1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Neurotoxicity

Description of key information

OECD 424, subchronic, rat, feed: NOAEL = 306.9 mg/kg bw/day (females); LOAEL = 580.9 mg/kg bw/day (females)
OECD 424, acute, rat, gavage: NOAEL = 100 mg/kg bw; LOAEL = 500 mg/kg bw; neuronal degeneration at 2000 mg/kg bw (males) 
OECD 426, developmental, rat, feed: NOAELmaternal = 83.8 mg/kg bw/day; LOAELmaternal = 568 mg/kg bw/day; NOAELoffspring = 83.8 mg/kg bw/day; LOAELoffspring= 432 mg/kg bw/day

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: J.M.A.F.F. Notification No 12 Nousan 8147

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Health Canada PMRA DACO No 4.5.11

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Han Crl:WI (HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., (Raleigh, NC)
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males 232.0-302.4 g, females 146.9-195.4 g
- Fasting period before study: not applivcable
- Housing: Individually housed in suspended stainless steel wire-mesh cages, each containing a feeder, a source of water (pressure-activated water lixits), and deotized cage board in the bedding tray.
- 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 neurobehavioral testing.
- Water: Tap water (Kansas City Missouri Municipal Water) was provided ad libitum except during neurobehavioral testing.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): min. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 October 2006 To: 18 January 2007; still within expiry date of test substance

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The diet was prepared every other week.
- Mixing appropriate amounts with (Type of food): Purina Mills Rodent Lab Chow 5002 in meal form
- Storage temperature of food: Ambient temperature, feed was available for ad libitum consumption for a period of one week prior to changing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of BCS-AA10717 in the ration was measured by LC-MS/MS 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 20 and 10000 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 20 and 10000 ppm had percent relative standard deviations (%RSD) of 3.2% and 2.9%, respectively.
Stability Analysis:
The stability of BCS-AA10717 in the ration was established at room temperature at dietary concentrations of 20 and 10000 ppm, with no appreciable decrease in concentration with seven days of storage. BCS-AA10717 was stable at freezer conditions for 63 days, with no appreciable decrease in concentration at 20 and 10000 ppm.
Concentration Analysis:
Actual (analytically-determined) concentrations of the active ingredient in the 200, 4000, 8000 and 10000 ppm dietary levels used in this study averaged 94% to 96% of the nominal concentrations. Based on these results, the mean analytically confirmed dietary levels for this study were 191, 3847, 7589 and 9394 ppm for males and females, respectively.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously in feed

Dose / conc.:

200 ppm

Remarks:

corresponding to 12.2 mg/kg bw/day for males and 15.1 mg/kg bw/day for females

Dose / conc.:

4 000 ppm

Remarks:

corresponding to 243.6 mg/kg bw/day for males and 306.9 mg/kg bw/day for females

Dose / conc.:

8 000 ppm

Remarks:

females only; corresponding to 580.9 mg/kg bw/day

Dose / conc.:

10 000 ppm

Remarks:

males only; corresponding to 585.7 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The rationale for dose selection was based primarily on the results of 3 previously performed studies: 1) A 90-day toxicity study, with the test substance administered via the diet at nominal concentrations of 0, 200, 5000 and 10000 ppm to male and female Wistar rats (10/sex/dietary level); 2) The preliminary results from an ongoing chronic toxicity study with the test substance administered via the diet at nominal concentrations of 0, 300, 3000 and 10000 ppm to male and female Wistar rats (70/sex/dietary level); 3) The preliminary results from a reproductive toxicity range-finding pilot with the test substance administered via the diet at nominal concentrations of 0, 200, 1000, 3000 and 8000 ppm to male and female Wistar rats (10/sex/dietary level).

Based on these results, the dietary levels selected for the present subchronic neurotoxicity study were 0, 200, 4000 and 10000 ppm for males and 0, 200, 4000 and 8000 ppm for females. The 8000 and 10000 ppm dietary levels were selected as maximum-tolerated doses (MTD) in females and males, respectively, following subchronic exposure. The 200 ppm dietary level was selected to produce no evidence of toxicity for endpoints measured in this neurotoxicity study and the 4000 ppm dietary concentration was selected as an intermediate dietary level.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (once daily on holidays and weekends)
- Cage side observations included: Mortality or clinical signs of moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once each week

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; additionally on day of sacrifice for determination of terminal body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Individual food consumption was measured weekly.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as average daily intake from the consumption and body weight gain data: Yes, the general relationship used for this calculation was: [AI in feed (ppm)/1000] x [feed consumed (g/kg bw/day)] = mg AI/kg bw/day

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-exposure and pre-terminal (week 12)
- Dose groups that were examined: all animals

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined:
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-to-touch).
Open field (2 min.) observations included: number of rears, piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy (excessive or repetitive behavior), bizarre behavior, 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 [Chatillon, Model DFIS-10 and DFS-010 digital strain gauges (5.0 kg capacity), which were both equipped with a grid system attached to an extension arm], body weight, body temperature, and landing foot splay.
- Description of procedures: On the day prior to each test day, the appropriate animals were placed in the correct sequence that had been established for testing on that day. Animals were then transferred to the room where testing took place and allowed to acclimate with minimal disturbance until testing on the following day. Data were collected while the rats were in their home cage, during handling, and in an open field for 2 minutes (in the center 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 fieldSets 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.
Each week, testing was staggered over two days for each sex to accommodate the schedule for behavioral testing. Males and females were tested on separate days, with the open field and mazes cleaned during the ensuing interval to reduce the residual scent from the other sex.
This FOB closely follows the battery of tests described by Moser, with each animal tested individually. Scoring criteria and explicitly-defined scales were used to rank the severity of observations that do not readily lend themselves to quantitation. The procedures used to determine landing foot splay and grip strength are based on established methods.
- Minimization of bias: 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. The dose group identification was concealed before animals were transferred to the testing room to ensure that testing would be performed without knowledge of the group assignment.
- Same technicians used throughout testing: No, but inter-observer reliability has been established in order to allow multiple persons to perform either the observations and/or measurements, ensuring the consistency of the results of each technician.
- Technicians were blind to treatment status of animals: Yes, the dose group identification was concealed prior to testing to ensure that testing would be conducted without knowledge of the group assignment.
- Site of testing: The test room was a standard animal room that was maintained on the same light:dark cycle and settings for temperature and relative humidity as the animal room, with tests conducted during the light phase.
- Time schedule for examinations: Once during the week prior to initiating the exposure and again during weeks 2, 4, 8 and 13.
- Environmental conditions: Same light:dark cycle and settings for temperature and relative humidity as the animal room.
- Scoring criteria (if any): When applicable, observations were scored on intensity as follows: 1) slight (barely perceptible or infrequent) or 2) moderate to severe. For most of the parameters more detailed scores were used which are specified in an internal SOP.
- Duration of observation period for open field observations: 2 minutes

LOCOMOTOR ACTIVITY: Yes, approximately 30 minutes after the last animal in the set (8 rats maximum) had finished the FOB.
- Type of equipment used: Figure-eight maze, each consisting of a series of inter-connected alleys, converging on a central arena and covered by transparent plastic. 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. The floor of each maze rested above absorbent paper which was changed at the end of each day. A Columbus Instruments (Columbus, OH) Universal Maze Monitoring System and a personal computer were used for automated data collection. Broadspectrum background noise was provided throughout the test to minimize acoustical variations during testing. The uniformity of light intensity (100±70 lux) over each of the mazes was verified daily.
- Length of session, number and length of subsessions: 60 minutes, 6 subsessions of 10-minute length
- Noise level: 74±2 dB(A)
- Parameters measured: Motor activity, locomotor activity, habituation.
- Total activity: Motor activity was measured as the number of beam interruptions that occurred during the test session.
- Ambulatory activity: 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.
- Other: Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and (histo)pathology:

- Time point of sacrifice: In week 13.
- Number of animals sacrificed: All animals; 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 inadequate.
- Parameters measured: Examination of all organs, body cavities, cut surfaces, external orifices and surfaces. 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 formalin.
- Brain weight: The brain was weighed upon removal from the skull, prior to placement into formalin, and the brain:body weight ratio was calculated.
- Length and width of brain: Not determined.
- Procedures for perfusion: Animals were deeply anesthetized using an intraperitoneal dose (50 mg/kg) 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 formaldehyde) in phosphate buffer.
- Number of animals perfused: 6 animals/sex/dose
- Tissues evaluated: Parameters examined: Olfactory bulbs, cerebral cortex, caudate-putamen/globus pallidus, hippocampus, thalamus, hypothalamus, midbrain (tectum, tegmentum, and cerebral peduncles), cerebellum, medulla oblongata, spinal cord (cervical, thoracic and lumbar swelling), cauda equina, Gasserian ganglion, optic nerve, eyes, gastrocnemius muscle, sciatic nerve (bilateral), tibial nerve (bilateral), sural nerve (bilateral), lumbar dorsal root ganglion, lumbar dorsal root fibers, lumbar ventral root fibers, cervical dorsal root ganglion, cervical dorsal root fibers, cervical ventral root fibers and any gross lesions collected at necropsy.
- Type of staining: Hematoxylin and eosin, modified Lee's stain.
- Methodology of preparation of sections: Micropathology examinations were performed on a comprehensive battery of neural tissues from perfusion-fixed control and high-dose rats of both sexes. Eight coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar), the cauda equina, eyes, optic nerves and gastrocnemius muscle were embedded in paraffin and stained utilising hematoxylin 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). Peripheral nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA, as well, and cut in cross/transverse-section as well as longitudinal section. GMA-embedded tissues were sectioned at 2-3 µm and stained using a modified Lee's stain. The sciatic nerve was also cross-sectioned at approximately 2-3 µm and stained with a modified Lees stain.
- Thickness: Paraffin-embedded 5 µm, GMA-embedded 2-3 µm
- Embedding media: Paraffin or glycol methacrylate
- Number of sections: not specified
- Number of animals evaulated from each sex and treatment group: 12/sex/dose for gross necropsy, 6/sex/dose (control and high dose) for micropathology. Perfusion-fixed animals at low- and mid-dose levels were not evaluated unless treatment-related lesions were present in the high-dose group.

Positive control:

The study did not include concurrent positive controls, but references are made to studies conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of the test procedures, the adequacy of training of technical personnel and to serve as a historical control for the FOB.
To assess motor activity, references have been made to previous studies with untreated animals and with rats treated with reference substances increasing (triadimefon) and decreasing (chlorpromazine) activity, that have established the sensitivity, reliability and validity of the test procedures used. Finally, previous studies performed at Bayer's Toxicology laboratory with trimethyltin and acrylamide have established the sensitivity and reliability of the micropathology procedures for detecting lesions in peripheral nerves and the central nervous system.

Statistics:

In general, statistical evaluations were performed using software from either INSTEM Computer Systems or SAS. With the exception of Bartlett's test, which was tested at p≤0.001, the level used to establish statistical significance was p≤0.05.
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 of Contrasts, respectively.
Motor and locomotor activity (activity for the entire session and activity for each 10-minute interval) were analyzed using ANOVA procedures. Session activity data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA if there was a significant interaction with test occasion. For weeks in which there was a significant treatment effect, Dunnett's test was used to determine which groups, if any, 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 the repeated measures, followed by a Repeated Measures ANOVA to determine on which weeks there was a significant treatment by interval interaction.

Continued under "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality:

mortality observed, treatment-related

Description (incidence):

adverse

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

adverse

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

adverse

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

adverse (clinical signs at sacrifice)

Neuropathological findings:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
High-dose:
In males, tremors and repetitive chewing movements were observed in 1/12 animals, perianal stain in 9/12 animals, red lacrimal stain in 2/12 and red nasal stain in 3/12 animals. Observations in females included tremors in 5/12 animals and perianal and urine stain in 1/12 animals, each.

No treatment-related findings were observed at lower dietary levels in any animal.

There were no treatment-related deaths in males or females at any dietary level. Three females (one assigned to the control group and two assigned to the mid-dose were found dead after pretreatment week FOB testing.

BODY WEIGHT AND WEIGHT GAIN
High-dose:
For high-dose males, body weight was non-statistically decreased (8-10%), compared to control, on day 28 through study termination. For high-dose females, body weight was statistically decreased (9-12%), compared to control, on day 7 through study termination. In addition, total body weight gain in high-dose males was non-statistically decreased (20%), compared to controls. In high-dose females, total body weight gain was statistically decreased (30%), compared to controls.

Mid-dose:
For mid-dose females, body weight was slightly (non-statistical) decreased (6%) on days 21-28, and total body weight gain for the duration of the study was slightly (non-statistical) decreased (14%). In addition, this trend [i.e., slight decrease (4-5%) in body weight] in mid-dose females was evident for all subsequent weeks of the study.

Body weight and total body weight gain were not different from controls in low- and mid-dose males or in low-dose females.

FOOD CONSUMPTION AND COMPOUND INTAKE (IF FEEDING STUDY)
High-dose:
For high-dose males, food consumption was statistically decreased on days 0-7, 21-28 and 28-35 (18%, 12% and 12%, respectively). Also, food consumption was non-statistically decreased (6-9%) in high-dose males on days 35-42 through days 63-70.
For high-dose females, food consumption was non-statistically decreased (11%) the first week of the study and non-statistically increased (15%) on days 14-21. Food consumption was statistically decreased (13-19%) beginning on days 21-28 and continuing for all remaining weeks measured. The observed increase on days 14-21 was not thought to be related to treatment, since it was an isolated occurrence that involved an increase instead of a decrease in food consumed, it was not statistically different from control and it occurred in only one sex.

Food consumption was not affected by treatment at the two lowest dietary levels in either sex.

No calculation of food consumption was possible for study weeks 10 and 11 because weighing of food was inadvertantly neglected at the end of week 10/beginning of week 11.

OPHTHALMOSCOPIC EXAMINATION
All ophthalmologic findings were considered incidental and unrelated to test substance exposure.

NEUROBEHAVIOUR
For the overall 60-minute test session, high-dose females had measures of motor and locomotor activity that were statistically decreased for week 2 (34% and 42%, respectively). In addition, measures of locomotor activity for high-dose females were statistically decreased (43%) for week 4. Lastly, there were non-statistical treatment-related decreases (27% each) in measures of motor activity for week 4 and in measures of locomotor activity for week 8 in high-dose females. Motor and locomotor activity was not affected by treatment in males at any dietary level or in low- and mid-dose females.

Motor and locomotor activity data were also analysed 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 the test substance BCS-AA10717 in males or females at any dietary level.

GROSS PATHOLOGY
There were no compound-related gross lesions evident at terminal sacrifice in males or females at any dietary level. There were a few gross observations seen in high-dose males that are thought to be related to treatment and included a wet/stained ventrum and brown perianal stain in 2/12 animals and lacrimation in another 2/12 animals.

The remaining findings were considered incidental and unrelated to treatment. This included alopecia of the forelimbs observed in a few control and treated females and unilateral (right) kidney pelvic dilation seen in one control male. None of these findings were considered treatment-related.

Terminal body weight was statistically decreased (9%) in high-dose females and non-statistically decreased (9%) in high-dose males. Terminal body weight was not different from control in mid- and low-dose males and females. Absolute and relative brain weights were not different from control at any dietary level in either sex.

NEUROPATHOLOGY
There were no treatment-related findings in neural and/or non-neural tissues from perfusion-fixed high-dose males or females that were related to administration of the test substance. Tissues from animals at lower dose levels were, therefore, not examined.

Dose descriptor:

NOAEL

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: corresponding to 243.6 and 306.9 mg/kg bw/day in males and females, respectively

Dose descriptor:

LOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: neurobehaviour (tremors, repetitive chewing movements, perianal, red lacrimal and red nasal stain, wet/stained ventrum, lacrimation)

Remarks on result:

other: corresponding to 585.7 mg/kg bw/day

Dose descriptor:

LOAEL

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: neurobehaviour (motor and locomotor activity, tremors, perianal and urine stain)

Remarks on result:

other: corresponding to 580.9 mg/kg bw/day

CONCLUSION:

Treatment-related findings included statistical and/or non-statistical decreases in body weight, total body weight gain and food consumption in high-dose males and females. In addition, measures of motor and locomotor activity were statistically and non-statistically decreased, compared to controls, in high-dose females. There were various clinical signs in one or both sexes at the highest dietary level that included tremors, repetitive chewing movements, perianal stain, diarrhea, wet/stained ventrum, lacrimation, urine stain, red lacrimal stain and red nasal stain. Lastly, body weight and total body weight gain were slightly (non-statistically) decreased in mid-dose females.

These results establish a NOAEL for neurotoxicity endpoints of 243.6 mg/kg bw/day in males and 306.9 mg/kg bw/day in females (corresponding to nominal 4000 ppm in the diet).

Reference 2

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions. Contradictions between summary and raw data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

yes

Remarks:

adapted to single exposure

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Deviations:

yes

Remarks:

adapted to single exposure

Qualifier:

according to guideline

Guideline:

other: J.M.A.F.F. Notification No 12 Nousan 8147

Deviations:

yes

Remarks:

adapted to single exposure

Qualifier:

according to guideline

Guideline:

other: Health Canada PMRA DACO No 4.5.12

Deviations:

yes

Remarks:

adapted to single exposure

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar HAN CRL:WI (HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., (Raleigh, NC)
- Age at study initiation: at least 9 weeks
- Weight at study initiation: Initial study: males 247.9-319.4 g, females 162.2-203.8 g; Follow-up study: females 168.0-203.8 g
- Fasting period before study: Not fasted before treatment
- Housing: Individually housed in suspended stainless steel wire-mesh cages, each containing a feeder, a source of water (pressure-activated water lixits), and deotized cage board in the bedding tray.
- Diet (e.g. ad libitum): Purina Mills Rodent Lab Chow 5002 in meal form provided for ad libitum consumption during the acclimation period and throughout the study except during neurobehavioral testing.
- Water (e.g. ad libitum): Tap water (Kansas City Missouri Municipal Water) was provided ad libitum except during neurobehavioral testing.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): Minimum of 10.17 (initial study) or 12.77 (follow-up study)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 May 2007 To: 13 September 2007; still within expiry date of test substance

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Doses were prepared by suspending the test substance in 0.5% (w/v) methylcellulose in deionized water.

VEHICLE
- Justification for use and choice of vehicle (if other than water): commonly used vehicle for oral dosing by gavage
- Concentration in vehicle: 10, 50, 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): Lot #125K0196

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of BCS-AA10717 in the vehicle was measured using high-performance liquid chromatographic/ultra violet (HPLC/UV) analysis. The homogeneity and stability of BCS-AA10717 in the vehicle were established during the analytical method development using samples at nominal concentrations (1 and 200 mg/mL) that bracketed the range of concentrations used in the present study (nominal 2.5 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 BCS-AA10717.
Homogeneity Analysis: Homogeneity of the test substance in the vehicle was accepted for the range of concentrations used here, as the 1.0 and 200 mg/mL concentrations had percent relative standard deviations (%RSD) of 1.0% and 1.4%, respectively.
Stability Analysis: The stability (at room temperature conditions) of BCS-AA10717 in the vehicle was established, with no appreciable decrease in concentration with seven or eight days at room temperature storage for nominal concentrations of 1.0 or 200 mg/mL, respectively (equivalent to doses of 10 or 2000 mg/kg, respectively).
Concentration Analysis: For the initial study, doses of 0, 100, 500 and 2000 mg/kg for males and females ranged from 97% to 101% of the nominal concentrations. Based on these results, the analytically-confirmed doses for males and females were 0, 97, 501 and 2020 mg/kg. For the follow-up study, doses of 0, 25 and 50 mg/kg for females were 96% and 93%, respectively of the nominal concentrations. Based on these results, the analytically confirmed doses for females were 0, 24 and 47 mg/kg.

Duration of treatment / exposure:

Single exposure by gavage

Frequency of treatment:

Single exposure

Remarks:

Doses / Concentrations:
100, 500, 2000 mg/kg bw
Basis:
actual ingested
Initial study

Remarks:

Doses / Concentrations:
25, 50 mg/kg bw
Basis:
actual ingested
Follow-up study

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of an acute oral toxicity study in young adult female Wistar rats. In that study, six fasted female Wistar rats were administered an acute oral (gavage) dose of 2000 mg/kg as an aqueous suspension in 2% cremophor EL in tap water, at a dosing volume of 10 mL/kg. Animals were observed for mortality and clinical signs for at least 14 days after treatment. The test substance produced no mortality and no clinical signs at the limit dose. These results supported the use of a limit dose (2000 mg/kg) in the neurotoxicity study but provided no information to establish the time of peak effect.
Thus, the results from a study using radio-labeled BCS-AA10717 were examined to estimate the time of peak effect. In that study, fasted adult male and female Wistar rats (4/sex/dose) received a single oral (gavage) dose of an aqueous 1% (w/w) carboxymethylcellulose and 0.5% (w/w) Tween 80 (ca. 1.0 mL) solution/suspension with BCS-AA10717 at a target rate of 14 mg/kg body weight. The tmax in plasma in males and females from the indane-3 and the triazine-2,4 radiolabel groups occurred at approximately 40 and 60 minutes, respectively following administration of the single dose.

Based on these collective results, the doses selected for the initial study were 0, 100, 500 and 2000 mg/kg for both sexes. The 2000 mg/kg dose was selected as a limit dose that may produce slight evidence of toxicity, the middle dose was selected to produce minimal or no effect and the low dose was expected to be an overall NOAEL in both sexes. Based on the initial findings, it was necessary to conduct a follow-up study to establish a NOAEL for measures of activity in females. In that study, doses selected were 0, 25 and 50 mg/kg. The 25 and 50 mg/kg doses were selected to establish an overall NOAEL. Based on the estimated time of peak blood concentrations at these dose ranges, the FOB began approximately 50 minutes (minimum) following dose administration, with the automated test of activity concluding at about 4 hours after treatment.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily (once daily on holidays and weekends)
- Cage side observations included: mortality, clinical signs of moribundity

DETAILED CLINICAL OBSERVATIONS: Yes, for clinical signs of toxicity.
- Time schedule: Once each day for 14 days following dosing (initial study) or for 1 or 2 days following dosing (follow-up study).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, as component of FOB. Additionally, all animals prior to dosing, and animals from the initial study on the day of sacrifice for terminal body weight measurement. Animals from follow-up study were not weighed after day 0.

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined:
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-to-touch).
Open field observations included: number of rears, piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy (excessive or repetitive behavior), bizarre behavior, gait abnormalities, vocalizations, arousal level, and amount of excretion.
Reflex and physiologic observations/measurements included: approach response, touch response, auditory response (clicker), tail pinch, pupil size at normal lighting, pupil response, righting reflex, grip strength [Chatillon, Model DFIS-10 (forelimb) and/or DFS-010 (hindlimb) digital strain gauges (5.0 kg capacity), which were both equipped with a grid system attached to an extension arm], body weight, body temperature, and landing foot splay.
- Description of procedures: 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. Animals were transferred to the testing room and were allowed to acclimate with minimal disturbance for approximately 30 minutes prior to testing. 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. It should be noted that animals from the follow-up study were treated the same as animals from the initial study for consistency. The NOAEL for clinical observations, FOB and body weight had already been established in the initial study.
- Minimization of bias: 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. The dose group identification was concealed before animals were transferred to the testing room to ensure that testing would be performed without knowledge of the group assignment.
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes, the dose group identification was concealed before animals were transferred to the testing room to ensure that testing would be performed without knowledge of the group assignment.
- Site of testing: The test room was a standard animal room that was maintained on the same light:dark cycle and settings for temperature and relative humidity as the animal room, with tests performed during the light phase.
- Time schedule for examinations: Initial study: one week prior to treatment, approx. 50 minutes (minimum) after administration of dose, and again 7 and 14 days following treatment; follow-up study: one week prior to treatment and approximately 50 minutes (minimum) after administration of dose.
- Environmental conditions: Same light:dark cycle and settings for temperature and relative humidity as the animal room.
- Scoring criteria (if any): When applicable, observations were scored as follows: 1) slight (barely perceptible or infrequent) or 2) moderate to severe. For most of the parameters more detailed scores were used which are specified in an internal SOP
- Duration of observation period for open field observations: 2 minutes

LOCOMOTOR ACTIVITY: Yes, approximately 30 minutes after the last animal in the set (8 rats maximum) had finished the FOB.
- Type of equipment used: Figure-eight maze, each consisting of a series of inter-connected alleys, converging on a central arena and covered by transparent plastic. 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. The floor of each maze rested above absorbent paper which was changed at the end of each day. A Columbus Instruments (Columbus, OH) Universal Maze Monitoring System and a personal computer were used for automated data collection. Broadspectrum background noise was provided throughout the test to minimize acoustical variations during testing. The uniformity of light intensity (100±70 lux) over each of the mazes was verified daily.
- Length of session, number and length of subsessions: 60 minutes, 6 subsessions of 10-minute length
- Noise level: 74 dB(A)
- Parameters measured: Motor activity, locomotor activity, habituation.
- Total activity: Motor activity was measured as the number of beam interruptions that occurred during the test session.
- Ambulatory activity: 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.
- Other: Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and (histo)pathology:

- Time point of sacrifice: On day 14 after administration (only animals from initial study)
- Number of animals sacrificed: all animals
- Parameters measured: 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 formalin.
- Brain weight: The brain was weighed upon removal from the skull, prior to placement into formalin, and the brain:body weight ratio was calculated.
- Length and width of brain: Not determined.
- Procedures for perfusion: Animals were deeply anesthetized using an intraperitoneal dose (50 mg/kg) 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 formaldehyde) in phosphate buffer.
- Number of animals perfused: 6 animals/sex/dose
- Tissues evaluated: Parameters examined: Olfactory bulbs, cerebral cortex, caudate-putamen/globus pallidus, hippocampus, thalamus, hypothalamus, midbrain (tectum, tegmentum, and cerebral peduncles), cerebellum, medulla oblongata, spinal cord (cervical, thoracic and lumbar swelling), cauda equina, Gasserian ganglion, optic nerve, eyes, gastrocnemius muscle, sciatic nerve (bilateral), tibial nerve (bilateral), sural nerve (bilateral), lumbar dorsal root ganglion, lumbar dorsal root fibers, lumbar ventral root fibers, cervical dorsal root ganglion, cervical dorsal root fibers, cervical ventral root fibers and any gross lesions collected at necropsy.
- Type of staining: Hematoxylin and eosin, modified Lee's stain.
- Methodology of preparation of sections: Micropathology examinations were performed on a comprehensive battery of neural tissues from perfusion-fixed control and high-dose rats of both sexes (2000 mg/kg males and 500 mg/kg females; 2000 mg/kg females were discontinued due to mortality on the day following treatment), as follows. Eight coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar), the cauda equina, eyes, optic nerves and gastrocnemius muscle were embedded in paraffin and stained utilising hematoxylin 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). Peripheral nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA, as well, and cut in cross/transverse-section as well as longitudinal section. GMA-embedded tissues were sectioned at 2-3 µm and stained using a modified Lee's stain. The sciatic nerve was also cross-sectioned at approximately 2-3 µm and stained with a modified Lees stain.
- Thickness: Paraffin-embedded 5 µm, GMA-embedded 2-3 µm
- Embedding media: Paraffin or glycol methacrylate
- Number of sections: not specified
- Number of animals evaluated from each sex and treatment group: 12/sex/dose for gross necropsy, 6/sex/dose (control and high dose) for micropathology. Perfusion-fixed animals at low- and mid-dose levels were not evaluated unless treatment-related lesions were present in the high-dose group. Instead of high-dose females the mid-dose females were evaluated because high-dose females were discontinued due to mortality on the day following treatment.

Positive control:

The study did not include concurrent positive controls, but references are made to studies conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of the test procedures, the adequacy of training of technical personnel and to serve as a historical control for the FOB.
To assess motor activity, references have been made to previous studies with untreated animals and with rats treated with reference substances increasing (triadimefon) and decreasing (chlorpromazine) activity, that have established the sensitivity, reliability and validity of the test procedures used. Finally, previous studies performed at Bayer's Toxicology laboratory with trimethyltin and acrylamide have established the sensitivity and reliability of the micropathology procedures for detecting lesions in peripheral nerves and the central nervous system.

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. 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 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 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 (CATMOD) 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 10-minute interval) was analyzed using ANOVA procedures. Session activity data were analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA 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 ANOVA 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 ANOVA 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.

See: "Any other information on materials and methods"!

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality:

mortality observed, treatment-related

Description (incidence):

adverse

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

adverse

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

adverse

Details on results:

CLINICAL SIGNS AND MORTALITY
2000 mg/kg:
In females, tremors and increased reactivity in 2/12 females, decreased activity and perianal stain in 1/12 females, and urine stain in 2/12 females. In addition, 3/12 females were found dead on the day following treatment. Upon necropsy, compound-related gross observations were limited to salivation in 4 high-dose females and ventrum wet/staining in all 3 high-dose females that were found dead. Due to the incidence of mortality, high-dose females were discontinued.
In males, compound-related signs included urine stain (7/12), oral stain (8/12) and perianal stain (3/12 males). These signs were first apparent on day 0 or day 1 and completely resolved by day 5.

500 mg/kg:
Clinical signs in mid-dose animals included urine stain (3/12 males) on day 0 only, oral stain (1/12 males) on day 2 only, and increased reactivity, decreased activity and tremors in 1/12 females on day 1. Additionally, tremors were seen in 2/12 females on day 0.

100 mg/kg:
There were no compound-related clinical signs in low-dose males or females.

Clinical signs that were not related to treatment included a dermal lesion described as a scab (one mid-dose male), areas of hair loss (one control and 2 mid-dose females and one high-dose male) and lacrimal stain (one control female).

In the follow-up study there were no treatment-related signs at any dose level.

BODY WEIGHT AND WEIGHT GAIN
For the initial study, body weight was not affected by treatment at any dose level in either sex.
For the follow-up study, body weight was not measured beyond day 0 since animals were terminated on days 1 or 2 post-treatment.

NEUROBEHAVIOUR
FOB:
For the initial study, treatment-related findings were limited to urine stain in mid- and high-dose males (1/12 mid- and 5/12 high-dose) on day 0 only. This finding was statistically significant in high-dose males. Due to mortality in 3 females on day 1, the high-dose females were discontinued and not included in the FOB analysis.
There were no compound-related findings in mid- and low-dose females or in low-dose males.
For the follow-up study, there were no compound-related findings at any dose level.

Motor activity:
For the overall 60-minute test session in the initial study, compound-related effects on session motor activity and locomotor activity were evident on the day of treatment in low- and mid-dose females and in high-dose males. Measures of motor and locomotor activity were not affected by treatment in low- and mid-dose males. On day 0, measures of motor and locomotor activity in high-dose males were significantly reduced, compared to controls (an average 40% and 46%, respectively). Measures of motor and locomotor activity were non-statistically reduced in low-dose (14% and 21%, respectively) and mid-dose (an average 30% and 31%, respectively) females. Although the difference from control that was evident in low-dose females was not statistically significant and was within the range normally considered to be within biological variability (i.e., approximately ±20%), it is considered to represent an effect based on consistency with the findings at the higher dose and other test occasions and the difference expressed across the six intervals of the test session on day 0. Thus, the follow-up study was performed to establish a clear no effect level. In the follow-up study, measures of motor and locomotor activity were not affected by treatment at either dose level.

For the initial study, motor and locomotor activity data were subjected to further analysis at each interval on each test day. Measures of motor and locomotor activity for high-dose males and mid- and low-dose females were statistically and/or non-statistically different from control on day 0. These differences from control are ascribed to treatment. On day 0, the high-dose males exhibited considerably lower levels of motor and locomotor activity during intervals 1 through 4 (32%, 53%, 56%, and 36%, respectively, for motor and 37%, 61%, 60% and 36%, respectively, for locomotor). Also on day 0, the mid-dose females exhibited modestly lower levels of motor and locomotor activity during intervals 1 through 6 (24%, 31%, 39%, 29%, 23% and 33%, respectively for motor and 24%, 32%, 39%, 37%, 19%, and 39%, respectively for locomotor). Lastly, the low-dose females exhibited slightly lower levels of motor and locomotor activity during intervals 3 through 6 (20%, 31%, 15% and 37%, respectively for motor and 25%, 47%, 16% and 50%, respectively for locomotor). In females, these differences from control were not statistically significant but are considered to be related to treatment. There were no differences ascribed to the test substance in males or females at any dose level after day 0, nor for males at the lowest dose level on any test occasion. For the follow-up study, there were no treatment-related effects on measures of interval motor or locomotor activity at any dose level.

Additionally, there were statistical differences from control for measures of interval motor and locomotor activity in both the initial and the follow-up study which were not attributed to treatment.

Habituation:
Not affected by test substance treatment at any dose level in either sex.

GROSS PATHOLOGY
There were no compound-related gross lesions in males at any dose level or in low- or mid-dose females.
Compound-related gross observations were limited to salivation in 4 high-dose females and ventrum wet/staining in all 3 high-dose females that were found dead (one was also noted with autolysis).

There was no compound-related effect on terminal body weight or absolute or relative brain weight in perfused males or females at any level of exposure. In low- and high-dose males, there was a statistically significant decrease in relative brain weight. This difference from control is most likely due to the higher mean body weight of males in all treated groups, compared to controls. Also, there was no dose relationship to indicate that this finding was treatment related, nor was there a similar trend in females.

NEUROPATHOLOGY
Compound-related microscopic lesions were evident in neural tissues from perfusion-fixed high-dose males that were related to treatment. There were statistically-significant and non-significant increases in focal to multi-focal individual nerve fiber degeneration in the gasserian ganglion and peripheral nerves (sciatic, sural and tibial) from several males related to test substance administration. Therefore, these tissues from mid-dose males were examined for lesions. The occurrence of these (or similar) findings in mid-dose males and/or females was similar incidence to that of the concurrent controls and historical controls and were considered to be incidental to treatment. Thus, lower dose levels were not examined microscopically. Other incidental findings consisted of retinal dysplasia in two control females and vacuolar degeneration of the dorsal root ganglion in a control male.

Dose descriptor:

NOEL

Effect level:

50 other: mg/kg bw

Based on:

test mat.

Sex:

male/female

Remarks on result:

other:

Dose descriptor:

LOEL

Effect level:

100 other: mg/kg bw

Based on:

test mat.

Sex:

female

Basis for effect level:

other: neurobehaviour (decrease in motor and locomotor activity)

Remarks on result:

other:

Dose descriptor:

NOAEL

Effect level:

100 other: mg/kg bw

Based on:

test mat.

Sex:

male/female

Remarks on result:

other:

Dose descriptor:

LOAEL

Effect level:

500 other: mg/kg bw

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: neurobehaviour (tremors, increased and decresed activity in females, urine and oral stains in males)

Remarks on result:

other:

CONCLUSION

The acute LOAEL for neurotoxicity was determined as 500 mg/kg bw, based on the observation of tremors, increased and decreasd activity in females and urine and oral stains in males. Motor and locomotor activity were non-statistically reduced in females on day 0 at this dose level.

A dose level of 100 mg/kg bw was determined as NOAEL, based on the lack of findings in males and only non-statistically reduced motor and locomotor activity in females; the observed signs of toxicity resolved by day 7. Therefore, this dose can also be considered as LOEL.

In order to determine a NOEL, a follow-up study was conducted. In this study no compound-related effects were found at a dose level of 50 mg/kg bw.

In addition to the clinical and neurobehavioural observations, which were fully reversible within 7 days after application the latest, compound-related microscopic lesions were found in neuronal tissues from high-dose males in the histopathological examination, comprising increases in focal to multi-focal individual nerve fiber degeneration in the Gasserian ganglion and the peripheral nerves. The incidence of these or similar findings in animals at 500 mg/kg bw was comparable to concurrent and historical controls. Thus, lower dose levels were not examined microscopically.

Reference 3

Endpoint:

neurotoxicity: oral

Remarks:

developmental

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 426 (Developmental Neurotoxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

Deviations:

yes

Remarks:

Period of exposure was extended to PND 21, the number of pups allocated to neuropathological evaluation on PND 11 was increased to 10/sex/dose, and the neuropathological examination was done on PND 21 instead of PND 11, with perfusion of the animals befor

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Han Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: Approximately 11 (males and females) weeks of age at co-housing (based on approximate birth date provided by vendor).
- Weight at study initiation: 187.2 – 240.2 g range for females (± 20% weight determination); Males had no specified weight requirements.
- Fasting period before study: Not applicable, feeding study.
- Housing: Suspended stainless steel cages; Individually, except with one male each during co-habitation with deotized cage board in the bedding tray. Individually in plastic cages with corn cob bedding during gestation and lactation. Each cage contained a feeder and a source of water (pressure-activated water lixits).
- Diet (e.g. ad libitum): Purina Mills Certified Rodent Diet 5002 in meal form provided for ad libitum consumption during the acclimation period and throughout the study except during neurobehavioral testing.
- Water (e.g. ad libitum): Tap water (Kansas City Missouri Municipal Water) was provided ad libitum except during neurobehavioral testing.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28-26
- Humidity (%): 30-70
- Air changes (per hr): Minimum daily average of 10.79
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 May 2007 To: 24 August 2007; still within expiry date of test substance.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prepared weekly by mixing appropriate amounts of the test substance in the diet.
- Mixing appropriate amounts with (Type of food): Purina Mills Certified Rodent Diet 5002 in meal form.
- Storage temperature of food: At freezer (-23.7 to -24.6 °C) conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of the test substance in the diet were measured by LC-MS/MS, using five batches of feed that were used in this study. The stability (at room temperature and freezer conditions) and homogeneity of the test substance in the feed were verified at dietary concentrations (20 and 10000 ppm) that bracket those used in the present study.
During gestation, the nominal 150, 1000 and 7000 ppm dietary levels averaged 97-104% of the nominal concentrations. Based on these results, the average dietary levels during gestation were 0, 156, 1025 and 6770 ppm, respectively.

Duration of treatment / exposure:

GD 6 to PND 21 (during gestation only pregnant females)

Frequency of treatment:

continuously in the feed

Dose / conc.:

150 ppm (nominal)

Remarks:

corresponding to 13.0 mg/kg bw/day during gestation and 13.0 mg/kg bw/day during lactation

Dose / conc.:

1 000 ppm (nominal)

Remarks:

corresponding to 83.8 mg/kg bw/day during gestation and 83.8 mg/kg bw/day during lactation

Remarks:

corresponding to 568 mg/kg bw/day during gestation and 432 mg/kg bw/day during lactation with the reduction on LD 4 to a nominal 4000 ppm

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Doses were selected on the basis of preliminary results that emerged in the rat over the course of a pilot reproductive toxicity testing study conducted with the test substance at doses of 0, 200, 1000, 3000 and 8000 ppm. In that study, clinical observations that were attributed to the test substance were not observed at any dietary level tested. Declines in body weight for the 8000 ppm females were evident throughout premating, gestation and lactation phases. The high-dose females also had declines in food consumption during the premating period, with initial declines during the first week of treatment and continuing through week eight. No test substance-related effect on body weight or food consumption was evident in the males at any dietary level. In the 8000 ppm dose group, declines in female terminal body weight (-7%) and uterine weight (absolute, -41%; relative, -37%) were evident, as well as, a trend toward increased thyroid weight (absolute, +29%; relative, +44%). Adult male organ weight effects included increased absolute and relative liver weight in both the 3000 and 8000 ppm dose groups and increased absolute and relative adrenal and kidney weight in the 8000 ppm dose group. Males exhibited no effect on terminal body weight at any dietary level tested. In the 8000 ppm dose group, female pup weight declines were evident on Day 21 (7%), with a decline in weight gain observed on Days 7-14 (8%) and Days 14-21 (14%). Organ weight declines were observed in the spleen (absolute, -21%; relative, -15%) and uterus (absolute, -19%; relative, -13%) of the female pups of this same dose group. Body and/or organ weight effects were not evident in the male offspring at any dietary level tested. Based on these combined results, the dietary levels selected for this developmental neurotoxicity study were 0, 150, 1000 and 7000 ppm. The 7000 ppm dietary level was selected as a maximum dose the animals would tolerate without excessive toxicity. The 1000 ppm dietary level was selected as an intermediate dose that might produce slight effects and the 150 ppm level was not expected to produce any compound-related effects and therefore was selected to establish an overall NOAEL.

- Rationale for animal assignment (if not random): Upon receipt, P-generation females were examined and those considered acceptable were placed into individual cages and acclimated to their ambient laboratory conditions. Male animals were randomly assigned an identification number as they were arbitrarily selected and removed from the shipping crates and placed into individual cages. Following at least six days of acclimation, P-generation females were weighed and those with a body weight more or less than 20% of the mean weight were rejected. The remaining females were assigned to the control or an exposure group in sequence, as they were determined to be inseminated. This approach was used to ensure unbiased assignment of the animals to dose groups and that there would be approximately an equal number of litters from each dose group available for testing on a given day. The P-generation females were mated and assigned to the study as they were determined to be sperm positive.

Offspring were assigned to testing subgroups at the time of litter standardization on PND 4. An animal allocation program written in SAS was used to assign offspring to the following four sets (designated A-D) for assessment at each age. One male and/or female per litter (approximately 16 - 20 (minimum 10)/sex/dietary level, representing at least 20 litters per level): Motor activity (Set A), Auditory Startle (Set B), Passive Avoidance, Water Maze and Functional Observational Battery (Set C). On PND 21, the whole brain was collected from a separate group of randomly selected offspring (Set D; 10/sex/dietary level; representing 20 litters per level) for micropathologic examination and morphometric analysis. The remaining pups assigned to Set D (~10/sex/dietary level) were reserved for possible use as replacement animals or were otherwise sacrificed on PND 21 without necropsy examination. The method of animal assignment minimised potential problems related to litter effects, by using at least one pup/litter.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: P-generation males and females at least once daily
- Cage side observations included: Mortality, moribundity, behavioural changes and overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily from GD 6 through lactation day 21.

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly during gestation and lactation: Gestation days 6-13, 13-20 and lactation days 0-7, 7-14 and 14-21. In addition, dams were weighed on GD 0 and LD 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Once weekly during gestation and lactation including consumption by the pups, especially during 3rd week of lactation.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: Yes
- Compound intake calculated as average daily intake of the active ingredient from the weekly food consumption and body weight gain data: Yes, according to the following formula: [AI in feed (ppm)/1,000] x [feed consumed (g/kg body wt/day)] = mg AI/kg body wt/day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at approx. 50-60 days of age
- Dose groups that were examined: Randomly selected animals (animals selected for perfusion at study termination on PND 75±5; a minimum of 10/sex/dietary level, representing at least 20 litters per level) from Sets A, B, and C. If needed to clarify the significance of findings, the animals reserved for adult brain weight measurements were also subjected to ophthalmologic examination. The exam took place in a semi-darkened room. The pupillary reflex was tested using a penlight or transilluminator, with a mydriatic agent applied to each eye to dilate the pupil. The conjunctiva, cornea and lens were examined with a slit lamp microscope either before or after pupillary dilatation. After mydriasis, the vitreous humor, retina, choroid, and optic disc were examined using an indirect ophthalmoscope equipped with a condensing lens.

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined: Observations in the home cage, during handling, and outside the home cage in an open field (one min), using standardized
procedures. This observational battery included, but was not limited to, assessments (with severity scoring) of lacrimation, salivation, piloerection,
exophthalmia, urination, defecation, pupillary function, palpebral closure, convulsions, tremor, abnormal movements, unusual behaviors (excessive or repetitive actions, stereotypies), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose or mouth, posture and gait abnormalities, and any other observations that may facilitate interpretation of the data.

- Description of procedures:
Animals that were presumed to be pregnant (approximately 30 per dietary level) were observed on GD 13 and GD 20 and a minimum of 10 dams/dietary level that were maintained on study with suitable litters were also observed on LD 11 and LD 21. This evaluation was performed under standard animal room conditions (temperature, relative humidity, etc.) and included observations in the home cage, during handling, and outside the home cage in an open field (1 min), using standardized procedures.

- Minimization of bias: The order of testing and assignment of animals to specific test devices was semi-random, such that groups were balanced across test times and devices and no animal was tested more than once in the same device.

- Same technicians used throughout testing: No, since it was not feasible for one person to evaluate all animals on all test occasions, the laboratory maintains evidence of inter-observer reliability (agreement) for individuals who were involved with performing these observations.

- Technicians were blind to treatment status of animals: Yes, all observations were performed by an individual who was unaware of each animal’s dose group assignment.

- Site of testing: The test room used for motor activity, auditory startle habituation and passive avoidance conditioning was a standard animal room that was set to be maintained on the same light:dark cycle as the room in which animals were housed, with tests conducted during the light phase.

- Time schedule for examinations: GD 13and GD 20; LD (lactation day) 11 and LD 21.
- Environmental conditions: Standard animal room conditions (temperature, relative humidity, etc.).
- Scoring criteria (if any): Degree of severity was considered.
- Duration of observation period for open field observations: 1 min

Additional information for developmental neurotoxicity study:
- Number of offspring/sex/group examined on postnatal days outside the home cage: On postnatal days 4, 11, 21, 35 (±1 day), 45 (±1day), and 60 (±2 days), approximately 20 offspring/sex/group (representing at least 20 litters per level).

- Description of procedures used for each age at which offspring were examined: Animals were examined outside the home cage in an FOB assessment, as appropriate for the developmental stage involved. FOB assessment was performed as described for maternal animals. The only difference was that neonates (i.e., PND 4 and 11) were not evaluated in the open field, since this is routinely done only if the observer considers it necessary for evaluation, and this was not the case in the present study. Motor activity, auditory startle habituation and passive avoidance conditioning were tested in a standard animal room that was set to be maintained on the same light:dark cycle as the room in which animals were housed, with tests conducted during the light phase. The water maze testing was performed in the room where animals were housed. The order of testing and assignment of animals to specific test devices was semi-random, such that groups were balanced across test times and devices and no animal was tested more than once in the same device. One planned exception was that animals were purposely tested in the same water maze on both occasions, as per standard procedure. Males and females were generally tested on the same days at the appropriate days of age. After sexual maturation, test devices were cleaned during the ensuing interval to reduce the residual scent from the other gender.

- Same offspring evaluated at each time point: yes
- Same parameters assessed in the maternal FOB examined for offspring: FOB assessment according to procedures described for maternal animals, but adapted to the developmental stage involved; eception: neonates (i.e., PND 4 and 11) were not evaluated in the open field.

LOCOMOTOR ACTIVITY: Yes
- Type of equipment used: Computer-automated figure-eight maze (Universal Maze Monitoring System, Version 1.41, ColumbusInstruments, Columbus, OH) and personal computer for automated data collection. Each maze consisted of a series of inter-connected alleys (approximately 10 x 10 cm in cross-section) converging on a central arena and covered by transparent acrylic plastic. Each maze had eight infrared emitter/detector pairs (three in each of the figure eight alleys and one in each of the blind alleys) to measure activity, and an activity count was registered each time a beam was interrupted. The floor of each maze rested above absorbent paper, which was changed routinely at the end of each day. A Columbus Instruments (Columbus, OH) Universal Maze Monitoring System and a personal computer were used for automated data collection. Broad-spectrum background noise [74±2dB(A)] was provided throughout the test to minimize acoustical variations during testing. The uniformity of light intensity (100±70 Lux) over each maze was verified daily.

- Length of session, number and length of subsessions: 60 min; 6 subsessions of 10 min length.
- Parameters measured: Increases and decreases in activity.
- Total activity: Number of beam interruptions that occurred during the test session.
- Ambulatory activity: 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 a different beam.

- Other: Habituation was evaluated as a decrement in activity over consecutive intervals of the test session.
Additional information for developmental neurotoxicity study:
- Number and age of offspring/sex/group examined (unless given in animal assignment table) and days of evaluation: approximately 20 rats/sex/dose (representing at least 20 litters per level) on PND 13, 17, 21 and 60 (±2 days).
- Description of procedures used for each age at which offspring were examined: As described above with adjustments for developmental age.
- Same offspring evaluated at each preweaning time point: yes
- Same parameters assessed in the maternal FOB examined for offspring: Maternal animals were not tested for locomotor activity.

Additional testing required for developmental neurotoxicity studies:

AUDITORY STARTLE REFLEX HABITUATION: Yes
- Number of animals: approximately 20 rats/sex/dose (representing at least 20 litters per level)
- Days of testing: postnatal days 23 and 60 (±2 days).
- Same offspring evaluated at each preweaning time point: yes
- Exact age: 23 and 60 (±2 days)
- Type of equipment used: A personal computer was used to control the operation of an integrated startle response test system (Coulbourn Acoustic Startle, Version 3.210-00, Coulbourn Instruments, Allentown, PA) and for automated data collection. Groups of four animals (maximum) were tested simultaneously within each of two startle system enclosures. Each enclosure was ventilated, lined with sound-attenuating and vibration-absorbing material, and houses a speaker mounted in a central position within the ceiling of the enclosure to provide the eliciting stimulus (S2) - a 50-msec burst (0 msec rise/fall) of broad-spectrum "white" noise [118±3 dB(lin)]. Each enclosure also houses four load cell/force transducer assemblies that
are designed to measure the startle response.
- Environmental conditions: During the test session, animals were placed into individual restraining cages that were positioned on top of each load cell.
- Number of trials performed: 50
- Length (msec) and intensity (dB) of sound: 50-msec burst (0 msec rise/fall) of broad-spectrum "white" noise [118±3 dB(lin)]
- Length of interval between trials: 10 seconds
- Other procedural details: The peak response amplitude was determined for each trial as described below. The average response amplitude and the magnitude of decrease (habituation) over blocks of ten trials were compared among the dosage groups. Data collection began with the presentation of S2 and continued thereafter for 200 msec. The analog signal for each response output (measured in mV) was digitized at one kHz (i.e., one sample/msec for 200 msec) and converted to grams using a previously determined calibration curve for each load cell. Peak response amplitude (g) and
latency (msec) measurements were taken from each animal’s individual response curve. Baseline was defined as the average force (g) exerted on the platform during the first 8 msec following the onset of S2, a time period that precedes response onset. This baseline value was taken to represent an approximate body weight measurement that was used to verify that the equipment used to measure the response amplitude was functioning properly. Response amplitude is defined as the maximum value of the average curve, minus the baseline (i.e., removing the animal’s body weight from the measurement). Latency to peak is the time (msec) following the onset of S2 when the peak response amplitude occurs.

LEARNING AND MEMORY TESTING: Yes
(1) Overall testing design: Passive avoidance testing
- Number of animals: approximately 16 rats/sex/dose (minimum 10 offspring/sex/dose)
- Days of testing: passive avoidance (acquisition on PND 23 and for retention 30) and water maze (PND 60 ±2 days and again seven days later).
- Evaluation of both short and long term recall: yes
(2) Equipment used
- Type of equipment (including manufacturer, if available):
Passive avoidance testing was conducted using equipment and computer programs from Coulbourn Instruments (Graphic State Notation 2 Version 2.002-00, Allentown, PA). A personal computer was used to control the operation of the equipment and for automated data collection. Testing took place in individual isolation cubicles, each housing a single shuttle cage. Each isolation cubicle was lined with foam insulation to attenuate sound in the chamber and had a fan with a baffled air intake and exhaust system for ventilation. The shuttle cage consisted of a Plexiglas and stainless-steel rectangular chamber fitted with front-loading access. Each shuttle cage (15 inches wide x 7.25 inches deep) was separated into two compartments of equal size (approximately 7 x 7 inches) by a wall that supported a centrally-located sliding (guillotine-type) door. The two compartments were identical, except that the walls in one compartment were lined with black film (dark-side) and the walls in the other compartment were not lined and it was illuminated during the test with a high-intensity lamp. The floor of the cage consisted of a grid of stainless-steel bars. The movement of the animal from the starting (light) side to the dark compartment was detected by a photocell system. A Coulbourn solid-state scanning shock generator was used to deliver a brief (0.5 sec) pulse of mild (0.5 mA) distributed shock to the grid floor when the animal crossed to the dark compartment.

- Environmental conditions: The lamp was switched on to illuminate the light compartment at the start of each trial and remained on until either the animal crossed to the dark compartment or the trial ended. The floor of the cage consisted of a grid of stainless-steel bars. The movement of the animal from the starting (light) side to the dark compartment was detected by a photocell system. A Coulbourn solid-state scanning shock generator was used to deliver a brief (0.5 sec) pulse of mild (0.5 mA) distributed shock to the grid floor when the animal crossed to the dark compartment.

(3) Testing and training procedures
The passive avoidance test was conducted according to established procedures. After adaptation, individual animals were placed individually into the "lighted" compartment of a conditioning apparatus (the shuttle cage), facing toward the light. After approximately 60 seconds, the trial began with the light being illuminated to signal the beginning of the trial and the door separating the two compartments opening, so that each rat was provided access to the non-illuminated side of the cage. When the rat crossed into the dark compartment, the door automatically closed, the shock was delivered, and the light switched off - signaling the end of that trial. At that time, the animal was returned promptly to the holding cage to wait for the next trial. If the rat failed to cross within 180 sec, it was returned to the holding cage and the latency assigned an arbitrary score of 180. This restriction dictated the use of nonparametric statistical analyses. The procedure was repeated until either the rat remained in the lighted compartment for 180 sec on two consecutive trials or until 15 trials had elapsed, whichever occurred first. Rats that failed to meet the criterion during the learning phase were assigned a value of 15 for the trials-to-criterion variable. The test was repeated one week later. For this second trial, rats were placed in the illuminated side of the apparatus, given a 20-sec acclimation period, and the latency to enter the dark side recorded. Animals that either failed to reach criterion performance within 15 trials or failed to cross during the first two trials during acquisition were excluded from the retention phase of the experiment. The dependent measures were the number of trials-to-criterion, latency to cross on Trial 1 and Trial 2 (learning phase only) and the number of rats/group that failed to reach criterion within 15 trials (learning phase only).
- Number of trials per day: 15
- Number of days of testing: 1 day learning phase, 1 day retention phase
- Inter-trial intervals: not specified
- Stimulus parameters: mild shock (0.5 mA) for 0.5 sec
- Learning criteria: suppression of the natural tendency to enter a dark chamber

(4) Performance measures
- Number of trials to criterion: yes
- Time or latency to reach goal: yes
- Performance on "probe trials": yes

LEARNING AND MEMORY TESTING: Yes
(1) Overall testing design: Water maze (M maze)
- Number of animals: approximately 20 rats/sex/dose (representing at least 20 litters per level)
- Days of testing: PND 60 (±2 days), and again 7 days later
- Evaluation of both short and long term recall: yes

(2) Equipment used
- Type of equipment (including manufacturer, if available): M-maze, constructed of opaque Plexiglas, with corridors approximately five inches wide and walls approximately 16 inches high with approximately 7.5 inches of water. This maze was selected as an established and widely used device that can be used to measure associative learning and memory.
- Environmental conditions: The water in the M-maze was maintained at 22 ± 1°C.

(3) Testing and training procedures
On each test trial, the rat was placed into the starting position at the base of the M-maze stem, located between the two lateral arms. On the first (learning) trial, the rat was required to enter both arms of the maze before being provided access to the exit ramp to escape the water and then removed from the maze. The initial arm chosen on this learning trial was designated the incorrect goal during the subsequent 15 trials (maximum). Rats that failed to make a correct goal choice within 60 seconds in any given trial were guided to the correct goal with the exit ramp and then removed from the water. Between trials, the animal was returned to a transport cage to wait for the next trial. The inter-trial interval was approximately 15 (±5) seconds. Each rat was required to reach a criterion of five consecutive errorless trials to terminate the test session. The maximum number of trials in any test session was fifteen. Latency (in seconds) to choose the correct goal or the maximum 60-second interval was recorded for each trial, as was the number of errors (incorrect turns in the maze) during each trial. Animals that satisfied the above criteria within the 15-trial limit were tested for retention seven days following acquisition (animals that failed to reach criterion during acquisition were excluded from the retention phase of the experiment). The correct goal and the criterion were the same for both sessions. Dosage groups were compared for the following dependent measures: Measures for acquisition included the number of trials-to-criterion, the average number of errors (incorrect turns in the maze) for each trial and the latency (in seconds) to reach the correct goal on trial 2 (a measure of short-term retention). Measures for retention included the number of
trials-to-criterion, the average number of errors for each trial, and the latency (in seconds) to reach the correct goal on trial 1 (a measure of long-term retention).
- Number of trials per day: 15
- Number of days of testing: 1 day acquisition, 1 day retention
- Inter-trial intervals: 15 ± 5 seconds
- Stimulus parameters: Escape from cold water.
- Use of cut-off times or error correction procedures: Animals that failed to reach criterion during acquisition were excluded from the retention phase of the experiment.
- Definition of errors: Incorrect turns in maze.
- Learning criteria: Latency in seconds to choose the correct goal with the exit ramp from the water. Criterion of five consecutive errorless trials.

(4) Performance measures
- Number of errors or trials to criterion: yes, both
- Time or latency to reach goal: yes (in seconds)
- Performance on "probe trials": yes

Sacrifice and (histo)pathology:

MATERNAL ANIMALS
- Time point of sacrifice or discard without postmortem examination: Dams that were found dead or moribund (if any) underwent a gross necropsy examination, with possible collection of tissues, if approved by the Study Director to assist in determining the cause of death. P-generation males that were found dead or moribund (if any) did not undergo a necropsy examination and were discarded without routine collection of tissues.
On day 21 of lactation following weaning of their respective litters dams were discarded without postmortem examination. Females that were sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed on GD 24 without necropsy examination.
- Method of sacrifice: CO2 asphyxiation
- Description of any postmortem evaluations conducted: gross necropsy


OFFSPRING
- Time point of sacrifice of offspring selected for brain weight or neuropathological evaluation: Postnatal day 21 (neuropathology, brain weight; Set D; 10/sex/dietary level; representing 20 litters per level) and 75 (± 5 days) (brain weight; from additional randomly selected animals from Set A, B and C; 10/sex/dose group; representing 20 litters per level). The remaining pups assigned to Set D (~10/sex/dietary level) were reserved for possible use as replacement animals or were otherwise sacrificed on PND 21 without necropsy examination. F1-generation animals that were found moribund (if any) while on study were also sacrificed by CO2 asphyxiation.

- Description of postmortem examinations: F1-generation animals that were found moribund, animals from Sets A, B, and C randomly selected for brain weight determination and from Set D selected for neuropathology underwent a gross necropsy examination. Tissues were collected at the discretion of the Study Director. The remaining animals assigned to sets A-C were sacrificed without routine gross necropsy examination or collection of tissues. Where required, the necropsy involved an examination of all organs (including the brain), body cavities, cut surfaces, external orifices and surfaces, with all gross abnormalities recorded. Gross lesions in neural tissues or skeletal muscle were appropriately sampled for microscopic examination. Other gross lesions were generally not collected for microscopic examination. Animals found dead (if any) underwent a necropsy examination and were disposed of without the routine collection of tissues.

- At postnatal day 21, ten pups/sex/group were selected for brain weight measurements and neuropathology (from Set D), deeply anesthetised and then perfused via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by in situ fixation using universal fixative (1.0% (w/v) glutaraldehyde and 4% (w/v) EM-grade formaldehyde) in phosphate buffer. On PND 21 examinations only the brain (with olfactory bulbs) was collected. The brain was weighed upon removal from the skull, prior to placement into 10% buffered formalin for post-fixation, and the brain:body weight ratio was calculated.
Prior to sectioning the brain for histology, a Vernier caliper was used to obtain two measurements: 1. Anterior-to-posterior (AP) length of the cerebrum, extending from the anterior pole to the posterior pole, exclusive of the olfactory bulbs; and 2. Anterior-to-posterior (AP) length of the cerebellum, extending from the anterior edge of the cortex to the posterior pole. These gross measurements were performed by an individual who was aware of dose group assignments.

- After measurement, the brains from these 10 pups/sex/group perfused on PND 21 were post-fixed in 10% buffered formalin and divided into eight coronal sections for microscopic examination. The eight brain sections were processed according to standard procedures for paraffin embedding, sectioned at approximately 5 µm, and examined after staining with hematoxylin and eosin (H&E). In addition, the brain sections reserved for morphometric measurements (levels 3-5 and 7) were stained using luxol fast blue/cresyl violet.

- Histopathological examination was performed on tissues from control and high-dose pups.

- At termination (PND 75 (±5 days)), 10 randomly selected animals/sex/group from Sets A, B and C (those subjected to ophthalmoscopic examination) were anesthetized and sacrificed by perfusion with via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by in situ fixation using universal fixative (1.0% (w/v) glutaraldehyde and 4% (w/v) EM-grade formaldehyde) in phosphate buffer, with neural and muscle tissues collected for microscopic examination. Also at termination on PND 75 (±5 days), brains were collected from additional randomly selected animals (10/sex/dose group; representing 20 litters per level). These brains were weighed (fresh tissue weight) and then discarded.

Prior to sectioning for histology, the brains from the perfused animals were weighed upon removal from the skull, and a Vernier caliper was used to obtain two measurements:
1. Anterior-to-posterior (AP) length of the cerebrum, extending from the anterior pole to the posterior pole, exclusive of the olfactory bulbs; and
2. Anterior-to-posterior (AP) length of the cerebellum, extending from the anterior edge of the cortex to the posterior pole.
These gross measurements were performed by an individual who was aware of dose group assignments.

After the gross measurements were taken, the brain was placed into formalin, and the brain:body weight ratio calculated. Then the brain was divided into eight coronal sections for microscopic examination (including forebrain, center of cerebrum, midbrain, cerebellum, pons, medulla oblongata). The eight brain sections were processed according to standard procedures for paraffin embedding, sectioned at approximately 5 µm, and examined after staining with hematoxylin and eosin (H&E). In addition, the brain sections reserved for morphometric measurements (levels 3-5 and 7) were stained using luxol fast blue/cresyl violet. Additional tissues were collected for microscopic examination from animals that were perfused at study termination and post-fixed in 10% buffered formalin. These included three levels of the spinal cord (cervical, thoracic and lumbar), the cauda equina, eyes, optic nerves and gastrocnemius muscle were embedded in paraffin and stained with H&E. Dorsal root ganglia (including dorsal and ventral root fibers) from the cervical and lumbar swellings and gasserian ganglia were embedded in glycol methacrylate (GMA). GMA-embedded tissues were sectioned at 2 µm - µ3 m and stained using a modified Lee’s stain. Peripheral (bilateral) nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA resin and sectioned longitudinally. The sciatic nerve was also cut in cross section. Additionally, both forelimbs and the physical identifier were collected.

- Histopathological evaluation was performed on tissues from males and females in the control and high dose groups. If no treatment-related lesion was evident, further analysis was not performed. Any region where treatment-related neuropathology was observed underwent the following semi-quantitative analysis in a blind fashion. The frequency of each type of lesion was determined with the severity of each lesion graded and the data evaluated for dose-effect relationships.

- Detailed morphometric evaluation: Selected brain regions underwent the following quantitative analysis, with the individual performing the measurements aware of dose assignments. Initially, eight linear measurements were taken. If treatment-related effects were evident following this initial evaluation, then additional measurements may have been undertaken. Two of the seven measurements involved gross measurements of the intact brain, as described above. The other five were taken from the histologic sections using software calibrated with an ocular micrometer. These five measurements are described as follows:
1. Frontal cortex thickness (Forebrain): This measurement was of the dorsal portion of the cerebral cortex within the coronal section passing through the region of the optic chiasm.
2. Parietal cortex thickness (Forebrain): This measurement was of the dorsolateral portion of the cerebral cortex within the coronal section taken through the optic chiasm.
3. Caudate putamen horizontal width (Forebrain; maximum cross-sectional width). This measurement was performed on the coronal section taken at the level of the optic chiasm.
4. Hippocampal gyrus thickness (Midbrain): This measurement was of the full width of the hippocampal gyrus from the ventral tail of the dentate gyrus to the overlying subcortical white matter. Measurements were taken from the hippocampus from both sides of this section, and the mean value was recorded.
5. Cerebellum height (Cerebellum / Pons): This measurement extended from the roof of the fourth ventricle to the dorsal surface.

In addition to these measurements, all brain sections from these control and high-dose male and female offspring underwent an extensive micropathologic evaluation.

Other examinations:

Reproductive indices: Mating, fertility.
Offspring viability indices: Live Birth, viability, lactation.

Positive control:

The study did not include concurrent positive controls, but references are made to previous studies to serve that purpose. Positive control data demonstrate the sensitivity of the test method to detect changes in the measured parameters. Some of these data were generated from studies using prenatal exposure while others used young-adult rats. Collectively, these studies verify the laboratory competence in evaluation of effects in neonatal animals perinatally exposed to chemicals and establish test norms for the appropriate age group. The laboratory maintains evidence of inter-observer reliability (agreement) for individuals who were involved with performing these observations, since it is not feasible for one person to evaluate all animals on all test occasions. Positive control chemicals included acrylamide, trimethyltin, triadimefon, chlorpromazine, methimazole, 8OHDPAT, mCPP and scopolamine.

Statistics:

Statistical evaluations were generally performed using software from INSTEM Computer Systems, SAS or TASC. The level of significance was set at p≤0.05, with the exception of Bartlett’s test, which was tested at p≤0.001. 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 ANOVA, followed by a Dunnett's test if a significant F-value was determined in the ANOVA. In the event of unequal variances, these data were analyzed using nonparametric statistical procedures (Kruskal-Wallis ANOVA followed by Mann-Whitney U test for between-group comparisons). Functional Observational Battery. Continuous data were analyzed using an ANOVA, with post-hoc comparisons using Dunnett's test. Categorical data were analyzed using General Linear Modeling and Categorical Modeling (CATMOD) 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 10-min interval) were analyzed using ANOVA procedures. Session activity data for the four test occasions were first analyzed using an ANOVA to determine if there was a significant day by treatment interaction. For days on which there was a significant treatment effect, Dunnett's test was used to determine whether the treated group was significantly different from the control. Interval data were subjected to a Repeated-Measures ANOVA, using both test interval and test occasion as repeated measures, followed by an ANOVA to determine whether there was a significant treatment by interval interaction on each test occasion. For those test days, the data for each interval was subjected to analysis using Dunnett's test to determine whether the treated group was significantly different from the control. Further details in "Any other information on Materials & Methods incl. tables".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality:

mortality observed, treatment-related

Description (incidence):

adverse

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

adverse

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect on reproduction parameters at any dietary level.

VIABILITY (OFFSPRING)
Litter parameters and pup viability were not affected by treatment at any dietary level.

SEXUAL MATURATION (OFFSPRING)

DEVELOPMENTAL LANDMARKS (OFFSPRING)
The ages for onset of balanopreputial separation, vaginal patency and surface righting were not affected by treatment at any dietary level. Pupil constriction in response to a penlight was apparent in all control and treated pups on PND 21. Therefore, there was no indication of a compound-related effect at any dietary level.

OTHER FINDINGS (migrated information)

Details on results:

CLINICAL SIGNS AND MORTALITY
No P-generation females were found dead during gestation or lactation. There were also no P-generation males found moribund or dead after initiation of the study (males did not receive the test substance).

Gestation:
Compound-related clinical signs were evident in high-dose animals as coarse tremor in 4/30 females, beginning on GD 14, dilated pupils in 7/30 females, beginning on GD 15, and nasal stain in 3/30 females, beginning on GD 6. There were no compound-related effects at lower dietary levels. Findings considered incidental and unrelated to treatment included hair loss (1-3/30 animals from each dose group, including controls), which is a common finding associated with nest-building behavior in pregnant rats.

Lactation:
Compound-related clinical signs were limited to dilated pupils in 4/29 high-dose females, which were apparent during gestation and resolved in all animals by LD 4, when the dose was reduced to 4000 ppm (from 7000 ppm on LD4), and repetitive chewing movements in 1/29 animals. Although chewing movements were apparent in only one animal as a clinical observation, it was evident by FOB in 3/30 high-dose animals (GD 20; see below), which further supports this finding being a compound-related effect. There were no compound-related signs at lower dietary levels. Findings that are considered incidental and unrelated to treatment included lacrimal stain.

FOB:
Compound-related findings were evident in high-dose females but not at lower dietary levels. Compound-related effects were first evident on GD 13, with dilated pupils in three high-dose animals. On GD 20, high-dose females had dilated pupils (6/30), dilated pupils that were unresponsive to penlight (2/30), tremor (1/30) and repetitive chewing movements (3/30). There were no compound-related effects during lactation at any dietary level – noting that the high dose was reduced after LD 4.

CLINICAL SIGNS OFFSPRING:
Postpartum (PND 0-21). There were no compound-related clinical signs during lactation in males or females at any dietary level. Incidental findings that were evident on occasion in a few (1-4) individuals from various dose groups, including controls, were limited to various bruises (face, head or tail), cuts (neck), swelling (head), and evidence of cannibalization (missing limb or tail). These findings are common in such young rats and did not occur in a pattern (e.g., doserelated or sustained) to indicate a relationship with exposure to the test substance.

Post Weaning: There were no compound-related effects after weaning (when exposure was discontinued) at any dietary level. Incidental findings in control and/or treated animals from various dose groups included urine and nasal stains, areas of alopecia, exophthalmia and minor dermal lesions at various locations. These findings are not thought to be related to treatment since they occurred after treatment had been discontinued, generally also occurred in controls and did not occur in a dose-related pattern.

Animals Found Dead or Moribund (Post-Culling): The number of offspring (males and females combined) that were missing, found dead or moribund after culling litters on PND 4 was 0, 1, 1 and 0 for the control, low-, mid- and high-dietary levels, respectively. Thus, there was no correspondence with dietary level.

BODY WEIGHT AND WEIGHT GAIN
Gestation:
Body weight gain for high-dose females was reduced an average 42%, relative to control, from the initiation of exposure (GD 6) to GD 20, with differences in body weight, relative to controls, of 7.4% and 13% on GD 13 and 20, respectively. Body weight and weight gain were not affected by treatment at lower dietary levels.

Lactation:
Body weight for high-dose females was reduced an average 14% and 11%, compared to controls, on lactation Days 0 and 4, respectively. Based on substantively reduced body weight, with associated concerns for the offspring, the high-dose was reduced on LD 4. After LD 4, the difference in body weight for high-dose animals progressively decreased, relative to control, with differences of 8.7%, 4.1% and 3.7% on lactation Days 7, 14 and 21, respectively. Body weight was not affected by treatment at lower dietary levels.

BODY WEIGHT OFFSPRING
Preweaning:
Body weight was severely reduced for high-dose males and females, combined, relative to controls, at birth (-13%) and PND 4 (-16%). The high dose was reduced beginning on PND 4, to avoid excessive toxicity that would interfere with achieving the objectives of the study. With the reduction in the high dose, weight gain recovered, such that differences from control in males and females, combined averaged 9.2%, 6.2% and 6.1% on PND 11, 17 and 21, respectively. There was no difference in body weight for males or females at lower dietary levels.

Body weight gain for PND 0-4 was statistically decreased an average 22% in high-dose males and females, combined (3.2 g), compared to controls (4.1 g), with minimal (non-statistical) differences in weight gain for other time intervals, after the high dose was reduced, such that weight gain from PND 4 – 21 averaged only 3.3% (1.3 g) less than control. Body weight gain was not different from control for males or females at lower dietary levels.

Post-Weaning:
After weaning (and discontinuation of treatment), the body weight of high-dose males gradually recovered from the 7.1% difference in weight evident on PND 21 (see above). The body weight for high-dose males averaged 6.6% and 7.9% less than control for the first two weeks measured after weaning, respectively, with recovery thereafter to approximately 3-6% less than control for the remaining weeks. The weight of high-dose males was not different (statistically or biologically) from control during the last week measured (week 7). There was no difference in body weight after weaning for high-dose females or for males or females at lower dietary levels.


FOOD CONSUMPTION AND COMPOUND INTAKE (IF FEEDING STUDY)
Gestation:
There was no effect on food consumption during gestation at any dietary level. Excessive feed spillage at the high dose during the first week of treatment (GD 6-13) resulted in an unreliable measure of feed consumption for that week. Beginning on GD 13, grated feeders were given to all high-dose females to reduce feed spillage and thereby allow more accurate measurements of food consumption.

Lactation:
Beginning on LD 7, all low- and mid-dose females were given grated feeders due to excessive feed spillage during the first week of lactation. Food consumption was 15% decreased compared to controls on lactation day 0-7 for the high-dose females. There was no effect on food consumption after the reduction of the high-dose for the remainder of lactation at any other time point or at any other dietary level.

Compound Intake:
The average daily intake of active ingredient during gestation and lactation for the low- and mid-dose animals was 13.0 and 83.8 mg/kg bw/day, respectively. These results verify that the adjustments in dietary concentrations during lactation effectively maintained a constant
dosage (mg/kg/day) throughout the period of exposure. For high-dose females, spillage precluded a determination of test substance intake during the first week of exposure. However, the 568 mg/kg bw/day value for GD 13-20 is considered to be a representative measure of test substance intake for both weeks of exposure during gestation. For high-dose animals, the average daily intake for LD 0-4 and LD 4-7 was 559.4 and 437.9 mg/kg bw/day, respectively, as the dietary concentration was reduced, with relatively higher food consumption with the reduction in dietary concentration. Taking the average of these two values for LD 0-7 along with the two values for LD 7-14 and LD 14-21, the average daily intake for high-dose animals was 432 mg/kg/day.

OPHTHALMOSCOPIC EXAMINATION
There were no compound-related lesions in males or females at any dietary level. The findings in males and females from various dose groups (including control) were considered to be incidental and unrelated to treatment, due to lack of dose response, consistency by gender and/or because the incidence was within the range of historical control. The only ophthalmic findings in this study were as follows:
Opacity, corneal (M: 6, 0, 1, 1; F: 0, 0, 0, 0)
Degeneration, retinal (M: 1, 0, 0, 0; F: 0, 0, 0, 2)
(gender: control, 150, 1000, 7000/4000 ppm)

NEUROBEHAVIOUR
a) FOB:
Only 19 suitable litters at the high-dose were available to allocate for testing so only 19 males and females were tested at this level. In addition, one low-dose female was missing in the afternoon after testing on PND 4 and was not replaced. The remaining sample size of 19 high-dose males and females and 19 low-dose females, each was considered sufficient to establish whether there were compound-related effects.
There were no compound-related findings in males or females at any dietary level.

There were minimal findings in single males or females for all dietary groups, on all test occasions, and none that was dose-related or otherwise consistent with exposure to the test substance. Incidental findings included yellow urine stain, red nasal stain, a dermal lesion described as a scab, dehydration, areas of alopecia.

b) Motor and locomotor activity (Summary session):
Only 19 suitable litters at the high-dose were available to allocate for testing so only 19 males and females were tested at this level. The only potential compound-related effect was a decrease in motor activity in high-dose males on PND 21, where the level of activity was slightly (29%), but statistically, less than control. This difference from control is considered to represent a potential effect because it was statistically significant and the average results for the high-dose males (mean 228) was below the range of historical control from four developmental neurotoxicity studies (mean 261-341) conducted during the same time period (2005-2007), while the results for controls was well within this range. On the other hand, this difference is relatively small and there was no compound-related effect on any measure of activity in high-dose males at other ages or in females at any age or dietary level. Differences from control that are considered to be unrelated to treatment occurred on PND 17, with statistical and non-statistical differences in motor activity for males (34-39% lower) and females (21-32% lower), respectively, at all dietary levels. These differences are attributed to high levels of motor activity for control males (284) and females (276), relative to the treated animals and historical controls. By comparison, motor activity for controls at PND 17 in the aforementioned DNT studies ranged from 126-222 for males and 143-207 for females, which compares with values for treated males (174-188) and females (187-218) that were comparable or slightly above the range of these historical controls. Locomotor activity was similarly lower than control on PND 17 in males (33-41%) and females (20-32%) at all levels, without statistical significance. Combining this information for controls and treated animals with the lack of a dose-response over this large range of dietary levels supports that these differences are incidental and unrelated to treatment.

Interval Motor and Locomotor Activity:
An analysis of the data by test interval provided no further evidence of a compound-related effect, relative to the results for the entire session. The levels of motor and locomotor activity were generally comparable to control for all test intervals on all test occasions. One minor exception occurred on PND 17 where activity levels were significantly decreased during interval 3 for motor activity in mid-dose females. This difference from control was considered to be incidental and unrelated to treatment because it was not related to dose and occurred during only one interval.

The differences discussed above (for the entire session) involving the PND 17 (all levels) and PND 21 (high-dose) males were evident here as slightly lower levels of activity throughout the session, with the magnitude of habituation comparable to that of control.

c) Auditory Startle Habituation:
Only 19 suitable litters at the high-dose were available to allocate for testing so only 19 males and females were tested at this level. There were no differences in startle amplitude for treated males or females, relative to control, at any dietary level. The average response amplitude for treated animals for all 50 trials and the response amplitude for the five blocks of trials, which is used to assess habituation, were comparable to control at all dietary levels.

d) Learning and memory testing:
Postweaning - Passive Avoidance:
For treated males and females, there were no statistical or non-statistical differences in acquisition or retention, relative to controls, at any dietary level.

Adult Offspring - Water Maze:
Males and females at all dietary levels demonstrated acquisition and retention, with no compound-related differences from control at any dietary level. The time to escape (duration) for the first trial of acquisition was statistically increased for low-dose males, compared to controls, and the average number of trials to criterion (acquisition / learning phase) was non-statistically higher for this group (9.1 trials), compared to control (7.4 trials). This difference from control is not considered a compound-related effect since it only occurred at the low dose and only in males. Moreover, differences from control for the first trial would not reflect an effect on either acquisition or retention, since the animals were completely naïve to the test during that trial.

GROSS PATHOLOGY
Gross observations were not noted in perfused Day 21 animals. The only gross lesion in termination animals was a brain malformation, which consisted of a cystic opening of the left cerebral cortex in 1 control male. Gross observations were not noted in non-perfused termination animals.

Terminal body weight for perfused day 21 high-dose males and females was 11% (statistical) and 6% (non-statistical) less than controls, respectively. These differences from control for this set of high-dose males and females compares with an average 6.1% difference from control for all males and females, combined (see above). There were no differences from control for males or females at lower dietary levels. Terminal body weight for perfused and non-perfused termination (PND 75) males and females were not different from control at any dietary level.

Absolute and relative fixed brain weights for perfused day 21 males and females were not affected by compound administration at any dietary level. Absolute and relative fixed brain weight for terminal (day 75) perfused females and non-perfused terminal (PND 75) males and females at all dietary levels were not affected by compound administration.

NEUROPATHOLOGY
There were no differences in gross measurements of cerebrum or cerebellum length in male or female pups at any dietary level on PND 21.
For perfused termination (PND 75) high-dose females, there was a statistically significant decrease in the cerebrum length, compared to controls. This 2% difference from control is not thought to be compound-related, since this difference from control was minimal, there was no similar difference in high-dose males, and the measurements were well within the historical control range for this laboratory. At all other dietary levels, the cerebrum and cerebellum lengths were comparable to control for perfused termination (PND 75) males and females.

There were no differences in micropathology brain measurements in day 21 high-dose males or females. There were no compound-related differences in micropathology brain measurements in high-dose termination males or females at PND 75.

There were no compound-related microscopic findings in brain tissues from perfused PND 21 high-dose males or females. There were no compound-related microscopic findings in brain tissues from perfused termination high-dose males or females at PND 75.

Additional Non-Brain Termination Animal Tissues:
Spinal cord (cervical, thoracic, and lumbar), cauda equina, spinal nerve roots and dorsal root ganglia (cervical and lumbar), gasserian ganglion, eyes, optic nerves, gastrocnemius muscle, sciatic nerve, tibial nerve, and sural nerves were also collected from perfused termination animals (PND 75) and evaluated microscopically. There were no compound-related microscopic lesions evident in any tissue from the termination high-dose males or females.

Dose descriptor:

NOEL

Remarks:

maternal

Effect level:

83.8 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Remarks on result:

other: calculated from 1000 ppm in feed

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

83.8 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: calculated from 1000 ppm in feed

Dose descriptor:

LOEL

Remarks:

maternal

Effect level:

432 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: calculated from 4000 ppm in feed

Dose descriptor:

LOAEL

Remarks:

maternal

Effect level:

568 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

Remarks on result:

other: calculated from 7000 ppm in feed

Dose descriptor:

NOAEL

Remarks:

offspring

Effect level:

83.8 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: calculated from 1000 ppm in the feed

Dose descriptor:

LOAEL

Remarks:

offspring

Effect level:

432 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Remarks on result:

other: calculated from 4000 ppm in feed

CONCLUSION

The average mean daily intake of the test substance, based on the average dietary consumption for the last two weeks of gestation and three weeks of lactation at nominal dietary concentrations of 150 and 1000 ppm was 13.0 and 83.8 mg/kg/day, respectively. For the high-dose animals, exposure during gestation and lactation was 568 and 432 mg/kg/day, respectively, with the reduction in dose on LD 4.

The maternal NOEL was determined to be 1000 ppm (corresponding to 83.8 mg/kg bw/day). At the high-dose of 7000/4000 ppm clinical signs of toxicity (tremor, dilated pupils, nasal stain and repetitive chewing movements), a marked decrease in weight gain, decreased body weight and food consumption were observed. Therefore, 7000 ppm was considered as maternal LOAEL. Clinical signs and body weight differences (-3.7% on PND 21) recovered after the dose was reduced to 4000 ppm on LD 4, therefore this dose was considered as maternal LOEL.

The NOAEL for offspring was also determined to be 1000 ppm. At 7000 ppm body weight was decreased in both sexes on PND 0 and PND 4, and body weight gain from PND 0-4 was reduced, too. Recovery was evident after reduction of the high dose to 4000 ppm on PND 4. A slight difference in weight persisted after weaning in males only. A statistical significant difference in motor activity (29% lower) was observed for the high-dose males on PND 21 only. Since this effect only occurred on one day and in one sex it is unclear whether it was actually compound-related or not. However, as this possibility cannot be finally excluded 4000 ppm was considered as LOAEL for the offspring.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

306.9 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are an acute, a subchronic and a developmental study available addressing neurotoxic effects in rats.

In the subchronic study which was conducted according to OECD guideline 424 under GLP-conditions, the animals were exposed to dietary levels of 200, 4000, 8000 (females) and 10000 (males) ppm of the test substance over a period of 13 weeks. Treatment-related findings included statistical and/or non-statistical decreases in body weight, total body weight gain and food consumption in high-dose males and females. In addition, measures of motor and locomotor activity were statistically and non-statistically decreased, compared to controls, in high-dose females. There were various clinical signs in one or both sexes at the highest dietary level that included tremors, repetitive chewing movements, perianal stain, diarrhea, wet and stained ventrum, lacrimation, urine stain, red lacrimal and nasal stain. In mid-dose females body weight and total body weight gain were non-statistically decreased. Based on these results a NOAEL of 4000 ppm in the diet, corresponding to 243.6 and 306.9 mg/kg bw/day for males and females, respectively, was established for neurotoxicological endpoints. The highest dose level of 8000/10000 ppm in females/males, which corresponds to a dose of 580.9 mg/kg bw/ day in females, represents the LOAEL for neurotoxicity under the conditions of the study. There were no treatment-related findings in neuronal and non-neuronal tissues in high-dose males or females after subchronic treatment; therefore, tissues from animals at lower dose levels were not examined.

In the acute neurotoxicity study which was also conducted according to OECD guideline 424 under GLP-conditions the animals received a single administration of 100, 500 and 2000 mg/kg bw of the test substance in the initial study. Due to the observation of non-statistically significant reductions in motor and locomotor activity at the low dose of 100 mg/kg bw a follow-up study with dose levels of 25 and 50 mg/kg bw was subsequently conducted to establish a NOEL. Mortality and signs of neurotoxicity were observed at a dose of 2000 mg/kg bw in females; due to mortality this dose level was dicontinued in females. The signs of neurotoxicity observed in males completely resolved by day 5. The acute LOAEL for neurotoxicity was determined as 500 mg/kg bw, based on the observation of tremors in females on day 0 and 1, increased reactivity and decreased activity in females on day 1 only, and urine and oral stains in males on day 0 and day 2, respectively. The dose level of 100 mg/kg bw was determined as NOAEL, based on the lack of findings in males and only non-statistically reduced motor and locomotor activity in females, which resolved by day 7. Therefore, a dose of 100 mg/kg bw can also be considered as LOEL. In the follow-up study no compound-related effects were found at any dose level tested, therefore the NOEL is 50 mg/kg bw.

In contrast to the clinical and neurobehavioural observations, which were completely reversible within 7 days after application the latest, compound-related microscopic lesions were found in neuronal tissues from high-dose males in the histopathological examination, comprising increases in focal to multi-focal individual nerve fiber degeneration in the Gasserian ganglion and the peripheral nerves. However, these findings only occurred at the limit dose of 2000 mg/kg bw and in the presence of severe acute toxicity, characterised by mortality occurring in the high dose females, which were discontinued accordingly. The effects were only of mild characteristics and lacked dose-dependency, as the incidence of these or similar findings in animals at 500 mg/kg bw was comparable to concurrent and historical controls. Thus, lower dose levels were not examined microscopically. In conclusion, the findings were not considered sufficient to allocate a classification for Specific Target Organ Systemic Toxicity after Single Exposure.

The developmental neurotoxicity study was conducted under GLP-conditions according to OECD guideline 426. Mated females were exposed to dietary levels of 150, 1000 and 7000 ppm of the test substance (corresponding to 13.0, 83.8 and 568 mg/kg bw/day) starting on gestation day 6 until lactation day 4, when the dietary concentration of the high-dose group was reduced to 4000 ppm, which corresponds to a dose of 432 mg/kg bw/day during lactation. Test substance exposure was stopped on postnatal day 21 after weaning.

Dams were observed for neurotoxicological effects during gestation and were sacrificed on day 21. Pups assigned to various subsets were sacrificed on PND 21 and 75 and subjected to neuropathological examination. Pups of other subsets were tested in functional observational batteries on postnatal days 23 and 60; learning and memory were tested around postnatal day 60 and again 7 days later.

At the high dose of 7000/4000 ppm clinical signs of toxicity (tremor, dilated pupils, nasal stain and repetitive chewing movements), a marked decrease in weight gain and decreased body weight during gestation and lactation, and reduced food consumption during lactation were observed. Therefore, 7000 ppm was considered as maternal LOAEL. Clinical signs and body weight differences recovered (-3.7% on postnatal day 21) after the dose was reduced to 4000 ppm (432 mg/kg bw/day) on lactation day 4, therefore this dose can be considered as maternal LOEL. Based on these findings the maternal NOEL was determined to be 1000 ppm (83.8 mg/kg bw/day) (referred to as NOAEL by the study authors).

The offspring did not show any clinical signs of neurotoxicity. There were no compound-related microscopic lesions observed in the neuropathological examination. At 7000 ppm body weight was severely decreased in both sexes on postnatal day 0 and 4, and body weight gain for the interval of postnatal day 0 to 4 was reduced, too. Recovery was evident after reduction of the high dose to 4000 ppm on postnatal day 4. A slight difference in weight persisted after weaning in males only. A statistical significant decrease in motor activity (29%) was observed for the high-dose males on postnatal day 21 only. Since this effect only occurred on one day and in one sex it is unclear whether it was actually compound-related or not. However, as this possibility cannot be finally excluded, and the finding was considered relevant by the study authors, 4000 ppm (432 mg/kg bw/day) was considered as LOAEL for the offspring. Therefore, the NOAEL for the offspring was determined to be 1000 ppm (83.8 mg/kg bw/day), as well.

Justification for classification or non-classification

According to the criteria of Regulation (EC) No 1272/2008, the test substance does not have to be classified as exerting irreversible neurotoxic effects after single or repeated exposure, based on the results of the available neurotoxicity studies, although signs of neuronal degeneration were observed in male rats after single exposure to a dose of 2000 mg/kg bw.

However, this effect has only been observed in the males at the highest dose level of 2000 mg/kg bw, which is the limit dose for classification. At the same dose mortality had been observed in the females, therefore the high-dose females had been discontinued, and only high-dose males were investigated at the end of the observation period. The finding lacked dose-dependency, as no indication of a comparable effect was found in males or females treated with 500 mg/kg bw. Furthermore, the observations were only of mild characteristics and did not occur in all high-dose males investigated. In conclusion, a classification for STOT-SE is not considered as justified.