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3. Test substance dosage formulations and analysis 

The appropriate amount of test 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 of methylcellulose 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% of nominal values, Stability of the compound in suspension in the vehicle was 

determined before· the start of the 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 similar conditions to those of the 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. 	Body weight 


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


3. Food consumption 

Full feeder weights were measured on GD: 3,4,5,6,8, 10, 12, 14, 16, 18,20,22,24,26 and 28. 

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 of no 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 were then discarded. 

On GD 29, surviving females were killed by intravenous injection of Dolethal® for examination of 

their uterine content. Each female was first subjected to macroscopic examination of the visceral 

organs and the number of ribs was recorded. In the case of no visible uterine implants, but with 

corpora lutea, uterine horn(s) were immersed in a 20% solution of ammonium 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 were then discarded. 

The reproductive tract was weighed (gravid uterine weight), dissected out and the following 

parameters recorded: number of corpora lutea, number of implantation sites, number of resorption 

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. 	 Fretal examination 

All data were recorded without knowledge of treatment group. After internal examination of the 

neck, the head of fretuses from approximately half of each 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 

examination was performed. 

6. Statistics 

Means and standard deviations (STD) for all maternal and litter parameters were calculated for each 

group. Statistical analyses were performed on the following variables using TERA TEST Version 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 of corpora lutea, number of implantation 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. Maternal Observations 

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 poor food consumption prior to death. At autopsy the principal findings were 

as follows: 

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Table 5.6.11-2 Macroscopic findings in decedent animals at 500 mg!kglday 

I 

Macroscopic findings 

I 

Organs I Number of decedents affected at 500 

mg/kg/day 

Kidney Yellow sediments 417 

Marbled pattern 217 

Urinary bladder Yellow sediments 217 

Red contents 117 

Liver I 

Prominent lobulation 517 

Uterus Red contents 217 

As this dose level had clearly exceeded a Maximum Tolerated Dose (MTO), treatment at 


500 mglkg/day was terminated. Surviving females were killed prior to scheduled autopsy with no 


macroscopic examination performed and no tissues taken. 


At 125 mglkg/day, one female was found dead on GO 19 due to gavage dosing error. 


At 50 mg/kg/day, one female was found dead on GD 15. The cause of death was also judged to 


have been gavage error. There were no other mortalities during the course of the study at 125 or 


50 mglkg/day. 


2. Abortions 

One female from the control group aborted on GO 28. There were no other abortions on the 

study. 

3. Clinical signs 

Of the surviving females at 500 mglkg/day, treatment-related clinical signs consisted of yellow 

sediment in the urine 00/18 dams and few faeces for 4/18 dams, prior to termination ofthis dose 

group. At 125 mglkg/day, treatment-related clinical signs consisted of yellow sediment in the 

urine of 2/25 dams. There were no treatment-related clinical signs observed at 50 mglkg/day. 

4. Pregnancy rate 

The pregnancy rate was not established at 500 mglkglday as the majority of females 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 of between 

0.21 and 0.29 kg prior to termination ofthis dose level. No effect on mean body weight change or 

mean corrected body weight change (maternal body weight change independent of the uterine 

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weight) was noted at 125 or 50 mglkg/day. 

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 of the group. 


Food consumption was similar to the control group at 125 and 50 mglkg/day. 


6. Maternal necropsy and microscopic findings 


At autopsy, yellow sediment in the kidney was noted in 1125 females at 125 mglkg/day, whilst the 


decedent female at 125 mglkg/day and 1/25 females at 50 mglkg/day had a kidney with a marbled 


pattern appearance. In the absence of microscopic findings in this target organ at 125 mg/kg/day, 


these findings were considered not toxicologically relevant. 


At the microscopic examination of the urinary bladder and kidney at 125 mglkg/day, no 


treatment-related fmdings were noted. 


B. Litter data 


There was no effect on litter parameters including early and late resorptions, feetal death status, 


percentage of male feetuses and mean feetal body weight, at either remaining dose level. At 50 mglkg/day, 


mean feetal weights for the combined sexes and for females only were slightly reduced compared to the 


controls. However, in the absence of an effect at the higher dose level of 125 mglkg/day, this finding was 


considered to be incidental. 


C. Fretal necropsy findings 

1. Fretal evaluation: external observations 


The few anomalies observed occurred in a non dose-related manner, in isolation, or in the case of the 


finding short tail, where the incidence at 125 mglkg/day was slightly higher than in the control group, 


both in terms of mean percentage offretuses affected (U%) and percentage of Iitters affected (4.2%), 


the incidence was within the in-house historical control range (0.0-3.1 % and 0.0-4.3%, respectively) 


and was therefore considered to have occurred by chance. 


No variant findings were noted. 


The incidence of runt feetuses was similar at 125 and 50 mglkg/day, and only slightly higher than 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, in terms of mean percentage offeetuses affected (1.9%) and 


percentage of litters affected (12.5%), however, the incidence was comparable to the in-house 


historical control range of 0.0-4.4% 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 considered to 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, 

including the controls. 

3. Fretal evaluation: skeletal observations 

Two fretuses from separate litters at 125 mglkglday had hemivertebrae in at least one lumbar 

vertebrae. The mean percentage of fretuses affected (0.9%) and percentage of litters affected (8.3%) 

was slightly outside the historical control range of 0.0-0.5% 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 either the thoracic or lumbar region. Therefore this difference 

was considered to 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 of both fretal and litter parameters. This condition was 

associated with the absence of some caudal vertebrae in 3/4 cases. It should be noted however, 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% 

compared to a range of 0.0-1.0% for the historical data, but was within the historical control range for 

percentage of litters 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 

considered to 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 

considered to be fortuitous. 

III. CONCLUSION 

In conclusion, a dose level of 500 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 the New Zealand White Rabbit. 

(Wason S., 2005b) 

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Report: KIIA 5.6.11/02, Wason, S.; 2oo5c 

Title: AE 0001789 - Developmental toxicity study in the rabbit by gavage 

(complementary study) 

Report No & SA 04196 

Document No 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 of 0.5% methylcellulose 400. 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 of 500 mglkg/day was found to be too toxic 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 of ribs were recorded and the dams were 

evaluated for number of corpora lutea, number and status of implantations (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 half of each 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 of the 

Staples and Schnell technique 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 of few/mucoid faeces on several occasions, yellow sediment 

in the urine on GD 24-26 and 28, and anogenital soiling, red traces on tray and placental 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 

the macroscopic 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 or skeletal 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: white powder 

Lot/Batch: 08466/0013 

Purity: 97.4 % 

CAS: 221667-31-8 

Stability oftest compound: Stable in suspension in the vehicle (aqueous solution of 

methylcellulose 400 at 0.5%) at concentrations of 0.1 and 250 gil 

for a period of 28 days under similar conditions to those of the 

current study. 

2. Vehicle and lor positive control: The vehicle was an aqueous solution of methylcellulose 400 at 

0.5% 

3. Test animals: 

Species: Rabbit 

Strain: New Zealand White Crl:KBL (NZW) 

Age: 18 weeks approximately 

Weight at dosing: 3.12 to 3.70 kg for the females 

Source: Charles River laboratories, Chiitillon-sur-Chalaronne, France 

Acclimation period: At least 4 days prior to dosing 

Diet: Laboratory animal pellets 110C-IO from S.A.F.E. (Scientific 

Animal Food and Engineering, Augy, France), ad libitum 

Water: Tap water, ad libitum 

Housing: Animals were caged individually in polycarbonate cages on a 

perforated cage floor 


Environmental conditions ­

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Temperature: 19 ± 2°C 


Humidity: 55 ± 15% 


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


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


B. STUDY DESIGN: 

1. In life dates: November 02 to December 07, 2004 at Bayer CropScience, 

Sophia Antipolis, France. 

2. Animal assignment and 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 

of insemination was designated as GD O. The animals were approximately 18 weeks of age on arrival 

and were received on GD 1 or 2. On each day of mating, 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 equivalent volume of vehicle alone (methylcellulose). 

The experimental groups were as follows: 

Tbl56113Sdd'd a e •• - tu ly eS)2D an amma aSS)2Dment 

Test group 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 substance dosage formulations and analysis 

The appropriate amount of test substance was periodically (six formulations at 62.5 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. The concentration was checked for all formulations. Homogeneity and concentration 

checks of AE 0001789 were between 92 and 107% of nominal values. Stability of AE 0001789 in 

suspension in the vehicle was determined before the start of the 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 

to those 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, 18,20,22,24,26 and 29. 

3. Food consumption 

Full feeder weights were measured on GD: 3,4,5,6,8, 10, 12, 14, 16, 18,20,22,24,26 and 28. 

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 killed for human reasons 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 of no 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 were then discarded. 

On GD 29, surviving females were killed by intravenous injection of Dolethal® for examination of 

their uterine content. Each female was first subjected to macroscopic examination of the visceral 

organs and the number of ribs was recorded. In the case of no visible uterine implants, but with 

corpora lutea, uterine horn(s) were immersed in a 20% solution of ammonium 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 of corpora lutea, number of implantation sites, number of resorption 

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. 	 Fretal examination 

All data were recorded without knowledge of treatment group. After internal examination of the 

neck, the head of fretuses from approximately half of each 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 

examination was perfonned. 

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6. Statistics 

Means and standard deviations (STD) for all maternal and litter parameters were calculated for each 

group. Statistical analyses were performed on the following variables using TERA TEST Version 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 of corpora lute a, number of implantation 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. MATERNAL OBSEV ATIONS 

1. Mortality 


At 250 mg/kglday, two dams were sent to necropsy on GD 28 after aborting. Clinical signs for the 


first female consisted of few faeces on several occasions, mucoid faeces on one occasion, yellow 


sediment in the urine on GD 24-26 and 28 and anogenital soiling, red traces on tray and placental 


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 GD 28. 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 in body weight. In both cases, the body weight loss was associated 


with a marked 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 


considered to be incidental findings. 


3. Pregnancy rate 


The pregnancy rate was unaffected by treatment, with a pregnancy rate of 100% at 250 


mg/kg/day and 96% in the control group. 


4. Body weight 


No effect on mean maternal body weight change or mean maternal corrected body weight change 


(maternal body weight change independent of the uterine weight) was observed at 


250 mg/kglday. 


5. Food consumption 


Mean maternal food consumption was comparable to the controls at 250 mglkglday. 


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6. Maternal necropsy and microscopic findings 


No treatment-related maternal necropsy findings were observed at autopsy. 


B. LITTER DATA 

There was no effect on litter parameters including early and late resorptions, fretal death status, 

percentage of male fretuses and mean fretal body weight. The percentage of male 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 of males controls as the percentage of male 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<ET AL NECROPSY FINDINGS 

1. Fretal evaluation: external observations 


There were no treatment-related malformations. 


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 


occurred by chance. 


There were no variant findings at the external fretal examination. 


2. Fretal evaluation: visceral observations 


There were no treatment-related malformations. 


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 at the fretal or litter level, these findings were considered to have occurred by chance. 


3. Fretal evaluation: skeletal observations 


There were no treatment-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 


considered to have occurred by 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) of pelvic girdle 


unossified. But the differences were not statistically significant for these findings, and were therefore 


considered to be fortuitous. 


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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) 

IIA 5.7 Neurotoxicity 

All studies presented in this section were conducted between 2005 and 2006 and complied with the EU, 

OEeD, USEPA and Japanese MAFF testing guidelines and Good Laboratory 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 of neurotoxicity at any dose level and no compound-related 

gross or microscopic lesions at the high dose of 2060 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 

0001 789/kg body wt/day for males and females, respectively). 

Table 5.7-1 Summary ofneurotoxicity with AE 0001789 

Type ofstudy 

Doses 

NO(A)EL 

(mglkgld) 

LOAEL 

(mglkgld) Adverse effects / target organs 

Acute neurotoxicity in the rat 

M-283196-0 1-1 

0, 126, 508, 2060 mglkg bw 

508 2060 Urine staining in both sexes 

90-day neurotoxicity in the rat IM-283218-01-1 

0,1000,3000,9000ppm 

592/748 

(M/F) 

>5921>748 

(MIF) 

None 

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IIA 5.7.1 Acute neurotoxicity - rat 

Report: KIIA 5.7.1101, Gilmore RG., 2006 

Title: An acute Oral Neurotoxicity Screening Study with Technical Grade AE 0001789 in 

Wistar Rats 

Report No & 201558 

Document No 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 of 0 (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 of AE 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 of the 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 during the week prior to treatment and on days 0 (the day of 

treatment, at the time of peak effect), 7 and 14. All animals placed on study were subjected to a gross 

necropsy. Selected animals (six/sex/dose level) were perfused, the brain was weighed in order to 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 of AE 0001789 used in the study were 0, 126, 508 and 2060 

mglkg for males and females. 

Findings attributed to exposure to AE 0001789 were as follows: 

2060 mg/kg: Urine staining was evident in both sexes on day 0 and resolved by the following day. 

508 mg/kg: There were no signs related to treatment in either sex. 

126 mg/kg: There were no signs related to treatment in either sex. 

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In conclusion, compound-related effects following a single oral dose of the test substance were limited to 

the high dose (2060 mglkg) males and females. Effects in high-dose males and females consisted of urine 

staining, which resolved the following day after treatment. Based on these findings, a NOEL of 508 mg 

AE 0001789/kg body weight was established in both sexes. There was no evidence of neurotoxicity at 

any dose level and there were no compound-related gross or microscopic lesions at the high dose of2060 

mglkg. 

I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test Material: AE 0001789 

Description: white powder 

Lot/Batch: 08466/0013 

Purity: 97.4% (October 2003, certified to October 20,2005) 

CAS: 221667-31-8 

Stability oftest compound: Stable in the vehicle at the room temperature storage conditions 

used in this study. 

2. Vehicle and lor positive control:0.5% methylcellulose/O.4% Tween 80 in deionized water 

3. Test animals: 

Species: Rat 

Strain: Wistar HAN CRL: WI (HAN) 

Age: at least 9 weeks 

Weight at dosing: 232.8 to 309.0 g for the males - 167.4 to 215.4 g for the females 

Source: Charles River Laboratories, 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 

throughout the study except during neurobiavioral testing 

Water: Tap water, ad libitum 

Housing: Animals were caged individually in suspended stainless steel 

wire-mesh cages. 

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Environmental conditions ­

Temperature: 22 ± 4°C 

Humidity: 50 ±20% 

Air changes: Approximately 10 changes per hour 

Photoperiod: Alternating 12-hour light and dark cycles 

B. 	 STUDY DESIGN: 

1. In life dates: March 7, 2005 to April 7, 2005 at Bayer Crop Science LP, 

Stilwell, KS, United States. 

2. Animal assignment and treatment 

There were 12 animals of each 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 of 0 (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. 

Table d 	 • 5.7.1.-1: Study eSi2n and animal aSSi2ment 

Dose Group mWk2 bwt) 

~AI'''' m.ental Parameter Control 125 500 2000

Total number of animals/sex/group 12 12 12 12

Behavioral Testing (FOB, Motor Activity) • 12/sex 12/sex 12/sex 12/sex 

Neuropathology· 6/sex O/sex O/sex 6/sex 

. FOB and motor actIVIty were assessed prior to dosmg and agam durmg days 0, 7 and 14. 

h 	 Tissues from the mid- and low-dose groups were not examined, as no treatment-related neuropathology was noted at the 

highest dose level, 

The rationale for dose selection was based on the results of an 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, at a dosing volume of 10 mllkg. Animals were 

observed for mortality and clinical signs for 13 days after treatment. The test substance produced no 

clinical signs or mortality at the limit dose. These results support the use of a limit dose (2000 

mglkg) in the neurotoxicity study but provided no information to establish the time of peak effect. 

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Thus, the results from a study using radio-labeled AE 0001789 were examined to estimate the time 

of peak effect. In that study, adult Wistar male and female rats received a single oral (gavage) dose 

of2 or 200 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 after treatment 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 estimated time of peak blood concentrations within this dose range, the 

FOB began approximately 50 minutes (minimum) following dose administration, with the automated 

test of activity concluding at about three hours after treatment. 

3. Diet preparation and analysis 

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 of AE 0001789 in the vehicle were established using samples at nominal concentrations (l0 

and 200 mg/m!) that bracketed the range of concentrations used in the present study (nominal 12.5 

and 200 mg/ml). Homogeneity was accepted if the percent relative standard deviation (%RSD) was 

<5%. Each dosing suspension was also analyzed to measure the concentration of AE 0001789. 

Homogeneity Analysis: Homogeneity of the 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 standard deviations (%RSD) of 0.39% and 1.33%, respectively. 

Stability Analysis: The stability (at room temperature conditions) of AE 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 or 2000 mg/kg. respectively). 

Concentration Analysis: Doses of 0, 125, 500 and 2000 mg/kg for males and females ranged from 

101% to 103% of the 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 of significance was set at p ~ 0.05, with the exception of Bartlett's test, 


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which was tested at p S 0.00 I. 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 of variance using Bartlett's test. Group means with equal variances were analyzed further 

using an Analysis of Variance (ANOVA), followed by a Dunnett's test if a significant F-value was 

determined in the ANOV A. In the event of unequal variances, these data were analyzed using 

nonparametric statistical procedures (Kruskal-Wallis ANOVA followed by the Mann-Whitney U test 

for between-group comparisons). For the FOB, continuous data were first analyzed using a 

Repeated-Measures ANOVA, followed by a one-way ANOVA if there 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, if any, 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 (CA TMOD) Procedures, with post-hoc 

comparisons using Dunnett's test and an Analysis of Contrasts, respectively. 

Motor and locomotor activity (total session activity and activity for each lO-minute interval) was 

analyzed using ANOV A procedures. Session activity data were analyzed using a Repeated-Measures 

ANOV A, followed by a one-way ANOV A if there was a significant interaction with test occasion. 

For days on which there is a significant treatment effect, Dunnett's test was used to determine which, 

if any, 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 ANOV A to determine on which days there was a 

significant treatment by interval interaction. For those days, the data for each interval were subjected 

to analysis using a one-way ANOV A to determine at which intervals there was a significant 

treatment effect. For those intervals, Dunnett's test was used to determine which groups, if any, were 

significantly different from the control group. 

Continuous pathology data (e.g., brain weight) was initially evaluated using Bartlett's test to analyze 

for homogeneity of variance among groups. Groups with homogeneous variances were analyzed 

further using an ANOV A followed by Dunnett's test for pair-wise comparisons. In the event of nonhomogeneous 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-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 

statistical tests 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 of moribundity. Detailed physical examinations for clinical signs of 

toxicity were carried out and recorded daily. 

2. Body weight: 

Animals were weighed weekly as a component of the FOB. Additionally, all animals were 

weighed on the day of sacrifice for tenninal body weight measurement. 

3. Food Consumption: 

Food consumption was not measured in this study. 

4. Neurobehavioral Assessment: 

All animals that were assigned to the study were tested using the FOB and motor activity on four 

occasions - one week prior to treatment, approximately 50 minutes after administration of the 

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 balanced across test 

times and test devices, and no animal would be tested more than once in the same maze. On the 

day of FOB 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 of the 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 into the mazes to measure activity. 

a. Functional Observational Battery (FOB): 

The FOB closely follows the battery of tests 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) 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 of a 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 of removal 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. Motor and Locomotor Activity: 

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 of the figure-eight alleys and one in each of the 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 of the six, ten-minute intervals. 

Motor activity was measured as the number of beam 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 of a 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 of all organs, body cavities, cut surfaces, external orifices and surfaces. On day 

14, a minimum of six males and six females at each dose level were selected for perfusion and 

collection of tissues, with replacement, as necessary, if the 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 without perfusion. 

Micropathology examinations were perfonned on a comprehensive battery of neural 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) 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. Clinical signs 

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 attributed to 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 of historical 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 

after treatment was administered. The remaining incidental fmding consisted of areas of alopecia 

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). 

Table 5.7.1.-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 total number ofanimals exhibiting the sign at least once/number ofanimals in each group 

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 of the study. 

B. BODY WEIGHT 


Body weight was not affected by treatment in either sex at any dose level. 


Table 5 ... 71-3· Body Weight (g ± s.d.)

Dose Level (mg/kg bw) ·-----------......----1....-------------- -------------------­

Observation (g ± s.d.) Control 125 500 2000 

Males 

Day 0 264±20 260±19 257±18 263±14

Day 7 280±30 284±23 281±22 285±19

Day 14 311±27 311±33 302±30 305±24

Females 

Day 0 188±14 184±12 181±9 185±14 

Day 7 201±13 195±1l 194±14 201±13 

Day 14 214±14 207±13 206±15 210±13 

Values represent mean ± s.d. 


n=12 *=p :=; 0.05, **=p :=; 0.01, when compared to control means. 


C. NEUROBEHA VIORAL RESULTS 

1. FOB findings 


There were no treatment-related findings at any dose level in either sex. 


The only finding, areas of alopecia, was considered not to be related to treatment 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. 

Table 5.7.1.-4: Functional Observational Battery Results 

Males 

---------------------------~~~~~:~;::-------------------------

Dose Level (mg/kg bw) -----t-c-on-t-ro-I---rl-----~;~----r---~~~-------l---------;~~~-------

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Day 14 

Handling - Other: 

Not Observed 

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: 

Not Observed 

Alopecia. Present 

12(100) 

0(0) 

11(92) 

1(8) 

12(100) 

0(0) 

12(100) 

0(0) 

Day 14 

Handling - Other: 

Not Observed 

Alopecia. Present 

12(100) 

O{O) 

11(92) 

)(8) 

12(100) 

0(0) 

12(100) 

0(0) 

Values represent the number ofanimals and % incidence (in parentheses) with observation. 

n=12 animals/dose level *=p ~ 0.05 . ..P 0.01 comparedwilh controls 

2. Motor activity 

An examination of inherent variability, using the average pre-treatment values among the four 

groups of males and females, confinn that differences of ± 20% are within the range of normal 

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 locomotor activity at any dose level in either sex. 

Differences from control that were considered incidental and unrelated to treatment included 

measures of motor (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 considered to be related to treatment because the 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 of activity 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 

referenced historical controls) activity were well within the range of historical control. 

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Table 5.7.1.-5: Summary Session Motor Activity Results (Percent Difference from Control)' 

MALES 

Nominal Dose Pre-treatment Day 0 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 

a Percent greater (+j or less (-j than concurrent controlfor N=12. 

Summary session motor activity was not statistically different from control (* = p :$; 0.05; ANDVA). 

Summary Session Locomotor Activity Results (Percent Difference from 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 Percent weater (+j or less (-j than concurrent controlfor N=12. 


Summary session locomotor activity was not statistically different 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 left uterine hom in one mid-dose female. 

2. Terminal body and brain weights 

There was no compound-related effect on terminal body weight or brain weight in perfused 

males or females at any level of exposure. 

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Table 5.7.1.-6: Absolute and Relative Brain Weights 

Dose Level (mglkg bw) ,-­ -_._---_..__._----------_._-]--_._-_._---------------------­

Weights (g) Control 125 500 2000

Males 

Bodywt • 308.7±28.6 305.8±27.9 298.0±28.5 303.6±22.8

Brain wt b 1.819±0.074 1.817±0.070 1.858±0.096 1.848±0.068

b 0.583±0.036 O.620±0.O52 0.629±0.082 0.622±0.055

Female 

212.6±12.8 205.4±11.9 204.5±14.6 209.8±13.3 

Brain wt 1.732±0.OS7 1.669±O.032 1.75O±O.077 1.733±.049 b 

Brainlbody wt b 0.810±0.055 0.822±O.048 62±O.048 0.838±0.045 

a N=12 

b N=6 

*Statistically different (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 lower dose of AE 0001789 were not examined. 

III. CONCLUSION 

In conclusion, compound-related findings following a single oral dose of the test substance were limited 

to the high dose (2060 mglkg) males and females. Effects in males and females consisted of urine 

staining, which resolved the day after treatment. There was no evidence of neurotoxicity at any dose level 

and no compound-related gross or microscopic lesions at the high dose of 2060 mg/kg. These results 

establish a NOEL of 508 mg AE 000 1 789/kg body weight. 

(Gilmore R.G., 2006) 

IIA 5.7.2 Delayed neurotoxicity following acute exposure 

AE 0001789 is not an organophosporous insecticide and there are no indication of neurotoxicity in any of 

the other studies, therefore this type of study is not triggered. 

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IIA 5.7.3 28-day delayed neurotoxicity 

AE 0001789 is not an organophosporous insecticide and there are no indication of neurotoxicity in any of 

the other studies" therefore this type of study is not triggered. 

IJA 5.7.4 Subchronic neurotoxicity - rat - 90-day 

Report: KIIA 5.7.4/01, Gilmore R.G., 2006 

Title: A Subchronic Neurotoxicity Screening Study with Technical Grade AE 0001789 in 

WistarRats 

Report No & 201573 

Document No 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 of 0, 

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 ad libitum consumption throughout the study 

except during neurobehavioural testing. All 12 rats/sexldietary level were used for neurobehavioural 

evaluation, with micropathology performed on selected tissues from 6 rats/sex from control and high-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 prior to treatment and during weeks 2, 4, 

8 and 13. Ophthalmologic examinations were conducted on all animals prior to 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 examined microscopically for lesions. 

The mean daily intake of the test substance (mg AE 0001789/kg body wVday) 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 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 of 9000 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 oftest compound: 

AE 0001789 

white powder 

08466/0013 

97.4% (October 2003) ; 97.5% (October 2005) 


221667-31-8 


Stable in the diet at 10 and 10 000 ppm at the room temperature 


for 7 days or at freezing storage after 28 days. 


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

3. Test animals: 

Species: 

Strain: 

Age: 

Weight at dosing: 

Source: 

Acclimation period: 

Diet: 

Water: 


Housing: 


Environmental conditions ­

Temperature: 

Humidity: 

Air changes: 

was allowed to evaporate prior to administration 

Rat 

Wistar HAN CRL: WI (HAN) 

approximately 8 weeks 

210.0 to 285.1 g for the males 141.4 to 179.5 g for the females 

Charles River Laboratories, Inc., (Raleigh, NC) 

7 days 

Purina Mills Rodent Lab Chow 5002 in meal form provided for 

ad libitum consumption during the acclimation period and 

throughout the study except during neurobiavioral testing 

Tap water, ad libitum 

Animals were caged individually in suspended stainless steel 

wire-mesh cages. 

22 ± 4°C 

50 ±20% 

Approximately 10 changes per hour 

925 
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Photoperiod: Alternating 12·hour light and dark cycles 

B. STUDY DESIGN: 

1. In life dates: August 22, 2005 to December 8, 2005 at Bayer Crop Science LP, 

Stilwell, KS, United States. 

2. Animal assignment and treatment 

There were 12 animals of each 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 

administered the test substance in the diet at nominal concentrations of 0, 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. 

Table 5.7.4.-1 Study DesilD 

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 number ofanimals!sexlgroup 12 12 12 12 

Behavioural Testing (FOB, Motor Activity) • 12/sex 12/sex 12/sex 12/sex 

Neuropathology b 6/sex O/sex O/sex 6/sex 

Ophthalmic Examination C 12/sex 12!sex 12/sex 12/sex . ,

FOB and motor actIVIty was assessed prIOr to dOSing and again during weeks 2. 4, 8 and 13. 

b 	 Tissues from the mid- and low-dose groups were not examined. as no treatment-related neuropathology was noted at the 

highest dietary level. 

OphthalmiC exams were performed prior to dosing and at week 12, 

The rationale for dose selection was based primarily on the results of a 90-day toxicity study, with 

the test substance administered via the diet at nominal concentrations of 0, 1000, 4000, and 12,000 

ppm to male and female Wistar rats (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 of the urothelium in the urinary 

bladder of one male and one female. Findings in animals treated at the 12,000 ppm dietary level 

included the death of two females. Animals that survived until study termination were observed with 

soiled fur around the anogenital area (two males) and hair loss (two males and one 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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kidney weights in both sexes. Microscopic findings included slight centrilobular hepatocellular 

hypertrophy in males and simple hyperplasia of the urothelium in the urinary bladder of males and 

females. In both sexes, a slight increase in the incidence and/or severity of basophilic 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 evident in both sexes. The incidence of brown pigment in 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 in the severity of decreased 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 9000 ppm in both sexes. 

3. 	 Diet preparation and analysis 

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 ad libitum consumption for a period of one week prior to changing, at which time any 

uneaten feed was collected and disposed of by incineration. 

The concentration of AE 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 of the 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 of the 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 

percent relative standard deviations (%RSD) of 1.98% and 1.81%, respectively. 

Stability Analysis: The stability ofAE 0001789 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 of storage. 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 of the active ingredient in 

the 0, 1000, 3000 and 9000 ppm dietary levels used in this study averaged 97% to 98% of the 

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 of significance was set at p :s; 0.05, with the exception of Bartlett's test, 

which was tested at p:s; 0.00 I. 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 if there 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, if any, were significantly different from the control group. Categorical data 

collected in the FOB were analyzed in a similar manner, 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 

analyzed using ANOV A procedures. Session activity data were analyzed using a Repeated-Measures 

ANOVA, followed by a one-way ANOV A if there was a significant interaction with test occasion. 

For days on which there is a significant treatment effect, Dunnett's test was used to determine which, 

if any, 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 ANOV A to determine on which days there was a 

significant treatment by interval interaction. For those days, the data for each interval were subjected 

to analysis using a one-way ANOV A to determine at which intervals there was a significant 

treatment effect. For those intervals, Dunnett's test was used to determine which groups, if any, were 

significantly different from the control group. 

Continuous pathology data (e.g., brain weight) was initially evaluated using Bartlett's test to analyze 

for homogeneity of variance among groups. Groups with homogeneous variances were analyzed 

further using an ANOV A followed by Dunnett's test for pair-wise comparisons. In the event of nonhomogeneous 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 Exact Test in cases of significant 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 of moribundity. Detailed physical examinations for clinical signs of 

toxicity were carried out and recorded daily. 

2. Body weight: 

Animals were weighed weekly. Additionally, all animals were weighed on the day of sacrifice for 

terminal body weight measurement. 

3. Food Consumption: 

Individual food consumption was measured weekly. Daily food consumption was averaged over 

the duration of the study, based on a per kg 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 of A.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 to the 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 of animals to mazes were done in a semi-random manner, such that groups 

were balanced across test times and test devices, and no animal would be tested more than 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 that day. The dose group identification was 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 

behavioural testing. 

a. Functional Observational Battery (FOB): 

The FOB closely follows the battery of tests 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, ensuring the consistency of the results of each technician. 

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 of a 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 

reacti vity . 

Observations during handling included: ease of removal 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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Tier 2, llA, Sec. 3, Point 5: Cyprosulfamide 

response, auditory response, tail pinch, pupil size at nonnal lighting, pupil response, righting 

reflex, grip strength body weight, body temperature, and landing foot splay. 

b. Motor and Locomotor Activity: 

Motor and locomotor activities were measured approximately 30 minutes after the last animal 

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 of the figure-eight alleys and one in each of the 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 of the six, ten-minute intervals. 

Motor activity was measured as the number of beam 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 of a 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-darkened room. The pre-exposure examination was used to select animals which did 

not have ophthalmological defects that could interfere with the interpretation of study results. 

Animals with such defects were used for vendor surveillance or were sacrificed without 

necropsy. The pupillary reflex was tested using a penlight or transilluminator with Finnoff and 

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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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) 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 to allow for calculation of organ to body weight ratios. 

Micropathology examinations were performed on a comprehensive battery of neural 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) and the cauda equina were embedded in paraffm and examined utilizing haematoxylin 

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 were present in the high-dose group. 

II. RESULTS AND DISCUSSION 


A. OBSERV A nONS 

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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Tier 2, (lA, Sec. 3, Point 5: Cyprosulfamide 

range of historical control and considered incidental to treatment. 

Remaining findings were also considered incidental and unrelated to treatment and consisted 

of areas of alopecia (1 low-dose female), maloccluded teeth (lmid-dose female) and dermal 

lesions described as a scabs on cheek and shoulder (one mid-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 

ITeeth, Maloccluded -Incisors-Lower, Both 0/12 0/12 1112 0/12 

Numbers represent the total number ofanimals exhibiting sign at least once/number ofanimals in each group 

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 of the study. 

B. BODY WEIGHT 


Body weight and body weight gain were not affected by treatment in either sex at any dietary level. 


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Table 5 ... 74-3 : Bodv Weigbt and body weigbt gain (g:l:: s.d.) . 

Dose Level ppm (mg/kg bw/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 

Day 42 213.2±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 Body weight gain 

(Dav 0-91) -Males 

160.9±24.0 176.2±21.2 1 69.0±31.2 167.5±23.4 

Total Body weight gain 

(Day 0-91) -Females 

79.5±10.0 75.7±I3.S 7S.4±1O.0 69.3±9.8 

Values represent mean ± s.d. 


n=12 *=pS05, when compared to control. 


C. FOOD CONCUMPTION 

There were no compound-related effects on food consumption in either sex, at any dietary level. 

Table 5.7.4.-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 represent mean ± s.d *~pS05, when compared to control. noll 12 

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D. NEUROBEHA VIOURAL RESULTS 

1. FOB findings 

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 of one at 3000 

and 9000 ppm, respectively). These findings were within the range of historical controls and 

considered incidental to treatment. 

Additional observations considered incidental and unrelated to treatment 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 - : FunctionalObserva f lona I B att ery R esuIts

I 	 I 

I Dose Level ppm (mg/kg bw/day) 

I Observation 1000 3000 9000

Males 	 Control (6' 65.2) (6 193) I ~~ 592) i (2~_ {fi' 2SI} 748) - i 

~atment No Findings 12 (100) 12 (100) 12 (100) 12 (l00)

Week 2 No Findings 12(100) ! 12 (100) 12 (100) 12 (100) 

Week 4 No Findings 12 (tOO) I 12 (100) 12 (I DO} 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 I No Findings 12 (100) I 12 (100) 12(100) , 12 (\ 00) 

Values represent the number ofammals and % mCldence (m parentheses) With observation. 

n=12 animals/dietary level *=p505, compared with controls 

Table 5.7.4.-5 : Functional Observational Battery Results (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 • 	 Not Observed 


Urine, Yellow 
 Week 2 12 (I 00) 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: 

Not Observed 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 Not Observed 

Urine, YeHow I 0(0) 0(0) 0(0) 1 (8) 

Values represent the number ofanimals and % incidence (in parenthesesj with observation. 

n= ]2 animals/dietary level *=p'iO, 05, compared with controls 

2. Motor activity 

An examination of the inherent variability, using the average pretreatment values among the 

four groups of males 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 

and there was no relationship to dose or direction (relatively higher and lower than control). 

Table 5.7.4.-6 Summary Session Motor Activity Results (Percent Difference from Control) 

MALES ! 

Nominal Dose Pretreatment Week 2 Week 4 Week 8 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 Week 2 Week 4 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 Percent greater (-,-) or less (-j than concurrent controlfor n=] 2, 


Summary session motor activity was not statistically different from control (p'!OO,05: ANOVAj at any time for any 


dietary level. 


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Table 5.7.4.-7 Summary Session Locomotor Activity Results (Percent Difference from 

Control) 

MALES 

Nominal Dose Pretreatment Week 2 Week 4 Week 8 Week 13 

1000 ppm - 19 - 2 - 9 - 3 - 12 

3000 ppm +11 +20 - 4 + 24 + 9 

9000 ppm + 7 + 1 + 3 + 10 + 14 

FEMALES 

Nominal Dose Pretreatment Week 2 Week 4 Week 8 Week 13 

1000 ppm + 3 - 18 - 35 - 13 -0.4 

3000 ppm + 6 - 2 -10 - 3 -4 

9000 ppm + 2 - 6 - 11 + 12 + 22 

a Percent greater (+) or less (-) than concurrent controlfor n=12. 


Summary session motor activity was not statistically different from control (pg).05; ANOVA) at any time for any 


dietary level. 


Motor and locomotor activity data were also analyzed for differences at each 10-minute 

interval of each test session. Measures of motor and locomotor activity were not statistically 

different from controls at any dietary level, on any test occasion, in either sex. 

Habituation was not affected by treatment with AE 0001789 in males or females at any dietary 

level. 

E. OPHTHALMOLOGY 


All ophthalmologic findings were considered incidental and unrelated to exposure to AE 0001789. 


F. SACRIFICE AND PATHOLOGY 

1. Gross pathology 

There were no compound-related gross lesions evident at terminal sacrifice in males or 

females at any dietary level. 

2. Terminal body and brain weights 

There was no compound-related effect on terminal body weight or brain weight in perfused 

males or females at any level of exposure. 

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Table 5 7 . . . 1 -8' Absolute and Relative Brain Weights 

Weights (g) 

Dose Level ppm (mglkg bw/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 

Brain Wt.b LS9S±0.IOS L925±O.073 LS36±0.247 L933±O.041 

BrainlBody Wt.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/Body Wt.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 

i Kidney/Body wt.b O.851±O.O49 O.849±O.043 0.846±O.O34 O.852±O.045 

Values represent mean ± s.d 

a N= 12for remaining animals. * Statistically different (p ~O.05)from the control. 

b N=6 

3. Neuropathology 

There were no histopathologic changes in neural tissues from perfusion-fixed control and 

high-dose rats of both sexes that were related to administration of the 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 either sex. 

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III. CONCLUSION 

In conclusion, there was no evidence of toxicity or neurotoxicity at any dietary level and no compoundrelated gross or microscopic lesions at the highest dietary level of 9000 mglkg. For both 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) 

IIA 5.7.5 Postnatal developmental 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 studies on metabolites 

The toxicological properties of two metabolites of AE 0001789 were investigated in a series of studies 

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 have the potential to leach to shallow groundwater with 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 to the rat. 

The metabolite AE 0852999 (M 02, 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 groundwater resulting in concentrations close to 0.1 Ilg/L. This 

metabolite was devoid of genotoxic potential and had a similar toxicological profile to the parent 

compound after subacute administration to the rat. 

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Table 5.8-1 Results oftoxici studies witb tbe metabolites 

Stud S ecies Results 


AE 0467398 M 03) 


Ames test 
 Negative 


M-278703-0 1-2 
 Salmonella T h. 

· In vitro V79!HPRT Locus Gene Negative 


Mutation Assay 
 hamster 

cells 

In Vitro Chromosome Aberration 

•M-273551-0 1-2 

Negative 

Chinese hamster 

M-273607-0 1-2 

•test 

V79 lun cells 

I 90-day dietary study: NOEL 15 000 ppm ( 1094 mglkglday in . 

·M282911012 - - - 0, 400, 2000 and 15000 ppm I 

Rtf, a ema I es, 888 m glkgld ay III rna I ) es 

I 

. AE 0852999 (M 02) 

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-274272-0 1-2 V79 lung cells 

i 28-day dietary study: NOEL 300 ppm (25.5 mglkg bw/day in • 

I0, 40, 300, 800, 3000 ppm females, 22.8 mglkglday in males) IRat 

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 and pre-incubation method 

Report No & A T03259 

Document No M-278703-01-2 

Guidelines: OECD 471 (1997); EEC Directive 2000/32/EC Method B13/14 (2000); EPA 

Health Effects Test Guideline (OPPTS 870.5100; 1998) 

GLP Yes (certified laboratory) 

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Executive summary: 

In this in vitro assessment of the mutagenic potential of AE 0467398 (Batch KTS 10207-1-1,96.0% of 

purity), histidine dependent auxotrophic mutants of Salmonella 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 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 of 

incubation at 37°e, the numbers of revertant colonies were scored using an automated colony 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 of this effect, 

this range could nevertheless be used for assessment purposes. 

AE 0467398 did not cause any significant increase in the number of revertant colonies in either the presence 

or absence of metabolic 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 or without S9 mix in the plate incorporation as well as in 

the pre-incubation modification ofthe Salmonella/microsome test. 

I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test Material: AE 0467398 

Description: fine white powder 

Lot/Batch: KTS 10207-1-1 

Purity: 96.0% 

CAS: 138-41-0 

Stability of test compound: Stable for the duration of the 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 at 0.2 Ilg!plate, 


4-Nitro-I,2-phenylene diamine (Merck-Schuchardt) for TA 1537 


at 10 Ilg!plate and TA 98 at 0.5 Ilg!plate, 


Mitomycin C (Fluka) for TAl 02 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 of the S9 


mix in all strains at 3 Ilg!plate. 


3. Test organisms: 

Species: Salmonella typhimurium L T2 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 since then. 

4. Test compound concentrations: 

Plate incorporation 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 of the 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 of reversion to prototrophy is evaluated in negative control and 

treated groups. 

1. Plate incorporation assay: 

AE 0467398 or the control material was dissolved in 0.1 ml of DMSO. 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 of S9 mix or buffer and 2 ml of soft 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 of a standard protocol with a maximum dose 

of 5000 Ilg/plate and at least 5 additional doses. If less than three doses were used for assessment, at 

least two repeats were performed. 

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 of the pre-incubation period. 2 ml of molten soft agar 

were added to the tubes, the content mixed and plated onto Petri dishes with solid agar. After 48 

hours of incubation at 37°C, the numbers of revertant colonies were also scored using an automated 

colony counter. 

3. Acceptance criteria: 

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 studied with S9 mix. 

4. Assessment criteria 

A reproducible and dose-related increase in mutant colonies of at least one strain was considered to 

be positive. For TA 1535, TA 100 and TA 98, this increase should be about twice that of negative 

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controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 and 

increase of about 100 mutants should be reached. Otherwise, the result was considered as negative. 

II RESULTS AND DISCUSSION 

There was no indication of a bacteriotoxic effect of AE 0467398 at doses of up to 158 J.lg!plate. The total 

bacterial counts consistently produced results comparable to the negative controls, or differed only 

insignificantly. No inhibition of growth was noted either. Higher doses had only in the plate incorporation 

trial a weak, strain-specific bacteriotoxic-effect. Therefore they could be used for assessment purposes. 

None of the five strains concerned showed in the plate incorporation test a dose-related and biologically 

relevant increase in mutant colonies over those of the negative controls. This applied both to the tests with 

and without S9 mix and was confirmed by the results of the pre-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, and thus demonstrated the system's sensitivity and the activity of the S9 mix. 

III CONCLUSION 

No indication of mutagenic effects of AE 0467398 were found at doses of up to 5000 J.lg!plate in any of 

the Salmonella typhimurium strains used in the assay. 

(Herbold B., 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 Hamster V79 cells 


Report No & AT03096 


Document No 
 M-273607-01-2 


Guidelines: OECD 473 (1997); EEC Directive 2000/32IEC Method BI0 (2000); EPA Health 


Effects Test Guideline (OPPTS 870.5375; 1998) 


GLP Yes (certified laboratory) 


Executive summary: 

In this in vitro assessment of the clastogenic potential of AE 0467398 (batch KTS 10207-1-1,96.0% of 

purity), Chinese Hamster V79 cells were 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. After 4 hours treatment, the medium was changed and the cells were harvested 

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14 hours later. In addition, cells treated at 2100 Ilglml were harvested 30 hours after the 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 0467398 concentrations of 525, 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 prior to harvest to arrest the cells in a metaphase-like stage 

of mitosis. 

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 of the 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 mitomycin C and cyclophosphamide induced clastogenic effects and demonstrated 

the sensitivity ofthe test system and the activity ofthe used S9 mix. 

Based on this test, AE 0467398 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 oftest compound: Stable for the duration of the study 


2. Control materials: 	 Negative: Culture medium 

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 of 4 hours and 0.03 Ilglml for 

a treatment period of 18 hours. 

Cyclophosphamide (Endoxan 100 mg injection vials of dry 

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substance, Baxter Oncology GmbH) with S9 mix at 2 Ilglml. 

3. Test organisms: 

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. 	 Test compound concentrations: 

AE 0467398 was used with or without S9 mix at 525, 1050 and 

2100 Ilglml. 

B. STUDY DESIGN AND METHODS: 

The experimental phase of the study was performed from March 9 to April 4, 2006 at Bayer HealthCare 

AG (PH-PO P Health Care Toxicology) .. 

The in vitro cytogenetic test is a mutagenicity test system for the detection of chromosome aberrations in 

cultured mammalian cells. The test is designed to detect structural aberrations (chromatid and 

chromosome aberrations) in cells at their first post-treatment mitosis. 

1. Determination of cytotoxicity: 

Duplicate cultures were exposed in a pre-test to AE 0467398 at concentrations ranging from I to 

2100 Ilg/ml with or without S9 mix for 4 hours. In addition, ceIls were exposed without S9 mix for 18 

hours to concentrations ranging from 50 to 2100 Ilglml. The mitotic index was determined for all 

cultures. The number of mitotic cells among a total of 1000 cells per culture was determined using a 

light microscope at a magnification of about 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 of AE 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 at the end of the treatment period. 

AE 0467398 at 500 Ilglml and above slightly decreased the pH in the medium (reduction to 6.3). The 

osmolality in the medium of the pre-test was not changed by concentrations of up to 2100 Ilglml of 

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AE 0467398. 

2. Treatment protocol: 

Chinese hamster V79 cells were passaged on the day prior to treatment. Approximately Ixl06 cells 

were seeded in 20 ml of medium 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 calf serum. Immediately before treatment, the medium was removed from the 

cultures. For the trials without S9 mix, 20 ml of medium containing 2% foetal calf serum and 0.2 ml 

of test substance solution were added to each flask. For the trials with S9 mix, 19 ml of medium 

containing 2% foetal calf serum, 1 ml of S9 mix and 0.2 ml of test substance solution were added to 

each flask. The cells were incubated for 4 hours at 37°C. After 4 hours of treatment, the medium was 

removed, the cells were washed with PBS and 20 ml of fresh medium containing 10% foetal calf 

serum was added to the flasks. The flasks were placed in a COrincubator for the remaining 

incubation time. In the cultures treated for 18 hours, the medium was not removed. 

To each flask 0.2 ml of Colcemid-solution (40 f.lglml) was added to each flask 2 hours prior to the 

end of the incubation period to arrest the 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. Chromosome preparations: 

After the removal of the 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 

of a hypotonic solution (0.4% KCI; 37°C) was added to each tube. 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 of fixative 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 least two slides were generated per culture. 

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4. Evaluation criteria: 

Coded slides were evaluated using a light microscope at a magnification of about 1000. 

Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two 

parallel cultures, were examined. 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 of one chromatid with dislocation of the chromatid end), fragment (part of chromosome 

without centromer), deletion (result of a break with the terminal chromatid part of the 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. Assessment criteria: 

An assay was acceptable, if there was a biologically relevant increase in chromosome aberrations 


induced by positive controls and if the numbers of aberrations for the negative controls were in the 


expected range. 


An increased incidence of gaps of both types without a concomitant increase of other aberration types 


was considered not to be an indication of a clastogenic effect. 


A test was considered positive, if there was a relevant and statistically significant increase in the 


aberration rate. 


A test was considered negative, if there was no such increase at any time interval or if there were 


statistically significant values, which were, however, within the range of historical 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 of AE 0467398-treated and positive 

control groups to the respective solvent control group. 

The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of 

the corresponding solvent control) using the one-sided Chi2-test. 

The numbers of metaphases with aberrations (including and excluding gaps) and of metaphases with 

exchanges were compared (provided that these data superceded the respective solvent control). The 

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one-side Che-test was used for the statistical evaluation. 

A difference was considered to be significant, if the probability of error 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 

0467398 at concentrations of up to and including 21 00 ~g/ml and, in addition, without S9 mix for 18 

hours to AE 0467398 at concentrations of up to and including 2100 I-lg/ml. Without and with S9 mix 

substance precipitation did not occur in the medium. 

B. MITOTIC INDEX 

Mitotic index without S9 mix: In comparison to the solvent control, the mitotic indices were only reduced 

in the cultures treated for 18 hours, starting at 1050 I-lg/ml. The cultures treated 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 of the mitosis rate. The positive control cyclophosphamide reduced the mitosis rate. 

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 after 4 hours treatment. 

Survival index with S9 mix: In comparison to the solvent control, the treated cultures showed no relevant 

reduction of the survival rate. The positive control cyclophosphamide reduced the survival rate. 

D. CHROMOSOME ABERRATION 

AE 0467398 without S9 mix: No biologically relevant and statistically significant increases of numbers of 

metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 

hours. The same was true for a treatment period and total 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 of metaphases with aberrations and demonstrated the sensitivity of the test system. 

AE 0467398 with S9 mix: No biologically relevant and statistically significant increases of numbers 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 of Chinese hamster V79 cells with AE 0467398 concentrations of 525, 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 of 525, 1050 and 2100 

flglml were selected for reading without S9 mix. 

None of these cultures treated with AE 0467398 in the absence or presence of S9 mix showed statistically 

significant and biologically relevant increases of numbers of meta phases with aberrations. 

The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and 

demonstrated the sensitivity of the test system and in the case of cyclophosphamide the activity of the 

used S9 mix. 

Based on the results of this test, AE 0467398 was considered not to be clastogenic for mammalian cells in 

vitro. 

(Thurn M., 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 of induced forward mutations 


Report No & AT03095 


Document No M-273551-01-2 


Guidelines: OECD 476 (1997); EEC Directive 2000/321EC Method B17 (2000); EPA 


Health Effects Test Guideline (OPPTS 870.5300; 1998) 


GLP Yes (certified laboratory) 


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Executive summary: 

The purpose of the study was to assess the point mutagenic potential of AE 0467398 (batch KTS 10207­

1-1,96 % of purity) at the hypoxanthine-guanine phosphoribosyl 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 of AE 0467398 ranging from 

12.5 to 2100 Ilg/ml. Precipitation of AE 0467398 in the culture medium was not observed. 2100 Ilg/ml 

AE 0467398 reduced the pH in the medium of the pre-test from 7.6 for the vehicle control to 6.3 for 2100 

!Jog/ml AE 0467398. The osmolality in the medium of the 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 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 or to a range of concentrations 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 

each test substance directly after treatment (survival to treatment). 

Cells in 75 cm2 flasks were incubated to permit growth and expression of induced mutations. Cells were 

subcultured (= count I, normally after 3 days) by reseeding cells in 75 cm2 flasks. At the end of the 

expression period (=count 2, normally a total of 6 days), cultures were reseeded in Petri dishes without 

hypoxanthine but containing 6-TG for selection of mutants. In addition, 200 cells per dish were seeded in 

5 ml culture medium to determine the absolute cloning efficiency for each concentration. After incubation 

for 6 to 8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of 6­

TG resistant colonies in the mutation assay dishes and the number of colonies in the cloning efficiency 

dishes. Two trials were performed. 

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 of the vehicle controls. Ethyl methanesulfonate and 

dimethylbenzanthracene induced clear mutagenic effects demonstrating the sensitivity of the test system 

and the activity of the S9 mix. 

Based on these results, AE 0467398 was considered to be non-mutagenic in the V79IHPRT forward 

951 
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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 


Stability of test compound: Stable for the duration ofthe study 


2. Control materials: 	 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 

for ethyl methane sulfonate as it is a liquid. 

Positive: 	 Ethyl methanesulfonate (EMS), a directly alkylating agent, used 

at a final concentration of 900 Ilglml in non-activation trials. 

Dimethylbenzanthracene (DMBA), promutagen requiring a 

metabolic activation, used at a final concentration of 20 Ilglml 

for trials with S9 mix. 

3. 	 Test organisms: 

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 

rapid population doubling time (10 to 14 hours) 

Culture condition: 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 in the 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 23.9 mg/ml) and was 

kept frozen at -80"C. The batch was tested for contamination and 

cytotoxicity prior to 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 unable to use 6-TG for DNA synthesis. Therefore, cell colonies formed in 

the presence of 6· TG are considered to represent mutants at the HPR T gene. 

1. Determination of cytotoxicity: 

Exponentially growing V79 cells were plated in 20 ml culture medium in a 75 cm2 flask with a total 

volume of 275 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 or to a range of 

concentrations ofthe test substance for 5 hours in 20 ml medium containing 2% FCS. In experiments 

with metabolic activation 1 ml of medium 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 of the plates. 

Cytotoxicity was expressed by comparison of colonies in treated cultures versus vehicle control 

cultures (relative cloning efficiency). 

2. Treatment protocol without metabolic 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 cells were exposed to vehicle alone or to a range of concentrations of the test substance for 

5 hours 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 1.5xl06 cells per 75 cm2 

flask and in 5 ml culture medium using 200 cells per Petri dish (diameter of 60 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 of induced mutations. Cells 

were subcultured count I, normally after 3 days) by reseeding 1.5x 106 cells into 20 ml of medium 

in 75 cm2 flasks. At the end of the expression period (""count 2, normally a total of 6 days), cultures 

were reseeded in Petri dishes (diameter of 100 mm) at 3xl05 cells per dish (8 dishes per culture) in 

20 ml culture medium without hypoxanthine but containing 10 Ilglml 6-TG for selection of mutants. 

In addition, 200 cells per dish (diameter of 60 mm, 3 dishes per culture) were seeded in 5 ml culture 

medium to determine the absolute cloning efficiency for each concentration. After incubation for 6 to 

8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of 6-TG 

resistant colonies in the mutation assay dishes and the number of colonies in the cloning efficiency 

dishes. 

Two trials 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 ml 

culture medium and additonally \ ml of S9 mix were added to the flasks for the treatment period, 

resulting in a concentration of 5% S9 mix in the cultures. The number of 6-TG resistant mutants and 

viability were determined as in the non-activation assay. 

Two trials were performed. 

4. Parameters assessed: 

The parameter "survival to treatment" in % was determined on the basis of the following calculation: 

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Mean number of colonies (treated cultures) x 100 

Mean number of colonies (vehicle control cultures) 

The "absolute population growth" was calculated using the following fonnula: 


Absolute population growth (for each culture) = cell count I x cell count 2 


The parameter "relative population growth" shows the cumulative growth of the treated cell 

populations, relative to the vehicle control. 

Absolute population growth treated culture x 100 


Absolute population growth of corresponding vehicle control culture 


The ability of cells to fonn colonies at the time of mutant selection is measured by the parameter 

"absolute cloning efficiency". It is expressed in %. 

Mean number of colonies per dish x 100 


200 


The "mutant frequency" is calculated for each group by dividing the total number ofmutant colonies 

by the number of cells seeded (usually 8-10 plates at 3 x 1 05 cells per plate), corrected for the absolute 

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

cells. 

Total number of mutant colonies x 100 

Number of evaluated dishes x 3 x 105 x C.E. 

5. Acceptance criteria: 

The average cloning efficiency ofthe negative and vehicle controls should be at least 50%. 

The average mutant frequency ofthe vehicle controls should not exceed 25 x 10-6 cells. 

The mutant frequency ofthe two cultures of the vehicle and lor the negative control should differ 

only to an acceptable extent. As a rule ofthumb, the difference of mutant frequencies should not 

be greater than 5 x 10-6

. 

The positive control should induce an average mutant frequency of at least three times that ofthe 

vehicle control. 

1fnot limited by the solubility of the test substance in the vehicle the highest concentration should 

induce cytotoxicity of about 80 to 90% or should be a concentration where precipitation 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 should be 10% or greater. 

However, these criteria may be overruled by good scientific judgement. 

7. Assessment criteria: 

Mutant frequencies were only used for assessment, if at 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 least two to three times that of the highest negative or vehicle control value observed 

in the respective trial. If this result was reproduced in a second trial, the test substance was 

considered to be mutagenic. 

Despite these criteria, a positive result was only considered relevant, if no significant change in 

osmolality compared to the vehicle control was observed. Otherwise, unphysiological culture 

conditions may have be the reason for the positive result. 

A test substance was judged as equivocal if there was no strictly concentration related increase in 

mutation frequencies but if one or more concentrations induced a reproducible and biologically 

relevant increase in mutant frequencies in all trials. 

An assay was considered negative if no reproducible and relevant increases of mutant frequencies 

were observed. 

However, these criteria may be overruled by good scientific judgement. 

7. Statistical analysis: 

The statistical analysis relied on the mutant frequencies which were submitted to a weighted analysis 

of variance as well as to a weighted recursive regression, both with Poisson derived weights. 

The two mutant frequency values obtained per group were, although somewhat related, 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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respectively with activation was the most important one for classitying substances into mutagens and 

non-mutagens. However, separate analyses were run for each trial in order to examine the 

consistency of the results. 

All acceptable groups were included in the weighted analysis of variance followed by pairwise 

comparisons to the vehicle control on a nominal significance level of ex. =:0 0.05 using the Dunnett test. 

The regression analysis part was performed on the basis of the actual concentrations thereby omitting 

the positive, 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. GENERAL REMARKS: 

In the presence and in the absence of S9 mix Chinese hamster V79 cells were exposed to AE 0467398 at 

concentrations of up to and including 21 00 ~glml. With and without S9 mix no substance precipitation 

occurred in the medium. 

The means of the 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 for the experiments. 

B. MUTATION ASSAY WITHOUT METABOLIC ACTIVATION: 

Two trials were performed. The mutant frequencies of the negative controls (means of 3.65 x 10'6 and 

1.75 x 10,6, respectively) and of the 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 

significant effects in all trials (means of253.7 x 10,6 and 352.95 x 10'6, respectively). 

For AE 0467398 treated cultures no concentration-related decreases were observed neither in relative 

survival to treatment nor in relative population growth. Relevant AE 0467398 induced increases in mutant 

frequencies could not be found (ranging from 0.0 x 10'6 to 4.2 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-activation trial. 

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C. MUT A nON ASSAY WITH METABOLIC ACnVAnON: 

Two trials were perfonned with S9 mix. In all experiments, cytotoxic effects were induced. The mutant 

frequencies of the negative controls (means of 8.2 x 10,6 and 2.75 x 10-6, respectively) and of the vehicle 

controls (means of 6.85 x 10,6 and 1.1 x 10.6

, respectively) were all within the nonnal range. The positive 

control DMBA induced clear mutagenic and statistically significant effects in all trials (means of 103.55 x 

10.6 and 61.3 x 10'6, respectively). 

The AE 0467398 treated cultures showed no concentration-related decreases neither in relative survival to 

treatment nor in relative population growth. Relevant AE 0001789 induced increases in mutant 

frequencies could not be found (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. 

The positive controls EMS and DMBA had a marked mutagenic effect. 

Based on these results, AE 0467398 was considered not to be mutagenic in the V79IHPRT forward 

mutation assay. 

(Herbold B., 2006) 

Oral 90-day toxicity in tbe rat 

Report: KIIA 5.8/04, Blanck 0., 2007 

Title: AE 0001789-Sulfamoylbenzoic acid (AE 0467398), 90-day toxicity study in the 

rat by dietary administration 

Report No & SA 06036 

Document No M-2829 I 1-0 1-2 

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 of Wi star rats (lO/sex/group) at concentrations of 400,2000 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 constituted group 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 of the study. 


Ophthalmological examinations were performed on all animals during the acclimatization phase and on 


all animals of the 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 of each surviving animal for haematology and clinical chemistry 


determinations. All animals were necropsied, selected organs weighed and a range of tissues were taken, 


fixed and examined microscopically. 


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 000 1789-sulfamoy lbenzoic acid (AE 


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 mg/kglday and 1094 mg/kglday respectively). 


I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test Material: AE 0467398 

Description: off-white powder 

Lot/Batch: KTS 10207-1-1 

Purity: 96.0% 

CAS: 138-41-0 

Stability oftest compound: Stable in rodent diet 400 and 20000 ppm over at least a 67-day 

period at ambient temperature 

2. Vehicle and lor positive control: None 

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3. Test animals: 

Species: Rat 

Strain: Wistar Rj: WI (lOPS HAN) 

Age: 7 weeks approximately 

Weight at dosing: 230 to 269 g for the 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 A04CP 1-1 0 from 

S.A.F.E. (Scientific Animal Food and Engineering, Augy, 

France), ad libitum except before urine and blood collection 

Water: Tap water, ad libitum except before urine collection 

Housing: Animals were caged individually in suspended stainless steel 

wire mesh cages. 

Environmental conditions ­

Temperature: 22 ±2°C 

Humidity: 55 ± 15% 

Air changes: Approximately 15 changes per hour 

Photoperiod: Alternating 12-hour light and dark cycles (7 am- 7 pm) 

B. STUDY DESIGN: 

1. In life dates: April 5 to November 3, 2006 at Bayer Crop Science, 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 0467398 was administered in the diet for at least 90 days to Wistar rats 

at the following doses - 0, 400, 2000 and 15000 ppm (equating to 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 of the Guide for 

the Care and Use of Laboratory 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, W86/609!CEE du 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 or a 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 concentrations to demonstrate adequate formulation procedures. The homogeneity in the diet 

ranged from 99 to 111% of the nominal concentrations. Mean values obtained from the homogeneity 

check were taken as measured concentration. The dietary levels of the test substance were verified 

for each concentration on each preparation and ranged from 91 to 105% of the nominal 

concentrations. All results were within the in-house range of 85 to 115% of nominal concentration. 

Table lIA582 .- StudIY d . eS)2D 

Test group Concentration in 

diet (ppm) 

Dose per animal 

(study averages) 

Animals assigned 

Male 

(mg/kg bw/day) 

Female 

(mg/kg bwiday) 

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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Table IIA 5.8-3 Homo2eneity and concentration oftest material 

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 


(first preparation) 2000 (load 2) 2061 
 103 


15 000 (load I) 
 15772 
 105 


15 000 (load 2) 
 15655 
 104 


400 (load 1) 
 391 
 98 


I 400 (load 2) 
 403 
 101 


Concentration 2 000 (load ]) 
 2072 
 104 


(second preparation) 2 000 (load 2) 2074 
 104 


15000 (load 1) 
 15351 
 102 


15 000 (load 2) 
 15257 
 102 


400 (load I) 
 370 
 93 


400 (load 2) 
 371 
 93 


Concentration 2 000 (load 1) 1827 
 91 


(third preparation) 2000 (load 2) 1843 
 92 


15000 (load 1) 
 14038 
 94 


15000 (load 2) 
 14117 
 94 


R = surface; M middle; B bottom of the preparation. Three different samples 1,2 and 3 were taken at 

the each level ofthe preparation (R, M, B); only two were 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 ANOV A. When the data were not homogeneous even after transformation, a 

Kruskal-Wallis AN OVA was performed followed by the Dunn's test if the 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. (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 

detailed physical examinations were performed once during the acclimatization phase and at least 

weekly during the treatment period. 

2. Neurotoxicity assessment 

During study Weeks 11 to 12, a neurotoxicity assessment was performed for all surviving animals by 

observers who were blind with respect to the dose level. 

a. Exploratory locomotor 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 intervals throughout the session. 

b. Sensory reactivity 

The following reflexes and responses were recorded: 

- Pupillary reflex (by covering the eyes of the animal for a few seconds and then observing 

pupillary constriction by focusing a narrow beam oflight in the eyes). 

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- Surface righting reflex (by putting the animal on its back and measuring its ability/rapidity to 


reassume a normal standing position). 


- Corneal reflex (by touching the medial canthus with a fine object and observing the quick 


and complete closure of the eyelids). 


- Flexor reflex (by pinching the toes and measuring the presence/strength of the flexor 


response of each hindlimb). 


- Auditory startle response (by measuring the animal response to an auditory stimulus). 


- Tail pinch response (by pinching the tail with a forceps and measuring the animal reaction). 


c. Grip strength 

The fore- and hindlimb grip strength of all animals were measured quantitatively using a grip 

strength tester equipped with one pull or one push strain gauge (Bioseb, Chaville, France). The 

mean of three successive measurements was noted for 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. Body weight 

Body weights were recorded three times during the acclimatization phase, on the first day of test 

substance administration, then at weekly intervals throughout the treatment. Additionally, fasted 

animals were weighed before necropsy. 

4. Food consumption and compound intake 

Food consumption was recorded weekly for the first two weeks of the study, then twice weekly 


thereafter due to spillage problems for all animals during the treatment period. 


The weekly mean achieved dosage intake in mglkglday for each week and for Weeks 1 to 13 was 


calculated for each sex using the formula: 


Test substance intake Dose level (ppm) x Group mean food consumption (glday) 

(mglkglday) Group mean body weight (g) at end of the food consumption period 

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5. Ophthalmological examination 

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 and high 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 of the 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 and platelet count. 

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 of the 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 water were not accessible during urine collection. 

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Tier 2, llA, Sec. 3, Point 5: Cyprosulfamide 

Any significant change in the general appearance of the 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 of the urinary sediment was performed after centrifugation of the urine. The presence of 

red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals was graded. 

8. Sacrifice and 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, 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 only. Paired organs were weighed together. 

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 of the 

eye, optic nerve, Harderian gland, epididymis and testis that were fixed in Davidson's fixative. 

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, 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 was judged not to be treatment-related. 

B. Clinical signs 

No treatment-related clinical signs were observed during the study in either sex. 

C. Neurotoxicity assessment 

l. 	Exploratory locomotor activity 


Overall mean exploratory locomotor activity was unaffected by the treatment at any dose level in 


either sex. In addition, the pattern of the 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 


observed with no dose relationship and was thus considered to 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 


All reflexes and responses evaluated were unaffected by the treatment at any dose level in either sex. 


The increased incidence of exaggerated tail pinch response in the high dose males was considered 


not to be treatment-related, due to the limited magnitude of the change and interindividual variation 


of this parameter. 


4. Grip strength 


The fore- and hindlimb grip strength were unaffected by the treatment at any dose level in either sex. 


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D. Body weight 


No effect on mean body weight or body weight gain was noted in either sex. 


E. Food consumption 


No effect on food consumption was noted in either sex. 


F. Achieved dosages 

The mean achieved dosage intake of AE 000 1789-sulfamoylbenzoic acid (AE 0467398) per group was as 

follows: 

Table IIA 5.8-4: Mean achieved dietary intake of AE 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, one male was noted with pupillary reflex not observable (left eye) and one male was noted 

with haemorrhage of the vitreus and hyaloid remnant (right eye). In isolation, these findings were 

considered not to be treatment-related. 

H. Clinical pathology 

1. Haematology 


No treatment-related change was noted at any dietary level in either sex. 


2. Clinical chemistry 


No treatment-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 isolation this 

change was considered not to be 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 not treatment-related. 

2. 	 Gross pathology 

Unscheduled death: 

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 

secondary to an accidental trauma. 

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) of AE 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). 

(Blanck 0.,2007) 

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• AE 0852999 (AE 0001789-cylopropylsulfamoylbenzamide) 

In vitro genotoxicity - Bacterial assay for gene mutation 

Report: KIIA 5.8/05, Herbold B., 2006 

Title: AE 000 1789-cyclopropylsulfamoylbenzamide (AE 0852999), 

Salmonella/microsome test, plate incorporation and preincubation method 

Report No & AT03ll 0 

Document No M-27423 0-0 1-2 

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 of AE 0852999 (M 02)(Batch KTS 10218-3-2, 


98.8% of purity), histidine dependent auxotrophic mutants of Salmonella typhimurium, 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 of incubation at 37°C, the numbers of revertant 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 of growth was observed. 


AE 0852999 did not cause any significant increase in the number of revertant colonies in either the presence 


or absence of metabolic activation. 


All the positive control compounds produced expected increases in the number of revertant colonies, 


thereby demonstrating the sensitivity of the assay and the efficacy ofthe S9 mix. 


Therefore, AE 0852999 was non-mutagenic with or without S9 mix in the plate incorporation as well as in 


the pre-incubation modification ofthe Salmonella/microsome test. 


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Tier 1, (lA, See. 3, Point 5: Cyprosulfamide 

I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test Material: 

Description: 

Lot/Batch: 

Purity: 

CAS: 

Stability of test compound: 

2. Control materials: Negative: 

Solvent: 

Positive: 

3. Test organisms: 


Species: 


Strain: 


Source: 

4. Test compound concentrations: 

Plate incorporation assay: 

AE 0852999 

fine white powder 

KTS 10218-3-2 

98.8% 

No CAS entry yet 

Stable for the duration ofthe study 

Culture medium 

OMSO 

Sodium azide (Serva) for TA 1535 at 10 Jlg/plate, Nitrofurantoin 

(Sigma) for TAl 00 at 0.2 Jlg/plate, 

4-Nitro-l,2-phenylene diamine (Merck-Schuchardt) for TA 1537 

at 10 Jlg/plate and T A 98 at 0.5 Jlg/plate, 

Mitomycin C (Fluka) for TAl 02 at 0.2 Jlg/plate only in plate 

incorporation plate, 

Cumene hydroperoxide (Sigma) for TAl 02 in pre-incubation 

trials only at 50 Jlg/plate, 

2-Aminoanthracene (Aldrich) for the activating effect of the S9 

mix in all strains at 3 Jlglplate. 

Salmonella typhimurium L T2 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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Tier 2, lIA, Sec. 3, Point 5: Cyprosulfamide 

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 of the 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 of Salmonella typhimurium are used to demonstrate 

this effect. For this purpose, the rate of reversion to prototrophy is evaluated in negative control and 

treated groups. 

1. Plate incorporation assay: 

AE 0852999 or the control material was dissolved in 0.1 ml of DMSO. 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 of S9 mix or buffer and 2 ml of soft 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 of a standard protocol with a maximum dose 

of 5000 llg/plate and at least 5 additional doses. If less than three doses were used for assessment, at 

least two repeats were performed. 

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 of the preincubation period, 2 ml of molten soft agar 

were added to the tubes, the content mixed and plated onto Petri dishes with solid agar. After 48 

hours of incubation at 37°C, the numbers of revertant 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. Assessment criteria 

A reproducible and dose-related increase in mutant colonies of at least one strain was considered to 

be positive. For TA 1535, TA 100 and TA 98, this increase should be about twice that of negative 

controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an 

increase of about 100 mutants should be reached. Otherwise, the result was considered as negative. 

II RESULTS AND DISCUSSION 

There was no indication of a bacteriotoxic effect of AE 0852999 at doses of up 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 of growth was noted either. 

None of the five strains concerned showed in the plate incorporation test a dose-related and biologically 

relevant increase in mutant colonies over those of the negative controls. This applied both to the tests with 

and without S9 mix and was confinned by the results of the pre-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, and thus demonstrated the system's sensitivity and the activity of the S9 mix. 

III CONCLUSION 

No indication of mutagenic effects of AE 0852999 were found at doses of up to 5000 I-lg/plate in any of 

the Salmonella typhimurium strains used in the assay. 

(Herbold B., 2006) 

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Tier 2, IIA, Sec:. 3, Point 5: Cyprosulfamide 

In vitro genotoxicity - Test for c1astogenicity in mammalian cells 

Report: KIIA 5.8/06 Thum M., 2006 

Title: AE 0001789-cyclopropylsulfamoylbenzamide (AE 0852999 carboxamide), In 

vitro chromosome aberration test with Chinese Hamster V79 cells 


Report No & AT03121 


Document No M·274272·01·2 


Guidelines: OECD 473 (1997); EEC Direc:tive 2000/32IEC Metbod 810 (2000); EPA Healtb 


Effects Test Guideline (OPPTS 870.5375; 1998) 


GLP Yes (certified laboratory) 


Executive summary: 

In this in vitro assessment of the clastogenic potential of AE 0852999 (M02) (batch KTS 10218·3-2, 

98.8% of purity), 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 of an Aroclor 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. 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 after the 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 of 250, 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 

of mitosis. 

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 of the cultures treated with AE 0852999 in the presence and in the absence of S9 mix 

showed biologically relevant and statistically significant increased numbers of aberrant metaphases. 

The positive controls mitomycin C and cyclophosphamide induced clastogenic effects and demonstrated 

the sensitivity of the test system and the activity of the used S9 mix. 

Based on this test, AE 0852999 was considered not to be clastogenic for mammalian cells in vitro. 

I. MATERIALS AND METHODS 

A. MATERIALS: 

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Tier 2, IlA, Sec. 3, Point 5: Cyprosulfamide 

1. Test Material: AE 0852999 


Description: fine white powder 


LotlBatch: KTS 10218-3-2 


Purity: 98.8% 


CAS: No CAS entry yet 


Stability oftest compound: Stable for the duration of the study 


2. Control materials: Negative: Culture medium 

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 of 4 hours and 0.03 Ilglml for 

a treatment period of 18 hours. 

Cyclophosphamide (Endoxan 100 mg injection vials of dry 

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

3. Test organisms: 

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. 	 Test compound concentrations: 

AE 0852999 was used with or without S9 mix at 250, 500 and 

1000 Ilglm\. 

B. STUDY DESIGN AND METHODS: 

The experimental phase of the study was performed from March 15 to April 27, 2006 at Bayer Healthcare 

AG (PH-PD P Health Care Toxicology). 

The in vitro cytogenetic test is a mutagenicity test system for the detection of chromosome aberrations in 

cultured mammalian cells. The test is designed to detect structural aberrations (chromatid and 

chromosome aberrations) in cells at their first post-treatment mitosis. 

1. Determination of cytotoxicity: 

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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 or without 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 of mitotic cells among a total of 1000 cells per culture was determined using a 

light microscope at a magnification of about 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 of AE 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 ofthe treatment period. 

AE 0852999 at up to 2500 f.l.g/ml did not changed the pH in the medium. The osmolality in the 

medium of the pre-test was not changed by concentrations of up to 2500 Ilg/ml of AE 0852999. 

2. Treatment protocol: 

Chinese hamster V79 cells were passaged on the day prior to treatment. Approximately Ixl06 cells 

were seeded in 20 ml of medium 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 calf serum. Immediately before treatment, the medium was removed from the 

cultures. For the trials without S9 mix, 20 ml of medium containing 2% foetal calf serum and 0.2 ml 

of test substance solution were added to each flask. For the trials with S9 mix, 19 ml of medium 

containing 2% foetal calf serum, I ml of S9 mix and 0.2 ml of test substance solution were added to 

each flask. The cells were incubated for 4 hours at 37°C. After 4 hours of treatment, the medium was 

removed, the cells were washed with PBS and 20 ml of fresh medium containing 10% foetal calf 

serum was added to the flasks. The flasks were placed in a COrincubator for the remaining 

incubation time. 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 of the 

incubation period to arrest the cells in a metaphase-like stage of mitosis( 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. Chromosome preparations: 

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After the removal of the 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 1 to 2 ml 

of a hypotonic solution (0.4% KCI; 37°C) was added to each tube. 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 of fixative 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 least two slides were generated per culture. 

4. Evaluation criteria: 

Coded slides were evaluated using a light microscope at a magnification of about 1000. 

Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two 

parallel cultures, were examined. 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 of one chromatid with dislocation of the chromatid end), fragment (part of chromosome 

without centromer), deletion (result of a break with the terminal chromatid part of the 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. Assessment criteria: 

An assay was acceptable, if there was a biologically relevant increase in chromosome aberrations 

induced by positive controls and if the numbers of aberrations for the negative controls were in the 

expected range. 

An increased incidence of gaps of both types without a concomitant increase of other aberration types 

was considered not to be an indication of a clastogenic effect. 

A test was considered positive, if there was a relevant and statistically significant increase in the 

aberration rate. 

A test was considered negative, if there was no such increase at any time interval or if there were 

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statistically significant values, which were, however, within the range of historical 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 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) using the one-sided Chi2-test. 


The numbers of metaphases with aberrations (including and excluding gaps) and of metaphases with 


exchanges were compared (provided that these data superceded the respective solvent control). The 


one-side Che-test was used for the statistical evaluation. 


A difference was considered to be significant, if the probability of error 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 of up to and including 1000 Ilg/ml and, in addition, without S9 mix for 18 

hours to AE 0852999 at concentrations of up 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 after 4 hours treatment. 

Mitotic index with S9 mix: In comparison to the solvent control, the treated cultures showed a relevant 

reduction of the mitosis rate at 1000 Ilg/ml. The positive control cyclophosphamide also reduced the 

mitosis rate. 

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C. SURVIV AL 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. 

Survival index with S9 mix: In comparison to the solvent control, the treated cultures showed no relevant 

reduction of the survival rate. The positive control cyclophosphamide reduced the survival rate. 

D. CHROMOSOME ABERRATION 

AE 0852999 without S9 mix: No biologically relevant and statistically significant increases of numbers of 

metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 

hours. The same was true for a treatment period and total culture time of 18 hours. 

The treatment with the positive control mitomycin C resulted in a clear and statistically significant 

increase of meta phases with aberrations and demonstrated the sensitivity of the test system. 

AE 0852999 with S9 mix: No biologically relevant and statistically significant increases of numbers 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 of the used S9 mix. 

III. CONCLUSION 

After 4 hours treatment of Chinese hamster V79 cells with AE 0852999 concentrations of 250, 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. 

N one of these cultures treated with AE 0852999 in the absence or presence of S9 mix showed statistically 

significant and biologically relevant increases of numbers of meta phases with aberrations. 

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The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and 

demonstrated the sensitivity of the test system and in the case of cyclophosphamide the activity of the 

used S9 mix. 

Based on the results of this test, AE 0852999 was considered not to be clastogenic for mammalian cells in 

vitro. 

(Thurn M., 2006) 

In vitro genotoxicity - Test for gene mutation in mammalian cells 

Report: IIA 5.8/07, Herbold B., 2006 

Title: AE 0001789-cyclopropylsulfamoylbenzamide (AE 0852999 carboxamide), 

V791HPRT-test in vitro for the detection of induced forward mutations 


Report No & AT03164 


Document No M-274735-01-2 


Guidelines: OECD 476 (1997); EEC Directive 2000/32IEC Method B17 (2000); EPA 


Health Effects Test Guideline (OPPTS 870.5300; 1998) 


GLP Yes (certified laboratory) 


Executive summary: 

The purpose of the study was to assess the point mutagenic potential of AE 0852999 (batch KTS 10218­

3-2,98.8 % of purity) at the hypoxanthine-guanine phosphoribosyl 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 of AE 0852999 ranging from 

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

Concentrations of up to 650 Ilglml did not change the pH in the medium of the pre-test. The osmolality in 

the medium of the 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 or to a range of concentrations 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 of induced mutations. Cells were 

subcultured count I, nonnally after 3 days) by reseeding cells in 75 cm2 flasks. At the end of the 

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expression period (=count 2, nonnally a total of 6 days), cultures were reseeded in Petri dishes without 

hypoxanthine but containing 6-TG for selection of mutants. In addition, 200 cells per dish were seeded in 

5 ml culture medium to detennine 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 of 6­

TG resistant colonies in the mutation assay dishes and the number of colonies 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 of AE 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 of the vehicle controls. Ethyl methanesulfonate and dimethylbenzanthracene 

induced clear mutagenic effects demonstrating the sensitivity of the test system and the activity of the 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. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test Material: AE 0852999 

Description: white powder 

Lot/Batch: KTS 10218-3-2 

Purity: 98.8% 

CAS: No CAS entry yet 

Stability oftest compound: Stable forthe duration of the study 

2. Control materials: 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-activation trials. 

Dimethylbenzanthracene (DMBA), promutagen requiring a 

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metabolic activation, used at a final concentration of 20 Jlglml 

for trials with S9 mix. 

3. Test organisms: 

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 

rapid population doubling time (10 to 14 hours) 

Culture condition: 	 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 and 900 Jlg/ml in the 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 prior to 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 of 6-TG are considered to represent mutants at the HPRT gene. 

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1. Determination of cytotoxicity: 

Exponentially growing V79 cells were plated in 20 ml culture medium in a 75 cm2 flask with a total 

volume of 275 m] (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 or to a range of 

concentrations of the test substance for 5 hours in 20 ml medium containing 2% FCS. In experiments 

with metabolic activation 1 ml of medium 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 of the plates. 

Cytotoxicity was expressed by comparison of colonies in treated cultures versus vehicle control 

cultures (relative cloning efficiency). 

2. Treatment protocol without metabolic 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 or to a range of concentrations ofthe test substance for 

5 hours 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 of 60 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 of induced mutations. Cells 

were subcultured (= count 1, normally after 3 days) by reseeding 1.5x 106 cells into 20 ml of medium 

in 75 cm2 flasks. At the end of the expression period (=count 2, normally a total of 6 days), cultures 

were reseeded in Petri dishes (diameter of 100 mm) at 3xl05 cells per dish (8 dishes per culture) in 

20 ml culture medium without hypoxanthine but containing 10 Ilg/ml 6-TG for selection of mutants. 

In addition, 200 cells per dish (diameter of 60 mm, 3 dishes per culture) were seeded in 5 ml culture 

medium to determine the absolute cloning efficiency for each concentration. After incubation for 6 to 

8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of 6-TG 

resistant colonies in the mutation assay dishes and the number of colonies in the cloning efficiency 

dishes. 

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Two trials 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 of S9 mix were added to the flasks for the treatment period, 

resulting in a concentration of 5% S9 mix in the cultures. The number of 6-TG resistant mutants and 

viability were determined as in the non-activation assay. 

Two trials were performed. 

4. Parameters assessed: 

The parameter "survival to treatment" in % was determined on the basis of the following calculation: 

Mean number of colonies (treated cultures) x 100 

Mean number of colonies (vehicle control cultures) 

The "absolute population growth" was calculated using 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, relative to the vehicle control. 

Absolute population growth treated culture x 100 

Absolute population growth of corresponding vehicle control culture 

The ability of cells to form colonies at the time of mutant selection is measured by the parameter 

"absolute cloning efficiency". It is expressed in %. 

Mean number of colonies per dish x 100 

200 

The "mutant frequency" is calculated for each group by dividing the total number of mutant colonies 

by the number of cells seeded (usually 8-10 plates at 3x 105 cells per plate), 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 of mutant colonies x 100 


Number of evaluated dishes x 3 x 105 x C.E. 


5. Acceptance criteria: 

The average cloning efficiency of the negative and vehicle controls should be at least 50%. 

The average mutant frequency of the vehicle controls should not exceed 25 x 10-6 cells. 

The mutant frequency of the two cultures of the vehicle and lor the negative control should differ 

only to an acceptable extent. As a rule of thumb, the difference of mutant frequencies should not 

be greater than 5 x 10-6• 

The positive control should induce an average mutant frequency of at least three times that of the 

vehicle controL 

If not limited by the solubility of the test substance in the vehicle the highest concentration should 

induce cytotoxicity of about 80 to 90% or should be 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% or greater. 

However, these criteria may be overruled by good scientific judgement. 

6. Assessment criteria: 

Mutant frequencies was only used for assessment, if at 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 least two to three times that of the highest negative or vehicle control value observed 

in the respective triaL If this result was reproduced in a second trial, the test substance was 

considered to be mutagenic. 

Despite these criteria, a positive result was only considered relevant, if no significant change in 

osmolality compared to the vehicle control was observed. Otherwise, unphysiological culture 

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conditions may be the reason for the positive result. 


A test substance was judged as equivocal if there was no strictly concentration related increase in 


mutation frequencies but if one or more concentrations induced a reproducible and biologically 


relevant increase in mutant frequencies in all trials. 


An assay was considered negative ifno reproducible and relevant increases ofmutant frequencies 


were observed. 


However, these criteria may be overruled by good scientific judgement. 

7. Statistical analysis: 

The statistical analysis relied on the mutant frequencies which were submitted to a weighted analysis 

of variance as well as to a weighted recursive regression, both with Poisson derived weights. 

The two mutant frequency values obtained per group were, although somewhat related, 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 

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 of the results. 

All acceptable groups were included in the weighted analysis of variance followed by pairwise 

comparisons to the vehicle control on a nominal significance level of a = 0.05 using the Dunnett test. 

The regression analysis part was performed on the basis ofthe actual concentrations thereby omitting 

the positive, negative and vehicle controls. If there was a significant concentration 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. RESULTS AND DISCUSSION 

A. GENERAL REMARKS: 

In the presence and in the absence of S9 mix Chinese hamster V79 cells were exposed to AE 0852999 at 

concentrations of up to and including 900 I-lglml. With and without S9 mix substance precipitation 

occurred in the medium of the main study at 750 I-lglml and above. No changes in osmolality or pH were 

observed. 

The means of the 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.These results demonstrated good cloning conditions for the experiments. 

B. MUTATION ASSAY WITHOUT METABOLIC ACTIVATION: 

Two trials were performed. The mutant frequencies of the negative controls (means of 2.6 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 10'6 and 426.2 x 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 10,6 to 9.7 x 10,6 in the second trial). In addition, the overall statistical analysis revealed no 

statistically significant increases. Therefore, AE 0852999 was evaluated as non-mutagenic in the nonactivation trial. 

C. MUTATION ASSAY WITH METABOLIC 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 10-6 and 0.8 x 10'6, respectively) and of the 

vehicle controls (means of 3.6 x 10'6 , 1.2 X 10'6 and 2.0 x 10'6, respectively) were all within the normal 

range. The positive control DMBA induced clear mutagenic and statistically significant effects in all trials 

(means of 19.9 x 10'6,69.8 x 10'6 and 45.0 x 10-6, respectively). 

The AE 0852999 treated cultures showed concentration-related decreases in relative survival to treatment 

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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 or pH were observed. AE 0852999 induced no biologically relevant increases in 

mutant frequencies 

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) 

Oral 28-day toxicity in the rat 

Report: KIIA 5.8/08, Odin-Feurtet M., 2007 


Title: AE 0852999, 28-day toxicity study in the rat by dietary administration 


Report No & SA06281 


Document No M-284847-0 1-2 


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 of the safener AE 0001789 (batch number NLL 7793-9: a white powder, 

99.3% purity) was administered continuously via the diet to groups of Wistar rats (to/sex/group) for 29 

days at concentrations of 40, 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 of the control and high dose groups during Week 4. Haematology, clinical chemistry 

and urine parameters were determined at the end of the study. AI! animals were necropsied, selected 

organs weighed and a range of tissues were taken, fixed and examined microscopically. 

988 
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No mortalities or treatment-related clinical signs occurred during the study. 

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 in males at each interval but only on the interval Day 

1 to Day 8 in females. Mean body weight gain/day was reduced between Study Day 1 and 15, in males 

and between Study Day 1 and 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. 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 of lower 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 compared to 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 of pelvic 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 one male. 

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 211 0 males and 411 0 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. 

989 
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At 300 and 40 ppm, no treatment-related effects 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, 

based on reduced body 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 

Stability of test compound: 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 lor positive control:None 

3. Test animals: 

Species: Rat 

Strain: Wistar Rj: WI (lOPS HAN) 

Age: 7 weeks approximately 

Weight at dosing: 237 to 267 g for the males - 163 to 192 g for the females 

Source: R. Janvier, Le Genest St Isle, 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), ad libitum except before urine and blood collection 

Water: Tap water, ad libitum except before urine collection 

Housing: Animals were caged individually in suspended stainless steel 

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wire mesh cages. 

Environmental conditions ­

Temperature: 22 ±2°C 

Humidity: 55 ± 15% 

Air changes: 10 to 15 changes per hour 

Photoperiod: Alternating 12-hour light and dark cycles (7 am- 7 pm) 

B. STUDY DESIGN: 

1. In life dates: September 20 to October 27,2006 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 Wistar rats 

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 of Laboratory 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 preparation and 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 20000 ppm, 

AE 0852999 was found to be stable in the diet over a 62-day period at room 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% of the nominal concentrations. Mean values obtained from the homogeneity 

check were taken as measured concentration. The dietary levels of the 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 were within the in-house range of 85 to 115% of nominal concentration. 

Table I A I 58 . -5 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 IIA 5.8-6 Homogeneity and concentration oftest material 

Nominal concentrations Analysed concentrations i % of nominal 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 of the preparation. Three different samples 1,2 and 3 were taken at 

the each level ofthe preparation (H, M, B); only two were 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 if the 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. 

Statistical analyses were carried out using 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 

detailed physical examinations were performed at least weekly during the treatment period. 

2. Body weight 

Body weights were recorded twice during the acclimatization phase, on the first day of test substance 

administration, then at weekly intervals throughout the treatment. Additionally, fasted animals were 

weighed before necropsy. 

3. Food consumption and compound intake 

Food consumption was recorded weekly for all animals during the treatment period. 


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 mean body weight (g) at end ofthe food consumption period 

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4. Ophthalmological examination 

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 of the 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 and platelet count. 

A blood smear was prepared and stained with May-Grunwald-Giemsa method. It was 

examined when results of Advia 120 determinations were abnormal. 

Prothrombin time was assayed on an ACL 3000. 

b. Clinical chemistry 

Any significant change in the general appearance of the 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. 

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 water were not accessible during urine collection. 

Any significant change in the general appearance of the 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 of the urinary sediment was performed after centrifugation of the urine. The presence of 

red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals was graded. 

7. Sacrifice and pathology 

On study days 30, 31 or 32, a complete necropsy was performed on all animals. Animals were deeply 

anaesthetized by inhalation of lsoflurane, then exsanguinated before necropsy. All animals were diet 

fasted prior to scheduled sacrifice. The necropsy included the examination of all major organs, tissues 

and body cavities. Macroscopic abnormalities were recorded, 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 weighed together. 

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 of the 

eye, optic nerve, harderian gland, epididymis and testis that were fixed in Davidson's fixative. 

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 

bladder 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. RESUL TS AND DISCUSSION 

A. Mortality 

There were no mortalities during the course ofthe study. 

B. Clinical signs 

No treatment-related clinical signs were observed throughout the course of the study. The few clinical 

signs observed were considered not to be related to AE 0852999 administration. As these findings 

occurred as isolated incidences and at the low and intermediate dose levels only, they are considered to be 

spontaneous findings. 

C. Body weight 

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 1 and 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 significant p<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 1 or p<0.05) 

between Study Days I and 15. 

At 300 and 40 ppm, body weight and body weight gain in male and female animals were unaffected by 

treatment with AE 0852999. 

D. Food consumption 

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

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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 unaffected by treatment in either sex. 

E. Achieved dosages 

The mean achieved dosage intake of AE 0852999 expressed in mg/kg body weight/day per group was as 

follows: 

Table llA 5.8-7: Mean achieved dietary intake of AE 0852999 (Weeks 1-4) 

Diet concentration in 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. Ophthalmological examination 


There were no treatment-related ophthalmological findings. 


G. Clinical pathology 


1. Haematology 

Lower mean erythrocyte counts, hemoglobin concentrations and hematocrit were noted at 3000 ppm 

in both sexes, together with higher mean reticulocyte counts. 

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Table lIA 5.8-8: Haematoloaical parameters variations (% chanl!e when compared to controls) 

• Dosage level of AE 0852999 (ppm) 0 3000 0 3000 

Sex Males Females 

Red blood cell 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 

No treatment-related change was noted at 800, 300 and 40 ppm for the parameters assayed. 

2. Clinical chemistry 

Higher mean urea, creatinine and total cholesterol concentrations were observed at 3000 ppm in both 

sexes. 

Table IIA 5.8-9: Clinical chemistry parameter variations (% chanl!e when compared to controls) 

Dosage level of AE 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 of the individual variation. No treatment-related change was 

noted at 800, 300 and 40 ppm for the 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 of the 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 to 2) 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 of parent, 

but due to the small amount of these 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. 

No treatment-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 compared to 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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Table IIA 5.8-10: Kidney weight changes at terminal sacrifice (% change when 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%) 

Mean kidney 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 significant histopathological 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 of the kidneys at 3000 ppm. 

Table IIA 5.8-11: Incidence of macroscopic 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 (211 0) and gritty content (2/10) were found in the urinary bladder in males and 

dilatation of the ureter was found in 2/10 males at 3000 ppm. Other changes were considered as 

incidental and not treatment-related. 

3. 	 Microscopic pathology 

At tenninal sacrifice, treatment-related changes were observed in the kidney, urinary bladder and 

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ureter at 3000 ppm. No treatment-related effects were seen at 40,300 and 800 ppm. 


Treatment-related changes observed at 3 000 ppm were considered to be adverse and associated with 


clear clinical 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 tubular dilatation proximal to the sites of obstruction. 


Intrapelvic crystals were observed. 


These changes were associated with an increase in incidence and severity of pelvic epithelium 


hyperplasia, collecting duct hyperplasia and basophilic tubules. 


Table IIA 5.8-12: Incidence and/or severity of microscopic changes in the kidney - 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 

Number of animals 

examined 

10 10 10 10 10 10 10 10 10 10 

ntratubular crystals 

TOTAL 0 0 0 0 6 0 0 0 0 5 

ntrapelvic crystals 

TOTAL 0 0 0 0 3 0 0 0 0 2 

Obstructive nephropathy 

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 

rollecting ducts hyperplasia: 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 

!Basophilic tubules 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 (3/1 0) and in female 

(1/10). Intraluminal crystals were observed in the urinary bladder in two males. 

Table llA 5.8-13: Incidence and severity of microscopic cban2es in tbe urinal)' bladder - terminal sacrifice 

Incidence and severity of microscopic changes in the urinary bladder - terminal sacrifice 

Sex Male Female 

Dose level ofAE 

0 40 300 800 3000 0 40 300 800 

0852999 (ppm) 

Number of animals 

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 

Slight diffuse urothelial hyperplasia was observed in one ofthe two sampled ureters in males. 

III. CONCLUSION 

In conclusion, the No Observed Effect Level (NOEL) of AE 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 Report on medical surveillance on 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 to the campaigns, did not show any effects. 

IIA 5.9.2 Report on clinical cases and poisoning incidents 

No poisoning incidents or clinical cases have occurred. 

IIA 5.9.3 Observations on general population exposure and epidemiological studies 

So far there has been no exposure of the general public. 

IIA 5.9.4 Clinical signs and symptoms of poisoning and details of clinical tests 


There are no experiences with human poisonings, animal experiments did not show any symptoms. 


IIA5.9.5 First aid measures and therapeutic regimes 


Remove patient from exposure/tenninate exposure. Thorough skin decontamination with copious 


amounts of water and soap. Flushing of the 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 of the very low acute toxicity of the compound. However, the 

application of activated charcoal and sodium sulphate is always advisable in significant ingestions. 

As there is no antidote, treatment has to be symptomatic and supportive. 

IIA 5.9.7 Expected effects & duration of poisoning as a function of exposure 

There are no experiences with human poisonings, animal experiments did not show any symptoms. 

IIA 5.9.8 Effects & duration of poisoning as a function oftime 

There are no experiences with human poisonings, animal experiments did not show any symptoms 

IIA 5.9.9 Dermal penetration 

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A dermal penetration study with the plant protection product has been conducted and is reported in Annex 

IlIAI point 7.6. 

IIA 5.10 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 & 


Document No M-285709-01-1 


Guidelines: EU DG SANCO/22112000-rev.l0, 25th February 2003 


GLP Not applicable 

Executive summary: 

The behaviour of cyprosulfamide in various environmental compartments is well described by a wide 

variety of studies. The potential relevance of the 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. 

Summary of non-relevance of tbe metabolites of Cyprosulfamide 

• 	 The degradation of cyprosulfamide under aerobic soil conditions is rapid with half-life values 

in the range of2.3 to 10.6 days at 20°C. This degradation results in the formation of four 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 postulated to be present. 

• 	 Under sterile aqueous conditions cyprosulfamide is hydrolytically stable but in natural waters 

is slowly partitioned from water to sediment with a DT 50 of around 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 aqueous photolysis. 

• 	 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 of M03 

(AE 0467398), 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 (AE 0467398) nor M02 (AE 0852999) were found to be genotoxic in three tests. 

• 	 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 similar toxicological profile as AE 0001789. 

• 	 Conducting a consumer risk assessment for the M03 (AE 0467398) and M02 (AE 0852999) 

for the extreme worst case conditions of the 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) 

would be less than 0.1 %. 

• 	 The metabolites present in soil including M03 (AE 0467398) and M02 (AE 0852999) were 

assessed for their toxicity to earthwonns (Eisenia Jetida), Daphnia (Daphnia magna), Green 

algae (Pseudokirchneriella subcapitata) 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 other metabolites 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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Tier 2, IIA, Sec. 3, Point 5: Cyprosulfamide 

the risk assessment cyprosulfamide can be safely used on maize. 

IIA 5.11 Summary of mammalian toxicity and overall evaluation 

• Absorption, distribution, excretion and metabolism 

The biokinetic behaviour and metabolism of AE 0001789 in the rat was investigated following a single 

oral dose to male and female animals, employing a low dose level of 2 mglkg b.w. and a high dose of 200 

mg/kg b.w. and using two different label positions with 14C uniformly labeled in the sulfonylbenzamide 

and the methoxy benzoyl ring. 

The gastro-intestinal absorption of AE 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) or even more than 90 % (sulfonylbenzamide 

label) of the 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 of the 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 of 2 - 3 

compared to the low-dose animals. 

The excretion was fast and almost complete after 72 hours as 82 - 84 % of the administered dose 

(corresponding to 98 % of the recovered radioactivity) was excreted 72 hours after administration. The 

predominant route of excretion was via urine, accounting for 69 to 90 % of the dose administered. There 

was an insignificant quantity expired as radio labeled 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:5 0.06 % of the 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 of bioaccumulation. A particular concentration of radioactivity 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 concluded that residues of AE 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, or thyroid gland) were rapidly depleted in parallel with depletion from 

the other organs and tissues. 

A delayed gastric emptying was observed particularly with some of the high-dose animals, resulting in a 

double peak in the plasma concentration-time curves and in a late excretion of significant portions of the 

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 andlor delayed gastric emptying of a portion of the dose. 

From the metabolites identified in urine and faeces three metabolic steps can be concluded:. 

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(4) Elimination 	of the cyclopropylamine moiety by hydrolysis of carboxamide bond in the sulfonylbenzamide part forming AE 000 I 789-descyclopropylamino. 

(5) Desmethylation ofthe methoxybenzoyl moiety leading to AE 0001 789-desmethyl 

(6) Hydrolytic cleavage 	of carboxamide bond in the methoxybenzoyl group of the parent molecule 

resulting in AE 000 1 789-cyclopropyl-sulfamoylbenzamide and AE 000 I 789-anisic acid. 

These metabolic reactions result in the formation of the following metabolites 

• 	 M04, AE 1448796 (AE 000 1789-desmethyl) 

• 	 MOl, AE 0893016 (AE 000 1 789-descyclopropylamino) 

• 	 M02, AE 0852999 (AE 000 1 789-cyclopropyl-sulfamoylbenzarnide) 

• 	 M07, AE 0854787 (AE 0001789-anisic acid) 

• 	 M06, AE 2300015 (cycIopropylamine (postulated») 

Sex specific differences of absorption, distribution, metabolism and excretion of AE 0001789 were not 

observed. 

The proposed metabolic pathway of AE 0001789 in the rat composed from the partial pathways from 

each of the different label positions is shown in Figure 5.1-0 I. 

Figure 5.11-01: Proposed metabolic pathway of AE 0001789 in the rat. 

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Tier 2, IlA, Sec. 3, Point 5: CyprosuJfamide 

H,C, 0 0 

o 0 ~//

V· ~HN"'S'0y*~ 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 

"AE 0001789-anisic acid" "AE 0001789·cyclopropyl-sulfamoylbenzamide" 

* denotes the two label positions postulated, not observed as the [ 
 radiolabel was outside this group ] 

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Tier 2, llA, Sec. 3, Point 5: Cyprosulfamide 

• Acute toxicity, local tolerance and sensitisation 

The acute toxicity of AE 0001789 (97.4% of purity) 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 

were recorded in females on days I and lor 2. 

AE 0001789 was not an irritant to rabbit skin and caused only redness of the conjunctivae which reversed 

within 72 hours. No evidence of skin sensitisation (delayed contact hypersensitivity) was seen in a guinea 

pig Magnusson and Kligman maximisation test. 

Table llA 5.11-2:Summary ofacute toxicity data for AE 0001789 

Type ofstudy ISpecies Results OECD Classification 

(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) 

Dermal route 

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 

Category 5 I Unclassified. C III • ategory 

Skin sensitization 

M-249757-01-2 

Guinea pig Not sensitising . Category 5 / Unclassified IN A I' bl • ot pp Ica e 

• Short term 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 of stones in the urinary tract. No 

effects in the urinary tract were observed in the mouse after short term exposure. 

In rats, continuous dietary administration for 90 days at the high dose of 12 000 ppm induced mortalities 

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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 of both 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 of the urothelium in the urinary bladder, of collecting 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 12000 ppm. Sulfonamide-like crystals and hyperplasia ofthe urothelium in the urinary bladder 

were also seen in both sexes at 4000 ppm. The NOEL in the Wistar rat when administered AE 0001789 

in the diet over a 90-day period was 1 000 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 of lymphocytolysis in the thymus of female 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: elevated urine 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. The NOAEL for this study was 7 500 ppm (equivalent to 221 mglkglday for males and 

females) as there were no microscopic pathology observations to correlate with the decrease in urine 

specific gravity in the 7 500 ppm 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 or in the 90­

day rat study, the findings (haemorrhages, inflammation and hyperplasia) were consistent with the 

fonnation of stones in the urinary tract. The NOEL for this study was 2500 ppm (equivalent to 66 and 67 

mglkglday for males and females, respectively). 

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Table 5.11-3: Summary ofsbort-term toxicit of AE 0001789 

Type ofstudy 

Doses 

NOAEUNOEL 

ppm mglkg/d 

LOAEL 

ppm mglkg/d 

Treatment-related effects 

90-day rat 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) 

2 000 

(F) 

1110 

(M) 

398 

(F) 

>7000 

(M) 

7000 

(F) 

>1110 

(M) 

1297 

(F) 

Lymphocytolysis in thymus of females 

only 

90-day dog feeding study 

M-263605-0 I-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-283 I 94-01-1 

(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 of genotoxicity 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 of gene mutation either in the presence or absence of metabolic 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 no genotoxic potential. 

Table 5.11-4 Summary of e:enotoxicity test 

I Mutagenicity tests with AE 0001789 Results I 

I A. In vitro tests I Metabolic Activation I 

I Ames Test I 

+1­ Negative I 

: M-080265-02-2 i 

i Ames Test (on new technical) +1­ JNegative i 

I M-283435-01-2 

I Chromosome aberrations (V79 cells) i +1­ iNegative 

M-129315-02-2 I 

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I 

I 

· HPRT Test (V79 cells) 

M-266527 -01-2 

+1­

i 

Negative 

i B. In vivo tests Dose levels I 

Micronucleus Test in mice - oral administration 

M-077 491-02-2 

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 of stones in the urinary tract induced hyperplastic and inflammatory changes in the 

kidney, urinary bladder and/or ureters. It was demonstrated that these stones were mainly formed from the 

parent compound, which is poorly metabolized and precipitates in the urine at high doses. 

AE 0001789 was administered to groups of 60 male and 60 female Wistar rats by continuous dietary 

treatment at 1000,4000 and 8000 ppm, corresponding to 39, 159 and 321 mglkgJday in males and 56, 220 

and 447 mglkgJday in females, respectively, over a 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 after 24 months of treatment and was largely due to 

secondary treatment-related nephropathy following product administration. Mean cumulative body 

weight gain was reduced during the first week of treatment by 7 and 12% in males and females treated at 

8000 ppm, respectively, compared to the controls. Urinalysis revealed the presence of sulfonamide-like 

crystals throughout the study in both sexes, the effect being more pronounced in females than in males. At 

the 12-month interim sacrifice, treatment-related non-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 of the commonly occurring 

lesion diffuse bilateral tubular degeneration of the testis and bilateral oligospermia of the epididymis was 

observed at the carcinogenicity phase. 

Neoplastic changes comprised of a high dose-related transitional cell carcinoma in the kidney of one male 

and a transitional cell carcinoma in the urinary bladder of one 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 with the presence of stones. 

In conclusion, dietary administration of AE 0001789 over a 24-month period to the Wistar rat, at dose 

levels up to 8000 ppm (corresponding to 321 mglkglday in males and 447 mglkglday in females), 

produced a transitional cell carcinoma in the kidney of one male and a transitional cell carcinoma in the 

urinary bladder of one female. The incidence of these 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 not to be directly carcinogenic in the rat. 

The No Observed Adverse Effect Level (NOAEL) over a 12-month period of dietary 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 mglkg body weight/day in females). 

The NOAEL over a 24-month period of dietary administration with AE 0001789 to the Wistar rat was 

1000 ppm in both sexes (equivalent to 39 mglkglday in males and 56 mglkglday in females). 

Groups of60 male and 60 female C57BLl6J mice were initially intended to be fed diet containing 0,350, 

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 of treatment, an additional treatment group (60 

animals/sex) at 3500 ppm was added to the study approximately one month after the start of treatment. 

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 Week 46 (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 or due to secondary treatment-related nephropathy following product administration. 

At the 18-month haematology determination, a tendency 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 of urinary stones or to 

treatment-related nephropathy. These changes were located in the urinary bladder and to a lesser extent 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 of hepatocellular single cell necrosis were observed in males. All the effects seen in 

the urinary tract were considered to be due to 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 considered to be an indirect and non-adverse response to the treatment. 

Neoplastic changes comprised transitional cell papilloma in the urinary bladder of 2/49 females at 3500 

ppm. This finding was considered to be secondary to the chronic hyperplastic changes due to the presence 

of calculi. Analysis of urinary bladder stones taken from one male and one female at terminal sacrifice 

revealed that the composition of the stones consisted of approximately 90 to 95% of test material. 

In conclusion, dietary administration of AE 0001789 over an 18-month period to 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 of these tumours 

was very low and was considered to be secondary to the chronic hyperplastic changes resulting from 

chronic irritation due to the presence of calculi. Thus AE 0001789 was considered not to be directly 

carcinogenic in the mouse. The No Observed Effect Level (NOEL) was 350 ppm for males (equivalent to 

50 mglkglday) and 2000 ppm for females (equivalent to 354 mg/kglday). 

Tab)e 511 -5: Summaryoflong-term toXlcnYlcarcmogemcuy . 't / "t WI'th 

Type of study 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 & 8000 ppm in the kidney and urinary 

bladder 

Mouse - 78 week- Chronic! Treatment-related Oncogenicity 50/354 287/616 nephropathy 

M-28 1276-01-2 (M/F) (M/F) 

in the urinary tract 

350, 2000, 3500 & 7000 ppm Hepatocellular hypertrophy 

• Reproductive and embryonic toxicity 

Histopathological findings due to the precipitation of foreign 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 of 25 male and 25 female rats at concentrations of 0 (control), 480, 2400 and 

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12000 ppm in their diet. At 12000 ppm, 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 a reduced 

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 of the FO and F 1 rats. At 480 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 and 260 mg/kg bw/day in females), and the parental systemic NOAEL 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) due to pup weight decrease. 

In a developmental toxicity study AE 0001789 was administered daily by gavage from gestation day 

(GD) 6 to 20 to groups of25 pregnant Sprague-Dawley female rats per dose-group. The doses given were 

0, 60, 250 and 1000 mg/kglday in suspension in an aqueous solution of 0.5% 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 of this 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, litter based or fretal 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 

exposed to 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 of 0.5% methylcellulose 400. The high dose level 

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of 500 mg/kglday was terminated prior to commencement of scheduled 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 of the kidney or the urinary 


bladder. No adverse effects were observed at 50 mg/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 per group) were exposed to AE 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. 


No treatment-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 


developmental toxicity was 250 mglkglday. 


The NOELs for reproductive effects are summarized in the following table: 


Table 5.11-6 Summary of reproductive toxicit) with AE 0001789 

Type of study NO(A)EL LOAEL Adverse effects / target organs (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 

and urinary tract 

M-28351O-01-2 113 840 Reduced rearing index 

0,480,2400, 12000 ppm 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, 250 mglkg bw/day 

Developmental toxicity rabbit - 250 

250 I >250 • fretus 

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• Neurotoxicity 

AE 0001789 did not induce any neurotoxicological findings after acute, short or long tenn 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 of neurotoxicity at any dose level and no compound-related 

gross or microscopic lesions at the high dose of 2060 mglkg/day. These results establish a NOEL of 508 

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 or 9000 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 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 

000 1789lkg body wt/day for males and females, respectively). 

Table 5.11-7 SummarY of neurotoxicity with AE 0001789 

Type of study 

Doses 

NO(A)EL 

(mglkg/d) 

LOAEL 

(mg/kg/d) 

! 

I Adverse effects / target organs 

Acute neurotoxicity in the rat 

M-283196-0 1-1 508 2060 Urine staining in both sexes 

.0, 126,508,2060 mg/kg bw I 

90-day neurotoxicity in the rat 

M-2832 18-0 1-1 

0, 1000, 3000, 9000 ppm I 

5921748 

(M/F) I 

>592/>748 

(MlF) 

None 

• Toxicity on metabolites 

The toxicological properties of two metabolites of AE 0001789 were investigated in a series of studies 

carried out according to the 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 predicted by the EU FOCUS leaching modelling to have the potential, to leach to 

shallow groundwater with a concentration of> 0.75 IlgIL. This metabolite was devoid of genotoxic 

potential and did not cause any treatment-related effect after subchronic administration. 

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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 and had a similar toxicological profile to the parent compound after subacute administration. 

Table 5.11-8 Results oftoxicity studies with the metabolites 

Study Species Results 

AE 0467398 (M 03) 

Ames test Negative 

M-278703-0 1-2 Salmonella Typ. 

In vitro V79IHPRT Locus Gene Negative 

Mutation Assay Chinese hamster 

M-273551-01-2 V79 lung cells I 

I In 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 (M 02) 

I Ames Test Negative 

IM-274230-01-2 Salmonella typh IIn vitro V79/HPR T Locus Gene Negative 

•Mutation Assay 	 · Chinese hamster I 

•M-274735-01-2 · 

i V79 lung cells 

I In Vitro Chromosome Aberration· Negative i 

test 

Chinese hamster 

V79 lung cells 

28-day dietary study: 

• M-274272-01-2 

NOEL 300 ppm (25.5 mglkg bw/day in 

0, 40, 300, 800, 3000 ppm 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 of mammalian 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 of Cyprosulfamide to rat and other laboratory animals indicates that the compound 

would present little if any risk to wild mammal populations. 

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Following acute exposure of rats, no mortality or other signs of toxicity were observed at the regulatory 

top dose of 2000 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 of the LD50 cut-off of 5000 mg a.s.lkg bw. 

Typical effects of high doses of Cyprosulfamide administered over prolonged periods 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 of stones, which are mainly 

formed of parent compound. Such effects are considered not to be relevant for the ecotoxicological 

assessment (see below). Genuine effects on reproductive parameters were not observed up to the top dose 

levels in any species tested. 

With regard to the selection of endpoints for the long-term assessment, it is considered that effects on 

populations will not occur if the survival rate, reproduction rate and development of individuals are not 

affected. Therefore, in principle, only endpoints in toxicity tests which are related to these key factors of 

population dynamics are ecotoxicologically relevant. 

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 or growth rate of the offspring, or behavioural parameters 

in adults or young. 

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 in parents or offspring at the medium dose level, 2400 ppm. At this level, effects were limited to 

(histo-) pathological changes in kidneys, urinary bladder and spleen. Survival, growth, development and 

reproduction of all animals were not impaired at this treatment level over the entire 2-generation study. 

For the ecotoxicological assessment ofCyprosulfamide, the LOAEL of this rat multigeneration study was 

12000 ppm, and the NOAEL was 2400 ppm (202 mg a.s.lkg bw/d). 

In the developmental toxicity studies in rat and rabbit, effects of ecotoxicological relevance were only 

observed at higher doses. 

In the rat, treatment related effects were only observed in dams treated at 1000 mg a.s.lkg bw/day (no 

developmental toxicity up to 1000 mg a.s.lkg bw/day). In rabbits, treatment related effects (two abortions) 

were observed at 250 mg a.s.lkg bw/day, but no other ecotoxicologically relevant effects were observed at 

this dose level. 

These effects were observed after gavage exposure which is of low relevance to long-term exposure of 

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wild mammals to Cyprosulfarnide in the field, and the LOELs from the developmental toxicity studies are 

well above the NOAEL at 202 mg a.s.ikg bw/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 could be an endocrine disruptor. 

• Conclusions - ADI, AOEL, maximum concentration in drinking water 

Proposed Acceptable Daily Intake (ADI) 

The potential risk for consumer is mainly linked to chronic exposure to possible residue of AE 0001789 

in the food. This is the reason why the ADI should be based on the results of the following chronic 

studies: 

T a . bl e 511 -9 S : ummaryofIon2 term S t u d'16 

, 

Study 

(Dose levels) 

NOELfNOAEL LOEL Target Organ(s) and 

Itreatment-related effects! 

ppm mg/kg/day ppm mg/kg/day 

One year dog study 

M-283194-02-1 

(0, 1000,2500, 8000 ppm) 

2500 66/67 

(MlF) 

8000 226/242 

(MIF) 

Kidney and urinary 

bladder 

2 year rat carcinogenicity 

M-281767-01-2 

(0, [200], 1000,4000,8000 ppm) 

1000 39/56 

(MIF) 

4000 159/220 

(MlF) 

Kidney and urinary 

bladder 

Mouse oncogenicity 

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-generation reproduction 

Study 

M-2835 10-01-2 

(0,480,2400, 12000 ppm) 

Parents 480 39/55 2400 202/260 Organ changes in 

spleen & urinary tract 

Reproduction 2400 173 12000 840 Reduced rearinj;!; index 

Pups 2400 173 12000 840 Body weif;!;ht decrease 

1020 
Page 438 of 441 EBBayer CropScience 

2007-03-28 

Tier 2, IlA, Sec. 3, Point 5: Cyprosulfamide 

In accordance with internationally accepted procedures the ADI (or Reference Dose RID) is estimated on 

the basis of the 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 therefore the most sensitive species. The lowest NOAEL of39 mglkg/day was obtained in the 

2-year rat study. Treatment-related nephropathy was observed at 159/220 mg /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 of safety (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 mg Ikg/day 

For the USA, no additional FQPA safety factor seems necessary. There is no indication of an increased 


sensitivity of the young. Additionally, the toxicological database of AE 0001789 is considered complete. 


Therefore, for the USA the suggested chronic Population Adjusted Dose (cPAD) is the same as the RID 


(cPAD = RID = 0.39 mg/kg/day) 


Proposed Acute Dietary Exposure ARID (Acute reference dose) 


On the basis of the low order of acute toxicity of AE 0001789, following exposure by oral, dermal and 


inhalation routes, it is not necessary to propose 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 not need to be established. 


Proposed Acceptable Operator Exposure Level (AOEL) 


Since health risks to operators relate to short term rather than to chronic exposure, the NOAELs derived 


from short term and developmental toxicity studies and the rat 2-generation reproduction study should be 


taken into account for the purposes of establishing an AOEL for the EU and for the estimation of the 


operator exposure in the USA and Canada. 


AE 0001789 has low acute toxicity to mammals irrespective of the route of exposure (oral, percutaneous 


or inhalation exposure). It is not a skin sensitizer, it is not irritating to skin and causes only a slight 


redness of the conjunctivae in the rabbit. 


Subchronic studies showed that the kidney and the urinary bladder are the major target organs in both rats 


and dogs. The effects observed are consistent with the formation of stones in the urinary tract. The mice 


1021 
Page 439 of 441 EEYBayer CropScience 

2007-03-28 

Tier 2, llA, Sec. 3, Point 5: 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 maternal toxicity) in a rat reproductive study but at a 


dose level causing overt maternal toxicity such as increased mortality. No embryotoxic effects were 


observed in the rat or rabbit teratogenicity studies. 


The relevant NOAELs to be considered for calculation of the operator exposure are summarised in the 


following table. 


Table • 511-10 : S ummary 0 f re I evantdt'tb aa or e setfID~ 0 f operator exposure

Study 

(Dose levels) 

NOEL0JOAEL LOAEL Target Organ(s) 

ppm mg/kglday ppm mg/kglday 

90-day dog study 

M-263605-0 I-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, 12000 ppm) 

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-261462-0 1-2 

(0.60,250 1000 mglkg/day) 

Dams - 250 - 1000 Kidney 

Pups - 1000 - >1000 

Rabbit teratology 

M-261627-01-2 

M-273153-01-2 

(0, 50,125. [250], 500 mglkg/day) 

Dams - 125 - 250 . Kidney 

Pups . 250 - >250 

I 

Rat 2-generation reproduction 

Study 

M-28351O-01-2 

(0, 480. 2400. 12000 ppm) 

Parents 480 39155 2400 202/260 Organ changes in spleen & 

urinary tract 

Reproduction 2400 173 12000 840 Reduced rearing index 

Pups 2400 173 12000 840 Body weight decrease 

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 

1022 
Page 440 of 441 EBBayer CropScience 

2007-03-28 

Tier 2, IIA, Sec. 3, Point 5: Cyprosulfamide 

in the rat 2-generation reproduction study: 39 mg:kglday. The target organs observed in this study (spleen 

and the 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 considered the appropriate NOAEL to be used for the 

setting ofthe AOEL. 

A vailable studies indicate that AE 0001789 is rapidly absorbed by rats following oral administration by 

gavage. More than 82 to 90% of the administered dose was absorbed in rats receiving 2 mglkg bw of AE 

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 

considered to be appropriate for setting AOEL. For this reason the following systemic AOEL is proposed 

AOEL systemic = 58 (mg/kg/day)/100 = 0.58 mg /kg/day 

Proposed maximum acceptable concentration in drinking water 

No suitable human data are available and chronic exposure animal studies involving administration of AE 

0001789 drinking water were not carried out. It is therefore proposed that for use in EU the Maximum 

Allowable Concentration (MAC) in drinking water be 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) proposed that for risk assessment purposes a 

reference human be considered to weigh 64 kg and be considered to have a daily intake of drinking water 

of].4 litres. Thus the MAC value for AE 0001789 for use in EU can be estimated as follows ­

MAC = 0.039 x 64 kg = 1.78 mg of AE 0OO178911itre = 1.78 mglL 

].4 

For the USA and Canada., theoritical concentrations of the 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 of Concern (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: 

1023 
Page 441 of 441 EBBayer CropScience 

2007-03-28 

Tier 2, IIA, Sec. 3, Point 5: Cyprosulfamide 

T a e bl 51111 - : P c roposed I assl 'fi Ication Iior USA an d C d ana a

Guideline No. Study Type Results Toxicity Catlllory I 

LDso > 2000 I 

81-1 Acute Oral · mglkg in males & III 

i females 

I 

· LDso >2000 mglkg 


81-2 
 Acute Dermal 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 81·6 i N/A

Sensitization , sensitizer 

Based on its toxicological and pharrnacokinetic metabolism profile no additional classification or 

labelling is considered to be appropriate. 

1024 
".r'-"'::-' 

I' -' 

-: ;. . , .. '- ~ .. 

Company Official: 

Company Name: 

Company Contact: 

Company Telephone: 

TRANSMITTAL DOCUMENT 

Cyprosulfamide 

OECD Annex II I Joint Review 

Transmittal Date 

April 16, 2007 

Bayer Crop Science 

R.M. See 


919-549-2913 


1025 
BIBLIOGRAPHY OF SUBMITTED DATA 


Cain. P.;TransmittBl DocLJment for cyprosulfamic:le 

(17891;8ayer CropScience. 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 of AE 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 of AE 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 

MilA Sec 2 

CA47069601 

:3 Fontaine. L. D.;Proouct chemistry of AE 0001789 

tecl1nical;8ayer CropScience. Kansas City. MO. USA; 

Report No. BR2561: Document No. M-28618C-Ol1;311312007: Pages: 308 

830 Group B MilA Sec 1 

MilA Sec 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 of the QUllnturT' 

yield and assessment of the environmental half-life of the 

direct photodegradation in water;Bayer CropScience 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 of the 

quantum yield and assessment of the environmental halflife of the direct phOlo-oegradatiol1 in water;Bayer 

CropScience AG, 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 of the Quantum yield and assessment of 

the environmental half-life of the direct 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 !he chemical Nfelime 

in the 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.;Aerobic degradation and metabolism of AE 

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;Bayer Crop$cience. Stilwell. 1<$, 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 under aerobic 

laboratory conditions; Bayer CropSc1ence 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 of degradation in three european soils under aerobic 

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;Bayer CropScience AG. Monheim, 

Germany; Report No.: MEF.061103; Document No.: M274945-01-2;211512007; Pages: 86 

162-1 KIIA 7.2.3 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;Bayer CropScience. 

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;Bayar CropScience AG, 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;Bayer CropScience. 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;Bayer Croo$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 

by column feachlng;Bayer CropSCience 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 of AE 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 of AE 0001789 in 

Illinois soil. 2oo5;Bayer CropScience. 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 field dissipation of AE 0001789 in 

Ontario. Canada soil. 2005;Sayer CropScience. 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 of AE 0001789 in 

California soil. 2005;Sayer CropScience. 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 toxicity of AE 0001789 

technical to the 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.:AaJte tOXicity of AE 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 of AE0001789· 

cyCIopropyf..sulfamoylbenzamide (AE 0852999) to the 

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 of 2ft 

1029 
BIBLIOGRAPHY OF SUBMITTED DATA 


32 Banman. C. S.: Lam, C. V.:Acute toxicity of AE 0001789· 

anisic acid (AE 0854787) to the Daphnia magna under 

static conditions,Bayer CropScience. 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.;Acute tQJ(icity of AE 0001789­

descyclopropylamillo (AE 0893016) to the Daphnia magna 

under static conditions;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 of AE 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 KIIA 8.3.1,1 41069633 

35 Banman, C. S.; Lam. C. V.;Acute tOXicity of AE 0001789· 

cyclopropyl-sulfonic acid (AE 2158927) to the Daphnia 

magna under static conditions;Bayer CropScience. 

Stilwell. KS. USA: Report No.: EBUBP096: Document No.. 

M-277112·01-1 ;612812006: Pages' 36 

850.1010 41069634 

36 Weyers. A.:PE 0001789-1!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 0001789 technical - Acute toxicity to 

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· acute toxicity to 

eastern oyster (CraSSOSb'ea virginal under flow-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 of AE 000~789 

to \he ra.nbow trout (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 Toxicity 0( 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 of AE 0001789 

technical to the Daphnia magna· under static-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.:AE 0001789 ted'1nical· Ufe-cycle toxicity test 

with myslds (Americamysis bahla) under flow-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 of AE 0001789 

\ectmicaJ 10 the Fathead Minnow (Pimephales promelas) 

under flow-through conditions;Bayer CropScience, 

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 toxicity for bobwhite quail 

(Colinus virginianus) with AE 0001789 a.s.;Bayer 

CropScience AG, 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;Bayer CropScience, 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:Bayer CropScience, 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 subacute dietary 

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.:ENect of technical AE 0001789 on northern 

bobwhite reproduction;Bayer CropScience, 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.;Effects of AE 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 - Effects on eleven species of norl-target terrestrial 

plants: seedling emergence and growth test (tie~ 1 and 

2);6ayer CropScience 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 species of non·target terrestnal 

plants: vegetative vigour test (tier 1 );Bayer CropScience 

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 of AE 

0001789 technical 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: 8 of20 

1032 
BIBLIOGRAPHY OF SUBMITIED DATA 


56 Christ, M. T.; Holfmann, J. M.: lam, C. V.;Toxicity of AE 

0OO1789-cyclopropyl-sulfamoylbetU:amide (AE 0852999) 

to duCkweed (Lemna 91bba G3) under stallc-renewal 

conditions;8ayer CropSc: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 of AE 

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.;Toxicityof AE 

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 of AE 

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 of AE 0001789­

descyclopropyl (AE 1272799) to duckweed (Lemna gibba 

G3) under static-renewal condnions;Bayer CropScience, 

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 of AE 0001789 technical to 

the green alga Pseudokirchneriella subcapitata;Bayer 

CropScience. Stilwell. KS. USA; Report No: EBUBP012; 

Document No.: M·25201 S-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 of AE 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.;Toxicity d 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;Bayer CropScience, 

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 or AE 0001789­

descyclOpropyl (AE 1272799) to the green alga 

PSi!Udokirchneriella subcapitata;Bayer CrapScience, 

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 of AE 0001789· 

cycIOpropyl-sutron;c ilc.d (AE 2158927) 10 the green alga 

Pseudokirchnerieila sUbcapitata;Bayer CropScience. 

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 

substance technical Acute toxicity to earthworms (EisenJa 

felida) tested in artifical soil:Bayer CropScience AG, 

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 (Eisenia fetida) tested in artificial soil:Bayer 

CropScience AG. 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-27711 5­

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 of the total radioactive residue in 

sorghum raw agricultural commodities after seed 

treatment with ISu!fonylbenzamide·UL·14C] AE 

000 1789; 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; Report No.: 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 KIIA 6.2,3 47069732 

84 Timberlake. 8, C,;Validation of Bayer CropScience method 

US-008-P06-01 • M analytical method tor the 

delerminatiOn of residues 01 AE 0001789 in crop matrices 

using LCIMS/MS;Bayer CropScience, Slilwell, KS, USA: 

Report No., RAUBP046; Document No: M-286209-01. 

1;3122/2007; Pages: 170 

860.1340 KlIA 4,3 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 of AE 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 

determination of residues of AE 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 
BmLIOGRAPHY OF SUBMITTED DATA 

88 S~lian, p,; Wolters, A.;SlOrage stability of c:yclopropyl· 

sulfamoylbenzamide, suifonamlC!e-lactale and 

sulfonamide-alanine (metabolites of AE 0001189) in plam 

mat/'lces fOl' 18 montns· Results for an interval of 0 \0 12 

months;6ayer CropScience 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 

of Bayer method no. UB·006-A06.(Jl • AE 0001189: An 

analytical method for the detemination of AE 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 of AE 

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 of the method of analysis for the determination 

of AE 0001789 and its metabolites 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,;Stabitity of AE 0001789 and its 

metabolites AE 1272799, AE 0852999, AE 0893016. AE 

0467398 and AE 1448796 in soil ruling frozen storage 

(reported through a maximum cl383 days storage);Bayer 

CropScience. Stilwell. KS. USA; Report No.: RAUBP005; 

Document No.: M-285691.(Jl·1;319/2007: Pages: 49 

850.1340 KIIA 7,3,' 47069741 

93 Nandihalli. U, B,;Analy1lcal method for the determination of 

residues of AE 0001789 and ~s metabolite AE 0652999 in 

water using LClMSIMS;Bayer CropScience. 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 the determination of AE 

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 
BIBLIOGRAPHY OF SUBMITIED DATA 


96 	 Lam, C. K.;AE 000 1789 - Magnitude of the 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 - Magnitude 01 the residue Irv'on field com. _t com, 

and pop com;Bayer CropScience, 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 - Request for waiver of 

the study of the magnitude of the residue 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 accumulation of [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;AEOO01789 500 SCMagnitude or the 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 of the residue in field 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 • Acute tOXicity 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 - Acute toxiCity in the rat after 

dermal applicatior.;Bayer HealthCare 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 SUBMITTED DATA 


, 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 - Acute eye irritation on 

rabbits;Bayer Healtl<jCare AG, Wuppertal. Germany; Report 

No.: AT02436; Document No.: M-258984-01-2;211912007: 

Pages: 24 

87 0.2400 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.: A T01649: 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.;AE 0852999 - 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.: SA 02352; 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 dietary administration:6ayer Cr0p5C1ence. 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;Bayer CropScience 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 with technical grade AE 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· Developmental toxicity study in 

the rabbit by ga\lage {Complementary study);Bayer 

CropScience SA. 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 by gavage;Bayer CropSc::ience SA, Sophia Antipolis. 

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 KIIA 5.61 47069815 

1>7 Eigenberg, D. A.;A chronic toxic::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,;Chronic toxicity and carcinogenicity 

study of AE 0001789 ill the wister rat by dietary 

administration;Bayer CropScience 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 of AE 0001789 

in the C57BLI6J mouse by dietary 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 incorporation and 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: AE 0001789)­

SalmoneUalmicrosome test - 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 000 1789-sulfamoybenzoic acid (AE 

0467398) - (Project: AE (01789) - Salmonellalmicrosome 

test plate incorporation and preincubation method;Bayer 

HcallhCareAG, Wuppertal. Germany; Report No.: 

AT03259; Document No,: M-278703-01-2:212212007; 

Pages: 62 

870,5100 KilA5.8 47069821 

123 Herbold, B.;AE oo01789-cyclopropyl-sulfamoybenzamide 

(tested as AE. 0852999 carboxamide) 

Salmonellalmicrosome test plate incorporation and 

prei,cubation method:6ayer HealthCare AG. 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 for the 

detection of induced forward 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 

for the detection or induced forward 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 as AE. 0052999 carbox2I'nide) {project: AE 

0001189) - V79/HPRT-Iest in vitro for the detection of 

induced forward 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 HeafthCare AG, 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 Ii or20 

1041 
BffiLIOGRAPHY OF SUBMITTED DATA 

128 Thurn. M.;AE 0001789-Sulfamoylbenzoic acid· Project: AE 

0001789 - In vitro 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 000 1789-cyciopropyl-sulfamoybenzamide 

(Project AE 0001789) - In 'litro chromosome aberration 

test with 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-test on 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;Bayer CropScience, 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 CropScience AG. 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 in the rat:Bayer CropScience AG. 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:A 28-day toxicity feeding study ir the 

beagle dog wi: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 8 of 2() 

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 assessment for 

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 assessment for cyprosulfamide (AE 

0001789) for the 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 of the 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 • 

List of endpoints for the active substance cyprosulfamide 

(AE 0OO1789):Bayer CropScience; 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 of AE0001789 SOO Document H 47069841 C 

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 

K111A 1 2.10.1 

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 

KUlA1 2.8.8.1 

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 of AE 8702600 KIl1A17.1.7 47069842 

0001189 FS 500 formulation;Bayer CropScience. 

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 
Bayer Crop Science 


Material Safety Data Sheet MSDS Number: 102000006398 

CYPROSULFAMIDE TECHNICAL MSDS Version 1.0 

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 refer to Section 11 for detailed toxicological information. 

Emergency Overview Caution! Dust may form explosive mixtures with air. 

Physical State powder 

Appearance white 

Routes of Exposure Inhalation, Ingestion 

Eye 

Skin 

Ingestion 

Inhalation 

Wash off immediately with plenty of water for at least 15 minutes. Remove 

contact lenses, if present. after the first 5 minutes, then continue rinsing eye. Get 

medical attention if irritation develops and persists. 

Wash off immediately with soap and plenty of water. If symptoms persist. call a 

physician. 

Rinse mouth. Keep patient warm and at rest. Do NOT induce vomiting. If 

symptoms persist. call a physician. 

Move to fresh air. Keep patient warm and at rest. If symptoms persist, call a 

physician. 

Page 1 of7 

1045 
Bayer CropScience 


Material Safety Data Sheet MSDS Number: 102000006398 

CYPROSULFAMIDE TECHNICAL MSDS Version 1.0 

Notes to Physician 

Signs and 

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 

Not explosive. 


92/69/EEC, A.14 IOECD 113 


In the event of fire the following can be released: 


Nitrogen oxides (NOx) 


Sulphur oxides 


Carbon monoxide (CO) 


Water spray, Foam 


Remove product from areas of fire, or otherwise cool containers with water in 


order to avoid pressure being built up due to heat. Do not allow run-off from fire 


fighting to enter drains or water courses. 


Firefighters should wear NIOSH approved self-contained breathing apparatus 


and full protective clothing. 


Personal Precautions 

Methods for Cleaning Up 

Additional Advice 

Use personal protective equipment. Remove all sources of ignition. 

Avoid dust formation. Keep unauthorized people away. Isolate hazard area. 

Avoid contact with spilled product or contaminated surfaces. 

Avoid dust formation and electrical charging (sparking) because dust explosion 

might occur. 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. HANDLING AND 

Page 2 of7 

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Bayer CropScience 


Material Safety Data Sheet MSDS Number 102000006398 

CYPROSULFAMIDE TECHNICAL MSDS Version 1.0 

Handling Procedures 

Storing Procedures 

Work/Hygienic 

Procedures 

Min/Max Storage 

Temperatures 

Ensure adequate ventilation. Avoid dust formation. 


Dust may form explosive mixtures with air. Keep away from heat and sources of 


ignition. Take measures to prevent the build up of electrostatic 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 water after handling and before eating, 

drinking, chewing gum, using tobacco, using the toilet or applying 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 contact with skin and eyes. Do not breathe dust. 

Tightly fitting 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 extract ventilation. 


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 

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Bayer CropScience 


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 

Water solubility 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 

Molecular Weight 374.4 g/mol 

Decomposition 245 - 355°C 

Temperature DSC , Heating rate: 10 Klmin 

Partition coefficient: n­ 0.158 log Pow: -0,8 at 23°C 

octanollwater measured at pH 7 

SECTION 10. STABILITY AND 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 Dermal Toxicity rat: LD50: > 2,000 mgtkg 

Acute Inhalation Toxicity rat: LC50: > 3.51 mgtl 

Highest attainable concentration. 

Determined in the form of a 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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Material Safety Data Sheet MSDS Number: 102000006398 

CYPROSULFAMIDE TECHNICAL MSDS Version 1.0 

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 mutagenicity tests do not indicate genotoxic activity. 

SECTION 12. ECOLOGICAL INFORMATION 

Toxicity to Fish Cyprinodon variegatus (sheepshead minnow) 

LC50: > 106 mgtl 

Toxicity to Aquatic Plants Lemna gibba (duckweed) 

EC50: > 104 mgtl 

Exposure time: 7 d 

Pseodokirchneriella subcapitata 

EC50: > 99.7 mgtl 

Exposure time: 96 h 

Acute Toxicity to Aquatic 

Invertebrates 

Water flea (Daphnia magna) 

static test 

EC50: > 102 mgll 

Exposure time: 48 h 

Toxicity to 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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Bayer CropScience 


Material Safety Data Sheet MSDS Number: 102000006398 

CYPROSULFAMIDE rECHNICAL MSDS Version 1.0 

LD50: > 1 00 ~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 allow to get into surface water, drains and ground water. 

Precautions 

General Disposal 

Guidance 

Container Disposal 

In accordance with current regulations and, if necessary, consultation with 

the site operator and/or with 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 Substances Control Act (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 

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Bayer CropScience 


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 of California to cause cancer. 

This product does not contain any substances known to the State of California 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 Water Act 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 or implied 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 

1051