Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Neurotoxicity

Description of key information

Oral (OECD 424), acute, rat: NOEL systemic (males/females) = 508 mg/kg bw; NOEL neurotoxicity (males/females) = 2060 mg/kg bw. 
Oral (OECD 424), subchronic, rat: NOEL systemic (males/females) = 592/748 mg/kg bw/day; NOEL neurotoxicity (males/females) = 592/748 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: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Mar - 7 Apr 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

adopted 21 Jul 1997

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: At least 9 weeks
- Weight at study initiation: Males: 232.8 - 309.0 g; females: 167.4 - 215.4 g
- Housing: Individually housed in suspended stainless steel wire-mesh cages, each containing a feeder, a source of water (pressure-activated water nipples), and deotized (sanitized) cage board in the bedding tray.
- Diet: Purina Mills Rodent Lab Chow 5002 in meal form, ad libitum, except during neurobehavioral testing,
- Water: Tap water (Kansas City Missouri Municipal Water), ad libitum, except during neurobehavioral testing,
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 (4 interruptions were noted including adjustment for daylight savings time).

IN-LIFE DATES: 21 Mar - 07 Apr 2005

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose/0.4% Tween 80 in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Doses were prepared by suspending the test substance in 0.5% methylcellulose/0.4 % Tween 80 in deionized water. The dose volume was 10 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test substance in the vehicle was measured using high performance liquid chromatographic /ultra violet (HPLC/UV) analysis. The homogeneity and stability of the test substance in the vehicle were established using samples at nominal concentrations of 10 and 200 mg/mL which covered the dosing range. Homogeneity of the test substance in the vehicle was accepted for the range of concentrations as the 10 mg/mL and 200 mg/mL concentrations had percent relative standard deviations (%RSD) of 0.39% and 1.33%, respectively. Stability analysis of the test substance in the vehicle showed no appreciable decrease in concentration over seven or eight days at room temperature storage for the nominal concentrations of 10 mg/mL or 200 mg/mL, respectively (equivalent to doses of 100 or 2000 mg/kg, respectively). Concentration analysis showed doses of 125, 500 and 2000 mg/kg for males and females ranged from 101% to 103% of the nominal concentrations. Based on these results, the analytically confirmed doses for males and females were 126, 508 and 2060 mg/kg.
The homogeneity and stability of the test substance in the vehicle were established using samples at nominal concentrations (10 and 200 mg/ml) that bracketed the range of concentrations used in the present study (nominal 12.5 and 200 mg/ml). Homogeneity was accepted if the percent relative standard deviation (%RSD) was <5%. Each dosing suspension was also analyzed to measure the concentration of test substance.

Duration of treatment / exposure:

Single exposure by gavage

Frequency of treatment:

Single exposure

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

126 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

508 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

2060 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

The rationale for dose selection was based on the results of an acute oral toxicity study in young-adult male and female Wistar rats [4]. In that study, two fasted male and female Wistar rats were administered an acute oral (gavage) dose of either 500, 1000 or 2000 mg/kg as an aqueous suspension in 0.5% methylcellulose in distilled water, at a dosing volume of 10 mL/kg bw. Animals were observed for mortality and clinical signs for 13 days after treatment. The test substance produced no clinical signs or mortality at the limit dose. These results support the use of a limit dose (2000 mg/kg) in the neurotoxicity study but provided no information to establish the time of peak effect.

The results from a study using radio-labeled test substance were examined to estimate the time of peak effect. In that study, adult Wistar male and female rats received a single oral (gavage) dose of 2 or 200 mg/kg test substance in 0.5% aqueous tragacanth, at a dosing volume of 10 ml/kg. In that study, the tmax in plasma occurred 40 - 60 minutes after treatment in both sexes following administration at 200 mg/kg. For the low dose (2 mg/kg), the tmax in plasma occurred 10-40 minutes after treatment in both sexes.

Based on these collective results, the doses selected for this study were 125, 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 NOEL in both sexes. Based on the estimated time of peak blood concentrations within this dose range, the FOB began approximately 50 minutes (minimum) following dose administration, with the automated test of activity concluding at about three hours after treatment.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily.
- Cage side observations checked included: Mortality, moribundity and clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly as a component of the FOB and on the day of sacrifice for terminal body weight measurement.

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 behaviour), bizarre behaviour, gait abnormalities, vocalizations, arousal level, and amount of excretion.
Reflex and physiologic observations/measurements included: Approach response, touch response, auditory response, tail pinch, pupil size at normal lighting, pupil response, righting reflex, grip strength [Chatillon, Model DFIS-10 (forelimb) and DPI-10 (hind limb) 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:
All animals that were assigned to the study were tested using the FOB and motor activity on four occasions - one week prior to treatment, approximately 50 minutes after administration of the dose, and again 7 and 14 days following treatment. The order of testing and assignment of animals to mazes were done in a semi-random manner, such that groups were balanced across test times and test devices, and no animal would be tested more than once in the same maze. On the day of FOB and motor activity testing, the appropriate animals were placed in the sequence that was established for testing on that day. The dose group identification was concealed at that time to ensure that testing would be performed without knowledge of the group assignment. Animals were then transferred to the room where the testing took place and were allowed to acclimate with minimal disturbance for approximately 30 minutes prior to 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. 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.
An additional sixteen animals (eight males and eight females) were tested (FOB and motor activity) during the pretreatment week and reserved for use in case they were needed to replace animals (e.g., if mis-dosed) that were assigned to the study.
- Minimization of bias: Dose group identification was concealed to ensure that testing would be performed without knowledge of the group assignment. 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
- Same technicians used throughout testing: Yes, However, inter-observer reliability has been established in order to allow a second person to perform either the observations or measurements, ensuring the consistency of the results of each technician. Studies have been conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of these test procedures, the adequacy of training of technical personnel and to serve as a historical control
- Technicians were blind to treatment status of animals: Yes
- Site of testing: Data were collected while the rats were in their home cage, during handling, and in an open field for 2 minutes (in the centre of a flat surface with a perimeter barrier, such as a cart). In addition, reflex and physiologic observations and measurements were made while the animals were sitting on the cart surface following open field observations.
- Time schedule for examinations: During the light phase of the day/night cycle
- Environmental conditions: 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.
- Noise level: Not stated for FOB test.
- Scoring criteria: Observations were scored on intensity as follows: 1) slight (barely perceptible or infrequent) or 2) moderate to severe.
- Duration of observation period for open field observations: 2 minutes
- Description of equipment where required: Grip strength [Chatillon, Model DFIS-10 (forelimb) and DPI-10 (hindlimb) digital strain gauges (5.0 kg capacity), which were both equipped with a grid system attached to an extension arm].

LOCOMOTOR ACTIVITY: Yes
- Replicates used: Sets of a maximum of 8 rats
- Type of equipment used: Figure-8 maze was selected as an established and widely-used automated activity-measuring device that can be used to detect both increases and decreases in activity. Each maze consisted of a series of inter-connected alleys, converging on a central arena and was 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. Broad spectrum background noise (approximately 74 dB(A)) 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: Motor and locomotor activities were examined during each of the six, ten-minute intervals.
- Noise level: 74 dB (A)
- Parameters measured:
- 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
- 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
- Brain weight: Yes
- Length and width of brain: No
- Other: Brain:bodyweight ratio.
- 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: A minimum of six males and six females at each dose level.
- Tissues evaluated: Central nervous tissue and brain; Olfactory bulbs, Cerebral cortex, Caudate-putamen/globus pallidus, Hippocampus, Thalamus, Hypothalamus, Midbrain (tectum, tegmentum, and cerebral peduncles), Cerebellum, Medulla oblongata. Spinal cord including cervical, thoracic, lumbar and cauda equina, and the gasserian ganglion, optic nerve, eyes, Gastrocnemius muscle; and the peripheral nervous system including the sciatic nerve (bilateral);tibial nerve (bilateral) and sural nerve (bilateral), the lumbar dorsal root ganglion, lumbar dorsal root fibres and lumbar ventral root fibers, the cervical dorsal root ganglion, the cervical dorsal root fibres and the cervical ventral root fibres.
- Type of staining: Haematoxylin and eosin (H&E) for paraffin-embedded tissues and modified Lee’s stain for glycol methacrylate (GMA)-embedded tissues.
- Thickness: 2-3 μm
- Embedding media: Paraffin, GMA.
- 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 group) for micropathology. Perfusion-fixed animals from low and mid dose were examined only when there were findings at the high dose.

Positive control:

Concurrent positive controls were not included in this study. However, for the Functional Observational Battery (FOB), previous studies were conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of these test procedures, the adequacy of training of technical personnel and to serve as a historical control. To assess motor activity, previous studies with untreated animals and with rats treated with reference substances that increase (triadimefon) and decrease (chlorpromazine) activity have established the sensitivity, reliability and validity of the test procedures used. Finally, studies performed at the 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. 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 Analysis of Variance (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
the Mann-Whitney U test for between-group comparisons). For the FOB, continuous data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA if there was a significant interaction between dose group and test day. For days on which there was a significant treatment effect, Dunnett's test was applied to determine which groups, if any, were significantly different from the control group. Categorical data collected in the FOB was analyzed in a similar manner, using General Linear Modeling and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively.

For additional information on statistics, see Any other information on materials and methods incl. tables.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Urine stain at 2000 mg/kg in three males and five females on Day 0 only. The effect is considered treatment-related but not adverse and not corroborated by any other finding indicative of systemic toxicity.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Not applicable.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity:
An examination of inherent variability, using the average pre-treatment values among the four groups of males and females, provides a measure of the magnitude of the difference that should be considered biologically significant. For motor activity, the pre-treatment values for groups that later received the test substance averaged from 9% to 17% lower than controls for males and from 1% lower to 3% higher than controls for females. For locomotor activity, the pre-treatment values for groups that later received the test substance averaged from 12% to 21% lower than controls for males and from 1% to 17% higher than controls for females. As a general guide, these results confirm that differences of ± 20% are within the range of normal variability in this laboratory for groups of 10-12 rats/sex/dose level and, therefore, are not biologically significant.

For the overall 60-minute test session, there were no compound-related effects in session motor or locomotor activity at any dose level in either sex.

Differences from control that were considered incidental and unrelated to treatment included measures of motor (23%) and locomotor (27%) activity that were slightly below the standard (±20%) range of normal variability in high-dose males on day 0. These differences from control are not considered to be related to treatment because the differences were small, only seen in one sex (no trend in females), and were not statistically significant. Moreover, the high-dose males exhibited consistently lower levels of activity than controls at all time-points, including prior to treatment. In addition, these measures of motor (total activity counts of 295-516 for referenced historical controls) and locomotor (total activity counts of 192-312 for referenced historical controls) activity are well within the range of historical control.

Motor and locomotor activity data were subjected to further analysis at each interval on each test day. There were no compound-related effects at any dose level in either sex.

Habituation was not affected by treatment any dose level in either sex.

A summary of motor activity and locomotor activity is presented in Attachment 1.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOEL

Remarks:

Neurotoxicity

Effect level:

> 2 060 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen at this dose level.

Key result

Dose descriptor:

LOEL

Remarks:

systemic toxicity

Effect level:

2 060 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

508 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects at this dose level

Conclusions:

The study was performed in accordance to OECD TG 424 under GLP conditions and is considered reliable. Non-fasted young adult Wistar rats were administered doses 0, 126, 508 and 2060 mg/kg bw via oral gavage. Urine stain was evident in both sexes at 2060 mg/kg bw on Day 0 and resolved by the following day. No treatment-related findings were observed at 508 and 126 mg/kg bw. There was no evidence of neurotoxicity at any dose level and no compound-related gross or microscopic lesions at the high dose of 2060 mg/kg bw. These results establish a NOEL of 508 mg/kg bw for systemic effects.

Reference 2

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Aug - 08 Dec 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

adopted 21 Jul 1997

Deviations:

yes

Remarks:

The highest dose level did not induce neurotoxic effects or clear systemic toxic effects.

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 210-285.1 g, females 141.4-179.5 g
- 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, ad libitum. Provided for consumption during the acclimation period and throughout the study except during neurobehavioral testing.
- Water: Tap water, ad libitum (Kansas City Missouri Municipal Water), except during neurobehavioral testing.
- Acclimation period: 7 days

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

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Every other week.
- Mixing appropriate amounts with (Type of food): Acetone was used to dissolve/suspend the test substance. The test substance/acetone mixture was added to the feed (Purina Mills Rodent Lab Chow 5002), mixed and the acetone was allowed to evaporate. The control diet was prepared the same way, excluding the test substance. Feed was available for ad libitum consumption for a period of one week prior to changing, at which time any uneaten feed was collected and disposed of by incineration.
- Storage temperature of food: At room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A High Performance Liquid Chromatographic / mass spectrometry/ mass spectrometry method was used to measure test substance concentrations in rodent ration. The stability [following both room temperature (~22 °C) and freezer (~-23 °C) exposure] and homogeneity of the test substance in the feed were established by analysis of samples at nominal concentrations of 10 and 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 of the test substance in the ration was accepted for concentrations that bracket those used in this study. These concentrations of 10 and 10000
ppm had percent relative standard deviations (%RSD) of 1.98% and 1.81%, respectively.
The stability of the test substance in the ration was established at room temperature at dietary concentrations of 10 and 10000 ppm, with no appreciable decrease in concentration with seven days of storage. The test substance was stable at freezer conditions for 28 days, with no appreciable decrease in concentration at 10 and 10000 ppm.
Analytically-determined concentrations of the active ingredient in the 1000, 3000 and 9000 ppm dietary levels used in this study averaged 97% to 98% of the nominal concentration. Based on these results, the mean analytically-confirmed dietary levels for this study were 976, 2904 and 8698 ppm for males and females.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily, 7 days/week

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 65.2 and 84.5 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

3 000 ppm

Remarks:

corresponding to 193 and 251 mg/kg bw/day actual dose ingested for males and females, respectively.

Dose / conc.:

9 000 ppm

Remarks:

corresponding to 592 and 748 mg/kg bw/day actual dose ingested for males and females, respectively.

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 a 90-day toxicity study, with the test substance administered via the diet at nominal concentrations of 1000, 4000, and 12000 ppm to male and female Wistar rats (10/sex/dietary level; see chapter 7.5.1). Based on the results of this study, the doses selected for the present subchronic neurotoxicity study were 1000, 3000 and 9000 ppm in both sexes. The 9000 ppm dietary level was selected as a maximum-tolerated dose (MTD) following subchronic exposure, since effects at the 12000 ppm dietary level demonstrated excessive toxicity (e.g., two deaths) for that study. The 1000 ppm dietary level was selected to produce no evidence of toxicity for endpoints measured in this neurotoxicity study and the 3000 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: 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: [Active ingredient (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 field. 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.
Each week, testing was staggered over two days for each sex to accommodate the schedule for behavioural 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 (1989), 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: Yes, and inter-observer reliability was 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.
- 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: Brain: 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; Other: 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; non-perfused: Kidney, urinary bladder.
- 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 examined 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 Lee's 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 were 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 the testing 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 and common statistical test were used (Bartlett's test, ANOVA, Dunnett's test, Chi-Square Test, Fisher's Exact Test).

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related findings in either sex at any dietary level. One high-dose female had a urine stain (observed on Days 4, 18-88). This finding in one animal is within the range of historical control and considered incidental to treatment.

Remaining findings were also considered incidental and unrelated to treatment and consisted of areas of alopecia (1 low-dose female), maloccluded teeth (Imid-dose female) and dermal lesions described as a scabs on cheek and shoulder (one mid-dose male).

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor Activity:
An examination of inherent variability, using the average pre-treatment values among the four groups of males and females, provides a measure of the magnitude of the difference that should be considered biologically significant. For motor activity, the pre-treatment values for groups that later received the test substance averaged from 16% lower to 6% higher than animals assigned to the control group for males and from 3% lower to 9% higher than controls for females. For locomotor activity, the pre-treatment values for groups that later received the test substance averaged from 19% lower to 11% higher than controls for males and from 2% to 6% higher than controls for females. As a general guide, these results confirm that differences of + 20% are within the range of normal variability in this laboratory for groups of 10-12 rats/sex/dietary level and, therefore, are not biologically significant.

For the overall 60-minute test session, there were no compound-related differences in session motor or locomotor activity for males or females at any dietary level. There were a few occasions on which measures of motor (week 8 mid- and high-dose males (+33% and +23%, respectively), week 13 high-dose males (+21%) and week 4 low-dose females (-39%)) and locomotor (week 8 mid-dose males +24%), week 4 low-dose females (-35%) and week 13 high-dose females (+22%)) activities were slightly beyond the standard (+20%) range of normal variability. These differences from control are considered incidental and unrelated to treatment, since they were relatively small and there was no relationship to dose or direction (relatively higher and lower than control).

A summary of motor activity and locomotor activity is presented in Attachment 1.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no histopathologic changes in neural tissues from perfusion-fixed control and highdose rats of both sexes that were related to administration of the test compound. Tissues from animals at lower dose levels were, therefore, not examined.

Incidental microscopic changes not related to treatment included retinal degeneration or dysplasia in control and/or treated high-dose rat eyes. In addition, there were isolated degenerative nerve fibers seen in control and treated rats. In non-perfused rats kidney congestion was present in control and all treated groups. Other incidental kidney changes included pelvic dilatation, mineralization, lymphocytic inflammation and haemorrhage in individual rats.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

9 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects at this dose level.

Remarks on result:

other: corresponding to 592 and 748 mg/kg bw/day actual dose ingested for males and females, respectively.

Key result

Dose descriptor:

NOEL

Remarks:

neurotoxicity

Effect level:

9 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects at this dose level.

Remarks on result:

other: corresponding to 592 and 748 mg/kg bw/day actual dose ingested for males and females, respectively.

Critical effects observed:

no

Conclusions:

The study was performed in accordance to OECD TG 424 under GLP conditions and is considered reliable. In a 90-day neurotoxicity study, the test substance was administered in the diet for 13 weeks to young adult Wistar rats. The mean daily intake of the test substance over approximately 13 weeks at nominal dietary concentrations of 1000, 3000 or 9000 ppm, respectively, was 65.2, 193 and 592 mg/kg bw/day for males and 84.5, 251 and 748 mg/kg bw/day for females. There were no treatment-related effects attributed to exposure to the test substance at any dietary level in either sex. Based on these findings, a NOEL (systemic and neurotoxicity) of 9000 ppm was established for the rat (specifically, 592 and 748 mg /kg bw/day for males and females, respectively).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The quality of the database is good comprising two neurotoxicity studies in rat (acute and sub-chronic). Both studies were conducted according to OECD TG 424 and under GLP conditions. Both studies are considered of reliable quality and validity, fulfilling the criteria of a key study. Thus, both are suitable for assessment of the present endpoint.

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

An acute neurotoxicity study (M-283196-01-1) was performed in Wistar rats under GLP condition and in accordance with OECD TG 424 (adopted 1997).

The test substance was administered by gavage to groups of 12 rats/sex at doses of 126, 508 and 2060 mg/kg bw (analytical results). The test substance was formulated in 0.5% methylcellulose / 0.4% Tween 80 in deionised water.

After treatment, urine stain was evident in both sexes at 2060 mg/kg bw on Day 0 and resolved by the following day. No treatment-related findings were observed at 508 and 126 mg/kg bw. There was no evidence of neurotoxicity at any dose level and no compound-related gross or microscopic lesions at the high dose of 2060 mg/kg/day. These results establish a general NOEL of 508 mg/kg bw and a NOEL for neurotoxicity of 2060 mg/kg bw/day.

A sub-chronic neurotoxicity study (M-283218-01-1) was performed in Wistar rats under GLP condition and in accordance with OECD TG 424 (adopted 1997).

Groups of 12 rats/sex/dose received the test substance dissolved in acetone in their diet over a period of 13 weeks. The following doses were administered: 1000, 3000 and 9000 ppm of the test substance, corresponding to 65.2, 193 and 592 mg/kg bw/day for males and 84.5, 251 and 748 mg/kg bw/day for females.

There were no treatment-related effects attributed to exposure to the test substance at any dietary level in either sex. Based on these findings, a NOEL of 9000 ppm was established for the rat (specifically, 592 and 748 mg/kg bw/day for males and females, respectively).

Justification for classification or non-classification

The available data on neurotoxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.