thiacloprid (ISO);(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyanamide;{(2Z)-3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide

Neurotoxicity

Description of key information

Key, M-000894-03-1, EPA OPP 81-8, rat, acute:

NOAEL: 11 mg/kg bw (males), 3.1 mg/kg bw (females)

LOAEL: 20 mg/kg bw (males), 11 mg/kg bw (females)

Key, M-003815-01-1, S-EPA-FIFRA Guideline 82-5(b), rat, 90 d:

NOAEL (systemic): 50 ppm (corresponding to 2.94 mg/kg bw/day in males and 3.41 mg/kg bw/day in females)

LOAEL (systemic): 400 ppm (corresponding to 24.2 mg/kg bw/day in males and 27.9 mg/kg bw/day in females)

NOAEL (neurotoxicity): 1600 ppm (corresponding to 101 mg/kg bw/day in males and 115 mg/kg bw/day in females, highest dose tested)

Key, M-088059-01-2, EPA OPPTS 870.6300, rat, developmental:

NOAEL (maternal neurotoxicity): 500 ppm (corresponding to 40.8 mg/kg bw/day during gestation (DGs 0 to 21) and 82.8 mg/kg bw/day during lactation (DL 1 to 14); highest dose tested)

NOAEL (neurotoxicity, F1 generation): 500 ppm (corresponding to 40.8 mg/kg bw/day during gestation (DGs 0 to 21) and 82.8 mg/kg bw/day during lactation (DL 1 to 14) of dams; highest dose tested)

NOAEL (maternal systemic toxicity): 50 ppm (corresponding to 4.4 mg/kg bw/day during gestation (DGs 0 to 21) and 8.2 mg/kg bw/day during lactation (DL 1 to 14))

NOAEL (systemic and developmental toxicity, F1 generation): 50 ppm (corresponding to 4.4 mg/kg bw/day during gestation (DGs 0 to 21) and 8.2 mg/kg bw/day during lactation (DL 1 to 14) of dams)

LOAEL (maternal systemic toxicity): 300 ppm (corresponding to 25.6 mg/kg bw/day during gestation (DGs 0 to 21) and 49.4 mg/kg bw/day during lactation (DL 1 to 14))

LOAEL (systemic and developmental toxicity, F1 generation): 300 ppm (corresponding to 25.6 mg/kg bw/day during gestation (DGs 0 to 21) and 49.4 mg/kg bw/day during lactation (DL 1 to 14) of dams)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jan - 16 Feb 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-8 (Neurotoxicity Screening Battery)

Version / remarks:

adopted 1991

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SASCO, Inc., Wisconsin, USA
- Age at study initiation: 9 weeks
- Weight at study initiation: 172 - 177 g for males and 121 - 124 g for females
- Fasting period before study: overnight prior to dosing and during neurobehavioral assessment when only water was available
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 25.6
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Jan 1996 To: 06 Feb 1996

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered by gavage as a single dose in 0.5% methylcellulose and 0.4% Tween 80 in deionized water, at a dosing volume of 10 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration:
The concentration of the test substance in the vehicle was measured using liquid chromatographic (LC) analysis. Each dosing suspension was analyzed to measure the concentration. Nominal doses of 0, 20, 50 and 100 mg/kg for both sexes were determined to be 106% to 110% of the nominal dose level.

Homogeneity:
The stability of the test substance in the vehicle, following room temperature exposure, was established using samples at nominal concentrations (2.0 and 100 mg/mL) that covered the range of test concentrations (nominal 2 mg/mL to 10 mg/mL) used in the present study. Homogeneity of the test substance in the vehicle was verified, with a coefficient of variation (C.V.) of less than 6% for the nominal 2.0 and 100 mg/mL (equivalent to dose levels of 20 and 1000 mg/kg, respectively) dosing suspensions.

Stability:
The stability of the test substance in the vehicle, following room temperature exposure, was established using samples at nominal concentrations (2.0 and 100 mg/mL) that covered the range of test concentrations (nominal 2 mg/mL to 10 mg/mL) used in the present study. The stability of the test item in the dosing suspension was established, with no appreciable decrease in concentration with eight days of storage for the 100 mg/mL dose and eleven days of storage for the 2 mg/mL dose.

Duration of treatment / exposure:

not applicable since single treatment

Frequency of treatment:

single treatment

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

corresponding to 22 mg/kg bw/day (actual dose received) for males and females

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

corresponding to 53 mg/kg bw/day (actual dose received) for males and females

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

corresponding to 109 mg/kg bw/day (actual dose received) for males and females

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were selected on the basis of the results from an acute oral dose range-finding study, using analytically-confirmed doses of 27, 36, 85, 244 and 526 mg/kg bw for both sexes (five rats/sex/dose level). The NOEL for clinical signs was 27 mg/kg bw for males and 36 mg/kg bw for females. Clinical signs were observed starting at 36 mg/kg bw in males (repetitive chewing movements). This was also observed in both sexes at 85 mg/kg bw together with tremor in males and stains (oral and nasal) in one female each. Higher doses produced progressively more evidence of toxicity (tremor, decreased activity, repetitive chewing movements, cool-to-touch body, dilated pupils, clear lacrimation, and 100% mortality in both sexes). Based on these results, doses selected for the acute neurotoxicity study are 0, 20, 50 and 100 mg/kg bw for both sexes.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for detailed physical examinations for clinical signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: 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 (FOB): Yes
- Parameters checked: home cage observations (posture, piloerection, gait abnormalities, involuntary motor movements, vocalizations and activity), observations during handling (resistance, muscle tone, palpebral closure, pupil reactions, lacrimation, salivation and stains), open field observations (piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, behavior, gait abnormalities, vocalizations, arousal, rearing and excretion), reflex and physiologic observations and measurements (approach, touch, auditory and tail pinch response, righting reflex, grip strength, landing foot splay, body weight and body temperature)
- Description of procedures: FOB closely follows the battery of tests described by Moser (Moser 1989). Animals were transferred to the room where the testing took place and were allowed to acclimate with minimal disturbance for at least 30 minutes prior to testing. The conditions in the testing room were the same as for the standard animal room. Sets of eight animals (maximum) were evaluated individually using the FOB.
- Minimization of bias: Observations for all animals were performed by the same observer throughout the study, with a second person performing the measurements. Inter-observer reliability has been established in order to allow a second person to perform either the observations or measurements, ensuring the consistency of the results of each technician. Studies have been conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of these test procedures, the adequacy of training of technical personnel and to serve as a historical control.
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: one week prior to treatment, approximately 4 hours (minimum) after administration of the dose, and again seven and 14 days following treatment.
- Environmental conditions: same conditions as for the standard animal room
- Noise level: Broad-spectrum background noise (approximately 70dB(A))

LOCOMOTOR ACTIVITY: Yes
- Replicates used: All animals that were assigned to the study were tested.
- Type of equipment used: Figure-eight maze consisting of a series of interconnected alleys (approximately 10x10 cm in cross-section) converging on a central arena and covered by transparent acrylic plastic. Each maze had eight infrared emitter/ detector pairs (three in each of the figure-eight alleys and one in each of the blind alleys) to measure activity; 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: Motor and locomotor activity were examined for the 90-minute session and during each ten-minute interval.
- Parameters measured: motor activity (measured as the number of beam interruptions), locomotor activity (measured by eliminating consecutive counts for a given beam) and habituation (evaluated as a decrement in activity)

Sacrifice and (histo)pathology:

- Time point of sacrifice: 15 days post-treatment (after the final FOB and motor activity tests)
- Number of animals sacrificed: All animals were subjected to a complete gross necropsy. Starting after FOB and motor activity testing, the first six males and six females at each dose level were selected for perfusion and collection of tissues. All other surviving animals were sacrificed by CO2 asphyxiation without perfusion or collection of tissues.
- Parameters measured: examination of all organs, body cavities, cut surfaces, external orifices and surfaces
- Brain weight: yes
- Length and width of brain:
- Procedures for perfusion: Each animals was 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 in situ fixation.
- Number of animals perfused: 6 of each dose/sex
- Tissues evaluated: brain and spinal cord, both eyes (with optic nerves) and selected (bilateral) peripheral nerves (sciatic, tibial and sural), the gasserian ganglion, and gastrocnemius muscle
- Methodology of preparation of sections:

Hematoxylin and Eosin (H&E) staining:
- Type of staining: Hematoxylin and Eosin (H&E)
- Embedding media: paraffin
- Tissues: Eight coronal sections of the brain and sections from four levels of the spinal cord (cervical, thoracic, lumbar and cauda equina)
- Thickness: not reported

Luxol Fast Blue/Cresyl Violet and Sevier-Munger stains
- Type of staining: Luxol Fast Blue/Cresyl Violet and Sevier-Munger stains
- Embedding media: paraffin
- Tissues: Eight coronal sections of the brain and sections from four levels of the spinal cord (cervical, thoracic, lumbar and cauda equina)
- Thickness: not reported

Modified Lee's staining
- Type of staining: Modified Lee's
- Embedding media: glycol methacrylate (GMA)
- Tissues: Dorsal root ganglia (including dorsal and ventral root fibers) from the cervical and lumbar swellings, gasserian ganglion, eyes, optic nerves and gastrocnemius muscle
- Thickness: 2 - 3 µm

Toluidine Blue staining
- Type of staining: Toluidine Blue
- Embedding media: Epoxy resin
- Tissues: Peripheral nerve tissues (sciatic, tibial and sural nerves) and sciatic nerve
- Thickness: 1 µm

- Number of animals evaluated histopathologically from each sex and treatment group: tissues from control and high-dose animals, and any gross lesions collected at necropsy

Positive control:

Previous studies with trimethyltin and acrylamide have established the sensitivity and reliability of the used procedures.

Statistics:

With the exception of Bartlett's test, which was tested at p ≤ 0.001, the level used to establish statistical significance was p ≤ 0.05. Continuous data were analyzed using an Analysis of Variance (ANOVA), followed by a Dunnet's test if a significant F-value was determined in the ANOVA. For the FOB, continuous data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA if there was a significant interaction between dose group and test week. For weeks on which there was a significant treatment effect, Dunnet's test was applied to determine which groups, if any, were significantly different from the control group. Categorical data collected in the FOB were analyzed in a similar manner, using General Linear Modeling (GLM) and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively. Motor and locomotor activity (activity for the entire session and activity for each 10-minute interval) were analyzed using ANOVA procedures. Session activity data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA if there was a significant interaction with test occasion. For weeks on which there was a significant treatment effect, Dunnett's test was used to determine which groups, if any, were significantly different from the control group. Interval data were subjected to a two-way Repeated-Measures ANOVA, using both test interval and test occasion as the repeated measures, followed by a Repeated Measures ANOVA to determine on which weeks there was a significant treatment by interval interaction. For those weeks, the data for each interval were subjected to analysis using a one-way ANOVA to determine at which intervals there was a significant treatment effect. For those intervals, Dunnett's test was used to determine which groups, if any, were significantly different from the control group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The following clinical signs were considered to be related to the treatment:
20 mg/kg bw: no treatment-related clinical signs observed
50 mg/kg bw: dilated pupils in 1/12 females
100 mg/kg bw: tremors (all animals), decreased activity (12/12 males and 11/12 females), locomotor incoordination (ataxia 8/12 males and 8/12 females), dilated pupils (6/12 males and 11/12 females), cool-to-touch body (all animals), urine stain (6/12 males and 7/12 females), ptosis of the eyelids (5/12 males and 11/12 females), red nasal stain (8/12 females), oral stain (7/12 females), clear nasal discharge (5/12 females) and clear lacrimation (2/12 females)
Compound-related clinical signs were generally apparent only on the day of treatment (Day 0) and resolved for most signs by Day 1 following treatment. All compound-related clinical signs resolved by Day 5.
The remaining clinical signs in control and low-dose animals (perianal, red lacrimal and urine stain) are considered incidental and not related to treatment.
For details, please refer to the attached background material (attachment 1).

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

There were no deaths prior to terminal sacrifice, 15 days following treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The following body weight changes were considered to be related to the treatment:
20 and 50 mg/kg bw: no treatment-related changes in body weights were observed
100 mg/kg bw: A slight but statistically significant decrease of the mean body weights (< 10%) were observed on Day 7 for males only when compared to the control animals. By day 14, there was no difference in weight for these males relative to controls.
For details, please refer to the attached background material (attachment 2).

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 observations:
The following FOB observations were considered to be related to the treatment and occurred after treatment on Day 0:
20 mg/kg bw: eyelid ptosis in 1/12 males (open field observations), slight tremors in 3/12 males (open field observations) and impaired aerial righting response in 1/12 females
50 mg/kg bw: slight tremors in 6/12 males and 3/12 females (open field observations), eyelid ptosis in 1/12 males (open field and home cage observations, respectively), a slightly impaired righting response in 1/12 males and 1 /12 females, no approach response in 3/12 females and dilated pupils in 4/12 females
100 mg/kg bw: incoordinated gait in 9/12 males and 10/12 females, tremors in 10/12 males and 11/12 females (home cage observations) and in all animals (open field observations), decreased activity in 10/12 males and in all females, eyelid ptosis in 7/7 males and 9/10 females (home cage observations) and in 7/7 males and 10/12 females (open field observations), dilated pupils in 9/12 males and 11/12 females, impaired righting response in 5/12 males and 9/12 females, decreased body temperature in 5/12 males and 1/12 females, a general dose-related shift from a slight reaction to no reaction to various stimuli (i.e., approach, touch, auditory and tail pinch), clear lacrimation in 2/12 females, females had fewer rears and a higher incidence of sitting or lying during open field observation compared to control animals.
Even though the effects observed at the low- and/or mid-dose levels were generally not statistically significant, they are ascribed to treatment based on their absence in control animals, as well as their occurrence in relationship to dose level and day of exposure. All compound-related effects resolved at all dose levels by the next test occasion, seven days following treatment.
All other observations (repetitive chewing and red lacrimal stain) were considered incidental and not related to treatment.
For details, please refer to the attached background material (attachment 3).

Motor and Locomotor Activity:
In general, differences of less than 20% are within the range of normal variability in the performing laboratory for groups of 10-12 rats/sex/dose level and, therefore, are not considered biologically significant.
Treatment-related findings with respect to motor and locomotor activity are as follows:
20 mg/kg bw: A statistically significant reduction in locomotor activity of 63% was observed in females on Day 0. Motor activity was also reduced in females, but this decrease did not reach statistical significance.
50 and 100 mg/kg bw: Statistically significant differences in activity for females were observed when compared to the control animals. This outcome reflects reductions in motor activity of 41% and 71% and in locomotor activity of 40% and 99% for mid- and high-dose females, respectively on Day 0. Compound-related decreases in motor activity of 56% and locomotor activity of 95% were also observed on Day 0 in males of the high-dose group. Complete recovery occurred in all males and females within seven days following treatment.
For details, please refer to the attached background material (attachment 4).

Habituation:
Habituation was not affected by treatment with the test substance.

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):

There was no compound-related effect on brain weight in males or females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no compound-related gross lesions evident at terminal sacrifice in males or females at any dose level.

Neuropathological findings:

no effects observed

Description (incidence and severity):

please refer to histopathological findings

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no compound-related microscopic lesions in the high-dose males or females within the examined organs/tissues (brain and spinal cord, both eyes with optic nerves, peripheral nerves (sciatic, tibial and sural), the gasserian ganglion, and gastrocnemius muscle).

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:

< 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: adverse effects observed at the lowest dose tested

Key result

Dose descriptor:

LOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

clinical signs

Key result

Critical effects observed:

no

Conclusions:

The study was performed under GLP conditions and is in accordance with US-EPA-FIFRA 81-8 (neurotoxicity screening battery, adopted 1991). Therefore, the study is considered reliable and valid. Under the conditions of the test, an overall NOAEL was not established due to clinical signs and functional findings (FOB) at the lowest dose tested. Therefore, the LOAEL was set to 20 mg/kg bw and the NOAEL was assumed to be < 20 mg/kg bw for both sexes. Since an overall NOAEL was not established in this study, a follow-up study was conducted at lower dose levels to establish a NOAEL for the endpoints that were affected at a dose of 20 mg/kg bw.

Reference 2

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 06 Aug 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-8 (Neurotoxicity Screening Battery)

Version / remarks:

adopted 1991

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SASCO, Inc., Wisconsin, USA
- Age at study initiation: 9 weeks
- Fasting period before study: during the overnight fast prior to dosing and during neurobehavioral assessment when only water was available
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Aug 1997 To: 06 Aug 1997

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered by gavage as a single dose in 0.5% methylcellulose and 0.4% Tween 80 in deionized water, at a dosing volume of 10 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration:
The concentration of the test substance in the vehicle was measured using liquid chromatographic (LC) analysis. Each dosing suspension was analyzed to measure the concentration. Nominal doses of 2.5, and 10 mg/kg bw for both sexes were determined to be 106% to 125% of the nominal dose level.

Homogeneity:
The homogeneity of the test substance in the vehicle was verified by analysis of three samples each of the nominal 2.0 and 100 mg/mL dosing suspensions in the previous study. In addition, in the present study, homogeneity was verified for three samples of the dosing suspension of 0.25 mg/mL (equivalent to the dose level of 2.5 mg/kg bw). In total, homogeneity of the test substance in the vehicle was verified, with a coefficient of variation (C.V.) of less than 6% for the nominal 0.25, 2.0, and 100 mg/mL (equivalent to dose levels of 2.5, 20, and 100 mg/kg bw, respectively) dosing suspensions.

Stability:
The stability of the test substance in the vehicle, following room temperature exposure, has been established in the previous study using samples at nominal concentrations (2.0 and 100 mg/mL). In addition, the stability of a dosing suspension of 0.25 mg/mL was verified. Thus, the range of test concentrations (nominal 2.5 mg/mL to 10 mg/mL) used in the present study was covered. The stability of the test substance in the dosing suspension was established, with no appreciable decrease in concentration after eight days of storage for the 0.25, 2.0, or 100 mg/mL doses.

Duration of treatment / exposure:

not applicable since single treatment

Frequency of treatment:

not applicable since single treatment

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Remarks:

corresponding to 3.1 mg/kg bw/day (actual dose received) for males and females

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

corresponding to 11 mg/kg bw/day (actual dose received) for males and females

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 is based on the results of an acute oral neurotoxicity screening study with the test substance at dose levels of 0, 20, 50, and 100 mg/kg bw for males and females (12/sex/dose level). Evidence of exposure in males and females that received the low dose of 20 mg/kg bw was limited to minimal compound-related effects at the time of the FOB and, in females only, compound-related decreases in motor and locomotor activity in the figure-eight maze. Based on these results, the doses selected for the present study were 0, 2.5 and 10 mg/kg bw for males and females to establish a NOAEL.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for detailed physical examinations

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed just prior to treatment to determine individual dose volumes.

FOOD CONSUMPTION: 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 (FOB): Yes
- Parameters checked: home cage observations (posture, piloerection, gait abnormalities, involuntary motor movements, vocalizations and activity), observations during handling (resistance, muscle tone, palpebral closure, pupil reactions, lacrimation, salivation and stains), open field observations (piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, behavior, gait abnormalities, vocalizations, arousal, rearing and excretion), reflex and physiologic observations and measurements (approach, touch, auditory and tail pinch response, righting reflex, grip strength, landing foot splay, body weight and body temperature)
- Description of procedures: FOB closely follows the battery of tests described by Moser (Moser 1989). Animals were transferred to the room where the testing took place and were allowed to acclimate with minimal disturbance for at least 30 minutes prior to testing. The conditions in the testing room were the same as for the standard animal room. Sets of nine animals were evaluated individually using the FOB.
- Minimization of bias:
Observations for all animals were performed by the same observer throughout the study, with a second person performing the measurements. Inter-observer reliability has been established in order to allow a second person to perform either the observations or measurements, ensuring the consistency of the results of each technician. Studies have been conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of these test procedures, the adequacy of training of technical personnel and to serve as a historical control.
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations:
on one occasion, which was at the time of peak neurobehavioral effects, approximately 4 hours (minimum) after administration of the dose
- Environmental conditions:
same conditions as for the standard animal room
- Noise level:
Broad-spectrum background noise (approximately 70dB(A))

LOCOMOTOR ACTIVITY: Yes
- Replicates used:
Only females that were assigned to the study were tested.
- Type of equipment used: Figure-eight maze consisting of a series of interconnected alleys (approximately 10x10 cm in cross-section) converging on a central arena and covered by transparent acrylic plastic. Each maze has eight infrared emitter/ detector pairs (three in each of the figure-eight alleys and one in each of the blind alleys) to measure activity; each time a beam is interrupted, an activity count is 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:
Motor and locomotor activity were examined for the 90-minute session and during each ten-minute interval.
- Parameters measured:
motor activity (measured as the number of beam interruptions), locomotor activity (measured by eliminating consecutive counts for a given beam) and habituation (evaluated as a decrement in activity)

Sacrifice and (histo)pathology:

Gross pathology and histopathology were not included in the present study, since no effects were observed at higher doses (up to 100 mg/kg bw) in the previous study.

Positive control:

Previous studies with trimethyltin and acrylamide have established the sensitivity and reliability of the used procedures.

Statistics:

In general, continuous data were analyzed using an Analysis of Variance (ANOVA), followed by a Dunnett's test if a significant F-value was determined in the ANOVA. For the FOB, continuous data were first analyzed using an ANOVA to determine whether there was a significant treatment effect. If there was a significant treatment effect, Dunnett's test was applied to determine which groups, if any, were significantly different from the control group. Categorical data collected in the FOB were analyzed in a similar manner, using General Linear Modeling (GLM) and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively. Motor and locomotor activity (activity for the entire session and activity for each 10-minute interval) were analyzed using ANOVA procedures. Session activity data were first analyzed using a oneway ANOVA. If there was a significant treatment effect, Dunnett's test was used to determine which groups, if any, were significantly different from the control group. Interval data were subjected to a one-way ANOVA, using test interval as the repeated measure. If there was a significant treatment by interval interaction, the data for each interval were subjected to analysis using a one-way ANOVA to determine at which intervals there was a significant treatment effect. For those intervals, Dunnett's test was used to determine which groups, if any, were significantly different from the control group.

Clinical signs:

no effects observed

Description (incidence and severity):

Compound-related clinical signs were not evident in males or females treated with 2.5 or 10 mg/kg bw. In 1/12 males of the control group, perianal stain was observed on the day of treatment.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

There were no deaths prior to terminal sacrifice, one day following treatment.

Body weight and weight changes:

not examined

Description (incidence and severity):

Animals were weighed just prior to treatment to determine individual dose volumes.

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 observations:
No compound-related effects were evident at the time of peak effect on the day of treatment (day 0) in males or females at either dose level.
Various observations on day 0 are considered incidental and not related to treatment with the test substance. These statistically significant effects included lower body temperature (females only), increased number of urine pools (females only) and decreases in defecation (males only). All these effects are considered incidental to exposure since these were not evident as compound-related effects at higher dose levels (increased urine pools and decreased defecation) or since a NOAEL was established in both males and females that received 50 mg/kg bw (decreased body temperature) in the previous study.

Motor and Locomotor Activity:
In general, differences of less than 20% are within the range of normal variability in the performing laboratory for groups of 10-12 rats/sex/dose level and, therefore, are not biologically significant. For the overall 90-minute test session, there were no statistically significant differences in activity for females at the 2.5 or 10 mg/kg bw dose levels when compared to the controls. However, a decrease in motor (21%) and locomotor activity (27%) occurred at the high dose of 10 mg/kg bw, which did not reach statistical significance. Since the reduction in motor and locomotor activity exceeded 20%, these effects are considered biologically significant. Smaller apparent differences at the low dose (1% and 18%) are considered incidental and not related to treatment with the test substance. For details, please refer to the attached background material (attachment 1).

Habituation:
Habituation was not affected by treatment with the test substance.

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

not examined

Description (incidence and severity):

not applicable

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

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:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse affects observed at this dose level

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduction in motor and locomotor acitivity

Key result

Critical effects observed:

no

Conclusions:

The study was performed under GLP conditions and is in accordance with US-EPA-FIFRA 81-8 (neurotoxicity screening battery, adopted 1991). Therefore, the study is considered reliable and valid. The study was performed as supplementary study to the main study to establish a NOAEL for acute neurotoxicity. Therefore, rats were orally treated with nominal doses of 2.5 and 10 mg/kg bw. Under the conditions of the test, the overall NOAEL was 3.1 mg/kg bw (actual dose received) for females due to decreased motor and locomotor activities at 11 mg/kg bw. For male rats, the NOAEL was 11 mg/kg bw (actual doses received).

Reference 3

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Aug - 30 Nov 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: S-EPA-FIFRA, Guideline 82-5(b)

Version / remarks:

adopted 1991

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SASCO, Inc., Wisconsin, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: 193.4 - 196.0 g for males and 131.1 - 131.8 g for females
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, ad libitum except during neurobehavioral testing, when only water was provided
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 25.6
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 Aug 1995 To: 30 Nov 1995

Route of administration:

oral: feed

Vehicle:

corn oil

Remarks:

Corn oil was used as the vehicle for the test article at 1% by weight of the diet.

Details on exposure:

DIET PREPARATION
Corn oil was used as the vehicle for the test article at 1% by weight of the diet; a small amount of acetone served as a solvent in the diet preparation process and was allowed to evaporate. The control diet was prepared the same way, excluding the test substance. A given batch of feed was available for ad libitum consumption for a period of one week prior to changing, at which time any uneaten feed was collected.
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: basal diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chemical identity and structure of the test substance were confirmed by nuclear magnetic resonance and mass spectroscopy. The concentration of active ingredient in the test substance was measured within six months of initiation of dietary exposure, during the study, and then again after terminal sacrifice.

Concentration:
The concentration of the test substance in the ration was measured by liquid chromatographic analysis. The concentration of the test article in the ration was measured for the ration that was used during weeks 1, 5, 10 and 14 of the study. Actual concentrations of the active ingredient in the 0, 50, 400, and 1600 ppm dietary levels used in this study averaged 86.6% to 95.4% of the nominal concentration.

Homogeneity and stability:
The stability (following both room temperature and freezer exposure) and homogeneity of the test substance in the feed were established by analysis of samples at nominal concentrations (20 and 2000 ppm) that bracketed the range of test concentrations in diet that were used in the study. At these nominal concentrations, the test substance was determined to be homogeneous, with a coefficient of variation of approximately 5%, and stable for a minimum of one week at room temperature and four weeks at freezer (approximately -23°C) storage conditions.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously via diet

Dose / conc.:

50 ppm (nominal)

Remarks:

corresponding to 2.94 mg/kg bw/day for males and 3.41 mg/kg bw/day for females

Dose / conc.:

400 ppm (nominal)

Remarks:

corresponding to 24.2 mg/kg bw/day for males and 27.9 mg/kg bw/day for females

Dose / conc.:

1 600 ppm (nominal)

Remarks:

corresponding to 101 mg/kg bw/day for males and 115 mg/kg bw/day for females

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 on the results of a subchronic (13-week) feeding study with the test substance at dietary levels of 0, 25, 100, 400 and 1600 ppm. Males that received the 100 ppm dietary level had minimal changes in thyroid parameters and increased hepatic microsomal enzyme activity. At both 400 and 1600 ppm, there was further elevation of metabolic enzyme activities, increased liver weight and hepatocellular hypertrophy.
Additional effects involving the thyroid (increased triiodothyronine levels, thyroid binding capacity and organ weight) were also evident at the high dose. Body weight gain was reduced by 14-17% in high-dose males and females but was not affected at lower doses. Clinical signs of toxicity were not evident at any dietary level. Based on these results, the proposed doses for the present subchronic neurotoxicity study were 0, 50, 400 and 1600 ppm. The 1600 ppm dietary level was selected to produce evidence of toxicity following subchronic exposure, based on evidence that this dose level exceeds the maximum tolerated dose (MTD; > 10% reduction in body weight gain). The 50 ppm dietary level was selected to produce no evidence of toxicity and the 400 ppm dietary concentration was selected to produce an intermediate level of toxicity.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations checked: for mortality or clinical signs of moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and pre-terminal (week 12)
- Dose groups that were examined: all animals
- Parameters checked: The pupillary reflex was tested using a penlight or transilluminator. After mydriasis by application of a mydriatic agent, the eyelids, conjunctiva, cornea, aqueous humor, and lens were examined with a slit lamp microscope. Subsequently, the vitreous humor, retina, choroid, and optic disc were examined using an indirect ophthalmoscope and a condensing lens.

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY (FOB): Yes
- Parameters checked: home cage observations (posture, piloerection, gait abnormalities, involuntary motor movements, vocalizations and activity), observations during handling (resistance, muscle tone, palpebral closure, pupil reactions, lacrimation, salivation and stains), open field observations (piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, behavior, gait abnormalities, vocalizations, arousal, rearing and excretion), reflex and physiologic observations and measurements (approach, touch, auditory and tail pinch response, righting reflex, grip strength, landing foot splay, body weight and body temperature)
- Description of procedures: FOB closely follows the battery of tests described by Moser (Moser 1989). On the day prior to each test day, the appropriate animals were placed in the correct sequence that had been established for testing and were then transferred to the room where testing took place. Animals were allowed to acclimate with minimal disturbance until testing on the following day. The conditions in the testing room were the same as for the standard animal room. Sets of eight animals (maximum) were evaluated individually using the FOB.
- Minimization of bias:
Observations for all animals were performed by the same observer throughout the study, with a second person performing the measurements. Inter-observer reliability has been established in order to allow a second person to perform either the observations or measurements, ensuring the consistency of the results of each technician. Studies have been conducted with acrylamide, carbaryl and untreated rats to establish the sensitivity, reliability, and validity of these test procedures, the adequacy of training of technical personnel and to serve as a historical control.
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: once during the week prior to initiating the exposure and again during weeks 4, 8 and 13
- Environmental conditions: same conditions as for the standard animal room
- Noise level: Broad-spectrum background noise (approximately 70dB(A))

LOCOMOTOR ACTIVITY: Yes
- Replicates used:
All animals that were assigned to the study were tested.
- Type of equipment used: Figure-eight maze consisting of a series of interconnected alleys (approximately 10x10 cm in cross-section) converging on a central arena and covered by transparent acrylic plastic. Each maze had eight infrared emitter/ detector pairs (three in each of the figure-eight alleys and one in each of the blind alleys) to measure activity; 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:
Motor and locomotor activity were examined for the 90-minute session and during each ten-minute interval.
- Parameters measured:
motor activity (measured as the number of beam interruptions), locomotor activity (measured by eliminating consecutive counts for a given beam) and habituation (evaluated as a decrement in activity)

Sacrifice and (histo)pathology:

- Time point of sacrifice: at the end of the 13 weeks treatment period
- Number of animals sacrificed: all animals were subjected to a complete gross necropsy. The first six males and six females of each dose group were selected for perfusion and collection of tissues. The remaining animals were sacrificed by CO2 asphyxiation, without perfusion.
- Parameters measured: examination of all organs (including the brain), body cavities, cut surfaces, external orifices and surfaces
- Brain weight: yes
- Procedures for perfusion: animals were deeply anesthetized using an intraperitoneal dose (approximately 50 mg/kg bw) of sodium pentobarbital and then perfused via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by insitu fixation using Universal fixative (4%(w/v) glutaraldehyde and 4%(w/v) EM grade formaldehyde) in phosphate buffer
- Number of animals perfused: first six males and six females at each dose level
- Tissues evaluated: eight sequential levels of the brain, four levels of the spinal cord (cervical, thoracic, lumbar: transverse and longitudinal section; and cauda equina: longitudinal section including conus medullaris), gasserian ganglia (longitudinal sections), spinal nerve roots and dorsal root ganglia (cervical and lumbar enlargements), sciatic nerves (transverse and longitudinal sections), tibial nerves (transverse sections), sural nerves (transverse sections), optic nerves (transverse sections), left and right eyes, and gastrocnemius muscle (unilateral transverse section).
- Type of staining:

Hematoxylin and Eosin (H&E) staining:
- Type of staining: Hematoxylin and Eosin (H&E)
- Embedding media: paraffin
- tissues: coronal sections from eight levels of the brain, cross- and longitudinal-sections from three levels of the spinal cord (cervical, thoracic and lumbar) and the cauda equina
(longitudinal section only)
- Thickness: not reported

Luxol Fast Blue/Cresyl Violet and Sevier-Munger stains
- Type of staining: Luxol Fast Blue/Cresyl Violet and Sevier-Munger stains
- Embedding media: paraffin
- tissues: additional sections from each region of the brain and spinal cord
- Thickness: not reported

Modified Lee's staining
- Type of staining: Modified Lee's
- Embedding media: glycol methacrylate (GMA)
- tissues: Dorsal root ganglia (including dorsal and ventral root fibers), gasserian ganglion, eyes, optic nerves, and gastrocnemius muscle
- Thickness: 2 - 3 µm

Toluidine Blue staining
- Type of staining: Toluidine Blue
- Embedding media: epoxy resin
- tissues: peripheral nerve tissues
- Thickness: 1 µm

- Number of animals evaluated from each sex and treatment group: tissues from control and high-dose animals were further processed for histopathological examination

Positive control:

Previous studies with trimethyltin and acrylamide established the sensitivity and reliability of these procedures for detecting lesions in peripheral nerves and the central nervous system.

Statistics:

With the exception of Bartlett's test for homogeneity of variance, which was tested at p ≤ 0.001, the level used to establish statistical significance was p ≤ 0.05. In the event of unequal variances, data were analyzed using nonparametric statistical procedures (Kruskal-Wallis ANOVA followed by the Mann-Whitney U test). In general, continuous data were analyzed using an Analysis of Variance (ANOVA), followed by a Dunnett's test ifa significant F-value was determined in the ANOVA. For the FOB, continuous data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA if there was a significant interaction between dose group and test week. For weeks on which there was a significant treatment effect, Dunnett's test was applied to determine which groups were significantly different from the control group. Categorical data collected in the FOB were analyzed in a similar manner, using General Linear Modeling and Categorical Modeling Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively. Motor and locomotor activity were analyzed using ANOVA. Session activity data were first analyzed using a Repeated-Measures ANOVA, followed by a one-way ANOVA if there was a significant interaction with test occasion. For weeks on which there was a significant treatment effect, Dunnett's test was used to determine which groups were significantly different from the control group. Interval data were subjected to a two way Repeated-Measures ANOVA, using both test interval and test occasion as the repeated measures, followed by a Repeated Measures ANOVA to determine on which weeks there was a significant treatment by interval interaction. For those weeks, the data for each interval were subjected to analysis using a one-way ANOVA to determine at which intervals there was a significant treatment effect. For those intervals, Dunnett's test was used to determine which groups, were significantly different from the control group.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related clinical signs in males and females at any dose level.
Clinical signs that occurred in females at various dose levels on various occasions are considered incidental and not related to treatment with the test substance. These included red lacrimal stain and red lacrimation in females (at all three dose levels), urine stain in females (control and 50 ppm level) and exophthalmos in one female (50 ppm group). All these incidental clinical signs were considered non-treatment-related as they occurred within the range of historical control for untreated animals or as it was ascribed to injury rather than exposure to the test substance (exophthalmos).

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

There were no deaths before the scheduled terminal sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The following body weight changes were considered to be related to the treatment:
50 ppm: no treatment-related changes in body weights were observed
400 ppm: no treatment-related changes in body weights were observed
1600 ppm: A statistically significant reduction in body weight of approximately 12% was observed in male rats on Day 7 of exposure when compared to the control animals. The difference was less thereafter, but the average body weight remained significantly lower than controls for most weeks of the study. The body weight of females was reduced by 6% on average on Day 7 when compared to the control group, recovered thereafter and was comparable to controls for the remainder of the study.
For details, please refer to the attached background material (attachment 1).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The following changes in food consumption were considered to be related to the treatment:
50 ppm: no treatment-related changes in food consumption were observed
400 ppm: A statistically significant reduction in food consumption was observed in both sexes during the first week of exposure relative to the control group. During this week, males and females both consumed on average 8-10% less food than control animals. For the remainder of the study, the animals consumed an amount of food comparable to that of the control animals.
1600 ppm: Food consumption was statistically significantly reduced by 34-37% on average, compared to controls, during the first week of treatment and was reduced approximately by 6-15% during many weeks of the remainder of the study.
For details, please refer to the attached background material (attachment 2).

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, non-treatment-related

Description (incidence and severity):

All ophthalmologic findings (corneal opacity, corneal scar, iris coloboma, retinal degradation and myosis) were considered incidental and not related to exposure to the test substance.

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):

FOB observations:
Compound-related effects were not observed in males or females at any dietary level.
Observations that occurred in various dietary groups are considered incidental and not related to treatment. These findings included lacrimal stain, reduced hind-limb grip strength and evidence of a slightly impaired aerial righting response. These effects were considered incidental due to the lack of a dose-response and/or due to the minimal difference that was evident when compared to the control animals.

Motor and Locomotor Activity:
In general, differences of less than 20% are within the range of normal variability in the performing laboratory for groups of 12 rats/sex/dose level and, therefore, are not biologically significant. Motor and locomotor activity for the entire test session were not affected by treatment in males or females at any dietary level. Observed differences from control for males and females of various dietary groups on various test occasions were not statistically significant and are not considered biologically-significant. Motor and locomotor activities from females increased more than 20% when compared to the controls, however, this did not reach statistical significance and was not systematically related to dietary level or duration of exposure.
For details, please refer to the attached background material (attachment 3).

Habituation:
Habituation was not affected by treatment with the test substance.

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):

There were no significant differences in brain weight or terminal body weight at any dietary exposure level.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related gross lesions observed at terminal sacrifice in either sex.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no microscopic lesions in brain, spinal cord, the gasserian ganglion, spinal nerve roots and dorsal root ganglia, sciatic nerves, tibial nerves, sural nerves, and optic nerves in high-dose animals that were ascribed to treatment with the test substance.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no microscopic lesions in eye and gastrocnemius muscle in high-dose animals that were ascribed to treatment with the test substance.

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

Remarks:

neurotoxicity

Effect level:

1 600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose level tested

Remarks on result:

other: corresponding to 101 mg/kg bw/day for males and 115 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 2.94 mg/kg bw/day for males and 3.41 mg/kg bw/day for females

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 24.2 mg/kg bw/day for males and 27.9 mg/kg bw/day for females

Key result

Critical effects observed:

no

Conclusions:

The present guideline study was conducted in compliance with GLP. Under the test conditions used, the NOAEL for male and female rats regarding general toxicity was set at 50 ppm which was the lowest dose tested, corresponding to an actual ingested dose level of 3.41 mg/kg bw/day for males and 2.94 mg/kg bw/day for females. Since no obvious treatment-related effects indicative of neurotoxicity were seen up to the highest dose tested of 1600 ppm, this dose was set as NOAEL for male and female rats with respect to neurotoxicity, corresponding to an actual ingested dose level of 101 mg/kg bw/day for males and 115 mg/kg bw/day for the females.

Reference 4

Endpoint:

neurotoxicity, other

Remarks:

Developmental Neurotoxicity Screening Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Sep - 22 Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

Version / remarks:

adopted 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Michigan, USA
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: approximately 216 - 260 g
- Housing: Rats were individually housed in stainless steel wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of rats were housed in the male rat's cage. Not later than gestation Day 20, F generation female rats were individually housed in nesting boxes. Each dam and delivered litter were housed in a common nesting box during the postpartum period.
- Diet: Certified Rodent Diet® #5001-4 (PMI Nutrition International, St. Louis, Missouri, USA), ad libitum
- Water: local water processed by passage through a reverse osmosis membrane, ad libitum
- Acclimation period: approximately 2 weeks

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

IN-LIFE DATES: From: 11 Sep 2000 To: 22 Dec 2000

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
A constant concentration of the test substance in the diet was offered to the rats. Diet admixtures containing the test substance at each of three dose concentrations, plus the carrier control diet, were prepared by the Diet Preparation Lab at the Sponsor's facility for use at the testing facility. Batches of feed were prepared for weekly usage. Prepared diet admixtures and the plain diet were retained frozen (-20°C) and stored at room temperature during each week of use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration:
The concentrations of the test substance in the diet were measured by liquid chromatography for batches of feed that were used in the study. The measured treatment level concentrations for dietary samples used in the study were 47.6, 289 and 495 ppm, respectively, for the nominal levels of 50, 300 and 500 ppm.

Homogeneity and stability:
The stability (at room temperature and freezer conditions) and homogeneity of the test substance in the feed have been verified at concentrations of 20 and 2000 ppm in a previous study, which bracket the range of dietary concentrations used in the present study.

Duration of treatment / exposure:

F0: From beginning of gestation (day of gestation 0, DG 0) and continuing through DG 24 for rats that did not deliver a litter or during the postpartum period (day postpartum, DP 22) for dams that delivered a litter. The latter equals the lactation period (day of lactation, DL 22).
F1: Pups were not directly offered the test substance, but may have been exposed in utero during gestation and via maternal milk and maternal feed during the postpartum period.

Frequency of treatment:

continuously via diet

Dose / conc.:

50 ppm

Remarks:

corresponding to 4.4 mg/kg bw/day during gestation (DGs 0 to 21) and 8.2 mg/kg bw/day during lactation (DL 1 to 14)

Dose / conc.:

300 ppm

Remarks:

corresponding to 25.6 mg/kg bw/day (DGs 0 to 21) and 49.4 mg/kg bw/day during lactation (DL 1 to 14)

Dose / conc.:

500 ppm

Remarks:

corresponding to 40.8 mg/kg bw/day (DGs 0 to 21) and 82.8 mg/kg bw/day during lactation (DL 1 to 14)

No. of animals per sex per dose:

25 P females/dose
80 F1 males and 80 F1 females (approximately 20 F1 males and females per subset (subsets 1 to 4) and dose, respectively)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose was selected based on results of a two-generation reproduction study. In this study, separate groups of young adult male and female Sprague-Dawley rats (30/sex/dietary level) were exposed to the test substance via the diet at nominal concentrations of 0, 50, 300 and 600 ppm. The 50 ppm dietary concentration was considered as the overall NOAEL (systemic). Compound-related effects at 300 and 600 ppm dietary levels included dystocia, decreased pup body weight during lactation and effects on the liver and thyroid. Additional effects at 600 ppm consisted of decreased body weight in P generation females during the premating phase, decreased live-birth index, and lower pup viability index in F1 and F2 generations. Based on these results, the dietary concentrations of 0, 50, 300 and 500 ppm of the test substance were selected to assess developmental neurotoxicity in Sprague-Dawley rats. The 50 ppm dose was selected to be an overall NOAEL, the 500 ppm dietary level was selected as a dose that produces toxicity in P and F1 generation animals, and the 300 ppm level was selected to produce intermediate toxicity to assist in establishing compound related effects. For a detailed schematic illustration of the study design, please refer to attachment 8.

- Details on mating procedure:
Impregnation procedure: cohoused
M/F ratio per cage: 1/1
Length of cohabitation: up to five consecutive days
Proof of pregnancy: smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0

Observations and clinical examinations performed and frequency:

1. Parental generation

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- clinical observations checked: signs of autonomic dysfunction, abnormal postures, abnormal movements or abnormal behavior patterns, and unusual
appearance daily beginning on GD 0 at approximately the same time each day, duration of gestation, litter size, live litter size, pup viability at birth and maternal behavior of the dams on DPs 1, 5, 8, 14 and 22

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly and on DG 0 during the pre-exposure period, daily during the exposure period and on the day sacrificed

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
Food consumption values were recorded on DG 0 and daily during the exposure period. They were no longer tabulated after DP 14, when it was expected that pups began to consume maternal feed.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

2. F1 generation

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the preweaning period

BODY WEIGHT: Yes
- Time schedule for examinations: on DPs 1 (birth), 5 (values cited both pre- and poststandardization), 8, 12, 14, 18 and 22, weekly during the post weaning period and at sacrifice

FOOD CONSUMPTION: yes
- Time schedule: recorded weekly during the postweaning period

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

OTHER:
On DP 5, litters were reduced to ten pups each. On DP 12, 20 appropriately sized litters per exposure group were selected for continued examination on study. Observations conducted in each of four subsets were as follows: brain weights and histopathological examinations (subset 1), passive avoidance and water maze (subset 2), motor activity and auditory startle habituation (subset 3) and neurohistological examination (subset 4). For details, please refer to neurobehavioral examinations performed and frequency.

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

1. P generation

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Number of animals: all rats
- Days of observations: appearance daily beginning on DG 0
- Parameters checked: signs of autonomic dysfunction, abnormal postures, abnormal movements or abnormal behavior patterns, unusual appearance and maternal behavior
- Description of procedures: An individual unaware of each rat's exposure group examined the rats daily beginning on DG 0.
- Same technicians used throughout testing: No data
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: daily approximately at the same time

2. F1 generation

On DP 5, litters were reduced to ten pups each. On DP 12, 20 appropriately sized litters per exposure group were selected for continued examination on study.
subset 1: brain weights and histopathological examinations
Subset 2: passive avoidance and water maze
Subset 3: motor activity and auditory startle habituation
Subset 4: FOB and neurohistological examination
For details, please refer to the attached background material (attachment 8).

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Number of animals: all rats assigned to subset 4
- Days of observations: on DP 12 and weekly during the postweaning period
- Parameters checked: autonomic dysfunction, abnormal postures, abnormal movements or abnormal behavior patterns and unusual appearance
- Description of procedures: An individual unaware of each rat's exposure group examined the rats.
- Same technicians used throughout testing: No data
- Technicians were blind to treatment status of animals: Yes.
In addition, on DP 5, one male and one female pup from ten randomly selected litters per exposure group were subjected to observations outside the home cage as described under FOB.

Additional information for developmental neurotoxicity study:
Animals of subset 2 through 4 were further examined with regard to physical developmental landmarks (evaluation of pupil constriction was performed once on DP 21) and for sexual maturation (evaluated for the age of vaginal patency beginning on DP 28; male rats were evaluated for the age of preputial separation beginning on DP 39)

PASSIVE AVOIDANCE TESTING (subset 2)
- Number of animals: one male rat and one female rat (when possible) from each litter
- Days of testing: on DPs 23 to 25 and 30 to 32
- Parameters measured: learning, short-term retention, long-term retention and hyperactivity
- Type of equipment used:
The passive avoidance apparatus consisted of a two-compartment chamber with hinged Plexiglas® lids. One compartment was fitted with a bright light and Plexiglas® floor. The other compartment was fitted with a grid floor to which a brief (1 sec) pulse of mild electric current (1 mA) was delivered. The two compartments were separated by a sliding door.
- Description of procedures:
On each test trial, the rat was placed into the "bright" compartment, the sliding door was opened and the light was turned on. The rat was allowed to explore the apparatus until it entered the "dark" compartment. The sliding door was then immediately closed, the light was turned off and the brief pulse of current was delivered to the grid floor. The rat was then removed from the apparatus and placed into a holding cage for 30 seconds before the start of the next trial. Trials were repeated until the rat remained in the "bright" compartment for 60 seconds on two consecutive trials (the criterion for learning) or until 15 trials had been completed. The latency to enter the dark compartment or the maximum 60-second interval was recorded for each trial. Each rat was tested twice. The test sessions were separated by a one-week interval, and the criterion was the same for both days of testing.

WATERMAZE TESTING (subset 2)
- Number of animals: one male rat and one female rat (when possible) from each litter
- Days of testing: on DPs 59 to 63 and 66 to 70
- Parameters measured: coordination, swimming ability, learning and memory
- Type of equipment used:
A watertight 16-gauge stainless-steel modified M-maze was used for the testing. The maze was filled with water to a depth of approximately nine inches, and the water was monitored for temperature (range of 21 °C ± 1 °C).
- Description of procedures:
On each test trial, the rat was placed into the starting position and required to swim to one of the two goals of the M-maze, in order to be removed from the water. On the first trial, the rat was required to enter both arms of the maze before being removed from the water. The initial arm chosen on trial 1 was designated the incorrect goal during the remaining trials. Rats that failed to make a correct goal choice within 60 seconds in any given trial were guided to the correct
goal and were then removed from the water. A 15-second intertrial interval separated each trial. Each rat was required to reach a criterion of five consecutive errorless trials to terminate the test session. The maximum number of trials in any test session was 15. Latency (measured in seconds) to choose the correct goal or the maximum 60-second interval was recorded for each trial, as was the number of errors (incorrect turns in the maze) during each trial.
Each rat was tested twice. The test sessions were separated by a one-week interval, and the correct goal and the criterion were the same for both test sessions.

MOTOR ACTIVITY (subset 3)
- Number of animals: one male and one female rat (when possible) from each litter
- Days of testing: on DPs 14, 18, 22 and 58 to 60
- Type of equipment used:
A passive infrared sensor mounted outside a stainless-steel cage (40.6 cm x 25.4 cm x 17.8 cm) with Plexiglas® flooring was used.
- Description of procedures:
The movements of each pup were monitored by a passive infrared sensor. During preweaning sessions each pup was tested on Plexiglas® flooring. Each test session was one hour in duration with the number of movements and time spent in movement tabulated at each ten-minute interval. The apparatus monitored a rack of up to 32 cages and sensors during each session, with each rat tested in the same location on the rack across test sessions. Groups were counterbalanced (sex, exposure group) across testing sessions and cages, where possible.

AUDITORY STARTLE HABITUATION (subset 4)
- Number of animals: one male and one female rat (when possible) from each litter
- Days of testing: on DPs 23 and 59 to 61
- Parameters measured: reactivity to auditory stimuli and habituation of responses with repeated presentation of stimuli
- Type of equipment used:
A sound-attenuated chamber with a force transducer and a microcomputer that sampled the output of the force transducer were used.
- Description of procedure:
The rats were tested in sets of four within a sound-attenuated chamber. Each rat was placed inside a small cage situated above a platform containing a force transducer in its base. The rats were initially given an adaptation period of five minutes. During the last minute of this period, ten "blank" trials were given to sample the baseline force in the absence of a stimulus. The rats were then presented with 30 msec, 120 dB bursts of noise at ten-second intervals for 50 trials. An additional ten "blank" trials followed. The peak amplitude of each response was recorded and the average response on baseline trials subtracted to calculate the response magnitude. The average response magnitude and the pattern of responses over ten trial blocks were compared among the exposure groups.

Sacrifice and (histo)pathology:

1. P generation
- Procedure of sacrifice: by carbon dioxide asphyxiation
- Time point of sacrifice:
Rats that did not deliver a litter: sacrificed on DG 25
Dams that delivered a litter: sacrificed on DP 12 (when not selected for continued observation otherwise on DP 22
Dam with no surviving pups: sacrificed after the last pup was found dead or missing and presumed cannibalized
- Parameters measured:
Rats that did not deliver a litter: gross lesions including uteri to confirm the absence of implantation sites
Dams that delivered a litter: gross necropsy of thoracic, abdominal and pelvic viscera; number and distribution of implantation sites
Dam with no surviving pups: gross necropsy of the thoracic, abdominal and pelvic viscera
Dam found dead: examined for the cause of death on the day the observation of death was made, gross lesions, pregnancy status and uterine contents

2. F1 generation
- Procedure of sacrifice: Rats were sacrificed by carbon dioxide asphyxiation, with the exception of rats from the F1 generation selected for neurohistopathological evaluation (subset 4). These rats were sacrificed by exsanguination under anesthesia.
- Time point of sacrifice:
Pups not selected for continued observation: on DP5 (all offspring selected for standardization) and on DP 22 (F1 generation pups not selected for continued observation)
Subset 1: on Day 12
Subset 2: after completion of all the postweaning behavioral evaluations (DPs 70 to 75)
Subset 3: after completion of all the postweaning behavioral evaluations (DPs 67 to 72)
Subset 4: on DPs 68 - 79
- Parameters measured:
Pups found dead on Day 1 postpartum: gross lesions, lung immersed in water for determination of stillbirth
Pups found dead on Day 12 - 22 postpartum: gross lesions and examined for cause of death or the moribund condition
Pups not selected for continued observation: gross lesions
Subset 1: gross lesions (all animals), morphometric measurements and neurohistological examination of the brain (10 animals/sex/dose)
Subset 2: gross lesions (all animals)
Subset 3: gross lesions (all animals)
Subset 4: gross lesions, neurohistological examination of brains, heads, spinal columns and hindlimbs (10 animals/sex/dose)

Neurohistological examinations were performed by a different testing facility, therefore the samples were shipped in neutral buffered 10% formalin.

Other examinations:

not applicable

Positive control:

no

Statistics:

Variables with interval or ratio scales of measurement, such as body weights, feed consumption values, latency and errors per trial scores in behavioral tests and percent mortality per litter was analyzed as described under the Parametric heading of the schematic. Bartlett's Test of Homogeneity of Variances was used to estimate the probability that the exposure groups have different variances. If Bartlett's Test was not significant (p > 0.001) the data were compared using the Analysis of Variance Test. If that test was significant (p ≤ 0.05), the groups offered the test substance were compared with the control group using Dunnett's Test. If Bartlett's Test was significant (p ≤ 0.001), the Analysis of Variance Test was not appropriate, and the data were analyzed with a nonparametric test. When 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis Test was used to analyze the data, and in the event of a significant result (p ≤ 0.05), Dunn's Test was used to compare the groups offered the test substance with the control group. When more than 75% of the scores in any exposure group were tied, Fisher's Exact Test was used to compare the proportion of ties in the exposure groups. Data from the motor activity evaluation were analyzed using an Analysis of Variance with Repeated Measures. A significant result (p ≤ 0.05) in that test can appear as effect of exposure or as an interaction between concentration and Block. If the exposure effect was significant, the totals for the control group and the groups given the test substance were compared using Dunnett's test. If the Exposure x Block interaction was significant, an Analysis of Variance Test was used to evaluate the data at each measurement period, and a significant result (p ≤ 0.05) was followed by a comparison of the exposure groups using Dunnett's Test. Continued under "Any other information on materials and methods including tables".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

P generation
No clinical observations related to the test substance occurred in the P generation.

Observation during gestation, which were not considered related to the test substance included:
Control: localized alopecia (limbs) in 2/25 females, chromorhinorrhea in 1/25 females
50 ppm: localized alopecia (limbs) in 1/25 females
300 ppm: localized alopecia (limbs) in 4/25 females
500 ppm: localized alopecia (limbs) in 2/25 females, reddish-brown perivaginal substance 1/25 females

Observation during lactation in the P generation, which were not considered related to the test substance included:
Control: abnormal autonomic functions in 2/24 females, localized alopecia (limbs) in 5/24 females, chromorhinorrhea in 1/24 females, liquid feces in 2/24 females, swollen snout 1/24 females
50 ppm: abnormal autonomic functions in 1/25 females, localized alopecia (limbs) in 2/25 females, liquid feces in 1/25 females, cold to touch in 1/25 females, pale extremities in 1/25 females
300 ppm: abnormal autonomic functions in 1/24 females, localized alopecia (limbs and back) in 5/24 females, emaciation in 1/24 females, reddish-brown urine in 1/24 females
500 ppm: abnormal autonomic functions in 1/25 females, localized alopecia (limbs) in 2/25 females, chromodacryorrhea in 1/25 females, lacrimation in 1/25 females, missing or broken incisors in 1/25 females

All of these observations were not considered related to the treatment as the incidences were either not exposure-dependent or they did not persist and/or the observation occurred in a single rat and/or the observation commonly occurs in this strain of rat. For details, please refer to the attached background material (attachment 1).

F1 generation
No clinical observations related to maternal exposure to the test substance occurred.

Clinical observations of the pups from birth to Day 5 postpartum (preculling) included:
Control: tip of tail missing in 1/24 litters, tip of tail black in 4/24 litters
50 ppm: tip of tail missing in 5/24 litters
300 ppm: tail missing in 5/24 litters
500 ppm: cold to touch in 1/24 litters, anal opening not apparent in 5/24 litters, tail missing in 5/24 litters
All these observations were considered unrelated to the test substance as the incidences were not exposure-dependent or the observations occurred in only one or two litters.

Clinical observations in F1 generation (subset 2, 3 and 4) included:
Control: missing/ broken incisors in 1/80 males, scab on head, back or neck in 2/80 males, ringed tail in 1/80 males, chromorhinorrhea in 1/80 males, dehydration in 1/80 females
50 ppm: misaligned incisors in 1/80 males, chromodacryorrhea in 1/80 males, abrasion on left axilla or neck in 2/80 males, scab on head, back or neck in 1/80 males, cold to touch in 1/80 males, autonomic functions abnormal in 2/80 females, excess salivation in 1/80 females, bent tail in 1/80 females, soft or liquid feces in 1/80 females
300 ppm: misaligned incisors in 1/80 males, chromodacryorrhea in 1/80 males, abrasion on left axilla or neck in 1/80 males, tip of tail missing in 1/80 males, red substance on penis in 1/80 males, autonomic functions abnormal in 1/80 females, excess salivation in 1/80 females, dehydration in 1/80 females
500 ppm: misaligned incisors in 5/80 males, missing incisors in 1/80 males, chromodacryorrhea in 5/80 males, localized alopecia in 1/80 males (head) and in 1/80 females (limbs)
Evaluation of autonomic functions appeared normal except for three females, two of the 50 ppm group and one of the 300 ppm group. The observed clinical signs included soft or liquid feces (one F1 female in the 50 ppm group), excess salivation (one F1 female in the 50 ppm group and 300 ppm group, respectively) and misaligned incisors and chromodacryorrhea (F1 males in the 500 ppm group). All these clinical observations were not considered to be treatment-related, as the findings were not exposure-dependent or occurred in only one or two rats or were common for the strain of rats used.
For details, please refer to the attached background material (attachment 2).

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

P generation
No deaths related to the test substance occurred.
Control: One dam was found dead on DL 21. The dam had no adverse clinical observations prior to death. The animal generally gained weight until DL 21 and feed consumption values were unremarkable. The dam delivered 17 normal pups. All tissues examined at necropsy appeared normal. Therefore, no specific cause of death could be determined.
50 ppm: One Dam was sacrificed on DL1, because it had no surviving pups. The animal was cold to touch, had pale extremities and localized alopecia on the limbs on DL 1. The dam lost weight on DG 23 and feed consumption values were reduced on DGs 22 to 23. The duration of gestation for this dam was 24 days, which was increased over the average duration of gestation for the control group (22.8 days). The litter consisted of seven stillborn pups and one pup in which cannibalization precluded the determination of viability.
All other rats survived until scheduled sacrifice.

F1 generation
No deaths related to the test substance occurred. However, deaths occurred during the study as follows:
Control: 3/80 males (DP 15, 23 and 37)
50 ppm: 1/80 males (DP 15) and 1/80 females (DP 28)
300 ppm: 2/80 males (DP 17)
500 ppm: 1/80 males (DP 61)
One F1 female of the control group was found missing on DP 30 and one female of the 500 ppm dose group was inadvertently sacrificed on DP 22.
All these deaths were considered unrelated to the maternal consumption of the test substance because the incidences did not differ significantly among groups, including the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

P generation
Treatment-related changes in body weight of the P generation females were as follows:
50 ppm: Body weight gains and body weights during the gestation and lactation periods were unaffected when compared to the control group.
300 ppm: Maternal body weight gains during the gestation period were statistically significantly reduced on DGs 0 to 6 (-30.6%) when compared to the control animals. Average body weights were also significantly reduced during the gestation period on DGs 2 to 8, 12 and 14 (< 10%) relative to the control animals. Similar to that, also the average body weight during lactation period was significantly reduced on DLs 13 and 22 (< 10%) compared to the control group.
500 ppm: Maternal body weight gains during the gestation period were statistically significantly reduced on DGs 0 to 6 (-55.9%) when compared to the control animals. Average body weights were also significantly reduced during the gestation period on DGs 2 to 16 (< 10%) relative to the control. Maternal body weight gains during lactation period were significantly reduced on DLs 1 to 4 (-67.2%) when compared to the control group. Average body weight during lactation period was also significantly reduced on DLs 3 to 8, 11, 13, 17, 21 and 22 (< 10%).

F1 generation (subset 1, 2, 3, and 4):
Treatment-related changes in body weight of the F1 generation were as follows:
50 ppm: The body weight reduction and subsequent increase compared to the control group was not considered test substance related because it did not persist throughout the observation period. Male and female body weight gains during the postpartum period were unaffected by maternal exposure to the test substance relative to the control values.
300 and 500 ppm: Average body weights in males and females were significantly reduced from DPs 8 to 65 and at sacrifice when compared to the control animals. Body weight gains in males were significantly reduced for the entire postpartum period (DPs 5 to 65) and at most tabulated intervals during the postpartum period. Body weight gains in females were also significantly reduced on DPs 5 to 8, 8 to 12, 12 to 14 and 18 to 22 relative to the control values.

For details, please refer to the attached background material (attachment 3).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

P generation
Treatment-related changes in feed intake of the P generation were as follows:
50 ppm: Absolute and relative feed consumption values during the gestation and lactation periods were unaffected when compared to the control animals.
300 ppm: Absolute and relative feed consumption values during the gestation period were statistically significantly reduced on DGs 0 to 21 (< 10%) compared to the control group.
500 ppm: Both absolute and relative feed consumption values during the gestation period were statistically significantly reduced on DGs 0 to 21 (-12.5% and -8.9%, respectively) and absolute feed consumption values were also significantly reduced on DGs 0 to 6, 6 to 9, 9 to 12 and 12 to 15 in relation to the control group. Further, absolute feed consumption values during lactation were significantly reduced on DLs 4 to 7 (-10.9%) when compared to the control animals.

F1 generation
Treatment-related changes in feed intake of the F1 generation were as follows:
50 ppm: No treatment-related changes in feed intake observed.
300 ppm and 500 ppm: Absolute (g/day) feed consumption values for males in the 300 ppm and 500 ppm maternal exposure groups were significantly reduced (< 10%) for the entire postpartum period (calculated as DPs 30 to 65) and at most tabulated intervals during the postpartum period when compared to the control animals. However, the relative (g/kg bw/day) feed consumption values were significantly increased during this period, because of the lower pup body weights. Absolute (g/day) feed consumption values for females were comparable between the maternal exposure groups. However, because of the lower body weights, the relative (g/kg bw/day) feed consumption values were significantly increased for the entire postpartum period (calculated as DPs 30 to 65) and at most tabulated intervals during the postpartum period.

For details, please refer to the attached background material (attachment 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:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation (subset 2):

Passive avoidance testing
There were no biologically important differences in the values for learning, short-term retention, long-term retention or response inhibition in the F1 generation male or female rats, as evaluated by performance in a passive avoidance paradigm.
Latency (trial 2) was significantly increased in the first session of passive avoidance testing for the females of the 300 and 500 ppm maternal exposure groups compared to the control animals. However, this increase was not considered to be biologically or toxicologically important, since a reduction in the latency in Trial 2 would be the result of a toxicant rather than an increase.

Water maze Perfomance:
There were no biologically important differences in the values for learning, short-term retention, long-term retention or response inhibition in male or female rats, as evaluated by performance in a water maze paradigm. No statistically significant differences in either of the sexes occurred in the number of trials to criterion, the number of errors per trial, trial latencies or numbers of rats that failed to learn.

F1 generation (subset 3):

Motor activity:
Maternal exposure to the test substance as high as 500 ppm did not affect the average number of movements or the time spent in movement for the male or female rats as evaluated in a 1 hour session (6 ten-minute blocks) for all four ages (DPs 14, 18, 22 and Adult [DPs 58 to 60]). The average number of movements and the time spent in movement were comparable among the exposure groups and did not differ significantly.

Auditory Startle Habituation:
Maternal exposure to the test substance up to and including 500 ppm did not affect the auditory startle response magnitude for male or female rats at either time period (DPs 23 and Adult [DPs 59 to 61]).

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation (subset 1):
Brain weights revealed no statistically significant differences in males and females compared to the control. However, because of the lower body weights in the 500 ppm maternal exposure group, the brain to body weight ratio was significantly increased.

F1 generation (subset 4):
No statistically significant difference occurred in male and female rats in the average brain weights and the ratios of the brain weight to the terminal body weight when compared to the control animals.

For details, please refer to the attached background material (attachment 5).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1. P generation
All gross pathological findings were considered unrelated to the treatment, as the incidences were not exposure-dependent and the observations occurred in only one or two animals. These findings included the following:
300 ppm: One dam had a red fluid (presumably blood) in the urinary bladder, which correlated with the histopathological finding of mild distention of the urinary bladder.
500 ppm: One dam had moderate dilation of the pelvis of both kidneys, which is a common observation in this species and strain. In addition, another dam had a moderate dilation of the pelvis of the right kidney and localized alopecia (right axilla and right hindlimb). The gross pathological findings were also confirmed by histopathological examination.

2. F1 generation
All gross pathological findings of the pups were considered unrelated to the test substance as the incidences were not exposure-dependent or the observations occurred in only one pup. These observations included the following:
Control: One pup had a missing tip of tail.
300 ppm: Two pups that were found dead, had no milk in the stomach.
500 ppm: One pup that was found dead, had no milk in the stomach. One pup had no apparent anal opening and a missing tail.

F1 generation:
No treatment-related gross pathological findings were observed in the F1 generation after test substance exposure to the dams. Observations observed were as follows:
Control: slight or moderate dilation of the renal pelvis (3/80 males)
50 ppm: slight or moderate dilation of the renal pelvis (2/80 males)
300 ppm: slight or moderate dilation of the renal pelvis (3/80 males and 1/80 females), indented calvaria over the right cerebral hemisphere (1/80 males) and indentation in the cerebrum (1/80 males)
500 ppm: slight or moderate dilation of the renal pelvis (2/80 males and 1/80 females)
All these gross pathological findings were considered unrelated to the test substance as the findings were not exposure-dependent or occurred in only one or two rats or were common for the strain of rats used.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation (subset 1):
There was no microscopic evidence of any treatment-related neuropathologic processes. Neurohistological findings, which were not considered to be treatment-related included significant decrease in the diagonal measurement of the corpus striatum and the thickness of the corpus callosum in the 500 ppm maternal exposure group when compared to the control group. However, the value for the caudate-putamen was within the historical control range for that measurement, but the value for the corpus callosum was below the historical control range for that measurement. This decrease is not considered to be test substance-related because the corresponding measurement for the female rats in the same exposure group was not different from the concurrent control group. These differences are considered to be the result of slightly lower brain weights for the males. These lower brain weights are, in turn, related to lower body weights in this exposure group. These lower body weights are most likely the result of nutritional factors related to toxicity of the test substance to the dams. Morphometric measurements of the DP 12 F1 generation female pups revealed no statistically significant difference.

F1 generation (subset 4):
There was no microscopic evidence of any treatment-related neuropathologic processes. Neurohistological findings considered non-treatment-related included a significant decrease in the diagonal measurement across the corpus striatum (caudate putamen) and the width of the dentate gyrus of the hippocampus. However, these values were within the historical control range for the respective measurement and were considered to be the result of slightly lower brain weights in the 500 ppm maternal exposure group males. Morphometric measurements of the adult female rats revealed no statistically significant difference to control.

For detail on brain weights and morphometry data, please refer to attachment 10.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to the results under "Neuropathological findings".

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Natural Delivery Observations
Up to 500 ppm, natural delivery observations were unaffected by the treatment with the test substance. The parameters number of litters, the duration of gestation, implantation sites per litter, dams with stillborn pups, dams with no liveborn pups and the gestation index were comparable among the four exposure groups and did not significantly differ compared to the control animals. One dam treated with 50 ppm had no liveborn pups. This observation was not considered test substance related because it showed no exposure-dependency. For details, please refer to the attached background material (attachment 6).

Litter Observations
Up to 500 ppm, litter observations were unaffected by the test substance. Values for the average number of pups delivered, liveborn and stillborn pups, number of pups found dead or presumed cannibalized (DL 1 and DLs 2 to 5), viability index, the lactation index, sex ratio and pup body weights for DLs 1 or 5 (pre- or postculling) did not differ among the groups. For details, please refer to the attached background material (attachment 6).

Sexual maturation of F1 generation (subset 2, 3 and 4)
50 ppm: Maternal exposure to the test substance did not affect the day of preputial separation or of vaginal patency in the F1 generation.
300 and 500 ppm: The average day postpartum that the prepuce was observed to be separated (preputial separation) was significantly increased from 48.2 days postpartum (in the 300 and 500 ppm groups) to 46.7 days postpartum (in the control group). The average day postpartum that the vagina was observed to be patent (vaginal patency) was significantly increased in the 500 ppm maternal exposure group only (34.7 days postpartum versus 33.4 days postpartum in the control group). The delay in preputial separation and vaginal patency is considered to be related to the reduced body weights in these exposure groups. For details, please refer to the attached background material (attachment 7).

Key result

Dose descriptor:

NOAEL

Remarks:

maternal neurotoxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse neurotoxic effects observed at this dose level

Remarks on result:

other: corresponding to 40.8 mg/kg bw/day during gestation (DGs 0 to 21) and 82.8 mg/kg bw/day during lactation (DL 1 to 14)

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxicity F1 generation

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse neurotoxic effects observed at this dose level

Remarks on result:

other: corresponding to 40.8 mg/kg bw/day during gestation (DGs 0 to 21) and 82.8 mg/kg bw/day during lactation (DL 1 to 14) of P dams

Key result

Dose descriptor:

NOAEL

Remarks:

maternal systemic toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 4.4 mg/kg bw/day during gestation (DGs 0 to 21) and 8.2 mg/kg bw/day during lactation (DL 1 to 14)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic and developmental toxicity (F1 generation)

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 4.4 mg/kg bw/day during gestation (DGs 0 to 21) and 8.2 mg/kg bw/day during lactation (DL 1 to 14) of P dams

Key result

Dose descriptor:

LOAEL

Remarks:

maternal systemic toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 25.6 mg/kg bw/day during gestation (DGs 0 to 21) and 49.4 mg/kg bw/day during lactation (DL 1 to 14)

Key result

Dose descriptor:

LOAEL

Remarks:

systemic and developmental toxicity (F1 generation)

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: sexual maturation

Remarks on result:

other: corresponding to 25.6 mg/kg bw/day during gestation (DGs 0 to 21) and 49.4 mg/kg bw/day during lactation (DL 1 to 14) of P dams

Key result

Critical effects observed:

no

Conclusions:

The present developmental neurotoxicity guideline study was conducted in compliance with GLP. Under the test conditions used, dietary exposure to the test substance did not cause any neurotoxic effects in parental as well as offspring animals up to the high dose of 500 ppm in the diet (corresponding to 40.8 mg/kg bw/day (DGs 0 to 21) and 82.8 mg/kg bw/day during lactation (DL 1 to 14) of P dams). Both, the maternal as well as the offspring NOAEL with regards to effects indicative of general systemic toxicity were set to 50 ppm or 4.4 mg/kg bw/day, based on reduced maternal and offspring body weight parameters and food consumption as well as a delay in sexual maturation (preputial separation and vaginal patency) of the F1 pups, which was considered to be secondary to body weight reduction.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

With respect to neurotoxicity, an acute, a subchronic as well as a developmental neurotoxicity study are available. All of these studies are guideline studies, conducted under GLP conditions. Thus, they all are considered of reliable quality and validity, fulfilling the criteria of key studies.

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

No data available.

Additional information

Neurotoxicity: oral administration

Data on neurotoxicity are available after single and repeated exposure to characterize the potency of the test substance to induce neurotoxicity after short-term and subchronic exposure. Moreover, a developmental neurotoxicity study is available to assess the toxicity to offspring.

Acute

The acute neurotoxicity study on rats was conducted according to GLP and the US-EPA-FIFRA guideline EPA OPP 81 - 8 (M-000894-03-1) and was considered as key study. The test substance was administered by gavage in a single dose to Fischer 344 rats (12/sex/dose) using nominal doses of 20, 50 and 100 mg/kg bw for both sexes. The test substance was suspended in 0.5% methylcellulose/0.4% Tween 80 in deionized water and administered at a dosing volume of 10 mL/kg bw. The control animals were treated with vehicle only. Clinical observations, mortality, body weight, automated measurements of activity (figure-eight maze), a functional observation battery (FOB), brain weight, gross pathology and microscopic examination of the skeletal muscles, peripheral nerves, eyes and tissues from the central nervous system were included in the study. The actual doses of the test substance were 22, 53 and 109 mg/kg bw for male and female rats. During the study, no deaths occurred at any dose level prior to scheduled terminal sacrifice. Observed clinical signs, which were attributed to the highest dose of the test substance, included tremor, decreased activity, locomotor incoordination (ataxia), cool-to-touch-body, dilated pupils, urine staining and eyelid ptosis in both sexes. In females given the highest dose level, further clinical signs were red nasal and oral staining, clear nasal discharge, and clear lacrimation. The mid-dose only caused dilated pupils in one female rat. All these clinical signs were evident only at the day of treatment and were typically resolved by Day 1, latest by Day 4 post-treatment. With regards to body weight, a slight (< 10%) but statistically significant reduction of male body weights was observed for the high-dose group when compared to the control group. By Day 14, there was no difference in weight for these males relative to controls. For the FOB measurements, compound-related effects showing dose-dependency were noted at the time of peak neurobehavioral effects on Day 0 after treatment with all three doses. All compound-related effects rapidly resolved at all dose levels by the next test occasion, 7 days post-treatment. Compound-related and statistically significant decreases in motor activity (41% and 71%) and locomotor activity (40% and 99%) were observed for females of the mid- and high-dose groups, respectively, when compared to the control animals. Compound-related decreases in motor activity of 56% and locomotor activity of 95% were also observed on Day 0 in males of the high-dose group. Complete recovery occurred in all males and females within seven days following treatment. Habituation was not affected by treatment with the test substance. No compound-related effects were observed for brain weights, gross pathology or histopathological examinations. Based on the results of the study, an overall NOAEL was not established due to clinical signs and functional findings (FOB) at the lowest dose tested. Therefore, the LOAEL was set to 20 mg/kg bw and the NOAEL was assumed to be < 20 mg/kg bw for both sexes. Since an overall NOAEL was not established in this study, a follow-up study was conducted at lower dose levels to establish a NOAEL for the endpoints that were affected at a dose of 20 mg/kg bw.

In this follow-up study, fasted male and female Fischer 344 rats (12/sex/dose level) were treated with a single dose of 2.5 and 10 mg/kg bw (nominal) by gavage in the same manner as described above. Control animals received vehicle only. Based on the analytics, the actual doses were 3.1 and 11 mg/kg bw for male and female rats. The study design was identical to that described above with the exception that body weights, brain weight, gross necropsy and histopathology were not included, since no effects were observed at higher doses in the aforementioned study. No deaths and no clinical signs were observed at any dose level prior to scheduled terminal sacrifice. No compound-related effects were evident at the time of peak neurobehavioral effects on the day of treatment (day 0) in males or females at either dose level. In females, a decrease in motor (21%) and locomotor activity (27%) occurred at the high dose of 10 mg/kg bw, which did not reach statistical significance. However, since the reduction in motor and locomotor activity exceeded 20%, these effects are considered biologically significant. Habituation was not affected by treatment with the test substance. The overall NOAEL was 3.1 mg/kg bw (actual dose received) for females due to decreased motor and locomotor activities at 11 mg/kg bw. For male rats, the NOAEL was 11 mg/kg bw (actual doses received).

 

Subchronic

To assess the potential of the test substance with regards to subchronic neurotoxicity, a GLP- and guideline-compliant rat study (US-EPA-FIFRA 82 -5(b)) was conducted (M-003815-01-1). The test item was administered via the diet for 13 weeks to Fischer 344 rats (12/sex/dietary level) at nominal dose levels of 0, 50, 400 and 1600 ppm (corresponding to 0, 2.94, 24.2 and 101 mg/kg bw/day in males and to 0, 3.41, 27.9 and 115 mg/kg bw/day in females). Clinical observations, mortality, body weight, automated measurements of activity (figure-eight maze), FOB, brain weight, gross pathology and microscopic examination of the skeletal muscles, peripheral nerves, eyes and tissues from the central nervous system were included in the study. All rats were used for neurobehavioral evaluation and half of them for neuropathology. There were no compound-related clinical signs in males and females at any dose level and no deaths occurred before the scheduled terminal sacrifice. At the highest dose tested, a statistically significant reduction in body weight of approximately 12% was observed in male rats on Day 7 of exposure when compared to the control animals. The difference diminished thereafter, but the average body weight remained significantly lower than controls for most weeks of the study. The body weight of females was reduced by 6% on average on Day 7 when compared to the control group, recovered thereafter and was comparable to controls for the remainder of the study. Food consumption was statistically significantly reduced in the mid- (8-10%) and high-dose groups (34-37%) compared to controls, during the first week of treatment. For the remainder of the study, the animals of the mid-dose group consumed a comparable amount of food to that of the control animals, whereas for animals treated with 1600 ppm a reduced food consumption of approximately 6-15% was observed during many weeks of the remainder of the study. There were no compound-related ophthalmological findings at any dose level. With regards to the FOB measurements, no compound-related effects were observed in males or females at any dietary level. In addition, automated measures of motor activity, locomotor activity and habituation were not affected by any dietary dose level. Brain weight was not affected by treatment in either sex. There were no compound-related gross or histopathological lesions observed at terminal sacrifice in either sex. Under the test conditions used, the NOAEL for male and female rats regarding general systemic toxicity was set at 50 ppm, which was the lowest dose tested, corresponding to an actual ingested dose level of 2.94 mg/kg bw/day for males and 3.41 mg/kg bw/day for females. Since no obvious treatment-related effects indicative of neurotoxicity were seen up to the highest dose tested of 1600 ppm, this dose was set as NOAEL for male and female rats with respect to neurotoxicity, corresponding to an actual ingested dose level of 101 mg/kg bw/day for males and 115 mg/kg bw/day for the females.

 

Developmental neurotoxicity

A developmental neurotoxicity study, considered as key study was conducted according to GLP and the EPA OPPTS 870.6300 guideline (M-088059-01-1). The test substance or basal diet (control) were offered to the parental generation rats (25 female rats/dose) beginning on Day 0 of presumed gestation (DG 0) and continuing through DG 24 (rats that did not deliver a litter) or day 22 postpartum (DP 22) (dams that delivered a litter) at doses of 50, 300 and 500 ppm. During the exposure and postexposure periods, the rats were examined for clinical observations, signs of autonomic dysfunction, abnormal postures, abnormal movements or abnormal behavioral patterns, and unusual appearance daily beginning on DG 0 at approximately the same time each day. During gestation period, dams were evaluated for litter size, live litter size and pup viability at birth. Further, maternal behavior of the dams was evaluated on DPs 1, 5, 8, 14 and 22. Body weights and feed consumption were recorded daily during the exposure period. Pups were observed for viability (twice daily) and for clinical signs (daily during preweaning and weekly during postweaning period). On DP 5, litters were reduced to ten pups each. On DP 12, 20 appropriately sized litters per exposure group were selected for continued examination in four different subsets: Subset 1 was examined with regards to brain weights and neurohistology; subset 2 was used for passive avoidance and water maze testing; subset 3 was used for motor activity and auditory startle habituation testing; and subset 4 was examined for adult brain weights and neurohistology. In addition, one pup per sex was used to standardize litter size to eight pups per litter on DPs 12 to 22 and was assigned to subset 5. The F1 generation pups/rats selected for continued observation were sacrificed after completion of all postweaning behavioral evaluations. All pups/rats were necropsied and gross lesions were retained. The average mean daily intake of the test item was approximately 0, 4.4, 26 or 41 mg/kg bw/day during gestation and 0, 8.2, 49 and 83 mg/kg bw/day postpartum/during lactation. No deaths and clinical observations related to the test substance occurred in the P or F1 generation. Treatment-related changes in body weights were observed in parental (P) females at 300 and 500 ppm. Statistically significantly reduced body weight gains were observed in P females at 300 ppm and 500 ppm during the gestation period DG 0 to 6 (-30.6% and -55.9%) and additionally during the lactation period DL 1 to 4 at 500 ppm (-67.2%) when compared to the control animals. Average body weights were also significantly reduced during the gestation and lactation period in the mid- and high-dose group P females relative to the control group. Similarly, absolute and relative feed consumption was statistically decreased during the gestation period at 300 ppm (-4.5 to -6.3%) and 500 ppm (12.5% and 8.9%, respectively) compared to the control. Further, during lactation period P females treated with 500 ppm showed significantly reduced absolute food consumption values when compared to the control animals. Regarding the F1 generation, body weights as well as body weight gain were also statistically significantly reduced in mid- and high-dose F1 males and females and absolute food consumption was also reduced in mid- and high-dose F1 males when compared to the respective control animals. Relative food consumption was increased in mid- and high-dose F1 rats of both sexes due to the reduced body weights. Terminal body weights were also significantly decreased in males at 300 and 500 ppm and in females at 500 ppm of the F1 generation relative to the control group. There were not treatment-related effects on behavioral and neurological parameters (passive avoidance testing, water maze performance, motor activity and auditory startle habituation) in the F1 generation. Further, brain weights of the F1 generation revealed no statistically significant differences in males and females compared to the control. In addition, there was no microscopic evidence of any treatment-related neuropathologic processes in the F1 generation. F1 offspring of the mid- and high-dose groups exhibited a delay in sexual development (preputial separation in mid- and high-dose males and vaginal patency in high-dose females) which is considered to be secondary to body weight changes. In total, dietary exposure to the test item did not cause any neurotoxic effects in parental animals as well as their offspring up to the high dose of 500 ppm in the diet ( corresponding to 40.8 mg/kg bw/day and 82.8 mg/kg bw/day during lactation phase of dams). Both, the maternal as well as the offspring NOAEL were 50 ppm in the diet (corresponding to 4.4 mg/kg bw/day during gestation and 8.2 mg/kg bw/day during lactation phase of dams), based on reduced maternal and offspring body weight parameters and food consumption as well as a delay in sexual maturation (preputial separation and vaginal patency) of the F1 pups, which was considered to be secondary to body weight reduction.

Justification for classification or non-classification

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