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### 3. Test substancedosage formulations and analysis The appropriate amount oftest substance was periodically (six formulations at 12.5 and 31.25 gil and four formulations at 125 gil) suspended (w/v) 

### in an aqueous solution ofmethylcellulose 400 (Fluka, Mulhouse, France) at 0.5% and stored at approximately 5°C (± 

### 3°C). Homogeneity of the suspensions was checked during the first formulation for the lowest and highest concentrations. All concentrations were checked for all formulations. Homogeneity and concentration checks were between 94 and 108% ofnominal values, Stability ofthe compound in suspension in the vehicle was determined before·the start ofthe study at 0.1 and 250 gil in 0.5% aqueous methylcellulose in a previous study and was found to be stable for 28 days under similarconditionsto those ofthe current study. C. 

### METHOD: 1. Observations All rabbits were observed daily for clinical signs and twice daily for mortality (except once daily on weekends and public holidays), 2. 

### Bodyweight Body weights weremeasured on GD: 3,6,8, 10, 12, 14, 16, 18,20,22,24,26and 29, 

### 3. Food consumption Full feeder weights were measured onGD: 3,4,5,6,8, 10, 12, 14, 16, 18,20,22,24,26and28. Empty feeder weights were measured on GD: 4, 5, 6, 8, 10, 12, 14, 16, 18,20,22,24,26,28 and 29. 

### 4. Caesarian sections Animals ki\led 

### in extremis by intravenous injection of Dolethal® (Sanofi, Libourne, France) (including females which aborted) or found dead, were autopsied. The number and type of implantations and corpora lutea were noted when present. In the case ofno visible uterine implants, but with corpora lutea, uterine horn(s) were immersed in a 20% solution of ammonium sulfide according to the Salewski method (1964). The urinary bladder and kidney were taken and preserved in 10% neutral buffered formalin. Both organs were subsequently examined microscopically at 125 mg/kglday and from the control females. Tissues and carcasses werethen discarded. On GD 29, surviving females were killed by intravenous injection ofDolethal® for examination of their uterine content. Each female was first subjected to macroscopic examination ofthe visceral organs and the number of ribs was recorded. In the case ofno visible uterine implants, but with corpora lutea, uterine horn(s) were immersed in a 20% solution ofammonium sulfide according to the Salewski method. The urinary bladder and kidney were taken and preserved in 10% neutral buffered formalin. Both organs were subsequently examined microscopically at 125 mg/kglday and from the control females. Tissues and carcasses werethen discarded. The reproductive tract was weighed (gravid uterine weight), dissected out and the following parameters recorded: number ofcorpora lutea, number of implantation sites, number ofresorption sites (classified as early and late), number of live and dead fretuses, individual weights of live

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### fretuses. Dead fretuses: were defined as fretuses showings distinct digits visible on fore and hindpaws. All the live fretuses were and subjected to an external examination. Resorption death was classified as early resorptions when macroscopic discrimination between fretal residues and placental material was not possible and late resorption when distinct macroscopic discrimination between fretal residues and placental material was possible.


### 5. Fretalexamination All data were recorded without knowledge oftreatment group. After internal examination of the neck, the head offretuses from approximately halfofeach litter was immersed in Bouin's fluid and the internal structures examined after fixation. The bodies of all fretuses were dissected for soft tissue anormalies and sexed. Then the fretuses were fixed in absolute ethanol before staining. A modification of the Staples and Schnell staining technique was used and a subsequent skeletal examinationwas performed.


### 6. Statistics Means and standard deviations (STD) for all maternal and litter parameters were calculated for each group. Statistical analyses wereperformedon the following variables using TERATESTVersion 1.0. Maternal endpoints: body weight change calculated according to time periods, calculated corrected body weight change, average food consumption calculated according to time periods. Litter based and fretal endpoints: number ofcorpora lutea, number ofimplantation sites, number of resorption (early and late), pre- and post-implantation loss percentages, fretal sex, fretal death status, fretal body weight


### II. RESULTS AND DISCUSSION A. 

### MaternalObservations 1. 

### Mortality At 500 mglkglday, 

### 7/25 dams died between GD 9 and 20, four were killed for humane reasons and three were found dead. The principal clinical signs observed were yellow sediment in the urine in all animals, associated with red traces on the cage tray in five cases. All animals had fewlno feces on one or more occasion, and one female had reduced motor activity, tremors and was warm to touch on the day of death. All animals showed a marked body weight loss, associated with very poorfood consumption priorto death. At autopsy the principal findings were as follows:

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Table5.6.11-2 Macroscopicfindings in decedentanimals at500 mg!kglday I Macroscopic findings I Organs I Numberofdecedents affected at 500 mg/kg/day Kidney Yellow sediments 417 Marbledpattern 217 Urinary bladder Yellow sediments 217 Red contents 117 Liver I Prominent lobulation 517 Uterus Redcontents 217 As this dose level had clearly exceeded a Maximum Tolerated Dose (MTO), treatment at 500mglkg/day was terminated. Surviving females were killed prior to scheduled autopsy with no macroscopic examination performed andnotissuestaken. At 125 mglkg/day, one female was found dead on GO 19 due togavage dosing error. At 50 mg/kg/day, one female was found dead on GD 15. The cause ofdeath was alsojudged to have been gavage error. There were no other mortalities during the course ofthe study at 125 or 50 mglkg/day. 2. Abortions One female from the control group aborted on GO28. There were no other abortions on the study. 3. Clinical signs Ofthe surviving females at 500 mglkg/day, treatment-related clinical signs consisted ofyellow sediment in the urine 00/18 dams and few faeces for 4/18 dams, priorto termination ofthis dose group. At 125 mglkg/day, treatment-related clinical signs consisted of yellow sediment in the urine of2/25 dams. There werenotreatment-related clinical signs observedat 50mglkg/day. 4. Pregnancy rate The pregnancy rate was not established at 500mglkglday as the majority offemales were culled with no macroscopic examination. There were no effects on pregnancy rate in any evaluated group. The pregnancy rate was 100% at 125 mglkg/day, 96% at 50 mglkg/day and 92% in the control group. 

### 5. Body weight In surviving females at 500 mglkg/day, 2118 showed a significant body weight loss ofbetween 0.21 and 0.29 kgpriorto termination ofthis dose level. Noeffect on mean body weight change or mean corrected body weight change (maternal body weight change independent ofthe uterine

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### 5. Food consumption Poor food consumption, which was associated with significant body weight loss was observed in 2118 surviving females at 500 mg/kg/day, prior to termination ofthe group. Food consumption was similarto the control group at 125 and 50 mglkg/day. 

### 6. Maternal necropsy and microscopic findings At autopsy, yellow sediment in the kidney wasnoted in 1125 females at 125 mglkg/day, whilstthe decedent female at 125 mglkg/day and 1/25females at 50 mglkg/day had a kidney with a marbled pattern appearance. In the absence ofmicroscopic findings in this target organ at 125 mg/kg/day, these findings were considerednot toxicologically relevant. At the microscopic examination of the urinary bladder and kidney at 125 mglkg/day, no treatment-related fmdings were noted. 

### B. Litterdata There was no effect on litter parameters including early and late resorptions, feetal death status, percentage ofmale feetuses and mean feetal body weight, at eitherremaining dose level. At 50mglkg/day, mean feetal weights for the combined sexes and for females only were slightly reduced compared to the controls. However, in the absence ofan effect at the higher dose level of 125 mglkg/day, this finding was consideredto be incidental. 

### C. Fretal necropsy findings 1. 

### Fretal evaluation: external observations The few anomalies observed occurred in a non dose-related manner, in isolation, orin the case ofthe finding shorttail, where the incidence at 125 mglkg/day was slightly higherthan in the control group, both in terms ofmean percentage offretuses affected (U%)and percentage ofIitters affected (4.2%), the incidence was within the in-house historical control range (0.0-3.1% and 0.0-4.3%, respectively) andwas therefore consideredto have occurredby chance. No variantfindings were noted. The incidence ofrunt feetuses was similar at 125 and 50mglkg/day, and only slightly higherthan the control group. Since these incidences were within the in-house historical control range, did not occur in a dose-related manner and there was no obvious effect on mean feetal weights, this was considered to be fortuitous. 

### 2. Fretal evaluation: visceral observations The incidence of the anomaly retroesophageal subclavian artery, was slightly higher at 125 mglkg/day compared to the controls, interms ofmean percentage offeetuses affected (1.9%) and percentage of litters affected (12.5%), however, the incidence was comparable to the in-house historical control range of0.0-4.4% and 0.0-12.5% for the corresponding figures, and was therefore considered to be a chance fmding. The only variant fmding which was slightly higher in the treated groups compared with the controls

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### for both mean percentage of fretuses affected (3.3% at 125 mglkglday) and percentage of litters affected (12.5% at 125 mglkglday) was short innominate artery, but as the increase was not doserelated and was within the in-house historical control range of 1.0-3.9% for the fretal incidence and 9.1-13.0% for the litter incidence, at 125 mglkglday, this finding was consideredto be incidental. All other anomalies or variant findings occurred at a similar or higher incidence in the controls compared with the treated groups or as isolated findings occurring randomly across the groups, includingthe controls. 3. Fretal evaluation: skeletalobservations Two fretuses from separate litters at 125 mglkglday had hemivertebrae in at least one lumbar vertebrae. The mean percentage offretuses affected (0.9%) and percentage oflitters affected (8.3%) was slightly outside the historical control range of0.0-0.5% and 0.0-4.2%, respectively. However, in the complementary study (SA 04196, see below) where dams were dosed at 250 mglkglday, there were no fretuses with hemivertebrae in eitherthe thoracic or lumbar region. Therefore this difference was consideredto be incidental. The following anomaly: at least two caudal vertebrae fused, occurred at a higher frequency at 125 mglkglday than in the controls in terms ofboth fretal and litter parameters. This condition was associated with the absence ofsome caudal vertebrae in 3/4 cases. It

### shouldbe notedhowever, that all four fretuses were from the same litter and the incidence was only slightly outside the in-house historical control range in terms of mean percentage of fretuses affected with a figure of 1.5% comparedto a range of0.0-1.0% for the historical data, butwas within the historical control range for percentage oflitters affected, where the figure was 4.2% compared to the historical control range of 0.0-4.2%. In addition, in the complementary study (SA 04196) where pregnant rabbits were dosed at 250 mglkglday AE 0001789, there were no fretuses with this finding. This finding was therefore consideredto be fortuitous. A few variant findings occurred at a higher frequency in the treated groups or at 125 mglkg/day alone, compared to the controls. However, as all findings were within the historical control range for both fretal and litter parameters at the higher dose level of 125 mglkglday, these findings were consideredtobe fortuitous. 

### III. CONCLUSION In conclusion, a dose level of500 mglkg/day clearly exceeded a Maximum Tolerated Dose (MTD) in the pregnant rabbit. A dose level of 125 mglkglday was a No Observed Adverse Effect Level (NOAEL) in the dam due to the findings observed in the kidney, whilst being a No Observed Effect Level (NOEL) in the fretus, in theNewZealand White Rabbit. (Wason S., 2005b)

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### Report: KIIA5.6.11/02,Wason, S.; 2oo5c Title: AE 0001789
- Developmental toxicity study in the rabbit by gavage (complementary study) Report No & 

### SA 04196 DocumentNo M-2731S3-01-2 Guidelines: OECD 414 (2001); EPA Health Effects Test Guideline (OPPTS 870.3700; 1998); M.A.F.F. in Japan notification 12 Nousan N°8147 (2000) guidelines. GLP Yes (certified laboratory) Executive summary Twenty-five time-mated female New Zealand White Rabbits per group were exposed to AE 0001789. a herbicide safener belonging to the reverse sulfamide family, (batch number 08466/0013, a white powder, 97.4% w/w purity) by gavage from Gestation Day (GD) 6 to 28. The doses given were 0 and 250 mglkg/day in suspension in aqueous solution of0.5%methylcellulose400. This study was conducted as a complementary study to SA 03349 developmental toxicity study in the rabbit with the same compound, where the high dose level of500mglkg/day was found to be tootoxic to the dams. Maternal body weights were recorded for all surviving females on GD 3, 6, 8, 10, 12, 14, 16, 18,20,22, 24,26 and 29. Food consumption was measured for all surviving females during the intervals GD 3-6, 6­ 8,8-10, 10-14, 14-18, 18-22,22-26 and 26·29. Clinical observations were recorded daily. At scheduled sacrifice, on GD 29, the gravid uterine weight and the number ofribs were recorded and the dams were evaluated for number ofcorpora lutea, number and status ofimplantations (resorptions, dead and live fretuses). The urinary bladder and kidney were retained from all females but not histologically examined. Live fretuses were removed from the uteri, counted, weighed and examined externally. The heads of fretuses from approximately halfofeach litter were immersed in Bouin's fluid and the internal structures examined after fixation. The bodies of all fretuses were dissected for soft tissue anomalies and sexed. Fretuses were eviscerated, skinned and fixed in absolute ethanol before staining. A modification ofthe Staples and Schnelltechnique was applied and a subsequent skeletal examination was performed. In the treatment group at 250 mg/kg/day, two dams were sent to necropsy on GD 28 after aborting. Clinical signs for the first female consisted offew/mucoid faeces on several occasions, yellow sediment in the urine on GD 24-26 and 28, and anogenital soiling, red traces on tray andplacental tissue on tray on GD 28. Clinical signs for the second female included no/few faeces on several occasions, yellow sediment in the urine on GD 23·28, red traces on tray on GD 26·28 and placental/fretoplacental tissue on tray on GD 28. Between GD 10 and 28 the first female lost 0.49 kg, whilst between GD 20 and 28 the second female lost 0.57 kg in body weight. In both cases; the body weight loss was associated with a marked reduction in food consumption. No treatment-related findings were observed in either female at themacroscopic examination. In surviving females, 

### 5/23 animals had yellow sediment in the urine on one or more occasions. In addition, one female had red traces on tray on GD 28. No effect on mean maternal body weight change, mean maternal corrected body weight change or mean maternal food consumption was observed at this

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### dosage. No treatment-related necropsy findings were observed in the dams at the macroscopic examination. At Caesarean section, no effect on litter parameters was observed. No treatment-related findings were observed at the external, visceral orskeletal fretal examination. In conclusion, a dose level of 250 mglkglday, was considered to cause clear maternal toxicity but no developmental toxicity. 

### I. MATERIALS AND METHODS A. MATERIALS: 1. 

### Test Material: AE 0001789 

### Description: whitepowder 

### Lot/Batch: 08466/0013 

### Purity: 97.4 % 

### CAS: 221667-31-8 

### Stabilityoftestcompound: Stable in suspension in the vehicle (aqueous solution of methylcellulose400 at 0.5%) at concentrations of0.1 and 250 gil for a period of28 days under similar conditions to those ofthe current study. 2. 

### Vehicle and lor

### positive control:The vehicle was an aqueous solution ofmethylcellulose 400 at 0.5% 3. 

### Testanimals: Species: Rabbit 

### Strain: New Zealand White Crl:KBL (NZW) 

### Age: 18 weeks approximately 

### Weightatdosing: 3.12 to 3.70 kg for the females 

### Source: CharlesRiverlaboratories, Chiitillon-sur-Chalaronne, France 

### Acclimation period: At least 4 dayspriorto dosing 

### Diet: Laboratory animal pellets 110C-IO from S.A.F.E. (Scientific Animal Food andEngineering, Augy, France), adlibitum 

### Water: Tap water, adlibitum 

### Housing: Animals were caged individually in polycarbonate cages on a perforated cage floor 

### Environmentalconditions

­

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### Temperature: 19 

### ± 2°C 

### Humidity: 55 

### ± 15% 

### Air changes: Approximately 10tol5 changes per hour (not monitored) 

### Photoperiod: Alternating 16-hourlight and 8-hour dark cycles (5 am- 9pm) 

### B. STUDY DESIGN: 1. In life dates: November 02 to December 07, 2004 at Bayer CropScience, SophiaAntipolis, France.

2. 

### Animal assignmentand treatment Time-mated female New Zealand White Crl:KBL (NZW) rabbits were used in this study. Stock males from the same strain were used by the supplier to naturally mate nulliparus females. The day ofinsemination was designated as GD 

### O. The animals were approximately 18 weeks ofage on arrival and were received on GD 1or 2. On each day ofmating, the females were allocated to control and treated group using a computerized randomization procedure. Body weight means were checked to ensure similar means among all groups. The doses were administered daily by gavage at a volume of 4 mUkg to each female from GD 6 to GD 28 inclusive, based on the animal's most recent body weight. Control animals received an equivalentvolume ofvehicle alone (methylcellulose). The experimental groups were as follows: 

### Tbl56113Sdd'd

a e •• 

# - tu ly eS)2D an amma aSS)2Dment Testgroup 

# ITest substance Dose levels 

# IConcentrations Volume Number of mglkglday I 

## gIL (mL/kg) animals I 1 0 0 0 4 25 2 AE 0001789 250 62.50 4 25 

### 3. Test substancedosage formulations andanalysis The appropriate amount oftest substance was periodically (six formulations at 62.5 gil) suspended (w/v) in an aqueous solution ofmethylcellulose400 (Fluka, Mulhouse, France) at 0.5% and stored at approximately 5°C (± 3°C). Homogeneity of the suspensions was checked during the first formulation. The concentration was checked for all formulations. Homogeneity and concentration checks ofAE 0001789 were between 92 and 107% ofnominal values. Stability ofAE 0001789 in suspension in the vehicle was determined before the start ofthe study at 0.1 and 250 gil in aqueous methylcelJulose in a previous study and was found to be stable for 28 days under similar conditions tothose ofthe current study. C. 

### METHOD:

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### 1. 

### Observations All rabbits were observed daily for clinical signs and twice daily for mortality (except once daily on weekends and public holidays). 

### 2. Body weight Body weights were measured on GD: 3,6, 8, 10, 12, 14, 16, 18,20,22,24,26and29. 

### 3. Food consumption Full feeder weights were measured on GD: 3,4,5,6,8, 10, 12, 14, 16, 18,20,22,24,26and 28. Empty feeder weights weremeasured onGD: 4, 5,6, 8, 10, 12, 14, 16, 18, 20,22,24,26,28 and 29. 

### 4. Caesarian sections Animals killed for human reasons by intravenous injection ofDolethal® (Sanofi, Libourne, France) including females which aborted or found dead, were autopsied. The number and type of implantations and corpora lutea were noted when present. In the case ofno visible uterine implants, but with corpora lutea, uterine horn(s) were immersed in a 20% solution of ammonium sulfide according to the Salewski method (1964). The urinary bladder and kidney were taken and preserved in 10% neutral buffered fonnalin. Tissues and carcasses werethen discarded. On GD 29, surviving females were killed by intravenous injection ofDolethal® for examination of their uterine content. Each female was first subjected to macroscopic examination ofthe visceral organs and the number of ribs was recorded. In 

### the case ofno visible uterine implants, but with corpora lutea, uterine horn(s) were immersed in a 20% solution ofammonium sulfide according to the Salewski method. The urinary bladder and kidney were taken and preserved in 10% neutral buffered fonnalin. Tissues and carcasses were then discarded. The reproductive tract was weighed (gravid uterine weight), dissected out and the following parameters recorded: number ofcorpora lutea, number ofimplantation sites, number ofresorption sites (classified as early and late), number of live and dead fretuses, individual weights of live fretuses. Dead fretuses: were defined as fretuses showings distinct digits visible on fore and hindpaws. All the live fretuses were and subjected to an external examination. Resorption death was classified as early resorptions when macroscopic discrimination between fretal residues and placental material was not possible and late resorption when distinct macroscopic discrimination between fretal residues and placental material was possible. 

### 5. Fretalexamination All data were recorded without knowledge of treatment group. After internal examination of the neck, the head offretuses from approximately halfofeach litter was immersed in Bouin's fluid and the internal structures examined after fixation. The bodies of all fretuses were dissected for soft tissue anonnalies and sexed. Then the fretuses were fixed in absolute ethanol before staining. A modification of the Staples and Schnell staining technique was used and a subsequent skeletal examinationwas perfonned.

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### 6. Statistics Means and standard deviations (STD) for all maternal and litterparameters were calculated for each group. Statistical analyses wereperformed onthe following variables using TERATEST Version 1.0. Maternal endpoints: body weight change calculated according to time periods, calculated corrected body weightchange, average food consumption calculated accordingto time periods. Litter based and fretal endpoints: number ofcorpora lutea, number ofimplantation sites, number of resorption (early and late), pre- and post-implantation loss percentages, fretal sex, fretal death status, fretal body weight. 

### II. RESULTSAND DISCUSSION A. MATERNALOBSEV ATIONS 1. 

### Mortality At 250 mg/kglday, twodams were senttonecropsy on GD 28 after aborting. Clinical signs for the first female consisted offew faeces on several occasions, mucoid faeces on one occasion, yellow sediment in the urine on GD 24-26 and 28 and anogenital soiling, redtraces on tray andplacental tissue on tray on GD 28. Clinical signs for the second female included no/few faeces on several occasions, yellow sediment in the urine on GD 23-28, red traces on tray on GD 26-28 and placentallfretoplacental tissue on tray on GD28. Between GD 10 and 28 (terminal body weight) the first female lost 0.49 kg in body weight, whilst between GD 20 and 28 (terminal body weight) the second female lost 0.57 kg inbody weight. In both cases, the body weight loss was associated with amarked reduction in food consumption. There were no treatment-related necropsy findings in either female. 

### 2. Clinical signs In surviving dams at 250 mglkglday, 5/23 females had yellow sediment in the urine on one or more occasions. In addition, one female had red traces on tray on GD 28. Other clinical signs occurred at a similar frequency in the control group or occurred in isolation and were therefore consideredtobe incidental findings. 

### 3. Pregnancyrate The pregnancy rate was unaffected by treatment, with a pregnancy rate of 100% at 250 mg/kg/day and 96%in the controlgroup. 

### 4. Body weight No effect on mean maternal body weight change ormean maternal corrected body weight change (maternal body weight change independent of the uterine weight) was observed at 250 mg/kglday. 

### 5. Foodconsumption Mean maternalfood consumption was comparabletothe controls at250 mglkglday.

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### 6. Maternal necropsy and microscopic findings Notreatment-related maternalnecropsy findings were observed at autopsy. B. LITTERDATA There was no effect on litter parameters including early and late resorptions, fretal death status, percentage ofmale fretuses and mean fretal body weight. The percentage ofmale fretuses was higher at 250 mglkg/day compared to the controls, the effect being statistically significant, but this finding was considered incidental and due to the low percentage ofmales controls as the percentage ofmale fretuses was closer to the norm of approximately 50% (46.3%) at 250 mglkg/day than was the case for the controls (45.2%). C. F<ETAL NECROPSY FINDINGS 1. 

### Fretal evaluation: external observations There were notreatment-relatedmalformations. The only anomalies observed in the treated group were short tail in one fretus and swollen abdomen in two fretuses from separate litters. At such low incidences these findings were considered to have occurredby chance. There were no variant findings at the external fretal examination. 

### 2. Fretal evaluation: visceral observations There were notreatment-relatedmalformations. Although the incidence of the variant finding misshapen thymus was higher at 250 mg/kg/day compared with the controls, it was well within in house historical data. The anomaly misshapen kidney(s) and the variant finding small gall bladder were marginally outside the historical control range in the group treated at 250 mglkg/day, when the percentages of litters affected and/or of fretuses affected were considered. However, as neither parameter was statistically significantly increased atthe fretal orlitter level, these findings wereconsideredto have occurredby chance. 

### 3. Fretalevaluation: skeletal observations There were notreatment-related malformations or anomalies. The variant findings of 5th distal phalanges of hindpaws unossified, and less than 15 caudal vertebrae, occurred at a slightly higher incidence in the treated group compared to the controls. However, as the incidence for both mean percentage of fretuses affected and percentage of litters affected for each parameter was within the in-house historical control range, these findings were consideredto have occurredby chance. The following variant findings occurred at a slightly higher incidence in the treated group compared to the controls: 13th thoracic ribs detached, pubis unossified, and insertion point(s) ofpelvic girdle unossified. Butthe differences were not statistically significant for these findings, and were therefore consideredtobefortuitous.

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Ill. CONCLUSION In conclusion, a dose level of 250 mglkglday, was considered to cause clear maternal toxicity but no developmental toxicity. (Wason S., 2005c) 

### IIA5.7 Neurotoxicity All studies presented in this section were conducted between 2005 and 2006 and complied with the EU, OEeD,USEPA and Japanese MAFFtesting guidelines andGoodLaboratory Practice (GLP). In an acute neurotoxicity study, AE 0001789 was administered by gavage to non-fasted young adult Wistar rats at 0, 126,508 and 2060 mglkg (analytical results). Urine stains were evident in both sexes at 2060 mglkg on day 0 and resolved by the following day. No treatment-related findings were observed at 508 and 126 mg/kg. There was no evidence ofneurotoxicity at any dose level and no compound-related gross ormicroscopic lesions at the high dose of2060 mglkglday. The NOEL was 508 mglkg. In a 90-day neurotoxicity study, AE 0001789 was administered in the diet for 13 weeks to young adult Wistar rats. The mean daily intake of the test substance (mg AE 0001789/kg body wt/day) over approximately 13 weeks at nominal dietary concentrations of 1000, 3000 or 9000 ppm, respectively, was 65.2, 193 and 592 mglkglday for males and 84.5, 251 and 748 mglkglday for females. There were no treatment-related effects attributed to exposure to AE 0001789 at any dietary level in either sex. Based on these findings, a NOEL of 9000 ppm was established for the rat (specifically, 592 and 748 mg AE 0001789/kgbodywt/day for males and females, respectively). Table5.7-1 Summaryofneurotoxicitywith AE0001789 Typeofstudy Doses NO(A)EL (mglkgld) LOAEL (mglkgld) Adverseeffects/target organs Acute neurotoxicity in therat M-283196-01-1 0, 126, 508, 2060 mglkg bw 508 2060 Urine staining in both sexes 90-day neurotoxicity inthe rat 

# I M-283218-01-1 0,1000,3000,9000ppm 592/748 (M/F) 

### >5921>748 (MIF) None

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### IIA5.7.1 Acute neurotoxicity

- rat Report: KIIA5.7.1101, GilmoreRG.,2006 Title: An acute Oral Neurotoxicity Screening Study with Technical Grade AE 0001789 in Wistar Rats Report No & 201558 DocumentNo M-283 196-01-1 

### Guidelines: OEeD 424 (1997); EPA Health Effects Test Guideline (OPPTS 870.6200; 1998); M.A.F.F. in Japan notification 12 Nousan N°8147 (2000) guidelines. GLP Yes (certified laboratory) 

### Executive summary: Technical grade AE 0001789 (batch number 08466/0013, 97.4% purity) was administered by gavage in a single dose to non fasted young-adult Wistar rats. Four dose groups (12 rats/sex/dose level) were administered the test substance at nominal doses of0 (vehicle), 125, 500 or 2000 mglkg for both sexes. The test substance was administered in 0.5% methylcellulose 

### 10.4% Tween 80 in deionized water, at a dosing volume of 10 ml/kg. Dose concentrations, as well as the homogeneity and stability ofAE 0001789 in the dosing suspensions, were confirmed. All 12 rats/sex/dose level were used for neurobehavioural evaluation, with six/sex/dose level used for micropathology. Body weight was measured weekly as a component ofthe functional observational battery (FOB). Detailed clinical observations for each animal were performed daily throughout the study. Observations for moribundity and mortality were performed at least once daily. Automated measurements of activity (figure-eight maze) and a functional observational battery (FOB)were conducted duringthe week priorto treatment and on days 0 (the day of treatment, at the time ofpeak effect), 7 and 14. All animals placed on study were subjected to a gross necropsy. Selectedanimals (six/sex/dose level) were perfused, the brain was weighed in orderto calculate the brainlhody weight ratio and skeletal muscle, peripheral nerves, eyes (with optic nerves) and tissues from the central nervous system were examined microscopically for lesions. Based on analytical results, the actual doses ofAE 0001789 used in the study were 0, 126, 508 and 2060 mglkg formales and females. Findings attributedto exposuretoAE 0001789wereas follows: 

### 2060 mg/kg: Urine staining was evident in both sexes on day 0 andresolvedby the following day. 

### 508 mg/kg: There were no signs relatedtotreatment in either sex. 

### 126 mg/kg: There wereno signs relatedtotreatment in either sex.

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### In conclusion,compound-related effects following a single oral dose ofthe test substance were limitedto the high dose (2060 mglkg) males and females. Effects in high-dose males and females consisted ofurine staining, which resolved the following day after treatment. Based on these findings, a NOEL of508 mg AE 0001789/kg body weight was established in both sexes. There was no evidence ofneurotoxicity at any dose level and there were no compound-related gross ormicroscopic lesions at the high dose of2060 mglkg.

I. MATERIALS AND METHODS

A. MATERIALS: 1. 

### Test Material: AE 0001789 Description: whitepowder Lot/Batch: 08466/0013 

### Purity: 97.4% (October 2003, certifiedto October20,2005) CAS: 221667-31-8 Stabilityoftestcompound: Stable in the vehicle at the room temperature storage conditions used in this study. 2. Vehicle and lor positivecontrol:0.5%methylcellulose/O.4% Tween 80 in deionized water 3. Testanimals: Species: Rat Strain: Wistar HAN CRL: WI (HAN) Age: at least 9 weeks Weight atdosing: 232.8 to 309.0 g for the males

- 167.4 to 215.4 g forthe females Source: Charles RiverLaboratories, Inc., (Raleigh, NC) Acclimation period: 7 days Diet: Purina Mills Rodent Lab Chow 5002 in meal form provided for ad libitum consumption during the acclimation period and throughoutthe study except during neurobiavioraltesting Water: Tap water, adlibitum Housing: Animals were caged individually in suspended stainless steel wire-mesh cages.

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Environmentalconditions

­ Temperature: 22 

### ±4°C Humidity: 50

## ±20% Airchanges: Approximately 10 changes perhour Photoperiod: Alternating 12-hourlight and dark cycles B. STUDY DESIGN: 1. In lifedates: March 7, 2005 to April 7, 2005 at Bayer CropScience LP, Stilwell, KS, United States. 2. Animalassignmentand treatment There were 12 animals ofeach sex per dose group. Animals were assigned to dose groups using randomization by weight. Four dose groups (12 rats/sex/dose level) were administered the test substance at nominal doses of0 (vehicle), 125, 500 or 2000 mglkg (actual doses; 126, 508 and 2060 mglkg) for both sexes. The test substance was administered by gavage as a single dose in 0.5% methylcellulose / 0.4% Tween 80 in deionized water, at a dosing volume of 10 mllkg. The table below summarizes the study design. d 	 • Table5.7.1.-1: Study eSi2n and animal aSSi2ment Dose Group mWk2 bwt) Control 125 500 2000**~AI''''**m.ental Parameter 12 12 12 12 Total numberofanimals/sex/group 12/sex 12/sex 12/sex Behavioral Testing (FOB,MotorActivity) • 12/sex 6/sex O/sex O/sex Neuropathology· 6/sex


# . FOBandmotoractIVIty were assessedpriorto dosmgandagamdurmgdays 0, 7and 14. h Tissuesfrom the mid- and low-dose groups were not examined, as no treatment-related neuropathology was noted at the highestdose level,

The rationale for dose selection was based on the results ofan acute oral toxicity study in youngadult male and female Wistar rats. In that study, two fasted male and female Wistar rats were administered an acute oral (gavage) dose of either 500, 1000 or 2000 mglkg as an aqueous suspension in 0.5% methylcellulose in distilled water, ata dosing volume of 10 mllkg. Animals were observed for mortality and clinical signs for 13 days aftertreatment. The test substance produced no clinical signs or mortality at the limit dose. These results support the use of a limit dose (2000 mglkg) in the neurotoxicity study butprovidedno information to establishthetime ofpeakeffect.

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### Thus, the results from a study using radio-labeled AE 0001789 were examined to estimate the time ofpeak effect. In that study, adult Wistar male and female rats received a single oral (gavage) dose of2 or200 mg/kg AE 0001789 in 0.5% aqueous tragacanth, at a dosing volume of 10 mllkg. In that study, the tmax in plasma occurred 40
- 60 minutes after treatment in both sexes following administration at 200 mglkg. For the low dose (2 mg/kg), the tmax in plasma occurred 10
- 40 minutes aftertreatment in both sexes. Based on these collective results, the doses selected for this study were 0, 125, 500 and 2000 mg/kg for both sexes. Based on the estimatedtime ofpeak blood concentrations within this dose range, the FOB began approximately 50 minutes (minimum) following dose administration, with the automated test ofactivity concluding at aboutthree hours after treatment. 3. Dietpreparation andanalysis Doses were prepared by suspending the test substance in 0.5% methylcellulose/0.4% Tween 80 in deionized water. The concentration of AE 0001789 in the vehicle was measured using highperformance liquid chromatographic lultra violet (HPLCIUV) analysis. The homogeneity and stability ofAE 0001789 in the vehicle were established using samples at nominal concentrations (l0 and 200 mg/m!) that bracketed the range ofconcentrations used in the present study (nominal 12.5 and 200 mg/ml). Homogeneity was accepted ifthe percent relative standard deviation (%RSD) was <5%. Each dosing suspension was also analyzedto measure the concentration ofAE 0001789. Homogeneity Analysis: Homogeneity ofthe test substance in the vehicle was accepted for the range of concentrations used here, as the 10 mg/ml and 200 mg/m] concentrations had percent relative standarddeviations (%RSD) of0.39% and 1.33%, respectively. Stability Analysis: The stability (atroom temperature conditions) ofAE 0001789 in the vehicle was established, with no appreciable decrease in concentration with seven or eight days at room temperature storage for nominal concentrations of 10 mg/ml or 200 mg/ml, respectively (equivalent to doses of 100 or2000 mg/kg. respectively). Concentration Analysis: Doses of0, 125, 500 and 2000 mg/kg for males and females ranged from 101% to 103% ofthe nominal concentrations. Based on these results, the analytically-confmned doses for males and females were 0, 126,508 and 2060 mg/kg. 4. Statistics: Statistical evaluations were generally performed using software from either INSTEM Computer Systems or SAS. The level ofsignificance was set at p**~** 

### 0.05, with the exception ofBartlett's test,

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### which was tested at p S 

### 0.00I. In some cases, data were visually screened for potential effects prior to being subjected to statistical analysis. In general, continuous data were initially assessed for equality ofvariance using Bartlett's test. Group means with equal variances were analyzed further using an Analysis ofVariance (ANOVA), followed by a Dunnett's test ifa significant F-value was determined in the ANOVA. In the event of unequal variances, these data were analyzed using nonparametric statistical procedures (Kruskal-Wallis ANOVA followed by the Mann-Whitney Utest for between-group comparisons). For the FOB, continuous data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA ifthere was a significant interaction between dose group and test day. For days on which there was a significant treatment effect, Dunnett's test was applied to determine which groups, ifany, were significantly different from the control group. Categorical data collected in the FOB was analyzed in a similar manner, using General Linear Modeling and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett'stest and an Analysis ofContrasts, respectively. Motor and locomotor activity (total session activity and activity for each lO-minute interval) was analyzed using ANOVA procedures. Session activity datawere analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOV A ifthere was a significant interaction with test occasion. For days on which there is a significant treatment effect, Dunnett'stest was used to determine which, ifany, groups were significantly different from the control group. Interval data were subjected to a two-way Repeated-Measures ANOVA, using both test interval and test occasion as repeated measures, followed by a Repeated Measures ANOVA to determine on which days there was a significant treatmentby interval interaction. Forthose days, the data for each interval were subjected to analysis using a one-way ANOVA to determine at which intervals there was a significant treatment effect. Forthose intervals, Dunnett's test was used to determine which groups, ifany, were significantly different from the control group. Continuous pathology data (e.g., brain weight) was initially evaluated using Bartlett'stest to analyze for homogeneity of variance among groups. Groups with homogeneous variances were analyzed further using an ANOVA followed by Dunnett's test for pair-wise comparisons. In the event ofnonhomogeneous variances, continuous data were analyzed using the nonparametric Kruskal-Wallis test followed by aMann-Whitney Utest for pair-wise comparisons. Micropathology frequency data were screened for potential effects and then evaluated using a Chi-Squared procedure, followed by a onetailed Fischer's Exact Test in cases of significant variation by the Chi-Square analysis. Micropathology severity grades were not routinely subjected to statistical analysis. Additional statisticaltests to assess continuous and frequency data were used when deemed appropriate.

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C. 

### METHODS: 1. 

### Mortality and Clinical Observations Cage-side observations were perfonned at least once daily (once daily on holidays and weekends) for mortality or clinical signs ofmoribundity. Detailed physical examinations for clinical signs of toxicity were carried outandrecorded daily. 

### 2. Body weight: Animals were weighed weekly as a component of the FOB. Additionally, all animals were weighed onthe day ofsacrifice fortenninalbody weightmeasurement. 

### 3. Food Consumption: Food consumptionwas not measured in this study. 

### 4. Neurobehavioral Assessment: All animals that were assigned to the study weretested using the FOB and motor activity on four occasions

- one week prior to treatment, approximately 50 minutes after administration ofthe dose, and again 7 and 14 days following treatment. The order of testing and assignment of animals to mazes were done in a semi-random manner, such that groups were balancedacross test times and test devices, and no animal would be tested more than once in the same maze. On the day ofFOB and motor activity testing, the appropriate animals were placed in the sequence that was established for testing on that day. The dose group identification was concealed at that time to ensure that testing would be perfonned without knowledge ofthe group assignment. Sets of eight animals (maximum) were evaluated individually using the FOB and then, approximately 30 minutes after the last animal in the set had finished being tested in the FOB, all eight rats were placed individually intothe mazes to measure activity.



### a. Functional Observational Battery(FOB): The FOB closely follows the battery oftests described by Moser, with each animal tested individually. The technicians who perfonned the FOB were "blind" with respect to the animal's group assignment. Observations and measurements (e.g., grip strength and foot splay)

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### for all animals were performed by the same person throughout the study. Inter-observer reliability has been established in order to allow a second person to perform either the observations or measurements, ensuring the consistency of the results of each technician. Studies have been conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of these test procedures, the adequacy of training of technical personnel and to serve as a historical control. When applicable, observations were scored on intensity as follows: 1) slight (barely perceptible or infrequent) or2) moderate to severe. Data were collected while the rats were in their home cage, during handling, and in an open field for 2 minutes (in the centre ofa flat surface with a perimeter barrier, such as a cart). In addition, reflex and physiologic observations and measurements were made while the animals were sitting on the cart surface following open field observations. Home cage observations included: posture, piloerection, involuntary motor movements (such as repetitive "chewing" movements of mouth and jaw, tremors, and convulsions), gait abnormalities, vocalizations, decreased activity, repetitive head bobbing, and increased reactivity. Observations during handling included: ease ofremoval from cage, reaction to being handled, muscle tone, palpebral closure, lacrimation, salivation, nasal discharge, stains (lacrimal, nasal, perianal, urine, oral), alopecia, emaciation, bite marks, exophthalmia, broken teeth/malocclusion, missing toe nail(s), dehydration, and temperature upon touching (cool-totouch). Open field (2 min.) observations included: number of rears, piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy (excessive or repetitive behavior), bizarre behaviour, gait abnormalities, vocalizations, arousal level, and amount of excretion. Reflex and physiologic observations/measurements included: approach response, touch response, auditory response, tail pinch, pupil size at normal lighting, pupil response, righting reflex, grip strength body weight, body temperature, and landing foot splay. 

### b. Motorand LocomotorActivity: Motor and locomotor activities were measured approximately 30 minutes after the last animal

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in the set (8 rats maximum) had finished the FOB. All rats in each set were placed individually into figure-eight mazes and activity was measured for a total of 60 minutes. Eight infrared emitter / detector pairs (three in each ofthe figure-eight alleys and one in each ofthe blind alleys) measured activity; each time a beam was interrupted, an activity count was registered. Broad-spectrum background noise (approximately 74 dB(A)) was provided throughout the test to minimize acoustical variations during testing. Motor and locomotor activities were examined during each ofthe six, ten-minute intervals. Motor activity was measured as the numberofbeam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption ofa given beam was counted until the rat relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session. 

### 5. Sacrifice and Pathology: All animals placed on study were subjected to a complete gross necropsy. The necropsy involved an examination ofall organs, body cavities, cut surfaces, external orifices and surfaces. On day 14, a minimum ofsix males and six females at each dose level were selected for perfusion and collection oftissues, with replacement, as necessary, ifthe perfusion was considered inadequate. These animals were deeply anaesthetized using an intraperitoneal dose (50 mglkg) of pentobarbital and then perfused via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by Universal fixative (1% (w/v) glutaraldehyde and 4% (w/v) EM-grade fonnaldehyde) 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, both forelimbs, gross lesions in neural tissues or skeletal muscle and physical identifier were dissected from each animal and post-fixed in 10% buffered fonnalin. The brain was weighed upon removal from the skull, prior to placement into fonnalin, and the brain:body weight ratio was calculated. Other animals that survived to tenn were sacrificed by CO2 asphyxiation withoutperfusion. Micropathology examinations were perfonned on a comprehensive battery ofneural tissues from perfusion-fixed control and high-dose rats ofboth sexes, as follows. Eight coronal sections ofthe brain and sections from three levels ofthe spinal cord (cervical, thoracic, lumbar) and the cauda equina were embedded in paraffin and examined utilizing haematoxylin and eosin (H&E). Dorsal root ganglia (including dorsal and ventral root fibres) from the cervical and lumbar swellings and gasserian ganglion were embedded in glycol methacrylate (GMA). Eyes, optic nerves and

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### gastrocnemius muscle were embedded in paraffin and stained using H&E. Peripheral nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA and cut in cross/transversesection as well as longitudinal section. GMA-embedded tissues were sectioned at 2-3**~m** 

### and stained using a modified Lee's stain. The sciatic nerve was also cross-sectioned at approximately 2-3**~m** 

### and stained with a modified Lee's stain. In addition, histopathology was performed on any gross lesions collected at necropsy. Tissues from perfusion-fixed animals at the low- and mid-dose levels were not subjected to micropathology unless compound-related lesions were present in the high-dose group. 

### II. RESULTS AND DISCUSSION A. OBSERVATIONS 1. Clinicalsigns Compound-related findings were limited to the highest dose and consisted of a urine stain, on day 0 only, in three males and five females. The remaining findings were not considered to be related to treatment. These findings included urine staining in two low- (one present only on day 0 and one present on days 3, 7-8) and one mid-dose (present only on day 0) females. The urine staining was attributedto treatment in highdose males and females but not in females at the mid- and low dose, due to the low incidence, which was within the range ofhistorical control, there was no relationship to dose, there was no similar fmding in males at these dose levels and, in one case, the staining first occurred 3 days aftertreatmentwas administered. The remaining incidental fmding consisted ofareas ofalopecia present in two low-dose males and one low-dose female (fITst present on day 9 in the males and on day 7 in

### the female continuing until study termination in all three) and in one high-dose male (present on day 12 and continuing until study termination).

Table5.7.1.-2: Clinical Observations

Observation Dose Level (mglkg bw) 

# ··-----------T-----------­

Control 125 500**~--~~-~-----------** 2000 Males 

### I Urine Staining 0112 0112 0/12 3112 Abdomen Left 0112 0112 0/12 1112 

## II Alopecia

- Limb

- Fore, Both 0/12 2/12 0112 0112 Females

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Urine Staining 0112 2/12 1112 5112 Alopecia

- Abdomen

- Ventral, Left 0112 1112 0112 0112 Numbers represent the totalnumber of animals exhibiting the signatleast once/number of animals in eachgroup 2. Mortality There were no compound-related deaths at any dose level in either sex. No animals were found dead or sacrificed in extremis during the course ofthe study.


### B. BODY WEIGHT Body weight was not affectedby treatment in either sex at any dose level.
Table 571-3· BodyWeight(g ± s.d.) 

## ... Dose Level (mg/kg bw) 

# ·-----------......----1....-------------- -------------------­ Control 125 Observation (g ± s.d.) 500 2000 Males 264±20 257±18 263±14 Day 0 260±19 284±23 280±30 281±22 285±19 Day 7 311±27 311±33 305±24 Day 14 302±30 Females Day 0 188±14 184±12 181±9 185±14 201±13 194±14 Day 7 195±1l 201±13 Day 14 214±14 207±13 206±15 210±13 Values representmean ± s.d. n=12 *=p 

### :=; 0.05, **=p 

### :=; 0.01, when comparedto controlmeans. C. 

### NEUROBEHAVIORAL RESULTS 1. 

### FOB findings There were no treatment-related findings at any dose level in either sex. The only finding, areas ofalopecia, was considerednot to be related totreatment and was seen in two low- and one high-dose males on day 14 and in one low-dose female on days 7 and 14.
Table5.7.1.-4: FunctionalObservational BatteryResults
Males**---------------------------~~~~~:~;::-------------------------** Dose Level (mg/kg bw)**-----t-c-on-t-ro-I---rl-----~;~----r---~~~-------l---------;~~~-------**

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Day 14 Handling

- Other: NotObserved**1l.1"nP";~** Present 12(100) 0(0) 10(83) 2(17) 12(100) 0(0) 11(92) 1(8)

Females 1-----------------------------.--.---.-.-....-.-.--.-.-.-...-­ Observation Control**.~~~.~~~** 

### (!!!W.!g.E!l. 125 500 2
Day 7 Handling

- Other: NotObserved Alopecia. Present 12(100) 0(0) 11(92) 1(8) 12(100) 0(0) 12(100) 0(0) Day 14 Handling

- Other: NotObserved Alopecia. Present 12(100) O{O) 11(92) )(8) 12(100) 0(0) 12(100) 0(0) Values represent the number of animals and%incidence (inparentheses) with observation. n=12animals/dose level *=p**~** 0.05. ..P 0.01 comparedwilh controls 

### 2. Motoractivity An examination ofinherent variability, using the average pre-treatment values among the four groups ofmales and females, confinn that differences of±20% are within the range ofnormal variability in this laboratory for groups of 10-12 rats/sex/dose level and, therefore, are not biologically significant. For the overall 60-minute test session, there were no compound-related effects in session motor or locomotoractivity at any dose level in either sex. Differences from control that were considered incidental and unrelated to treatment included measures ofmotor (23%) and locomotor (27%) activity that were slightly below the standard (±20%) range of nonnal variability in high-dose males on day O. These differences from control were not consideredtoberelatedto treatmentbecausethe differences were small, only seen in one sex (no trend in females), and were not statistically significant. Moreover, the high-dose males exhibited consistently lower levels ofactivity than controls at all time-points, including prior to treatment. In addition, these measures of motor (total activity counts of 295-516 for referenced historical controls) and locomotor (total activity counts of 192-312 for referencedhistorical controls) activity were well within the range ofhistorical control.

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Table 5.7.1.-5: SummarySession MotorActivity Results (PercentDifference from Control)' MALES Nominal Dose Pre-treatment Day0 Day 7 Day14

125 mg/kg

- 17 + 14 +11 +9 500 mg/kg - 9 + 2

- 12 + 1 2000 mg/kg
- 12
- 23
- 14 - 8 FEMALES 125 mg/kg - 1 + 1

- 16 +0.2 500 mg/kg 0 + 1 0 - 3 2000 mg/kg +3 +4 -4 -9
aPercentgreater (+j or less (-j than concurrentcontrolforN=12. Summarysession motoractivitywas notstatisticallydifferent from control (* = p :$; 0.05; ANDVA). SummarySession LocomotorActivity Results (PercentDifferencefrom Control) • MALES Nominal Dose Pre-treatment DavO Dav7 Dav14

125 mg/kg
- 21 + 1 - 3 - 2 500 mg/kg

- 12 - 3
- 18 - 4 2000 mg/kg
- 12

- 27
- 20
- 15 FEMALES 125 mg/kg + 1 0

- 17 +2 500 mg/kg +3 - 3 - 3 +2 2000 mg/kg
- 17 +4 + 6 -2

u Percentweater (+j or less (-j than concurrentcontrolforN=12. Summary session locomotoractivitywasnotstatisticallydifferent from control (* = p :$; 0.05; ANDVA.
Motor and locomotor activity data were subjected to further analysis at each interval on each test day. There were no compound-related effects at any dose level in either sex. Habituation was not affected by treatment with AE 0001789 any dose level in either sex. D. SACRIFICE AND PATHOLOGY 1. Gross pathology There were no compound-related gross lesions evident at terminal sacrifice in males or females at any dose level. There was one incidental finding described as an abcess located in the leftuterine hom in one mid-dose female.

2. Terminalbody and brain weights There was no compound-related effect on terminal body weight or brain weight in perfused males or females at any level ofexposure.

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Table5.7.1.-6: Absolute andRelative BrainWeights Dose Level (mglkg bw) 

## -_._---_..__._----------_._-]--_._-_._---------------------­ ,-­ Weights (g) 500 2000 125 Control Males 308.7±28.6 305.8±27.9 298.0±28.5 303.6±22.8 Bodywt• 1.819±0.074 1.817±0.070 1.858±0.096 1.848±0.068 Brain wt b b 0.622±0.055 0.583±0.036 O.620±0.O52 0.629±0.082 Female 204.5±14.6 212.6±12.8 205.4±11.9 209.8±13.3 1.733±.049 1.732±0.OS7 1.669±O.032 1.75O±O.077 Brain wt b Brainlbody wt b 0.810±0.055 0.822±O.048 62±O.048 0.838±0.045

aN=12 bN=6 

### *Statisticallydifferent (p::;; O.05)from the control.

3. Neuropathology There were no compound-related microscopic lesions in the high-dose males or females. Therefore, tissues from animals that received a lowerdose ofAE 0001789 were notexamined. 

### III. CONCLUSION
In conclusion, compound-related findings following a single oral dose ofthe test substance were limited to the high dose (2060 mglkg) males and females. Effects in males and females consisted of urine staining, whichresolvedthe day aftertreatment. There was no evidence ofneurotoxicity at any dose level and no compound-related gross or microscopic lesions at the high dose of 2060 mg/kg. These results establish aNOEL of508 mg AE 0001 789/kgbody weight. (Gilmore R.G., 2006) 

### IIA 5.7.2 Delayed neurotoxicity following acuteexposure AE 0001789 is not an organophosporous insecticide and there are no indication ofneurotoxicity in any of the other studies,thereforethis type ofstudy is not triggered.

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### IIA 5.7.3 28-daydelayed neurotoxicity AE 0001789 is not an organophosporous insecticide andthere are no indication ofneurotoxicity in any of the other studies" therefore this type ofstudy is not triggered.
IJA 5.7.4 Subchronic neurotoxicity

- rat- 90-day Report: KIIA5.7.4/01, GilmoreR.G., 2006 Title: A Subchronic Neurotoxicity Screening Study with Technical Grade AE 0001789 in WistarRats Report No & 201573 DocumentNo M-283218-01-1 Guidelines: OECD 424 (1997); EPA Health Effects Test Guideline (OPPTS 870.6200; 1998); M.A.F.F. in Japan notification 12 Nousan N°8147 (2000) guidelines. GLP Yes (certified laboratory) 

### Executive summary: Technical grade AE 0001789 (batch number 08466/0013, 97.4 to 97.5% purity) was administered in the diet for 13 weeks to young-adult Wistar rats (l2/sexldietary level), using nominal concentrations of0, 1000, 3000 and 9000 ppm for males and females. The top dose level for this study was chosen based on mortality observed in a 90-day toxicity study (M-I05691-01-2) at a higher dietary level (i.e., 12,000 ppm). All test diets (including control) were provided for adlibitum 

### consumption throughout the study except during neurobehavioural testing. All 12 rats/sexldietary level were used for neurobehavioural evaluation, with micropathologyperformed on selectedtissues from 6rats/sex from control andhigh-dose groups. Body weight and food consumption determinations, as well as a detailed clinical observation for each animal, were conducted weekly throughout the study. Observations for moribundity and mortality were performed at least once daily. Automated measurements of activity (figure-eight maze) and a functional observational battery (FOB) were conducted the week priorto treatment and duringweeks 2, 4, 8 and 13. Ophthalmologic examinations were conducted on all animals priorto shipment release and then again on all study animals during week 12. All animals placed on study were subjected to a gross necropsy. For selected animals, the brain was weighed in order to calculate the brain:body weight ratio and skeletal muscle, peripheral nerves, eyes (with optic nerves) and tissues from the central nervous system were also examinedmicroscopically for lesions. The mean daily intake ofthe test substance (mg AE 0001789/kg body wVday) over approximately 13 weeks at nominal dietary concentrations of 1000, 3000 or 9000ppm, respectively, was 65.2, 193 and 592 mglkglday for males and 84.5, 251 and748 mglkg/day for females. There were no treatment-related effects attributed to exposure to AE 0001789 at any dietary level in either sex. 

### Conclusions. Through approximately 13 weeks of continuous dietary exposure to the test substance,

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### there were no treatment-related fmdings apparent at any dietary level in either sex. Based on these findings, a NOEL of9000 ppm was established for the rat (equating to 592 and 748 mg AE 0001789/kg body wt/day for males and females, respectively).

I. MATERIALS AND METHODS A. MATERIALS: 1. 

### Test Material: Description: Lot/Batch: Purity: CAS: Stability oftestcompound: AE 0001789 white powder 08466/0013 

### 97.4% (October2003) ;97.5% (October2005) 221667-31-8 Stable in the diet at 10 and 10 000 ppm at the room temperature for 7 days orat freezing storage after28 days. 

### 2. Vehicle andlorpositive control:Acetone served as a solvent in the diet preparation process and



### 3. Testanimals: Species: Strain: Age: Weight atdosing: Source: Acclimation period: Diet:

Water: Housing: Environmentalconditions

­ Temperature: Humidity: Airchanges: was allowedtoevaporate priorto administration

Rat Wistar HAN CRL: WI 

### (HAN) approximately 8weeks 210.0 to 285.1 gfor themales 141.4to 179.5 g for the females Charles RiverLaboratories, Inc., (Raleigh, NC) 

### 7 days Purina Mills Rodent Lab Chow 5002 in meal form provided for 

### ad libitum consumption during the acclimation period and throughoutthe study except during neurobiavioraltesting Tap water, 

### adlibitum Animals were caged individually in suspended stainless steel wire-mesh cages. 22 

### ±4°C 50 ±20% Approximately 10 changes perhour

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### 2007-03·28 Tier2, llA, Sec. 3,Point5: Cyprosulfamide Photoperiod: Alternating 12·hourlight and dark cycles B. STUDY DESIGN: 

### 1. 

### In life dates: August 22, 2005 to December 8, 2005 at BayerCropScience LP, Stilwell, KS, United States. 2. Animalassignment and treatment There were 12 animals ofeach sex per dose group. Animals were assigned to dose groups using randomization by weight. For 13 weeks, four dose groups (12 rats/sex/dietary level) were administeredthe test substance in the diet at nominal concentrations of0, 1000, 3000 and 9000 ppm (65.2, 193 and 592 mg/kg/day for males and 84.5, 251 and 748 mg/kg/day for females). The table below summarizes the study design. Table5.7.4.-1 StudyDesilD Dose Group ppm (m1Uk2 bw/day)

Experimental Parameter 1000 3000 9000 Control (d' 65.2) 

### C:i 84.5) 

### (0' 193) 

### i§ 251) 

### (0' 592) (§ 748) Total numberofanimals!sexlgroup 12 12 12 12 Behavioural Testing (FOB, MotorActivity) • 12/sex 12/sex 12/sex 12/sex Neuropathology b 6/sex O/sex O/sex 6/sex OphthalmicExamination C 12/sex 12!sex 12/sex 12/sex

. , FOB andmotoractIVIty was assessedprIOr to dOSing andagainduringweeks 2. 4, 8 and 13. b Tissuesfrom the mid- and low-dose groups were not examined. as no treatment-related neuropathology was noted at the highestdietary level. OphthalmiCexams wereperformedprior to dosingandatweek 12,

The rationale for dose selection was based primarily on the results ofa 90-day toxicity study, with the test substance administered via the diet at nominal concentrations of0, 1000, 4000, and 12,000 ppm to male and female Wistarrats (lO/sex/dietary level). The principal findings from that study are as follows. There were no compound-related findings at the 1000 ppm dietary level. Treatmentrelated findings at 4000 ppm were limited to simple hyperplasia ofthe urothelium in the urinary bladder ofone male and one female. Findings in animals treated at the 12,000 ppm dietary level includedthe death oftwo females. Animals that survived until study termination were observed with soiled fur around the anogenital area (two males) and hair loss (two males andone female). In males, there was a reduction in body weight (5-7%) during weeks 5 through 13 and decreased weight gain (up to 64%). In addition, absolute body weight was reduced by 6% during weeks 4 through 5 in females. Food consumption was decreased by 9% and 13% in males during weeks 1 and 5, respectively and by 17% in females during week 5. Also, sulfonamide-like crystals were found in the urine in both sexes. Findings observed at study termination included increased absolute and relative

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# EBBayerCropScience 2007-03-28 Tier2, llA, See. 3,Point5: Cyprosulfamide kidney weights in both sexes. Microscopic findings included slight centrilobular hepatocellular hypertrophy in males and simple hyperplasia ofthe urothelium in the urinary bladder ofmales and females. In both sexes, a slight increase in the incidence and/or severity ofbasophilic tubules and hyperplasia of collecting ducts and pelvic epithelium was observed in the kidneys. Marked metaplasia of the collecting duct epithelium was observed in females and brown pigment in the proximal tubules was evidentin both sexes. The incidence ofbrown pigmentin the spleen (red pulp) was slightly greater at the highest dietary level in a few animals. Lastly, in the thymus, there was a slight increase inthe severity ofdecreased size ofthe cortex (involution) in a few males. Based on these results, the doses selected for the present subchronic neurotoxicity study were 0, 1000, 3000 and 9000ppm in both sexes. 3. Dietpreparation andanalysis The diet was prepared every other week. Acetone was used to dissolve/suspend the test substance. The test substance/acetone mixture was added to the feed, mixed and the acetone was allowed to evaporate. The control diet was prepared the same way, excluding the test substance. Feed was available for adlibitum consumption for a period ofone week prior to changing, at which time any uneaten feed was collected and disposed ofby incineration. The concentration ofAE 0001789 in the ration was measured by liquid chromatographic analysis. The stability [following both room temperature (- 22°C) and freezer**(~** -23°C) exposure] and homogeneity ofthe test substance in the feed were established by analysis of samples at nominal concentrations of 10 and 10,000 ppm. The concentration of the test substance in the ration was measured for the ration that was used during all weeks ofthe study. Homogeneity Analysis: Homogeneity of the test substance in the ration was accepted for concentrations that bracket those used in this study. These concentrations of 10 and 10,000 ppm had percentrelative standarddeviations (%RSD)of 1.98%and 1.81%,respectively. Stability Analysis: The stability ofAE0001789 in the ration was established at room temperature at dietary concentrations of 10 and 10,000 ppm, with no appreciable decrease in concentration with seven days ofstorage. AE 0001789 was stable at freezer conditions for 28 days, with no appreciable decrease in concentration at 10 and 10,000 ppm. Concentration Analysis: Actual (analytically-determined) concentrations ofthe active ingredient in the 0, 1000, 3000 and 9000 ppm dietary levels used in this study averaged 97% to 98% ofthe nominal concentration. Based on these results, the mean analytically-confirmed dietary levels for this

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### study were 0, 976, 2904 and 8698 ppm for males and females. 4. Statistics: Statistical evaluations were generally performed using software from either INSTEM Computer Systems or SAS. The level ofsignificance was set at p :s; 0.05, with the exception ofBartlett's test, which was tested at p:s; 0.00I. In general, continuous data were analyzed using an Analysis of Variance (ANOVA), followed by a Dunnett's test if a significant F-value was determined in the ANOVA. For the FOB, continuous data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA ifthere was a significant interaction between dose group and test week. For weeks in which there was a significant treatment effect, Dunnett's test was applied to determine which groups, ifany, were significantly different from the control group. Categorical data collected in the FOB were analyzed in a similarmanner, using General Linear Modeling (GLM) and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis ofContrasts, respectively. Motor and locomotor activity (total session activity and activity for each 10-minute interval) was analyzedusing ANOVA procedures. Session activity data were analyzedusing a Repeated-Measures ANOVA, followed by a one-way ANOVA ifthere was a significant interaction with test occasion. For days on which there is a significanttreatment effect, Dunnett's testwas used to determine which, ifany, groups were significantly different from the control group. Interval data were subjected to a two-way Repeated-Measures ANOVA, using both test interval and test occasion as repeated measures, followed by a Repeated Measures ANOVA to determine on which days there was a significanttreatment by interval interaction. Forthose days, the data for each interval were subjected to analysis using a one-way ANOVA to determine at which intervals there was a significant treatment effect. For those intervals, Dunnett'stest was used to determine which groups, ifany, were significantly different from the control group. Continuous pathology data (e.g., brain weight) was initially evaluated using Bartlett's testto analyze for homogeneity ofvariance among groups. Groups with homogeneous variances were analyzed further using an ANOVA followed by Dunnett's test for pair-wise comparisons. In the event ofnonhomogeneous variances, continuous data were analyzed using the nonparametric Kruskal-Wallis test followed by a Mann-Whitney U test for pair-wise comparisons. Micropathology frequency data were screened for potential effects and then evaluated using a Chi-Square procedure, followed by a onetailed Fischer's ExactTest in cases ofsignificant variation by the Chi-Square analysis.

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### C. METHODS: 1. 

### Mortality and Clinical Observation Cage-side observations were performed twice daily (once daily on holidays and weekends) for mortality or clinical signs ofmoribundity. Detailed physical examinations for clinical signs of toxicity were carried out and recorded daily. 2. 

### Bodyweight: Animals were weighed weekly. Additionally, all animals wereweighed on the day ofsacrifice for terminal body weightmeasurement. 

### 3. Food Consumption: Individual food consumption was measured weekly. Daily food consumption was averaged over the duration ofthe study, basedon a perkg body weight. The average daily intake of the active ingredient (A.I.) (mg A.I./kg body weight/day) was calculated using weekly body weight and food consumption data. The general relationship used for this calculation was: [AI in feed (ppm)/I,000] x [feed consumed (glkg body wt/day)) mg AI/kg body wtlday. Using this formula, the average consumption ofA.I. for males and females that received diets containing analytically-determined nominal concentrations of 0, 1000, 3000 and 9000 ppm AE 0001789 was as follows: 0,65.2,193 and 592 mglkglday, respectively, for males and 0, 84.5, 251 and 748 mglkglday, respectively, for females. 

### 4. Neurobehavioural Assessment: All animals that were assigned tothe study were tested using the FOB and motor activity on five occasions

- one week prior to treatment, and again during weeks 2, 4, 8 and 13. The order of testing and assignment ofanimals to mazes were done in a semi-random manner, suchthatgroups were balancedacross testtimes and test devices, and no animal would betestedmorethan once in the same maze. On the day prior of each test day, the appropriate animals were placed in the sequence that was established for testing on thatday. The dose group identificationwas concealed prior to testing to ensure that testing would be performed without knowledge of the group assignment. Sets of eight animals (maximum) were evaluated individually using the FOB and

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### then, approximately 30 minutes after the last animal in the set had finished being tested in the FOB, all eight rats were placed individually into the mazes to measure activity. Each week, testing was staggered over two days for each sex to accommodate the schedule for behaviouraltesting.



### a. Functional Observational Battery(FOB): The FOB closely follows the battery oftests described by Moser, with each animal tested individually. The technicians who performed the FOB were "blind" with respect to the animal's group assignment. Observations and measurements (e.g., grip strength and foot splay) for all animals were performed by the same person throughout the study. Inter-observer reliability has been established in order to allow a second person to perform either the observations or measurements, ensuringthe consistency ofthe results ofeachtechnician. When applicable, observations were scored on intensity as follows: I) slight (barely perceptible or infrequent) or 2) moderate to severe. Data were collected while the rats were in their home cage, during handling, and in an open field for 2 minutes (in the centre ofa flat surface with a perimeter barrier, such as a cart). In addition, reflex and physiologic observations and measurements were made while the animals were sitting on the cart surface following open field observations. Home cage observations included: posture, piloerection, involuntary motor movements (such as repetitive "chewing" movements of mouth and jaw, tremors, and convulsions), gait abnormalities, vocalizations, decreased activity, repetitive head bobbing, and increased reactivity
. Observations during handling included: ease ofremoval from cage, reaction to being handled, muscle tone, palpebral closure, lacrimation, salivation, nasal discharge, stains (lacrimal, nasal, perianal, urine, oral), alopecia, emaciation, bite marks, exophthalmia, broken teeth/malocclusion, missing toe nail(s), dehydration, and temperature upon touching (cool-totouch). Open field (2 min.) observations included: number of rears, piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy (excessive or repetitive behaviour), bizarre behaviour, gait abnormalities, vocalizations, arousal level, and amount of excretion. Reflex and physiologic observations/measurements included: approach response, touch

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response, auditory response, tail pinch, pupil size at nonnal lighting, pupil response, righting reflex, grip strengthbody weight, body temperature, and landing foot splay. 

### b. Motorand LocomotorActivity: Motor and locomotor activities were measured approximately 30 minutes after the last animal inthe set(8 rats maximum)had finished the FOB. Allrats in each set were placed individually into figure-eight mazes and activity was measured for a total of 60 minutes. Eight infrared emitter / detector pairs (three in each ofthe figure-eight alleys and one in each ofthe blind alleys) measured activity; each time a beam was interrupted, an activity count was registered. Broad-spectrum backgroundnoise (approximately 74 dB(A» was provided throughoutthe test to minimize acoustical variations during testing. Motor and locomotor activities were examined during each ofthe six, ten-minute intervals. Motor activity wasmeasured as the numberofbeam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption ofa given beam was counted until the rat relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session. 

### 5. Ophthalmology Pre-exposure and pre-tenninal (week 12) ophthalmic exams were conducted on study animals in a semi-darkenedroom. The pre-exposure examination was used to select animals which did not have ophthalmological defects that could interfere with the interpretation ofstudy results. Animals with such defects were used for vendor surveillance or were sacrificed without necropsy. The pupillary reflex was tested using apenlight ortransilluminatorwith Finnoffand then a mydriatic agent was applied to each eye to dilate the pupil. After mydriasis, the conjunctiva, cornea and lens were examined with a slit lamp microscope and the vitreous humor, retina, choroid, and optic disc were examined using an indirect ophthalmoscope HEINE OMEGA and a condensing lens (40 diopters).



### 6. Sacrifice and pathology All animals placed on study were subjected to a complete gross necropsy. The necropsy involved an examination of all organs, body cavities, cut surfaces, external orifices and surfaces. The first six males and six females at each dietary level were selected for perfusion and collection of tissues, with replacement, as necessary, if the perfusion was considered

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# EBBayerCropScience 2007-03-28 Tier2, llA, Sec. 3, Point5: Cyprosulfamide inadequate. These animals were deeply anaesthetized using an intraperitoneal dose (50 mglkg) ofpentobarbital and then perfused via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by Universal fixative (1% (w/v) glutaraldehyde and 4% (w/v) EMgrade formaldehyde) in phosphate buffer. The entire brain and spinal cord, both eyes (with optic nerves) and selected (bilateral) peripheral nerves (sciatic, tibial and sura!), the gasserian ganglion, gastrocnemius muscle, both forelimbs, gross lesions in neural tissues or skeletal 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. Other animals that survived to term were sacrificed by CO2 asphyxiation without perfusion. For all non-perfused animals (six/sex/dietary level) that remained on study until termination, the urinary bladder and kidneys were collected, weighed (kidneys only) and preserved in 10% buffered formalin for histopathologic evaluation. Terminal body weights were performed immediately prior to necropsy toallow for calculation oforgantobody weight ratios.
Micropathology examinations were performed on a comprehensive battery ofneural tissues from perfusion-fixed control and high-dose rats of both sexes, as follows. Eight coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar) andthe caudaequinawere embedded in paraffm and examinedutilizinghaematoxylin and eosin (H&E). Dorsal root ganglia (including dorsal and ventral root fibers) from the cervical and lumbar swellings and gasserian ganglion were embedded in glycol methacrylate (GMA). Eyes, optic nerves and gastrocnemius muscle were embedded in paraffin and stained using H&E. Peripheral nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA and cut in cross/transverse-section as well as longitudinal section. GMA-embedded tissues were sectioned at 2-3 !lm and stained using a modified Lee's stain. The sciatic nerve was also cross-sectioned at approximately 2-3 !lm and stained with a modified Lee's stain. In addition, histopathology was performed on any gross lesions collected at necropsy. Tissues from perfusion-fixed animals at the low- and mid-dose levels were not subjected to micropathology unless compound-related lesions werepresent in the high-dose group. 

### II. RESULTS AND DISCUSSION A. OBSERV AnONS 1. Clinical signs There were no treatment-related findings in either sex at any dietary level. One high-dose female had a urine stain (observed on days 4, 

### 18-88). This finding in one animal is within the

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range ofhistorical control and considered incidental totreatment. Remaining findings were also considered incidental and unrelated to treatment and consisted of areas ofalopecia (1 low-dose female), maloccluded teeth (lmid-dose female) and dermal lesions describedas a scabs on cheekand shoulder (onemid-dose male). Table 5 7 4-2 Clinical Observations 

# ... Dose Level ppm (m!Yk1!. bw/day) 1000 3000 9000 Observation Control (065.2) (0 193) (0'592) ('¥ 84.5) (Cjl 251)**(~** 748) Males Lesion

- Scab, Cheek. Left 0/12 0112 1/12 0112 Lesion

- Scab, Shoulder, Left 0112 0112 1/12 0112 Females Urine Stain

- Yellow 0/12 0112 0/12 1112 Alopecia

- Limbs-Fore, Both 0112 1112 0/12 0112 Alopecia

- Shoulder, Left 0112 1112 0/12 0112 Teeth, Maloccluded-Incisors-Upper, Both 0/12 0112 1112 0112 

# I Teeth, Maloccluded-Incisors-Lower, Both 0/12 0/12 1112 0/12 Numbers represent the totalnumberof animals exhibitingsignat least once/numberof animals in eachgroup 

### 2. Mortality There were no compound-related deaths at any dose level in either sex. No animals were found dead orsacrificed in extremis duringthe course ofthe study. 

### B. BODY WEIGHT Body weight andbody weightgain were not affectedby treatment in either sex at any dietary level.

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Table5 74-3 : BodvWeigbtand bodyweigbt gain (g:l:: s.d.) 

## ... . Dose Level ppm (mg/kgbw/dav) Observation (g ± s.d.) Control 1000 (665.2) (984.5) 3000 (d'193) (J 251) 9000 (6592) C? 748) Bodv wei2ht-Males DavO 247.2±22.4 2S0.3±16.0 2S0.4±20.5 252.6±13.5 Dav7 266.1±29.2 271.7±18.6 272.3±23.4**273.8±17~** Day 42 352.3±41.l 36S.5±21.4 361.0±39.3 360.3±29. Day 91 408.1±43.9 426.4±26.7 419.4±48.5 420.0±32.4 Body wei2bt-Females Day 0 161.5±9.6 161.4±10.3 IS9.8±9.5 162.5±9.1 Day 7 17S.1±9.8 1730±10S 171.5±S.S 176.2±12.1 Day42 213.2±9.5 211.9±14.1 211.1±1.3 212.3±14.6 Day91 241.0±IS.2 237.1±18.8 23S.2±12.3 231.8±15.8 Total Bodyweightgain (Dav0-91)-Males 160.9±24.0 176.2±21.2 1 69.0±31.2 167.5±23.4 Total Body weightgain (Day0-91)-Females 79.5±10.0 75.7±I3.S 7S.4±1O.0 69.3±9.8 Values representmean ± s.d. n=12 

### *=pS05, when comparedto control.

C. FOODCONCUMPTION Therewere no compound-relatedeffects on food consumption in either sex, at any dietary level. Table5.7.4.-4 

### :Food Consumption (21animallday ± 

### s.d.) Dose Level ppm (m21k2 bw/day) Week !'Io. 1000 3000 Control (665.2) (0 193) ('02 84.5) (2 251) 9000 (0592) ('j) 748) Males Day 0-7 24.33±1.91 24.23±2.56 23.04±2.39 23.91±1.61 Day 7-14 23.47±2.15 23.03±2.30 22.61±2.67 23.25±2.02 Dav 77-84 22.20±1.75 22.22±1.93 22.57±306 22.64±1.44 Day 84-91 20.95±1.l7 21.32±1.78 21.52±3.04 21.57±1.l7 Females Day 0-7 J8.63±1.55 18.20±1.64 18.46±2.05 19.23±1.81 Day 7-14 17.71±1.38 17.58±1.79 18.16±3.21 17.S6±1.38 Dav 77-84 17.24±1.30 17.48±1.83 17.35±1.30 17.01±1.86 Days 84-91 17.03±2.25 16.77±1.76 16.41±1.66 15.82±1. 1I Values representmean ± s.d***~pS05,** when comparedto control. noll 12

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### D. NEUROBEHAVIOURALRESULTS 1. FOBfindings For the functional observational battery (FOB), compound-related effects were not apparent at any dietary level in either sex. A urine stain was seen in two females (incidence ofone at 3000 and 9000 ppm, respectively). These findings were within the range ofhistorical controls and considered incidental totreatment. Additional observations considered incidental and unrelated totreatment included alopecia in one low-dose female (present on week 8) and a dermal lesion described as a scab in one middose male (present on week 8). Table 5.7.. 4 5

- :

### F

### uncti

### onalObserva lonaI Batt esuIt

### s f 

### ery R I I I Dose Level ppm (mg/kg bw/day) I 1000 3000 9000 Observation Males Control (6' 65.2) (6 193) I**~~** 592)**(2~_** 

### {fi' 2SI} 748) i

- i No Findings 12 (100) 12 (100) 12 (l00) 12 (100)**~atment** Week2 12(100) ! 12 (100) No Findings 12 (100) 12 (100) Week4 12 (tOO) I 12 (100) 12 (IDO} No Findings 12 (00)­ • Handling

- Scab: Week 8 12 (IOO) 12 (100) 11(92) 12 (100)

### ~~~~~served , I 0(0) 0(0) 1(8)
.. 0 (0) Week 13 12 (100) I 12 (100) I No Findings 12(100) , 12 (\00) Values representthe number of ammalsand% mCldence (mparentheses) With observation. n=12 animals/dietary level *=p505, comparedwith controls Table5.7.4.-5 

### :FunctionalObservationalBatteryResults (Continued) I Dose Level ppm (mg/kg bw/dav) 

## I 1000 3000 

## I 9000 Observation (665.2) 

### (0' 193) 

### (0' 592) (Cf' 84.5) ('£ 251) (2 748) Females I Pre-treatment ! No Findings 12 (100) 12 (100) 

### ! 12 (100) 12 (100) 

## I Handling

- Stains: I • NotObserved Urine, Yellow Week 2 12 (I00) 12 (100) 12 (100) 11 (92)
! 0(0) 0(0) 0(0) 1(8) Week 4 ! No Findings 

### i 12 (100) 12 (100) 12 (lOO) 12 (100) I

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Other

- Alopecia:

NotObserved 12 12 (100) 12(100) Present 0(0) l(8} 0(0) 0(0) Week 8 Handling

- Stains: Not Observed Urine. Yellow 12 (l00) 12 (100) II (92) 11 (92) I 0(0) 0(0) 1 (8) I (8) ! I I Handling

- Stains: 12 (l00) 12 (100) 12 (100) I 1l (92) Week 13 NotObserved Urine, YeHow I 0(0) 0(0) 0(0) 1 (8) Values representthe numberof animalsand% incidence (inparenthesesj withobservation. n= ]2animals/dietarylevel *=p'iO,05, comparedwith controls 

### 2. Motor activity An examination ofthe inherent variability, using the average pretreatment values among the four groups ofmales and females, confinn that differences of± 20% are within the range of nonnal variability in this laboratory for groups of 10-12 rats/sex/dose level and, therefore, are not biologically significant. For the overall 60-minute test session, there were no compound-related differences in session motor or locomotor activity for males or females at any dietary level (motor and locomotor activity results are summarized below). There were a few occasions on which measures of motor (week 8 mid- and high-dose males (+330 /0 and +23%, respectively), week 13 high-dose males (+21%) and week 4 low-dose females (-39%» and locomotor (week 8 mid-dose males +24%), week 4 low-dose females (-35%) and week 13 high-dose females (+22%» activities were slightly beyond the standard (±20%) range ofnonnal variability. These differences from control were considered incidental and unrelated to treatment, since they were relatively small andthere was no relationshiptodose ordirection (relatively higherand lowerthan control).
Table5.7.4.-6 SummarySession MotorActivityResults(PercentDifferencefrom Control) MALES ! NominalDose Pretreatment Week2 Week4 Week8 Week 13 1000 ppm • 16 I + 4 + 2 + 5 - 2 3000 ppm + 6 + 18 + 1 + 33 + 14 9000 ppm + 4 + 3 +11 + 23 + 21 FEMALES Nominal Dose I Pretreatment Week2 Week4 WeekS Week 13 1000 ppm - 3
- 17 ·39
- 18

- 0.2 3000 ppm + 9 • 8

- 14 - 3 +0.4 9000 ppm • 2 - 6 - 17 + 2 + 14

a Percentgreater (-,-) or less (-j than concurrentcontrolforn=]2, Summary session motor activity was not statistically differentfrom control (p'!OO,05: ANOVAj at any timefor any dietary level.

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Table5.7.4.-7 Summary Session Locomotor Activity Results (Percent Difference from Control) MALES Nominal Dose Pretreatment Week2 Week4 Week8 Week 13 1000 ppm - 19 - 2 - 9 - 3
- 12 3000 ppm +11 +20 - 4 + 24 + 9 9000 ppm + 7 + 1 + 3 + 10 + 14 FEMALES NominalDose Pretreatment Week2 Week4 Week8 Week 13 1000 ppm + 3

- 18
- 35
- 13 -0.4 3000 ppm + 6 - 2 -10 - 3 -4 9000 ppm + 2 - 6
- 11 + 12 + 22

aPercentgreater (+) or less (-) than concurrentcontrolforn=12. Summary session motor activity was not statistically differentfrom control (pg).05; ANOVA) at any timefor any dietary level.

Motor and locomotor activity data were also analyzed for differences at each 10-minute interval ofeach test session. Measures ofmotor and locomotor activity were not statistically different from controls at any dietary level, on any test occasion, in either sex. Habituation was not affectedby treatment with AE 0001789 in males or females at any dietary level.
E. OPHTHALMOLOGY All ophthalmologic findings were considered incidental andunrelatedto exposure to AE 0001789.
F. SACRIFICEAND PATHOLOGY 

### 1. Gross pathology There were no compound-related gross lesions evident at terminal sacrifice in males or females at any dietary level.
2. Terminalbody and brain weights There was no compound-related effect on terminal body weight or brain weight in perfused males or females at any level ofexposure.

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Table5 7 

# . . .

1-8' AbsoluteandRelative Brain Weights
Weights (g) Dose Level ppm (mglkgbw/day) Control 1000 (C 65.2) (984.5) 3000 (6193) (2251) 9000 (6592) (2748) i Males

BodyWt.• 407.5±43.9 42S.3±27.4 417.1±47.I 420.1±32.S BrainWt.b LS9S±0.IOS L925±O.073 LS36±0.247 L933±O.041 BrainlBodyWt.b O.469±O.O50 O.445±O.O17 O.436±O.O77 O.449±O.O36 Kidney Wt.b 2.903±O.291 3.I09±O.289 2.954±O.276 2.951±O.206 Kidney/Body Wt.b O.715±O.O57 O.745±O.O56 0.724±O.024 O.726±0.O61 Females BodyWt.· 23S.0±15.4 238.3±20.l 234.9±13.7 231.2±14.8 Brain Wt.b L838±0.058 1.792±O.O35 1.806±O.O74 1.815±O.O58 Brain/BodyWt.b 

## o750±O.035 O.729±0.060 O.763±O.O66 0.777±O.O32 KidneyWt. b L965±0.212 L944±O.I09 1.963±O.O66 L945±O.145 Kidney/Body

### wt.b i O.851±O.O49 O.849±O.043 0.846±O.O34 O.852±O.045 Values representmean ± s.d

aN= 12forremaininganimals. 

### * Statisticallydifferent (p**~O.05)from** the control. bN=6
3. Neuropathology 

### There were no histopathologic changes in neural tissues from perfusion-fixed control and high-dose rats ofboth sexes that were related to administration ofthe test compound. Tissues from animals at lower dose levels were, therefore, not examined. There were no treatment-related findings in the kidney or urinary bladder of non-perfused animals at any dietary level in eithersex.

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### III. CONCLUSION In conclusion, there was no evidence oftoxicity or neurotoxicity at any dietary level and no compoundrelated gross ormicroscopic lesions atthe highest dietary level of9000 mglkg. Forboth sexes, there were no treatment-related effects on mortality, clinical observations, FOB, body weight and weight gain, food consumption, motor activity, ophthalmology or brain weight. The NOEL was 592 mglkg/day in males and 748 mglkg/day in females. (Gilmore R.G., 2006) 

### IIA5.7.5 Postnataldevelopmental neurotoxicity
As AE 0001789 administration in rodents did not induce any neurotoxicological effect, a postnatal developmental neurotoxicity study is not triggered.


### IIA 5.8. Toxicity studiesonmetabolites
The toxicological properties oftwo metabolites ofAE 0001789 were investigated in a series ofstudies carried out according to the current OECD guidelines. All the studies were in compliance with GLP requirements. The metabolite AE 0467398 (M 03, AE 0001789-Sulfamoylbenzoic acid) is a major soil metabolite that has been investigated for its non relevance as it has been predicted by the EU FOCUS leaching modelling to havethe potential to leach to shallow groundwaterwith a concentration of>0.75 Ilg/L. This metabolite was devoid of genotoxic potential and did not cause any treatment-related effect after subchronic administration tothe rat. The metabolite AE 0852999 (M02, AE 0001789-cyclopropylsulfamoylbenzamide) is a soil and a plant metabolite that has been investigated for its non relevance as it has been predicted by the EU FOCUS leaching modelling to have the potential, under worst case conditions, with certain formulated mixtures and application rates, to leach to shallow groundwaterresulting in concentrations close to 0.1 Ilg/L. This metabolite was devoid of genotoxic potential and had a similar toxicological profile to the parent compoundafter subacute administration totherat.

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Table5.8-1 Results oftoxici studieswitbtbemetabolites 

### Stud S ecies Results AE 0467398 M 03) Ames test Negative M-278703-01-2 Salmonella T h.
·In vitro 

### V79!HPRT Locus Gene Negative MutationAssay hamster cells In Vitro 

### Chromosome Aberration •M-273551-01-2 Negative Chinese hamster M-273607-01-2 •test V79 lun cells I 

### 90-day dietary study: NOEL 15 000 ppm ( 1094 mglkglday in
. ·M282911012 

# - - - 0, 400, 2000 and 15000 ppm I 

### a ema es, m ay III

### rna es Rtf, I 

### 888 glkgld I ) I



### .AE 0852999 (M02) Ames Test Negative I 

### M-274230-01-2 Salmonella Typh. In vitro 

### V79/HPRT Locus Gene •Negative Mutation Assay Chinese hamster M-274735-01-2 V79 lung cells In Vitro 

### Chromosome Aberration Negative test Chinese hamster M-274272-01-2 V79 lung cells i 

### 28-day dietary study: NOEL 300 ppm (25.5 mglkg bw/day in • females, 22.8 mglkglday in males) I

### 0, 40, 300, 800, 3000ppm I

### Rat M-284847-01-2 

# I

• AE 0467398 (AE 0001789-Sulfamoylbenzoic acid)


### In vitro genotoxicity

- Bacterial assay for gene mutation Report: KIIA 5.8/01, 

### Herbold B., 2006 Title: AE 0001789-Sulfamoylbenzoic acid (AE 0467398), Salmonella/microsome test, plate incorporation andpre-incubation method Report No & 

### AT03259 DocumentNo M-278703-01-2 Guidelines: OECD 471 (1997); EEC Directive 2000/32/EC 

### Method B13/14 

### (2000); EPA Health EffectsTestGuideline (OPPTS 870.5100; 1998) GLP Yes (certified laboratory)

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### Executive summary: In this 

### in vitro assessment ofthe mutagenic potential ofAE 0467398 (Batch KTS 10207-1-1,96.0% of purity), histidine dependent auxotrophic mutants ofSalmonella typhimurium, strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to AE 0467398 up to 5000 J.lg/plate, diluted in dimethyl sulphoxide (DMSO). For each bacterial strain and dose level, triplicate plates were used in both the presence and absence ofan Aroclor 1254-inducedrat liver metabolic activation system (S9 mix). DMSO was also used as a negative control. Specific positive controlswere used for each strain. After48 hours of incubation at

### 37°e, the numbers ofrevertant colonieswere scored using an automatedcolony counter. Doses up to 158 J.lg/plate did not cause any bacteriotoxic effects. At higher doses the substance only had a weak, strain-specific bacteriotoxic effect in the plate incorporation trial. Due to the weakness ofthis effect, thisrange could neverthelessbe used for assessmentpurposes. AE0467398 didnot cause any significant increase in the number ofrevertant colonies in eitherthe presence orabsence ofmetabolic activation. All the positive control compounds produced expected increases in the number of revertant colonies, thereby demonstrating the sensitivity ofthe assay and the efficacy ofthe S9 mix. Therefore, AE 0467398 was non-mutagenic with orwithout S9 mix in the plate incorporation as well as in the pre-incubationmodification ofthe Salmonella/microsometest.

I. 

### MATERIALS AND METHODS A. MATERIALS: 1. 

### TestMaterial: AE 0467398 

### Description: fine white powder 

### Lot/Batch: KTS 10207-1-1 

### Purity: 96.0% 

### CAS: 138-41-0 

### Stability oftestcompound: Stable for the duration ofthe study

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2. Control materials: Negative: Culture medium Solvent: DMSO Positive: Sodium azide (Serva) for T A 1535 at 10 Ilg!piate, Nitrofurantoin (Sigma) for TA 100 at0.2 Ilg!plate, 4-Nitro-I,2-phenylene diamine (Merck-Schuchardt) for TA 1537 at 10 Ilg!plate and TA 98 at0.5 Ilg!plate, Mitomycin C (Fluka) for TAl02 at 0.2 Ilg!plate only in plate incorporation plate, Cumene hydroperoxide (Sigma) for TA 102 in pre-incubation trials only at 50 Ilg!plate, 2-Aminoanthracene (Aldrich) for the activating effect ofthe S9 mix in all strains at 3 Ilg!plate. 3. Testorganisms: Species: Salmonella typhimurium LT2 mutants Strain: Histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102 Source: Strains obtained from Prof. Bruce Ames in 1997 and stored in the laboratory sincethen. 4. Testcompound concentrations: Plateincorporation assay: For all strains with or without S9 mix: 16, 50, 158, 500, 1581 and 5000 Ilg/plate Pre-incubation assay: For all strains with or without S9 mix: 16, 50, 158, 500, 1581 and 5000 Ilg!plate 5. Metabolic activation: The S9 fraction was isolated from the livers of Aroclor 1254 induced male Sprague Dawley rats. The preparation dated from September 13, 2005 (protein content 23.4 mg per ml) and was kept frozen at -80°C. The batch was tested for contamination prior to use in the study. Cofactors were freshly dissolved in sodium phosphate buffer 

### (ISO mM, pH 7.4) B. STUDY DESIGN AND METHODS: The experimental phase ofthe study was performed between June 22 and July 3, 2006, at Bayer

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HealthCare AG (PH-PD P Health Care Toxicology). The Salmonella/microsome test is a screening method which detects point mutation caused by chemical agents 

### in vitro. Auxotrophic mutants of Salmonella typhimurium are used to demonstrate this effect. For this purpose, the rate ofreversion to prototrophy is evaluated in negative control and treated groups. 

### 1. Plateincorporation assay: AE 0467398 or the control material was dissolved in 0.1 ml ofDMSO. DMSO (0.1 mL) containing AE 0467398 or controls were added to glass vessels with 0.1 ml of bacterial cultures grown overnight, 0.5 ml ofS9 mix orbuffer and 2 ml ofsoft agar. The mixture was placed in a waterbath at 45°C for 30 seconds, shaken and overlaid onto Petri dishes containing solid agar. After 48 hours of incubation at 37°C, the numbers of revertant colonies were scored using an automated colony counter. Three plates were used, both with and without S9 mix, for each strain and dose. The doses for the fIrst trial were routinely determined on the basis ofa standard protocol with a maximum dose of5000 Ilg/plate and at least 5 additional doses. Ifless than three doses were used for assessment, at leasttwo repeats wereperformed. 2. Pre-incubation assay: An independent repeat was performed as pre-incubation of the previously described mixture in a water bath at 37°C for 20 minutes. At the end ofthe pre-incubation period. 2 ml ofmolten soft agar were added to the tubes, the content mixed and plated onto Petri dishes with solid agar. After 48 hours ofincubation at 37°C, the numbers ofrevertant colonies were also scored using an automated colony counter. 3. Acceptancecriteria: The negative controls had to be within the expected range. The positive controls had to show sufficient effects. Titre determinations had to demonstrate sufficient bacterial density in the suspension. The titre was determined by the total bacterial counts taken on two plates for each concentration studiedwith S9 mix. 4. Assessment criteria A reproducible and dose-related increase in mutant colonies ofat least one strain was considered to be positive. For TA 1535, TA 100 and TA 98, this increase should be about twice that ofnegative

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### II RESULTS AND DISCUSSION There was no indication ofa bacteriotoxic effect ofAE 0467398 at doses ofup to 158 J.lg!plate. The total bacterial counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition ofgrowth was noted either. Higherdoses had only in the plate incorporation trial a weak, strain-specific bacteriotoxic-effect. Therefore they could be used for assessmentpurposes. None ofthe five strains concerned showed in the plate incorporation test a dose-related and biologically relevant increase in mutant colonies overthose ofthe negative controls. This appliedbothtothe tests with andwithout S9 mix and was confirmedby the results ofthepre-incubation trials. The positive controls sodium azide, nitrofurantoin. 4-nitro-l,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant colonies to well over those of the negative controls, andthus demonstratedthe system's sensitivity and the activity ofthe S9 mix. III 

### CONCLUSION No indication ofmutagenic effects ofAE 0467398 were found at doses ofup to 5000 J.lg!plate in any of the Salmonellatyphimurium strains used in the assay. (HerboldB., 2006) 

### In vitro genotoxicity

- Test for clastogenicity in mammalian cells Report: KIIA 5.8/02, 

### Thum M., 2006 Title: AE 0001789-Sulfamoylbenzoic acid (AE 0467398), In vitro chromosome aberration test with Chinese HamsterV79 cells Report No & AT03096 DocumentNo M-273607-01-2 

### Guidelines: OECD 473 (1997); EEC Directive 2000/32IEC Method BI0 (2000); EPA Health EffectsTest Guideline (OPPTS 870.5375; 1998) GLP Yes (certified laboratory) 

### Executive summary: In this in vitro assessment ofthe clastogenic potential ofAE 0467398 (batch KTS 10207-1-1,96.0% of purity), Chinese Hamster V79 cellswere exposed to AE 0467398 at 525, 1050 and 2100 J.lg!ml, diluted in dimethyl sulphoxide (DMSO) in the presence or the absence of an Arodor 1254-induced rat liver metabolic activation system (S9 mix). For each dose level, duplicate cultures were used. DMSO was also used as a negative control. After4 hours treatment, the medium was changed andthe cells were harvested

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14 hours later. In addition, cells treated at 2100 Ilglml were harvested 30 hours afterthe start ofthe study. Without S9 mix, an additional experiment was performed using continuous treatment for 18 hours, harvest at the same time, and AE 0467398 concentrations of525, 1050 and 2100 Ilglml. Mitomycin C, which produces crosslinks in the DNA, and cyclophosphamide, which induces chromosomal damage after metabolic activation, were used as positive controls, diluted in Hanks' balanced salt solution. Colcemid was added to each flask two hours priorto harvest to arrest the cells in a metaphase-like stage ofmitosis. With and without S9 mix no cytotoxic effects were observed up to 2100 Ilglml after 4 hours treatment. After 18 hours treatment cytotoxic effects were observed at 1050 Ilglml and above. Precipitation in the medium did not occur. None ofthe cultures treated with AE 0467398 in the absence and in the presence of S9 mix showed biologically relevant and statistically significant increased numbers of aberrant metaphases. The positive controls mitomycinC and cyclophosphamide induced clastogenic effects and demonstrated the sensitivity ofthetest system and the activity ofthe used S9 mix. Based onthis test, AE0467398 was considered not to be clastogenic for mammalian cells in vitro.

I. 

### MATERIALS AND METHODS A. MATERIALS: 1. 

### Test Material: AE 0467398 

### Description: fme white powder 

### Lot/Batch: KTS 10207-1-1 

### Purity: 96.0% 

### CAS: 138-41-0 

### Stability oftestcompound: Stable for the duration ofthe study 

### 2. Control materials: Negative: Culturemedium 

### Solvent: DMSO for AE 0467398 and Hanks's balanced salt solution for positive controls (Seromed) 

### Positive: Mitomycin C (Fluka, batch 1149026 10605077) without S9 mix at 0.1 Ilglml for a treatment period of4 hours and 0.03 Ilglmlfor a treatmentperiod of 18 hours. Cyclophosphamide (Endoxan 100 mg injection vials of dry

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### substance, BaxterOncology GmbH) with S9 mix at 2 Ilglml. 

### 3. Test organisms: Cell line: Chinese hamsterV79 lung cells 

### Source: Cells obtained from Dr Utesch, Merck AG, Darmstadt in 1993, stored in the laboratory since then. 

### 4. Test compound concentrations: AE 0467398 was used with or without S9 mix at 525, 1050 and 2100 Ilglml.



### B. STUDY DESIGNAND METHODS: The experimental phase ofthe study was performed from March 9 to April 4, 2006 at Bayer HealthCare AG (PH-PO PHealth Care Toxicology)
.. The in vitro 

### cytogenetic test is a mutagenicity test system for the detection ofchromosome aberrations in cultured mammalian cells. The test is designed to detect structural aberrations (chromatid and chromosome aberrations) in cells at their first post-treatmentmitosis. 1. 

### Determination ofcytotoxicity: Duplicate cultures were exposed in a pre-test to AE 0467398 at concentrations ranging from I to 2100 Ilg/ml with orwithout S9 mix for 4 hours. In addition, ceIls were exposedwithout S9 mix for 18 hours to concentrations ranging from 50 to 2100 Ilglml. The mitotic index was determined for all cultures. The number ofmitotic cells among a total of 1000 cells per culture was determined using a light microscope at a magnification ofabout 630. All cells which were not in interphase were defined as mitotic. In the main study, cultures with a total incubation period 

### of 4 hours were additionally used to determine the cytotoxicity ofAE 0467398 at concentrations ranging from 525 to 2100 Ilg/ml 

### with a harvest time of 18 hours and at concentrations of 2100 Ilglml with an harvest time of 30 hours. Additional cultures were exposed without S9 mix for 18 hours to concentrations of AE 0001789 ranging from 525 to 2100 Ilglml. These cultures were harvested atthe end ofthe treatmentperiod. AE 0467398 at 500 Ilglml and above slightly decreasedthepH in the medium (reduction to 6.3). The osmolality in the medium ofthe pre-test was not changed by concentrations ofup to 2100 Ilglml of

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### AE 0467398. 

### 2. Treatmentprotocol: Chinese hamster V79 cells were passaged on the day prior to treatment. Approximately Ixl06 

### cells were seeded in 20 ml ofmedium per 75 m2 

### flasks and incubated at 37°C in a CO2

### -incubator (5% CO2). 

### Unless reported otherwise, the cells were grown in Eagle's minimal essential medium containing 10% foetal calfserum. Immediately before treatment, the medium was removed from the cultures. Forthe trials without S9 mix, 20 ml ofmedium containing 2% foetal calfserum and 0.2 ml oftest substance solution were added to each flask. For the trials with S9 mix, 19 ml ofmedium containing 2% foetal calfserum, 1ml ofS9 mix and 0.2 ml oftest substance solution were added to each flask. The cells were incubated for 4 hours at 37°C. After 4 hours oftreatment, the medium was removed, the cells were washed with PBS and 20 ml offresh medium containing 10% foetal calf serum was added to the flasks. The flasks were placed in a COrincubator for the remaining incubationtime. In the culturestreated for 18 hours, themedium was notremoved. To each flask 0.2 ml ofColcemid-solution (40 f.lglml) was added to each flask 2 hours prior to the endofthe incubationperiodto arrestthe cells in a metaphase-like stage ofmitosis(c-metaphase). Positive controls and solvent controls (0.2 ml of solvent per culture) were set up in parallel and handled as described for AE 0467398-treated cultures. Untreated controls and solvent controls were used as negative controls. 

### 3. Chromosomepreparations: After the removal ofthe medium from each flask, the cells were trypsinized, suspended in medium and centrifuged for approximately 5 minutes at 700 rpm. The supernatant was removed and I to 2 ml ofa hypotonic solution (0.4% KCI; 37°C) was added to each tube. Within4 minutes, the volume was brought to 6 ml with additional hypotonic solution and cells were resuspended. The cells were centrifuged again and fixed with cold (4°C) fixative (ethanol/acetic acid 3:1) for 20 minutes at room temperature. Cells were pelleted and resuspended in fixative as before and centrifuged again. The pelleted cells were resuspended in a small volume offixative and the suspension was dropped onto clean slides. The slides were allowed to dry for at least 2 hours. Thereafter, they were submerged in pure methanol for 3 minutes and stained for 15-20 minutes in 3% 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 leasttwo slides were generatedper culture.

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### 4. Evaluationcriteria: Coded slides were evaluated using a light microscope at a magnification of about 1000. Chromosomes ofapproximately 200 metaphases per concentration, 100 metaphases from each oftwo parallel cultures, were examined. Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges were detected. The following aberrations were recorded: gaps (an achromatic lesion within a chromatid arm without dislocation of the chromatid end), break (a discontinuity ofone chromatid with dislocation ofthe chromatid end), fragment (part ofchromosome without centromer), deletion (result ofa break with the terminal chromatid part ofthe chromosome missing within the metaphase under assessment), exchange (exchange of chromatid-parts between different chromosomes or within the same chromosome), multiple aberration (when five or more structural changes occur within one metaphase). Observed polyploidy metaphases were recorded but not used for assessment. 

### 5. Assessmentcriteria: An assay was acceptable, ifthere was a biologically relevant increase in chromosome aberrations induced by positive controls and ifthe numbers ofaberrations for the negative controls were in the expected range. An increased incidence ofgaps ofbothtypes without a concomitant increase ofotheraberrationtypes was considerednot tobe an indication ofa clastogenic effect. A test was considered positive, ifthere was a relevant and statistically significant increase in the aberration rate. A test was considered negative, ifthere was no such increase at any time interval or ifthere were statistically significant values, which were, however, within the range ofhistorical negative controls. A test was considered equivocal, if there was an increase above the range of historical negative controls which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statistically significant. 

### 6. Statistics: The statistical analysis was performed by pair-wise comparison ofAE 0467398-treated and positive control groups totherespective 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-sidedChi2

### -test. The numbers ofmetaphases with aberrations (including and excluding gaps) and ofmetaphases with exchanges were compared (provided that these data superceded the respective solvent control). The

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one-side Che-testwas used for the statistical evaluation. A difference was consideredtobe significant, ifthe probabilityoferrorwas below 5%. 

### II RESULTS AND DISCUSSION A. GENERAL REMARKS In the the presence or absence of S9 mix Chinese hamster V79 cells were exposed for 4 hours to AE 0467398 at concentrations ofup to and including 2100**~g/ml** and, in addition, without S9 mix for 18 hours to AE 0467398 at concentrations ofup to and including 2100 I-lg/ml. Without and with S9 mix substance precipitation did not occurin the medium. 

### B. MITOTIC INDEX Mitotic index without S9 mix: In comparisontothe solvent control, the mitotic indices were only reduced in the cultures treated for 18 hours, starting at 1050 I-lg/ml. The culturestreated with mitomycin C showed no reduction in mitosis rate. Mitotic index with S9 mix: In comparison to the solvent control, the treated cultures showed no relevant reduction ofthe mitosis rate. The positive control cyclophosphamidereducedthe mitosisrate. C. 

### SURVIVAL INDEX Survival index without S9 mix: In comparison to the solvent control, the survival indices in the treated cultures were not relevantly reduced. The cultures treated with mitomycin C showed a reduction in survival rate only after4 hours treatment. Survival index with S9 mix: In comparison to the solvent control, the treated cultures showed no relevant reduction ofthe survival rate. The positive control cyclophosphamidereducedthe survival rate. 

### D. CHROMOSOME ABERRATION AE 0467398 without S9 mix: No biologically relevant and statistically significant increases ofnumbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The same wastrue for a treatmentperiod andtotal culture time of 18 hours. The treatment with the positive control mitomycin C resulted in a clear and statistically significant

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### increase ofmetaphases with aberrations and demonstratedthe sensitivity ofthe test system. AE 0467398 with S9 mix: No biologically relevant and statistically significant increases ofnumbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The positive control cyclophosphamide induced statistically significant and biologically relevant increases of metaphases with aberrations and demonstrated the sensitivity of the test system and the activity ofthe used S9 mix.



### III. CONCLUSION After 4 hours treatment ofChinese hamster V79 cells with AE 0467398 concentrations of525, 1050 and 2100 flglml were used without and with S9 mix for assessment of the clastogenic potential of AE 0467398. In addition, after 18 hours treatment with AE 0467398 concentrations of525, 1050 and 2100 flglml were selectedfor reading without S9 mix. None ofthese cultures treated with AE 0467398 in the absence orpresence ofS9 mix showed statistically significant and biologically relevant increases ofnumbers ofmetaphases with aberrations. The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and demonstrated the sensitivity ofthe test system and in the case ofcyclophosphamide the activity ofthe used S9 mix. Based on the results ofthistest, AE 0467398 was considerednotto be clastogenic for mammalian cells in vitro. (ThurnM., 2006) 

### In vitro 

### genotoxicity

- Test for gene mutation in mammalian cells Report: KIIA 5.8/03, Herbold B., 2006 Title: AE 0001789-Sulfamoylbenzoic acid (AE 0467398), V791HPRT-test 

### in vitro for the detection ofinduced forward mutations Report No & 

### AT03095 DocumentNo M-273551-01-2 

### Guidelines: OECD 476 (1997); EEC Directive 2000/321EC Method B17 (2000); EPA Health Effects TestGuideline(OPPTS 870.5300; 1998) GLP Yes (certified laboratory)

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Executive summary: 

### The purpose ofthe study was to assess the point mutagenic potential ofAE 0467398 (batch KTS 10207­ 1-1,96 % 

### ofpurity) atthehypoxanthine-guaninephosphoribosyltransferase (HPRT) locus in V79 cells. For dose selection, a preliminary cytotoxicity test was conducted with and without an Aroclor 1254 induced rat liver metabolic activation system (S9 mix) using concentrations ofAE 0467398 ranging from 12.5 to 2100 Ilg/ml. Precipitation ofAE 0467398 in the culture medium was not observed. 2100 Ilg/ml AE 0467398 reducedthe pH in the medium ofthe pre-test from 7.6 for the vehicle control to 6.3 for 2100 !Jog/ml AE 0467398. The osmolality in the medium ofthe pre-test was also slightly reduced from 445 mOsmollkg to 428 mOsmol/kg. A slight cytotoxicity was observed. AE 0467398 was tested in the first mutation experiment at concentrations ranging from 175 to 2100Ilg/ml with or without metabolic activation. The same concentrations were used forthe independentrepeats. Exponentially growing V79 cells were plated in 20 ml culture medium in two 75 cm2 

### flasks. After attachment, cells were exposed with or without S9 mix to vehicle alone orto a range ofconcentrations of the test substance for 5 hours. Thereafter, each cell monolayer was washed with PBS, trypsinized and replated into 3 Petri dishes. Per culture one flask and 3 Petri dishes were used. These dishes were incubated for 6 to 8 days to allow colony development and to determine the cytotoxicity associated with eachtest substance directly aftertreatment (survival to treatment). Cells in 75 cm2 

### flasks were incubated to permit growth and expression ofinduced mutations. Cells were subcultured (= 

### count I, normally after 3 days) by reseeding cells in 75 cm2 

### flasks. At the end ofthe expression period (=count 2, normally a total of6 days), cultures were reseeded in Petri dishes without hypoxanthine but containing 6-TG for selection ofmutants. In addition, 200 cells perdish were seeded in 5ml culture medium to determine the absolute cloningefficiency for each concentration. After incubation for 6 to 8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of6­ TG resistant colonies in the mutation assay dishes and the number ofcolonies in the cloning efficiency dishes. Two trials wereperformed. Without and with S9 mix AE 0467398 induced neither decreases in survival to treatment nor decreases in relative population growth. Precipitation of AE 0467398 in the culture medium was not observed. However, AE 0467398 was tested up to 2100 !Jog/ml, which is slightly above the requested limit concentration of 10mM (equal to 2012 llg/m1). With and without S9 mix there was no biologically relevant increase in mutant frequency above that ofthe vehicle controls. Ethyl methanesulfonate and dimethylbenzanthracene induced clear mutagenic effects demonstrating the sensitivity ofthe test system and the activity ofthe S9 mix. Based on these results, AE 0467398 was considered to be non-mutagenic in the V79IHPRT forward

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### mutation assay, both with and without metabolic activation. I. MATERIALS AND METHODS A. MATERlALS: 1. 

### Test Material: AE 0467398 Description: white powder Lot/Batch: KTS 10207-1-1 Purity: 96% CAS: 138-41-0 Stabilityoftest compound: Stable for the duration ofthe study 2. Controlmaterials: Negative: Culture medium [Eagle's minimal essential medium supplemented with 1% L-glutamine, 1% MEM-vitamins, 1% MEM NEAA, 1 % penicillin/streptomycin and 10% foetal calf serum (FCS)] Solvent: DMSO for AE 0467398 and Dimethylbenzanthracene not exceeding 1% (v/v) in the culture medium. No solvent needed forethyl methanesulfonate as it is a liquid. Positive: Ethyl methanesulfonate (EMS), a directly alkylating agent, used at afinal concentration of900 Ilglml in non-activationtrials. Dimethylbenzanthracene (DMBA), promutagen requiring a metabolic activation, used at a final concentration of20 Ilglml fortrials with S9 mix. 3. Testorganisms: Cell line: Chinese hamster V79 lung cells Source: Cells obtained from Prof. G. Speit, University ofUlm, Gennany. These cells have since been recloned to maintain karyotypic stability. They have a modal chromosome number of22 and a rapid population doubling time (10 to 14 hours) Culturecondition: Incubation perfonned at 37°C in a humidified atmosphere with about 5%CO2• 

### 4. Test compound concentrations: AE 0467398 was used at 12.5,25,50, 100,250,500, 1500 and

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### 2100 Ilg/ml in the clonal cytotoxicity assay and at 175,350, 700, 1050, 1400, 1750 and 2100 Ilg/ml inthemutagenic assays. 

### 5. Metabolic activation: The S9 fraction was isolated from the livers of Aroclor 1254 induced male Sprague Dawley rats. The preparation dated from September 13, 2005 (protein content from 23.9 mg/ml) and was kept frozen at -80"C. The batch was tested for contamination and cytotoxicity priorto use in the first study. Cofactors were freshly dissolved in sodium phosphate buffer (150 mM, pH 7.4)



### B. STUDY DESIGN AND METHODS: The experimental phase of the study was performed from February 14 to April 4, 2006 at Bayer Healthcare AG. The selection of V79 forward mutations is based on the resistance of induced mutants to the purine analogue 6-thioguanine (6-TG). This resistance is a result of a mutation at the X-chromosome-linked HPRT locus rendering the cells unableto use 6-TG for DNA synthesis. Therefore, cell colonies formed in the presence of6·TG are consideredto represent mutants atthe HPRTgene. 

### 1. Determinationofcytotoxicity: Exponentially growing V79 cells were plated in 20 ml culture medium in a 75 cm2 

### flask with a total volume of275 ml (4x106 

### cells per flasks). For each concentration, one culture was available. After attachment (16 to 24 hours later), cells were exposed without S9 mix to vehicle alone orto arange of concentrations ofthetest substance for 5hours in 20 ml medium containing2% FCS. In experiments with metabolic activation 1 ml ofmedium was replaced by 1 ml of S9 mix. Thereafter, each cell monolayer was washed with PBS, trypsinized and replated in 5 ml culture medium at a density of 200 cells into 3 Petri dishes (diameter of60 mm). These dishes were incubated for 6 to 8 days to allow colony development. Thereafter, colonies were fixed with 95% methanol, stainedwith Giemsa (Merck; stock solution diluted 1:5 with deionized water) and counted automatically using an Artek counter, when there was no interference by precipitation on the plates or colouration ofthe plates. Cytotoxicity was expressed by comparison of colonies in treated cultures versus vehicle control cultures (relative cloningefficiency). 

### 2. Treatmentprotocolwithoutmetabolic activation:

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Exponentially growing V79 cells were plated in 20 ml culture medium in two 75 cm2 flasks per concentration (4x106 cells per flask) including all control groups. After attachment (16 to 24 hours later), the cellswere exposedto vehicle alone orto a range ofconcentrations ofthe test substance for 5 hours in 20 ml culture medium with reduced serum content(2%). Thereafter, cellmonolayers were washed with PBS, trypsinized and replated in 20 ml culture medium using 1.5xl06 cells per 75 cm2 flask and in 5 ml culture medium using 200 cells per Petri dish (diameter of60 mm). One flask and 3 Petri dishes were used per culture. These dishes were incubated for 6 to 8 days to allow colony development and to determine the cytotoxicity associated with each test substance directly after treatment (survival to treatment). Cells in 75 cm2 flasks were incubated to permit growth and expression ofinduced mutations. Cells were subcultured count I, normally after 3 days) by reseeding 

### 1.5x106 cells into 20 ml ofmedium in 75 cm2 flasks. Atthe end ofthe expression period (""count 2, normally a total of6 days), cultures were reseeded in Petri dishes (diameter of 100 mm) at 3xl05 cells perdish (8 dishes per culture) in 20 ml culture medium without hypoxanthine but containing 10 Ilglml 6-TG for selection ofmutants. In addition, 200 cells perdish (diameter of60 mm, 3 dishes per culture) were seeded in 5 ml culture medium to determine the absolutecloningefficiency for each concentration. Afterincubation for 6 to 8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of6-TG resistant colonies in the mutation assay dishes and the number ofcolonies in the cloning efficiency dishes. Twotrials were performed. 

### 3. Treatmentprotocol with metabolic activation: The activation assay was performed independantly. The procedure was identical to the nonactivation assay except for the addition of S9 mix. In these experiments 19 ml instead of 20 ml culture medium and additonally \ ml of S9 mix were added to the flasks for the treatment period, resulting in a concentration of5% S9 mix in the cultures. The number of6-TG resistantmutants and viability were determined as in the non-activation assay. Twotrials were performed. 

### 4. Parameters assessed: The parameter "survival to treatment" in % was determinedonthe basis ofthe following calculation:

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Meannumber ofcolonies (treatedcultures) x 100 Meannumberofcolonies (vehicle control cultures) The "absolute population growth" was calculatedusingthe following fonnula: Absolute populationgrowth (foreach culture) 

### =cell count I x cell count2 The parameter "relative population growth" shows the cumulative growth of the treated cell populations, relativetothe vehicle control. Absolute population growth treated culture x 100 Absolute population growth ofcorrespondingvehicle control culture The ability of cells to fonn colonies at the time ofmutant selection is measured by the parameter "absolutecloningefficiency". Itis expressed in 

### %. Meannumberofcoloniesperdish x 100 200 The "mutant frequency" is calculated for each groupby dividingthetotal numberofmutant colonies bythe numberofcells seeded (usually 8-10plates at 3 x 1 05 cells perplate), corrected for the absolute cloning efficiency. The mutant frequency is expressed as 6-TG resistant mutants par 106 clonable cells. Total numberofmutant colonies x 100 Numberofevaluated dishes x 3 x 105 x C.E. 

### 5. Acceptance criteria: Theaverage cloningefficiency ofthe negative and vehicle controls shouldbe at least 50%. The average mutant frequency ofthe vehicle controls shouldnotexceed25 x 10-6 cells. The mutant frequency ofthe two cultures ofthe vehicle and 

### lor the negative control should differ only to an acceptable extent. As a rule ofthumb, the difference ofmutant frequencies should not be greater than 5 x 10-6

. Thepositive control should induce an average mutant frequency ofat least three times thatofthe vehicle control. 1fnotlimitedbythe solubility ofthetest substance in the vehiclethe highest concentration should induce cytotoxicity ofabout 80 to 90% orshouldbe a concentration whereprecipitation occurs in

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### the medium. The survival at the lowest concentration should be in the range of the negative control. For the calculation of an acceptable mutant frequency at least 5 dishes per culture should be available and relative survival to treatment, relative population growth and absolute cloning efficiency shouldbe 10% orgreater. However, these criteriamay be overruled by good scientificjudgement. 

### 7. Assessment criteria: Mutant frequencies were only used for assessment, ifat least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10%orgreater. A trial was considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies was observed. To be relevant, the increase in mutant frequencies should be at leasttwotothreetimes that ofthe highest negative or vehicle control value observed in the respective trial. If this result was reproduced in a second trial, the test substance was consideredtobemutagenic. Despite these criteria, a positive result was only considered relevant, ifno significant change in osmolality compared to the vehicle control was observed. Otherwise, unphysiological culture conditions may have be the reason for the positive result. Atest substance wasjudged as equivocal ifthere was no strictly concentration related increase in mutation frequencies but ifone or more concentrations induced a reproducible and biologically relevant increase in mutant frequencies in all trials. An assay was considered negative ifno reproducible andrelevant increases ofmutant frequencies were observed. However, these criteriamay be overruledby good scientificjudgement. 

### 7. Statistical analysis: The statistical analysisrelied on the mutant frequencies which were submittedto a weighted analysis ofvariance as well as to a weightedrecursive regression, both with Poisson derived weights. The two mutantfrequency values obtainedper group were, although somewhatrelated, considered as independent measurements thus increasing the power of the statistical tests applied. Since the protocol of the HPRT assay required at least two independent trials, the overall analysis without

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# ffiBayerCropScience 2007-03-28 Tier2, 11A, Sec. 3,Point5: Cyprosulfamide respectively with activation was the most importantone for classitying substances into mutagens and non-mutagens. However, separate analyses were run for each trial in order to examine the consistency ofthe results. All acceptable groups were included in the weighted analysis of variance followed by pairwise comparisonstothe vehicle control on anominal significance level of

### ex. 0.05 using the Dunnett test. =:0 Theregression analysis partwasperformed onthe basis ofthe actual concentrations thereby omitting thepositive, negative and vehicle controls. Ifthere was a significant concentration related increase of the mutant frequency 

### (ex. 

### =0.05) in the main analysis the highest concentration was dropped and the analysis repeated. This procedure was repeated until p > 0.05. In that way eliminated concentrations were flagged correspondingly. 

### II. RESULTS AND DISCUSSION A. GENERALREMARKS: In the presence and in the absence ofS9 mix Chinese hamster V79 cells were exposed to AE 0467398 at concentrations ofup to and including 2100**~glml.** With and without S9 mix no substance precipitation occurred in the medium. The means ofthe absolute cloning efficiency for the vehicle controls in the mutation experiments were 50.6% and 64.4% in the experiments without activation, and 55.7% and 57.0% in the experiments with activation. These results demonstrated good cloning conditions forthe experiments. 

### B. MUTATIONASSAY WITHOUTMETABOLIC ACTIVATION: Two trials were performed. The mutant frequencies ofthe negative controls (means of3.65 x 

### 10'6 and 1.75 x 10,6, respectively) and ofthe vehicle controls (means of 1.65 x 

### 10'6 and 1.55 x 

### 10,6, respectively) were all within the normal range. The positive control EMS induced clear mutagenic and statistically significanteffects in alltrials (means of253.7x 

### 10,6and 352.95x 

### 10'6, respectively). For AE 0467398 treated cultures no concentration-related decreases were observed neither in relative survival to treatment nor inrelative population growth. Relevant AE 0467398 induced increases in mutant frequencies could not 

### be found (ranging from 0.0 x 10'6to4.2 X 

### 10'6 in the first trial, and from 0.5 x 10'6 to 3.5 X 

### 10'6 in the second trial). In addition, the overall statistical analysis revealed no statistically significant increases. Therefore, AE 0467398 was evaluated as non-mutagenic in the non-activationtrial.

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### C. MUTAnONASSAY WITHMETABOLIC ACnVAnON: Two trials were perfonned with S9 mix. In all experiments, cytotoxic effects were induced. The mutant frequencies ofthe negative controls (means of8.2 x 10,6 and 2.75 x 10-6, respectively) and ofthe vehicle controls (means of6.85 x 10,6 and 1.1 x 10.6 , respectively) were all within the nonnal range. The positive control DMBA inducedclearmutagenic and statistically significant effects in all trials (means of 103.55 x 10.6and 61.3 x 10'6, respectively). The AE0467398 treated cultures showedno concentration-related decreases neither in relative survival to treatment nor in relative population growth. Relevant AE 0001789 induced increases in mutant frequencies couldnotbefound (ranging from 0.7 x 10,6 to 5.3 x 10'6 in the first trial and from 0.0 x 10,6 to 5.0 x 10.6 in the second trial). In addition, the overall statistical analysis revealed no statistically significant increases. Therefore, AE 0467398 was evaluated as non-mutagenic in the activation trial. 

### III. CONCLUSION Under both activation conditions, neither cytotoxic effects nor precipitation of AE 0467398 in the medium were found. However, AE 0467398 was tested up to 2100**~g/ml,** a concentration which slightly exceeds the requested limit concentration of 10 mM (equal to 2012**~g/ml).** AE 0467398 induced no biologically relevant increases in mutant frequencies. Thepositive controlsEMS and DMBAhad a markedmutagenic effect. Based on these results, AE 0467398 was considered not to be mutagenic in the V79IHPRT forward mutation assay. (HerboldB., 2006) 

### Oral90-daytoxicity in tbe rat Report: KIIA 5.8/04, 

### Blanck 0.,2007 Title: AE 0001789-Sulfamoylbenzoic acid (AE 0467398), 90-day toxicity study in the ratby dietary administration Report No & SA 06036 DocumentNo M-2829I 1-01-2 

### Guidelines: OECD 408 (1998); EEC Directive 200l/591EC Metbod B26 (2001); EPA Healtb Effects Test Guideline (OPPTS 870.3100; 1998) GLP Yes (certified laboratory)

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### Executive summary: AE 0001789-sulfamoylbenzoic acid (AE 0467398), a soil metabolite of the herbicide safener AE 0001789, (batch number KTS 10207-1-1, 96% purity) was administered continuously via dietary administration to separate groups ofWistarrats (lO/sex/group) at concentrations of400,2000 and 15000 ppm (equating approximately to 24, 116 and 888 mg/kglday in males and 29, 142 and lO94 mg/kg/day in females) for at least 90 days. A similarly constitutedgroup of 10 males and lO females received untreated diet and acted as a control. Clinical signs were recorded daily, body weight was measured weekly and food consumption was measured weekly for the first two weeks and twice weekly thereafter. A detailed physical examination was performed once during the acclimatization phase and weekly throughout the study. All surviving animals were subjected to a neurotoxicity assessment (exploratory locomotor activity, open field observations, sensory reactivity and grip strength) during Weeks 11 to 12 ofthe study. Ophthalmological examinations were performed on all animals during the acclimatization phase and on all animals ofthe control and high dose groups during Week 13. Urine samples were collected overnight on the week before necropsy from all surviving animals. Before necropsy, a blood sample was collected from the retro-orbital venous plexus ofeach surviving animal for haematology and clinical chemistry determinations. All animals were necropsied, selected organs weighed and a range oftissues were taken, fixed and examinedmicroscopically. Up to and including the highest dose level tested of 15000 ppm, no treatment-related effects were observed in either sex. In conclusion, the No Observed Effect Level (NOEL) of AE 0001789-sulfamoylbenzoic acid (AE 0467398), when administered via the diet to the Wistar rat for at least 90 days, was 15000 ppm in males and females (equatingto approximately 888 mg/kglday and 1094 mg/kglday respectively). I. 

### MATERIALSAND METHODS A. MATERIALS: 

### 1. TestMaterial: AE 0467398 Description: off-white powder Lot/Batch: KTS 10207-1-1 Purity: 96.0% CAS: 138-41-0 Stabilityoftestcompound: Stable in rodent diet 400 and 20000 ppm over at least a 67-day periodat ambient temperature 2. Vehicle and lorpositive control:None

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### 3. Test animals: Species: Rat 

### Strain: Wistar Rj: WI (lOPS HAN) 

### Age: 7weeks approximately 

### Weightatdosing: 230 to 269 g forthe males 175 to 209 g for the females 

### Source: R. Janvier, Le Genest St Isle, France 

### Acclimation period: 13 days 

### Diet: Certified rodent powdered and irradiated diet A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), adlibitum except before urine andblood collection 

### Water: Tap water, adlibitum exceptbefore urine collection 

### Housing: Animals were caged individually in suspended stainless steel wire mesh cages. 

### Environmentalconditions

­ Temperature: 22 ±2°C 

### Humidity: 55 ± 15% 

### Airchanges: Approximately 15 changes perhour 

### Photoperiod: Alternating 12-hourlightand dark cycles (7 am- 7pm) 

### B. STUDY DESIGN: 1. In life dates: April 5 to November 3, 2006 at Bayer CropScience, Sophia Antipolis Cedex, France 

### 2. Animal assignment andtreatment There were 10 animals of each sex per dose group. Animals were assigned using computer randomization by weight. AE 0467398 was administered in the diet for atleast 90 days to Wistarrats at the following doses

- 0, 400, 2000and 15000ppm (equatingto approximately to 24, 116 and 888 mg/kglday in males and 29, 142 and 1094 mg/kglday in females). A negative control group received plain diet. Animal housing and husbandry were in accordance with the regulations ofthe Guide for the Care and Use ofLaboratory Animals (Public Health Service, National Institute ofHealth, NIH publication N°86-23, revised 1985) and "Le Guide du Journal Officiel des Communautes Europeennes L358, 18 Decembre 1986, W86/609!CEEdu 24 Novembre 1986".

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### 3. Diet preparation and analysis AE 0467398 was ground to a fme powder before being incorporated into the diet by dry mixing to provide the required concentrations. There were three preparations of each concentration for the whole study. The stability was demonstrated in a pre-study. At 400 ppm, AE 0467398 was found to be stable in the diet over a 77 day period at room temperature or a 67-day freezing period followed by 10 days at ambient temperature. At 20000 ppm, AE 0467398 was found to be stable in the diet over a 67 day period at room temperature ora 57-day freezing period followed by 10 days at ambient temperature. The homogeneity of AE 0467398 in diet was verified on the first preparation at the lowest and highest concentrationsto demonstrate adequate formulation procedures. The homogeneity in the diet rangedfrom 99 to 111% ofthe nominal concentrations. Mean values obtained from the homogeneity check were taken as measured concentration. The dietary levels ofthe test substance were verified for each concentration on each preparation and ranged from 91 to 105% of the nominal concentrations. All results were withinthe in-house range of85 to 115%ofnominal concentration.
Table lIA582 Stud IYd . 

# .- eS)2D Test group Concentration in diet (ppm) Dose per animal (study averages) Animals assigned
Male (mg/kg bw/day) Female (mg/kgbwiday) Male Female 

### 1 0 0 0 10 10 2 400 24 29 10 10 3 

### 2000 116 

### 142 10 

### 10 4 15000 888 

### 1094 10 

### 10

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TableIIA5.8-3 Homo2eneity and concentration oftestmaterial Nominal concentrations Analysed concentrations % ofnominal concentration (ppm) (ppm) HI 407 102 400 100 Ml 395 H3 398 99 Homogeneity M3 399 100 (first preparation) Bl 409 102 B3 401 100 HI 15702 105 15000 H3 15492 103 Ml 15582 104 M3 15676 105 Bl 15580 104 B3 16601 III 400 (load 1) 401.5 100 400 (load 2) 396 99 Concentration 2 000 (load 1) 2079 104 (firstpreparation) 2000(load 2) 2061 103 15 000 (load I) 15772 105 15 000 (load2) 15655 104

400 (load 1) 391 98
I 400 (load 2) 403 101 Concentration 2 000 (load ]) 2072 104 (secondpreparation) 2 000 (load 2) 2074 104 15000(load 1) 15351 102 15 000 (load 2) 15257 102

400 (load I) 370 93 400 (load2) 371 93 Concentration 2 000 (load 1) 1827 91 (thirdpreparation) 2000(load2) 1843 92 15000(load 1) 14038 94 15000 (load 2) 14117 94 R = surface; M middle; B bottom ofthe preparation. Three different samples 1,2 and 3 were taken at the each level ofthe preparation (R, M, B); onlytwowere analyzed.

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### 4. Statistics

Data were analyzed by the Bartlett's test for homogeneity of variances. When the data were homogeneous, an ANOVA was performed followed by Dunnett's test on parameters showing a significant effect by ANOVA. 

### When the data were not homogeneous even after transformation, a Kruskal-Wallis ANOVA was performed followed by the Dunn's test ifthe Kruskal-Wallis was significant. When one or more group variance(s) equaled 0, means were compared using nonparametric procedures. Group means were compared at the 5% and 1 % levels of significance. Statistical analyses were carried out using PathJTox System V4.2.2. (ModuleEnhanced Statistics).



### C. METHODS: 1. 

### Observations The animals were observed twice daily for moribundity and mortality (once daily on weekends or public holidays). Observed clinical signs were recorded at least once daily for all animals and detailed physical examinations were performed once during the acclimatization phase and at least weekly during the treatmentperiod. 

### 2. Neurotoxicity assessment During study Weeks 11 to 12, a neurotoxicity assessment was performed for all surviving animals by observers who were blindwith respect tothe dose level.



### a. Exploratorylocomotor activity Animals were tested individually using an automated photocell recording apparatus designed to measure quantitatively spontaneous exploratory locomotor activity in a novel environment. Exploratory locomotor activity was recorded for the first 90 minutes with data being collected at 15-minute intervalsthroughoutthe session. 

### b. Sensoryreactivity The following reflexes and responses were recorded:

- Pupillary reflex (by covering the eyes ofthe animal for a few seconds and then observing pupillary constrictionby focusing a narrow beam oflightin the eyes).

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- Surfacerighting reflex (by putting the animal on its back andmeasuring its ability/rapidityto reassume a normal standingposition).

- Corneal reflex (by touching the medial canthus with a fine object and observing the quick andcomplete closure ofthe eyelids).

- Flexor reflex (by pinching the toes and measuring the presence/strength of the flexor response ofeach hindlimb).

- Auditory startle response (bymeasuringthe animal response to an auditory stimulus).

- Tailpinch response (by pinchingthe tail with a forceps and measuringthe animal reaction).



### c. Grip strength The fore- and hindlimb grip strength ofall animals were measured quantitatively using a grip strengthtesterequippedwith onepull or onepush strain gauge (Bioseb, Chaville, France). The mean ofthree successive measurements was notedfor both fore- and hindlimb grip strength.



### d. Open field observation Changes in gait, posture, as well as presence of clonic or tonic movements, stereotypic behaviour (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. selfmutilation, walking backward) and other neurological-related changes were recorded for all surviving animals. 

### 3. Bodyweight Body weights were recorded three times during the acclimatization phase, on the first day oftest substance administration, then at weekly intervals throughout the treatment. Additionally, fasted animals wereweighedbefore necropsy. 

### 4. Food consumption and compound intake Food consumption was recorded weekly for the first two weeks ofthe study, then twice weekly thereafter due to spillage problems for all animals during the treatmentperiod. The weekly mean achieved dosage intake in mglkglday for each week and for Weeks 1to 13 was calculated for each sex usingthe formula: Test substance intake Dose level (ppm) x Group mean food consumption (glday) (mglkglday) Group mean body weight (g) at endofthe food consumption period

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### 5. Ophthalmologicalexamination During the acclimatization period all animals were subjected to an ophthalmological examination. After instillation of an atropinic agent (Mydriaticum, Merck Sharp and Dohme) each eye was examined by means of an indirect ophthalmoscope. During Week 13, all surviving animals from control andhigh dose groups were re-examined. 

### 6. Clinical chemistry
On 

### study days 93, 94 or 95, prior to necropsy, blood samples were taken from all surviving animals in all groups by puncture oftheretro-orbital venous plexus. Animals were diet fasted overnight prior to bleeding and anesthetized by inhalation of isoflurane. Blood was collected on EDTA for haematology, on lithium heparin for plasma and clot activator for serum for clinical chemistry and on sodium citrate for coagulation parameters.



### a. Haematology The following haematology parameters were assayed using an Advia 120; red blood cell count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin concentration, reticulocyte count, white blood cell count and differential count evaluation and plateletcount. A blood smear was prepared and stained with Wright stain. It was examined when results of Advia 120 detenninations were abnonnal. Prothrombin time was assayed on an ACL 3000.



### b. Clinical chemistry Any significant change in the general appearance ofthe plasma and the serum was recorded. Total bilirubin, glucose, urea, creatinine, total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations and aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyltransferase activities were assayed on plasma samples, total protein and albumin concentrations were assayed on serum samples using an Advia 1650 (Bayer Diagnostics, Puteaux, France). Globulin and albumin/globulin ratio values were calculated. 

### 7. Urinalysis In the morning, overnight urine samples were collected on study Days 86 or 87 from all surviving animals from all groups. Food and waterwerenot accessible during urine collection.

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### Any significant change in the general appearance ofthe urine was recorded. The urine volume was measured. pH was assayed using a Clinitek 200+ and Ames Multistix dipsticks (Bayer Diagnostics, Puteaux, France). Urinary refractive index was measured using a RFM320 refractometer (Bioblock Scientific, Illkirch, France). The following semi-quantitative parameters were assayed using a Clinitek 200+ and Multistix dipsticks: glucose, bilirubin, ketone bodies, occult blood, protein and urobilinogen. Microscopic examination ofthe urinary sediment was performed after centrifugation ofthe urine. The presence of redblood cells, whiteblood cells, epithelial cells, bacteria, casts and crystals was graded.


### 8. Sacrificeand 

### pathology On study days 93, 94 or 95, a complete necropsy was performed on all surviving animals. Animals were deeply anaesthetized by inhalation of lsoflurane, then exsanguinated before necropsy. All animals were diet fasted prior to scheduled sacrifice. The animal found dead during the study was necropsied. The necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded, sampledand examined microscopically. Adrenal gland, brain, epididymides, heart, kidney, liver, ovary, pituitary gland, prostate gland, spleen, testis, thymus, thyroid gland (with parathyroid gland) and uterus (including cervix) were weighed fresh at scheduled sacrifice only. Pairedorgans were weighedtogether. The following organs or tissues were sampled: adrenal gland, aorta, articular surface (femoro-tibial), bone (sternum), bone marrow (sternum), brain, epididymis, oesophagus, exorbital (lachrymal) gland, eye and optic nerve, Harderian gland, heart, intestine (duodenum, jejunum, ileum, caecum, colon, rectum), kidney, larynx/pharynx, liver, lung, lymph nodes (submaxillary, mesenteric), mammary gland, nasal cavities, ovary, pancreas, pituitary gland, prostate gland, sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, submaxillary (salivary) gland, testis, thymus, thyroid gland (with parathyroid), tongue, trachea, urinary bladder, uterus (with cervix), vagina. For sacrificed animals, a bone marrow smear was prepared from one femur, stained with May-Grunwald Giemsa, but not examined. Tissues samples were fixed by immersion in neutral buffered 10% formalin withthe exception ofthe eye, optic nerve, Harderian gland, epididymis andtestisthat were fixed in Davidson'sfixative. Histopathological examinations were performed on alltissues from all the animals in the control and high dose groups and all decedents in all groups. The liver, kidney, lung, spleen, thyroid gland and macroscopic findings were examined in all animals in the study. All histopathological examinations were performed by the Study Pathologist. Following the initial

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### histopathological examination, a review of representative slides was performed by a reviewing pathologist. The diagnoses presented in this report represent the consensus opinion of the two pathologists. 

### II. RESULTS AND DISCUSSION A. Mortality: One male from the 400 ppm group died prematurely on Study Day 67 due to thoracic haemorrhage. This death wasjudgednot to be treatment-related. 

### B. Clinical signs Notreatment-related clinical signs were observed during the study in either sex. 

### C. Neurotoxicity assessment l. 

### Exploratorylocomotoractivity Overall mean exploratory locomotor activity was unaffected by the treatment at any dose level in either sex. In addition, the pattern ofthe locomotor activity over time was similar to controls. A slight decrease in overall mean exploratory locomotor activity was noted at 15000 ppm (-15%, not statistically significant) and 400 ppm (-28%, not statistically significant) in females, but it was observedwith no dose relationship and was thus consideredto be 

### fortuitous. 

### 2. Open field observation No treatment-related changes were recorded during the open field observation at any dose level in either sex. 

### 3. Sensory reactivity Allreflexes and responses evaluatedwere unaffected by the treatment at any dose level in either sex. The increased incidence ofexaggerated tail pinch response in the high dose males was considered not to be treatment-related, due to the limited magnitude ofthe change and interindividual variation ofthis parameter. 

### 4. Grip strength The fore- andhindlimb grip strength were unaffected by the treatment at any dose level in either sex.

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### D. Body weight Noeffect onmean body weight orbody weight gainwas noted in either sex. 

### E. Food consumption Noeffect on food consumption was noted in either sex. 

### F. Achieved dosages The mean achieved dosage intake ofAE 0001789-sulfamoylbenzoic acid (AE 0467398)pergroup was as follows:
Table IIA5.8-4: Mean achieved dietary intakeofAE 0467398 (Weeks 1-13)

Diet concentration in ppm Males mglkglday Females mglkglday 400 24 29 2000 116 142 15000 888 1094


### G. Ophthalmological examination At 15000 ppm, onemale was noted with pupillaryreflexnotobservable (left eye)andonemale was noted with haemorrhage of the vitreus and hyaloid remnant (right eye). In isolation, these findings were considerednottobe treatment-related. 

### H. Clinicalpathology 1. 

### Haematology Notreatment-related change was noted at any dietary level in either sex. 

### 2. Clinical chemistry Notreatment-related change was noted at any dietary level in either sex.

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### 3. Urinalysis A tendency towards lower pH values was noted at 15000 ppm in both sexes, when compared to the control groups, -11% (p<O.OI) and -10% (p<0.05) in males and females respectively. In isolationthis change was considerednottobe adverse. I. 

### Post-mortem examination 1. 

### Organ weights

There was no relevant change in mean terminal body weights when compared to controls. All the organ weight differences between treated and control animals were minimal and were considered to be incidental and nottreatment-related. 

### 2. Gross pathology

Unscheduleddeath: One male from the 400 ppm group was found dead on study Day 67, with moderate red liquid content in the thoracic cavity, haemorrhages in the mesentery, slight congested/red pancreas and mottled red thymus and lung. At the microscopic examination, these findings were correlated with slight interstitial haemorrhage in the thymus and moderate agonal congestion in the lung. The thoracic haemorrhage was considered to be the probable cause of death and was judged to be secondaryto an accidentaltrauma. Terminal sacrifice: All the macroscopic changes observed at terminal sacrifice were considered as incidental and not treatment-related. 

### 3. Microscopic pathology

No treatment-related microscopic findings were observed in the unscheduled animal or at terminal sacrifice. 

### III. CONCLUSION In conclusion, the No Observed Effect Level (NOEL) ofAE 0467398, when administered via the diet to the Wistar rat for at least 90 days, was 15000 ppm in males and females (equating to approximately 888 mglkgldayand 1094 mglkg/day in males and females, respectively). (Blanck0.,2007)

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### • AE 0852999 (AE 0001789-cylopropylsulfamoylbenzamide)

In vitrogenotoxicity

- Bacterialassay for gene mutation Report: KIIA 5.8/05, Herbold B., 2006 Title: AE 0001789-cyclopropylsulfamoylbenzamide (AE 0852999), Salmonella/microsometest, plate incorporation andpreincubation method Report No & AT03ll0 DocumentNo M-274230-01-2 

### Guidelines: OECD 471 (1997); EEC Directive 2000/321EC Method B13/14 (2000); EPA Health Effects Test Guideline(OPPTS 870.5100; 1998) GLP Yes (certified laboratory) Executive summary: In this in vitro assessment of the mutagenic potential ofAE 0852999 (M 02)(Batch KTS 10218-3-2, 98.8% ofpurity), histidine dependent auxotrophic mutants ofSalmonellatyphimurium, strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to AE 0852999 up to 5000 lAg/plate, diluted in dimethyl sulphoxide (DMSO). For each bacterial strain and dose level, triplicate plates were used in both the presence and absence of an Aroclor 1254-induced rat liver metabolic activation system (S9 mix). DMSO was also used as a negative control. Specific positive controls were used for each strain. After 48 hours ofincubation at 37°C, the numbers ofrevertant colonies were scored using an automated colony counter. Doses up to and including 5000**~plate** did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition ofgrowth was observed. AE 0852999 did not cause any significant increase in the number ofrevertantcolonies in eitherthe presence orabsence ofmetabolic activation. All the positive control compounds produced expected increases in the number of revertant colonies, thereby demonstratingthe sensitivity ofthe assay andthe efficacy ofthe S9 mix. Therefore, AE 0852999 was non-mutagenic with orwithout S9 mix in the plate incorporation as well as in thepre-incubationmodification ofthe Salmonella/microsometest.

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I. 

### MATERIALSAND METHODS A. MATERIALS: 

### 1. Test Material: Description: Lot/Batch: Purity: CAS: Stability oftest compound: 

### 2. Controlmaterials: Negative: Solvent: Positive:

3. Test organisms: Species: Strain: Source:

4. Testcompound concentrations: Plateincorporation assay: AE 0852999 fine whitepowder KTS 10218-3-2 98.8% No CAS entry yet Stable forthe duration ofthe study Culture medium OMSO Sodium azide (Serva) for TA 1535 at 10 Jlg/plate, Nitrofurantoin (Sigma) for TAl00 at 0.2 Jlg/plate, 4-Nitro-l,2-phenylene diamine (Merck-Schuchardt) for TA 1537 at 10 Jlg/plate and T A98 at 0.5 Jlg/plate, Mitomycin C (Fluka) for TAl02 at 0.2 Jlg/plate only in plate incorporation plate, Cumene hydroperoxide (Sigma) for TAl02 in pre-incubation trials only at 50 Jlg/plate, 2-Aminoanthracene (Aldrich) for the activating effect ofthe S9 mix in all strains at 3 Jlglplate.

Salmonellatyphimurium LT2 mutants Histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102 Strains obtained from Prof. Bruce Ames in 1997 and stored in the laboratory since then.

For all strains with or without S9 mix: 16, 50, 158, 500, 1581 and 5000 Jlg/plate Pre-incubation assay: For all strains with or without S9 mix: 16, 50, 158, 500, 1581

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### and 5000 llg/plate 5. Metabolic activation: The S9 fraction was isolated from the livers of Aroclor 1254 induced male Sprague Dawley rats. The preparation dated from September 13, 2005 (protein content 23.4 mg per ml) and was kept frozen at -80°C. The batch was tested for contamination prior to use in the study. Cofactors were freshly dissolved in sodium phosphate buffer (150 mM, pH 7.4) B. STUDY DESIGN AND METHODS: The experimental phase ofthe study was performed between April 13 and April 28, 2006, at Bayer HealthCare AG (PH-PD P Health Care Toxicology). The Salmonella/microsome test is a screening method which detects point mutation caused by chemical agents 

### in vitro. Auxotrophic mutants ofSalmonella typhimurium are used to demonstrate this effect. Forthis purpose, the rate ofreversion to prototrophy is evaluated in negative control and treatedgroups. 1. 

### Plateincorporation assay: AE 0852999 or the control material was dissolved in 0.1 ml ofDMSO. DMSO (0.1 ml) containing AE 0852999 or controls were added to glass vessels with 0.1 ml of bacterial cultures grown overnight, 0.5 ml ofS9 mix or buffer and 2 ml ofsoft agar. The mixture was placed in a waterbath at 45°C for 30 seconds, shaken and overlaid onto Petri dishes containing solid agar. After 48 hours of incubation at 37°C, the numbers of revertant colonies were scored using an automated colony counter. Three plates were used, both with and without S9 mix, for each strain and dose. The doses for the first trial were routinely determined on the basis ofa standard protocol with a maximum dose of5000 llg/plate and at least 5 additional doses. Ifless than three doses were used for assessment, at leasttwo repeats were performed. 2. Pre-incubation assay: An independent repeat was performed as pre-incubation ofthe previously described mixture in a water bath at 37°C for 20 minutes. At the end ofthe preincubation period, 2 ml ofmolten soft agar were added to the tubes, the content mixed and plated onto Petri dishes with solid agar. After 48 hours ofincubation at 37°C, the numbers ofrevertant colonies were also scored using an automated colony counter.

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### 3. Acceptance criteria: The negative controls had to be within the expected range. The positive controls had to show sufficient effects. Titre detenninations had to demonstrate sufficient bacterial density in the suspension. The titre was detennined by the total bacterial counts taken on two plates for each concentration studied with S9 mix. 4. Assessmentcriteria A reproducible and dose-related increase in mutant colonies ofat least one strain was considered to be positive. For TA 1535, TA 100 and TA 98, this increase should be about twice that ofnegative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase ofabout 100 mutants should be reached. Otherwise, the resultwas considered as negative. II RESULTS AND DISCUSSION There was no indication of a bacteriotoxic effect ofAE 0852999 at doses ofup to and including 5000 I-lg/plate. The total bacterial counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition ofgrowth was noted either. None ofthe five strains concerned showed in the plate incorporation test a dose-related and biologically relevant increase in mutant colonies overthose ofthe negative controls. This applied both tothetests with and without S9 mix and was confinnedby the results ofthe pre-incubationtrials. The positive controls sodium azide, nitrofurantoin, 4-nitro-l,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant colonies to well over those of the negative controls, and thus demonstratedthe system's sensitivity and the activity ofthe S9 mix. III 

### CONCLUSION No indication ofmutagenic effects ofAE 0852999 were found at doses ofup to 5000 I-lg/plate in any of the Salmonellatyphimurium strains used in the assay. (HerboldB., 2006)

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In vitrogenotoxicity

- Test for c1astogenicity in mammalian cells Report: KIIA 5.8/06Thum M., 2006 Title: AE 0001789-cyclopropylsulfamoylbenzamide (AE 0852999 carboxamide), 

### In vitro chromosome aberrationtestwith Chinese HamsterV79 cells Report No & AT03121 DocumentNo M·274272·01·2 Guidelines: OECD 473 (1997); EEC Direc:tive 2000/32IEC Metbod 810 (2000); EPA Healtb Effects TestGuideline (OPPTS 870.5375; 1998) GLP Yes (certified laboratory)
Executivesummary: In this in vitro assessment ofthe clastogenic potential ofAE 0852999 (M02) (batch KTS 10218·3-2, 98.8% ofpurity), Chinese Hamster V79 cells were exposed to AE 0852999 at 250, 500 and 1000 Ilg/ml, diluted in dimethyl sulphoxide (DMSO) in the presence or the absence ofan Aroclor 1254·induced rat liver metabolic activation system (S9 mix). Foreach dose level, duplicate cultures were used. DMSO was also used as a negative control. After 4 hours treatment, the medium was changed and the cells were harvested 14 hours later. In addition, cells treated at 1000 /lglml were harvested 30 hours afterthe start of the study. Without S9 mix, an additional experiment was performed using continuous treatment for 18 hours, harvest at the same time, and AE 0852999 concentrations of250, 500 and 1000 /lg/ml. Mitomycin C, which produces crosslinks in the DNA, and cyclophosphamide, which induces chromosomal damage after metabolic activation, were used as positive controls, diluted 

### in Hanks' balanced salt solution. Colcemid was added to each flask two hours prior to harvest to arrest the cells 

### in a metaphase-like stage ofmitosis. Without S9 mix no cytotoxic effects were observed up to 1000 Ilglml after 4 and 18 hours treatment. With S9 mix cytotoxic effects were observed at 1000 /lglml. Precipitation in the medium occurred at 1000 /lglml. None ofthe cultures treated with AE 0852999 in the presence and in the absence ofS9 mix showed biologically relevant and statistically significant increasednumbers ofaberrant metaphases. The positive controls mitomycin C and cyclophosphamide induced clastogenic effects and demonstrated the sensitivity ofthe test system andthe activity ofthe used S9 mix. Based on this test, AE0852999was considered notto be clastogenic for mammalian cells in vitro.

I. MATERIALSAND METHODS A. MATERIALS:

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### 1. Test Material: AE 0852999 Description: fine white powder LotlBatch: KTS 10218-3-2 Purity: 98.8% CAS: No CAS entryyet Stability oftestcompound: Stable for the duration ofthe study 2. Controlmaterials: Negative: Culture medium Solvent: DMSO for AE 0467398 and Hanks's balanced salt solution for positivecontrols (Seromed) Positive: Mitomycin C (Fluka, batch 1149026 10605077) without S9 mix at 0.1 Ilglml for a treatment period of4 hours and 0.03 Ilglml for atreatment periodof 18 hours. Cyclophosphamide (Endoxan 100 mg injection vials of 

### dry substance, Baxter Oncology GmbH) with S9 mix at 2 Ilglm\. 

### 3. Testorganisms: Cell line: Chinese hamster V79 lung cells Source: Cells obtained from Dr Utesch, Merck AG, Darmstadt in 1993, stored in the laboratory since then. 4. Testcompoundconcentrations: AE 0852999 was used with or without S9 mix at 250, 500 and 1000 Ilglm\.



### B. STUDY DESIGN AND METHODS: The experimental phase ofthe study was performed from March 15 to April 27, 2006 at Bayer Healthcare AG (PH-PD P HealthCare Toxicology). The in vitro cytogenetic test is a mutagenicity test system for the detection ofchromosome aberrations in cultured mammalian cells. The test is designed to detect structural aberrations (chromatid and chromosome aberrations) in cells at theirfirst post-treatmentmitosis. 

### 1. Determination ofcytotoxicity:

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### Duplicate cultures were exposed in a pre-test to AE 0852999 at concentrations ranging from 10 to 2500 Ilg/ml with orwithout S9 mix for 4 hours. In addition, cells were exposed without S9 mix for 18 hours to concentrations ranging from I to 1500 Ilg/ml. The mitotic index was determined for all cultures. The number ofmitotic cells among a total of 1000 cells per culture was determined using a light microscope at a magnification ofabout 630. All cells which were not in interphase were defined as mitotic. In the main study, cultures with a total incubation period of 4 hours were additionally used to determine the cytotoxicity ofAE 0852999 at concentrations ranging from 250 to 1000 Ilg/ml with a harvest time of 18 hours and at concentrations of 1000 Ilg/ml with an harvest time of 30 hours. Additional cultures were exposed without S9 mix for 18 hours to concentrations of AE 0852999 ranging from 250 to 1000 Ilg/ml. These cultures were harvested at the end ofthetreatmentperiod. AE 0852999 at up to 2500 f.l.g/ml did not changed the pH in the medium. The osmolality in the medium ofthepre-testwas not changedby concentrationsofup to 2500 Ilg/ml ofAE0852999. 

### 2. Treatment protocol: Chinese hamster V79 cells were passaged on the day prior to treatment. Approximately Ixl06 

### cells were seeded in 20 ml ofmedium per 75 m2 

### flasks and incubated at 37°C in a CO2

### -incubator (5% CO2). 

### Unless reported otherwise, the cells were grown in Eagle's minimal essential medium containing 10% foetal calfserum. Immediately before treatment, the medium was removed from the cultures. For the trials without S9 mix, 20 ml ofmedium containing 2% foetal calfserum and 0.2 ml oftest substance solution were added to each flask. For the trials with S9 mix, 19 ml ofmedium containing 2% foetal calfserum, I ml ofS9 mix and 0.2 ml oftest substance solution were added to each flask. The cells were incubated for 4 hours at 37°C. After4 hours oftreatment, the medium was removed, the cells were washed with PBS and 20 ml offresh medium containing 10% foetal calf serum was added to the flasks. The flasks were placed in a COrincubator for the remaining incubationtime. In the cultures treated for 18 hours, the medium was not removed. 0.2 ml of Colcemid-solution (40 f.l.g/ml) was added to each flask 2 hours prior to the end ofthe incubation periodto arrestthe cells in ametaphase-like stage ofmitosis(c-metaphase). Positive controls and solvent controls (0.2 ml of solvent per culture) were set up in parallel and handled as described for AE 0467398-treated cultures. Untreated controls and solvent controls were used as negative controls. 

### 3. Chromosomepreparations:

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### After the removal ofthe medium from each flask, the cells were trypsinized, suspended in medium and centrifuged for approximately 5 minutes at 700 rpm. The supernatantwas removed and 1to 2 ml ofa hypotonic solution (0.4% KCI; 37°C)was addedto eachtube. Within 4 minutes, the volume was brought to 6 ml with additional hypotonic solution and cells were resuspended. The cells were centrifuged again and fixed with cold (4°C) fixative (ethanol/acetic acid 3:1) for 20 minutes at room temperature. Cells were pelleted and resuspended in fixative as before and centrifuged again. The pelleted cells were resuspended in a small volume offixative and the suspension was dropped onto clean slides. The slides were allowed to dry 

### for at least 2 hours. Thereafter, they were submerged in pure methanol for 3minutes and stained for 15-20 minutes in 3% Giemsa solution. Slideswere rinsed twice in water and once in acetone and were then kept in xylene for about 30 minutes. The slides were allowedto dry completely andcovered. At leasttwo slides were generatedperculture. 

### 4. Evaluation criteria: Coded slides were evaluated using a light microscope at a magnification of about 1000. Chromosomes ofapproximately 200 metaphases perconcentration, 100 metaphases from each oftwo parallel cultures, were examined. Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges were detected. The following aberrations were recorded: gaps (an achromatic lesion within a chromatid arm without dislocation of the chromatid end), break (a discontinuity ofone chromatid with dislocation ofthe chromatid end), fragment (part ofchromosome without centromer), deletion (result ofa break with the terminal chromatid part ofthe chromosome missing within the metaphase under assessment), exchange (exchange of chromatid-parts between different chromosomes or within the same chromosome), multiple aberration (when five or more structural changes occur within one metaphase). Observed polyploidy metaphases were recorded but notused for assessment. 

### 5. Assessment criteria: An assay was acceptable, ifthere was a biologically relevant increase in chromosome aberrations induced by positive controls and ifthe numbers ofaberrations for the negative controls were in the expectedrange. An increased incidence ofgaps ofboth types without a concomitant increase ofother aberration types was considerednottobe an indication ofa clastogenic effect. A test was considered positive, ifthere was a relevant and statistically significant increase in the aberrationrate. A test was considered negative, ifthere was no such increase at any time interval or ifthere were

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# EBBayerCropScience 2007-03-28 Tier2, IIA, Sec. 3, Point5: Cyprosulfamide statistically significant values, which were, however, withinthe range ofhistorical negative controls. A test was considered equivocal, if there was an increase above the range of historical negative controls which was statistically significant but not considered relevant, or if an increase occurred, which was consideredrelevant, but which was not statistically significant. 

### 6. Statistics: The statistical analysis was performed by pair-wise comparison of AE 0852999-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) usingthe one-sided Chi2-test. The numbers ofmetaphases with aberrations (including and excluding gaps) and ofmetaphases with exchanges were compared (provided that these data superceded the respective solvent control). The one-side Che-testwas used for the statistical evaluation. A difference was consideredto be significant, ifthe probability oferror was below 5%. 

### II RESULTS AND DISCUSSION A. GENERAL REMARKS In the the presence or absence of S9 mix Chinese hamster V79 cells were exposed for 4 hours to AE 0852999 at concentrations ofup to and including 1000 Ilg/ml and, in addition, without S9 mix for 18 hours to AE 0852999 at concentrations ofup to and including 1000 Ilg/ml. With and without S9 mix substance precipitation occurred in the medium at 1000 Ilg/ml. 

### B. MITOTIC INDEX Mitotic index without S9 mix: In comparison to the solvent control, the mitotic indices in the treated cultures were not relevantly reduced. The cultures treated with mitomycin C showed a reduction in mitosis rate only after4 hours treatment. Mitotic index with S9 mix: In comparison to the solvent control, the treated cultures showed a relevant reduction ofthe mitosis rate at 1000 Ilg/ml. The positive control cyclophosphamide also reduced the mitosis rate.

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C. SURVIVALINDEX Survival index without S9 mix: In comparison to the solvent control, the survival indices in the treated cultures were not relevantly reduced. The cultures treated with mitomycin C showed a reduction in survival rate. Survival index with S9 mix: In comparison to the solvent control, the treated cultures showed no relevant reduction ofthe survival rate. The positive control cyclophosphamidereducedthe survival rate. D. CHROMOSOME ABERRATION AE 0852999 without S9 mix: No biologically relevant and statistically significant increases ofnumbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The same wastrue for atreatmentperiod and total culture time of 18 hours. The treatment with the positive control mitomycin C resulted in a clear and statistically significant increase ofmeta phases with aberrations and demonstratedthe sensitivity ofthe test system. AE 0852999 with S9 mix: No biologically relevant and statistically significant increases ofnumbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The positive control cyclophosphamide induced statistically significant and biologically relevant increases of metaphases with aberrations and demonstrated the sensitivity of the test system and the activity ofthe used S9 mix.

III. CONCLUSION After 4 hours treatment ofChinese hamster V79 cells with AE 0852999 concentrations of250, 500 and 1000 J..lglml were used without and with S9 mix for assessment of the clastogenic potential of AE 0852999. In addition, after 18 hours treatment with AE 0852999 concentrations of 250, 500 and 1000 J..lglml were selected for reading without S9 mix. None ofthese cultures treated with AE 0852999 in the absence orpresence ofS9 mix showed statistically significant and biologically relevant increases ofnumbers ofmeta phases with aberrations.

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### The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and demonstrated the sensitivity ofthe test system and in the case ofcyclophosphamide the activity ofthe used S9 mix. Based on the results ofthis test, AE 0852999 was considerednottobe clastogenic for mammalian cells in vitro. (Thurn M., 2006) Invitro genotoxicity

- Testfor gene mutation in mammalian cells Report: IIA5.8/07, 

### Herbold B., 2006 Title: AE 0001789-cyclopropylsulfamoylbenzamide (AE 0852999 carboxamide), V791HPRT-test in vitro 

### for the detection ofinduced forward mutations Report No & 

### AT03164 DocumentNo M-274735-01-2 Guidelines: OECD 476 (1997); EEC Directive 2000/32IEC Method B17 (2000); EPA Health Effects TestGuideline(OPPTS 870.5300; 1998) GLP Yes (certified laboratory) Executive summary: The purpose ofthe study was to assess the point mutagenic potential ofAE 0852999 (batch KTS 10218­ 3-2,98.8 % ofpurity) atthe hypoxanthine-guaninephosphoribosyl transferase (HPRT) locus in V79 cells. For dose selection, a preliminary cytotoxicity test was conducted with and without an Aroclor 1254 induced rat liver metabolic activation system (S9 mix) using concentrations ofAE 0852999 ranging from 6.5 to 650 Ilglml. 

### Precipitation of AE 0852999 in the culture medium occurred at 650 Ilglml. 

### Concentrations ofup to 650 Ilglml did not change the pH in the medium ofthe pre-test. The osmolality in the medium ofthe pre-test was not changed up to 650 Ilg/ml. 

### No cytotoxicity was observed with or without S9. AE 0852999 was tested in the first mutation experiment at concentrations ranging from 75 to 900 Ilglml with or without metabolic activation. The same concentrations were used for the independent repeats. Exponentially growing V79 cells were plated in 20 ml culture medium in two 75 cm2 

### flasks. After attachment, cells were exposed with or without S9 mix to vehicle alone orto arange ofconcentrations of the test substance for 5 hours. Thereafter, each cell monolayer was washed with PBS, trypsinized and replated into 3 Petri dishes. Per culture one flask and 3 Petri dishes were used. These dishes were incubated for 6 to 8 days to allow colony development and to detennine the cytotoxicity associated with each test substance directly after treatment (survival to

### treatment). Cells in 75 cm2 

### flasks were incubated to pennit growth and expression ofinduced mutations. Cells were subcultured count I, nonnally after 3 days) by reseeding cells in 75 cm2 

### flasks. At the end ofthe

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### expression period (=count 2, nonnally a total of6 days), cultures were reseeded in Petri dishes without hypoxanthine but containing 6-TG for selection ofmutants. In addition, 200 cells per dish were seeded in 5ml culture mediumtodetennine the absolute cloning efficiency for each concentration. After incubation for 6 to 8 days, the colonies were fixed, stained with Giemsa and counted to detennine the number of6­ TG resistant colonies in the mutation assay dishes and the number ofcolonies in the cloning efficiency dishes. Two trials were perfonned. With and without S9 mix AE 0852999 induced decreases in survival to treatment and decreases in relative population growth. Precipitation ofAE 0852999 in the culture medium was observed in the main study at 750 Ilglml and above. With and without S9 mix there was no biologically relevant increase in mutant frequency above that ofthe vehicle controls. Ethyl methanesulfonate and dimethylbenzanthracene induced clear mutagenic effects demonstrating the sensitivity ofthe test system and the activity ofthe S9 mix. Based on these results, AE 0852999 was considered to be non-mutagenic in the V79IHPRT forward mutation assay, both with and without metabolic activation. I. MATERIALSAND METHODS
A. MATERIALS: 1. 

### TestMaterial: AE 0852999 Description: white powder Lot/Batch: KTS 10218-3-2 Purity: 98.8% CAS: No CAS entry yet Stabilityoftestcompound: Stable forthe duration ofthe study 2. Controlmaterials: Negative: Culture medium [Eagle's minimal essential medium supplemented with 1% L-glutamine, 1% MEM-vitamins, 1% MEM NEAA, 1% 

### penicillin/streptomycin and 10% foetal calf serum (FCS)] Solvent: DMSO for AE 0852999 and Dimethylbenzanthracene not exceeding 1% (v/v) in the culture medium. No solvent needed for ethyl methanesulfonate as 

### it is a liquid. Positive: Ethyl methanesulfonate (EMS), a directly alkylating agent, used at a final concentration of900 Ilglml in non-activationtrials. Dimethylbenzanthracene (DMBA), promutagen requiring a

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metabolic activation, used at a final concentration of 20 Jlglml for trials with S9mix. 

### 3. Testorganisms: Cell line: Chinese hamster V79 lung cells 

### Source: Cells obtained from Prof. G. Speit, University ofUlm, Gennany. These cells have since been recloned to maintain karyotypic stability. They have a modal chromosome number of 22 and a rapidpopulation doubling time (10 to 14hours) 

### Culturecondition: Incubation perfonned at 37°C in a humidified atmosphere with about 5% CO2. 

### 4. Test compound concentrations: AE 0852999 was used at 6.5, 13,26,52, 104,208,416 and 650 Jlglml in the clonal cytotoxicity assay and at 75, 150, 300, 450, 600, 750 and900 Jlg/ml inthe mutagenic assays. 

### 5. Metabolic activation: The S9 fraction was isolated from the livers of Aroclor 1254 induced male Sprague Dawley rats. The preparation dated from September 13, 2005 (protein content from 24.0 mglml) and was kept frozen at -80°C. The batch was tested for contamination and cytotoxicity priorto use in the first study. Cofactors were freshly dissolved in sodium phosphate buffer(I50 mM, pH 7.4)



### B. STUDY DESIGN AND METHODS: The experimental phase of the study was perfonned from February 14 to April 19, 2006 at Bayer HealthCare AG (PH-PD P Health Care Toxicology). The selection of V79 forward mutations is based on the resistance of induced mutants to the purine analogue 6-thioguanine (6-TG). This resistance is a result of a mutation at the X-chromosome-linked HPRT locus rendering the cells unable to use 6-TG for DNA synthesis. Therefore, cell colonies fonned in the presence of6-TG are consideredto represent mutants atthe HPRT gene.

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1. 

### Determination ofcytotoxicity: Exponentially growing V79 cells were plated in 20 ml culture medium in a 75 cm2 flask with a total volume of275 m] (4x106cells per flasks). For each concentration, one culture was available. After attachment (16 to 24hours later), cells were exposed without S9 mixto vehicle alone orto arange of concentrations ofthe test substance for 5 hours in 20 ml medium containing2% FCS. In experiments with metabolic activation 1 ml ofmedium was replaced by 1 ml of S9 mix. Thereafter, each cell monolayer was washed with PBS, trypsinized and replated in 5 ml culture medium at a density of 200 cells into 3 Petri dishes (diameter of60 mm). These dishes were incubated for 6 to 8 days to allow colony development. Thereafter, colonies were fixed with 95% methanol, stained with Giemsa (Merck; stock solution diluted 1:5 with deionized water) and counted automatically using an Artek counter, when there was no interference by precipitation on the plates or colouration ofthe plates. Cytotoxicity was expressed by comparison of colonies in treated cultures versus vehicle control cultures (relative cloningefficiency). 

### 2. Treatmentprotocol withoutmetabolic activation: Exponentially growing V79 cells were plated in 20 ml culture medium in two 75 cm2 flasks per concentration (4x106 cells per flask) including all control groups. After attachment (16 to 24 hours later), the cells were exposed to vehicle alone orto a range ofconcentrations ofthetest substance for 5hours in 20 ml culture medium with reduced serum content (2%). Thereafter, cell monolayers were washed with PBS, trypsinized and replated in 20 ml culture medium using I.5x106 cells per 75 cm2 flask and in 5 ml culture medium using 200 cells per Petri dish (diameter of60 mm). One flask and 3 Petri dishes were used per culture. These dishes were incubated for 6 to 8 days to allow colony development and to determine the cytotoxicity associated with each test substance directly after treatment (survivalto treatment). Cells in 75 cm2 flasks were incubated to permit growth and expression ofinduced mutations. Cells were subcultured (= count 1, normally after 3 days) byreseeding 1.5x106 cells into 20 ml ofmedium in 75 cm2 flasks. Atthe end ofthe expression period (=count 2, normally a total of6 days), cultures were reseeded in Petri dishes (diameter of 100 mm) at 3xl05 cells per dish (8 dishes per culture) in 20ml culture medium without hypoxanthine but containing 10 Ilg/ml 6-TG for selection ofmutants. In addition, 200 cells per dish (diameter of60 mm, 3 dishes per culture) were seeded in 5 ml culture medium to determinethe absolute cloningefficiency for each concentration. Afterincubation for 6to 8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of6-TG resistant colonies in the mutation assay dishes and the number ofcolonies in the cloning efficiency dishes.

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Twotrials were performed. 

### 3. Treatment protocol with metabolic activation: The activation assay was performed independantly. The procedure was identical to the nonactivation assay except for the addition of S9 mix. In these experiments 19 ml instead of 20 m] culture medium and additonally 1 ml ofS9 mix were added to the flasks for the treatment period, resulting in a concentration of5% S9 mix in the cultures. The numberof6-TG resistant mutants and viability were determined as in thenon-activation assay. Twotrials were performed. 

### 4. Parameters assessed: The parameter "survivalto treatment" in 

### % was determined onthebasis ofthe following calculation: Mean number ofcolonies (treated cultures) x 100 Mean number ofcolonies (vehicle control cultures) The "absolute population growth" was calculatedusing the following formula: Absolute population growth (for each culture) cell count 1 x cell count 2 The parameter "relative population growth" shows the cumulative growth of the treated cell populations, relativeto the vehicle control. Absolute population growthtreated culture x 100 Absolute population growth ofcorrespondingvehiclecontrol culture The ability ofcells to form colonies at the time ofmutant selection is measured by the parameter "absolutecloning efficiency". Itis expressed in %. Mean numberofcolonies per dish x 100 200 The "mutant frequency" is calculated for each group by dividing the total number ofmutant colonies by the number ofcells seeded (usually 8-10 plates at 3x105cells perplate), corrected for the absolute

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### cloning efficiency. The mutant frequency is expressed as 6-TG resistant mutants par 106 

### clonable cells. Total number ofmutant colonies x 100 Numberofevaluateddishes x 3 x 105

### x C.E. 

### 5. Acceptancecriteria: The average cloning efficiency ofthe negative and vehicle controls should be at least 50%. The average mutant frequency ofthe vehicle controls shouldnot exceed25 x 10-6 

### cells. The mutant frequency ofthe two cultures ofthe vehicle and lor

### the negative control shoulddiffer only to an acceptable extent. As a rule ofthumb, the difference ofmutant frequencies should not be greaterthan 5 x 10-6• 

### The positive control should induce an average mutant frequency ofat least three times that ofthe vehicle controL Ifnot limitedby the solubility ofthe test substance in the vehiclethehighest concentration should induce cytotoxicity ofabout 80 to 90% or shouldbe a concentration where precipitation occurs in the medium. The survival at the lowest concentration should be in the range of the negative controL For the calculation of an acceptable mutant frequency at least 5 dishes per culture should be available and relative survival to treatment, relative population growth and absolute cloning efficiency should be 10%orgreater. However, these criteriamay be overruledby good scientificjudgement. 

### 6. Assessment criteria: Mutant frequencies was only used for assessment, ifat least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater. A trial was considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies was observed. To be relevant, the increase in mutant frequencies should be at leasttwoto three times that ofthe highestnegative orvehicle control value observed in the respective triaL If

### this result was reproduced in a second trial, the test substance was consideredtobemutagenic. Despite these criteria, a positive result was only considered relevant, ifno significant change in osmolality compared to the vehicle control was observed. Otherwise, unphysiological culture

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conditions may bethe reason forthe positive result. A test substance wasjudged as equivocal ifthere was no strictly concentration related increase in mutation frequencies but ifone or more concentrations induced a reproducible and biologically relevant increase in mutant frequencies in all trials. An assay was considerednegative ifnoreproducible and relevant increases ofmutant frequencies were observed. However, these criteriamay be overruledby good scientificjudgement. 

### 7. Statistical analysis: The statistical analysis relied on the mutant frequencies which were submittedto a weighted analysis ofvariance as well astoa weighted recursiveregression, bothwithPoisson derived weights. The twomutant frequency values obtainedpergroupwere, although somewhat related, consideredas independent measurements thus increasing the power of the statistical tests applied. Since the protocol ofthe HPRT assay required at least two independent trials, the overall analysis without respectively with activation was the most important one for classifying substances into mutagens and non-mutagens. However, separate analyses were run for each trial in order to examine the consistency ofthe results. All acceptable groups were included in the weighted analysis of variance followed by pairwise comparisons tothe vehicle control on a nominal significance level of

### a = 0.05 usingthe Dunnett test. The regression analysispartwasperformedonthe basis ofthe actual concentrationsthereby omitting the positive, negative and vehicle controls. Ifthere was a significantconcentration related increase of the mutant frequency (a 0.05) in the main analysis the highest concentration was dropped and the analysis repeated. This procedure was repeated until p 

### > 0.05. In that way eliminated concentrations were flagged correspondingly.

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### II. RESULTSAND DISCUSSION A. GENERALREMARKS: In the presence and in the absence ofS9 mix Chinese hamster V79 cells were exposedto AE 0852999 at concentrations of up to and including 900 I-lglml. With and without S9 mix substance precipitation occurred in the medium ofthe main study at 750 I-lglml and above. No changes in osmolality or pH were observed. The means ofthe absolute cloning efficiency for the vehicle controls in the mutation experiments were 53.8% and 68.0% in the experiments without activation, and 51.0% and 90.7% in the experiments with activation.Theseresults demonstrated good cloning conditions for the experiments. 

### B. MUTATIONASSAY WITHOUTMETABOLIC ACTIVATION: Twotrials were performed. The mutant frequencies ofthe negative controls (means of2.6 x 10'6 and 2.75 x 10'6, respectively) and ofthe vehicle controls (means of2.5 x 10'6 and 3.6 x 10'6, respectively) were all within the normal range. The positive control EMS induced clear mutagenic and statistically significant effects in all trials (means of 105.2 x 10'6and426.2x 10''', respectively). For AE 0852999 treated cultures concentration-related decreases were observed in relative survival to treatment and/or in relative population growth, starting at 750 I-lglml. Relevant AE 0852999 induced increases in mutant frequencies could not be found (ranging from 3.2 x 10'6 to 8.7 X 10'6 in the first trial, and from 0.4 x 10,6to9.7 x 10,6 in the secondtrial). In addition, the overall statistical analysis revealedno statistically significant increases. Therefore, AE 0852999 was evaluated as non-mutagenic in the nonactivationtrial. 

### C. MUTATION ASSAY WITHMETABOLIC ACTIVATION: Three trials were performed with S9 mix. In all experiments, cytotoxic effects were induced. The mutant frequencies ofthe negative controls (means of3.5 x 10,6,0.8 X 10-6and 0.8 x 10'6, respectively) and ofthe vehicle controls (means of3.6 x 10'6 , 1.2 X 10'6 and 2.0 x 10'6, respectively) were all within the normal range. Thepositive control DMBA induced clear mutagenic and statistically significant effects in all trials (means of 19.9 x 10'6,69.8 x 10'6and 45.0 x 10-6, respectively). The AE 0852999 treated cultures showed concentration-related decreases in relative survival to treatment

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### Cyprosulfamide
and/or in relative population growth. Relevant AE 0852999 induced increases in mutant frequencies could not be found (ranging from 3.4 x 10.6 

### to 13.7 x 10-6 in the first trial, from 0.7 x 10,6 to 3.9 x 10,6 in the second trial and from 1.5 x 10,6 to 2.1 X 

### 10,6 in the third trial). In addition, the overall statistical analysis revealed no statistically significant increases. Therefore, AE 0852999 was evaluated as nonmutagenic in the activation trial. 

### III. CONCLUSION Under both activation conditions, cytotoxic effects were induced. In addition, precipitation of AE 0852999 in the medium was observed in the main study under both activation conditions at 750 !-tg/m\.. No changes in osmolality orpH were observed. AE 0852999 induced no biologically relevant increases in mutantfrequencies The positive controls EMS and DMBA had a marked mutagenic effect. Based on these results, AE 0852999 was considered not to be mutagenic in the V79IHPRT forward mutation assay. (Herbold B., 

### 2006) 

### Oral28-daytoxicity in therat Report: KIIA5.8/08, Odin-Feurtet M., 2007 Title: AE 0852999, 28-daytoxicity study in the rat by dietary administration Report No & 

### SA06281 DocumentNo M-284847-01-2 

### Guidelines: OECD 407 (1995); EEC Directive 96/54IEC 

### Method B7 (1996); EPA Health Effects Test Guideline (OPPTS 870.3050; 2000) except neurotoxicological evaluation GLP Yes (certified laboratory) Executive summary: AE 0852999, a soil metabolite ofthe safener AE 0001789 (batch number NLL 7793-9: a white powder, 99.3% purity) was administered continuously via 

### the diet to groups ofWistar rats (to/sex/group) for 29 days at concentrations of40, 300, 800 and 3000 ppm. A similarly constituted group received untreated diet and acted as a control group. Animals were observed daily for mortality and clinical signs. Detailed physical examinations were performed weekly. Body weight and food consumption were recorded once weekly. Ophthalmological examinations were performed on all animals during the acclimatization phase and on all animals ofthe control and high dose groups during Week 4. Haematology, clinical chemistry and urine parameters were determined at the end ofthe study. AI! animals were necropsied, selected organs weighed and arange oftissues weretaken, fixed and examinedmicroscopically.

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No mortalities ortreatment-related clinical signs occurred duringthe study. At3000 ppm: Mean body weight was reduced between 5 and 13% in males and around 7.5% in females from Study Day 8to 29. The effect was statistically significant in males at each interval but only on the interval Day 1to Day 8 in females. Mean body weight gain/day was reduced between Study Day 1and 15, in males and between Study Day 1 and 8 in females. Between Study Days 1and 29, the cumulative body weight gain was reduced by 35% in males and by 26% in females. Mean food consumption was reduced by 11

### % between Study Day I and 8 in male and female animals. Between Study Days 15 and 22, mean food consumption was reduced by 17% in male animals. Haematological evaluation revealed lower mean erythrocyte counts, haemoglobin concentrations and haematocrit in both sexes, together with higher mean reticulocyte counts. Clinical chemistry evaluation revealed higher mean urea, creatinine and total cholesterol concentrations in both sexes. Additionally, triglycerides were higher in females only. Urinary analysis revealed higher urinary volume in both sexes (+42% in males and +360% in females). As a consequence, a lower mean refractive index was observed in females and lower ketone levels were observed in males. Microscopic examination of the urinary sediments revealed the presence of erythrocytes, leucocytes and epithelial cells in both sexes. In addition, unusual crystals (fme sticks often arranged in sheaves and/or needles, fan-shaped or clump-arranged) were observed in both sexes. Mean pH values were lower in males. This variation was associated with the observation oflower amounts of usual crystals in the sediments. At necropsy, mean terminal body weights were lower in both males and females when compared to controls. Mean absolute and relative kidney weights were statistically significantly higher when comparedto controls in males. At the histopathological examination, a minimal to marked obstructive nephropathy in the kidney was observed in 7110 

### males and in 5/10 females. The term obstructive nephropathy included different degenerative/regenerative lesions involving a granulomatous type of inflammation, with tubular dilatation proximal to the sites of obstruction. Intratubular crystal deposition and intrapelvic crystals were observed. An increase in incidence and severity ofpelvic epithelium hyperplasia, collecting duct hyperplasia and basophilic tubules was noted. Diffuse urothelial hyperplasia was observed in the urinary bladder in males (3110) and in females (1/10). Intraluminal crystals were observed in the urinary bladder in two males. Slight diffuse urothelial hyperplasia (1110) was observed in the ureter in onemale. At 800 ppm, no treatment-related clinical signs were observed in either sex. A reduction of 16-17% in cumulative mean body weight gain was observed in males during the flISt two weeks of the study. Unusual urinary crystals were observed in 2110 males and 4110 females. They were not associated with any clinical pathology changes or histopathological findings and were therefore considered as not adverse. Mean pH values were lower in males.

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### At 300 and40 ppm, no treatment-relatedeffects were observed in either sex. The No Observed Effect Level (NOEL) in this study was found to be 300 ppm in both sexes equating approximately to 22.8 mgikg body weight/day in males and 25.5 mgikg body weight/day in females, basedon reducedbody weight gain in males at 800 ppm. I. 

### MATERIALS AND METHODS A. MATERIALS: 1. 

### Test Material: AE 0852999 Description: white powder Lot/Batch: NLL 7793-9 Purity: 99.3 % CAS: No CAS entry yet Stabilityoftestcompound: Stable in rodent diet 100 and 20000 ppm over at least a 62-day period at ambient temperature and after 52 days frozen storage followed by 10 days at room temperature. Stable in rodent diet at 40 ppm over at least a 40-day period at ambient temperature and after 29 days frozen storage followed by II days at room temperature. 2. Vehicle and lorpositive control:None 3. 

### Test animals: Species: Rat Strain: WistarRj: WI (lOPS HAN) Age: 7weeks approximately Weightatdosing: 237 to 267 gforthemales

- 163 to 192 gforthe females Source: R. 

### Janvier, Le Genest StIsle, France Acclimation period: 6 days Diet: Certified rodent powdered and irradiated diet A04CPI-IO from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), adlibitum 

### exceptbefore urine and bloodcollection Water: Tapwater, adlibitum 

### except beforeurine collection Housing: Animals were caged individually in suspended stainless steel

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### wire mesh cages. Environmentalconditions

­ Temperature: 22 ±2°C Humidity: 55 ± 15% Airchanges: 10to 15 changesperhour Photoperiod: Alternating 12-hourlight and dark cycles (7 am- 7 pm) B. STUDY DESIGN: 

### 1. In life dates: September20 to October27,2006 at Bayer CropScience, Sophia Antipolis Cedex, France 2. Animal assignment and treatment There were 10 animals of each sex per dose group. Animals were assigned using computer randomization by weight. AE 0852999 was administered in the diet for at least 29 days to Wistarrats at the following doses

- 0, 40, 300, 800 and 3000 ppm (equating to approximately to 3,22.8, 59.5 and 219 mglkg/day 

### in males and 3.4,25.5,67.2 and 246 mglkglday in females). A negative control group received plain diet. Animal housing and husbandry were in accordance with the regulations of the Guide for the Care and Use ofLaboratory Animals (Public Health Service, National Institute of Health, NIH publication N°86-23, revised 1985) and "Le Guide du Journal Officiel des Communautes Europeennes L358, 18 Decembre 1986,

### N°86/609/CEE du 24 Novembre 1986".

3. Diet preparationand analysis AE 0852999 was ground to a fine powder before being incorporated into the diet by 

### dry mixing to provide the required concentrations. There were two preparations of each concentration for the whole study. The stability was demonstrated before and during the study. At 100 and 20000ppm, AE 0852999 was found to be stable in the diet over a 62-day period atroom temperature or a 52-day freezing period followed by 10 days at room temperature. At 40 ppm, AE 0852999 was found to be stable in the diet over a 40-day period at room temperature or a 29-day freezing period followed by 11 days at room temperature. The homogeneity of AE 0852999 in diet was verified on the first preparation at the lowest and highest concentrations to demonstrate adequate formulation procedures. The homogeneity in the diet ranged from 93 to 99% ofthe nominal concentrations. Mean values obtained from the homogeneity check were taken as measured concentration. The dietary levels ofthe test substance were verified

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for each concentration on each preparation and ranged from 93 to 99% of the nominal concentrations. All results werewithinthe in-house range of85 to 115%ofnominal concentration. TableI A . -5 I 58 

## s d . tudy eSign Test group Concentration in Dose per animal Animals assigned diet (ppm) (studv averages) Male Female Male Female (mWkg bw/day) (mWkg bw/day) 1 0 0 0 10 10 2 40 3.0 3.4 10 10 3 300 22.8 25.5 10 10 4 800 59.5 67.2 10 10 5 3000 219 246 10 Table IIA5.8-6 Homogeneity and concentration oftestmaterial Nominal concentrations Analysed concentrations 

### i % ofnominal concentration (ppm) (ppm) HI 39.0 98 40 H3 38.2 96 MI 37.7 94 Homogeneity M3 38.8 97 (first preparation) 81 37.6 94 83 37.3 93 HI 2927 98 3000 H3 2981 99 Ml 2949 98 M3 2932 98 81 2908 97 83 2897 97 40 38.1 95 Concentration 300 297 99 (first preparation) 800 789 99 3000 2932 98

40 37.2 93 Concentration 300 288 96 (second preparation) 800 785 98 3000 2935 98

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H surface; M middle; B = bottom ofthe preparation. Three different samples 1,2 and 3 were taken at the each level ofthepreparation (H, M, B); only twowere analyzed. 

### 4. Statistics

Data were analyzed by the Bartlett's test for homogeneity of variances. When the data were homogeneous, an ANOVA was performed followed by Dunnett's test on parameters showing a significant effect by ANOVA. When the data were not homogeneous even after transformation, a Kruskal-Wallis ANOVA was performed followed by the Dunn's test ifthe Kruskal-Wallis was significant. When one or more group variance(s) equaled 0, means were compared using nonparametric procedures. Group means were compared at the 5% and I % levels of significance. Statisticalanalyseswere carriedoutusing Path/Tox System V4.2.2. (Module Enhanced Statistics).



### C. METHODS: 1. Observations The animals were observed twice daily for moribundity and mortality (once daily on weekends or public holidays). Observed clinical signs were recorded at least once daily for all animals and detailedphysical examinations wereperformedat least weekly duringthetreatmentperiod. 

### 2. Body weight Body weights were recordedtwice during the acclimatization phase, on the first day oftest substance administration, then at weekly intervals throughout the treatment. Additionally, fasted animals were weighed beforenecropsy. 

### 3. Food consumption and compound intake Food consumptionwas recorded weeklyfor all animals during the treatmentperiod. The weekly mean achieved dosage intake in mglkglday for each week and for Weeks I to 4 was calculated for each sex using the formula: Test substance intake Dose level (ppm)x Group mean food consumption (glday) (mglkg/day) Group meanbodyweight (g) at end ofthe food consumption period

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### 4. Ophthalmologicalexamination During the acclimatization period all animals were subjected to an ophthalmological examination. After instillation of an atropinic agent (Mydriaticum, Merck Sharp and Dohme) each eye was examined by means of an indirect ophthalmoscope. During Week 4, all surviving animals from control and high dose groups were re-examined. 

### 5. Clinical chemistry On study days 30, 31 or 32, prior to necropsy, blood samples were taken from all animals in all groups by puncture ofthe retro-orbital venous plexus. Animals were diet fasted overnight prior to bleeding and anesthetized by inhalation of isoflurane. Blood was collected on EDTA for haematology, on lithium heparin for plasma and clot activator for serum for clinical chemistry and on sodium citrate for coagulation parameters.



### a. Haematology The following haematology parameters were assayed using an Advia 120: red blood cell count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin concentration, reticulocyte count, white blood cell count and differential count evaluation andplateletcount. A blood smear was prepared and stained with May-Grunwald-Giemsa method. It 

### was examined when results ofAdvia 120 determinations were abnormal. Prothrombin time was assayed on an ACL 3000.



### b. Clinicalchemistry Any significant change in the general appearance ofthe plasma and the serum was recorded. Total bilirubin, glucose, urea, creatinine, total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations and aspartate aminotransferase, alanine aminotransferase, alkalinephosphatase and gamma-glutamyltransferase activities were assayed on plasma samples, total protein and albumin concentrations were assayed on serum samples using an Advia 1650 (Bayer Diagnostics, Puteaux, France). Globulin and albumin/globulin ratio values were calculated. 

### 6. Urinalysis Overnight urine samples were collected on study Days 24 or 25 from all animals from all groups.

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Food and waterwere not accessible during urine collection. Any significant change in the general appearance ofthe urine was recorded. The urine volume was measured. pH was assayed using a Clinitek 200+ and Ames Multistix dipsticks (Bayer Diagnostics, Puteaux, France). Urinary refractive index was measured using a RFM320 refractometer (Bioblock Scientific, Illkirch, France). The following semi-quantitative parameters were assayed using a Clinitek 200+ and Multistix dipsticks: glucose, bilirubin, ketone bodies, occult blood, protein and urobilinogen. Microscopic examination ofthe urinary sediment was performed after centrifugation ofthe urine. The presence of red blood cells, white blood cells, epithelial cells,bacteria, casts and crystals was graded.


### 7. Sacrificeand pathology On study days 30, 31 or32, a complete necropsy was performed on all animals. Animals were deeply anaesthetized by inhalation oflsoflurane, then exsanguinated before necropsy. All animals were diet fasted prior to scheduled sacrifice. The necropsy includedthe examination ofall major organs, tissues andbody cavities. Macroscopic abnormalities wererecorded, sampled and examined microscopically. Adrenal gland, brain, epididymides, heart, kidney, liver, ovary, pituitary gland, prostate gland, spleen, testis, thymus, thyroid gland (with parathyroid gland) and uterus (including cervix) were weighed fresh at scheduled sacrifice. Paired organs were weighedtogether. The following organs or tissues were sampled: adrenal gland, aorta, articular surface (femoro-tibial), bone (sternum), bone marrow (sternum), brain, epididymis, oesophagus, exorbital (lachrymal) gland, eye and optic nerve, harderian gland, heart, intestine (duodenum, jejunum, ileum, caecum, colon, rectum), kidney, larynx/pharynx, liver, lung, lymph nodes (submaxillary, mesenteric), mammary gland, nasal cavities, ovary, pancreas, pituitary gland, prostate gland, sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, submaxillary (salivary) gland, testis, thymus, thyroid gland (with parathyroid), tongue, trachea, urinary bladder, uterus (with cervix), vagina. For sacrificed animals, a bone marrow smear was prepared from one femur, stained with May-Grunwald Giemsa, but not examined. Tissues samples were fixed by immersion in neutral buffered 10% formalin with the exception ofthe eye, opticnerve, harderian gland, epididymis andtestis that were fixed in Davidson'sfixative. Histopathological examinations were performed on all tissues from all the animals in the control and high dose groups and all decedents in all groups. The liver, kidney, lung, thyroid gland, urinary bladderand macroscopic findings were examined in all animals in the study. All histopathological examinations were performed by the Study Pathologist. Following the initial

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# ffiBayerCropScience 2007-03-28 Tier2, lIA, Sec. 3,Point5: Cyprosulfamide histopathological examination, a review of representative slides was performed by a reviewing pathologist. The diagnoses presented in this report represent the consensus opinion of the two pathologists. II. RESULTS AND DISCUSSION A. Mortality There were no mortalities during the courseofthe study. B. Clinical signs No treatment-related clinical signs were observed throughout the course ofthe study. The few clinical signs observed were considered not to be related to AE 0852999 administration. As these findings occurred as isolatedincidences andat the low andintermediate dose levels only, they are consideredtobe spontaneousfindings.



### C. Bodyweight

At 3000 ppm, mean body weight was reduced between 5 and 13% in males and around 7.5% in females from Study Day 8 to 29. The effect was statistically significant (p<O.OI) in males at each interval and only on the interval Day I to Day 8 (p<0.05) in females. Mean body weight gain/day was reduced between Study Days I and 15 in males and between Study Day 1and 8 in females. Between Study Days 1 and 29, the cumulative body weight gain was reduced by 35% in males and by 26% in females (statistically significantp<O.O 1). At 800 ppm, body weight in male and female animals was unaffected by treatment with AE 0852999. 

### In males, cumulative body weight gain was reduced by 16
- 17% (statistically significant p<O.O 1or p<0.05) between StudyDays I and 15. At 300 and 40 ppm, body weight and body weight gain in male and female animals were unaffected by treatmentwith AE0852999. D. Food consumption

At 3000 ppm, mean food consumption was reduced by 11% (p<O.05) between Study Day I and 8 inmale

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and female animals. Between Study Days 15 and 22, mean food consumption was reduced by 17% in male animals. At 800, 300 and 40 ppm, mean food consumption was unaffectedbytreatment in either sex. E. Achieved dosages The mean achieved dosage intake ofAE 0852999 expressed in mg/kg body weight/day per group was as follows: TablellA5.8-7: Mean achieved dietaryintakeofAE0852999 (Weeks 1-4)

Dietconcentrationin ppm Males mg/kglday Females mg/kglday 40 3.0 3.4 300 22.8 25.5 800 59.5 67.2 3000 219 246

F. Ophthalmologicalexamination There were notreatment-related ophthalmological findings. G. Clinicalpathology 

### 1. Haematology Lower mean erythrocyte counts, hemoglobin concentrations and hematocrit were noted at 3000 ppm in both sexes, togetherwithhighermean reticulocyte counts.

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TablelIA5.8-8: Haematoloaical parametersvariations (%chanl!ewhen compared to controls) • Dosage level ofAE 0852999 (ppm) 0 3000 0 3000 Sex Males Females Red bloodcell count (10 12/1) 8.65±O.35



# - 8.08±0.37

## ** 8.68±0.28 ! 8.02±0.55

## ** (-7%) 

## - (-8%) Hemoglobin concentration (gldl) l5.5±0.5 14.1±0.6

### **



## - (-9%) 15.6±0.4



# - 14.3±1.2 n (-8%) Hematocrit (111) 0.468±0.016



# - 0.433±0.017

### ** (-7%) 0.460±0.09



# - 0.424±0.025

## ** (-8%) Reticulocyte count absolute (1012/1) 0.21±0.03



## - 0.27±0.10 (+29%) 0.19±0.05



# - 0.26±0.08

## * (+37%) percentage (%) 2.4±0,4



## - 3,4±1,4 (+42%) 2.2±0.6



# - 3.3±1.2 (+50%) 

### *:**p~0.05** 

## ** :**p~O.Ol** 

### Notreatment-related change was noted at 800, 300 and 40 ppm for theparameters assayed. 2. Clinical chemistry Highermean urea, creatinine and total cholesterol concentrations were observed at 3000 ppm inboth sexes. Table IIA 5.8-9: Clinical chemistryparametervariations (%chanl!e when compared to controls) Dosage level ofAE 0852999 (ppm) 0 3000 0 

## I 3000 Sex Males Females Urea(mmolll) 4.72±O.64



# - 8.93±3.94

## * (+89%) 5.06±0.74



# - 7.97±3.31 (+58%) Creatinine (llmol/l) 49±1



## - 55±7 (+12%) 51±3



# - 61±14 (+20%) Total cholesterol (mmol/l) 1.49±0.23



# - 2.03±0.32

## ** (+36%) 1.62±0.23



# - 2.37±0.48

### ** (+46%) 

### *:**p~0.05** 

## ** :**p~O.Ol** 

### In addition, a tendency towards higher triglycerides concentrations was noted at 3000 ppm in females (+61%,**p~O.Ol).** 

### Other differences, even if statistically significant (total bilirubin in males at 3000 ppm), were

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### considered not to be relevant in view ofthe individual variation. No treatment-related change was noted at 800, 300 and 40 ppm forthe parameters assayed. 3. Urinalysis Higher mean urinary volumes were noted at 3000 ppm (+42%, p:S0.05 in 

### males and +360%, p:sD.OI in females, relative to the controls) with lower ketone levels in males and lower mean refractive index in females. The microscopic examination of the sediments revealed the presence of erythrocytes (confirming the semi-quantitative chemical evaluation), leucocytes and epithelial cells in most ofthe samples from both sexes at 3000 ppm. In addition, unusual crystals (fme sticks often arranged in sheaves and/or fine needles, fan-shaped or clump-arranged) were observed at 3000 ppm in all males (grade 2 to 4) and 6 out of9 females (grade I to2) and at 800 ppm in 2 out of 10 males and 4 out of 10 females (grade I). It 

### is considered that this is may be parent or metabolites ofparent, but due tothe small amount ofthese crystals in urine it was not possible to analyze them. Mean pH values were lower in males at 3000 and 800 ppm (relative to the controls, -16% and -II% respectively, p:SO.OI). At 3000 ppm, this variation was associated with the observation of lower amounts of usual crystals in the sediments and a cloudy appearance was noted for 4 out of 10 samples. The statistically significant difference noted in urinary volumes in males at 40 ppm was considered to be incidental. Notreatment-related change was noted at 300 and 40 ppm for the parameters assayed. H. Post-mortem examination 1. 

### Organ weights Mean terminal body weights were lower in both males (-13%, p<O.OI) and females (-6%, not statistically significant) at 3000 ppm, when comparedto controls. Mean absolute and relative kidney weights were higher in both sexes at 3000 ppm (statistically significant in males). Kidney weight changes were correlated with gross and histopathological changes.

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TableIIA5.8-10: Kidneyweightchanges atterminal sacrifice (% changewhen compared to controls) Sex Male Female Dose level of 0 40 300 800 3000 0 40 300 800 3000 AE 0852999 (ppm) Mean absolute kidney weight 2.63 2.69 (+2%) 2.63 (0%) 2.71 (+3%) 3.23* (+23%) 1.66 1.60 (-4%) 1.68 (+1%) 1.71 (+3%) 2.00 (+20%) Mean kidney to body weight ratio 0.677 0.689 (+2%) 0.679 (0%) 0.726 (+7%) 0.957** (+41%) 0.750 0.726 (-3%) 0.768 (+2%) 0.780 (+4%) 0.968 (+29%) Meankidney to brain weight ratio 128.040 129.751 (+1%) 128.292 (0%) 134.171 (+5%) 158.855** (+24%) 88.282 82.775 (-6%) 87.822 (-1%) 88.830 (+1%) 107.586 (+22%) *: p:;:0.05, **:**p~.OI** 

### All other organ weight differences were considered to be incidental since they were not associated with significanthistopathological fmdings. 

### 2. Gross pathology Tenninal sacrifice: Treatment-related changes (enlargement, irregular surface, gritty content andlor mottled surface) were observed in both sexes in a proportion ofthe kidneys at 3000ppm. Table IIA5.8-11: Incidence ofmacroscopic changes in the kidney

- terminal sacrifice Sex Male Female Dose level of AE 0852999 (ppm) 0 40 300 800 3000 0 40 300 800 3000 Enlarged 0110 1110 0110 0110 6/10 0110 0110 0110 OlIO 4110 Irregular surface 0110 0110 0110 0110 4110 0110 0110 0110 OlIO 3110

Gritty content 0/10 0/10 0/10 0110 7110 0/10 0110 OliO OlIO 2110 Mottled OlIO 0110 0/10 0110 7110 0110 0/10 0110 0110 5110 

### In addition, stones (2110) and gritty content (2/10) 

### were found in the urinary bladder in males and dilatation ofthe ureter was found in 2/10 males at 3000 ppm. Other changes were considered as incidental and nottreatment-related. 

### 3. Microscopic pathology At tenninal sacrifice, treatment-related changes were observed in the kidney, urinary bladder and

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ureter at 3000ppm. Notreatment-relatedeffects were seen at40,300and 800 ppm. Treatment-related changes observed at 3 000 ppm were consideredtobe adverse and associated with clearclinical pathological changes. A minimal to marked obstructive nephropathy was observed in the kidney in 

### 7110 males and in 

### 5/10 females. This change was induced by the deposition of intratubular crystals. The term obstructive nephropathy included different degenerativelregenerative lesions involving a granulomatous type of inflammation, with tubulardilatation proximal to the sites ofobstruction. Intrapelvic crystalswere observed. These changes were associated with an increase in incidence and severity of pelvic epithelium hyperplasia, collectingducthyperplasia andbasophilic tubules. Table IIA 5.8-12: Incidence and/orseverity ofmicroscopic changesin thekidney

- terminal sacrifice



### I !
I 

## I
I 

## i

I

I I Sex Male Female Dose level ofAE 0852999 (ppm) 0 40 300 800 3000 0 40 300 800 3000 Numberofanimals examined 

### 10 10 10 10 

### 10 10 10 10 10 10 ntratubularcrystals 

### TOTAL 0 0 0 0 6 0 0 0 0 

### 5 ntrapelvic crystals 

### TOTAL 0 0 0 0 3 0 0 0 0 2 Obstructivenephropathy Minimal 0 0 0 0 2 0 0 0 0 0

Slight 0 0 0 0 5 0 0 0 0 3 i Moderate 0 0 0 0 0 0 0 0 0 I Marked 0 0 0 0 0 0 0 0 0 

### 1 

### TOTAL 0 0 0 0 7 0 0 0 0 

### 5 I Pelvic epithelium hyperplasia: focallmultifocal i Minimal 2 2 

### 1 1 3 0 

### 1 0 1 0

Slight 

### 1 0 0 0 6 1 0 0 0 4 Moderate 0 0 0 0 1 0 0 0 0 2 I 

### TOTAL 3 2 

### 1 1 10 1 1 0 1 6 rollectingductshyperplasia: bilateral Minimal 0 0 0 0 5 0 0 0 0 0

Slight 0 0 0 0 3 0 0 0 0 4 Moderate 0 0 0 0 0 0 0 0 0 

### 1

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TOTAL 

### 0 0 0 0 8 

### 0 0 0 0 

### 5 !Basophilictubules focaVmultifocal Minimal 0 5 1 0 3 0 1 0 

### 1 0

Slight 0 0 0 0 5 0 0 0 0 3 Moderate 0 0 0 0 

### 1 0 0 0 0 2 TOTAL 

### 0 

### 5 1 

### 0 9 

### 0 1 

### 0 1 

### 5 Diffuse urothelial hyperplasia was observed in the urinary bladder in males (3/10) and in female (1/10). Intraluminal crystals were observed in the urinary bladder in two males. Table llA5.8-13: Incidence and severity ofmicroscopic cban2esin tbe urinal)' bladder

- terminal sacrifice Incidence and severity ofmicroscopic changes in the urinary bladder

- terminal sacrifice Sex Male Female Dose level ofAE 0 40 300 800 3000 0 40 300 800 0852999 (ppm) Numberofanimals 

### to to to 10 10 

### to 10 10 

### to examined ntraluminal crystals TOTAL 

### 0 0 0 

### 0 2 0 

### 0 0 

### 0 Urothelial hyperplasia: diffuse Minimal 0 0 0 0 2 0 0 0 0

Slight 0 0 0 0 I 0 0 0 0 TOTAL 

### 0 0 0 

### 0 3 0 0 0 

### 0 3000 10


### 0
1 0 

### 1 Slightdiffuse urothelial hyperplasiawas observedin oneofthetwo sampled ureters inmales. III. CONCLUSION In conclusion, the No Observed Effect Level (NOEL) ofAE 0852999, when administered via the diet to the Wistar rat for at least 28 days, was 300 ppm in males and females (equating to approximately 22.8 mglkglday and 25.5 mglkglday in males and females, respectively). (Odin-Feurtet M., 2007)

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### IIA5.9 Medical data
IIA 5.9.1 Reporton medical surveillanceon manufacturing plant personnel So far only two sample production campaigns have been perfonned with AE 0001789 in 2003 and 2005, a third campaign is ongoing. During these campaigns no untoward effects in the workers have been observed. Medical surveillance, not directly related tothe campaigns, did not show any effects. 

### IIA 5.9.2 Reporton clinical cases and poisoningincidents Nopoisoning incidents or clinicalcases have occurred. 

### IIA 5.9.3 Observationson general populationexposure and epidemiological studies So far therehas been noexposure ofthe general public. 

### IIA5.9.4 Clinical signs and symptoms ofpoisoning and details ofclinical tests There are no experiences withhuman poisonings, animal experiments did not show any symptoms. 

### IIA5.9.5 Firstaid measures and therapeutic regimes Remove patient from exposure/tenninate exposure. Thorough skin decontamination with copious amounts of water and soap. Flushing ofthe eyes with lukewann water for 15 minutes. Iinduction of vomiting is not indicated. 

### IIA 5.9.6 Therapeutic regimes Gastric lavage is not required in regard ofthe very low acute toxicity ofthe compound. However, the application ofactivatedcharcoal and sodium sulphate is always advisable in significant ingestions. As there is no antidote, treatmenthas tobe symptomatic and supportive. 

### IIA 5.9.7 Expected effects & 

### duration ofpoisoning as a function ofexposure There are noexperiences with human poisonings, animal experiments did not show any symptoms. 

### IIA5.9.8 Effects & 

### duration ofpoisoning as a function oftime There are no experiences with humanpoisonings, animal experiments didnot show any symptoms 

### IIA5.9.9 Dermalpenetration

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A dermal penetration study withthe plantprotection product has been conducted and is reported in Annex IlIAI point 7.6. 

### IIA5.10 Other/special studies Report: KIIA 5.10/01, Leake, 

## c.R., BDrkle, 

### Lo, Lasserre-Bigot, D., Sowig, P. and Cbapple, A. 2007 Title: 

### The Non-Relevance of the Environmental Metabolites of Cyprosulfamide (AE 0001789) Report No & 

### DocumentNo M-285709-01-1 Guidelines: EU DG SANCO/22112000-rev.l0, 25th February2003 GLP Not applicable Executivesummary: The behaviour of cyprosulfamide in various environmental compartments is well described by a wide variety ofstudies. The potential relevance ofthe environmental metabolites has been investigated with a combination of environmental fate studies followed by environmental exposure studies in which predicted environmental concentrations have been calculated. For those metabolites which under certain application scenarios have the potential to reach groundwater, biological activity testing followed by genotoxicity testing and subchronic testing has been conducted. In addition ecotoxicological tests with three aquatic organisms, 

### Daphnia magna, Algae and Aquatic plants 

### (Lemna gibba) and a terrestrial organism

- Earthworm 

### (Eiseniafetida) have been conducted. All these results have been combined in ajoint summary. Summaryofnon-relevance oftbemetabolites ofCyprosulfamide

• The degradation ofcyprosulfamide under aerobic soil conditions is rapid with half-life values in the range of2.3to 10.6 days at 20°C. This degradation results in the formation offour major metabolites MOl (AE 0893016), M02 (AE 0852999), M03 (AE 0467398) and M04 (AE 1448796). In addition M05 (AE 1272799) was formed by photolysis and M06 (AE 2300015) can be postulatedto bepresent.

• Under sterile aqueous conditions cyprosulfamide is hydrolytically stable but in natural waters is slowly partitioned from waterto sediment with a DT50 

### ofaround 80-95 days and an overall degradation half-life in the range of 80-160 days. Two major metabolites are present in the water MOl (AE 0893016) and M02 (AE 0852999). In addition M07 (AE 0854787) and M08

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### (AE 2158927)were fonned by aqueousphotolysis.

• A groundwater leaching assessment has been conducted using two models and all the metabolites have been shown to have concentrations below 0.1 fl/L 

### with the exception ofM03 (AE0467398), which could reach up to 2.6 flglL with the maximum application rate of 100 g cyprosulfamide/ha.

• Both M03 (AE 0467398) and M02 (AE 0852999) have been shown to have (in comparison with the parent cyprosulfamide) no herbicide safening properties and no plant growth regulatory properties.

• Neither M03 (AE0467398) norM02 (AE 0852999)were found tobe genotoxic in threetests.

• A subchronic 90 day toxicity studies with M03 (AE 0467398) gave NOEL values of 1094 and 888 mglkglday for females and males respectively, and a subchronic 28-day toxicity study with M02 (AE 0852999) gave NOEL values of 25.5 and 22.8 mglkglday for females and males respectively, with a similartoxicological profile as AE 0001789.

• Conducting a consumer risk assessment for the M03 (AE0467398) and M02 (AE 0852999) for the extreme worst case conditions ofthe pore water at 1 m depth being taken unprocessed as drinking water showed that Margins of Safety (MOS) were greater than ca. 5,000,000 for M03 (AE 0467398) and greater than ca. 330,000 for M02 (AE 0852999). In addition a dietary risk assessment for both metabolites showed the % 

### of the Acceptable Daily Intake (ADI) wouldbe less than 0.1 %.



### • The metabolites present in soil including M03 (AE 0467398) and M02 (AE 0852999) were assessed for their toxicity to earthwonns (EiseniaJetida), 

### Daphnia (Daphnia magna), 

### Green algae (Pseudokirchneriellasubcapitata) 

### and an aquatic plant duck weed (Lemna gibba) 

### and all were found to be non-relevant in the terrestrial environmental compartment and should they run-off or leach to the aquatic compartment were found to be non-relevant in the aquatic environment. In summary the metabolites M3 (AE 0456398) and M2 (AE 0852999) of cyprosulfamide are biologically, toxicologically and eco-toxicologically non relevant. In addition the othermetabolites found in the terrestrial and aquatic compartments have also been shown to be eco-toxicologically non-relevant. It 

### can be concluded that even under the worst case conditions taking conservative approaches to

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### the risk assessment cyprosulfamidecan be safely used on maize.


### IIA5.11 Summaryofmammalian toxicityand overallevaluation
• Absorption,distribution,excretion and metabolism The biokinetic behaviour and metabolism ofAE 0001789 in the rat was investigated following a single oral dose to male and female animals, employing a low dose level of2 mglkg b.w. and a high dose of200 mg/kg b.w. and using two different label positions with 14C 

### uniformly labeled in the sulfonylbenzamide andthe methoxy benzoyl ring. The gastro-intestinal absorption ofAE 0001789 started immediately after dosing. The maximum plasma levels were reached already after 10
- 60 minutes. The absorption was complete at the low dose (2 mglkg b.w.) in both sexes; between 82 

### % (methoxy benzoyl label) oreven more than 90 

### % (sulfonylbenzamide label) ofthe dose recovered was renally excreted or remained in the body at sacrifice, 96 hours after dosing (without GIT). In the high dose group, a certain degree of saturation ofthe absorption process could be observed as shown by the maximum dose-normalized plasma levels. They were lower in the high-dose than in the low-dose animals and the faecal excretion was increased in the high-dose animals by a factor of2 - 3 comparedto the low-dose animals. The excretion was fast and almost complete after 72 hours as 82
- 84 % 

### of the administered dose (corresponding to 98 % 

### ofthe recovered radioactivity) was excreted 72 hours after administration. The predominant route ofexcretion was via urine, accounting for 69 to 90 

### % ofthe dose administered. There was an insignificant quantity expired as radiolabeled carbon dioxide or organic volatile. Excretion was complete at the end of the studies, 96 hour after administration. After sacrifice the sum of the total residues in organs, tissues and the carcass amounted to:50.06 

### % ofthe administered radioactivity. The distribution of AE 0001789-related radioactivity within the body was uniform. The quantity of radioactivity in all organs and tissues was low compared to administered dose and always lower than the level in blood, indicating no tendency ofbioaccumulation. A particular concentration ofradioactivity in certain organs and tissues was not observed. The highest residue levels were detected in kidney (renal medulla) acting as main organ for excretion. Therefore, 

### it can be concludedthat residues ofAE 0001789 did not accumulate in any of the organs, tissues or glands. As observed in the whole body autoradiography studies particularly residues from glandular organs or tissues responsible for hormonal regulation (such as adrenal, testis, orthyroid gland) were rapidly depleted in parallel with depletion from the otherorgans and tissues. A delayed gastric emptying was observed particularly with some ofthe high-dose animals, resulting in a double peak in the plasma concentration-time curves and in a late excretion ofsignificant portions ofthe administered dose between 24 and 72 hours after the administration. These double peak phenomena in plasma profiles already have been described in the pharmacokinetic literature as effects caused by discontinuous absorption andlordelayed gastric emptying ofaportion ofthe dose. From the metabolites identified in urine and faeces three metabolic steps can be concluded:.

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# EBBayerCropScience 2007-03-28 Tier2, llA, Sec. 3, Point5: Cyprosulfamide (4) Elimination of the cyclopropylamine moiety by hydrolysis of carboxamide bond in the sulfonylbenzamide partforming AE 000I789-descyclopropylamino. (5) Desmethylation ofthe methoxybenzoylmoiety leadingto AE 0001789-desmethyl (6) Hydrolytic cleavageof carboxamide bond in the methoxybenzoyl group of the parent molecule resulting in AE 0001 789-cyclopropyl-sulfamoylbenzamide andAE000I789-anisic acid. Thesemetabolic reactions result in the formation ofthe following metabolites • M04, AE 1448796 (AE 0001789-desmethyl) • MOl, AE0893016 (AE0001 789-descyclopropylamino) • M02, AE0852999 (AE0001 789-cyclopropyl-sulfamoylbenzarnide) • M07, AE0854787 (AE 0001789-anisic acid) • M06, AE 2300015 (cycIopropylamine (postulated») Sex specific differences ofabsorption, distribution, metabolism and excretion ofAE 0001789 were not observed. The proposed metabolic pathway ofAE 0001789 in the rat composed from the partial pathways from each ofthe different label positions is shown in Figure 5.1-0I. Figure5.11-01: Proposed metabolic pathwayofAE0001789 in the rat.

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H,C, 0 0 

### o 0**~//**

# ~HN"'S'0y*~ 

# V· A NH 

# V AE 2300015 AE 0001789 (a.i.) 0 '" CYCloproPYlami'/

### ~ H,C, 0 0

# &?~OH o AE 0893016 "AE 0001789-descycloproPylamino"

# 2lH:~~~ oV
AE 1448796 "AE 0001789-desmethyl"

AE0854787 AE 0852999 "AE0001789-anisic acid" "AE0001789·cyclopropyl-sulfamoylbenzamide" 

### * denotes the two label positions postulated, notobserved asthe radiolabel was outside this group 

# [ ]

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• Acutetoxicity, local tolerance and sensitisation The acute toxicity ofAE 

### 0001789 (97.4% ofpurity) was low for all routes evaluated (oral, dermal and inhalation). The oral LD50 cut-off per OECD guidance for rats was greater than S 

### 000 mglkg and no mortality, clinical signs or abnormalities at necropsy were reported at 

### 2000 mglkg. The rat acute dermal LDso was> 

### 2000 mglkg body weight. A dermal dose of

### 2000 mglkg body weight was tolerated by male and female rats without mortalities, clinical signs, effects on weight development or gross pathological findings. The rat acute inhalation LCso (4-hour) was> 3S 13 mglm3, which was the highest achievable concentration and did not cause mortality, effects on body weight evolution or findings at necropsy. Although male rats tolerated the exposure without specific signs, piloerection and ungroomed hair-coat wererecorded in females on days I and lor2. AE 

### 0001789 was not an irritantto rabbit skin and caused only redness ofthe conjunctivae which reversed within 72 hours. No evidence ofskin sensitisation (delayed contact hypersensitivity) was seen in a guinea pigMagnusson and Kligman maximisationtest. TablellA5.11-2:SummaryofacutetoxicitydataforAE0001789

Type ofstudy 

# ISpecies Results OECDClassification (proposed) EPA Classification 

## I(proposed) Oral route M-249800-01-2 Rat No mortalities observed at 2000 mglkg Category 5 I Unclassified Category III

### ~:~) cut off >= 5000.(LDSO >=2000 mglkg) Dermalroute M-247221-01-2 Rat LDSO > 2 000 mglkg. ICategory 5 / Unclassified Category III Inhalation M-076020-02-2 Rat LC50 at 4 hours> 3513 Category 5 /Unclassified mg/m3 • Category IV Primary skin irritation M-129076-01-2 Rabbit Non irritating Category 5 I Unclassified Category IV

· Eye irritation

M-258984-01-2 Rabbit Redness of the conjunctivae Category5 IUnclassified.C III • ategory Skin sensitization M-249757-01-2 Guineapig Not sensitising
.Category 5 / Unclassified IN A I' bl • ot pp Ica e

• Shortterm toxicity The main target organs observed after short term exposure in rats and dogs were the kidney and the urinary bladder. The effects observed were consistent with the formation ofstones in the urinary tract. No effects in the urinary tract were observed in themouse after shortterm exposure. In rats, continuous dietary administration for 

### 90 days at the high dose of 

### 12 000 ppm inducedmortalities

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in 2 females during week 5. Body weight gain decrease was most pronounced between weeks 4 and 5 in both sexes with reduced food consumption. Urinalysis revealed sulfonamide-like crystals, radially striated sperical structures, in the urine ofboth sexes. Higher absolute and relative kidney weights were observed at tenninal sacrifice in both sexes. Slight centrilobular hepatocyte hypertrophy and thymus involution were observed in males. Hyperplasia ofthe urothelium in the urinary bladder, ofcollecting ducts and pelvic epithelium in the kidneys and increased incidence and/or severity of basophilic tubules in the kidneys were observed in both sexes. In the spleen brown pigment in the red pulp was seen in males and females at 12000ppm. Sulfonamide-like crystals andhyperplasia ofthe urothelium in the urinary bladder were also seen in both sexes at4000 ppm. The NOEL in the Wistar rat when administered AE 0001789 in the diet over a 90-day period was 1000 ppm (equivalent to 58 mglkglday in males and 70 mg/kglday in females). The mouse was less sensitive than the rat. The only treatment-related finding observed at 7 000 ppm was an increased incidence and severity oflymphocytolysis in the thymus offemale mice only. The NOEL was 2000 ppm in females (equivalent to 398 mglkglday) and 7000 ppm in males (equivalent to 1110 mglkg/day). In the dog. dietary administration of AE 0001789 for 90 days at 15000 ppm caused mortality in one female due to kidney calculi and abscessation. Body weight loss was observed in the other females and reduced food consumption was obtained in both sexes. Elevated urea nitrogen and serum creatinine was observed in both sexes, whereas elevated serum phosphorus was only seen in females. Treatment-related urinalysis findings were: elevatedurine volume in males, a decrease in urine specific gravity in males and females, decreased pH in females. Calculi in the renal pelvis, ureter and/or urinary bladder were seen in most animals. Correlated microscopic changes of the urinary tract were inflammatory changes, renal pelvis epithelium hyperplasia and necrosis and associated changes in the ureters and urinary bladder of males and females. At 7 500 ppm the only treatment-related finding was a decrease in urine specific gravity in females. TheNOAEL for this study was 7 500 ppm (equivalentto 221 mglkglday for males and females) as there were no microscopic pathology observations to correlate with the decrease in urine specific gravity in the 7 500ppm females. Dietary administration of AE 0001789 for a year at 8000 ppm caused macroscopic and microscopic fmdings in the kidney and the urinary bladder in both sexes. Like in the previous dog studies orin the 90­ day rat study, the findings (haemorrhages, inflammation and hyperplasia) were consistent with the fonnation ofstones in the urinary tract. The NOEL for this study was 2500 ppm (equivalentto 66 and 67 mglkglday for males and females, respectively).

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ofAE 0001789 Table5.11-3: Summaryofsbort-termtoxicit Type ofstudy Doses NOAEUNOEL ppm mglkg/d LOAEL ppm mglkg/d Treatment-related effects 90-dayrat feeding study M-105691-01-2 (0, 1000,4000& 12000 ppm) 1000 58170 (MIF) 4000 240/281 (MIF) Hyperplasia in the urinary bladder
90-day mouse feeding study M-104791-01-2 (0,500,2000 & 7000 ppm) 7000 (M) 2000

(F) 1110 (M) 398 (F) >7000 (M) 7000

(F) >1110 (M) 1297 (F) Lymphocytolysis in thymus offemales only
90-day dog feeding study M-263605-0I-I 0,2500, 7500 & 15000 ppm) 7500 221/221 (MIF) 15 000 416/341 (MIF) Calculi in the urinary tract. inflammatory changes, hyperplasia and necrosis in ureters and urinary bladder I year dog feeding study M-283I94-01-1 (0, 1000,2500& 8000 ppm) 2500 66/67 (MIF) 8000 2261242 (MIF) Calculi in the urinary tract, inflammatory changes. hyperplasia and haemorrhages

• Genotoxicity AE 0001789 was tested in a standard battery ofgenotoxicity and mutagenicity tests in 

### vitro and 

### in vivo carried out according to the current OECD EU, USEPA and Japanese MAFF testing guidelines. There was no indication ofgene mutation either in the presence or absence ofmetabolic activation in both the bacterial reverse mutation and mammalian gene mutation tests. The 

### in vitro chromosome aberration test and the 

### in vivo mouse micronucleus test were also both negative. These studies demonstrate that AE 0001789 has nogenotoxicpotential. Table5.11-4 Summaryofe:enotoxicitytest 

# I Mutagenicitytests with AE0001789 Results I I A. 

### In vitro tests I MetabolicActivation I 

## I Ames Test I +1­ Negative I : M-080265-02-2 i
i Ames Test (onnewtechnical) +1­ 

# JNegative i I M-283435-01-2 

## I Chromosome aberrations (V79 cells) i +1­ 

# i Negative

M-129315-02-2 

## I

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I 

## I ·HPRTTest (V79 cells)

M-266527-01-2 

### +1­ 

# i Negative

i 

### B. In vivo tests Dose levels I



### Micronucleus Test in mice

- oral administration M-077491-02-2 500, 1000 & 

### 2000 mglkg 

## I Negative 

## I I 

## I

• Long-term toxicity and carcinogenicity After long term exposure, treatment-related nephropathy was observed in both rats and mice at high doses. The presence ofstones in the urinary tract induced hyperplastic and inflammatory changes in the kidney, urinary bladderand/orureters. It was demonstratedthat these stones were mainly formed from the parent compound, which is poorly metabolized and precipitates inthe urine athigh doses. AE 0001789 was administered to groups of 60 male and 60 female Wistarrats by continuous dietary treatment at 1000,4000 and 8000 ppm, correspondingto 39, 159 and 321 mglkgJday in males and 56, 220 and 447 mglkgJday in females, respectively, overa 24-month period. Additionally, groups of 10 male and 10 female rats were treated at 200, 1000,4000 and 8000 ppm AE 0001789, corresponding to 9, 45, 181 and 364 

### mglkg/day in males and 13,62,249 and 491 mglkgJday in females, over a 12-month period to investigate chronic toxicity only. The mortality rate was higher in females at 8000 ppm after24 months oftreatment andwas largely due to secondary treatment-related nephropathy following product administration. Mean cumulative body weight gain was reduced during the first week oftreatment by 7 and 12% in males and females treated at 8000 ppm, respectively, compared to the controls. Urinalysis revealed the presence ofsulfonamide-like crystalsthroughoutthe study in both sexes, the effect beingmore pronounced in females than in males. At the 12-month interim sacrifice, treatment-relatednon-neoplastic findings were seen microscopically in the kidney and the urinary bladder. At the carcinogenicity phase (24-month) sacrifice, treatment-related effects were found in the urinary tract, i.e. kidney, urinary bladder, and ureters. These changes were due to treatment-induced nephropathy, characterized in the kidney by a combination of hyperplastic and inflammatory changes associated with the presence of stones. Urothelial hyperplasia was noted in the urinary bladder in both sexes at the 12-month interim sacrifice, whereas intraluminal stones and multifocal/diffuse urothelial hyperplasia were also observed in the urinary bladder and in a number of ureters in both sexes at the carcinogenicity phase. A slight higher incidence ofthe commonly occurring lesion diffuse bilateral tubular degeneration ofthe testis and bilateral oligospermia ofthe epididymis was observed atthe carcinogenicity phase. Neoplastic changes comprisedofa high dose-relatedtransitional cell carcinoma in the kidney ofone male and a transitional cell carcinoma in the urinary bladder ofone female. These findings, seen only at 8000 ppm, were considered to be secondary to the combination of hyperplastic and inflammatory changes

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associated withthepresence ofstones. In conclusion, dietary administration ofAE 0001789 over a 24-month period to the Wistar rat, at dose levels up to 8000ppm (corresponding to 321 mglkglday in males and 447 mglkglday in females), produced a transitional cell carcinoma in the kidney ofone male and a transitional cell carcinoma in the urinary bladder ofone female. The incidence ofthese tumours was very low and was considered to be secondary to the chronic hyperplastic changes resulting from chronic irritation due to the presence of stones. Thus, AE 0001789 was considered notto 

### be directly carcinogenic in the rat. The No Observed Adverse Effect Level (NOAEL) over a 12-month period ofdietary administration with AE 0001789 to the Wistar rat was 4000 ppm in both sexes (equivalent to 181 mglkg body weight/day in males and 249 mglkgbody weight/day in females). The NOAEL over a 24-month period ofdietary administration with AE 0001789 to the Wistar rat was 1000 ppm in both sexes (equivalentto 39 mglkglday in males and 56 mglkglday in females). Groups of60 male and 60 female C57BLl6J mice were initially intended to be fed diet containing0,350, 2000 or 7000 ppm of AE 0001789 for at least 78 weeks. Since the Maximum Tolerated Dose was exceeded in females at 7000 ppm during the first weeks oftreatment, an additional treatment group (60 animals/sex) at 3500 ppm was added to the study approximately one month after the start oftreatment. The high rate of mortality observed in both sexes at 7000 ppm led to the early termination of this treatment group, with all surviving females being sacrificed during Week 6 (study Day 40) and all males being sacrificed during Week46 (study Day 320). The mortality incidence in males was higher at 3500 ppm than in the control group after 12 and 18 months of treatment, largely due to the presence of stones within the urinary tract, causing acute or chronic renal failure ordue to secondary treatment-related nephropathy following product administration. Atthe 18-month haematology determination, atendency towards lower red blood cell count was noted in both sexes. This change was associated with slightly lower haemoglobin concentration, haematocrit and mean corpuscular haemoglobin concentration and slightly higher mean corpuscular volume. Histopathological treatment-related changes were mainly attributed to the presence ofurinary stones orto treatment-relatednephropathy. These changes were located in the urinary bladder and to a lesserextent in the kidney at the 12-month interim sacrifice. At the carcinogenicity phase (l8-month) sacrifice, changes due to treatment-related nephropathy were seen in the kidney, urinary bladder, prostatic urethra and ureters. In the liver, minimal to slight centrilobular to panlobular hepatocellular hypertrophy and an increased incidence ofhepatocellular single cell necrosis were observed in males. All the effects seen in the urinary tract were consideredto be dueto a chronic irritative mechanism, resulting from the formation of stones or crystals. In addition, indirect treatment-related fmdings were observed in the lymphoid

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### system in males (apoptotic bodies in the spleen and mesenteric lymph nodes, and lymphoid atrophy in the spleen) and were considered to be secondary to the stress induced by the stone deposition and/or the treatment-related nephropathy. In the heart, a higher incidence of epicardial mixed cell infiltrates was noted in 5/50 males at 3500 ppm, when compared to no cases in controls. This minimal change only observed in males was consideredto 

### be an indirect and non-adverseresponse to the treatment. Neoplastic changes comprised transitional cell papilloma in the urinary bladder of2/49 females at 3500 ppm. This finding was consideredtobe secondary to the chronic hyperplastic changes due tothe presence ofcalculi. Analysis ofurinary bladder stones taken from one male and one female at terminal sacrifice revealedthat the composition ofthe stones consisted ofapproximately 90 to 95% oftest material. In conclusion, dietary administration ofAE 0001789 over an 18-monthperiodto the C57BLl6J mouse, at dose levels up to 3500 ppm (corresponding to 506 mglkglday in males and 616 mglkglday in females), produced transitional cell papilloma in the urinary bladder of females. The incidence ofthese tumours was very low and was considered to be secondary to the chronic hyperplastic changes resulting from chronic irritation due to the presence ofcalculi. Thus AE 0001789 was considered not to be directly carcinogenic in the mouse. TheNo Observed Effect Level (NOEL) was 350 ppm for males (equivalent to 50 mglkglday) and2000 ppm for females (equivalentto 354 mg/kglday). Tab)e 511

# - 5: 

### Summaryofl

### ong-

### term t

### oXlcnYlcarcmogemcuy . 't / "t WI 'th 

### Typeofstudy NOAEL LOAEL Effects \ 

### Doses mg/kg/day mglkg/day Sulfonamide like crystals in Rat \04-week Chronic Toxicity/ 

### urine Oncogenicity 

### 39/56 159/220 M-281767-01-2 

### Treatment-related nephropathy (M/F) (MIF) 200, 1000,4000 & 8000ppm 

### in the kidney and urinary bladder Mouse 

## - 78 week- Chronic! 

### Treatment-related Oncogenicity 

### 50/354 287/616 nephropathy M-281276-01-2 (M/F) (M/F) 

### in the urinary tract 350, 2000, 3500 & 7000ppm Hepatocellularhypertrophy



### • Reproductive andembryonic toxicity Histopathological findings due to the precipitation offoreign material were also observed in the urinary tract of animals treated at the high doses in the rat 2-generation study. In this study, AE 0001789 was administered to groups of25 male and 25 female rats at concentrations of0 (control), 480, 2400 and

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12000ppm in their diet. At 12000ppm, the principal findings were as follows. Mortality was increased in FO and Fl females. Body weight gain was decreased in FI males and females during the premating phase and in FO and Fl females during gestation and lactation. Kidneys of Fl females were found to be discoloured, enlarged and/or changed at their surface. Histopathological findings due to precipitates of foreign material were seen in the kidney, ureters and/or urinary bladder. The spleen was also a target organ for males and females with histopathological fmdings and absolute and relative spleen weight increases. Changes were also noted in females only in the adrenal cortex, ovaries, uterus, vagina and liver. FO females exhibited a reduced rearing index due to maternal death during lactation. There was areduced viability index in Fl pups and a reduced lactation index in Fl and F2 pups. More pups showing clinical findings (paleness, cold to touch, missing milk spot, thin) were evident. The pup/litter weights were depressed at 12000 ppm. Due to depressed body weight, vaginal opening in Fl females was delayed. Ano-genital distance measurements in F2 pups did not indicate any treatment-related effect. At 2400 ppm, treatment-related changes were limited to histopathological changes in the kidneys, urinary bladder and spleen oftheFO and F 1rats. At480 ppm, no treatment-related findings were reported. Thus, under the conditions described, the parental systemic LOAEL in rats was 2400 ppm (corresponding to 202 mg/kg bw/day in males and260mg/kg bw/day in females), and the parental systemicNOAEL was 480 ppm (39 mglkg bw/day in males and 55 mglkg bw/day in females), based on organ changes in the spleen and urinary tract. The NOAEL for parental reproductive parameters was 2400 ppm (173 mglkg bw/day) due to a reduced rearing index. The offspring NOAEL (=NOEL) was at 2400 ppm (173 mg/kg bw/day) dueto pupweightdecrease. In a developmental toxicity study AE 0001789 was administered daily by gavage from gestation day (GD) 6to 20 to groups of25 pregnant Sprague-Dawley female rats perdose-group. The doses given were 0, 60, 250 and 1000 mg/kglday in suspension in an aqueous solution of0.5%methylcellulose 400. Body weight change was reduced in the dams treated at 1000 mg/kglday between GD 6 to 8. At microscopic examination, one female was found to have a moderate bilateral papillary necrosis in the kidney, associated with mixed cell infiltrate. The change correlated with the macroscopic fmding of yellow bilateral sediment in the kidney ofthis female. No litter parameters were affected. At external, visceral and skeletal fretal examination, no treatment-related findings were observed. At 250 and 60 mglkglday, there were no treatment-related maternal, litterbasedorfretal findings at either dosage. The NOEL for maternal toxicity was 250 mglkg/day, while 1000 mg/kglday was a NOEL in terms of fretal development.
In a developmental toxicity study time-mated female New Zealand White rabbits (25 per group) were exposedto AE 0001789, by gavage from Gestation Days (GD) 6 to 28. The doses given were 0,50, 125 and 500 mglkglday in suspension in aqueous solution of0.5% methylcellulose 400. The high dose level

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# EBBayerCropScience 2007-03-28 Tier2, lIA, Sec. 3, Point5: Cyprosulfamide
of500 mg/kglday was terminated priorto commencement ofscheduled autopsy, due to the high mortality rate observed at this dosage. At 125 mglkg/day, treatment-related clinical signs consisted of yellow sediment in urine in 

### 2/25 dams. Yellow sediment in the kidney was also noted at autopsy in 1125 females. No treatment-related changes were detected at the microscopic examination ofthe kidney or the urinary bladder. No adverse effects were observedat 50mg/kglday. As only two treated groups were fully evaluated in this study, a second study was performed where time­ mated female New Zealand White rabbits (25 pergroup) were exposedtoAE 0001189 at 250 mglkglday, by gavage from Gestation Days (GD) 6 to 28. A concurrent control group was added. The dose level of 250 mglkglday induced clear maternal toxicity with two abortions on GD 28. Both females showed yellow sediments in urine on several occasions and lost weight. In the surviving females, 

### 5/23 animals had yellow sediment in the urine on one or more occasions. No effect on litter parameters was observed. Notreatment-related findings were observed at external, visceral and skeletal fretal examinations. Based on these two studies, the NOAEL for maternal toxicity was 125 mglkglday, as no microscopic findings were detected in the kidney or the urinary bladder at this dose level and the NOEL for developmentaltoxicity was 250 mglkglday. TheNOELs for reproductive effects are summarized in the following table: Table5.11-6 Summaryofreproductivetoxicit) with AE0001789 NO(A)EL Typeofstudy LOAEL Adverseeffects/ targetorgans (mglkgld) (mglkgld) 39 in males I 202 in males •Parents 55 in females i 260 in females Reproduction Multi2eneration study Organ changes in spleen Two-generation rat Doses
andurinary tract M-28351O-01-2 113 840 Reducedrearing index 0,480,2400, 12000ppm 840 Offspring Body weight decrease 113

Develol mental toxicity studies 250 1000 dams Reduced body weight, Developmental toxicity rat kidney M-261462-01-2 0, 60, 250, 1000 mglkg > 1000 1000 fretus bw/day 125 

### I 500 Dams Mortality, kidney Developmental toxicity rabbit M-261621-01-2 

## I 125 >125 fretus 0, 50, 125, 500 mglkg 

### bw/day I dam .kidney M-273153-01-2 0, 250mglkg bw/day Developmental toxicity rabbit 

# - 250 250 

## I >250 •fretus

1016

# Ei'BayerCropScience Page434 of441 2007-03-28 Tier2, IIA, Sec. 3,Point5: Cyprosulfamide

• Neurotoxicity AE 0001789 did not induce any neurotoxicological findings afteracute, short or longtenn exposure. In an acute neurotoxicity study, AE 0001789 was administered by gavage to non-fasted young adult Wister rats at 0, 126, 508 and 2060 mglkg (analytical results). Urine stain was evident in both sexes at 2060 mg/kg on day 0 and resolved by the following day. No treatment-related findings were observed at 508 and 126 mglkg. There was no evidence ofneurotoxicity at any dose level and no compound-related gross or microscopic lesions at the high dose of2060 mglkg/day. These results establish a NOEL of508 mg/kg. In a 90-day neurotoxicity study, AE 0001789 was administered in the diet for 13 weeks to young adult Wistar rats. The mean daily intake of the test substance (mg AE 0001789lkg body wt/day) over approximately 13 weeks at nominal dietary concentrations of 1000,3000 or9000 ppm, respectively, was 65.2, 193 and 592 mglkg/day for males and 84.5, 251 and 748 mglkg/day for females. There were no treatment-related effects attributed to exposuretoAE 0001789 at any dietary level in either sex. Basedon these findings, a NOEL of 9000 ppm was established for the rat (specifically, 592 and 748 mg AE 0001789lkgbody wt/day for males and females, respectively). Table5.11-7 SummarYofneurotoxicitywith AE0001789 Typeofstudy Doses NO(A)EL (mglkg/d) LOAEL (mg/kg/d) ! I Adverseeffects/target organs Acuteneurotoxicity in therat M-283196-01-1 508 2060 Urine staining in both sexes .0, 126,508,2060mg/kgbw I 90-day neurotoxicity intherat M-2832 18-01-1 0, 1000, 3000, 9000 ppm 

# I 5921748 (M/F) 

## I >592/>748 (MlF) None

• Toxicityon metabolites The toxicological properties oftwo metabolites ofAE 0001789 were investigated in a series ofstudies carried out accordingtothe current OECD guidelines. The metabolite AE 0467398 (M 03) is a major soil metabolite that has been investigated for its non relevance as it has been predictedbythe EU FOCUS leachingmodelling to have the potential, to leach to shallow groundwater with a concentration of> 0.75 IlgIL. This metabolite was devoid of genotoxic potential anddid not cause any treatment-related effect aftersubchronic administration.

1017

# EBBayerCropScience Page 435 of441 2007-03-28 Tier2, IlA, Sec. 3,Point5: Cyprosulfamide
The metabolite AE 0852999 (M 02) is a soil and a plant metabolite that has been investigated for its non relevance as it has been predicted by the EU FOCUS leaching modelling to have the potential, under worst case conditions, with certain formulated mixtures and application rates. to leach to shallow groundwater resulting in concentrations close to 0.1 Ilg/L. This metabolite was devoid of genotoxic potential andhad a similartoxicologicalprofile to theparent compound after subacute administration. Table5.11-8 Results oftoxicitystudies with themetabolites Study Species Results AE 0467398 (M03) Amestest Negative M-278703-01-2 Salmonella Typ. In vitro V79IHPRT Locus Gene Negative Mutation Assay Chinese hamster M-273551-01-2 V79 lung cells I IIn Vitro Chromosome Aberration. Negative test 

## IChinese hamster M-273607-01-2 V79 lung cells
•NOEL 15 000 ppm ( 1094 mglkg/day in M-282911-01-2 90-day dietary study: Rat females, 888 mglkg/day in males) 0, 400, 2000 and 15000 ppm

•AE 0852999 (M02) IAmes Test Negative IM-274230-01-2 Salmonella typh 

# IIn vitro V79/HPRT Locus Gene Negative
•Mutation Assay ·Chinese hamster I •M-274735-01-2 · iV79 lung cells IIn Vitro Chromosome Aberration· Negative i test Chinese hamster V79 lungcells 28-day dietary study: 

## • M-274272-01-2 NOEL 300 ppm (25.5 mglkg bw/day in 0, 40, 300, 800, 3000ppm females, 22.8 mglkg/day in males) i M-284847-01-2 i ·Rat

• Toxicological endpoints in rodents for ecotoxicological wild mammals risk assessment purposes Use ofmammalian toxicity study results in the ecotoxicological assessment (Annex III 10.3.), based on recommendations given in SANC0/4145/2000 (final rev6, 2002) The toxicity profile ofCyprosulfamide to rat and other laboratory animals indicates that the compound would present little ifany risk towildmammal populations.

1018Page436 of441 

# EBBayerCropScience 2007-03-28 Tier2, llA, Sec. 3, Point5: Cyprosulfamide
Following acute exposure ofrats, no mortality or other signs oftoxicity were observed at the regulatory top dose of2000 mg a.s.lkg bw. This suggests that the LD50 would in fact be much higher than 2000 mg a.s.lkg bw. Therefore the acute oral study results can 

### be expressed according to the proposed OECD classification system in terms ofthe LD50cut-offof5000mg a.s.lkgbw. Typical effects ofhigh doses ofCyprosulfamide administered overprolongedperiods to the rat and other laboratory animals are histopathological findings particularly in the urinary tract and the spleen. Histopathological changes in the urinary tract are secondary to the formation ofstones, which are mainly formed of parent compound. Such effects are considered not to 

### be relevant for the ecotoxicological assessment (see below). Genuine effects on reproductiveparameters were not observed up to the top dose levels in any speciestested. With regard to the selection of endpoints for the long-term assessment, 

### it is considered that effects on populations will not occur ifthe survival rate, reproduction rate and development ofindividuals are not affected. Therefore, in principle, only endpoints in toxicity tests which are related to these key factors of population dynamics are ecotoxicologicallyrelevant. If not indicated otherwise by the overall toxicological data available, an endpoint relating to overall reproductive success should be selected to define the long term endpoint for wild mammals. These would be effects on the reproduction rate, the survival orgrowth rate ofthe offspring, orbehaviouralparameters in adults oryoung. In the rat multigeneration study, mortality, reduced body weight gain and other effects indicating poor general conditions were observed in the high dose treatment level (12000 ppm). No such effects were observed 

### inparents oroffspring atthe medium dose level, 2400 ppm. Atthis level, effects were limited to (histo-) pathological changes in kidneys, urinary bladder and spleen. Survival, growth, development and reproduction ofall animals were not impaired at this treatment level overthe entire 2-generation study. For the ecotoxicological assessment ofCyprosulfamide, the LOAEL ofthis rat multigeneration study was 12000ppm, andthe NOAEL was 2400ppm (202 mg a.s.lkg bw/d). In the developmental toxicity studies in rat and rabbit, effects of ecotoxicological relevance were only observed athigher doses. In the rat, treatment related effects were only observed in dams treated at 1000 mg a.s.lkg bw/day (no developmentaltoxicity up to 1000mg a.s.lkg bw/day). In rabbits, treatment related effects (two abortions) were observed at 250 mg a.s.lkg bw/day, but no otherecotoxicologically relevanteffectswere observed at this dose level. These effects were observed after gavage exposure which is oflow relevance to long-term exposure of

1019

### Page437 of441 

# EBBayerCropScience 2007-03-28 Tier2, IIA, Sec. 3, Point5: Cyprosulfamide
wildmammals to Cyprosulfarnide in the field, and the LOELs from the developmental toxicity studies are well above the NOAEL at 202 mg a.s.ikgbw/day as derived from the rat multigeneration study. 

### Therefore it is proposed to establish the long-term ecotoxicological endpoint for wild mammals at 2400 ppm (202 mg a.s.lkg bw/d). Furthermore, based on the toxicological profile obtained in rodents, there was no indication in any study that AE 0001789 couldbe an endocrinedisruptor.



### • Conclusions

- ADI, AOEL, maximum concentration in drinking water Proposed Acceptable DailyIntake(ADI) The potential risk for consumer is mainly linked to chronic exposure to possible residue ofAE 0001789 in the food. This is the reason why the ADI should be based on the results ofthe following chronic studies:

a . 

# - : t d' T ble511 9 SummaryofIon2term S u 16 

# , Study (Dose levels) NOELfNOAEL LOEL TargetOrgan(s) and Itreatment-related effects! ppm mg/kg/day ppm mg/kg/day Oneyear dog study

M-283194-02-1 (0, 1000,2500, 8000 ppm) 2500 66/67 (MlF) 8000 226/242 (MIF) Kidney and urinary bladder
2 yearrat carcinogenicity M-281767-01-2 (0, [200], 1000,4000,8000ppm) 1000 39/56 (MIF) 4000 159/220 (MlF) Kidney and urinary bladder
Mouseoncogenicity

M-281276-01-2 (0, 350,2000. 3500, 7000 ppm) 3501 2000 (MIF) 50/354 (MlF) 20001 3500 (MIF) 2871616 (MIF) liver, kidney and urinary bladder
Rat 2-generationreproduction Study M-2835 10-01-2 (0,480,2400, 12000ppm) Parents 480 39/55 2400 202/260 Organ changes in spleen & urinary tract Reproduction 2400 173 12000 840 Reducedrearinj;!; index Pups 2400 173 12000 840 Body weif;!;htdecrease

1020Page438 of441 

# EBBayerCropScience 2007-03-28 Tier2, IlA, Sec. 3,Point5: Cyprosulfamide
In accordance with internationally accepted procedures the ADI (or Reference Dose RID) is estimated on the basis ofthe No-Observed-Adverse-Effect Level (NOAEL) obtained in a chronic toxicity study with the most sensitive species. The lowest Low Observed Adverse Effect Level (LOAEL) is obtained in the rat, which is thereforethemost sensitive species. The lowestNOAEL of39mglkg/day was obtained in the 2-yearrat study. Treatment-relatednephropathy was observed at 159/220mg/kg/day in males and females, respectively. Since none of the other toxicological studies showed any indication of mutagenicity or reproductive toxicity or any other special hazard potential, and taking into account the toxicological profile, a margin ofsafety (MOS) of 100 is considered to be appropriate. Based on these considerations, the following ADI value (=chronic Reference Dose cRID) is proposed for AE 0001789:

ADI

## =39 (mglkglday)/100 = 0.39 mgIkg/day For the USA, no additional FQPA safety factor seems necessary. There is no indication ofan increased sensitivity ofthe young. Additionally, the toxicological database ofAE 0001789 is considered complete. Therefore, for the USA the suggested chronic Population Adjusted Dose (cPAD) is the same as the RID (cPAD = RID = 0.39mg/kg/day)
Proposed AcuteDietaryExposure ARID (Acute reference dose) On the basis ofthe low order ofacute toxicity ofAE 0001789, following exposure by oral, dermal and inhalation routes, it is notnecessarytopropose an acute reference dose for the EU. For the USA and Canada, an endpoint of concern attributable to a single dose (exposure) was not identified from the available studies. An acute RID does notneedto be established. Proposed Acceptable OperatorExposure Level (AOEL) Since health risks to operators relate to short term rather than to chronic exposure, the NOAELs derived from shortterm and developmental toxicity studies andthe rat 2-generation reproduction study should be taken into account for the purposes ofestablishing an AOEL for the EU and for the estimation ofthe operatorexposure in the USA andCanada. AE 0001789 has low acute toxicity to mammals irrespective ofthe route ofexposure (oral, percutaneous or inhalation exposure). It is not a skin sensitizer, it is not irritating to skin and causes only a slight redness ofthe conjunctivae in the rabbit. Subchronic studies showedthat the kidney andthe urinary bladder are the majortarget organs in both rats and dogs. The effects observed are consistent with the formation ofstones in the urinary tract. The mice

1021Page439 of441 

# EEYBayerCropScience 2007-03-28 Tier2, llA, Sec. 3, Point5: Cyprosulfamide

are less sensitive after subchronic administration. Both in vivo and in vitro studies indicate that AE 0001789 is not genotoxic. AE 0001789 reduced the rearing index (secondary to maternaltoxicity) in aratreproductive study but at a dose level causing overt maternal toxicity such as increased mortality. No embryotoxic effects were observed in the rat orrabbit teratogenicity studies. The relevant NOAELs to be considered for calculation ofthe operator exposure are summarised in the following table.

Table • : ummary 0f I aa or e setf**ID~** 0 operatorexposure 511

# - 10 S re evantdt'tb f Study (Dose levels) NOEL0JOAEL LOAEL TargetOrgan(s) ppm mg/kglday ppm mg/kglday 90-day dog study M-263605-0I-I (0,2500,7500. 15000ppm) 2500 75179 (M/F) 7500 226/226 (M/F) Kidney Urinary bladder Ureters 90-day rat study M-I05691-01-2 (0. 1000.4000, 12000ppm) 1000 58/70 (M/F) 4000 240/280 (MlF) Urinary bladder
90-day mouse study

M-104791-01-2 (0. 500.2000, 7000 ppm) 7000 (M) 2000 (F) 1110 (M) 398 (F) >7000 (M) 7000 (F) >1110 (M) 1297 (FJ Thymus in females only

Rat teratology M-261462-01-2 (0.60,250 1000mglkg/day) Dams 

# - 250 

## - 1000 Kidney Pups 

## - 1000 

# - >1000
Rabbitteratology M-261627-01-2 M-273153-01-2 (0, 50,125. [250], 500 mglkg/day) Dams 

## - 125 

# - 250 .Kidney Pups 

## . 250 

## - >250



## IRat 2-generationreproduction Study M-28351O-01-2 (0, 480. 2400. 12000ppm) Parents 480 39155 2400 202/260 Organ changes in spleen & urinary tract Reproduction 2400 173 12000 840 Reducedrearing index Pups 2400 173 12000 840 Bodyweightdecrease The most sensitive species is the rat with the lowest Low Observed Adverse Effect Level (LOAEL) obtained in the rat 2-generation reproduction study: 202 mglkglday. The lowest NOAEL is also observed

1022Page440 of441 

# EBBayerCropScience 2007-03-28 Tier2, IIA, Sec. 3,Point5: Cyprosulfamide
in the rat 2-generation reproduction study: 39 mg:kglday. The target organs observed in this study (spleen andthe urinary tract) were also extensively evaluated in the 90-day rat study. Therefore the NOAEL from the 90-day rat study, which is 58 mglkglday, can be consideredthe appropriate NOAEL to be used for the setting ofthe AOEL. Available studies indicate that AE 0001789 is rapidly absorbed by rats following oral administration by gavage. More than 82 to 90% ofthe administered dose was absorbed in rats receiving 2 mglkg bw ofAE 0001789. Therefore, no adjustment for oral absorption is considered necessary when calculating systemic AOEL. Based on this toxicological profile, the current conventional (EU) Uncertainty Factor (UF) of 100 is consideredtobe appropriate for settingAOEL. Forthis reason the following systemicAOEL is proposed
AOEL systemic= 58 (mg/kg/day)/100 = 0.58 mg/kg/day
Proposed maximum acceptableconcentration in drinkingwater No suitable human data are available and chronic exposure animal studies involving administration ofAE 0001789 drinking water were not carried out. It is therefore proposed that for use in EU the Maximum Allowable Concentration (MAC) in drinking waterbe based on the ADI derived from dietary studies. In order to calculate the MAC for drinking water it is appropriate to divide the ADI by an additional uncertainty factor of 10 and thus derive an allowable intake level of 0.039 mglkg bw/day. The International Programme on Chemical Safety (WHO, 1994) proposedthat for risk assessment purposes a reference human be consideredtoweigh 64 kg and be consideredto have a daily intake ofdrinking water of].4 litres. Thus the MAC value forAE 0001789 for use in EU can be estimatedas follows

­ MAC = 0.039 x 64 kg = 1.78 mg ofAE 0OO178911itre = 1.78 mglL ].4 For the USA and Canada., theoritical concentrations ofthe compound in drinking water from surface and groundwater sources are calculated and directly used in the dietary risk assessment on consumption basis. No additional Drinking Water Levels ofConcern (DWLOC) are calculated. Proposal for classification and labelling According to the criteria for classification and labelling as defined in EEC Directive 93/211EEC AE 0001789 is not classified for acute toxicity in the EU. For the USA and Canada the following classification is proposed:

1023Page441 of441 

# EBBayerCropScience 2007-03-28 Tier2, IIA, Sec. 3,Point5: Cyprosulfamide

a e 

# - : roposed I 'fi an ana a T bl 51111 P c assl Ication Ii orUSA d C d StudyType GuidelineNo. Results ToxicityCatlllory I LDso > 2000 I 81-1 Acute Oral · mglkg in males & III i females I · LDso >2000 mglkg 81-2 AcuteDermal III in males &, females I Acute Inhalation LCso >3.513 mgIL I IV (4 h) 81·3 in males & females I i Eye Temporary redness \ Primary , 81·4 III i Irritation ofthe coniunctivae Primary Skin 81·5 not a dermal irritant IV Irritation i I Dermal a dermal • not N/A 81·6 i Sensitization , sensitizer

Based on its toxicological and pharrnacokinetic metabolism profile no additional classification or labellingis considered to be appropriate.

1024".r'-"'::-' I' -'
-: ;. . ,.. '-**~**

### ..

Company Official: CompanyName: CompanyContact: CompanyTelephone: 

## TRANSMITTAL DOCUMENT

Cyprosulfamide

OECD Annex II I

### Joint Review



### TransmittalDate April 16, 2007

BayerCropScience R.M. See 

### 919-549-2913

1025

## BIBLIOGRAPHYOF SUBMITTED DATA

Cain. P.;TransmittBl DocLJment for cyprosulfamic:le (17891;8ayerCropScience. Research Triangle Park. NC, USA; Report No.: M-286547-0H; Document No
.. M286547-01-1 :411112007: Pages: 0 . 47069600

2 Fontaine. L. D.;Product chemistry ofAE 0001789;Bayer CropScience. Kansas City. MO. USA; Report No
.. BR 2560: Document No.: M-286067-0l-1;:311512007; Pages. 11 830 Group A MilA Sec 1 47069601C

2 Fontaine. L. D.:Confidenlial business informalionlor product chemistry ofAE 0001789;Bayer CropScience. Kansas City, MO. USA: Report No.: BR 2560; Document No.: M-286065-01-1;3I1512007; Pages: 381 830 Group A MilA Sec 1 MilASec 2 CA47069601

:3 Fontaine. L. D.;Proouct chemistry ofAE 0001789 tecl1nical;8ayer CropScience. Kansas City. MO. USA; Report No. BR2561: Document No. M-28618C-Ol1;311312007: Pages: 308 830 Group B MilASec 1 MilASec 2 47069602

4 Shepard. J.;[MethoxybenzoyI-Ul-14CJ and [sulfonylbenzamide-UL-14CJAE 0001789: Hydrolytic degradation;Bayer CropScience. Stilwell. KS, USA; Report No.: MEUBXOOO; Document No.: M-279728-01­ 1:8117/2005: Pages: 98 161-1 KIIA2.9.1 KIIA 7.5 47069603

5 Sneikus. ':.: Dallstream. K; Mislanker, S.;[MethoXybenzoyl· ring-UL-14C) and [SLJIfonylbenzamide-ring-UL-14Cj AE 0001789: Phototransfonnation in pH 7 phosphate buffer:BayerCropScience. Stilwell. KS, USA: Report No
.. MEUBX010, Document No.: M-281558-01-1;612712006, Pages: 93 161-2 KIIA 2.9.2 KIIA 7.6 47069604

6 Desmarteau, D. A.;[Methoxybenzoyl-nng-UL-14C and Sullonylbenzamide-ring-Ul-14CJAE 0001789: Phototransformation on soil;8ayer CropScience, Environmental Research Section. Stilwell, KS. USA: Report No.: O4MEUBX012; Document No.: M-254211-01· 1;6/2112005; Pages: 1:J8 161-3 KIIA 71.3 47069605

7 Sneikus. J.;AEOOO1789: Detennination oftheQUllnturT' yield and assessment ofthe environmental half-lifeofthe direct photodegradation in water;BayerCropScience AG. Monheim, Gennany; Report No.: MEF-05f146; Document No.: M-256709-01-2;2115/2007: Pages: 36 lS1·Suppl KIIA2.9.3 KIlA 7.6 ;:-C69606

Pag'" 2 or20 1026

## BIBLIOGRAPHY OF SUBMITTED DATA 8 Stupp, H. P,;AE 0001789-anisic acld: Determination ofthe quantum yield andassessment oftheenvironmental halflifeofthe direct phOlo-oegradatiol1 in water;Bayer CropScienceAG, Manheim, Germany; Repon No
.. MEF· 06(012; Document No.: M·274377-01-2:11f712006; Pages 43 161·Suppl KIiA 7.6 47069607

9 Snelkus, J.;AE000178!J.cyClopropyl-sulfonic acid' Determination ofthe Quantum yield and assessment of the environmental half-life ofthedirect Photodegradation in water;Bayer CropScience AG, Monheim, Germany: Report No.: MEF-05I543; Document No.: M-26B514-01­ 2;11nt2006; Pages 37 161·Suppl KIIA 7.6 47069608

10 Fliege, R.;AE ooo17B9: Calculation of !hechemical Nfelime inthe tncposphere;Bayer CropScience AG, Manheim. Germany; Report No.: MEF.Q5I092; Document No.: M248810.01-2:1/4/2007; Pages: 18 161-Suppl. KItA2.10 KIIA 710 47069609

11 Sneikus, J.;Aerobicdegradation and metabolism ofAE 0001789 in different soils;Bayer CropScience AG. Monhelm, Germany; Report No.: MEF-04/3SO; Documenl No.: M-25789Hll.2;1111712006: Pages: 91 162-1 KIIA 7.11 KIIA 7.2.1 47069610

12 Mislankar. S. G.; Dallstream. K.;[MethoXybenzoyl·ring-UL· 14C)AEoo01789: Aerobic soil metabolism in 3 EU SOils. 1 US soil;BayerCrop$cience. Stilwell. 1<$, USA; Report No.: MEUBXQ04-2; Document No.: M-279303-02-1;912512006; Pages: 96 162·1 KIIA7.1.1 KIIA 7.2.1 47069611

13 Fliege. R:AE 0001789-oescyclopropyl (AE 1272799): Rate of degradation in three European soils underaerobic laboratory conditions;BayerCropSc1ence AG. Monheim. Germany; Report No.: MEF-061027: Document No.: M· 274921.01-2;11/712006; Pages: S2 162-' KUA 7.2.3 47069612

14 Riege, R;AE 0001789-<::yclopropylamine (AE 2300015)' Rate ofdegradation in three european soils underaerobic laboratory conditions:Bayer Crop$cience AG. Monheim. Germany; Report No,: MEF.061081; Document No.: M· 274613-01-2;2113/2007; Pages: 84 162-1 KlIA 7.2.3 47069613

15 Filege. RAE 0001789-oesmethyl (AE 1448796): Rate of degradation in three European soils under aerobic laboratory condltions;BayerCropScience AG. Monheim, Germany; Report No.: MEF.061103; Document No.: M274945-01-2;211512007; Pages: 86 162-1 KIIA7.2.3 47069614

Page: 3 ofl0 1027

## BIBUOGRAPHY OF SUBMITTED DATA 16 Mislankar, S. G.; Snelkus, J.; Oallslream. K. :[Methoxybenzoyl-Ul-14Cj and {Sulfonylbenzamide-UL14Cj AE 0001789 Anaerobic SOil metabolism;Baye" CropScience. Stilwell. KS. USA; Report No.: MEUBXOO5: Oocumem No.: M-279306-01.1;1012/2006.: Pages 102 162-2 K!1A 7 1.2 KIIA 7.2.4 47069615

17 Desmarteau. D. A.; Mathew. A. E.;[14C}AE 0001789: Anaerobic aquatic metabolism;BayerCropScience. Stilwell, KS, USA; Report No' MEUBX019; Documenl No.: M-279716-01-1;7/512006; Pages: 100 162-3 KIIA 7.8.2 47069616

18 Allan, J. G.;[methoxybenzoyl-ring-UL-14Cj-AE 0001789 anc [sulfonylbenzamide-nng-UL-14Cj-AE 0001789: Aerobic aquatic metabolism;BayerCropScience, Stilwell. KS. USA; Report No.: MEUBX018; Document No.; M·281233-01· 1;1011912006; Pages: 127 162-4 KIIA 7.8.3 47069617

19 Fllege, R;AE 0001789; Adsorption/desorption on seven soils;BayarCropScienceAG, Monheim, Germany; Report No
.. MEF-041301: Document No. M-123114-01­ 2;811712006; Pages: 127 163-1 KIIA 7.4.1 47069618

20 Allan, J. G.;AE 0467398: AdsorptionIdesorption on five soils;BayerCropScience. Stilwell. KS, USA; Report No. MEUBX024; Document No
.. M-284877-01-1;2116/2007; Pages: 104**163-~** KIIA 7.4.2 47069619

21 Allan, J. G.;AE 0852999: Adso1)bon/desorption on fIVe soUs;BayerCroo$cience, Stilwell, KS, USA: Report No.: MEUBX023; Document No. M-264874-01-1;211612007: Pages: 114 163-1 KIIA 7.4.2 47069820

22 Shepard, J.; Allan, J.;AE 0001789-descyclopropylamlno (AE 0893016): Adsorption/desorption on fIVe sOils;Bayer CropSclence, Stilwell, KS, USA; Report No.: MEUBX056: Document No.: M-280497-01-1;10117/2006; Pages 93 163-1 KJlA 7.4.2 47069621

23 Shepard, J.: Allan. J. G
.. A1hur, E. L;AE 0001789 tmsmethyl (AE 1448796) Adsorption/desorption on five solls;BayerCropScience, Stilwell. KS. USA; Report No.: MEUBX025; Document No M-279817-01-1;912912006: Pages: 83 163-1 KIIA 7 4.2 47069622

Page: 4or2C 1028

## BIBLIOGRAPHY OF SUBMITTED DATA
24 Fliege, R.;AE 0OO1789-descycloprq;yl (AE 1272799) Determination of the adsorplion coefficient (KOG) on &Oil bycolumn feachlng;BayerCropSCience AG, Monheim, Germany; Report No.: MEF-06/00ll; Document No. M274606-01-2;2116/2007: Pages: 80 163-1 Suppl. KIIA 7.4.4 470£9623

25 Wyatt, D. R;Terreslrial field dissipation ofAE 0001789 in Nebraska soil. 2oo5;Bayer CropScience. Stilwell. KS. USA; Report No.: MEUBPOO3; Document No.: M·285787-01· 1;3/15/2007; Pages: 232 164.1 KIIA 7.3.1 47069624

26 Wyatt. D. R;Terrestrial field dissipation ofAE 0001789 in Illinois soil. 2oo5;BayerCropScience. Stilwell. KS. USA; Report No.; MEUSP004; Document No.: M·285173·01· 1 ;3/1512007; Pages: 233 164-1 KIIA 7.3.1 47069625

27 Wyatt. D. R;Terrestrial fielddissipationofAE 0001789 in Ontario. Canada soil. 2005;SayerCropScience. Stilwell, KS. USA; Report No.: MEllBP005; Document No
.. W· 285770-01-1;311512007; Pages: 255**164·~** KIIA 7.3.1 47069626

28 Wyatt. D. R,;Terrestrial field dissipation ofAE 0001789 in California soil. 2005;SayerCropScience. SbiweR. KS. USA; Report No.: MEUBX021: Document No.: M-285766-01. 1:3115/2007; Pages: 240 164-1 KIIA 7.3.1 47069627

29 Roberts. J.; Kem. M.: Lam, C ;Acute toxicityof AE 0001789 technical tothe Daphnia magna under static conditions;Sayer CropScience. Kansas City. MO, USA Report No.: 200982: Document No.: M·2402t32-01· 2;1011/2004; Pages: 34 850.1010 KIIA 8.3.11 47069628

30 Banman. C. S.; Lam, C. V.:AaJtetOXicity ofAE 0001789­ sulfamoylbenzoic acid (AE 0467398) 10 the Daphnia magna under static conditions;Bayer CropSCience. Stilwell, KS, USA; Report No.: EBUBP076; Document No.: M-277696-01-1 ;7/17/2006; Pages: 36 850 1010 KIIA 8.3.1.1 47069629

31 Banman. C.S., Lam, CY;Acute lOxicity ofAE0001789· cyCIopropyf..sulfamoylbenzamide (AE 0852999) tothe Dapl1nia magna under static conditions;Bayer CropScience. Stilwell, KS, USA; Report No.: EBUBP084; Document No
.. M·26890S-01·1;312212006; Pages: 36 850.1010 KIIA 8.3.1.1 47069630

Page: 5 of2ft 1029

## BIBLIOGRAPHY OF SUBMITTED DATA 32 Banman. C. S.: Lam, C. V.:Acute toxicity ofAE 0001789· anisic acid (AE 0854787)to the Daphnia magna under staticconditions,BayerCropScience. Stilwell. KS. USA; Report No.: EBUBP088; Document No: M-277899-01. 1;9/1912006; Pages: 35 850,1010 KIIA 8,31.1 47069631

33 Bam'nan, C. S: Lam. C. V.;AcutetQJ(icity ofAE 0001789­ descyclopropylamillo (AE 0893016)to the Daphnia magna under staticconditions;Bayer CropScience, Stilwell. KS. USA; Report No.: EBUBP091; Document No
.. M-277502­ 01·1;9/1512006: Pages: 37 650.1010 KIIA8,3.e 47069632

34 Banman, C. S.; Lam, C. V ..Acule toxicity ofAE 0001769· descyclopropyl (AE 1272799)to the Daphnia magna under static conditlons;Bayer CropScJence, Stilwell, KS. USA Report No.: EBUBP094; Document No.: M-277707-D1­ 1;512112006; Pages: 36 650.1010 KIIA8.3.1,1 41069633

35 Banman, C. S.; Lam. C. V.;Acute tOXicityof AE 0001789· cyclopropyl-sulfonic acid (AE 2158927)to the Daphnia magna understatic conditions;Bayer CropScience. Stilwell. KS. USA: ReportNo.: EBUBP096: Document No
.. M-277112·01-1;612812006: Pages' 36 850.1010 41069634

36 Weyers. A.:PE0001789-1!midclliorid (AE 1379884) • Acute daphnia toxicily;Bayer Industry Services GmbH, Leve!kusen. Germany; Report No.: 2006J0021102: Document No.: M·27371Cf-01-2:3f912007: Pages: 21 850.1010 KfiA 8.3,1.1 47069635

37 Sayers. L. E.:AE 0001789technical - Acutetoxicityto mysids (Amencamysis bahia) under static conditions;Springbom Smithers Laboratories. Wareham. MA, USA: Report No.: EBUBP013; Oocl.l"!lent No.: M· 277521-01-1;91512006: Pages: 49 850.1035 KI:A 8.11.1 47059636

36 Cafarella. M-A.:AE 0001769 Technical· acutetoxicity to eastern oyster (CraSSOSb'ea virginal underflow-through conditions:Springborn Smithers Laboratories, Wareham, MA, USA; Report No.: EBUBP014: Document Nc,: M· 278506-D1-1:912012006: Pages: 54 650.1055 KIIA 8.11.1 47069637

39 Banman, C. S,: Lam. C. V.:Acute toxicity of AE 0001789 technical to the sheepshead minnow (Cyprinodon variegatus):Bayer CropScience, Stilwell, Kansas. USA; Report No. EBU6P007; Document No.: M-251501-D1­ 1;511312005: Pages: 32 850,1075 KllA 6,11.1 47069638

Page. 60[20 1030

## BIBLIOGRAPHY OF SUBMITTED DATA 40 Roberts. J.; Kern. M.; lam. C.;AcutEi toxicity ofAE**000~789** to\he ra.nbowtrout (Oncorhynchus mykiss) under statiC cond!tions;Bayer CropScience. Ka"lSaS City, MO, USA: Report No
.. 201011: Document No
.. M-240253-01­ 2:913012004: Pages: 36 850.**~075** KIIA 82.1.1 47069639

4< Roberts.J.; Kern, 1.4.; Lam, C.:Acute Toxicity0(AE 0001789 to the Bluegill Sunfish (Lepomis macrochirus) Under Static Conditions:Bayer CropScience. Stilwell, !<S. USA; Report No.: 201020 (EBUBXoo)7; Document No.: M-240252­ 01· 1 ;9/30/2004: Pages: 36 850.1075 KIIA8.2.1.2 47069640

42 Weyers, A.:AE 00017B9-amidchlorid (AE 1379884) - Acute fISh loxiclty;Bayerlndustry Services GmbH, Leverkusen. Germany; Report No.: 2006/0021103; Document No.; M273628-01·3;2121/2007; Pages: 21 850.1075 KIIA8.2.1.1 47009641

43 Kem, M.E.; Lam. C.V.;Chronic toxicity ofAE 0001789 technicalto the Daphnia magna· understatic-renewal conditions;Bayer CropScience, Kansas City, MO, USA; Report No.: EBUBP011; Document No.: M·255076·01· 1;7/1812005; Pages: 61 850.1300 KIIA 6.3.21 47069642

44 Putt. A. E.:AE0001789ted'1nical· Ufe-cycle toxicity test with myslds (Americamysis bahla) underflow-through condltlons;Springbom Smithers Laboratories. Wareham. MA. USA: Report No.: EBUBPOO4; Document No.: M284852-01-1;111612007: Pages: 100 850.1350 KIIAS.111 41069643

45 Kern. M.E
.. Lam, C.V.:Early life stage toxicity ofAE 0001789 \ectmicaJ 10the Fathead Minnow(Pimephales promelas) underflow-through conditions;BayerCropScience, Kansas City, MO, USA; Report No.: EBUBPOO9; Document No,: M-254985-01-1;7J1812005; Pages' 77 850,1400 KIIA8.2.4 41069644

46 Barfknecht, R.;A:cute oral toxicityfor bobwhite quail (Colinus virginianus) with AE 0001789 a.s.;Bayer CropScienceAG, Manheim, Germany; Report No.: BARiLD 061 ; Document No.: M-129362-01-2;9/1312000: Pages: 29 850.2100 KIIA 8.1.1 41069645

47 Stoughton 1. L.;Technical AE 0001789 a subacute dietary Le50 with mallards;BayerCropScience, Stilwell. KS, USA; Report No: EBUBP125. Documem No.: 1.4-278425-01· 1:812212006: F'ages: 48 850.2200 KIIA 8.1.3 41069646

Page: 7 nnD 1031

## BIBLIOGRAPHY OF SUBMITTED DATA 48 Stoughton, T. L..Technicai AE 0001769: II subacute dietary LC50 with mallards:BayerCropScience, Stil_II, KS, USA; Report No.: EBU8X002: Document No.:**M·278433-0~·** 1:5/11/2000: Pages: 49 850.2200 KIIA 6.1.3 47009647

49 Stoughton. T. L.;TechnicaJ AE 0001789: a subacutedietary LC50 with northern bobWhite;8ayer CropScience. Stilwell KS. USA: Report No.: EBUBX004; Ooeument No.: M271799-01·1:511/2000: Pages: 48 850.2200 KIIA8,1,2 47069548

50 Stoughton, T. L.:ENectof technical AE 0001789 on northern bobwhite reproduction;BayerCropScience, Stilwell, KS, USA; RepOrt No.: EBUBP015; Document No.: M·284854· 01-1;2/2812007; Pages: 153 650.2300 KIIA 8.1.4 47069649

51 Temple, D.L; Martin, K H.; Beavers, J B.; Jaber, M.:AE 0001789:A reproduction study with the mallard;WildUfe Intemational Ltd., Easton, MD, USA; Report No
.. EBUBPQ16: Document No.: M-283926-01·1;1f10/2007; Pages:222 850.2300 KlIA 8.1.4 47069650

52 Schmitzer, S.;EffectsofAE 0001789 00 1CII7 0005 (acute contact and oral) on hooey bees (Apis mellifera L.) In the laboratory;lbacon GmbH, Rossdorf, Germany; Report No.: 25111035; Document NO.: M·262566-01-2;4f2l2007; Pages: 29 850.3020 KIIA 8.7,1 KIIA8.7.2 47069701

53 Gosch. H.; Pallett, K.; Nguyen, 0, H.;AE 0001789 SC 500 giL - Effectson eleven species of norl-target terrestrial plants: seedling emergence and growth test**(tie~** 1 and 2);6ayerCropScience GmbH. Frankfurt am Main, Germany; RepOrt No.: SE 061027; Document No.: M· 283760-01·2;3f14J2007; Pages: 105 850.4225 KIIA 8.12 47069702

54 Gosch, H.: Pallett, K
.. Nguyen, O. H.;AE 0001789 SC 500 gIL; Effects on eleven speciesof non·targetterrestnal plants: vegetative vigourtest (tier 1);BayerCropScience GmbH, Frankrun am Main, Germany; RepOrt No.: W 06/026; Document No : M·283542-01·2;211612OO7; Pages. 50 850.4250 KIIA8.12 47069703

55 Kern, M. E.; Roberts, J. A
.. Lam, C.**V.;ToxiC~y** ofAE 0001789technical to duckweed (Lemna gibba G3) under static-renewal conditions:Bayer CropScience. Stilwell, Kansas, USA; Report No,; EBUBPOO5; Document No.: M251602-01·1;4126/2005; Pages: 42 850.4400 KIIA8.5 47069704

Page: 8of20 1032

### BIBLIOGRAPHY OF SUBMITIEDDATA 56 Christ, M. T.; Holfmann, J. M.: lam, C. V.;Toxicity of AE 0OO1789-cyclopropyl-sulfamoylbetU:amide (AE 0852999) to duCkweed (Lemna 91bba G3) understallc-renewal conditions;8ayerCropSc:ience. SIDwell, KS, USA; Report No.: E8UBP086; Document No.: M-284345-Q1· 1;2/1412007; Pages: 43 850.4400 KIIA8.6 47069705

57 Christ, M. T
.. Hoffmann, J. M.; Lam, C. V.;ToXici!y ofAE 0001789-sulfamoylben:z:oic add (AE 0467398) to dUCkweed (Lemna gibba G3) under static-renewal conditions;Bayer CropScience, Stilwell, KS, USA. Report No.: E8UBPOB2-1; Document No.: M-284335-02­ 1;2113/2007; Pages: 42 850.4400 KIIA8.6 47069706

58 Christ, M. T., Hoffmann. J. M.; lam. C. V.;ToxicityofAE 0001789-anisic acid (AE 0854787) to duckweed (lemOS gibba G3) under statlc-renewal conditions:Bayer CropScience. Stilwell, KS. USA; Report No.: EBUBP067 Document No.: M-284359-01-1;2I1Si2007; Pages: 43 850.4400 KIIA8.6 47069707

59 Christ. M. T.; Roberts, J A.; Lam, C. V.;Toxicity ofAE 000H89-descyciopropyiamino (AE 0893016) to duckweed (Lemna 9ibba G3) under static-renewal conditions;Bayer CropScience, Stilwell, KS, USA; Report No. EBUBP093; Document No.: M-28433O-01­ 1;211912007; Pages' 42 850.4400 KJlA8.6 47069706

60 Banman, C. S.; Lam. C. V.;Toxiclty ofAE 0001789­ descyclopropyl (AE 1272799) to duckweed (Lemna gibba G3) understatic-renewal condnions;BayerCropScience, sm_lI. KS. USA; Report No.: EBUBP095; Document No.. M-279707-01-2;11/312oo6; Pages: 43 850.4400 KIIA8.6 47069709

61 Christ, M T.: Hoffmann. J. M
.. Lam. C. V.;Toxicny of AE oo01789-cyclopropyl-suIfOnic acid (AE 2158927) to duckweed (Lemna glbba G3) under static-renewal conditioos;Bayer CropScience,**S~IweII.** KS, USA; Report No.: EBUBP099; Document No
.. M-284341-Q1­ 1;2114/2007: Pages' 42 850.4400 KIIA8.6 47069710

62 Kern, M. E
.. Lam, C. V.;TOXiCjty ofAE 0001789 technical to the green alga Pseudokirchneriella subcapitata;Bayer CropScience. Stilwell. KS. USA; Report No: EBUBP012; Document No.: M·25201S-01·1 .5/2512005; Pages: 42 850.5400 KIIA 8.4 <17069711

63 Banman, C. S., Lam. C. V.:Toxicity 0:AEOO01189­ C)lclopropyi-sulfamoylbcnzamide (AE 0852999) to the green alga Pseudokirchnenella sulX:apitata;Bayer CropScience, Stilwell, KS, USA; Report No.: EBUBP085, Document No
.. M-271702-D1-1 ;6/19f2006; Pages. 47 850,5400 KIIA8A 47069712

Page. 9 of20 1033

## BIBLIOGRAPHY OF SUBMITTED DATA 64 Banman, C. S.; Lam. C. V.;Toxicity ofAE 0001789­ sulfamoylbenzoiC acid (AE 0467396)to the green alga Psaudokirchneriella subcapitata;Bayer CropScience. Stilwell. 1<5. USA; Report No.: EBUBP081; Documenl No. M·27769B-01·1 ;91812006; Pages: 48 850.5400 KIIA S.4 47069713

65 Banman. C.S.; Lam. C.V.;Toxicityd AE 0001769-anis;c acid CAE 0854787) to the green alga P$eudokirchneriella subcapitata.;Bayer CropScience. Stilwell. KS. USA; Report No.: E8UBP089; Document No.: M-279156-01· 1;10/23/2006: Pages: 48 850,5400 KIIAS.4 47069714

6I:l Banman. C.S.; Lam, C.V.;Toxicity of AE 0001789­ descyclopropylamino (AE 0893016) to the green alga Pseudokirchneriella subcapilala;BayerCropScience, Stilwell. 1<5, USA; Report Nc.: EBUBP092; Document No,: M-279706·01·2;11/312006; Pages: 47 850,5400 KIIA 8.4 47069715

67 Banman, C. 5.; Lam. C. V,;Toxicity orAE 0001789­ descyclOpropyl (AE 1272799)to the green alga PSi!Udokirchneriella subcapitata;BayerCrapScience, Stilwell, 1<5. USA; Report No.: EBUBP096; Document No, M-277709-01-1;6127/2006: Pages: 50 850.5400 KIIA 8,4 47069716

66 Banman. C. S.; Lam. C, V.;Toxicity ofAE 0001789· cycIOpropyl-sutron;c ilc.d (AE 2158927) 10 thegreen alga Pseudokirchnerieila sUbcapitata;BayerCropScience. Stilwell. KS. USA; Repo'1 No.; EBUBP1oo; Document No,: M-277715-01-1;81412006: Pages; 47 850.5400 KIIA 8.4 47069717

69 Neuhahn. A.;AE oo01789-amidchlorid CAE 1379884)- Alga growth inhibition test;Bayer Industry SeNices GmbH. LeverkuSiln. Germany: Report No,: 200610021/01: Document No.: M-274307-01·3;2119/2oo7: Pages: 39 850Suppl. KIIA8,4 47069718

70 HeImbach. F.:AE 000178900 1e97 0005: AE OOCHa9 substancetechnical Acute toxicity to earthworms (EisenJa felida) tested inartifical soil:Bayer CropScienceAG, Monheim. Germany; Report No.: LKCIRG-A-61106: Document No,: M-266322-01-2;211612oo7; Pages: 24 850 Supp/**KI1A8,9,~** 47069719

71 Heimbach, F,;AE 000H89-suifamoylbenzoic acid (AE 0467398): Acute loxicity to earthworms (Eisen;a fetida) tested In artifiCial soil;Bayer CropScience AG, Manheim, Germany; Report Nc.: LKCIRG-A-62106; Document No. M· 268093-01-2;211612007; Pages: 24 85OSuppi. KIIA8,9,1 47069720

Page: 10 of2U 1034

## BIBLIOGRAPHY OF SUBMITTED DATA
72 Leicher. T.;AE ooOl789-descyclopropylamino (AE 0893016): acule toxicity to eanhworms (Eisenia letlda) tested in artificial soil;Bayer CropScience AG. Monheim, Gennany; Report No.: LRTIRG-A-70/06. Document No.: M· 276674-01-2;211612007; Pages: 24 850 Suppl. KIIA 8.9.1 47069721

73 Heimbach. F.;AE 0001789-cydOpropylsullamoylbenzamide (AE 0852999): Acute toxicity to earthworms (Eiseniafetida) tested in artificial soil:Bayer CropScienceAG. Monheim. Gennany; Report No. LKC/RG-A-63f06; Document No.: M-269348-01­ 2;211612007; Pages: 24 850 SuPP!. KIIA 8.9.1 47069722

74 Leicher. T.;AE 0001789-descyclopropyl (AE 1272799): Acute toxicity to earthworms (Eisenia fetida)tested in artificial SOiI;Bayer CropScience AG. Monheim. Germany; Report No.: LRT/RG·A·71/06; Document No.: M-277115­ 01-2;211612007; Pages: 24 850 Supp!. KIlA8.9.1 47069723

75 Rupprecht. K. J.; Ying, S. L.;The metabOlism of [suffonylbenzamide-ring.UL·14Cj-AE 0001789 in cern (foliar**post~ergence** applicaUon);Bayer CropScience, Stllwell, KS. USA: Report No
.. MEUBX047; Document No.. M-285882-o1-1;3113/2007; Pages: 85 8601300 KIIA6.2.' 47069724

76 Rupprecht, K. J.: Ying, S. L..The metabOlism of [methOXybenzoyl-ring·UL-I4C)-AE 0001789 in com (foliar post-emergence application);Bayer CropScieoce. Stilwell KS. USA; Report No.: MEUBX048; Document No.: M285883-01-1 ;311312007; Pages: 78 8601300 KliA6.2.1 47069725

77 Fischer. D. R., Roensch, W. L.;The metabolism of [sulfonylbenzamide-ring-UL·14Cl and [methoxybenzoy\­ ring-UL-14C] AE 0001789 in cem (pre-emergence application):BayerCropScience, Stilwell, KS, USA; Report No.: MEUBX052: Documenl No.: M-284865001­ 1;2/15/2007; Pages 152 860.1300 KIIA 6.2.1 47069726

78 Fischer, D. R.: Roensch, W L.:The metabolism 0' [sulfonylbenzamide-ring-UL-14Cl and [methoxybenzoylring-UL-14C] 0001789 in com (seed treatment application):Bayer CropScience, Stllwel', KS, USA; Report No.: MEUBPool;**Oocume~** No.: M·284890-o1­ 1;2115/2007, Pages: 125 860.1300 KIIA6.2.1 47069727

79 Nguyen, T.;Determination ofthe total radioactive residue in sorghum raw agricultural commodities after seed treatmentwith ISu!fonylbenzamide·UL·14C] AE 0001789;Bayer CropScience. Stilwell. KS, USA: Report No.: MEUBPOI6; Document No.: M-284863-01· 1;2/21/2007; Pages: 48 860.1300 KIIA 6.2.1 47069728

Page: I ! 0[20 1035

## BIBLIOGRAPHY OF Sli'BMITTED DATA 80 Duah. F K., Lopez. R. T.: Beedle, E. C,;Metabo/ism of [sulfonylbenzamide-UL·l4Cj AE 0001789 in the lay;ng hen;Bayer CropScience. Slllwell. KS. USA: Report No: MEUBX041: Document No M·285276·01·1:311212007; Pages: 120 860.1300 Kl!A 6.2,2 47069729

81 Duah, F. K; Lopez. R. T,;Metabolism of[methoxybenzoyi. UL·14Cj AE 0001789 in laying hens;Bayer CropScience, Stilwell. KS, USA; ReportNo.: MEUElX042; Document Nc: M-285275-01·';319/2007: Pages: 103 860.1300 KIIA6.2.2 47069730

82 Duah, F. K,; Lopez. R. T.;Metabolism of [sulfonylbenzamide-UL-14Cj AE 0001789 in the lactating goat;8ayer CropScience, Stilwell, KS. USA; Report No,: MEU8X043: Document No.: M-28S269-01-1;3/9f2007: Pages: lS5 860,1300 KIIA6.2.3 47069731

83 Duah. F. K..lope:z, R. T,;MetaboUsm of [methoxybenzoyl· Ul-14Cl AE 0001789 in the lactating goat;Bayer CropSCience. Stllwell. KS. USA: Report No.: MEUBX044: Document No.: M-285267-01-1;3I1212oo7; Pages: 144 860,1300 KIIA6.2,3 47069732

84 Timberlake. 8, C,;Validation of BayerCropScience method US-008-P06-01 • M analytical methodtor the delerminatiOn of residues 01 AE 0001789 in crop matrices using LCIMS/MS;Bayer CropScience, Slilwell, KS, USA: Report No., RAUBP046; DocumentNo: M-286209-01. 1;3122/2007; Pages: 170 860.1340 KlIA4,3 47069733

85 Ennis, D.; Mclean, N.;lndependent laboratory validation of Bayer method U8..Q08-P06-01 • M analytical method for lIle determination of residves of AE 0001789 in crop matrices using LCIMSfMS;ALS laboratory Group, Edmonton, canada; Report No.: RAUBP022; Documen: No.: M-286201-01-1 ;1/2612007: Pages: 10' 86,:),1340 KIIA4.3 47069734

86 8illian. P.; Wolters, A;Analyticaf method 00961 for lIle determination of residues ofAE 0001789 and its metabolites cyclopropyl-sulfamoylbenzamide, sulfonamide-lactate and sulfonamide-alanine inion plant matrices by HPlC·MS/MS using stable-labelled internal ;Bayer CropScience AG. Monheim, Germany: Report No. MR·124105: 860,1340 KIIA4.3 47069735

87 Nguyen, T.;Exlraction efficiency of AE 0001789. AE 2300002. AE 2300003, and AE 0852999 used in the determinationofresidues ofAE 0001789 in plant matrices;Bayer CropScience, Stilwell. KS, USA; Report No.: RAUBP031; Document No,: M·285892-01· 1;311512007; Pages: 55 860,1340 KlIA 4.3 47069736

Page: :2 0[:0 1036

## BmLIOGRAPHYOF SUBMITTED DATA 88**S~lian,** p,; Wolters, A.;SlOrage stability of c:yclopropyl· sulfamoylbenzamide, suifonamlC!e-lactale and sulfonamide-alanine (metabolites ofAE 0001189) in plam mat/'lces fOl' 18 montns· Resultsfor an interval of0\0 12 months;6ayerCropScience AQ, Manheim, Germany, Report No.: MR-145105; Document No,: M-281141-01­ 860.1340 KIIA 6.1,1 47069737

89 Ibrahim, A: Perez. S, R:lndependentlabOratory validation ofBayer method no. UB·006-A06.(Jl • AE 0001189: An analytical method for the detemination ofAE 0001789 and AE 0852999 in caWe and biota uSing LG-MS/MS and stable isotopic intemal standan:ls;AOPEN Laboratones. Inc, Jacksonville, FL. USA; Report No.: AOPEN-2K6-982· (RAUBP025) 860.1340 KIIA4.3 41069738

90 lam, C. K.;Radiovalidallon of Bayer method US·006-A-06­ 01 - An analylicaf methOd for the determination ofAE 0001789 and AE 0852999 in catUe and biota using LCMS/MS and stable isotopic internal standan:ls;Bayer CropScience, Stilwell, KS. USA; Report No.: RAUBP028, Document No,: M-285782-01-1 ;3/9/2007: Pages: 52 860,1340 KIIA4.3 47069739

91 Nandihalti. U, B,: Woodard, D L.;In house labOl'atory validation ofthe method ofanalysis for the determination ofAE 0001789 and itsmetabolites in soil and sediment;Bayer CropScience. Stii'NeIl, KS, USA; Report No.: RAUBP007; Document No.: M-285696-01-1 ;319/2007: Pages: 111 860,1340 KIIA4,4 47069740

92 Nandihalli. U, e,; Boyle, S,;StabitityofAE 0001789 and its metabolites AE 1272799, AE 0852999, AE 0893016. AE 0467398 and AE 1448796 in soil rulingfrozen storage (reported through a maximum cl383 days storage);Bayer CropScience. Stilwell. KS. USA; Report No.: RAUBP005; DocumentNo.: M-285691.(Jl·1;319/2007: Pages: 49 850.1340 KIIA 7,3,' 47069741

93 Nandihalli. U, B,;Analy1lcal methodforthedetermination of residues ofAE 0001789 and**~s** metabolite AE 0652999 in water using LClMSIMS;BayerCropScience. Stii'Nell. KS, USA; Report No,: UB-005-W06-02; Document No,: M· 285011-01-1:1/1812007; Pages: 31 850,1340 KIIA4,5 47069742

94 Krebber. R; LeppeR, L;lndeoendent laboratory validation of method Ue-005-W06-01 for thedetermination ofAE 0001789 and its metabclites AE 0852999 in water uslna LClMS/MS;Bayer CropScience AG. Monheim, Germany; Report No.: MR.Q6/137; Oocument No
.. M-286048-01· 2:4/412007: Pages: 32 860.1340 K!tA4.5 47069743

95 Pruitt, W. E.;FDA PAM Mulliresidue method (MRM) testing for AE 0001789 (cyprosulfamide) and three metaboliles;EN-CAS Analytical Laboratories. W:nston· Salem.NC. USA; Report No,: RAUBX010; Document No.: M-285272-01-1;31712OO7; Pages: 72 860.1360 KIIA4.3 47069744

Page: 13 of:!O 1037

## BIBLIOGRAPHYOF SUBMITIED DATA 96 Lam, C. K.;AE 0001789- Magnitudeofthe residue in 860.1480 KIIA642 47069745 lactating cows;Bayer CropScience, Stilwell, KS, USA; Report No.: RAUBP019; Document No.: M-28526S-Q1­ 1:311412007: Pages: 326

97 Beedle, E. C,; Harbin, A. M,;AE 0001789 500 SC and 500 FS - Magnitude01 the residue Irv'on field com. _t com, and pop com;BayerCropScience, Stilwell, KS, USA: Report No.: RAUBX012; Document No: M-285793-01· 1;3f1512007; Pages: 609 860.1500 KIIAS,3.1 47069746

98 Beedle, E. C.;AE 0001789500 SC- Requestfor waiver of the study ofthe magnitudeoftheresidue inion field corn processed commodities;BayerCropScience, Stilwell, KS, USA; Report No.: RAUBP010; Document No.: M-285699­ 01-1;31812007; Pages: 64 860.1520 KIIA 6.5.4 47069747

99 Krolski. M. E.; Nguyen, T.;The accumulationof [14C] AE 0001789 residues in confined rotational crops;Bayer CropScience, Stilwell, KS, USA; Report No,: MEUBX050; Document No.: M-28443S-02·1;2I8I2oo7; Pages: 116 860.1850 KIIA 6.6.2 47069746

100 Beedle, E. C; Dominic, A. R;AEOO01789500 SCMagnitude

### orthe residue in field rotational crops ­ soybeans, turnips, and wheat (2-month plantback .nterval)

- Limited rotational crop;Bayer CrepScience, SUlwell, KS, USA; Report No.: RAUBP001; Document No.: M-285811· 01-1;3/1512007: Pages: 223 660.1900 K1IA6.6.3 47009749

101 Beedle, E. C., Dominic, A. R.;AEOOO1789 500 FS
. Magnitude ofthe residue infield rotational crops ­ soybeans, turnips, and wheat (1-month plant-back i:'!tervs!) • limited rotational crop;Bayer Crop Science. Stilwell, KS, USA; Report No.: RAUBP017; Document No. M·285264-01-1;3/1412007; i='ages 232 860.1900 KIIA6,S.3 47069750

102 Schuengel. M.;AE 0001789 • AcutetOXicity in t'le rat after orel administration;Bayer HealthCare AG, Wuppertal, Germany; Report No.: ATOl958; Document No.: M· 249800-01-2;1011912008; Pages: 27 870.1100 KIIA 5.21 KIIIA17.1.1 47009801

tQ3 SChuengel, M.;AE 0001789- AcutetoxiCity in the rat after dermal applicatior.;BayerHealthCare AG, Wuppertal, Germany: Report No.: ATOl909; Document No.' M· 247221.01-2;1011912006; Pages: 28 670.1200 KIIA5,2,2 KIIIA17.1.2 47069802

Page: !4 of20 1038

## BIBLIOGRAPHY OF SUBMITTEDDATA

, 04 Pauluhn. J.;AE 0001789 - (1st revision of report AT 01279, dated June 23. 2004) - Acute inhalation toxicity in rats:Bayer HealthCare AG.Wuppertal. Germany; Report No. AT01392; Document No.: M-Q75020-Q2-2;811912006; Pages: 70 870.1300 KIIAS.2,3 KIIIA17.1.3 47069803

105 Sclluenget. M.;AE 0001789 - Acuteeye irritation on rabbits;Bayer Healtl<jCare AG, Wuppertal. Germany; Report No.: AT02436; Document No.: M-258984-01-2;211912007: Pages: 24 870.2400 KIIA5.2,5 KIIIA17.1.5 4706980&

106 Schuenget. M.;AE**OOO~** 789 - Acute skin irritation/corrosion ill rabbils;Bayer HeafthCare AG. Wuppertal, Germany; Report No.:AT01649: Document No.: M-129076-Q,­ 2;1011912006; Pages: 22 870.2500 KIIA 5.24 KIIIA1 7.1.4 47069805

107 Vohr, H. W.;AEOO01789 - Study fortne skin sensitization effect in guinea pigs (guinea pig maximization tesl according to Magnussor. and Kligman);Bayer HealthCare AG.Wuppertal, Germany; Report No. AT01944; DocumeClt No.: M-249757-01-2;10119/2006; Pages: 29 870.2600 KIIAS.2.6 47069806

108 Odin-Feurtet. M.;AE0852999 - 28-day toxicity study in the rat by dietary administralion;Bayer CropScience S.A.,Sophla Antipolis, France; Report No.: SA 06281; Document No.: M-284847-o1-2;312712007; Pages: 387 870.3050 KIiAS.8 47069807

109 Me Elligott, A.;AE 0001789 - 9O-day toxiCity study In the rat by dietary administralion;Bayer Cr0p5cience, Sophia AntiPOlis. France; RepOrt No.: SA02352; Document No : M105691-01-2;6/212006; Pages: 374 870.3100 KIIA5.3.2 47069808

110 Kennel. P.;AE 0001789 - 9O-day toxicity study in the 'Tlouse by dietaryadministration:6ayerCr0p5C1ence. Sophia AntipOlis. France; Report No.: SA 03005; Document No.: M104791-01·2;513012006; Pages: 275 870.3100 KIIAS.3.2 47069809

111 Blanck. O.:AE 0001789-sulfamoylbenzoic acid (AE 0467398) - flQ.day toxicity study in the rat by dietary administration;BayerCropScience SA. Sophia AnUpoIis, France: Report No.: SA 06036: Document No.: M-28291'­ 01-2;3/23/2007. Pages: 438 870.31OC KIIAS.8 47069810**P~e:** 15000
1039

## BIBLIOGRAPHY OF SUBMITTED DATA 112 Eigenberg, D. A.;A90-day toxicilyfeeding study in the beagle dOg withtechnical gradeAE 0001789;3ayer CropScience. Stilwell, KS, USA; Report No.: 201279: Document No
.. M·263605-01·1 ;10f712005: Pages: 631 8703150 KIIA 5.3.3 47069811

'13 Wason. S.;AE 0001789· Developmental tOlticity study in the rabbil by gavage;Bayer CropScience SA. Sophia Antipolis, France; Report No,: SA 03349; Document No,: M· 261627-01·2:11/15/2006: Pages: 300 870.3700 KIIA 5.6.11 47069812

114 Wason. S.:AE 0001789· Developmentaltoxicity study in the rabbit by ga\lage {Complementary study);Bayer CropScienceSA. Sophia Antipolis. France; Report No: SA04196; Document No.: "'·273153-01·2:312612007, Pages: 186 87::),3700 KIIA5.6.11 47069813

115 Wason. S.;AE 0001789· Developmental toxicity study in the rat bygavage;Bayer CropSc::ience SA, SophiaAntipolis. France; Report No
.. SA 03348; Document No.: M·261462· 01·2;11/3012006; Pages: 310 870.3700 KIIA 5.6.10 47069614

116 Eiben. R:AE 0001789·T......a-generation reproduction study in wistar rats by adminisltation in the diet;Bayer HealthCare AG. Wuppertal. Germany; Report No, AT03567; Document No.: M·283510-01·2;3127/2007; Pages: 1231 870.3800 KIIA5.61 47069815

1>7 Eigenberg, D. A.;A chronictoxic::i1y feeding study in the beagle dog with technical grade AE oo01789;Bayer CropScience. Stilwell, KS, USA; Report No.: 201496-1; Document No.: M·283194-02·1;3J912007; Pages; 1034 870.4100 Kl1A 5.34 47069816

116 Langrand·Lerche, C,;Chronictoxicity and carcinogenicity studyofAE 0001789 ill the wister rat bydietary administration;BayerCropScience SA. Sophia Antipolis, France; Report No.: SA03277; Document No.: M-281767· 01·2;3/2612007; Pages. 3183 670.4200 KIIA5.5.1 KIIA 5.5.2 47069817

119 \"angrand·Lerche, C.;CarcinogeniC:ity study ofAE 0001789 in the C57BLI6J mouse bydietary administration;Baye' CropScience SA. Sophia Antipolis. France; Repol1 No
.. SA04065; Document No.: M-281276·01·2;11812007: Pages: 2591 870.4200 KIIA 5.5.3 47069818

Page 16or20 1040

## BIBLIOGRAPHY OF SUBMITTED DATA 120 Herbold. B.;AE 0001789 - Salmonella/microsome lesl Plate incorporationand preincubation melhod:Bayer Healt,,"Care AG, Wuppertal, Germany; Report No
.. AT03534; Document No.: M-283435-01-2:212212oo7: Pages: 53 8705100 KIIA 5 4.1 47069819

!21 Herbold, B.;AE 0001789 (Project: AE0001789)­ SalmoneUalmicrosometest - Plate incorporation and preincubation method ­ 1 sl amendmenl;Bayer HeallhCare AG. Wuppertal. Germany; Report No.: ATOI348; Documen1 No.: M-080265-02-2;10/1912006. Pages: 62 870.5100 KlIA 5.4.1 47069820

122 Herbold. B.;AE 0001789-sulfamoybenzoic acid (AE 0467398) - (Project: AE (01789)- Salmonellalmicrosome test plate incorporation and preincubation method;Bayer HcallhCareAG, Wuppertal. Germany; Report No.: AT03259; DocumentNo,: M-278703-01-2:212212007; Pages: 62 870,5100 KilA5.8 47069821

123 Herbold, B.;AE oo01789-cyclopropyl-sulfamoybenzamide (tested asAE. 0852999carboxamide) Salmonellalmicrosometest plate incorporation and prei,cubation method:6ayer HealthCareAG. Wuppertal, Germany: Report No.: AT03110; Document No.: M274230..01-2;512412006: Pages: 61 870.5100 KItAS,8 47069822

124 Herbold. B.:AE 0001789 - V79/HPRT-Test in**v~ro** forthe detection ofinducedforward mutalions;Bayer HealthCare AG, Wuppertat, Germany; Report No,: AT02816; Document No,: M-256527-Q1-2:10119/2006: Pages: 39 870.5300 KItA 5,4,3 47069823

125 Herl:>old, B,;AE 0001789-sulfamoylbenzoic acid (AE 0457398)- Project AE 0001789 V7MiPRT-teSlln vitro forthe detection

### or inducedforward mulaUons;Bayer HealthCere AG. Wuppertal, Germany; Report No
.. AT03095; Document No.: M-273551-Q1-2:311312oo7: Pages: 41 870.5300 KIIA5.8 47069824

126 Herbold, B.;AE 00OI789-cycIOpropyl-sulfamoylbenzamide (tested asAE. 0052999 carbox2I'nide) {project: AE 0001189) - V79/HPRT-Iestinvitro for the detection of inducedforward mutations;Bayer HealthCare AG, Wuppertal. Germany: Report No.: AT03164; Document No. M-274735-01-2;212112oo7; Pages: 42 870.5300 KIIA5,8 47069825

127 Herbold, 6.;AE 0001789 (Project: AE 0001789) • Ir vitro chromosome aberration test with Chinese hamster V79 cells - 1s1 amendment;Bayer HeafthCareAG, Wuppertal. Germany: Report No,: AT01574; Document No.: M· 129315-02-2;10/19/2006: Pages: 54 870.5375 K/iA 5.4.2 47069826

Page Iior20 1041

## BffiLIOGRAPHY OF SUBMITTED DATA 128 Thurn. M.;AE 0001789-Sulfamoylbenzoic acid· Project: AE 0001789 - Invitro chromosome aberration test with chinese hamster V79 cells;Bayer HealthCare AG. Wuppertal. Germany; Report No,: AT03096: Document No:**M·273607·0~-2;212212oo7:** Pages: 50 870.5375 KIIA 5.8 47009827

129 Thum, M.:AE 0001789-cyciopropyl-sulfamoybenzamide (Project AE 0001789) - In 'litro chromosome aberration testwith chinese hamster V79 cells;Bayer HealthCare AG. Wuppertal, Germany; Report No,: AT03121; Document No.: M·274272..{)1-2:212212007: Pages: 53 870.5375 KltA 5.8 47069828

130 Herbold. B.;AE 0001789 (Project: AE 0(01789)­ Micronucleus-teston the male**mo~** - 1st amendment;Bayer HealthCare AG. Wuppertal, Germany. Report No.; AT01302; Document No.: M"{)77491-02­ 2;10/1912006; Pages: 56 870.538515395 KIIA 5.4.4 47069829

131 Gilmore, R. G.: Stuart. B, P.;An acute oral neurotoxicity screening study with technical grade AE 0001789 in Wislar rats;Bayer C!opScience, Stllwell. 1<8, USA; Report No.: 201558; Document No
.. M-28319S-01-1;12f5l2006; Pages: 456 870.6200 KIIA 5.7.1 47069830

132 Gilmore,R G.: Hess, H. E.;A sutx;hronic neurotoxicity screening study with technical grade AE 0001789 in Wistar rats;BayerCropScience, Stilwell. KS, USA; Repon No,: 201573; Document No,: M-283218..{)1-1;1112812006; Pages: 644 870.6200 KIIA 5.7.4 47069831

133 Preu, M.; Spiegel. K.;[Sulfooylbenzamide-UL-14C)AE 0001789: Absorption, distribution. excre!lon and metabolism in the rat:Bayer CropScienceAG. Monheim, Germany: Report No.: MEF-05l326; Document No: M276053-01-2;111712006: Pages: 171 870.7485 KJIA 5.1.1 47069832

'34 Preu. M,: Spiegel. K.;[Methoxy-benzoyl-ring-UL-14C)AE 0001789: Absorption. distribution. excretion and metabolism inthe rat:BayerCropScienceAG. Monheim, Germany: Report No,: MEF..{)5/327; Document No. M· 274975-01-2;10/2312006; Pages: 100 870.7485 KIlAS,1.2 47069833

135 Eigenberg. DA:A28-day toxicity feeding study ir the beagle dogwi:h technical grade AEOOO1789;Mobay Chemical Corporation, StilweU, 1<5, USA; Report No.: 201146; Document No
.. M·254188-01·1;512312005: Pages: 257 870 Suppl. KIIA 5.3,1 47069834

Page: 1 8of2() 1042

## BffiLIOGRAPHY OF SUBMITTED DATA 130 Lunchick. C,;Assessment of the non-dietary exposure 10 cyprosulfamide from use on com. non·crop areas. sorghum seed treatment, and turf and omamentalS in the United States;Bayer Crop Science, RTP. NC. USA: Report No.: G201671; Document No,: M·285875-01-1;311212007: Pages: 17 None DocumentN 47069835

137 Zhenxu.T.;Aquatic ecological exposure assessmentfor AE 0001789 from use on com, tur., ornamentals, and sorghum in the UnHed States;Bayer CropScience. Stilwell, KS. USA; Report No.: MEUBX033; Document No
.. M· 286196-01-< ;312312007; Pages: 81 None DocumentN 47069830

138 Tang. Z.;Drinking water exposure assessment for AE 0001789 nom use on com, turf. ornamentals, and sorghum in \he United States;Sayer CropScience, Stilwell, 1<8, USA; Report No.: MEUBX032US; Document No.: M· 286195-01.1;3123(2007; Pages: 119 None Document N KIIA6.9.1 47069837

139 Bowers, L.: DaclJs, 5.;Tang, X.;Environmenlalfate and ecological risk assessmentfor cyprosulfamide (AE 0001789)forthe USA and CanaOa;Bayer Crop Science, RTP. NC. USA: Report No.: EBUBP023; Document No.: M· 286198-01·1 ;312812007; Pages: 76 None DocumentN 47069838

140 Lemke, V. J.:Evaluallon ofthe dietary exposure 10 cyprosulfamide (AE 0001789) and assessment.of potential risk for the U.S. and Canada;Bayer CropScience Stilwell. 1<8, USA: Report No.: RAUBP053; Document No. M-286231-01.1:3114r.2007; Pages: 52 None DocumentN KJIA 6.9.1 47069839

141 Buerkle. L. 'N.; i..asserre-Bigot. D.: Leake. C.; Linke-Ritzer. P.; McMillan-Staff. S. L.; Sowig. P.; Sutor, P.:Document N • Listofendpoints for the active substance cyprosulfamide (AE 0OO1789):BayerCropScience; Report No.' M-286406­ 01·1; Document No.: M·286406-01-1;41312007; Pages; 40 DocumentN 47069840

Page: 19 of20 1043

## BIBLIOGRAPHY OF S{;BMITTED DATA

142 Hines. D.;The phySical characterization ofAE0001789 SOO Document H 47069841C FS neutral end-use product;Bayer Crop Science. RTP, NC. Document J CA47069841 USA; Report No.: PCl06-005; Document No., M·2858B5­ KIIIA1 1.2.1 01·1:2116/2001' Pages: 129 KlllAl 1.2.2 K1I1A11.2.3 K1I1Al1.4.1 KIIlA11.4.2 KIIiA1 1.4.3.1 KIlIA1 1.4.:1.3 KUIA1 1.4.5.1 KJllAl 1.5 KllIAl 11.6 K1I1A12.1 K111A12.10.1 KIIIA12.10.2 KIIIA12.11 KIIlA12.13 KIlIM 214 KIIIA12.2.1 KIIIA12.2.2 KIIIA12.3.1 KIIIA12.3.2 KIIIA12.3.3 KIIIA12.4.1 K1I1A12.4.2 KIlIA12.5.1 KIIIA12.5.2 KIIIA12.53 KIIIA12.6.1 KIIIA12.6.2 KIlIA12.7.1 KIlIA12.7.2 KIIIA12.7.3 KIIIA12.7.4 KJllAl 2.7.5 KIIIA12.76 KIIIA12.B.1 KIIIA12.8.2 KIIIA1 2.8.3.1 KmA1 2.B.3.2 KIIIA12.8.4 KUIA12.S.S.1 KIIIA12.S.52 KIllAl 2.S.6.1 KIIIA12.S.6.5 KUlA12.8.8.1 KIIIA1 2.8.8.2 KIIIA12,9.1 KllIA12.9.2 KltIA15.2.1 KIIlA1S.2.5 KIl1A17.9.1 KIIIA17,9.2 143 Cunny. H. C.;Bridgiog rationale for acute toxiCity ofAE 8702600 KIl1A17.1.7 47069842 0001189 FS 500formulation;BayerCropScience. Research Triangle Par1(. NC. USA; RE!I)OI"l No.: HC-12007. Document No. M-285317-01-1;212312007; Pages: 12

Page: 20 oflO 1044

# BayerCropScience Material Safety Data Sheet MSDS Number: 102000006398 MSDS Version 1.0 

### CYPROSULFAMIDE TECHNICAL Revision Date: 08/01/2008

Product Name ULFAMIDE TECHNICAL Chemical Name benzamide, N-[[4-[(cyclopropylamino)carbonyljphenyljsulfonyl]-2-methoxy Common Name Cyprosulfamide MSDS Number 102000006398 Chemical Formulation C18H18N205S Bayer CropScience 2 T.W. Alexander Drive Research Triangle PK. NC 27709 USA For MEDICAL, TRANSPORTATION or other EMERGENCY call: 1-800-334-7577 (24 hours/day) For Product Information call: 1-866-99BAYER (1-866-992-2937)

Hazardous Component Name Cyprosulfamide 221667-31-8



### NOTE: Please referto Section 

### 11 fordetailed toxicologicalinformation.

Emergency Overview Caution! Dust may form explosive mixtureswith air. Physical State powder Appearance white Routes of Exposure Inhalation, Ingestion
Eye
Skin Ingestion Inhalation Wash off immediatelywith plenty ofwaterfor at least 15 minutes. Remove contact lenses, ifpresent. afterthe first 5 minutes, then continue rinsing eye. Get medical attention if irritation develops and persists. Wash off immediately with soap and plenty ofwater. Ifsymptoms persist. call a physician. Rinse mouth. Keep patientwarm and at rest. Do NOT induce vomiting. If symptoms persist. call a physician. Move to fresh air. Keep patient warm and at rest. Ifsymptoms persist, call a physician. Page 1of7

1045

# BayerCropScience Material Safety Data Sheet MSDS Number: 102000006398 MSDS Version 1.0 

### CYPROSULFAMIDE TECHNICAL Notes to Physician Signsand Symptoms Treatment To date no symptoms are known. Initial treatment: symptomatic. There is no specific antidote. SECTION 5. FIRE FIGHTING MEASURES

Autoignition Temperature Explosiveness Fire and Explosion Hazards
Suitable Extinguishing Media Fire Fighting Instructions 630 

### °C I 1166 of Notexplosive. 

### 92/69/EEC, A.14 

### IOECD 113 In the eventoffire the following can be released: Nitrogen oxides (NOx) Sulphur oxides Carbon monoxide (CO) Water spray, Foam Remove productfrom areas offire, orotherwise cool containers with water in orderto avoid pressure being built up dueto heat. Do not allow run-offfrom fire fighting to enter drains orwater courses. Firefighters should wear NIOSH approved self-contained breathing apparatus and full protective clothing.

Personal Precautions

MethodsforCleaning Up
Additional Advice Use personal protective equipment. Remove all sources of ignition. Avoid dustformation. Keep unauthorized people away. Isolate hazard area. Avoid contact with spilled product orcontaminated surfaces. Avoid dust formation and electrical charging (sparking) because dust explosion mightoccur. Use mechanical handling equipment. Keep in suitable, closed containers for disposal. Information regarding safe handling, see section 7. Information regarding personal protective equipment, see section 8. Information regarding waste disposal, see section 13. 7. HANDLINGAND Page 2 of7

1046

# Bayer CropScience Material Safety Data Sheet MSDS Number 102000006398 MSDSVersion 1.0 

### CYPROSULFAMIDE TECHNICAL Handling Procedures

Storing Procedures
Work/Hygienic Procedures

Min/MaxStorage Temperatures Ensure adequate ventilation. Avoid dustformation. Dust mayform explosive mixtures with air. Keep away from heat and sources of ignition. Take measures to prevent the build up ofelectrostatic charge. Handle under inert gas. Keep containers tightly closed in a dry, cool and well-ventilated place. Keep away from food, drink and animal feedingstuffs. Wash hands thoroughly with soap and waterafter handling and before eating, drinking, chewing gum, using tobacco, using thetoilet orapplying cosmetics. Remove Personal Protective Equipment (PPE) immediately after handling this product. Remove soiled clothing immediately and clean thoroughly before using again. Wash thoroughly and put on clean clothing. Recommended minimum transport/storage temperature: -10 DC /14 

### OF Recommended maximum transport/storage temperature: 40°C/104 

### OF

General Protection Eye/Face Protection Hand Protection Body Protection Respiratory Protection Avoid contactwith skin and eyes. Do not breathe dust. Tightlyfitting safety goggles Chemical resistant nitrile rubber gloves Wear long-sleeved shirt and long pants and shoes plus socks. No personal respiratory protective equipment normally required. Respiratory protection should only be used to control residual risk of short duration activities, when all reasonably practicable steps have been taken to reduce exposure at source e.g. containment and/or local extractventilation. Always follow respirator manufacturer's instructions regarding wearing and maintenance.

Appearance Physical State powder pH approx. 3.9 (1.0 %) at 23 DC suspension in water Page 3 of 7

1047

# BayerCropScience Material Safety Data Sheet CYPROSULFAMIDE TECHNICAL MSDS Number 102000006398 MSDS Version 1,0 Vapor Pressure < 21.9 JJPa at 50°C Density 1.51 g/cm3 at 20°C Boiling Point The substance decomposes before reaching the boiling point. Melting I Freezing Point 217°C 1423 

### of Watersolubility 3.4 mgll measured 

### at pH 4 approx. 1,1 gIl measured at pH 7 approx. 26 gIl measured 

### at pH 8 Minimum Ignition Energy < 3 mJ MolecularWeight 374.4 g/mol Decomposition 245 - 355°C Temperature DSC , Heating rate: 10 Klmin Partition coefficient: n­ 0.158 log Pow: -0,8 at23°C octanollwater measured at pH 7 SECTION 10. STABILITYAND REACTIVITY

Hazardous Reactions incapable 

### of deflagration Dust may form explosive mixtures with air. Stable under recommended storage conditions.

Acute Oral Toxicity rat: LD50: > 2.000 mglkg Acute DermalToxicity rat: LD50: > 2,000 mgtkg Acute Inhalation Toxicity rat: LC50: > 3.51 mgtl Highest attainable concentration. Determined in the form ofa respirable aerosol. Skin Irritation rabbit: No skin irritation. Eye Irritation rabbit: No eye irritation. Sensitization guinea pig: Non-sensitizing. Page 4 of7

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# BayerCropScience Material Safety Data Sheet MSDS Number: 102000006398 MSDS Version 1.0 

### CYPROSULFAMIDE TECHNICAL Assessment Carcinogenicity Did not show carcinogenic effects in animal experiments. ACGIH None. NTP

None. IARC

None. OSHA None. Reproductive & No indications of reprotoxic effects were observed in reproduction studies in Developmental Toxicity animals. Neurotoxicity Did not show neurotoxic effects in animal experiments. Mutagenicity In vitro and in vivo mutagenicitytests do not indicate genotoxic activity. SECTION 12. ECOLOGICAL INFORMATION Toxicityto Fish Cyprinodon variegatus (sheepshead minnow) LC50: > 106 mgtl ToxicitytoAquatic Plants Lemna gibba (duckweed) EC50: > 104 mgtl Exposure time: 7 d Pseodokirchneriella subcapitata EC50: > 99.7 mgtl Exposure time: 96 h Acute ToxicitytoAquatic Invertebrates Waterflea (Daphnia magna) statictest EC50: > 102 mgll Exposure time: 48 h Toxicityto other organisms Acute Dietary Bobwhite quail (Colinus virginianus) LD50: > 2,000 mgtkg OECD Test Guideline 205 Avian Dietary Toxicity Test Mallard duck LD50: > 4,959 mgtkg Exposure time: 5 d OECD Test Guideline 213/214 Honeybees contact/oral toxicity bees Page 50f7

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# BayerCropScience Material Safety Data Sheet MSDS Number: 102000006398 

### CYPROSULFAMIDE rECHNICAL MSDS Version 1.0 LD50: > 100**~g/bee** Exposure time: 48 d Non-hazardous for bees. OECD Test Guideline 207 Earth worm acute toxicity earthworm (Eisenia fetida) LC50: > 1,000 rng/kg Exposure time: 14 d Environmental Do not allowtoget into surface water, drains and ground water. Precautions

General Disposal Guidance Container Disposal In accordance with current regulations and, ifnecessary, consultation with the site operator and/orwith the responsible authority, the product may be taken to a waste disposal site or incineration plant. Rinsed packaging may be acceptable for landfill, otherwise incineration will be required in accordance with local regulations. SECTION 14. TRANSPORT INFORMATION

US DOT: Not Regulated IMDG: Not Regulated IATA/ICAO: Not Regulated Freight Classification: Chemicals, N.O.1.

US Federal Regulations TSCA list None. US. Toxic SubstancesControlAct (TSCA) Section 12(b) Export Notification (40 CFR 707, Subpt D)

None. SARA Title III • Section 302 - Notification and Information None. SARA Title III - Section 313 - Toxic Chemical Release Reporting None. Page 6 of7

1050

# BayerCropScience Material Safety Data Sheet MSDS Number: 102000006398 

### CYPROSULFAMIDE "rECHNICAL MSDS Version 1.0 

### US States Regulatory Reporting CA Prop65

This product does not contain any substances known to the State ofCalifornia to cause cancer. This product does not contain any substances known to the State ofCalifornia to cause reproductive harm. 

### US State Right-To-Know Ingredients

None. 

### Canadian Regulations Canadian Domestic Substance List None. 

### Environmental CERCLA None. 

### Clean Water Section 307 Priority Pollutants None. 

### Safe Drinking WaterAct Maximum Contaminant Levels None.


### SECTION 16. OTHER INFORMATION 

# ...................... NFPA 704 (National Fire Protection Association): Health - 2 Flammability - 3 Reactivity - 1 Others - none 0= minimal hazard. 1 = slight hazard. 2 

### = moderate hazard. 3 = severe hazard. 4 = extreme hazard Reason to Revise: New Material Safety Data Sheet Revision Date: 

### 08/01/2008 This information is provided in good faith but without express orimplied warranty. The customer assumes all responsibility for safety and use not in accordance with label instructions. The product names are registered trademarks of Bayer.

Page 7 of7

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