for these findings up to and including the 800 mg/kg 

bw/day dose level. Final evaluation was not possible at the 1000 mg/kg bw/day level in this pilot study. 

External and visceral fetal evaluation gave no further indication for toxicologically relevant effects up to 

and including 1000 mg/kg bw/day. Retarded fetal ossification together with marginally to slightly 

increased incidence of wavy ribs (variation) occurred at a dose level of 800 mg/kg bw/day and above. 

Incidence of additional 14th ribs (variation) was as well slightly increased at the 1000 mg/kg bw/day 

level. The developmental toxicity LOAEL was 200 mg/kg bw/day 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 dose level of 1000 mg/kg bw/day is suitable for the high 

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 20 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 Study 

CITATION: Klaus, A.M. (2004). BYI 08330: Supplementary developmental toxicity study in rats after oral 

administration. Bayer HealthCare AG (Wuppertal, Germany). Laboratory report number 

AT01512, October 7, 2004. Unpublished. 

A supplementary developmental toxicity study was performed for clarification of results of the main 

developmental toxicity study with BYI 08330 in rats (Lab Report AT01413). In the main study, equivocal 

retarded ossification of single localisations and equivocal increased incidence of wavy ribs were noted in 

the 140 and in the 20 mg/kg bw/day dose group without 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 following exceptions: the liver of the dams were weighed and 

those of the control and high dose groups were subjected 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 parameters (erythrocytes, hemoglobin, hematocrit, mean 

corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular hemoglobin concentration) 

and clinical chemistry parameters (aspartate aminotransferase, alanine aminotransferase, alkaline 

phosphatase, -glutamyltransferase, cholesterol, triglycerides, creatine, urea, calcium, and 

phosphorus). 

The total numbers of fetuses examined (number of litters) 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, sexed, 

and examined for external abnormalities. Every other fetus within a litter was prepared for either 

skeletal or visceral evaluation. During fetal evaluation, it was observed that offspring of one male 

revealed a specific type of malformation (missing head of 1st rib or microphthalmia or both) irrespective 

of dose group, indicating a paternally mediated effect. The litters (fetuses) sired by this male were 

excluded from calculation of the mean values for fetal external, visceral and skeletal findings as well as 

from calculation of mean values for incidence of malformations 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 fetuses (litters) available for evaluation were still 

regarded adequate to fulfil the guideline requirements of approximately 20 litters per group. 

Maternal toxicity: No treatment related maternal effects were observed at any dose level with respect 

to mortality, clinical signs, food consumption, body weight development, hematology and clinical 

chemistry parameters, liver weight, necropsy findings 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 rats 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 effects 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 and type of fetal malformations were unaffected by 
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treatment at levels up to and including 140 mg/kg bw/day. Meaningful fetal external or visceral 

deviations were not evident at a dose level up to and including 140 mg/kg bw/day in this study. Fetal 

skeletal ossification and incidence of skeletal variations including evaluation of cartilaginous structures 

revealed no evidence for treatment related effects at a dose level up to and including 140 mg/kg bw/day. 

The incidence of wavy ribs was not affected at a dose level up to and including 140 mg/kg bw/day. No 

evidence for a primary embryotoxic or teratogenic potential 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 toxicity NOAEL was 140 mg/kg bw/day, based 

on the absence of treatment-related deviations at 140 mg/kg bw/day. 

More detailed results of developmental toxicity are included below: 

Malformations: Table 6-99 summarizes fetal malformations, categorized as external, visceral, or 

skeletal. External, visceral and skeletal evaluation of fetuses revealed no treatment related 

malformations at a dose level up to and including 140 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 fetal and/or litter basis either in comparison to the

concurrent control or to the results of the preceding study. 

Isolated malformations of the eyes (uni- and/or bilateral microphthalmia) was the most common 

malformation in this study (35 mg/kg bw/day: 5 cases; 140 mg/kg bw/day: 1 case), but displayed no 

dose relationship in the actual and in comparison to the preceding study (Lab Report AT01413). Thus 

the incidence of microphthalmia in this study was regarded 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 findings 

due to lack of dose relationship in comparison to the preceding study with dose levels up to 1000 mg/kg 

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 the treated groups, i.e. atrial septal defect of the

heart, displaced azygos vein, right-sided aortic spur and dysplastic forelimb bones were single findings 

without dose response relationship and were comparable to the actual or historical controls so that 

treatment relationship was not evident for these findings. 
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Table B.6-99: Malformations a

Observations Dose (mg/kg bw/day) 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 ossification 

center missing and cartilage bipartite; bone and cartilage 

of 3rd and 5th thoracic vertebral bodies asymmetric 

1 0 0 0 

4

th and 5th thoracic vertebral bodies fused 1 0 0 0 

Dysplasia of forelimb bones (scapula, humerus, radius 

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 no 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 deviations showed considerably lower incidences of wavy ribs at 

all dose levels tested, when compared to the main study. Furthermore all incidences were comparable 

to the control group [13 (8), 12 (5), 19 (6), and 11 (4) fetuses (litters) in the 0, 10, 35, and 140 mg/kg 

bw/day dose groups, respectively] and thus revealed no indication for a treatment related effect. This

also renders a treatment related effect for occurrence 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 bones on a 

fetal basis, scattered across all dose groups tested. However, a relationship to treatment was not 

assumed, based on the lack of reproducibility in almost all cases when compared with the preceding 

study. Moreover, none of the fetal means of the dose 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 treatment 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. Statistically significantly retarded ossification of digits of 

the forelimbs was noted on a fetal basis. The statistically significant findings at the 5th right digit showed 

no statistical significance in the preceding study at a dose level up to and including 140 mg/kg bw/day 

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 4th digit left). Retarded ossification of distal 

phalanges of toes was seen with statistical significance on a fetal basis for distal phalanx of 3rd toe right 

and 2nd and 5th toe left. In the preceding study, only the 2nd 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%). Incomplete ossification of 4th sacral vertebral arch left 

occurred in the 35 and 140 mg/kg bw/day groups with statistical significance on a fetal basis but without 

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 statistically significantly retarded ossification at the 140 

mg/kg bw/day dose level on a fetal basis while ossification 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 statistical 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 punctiform and comma-shaped supernumerary ribs (14th

ribs) were observed in the 140 mg/kg bw/day dose group. However, statistical significance was not 

observed for percentage of overall number of 14th ribs on a fetal or litter basis. The highest overall 

percentage of 14th 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 14th ribs 

was not evident in the preceding study with BYI 08330 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 

 -2nd

 -4th

 -5th 

Distal phalanx digits, left

 -2nd

 -3rd

 -4th

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, 3rd

Distal phalanx toes, left, 2nd

35 (14) 

63 (18) 

55* (19) 

81 (20) 

44 (14) 

75 (20) 

52** (16) 

73* (18) 

Sternebra, 5th 18 (9) 37* (15) 25 (12) 23 (12)

Sacral vertebral arch(es), left, 4th 18 (10) 33 (14) 38* (14) 30* (12) 

Sphenoid bone 1 (1) 8 (7) 4 (4) 9* (6) 

No ossification

Proximal phalanx digits, right, 4th 

Proximal phalanx digits, left, 3rd 

104 (21) 

105 (22) 

114 (22) 

116 (22) 

119 (22) 

126* (22) 

103* (19) 

100 (19) 

Distal phalanx toes, left, 5th 10 (22) 13 (22) 17 (22) 22* (19) 

Wavy ribs, sum of fetal incidence 13 (8) 12 (5) 19 (6) 11 (4) 

14th rib, sum of fetal incidence 20 (12) 18 (9) 21 (8) 31 (9) 

14th rib, punctiform, bilateral 0 3 (2) 1 (1) 8** (3) 

14th 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 available. 

d

 Fetal (litter) incidence.

* Statistically different (p <0.05) from the control. 

** Statistically different (p <0.01) from the control
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B.6.6.4 Teratogenicity test by the oral route in the rabbit (OECD Annex IIA 5.6.11) 

CITATION: Klaus, A.M. (2004). BYI 08330: Developmental toxicity study in rabbits after oral 

administration. Bayer HealthCare AG (Wuppertal, Germany). Laboratory report number 

AT01003, February 17, 2004. Unpublished. 

Holzum, B. (2001). BYI 08330: Pilot developmental toxicity study in rabbits after oral 

administration. Bayer HealthCare AG (Wuppertal, Germany). 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 demineralized 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 ± 2oC 

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 CET. One male rabbit was mated with 

one female rabbit under observation. About one hour after the first mating had occurred the same 

animals were mated again. It was recorded which female was mated with which male. The day on 

which the copulation was observed was considered as day 0 of gestation (GD 0). 
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3. Animal assignment: After copulation, 88 females were sequentially allocated to 4 experimental groups 

according to a computer-generated randomization procedure (see Table 6-101). During the conduct of 

the study, 10 additional females were allocated to the study without randomization: 2 females to the 40 

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 were 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, convulsion, 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 mg/kg bw/day and in one animal of the 250 mg/kg 

bw/day dose level. Necropsy revealed in single animals hardened abdominal fatty tissue, round 

depressions in the gastric mucosa, enlarged gall bladder, or gaseous content in the intestine and 

hemorrhages in the renal capsule. The gestation rate 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 groups the evaluation of the remaining reproduction 

parameters was limited to dose levels up to and including 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 has more detail on the pilot range-finding study. Based 

on these results, it is concluded that the dose level of 160 mg/kg bw/day is suitable for the high dose 

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 middle and low dose levels, respectively. 

6. Dosage preparation and analysis: Test material-vehicle suspensions for each concentration were 

prepared by mixing appropriate amounts of test substance with 0.5% carboxymethyl-cellulose in 

demineralised water with storage at room temperature. Investigations on the stability and homogeneity 

and a content check of the active ingredient in samples of 1.0 and 100 mg/ml revealed no significant 

deviations from the content determined on the day of 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 deviation of the active ingredient content from the nominal 

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 storage 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 detectable - - 

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 detectable - - 

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 procedure 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 weight. 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 each 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 gestation 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 provided and feed which remained unconsumed. 

Dams were sacrificed on GD 29 by intravenous injection of 2 ml T61®

 ad us.vet. (Hoechst Roussel Vet.) 

and subjected to cesarean section and necropsy. Examinations 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 implantations were determined along with early 

resorptions, late resorptions, and live and dead fetuses. In females without visible implantation sites, the 

number of implantations was determined after staining 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 abnormalities, and visceral and skeletal varitions and 

malformations. Fetuses were killed on dry ice. In the visceral examination, the fetuses were sectioned

according to the modified Staples technique and the organs in the thoracic, abdominal and pelvic regions 

and in the brain were evaluated for abnormalities. For skeletal examination, fetuses were eviscerated, 

treated with the cartilage staining technique of Dawson/Inouye, using alcian blue, cleared with a dilute 

potassium hydroxide solution and stained with Alizarin 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 weight 

gain and corrected body weight gain), uterine weight, number of corpora lutea per female, number of 

implantations per female, number of live fetuses per female and as a percentage of implantations per 

female, placental weights per female and fetal weights per female. The Chi square test was used to 

evaluate fertility and gestation rates, number of implantations per group, number of preimplantation 

losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses 

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 group, number of placentas with findings or litters with 

placental findings per group, number of fetuses or litters with external, visceral or skeletal findings, and 

number of fetuses or litters with malformations. If a significant difference existed, Fisher’s exact test with 

the Bonferroni correction was used for pairwise comparison. The Chi square test also was used to 

evaluate the number of fetuses or litters with visceral or cartilaginous tissue observations. The KruskalWallis test was used to evaluate the number of preimplantation losses per female, the number of 

postimplantation losses, early resorptions, late resorptions, or dead fetuses per female, number of male 

or female fetuses or fetuses of undeterminable sex per female, and the proportion of placental, fetal 

external or visceral findings and fetal malformations and external deviations per female. If a significant 

difference existed, Dunn’s test was used for pairwise comparisons. Females without implantation sites

or females which aborted, died or were sacrificed in moribund condition were excluded from any 

statistical evaluation. Cases of skeletal localizations with mechanical damage were excluded from the 

calculation of percentages of affected localizations. Differences were considered significant when 

p<0.05. 

2. Indices: The following indices were calculated from cesarean section data of study animals: 

Corrected body weight gain = Body weight gain GD0-29 - uterus weightGD29 

100

No. of corpora lutea

No. of corpora lutea - No. of implantations Preimplantation loss = ×

100

No. of implantations

No. of late resorption s No. of dead fetuses Post implantation loss ×+

=
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100

Total No. of fetuses

No. of male fetuses Fetal sex ratio = ×

3. Historical control data: Historical control data from studies conducted between 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 maternal toxicity was seen at the 40 mg/kg and 160 

mg/kg dose levels. Death, sacrifice in moribund condition, cold ears, alopecia, altered fecal 

amount/appearance and reddish excretions occurred at the 160 mg/kg level. One female of the 

160 mg/kg group was found dead after having shown reddish excretion and soft feces and 5 females 

had to be sacrificed in moribund condition after having shown severely reduced to no food consumption,

severe body weight loss, cold ears, alopecia, reduced amount or no feces, diarrhea (in one female) and

soft and light colored feces, reddish excretion, decreased 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 females of the 160 mg/kg group and in one female of the 

40 mg/kg group showing clinical signs, impaired food consumption and body weight loss before abortion.

The remaining females of the 160 mg/kg dose group revealed cold ears, alopecia and soft, mucoid and 

light colored feces. A summary of maternal clinical signs is presented in Table 6-102. 

Table B.6-102: Maternal clinical observations during 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 160 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 meaningful treatment related effects. Statistically 

significantly reduced body weight gain during treatment (GD 6-29) and the overall gestation period (GD

0-29) was noted in the 10 mg/kg bw/day dose group which resulted in a marginal body weight loss when 

corrected body weight gain was taken into consideration. Impaired body weight development at the 

10 mg/kg bw/day dose level was regarded as an incidental finding and without relation to treatment due

to lack of dose dependency. Mortality or early sacrifice at the 40 and 160 mg/kg bw/day dose levels did 

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 negative impact of mortality on the evaluation of body 

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 weight 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 report (Lab Report No. AT01003). 

* Statistically different (p <0.05) from the control. 

** Statistically different (p <0.01) from the control. 

3. Food consumption: A statistically significant reduction in food consumption was noted in the 10 mg/kg 

bw/day dose group (GD 27-29). This finding was regarded 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 consumption was observed after start of treatment and/or

later on in females of the 160 mg/kg bw/day group which 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 decreased 19% from GD 18-20 to GD 20-21). The females 

which aborted or were sacrificed in moribund condition 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 sacrificed in moribund condition 

revealed gross pathological findings of the intestine (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/light 

discoloration). Treatment relationship was assumed for these findings. Incidental findings among all 

other females included cysts at the oviduct (0 mg/kg: 3 females; 10 mg/kg: 2 females; 160 mg/kg: 3 

females); whitish punctiform areas in the caecum wall (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 group 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 fetuses 

as well as placental and fetal weight and appearance, and fetal sex distribution. 

Table B.6-104: Cesarean section observations a [mean±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. littersb

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 report (Lab Report No. AT01003). 

b Including only females with viable litters were considered when calculating “per dam” or “per litter” values.

B. DEVELOPMENTAL TOXICITY: 
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Table 6-105 summarizes fetal malformations, categorized as external, visceral or skeletal. Malformations 

were not dose related and/or lay well within the range of normal scattering of the rabbit strain used 

and/or were also seen in the current control. Indeed, the highest total incidence of malformations was in 

the 10 mg/kg group (5.3% of fetuses, 31.8% of litters). 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 including 160 mg/kg bw/day. As some fetuses of the 10 

mg/kg and 160 mg/kg group revealed more than one malformation, the number of malformations is 

higher than the number of affected fetuses in these groups. 

1. External examination: External malformations included malpositioned forelimbs (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 doses 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 summarizes visceral deviations, including a possibly 

increased incidence of fetuses with distinct liver lobulation in the 160 mg/kg group. The toxicological 

significance of this finding was questioned, since the liver is normally lobed and abnormal liver lobulation 

was not reported. Other visceral deviations noted among dose groups included a slight dilation of lateral 

brain ventricles and whitish discoloration of the liver. The findings were comparable either to the current 

controls (slight dilation of brain ventricles) or to historical controls (up to 2.8% of fetuses and 14.3% of 

litters with whitish liver discoloration) and revealed no dose dependency. 

3. Skeletal examination: Skeletal malformations were observed in the cartilaginous 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 5th sternebra in the 10 

mg/kg dose group and of the 8th 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 of 

scattering for these findings in developmental toxicity studies in the strain of rabbits used. A dose 

dependency was not evident for these findings and statistical significance was not seen when calculation 

was done on a litter basis. 
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Table B.6-105: Malformations a,b

Observations Dose (mg/kg bw/day) 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 slight 

thickening of ribs with/without supernumerary 

ossification center above the 1st 

1 3(3) 1 1 

Sternal segment or at 8th rib cervical rib, 

supernumerary ossification center (with cartilage) 

above the 1st 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 report (Lab Report No. AT01003). 

b

 Fetal (litter) incidence. 

c

 Some observations may be grouped together 

Table B.6-106: Visceral deviations a, b

Observations Dose (mg/kg bw/day) 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 report (Lab Report No. AT01003). 

b

 Fetal (litter) incidence. 

c

 Some observations may be grouped together 

** Statistically different (p <0.01) from the control. 

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 consumption 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 
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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 body weight loss, cold ears, alopecia, reduced 

amount/altered appearance of feces, reddish excretion, decreased water intake and 

decreased/discolored urination, although the body weight and food consumption data for these animals 

were not included in the study report for review. Gross pathology of these females revealed treatmentrelated findings of the intestine (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/light discoloration). Body weight 

changes in the remaining females did not show meaningful treatment related effects. 

The oral maternal toxicity LOAEL for BYI 08330 in rabbits 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 viable 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 viable fetuses. 

 c. Developmental variations: Visceral deviations included an increased incidence of fetuses (8 

fetuses in 2 litters) with distinct liver lobulation in the 160 mg/kg group; statistical significance was 

evident on a fetal, but not litter, basis. The toxicological significance of this finding was questioned, 

since the liver is normally lobed and abnormal liver 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 evaluations revealed no treatment related effects at a 

dose level up to and including 160 mg/kg bw/day. Statistically significant retarded ossification of the 

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 considered 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 domed 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 microphthalmia and encephalomeningocele in the 160 

mg/kg dose group. A treatment related effect was not assumed for the malformations which 

appeared only once in the 160 mg/kg dose group (domed head/ encephalomeningocele, cleft palate, 
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and microphthalmia). Skeletal malformations were observed 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 including 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 condition was not provided for 

review. 
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APPENDIX 1: Oral Rabbit Developmental Toxicity Dose Ranging Study 

CITATION: Holzum, B. (2001). BYI 08330: Pilot developmental toxicity study in rabbits after oral 

administration. Bayer HealthCare AG (Wuppertal, Germany). Laboratory report number 

T3062735, November 14, 2001. Unpublished. 

In a range finding oral developmental toxicity study (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 (GD 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 females as well as on its effect on intrauterine 

development (including external, visceral and skeletal 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 consumption, cold ears) and were severe 

(hypoactivity, labored breathing, convulsion, 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 mg/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 GD10. The remaining two females of this group and 

one female of the 250 mg/kg bw/day group were killed 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 single 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 - 343 g) also occurred in the females of the 500 and 250 

mg/kg bw/day groups. Necropsy revealed hardened fatty 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 female and gaseous contents in the stomach and 

intestine as well as hemorrhages in the renal capsule in a further female of the 250 mg/kg bw/day group. 

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 also 

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 body weight development, decreased food 

consumption and cold ears. The maternal toxicity NOAEL was 100 mg/kg bw/day. 

Developmental toxicity: The gestation rate in the 250 mg/kg bw/day 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 groups 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 fetuses 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/kg 

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-related 

effect. A final assessment is, however, not possible due to the low number of females in a pilot study. 

The isolated malformations seen in one fetus each of 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 they are known as common findings in the strain of 

rabbits used. Thus, effects on intrauterine development could not be completely excluded at the 160 

mg/kg bw/day level (possibly marginally reduced fetal and placental weights). The developmental 

toxicity LOAEL was 160 mg/kg bw/day based on marginally reduced fetal and placental weights. 

The developmental toxicity NOAEL was 100 mg/kg bw/day. 

Based on these results, it is concluded that the dose level of 160 mg/kg bw/day is suitable for the high 

dose level in the main developmental study of BYI 08330 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 potentials of spirotetramat were tested in a multigeneration 

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 first (main) study. 

Reproductive toxicity 

In addition to testicular histopathology observed following subchronic and chronic exposure of male rats to 

spirotetramat, evidence of male reproductive toxicity was provided in the 2-generation reproductive toxicity 

study. Abnormal sperm cells were reported in F1-generation male rats treated with 6000 ppm (419 mg/kg

bw/day) spirotetramat in the diet, and decreased reproductive performance was also observed in one of 

these males. Similar results were obtained in the 1-generation reproductive toxicity range-finding study, 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 dose between the two 

studies was attributed to increased food consumption of parental animals in the 2-generation study. Renal 

toxicity was also observed in F1 adults in the 2-, but not 1-generation reproductive toxicity study. 

Offspring toxicity was limited to decreased body weights in F1 and F2 pups of both sexes during lactation at 

6000 ppm (≥419.3 mg/kg bw/day). Decreased body weights were also observed in parental animals at the 

same dose. Development of the sexual organs of offspring (balano-preputial separation, vaginal opening) 

was unaffected in either study. 

In an investigative study designed to explore the time of onset of testicular toxicity in rats, decreased 

epididymal sperm counts were recorded ≥10 days of treatment with 1000 mg/kg bw/day by gavage. 

Repeated dosing, therefore, is necessary to produce male reproductive toxicity in rats. In a second 

investigative study, male rats were treated by gavage with the enol metabolite of spirotetramat for 21 days at 

a dose of 800 mg/kg bw/day. Spermatotoxicity, abnormal sperm, and Sertoli cell vacuolation were observed 

in the testes-epididymides of treated animals. Therefore, male reproductive toxicity in rats is likely due to the 

enol metabolite of spirotetramat. 

Developmental toxicity 

Developmental toxicity in the absence of maternal toxicity was not observed in either the rat or rabbit. In the 

developmental toxicity study in rats, reduced fetal weight and increased incidences of malformations and 

skeletal deviations were observed at 1000 mg/kg bw/day. Malformations at the high dose included single 

cases of cleft palate, co-arctation of aortic arch, atrial septal defect of the heart, microphthalmia, and 

supernumerary lumbar vertebra, four cases of dysplastic forelimb bones, and three cases of malformed 

sacral vertebral arches with pelvic shift. Skeletal findings at this dose were primarily incomplete or delayed 

ossification and skeletal variations. Toxicity 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. Increased 

incidences of wavy ribs and retarded ossification at 20 and 140 mg/kg bw/day in the main study were 

considered equivocal, and when irreproducible at the same or lower doses in the supplementary rat 

developmental toxicity study, they were not considered treatment-related. 

In the developmental toxicity study in rabbits, offspring toxicity was not observed at any dose up to 160 

mg/kg bw/day. However, maternal toxicity was observed at ≥40 mg/kg bw/day, including a dose-dependent 

increase in abortion and other clinical signs of toxicity in affected animals. The maternal LOAEL of 40 mg/kg 

bw/day in the study was considered conservative, since late abortion, clinical signs, impaired food and 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 screening study with technical grade BYI 

08330 in Wistar rats. Bayer CropScience LP Toxicology, 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 temp 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-methoxy-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% Tween 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 suspended stainless steel siremesh cages 

Diet: Purina Mills Rodent Lab Chow 5002 was provided ad libitum

Water: Tap water was provided ad libitum

Environmental conditions: Temperature

: 

Humidity: 

Air changes: 

Photoperiod: 

18-26oC 

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 weights that were 

more or less than 20% of the mean weight for each sex 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 mortality or clinical signs of moribundity and were weighed 

(as part of the FOB) one week prior to treatment, approximately 4 hours after treatment, and 7 and 14 

days after treatment. 

The rationale for dose selection was based on the results of an acute oral toxicity (LD50) study in young 

adult female Wistar rats. In that study, fasted female Wistar rats were administered an acute oral 

(gavage) dose of 2,000 mg/kg as an aqueous suspension in 0.5% carboxymethylcellulose, at a dosing 

volume 10 ml/kg. Animals were observed for mortality and clinical signs for 14 days after treatment. The 

test substance produced no clinical signs or mortality at the limit dose. These results support the use of 

a limit dose (2,000 mg/kg) in the neurotoxicity study but provided no information to establish the time of 

peak effect. 

The results from a study using radio-labeled BYI 08330 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 sexes) or 

1,000 mg/kg (males only) BYI 08330 in 0.5% (w/v) tragacanth 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 to 

3.0 hours (males) or 0.67 to 1.0 hour (females) after treatment at 100 mg/kg and 1.0 to 8.0 hours (males 

only) following administration at 1,000 mg/kg. Plasma concentrations in males at a dose of 1,000 mg/kg 

(2% cremophor in water) changed very little at 1.0, 2.0, 4.0 and 8.0 hours following treatment (570, 600, 

770 and 700 μM, respectively). Based on these collective results, the doses selected for the initial study 

were 0, 200, 500 and 2,000 mg/kg for both sexes. The 2,000 mg/kg dose was selected as a limit dose 

that may produce slight evidence of toxicity, the middle dose was selected to produce minimal or no 

effect and the low dose was expected to be an overall 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 and 

500 mg/kg. The 500 mg/kg dose was selected to reproduce 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 test of activity concluding at about 4 hours after 

treatment. Animals were assigned to the test groups 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/sex 12/sex 

Neuropathology 6/sex – – 6/sex 6/sex 6/sex 

n=12 animals/dose group for each study; a total of 24 animals/sex were tested at 500 mg/kg. 

– = not examined at this dose level in the follow-up 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, concentration, and homogeneity analyses follow below.

Results: 
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Homogeneity Analysis: Homogeneity of the test substance in the vehicle was accepted for the range 

of concentrations used here, as the 4, 15 and 200 mg/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 concentrations of 4, 15 or 200 mg/ml levels 

were 100%, 103% and 97% of the initial concentrations, respectively. 

Concentration Analysis: For the initial study, doses of 0, 200, 500 and 2,000 mg/kg for males and 

females ranged from 91% to 103% of the nominal concentrations. 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, the analytically-confirmed doses for males and 

females were 0, 47.4, 99.8, and 523 mg/kg. 

The analytical data indicated that the mixing procedure was adequate and that the variance between 

nominal and actual dosage to the animals was acceptable. 

4. Statistics: Generally, continuous data were analyzed using ANOVA, followed by Dunnett’s test. For the 

FOB, continuous data were first analyzed using Repeated-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 using Dunnett’s test and an Analysis of Contrasts, 

respectively. Motor and locomotor activity were analyzed using ANOVA procedures followed by 

Dunnett’s test. Continuous homogeneous neuropathology 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 Test for pair-wise comparisons. Micropathology 

frequency data were analyzed using a Chi-Square Test 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 mortality and 

morbidity. Detailed clinical observations were recorded daily. 

2. Body weight: Animals were weighed weekly as a component of the FOB. 

3. Food consumption: Food consumption was not recorded. The test substance 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 were blind with respect to group assignment of an 

individual animal. The test room was a standard animal room. The animals were allowed to 

acclimate in this room for at least 30 minutes. This room was maintained on the same light/dark 

cycle and settings for temperature and relative humidity 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 following treatment. For the follow-up study, FOB 
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testing was conducted one week prior to treatment and approximately 90 minutes after dosing. 

Scoring criteria were given for the measured parameters. No information on the equipment used in 

measuring grip strength was presented in the test report. 

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 activity 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 / defecation* 

X Gait Abnormalities X Red/crusty deposits* Grooming 

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 / stereotypic 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 extensor 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 conducted 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 week 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 approximately 30 minutes after the last animal was 

tested in the FOB. All 8 rats were placed into figure-eight mazes for motor activity evaluations. 

Motor and locomotor activities were evaluated by testing the animals individually in a figure-eight 

maze that had 8 infrared emitter/detector pairs. Evaluations were conducted for 10-minute intervals 

for a total of 60 minutes. Motor activity was defined as the number of beam interruptions that 

occurred during the test session. Locomotor activity was measured by eliminating consecutive 

counts for a given beam. Habituation was defined as 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 not conducted since a NOAEL was established for postmortem pathology in the initial study. The necropsy involved an examination of all organs, body cavities, 

cut surfaces, external orifices, and surfaces. On day 14, the first 6 males and 6 females at each dose 

level were selected for perfusion and collection of tissues. If perfusion was inadequate, one of the 

remaining animals was used. Animals selected for perfusion were deeply anesthetized using an 

intraperitoneal dose of 50 mg/kg of pentobarbital and then perfused via the left ventricle with a sodium 

nitrite (in phosphate buffer) flush followed by universal fixative (4% EM-grade formalin and 1% 

glutaraldehyde) in phosphate buffer. The entire brain and spinal cord, both eyes with optic nerves, and 

selected (bilateral) peripheral nerves (sciatic, tibial, and sural), the gasserian ganglion, gastrocnemius 

muscle, and physical identifier were dissected from each animal and post-fixed in 10% buffered formalin. 

The brain was weighed upon removal from the skull, prior to placement into formalin, and the brain:body 

weight ratio was calculated. Surviving animals not perfused were sacrificed by CO2 asphyxiation. 

Tissues from the control and high-dose group animals and any gross lesions collected at necropsy were 

processed for microscopic examination. Eight coronal sections of the brain and sections from 3 levels of 

the spinal cord (cervical, thoracic, and lumbar) and the cauda equina were embedded in paraffin, 

sectioned and stained with hematoxylin and eosin. Dorsal root ganglia (including dorsal and ventral root 

fibers) from the cervical and lumbar swellings, gasserian ganglion, eyes, optic nerves, and 

gastrocnemius muscle were embedded in glycol methacrylate. Peripheral nerve tissues (sciatic, tibial,

and sural nerves) were embedded in glycol methacrylate 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 1993 and 2002; references are 

provided in the study. 

II. RESULTS: 

A. Observations: 

1. Clinical signs: Selected clinical observations from the initial study 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 considered to be related to treatment (1/12 males and 1/12

females in the 200 mg/kg dose group). Perianal stain was observed in males (one each) at the two 

highest dose levels but not at the lowest dose or in females at any dose level. These signs were evident 

on day 0 and generally resolved within 1 - 4 days after 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 level. 

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. 201283. 

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 related to treatment observed in high-dose males; 

therefore, the effect is not considered treatment-related. Females at any dose level were not affected. 

In the follow-up study, there were no treatment-related findings at any dose level in either sex. Based 

on these results, the NOAEL for functional observations 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) and 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 slightly 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 reduced 

in males at several intervals at 500 and 2,000 mg/kg; subsession locomotor activity (interval 3 only) was 

also reduced in males at 200 mg/kg on day 0. A similar trend was evident in females at 500 and 2,000 

mg/kg for motor activity and at 2000 mg/kg for locomotor activity, but the difference from control was not 

statistically significant. In the follow-up study, total motor and locomotor activity was slightly 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 activity 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 ± 116

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 ± 81

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, Report 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 activity (#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 ± 50 107 ± 30 93 ± 19 

3 77 ± 49 68 ± 39 80 ± 30 85 ± 25 98 ± 46 73 ± 42 104 ± 31 100 ± 24 

4 65 ± 44 65 ± 44 58 ± 29 65 ± 26 87 ± 48 65 ± 35 107 ± 35 94 ± 25 

5 58 ± 49 19 ± 21 52 ± 47 43 ± 38 70 ± 37 46 ± 50 80 ± 41 88 ± 35 

6 39 ± 28 4* ± 97 22 ± 31 22 ± 31 61 ± 49 45 ± 43 53 ± 44 59 ± 43 

Post-treatment 1 

1 

115 ± 

31 

118 ± 48 91 ± 29 80* ± 13 118 ± 36 114 ± 21 105 ± 24 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 50 ± 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 60 ± 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 67 ± 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 69 ± 38 77 ± 48 

4 59 ± 34 61 ± 30 41 ± 18 45 ± 17 52 ± 34 69 ± 53 58 ± 43 59 ± 40 

5 42 ± 27 38 ± 33 27 ± 32 30 ± 20 43 ± 26 47 ± 30 49 ± 37 57 ± 44 

6 33 ± 21 41 ± 35 21 ± 24 39 ± 34 36 ± 27 49 ± 18 36 ± 45 48 ± 32 

a

 Data obtained from pages 172-179, Report No. 201283. 

N = 12/sex/group. 

* Significantly different from control, p≤0.05. 

Pre-treatment = one week prior to treatment. 
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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 activity (#movements/10 minute interval) a

Dose (mg/kg) 

Interval 0 200 500 2000 0 200 500 2000 

Males Females 

Pretreatment 

1 62 ± 24 57 ± 14 63 ± 23 60 ± 12 70 ± 17 55 ± 18 77 ± 15 70 ± 13 

2 51 ± 39 46 ± 24 46 ± 14 48 ± 25 55 ± 21 44 ± 36 69 ± 28 54 ± 14 

3 52 ± 39 44 ± 27 52 ± 22 52 ± 21 64 ± 32 43 ± 34 64 ± 26 60 ± 23 

4 42 ± 34 41 ± 29 34 ± 19 42 ± 19 59 ± 35 36 ± 25 71 ± 31 66 ± 24 

5 38 ± 36 11 ± 14 31 ± 29 25 ± 24 45 ± 24 25 ± 26 51 ± 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 ± 24 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 99 ± 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 58 ± 20 45 ± 21 

4 41 ± 27 39 ± 14 36 ± 16 43 ± 25 36 ± 18 29 ± 34 38 ± 27 34 ± 20 

5 38 ± 21 29 ± 14 20 ± 16 35 ± 28 26 ± 23 23 ± 25 26 ± 22 21 ± 19 

6 30 ± 27 24 ± 15 13 ± 11 21 ± 21 18 ± 20 23 ± 30 20 ± 17 15 ± 15 

Post-treatment 3 

1 76 ± 21 72 ± 18 66 ± 14 71 ± 21 82 ± 35 92 ± 41 89 ± 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 41 ± 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 25 ± 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. 201283. 

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.6-112: Mean (±S.D.) motor and locomotor activity 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 ± 160

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 ± 151

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, Report No. 201283. 

N = 12/sex/group. 

Pre-treatment = one week prior to treatment. 

Post-treatment 1 = day of dosing. 
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Table B.6-113: Mean (±SD) sub-session motor activity (#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 ± 16 115 ± 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 88 ± 35 82 ± 39 

4 65 ± 43 68 ± 39 60 ± 32 49 ± 31 76 ± 48 94 ± 28 93 ± 37 75 ± 41 

5 47 ± 40 48 ± 34 45 ± 30 40 ± 34 73 ± 47 58 ± 29 78 ± 28 61 ± 38 

6 36 ± 30 35 ± 36 34 ± 24 29 ± 31 60 ± 50 52 ± 34 72 ± 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 88 ± 35 63 ± 38 

4 55 ± 21 60 ± 20 55 ± 25 38 ± 22 59 ± 43 71 ± 46 62 ± 48 58 ± 38 

5 41 ± 25 49 ± 22 51 ± 24 34 ± 18 45 ± 35 48 ± 40 63 ± 54 40 ± 34 

6 29 ± 24 32 ± 30 31 ± 24 35 ± 29 32 ± 29 41 ± 48 46 ± 37 50 ± 43 

a

 Data obtained from pages 181-184, Report No. 201283. 

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 activity (#movements/10 minute interval) a

Dose (mg/kg) 

Interval 0 50 100 500 0 50 100 500 

Males Females 

Pretreatment 

1 73 ± 28 81 ± 17 73 ± 15 70 ± 17 68 ± 19 65 ± 13 76 ± 13 77 ± 16 

2 56 ± 28 78 ± 27 49 ± 15 60 ± 27 57 ± 25 55 ± 21 57 ± 22 53 ± 17 

3 51 ± 22 72 ± 22 46 ± 19 48 ± 24 56 ± 37 64 ± 23 55 ± 24 57 ± 34 

4 44 ± 33 45 ± 26 36 ± 21 31 ± 22 54 ± 38 60 ± 25 64 ± 28 52 ± 30 

5 30 ± 30 28 ± 24 26 ± 20 20 ± 24 48 ± 31 34 ± 23 54 ± 22 45 ± 32 

6 21 ± 21 20 ± 26 13 ± 13 10 ± 17 37 ± 36 28 ± 24 43 ± 21 29 ± 26 

Post-treatment 1 

1 73 ± 23 76 ± 18 62 ± 12 61 ± 14 99 ± 39 89 ± 27 88 ± 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 61 ± 29 39 ± 28 

4 38 ± 19 42 ± 15 32 ± 18 23 ± 14 33 ± 30 45 ± 37 42 ± 39 33 ± 27 

5 22 ± 18 31 ± 17 28 ± 16 16 ± 10 23 ± 27 28 ± 28 44 ± 40 22 ± 22 

6 16 ± 15 17 ± 19 17 ± 16 12 ± 10 15 ± 15 23 ± 29 22 ± 22 30 ± 28 

a

 Data obtained from pages 195-198, Report No.201283. 
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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 males or females at any dose. 

2. Brain weight: Brain weight was not affected by treatment in either sex at any dose. 

3. Neuropathology: Compound-related microscopic lesions were not evident 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 toxicity was limited to clinical signs and decreased activity 

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 females. 

B. REVIEWER COMMENTS: 

In the initial study, evidence of acute oral toxicity was observed in both sexes at 200, 500 and 2,000

mg/kg bw. Evidence of toxicity was limited to clinical signs (urine stain at all doses, perianal stain in 

males at 500 and 2,000 mg/kg) and decreased activity in the figure-eight maze (in females at 500 and 

2,000 mg/kg and in males at 200, 500 and 2,000 mg/kg) beginning on the day of treatment and with 

complete recovery by day 7. A follow-up study confirmed 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 animals of each sex treated with 200 mg/kg showed the

clinical sign of urine stain, evidence of a dose-response at the higher doses combined with reduced 

motor activity in males (29% decrease) confirms choice 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 motor 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. 
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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 
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B.6.7.4 Summary on neurotoxicity 

Clinical signs of toxicity and/or decreased motor activity were observed following a single dose of 200 mg/kg 

bw spirotetramat to rats. These effects were observed with greater severity at doses higher than 200 mg/kg 

bw in this study, as well as in a follow-up study in rats. In the larger database, following one-year

administration of spirotetramat to dogs, brain dilation with dose-related severity was observed in males at 

≥600 ppm (20 mg/kg bw/day), while axonal degeneration in the hypothalamus was observed in one female at

1800 ppm (48 mg/kg bw/day). Clinical signs of neurotoxicity (dehydration, swelling, decreased activity and 

reactivity, seizures and ataxia) were also observed in one male with brain dilation at the highest dose tested. 

Similar effects were not observed in either rats or mice. 
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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 potential reproductive toxicity in the male rat 

following daily oral administration by gavage. Bayer CropScience, 355, rue Dostoievski, BP 

153, 06903 Sophia Antipolis Cedex, France. Laboratory 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 dosing volume was 5 mL/kg bw based on the most 

recent body weight. The dosing suspension was prepared three times during the study by suspending 

the test material in aqueous 0.5% methyl cellulose. 

Clinical observations were recorded daily; body weight was measured on the first day of treatment, 

weekly thereafter, and at necropsy. Food consumption was measured weekly. A detailed physical 

examination was performed at weekly intervals throughout the study. The animals were anesthetized 

and sacrificed by exsanguination 4 hours after administering the last dose. All sacrificed animals were 

necropsied; the testes, epididymis, and prostate were 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 sperm 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 increased 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 determined. Most clinical observations were seen in only one 

or two animals each, but localized soiled fur was observed in eight animals and generalized soiled fur, 

increased salivation, and wasted appearance were observed in three animals each. 

Body weight and weight gain data are presented in Table 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 days 1 and 10 and showed a continued decrease in 

weight gain for the remainder of the study resulting 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) 

Parameter Dose group (mg/kg) 

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 

aNumbers in parentheses are percent of control calculated by the reviewer. 

* p<0.05; **p<0.01, statistically significant compared 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 epididymis 

were significantly decreased by 10-11% on day 3; the relative weight (% bw) of the left testis was 

significantly increased by 12% and the absolute weight of the left epididymis was significantly 

decreased by 11% on day 10. No significant organ weight changes were observed on day 21, but 

absolute right and left testes and epididymides were significantly decreased by 22-26% and relative 

weights were decreased by 11-15% on day 41. No treatment-related effect was observed on prostate 

weight. The decreased organ weights were due in part 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 findings 

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 epididymis 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 testes 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 weight

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**
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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 weight

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 weight

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 No. SA 041181 

*p<0.05; **p< 0.01, statistically significant compared 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 for 3 but not after 10 days compared with 0/8 controls. 

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. Treatment-related changes were observed in the testes

(spermatid and Sertoli cell changes) and epididymides (aberrant spermatocytes) on days 21 and 41 

days of treatment. 

After treatment for 21 days, marked degenerating elongating spermatids (steps 9 to 14 of the 

maturation cycle) were observed in 8/8 treated animals, together with multinucleated giant spermatids 

in 2/8 rats and moderate to marked degenerating round spermatids in 4/8 rats. The investigators noted

that these testicular findings are consistent with a treatment-related effect on round spermatids or late 

stage spermatocytes. At final sacrifice on day 41, slight to severe degenerating elongating spermatids 

(steps 9 to 14) were observed in 7/7 treated animals, resulting in the loss of elongating spermatids 

(steps 9 to 19) in 5/7 treated rats. Multinucleated giant spermatids and marked degenerating round 

spermatids (around steps 7-8) were observed in 4/7 and 5/7 rats, respectively. The investigators noted 

that the microscopic findings in the testes at 41 days also are consistent with a treatment-related effect 

on round spermatids. Slight to moderate Sertoli cell vacuolation was observed in the testes of 6/7 rats 

on day 41. There was an increase of intraluminal abnormal 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. Oligospermia was not observed on day 21 but was 

observed in all treated rats on day 41. The investigators noted that the findings in the epididymides 

were a consequence of degenerating spermatids in the testis. 
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Table B.6-117: Incidence of treatment-related microscopic 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 1,000 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 (1.25) 

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, 

aNumbers in parentheses are average severity grade in affected animals: 1 = slight, 2 = moderate, 3 = marked, 4 

= severe. 

 

SPERM ANALYSIS: 

Sperm count and morphology data are presented in Table 6-118. Small statistically significant 

decreases (-13% to -15%) were observed in the absolute 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 decreased (P<0.01) by 77% and 68%, respectively, 

compared with that of controls. 

Analysis of sperm morphology showed that the percent abnormal sperm was significantly increased in 

the treated group on day 21. The greater-than-twofold increase in abnormal sperm on day 21 is 

considered toxicologically significant and is likely related to the increased incidences in degenerating 

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 106 

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 106 

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**
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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 compared 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 spermatocytes in male rats. This conclusion was based on 

decreased weight of the testes and epididymides, small testes and epididymides observed after 

treatment for 41 days, increased incidence of rats with degenerating round spermatids, degenerating 

elongating spermatids, and multinucleated giant spermatids on day 21 of treatment and a loss of 

elongating spermatids on day 41. In addition, an increased incidence of intraluminal aberrant cells was 

observed in the epididymis of treated rats on day 21 and oligospermia was observed on day 41. 

REVIEWER’S CONCLUSION: 

In male rats treated with BYI 05330 at 1000 mg/kg for up to 41 days, weight loss was observed early in

the study followed by decreased body weight gain for the remainder of the study resulting in a 12% 

decrease in body weight at the end of treatment. Food consumption was significantly reduced during the 

first week of treatment. The most prevalent clinical observations included soiling of fur along with 

increased salivation and wasting appearance in several rats. One treated rat died, but this animal was 

not necropsied to determine the cause of death; therefore, 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 epididymides. Microscopic findings confirming an effect of 

BYI 05330 treatment on the testes included degenerating round spermatids (Steps 7-8), degenerating 

elongating spermatid (Steps 9-14), and multinucleated 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 days 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 CropScience, 355, rue Dostoievski, BP 

153, 06903 Sophia Antipolis Cedex, France. Laboratory Report No. SA 06011, June 30, 

2006. Unpublished. 

METHODS AND MATERIALS: 

Groups of five Wistar rats Rj:WI (IOPS HAN) were administered 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 moribundity 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 animal was measured once during acclimation, daily from

day -1 to day 21, and at necropsy. Food consumption for each animal was measured weekly. Plasma 

was prepared from heparinized blood samples collected from the abdominal aorta at the time of sacrifice 

and stored frozen, but was not analyzed for metabolites. The animals were anesthetized with isoflurane 

and sacrificed by exsanguination 4 hours after administering the last dose. All sacrificed animals were 

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; the 

cauda epididymis was dissected and weighed. The epididymal sperm were counted and sperm 

morphology was assessed.

RESULTS AND DISCUSSION: 

GENERAL OBSERVATIONS: 

No mortalities were recorded during the course of this study. Treatment-related clinical observations 

included localized soiled fur on the anogenital region 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 weight gain was markedly reduced by 78%. Treated rats 

had a net weight gain of only 1 g during the last 10 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 consumption was similar to control levels during the last

week of exposure. 
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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. 

aNumber 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 testis of treated rats was increased by 8% (p<0.05 for 

left testis only) and the relative weight was increased 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 of 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 clinical observations. No other treatment-related gross 

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 diffuse sloughing of germ cells were found in all treated 

animals. Multinucleated giant spermatids were observed 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 cells 

was found in all treated animals. This finding is considered a consequence of degenerating spermatids

observed in the testes. 
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Table B.6-120: Incidence of treatment-related changes 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. 

aNumbers in parentheses are average severity grade in affected animals: 1 = slight, 2 = moderate, 3 = marked, 4 

= severe. 

SPERM ANALYSIS: 

The data for sperm count and morphology are summarized in Table 6-121. No statistically significant 

change in absolute or the relative (based on epididymal 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 756-1109 x 106

 spermatozoa/g tissue for controls and 

690-929 x 106 spermatozoa/g tissue for treated animals. 

The percentage of abnormal spermatozoa was significantly increased after 21 days of treatment with 

BYI 08330-ENOL when compared with control values (14.9% vs 3.2% in the control group). The majority 

of the abnormalities consisted of isolated heads with 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 ± SD) 

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 the control group. 
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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 weight, and effects in the testes and epididymides. 

Testicular and epididymal effects consisted primarily of degeneration and sloughing of spermatids 

(round and elongating) in the testes, exfoliation of germ cells in the epididymis, and an increase in the 

percentage of abnormal epididymal spermatozoa. These 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 unlikely due to the acyl chain but to the enol. 

REVIEWER’S CONCLUSION: 

This study was conducted to determine whether the acyl 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 was conducted with the enol. Male rats were 

administered BYI 08330-ENOL at 800 mg/kg bw by gavage for 21 days; this dose is equivalent to 1000 

mg/kg of the parent compound. Male rats administered the enol showed effects similar to those of male

rats administered the parent compound, BYI 08330 for 21 or 41 days. Effects in rats administered the 

enol consisted primarily of soiled fur and salivation (clinical observations), a marked decrease in body 

weight gain, a slight decrease in testes weight, and microscopic changes in the testes (degeneration of 

round and elongating spermatids, sloughing of germ cells, the presence of multinucleated giant 

spermatids, and Sertoli cell vacuolation) and epididymis (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 treatment-related change in sperm count. 

In conclusion, testicular/sperm toxicity in male rats administered BYI 08330 is due to the enol and is

unlikely due to the acyl chain alone. This conclusion in agreement with that of the investigators. 

B.6.8.2 Summary of mechanistic studies 

In an investigative study designed to explore the time of onset of testicular toxicity in rats, decreased 

epididymal sperm counts were recorded =10 days of treatment with 1000 mg/kg bw/day spirotetramat by 

gavage. Repeated dosing, therefore, is necessary to produce male reproductive toxicity in rats. In a second 

investigative study, male rats were treated by gavage with the enol metabolite of spirotetramat for 21 days at 

a dose of 800 mg/kg bw/day. Spermatotoxicity, abnormal sperm, and Sertoli cell vacuolation were observed 

in the testes-epididymides of treated animals. Based on these and similar results with the parent compound, 

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. 
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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 prepared 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 
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Fasting time: For administration, food was withheld from the animals for 

approximately 16 - 24 h before administration of the 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 necropsy 

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 death Mortality 

(%) 

Females 

2,000 1st 0 0 3 -- -- 0 

2,000 2nd 0 0 3 -- -- 0 

LD50 >2,000 mg/kg bw 

* 1st number = number of dead animals; 2nd 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 by the treatment 

D. Necropsy: No particular gross pathological changes were observed 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 of BYI 08330-CIS-Ketohydroxy 

is ≥ 5000 mg/kg bw (Category 5 / unclassified of the Globally Harmonized Classification System). 
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B. Reviewer Comments: In an acute oral toxicity study, two groups of three 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 of 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% Cremophor EL. The administration volume was 10 ml/kg 

body weight. Food was withheld from the animals for 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 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 during the study. No clinical signs or gross 

pathological findings were observed. 

Oral LD50 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 LD50 > 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 prepared 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 
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 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 animals for 

approximately 16 - 24 hr before administration of the 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 necropsy 

 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 death Mortality 

(%) 

Females 

2,000 1st 0 0 3 -- -- 0 

2,000 2nd 0 0 3 -- -- 0 

LD50 >2,000 mg/kg bw 

* 1st number = number of dead animals; 2nd number = number of animals with signs; 

3

rd number = number of animals used; 

B. Clinical observations: No clinical signs were observed. 
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C. Body weight: Body weight and body-weight gain was not affected by the treatment 

D. Necropsy: No particular gross pathological changes were observed 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 three 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% Cremophor EL. The administration volume was 10 

ml/kg body weight. Food was withheld from the animals 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 during the study. No clinical signs or gross 

pathological findings were observed. 

Oral LD50 Females > 2000 mg/kg bw 

Based on the lack of mortality at 2000 mg/kg, BYI 08330-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 LD50 > 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 prepared 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 
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 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 approximately 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 necropsy 

 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 death Mortality 

(%) 

Females 

2,000 1st 0 0 3 -- -- 0 
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2,000 2nd 0 0 3 -- -- 0 

LD50 >2,000 mg/kg bw 

* 1st number = number of dead animals; 2nd 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 by the treatment 

D. Necropsy: No particular gross pathological changes were observed 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 Classification System). 

B. Reviewer Comments: In an acute oral toxicity study, two groups of three 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 body 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 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 during the study. No clinical signs or gross 

pathological findings were observed. 

Oral LD50 Females > 2000 mg/kg bw 

Based on the lack of mortality at 2000 mg/kg, BYI 08330-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 LD50 > 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. Unpublished study prepared 

by Bayer Ag Inst. of Toxicology. 24 p. December 8, 2005. 
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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 animals for 

approximately 16 - 24 h before administration of the 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 necropsy 

 In life dates: 2005-10-05 – 2005-10-26 
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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 1st 0 0 3 -- -- 0 

2,000 2nd 0 0 3 -- -- 0 

LD50 >2,000 mg/kg bw 

* 1st number = number of dead animals; 2nd 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 by the treatment 

D. Necropsy: No particular gross pathological changes were observed 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 Globally Harmonized Classification System). 
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B. Reviewer Comments: In an acute oral toxicity study, two groups of three 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 of 2000 mg/kg body weight of BYI 08330-monohydroxy (Purity: 98.41%; Batch No. NLL 7635-2D; white 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 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 during the study. No clinical signs or gross 

pathological findings were observed. 

Oral LD50 Females > 2000 mg/kg bw 

Based on the lack of mortality at 2000 mg/kg, BYI 08330-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 LD50 > 2000 mg/kg in female rats for BYI 

08330-mono-hydroxy. 

D. Study Deficiencies: None.
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B.6.8.3.2 Salmonella/Escherichia/Mammalian Activation Gene Mutation Assay 

CITATION: Herbold, B. (2005). BYI 08330-CIS-Ketohydroxy (Project 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 incorporation) 

Cumene hydroperoxide 50 μg/plate TA 102 (preincubation)

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 

 MgCl2 x 6 H2O 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
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 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 strains, triplicate plating) 

Nonactivated conditions: 0, 16, 50, 158, 500, 1,581, 5,000 μg/plate 

Activated conditions: 0, 16, 50, 158, 500, 1,581, 5,000 μg/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 incorporation 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 without S9 mix. 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 without activation to 2 mL of molten 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 automatic 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 included in the mutagenicity assessment. If the second 

test confirmed the results 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 mixed 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, doses 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 determining (1) background growth on the plates of the 

mutation assay, (2) mutant counts per plate, compared to the negative controls, and looking for a dosedependent reduction, and (3) the bacterial titer, using a 1:1,000,000 dilution of the bacterial suspensions, 

except that the histidine concentration in the soft agar was increased five-fold to permit the complete 
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growth of bacteria. The tests were performed both with 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 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 determinations had to demonstrate sufficient bacterial density in the suspension. 

Only trials which complied 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 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) was 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 concentrations ranging from 16 to 5000 μg/plate using all 

five tester strains with and without S9-mix, in both a standard plate test and a 20-minute preincubation 

assay. Marked cytotoxicity was observed at 5,000 μg/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 DMSO control with strain TA98 were somewhat higher 

than the range of medians for 1996-2004 (Table 6-124) however, there was no difference in results 

between DMSO and any treatment dose. Results of the 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 summarized in Table 6-124. 
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Table B.6-122: Summary of the mutation assays with BYI 08330-CIS-Ketohydroxy without 

activation 

BYI 08330-CIS- Mean number of revertants/plate or tube ± standard deviation 

Ketohydroxy 

(μg/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 
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Table B.6-123: Summary of the mutation assays with BYI 08330-CIS-Ketohydroxy with activation 

BYI 08330-CIS- Mean number of revertants/plate or tube ± standard deviation 

Ketohydroxy 

(μg/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. 
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Table B.6-124: Historical control data, presented as 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 1117-1442 466-613 

Data summarized from Report No. AT02735, pages 32-45. 

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-ketohydroxy was non-mutagenic without or with 

metabolic activation under the conditions of the Salmonella/microsome test. 

B. REVIEWER COMMENTS: 

 The reviewer agrees with the investigator’s conclusion. BYI 08330-CIS-ketohydroxy was tested to the 

limit dose for the assay, proper experimental protocol 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 treatment 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 and bacteria for detection. Proc. Nat. Acad. Sci. 

(USA) 70, 2281-2285. 
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April 2008

Ames, B.N., J. McCann and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with 

the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31, 347-364. 
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April 2008

Salmonella/escherichia/mammalian activation gene mutation assay 

CITATION: Wirnitzer, U. 2006. BYI 08330-desmethyl-ketohydroxy (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 incorporation) 

Cumene hydroperoxide 50 μg/plate TA 102 (preincubation)

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 

MgCl2 x 6 H2O 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) 
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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 strains, triplicate plating) 

Nonactivated conditions: 0, 16, 50, 158, 500, 1,581, 5,000 μg/plate 

Activated conditions: 0, 16, 50, 158, 500, 1,581, 5,000 μg/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 incorporation 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 without activation to 2 mL of molten 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 automatic 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 included in the mutagenicity 

assessment. If the second test confirmed the results 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 mixed 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, doses were given as (μg/plate) and μg/tube, respectively, 

although the two terms could actually be used synonymously. 

All plating for both protocols was in triplicate. 

The toxicity of the substance was assessed by determining (1) background growth on the plates of the 

mutation assay, (2) mutant counts per plate, compared to the negative controls, and looking for a dosedependent reduction, and (3) the bacterial titer, using a 1:1,000,000 dilution of the bacterial suspensions, 

except that the histidine concentration in the soft agar was increased five-fold to permit the complete 

growth of bacteria. The tests were performed both with and without S9 mix. The count was made after 

the plates had been incubated for 48 hours at 37°C. 
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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 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 determinations had to demonstrate sufficient bacterial density in the suspension. 

Only trials which complied 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 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 Report 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 μg/plate 

using all five tester strains with and without S9-mix, in both a standard plate test and a 20-minute 

preincubation assay. Slight to moderate cytotoxicity (≤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 occurred. No evidence of a mutagenic effect was obtained in 

either assay. The solvent and positive controls induced the appropriate responses (essentially within the 

laboratory’s historical control ranges) in the respective strains. Results of the standard plate test and 

preincubation assay without S9 activation are summarized in Table 6-125, and with S9 activation are 

summarized in Table 6-126. Historical solvent control data are summarized in Table 6-127. 

Table B.6-125: Summary of the mutation assays with BYI 08330-desmethyl-ketohydroxy without 

activation 

BYI 08330-desmethyl- Mean number of revertants/plate or tube ∀ standard deviation 

ketohydroxy 

(μg/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 
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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 
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Table B.6-126: Summary of the mutation assays with BYI 08330-desmethyl-ketohydroxy with 

activation 

BYI 08330-CIS- Mean number of revertants/plate or tube ∀ standard deviation 

Ketohydroxy 

(μg/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 
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Table B.6-127: Historical control data, presented as 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 1117-1442 466-613 

Data summarized from Report No. AT02735, pages 32-45. 

4-NPDA = 4-Nitro-1,2-phenylene diamine 

Cumene HP = Cumene hydroperoxide 

III. DISCUSSION and CONCLUSIONS:

A. CONCLUSIONS: 

 The investigators concluded that BYI 08330-desmethyl-ketohydroxy was non-mutagenic without or with 

metabolic activation under the conditions of the Salmonella/microsome test. 

B. REVIEWER COMMENTS: 

 The reviewer agrees with the investigators’ conclusion. BYI 08330-desmethyl-ketohydroxy was tested to 

the limit dose for the assay, proper experimental protocol appears to have been followed, and the 

solvent and positive control values were appropriate. 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. 

STUDY DEFICIENCIES: Minor study deficiency is as follows: 

(1) The dose formulations were not analyzed for actual concentrations.

D. REFERENCES:
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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 and bacteria for detection. Proc. Nat. Acad. Sci. 

(USA) 70, 2281-2285. 

Ames, B.N., J. McCann and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with 

the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31, 347-364. 
– – 

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363

April 2008

Salmonella/escherichia/mammalian activation gene mutation assay 

CITATION: Herbold, B. (2005). BYI 08330-di-hydroxy (Project BYI 08330): Salmonella/microsome test 

– plate incorporation and preincubation method. Bayer HealthCare AG, PH-R&D 

Toxicology, 42096 Wuppertal, Germany. Report No. AT03069, 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-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) 

 MgCl2 x 6 H2O 162.6 mg 
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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 mutation, 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 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 count, three plates were 

used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the 

solvent minus the test substance, comprised the negative 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 routinely determined on the basis of a standard protocol: if not 

limited by solubility 5,000 μg or 5 μl per plate were used as the highest dose. At least five additional 

doses were routinely used. If less than three doses 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 least three doses could be used for assessment, the first 

trial was included in the assessment. If the second test confirmed the results of the first, no additional 

repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first 

experiment. The independent repeat was performed as preincubation in a water bath at 37°C for 20 

minutes. At the end of the preincubation period 2 ml of molten soft agar were added to the tubes, the 

content mixed and plated. For the mutant count, three plates were used for each strain and dose. An 

equal number of plates, filled with the solvent minus the test substance, comprised the negative control. 

Each positive control also contained three plates per 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 separation 

of plate incorporation and preincubation trials, despite the fact that μg/plate and μg/tube could be used 

synonymously. Following incubation for 48 hours at 37°C, revertants colonies were counted using an 
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Artek automatic colony counter. If precipitation interfered with automatic counting, colonies were 

counted manually. 

Cytotoxicity was assessed by evaluating the background lawn of bacteria, the bacterial titer and by 

checking for a reduction in the background number of revertants per plate. A toxic effect of the 

substance was assumed when there was a marked and dose-dependent reduction in the mutant count 

per plate, compared to the vehicle controls 

3. Statistical analysis: No statistical analysis was performed on the study data. 

4. Evaluation criteria: 

The following criteria determined the acceptance of an assay: 

a. The negative controls had to be within the expected 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 determinations had to demonstrate sufficient bacterial 

density in the suspension. Only trials which complied 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 test 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 per plate in at least one tester strain. The 

increase should be at least two-fold over the respective vehicle control value for strains TA98, TA100

and TA1535, at least a three-fold over the respective vehicle control value for strain TA1537 and an 

increase in the number of revertants per plate of at least 100 over the respective vehicle control for

TA102. 

II. REPORTED RESULTS:

A. PRELIMARY CYTOTOXICITY ASSAY: 

The results of the first experiment in the mutagenicity assay were considered as a pre-test for toxicity.

B. MUTAGENICITY ASSAY:

Doses up to and including 5,000 μg/plate did not cause any bacteriotoxic effects. Total bacteria counts 

remained unchanged and no inhibition of growth was observed. Precipitation did not occur at any 

concentration in any strain with or without metabolic activation. There was no evidence of mutagenic 

activity of BYI 08330-di-hydroxy in any tester strain with or without S9-mix using either the standard plate 

assay or the preincubation protocol. No biologically relevant increase in the mutant count, in comparison 

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 within the historical control ranges. Historical control 

values are summarized in Table 6-130. This is an Acceptable/Guideline study. 

III. DISCUSSION AND CONCLUSIONS: 

A. CONCLUSIONS: 
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B. REVIEWER’S COMMENTS: 

BYI 08330-di-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 vehicle 

control values observed at any test material concentration 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/microsome test. 

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for Test 

Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. 
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Table B.6-128: Summary of the mutation assays with BYI 08330-di-hydroxy without activation 

BYI 08330-mono- Mean number of revertants/plate + standard deviation 

hydroxy (μg/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 
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Table B.6-129: Summary of the mutation assays with BYI 08330-di-hydroxy with activation 

BYI 08330-CIS- Mean number of revertants/plate + standard deviation 

Ketohydroxy 

(μg/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 * 410 ± 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 actual 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 bacteria for detection. Proc. Nat. Acad. Sci. (USA) 70:

2281-2285. 

Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the 

Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31: 347-364. 
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April 2008

Table B.6-130: Historical control data, presented as 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 1117-1442 466-613 

Data summarized from Report No. AT02735, pages 32-45. 

4-NPDA = 4-Nitro-1,2-phenylene diamine 

Cumene HP = Cumene hydroperoxide 
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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. Unpublished. 

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 temperature 

 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 explanation 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% methylcellulose 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 toxicity and a significant reduction in mean body weight

as well as one death in rats (Bayer Study Report # SA 04181). 
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4. Dose preparation and analysis: The test compound was mixed with 0.5% methylcellulose 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 analyses of test compound in the dose formulations were 

performed. 

Results:

Homogeneity/Stability/Concentration analysis: Not available

5. Statistics: No statistical analyses were performed. Only means and standard deviations were 

calculated for body weight and food consumption using computer software. 

C. METHODS:

1. Observations: Animals were inspected twice daily for signs of toxicity and mortality. Observed signs 

(nature, onset, severity, reversibility and duration of clinical signs) were recorded at least once daily. 

Cageside inspections for signs of ill-health were conducted. 

2. Body weight: Animals were weighed daily. 

3. Food consumption and compound intake: The weight of the food supplied was recorded on Study 

Days 1 and 7. The remaining food was recorded on Days 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 were 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 comparison 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 remainder of the days, the mean body weight was the same 

as that recorded for Day 1. The mean overall gain for treated group was only 1g. For individual rats the 

body weight change varied from –3 g to +4 g. 
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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 reduced by 19% 

compared to that recorded on Study Day 7 (26±g). The 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 administered BYI 08330-enol daily by gavage at 

a dose level of 800 mg/kg/day for 10 days. The study 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 survival 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 significantly from the mean body weight on Day 1. The mean 

overall gain for treated group was only 1 g compared to 26 g for control rats of the similar age during a 

similar duration. The study author concluded that 800 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 conclusion that 10-day oral gavage administration of BYI 

08330-enol was not overtly toxic to male rats. The test compound caused a lack of body weight gain 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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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, employed only one dose group consisting of only 3 animals,

and measured limited parameters. Concentration analysis of the dosing solution was not available. No

rationale was provided for using only male rat. These deficiencies may affect interpretation of the study 

results. 
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Salmonella/escherichia/mammalian activation gene mutation assay 

CITATION: Herbold, B. (2005). BYI 08330-mono-hydroxy (Project BYI 08330): Salmonella/microsome 

test – plate incorporation and preincubation method. Bayer HealthCare AG, PH-R&D 

Toxicology, 42096 Wuppertal, Germany. Report No. AT02716, 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) 

 MgCl2 x 6 H2O 162.6 mg 

KCl 246.0 mg 

Glucose-6-phospate, disodium salt 179.1 mg 
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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 mutation, 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 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 count, three plates were 

used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the 

solvent minus the test substance, comprised the negative 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 routinely determined on the basis of a standard protocol: if not 

limited by solubility 5,000 μg or 5 μl per plate were used as the highest dose. At least five additional 

doses were routinely used. If less than three doses 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 least three doses could be used for assessment, the first 

trial was included in the assessment. If the second test confirmed the results of the first, no additional 

repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first 

experiment. The independent repeat was performed as preincubation in a water bath at 37°C for 20 

minutes. At the end of the preincubation period 2 ml of molten soft agar were added to the tubes, the 

content mixed and plated. For the mutant count, three plates were used for each strain and dose. An 

equal number of plates, filled with the solvent minus the test substance, comprised the negative control. 

Each positive control also contained three plates per 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 separation 

of plate incorporation and preincubation trials, despite the fact that μg/plate and μg/tube could be used 

synonymously. Following incubation for 48 hours at 37°C, revertants colonies were counted using an 

Artek automatic colony counter. If precipitation interfered with automatic counting, colonies were 

counted manually. 
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Cytotoxicity was assessed by evaluating the background lawn of bacteria, the bacterial titer and by 

checking for a reduction in the background number of revertants per plate. A toxic effect of the 

substance was assumed when there was a marked and dose-dependent reduction in the mutant count 

per plate, compared to the vehicle controls 

3. Statistical analysis: No statistical analysis was performed on the study data. 

4. Evaluation criteria: 

The following criteria determined the acceptance of an assay: 

a. The negative controls had to be within the expected 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 determinations had to demonstrate sufficient bacterial 

density in the suspension. Only trials which complied 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 test 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 per plate in at least one tester strain. The 

increase should be at least two-fold over the respective vehicle control value for strains TA98, TA100

and TA1535, at least a three-fold over the respective vehicle control value for strain TA1537 and an 

increase in the number of revertants per plate of at least 100 over the respective vehicle control for

TA102. 

II. REPORTED RESULTS:

A. PRELIMARY CYTOTOXICITY ASSAY: 

The results of the first experiment in the mutagenicity assay were considered as a pre-test for toxicity.

B. MUTAGENICITY ASSAY:

Doses up to 5,000 μg/plate did not cause any bacteriotoxic 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-mix 

using either the standard plate assay or the preincubation protocol. No biologically relevant increase in 

the mutant count, in comparison with the vehicle controls, was observed. Results of the mutation assays 

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 respective strains and were within the historical control 

ranges. Historical control values are summarized in 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 vehicle 

control values observed at any test material concentration in any tester strain with or without S9-mix in 

either assay. BYI 08330-mono-hydroxy was considered to be non-mutagenic without and with metabolic 

activation under the conditions of the Salmonella/microsome test. 
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April 2008

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for Test 

Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. 

C. STUDY DEFCIENCIES: The following minor deficiency was noted, but does not change the conclusions 

of this DER: 

- The dose formulations were not analyzed for actual concentrations. 

Table B.6-132: Summary of the mutation assays with BYI 08330-mono-hydroxy without activation 

BYI 08330-mono- Mean number of revertants/plate ∀ standard deviation 

hydroxy (μg/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 
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* Positive response 

Table B.6-133: Summary of the mutation assays with BYI 08330-CIS-Ketohydroxy with activation 

BYI 08330-CIS- Mean number of revertants/plate ∀ standard deviation 

Ketohydroxy 

(μg/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 bacteria for detection. Proc. Nat. Acad. Sci. (USA) 70:

2281-2285. 

Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the 

Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31: 347-364.
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Table B.6-134: Historical control data, presented as 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 1117-1442 466-613 

Data summarized from Report No. AT02735, pages 32-45. 

4-NPDA = 4-Nitro-1,2-phenylene diamine 

Cumene HP = Cumene hydroperoxide
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B.6.8.4 Summary of toxicological studies on metabolites 

Decreased body weight was observed following a 10-day exposure of rats by gavage to the enol metabolite 

of spirotetramat (800 mg/kg bw/day). Following a 21-day exposure of rats to the enol metabolite at the same 

dose by gavage, clinical signs of toxicity, decreased body weight gain, and testicular/spermatotoxicity were 

observed. The ketohydroxy, desmethyl-ketohydroxy, dihydroxy, and monohydroxy metabolites of 

spirotetramat were non-mutagenic when tested in the Ames test. 
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B.6.9 Medical data and information (OECD Annex IIA 5.9) 

B.6.9.1 Report on medical surveillance on manufacturing plant personnel (OECD Annex IIA 

5.9.1) 

Two cases of Type IV hypersensitivity (facial) were reported and confirmed by patch testing in spirotetramat 

manufacturing plant personnel in 2004. Due to the two cases of type 4 sensitization, a survey was 

conducted among all potentially exposed persons. It did not reveal any additional cases. 

B.6.9.2 Report on clinical cases and poisoning incidents (OECD Annex IIA 5.9.2) 

B.6.9.3 Observations on exposure of the general population and epidemiological studies 

(OECD Annex IIA 5.9.3) 

B.6.9.4 Clinical signs and symptoms of poisoning and details of clinical tests (OECD Annex 

IIA 5.9.4) 

The databases listed below were investigated via literature research to check whether 

incidences were reported globally regarding poisoning to cover following points in the EU 

Annex II dossier for spirotetramat: 

5.9.2 Report on clinical cases and poisoning incidents 

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 
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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. 
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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) [14C]-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, 2006. Unpublished. 

I. MATERIALS AND METHODS:

A. MATERIALS:

1. Test material: [

14C]-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 Chemistry, 

Wuppertal, Germany 

Other comments: None 

2. Relevance of test material to proposed formulation(s): Not relevant

3. Test animals: 

 
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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 during 

acclimation period and in metabolism cages (Jencon’s 

Metabowls or Radleys) during treatment phase. 

Diet: Certified rodent diet A04C-10, Scientific Animal Food 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 deposition of recommended field dilution.

Nominal doses: 100.0, 15.0 and 5.0 μg a.i./cm2

 skin. 

Actual doses: 1.2, 0.2, and 0.06 mg a.i./12 cm2 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 before, during, and after dosing. The homogeneity of 

each dose was also determined by assay of 5 aliquots of each dosing solution before administration of 

doses. 

Dose volume: 10 μL/cm2 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 exposed 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 period). 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-abraded areas were selected for application. The site of

application (approximately 6 cm2

/site; 2 sites/animal) was defined and protected by gluing plastic saddles 

to the unabraded skin using a cyanoacrylate-based glue. To protect the application site, after dosing, a 
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strip of perforated plastic was placed over the saddle and held in place with surgical tape. The cover 

prevented loss of material but allowed ventilation of the underlying skin. 

 

3. Dose preparation, administration and quantification: 

Preparation: Dosing suspensions of the three formulations of radiolabeled BYI 08330 were prepared by 

the Research and Development Centre of Bayer CropScience, Germany and stored at approximately -20 

C prior to dosing. 

Application: The dosing solutions were thawed and stirred continuously during dosing. The required 

volume (120 μL/rat) of the dose formulation was applied and spread 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. The perforated plastic cover was not in direct contact with 

the test material applied to the skin. Sixteen rats were dosed per exposure concentration. The pipette 

tip used to apply the dose was retained for subsequent 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 CO2 collection. 

Quantification: Radioactivity (dpm) and radiochemical purity were determined before dosing. 

Homogeneity of the doses was determined from radiochemical analysis of five dose-sized aliquots of the

dosing solutions taken before, during and after dosing. Pre-dose mid-dose and post-dose aliquots were

analyzed to confirm radioactivity levels and homogeneity. Data are summarized in Table 6-135. 

 

Table 6-135: Dosing 

Amount of formulation applied/animal 

(120 μL) 

Dose 

level 

(μg/cm2) 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. Four 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 swabs, to remove non-absorbed radioactivity. Swabbing

was continued until no radioactivity was detected in swabs using a Geiger counter. The skin was again

shaved and tape-stripped to remove the stratum corneum. 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 four 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./cm2 skin). At each termination 

period, 4 rats per dose level were exsanguinated under halothane anaesthesia and a blood sample was 

taken by cardiac puncture. Dressings (saddle, gauze, 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 wide) was also taken for analysis as well as an 

isolated control portion of untreated skin. The residual 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 tissues 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 correlation curves were prepared for each scintillation 

cocktail and checked using [14C]-n-hexadecane standards. The limit of detection (LOD) was taken to be 

two times the background count for blank samples prepared using the appropriate scintillation cocktail. 

Total amounts of radioactivity in samples were reported 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 14CO2 generated was 

trapped in CarboSorb and mixed with scintillant prior to analysis by liquid scintillation 

counting. 

Urine Duplicate aliquots of urine were added directly to scintillant and analyzed by liquid 

scintillation counting. 

Cardiac blood Triplicate weighed aliquots were dried on Combusto-pads, combusted in Combustocones and absorbed samples were added directly to scintillant 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 aliquots of the extractant were added 

to scintillant and analyzed by liquid scintillation counting. 

Carcass Solubilized in alcoholic potassium hydroxide and duplicate weighed samples analyzed 

by liquid scintillation counting. 

Fur samples Fur samples from treated skin (taken prior to tape stripping) were extracted with 

acetonitrile and duplicate aliquots of the extractant 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 directly to scintillant, and analyzed by 

liquid scintillation counting. 

II. RESULTS

A. SIGNS AND SYMPTOMS OF TOXICITY:

All animals were examined for mortality and signs of toxicity throughout the study period. The results 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/cm2 dose of [14C]-BYI 08330 

Residues in matrix (% of applied dose) 

Matrix analyzed 1 hr (Group 1) 10 hr (Group 2) 24 hr (Group 3) 10/168 (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 2.87±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 15.78±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 Tables 2 and 8, pp. 23 and 32; Report No. MEF-06/15. 

a 10 hours of exposure with observation period extended to termination at 168 hrs. 

Table 6-138: Amount of radiolabel recovered in each matrix at specified hours post-application of 

a 15 μg/cm2

 dose of [14C]-BYI 08330 

Residues in matrix (% of applied dose) 

Matrix analyzed 1 hr (Group 5) 10 hr (Group 6) 24 hr (Group 7) 10/168 (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 17.59±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; Report No. MEF-06/15. 

a

10 hours of exposure with observation period extended to termination at 168 hrs. 

Table 6-139: Amount of radiolabel recovered in each matrix at specified hours post-application of 

a 5 μg/cm2 dose of [14C]-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 12.64±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 Tables 6 and 8, pp. 29 and 32; Report No. MEF-06/15. 

a 10 hours of exposure with observation period extended 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/cm2 dose groups, respectively. The majority of the administered dose 

was recovered from skin washings, and as non-absorbed radiolabel (81.3-89.1, 75.3-87.1, and 87.6-

92.6% for the high, medium, and low dose groups, respectively). Of the applied dose, 2.87-11.39, 1.76-

15.6, and 1.82-10.6% was retained at the application 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 skin site in estimates of dermal absorption because the 

values declined over time (168 hrs) and could mask any increase of residues in the rest of the body over 

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 appeared to increase between 1 and 10 hrs exposure in the 

three dose groups, due to retention in the stratum corneum, but only significantly for the high and low 

dose groups and remained stable between 10 and 24 hrs exposure for all three dose groups. The mean 

potential dermal absorption values for the high dose group ranged from 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 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 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 

potentially absorbable radioactivity present in the stratum 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 during the 168-hr time period while the amount of radioactivity in excreta increased, thereby 

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 [14C]-BYI 08330 at dose levels of 100, 

15, or 5 μg a.i./cm2, the patterns of absorption, distribution (skin) and excretion were investigated. Male 

rats were sacrificed after each exposure period. Additional groups were exposed to the high, medium, 

and low doses for 10 hrs and sacrificed 168 hrs post-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 applied radiolabel in the high, medium, and low 

dose groups, respectively. 
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For the high dose group there was no significant difference 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 exposure, 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 were 0.88, 1.43, and 3.54% after 1, 10, and 24 hrs 

exposure, respectively. Generally there appeared to be a slight trend towards higher mean dermal 

absorption values with increasing duration of exposure. These increases were significant between 1 

and 24 hours exposure for the medium and low dose groups but not for the high dose group. There was 

a significant increase observed in the mean absorption levels at 10 and 24 hours for the high dose 

animals. Total direct dermal absorption did not appear to be significantly affected by the level of applied 

dose. 

The data from the 168-hour depuration groups indicate that only a portion of the radioactivity remaining 

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 the renewal of the stratum corneum. 

B. REVIEWER COMMENTS: 

The total recovered radioactivity for the 12 groups of male Wistar rats exposed dermally to [14C]-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 hour post-treatment sacrifice periods for the high, 

medium and low dose levels are given in Tables 6-137, 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 recovery values are also normally considered part of the 

estimated value for dermal absorption. The estimated value is the sum of the total radioactivity recovered 

from the skin at the application site, non-treated skin, surrounding skin, urine, feces, cage washes, 

blood, and carcass. This value is considered conservative since a portion of the active ingredient was

retained at the skin site and it is unlikely that all of the skin residues will become systemically available. 

This value appeared to increase between 1 and 10 hrs exposure in the three dose groups, due to 

retention in the stratum corneum, but only significantly 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 from 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 dose 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/indirect 

dermal absorption. 

As stated previously, based on the likely worker exposure 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. 
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C. STUDY DEFICIENCIES: 

There were no study deficiencies that would impact the integrity of the study results. Unlabeled parent 

material, if used, was not characterized. The results of examinations for mortality and clinical signs of 

toxicity were not reported. 
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B.6.9.9.2 In vitro dermal penetration study - rat/human 

CITATION: Capt A. (2006) [14C]-BYI 08330 in OD150 formulation: Comparative in vitro dermal 

absorption study using human and rat skin. Bayer CropScience, 355 rue Dostoievski, BP. 

153, 06903 Sophia Antipolis, France. Study No. SA 05254. 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 Chemistry, 

Wuppertal, Germany 

Other comments: None 

2. Relevance of test material to proposed formulation(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 (Biopredic, 

Rennes, France) 

Together (10) in wire-mesh bottomed, stainless steel cage 

during the acclimation period 

Pelleted; A04 C-10 . SAFE. Rte de St Bros. Aigu, France; ad 

libitum
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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 formulation concentrate 150 mg/mL) or 

anticipated dermal deposition of field spray dilutions (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 [14C]-BYI 08330 in the dosing solutions were 1.58, 0.013, 

or 0.0004 mg/10 μL dose (see Table 6-140 for details). Actual doses were calculated as the mean dose 

applied to each of 6 dermal membranes/dose rate/exposure 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: 10μL/cm2

 skin (2.54 cm2/membrane) 

Duration of exposures (time from dose to termination): 24 hours with skin wash after 8 hrs exposure. 

Termination periods (24 hrs): A reservoir was placed atop each membrane for receptor 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 tubing. Fractions were collected hourly following dosing and 

collected until termination at 24 hrs. The receptor 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 mass 

balance procedures.

Number of membranes /group: Six membranes (target number before exclusion of damaged 

membranes) were tested in individual diffusion cells/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 membrane 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-dermatome. Prior to use, the integrity of the selected 

membranes was assessed by measuring the trans-epidermal 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/m2

/hr). Human and rat skin with a 

TEWL of >40g/m2/hr were considered potentially damaged and were not used. 

3. Dose preparation, application to membranes and quantification: 
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Preparation: Dosing suspensions (three formulations) were prepared in the Monheim Research and 

Development Centre of Bayer CropScience, Germany, on 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 formulations 

were tested for solubility in the receptor fluid and found to be soluble for 24 hrs at 2.4mg in 3 mL of 

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 [14C]-BYI 08330 applied to the membranes of any cell. Doses were checked for 

radioactivity before during and after dosing. Homogeneity was checked by taking five aliquots of each

dose formulation before application. The formulations were considered to be homogeneous. 

Assembly of diffusion cells and application of dosing solutions: Glass flow-through diffusion cells 

(Franz cell modified, Gallas, France) were used with an exposed membrane area of 2.54cm2

. 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 pumped to a fraction collector at a flow rate of 1.5 mL/hr. 

The dosing solutions (high, medium, or low dose) were applied to the split-thickness skin samples with a 

pipette at 10 μL/cm2 skin surface. Each dosing solution was assayed for 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 purity was determined by TLC and HPLC analysis of the 

dosing preparations. Homogeneity of the doses was determined 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/cm2) 

Actual [14C]-BYI 

08330 

concentration 

(mg/mL) 

Applied dose (μg 

[

14C]-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; Study No. SA 05254. Means in columns 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 exposed to 

the high, medium, or low doses of [14C]-BYI 08330 for 8 hrs and washed by repeated swabbing after 

exposure to remove residual radioactivity. Tween 60 (1%) in phosphate buffered saline was used as the

swabbing agent. Swabbing was continued until no radioactivity could be detected on the sponges using 

a Geiger counter. Sampling of receptor fluid continued hourly to termination at 24 hrs. Membranes were 

swabbed again at termination and all swabs were 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 membranes were removed and saved for analysis by 

LSC. Receptor fluid at termination was removed from cells for analysis, the diffusion cell components
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were washed, and the washings saved for analysis for mass balance. Samples were stored at -20 C 

until analyzed. 

5. Sample analysis: Material balance samples, receptor fluid samples, and aliquots of dosing solutions 

were analyzed using Liquid Scintillation Counting (LSC) techniques. Receptor fluid samples were 

analyzed directly by addition to scintillant for counting. Skin swabs were solubilized in Soluene and

scintillation fluid was added for LSC analysis. Tape strips were solubilized using tetrahydrofuran and 

scintillation fluid was added for direct LSC analysis. Skin samples were solubilized in soluene, 

scintillation fluid was added and samples were counted by LSC. Diffusion cell components, including 

stirrers and tubing, were soaked in acetonitrile/distilled water (50:50 v/v) for 12 hrs and the washings 

were analyzed by LSC. All samples were counted using a Packard 1900 TR Liquid Scintillation 

Spectrometer with on-line computing. Quenching effects were determined using external 

standardization and spectral quench parameter (tSIE) methods. Efficiency correlation curves were 

prepared for each scintillation cocktail using radiolabeled n-hexadecane internal standards. The limit of 

detection was considered to be twice the background values for blank samples in scintillation cocktail. 

The calculation of % recovered dose, means and standard deviations were accomplished with a 

personal computer using Debra 5.2a software and Microsoft Excel. 

II. RESULTS:

A. MATERIAL BALANCE AND SPIROTETRAMAT DISTRIBUTION: 

The material balance for dermal penetration of [14C]-BYI 08330 through rat and human epidermis at the

high, medium, and low dose rates are shown in Tables 6-141, 6-142, and 6-143, respectively. Recovery 

of radioactive material was excellent with means ranging from 96.0-100.1% of the applied dose for rats

and 96.0-99.0% for humans. For the formulation concentrate, 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 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 with 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 with the epidermis after 24 hrs. In addition, a 

portion of the applied doses remained in the stratum corneum of both species. For the rat, 0.12, 1.7, and 

1.9% of the respective high, medium and low doses remained with the stratum corneum. For human 

skin, 0.1, 0.7, and 3.24% of the added radioactivity remained within the stratum corneum for the high,

medium, and low doses, respectively. At termination, <0.005% of the three applied doses remained in 

the diffusion chambers for the rat and <0.005, 0.005, 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 section. 

Table 6-141: Material balance and in vitro absorption of [14C]-BYI 08330 through human and rat 

epidermis expressed as percentage of the applied dose (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 
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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 absorption of [14C]-BYI 08330 through human and rat 

epidermis expressed as percentage of the applied dose (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 absorption of [14C]-BYI 08330 through human and rat 

epidermis expressed as percentage of the applied dose (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 
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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 doses, applied to rat and human skin membranes, 

are given in Tables 6-141, 6-142, and 6-143, respectively. The absorbed dose is expressed as the 

percentage of [C14]-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 chamber 

at termination (24 hrs). This amounted to 0.01% and 0.26% of the applied high dose in the human and 

rat skin, respectively. For the medium dose, the mean total amount of radioactivity directly absorbed

was 0.41 and 3.25% for human and rat skin, respectively. For the low dose, the mean total radioactivity 

directly absorbed was 0.56 and 12.22% for the human and rat skin, respectively. 

The study authors considered the potentially absorbable portion of the administered dose to be that 

found in the skin compartment excluding surface residual (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 was 

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 potentially absorbable in the human and rat skin, 

respectively. 

Given the uncertainty regarding actual deposition under actual field conditions, it is considered 

appropriate to derive an estimate of dermal absorption based on the results from the low dose group 

(0.05 mg a.i./mL dosing solution), since percent dermal absorption was greatest at this dose level. This 

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 cm2

). 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 to loss by desquamation. 

III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS: 

This study (No. SA 05254) was carried out to compare the in vitro dermal penetration of BYI 08330 

following single dermal application of [14C]-BYI 08330 to excised human skin and rat dermatomed skin. 

Dermal absorption was determined at three dose concentrations 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 epidermal membranes at a rate of 10μL/cm2. The 

membranes were held between chambers of flow through diffusion cells and receptor fluid was passed 

through the membranes at 1.5 mL/hr flow rate and collected from the effluent tubing using a fraction 

collector. After 8 hours exposure, the residual doses were washed off the skin and hourly sampling 
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was continued to termination at 24 hours. At termination the skin membranes were tape-stripped to 

remove residual surface dose and the stratum corneum. 

The percentage of [14C]-BYI 08330 directly absorbed over the 24 hr observation period was 0.01 and 

0.26% of the high dose, 0.41 and 3.25% of the medium dose and 0.56 and 12.22% of the low dose for 

human and rat skin, respectively. 

The percentage of [14C]-BYI 08330 considered potentially absorbable over the 24 hr observation 

period was 0.24 and 2.14% for the high dose, 2.74 and 23.21% for the medium dose and 8.45 and 

22.71% for the low dose for human and rat skin, respectively. 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 comparative in vitro percutaneous absorption of BYI 08330 

through rat and human epidermis following exposure to a [14C]-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 the 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 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 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 fraction 

collector were cycled every hour. Total radioactivity recovered from the chambers, skin wash, epidermal 

membranes, and receptor fluids was excellent, ranging 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 absorbable radioactivity ranged between 0.24-2.14%, 2.74-

23.21%, and 8.45-22.71% of the applied radioactivity 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 skin in vitro. Rat skin appeared to be more 

permeable than human skin. The potential absorption 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/cm2

). Although this omission does not affect the outcome of the experiments, data interpretation 

would have been more meaningful had the actual absorbed or potentially absorbable amounts of BYI 

08330 been calculated and reported. 

C. STUDY DEFICIENCIES: 

Data were reported as percent of applied radioactivity recovered and not as actual amounts of active 

ingredient by weight. Details of dose solution preparation were not given in the report. These omissions 

did not appear to affect the results of this in vitro dermal penetration study using membranes prepared 

from rat and human skin. 
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B.6.9.9.3 In vitro dermal penetration study–rat/human 

CITATION: Capt A. (2006) [14C]-BYI 08330 in SC240 formulation: Comparative in vitro dermal 

absorption study using human and rat skin. Bayer CropScience, 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 Chemistry, 

Wuppertal, Germany 

Other comments: None 

2. Relevance of test material to proposed formulation(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 (Biopredic, 

Rennes, France) 

Together (11 in wire-mesh bottomed, stainless steel cage 

during the acclimation period) 

Pelleted; A04 C-10 . SAFE. Rte de St Bros. Aigu, France; ad 
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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 formulation concentrate (240 mg/mL) or 

anticipated dermal deposition of field spray dilutions (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 [14C]-BYI 08330 in the dosing solutions were 2.68, 0.0088, 

or 0.0005 mg/10 μL dose (see Table 6-144 for details). Actual doses were calculated as the mean dose 

applied to each of 6 dermal membranes/dose rate/exposure 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: 10μL/cm2

 skin (2.54 cm2/membrane) 

Duration of exposures (time from dose to termination): 24 hours with skin wash after 8 hrs exposure. 

Termination periods (24 hrs): A reservoir was placed atop each membrane for receptor 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 tubing. Fractions were collected hourly following dosing and 

collected until termination at 24 hrs. The receptor 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 mass 

balance procedures.

Number of membranes /group: Six membranes (target number before exclusion of damaged 

membranes) were tested in individual diffusion cells/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 membrane 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-dermatome. Prior to use, the integrity of the selected 

membranes was assessed by measuring the trans-epidermal 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/m2/hr). Human and rat skin with a 

TEWL of >40g/m2/hr were considered potentially damaged and were not used. 

3. Dose preparation, application to membranes and quantification: 
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Preparation: Dosing suspensions (three formulations) were prepared in the Monheim Research and 

Development Centre of Bayer CropScience, Germany, and 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 receptor fluid and found to be soluble for 24 hrs at an

amount in 3 mL of 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 

exceeded the total quantity of {14C]-BYI 08330 applied to the membranes of any cell. Doses were 

checked for radioactivity before during and after dosing. Homogeneity was checked by taking five 

aliquots of each dose formulation before application. The formulations were considered to be 

homogeneous. 

Assembly of diffusion cells and application of dosing solutions: Glass flow-through diffusion cells 

(Franz cell modified, Gallas, France) were used with an exposed membrane area of 2.54 cm2. 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 pumped to a fraction collector at a flow rate of 1.5 mL/hr. 

The dosing solutions (high, medium, or low dose) were applied to the split-thickness skin samples with a 

pipette at 10 μL/cm2

 skin surface. Each dosing solution was assayed for 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 purity was determined by TLC and HPLC analysis of the 

dosing preparations. Homogeneity of the doses was determined 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/cm2) 

Actual [14C]-BYI 

08330 

concentration 

(mg/mL) 

Applied dose (μg 

[

14C]-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 Tables C4-C6, pp. 48-51; Study No. SA 05255. Means in columns 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 exposed to 

the high, medium, or low doses of [14C]-BYI 08330 for 8 hrs and washed by repeated swabbing after 

exposure to remove residual radioactivity. Tween 60 (1%) in phosphate buffered saline was used as the

swabbing agent. Swabbing was continued until no radioactivity could be detected on the sponges using 

a Geiger counter. Sampling of receptor fluid continued hourly until termination at 24 hrs. Membranes

were swabbed again at termination and all swabs were 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 membranes were removed and saved for analysis by 
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LSC. Receptor fluid at termination was removed from cells for analysis and the diffusion cell 

components were washed and the washings saved for analysis for mass balance. Samples were stored 

at -20 C until analyzed. 

5. Sample analysis: Material balance samples, receptor fluid samples, and aliquots of dosing solutions 

were analyzed using Liquid Scintillation Counting (LSC) techniques. Receptor fluid samples were 

analyzed directly by addition to scintillant for counting. Skin swabs were solubilized in Soluene and

scintillation fluid added for LSC analysis. Tape-strips were solubilized using tetrahydrofuran and 

scintillation fluid was added for direct LSC analysis. Skin samples were solubilized in soluene, 

scintillation fluid was added, and samples were counted by LSC. Diffusion cell components, including 

stirrers and tubing, were soaked in acetonitrile/distilled water (50:50 v/v) for 12 hrs and the washings 

analyzed by LSC. All samples were counted using a Packard 1900 TR Liquid Scintillation Spectrometer 

with on-line computing. Quenching effects were determined using external standardization and spectral

quench parameter (tSIE) methods. Efficiency correlation curves were prepared for each scintillation 

cocktail using radiolabeled n-hexadecane internal standards. The limit of detection was considered to be 

twice the background values for blank samples in scintillation 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 [14C]-BYI 08330 through rat and human epidermis at the

high, medium, and low dose rates are shown in Tables 6-145, 6-146, and 6-147, respectively. Recovery 

of radioactive material was excellent with means ranging from 92.88-99.46% of the applied dose for rats 

and 92.08-99.46% for humans. For the formulation concentrate, 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 high, medium, and low dose levels, respectively. 

Washing removed 91.37, 91.62, and 79.59% of the high, medium, and low doses, respectively, applied 

to human skin. The residual radioactivity remaining 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, and 

3.41% of the high, medium, and low doses remained with the epidermis after 24 hrs. In addition, a 

portion of the applied doses remained in the stratum corneum of both species. For the rat, 2.54, 9.33, 

and 6.73% of the respective high, medium and low doses remained with the stratum corneum. For 

human skin, 0.11, 0.87, and 7.82% of the added radioactivity remained in the stratum corneum for the 

high, medium, and low doses, respectively. At termination, <0.005% of the three applied doses 

remained in the diffusion chambers for the human and <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 absorption of [14C]-BYI 08330 through human and rat 

epidermis expressed as percentage of the applied dose (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 absorption of [14C]-BYI 08330 through human and rat 

epidermis expressed as percentage of the applied dose (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±17.96 

Total % recovery (material 

balance) 

94.73±4.70 99.46±7.34 

Data taken from Table 2, page 26; Study No. SA 05255 
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Table 6-147: Material balance and in vitro absorption of [14C]-BYI 08330 through human and rat 

epidermis expressed as percentage of the applied dose (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±3.29 

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 doses, applied to rat and human skin membranes, 

are given in Tables 6-145, 6-146, and 6-147, respectively. The absorbed dose is expressed as the 

percentage of [C14]-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 chamber 

at termination (24 hrs). This amounted to 0.01% and 0.29% of the applied high dose in the human and 

rat skin, respectively. For the medium dose, the mean total amount of radioactivity directly absorbed

was 0.07% and 1.49% for human and rat skin, respectively. 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 absorbable portion of the administered dose to be that 

found in the skin compartment excluding surface residual (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 medium dosage, 1.41% and 11.76% of the dose to 

human and rat skin, respectively, was considered potentially absorbable. For the low dose, 11.47% and

16.32% percent of the administered dose was considered potentially absorbable in the human and rat 

skin, respectively. 

Given the uncertainty regarding actual deposition under actual field conditions, it is considered 

appropriate to derive an estimate of dermal absorption based on the results from the low dose group 

(0.05 mg a.i./mL dosing solution) since percent dermal absorption was greatest at this dose level. This 

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 cm2

). 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 be labile to loss by desquamation. 
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III. DISCUSSION AND CONCLUSIONS:

A. CONCLUSIONS: 

This study (No. SA 05255) was carried out to compare the in vitro dermal penetration of BYI 08330 

following single dermal application of [14C]-BYI 08330 to excised human skin and rat dermatomed skin. 

Dermal absorption was determined at three dose concentrations 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 epidermal membranes at a rate of 10μL/cm2. The 

membranes were held between chambers of flow through diffusion cells, and receptor fluid was passed 

through the membranes at 1.5 mL/hr flow rate and collected from the effluent tubing using a fraction 

collector. After 8 hours exposure, the residual doses were washed off the skin and hourly sampling 

was continued to termination at 24 hours. At termination, the skin membranes were tape-stripped to 

remove residual surface dose and the stratum corneum. 

The percentage of [14C]-BYI 08330 directly absorbed over the 24 hr observation period was 0.1 and 

0.29% of the high dose, 0.07, and 1.49% of the medium dose and 0.24 and 6.99% of the low dose for 

human and rat skin, respectively. 

The percentage of [14C]-BYI 08330 considered potentially absorbable over the 24 hr observation 

period was 0.41 and 2.90% for the high dose, 1.41 and 11.76% for the medium dose and 11.47 and 

16.32% for the low dose for human and rat skin, respectively. 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 comparative in vitro percutaneous absorption of BYI 08330 

through rat and human epidermis following exposure to a [14C]-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 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 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 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 fraction collector were 

cycled every hour. Total radioactivity recovered from 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 the 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 0.24-6.99% of the applied dose for the high, medium, 

and low doses, respectively. The potentially absorbable radioactivity ranged between 0.41-2.90%, 

1.41-11.76%, and 11.47-16.32% of the applied radioactivity 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 skin in vitro. Rat skin appeared 

to be more permeable than human skin. The potential absorption differences for rat and human skin 

differed by factors of 7.1, 8.3, and 1.4 for the high, medium and low doses, respectively. 
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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/cm2

). Although this omission does not affect the outcome of the experiments, data interpretation 

would have been more meaningful had the actual absorbed or potentially absorbable amounts of BYI 

08330 been calculated and reported. 

C. STUDY DEFICIENCIES: 

Data were reported as percent of applied radioactivity recovered and not as actual amounts of active 

ingredient by weight. Details of dose solution preparation were not given in the report. These 

omissions did not appear to affect the results of this 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 was investigated in vivo in rats and in vitro on human and 

rat skin. In the in vivo study, male Wistar rats were administered nominal doses of 100, 15, or 5 μg a.i./cm2

of radiolabeled spirotetramat (OD150 formulation) to approximately 12 cm2

 of body surface for periods of 1, 

10, or 24 hours and monitored up to 168 hr post-dosing. Based on the likely worker exposure time frame 

and the increased absorption of spirotetramat from the site of application following exposure, it was 

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. 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 potentially absorbable radioactivity present in the stratum 

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 during the 168-hr time period while the amount of 

radioactivity in excreta increased, thereby implying that the compound continued to be absorbed after dosing 

was stopped. Dermal absorption values calculated from the 10hr exposure/168 hr post-exposure scenario, 

rather than from the 10 hr exposure scenario alone, were chosen for risk assessment, because the former 

scenario contained measures from excreta, which provided a more accurate estimate of actual and potential 

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 study with the OD 150 formulation, diffusion cells with 

epidermal membranes derived from rat or human skin were administered doses of 1.58, 0.013 or 0.0004 mg

of [14C]-BYI 08330 to 2.54cm2

 of epidermal surface and exposed for 8 hours, washed and monitored to 

termination at 24 hours post-dosing. The chosen doses were those from formulation concentrate and two

simulated aqueous spray dilutions of the OD150 formulation. Rat skin was more permeable to the 

treatments, and the potential 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. 

In the in vitro dermal absorption study with the SC 240 formulation, diffusion cells with epidermal membranes 

derived from rat or human skin were administered doses of 2.68, 0.088 or 0.0005 mg of [14C]-BYI 08330 to 

2.54cm2 of epidermal surface and exposed for 8 hours, washed, and monitored to termination at 24 hours 

post-dosing. Rat skin was more permeable to the treatments, and the potential 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.

In the US and Canada, in vitro data are not considered currently in the calculation of in vivo human dermal 

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 the in vivo study. The proposed dermal absorption value 

for spirotetramat is 10%, and this is based on the actual and potential absorption measured 168 hrs following 

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 hours 

and a period of 168 hours post application) together 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 demonstrate that no significant difference in dermal

absorption is detected between the OD 150 and SC 240 formulations. The same might be expected for the in 

vivo results. Therefore, the results of the in vivo study performed with the OD 150 formulation together with 

the results of the in vitro study performed with the SC 240 formulation are used to develop a proposal for 

dermal absorption of BYI 08330 in the SC 240 formulation for the EU. 
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B.6.10 Summary of mammalian toxicity and overall evaluation (OECD Annex IIA 5.11) 

The acute, short-term, and long-term toxicity of spirotetramat is well understood. Spirotetramat technical 

demonstrated moderate to low acute toxicity (Toxicity Category III or IV) via the oral, dermal, and inhalation 

routes. Spirotetramat is non-irritating to the skin (Toxicity Category IV), although it is an irritant to the eyes 

(Toxicity Category II) and exhibits a skin-sensitization 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 subchronic oral treatment of rats. Long-term toxicity studies 

reflected the short-term toxicological profile of spirotetramat with the thymus, thyroid, and brain as target 

organs following one-year oral exposure of dogs. Chronic exposure of rats to spirotetramat also reflected 

the subchronic pattern of testicular toxicity. No evidence of tumor formation was found following long-term 

studies of rodents, and spirotetramat was also negative for mutagenicity and clastogenicity in several

standard in vivo and in vitro assays. 

The reproductive and developmental toxicity potential of spirotetramat was tested in rats and rabbits. In 

addition to testicular histopathology observed following subchronic and chronic exposure of rats to 

spirotetramat, male reproductive toxicity was recorded in the 2-generation reproductive toxicity study. 

However, development of the sexual organs of offspring (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 with 

spirotetramat is necessary to produce male reproductive toxicity in rats. Similar effects were observed after 

repeated dosing with the enol metabolite of spirotetramat. Developmental toxicity was not observed with 

spirotetramat in the absence of maternal toxicity in either the rat or rabbit. 

Summary of mammalian toxicity and overall evaluation by AGES

ADME-behaviour 

The disposition of spirotetramat was investigated in three in vivo and one in vitro studies. Gastrointestinal 

absorption of spirotetramat was rapid following oral administration to male and female rats at a single dose 

of 2 or 100 mg/kg bw or repeated doses of 2 mg/kg bw for 14 days. The absorption rate in all tests was 

between 89 and 98% of the total recovered radioactivity, based upon recovery from the urine and carcass 

(without the gastrointestinal tract). No significant 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 was reached for all dose groups within 0.09 to 2.03 

hours after administration (values calculated by pharmacokinetic modelling). The radioactivity concentrations 

in plasma declined steadily by several orders of magnitude within 48 hours for all dose groups. Radioactivity 

in tissues and organs at the 48-hour termination were very low (<0.2% and below the limit of detection for 

some organs/tissues). Urinary excretion was very rapid (essentially complete in 24 hrs) and was the major 

excretion route at these doses. Faecal excretion accounted for 2-11% of the dose in male and female rats. 

The excretion behavior was similar for all dose groups. 

Spirotetramat (BYI 08330) was completely metabolized by the rat 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 the administered dose). All other identified metabolites could be derived from the enol 

intermediate. The second prominent metabolic transformation was oxidative demethylation of the 8-methoxy 

group to BYI 08330-desmethyl-enol (5-37% of the administered dose). Oxidation of the azaspiro moiety to 

BYI 08330-ketohydroxy and BYI 08330-desmethyl-ketohydroxy were detected as minor pathways. Other 
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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-related 

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 dose of 3 mg/kg bw in another disposition study using 

quantitative whole body autoradiography. The highest equivalent concentrations were observed in the liver, 

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 findings 

were consistent with saturation of cellular transport mechanisms. In addition, decline of tissue radioactivity 

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; however, BYI 08330-desmethyl-enol occurred at lower levels 

at 1000 mg/kg bw, and this may be a reflection of the decreased absorption at this dose. Similar to the low 

dose groups, BYI 08330-desmethyl-enol levels were greater in urine than in plasma and organs. The highest 

percentage of BYI 08330-desmethyl-enol was detected in liver and kidney. Results of this study indicate that 

saturation of active transport mechanisms occurs at a dose of 1000 mg/kg but not 2 mg/kg. This results in 

decreased excretion via urine and faeces and a potential for accumulation of BYI 08330 related residues in 

the body following repeated high doses. The results 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, resulting in a high body burden in multiple-dose toxicological 

studies. This assumes, however, that spirotetramat enters the systemic circulation as the metabolite BYI 

08330-enol. 

In a comparative in vitro metabolism study using hepatocytes from male rats, mice, and humans, differences 

in the proportions of several down-stream metabolites were observed; however, BYI 08330-enol was the first 

and most prominent metabolite and accounted for 66-100% of all metabolites across species. 

Acute toxicity 

Spirotetramat technical demonstrated moderate to low acute toxicity via the oral (LD50 > 2,000 mg/kg bw), 

dermal (LD50 > 2,000 mg/kg bw), and inhalation (LC50 > 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 potential under the 

conditions of the guinea pig maximization test and the local lymph node assay, but not the Buehler patch 

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 

LD50 > 2000mg/kg bw Eigenberg D.A. 

(2004) 

Acute Dermal rat Carboxymethylcellulose 

sodium salt solution 

LD50 > 2000mg/kg bw Eigenberg D.A. 

(2004) 

Acute Inhalation 

(solid aerosol) 

rat None LC50 > 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 Vohr H.W. 

(2002) 

Skin sensitisation Guinea pig Propylene glycol 400 Not a sensitiser Vohr H.W. 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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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 the short-term 

toxicological studies in rodents and dogs. Rats, mice, and dogs did not exhibit changes in plasma lipid 

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 thyroid hormones (T4 and/or T3) at ≥1200 ppm (32 mg/kg 

bw/day) were observed. However, correlative changes in thyroid weight, thyroid histopathology, or thyroid 

stimulating hormone (TSH) were not observed in either 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 following one-year oral exposure of dogs to spirotetramat. 

Thymus involution and brain dilation with dose-related severity were observed in males at ≥600 ppm (20 

mg/kg bw/day), while axonal degeneration in the hypothalamus was observed in one female at 1800 ppm 

(48 mg/kg bw/day). Brain dilation was observed in one 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 related 

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 indication 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 either sex. Clinical signs of neurotoxicity (dehydration, 

swelling, decreased activity and reactivity, seizures and ataxia) were observed in one male at the highest 

dose tested, a finding that was consistent with the multi-organ toxicity observed at this dose. No 

morphological changes in the testes were observed in 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 dose. 

Abnormal spermatozoa and hypospermia in the epididymis, 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 most animals after cessation of treatment. Other effects in 

subchronically treated rats were limited to declines in terminal body weight in 10000 ppm male rats and an 

increased incidence of accumulation of alveolar macrophages in both sexes at 10000 ppm. Thyroid and 

thymus were unaffected in rats at any dose. The NOAEL was considered to be 2500 ppm, equivalent to 148

mg/kg bw/d in male and 188 mg/kg bw/d in female rats. 

Unlike the rat, no adverse effects of any kind were observed in mice tested orally up to the limit dose. In vitro 

results from a comparative metabolism study using hepatocytes from male rats, mice, and humans revealed 

species differences in the metabolism of spirotetramat. Specifically, mouse hepatocytes were better able 

than rat or human liver cells to metabolize BYI 08330-enol via glucuronidation. Potentially lower levels of the 

enol metabolite in mice in vivo may account for the lack of testicular toxicity observed in this species. 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 yielded no evidence of systemic toxicity when spirotetramat 

was tested up to 1000 mg/kg bw/day. 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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Table B.6-136: Summarised results of subacute/subchronic toxicity studies with Spirotetramat 

Study; Reference Dose levels NOAEL Relevant effects at 

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 toxicologically 

significant changes at 

the highest dose tested 

Genotoxicity 

Overall, assays for point mutations and chromosomal aberrations (both in vivo and in vitro) were negative 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 Reference 

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 Reference 

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 for 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 results are consistent with the excretory and/or detoxification 

roles of the kidney and liver. Consistent with the subchronic study the lung demonstrated treatment-related 

presence of alveolar macrophages in mid and high dose males and high dose females. Abnormal sperm or 

spermatid degeneration in the testes and germ cell exfoliated debris in the epididymis were observed in male 

rats at the high dose (7500 ppm; 414 or 373 mg/kg bw/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 activity and responses to sensory stimuli. Treatment-related 

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 toxicity/cancerogenicity studies with Spirotetramat 

Study; Reference Dose levels NOAEL Relevant effects at 

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

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Study; Reference Dose levels NOAEL Relevant effects at 

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 following subchronic and chronic exposure of male rats to 

spirotetramat, evidence of male reproductive toxicity was provided in the 2-generation reproductive toxicity 

study. Abnormal sperm cells were reported in F1-generation male rats treated with 6000 ppm (419 mg/kg 

bw/day) spirotetramat in the diet, and decreased reproductive performance was also observed in one of 

these males. Similar results were obtained in the 1-generation reproductive toxicity range-finding study, 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 no fertility in parental generation animals. There were no 

implantation sites noted in the females due to treatment-related effects on sperm cells of males at this dose 

level (increased numbers of abnormal sperms, reduced epididymal sperm counts, decline in both motility and 

progression of epididymal sperm cells). Absolute and relative weight of the cauda epididymis was decreased 

in parental males. Histopathology showed abnormal sperm cells of minimal to moderate severity in the 

epididymis and the cauda epididymis. 

Renal toxicity was also observed in F1 adults in the 2-, but not 1-generation reproductive toxicity study. 

Offspring toxicity was limited to decreased body weights in both studies, observed in F1 and F2 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 parental 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 (balano-preputial separation, vaginal opening) was unaffected 

in both studies. Developmental toxicity in the absence 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 paper are: 

• Only in rats and only at very high doses the male reproductive system was affected (reproductive 

performance, sperm count, morphology and motility, weight of cauda epididymis, testicular and/or 

epididymal morphology). This is a high-dose phenomenon with a steep dose-response curve and 

reversibility after cessation of exposure. The overall 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 reproductive system in the subchronic and chronic studies 

even at doses exceeding the limit dose of 1000 mg/kg bw/day. 

• From multiple special mechanistic studies, the pathomechanism and toxicokinetics in rats and the 

relevance for humans are well understood: 

1. More than 10 days of treatment with repeated daily 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 dysregulation. 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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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 special 

toxicokinetic features: Due to the anionic state of the enol, hepatic and renal elimination of BYI 08330-

enol occur via very efficient active transport processes (against a concentration gradient) which can 

only be overwhelmed (saturated) by very high (approx. 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 study using hepatocytes from male rats, mice and 

humans revealed species differences in the proportion of spirotetramat metabolites formed. 

Specifically, mouse hepatocytes were better able than rat liver cells to metabolize BYI 08330-enol via

glucuronidation. The metabolic profile generated from 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 transformation noted in rats. Results obtained from rat 

hepatocytes corresponded to in vivo metabolism findings in the rat. 

8. This efficient metabolic elimination in mice is the obvious reason for the absence of testicular toxicity 

even at doses >1000 mg/kg bw. Based on these metabolic features, it can be expected that humans 

are, similar to mice, less sensitive than rats to effects of BYI 08330 on the male reproductive system. 

9. The rat is therefore not the relevant species for human risk assessment. But even if humans would be 

as sensitive as rats, humans will not develop testicular toxicity under practical conditions (>6000-fold 

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 repeated-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-finding study, decreased sperm motility and progression 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 generation 

animals. 

The NOAEL for reproductive toxicity was 70 mg/kg bw/day. So the effects on the male reproductive system 

are no “high dose phenomenon”.

The effects on testicular spermatogenesis were attributed to the BYI 08330-enol, because it is the main 

metabolite in the rat. But it can not be excluded that other metabolites can cause the same effects. 

BYI 08330-enol is further metabolised by oxidation reactions to BYI 08330-desmethylenol, BYI 08330-enolalcohol and BYI 08330-ketohydroxy. Oxidation products accounted for approximately 14%. Conjugation was

not detected. In the mouse, conjugation of BYI 08330-enol with glucuronic acid accounted for approximately 

30%. In human hepatozytes, conjugation to BYI 08330-enol-glucuronic acid was 6%. The in vitro conjugation 

rate is dependent on the concentration used and declined in mice from 30% to 9% and in humans from 6% 

to 2% at liver concentrations of 19 μg/g and 190 μg/g BYI 08330, respectively. Glucuronidation of the BYI 

08330-enol in mice leads to much lower systemic levels of free BYI 08330-enol when compared to the rat. 
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Spirotetramat – Volume 3, Annex B.6 Toxicology and metabolism 

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The conjugation enables the mouse to utilize separate active transport systems in the kidneys, thus avoiding 

a saturation of the elimination process. Thus, the utilization of different transport systems renders the mouse 

less sensitive to BYI 08330-mediated testicular toxicity 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 mouse 

(dependent on the concentration, in humans 6% respectively 2%, in mice 30% respectively 9%). Therefore a 

similarity in the metabolic pathway can not be followed. As for humans conjugation is only 2% at high doses, 

it can not be assumed that humans are less sensitive to Spirotetramat than rats. 

Due to abnormal sperm cells, decreased sperm motility 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 fertility“ according to Annex VI of the EC Council Directive 

67/548/EEC. 

In the developmental toxicity study in rats, toxicity 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 fetal 

weight and increased incidences of skeletal malformations and skeletal deviations were observed at 1000 

mg/kg bw/day. Malformations at the high dose included one case of supernumerary lumbar vertebra, one 

case of cleft palate and one case of co-arctation of aortic arch. One case of atrial septal defect of the heart 

and microphthalmia were observed in the control, low 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 with 

pelvic shift (1.1 %) were observed in the high dose. Historical control data of the performing laboratory 

(Bayer HealthCare AG) for dysplastic forelimb bones in studies conducted in the years 1999 – 2004 showed 

26 affected animals out of 1975 animals, a percentage 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 concurrent 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 in 

historical controls in studies conducted 1999 – 2006 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 observations 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 compared to 

concurrent and historical control values . Statistically significantly increased numbers of fetuses with 14th ribs 

were observed at a dose level of 1000 mg/kg/d spirotetramat. 

Table B.6-153: Percentage of observations at the ribs 

Dose (mg/kg/d) 

Observations 0 20 140 1000 

Historical control data (range) 
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Wavy ribs 5.5 17** 17.8** 57.5** (2.7 – 15.1) 

[1996 – 2001] 

14th rib 15 10.7 12.6 45.9** (0.0 – 13) 

[1996 – 2000] 

The maternal and the developmental NOAEL for this study was set at 140 mg/kg bw/d. 

Due to increased incidences of skeletal malformations and skeletal deviations and according to Annex VI 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 – Possible risk of harm to the unborn child“. 

In the developmental toxicity study in rabbits, offspring toxicity was not observed at any dose up to 160 

mg/kg bw/day. However, maternal toxicity was observed at ≥40 mg/kg bw/day, including a dose-dependent 

increase in abortion and clinical signs of toxicity in affected animals (severely reduced food consumption, 

body weight loss, alopecia, altered appearance of feces, discoloured urination). Gross pathology revealed 

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 toxicity/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 neurotoxicity in two acute neurotoxicity studies in the rat 

(Gilmore, 2005) and was shown to have no neurotoxic potential in these studies. 
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In the first acute neurotoxicity study, rats received single oral doses of 0, 200, 500 or 2000 mg/kg bw 

spirotetramat via gavage. Evidence of acute oral toxicity was observed in both sexes at all dose levels but 

was limited to clinical signs (urine and perianal stain) and decreased activity in the figure-eight maze 

beginning on the day of treatment and with complete recovery by day 7. 

In a follow-up study, rats received single oral doses 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 established at 100 mg/kg bw spirotetramat. Neurotoxic

effects were not observed up to a limit dose of 2000 mg/kg bw. 

Further toxicological studies 

Toxicity studies on metabolites

Acute oral (rat) and genotoxicity (bacterial reverse mutation) testing was conducted on the following four 

plant metabolites of Spirotetramat (BYI 08330) exceeding 0.01 mg/kg raw foodstuff for human consumption 

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 were 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 and 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 resulting in different mono-, di- and trioxygenated metabolites 

which are probably represented by isomers of mono-hydroxy, di-hydroxy and tri-hydroxy metabolites. Thus, 

there is no toxicological concern for the BYI 08330-cis-ketohydroxy residues and BYI 08330-desmethylketohydroxy residues found in plants because these plant metabolites were formed in relevant amounts in 

rats and have therefore been co-tested in the toxicity 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 mutagenicity) were equivalent to those observed with the parent 

compound and uncritical. 

Toxicity tests were not performed with the plant metabolite 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 in rats 

(Kennel P., 2005; Rep.Nr. SA 04181), decreased epididymal sperm counts were recorded ≥10 days of 
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treatment with 1000 mg/kg bw/day by gavage. Repeated dosing, therefore, is necessary to produce male 

reproductive toxicity in rats. 

In a second mechanistic study, male rats were treated by gavage with the enol metabolite of spirotetramat 

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 observed in the testes-epididymides of treated animals. 

Therefore, male reproductive toxicity in rats is likely due to the enol metabolite of spirotetramat. 

For the Toxicological Risk Assessment for the EU please see Appendix. 

B.6.10.1 Proposal for ADI 

EPA and AGES: The acceptable daily intake (ADI) was determined to be 0.05 mg/kg bw/day, based on the 

NOAEL of 5 mg/kg bw/day obtained in the one-year oral toxicity study in the dog and an uncertainty factor of 

100 (10 for intra-species variability and 10 for interspecies extrapolation). 

PMRA: In addition, it was considered appropriate to retain the PCPA factor, but that it could be reduced from 

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 be 1 mg/kg bw, based on the NOAEL of 100 mg/kg bw 

obtained in the acute neurotoxicity study in the rat and an uncertainty factor of 100 (10 for intra-species 

variability and 10 for interspecies extrapolation).

B.6.10.3 Proposal for drinking water limit 

The determination of a maximum allowable concentration (MAC) value in drinking water is not necessary,

because according to Directive 91/414/EC only the ADI, ARfD and AOEL values have to be determined. 

According to Directive 98/83/EC a drinking water limit of 0.1 μg spirotetramat/l is established. 

B.6.10.4 Proposal for AOEL 

The acceptable operator exposure level (AOEL) was determined to be 0.05 mg/kg bw/day, based on the 

NOAEL of 5 mg/kg bw/day obtained in the one-year oral toxicity study in the dog and an uncertainty factor of 

100 (10 for intra-species variability and 10 for interspecies extrapolation). 
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B.6.11 Acute toxicity of the preparations (OECD Annex 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 Toxicity in the Rat After Oral 

Administration. Project Number: T0075503, AT02161. 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 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 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: 
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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 animals for 

approximately 16 - 24 h before administration of the 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 necropsy 

 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 death Mortality 

(%) 

Females 

2,000 1st 0 0 3 -- -- 0 

2,000 2nd 0 0 3 -- -- 0 

acute oral LD50**: > 5,000 mg/kg bw 

* 1st number = number of dead animals; 2nd number = number of animals with signs; 

3

rd number = number of animals used; ** according to the 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 by the treatment 

D. Necropsy: No particular gross pathological changes were observed 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 three 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 
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water and the administration volume was 10 ml/kg body weight. For dosing, food was withheld from 

the animals for approximately 16-24 hours before dosing 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 days 

after dosing. A gross necropsy examination was performed on all animals at scheduled euthanasia. 

All animals survived and gained weight during the study. No clinical signs or gross pathological 

findings were observed. 

Oral LD50 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 LD50 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 Toxicity 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 
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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 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: 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 animals for 

approximately 16 - 24 h before administration of the 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 necropsy 

 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 1st 0 0 3 -- -- 0 

2,000 2nd 0 0 3 -- -- 0 

acute oral LD50**: > 5,000 mg/kg bw 
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April 2008

* 1st number = number of dead animals; 2nd number = number of animals with signs; 

3

rd number = number of animals used; ** according to the 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 by treatment. 

D. Necropsy: No particular gross pathological changes were observed 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 three 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 the 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 days after 

dosing. A gross necropsy examination was performed on all animals at scheduled euthanasia. All animals 

survived and gained weight during the study. No clinical signs or gross pathological findings were 

observed. 

Oral LD50 Females > 2000 mg/kg bw 

Based on the lack of mortality at 2000 mg/kg, BYI 08330 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 acute 

oral study (OPPTS 870.1100; OECD 425) in the rat. 

C. Reviewer Conclusions: All countries agree the oral LD50 for BYI 08330 240 SC for female rats was > 

2000 mg/kg. 

D. Study Deficiencies: None.
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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 Toxicity 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 polycarbonate 

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 
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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 weight, 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 LD50: > 4,000 mg/kg bw 

* 1st number = number of dead animals:; 2nd 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 by treatment in males. A slight 

decrease in body weight was observed on day 8 in two females and on day 15 in two other females. 

This effect is assumed to be caused by the stress of the occlusive dressing, and is not considered 

test compound related. 

D. Necropsy: No particular gross pathological changes were observed in animals sacrificed at the 

end of the study period. 

III. Discussion and Conclusions: 

A. Conclusions: The formulation Spirotetramat 150 OD is non-toxic after acute dermal 

administration. 

B. Reviewer Comments: In an acute dermal toxicity study, five young Wistar (HsdCpb:Wu) rats/sex 

(age: 9-13 weeks; weight: 213-232 g; 211-219 g females; source: Harlan/Winklemann GmbH, 

Borchen, Germany) were exposed to a single dermal application of BYI 08330 150 OD [Batch No. 

08030/0189(0152); light-brown suspension] for 24 hours. 
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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 days 

after dosing. A gross necropsy examination was performed on all animals at scheduled euthanasia. 

All animals survived the study. All male animals gained body weight during the study. A slight 

decreased in body weight was observed on Day 8 of the study in two females and on Day 15 in two 

other female animals. No clinical signs or gross pathological findings were observed. 

Dermal LD50 Males > 4000 mg/kg bw 

Dermal LD50 Females > 4000 mg/kg bw 

Dermal LD50 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 the rat. 

C. Reviewer Conclusions: All countries agree the dermal LD50 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 Toxicity in the Rat after Dermal 

Application. Project Number: AT02165, T3075524. Unpublished 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 
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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 polycarbonate 

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 weight, 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 LD50: > 4,000 mg/kg bw 
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April 2008

* 1

st number = number of dead animals:; 2nd 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 the 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 weight gain of the females in the first week is 

assumed to be caused by the stress of the application procedure, and is not considered test 

compound related. 

D. Necropsy: No particular gross pathological changes were observed 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 young Wistar (HsdCpb:Wu) 

rats/sex (age: 9-13 weeks; weight: 206-222 g for males; 208-225 g for females; source: 

Harlan/Winklemann GmbH, Borchen, Germany) were exposed to a single dermal application of BYI 

08330 240 SC [Batch No. 07218/0073(0020); white suspension] 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 days 

after dosing. A gross necropsy examination was performed on all animals at scheduled euthanasia. 

All animals survived the study. All male animals gained body weight during the study. A slight, 

transient decrease in body weight was observed at Day 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 the treatment area. No gross pathological findings 

were observed. 

Dermal LD50 Males > 4000 mg/kg bw 

Dermal LD50 Females > 4000 mg/kg bw 

Dermal LD50 Combined > 4000 mg/kg bw 

Based on no mortality at 4000 mg/kg bw, BYI 08330 240 SC 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 the rat.

C. Reviewer Conclusions: All countries agree the dermal LD50 for BYI 08330 240 SC for male and 

female rats was > 4000 mg/kg. 

D. Study Deficiencies: None. 
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B.6.11.3 Acute inhalation toxicity to rats (OECD Annex IIIA 7.1.3) 

CITATION: Pauluhn, J. (2005) BYI 08330 150 OD: Acute Inhalation Toxicity in Rats. Project 

Number: T6075301, AT02396. Unpublished study prepared 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, Switzerland 

 Water: tap water ad libitum

 Housing: During the acclimation and study periods, the animals 

were housed singly in conventional Makrolon® Type III 

cages (based on A. Spiegel and R. Gönnert, Zschr. 

Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. 

Versuchstierkunde, 7, 144-153 (1965)). Cages were 

changed twice a week while unconsumed feed and water 

bottles were changed once per week. The legal 

requirements for housing experimental animals (Directive 

86/609 EEC) were followed. Bedding consisted of type 

BK8/15 low-dust wood granulate from Ssniff, 

Soest/Westfalen, Germany. The wood granulate was 

randomly checked for harmful constituents at the request 

of the Laboratory Animal Services, Bayer Healthcare AG. 

B. Study Design and Methods: 

1. Animal assignment and treatment 
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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 temperature, 

reflex measurements, gross necropsy 

 In life dates: 2005-05-03 – 1005-05-18 

2. Generation of the test atmosphere / chamber description 

Generation and characterization of chamber atmosphere 

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 = Geometric Standard Deviation; 

-- not applicable. 

* Humidity was lower due to the use of dry air for the dispersion of the test substance. This deviation from the 

guideline had no apparent negative impact on the outcome of the study. 

Test atmosphere 

concentration: 

The nominal concentration was calculated from the ratio 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 concerning 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 evaporation of 

constituents contained in the formulation which also 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. 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 aluminum foil 

and glass fiber filter which were subjected to gravimetric analysis 
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as described in section 7.7. An adhesive stage coating (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 LC50: > 1,760 mg/m3 air 

* 1st number = number of dead animals; 2nd number = number of animals with signs; 

 3rd number = number of animals exposed 

B. Clinical observations: The following clinical signs were observed in the test substance groups: 

Males: labored breathing patterns, bradypnea, motility reduced, piloerection, hair-coat ungroomed, 

high-legged gait, nose: red encrustations, nasal discharge (serous). 

Females: labored breathing patterns, bradypnea, piloerection, 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 period: 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; weight: 168-199 g males; 168-175 g females; source: 

Harlan-Winkelmann GmbH, Borchen, Germany) were exposed via the inhalation (nose-only 

exposure) route to BYI 08330 150 OD [Batch No. 08030/0189(0152); light-brown suspension] for 4 

hours at a concentration of 1.76 mg/L (MMAD = 2.2 μm; GSD = 2.2). The liquid aerosol generated 

(undiluted test material) was respirable to rats. 
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Body weights were measured before the exposure period, on days 3 and 7, and weekly thereafter. 

The period of observation was for two weeks. On the day of exposure, the test animals were 

observed several times for clinical signs of toxicity and at the least once daily thereafter. A gross

necropsy examination was performed on all animals at the time of scheduled euthanasia. 

All animals survived the study. One male and two female animals had decreased body weights on 

Day 3 of study. By Day 7, these animals had surpassed their initial body weights and continued to 

gain weight till the end of the study. The remaining animals showed increased body weights at each 

weighing interval. Clinical signs observed in males 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-coat ungroomed, high-legged gait, nose: red 

encrustations, nose: reddened. All animals were without clinical signs on the fourth post-exposure 

day. No gross pathological findings were observed.

LC50 Males > 1.760 mg/L 

LC50 Females > 1.760 mg/L 

LC50 Combined > 1.760 mg/L 

Based on the lack of mortality at the LC50, BYI 08330 150 OD is classified as EPA Toxicity Category

III. 

This acute inhalation study is classified as Acceptable. 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 LC50 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 Inhalation Toxicity in Rats. Project 

Number: AT02374, T5075300. Unpublished study prepared 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 
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 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, Switzerland 

 Water: Tap water ad libitum

 Housing: During the acclimation and study periods, the animals 

were housed singly in conventional Makrolon® Type III 

cages (based on A. Spiegel and R. Gönnert, Zschr. 

Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. 

Versuchstierkunde, 7, 144-153 (1965)). Cages were 

changed twice a week while unconsumed feed and water 

bottles were changed once per week. The legal 

requirements for housing experimental animals (Directive 

86/609 EEC) were followed. Bedding consisted of type 

BK8/15 low-dust wood granulate from Ssniff, 

Soest/Westfalen, Germany. The wood granulate was 

randomly checked for harmful constituents at the request 

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 temperature, 

reflex measurements, gross necropsy 

 In life dates: 2005-05-10 – 2005-05-25 
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2. Generation of the test atmosphere / chamber description 

Generation and characterization of chamber atmosphere 

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 = Geometric Standard Deviation; -- not applicable. 

Test atmosphere 

concentration: 

The nominal concentration was calculated from the ratio 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 concerning 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 evaporation of 

constituents contained in the formulation which also 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. 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 aluminum foil 

and glass fiber filter which were subjected to gravimetric analysis 

as described in section 7.7. An adhesive stage coating (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 death Mortality 

[%] 

Males 
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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 LC50: > 3,013 mg/m3 air 

* 1st number = number of dead animals; 2nd number = number of animals with signs; 

 3rd number = number of animals exposed 

B. Clinical observations: Group 1: All rats tolerated the exposure without specific signs. 

Group 2/males: Piloerection, hair-coat ungroomed, bradypnea, 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 group did not reveal 

toxicologically significant decreases in body weights. 

D. Necropsy: Animals sacrificed at the end of the observation period: Macroscopic findings 

amongst the groups were indistinguishable.

III. Discussion and Conclusions: 

A. Conclusions: The formulation Spirotetramat 240 SC 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; weight: 174-199 g males; 171-175 g females; source: 

Harlan-Winkelmann GmbH, Borchen, Germany) were exposed via the inhalation (nose-only 

exposure) route to BYI 08330 240 SC [Batch No. 07218/0073(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 period, on days 3 and 7, and weekly thereafter. 

The period of observation was for two weeks. On the day of exposure, the test animals were 

observed several times for clinical signs of toxicity and at the least once daily thereafter. A gross

necropsy examination was performed on all animals at 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-coat ungroomed, bradypnea, labored breathing 

pattern, irregular breathing pattern, motility reduced and high-legged gait. All clinical signs resolved 

by fourth post-exposure day. No gross pathological findings were observed. 

LC50 Males > 3.013 mg/L 

LC50 Females > 3.013 mg/L 

LC50 Combined > 3.013 mg/L 

Based on no mortality at the limit dose, BYI 08330 240 SC is classified as EPA Toxicity Category IV. 
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April 2008

This acute inhalation study is classified as Acceptable. 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 LC50 for BYI 08330 240 SC for male 

and female rats was > 3.013 mg/L. 

D. Study Deficiencies: None. 
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April 2008

B.6.11.4 Skin irritation (OECD Annex IIIA 7.1.4) 

CITATION : Schungel, M. (2005) BYI 08330 150 OD: Acute Skin Irritation/Corrosion on Rabbits. 

Project Number: T6075365, AT02359. Unpublished study 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 cages 

contained low dust wood granulate bedding (J. Rettenmaier 

& Söhne, 73479 Ellwangen-Holzmühle, Germany). 

The wood granulate was changed at least twice weekly. The 

water bottles were changed weekly. 

The animals were regularly transferred to clean cages. 

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 
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April 2008

 Group size: 3 rabbits 

 Observations: Clinical signs, skin effects, body weight (at beginning of 

study) 

 In life dates: 2005-05-10 – 2005-05-17 

II. Results and Discussion: 

A. Findings: Under the present test conditions the following findings were noted: 

Animal no. one: erythema (grade 2) up to 72 hours examination time point. 

Animal no. two: erythema (grade 1) up to 72 hours examination time point. 

Animal no. three: erythema (grade 1) at 24 and 72 hours 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 = Erythema and eschar formation, O = Oedema formation; na = not 

applicable 

III. Discussion and Conclusions: 

A. Conclusions: The formulation Spirotetramat 150 OD is non-irritant to the skin. 
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April 2008

B. Reviewer Comments: In a primary dermal irritation study, 0.5 ml of BYI 08330 150 OD [Batch No. 

08030/0189(0152); light-brown suspension] was applied 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-lateral 

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 for 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 test patches applied to determine the extent of dermal

irritation. The first patch was removed after three minutes. As no serious dermal reactions were 

observed, the second patch was removed after one hour. At this stage the dermal observations 

indicated that the exposure could be allowed to extend to 4 hours, therefore the third patch was 

removed after four hours and the responses were graded one hour later. The study was completed 

using two additional animals, exposed for four hours. 

Dermal irritation was scored at 1, 24, 48 and 72 hours after patch removal. If no irritation was observed 

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 the changes had completely subsided. The degree of 

dermal irritation was recorded as specified by Draize and any serious lesions or toxic effects other than 

dermal irritation were also recorded. 

At one hour, all three animals were observed with very slight erythema (score 1). At 24 hours, two 

animals were observed with very slight erythema (score 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 observed with very slight erythema and one animal with

well defined erythema. By day 7, all dermal irritation was resolved. Primary Dermal Irritation Index 

(PDII) = 1.33. 

Based on the observation of very slight to well defined erythema observed at 72 hours, BYI 08330150 

OD is classified as EPA Toxicity Category IV. 

This study is classified as Acceptable. It does satisfy the guideline requirement for a primary dermal 

irritation study (OPPTS 870.2500; OECD 404) in the rabbit. 

C. Reviewer Conclusions: All countries agree BYI 08330 150 OD causes slight dermal irritation. 

D. Study Deficiencies: None. 

CITATION: Schungel, M. (2005) BYI 08330 240 SC: Acute Skin Irritation/Corrosion on Rabbits. 

Project Number: AT02291, T7075366. Unpublished study prepared by Bayer Ag 

Inst. of Toxicology. 20 p. August 19, 2005. 

I. Materials and Methods: 

A. Materials 
– – 

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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 cages 

contained low dust wood granulate bedding (J. Rettenmaier 

& Söhne, 73479 Ellwangen-Holzmühle, Germany). 

The wood granulate was changed at least twice weekly. The 

water bottles were changed weekly. 

The animals were regularly transferred to clean cages. 

The animal room had a standardized climate: 

Room temperature: 23 ± 3°C; Air humidity: 50 ± 25%; 

Light/ Dark cycle: 12 hour rhythm. 
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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 beginning of 

study) 

II. Results and Discussion: 

A. Findings: None of the three animals showed skin irritating effects. 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 = Erythema and eschar formation, O = Oedema formation; na = not 

applicable 

III. Discussion and Conclusions: 

A. Conclusions: The formulation Spirotetramat 240 SC is non-irritant to the skin. 
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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 for 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 test patches applied to determine the extent of 

dermal irritation. The first patch was removed after three minutes. As no serious dermal reactions 

were observed, the second patch was removed after one hour. At this stage the dermal observations 

indicated that the exposure could be allowed to extend to 4 hours, therefore the third patch was 

removed after four hours and the responses were graded one hour later. The study was completed 

using two additional animals, exposed for four hours. 

Dermal irritation was scored at 1, 24, 48 and 72 hours after patch removal. If no irritation was 

observed after 72 hours the study was completed. The degree of dermal irritation was recorded as 

specified by Draize and any serious lesions or toxic 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 satisfy the guideline requirement for a primary dermal 

irritation study (OPPTS 870.2500; OECD 404) in the rabbit. 

C. Reviewer Conclusions: All countries agree BYI 08330 240 SC is not an irritant to the skin. 

D. Study Deficiencies: None. 
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B.6.11.5 Eye irritation (OECD Annex IIIA 7.1.5) 

CITATION: Schungel, M. (2005) BYI 08330 150 OD: Acute Eye Irritation on Rabbits. Project 

Number: T2075370, AT02358. Unpublished study prepared 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 cages 

contained low dust wood granulate bedding (J. Rettenmaier 

& Söhne, 73479 Ellwangen-Holzmühle, Germany). The 

wood granulate was changed at least twice weekly. The 

animals were regularly transferred to clean cages. The 

animal room had a standardized climate: Room 

temperature: 20 ± 3°C; Air humidity: 50 ± 25%; Light/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 instillation. 
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 Group size: 3 rabbits 

 Observations: Clinical signs, eye effects, body weight (at beginning of 

study) 

 In life dates: 2005-05-24 – 2005-06-14 

II. Results and Discussion: 

A. Findings 

Under the present test conditions the following findings 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, grade 2 after 48 hours and grade 

1 after 72 hours. Conjunctival chemosis (grade 2) after 24 hours and grade 1 

after 48 hours. 

 Animal no. two: Corneal opacity (grade 1) after 24 hours and grade 1 up to 72 hours. 

Conjunctival redness (grade 2) after 24 hours and up to 72 hours. Conjunctival 

chemosis (grade 2) after 24 hours and up to 72 hours. 

 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 up 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 “positive” 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: 
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A. Conclusions: The formulation Spirotetramat 150 OD is non-irritant to the eyes. 

B. Reviewer Comments: In a primary eye irritation study, 0.1 ml of 08330150 OD [Batch No. 

08030/0189(0152); light-brown suspension] was instilled into the conjunctival sac of one eye in each 

of the three young healthy female Crl:KBL(NZW)BR rabbits (age: not reported; weight: 2.4-2.8 kg; 

source: Charles River, KiBlegg, Germany). The other untreated eye served as control. The eye was 

not rinsed for at least 24 hours following instillation. If one hour after instillation a severe irritation 

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 irritation 

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 recorded as specified by Draize. 

Corneal opacity 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. 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 satisfy 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 irritant. 

D. Study Deficiencies: None. 

CITATION: Schungel, M. (2005) BYI 08330 240 SC: Acute Eye Irritation on Rabbits. Project 

Number: AT02290, T0075369. Unpublished study prepared 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 
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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 cages 

contained low dust wood granulate bedding (J. Rettenmaier 

& Söhne, 73479 Ellwangen-Holzmühle, Germany). The 

wood granulate was changed at least twice weekly. The 

animals were regularly transferred to clean cages. The 

animal room had a standardized climate: Room 

temperature: 20 ± 3°C; Air humidity: 50 ± 25%; Light/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 beginning 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 24 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 
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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 “positive” 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-irritant to eyes. 

B. Reviewer Comments: In a primary eye irritation study, 0.1 ml of BYI 08330 240 SC [Batch No. 

07218/0073(0020); white suspension] was instilled into 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 least 

24 hours following instillation. 

Eye irritations were scored and recorded at 1, 24, 48 and 72 hours after instillation. If no irritation indices 

were observed after 72 hours, the study was completed. The degree of ocular irritation was recorded as 

specified by Draize. 

No corneal opacity or iritis was observed during the 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 Category III based on the conjunctival redness observed

at 24 hours. 

This study is classified as Acceptable. It does satisfy 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 240 SC is slightly irritating to the eyes based 

on the conjunctival redness (score of 2) observed at 24 hours 

D. Study Deficiencies: None. 
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B.6.11.6 Skin sensitisation (OECD Annex IIIA 7.1.6)

CITATION: Vohr, H. (2005) BYI 08330 150 OD: Study for the Skin Sensitization Effect in Guinea Pigs 

[Buehler Patch Test]. Project Number: T6075581, AT02570. 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 formulated (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 were 

exchanged for ones with clean bedding two times per week. 

Low-dust wood shavings supplied by Rettenmaier & Söhne, 

GmbH & Co, 73494 Rosenberg, Germany were used as 

bedding. The wood shavings were spot checked for 

contaminant levels. Records of these test results are 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 
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 Dose:

 1st to 3rd induction: The animals were dermally treated with the test material 3 

times at intervals of 7 days. The 1st to 3rd induction were 

performed with the 0.48% test item concentration. The animals 

in the test item group were treated with a hypoallergic patch 

loaded with the test item applied to the left flank and held in 

place on the skin with adhesive plaster. The volume applied 

per animal was 0.5 ml. The patches were removed after an 

exposure period of six hours, and any remaining test item was 

removed with physiological saline solution. The treatment 

areas were visually assessed 30 hours after initiation of 

exposure. For this, the treatment areas were not shorn or 

chemically depilated. 

Challenge: The challenge was performed 2 weeks after the last dermal 

induction. For the challenge the backs and right flanks 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 right 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. 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 six-hour exposure period, the patches were removed and 

the remaining test item was rinsed away with sterile 

physiological saline solution. 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 exposure period of six hours, 

and any remaining test item was removed with physiological 

saline solution. The volume applied per animal was 0.5 ml. The 

treatment areas were visually assessed 30 hours after 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 females 

Observations: Mortality, clinical signs, skin effects, body weight 

(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: 
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A. Findings: There were no skin effects in the animals of the test item group and the control group 

during the first to third induction treatment. The challenge with the 0.48 % test item concentration led to 

skin effect (grade 1) in 1 of 20 animals of the test item group (5 %) and to no skin effects in the control 

group. Single animals showing slight reaction (grade 1) can be detected from time to time not depending 

on whether they were treated with vehicle or test item. Thus, such skin effects of single animals might 

appear by chance. 

Number of animals exhibiting skin effects during challenge 

Test item group (20 animals) Control group (10 animals) 

Test item patch Control patch Test item patch Control 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 alpha hexyl cinnamic aldehyde showed that the study 

was able to detect sensitizing compounds in the 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 “ready to use” test formulation exhibits no skin sensitization potential 
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B. Reviewer Comments: The Buehler epicutaneous patch test was performed on 30 female Crl:HA 

guinea pigs (20 animals for the test Item group and 10 control animals) (age: 5-6 weeks; weight: 290-388 

g; source: Charles River Laboratory Animal Breeders, KiBlegg, Germany) to determine whether the test 

item “BYJ 08330 150 OD ready to use dilution” (content of the active ingredient: BYI 08330: 148.89 g/l) 

exhibits skin-sensitizing properties. 

The study was conducted with the following test item concentrations: 

1

st to 3rd induction: 0.48% 

Challenge: 0.48% 

The test item was diluted with Lewatit® water to the 0.48% ready to use concentration (suspension). 

There were no skin effects in the animals of the test item group and the control group during the first 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%) and 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 item group and in the control group it can be concluded 

that under the conditions of the Buehler Patch Test and with respect to the evaluation criteria the BYI 

08330 150 OD ready to use dilution exhibits no skin-sensitization potential. 

This study is classified as Acceptable. It does satisfy the guideline requirement for a dermal sensitization 

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 dermal sensitization potential. 

D. Study Deficiencies: None. 

CITATION: Vohr, H. (2005) BYI 08330 150 OD: Study for the Skin Sensitization Effect in Guinea 

Pigs. Project Number: T9074945, AT01873. Unpublished 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 
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 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 were 

exchanged for ones with clean bedding two times per week. 

Low-dust wood shavings supplied by Rettenmaier & Söhne, 

GmbH & Co, 73494 Rosenberg, Germany were used as 

bedding. The wood shavings were spot checked for 

contaminant levels. Records of these test results are 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:

 1st to 3rd induction: The animals were dermally treated with the test item 3 times at 

intervals of 7 days. The 1st to 3rd induction were performed with 

the 100% test item concentration. The animals in the test item 

group were treated with a hypo allergic patch loaded with the 

test material applied to the left flank and held in place on the 

skin with adhesive plaster. The patches were removed after an 

exposure period of six hours, and any remaining test item was 

removed under running water. The volume applied per animal 

was 0.5 ml. The treatment areas were visually assessed 30 

hours after initiation of exposure. For this, the treatment areas 

were not shorn or chemically depilated. 
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 Challenge: The challenge was performed 2 weeks after the last dermal 

induction. For the challenge the backs and right flanks of the 

animals were shorn one day prior to the challenge. During the 

challenge a hypoallergenic patch loaded with the 100% 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 flank, 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 six-hour 

exposure period, the patches were removed and the remaining 

test item was rinsed away with sterile physiological saline 

solution. Twenty-one hours later the skin of the animals was 

shorn in the region of the treatment sites. 

 Naive Controls: In the case of the control group animals, a hypo allergic dry 

patch 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 exposure period of six hours, 

and any remaining test item was removed under running water. 

The volume applied per animal was 0.5 ml. The treatment 

areas were visually assessed 30 hours after 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 females 

 Observations: Mortality, clinical signs, skin effects, body weight 

(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 test item group and the control group 

during the first induction treatment. After the second induction there were skin effects (grade 1) in 11 of 20 

animals and after the third induction (grade 1-3) in 20 of 20 animals of the test item group and no skin 

effects in 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 animals of the test item group (100 %) and no skin effects in the

control group. 

Number of animals exhibiting skin effects during challenge 

 Test item group (20 animals) Control group (10 animals) 

 Test item patch Control patch Test item patch Control patch 

Hours 30 54 Total 30 54 30 54 Total 30 54 

Challenge 

100 % 20 20 20 0 0 0 0 0 0 0 
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Positive Controls: The positive control study with alpha hexyl cinnamic aldehyde showed that the 

study was able to detect sensitizing compounds in the 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-sensitization potential. 

B. Reviewer Comments: The Buehler epicutaneous patch test was performed in 2005 on 30 female 

Crl:HA guinea pigs (20 animals for the test item group 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 active ingredient: BYI 08330: 144.65 g/l) exhibits skinsensitizing properties. Additional two animals were used for dose-finding for the challenge concentration. 

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 item concentrations: 

1

st to 3rd induction: 100% 

Challenge: 100% 

There were no skin effects in the animals of the test item group and the control group during the first 

induction treatment. After the second induction there were skin effects (grade 1) in 11 of 20 animals and 

after the third induction (grade 1-3) in 20 of 20 animals of the test item group and no skin effects in 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 animals of 

the test item group (100%). No skin effects were noted 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 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. 

This study is classified as Acceptable. It does satisfy the guideline requirement for a dermal sensitization 

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 potential. 

D. Study Deficiencies: None. 

CITATION: Vohr, H. (2005) BYI 08330 240 SC: Study for the Skin Sensitization Effect in Guinea Pigs 
– – 

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April 2008

(Buehler Patch Test). Project Number: AT01876, T8074944. 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 were 

exchanged for ones with clean bedding two times per week. 

Low-dust wood shavings supplied by Rettenmaier & Söhne, 

GmbH & Co, 73494 Rosenberg, Germany were used as 

bedding. The wood shavings were spot checked for 

contaminant levels. Records of these test results are 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:
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April 2008

 1st to 3rd induction: The animals were dermal treated with the test item three times 

at intervals of seven days. The 1st to 3rd induction was 

performed with the 100% test item concentration. The volume 

applied per animal was 0.5 ml. 

 Challenge: The challenge was performed two weeks after the last dermal 

induction. The volume applied per animal was 0.5 ml. 

 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 third 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 females 

 Observations: Mortality, clinical signs, skin effects, body weight 

(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 were not different from the control group. 

There were no skin effects in the animals of the test item group and the control group during the first and 

second induction treatments. After the third induction there were skin effects (grade 1) in 3 of 20 animals 

of the test item group and no skin effects in the animals of the control group. 

The challenge with the 100% test item concentration led to skin effects (grade 1) in 7 of 20 animals of the 

test item group (35%) and no skin effects in the control group.

Number of animals exhibiting skin effects during challenge 

 Test item group (20 animals) Control group (10 animals) 

 Test item patch Control patch Test item patch Control 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 cinnamic aldehyde showed that the study 

was able to detect sensitizing compounds in the 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-sensitization potential. 
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April 2008

B. Reviewer Comments: The Buehler epicutaneous patch test was performed in 2005 on 30 female 

Crl:HA guinea pigs (20 animals for the test item group 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 active ingredient: BYI 08330: 236.50 g/L) exhibits skinsensitizing properties. Additional two animals were used for dose-finding for the challenge concentration. 

The study was conducted with the following test item concentrations: 

1

st to 3rd induction: 100% 

Challenge: 100% 

The doses for the induction and challenge groups were based on the results of a range-finding study 

formulated into a suspension with sterile physiological saline solution. 

There were no skin effects in the animals of the test item group and the control group during the first and 

second induction treatments. After the third induction there were skin effects (grade 1) in 3 of 20 animals 

of the test item group and no skin effects in the animals of the control group. 

The challenge with the 100% test item concentration led to skin effects (grade 1) in 7 of 20 animals of the 

test item group (35%) and no skin effects in the control group. 

In summary, 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 and with respect to the evaluation criteria the test 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 control. (Report No. PH33529, October, 2004). 

This study is classified as Acceptable. It does satisfy the guideline requirement for a dermal sensitization 

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 potential. 

D. Study Deficiencies: None. 

CITATION: Vohr, H. (2005) BYI 08330 240 SC: Study for the Skin Sensitization Effect in 

Guinea Pigs. Project Number: AT02598, T5075616. Unpublished 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 
– – 

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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 were 

exchanged for ones with clean bedding two times per week. 

Low-dust wood shavings supplied by Rettenmaier & Söhne, 

GmbH & Co, 73494 Rosenberg, Germany were used as 

bedding. The wood shavings were spot checked for 

contaminant levels. Records of these test results are 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:

 1st to 3rd induction: The animals were dermally treated with the test item three 

times at intervals of seven days. The 1st to 3rd inductions were 

performed with the 0.3% test item concentration. The volume 

per animal was 0.5 ml. 

 Challenge: The challenge was performed two weeks after the last dermal 

induction. The volume per animal was 0.5 ml 

 Naïve Controls: For the control animals, a hypo allergic patch loaded only with 

the vehicle was applied to the left flank in the first to third 

inductions. The volume per animal was 0.5 ml. 

 Application route: Dermal (challenge: occlusive application) 

 Application volume: 0.5 ml/animal 
– – 

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April 2008

 Group size: Test item group: 20 females, control group: 10 females 

 Observations: Mortality, clinical signs, skin effects, body weight 

(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 test 

item group and the control group during the first 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.

Number of animals exhibiting skin effects during challenge 

 Test item group (18 animals) Control group (10 animals) 

 Test item patch Control patch Test item patch Control 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 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 “ready to use” test formulation exhibits no skin sensitisation potential.
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B. Reviewer Comments: The Buehler epicutaneous patch test was performed in 2005 on 30 female 

Crl:HA guinea pigs (20 animals for the test item group and 10 control animals) (age: 5-6 weeks; weight: 

313-385 grams; source: Charles River Laboratory Animal Breeders, KiBlegg, Germany) to determine 

whether the test item “BYI 08330 240 SC ready to use dilution” (content of the active ingredient: BYI 

08330: 244.48 g/l) exhibits skin-sensitizing properties. 

The study was conducted with the following test item concentrations: 

1

st to 3rd induction: 0.3% 

Challenge: 0.3% 

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. 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 control. (Report No. PH034010, August 2005.) 

There were no skin effects in the animals of the test item group and the control group during the first 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 item group and in the control group it can be concluded 

that under the conditions of the Buehler Patch Test, BYI 08330 240 SC ready to use dilution exhibits no 

skin-sensitization potential. 

This study is classified as Acceptable. It does satisfy the guideline requirement for a dermal sensitization 

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 dermal sensitization potential. 

D. Study Deficiencies: None. 
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B.6.11.7 Summary of toxicity studies with the formulation 

BYI 08330 150 OD (MOVENTO® 150 OD Insecticide) 

All of the BYI 08330 150 OD (MOVENTO® 150 OD Insecticide) 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 prevailing OECD and OPPTS guideline. Six of the studies 

were conducted using BYI 08330 150 OD [Batch No. 08030/0189 (0152); light-brown suspension] as the test 

material. The test material for the dermal sensitization study was identified as BYI 08330 150 OD ready to 

use dilution. An additional dermal sensitization study was conducted using BYI 08330 150 OD [Batch No. 

08030/180(0152); liquid brown suspension] as the test material. 

BYI 08330 150 OD was found to be of low acute toxicity to the rat by the oral, dermal and inhalation routes. 

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. Treatment-related findings in the acute inhalation toxicity 

study were limited to minor bodyweights and clinical signs (resolved by day 4) at the dose level of 1.760 

mg/L. 

In the primary eye irritation study, corneal opacity 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 animals were observed with very slight erythema at one hour. 

At 24 hours, two animals were observed with very slight erythema and one animal with well defined 

erythema. At 48 hours, two animals were observed with well defined erythema and one animal with very 

slight erythema. At 72 hours, two animals were observed 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 dilution. 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 and with 

respect to the evaluation criteria the BYI 08330 150 OD ready to use dilution exhibits no skin-sensitization 

potential. The test material for the second material was identified as BYI 08330 150 OD but with a different 

batch number than the other dermal sensitization and 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 Insecticide) 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 prevailing OECD and OPPTS guideline. Six of the studies 

were conducted using BYI 08330 240 SC [Batch No. 07218/0073(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 test material. 
– – 

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

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BYI 08330 240 SC was found to be of low acute toxicity to the rat by the oral, dermal and inhalation routes. 

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. Treatment-related findings in the acute inhalation toxicity 

study were limited to minor clinical signs (resolved 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 test material of the first study was identified as BYI 08330 

240 SC but with a different batch number than the other 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 and with respect to the evaluation criteria the test item exhibits a skin-sensitization potential. 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 group it can be concluded that under the conditions of the 

Buehler Patch Test, BYI 08330 240 SC ready to use dilution exhibits no skin-sensitization potential. 
– – 

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April 2008

B.6.12 Dermal absorption (OECD Annex IIIA 7.6) 

See B.6.9.9 
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April 2008

B.6.12.1 Summary on Dermal absorption (AGES) 

The results showed that the rate of absorption of [14C]-BYI 08330 through human and rat epidermis from the 

concentrate formulation and its v/v aqueous dilutions was slow. The majority of the applied dose (human: 85 

– 95%; rat: 65 – 92%) was removed by mild skin washing after an exposure period of 8 hours. These data

predict that the dermal absorption of BYI 08330 through human or rat skin from potential exposure to the OD 

formulation, either as the concentrate formulation or as the v/v aqueous spray strength dilutions, would 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 figures 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 potentially exposed to BYI 08330, the formulation concentrate 

was included to assess exposure to mixer/loaders and the spray strength dilutions are most relevant to spray 

operators. 

Following a 10 hour exposure period, the in vivo dermal absorption of [14C]-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 [14C]-BYI 08330 in 150 

OD formulation accounted for means of approximately 10.6%, 4.8% and 5.8% for the high, intermediate and 

low dose, respectively. 

In the in-vitro skin penetration study with human skin, an absorption 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 concentrate and 23.21% and 22.70% for the dilutions,

respectively, was obtained after the 8 hour exposure. 

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 concentrate. 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 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 
– – 

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

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April 2008

Species difference correction factor 8.9 8.5 2.7 

Dermal absorption: Human 1.19 0.56 2.15 
– – 

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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 

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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 pharmacokinetic 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-3-14C]-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 dermal 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 irritation 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 irritation 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 for 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 for 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 - Evaluation 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 - Subacute 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 grade 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 BYI 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 BYI 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 grade 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 toxicity 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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471

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 5.3.7 /01 Eigenberg A. D. 2006 A subacute dermal 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 - Salmonella/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 - Salmonella/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 vitro 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 - Cytogenetic 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/HPRT-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 - Micronucleus-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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472

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 5.4.4 /02 Honarvar, N. 2003 Chromosome aberration 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 with 

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 BYI 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 BYI 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 : Technical 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 homogeneity 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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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 5.5.4 /01 Kennel, P. 2005 BYI 08330 - Evaluation of the potential 

reproductive toxicity in the male rat following daily 

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 - Investigation of the 

testicular/sperm toxicity in the rat following 21 days 

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 BY1 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 BYI 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 developmental 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 
– – 

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

474

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 5.6.10 

/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 toxicity 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 toxicity 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 with 

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-Ketohydroxy - 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 
– – 

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

475

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 5.8 /02 Herbold, B. 2005 BYI 08330-CIS-Ketohydroxy (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-hydroxy - 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-hydroxy (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-hydroxy - 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 
– – 

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

476

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 5.8 /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 potential 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 (Spirotetramat) - 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 (Spirotetramat) - 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 (Spirotetramat) - 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 (syn. 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 
– – 

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

477

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 5.10 /02 Temerowski, M. 2008 Spirotetramat (syn. 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 organogenesis 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 abortion 

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 rabbits: 

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 
– – 

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

478

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 5.10 /07 Matsuoka, T.; 

Mizoguchi, Y.; 

Serizawa, K.; 

Ishikura, T.; 

Mizuguchi, H.; 

Asano, Y. 

2006 Effects of stage and degree of restricted feeding 

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 in 

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 toxicity 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 toxicity 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 
– – 

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

479

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 

KIIIA1 7.1.3 

/01 

Pauluhn, J. 2005 BYI 08330 150 OD - Acute inhalation 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 irritation/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 irritation 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 08330) - 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 dilution (Project: 

BYI 08330) - Study for the skin sensitization effect 

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 
– – 

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

480

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 

KIIIA1 7.3.1 

/02 

Anon. 1992 UK Predictive Operator Exposure Model (POEM): 

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 during 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 
– – 

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

481

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 

KIIIA1 7.5.1 

/01 

Hoernicke, E.; 

Nolting, H.G.; 

Westphal, D. 

1998 Label instructions for the protection of workers 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: techniques 

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 
– – 

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

482

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 

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 formulation: 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 toxicity 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 toxicity 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

nce 
– – 

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

483

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 

KIIIA1 7.1.3 

/01 

Pauluhn, J. 2005 BYI 08330 240 SC - Acute inhalation 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 irritation/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 irritation 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 08330) - Study for 

the skin sensitization effect in guinea pigs (Buehler 

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 dilution (Project: 

BYI 08330) - Study for the skin sensitization effect 

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 
– – 

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

484

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 

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 workers 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: techniques 

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 
– – 

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

485

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 

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 apple 

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 
– – 

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

486

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 

KIIIA1 7.5.1 

/07 

Urtizberea, M. 2002 Post-application worker exposure 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

nce 
– – 

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

487

April 2008

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 .....................................493

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 protective equipment is not used ......499

B.6.14.1.2 Estimation of operator exposure assuming personal protective equipment is used ............501

B.6.14.1.3 Measurement of operator exposure – (Mixer/Loader/Applicator).........................................501

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 protective equipment is not used.........504

B.6.14.3.2 Estimation of worker exposure assuming personal protective equipment is used...............505

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 ......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 Model) .....................................................................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 studies 

with spirotetramat. Given the results from all relevant 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 in dogs. Applying the standard uncertainty factor of 100 to 

account for intraspecies and interspecies variability to the NOAEL of 5.0 mg/kg bw/d from the dog study 

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 relevant. 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. Abortion 

due to systemic maternal toxicity occurred in one female 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 most appropriate one to derive the ARfD. For this purpose, 

the acute neurotoxicity study in rats is considered appropriate. In this study, a NOAEL of 100 mg/kg bw/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 factor 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 concentration (MAC) value in drinking water is not necessary,

because according to Directive 91/414/EC only the ADI, ARfD and AOEL values have to be determined. 

According to Directive 98/83/EC a drinking water limit of 0.1 μg spirotetramat/l is established. 

B.6.10.4 Proposal for AOEL 

According to the principles of Annex VI to Directive 91/414 EEC, the proposed acceptable operator exposure 

level should be established on the basis of the highest 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 studies 

(i.e. subacute/ subchronic and reproduction or developmental toxicity studies) since these studies in most 

cases can be considered a more appropriate model for 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 intraspecies and interspecies variability) is applied to the systemic 

NOAEL, a systemic AOEL of 0.05 mg/kg bw/d is proposed. 

Remark: The notifier proposed a systemic AOEL of 0.1 mg/kg bw/d based on the NOAEL of 10 mg/kg bw/d 

derived in the developmental toxicity study in rabbits. 
– – 

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

490

April 2008

Table 6.10-1: Summary of repeat oral dose studies with 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: [

14C]-BYI 08330 in OD150 formulation: In vivo dermal absorption 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 using [14C]-BYI 08330 in 150 OD formulation 

(Batch no. KML 2978-2; radiochemical purity > 99%) following single dermal application to male rats (strain: 

Wistar Rj: WI (IOPS HAN) derived, source: R. Janvier, France) at 3 concentrations: high dose (nominal value 

100 μg a.i./cm2

), intermediate dose (nominal value 15 μg a.i./cm2) and low dose (nominal value 5 μg 

a.i./cm2). 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./cm2) 

4 4 10 (168)* 

5 4 1 

6 4 10 

7 4 24 

Intermediate dose 

(15 μg a.i./cm2

) 

8 4 10 (168)* 

9 4 1 

10 4 10 

11 4 24 

Low dose 

(5 μg a.i./cm2

) 

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 cm2

 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 was identical with the time of sacrifice except for groups 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 swabs to 

remove non-absorbed radioactivity. Swabbing was continued until no radioactivity was detected in swabs

using a Geiger counter. The skin was shaved again and tape-stripped to remove the stratum corneum. 

Adhesive tape was applied for 5 seconds, then removed 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, blood 

samples were taken. 

The treated area of skin and a surrounding annular ring of untreated skin (approximately 1cm) were excised 

and taken for analysis as well as an isolated control portion of untreated skin. The residual carcass was 

retained for analysis also. 

The amounts detected in urine, faeces, cage wash, carcass, blood, stratum corneum (tape strips 3 to the last 

tape strip), treated skin (skin after tape-stripping), non-treated skin and surrounding skin were considered 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 measured 

(Sanco/222/2000 rev.7)]. 

Radioactivity recovered from the swabs, dressing (saddle, surgical tape, gauze and cover), fur and surface 

dose (tape strips 1 & 2) was considered to be unabsorbed. 

Radioactivity in samples was analysed by liquid scintillation counting (LSC). 

Findings:

Recovery of radiolabelled test material of the applied dose was acceptable, with recovery rates of 94.9 – 

99.0% for the high dose (100 μg a.i./cm2

), 92.3 – 95.5% for the intermediate dose (15 μg a.i./cm2) and 93.4 – 

100.6% for the low dose (5 μg a.i./cm2

). 

Based on the likely operator/worker exposure time frame, the most appropriate exposure period would be 10 

hours. So the dermal absorption values calculated for the groups of animals for which the skin was washed 

after 10 hours (groups 2, 6, and 10) and for those for which the skin was washed after 10 hours and which 

were sacrificed after 168 hours (groups 4, 8 and 12) were taken into account for determination of dermal 

absorption of BYI 08330 in vivo. 

After 10 hours exposure, the in vivo dermal absorption of [14C]-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 [14C]-BYI 08330 in 150 

OD formulation at the 3 concentrations accounted for means of approximately 10.6%, 4.8% and 5.8% of the 

dose respectively. At least more than 80% of the applied radioactivity was readily washed from the skin 

surface after the 10 hour exposure interval. 

Skin stripping showed that most radioactivity at the application site was in the stratum corneum. The amount 

of radioactivity in the stratum corneum, taken after the 10 hour application period and after skin wash, was 

14.26% for the high dose, 7.32% for the intermediate and 10.02% for the low dose, respectively. Termination 

after 168 hours showed that mean radioactivity in the stratum corneum decreased (2.6% for the high and

1.53% for the intermediate and low dose, respectively), indicating further resorption from this depots. 

The absorption and excretion pattern and the mean percentage 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 percutaneous administration of [14C]-BYI 08330 

(mean values expressed as % of the dose applied) 

High dose 

100 μg a.i./cm2

Intermediate dose 

15 μg a.i./cm2

Low dose 

5 μg a.i./cm2

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 0.02 0.02 0.0 
– – 

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

493

April 2008

High dose 

100 μg a.i./cm2

Intermediate dose 

15 μg a.i./cm2

Low dose 

5 μg a.i./cm2

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 12.03 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 0.13 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 1.52 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 corneum, treated, untreated and surrounding skin

Conclusion:

After topical application of [14C]-BYI 08330 in 150 OD formulation to male rats at 3 dose levels (100, 15 and 

5 μg a.i./cm2), the dermal absorption was considered to be low. Means of 80% of all doses were readily 

washed from the skin surface using mild soap solution and water washes. 

Following a 10 hour exposure period, the in vivo dermal absorption of [14C]-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 [14C]-BYI 08330 in 150 

OD formulation accounted for means of approximately 10.6%, 4.8% and 5.8% for the high, intermediate and 

low dose, respectively. 

B.6.12.2 Comparative dermal absorption, in vitro using human and rat skin 

Reference: [

14C]-BYI 08330 in OD150 formulation: Comparative In vitro 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 vitro through human and rat epidermis was determined by 

using [14C]-BYI 08330 (Batch no. KML 2978-2; radiochemical purity > 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 nominal 

doses were 150, 1.5 and 0.05 mg a.i./ml formulation) The dose volume was 10 l/cm2

 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. The skin was dermatomed by using a mini-dermatome. 

Dermal membranes 420 – 510 μM thick were prepared. 

Human skin membranes from 13 donors were prepared similarly using the mini-dermatome. 

Prior to use, the integrity of the selected membranes was assessed by measuring the trans-epidermal water 

loss (TEWL) from the stratum corneum. The membranes were assembled in diffusion cells and an 

evaporimeter probe was placed atop the receptor cell and water diffusion through the skin was measured

(g/m2

/hr). Human and rat skin with a TEWL > 40 g/m2/hr were considered potentially damaged and were not 

used. 

Dose preparation: The doses were applied as a 150 mg/ml OD formulation 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 splitthickness skin samples with a pipette at the rate of approximately 10μl/cm2 exposed skin. 

Sampling of receptor fluid: The receptor fluid passing through the receptor chamber was collected in glass 

vials held in a fraction collector which was started after dose application for each group was complete. 

Samples were then collected hourly for the duration of the experiment (24 hours). 

At 8 hours post-application, the skin was swabbed with 1% v/v Tween 80 in PBS (phosphate buffer saline) 

using sponge swabs, in order to remove and retain the non-absorbed dose, until no radioactivity was 

detected with a Geiger counter. 

24 hours after application, the treated skin cell was swabbed again prior to the tape-stripping. The tapestripping procedure was continued until a “shiny” appearance of the epidermis was evident, which indicated 

that the stratum corneum had been removed. The tape-strips were collected into scintillation vials. The 

remaining skin was removed and taken for analysis. 

Analysis: Receptor fluid, skin wash, donor chamber, tape strips (stratum corneum) and epidermis were 

analysed by LSC. 

Findings:

Mean recovery of radiolabelled test material for the concentrate and the dilutions was between 96 – 99% of 

the applied dose for human skin and 96 – 100% for rat skin. 

The majority (65 - 95%) of the applied dose was removed from the surface of human and rat skin by skin

washing. 

For the formulation concentrate (150 mg/ml formulation), the remaining proportion recovered from stratum 

corneum for human skin was 0.12% after the 24 hour exposure, 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 remaining proportion recovered from stratum corneum was 1.66% 

(human) and 15.62% (rat) leaving a mean of 0.67% and 4.34% respectively of the applied dose in the 

remaining epidermal tissue. 

For the 0.05 mg/ml formulation dilution, the remaining proportion recovered from stratum corneum was 

4.65% (human) and 9.22% (rat) leaving a mean of 3.24% and 1.26% respectively of the applied dose in the 

remaining epidermal tissue. 

The recovery of [14C]-BYI 08330 (expressed as % of dose applied) found in the different compartments after 

application of the concentrate and the dilutions are given in table1.5.2-1. 
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Table 6.12.2-1 Mean % of applied dose detected in different 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 formulation 

concentrate and the dilutions for human and rat skin. The majority of the applied dose was removed by skin 

washing (65 - 95%). 

Regarding the amounts of [14C]-BYI 08330 recovered in the receptor fluid, stratum corneum and epidermis 

as systemically available, the total penetration rate after 8 hour exposure was 0.24% (human) and 2.15% 

(rat) for the concentrate. For the dilutions, penetration 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 skin. 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 [14C]-BYI 08330 through human and rat epidermis from the 

concentrate formulation and its v/v aqueous dilutions was slow. The majority of the applied dose (human: 85 

– 95%; rat: 65 – 92%) was removed by mild skin washing after an exposure period of 8 hours. These data

predict that the dermal absorption of BYI 08330 through human or rat skin from potential exposure to the OD 

formulation, either as the concentrate formulation or as the v/v aqueous spray strength dilutions, would 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 figures 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 potentially exposed to BYI 08330, the formulation concentrate 

was included to assess exposure to mixer/loaders and the spray strength dilutions are most relevant to spray 

operators. 

Following a 10 hour exposure period, the in vivo dermal absorption of [14C]-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 [14C]-BYI 08330 in 150 

OD formulation accounted for means of approximately 10.6%, 4.8% and 5.8% for the high, intermediate and 

low dose, respectively. 

In the in-vitro skin penetration study with human skin, an absorption 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 concentrate and 23.21% and 22.70% for the dilutions,

respectively, was obtained after the 8 hour exposure. 

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 concentrate. 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 active ingredient Spirotetramat (150 g/l spirotetramat

technical) as well as an emulsifier, a surfactant, dispersing agents, an antifoam,an antioxidant, a buffer and a 

co-solvent. The notifier provided Material Safety Data Sheets (MSDS) for all these auxiliaries; only limited 

information is available concerning the toxicity of these substances. 

B.6.13.1 Material safety data sheet for each formulant 

The acute toxicity profile of all co-formulants in the representative formulation is sufficiently covered by 

studies performed with the preparation. On the basis 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 formulant 

Table 6.13.2-1: Toxicological data relating to the co-formulants 

Coformulants CAS Number Function MSDS date Labelling/Comments with respect to 

toxicology 

Genapol X-080 

(poly(oxy-1,2-

ethanediyl)αisotridecyl-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 150” (BYI 08330 150 OD) is an oil dispersion formulation 

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 on 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 the 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 sensitisation. Recommended protective clothing are gloves, a 

coverall and solid shoes. 

Operator exposure estimates for outdoor cultures are calculated using both the German model (Uniform 

Principles for Safeguarding the Health of Applicators of Plant Protection Products [Uniform Principles for 

Operator Protections]; Mitteilungen aus der Biologischen Bundesanstalt für Land- und Forstwirschaft, BerlinDahlem, no. 277, 1992) and the UK POEM (Scientific Subcommittee on Pesticides and British 

Agrochemicals Joint Medical Panel., Estimation of Exposure and Absorption of Pesticides by Spray 

Operators [UK MAFF] 1986 and the Predictive Operator Exposure Model [POEM], UK MAFF 1992). 

Exposure is calculated for each application technique with the maximum dose rate (German model and UK 

POEM) and minimum spray volume (UK POEM). Lower doses and higher spray volumes will be covered by 

this calculation. 

Hand applications in citrus are calculated using the 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 assisted 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 model 

60 kg for UK POEM 

Dermal absorption has been determined for “Spirotetramat 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 acceptable operator exposure level (AOEL) of 0.05 mg/kg bw/d 

was established from the 1 year toxicity study in dogs with a safety factor of 100 (NOAEL 5 mg/kg bw/d). No 

correction of oral absorption was made (96 % oral absorption). 

B.6.14.1.1 Estimation of operator exposure assuming personal protective equipment is not used 

Operator exposure values have been calculated according 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 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 0.0038 7.6 

Tractor broadcast air-assist applications – high crop 

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 crop 

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 Report: 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 und Gesundheitssicherheit GmbH)) the exposure of 

12 operators during mixing and loading and application were investigated for the two scenarios “low crops on 

tables” and “high crops on ground”. 

Dermal exposure of the body was determined via cellulose patches located on the outside of the operator’s 

clothing (potential dermal exposure), as well as by analysing whole body underwear (actual dermal 

exposure). 

Hand exposure was measured via glove rinsing (potential dermal exposure) and hand rinsing (actual dermal 

exposure). For determination of head exposure a patch 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 in 0.7 mg/person x kg a.s. handled and 7.5 mg/person x 

kg a.s. for the actual and potential exposure, respectively. 

The summary results of the geometric mean exposures are presented in the following table: 

Table 6.14.1.1-3: Surrogate glasshouse exposure study: 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 application. 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 the process of mixing/loading for both indoor and outdoor 

are similar. 

The exposure estimates for mixing/loading of an oil dispersion formulation such as “Spirotetramat OD 150” 

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 assessment 

presented assumes a work rate of 1 ha/d in the greenhouse. The spray gun/lance connected to a tank will be 

used for this scenario, especially if high water volumes 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 kg 

were estimated. 

Table 6.14.1.1-4: Glasshouse application - low crops: 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 0.00004 

 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% spray dilution, 100% inhalation 

**: body weight 60 kg 

According to the applied models the estimated operator 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 exposure assuming PPE is used are not required since model 

calculations without PPE predict the systemic exposure to be well within the acceptable exposure level. 

B.6.14.1.3 Measurement of operator exposure – (Mixer/Loader/Applicator) 

Measurement of operator exposure is not required since model calculations predict the systemic exposure 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 with a safety factor of 100). No correction of oral absorption 

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 no 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 use scenario that is considered to result in the highest level 

of spray drift which may contaminate the bystander – tractor-mounted air-assisted application in citrus. 

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 exposure 

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 provides 

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). Exposure to an incidental bystander is calculated based on 

the following parameters: 

Maximum application rate 

[mg/m2

] 

AR 0.288 kg a.i./ha = 28.8 mg/m2 

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 application) 

Exposed body surface [m2] BS 0.4250 m2 (corresponds to the unprotected surface of an adult 

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/m3 and a breathing rate of 1 m3/hr) 

Duration of exposure [min] T 5 min 

Dermal exposure calculates as follows: 

[ ]

BW

AR D BS Dermal Exposure mg / kg bw / day × ×

=

Inhalation exposure calculates as follows: 

[ ]

60

/ /

×

× ×

=

BW

C IE T

Inhalation Exposure mg kg bw day

Total systemic exposure calculates as follows: 

Total Systemic Exposure [mg / kg bw / day]= Dermal Exposure × DA + Inhalation Exposure

 

1

 Ganzelmeier et al.: Studies on the spray drift of plant protection products. Mitteilungen aus der Biologischen 

Bundesanstalt für Land- und Forstwirtschaft, Berlin-Dahlem, Heft 305, 1995. 

Updated: BBA (2000:1). Bekanntmachung über Abdriftwerte, die bei der Prüfung und Zulassung von 

Pflanzenschutzmitteln herangezogen werden. Bundesanzeiger 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 
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Based on this scenario, total systemic exposure for an incidental bystander calculates as follows: 

Table 6.14.2.1-1: Estimated bystander exposure value 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 assessment of the exposure risk for incidental bystanders. 

The calculated exposure to “Spirotetramat OD 150” is well below AOEL. 

B.6.14.2.2 Measurement of bystander exposure 

Measurement of bystander exposure is not required since model calculations predict the systemic exposure 

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 use scenario that is considered to result in the highest level 

of spray drift which may contaminate the bystander – tractor-mounted air-assisted application in citrus. 

These results indicate that short exposure of bystanders 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 of application of “Spirotetramat OD 150” may require reentry activities such as crop inspection, pruning and harvesting which may lead to worker exposure. An 8 

hour period of exposure is considered appropriate for 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 these 

crops. A foliar dislodgeable residue study was performed on grapes and hops, with a max. application rate of 

110g ai/ha (Fischer, 2006, see B.1.6).The worker re-entry exposure model published by Hoernicke et al 

(1998) has been used to provide a conservative estimate of exposure during the conduct of re-entry work 

activities4. 

For the foliar dislodgeable residues (FDR) the German re-entry model proposes the overly conservative 

transfer coefficient (TF) of 30,000 cm2/hour, which reflects tasks with the most intensive contact with the 

treated crop, such as pruning or harvesting. As a worst case Tier 1 approach this value is applied in the 

following calculations. 

B.6.14.3.1 Estimation of worker exposure assuming personal protective equipment is not used 

Worker exposure is calculated by the following approach: 

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 exposure levels are summarised in the following tables: 

Table 6.14.3.1-1: Re-entry worker calculations of exposure to “Spirotetramat OD 150” (no PPE) 

Symbol Definition Quantity Units 

DFR Foliar dislodgeable residues 0.943 μg/cm2

/kg a.i. 

TF Transferfactor 30000 cm2/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) 130,36 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. Nachrichtenbl. 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 personal protective equipment is used 

Estimation of worker exposure assuming PPE is used is not required since model calculations without PPE 

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 since 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 term activity such as pruning or harvesting has been 

calculated according to the German re-entry model to provide a conservative assessment of risk. The 

calculated exposure levels for “Spirotetramat OD 150” 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 Grapes 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: Group B 

GLP: Yes 

Deviations: -Cartridge samples from trial FN344-05D were not shipped according to shipment 

schedule outlined in the protocol. However, all samples were received safely at the 

analytical laboratory and field recovery samples were stored and transported with the 

samples to verify analyte stability. No adverse effect on the study. 

-Cation exchange capacity data for soil from plot from trial FN343-05D were not 

collected and are not available. Since these data are not critical for this type of study, 

there was no adverse effect. 

-Sample shipment chain of custody form for the cartridge samples from trial FN339-

05D shipped on October 5, 2005 was not available. These samples were reserve 

samples held at the field site, then shipped following safe receipt of the primary 

samples to the analytical lab. Since all primary samples were received safely at the 

laboratory, these samples were not needed for analysis, 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 the 

amount of spirotetramat residue that can be dislodged from grape and hops foliage, respectively, following 

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 to 

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. All applications were made using airblast equipment. 

Leaf punch sampling (400 cm2

 double-sided surface area) were collected prior to the first application (control 

samples) and at various time intervals after the first and second applications up to 35 days following the 

second application. Three treated samples were collected at each interval. Sample residue was dislodged 

from each sample with 200 ml of detergent solution. Aliquots of the dislodging solution were applied to solid 

phase extraction cartridges, and the cartridges were washed, dried, and frozen. Residues were eluted from 

the cartridges and quantitated by liquid chromatography/mass spectrometry-mass spectrometry (lc/ms-ms). 

The limit of quantitation was 0.01 μg/cm2

. The measured residues were corrected for any losses that 

occurred from sample collection through sample analysis. 

Findings: 

The following tables summarize the dislodgeable foliar 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/cm2

B 

μg/cm2

C 

μg/cm2

D 

μg/cm2 Half life (d) 

Pennsylvania 0.13 < LOQ 0.12 0.03 18 
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Trial location A 

μg/cm2

B 

μg/cm2

C 

μg/cm2

D 

μg/cm2 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 treatment 

B: Sampling interval – 30 days after first application; prior to 2nd treatment 

C: Sampling interval – immediately after 2nd treatment 

D: Sampling interval – 35 days after 2nd treatment 

Table 6.15.-2: Dislodgeable foliar residue values after one and two applications of spirotetramat to 

hops 

Trial location A 

μg/cm2

B 

μg/cm2

C 

μg/cm2

D 

μg/cm2 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 treatment 

B: Sampling interval – 14 days after first application; prior to 2nd treatment 

C: Sampling interval – immediately after 2nd treatment 

D: Sampling interval – 35 days after 2nd treatment 

Conclusion:

For the 4 trials conducted on grapes, the average dislodgeable 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/cm2 and 

from 0.12 to 0.36 μg/cm2, respectively. 35 days after the second application, the average DFR from the 4 

grape trials had declined to values ranging from 0.03 to 0.12 μg/cm2. 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 second 

application of BYI 08330 150 OD ranged from 0.10 to 0.13 μg/cm2 and from 0.09 to 0.20 μg/cm2, 

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/cm2. The half life of BYI 08330 150 OD on hop leaves at the 2 trial 

locations following the second application was 47 and 5 days, respectively. 

The recommended crops and use conditions of BYI 08330 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 residues for spirotetramat, application to citrus, 288 g 

ai/h/application, based on data for grapes (0.36 μg/cm2

 immediately after the second application of BYI 

08330 150 OD) were taken into account (worst case scenario). So for the estimation of worker exposure,

dislodgeable foliar residues of 0.943 μg/cm2 were assumed. 
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 Appendix I: Detailed Exposure Calculations (UK POEM) 

Table I-1: Tractor-mounted/trailed boom sprayer - low 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 nozzles 

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 crops (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 sprayer 

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 Model) 

Table II-1: Tractor mounted boom sprayer - low crops (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 Day (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 2.304 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 Day (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 22.1184 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 (citrus) 

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 Day (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 %