Pyrasulfotole

Neurotoxicity

Description of key information

Acute neurotoxicity in the rat (OECD 424), NOAEL = 500 mg/kg bw/day

Subchronic neurotoxicity in the rat (OECD 424), 90-days, NOAEL = 5000 ppm (highest dose tested), equivalent to doses of 345 and 416 mg/kg bw/day in males and females, respectively

Developmental neurotoxicity in the rat (OPPTS 870.6300), NOAEL for maternal animals = 450 ppm, equivalent to a dose of 37.1 mg/kg bw/day; NOAEL for offspring = 45 ppm, equivalent to a dose of 3.8 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:

key study

Study period:

6 Aug 2004 - 17 Nov 2004

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:

Wistar

Remarks:

HAN CRL:WI (GLX/BRL/HAN) IGSBR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC, USA)
- Age at study initiation: not more than 8 weeks
- Weight at study initiation: 194.0 - 261.7 g (males) and 137.0 - 183.2 g (females)
- Fasting period before study: no
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5002 in meal form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10 (minimum daily average)
- Photoperiod (hrs dark / hrs light): 12/12, lights toggled off during ophthalmic examinations

IN-LIFE DATES: From: 16 Aug 2004 To: 17 Nov 2004

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

(served as a solvent in the diet preparation process and was allowed to evaporate prior to administration)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every two weeks
- Mixing appropriate amounts with: Purina Mills Rodent Lab Chow 5002 in meal form

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the ration was measured by liquid chromatographic analysis. The stability [following both room temperature (~ 22°C) and freezer exposure (~ -23°C)] and homogeneity of the test substance in the feed were established by analysis of samples at nominal concentrations of 1000 and 16000 ppm. The concentration of the test substance in the ration was measured for the ration that was used during all weeks of the study.

RESULTS
Homogeneity Analysis: Homogeneity of the test substance in the ration was accepted for concentrations that bracketed those that were used on this study. These concentrations of 10 and 5000 ppm had percent relative standard deviations (% RSD) of 2.71% and 1.52%, respectively.
Stability Analysis: The stability of the test substance in the ration was established at room temperature at dietary concentrations of 10 and 5000 ppm, with no appreciable decrease in concentration with seven days of storage. The test substance was stable at freezer conditions for 77 days, with no appreciable decrease in concentration at 10 and 5000 ppm.
Concentration Analysis: Actual concentrations of the active ingredient in the 0, 500, 2500 and 5000 ppm dietary levels used in this study averaged 94% to 97% of the nominal concentration. Based on these results, the mean analytically confirmed dietary levels for this study were 0, 486, 2363 and 4772 ppm for males and females.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days a week

Dose / conc.:

500 ppm (nominal)

Remarks:

equivalent to doses of 32.3 and 41.9 mg/kg bw/day in males and females, respectively

Dose / conc.:

2 500 ppm (nominal)

Remarks:

equivalent to doses of 166 and 206 mg/kg bw/day in males and females, respectively

Dose / conc.:

5 000 ppm (nominal)

Remarks:

equivalent to doses of 345 and 416 mg/kg bw/day in 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 subchronic (13-week) study, with the test substance administered via the diet at nominal concentrations of 0, 2, 30,1000, 7000 and 12000 ppm to male and female Wistar rats (10/sex/dietary level) [M102924-01-2]. The principal findings from this study were as follows: There were no compound-related findings at the 2 and 30 ppm dietary levels. Compound-related effects at 1000 ppm included slightly reduced body weight gain during the first week of exposure, yellow (intense) urine in males and a bilateral corneal opacity (snowflake) in one female. In addition, mean absolute and relative liver weight in males and mean relative liver weight in females were increased. Finally, males at 1000 ppm had a slight to mild diffuse thyroid follicular cell hyperplasia. Animals that either died or were sacrificed for humane reasons consisted of two males at 7000 ppm and six males and one female at 12000 ppm and on Day 72, all remaining high-dose males were sacrificed for humane reasons.
Other findings ascribed to treatment in both sexes at the two highest dietary levels include yellow (intense) colored urine, soiled fur, labored or noisy respiration, increased salivation, wasted appearance, piloerection, general pallor, reduced motor activity, cold to touch, hunched posture and “snowflake” corneal opacity, which was generally associated with neovascularization of the cornea. In addition, body weight and food consumption were affected in both sexes. At study termination, effects related to treatment included yellow calculi in the urinary tract at 7000 ppm and in unscheduled sacrificed animals at the 12000 ppm dietary level. A histological examination was not performed at 12,000 ppm in either sex. Animals at the 7000 ppm level had increased kidney weight, pelvic dilatation, urinary epithelial hyperplasia and interstitial fibrosis of the urinary tract. This same group had increased mean absolute and/or relative liver weight, a slight to moderate diffuse centrilobular hepatocellular hypertophy and, in females only, periportal vacuolation. Lastly, males at the 7000 ppm dietary level had slight to moderate diffuse thyroid follicular cell hypertrophy / hyperplasia.

The results from an exploratory 14-day dietary-exposure study in Wistar rats were also considered for dose selection. In this study, the test substance was administered at dietary levels of 0, 400, 2000 and 7500 ppm to groups of five males and five females for 14 days. An additional three animals per sex was added to the high dose and control groups to be sacrificed 3 days after treatment initiation to evaluate liver weight, hepatic cellular proliferation and liver histopathology. There were no clinical signs or mortalities at any dietary level. The only finding related to treatment was a slight decrease in food consumption during the first week in high-dose females. There were no treatment-related changes at necropsy or in any hematology or clinical chemistry parameter.

Based on these combined results, the proposed doses for the present subchronic neurotoxicity study were 0, 500, 2500 and 5000 ppm in both sexes. The 5000 ppm dietary level was selected as a maximum-tolerated dose (MTD) following subchronic exposure. Effects at the 7000 ppm dietary level demonstrated excessive toxicity for the present study. The 500 ppm dietary level was selected to produce no evidence of toxicity for endpoints measured in this neurotoxicity study and the 2500 ppm dietary concentration was selected as an intermediate dietary level.

Other: All twelve rats/sex/dose level were used for neurobehavioral testing, with half used for histopathology. All animals were sacrificed after 13 weeks of exposure. Micropathology was performed on selected tissues from 6 rats/sex from the control and high dose groups.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on holidays and weekends)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly and on the day of sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

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

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined:
Home cage observations: 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, tail pinch, pupil size at normal lighting, pupil response, righting reflex, grip strength [Chatillon, Model DFIS-10 (forelimb) and DFI-10 (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.
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Site of testing: Test room was a separate standard animal room.
- Time schedule for examinations: 5 occasions: once during the week prior to initiating the exposure and again during Weeks 2, 4, 8 and 13.
- Environmental conditions: The test room 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.
- Scoring criteria (if any): Scoring criteria and explicitly-defined scales were used to rank the severity of observations that do not readily lend themselves to quantitation. 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 min

LOCOMOTOR ACTIVITY: Yes
- Replicates used: 5 occasions: once during the week prior to initiating the exposure and again during Weeks 2, 4, 8 and 13
- Type of equipment used:
Locomotor activity was monitored in figure-eight mazes, 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.
- Length of session, number and length of subsessions: 60 min (6 x 10 min)
- Parameters measured:
- Total activity: Yes
- Ambulatory activity: Yes
- Large movements: No
- Small movements: No
- Noise level: Broad-spectrum background noise (approximately 74 dB(A)) was provided throughout the locomotor activity test to minimize acoustical variations during testing.
- Other: The uniformity of light intensity (100 ± 70 lux) over each of the mazes was verified daily.

Sacrifice and (histo)pathology:

- Time point of sacrifice: Day 91
- Number of animals sacrificed: all
- Parameters measured: examination of all organs, body cavities, cut surfaces, external orifices and surfaces.
- Brain weight: Yes
- Length and width of brain: No
- Procedures for perfusion: animals were deeply anesthetized using an intraperitoneal dose (50 mg/kg bw) 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: the first 6 males and 6 females / group were selected for perfusion and collection of tissues, with replacement, as necessary, if the perfusion was considered inadequate.
- Tissues evaluated: 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.
Detailed list of tissues evaluated: olfactory bulbs, cerebral cortex, caudate-putamen / globus pallidus, hippocampus, thalamus, midbrain (tectum, tegmentum, cerebral peduncles), cerebellum, medulla oblongata, cervical swelling, thoracic swelling, 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.
- Type of staining and embedding media: Coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar) and the cauda equina were embedded in paraffin and examined utilizing 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). Eyes, optic nerves and gastrocnemius muscle were embedded in paraffin and stained using H&E. Peripheral nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA 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: 2 - 3 µm
- Number of sections: eight coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar) and the cauda equina
- Number of animals evaluated from each sex and treatment group: 6 males and 6 females from high dose and control group; tissues from perfusion-fixed animals at the low- and mid-dose levels were not subjected to micropathology unless compound-related lesions were present in the high-dose group.

Positive control:

The study did not include concurrent positive controls, but references are made to the following studies that serve this purpose. 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) motor activity have established the sensitivity, reliability and validity of the test procedures used. Finally, 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 performed using software from either INSTEM Computer Systems or SAS. The level of significance was set at p ≤ 0.05, with the exception of Bartlett’s test 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. 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.
Continuous pathology data (e.g. brain weight) was initially evaluated using Bartlett’s test to analyze for homogeneity of variance among groups. Groups with homogeneous variances were analyzed further using an ANOVA followed by Dunnett’s test for pair-wise comparisons. In the event of non-homogeneous variances, continuous data were analyzed using the nonparametric Kruskal-Wallis test followed by a Mann-Whitney U test for pair-wise comparisons. Micropathology frequency data were screened for potential effects and then evaluated using a Chi-Square procedure, followed by a one-tailed Fischer’s Exact Test in cases of significant variation by the Chi-Square analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Yellow and / or brownish-orange urine stain was the only evidence of exposure and was evident in one low- and seven high-dose males and in two mid- and seven high-dose females. These findings are related to the excretion of the unmetabolized compound and do not represent a systemic or adverse effect.
The remaining findings were considered incidental and unrelated to treatment. These findings consisted of areas of alopecia in one mid-dose male and a swollen perigenital area of one mid-dose female. The latter finding was determined to be a clitoral gland abscess at necropsy.

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

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:

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):

For the overall 60-minute test session, there were no compound-related differences in session motor or locomotor activity for males or females at any dietary level (motor and locomotor activity results are summarized below).
For mid- and / or high-dose males, there were five occasions on which measures of motor (Week 2 - +39% and +29%, respectively) and locomotor (Week 2 - + 34% and +27%, respectively and week 13 - +23% in highdose) activities were slightly above the standard (+20%) range of normal variability. By comparison, motor and locomotor activity for females was increased and outside of the standard range (+20%) of normal variability at all treated levels for all test weeks. These differences from control (26 - 65% higher for motor activity and 35 - 70% higher for locomotor activity) are considered incidental and unrelated to treatment, since (1) they were not statistically significant, (2) there was no relationship with dietary level, (3) this pattern was not evident in males and (4) the values were generally within the range of historical control for this laboratory. Furthermore, the opposite effect (i.e., a decrease in activity) was evident in the acute oral neurotoxicity study [M-263029-01-132]. In that study, there were significant decreases in motor (-25% and -37% in males and females, respectively) and locomotor (34% and 50%, in males and females, respectively) activity at the highest dose level (2000 mg/kg).
Motor and Locomotor activity data were also analyzed for differences at each 10-minute interval of each test session. For females, these measures of motor and locomotor activity were not statistically different from controls at any dietary level or on any test occasion.
For males, the only statistical difference from control was at the mid-dose, where locomotor activity was statistically greater than controls for the first interval during Week 2. This difference from control is considered incidental and unrelated to treatment since it only occurred on one occasion and was not dose-related. It should also be noted that interval 1 locomotor activity at all treated levels was at the upper limit of historical control. Lastly, locomotor activity during pretreatment week was significantly less than control for the last interval in males that were assigned to the low- and mid-dose groups. Since animals had not yet been treated, this finding is unrelated to treatment.
Habituation was not affected by treatment with the test substance at any dietary level.

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 compound-related microscopic lesions in the high-dose males or females. Therefore, tissues from animals that received a lower dose of the test substance were not examined.
There were a few incidental microscopic changes in control and/or treated high-dose animals that were considered to be unrelated to treatment. These findings consisted of retinal degeneration, keratitis and neovascularization of the cornea. In addition, there were a few incidences of isolated degenerative nerve fibers in control and treated rats. None of these findings were considered to be related to treatment.

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:

NOAEL

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed up to the highest dose tested.

Remarks on result:

other: equivalent to 345 and 416 mg/kg bw/day in males and females, respectively

Table 1: Mean achieved test item intake per group

Mean achieved test item intake (weeks 1-13)
Diet concentration [ppm]	Males [mg/kg bw/day]	Females [mg/kg bw/day]
500	32.3	41.9
2500	166	206
5000	345	416

 

Conclusions:

The study was performed under GLP conditions and according to OECD TG 424 (adopted 1997). There were no indications of a neurotoxic effect of the test substance after 90 days dietary administration, nor were there effects on body weight or food consumption. The NOAEL for this study was therefore 5000 ppm, or 345 mg/kg bw/day in males and 416 mg/kg bw/day in females.

Reference 2

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Sep - 15 Oct 2004

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:

Wistar

Remarks:

HAN CRL:WI (GLX/BRL/HAN) IGSBR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC, USA)
- Age at study initiation: at least 9 weeks
- Weight at study initiation: 225 - 307 g (males) and 159 - 201 g (females)
- Fasting period before study: no
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5002 in meal form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 27 Sep 2004 To: 15 Oct 2004

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification provided
- Amount of vehicle (if gavage): 10 mL/kg bw

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 0.5% methylcellulose / 0.4% Tween 80 in deionized water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the vehicle was measured using liquid chromatographic (LC) analysis. 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 20 and 200 mg/mL). Homogeneity was accepted if the percent relative standard deviation (%RSD) was < 6%. Each dosing suspension was also analyzed to measure the concentration oft he test substance.

Results:
Homogeneity Analysis: Homogeneity of the test substance in the vehicle was accepted for the range of concentrations used, as the 10 mg/mL and 200 mg/mL concentrations had percent relative standard deviations (% RSD) of 0.40% and 3.2%, respectively.
Stability Analysis: The stability (at room temperature conditions) of the test substance in the vehicle was established, with no appreciable decrease in concentration with eight days at room temperature storage for nominal concentrations of 10 or 200 mg/mL (equivalent to doses of 100 or 2000 mg/kg bw, respectively).
Concentration Analysis: Doses of 0, 200, 500 and 2000 mg/kg bw for males and females ranged from 96% to 107% of the nominal concentrations. Based on these results, the analytically confirmed doses for males and females were 0, 192, 504 and 2140 mg/kg bw.

Duration of treatment / exposure:

After a single treatment animals were observed for 14 days.

Frequency of treatment:

not applicable

Dose / conc.:

200 mg/kg bw/day

Remarks:

actual dose received: 192 mg/kg bw

Dose / conc.:

500 mg/kg bw/day

Remarks:

actual dose received: 504 mg/kg bw

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

actual dose received: 2140 mg/kg bw

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The rationale for dose selection was based on the results of an acute oral toxicity study in young-adult female Wistar rats. In that study, two sets of three fasted females were administered an acute oral (gavage) dose of 2000 mg/kg bw as an aqueous suspension in 2% cremophor in demineralized water, at a dosing volume of 10 mL/kg bw. Animals were observed for mortality and clinical signs for 14 days after treatment. The test substance produced no clinical signs or mortality. These results support the use of a limit dose (2000 mg/kg bw) in the neurotoxicity study but provided no information to establish the time of peak effect.
Thus, 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 100 mg/kg bw of the test substance in 0.75% (w/w) methyl cellulose in water, at a dosing rate of 1 g dose suspension per 200 g rat body weight. In this study, the group mean peak blood concentrations occurred 30 minutes after treatment in both sexes following administration at 100 mg/kg bw (75.5 and 56.8 μg [14C]-test substance equivalents per g whole blood in males and females, respectively).
Based on these collective results, the doses selected for this study were 0, 200, 500 and 2000 mg/kg bw for both sexes. The 2000 mg/kg bw 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 is expected to be an overall NOEL in both sexes. Based on the estimated time of peak blood concentrations at this dose range, the FOB began approximately 30 minutes (minimum) following dose administration, with the automated test of activity concluding at about 2.5 h after treatment.

Other: All twelve rats/sex/dose level were used for neurobehavioral testing, with half used for histopathology. All animals were sacrificed 14 or 15 days after exposure. Micropathology was performed on selected tissues from 6 rats/sex from the control and high dose groups.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (once daily on holidays and weekends)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

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

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined:
Home cage observations: 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, tail pinch, pupil size at normal lighting, pupil response, righting reflex, grip strength [Chatillon, Model DFIS-10 (forelimb) and DFI-10 (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.
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Site of testing: Test room was a separate standard animal room.
- Time schedule for examinations: 4 occasions (one week prior to treatment, approximately 30 minutes after administration of the dose, and again seven and 14 days following treatment)
- Environmental conditions: The test room 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.
- Scoring criteria (if any): Scoring criteria and explicitly-defined scales were used to rank the severity of observations that do not readily lend themselves to quantitation. 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 min

LOCOMOTOR ACTIVITY: Yes
- Replicates used: 4 (1 week prior to treatment, approximately 30 minutes after administration of the dose, and 7 and 14 days following treatment)
- Type of equipment used: Locomotor activity was monitored in figure-eight mazes, each consisting of a series of inter-connected alleys, converging on a central arena and covered by transparent plastic. 8 infrared emitter / detector pairs (3 in each of the figure-eight alleys and 1 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.
- Length of session, number and length of subsessions: 60 min (6 x 10 min)
- Total activity: Yes
- Ambulatory activity: Yes
- Large movements: No
- Small movements: No
- Noise level: Broad-spectrum background noise (approximately 74dB (A)) was provided throughout the locomotor activity test to minimize acoustical variations during testing.
- Other: The uniformity of light intensity (100±70 lux) over each of the mazes was verified daily.

Sacrifice and (histo)pathology:

- Time point of sacrifice: Day 14
- Number of animals sacrificed: all
- Parameters measured: examination of all organs, body cavities, cut surfaces, external orifices and surfaces
- Brain weight: Yes
- Length and width of brain: No
- Procedures for perfusion: animals were deeply anesthetized using an intraperitoneal dose (50 mg/kg bw) 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 males and 6 females/group were selected for perfusion and collection of tissues, with replacement, as necessary, if the perfusion was considered inadequate.
- Tissues evaluated: 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.
Detailed list of tissues evaluated: olfactory bulbs, cerebral cortex, caudate-putamen/globus pallidus, hippocampus, thalamus, midbrain (tectum, tegmentum, cerebral peduncles), cerebellum, medulla oblongata, cervical swelling, thoracic swelling, 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.
- Type of staining and embedding media: Coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar) and the cauda equina were embedded in paraffin and examined utilizing 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). Eyes, optic nerves and gastrocnemius muscle were embedded in paraffin and stained using H&E. Peripheral nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA 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: 2 - 3 µm
- Number of sections: eight coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar) and the cauda equina
- Number of animals evaluated from each sex and treatment group: 6 males and 6 females from high dose and control group; tissues from perfusion-fixed animals at the low- and mid-dose levels were not subjected to micropathology unless compound-related lesions were present in the high-dose group.

Positive control:

The study did not include concurrent positive controls, but references are made to the following studies that serve this purpose. 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) motor activity have established the sensitivity, reliability and validity of the test procedures used. Finally, 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 performed using software from either INSTEM Computer Systems or SAS. The level of significance was set at p ≤ 0.05, with the exception of Bartlett’s test 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. 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.
Continuous pathology data (e.g. brain weight) was initially evaluated using Bartlett’s test to analyze for homogeneity of variance among groups. Groups with homogeneous variances were analyzed further using an ANOVA followed by Dunnett’s test for pair-wise comparisons. In the event of non-homogeneous variances, continuous data were analyzed using the nonparametric Kruskal-Wallis test followed by a Mann-Whitney U test for pair-wise comparisons. Micropathology frequency data were screened for potential effects and then evaluated using a Chi-Square procedure, followed by a one-tailed Fischer’s Exact Test in cases of significant variation by the Chi-Square analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs were evident in males and females at all treated dose levels in both sexes. In males, clinical signs attributed to treatment included brownish-orange and/or yellow urine stains (8 low-, 8 mid- and 12 high-dose males), red nasal stain (2 mid- and 10 high-dose males), oral stain (1 low-, 1 mid- and 9 high-dose males) and red-stained fore paws (6 high-dose males).
In females, clinical signs attributed to treatment included brownish-orange and / or yellow urine stains (7 low-, 12 mid- and 12 high-dose females), red nasal stain (1 low-, 3 mid- and 8 high-dose females), oral stain (4 mid- and 9 high-dose females) and red-stained fore paws (1 mid- and 6 high-dose females). Urine stain was present on Day 14 prior to sacrifice in one low- and one high-dose female, and in three high-dose males. All remaining signs cleared prior to Day 14.
The urine stain observed in both sexes at all treated levels is most likely due to the excretion of the unmetabolized compound and does not represent a systemic or adverse effect. All of the remaining findings are either within the range of historical control (i.e., red nasal stain) and / or are attributed to grooming of the urine stain (i.e. oral stain and red stained fore paws). In all cases, a urine stain was present on the same day or before these stains occurred.

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, treatment-related

Description (incidence and severity):

FOB Findings: Treatment-related findings were evident in males at the highest dose level but not at lower dose levels. Treatment-related findings were limited to red nasal stain (one male) and red oral stain (two males) on Day 0.

Also urine stains as described under 'Clinical Signs' were noted during the FOB. As these findings are attributed to urinary excretion of the test substance rather than constituating a behavior change, details of these observations are not given in this section.

The remaining findings that were evident on occasion in one or both sexes at various dose levels were considered incidental and unrelated to treatment. There were two occasions where statistical differences from control occurred related to posture in the home cage for Day 0 low dose females and Day 14 mid-dose females. On Day 0, there were seven low-dose females standing normally and five rearing and on Day 14, there were eight mid-dose females standing normally and four rearing. Both instances compare with all twelve control females that were standing normally. Findings during pretreatment week included a minor lesion described as a “scab” in one control male. Lastly, one low-dose female had dilated pupils that did not respond to light (from a transilluminator) beginning at pretreatment week and continuing through Day 14. These findings are considered incidental and unrelated to treatment because there was no relationship with dose level or timing, relative to dose administration. In addition, standing normally and rearing are both considered common and normal postures in the home cage. The remaining tests and observations, including grip strength and landing foot splay, were not affected by treatment in males or females at any dose level.

Motor Activity Findings:
For the overall 60-min test session, there were no compound-related differences in session motor or locomotor activity in either sex at the low- and mid-dose levels (motor and locomotor activity results are summarized below). For high-dose males and females, there were compound-related decreases in motor (25% and 37%, respectively) and locomotor (34% and 50%, respectively) activity on Day 0. These differences from control for locomotor activity in high dose females were statistically significant.
For low dose males, there was one occasion on which measures of locomotor (Day 0: increased 22%) activity was slightly above the standard (± 20%) range of normal variability. By comparison, motor and locomotor activity for females was decreased and outside the standard range of normal variability at the low- (motor: 27% and locomotor: 33%) and mid-dose (locomotor: 29%) levels on Day 0. These decreases in locomotor activity at the two lowest dose levels in females were statistically significant. Motor (23 - 27%) and locomotor (24 - 30%) activity were decreased and outside of the standard range of normal variability in females at all treated levels on Day 7 and at the lowest dose level on Day 14 (locomotor only: 21%). These differences from control are not ascribed to treatment since there was no consistent relationship with dose and time or the nature of the difference from control (e.g., increased in low-dose males on Day 0 and decreased in females Day 0, 7 and 14).
Motor and locomotor activity data were subjected to further analysis at each interval on each test day. On Day 0, measures of motor (Table 6) and locomotor (Table 7) activity for high-dose females were statistically reduced, relative to controls, for intervals one through four and for locomotor activity during interval three for high-dose males. In addition, non-significant decreases in motor activity at all intervals and during the remaining intervals for locomotor activity were evident. After Day 0, there were no compound-related effects in males or females at any dose.
The remaining occurrences where interval motor or locomotor activity were statistically different from controls were not considered related to treatment.
Habituation was not affected by treatment with the test substance at any dose level.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Not applicable.

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related microscopic lesions in the high-dose males or females. Therefore, tissues from animals that received a lower dose of the test substance were not examined.

There were a few incidental findings such as focal nerve fiber degeneration in peripheral nerves of both control and treated rats. In addition, there was an optic nerve atrophy in one treated male and a focal retinal dysplasia and uveitis in one treated female, which were both considered to be incidental.

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:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Table 1: Summary of session motor activity results (% difference from control)

	MALES
Nominal dose (mg/kg bw)	Pretreatment	Day 0	Day 7	Day 14
200	+ 25	+ 13	+ 0.5	- 4
500	- 10	- 2	- 4	- 15
2000	+ 25	- 25	+ 3	+ 8
	FEMALES
200	- 15	- 27	- 27	- 17
500	+ 10	- 14	- 25	- 8
2000	- 2	- 37	- 23	- 12

% greater (+) or less (-) than concurrent control for N=12.

 Table 2: Summary of session locomotor activity results (% difference from control)

	MALES
Nominal dose (mg/kg bw)	Pretreatment	Day 0	Day 7	Day 14
200	+ 33	+ 22	+ 2	- 2
500	- 1	- 6	- 6	- 9
2000	+ 32	- 34	+ 0.3	+ 14
	FEMALES
200	- 13	- 33*	- 30	- 21
500	+ 14	- 29*	- 29	- 10
2000	+ 9	-50*	- 24	- 11

% greater (+) or less (-) than concurrent control for N=12).

Summary session locomotor activity was statistically different from control (* = p ≤ 0.05; ANOVA) .

Conclusions:

The study was performed under GLP conditions and according to OECD TG 424 (adopted 1997). Oral gavage administration of the test substance produced no clinical signs indicative of neurotoxicity. Motor activity and locomotor activity were decreased in both males and females at 2000 mg/kg bw, but there was no other effect on any neurotoxicity-related parameter at any time point. There was therefore no indication of neurotoxicity at any dose level. The NOAEL of the acute neurotoxicity study was therefore established at 504 mg/kg bw.

Reference 3

Endpoint:

neurotoxicity, other

Remarks:

developmental neurotoxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Dec 2004 - 02 Feb 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

other: review article

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

Version / remarks:

adopted in 1998

Deviations:

yes

Remarks:

as requested by the US EPA in September 1999 for developmental neurotoxicity studies (for details see 'Principles of method if other than guideline')

Principles of method if other than guideline:

In deviation to OPPTS guideline 870.6300 the following modifications were made to the study design:
(1) extend exposure to lactation / postnatal day 21 (rather than day 11) and (2) evaluate brains from 21-day-old (rather than 11-day-old) animals for morphometry and micropathology using (3) a sample size of 10 (rather than six) per sex per dietary level.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HAN CRL:WI (GLX/BRL/HAN) IGSBR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC, USA)
- Age at study initiation: at least 11 or 12 weeks (males and females, respectively) at co-housing
- Weight at study initiation: 172.2 - 198.5 g (females), males have no specified weight requirements (no data provided)
- Fasting period before study: no
- Housing: individually housed in suspended stainless steel wire-mesh cages, except during co-housing; individually in plastic cages with corn cob bedding during gestation and lactation
- Diet: Purina Mills Rodent Lab Chow 5002 in meal form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10 (minimum daily average)
- Photoperiod (hrs dark / hrs light): 12/12 (from 29 Nov through 11 Dec 2004, animals received a 11/13 hrs dark/light cycle)

IN-LIFE DATES: From: 19 Dec 2004 To: 18 Mar 2005

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

(served as a solvent in the diet preparation process and was allowed to evaporate prior to administration)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Mills Rodent Lab Chow 5002 in meal form
- Dose adjustment: Dietary concentrations were adjusted (reduced) during lactation, relative to gestation, to maintain a more constant level of exposure (mg/kg bw/day). After Day 21 of postnatal development, untreated feed was provided for consumption to all groups. A given batch of feed was available for ad libitum consumption for a period of one (GD0 - LD21) or two (post-weaning) weeks prior to changing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the ration was measured by LC-MS/MS. The stability (at room temperature and freezer conditions) and homogeneity of the test substance in the feed were verified at dietary levels of 10 and 5000 ppm.

RESULTS
Homogeneity Analysis: Homogeneity of the test substance in the ration was accepted for concentrations that bracketed those that were used on this study. These concentrations of 10 and 5000 ppm had percent relative standard deviations (% RSD) of 2.71% and 1.52%, respectively.
Stability Analysis: The stability of the test substance in the ration was established at room temperature at dietary concentrations of 10 and 5000 ppm, with no appreciable decrease in concentration with seven days of storage. The test substance was stable at freezer conditions for 77 days, with no appreciable decrease in concentration at 10 and 5000 ppm.
Concentration Analysis: For gestation, the nominal 45, 450 and 4500 ppm dietary levels averaged 100%, 101% and 97% of the nominal concentrations, respectively. Based on these results, the average dietary levels during gestation for this study were 44.9, 455 and 4375 ppm, respectively.
For lactation, dietary levels were adjusted to achieve a more consistent dosage (mg/kg bw/day) throughout the period of exposure, since food consumption increases during this time period. Nominal and analytically confirmed concentrations during the three weeks of lactation are detailed in Table 1.

Duration of treatment / exposure:

from gestation Day 6 through Day 21 of lactation / postnatal development

Frequency of treatment:

daily, 7 days a week

Dose / conc.:

45 ppm (nominal)

Remarks:

equivalent to a dose of 3.8 mg/kg bw/day

Dose / conc.:

450 ppm (nominal)

Remarks:

equivalent to a dose of 37.1 mg/kg bw/day

Dose / conc.:

4 500 ppm (nominal)

Remarks:

equivalent to a dose of 354 mg/kg bw/day

No. of animals per sex per dose:

30 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The rationale for dose selection is based on the results of one- and two-generation (M-263320-01-2) reproduction toxicology studies in Wistar rats. In the two-generation reproduction study, maternal toxicity was evident at the two highest dietary levels (300 and 3000 ppm). Findings attributed to treatment were generally in parameters not typically performed in a developmental neurotoxicity study. The findings in P-generation dams included reduced body weight during pre-mating at the highest dietary level and reduced body weight gain during lactation at 300 and 3000 ppm. In addition, there was a significant increase in absolute and relative adrenal and relative kidney weight at the highest dietary level. Based on those results, the dietary levels selected for the present study were 0, 45, 450 and 4500 ppm, with adjustments during lactation to maintain a more constant dosage throughout exposure. The 4500 ppm dietary level was selected as a maximum dose the animals will tolerate without excessive toxicity. The 450 ppm dietary level was selected as an intermediate dose that may produce effects that can be compared to the reproduction study and to assist in establishing compound-related effects. Finally, the 45 ppm dietary level was selected to establish an overall NOAEL in the offspring, with minimal or no effects evident in the dam.

Observations and clinical examinations performed and frequency:

PARENTAL ANIMALS

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Following acclimation and continuing until animals were removed from the study, P-generation males and females were observed (cage-side) for clinical signs at least once daily. These observations were sufficient to characterize mortality, moribundity, behavioral changes, and overt toxicity by viewing the animal in its cage. At the discretion of the observer, the animal was removed from the cage for a more detailed examination.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily from the initiation of exposure (gestation Day (GD) 6) through lactation Day (LD) 21

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly during gestation and lactation (GD 6-13, 13-20 and LD 0-7, 7-14 and 14-21), in addition, dams were weighed on LD 4

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

DELIVERY AND CULLING: Each dam was evaluated daily for evidence of delivery from GD 20 to the completion of delivery, which was designated LD 0 for the dam and postnatal Day 0 (PND 0) for the pups. Litter size (the number of pups delivered) and pup “status” at birth were recorded for each litter. If a dam delivered fewer than three pups per sex or if the litter size decreased to fewer than seven pups by PND 4, the dam and litter were sacrificed without necropsy examination. For litters that met the minimum size requirements, the size of each litter was adjusted on PND 4 to yield, as closely as possible, four males and four females. Adjustments of litters were made by random selection of the pups using SAS applications. If the number of male or female pups was less than four, a partial adjustment was made (e.g., three females and five males). If there were more than 23 acceptable litters for any dietary level, the surplus litters were sacrificed on PND 4 after weighing without routine necropsy, with preference given to retaining litters with a full complement of four males and four females. Culled dams and pups were sacrificed by carbon dioxide asphyxiation and decapitation, respectively. Dams with insufficient litters were also sacrificed by carbon dioxide asphyxiation.

OFFSPRING

LITTER OBSERVATIONS: The day of completion of parturition was designated as LD (PND) 0. As soon as possible following parturition, pups were examined for ano-genital distance to establish their gender, and then were tattooed and weighed. Live pups were counted and sexed individually for each litter on postnatal days 0, 4, 11, 17, and 21.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (a.m.) before weaning and once weekly thereafter

BODY WEIGHTS: Yes
- Time schedule for examinations: on PND 0, 4, 11, 17, and 21; once weekly after weaning as well as when vaginal patency or balanopreputial separation was first evident

DEVELOPMENTAL LANDMARKS:
Beginning on PND 38, male offspring were examined daily for balanopreputial separation. Beginning on PND 29, female offspring were examined daily for vaginal patency. All pups were also tested for the presence of pupil constriction on PND 21.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: approximately at 50 - 60 days of age
- Dose groups that were examined: all (a minimum of 10/sex/dietary level; representing at least 20 litters per level that were selected for perfusion at study termination)

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
MATERNAL ANIMALS:
- Parameters examined: lacrimation, salivation, piloerection, exophthalmia, urination, defecation, pupillary function, palpebral closure, convulsions, tremor, abnormal movements, unusual behaviors, posture and gait abnormalities
- Same technicians used throughout testing: No
- Technicians were blind to treatment status of animals: No data
- Time schedule for examinations: GD 13 and 20, LD 11 and 21
- Environmental conditions: under standard animal room conditions (temperature, relative humidity, etc.)
- Duration of observation period for open field observations: 1 min

OFFSPRING:
- Time schedule for examinations: on PND 4, 11, 21, 35 (+/-1 day), 45 (+/-1 day), and 60 (+/-2 days) (for detailed timing of neurobehavioral examinations see table 1)

Additional information for developmental neurotoxicity study:
- Number of offspring/sex/group examined on PND outside the home cage: approximately 16 offspring/sex/group (minimum one male or one female from each litter)
- Description of procedures used for each age at which offspring were examined: neonates (i.e. PND 4 and 11) were not evaluated in the open field
- Same offspring evaluated at each time point: yes
- Same parameters assessed in the maternal FOB examined for offspring: yes

LOCOMOTOR ACTIVITY: Yes, offspring only
- Replicates used: 4
- Type of equipment used: Locomotor activity was monitored in figure-eight mazes, 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.
- Length of session, number and length of subsessions: 60 (6 x 10 min)
- Parameters measured:
- Total activity: Yes
- Ambulatory activity: Yes
- Large movements: No
- Small movements: No
- Noise level reduction: Broad-spectrum background noise [74 +/- 2 dB(A)] was provided throughout the test to minimize acoustical variations during testing
- Number and age of offspring/sex/group examined and days of evaluation: 16 rats/sex/dose (minimum 10 offspring/sex/dose) on PND 13, 17, 21, and 60 (+/-2 days)
- Same offspring evaluated at each preweaning time point: yes

Additional testing required for developmental neurotoxicity studies:

AUDITORY STARTLE REFLEX HABITUATION: Yes
- Number of animals: 16 offspring per sex and dose
- Days of testing: PND 22 and 60 (+/- 2 days)
- Same offspring evaluated at each preweaning time point: yes
- Exact age: PND 22 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 Instruments, Allentown, PA, USA) 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 housed 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 [approximately 118 dB(lin)]. Each enclosure also housed four load cell/force transducer assemblies that were designed to measure the startle response.
- Environmental conditions: in ventilated cages, lined with sound-attenuating and vibration-absorbing material
- Number of trials performed: 50
- Length (msec) and intensity (dB) of sound: 50 msec and 118 dB
- Length of interval between trials: 10 sec

LEARNING AND MEMORY TESTING: Yes

PASSIVE AVOIDANCE:
(1) Overall testing design
- Number of animals: 16 offspring per sex and dose (minimum 10 offspring/sex/dose)
- Days of testing: PND 22 and 29
(2) Equipment used
- Type of equipment (including manufacturer, if available): Testing was conducted using equipment and computer programs from Coulbourn Instruments (Allentown, PA, USA). 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 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
- Number of trials per day: 15
- Number of days of testing: 2
- Stimulus parameters: 0.5 mA for 0.5 sec
- Use of cut-off times or error correction procedures: 180 sec
- Learning criteria: 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).

WATER MAZE:
(1) Overall testing design
- Number of animals: 16 offspring per sex and dose (minimum 10 offspring/sex/dose)
- Days of testing: PND 60 and 67
(2) Equipment used
- Type of equipment: 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
- Environmental conditions: water temperature: 21 - 23°C
(3) Testing and training procedures
- Number of trials per day: 1 learning trials, maximum 15 relearning trials
- Number of days of testing: 2
- Inter-trial intervals: 15 +/- 5 sec
- Use of cut-off times or error correction procedures: yes, 60 sec
- Definition of errors: incorrect turns in the maze
- Learning criteria: five consecutive errorless trials
(4) Control procedures
- Swim speed in straight alley: not examined
- Swimming angle development: not examined
(5) Performance measures
- Number of errors or trials to criterion: yes
- Time or latency to reach goal: yes
- Performance on "probe trials": no

Sacrifice and (histo)pathology:

MATERNAL ANIMALS:
- Time point of sacrifice: LD 21
- Number of animals sacrificed: all
- Parameters measured: The 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.

OFFSPRING:
- Time point of sacrifice: Offspring selected for brain weight or neuropathological evaluation were sacrificed on PND 21 or 75 (+/-5 days).
- Number of animals sacrificed: 10/sex/group per time point (selected for neuropathological examination), in addtition randomly-selected animals from that were used to measure fresh brain weight underwent a necropsy examination (10/sex/group per time point).
- Parameters measured: 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.
- Brain weight: Weight of fresh brain was recorded from 10 animals/sex/group/time point and of perfused brain from 10 animals/sex/group/time point.
- Length and width of brain: fixed brains: anterior-to-posterior (AP) length of the cerebrum (exclusive of the olfactory bulbs); and anterior-to-posterior (AP) length of the cerebellum
- Procedures for perfusion: Animals were 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.
- Number of animals perfused: 10/sex/group/time point
- Tissues evaluated: PD 21: brain; PD 75: brain and spinal cord, both eyes (with optic nerves) and (bilateral) peripheral nerves (sciatic, tibial and sural), gasserian ganglion, gastrocnemius muscle, both forelimbs.
Detailed list of tissues evaluated: forebrain, center of cerebrum, midbrain, medulla oblongata, cervical swelling, thoracic swelling, lumbar swelling, gasserian ganglion, optic nerve, eyes, cauda equina, 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, gastrocnemius muscle
- Type of staining and embedding media: Coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar) and the cauda equina were embedded in paraffin, sectioned at approximately 5 µm and examined utilizing 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). Eyes, optic nerves and gastrocnemius muscle were embedded in paraffin and stained using H&E. Peripheral nerve tissues (sciatic, tibial and sural nerves) were embedded in GMA cut in longitudinal sections. 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. Brain sections reserved for morphometric measurements were stained using luxol fast blue / cresyl violet;
- Embedding media: paraffin and glycol methacrylate
- Number of sections: eight coronal sections of the brain and sections from three levels of the spinal cord (cervical, thoracic, lumbar) and the cauda equina
- Number of animals evaluated from each sex and treatment group: 6 males and 6 females from high dose and control group; tissues from perfusion-fixed animals at the low- and mid-dose levels were not subjected to micropathology unless compound-related lesions were present in the high-dose group
Quantitative analysis: Selected brain regions underwent quantitative analysis: frontal cortex thickness, parietal cortex thickness, caudate putamen horizontal width, hippocampal gyrus thickness, cerebellum height

Positive control:

The study did not include positive controls, but references were made to previous studies to serve that purpose.

Statistics:

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. 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).
FOB: continuous data were analyzed using an ANOVA, with post-hoc comparisons using Dunnett’s test. Categorical data were analyzed using (CATMOD procedures, with post-hoc Dunnett’s test and an Analysis of Contrasts, respectively. Motor and locomotor activity: ANOVA procedures. For days on which there was a significant treatment effect, a post-hoc Dunnett’s test was used. Auditory startle response amplitude data were first analyzed using an ANOVA procedure. If there was a significant group effect, a post-hoc Dunnett’s test was used. The response amplitude data for each block of ten trials (five blocks/test session) were subjected to a Repeated-Measures ANOVA, using test block as the repeated measure. If there was a significant group by block interaction, the values for each block were subjected to analysis using an ad-hoc Dunnett’s test. Passive avoidance data: latency data were analyzed using a Wilcoxon Test for time to failure (i.e., time to cross). The number of trials-to-criterion was analyzed using Kruskal-Wallis and Wilcoxon tests for the acquisition phase and Fisher’s Exact Test for retention. Water maze: results were analyzed using parametric and non-parametric tests. Latency data were analyzed by a univariate ANOVA, with post-hoc analysis using Dunnett's test. The number of trials-to-criterion and the number of errors were analyzed using Kruskal-Wallis and Wilcoxon tests for the acquisition phase and Fisher’s Exact Test for retention.
Pathology data were screened for potential effects and then evaluated using different statistical approaches.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Maternal animals: General ocular opacities were noted during lactation in 5 mid- and 14 high-dose females.

During lactation there were no compound-related signs in male or female offspring at any dietary level. Treatment-related findings post weaning were limited to general ocular opacities in six high dose males and two mid- and one high-dose females.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortalities occurred in parental animals.

Litter parameters and pup viability were not affected by treatment at any dietary level. The number of offspring (males and females combined) found dead, moribund, or missing after culling litters on PND 4 was 0, 1, 0 and 2 for the control, low-, mid- and high-dose levels, respectively. Thus, there was no correspondence with dietary level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal animals: no effects on body weights were observed at any dietary level

Offspring:
Lactation: There was no difference in birth weight at any dietary level. The first indication of a difference in weight gain was evident on PND 4, with a statistically lower body weight for high-dose males (post-culling group) and a similar trend developing in high-dose females and, to a lesser extent, in the mid-dose males and females. Beginning on PND 11 and continuing through PND 21, body weight and weight gain (males and females combined) were statistically reduced compared to controls at the two highest dietary levels. Differences in body weight at the mid-dose averaged 7% less than control and 8 - 11% less than control at the highest dietary level. Body weight gain was reduced 8% and 12% in mid- and high-dose animals, respectively.

Post weaning: The reduced body weight that developed during lactation persisted in mid- and high-dose males until study termination and in females for the first two (high-dose) or three (mid-dose) weeks post-weaning. These differences from control were statistically significant in mid- and highdose males for all post-weaning weeks of the study (average 6 - 9% and 7 - 13%, respectively) and in mid- and high-dose females for post-weaning weeks 1 through 3 (average 4 - 8%, mid-dose) or weeks 1 and 2 (average 8 - 11%, high-dose).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Gestation: For high-dose animals, food spillage was evident during the first and second weeks of treatment, so the apparent increases (147% and 17%, respectively) in food consumption (an average 47.6 and 23.0 g/day, respectively versus 19.3 and 19.6 g/day, respectively for control animals) are attributed to wastage and the values are not an accurate measure of food consumption. After the first week, grates were placed in the feeders of high-dose animals to reduce food spillage from the animals wasting unpalatable food; thereafter, food consumption was more comparable to that of controls at all dietary levels (for details see table 4).

Lactation: Food consumption was significantly lower in mid- (12 - 19% on days 0 through 14) and high-dose (9 - 20% on days 0 through 21) animals. This modest decrease in food consumption at the two highest dietary levels is attributed to palatability, since there was no corresponding effect on body weight (for details see table 4).

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:

effects observed, treatment-related

Description (incidence and severity):

The incidence of retinal degeneration was significantly increased in both sexes at the highest dietary level and the same trend was evident in mid-dose females (for details see Table 7 "any other information on results incl. tables"). These findings suggest that the incidence of retinal degeneration in the 450 ppm (females only) and 4500 ppm (males and females) dietary groups is a compound-related effect. The occurrence of retinal degeneration in one low-dose male and female each is not considered a compound related effect because a single occurrence is within the range of historical control for this laboratory.

The remaining findings are considered to be incidental and unrelated to treatment, based on lack of dose response, consistency by gender and/or because the incidence is within the range of historical control.

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, treatment-related

Description (incidence and severity):

Maternal animals and offspring: no behavioral changes were noted during FOB testing

Offspring:
- Motor and locomotor activity: There were no compound-related effects at any dietary level. Moreover, there were no statistical differences from control at any dose level on any test occasion.
- Auditory startle habituation: Startle amplitude, latency and habituation were not affected by treatment at any dietary level, on any test occasion. Furthermore, there were no statistical differences from control at any dietary level on either test occasion.
- Learning and memory
- Passive Avoidance: For acquisition, there was no evidence of a compound-related effect in males or females at any dietary level. However, for retention there were two statistical differences from control in high dose males, and a similar non-statistical trend in high dose females, that are considered to represent compound-related effects. These differences from control consisted of a shorter latency to cross for trial 1 and a greater number of trials to criterion (for details see Table 6 "any other information on results incl. tables"). Although these results were statistically significant in males only, the occurrence at the highest dose, with a similar trend in females, supports the conclusion that these represent compound-related effects. There were no compound-related effects in males or females at lower dietary levels.
- Water maze: There were no compound-related effects in males or females at any dietary level.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Body Weight:
Day 21 - Terminal body weight for male and female pups was significantly decreased, relative to control, at the highest dietary level, but not at lower dietary levels in either sex.
Terminal - Terminal body weight for perfused mid-dose males was significantly decreased. In addition, terminal body weight for perfused and non-perfused males at the highest dietary level was non-statistically decreased. Terminal body weight for perfused and non-perfused females was not affected by treatment at any dietary level.

Brain Weight:
Day 21 - Absolute fixed brain weight was significantly decreased in high-dose males and females and in mid-dose females. Relative brain weight was not affected by treatment in either sex at any dietary level, reflecting that decreased absolute brain weight was associated with reduced body weight.
Terminal - Absolute fixed brain weight for high-dose perfused females and absolute brain weight for high-dose non-perfused females was significantly decreased, compared to controls.
Relative brain weight (perfused and non-perfused) was not affected by treatment at any dietary level in either sex, reflecting that decreased absolute brain weight was associated with reduced body weight.

Gross Necropsy Brain Measurements:
Day 21 - For perfused Day 21 pups, the cerebellum length was significantly decreased in high-dose males and females. Cerebrum length was also significantly decreased in mid- and high-dose females, but not in males at any dietary level.
Terminal - For perfused terminal adults, the cerebellum length was significantly decreased in high-dose males and non-statistically decreased in high-dose females. Cerebrum length was not affected by treatment at any dietary level in either sex.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Offspring: There were no compound-related necropsy findings in animals that were either found dead or sacrificed on PND 21 or at study termination (PND 75).

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Micropathology Brain Measurements
Day 21 - There was a compound-related decrease in cerebellum height for mid- and high-dose males and females that were associated with decreased body weight (8 - 15%) and absolute brain weight (8 - 11%). More specifically, there was a statistical decrease in cerebellum height (8 - 10%) in high-dose males and females and in mid-dose males (7%). For the mid-dose females, the cerebellum height was slightly (5%) less than control. Since there were statistical differences from control at the mid-dose, the cerebellum was measured at the lowest dietary level. There were no findings related to treatment in males or females at the low-dose.
Terminal - There was a statistically-significant decrease in the cerebellum height at the highest dietary level in both sexes (6% less than control), but not at the mid-dietary level in either sex. These differences from control are attributed to incomplete recovery from the differences that were evident on PND 21. There were no other findings attributed to treatment in males or females at any dietary level (for details see Table 8 "any other information on results incl. tables").

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Brain Tissue:
Day 21 - There were no compound-related microscopic findings in brain tissues from perfused PND 21 4500 ppm pups.
Terminal - There were no compound-related microscopic findings in brain tissues from perfused terminal 4500 ppm pups.

Additional Non-Brain Terminal Animal Tissues:
In terminal animals, minimal nerve fibre degeneration, generally limited to an individual fibre, occurred in an occasional control and 4500 ppm male and/or female rat sciatic nerve, sural nerves, tibial nerve and spinal nerve roots. There was no indication of treatment effect in the frequency or distribution of these changes, which are commonly recognized as a background occurrence. Cataract, synechia, corneal mineralization, dysplasia, inflammation and retinal degeneration occurred in an occasional control and 4500 ppm male and/or female rat. The dysplasia noted in 4500 ppm males and in control and 4500 ppm females were within historical control values for developmental neurotoxicity studies (see Attachment 1 for historical control data). The significant retinal degeneration noted ophthalmologically in both the 4500 ppm male and female rats was only confirmed microscopically in one 4500 ppm male. Nevertheless, this is considered to represent a compound-related effect, since there was a statistically-significant number of high-dose males and females with retinal degeneration noted ophthalmologically.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The onset of balanopreputial separation for treated mid- and high-dose males was delayed (statistically significant at the high-dose) compared to controls. The average age of onset was 44.1 days for controls, compared to 43.6, 46.0 and 46.7 days for 45, 450 and 4500 ppm dietary groups, respectively (for details see Table 5 "any other information on results incl. tables").
The onset of vaginal patency was not affected by treatment at any dietary level. The average age of onset was 32.8 days for controls, compared to 33.0, 34.7 and 33.7 days for 45, 450 and 4500 ppm dietary groups, respectively.
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.

Details on results:

Corneal opacities
Corneal opacities, occasionally accompanied by neovascularization and their histopathological correlates (keratitis, reactive epithelial hyperplasia, and vascularization) are considered a rat-specific phenomenon. Corneal changes were not seen in other species chronically treated with the test substance (i.e. mice and dogs). The test substance is an inhibitor of the HPPDase enzyme and induces increased plasma tyrosine levels. This effect is more pronounced in rats than in mice and dogs. Experimentally induced hypertyrosinemia has been shown to induce snow flake-like corneal lesions in rats but not in mice (M-210983-01-2). In mice and humans, even under conditions of strong HPPD inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99).

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal animals

Effect level:

450 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects were noted at 450 ppm.

Remarks on result:

other: equivalent to a dose of 37.1 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

Maternal animals

Effect level:

4 500 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased fertility index at 4500 ppm

Remarks on result:

other: equivalent to a dose of 354 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

Offspring

Effect level:

450 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: Developmental landmarks.

Remarks on result:

other: equivalent to a dose of 37.1 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

Offspring

Effect level:

45 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted at 45 ppm.

Remarks on result:

other: equivalent to a dose of 3.8 mg/kg bw/day

Critical effects observed:

yes

Lowest effective dose / conc.:

450 ppm

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

450 ppm

System:

eye

Organ:

cornea

retina

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 2: Nominal dietary levels and analytical results during lactation

Lactation Week	1			2			3
Dose group	low	mid	high	low	mid	high	low	mid	high
Nominal concentration (ppm)	24	237	2368	20	196	1957	16	161	1607
Mean concentration (ppm)#	25.5	244	2446	20.8	191	1904	16.4	158	1586
% Nominal	106	103	103	104	97	97	102	98	99

# Analytically confirmed

Table 3: Mean maternal test substance intake (mg/kg bw/day)

Period	Dose level (ppm)
	45	450	4500
	Gestation
GD 6 - 13	3.7 ± 0.09	42.9 ± 2.38	890.9 ± 103.7^a
GD 13 - 20	3.5 ± 0.06	36.9 ± 0.64)	402.9±14.32
	Lactation ^b
LD 0 - 7	3.8 ± 0.17	34.7 ± 1.22	343.2 ± 12.69
LD 7 - 14	4.2 ± 0.08	36.0 ± 0.54	360.9 ± 6.16
LD 14 - 21	3.6 ± 0.13	34.8 ± 0.66	309.7 ± 4.19

^a Associated with observed food spillage and considered an unreliable measure of test substance intake. This value was excluded from the mean average daily intake.

^b Dietary concentrations were reduced during weeks 1-3 of lactation (by factors of 1.9, 2.3 and 2.8, respectively), based on estimated increases in feed consumption (g consumed/kg body wt./day) during lactation).

Table 4: Mean (± SD) maternal body weight and food consumption

Period	Dose level (ppm)
	0	45	450	4500
	Gestation
Mean body weight gain (g) GD 0 - 20	102.3 ± 2.79	109.7 ± 2.42	97.8 ± 2.65	99.9 ± 2.36
Mean food consumption (g/animal/day) GD 6 - 13	19.3 ± 0.59	19.1 ± 0.49	21.4 ± 1.29	47.6** ± 5.70
Mean food consumption (g/animal/day) GD 13 - 20	19.6 ± 0.55	19.8 ± 0.43	19.9 ± 0.41	23.0** ± 0.90
	Lactation
Mean body weight (g) LD 0	235.6 ± 3.21	242.0 ± 2.83	233.8 ± 2.07	237.1 ± 3.31
Mean body weight (g) LD 21	269.0 ± 2.84	273.6 ± 3.11	267.7 ± 3.88	272.1 ± 3.48
Mean food consumption (g/animal/day) LD 0 - 7	41.7 ± 2.97	36.3 ± 1.51	33.6* ± 1.17	33.5* ± 1.06
Mean food consumption (g/animal/day) LD 7 - 14	56.0 ± 1.66	54.9 ± 1.06	49.5** ± 0.96	50.8* ± 1.05
Mean food consumption (g/animal/day) LD 14 - 21	66.7 ± 3.00	64.3 ± 2.13	61.4 ± 1.33	55.9** ± 1.07

* p < 0.05    ** p < 0.01

Table 5: Mean (± SD) age of sexual maturation (days)

Parameter	Dose level (ppm)
	0	45	450	4500
Preputial separation	44.1 ± 0.52	43.6 ± 0.31	46.0 ± 0.58	46.7** ± 0.63
Vaginal opening	32.8 ± 0.83	33.0 ± 0.65	34.7 ± 0.83	33.7 ± 0.30

** p < 0.01

Table 6: Passive avoidance performance at PND 22 offspring (mean ± S.D.)

Session/Parameter		Dose level (ppm)
		0	45	450	4500
Males
Session 1 (learning phase)	No. of animals tested	16	16	16	16
	Trials to criterion	2.9 ± 0.3	3.0 ± 0.0	3.5* ± 0.9	3.4* ± 0.6
	Latency trial 1 (sec)	49.0±58.7	39.6±35.8	31.2±22.6	27.5±22.9
	Latency trial 2 (sec)	180.0 ± 0.0	180.0 ± 0.0	162.5 ± 42.2	151.0* ± 56.9
	Failed to meet criterion	0 (0%)	0 (0%)	0 (0%)	0 (0%)
	Failed to cross during learning phase	2 (13%)	0 (0%)	0 (0%)	0 (0%)
Session 2 (retention phase)	No. of animals tested	14	16	16	16
	Trials to criterion	2.2 ± 0.6	2.0 ± 0.0	2.4 ± 0.8	2.8* ± 0.7
	Latency trial 1 (sec)	169.4 ± 39.6	180.0 ± 0.0	175.4 ± 18.5	137.7* ± 53.4
	Latency trial 2 (sec)	177.0 ± 11.3	180.0 ± 0.0	174.2 ± 18.9	169.7 ± 28.5
Females
Session 1 (learning phase)	No. of animals tested	16	16	16	16
	Trials to criterion	3.2 ± 0.5 3	3.5 ± 0.9	3.3 ± 0.8	3.1 ± 0.6
	Latency trial 1 (sec)	24.8 ± 25.2	20.8 ± 11.9	36.0 ± 42.6	41.8 ± 47.8
	Latency trial 2 (sec)	179.2 ± 3.1	171.8 ± 18.4	179.5 ± 2.1	180.0 ± 0.0
	Failed to meet criterion	0 (0%)	0 (0%)	0 (0%)	0 (0%)
	Failed to cross during learning phase	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Session 2 (retention phase)	No. of animals tested	16	16	16	16
	Trials to criterion	2.4 ± 0.6	2.7 ± 1.0	2.6 ± 0.8	2.9 ± 0.9
	Latency trial 1 (sec)	153.2 ± 48.4	162.3 ± 44.5	139.5 ± 66.9	125.1 ± 57.9
	Latency trial 2 (sec)	177.9 ± 8.2	166.9 ± 39.7	170.2 ± 24.8	169.4 ± 31.4

Trials to Criterion = Mean # Trials per Group ± SD

Latency to Trial 1 = Mean Session 1 duration (seconds) per Group ± SD

Latency to Trial 2 = Mean Session 2 duration (seconds) per Group ± SD

Failed to Meet Criterion = Number of Animals that received the shock but did not demonstrate acquisition

Failed to Cross = Number of Animals that never received the shock

* p < 0.05

Table 7: Ophthalmoscopic findings in offspring

Nominal dose (ppm)	0	45	450	4500
Findings	MALES
Synechia, anterior	1	0	0	1
Opacity, corneal	2	2	3	2
Coloboma	0	0	0	1
Degeneration, retinal	0	1	0	4*
Myosis	1	0	0	0
Cataract	0	0	0	2
	FEMALES
Synechia, anterior	0	0	0	0
Opacity, corneal	2	0	0	1
Coloboma	0	0	0	0
Degeneration, retinal	0	1	3	4*
Myosis	0	0	0	1
Cataract	0	0	1	0

* p < 0.05

Table 8: Histopathology findings (selected brain measurements, mm)

	Dose level (ppm)
	Males				Females
Parameter	0	45	450	4500	0	45	450	4500
PND 21
Ant/post cerebellum (gross measurement)	7.20 ± 0.32	7.03 ± 0.34	7.12 ± 0.30	6.80* ± 0.19	7.13 ± 0.56	7.06 ± 0.20	6.83 ± 0.30	6.65 ± 0.31
Parietal cortex (microscopic measurement)	2.008 ± 0.018	1.918 ± 0.004	1.901*± 0.005	1.922 ± 0.013	1.823 ± 0.019	--	1.763 ± 0.008	1.749 ± 0.011
Hippocampal gyrus (microscopic measurement)	1.723 ± 0.007	--	1.673 ± 0.003	1.541* ± 0.025	1.737 ± 0.008	--	1.696 ± 0.003	1.575* ± 0.005
Cerebellum (microscopic measurement)	4.367 ± 0.077	4.165 ± 0.046	4.057* ± 0.019	4.005* ± 0.033	4.292 ± 0.048	4.164 ± 0.072	4.086 ± 0.063	3.869* ± 0.022
PND 75
Ant/post cerebellum (gross measurement)	7.56 ± 0.38	7.69 ± 0.17	7.41 ± 0.31	7.21* ± 0.25	7.57 ± 0.31	7.52 ± 0.33	7.47 ± 0.41	7.25 ± 0.38
Frontal cortex (microscopic measurement)	1.760 ± 0.009	--	1.656* ± 0.010	1.713 ± 0.004	1.761 ± 0.004	--	1.662* ± 0.003	1.792 ± 0.005
Cerebellum (microscopic measurement)	3.831 ± 0.047	--	4.380* ± 0.052	3.618* ± 0.052	3.978 ± 0.033	--	4.488* ± 0.093	3.756* ± 0.057

* p < 0.05

-- not evaluated

Conclusions:

The study was performed under GLP conditions and according to EPA OPPTS 870.6300 (adopted 1998). The relevant maternal findings observed in this study were limited to decreased fertility index at 4500 ppm. The NOAEL for maternal animals administered the test substance by dietary incorporation was therefore 450 ppm (37.1 mg/kg bw/day). The treatment-related findings in the offspring at 450 and 4500 ppm included decreased body weight both prior to and after weaning, delay in preputial separation, ocular opacities and retinal degeneration, decreased terminal body weights and slight decreases in absolute but not relative fixed brain weight at postnatal day 21. There were also slight decreases in postnatal day 21 animals in cerebrum and / or cerebellum length, and cerebellum thickness was decreased in both postnatal day 21 and postnatal day 75 animals at 4500 ppm. The offspring NOAEL was 45 ppm (3.8 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3.8 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

Mode of Action Analysis / Human Relevance Framework

Data on the mode of action and human relevance for HPPDase inhibitors, specifically in relation to effects seen in the eye and thyroid gland are presented in the section Additional Toxicological Information, as well as being discussed in detail within the endpoint summary for Repeated Dose Toxicity (Additional Information field).

Additional information

Reliable oral studies on the potential for the test substance to cause acute, subchronic and developmental neurotoxicity are available.

 Acute oral neurotoxicity

In this GLP-compliant study that was performed according to OECD 424, the test substance administered by oral gavage once to four groups of 12 male and 12 female Wistar rats at doses of 0, 200, 500, and 2000 mg/kg bw (M-263029-01-1). In a preliminary ADME study (not reported in this dossier), the time after oral gavage dosing of the test substance to the peak blood concentration of the test substance was found to be 30 minutes in both males and females. Thus, conduct of the FOB and motor activity studies began approximately but no less than 30 minutes after dosing, and was concluded at approximately 2.5 hours after treatment. In total, the FOB and motor activity assessments were conducted one week prior to dosing, approximately 30 minutes after dosing, and on Days 7 and 14 after dosing. The FOB included standard parameters including home cage and open field observations, reflex testing, and determination of fore-limb and hind-limb grip strength, and landing foot splay. Motor and locomotor activities were measured according to standard methods over a 60-minute period for each animal. On Day 14 after dosing, all animals were sacrificed. Half of the animals at each dose level were anesthetized and perfused via the left ventricle. The brain was weighed, and the brain, spinal cord, eyes with optic nerve, sciatic, tibial, and sural nerves, gasserian ganglion, gastrocnemius muscle, both forelimbs, and any gross lesions were removed and preserved for examination. The other half of the animals at each dose level were sacrificed by CO2 asphyxiation and were not perfused. All animals were subjected to a gross necropsy. Histopathology was conducted on the nervous tissues from the six perfused animals in the control and high dose groups only.

There were no mortalities at any dose level in this study. Treatment-related clinical signs were limited to urine stains, nasal and oral stains, and stained forepaws. These stains were seen in all treatment groups, with a general dose-related incidence, and were attributed to the excretion of the test substance in the urine leading to intense yellow/orange coloration of the urine. The staining had reversed in almost all animals by the time of sacrifice. As the staining was due to compound excretion, it was considered not to be an adverse or toxicologically significant finding. There were no effects on body weight in either males or females at any dose level. There were no effects on any parameters of the functional observation battery, including grip strength and landing foot splay, in any dose group in either males or females. Motor and locomotor activity was biologically and / or statistically significantly decreased in both males and females at 2000 mg/kg bw/day on Day 0 only. There were no other treatment-related effects on either parameter at any other dose level, or at any dose level on Days 7 and 14 after dosing. There was no effect on brain weight (the only organ weight measured) at any dose in either males or females. There were no treatment-related macroscopic findings observed at any dose in either males or females. Microscopic findings: There were no treatment-related effects observed at 2000 mg/kg bw when compared to controls, thus histopathological sections were not made at either 200 or 500 mg/kg bw.

Conclusions: The oral gavage administration of the test substance produced no clinical signs indicative of neurotoxicity. Motor activity and locomotor activity were decreased in both males and females at 2000 mg/kg bw, but there was no other effect on any neurotoxicity-related parameter at any time point. There was therefore no indication of neurotoxicity at any dose level. The NOAEL of the acute neurotoxicity study was therefore established at 504 mg/kg bw.

Subchronic neurotoxicity

In this GLP-conform study that was performed according to OECD 424 (M-262680-01-1), the test item was incorporated into rodent meal at concentrations of 0, 500, 2500, and 5000 ppm and provided to 12 male and 12 female Wistar rats per group for 90 days. The dietary concentrations prepared provided doses of 0, 32.3, 166, and 345 mg/kg bw/day for males and 0, 41.9, 206, and 416 mg/kg bw/day for females. Clinical signs were assessed twice daily on weekdays and once daily on weekends and holidays. Body weight and food consumption were measured on a weekly basis. The FOB and motor activity were assessed on five occasions, once in the week prior to the start of the feeding period, and once each during Weeks 2, 4, 8, and 13. The FOB included standard parameters including home cage and open field observations, reflex testing, and determination of fore-limb and hind-limb grip strength, and landing foot splay. Motor and locomotor activity was measured according to standard methods over a 60-minute period for each animal. Ophthalmological examinations were conducted in all animals prior to the start of the study and during Week 12. After at least 90 days dietary administration, all animals were sacrificed. Half of the animals at each dose level were anesthetized and perfused via the left ventricle. The brain was weighed, and the brain, spinal cord, eyes with optic nerve, sciatic, tibial, and sural nerves, gasserian ganglion, gastrocnemius muscle, both forelimbs, and any gross lesions were removed and preserved for examination. The other half of the animals at each dose level were sacrificed by CO2 asphyxiation and were not perfused. All animals were subjected to a gross necropsy. Histopathology was conducted on the nervous tissues from the six perfused animals in the control and high dose groups only.

There were no mortalities in either males or females during the study. Treatment-related clinical signs observed in this study were limited to urine staining in both males and females, largely occurring in a dose-related manner. The yellow to brownish-orange stains observed were considered to be due to urinary excretion of the test substance and were considered not to be indicative of toxicity. There was no treatment-related effect on food consumption in either males or females at any dose level. Food consumption was statistically significantly increased in males at 5000 ppm on Days 21-28 through Days 28-35, and on Days 70-77 through Days 77-84. However, this was considered not to be indicative of a toxic effect as there was no corresponding increase in food consumption in females nor was body weight affected in males at 5000 ppm. There was no treatment-related effect on body weight in either males or females at any dose level. Neither males nor females revealed any effects at ophthalmological examination which were attributed to treatment. There was no effect on any parameter of the FOB, including grip strength and landing foot splay, in either males or females at any point during the study. There were no treatment-related alterations in either motor or locomotor activity in either males or females at any dose. There were occasional statistically nonsignificant increases in motor or locomotor activity at various time points, but they followed no dose-related pattern, were generally limited to one sex, and were opposite to the treatment-related change observed in the acute neurotoxicity study. Therefore, these increases were considered not to be treatment-related. There was no effect on brain weight in either males or females at any dose. No gross lesions or other macroscopic observations were present at necropsy in either male or female rats which could be attributed to treatment. There were no histopathological findings in male or female rats at 5000 ppm, relative to control, which were indicative of a treatment-related effect. In the absence of any treatment-related effect at 5000 ppm, tissues were not examined at lower doses.

Conclusions: There were no indications of a neurotoxic effect of the test substance after 90 days dietary administration, nor were there effects on body weight or food consumption. The NOAEL for this study was therefore 5000 ppm, or 345 mg/kg bw/day in males and 416 mg/kg bw/day in females.

Developmental neurotoxicity

In this GLP-compliant study that was performed according to EPA OPPTS 870.6300 (M-266434-02-1), the test substance was incorporated into rodent diet at concentrations of 0, 45, 450, and 4500 ppm and administered to groups of 30 sperm-positive female Wistar rats from gestation Day 6 through lactation Day 21. Dietary concentrations were adjusted during lactation to provide for a constant dosage throughout the treatment period. These concentrations provided an average daily intake of 0, 3.8, 37.1, and 354 mg/kg bw/day. Dams were observed for clinical signs at least once daily. Body weight and food consumption were measured on a weekly basis, on gestation Days 6, 13, and 20, and lactation Days 0, 7, 14, and 21. Maternal body weight was also measured on lactation Day 4. Litters were culled on lactation Day 4 to eight pups, with four male and four female pups wherever possible. Dams were sacrificed on lactation Day 21 following weaning of their litters, without post- mortem examinations.

As soon as possible after pup birth, anogenital distance was measured and pups were tattooed. Surviving pups were counted, sexed, and weighed individually on lactation Days 0, 4, 11, 17, and 21. Offspring were monitored daily throughout lactation for clinical signs or morbidity. After weaning on Day 21, the pups were monitored twice daily for morbidity and mortality, and were weighed on a weekly basis as well as on the day that vaginal patency or balanopreputial separation were achieved. Food consumption was not measured. Pups were examined daily starting from postnatal Day 29 (females) or postnatal Day 38 (males) for developmental landmarks, and pupil constriction was tested in all pups on postnatal Day 21. All tests used at least 10 offspring per sex per dose, and with the exception of learning and memory, no animal was tested more than once in the same test.

At least 10 male and 10 female offspring per dose group were selected for ophthalmological examination at approximately 50-60 days of age. On postnatal Day 21, 10 male and 10 female offspring were sacrificed and the brains were collected whole for micropathological examination and morphometric analysis. The animals were anesthetized (50 mg/kg bw pentobarbital, intraperitoneal injection) and perfused via the left ventricle with sodium nitrite in phosphate buffer followed by in situ fixation with universal fixative in phosphate buffer. On postnatal Day 75, a further 10 male and 10 female offspring were sacrificed and perfused, and brain, spinal cord, both eyes with optic nerves, bilateral sciatic, tibial, and sural nerves, gasserian ganglion, gastrocnemius muscle, and both forelimbs were collected and fixed. Brains were weighed. In both cases, prior to sectioning, the anterior-to-posterior lengths of the cerebrum and of the cerebellum were measured with Vernier calipers. Other measurements, made after histologic sectioning, were the thickness of the frontal cortex, parietal cortex, and hippocampal gyrus, horizontal width of the caudate putamen, and height of the cerebellum. The offspring not selected for neuropathological examination were sacrificed without examination.

 Findings for parental animals: There were no mortalities in parental females during either gestation or lactation. There were no treatment-related clinical signs observed during gestation. During lactation, ocular opacities were observed in five females at 450 ppm and in 14 females at 4500 ppm. Ocular opacities, evaluated as related to treatment, were observed during the FOB in three females at 450 ppm and seven females at 4500 ppm. This increase at 4500 ppm was statistically significant compared to controls. Ocular opacities are considered a rat-specific phenomenon of HPPDase inhibitors without relevance for humans. Accordingly, this finding was not used for setting a NOAEL (for a detailed justification see endpoint summary for repeated dose toxicity). There were no other treatment-related findings at FOB evaluation. Body weight and body weight gain were not affected during gestation or lactation at any dose. Food consumption was statistically significantly increased at 4500 ppm during gestation, although this was considered to be due to wastage from palatability issues. Installation of grates to reduce wastage in Week 2 of gestational treatment markedly reduced food consumption at 4500 ppm. During lactation, food consumption was statistically significantly reduced at both 450 ppm (lactation Days 0-7 and 7-14) and 4500 ppm (all periods). These reductions were attributed to unpalatability, as there was no effect on body weight at either dose. The fertility index was statistically non-significantly decreased at 4500 ppm compared to controls, and was evaluated as treatment related. There were no other effects on reproductive parameters.

 Findings for offspring: There were no effects of treatment on litter size, viability, or other litter parameters. There were no treatment-related signs observed during lactation in either males or females. In the post-weaning phase, treatment-related findings were restricted to ocular opacities in six males and one female at 4500 ppm, and two females at 450 ppm. There was no effect on the incidence of moribund or found-dead pups. Body weight at birth was similar at all doses. On postnatal Day 4 (post-culling), body weight was statistically significantly decreased in males at 4500 ppm, with statistically non-significant decreases in body weight in females at 4500 ppm and both sexes at 450 ppm. Body weight and body weight gain were statistically significantly reduced for males and females combined at 450 and 4500 ppm through weaning at postnatal Day 21. After weaning, statistically significantly decreased body weight continued in males at 450 and 4500 ppm through the end of the study, in females at 450 ppm for the first three weeks after weaning, and in females at 4500 ppm for the first two weeks after weaning.

Difference from control body weight during lactation reached a maximum of 7% at 450 ppm and 11% at 4500 ppm. Body weight gain was also reduced, by 8% relative to controls at 450 ppm and by 12% at 4500 ppm. After weaning, body weight reduction in males showed a maximum of 9% at 450 ppm and 13% at 4500 ppm. In females, body weight was reduced by a maximum of 8% at 450 ppm or 11% at 4500 ppm. These effects on body weight were considered to be treatment-related. Preputial separation was statistically significantly delayed at 4500 ppm (46.7 days, p < 0.01) and statistically non-significantly delayed at 450 ppm (46.0 days), compared to controls (44.1 days). This increase in time to preputial separation was considered to be related to treatment as a secondary effect of reduced body weight. There was no effect on time to vaginal patency. Pupillary constriction on Day 21 in both males and females was not affected by treatment. The only treatment-related changes at FOB assessment were ocular opacities in one female at 450 ppm and one female at 4500 ppm. These changes were first noted on postnatal Days 45 and 35, respectively, and persisted in both cases through postnatal Day 60. There were no treatment-related effects observed on either motor or locomotor activity in either males or females at any dose level during either lactation or post-weaning phases. There were no treatment-related effects on auditory startle at any time during the study or at any dose level. There was no effect of treatment during the acquisition phase of the passive avoidance testing. However, during trial 1 of the retention phase, males at 4500 ppm showed a statistically significantly decreased latency to crossing than controls. Females at 4500 ppm showed a similar, although statistically non-significantly, decreased latency to crossing. This effect is considered to be related to treatment. Although there were other statistically significant differences observed during the passive avoidance testing, these were considered not related to treatment as they were generally within historical control data, not related to treatment, and / or seen in one sex only. There were no treatment-related effects on acquisition and retention in either males or females during the water maze testing. Retinal atrophy was statistically significantly increased at 4500 ppm, with four males and four females affected at this dose. Retinal atrophy was also noted in three females at 450 ppm (although not statistically significant). Retinal atrophy was considered to be a treatment-related finding at both 450 and 4500 ppm. Absolute brain weight was statistically significantly decreased for males and females at 4500 ppm and females at 450 ppm on Day 21, and for females at 4500 ppm on Day 75. Relative brain weight in these groups was no different from controls, indicating that the decreased absolute brain weight was related to the reduced body weight at these doses. At Day 21, cerebellum length was statistically significantly decreased in males and females at 4500 ppm. Cerebrum length was also statistically significantly decreased in females at 450 and 4500 ppm. At Day 75, cerebellum length was statistically significantly decreased in males at 4500 ppm, and statistically non-significantly decreased in females at 4500 ppm. There was no effect on cerebrum length in either males or females at Day 75. These decreases observed on Day 21 and 75 were considered to be related to treatment.

On Day 21, males and females at 450 and 4500 ppm showed a treatment-related, statistically significant decrease in cerebellum height. On Day 75, cerebellar height was statistically significantly decreased at 4500 ppm in males and females. On histopathological examination of the brains and other nervous tissues, the only treatment-related finding was retinal degeneration in one male at 4500 ppm.

Conclusions:

The relevant maternal findings observed in this study were limited to decreased fertility index at 4500 ppm. The NOAEL for maternal animals administered the test substance by dietary incorporation was therefore 450 ppm (37.1 mg/kg bw/day).

The treatment-related findings in the offspring at 450 and 4500 ppm included decreased body weight both prior to and after weaning, delay in preputial separation, ocular opacities, and retinal degeneration, decreased terminal body weights, and slight decreases in absolute but not relative fixed brain weight at postnatal day 21. There were also slight decreases in postnatal day 21 animals in cerebrum and / or cerebellum length, and cerebellum thickness was decreased in both postnatal day 21 and postnatal day 75 animals at 4500 ppm. The offspring NOAEL was 45 ppm (3.8 mg/kg bw/day).

Justification for classification or non-classification

The available data on neurotoxicity with the test substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and is therefore conclusive but not sufficient for classification.