imidacloprid (ISO); 1-(6-chloropyridin-3-ylmethyl)-N-nitroimidazolidin-2-ylidenamine

Neurotoxicity

Description of key information

Oral neurotoxicity studies

 

Key, M-028815-02-1, EPA-FIFRA, rat, acute,
NOAEL (systemic): 42 mg/kg bw (males), 20 mg/kg bw (females)

LOAEL (systemic): 152 mg/kg bw (males), 42 mg/kg bw (females)
NOAEL (neurotoxicity): 307 mg/kg bw (both sexes, highest dose tested)

Key, M-0227944-01-1, EPA-FIFRA, rat, 90 d:
NOAEL (systemic): 150 ppm (corresponding to 10.5 mg/kg bw/day in males and 9.3 mg/kg bw/day in females)

LOAEL (systemic): 1000 ppm (corresponding to 69.5 mg/kg bw/day in males and 63.3 mg/kg bw/day in females)

NOAEL (neurotoxicity): 3000 ppm (corresponding to 213 mg/kg bw/day in males and 196 mg/kg bw/day in females, highest dose tested)

 

Key, M-084646-01-1, EPA-FIFRA, rat, developmental,

NOAEL (maternal, systemic): 250 ppm (corresponding to 32.4 mg/kg bw/day over the whole gestation and lactation period)
LOAEL (maternal, systemic): 700 ppm (corresponding to 209.7 mg/kg bw/day over the whole gestation and lactation period)

NOAEL (developmental): 250 ppm (corresponding to 32.4 mg/kg bw/day over the whole gestation and lactation period)

LOAEL (developmental): 700 ppm (corresponding to 209.7 mg/kg bw/day over the whole gestation and lactation period)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jan - 30 Apr 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US-EPA-FIFRA, Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Guideline Addendum 10, Neurotoxicity; NTIS, EPA 540/09-91-123, PB 91-154617

Version / remarks:

adopted 1991

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Remarks:

CDF(F-344)/BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sasco, Inc., Madison, WI, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: 136.0 - 169.4 g (males), 108.7 - 125.3 g (females)
- Fasting period before study: not applicable
- Housing: individually in in suspended stainless steel wire-mesh cages with deotized Animal Cage Board as bedding (changed three times a week)
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, during the study period with corn oil and test material, ad libitum
- Water: tap water (Kansas City Municipal Water), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 40 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: From 25 Jan to 29 Apr 1993

Route of administration:

oral: feed

Vehicle:

corn oil

Mass median aerodynamic diameter (MMAD):

other: not applicable

Remarks on MMAD:

not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Description of preparation: Corn oil was used as the vehicle for the test item. A small amount of acetone served as a solvent in the diet preparation process but was evaporated before administration. The diet was Purina Mills Rodent Lab Chow 5001-4 in "etts" form.
- Rate of preparation of diet: not reported, a given batch was available ad libitum for consumption for one week, after which any left overs were collected and disposed.
- Storage temperature of food: not reported

VEHICLE
- Justification for use and choice of vehicle: corn oil, no justification provided
- Concentration in vehicle: not provided
- concentration of vehicle in the diet: 1% (w/w)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of the test item in the feed as well as stability after room temperature and freezer exposure was measured by liquid chromatography (LC). To establish stability and homogenity, 50 and 4000 ppm of test material in the diet were used and proved to be stable in the diet for 14 days at room temperature and 28 days at approximately -23 °C.

The concentrations of the test item in the administered rations were measured during weeks 1, 5, 9 and 14 of the study. It was found that the nominal concentration in diet was 140, 963 and 3027 ppm.

Duration of treatment / exposure:

13 weeks (90 days)

Frequency of treatment:

feed including the test substance was available ad libitum

Dose / conc.:

150 ppm (nominal)

Remarks:

corresponding to 10.5 mg/kg bw/day for males and 9.3 mg/kg bw/day for females (actual dose received)

Dose / conc.:

1 000 ppm (nominal)

Remarks:

corresponding to 69.5 mg/kg bw/day for males and 63.3 mg/kg bw/day for females (actual dose received)

Dose / conc.:

3 000 ppm (nominal)

Remarks:

corresponding to 213 mg/kg bw/day for males and 196 mg/kg bw/day for females (actual dose received)

No. of animals per sex per dose:

12 (behavioral testing, with half used for neuropathology)
6 (satellite animals)

Control animals:

yes

Details on study design:

- Dose selection rationale: Doses were selected based on two subchronic dietary exposure studies. At 2400 and 3000 ppm (the highest doses administered), body weight gain was reduced by 13-21% in both sexes but no overt clinical signs were observed. At 600 ppm, males showed slightly decreased body weight gain and morphologic evidence of liver enzyme induction. At 2400 ppm males showed signs of hepatoxicity that were reversible after 4 weeks recovery. At 3000 ppm, similar hepatotoxic effects were observed in males and females. Males exhibited low-grade degenerative changes in the epithelium of the testicular tubules. The NOEL was set to 600 ppm for females and 150 ppm for males. Based on this results, the doses in this study were set to 0, 150, 1000 and 3000 ppm. 150 ppm should cause no effect, 1000 ppm an intermediate level of toxicity and the highest dose should cause clear signs of toxicity without being lethal.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, daily on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and pre-terminal (week 12)
- Dose groups that were examined: all study animals
- parameters assessed: pupillary reflex (using a penlight or transilluminator), mydriasis, examination of conjunctiva, cornea, and lens (with slit lamp microscope), vitreous humor, retina, choroid, and optic disc (with indirect ophthalmoscope and a condensing lens)

Specific biochemical examinations:

CLINICAL CHEMISTRY: Yes
-Time schedule for examination: weeks 4 and 13
- how many animals: 6 animals per sex/group (satellite animals)
- Animals fasted: No
- analysed parameters: sodium, potassium, chloride, urea nitrogen, glucose, creatinine, uric acid, triglycride, cholesterol, creatine kinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, total bilirubin, total protein, albumin, phosphorus, calcium, globulin

HEMATOLOGY: Yes
-Time schedule for examination: weeks 4 and 13
- how many animals: 6 animals per sex/group (satellite animals)
- Animals fasted: No
- analysed parameters: blood count (incl. platelet, leukocyte, erythrozyte count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration), differential blood smear (leukocyte differential, blood cell morphology)

Neurobehavioural examinations performed and frequency:

Acclimatisation: One day prior to testing, rats were transferred to the room where testing took place and allowed to acclimate with minimal disturbance.

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Home cage observations: posture, piloerection, involuntary motor movements (clonic, tonic), gait
abnormalities, vocalization
- Observations during handling: ease of removing the rat from its cage, reaction to being handled,
muscle tone (if abnormal), palpebral closure (if abnormal), lacrimation, salivation, nasal discharge, stains
- Open field observations: the rat was placed in the centre of a flat surface with a perimeter barrier, such as a cart, for 2 minutes. During this time, the number of rears was counted and other observations were made, including piloerection, respiratory abnormalities, posture, involuntary motormovements (clonic and tonic), stereotypy, bizarre behavior (if present), gait abnormalities, vocalization, arousal, rearing, excretion (defaecation and urination).
- Reflex/physiologic observations and measurements: following the open field observation, the fol
lowing parameters were analysed: Approach response, touch response, auditory response, tail pinch response, righting reflex, grip strength, landing foot splay, body weight and body temperature
- Description of procedures: The FOB closely followed the battery of tests described by Moser (Moser, C.V., 1989), with each animal tested individually. Scoring criteria and explicitly defined scales were used to rank the severity of observations that do not readily lend themselves to quantitation. All animals that were assigned to the study were tested using the FOB and motor activity. On the day of testing, the animals were transferred to the room where testing took place and allowed to acclimate with minimal disturbance for at least 30 minutes prior to testing. 30 min after the last animal had been testes, rats were transferred individually in figure-eight mazes to test motoractivity.
- Minimization of bias: rats were semi-randomly assigned, technicians were blind to treatment
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: - Time schedule: once during the week prior to initiating the exposure and again during weeks 4, 8 and 13
- Noise level: Broad-spectrum background noise (approximately 70dB(A)), generated by a white noise generator (Model S81-02, Coulbourn Instuments)

LOCOMOTOR ACTIVITY: Yes
- Replicates used: All animals that were assigned to the study were tested.
- Time schedule: once during the week prior to initiating the exposure and again during weeks 4, 8
and 13
- Type of equipment used: figure-eight mazes, 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. 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: 90 min and during each ten-minute interval
- Parameters measured: Motor activity was measured as the number of beam interruptions that occur red during the test session, Locomotor activity was measured by eliminating consecutive counts for a given beam.

Sacrifice and (histo)pathology:

- Time point of sacrifice: week 13
- Number of animals sacrificed: all surviving animals (6 with perfusion, see below, the rest without perfusion, complete necropsy or collection of tissues)
- Parameters measured for perfused animals: body weight, examination of all organs, body cavities, cut surfaces, external orifices and surfaces.
- Brain weight: yes
- Length and width of brain: yes

PERFUSION
- Procedures for perfusion: animals were deeply anesthetized with an intraperitoneal dose of sodium pentobarbital (approximately 50 mg/kg bw). Perfusion was performed via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by in situ fixation using Universal fixative (4% (w/v) glutaraldehyde and 4% (w/v) EM-grade formaldehyde) in phosphate buffer. All examined tissues (see below) were dissected from each animal and post-fixed in 10% buffered formalin.
- Number of animals perfused: 6 of each sex/dose
- Tissues evaluated: entire brain and spinal cord, both eyes (with optic nerves) and selected (bilateral) peripheral nerves (sciatic, tibial and sural), the gasserian ganglion, gastrocnemius muscle and physical identifier

HISTOLOGY
- Number of animals evaluated from each sex and treatment group: 6 of control and high dose group

H&E staining:
- Type of staining: Hematoxylin and Eosin (H&E)
- Embedding media: paraffin
- tissues: olfactory region of the brain, forebrain, midbrain, pons, medulla oblongata and cerebellum, cross- and longitudinal-sections from three levels of the spinal cord (cervical, thoracic and lumbar)
- Thickness: not reported
- Number of sections: 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), cauda equina, gasserian ganglion, eyes, optic nerves, gastrocnemius muscle and additional tissue from the hippocampus and cerebellar cortex
- Thickness: 2 - 3 μm
- Number of sections: not reported

Toluidine Blue staining
- Type of staining: Toluidine Blue
- Embedding media: Epon
- tissues: Peripheral nerve (sciatic, tibial and sural) tissues, sciatic nerve (longitudinally)
- Thickness: 1 μm
- Number of sections: 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: not reported
- tissues: Additional sections from each region of the brain and spinal cord

Other examinations:

The tissues from the high dose group and control animals were compared. Since no treatment-related lesions were evident, further examinations were not performed.

Positive control:

no

Statistics:

In general, statistical evaluations were performed using software from either INSTEM Compute Systems or SAS. Statistical signifance was set at p<0.05 or for Barlet's Test at p<0.001.

Continuous data was analysed using an Analysis of Variance (ANOVA). If a significant F-Test was
determined, a Dunnett's Test was performed. For the FOB, continuous data were first analysed using a Repeated-Measurers ANOVA, followed by a one-way ANOVA if there was a significant interaction between dose group and test week. To identify groups that were different from the control group, a Dunnett's test was performed. For motoractivity, the session activity was analysed in the same manner. Categorical data collected in the FOB were analysed 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.

For interval data of the motoractivity test, a two-way Repeated-Measures ANOVA, using both test interval and test occasion as the repeated measures was performed, followed by a Repeated Measures ANOVA to determine on which weeks there was a significant treatment by interval interaction. Thereafter, the data was subjected to a one-way ANOVA followed by Dunnett's test to determine which groups differed from the control.

If variance was unequal, nonparametric statistical procedures were used, namely the Kruskal-Wallis ANOVA followed by the Mann-Whitney U test for between-group comparisons.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical signs were considered non-treatment related since they were almost comparable in incidence and severity in all groups including the control, or occurred occasionally in single animals. Those signs included red lacrimal stain, nasal stain, urine stain, alopecia on forepaws and perianal stain.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

not applicable

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduction in body weight was seen with the following means:
- 1000 ppm: 8% (males), 4% (females)
- 3000 ppm: 15% (males), 7% (females)

Body weight in the lowest dosage comparable to controls; deviations at 1000 ppm was below 10% therefore irrelevant, and at 3000 ppm, deviation was relevant only for the male animals.

Summarized data can be found in Table 2 in Attachment 1.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced in males and females that received 1000 ppm and 3000 ppm test substance throughout the study (consistent with findings for body weight). Mean reduction of food consumtion over the exposure period were
- 1000 ppm: 8% (males), 6% (females)
- 3000 ppm: 16% (males), 11% (females)

Food consumtion was not changed in the low dose group. In the middle dose, changes were below 10% and therefore not relevant, in the 3000 ppm dose group, reduced food consumption was only relevantly in males. Summarized data can be found in Table 3 in Attachmend 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):

Incidental ophthalmic findings consisting of minimal to slightly severe corneal opacity and chromodacryorrhea were reported for all test groups including the control, for both, male and female rats. Corneal opacity has been described as part of spontaneous corneal dystrophy which is commonly seen in Fischer 344 rats.

Haematological findings:

no effects observed

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males
- 150 ppm: increased creatine kinase (CK) activity in week 4 (+154%)
- 1000 ppm: increased creatine kinase (CK) activity in week 4 (+145%), decreased CK activity in week 13 (-51%)
- 3000 ppm: increased CK activity in week 4 (+209%), reduction in CK activity in week 13 (-82%), lower triglyceride levels (-36% in week 4, -26% in week 13), reduced lactate dehydrogenase (LDH) activity (-51% in week 13), decreased phosphate levels (-9% in week 4, -10% in week 13)


Females
-150 ppm: increased glucose levels in week 13 (+10%)
- 1000 ppm: increased glucose levels in week 13 (+15%), reduced LDH activity (-58% in week 13), decreased CK activity(-47%), increased ALT levels (+38% in week 4)
- 3000 ppm: increased glucose levels in week 13 (+12%), lower triglyceride levels (-41% in week 4, -35% in week 13), reduced total protein (-5 % in week 4), reduced albumin (-6 % in week 4), increased ALT levels (+53% in week 4), decreased phosphate levels (-9% in week 4, -10% in week 13), reduced LDH level (-67% in week 13), reduced CK activity (-43% in week 13)

Details can be found in Table CC1-SUM in Attachment 3.

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):

Functional Observational Battery (FOB)
- 3000 ppm: slightly uncoordinated righting response (males, week 13), lower forelimb grip strength (males, week 8)
Since the lower forelimb grip strength appears to be in line with a reduction in body weight and was not seen at the end of the exposure period, the effect cannot be directly linked to neurotoxicity but is rather considered a secondary effect of reduced body weight.

Further incidental findings were reported for all groups including the control, comprising reduced exploratory movement in the open field and slightly uncoordinated righting response. The reduced exploratory movement in the open field was observed for all female groups and there was no dose-relationship for males, it is therefore considered incidental. The slightly uncoordinated righting response was comparable to controls and the incidents of high dose males sitting or lying in a normal position during observation was a single occuring event.

Summarized data can be found in Table 5 and 6 in the Attachment 4.

Motor and locomotor activity
In general, differences of less than 20% were considered to be within the range of normal variability in the testing laboratory for groups of 12 rats/sex/dose level.

Activity during sessions

- 3000 ppm: slightly higher motor activity (males, 17-26%), slightly higher locomotor activity (males, 23-33%), lower motoractivity (females, 1-11%), lower locomotor activity (9-21%)
All observations were considered incidental due to the lack of statistical significance.

Activity during intervals
- 3000 ppm: slightly higher activity in first test intervals (males, week 4), slightly lower activity (females, week 8)
The motor and locomotor activity during test intervals was related to age and sex differences and therefore not treatment-related. The described motor and locomotor activity were not considered treamtent relevant.

Summarized data can be found in Table 7 and 8 (sessions) and 9 and 10 (intervals) in the Attachment 4.

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):

No treatment-related changes were noticed regarding the absolute and relative brain weight of the treated male and female rats, as compared to the control animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy gross pathological examination revealed no treatment-related abnormalities in both, male and female rats.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Particular examination of brain, and spinal cord revealed no treatment-related abnormalities in both, male and female rats.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination revealed no treatment-related effects in skeletal muscle and nervous system tissue in both, male and female rats.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Histopathology revealed no neoplastic changes in both, male and female rats..

Description (incidence and severity):

not applicable

Details on results:

Historical control data can be found in Attachment 5.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

150 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

Remarks on result:

other: corresponding to 69.5 mg/kg bw/day for males and 63.3 mg/kg bw/day for females (actual dose received)

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxcity

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 196 mg/kg bw/day for the females and 213 mg/kg bw/day for the males (actual dose received)

All tables can be found in the attached background material. 

Conclusions:

The present guideline study was conducted in compliance with GLP. Under the test conditions used, the NOAEL for male and female rats regarding overall toxicity was set at 150 ppm which was the lowest dose tested, corresponding to an actual ingested dose level of 9.3 mg/kg bw/day for the females and 10.5 mg/kg bw/day for the males. Since no obvious treatment-related effects indicative of neurotoxicity were seen up to the highest dose tested of 3000 ppm, this dose was set as NOAEL for male and female rats with respect to neurotoxicity, corresponding to an actual ingested dose level of 196 mg/kg bw/day for the females and 213 mg/kg bw/day for the males.

Reference 2

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Main study: 30 Nov - 18 Dec 1992 Supplementary study: 13 - 28 Apr 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA-FIFRA, Addendum 10, EPA 540/09-91-123, PB 91-154617

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sas:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sasco, Inc., St. Louis, MO, USA
- Age at study initiation: 9 weeks
- Weight at study initiation: Main study: 167 - 304 g (males), 150 - 185 g (females); Supplementary study: 148 - 198 g (females)
- Fasting period before study: overnight
- Housing: individually in suspended stainless steel wire-mesh cages; bedding was deotized Animal Cage Board (changed three times per week)
- Diet: Purina Mills Rodent Lab Chow 5001-4, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 40 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: From 13 Apr to 28 Apr 1994

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) methylcellulose with 0.4% (w/v) Tween 80 in deionized water

Mass median aerodynamic diameter (MMAD):

other: not applicable

Remarks on MMAD:

not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test material was solubilized in in deionized water containing 0.5% methylcellulose and 0.4% Tween 80. The solution was administered via gavage
- Justification for use and choice of vehicle: not reported
- Concentration in vehicle: 5, 15 and 35 mg/mL (nominal)
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing suspension was analyzed to measure the concentration of the test material using liquid chromatography (LC).

Duration of treatment / exposure:

not applicable since single treatment

Frequency of treatment:

single exposure

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

corresponding to 20 mg/kg bw/day (actual dose received), only females

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

corresponding to 42 mg/kg bw/day (actual dose received), males and females

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

corresponding to 151 mg/kg bw/day (actual dose received), males and females

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

corresponding to 307 mg/kg bw/day (actual dose received), males and females

No. of animals per sex per dose:

Main test: 12 (for behavioral testing, including 6 for neuropathology)
Main test: 6 (satellite group animals for clinical pathology)
Supplementary test: 12

Control animals:

yes

Details on study design:

- Dose selection rationale: Doses were selected on the basis of an acute oral toxicity study and a dose range-finding study. In the acute toxicity study, 400 mg/kg bw caused mortality in both sexes and 50 mg/kg bw and 100 mg/kg bw were the NOEL for male and female rats, respectively. In the dose range-finding study, no mortality occured up to 417 mg/kg bw but severe signs of toxicity were observed in both sexes at 345 and 417 mg/kg bw. No toxicity was noticed at 52 or 109 mg/kg bw in males and at 52, 109 or 200 mg/kg bw in females.

Based on these results, 50, 150 and 350 mg/kg bw were selected for both sexes. Thereby, 50 mg/kg bw should not cause toxicity, while 150 mg/kg bw should produce intermediate toxicity and 350 mg/kg bw should cause clear signs of toxicity without mortality. The dose for the supplementaty study (20 mg/kg bw) was used since no NOEL could be found for females in the main study.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on week days, daily on weekends and holidays; once daily for the supplementary study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

CLINICAL CHEMISTRY: Yes
-Time schedule for examination: 24 h after administration
- how many animals: 6 animals per sex/group (excluding the animals of the supplementary test)
- Animals fasted: No
- analysed parameters: sodium, potassium, chloride, urea nitrogen, glucose, creatinine, uric acid, triglycride, cholesterol, creatine kinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, total bilirubin, total protein, albumin, phosphorus, calcium, globulin

HEMATOLOGY: Yes
-Time schedule for examination: 24 h after administration
- how many animals: 6 animals per sex/group (excluding the animals of the supplementary test)
- Animals fasted: No
- analysed parameters: blood count (incl. platelet, leukocyte, erythrozyte count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration), differential blood smear (leukocyte differential, blood cell morphology)

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Home cage observations: posture, piloerection, involuntary motor movements (clonic, tonic), gait abnormalities, vocalization
- Observations during handling: ease of removing the rat from its cage, reaction to being handled, muscle tone (if abnormal), palpebral closure (if abnormal), lacrimation, salivation, nasal discharge, stains
- Open field observations: the rat was placed in the centre of a flat surface with a perimeter barrier, such as a cart, for 2 minutes. During this time, the number of rears was counted and other observations were made, including piloerection, respiratory abnormalities, posture, involuntary motor movements (clonic and tonic), stereotypy, bizarre behavior (if present), gait abnormalities, vocalization, arousal, rearing, excretion (defaecation and urination)
- Reflex/physiologic observations and measurements: following the open field observation, the following parameters were analysed: Approach response, touch response, auditory response, tail pinch response, righting reflex, grip strength, landing foot splay, body weight and body temperature.
- Description of procedures: The FOB closely followed the battery of tests described by Moser (Moser, C.V., 1989), with each animal tested individually. Scoring criteria and explicitly defined scales were used to rank the severity of observations that do not readily lend themselves to quantitation. All animals that were assigned to the study were tested using the FOB and motor activity. On the day of testing, the animals were transferred to the room where testing took place and allowed to acclimate with minimal disturbance for at least 30 minutes prior to testing. 30 min after the last animal had been testes, rats were transferred individually in figure-eight mazes to test motoractivity.
- Minimization of bias: rats were semi-randomly assigned, technicians were blind to treatment
- Same technicians used throughout testing: Yes if possible
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: one week prior to treatment, approximatly 90 min after administration, 7 and 14 days after administration. Each session took 90 min.
- Noise level: Broad-spectrum background noise (approximately 70dB(A)), generated by a white noise generator (Model S81-02, Coulbourn Instuments)

LOCOMOTOR ACTIVITY: Yes
- Replicates used: All animals that were assigned to the study were tested.
- Type of equipment used: figure-eight mazes, 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. 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: 90 min and during each ten-minute interval
- Parameters measured: Motor activity was measured as the number of beam interruptions that occurred during the test session, Locomotor activity was measured by eliminating consecutive counts for a given beam.

Sacrifice and (histo)pathology:

- Time point of sacrifice: 15 days post-treatment (after the final FOB and motor activity tests)
- Number of animals sacrificed: 12 (all surviving except satellite animals)
- Parameters measured: examination of all organs, body cavities, cut surfaces, external orifices and surfaces
- Brain weight: yes
- Length and width of brain: yes

PERFUSION
- Procedures for perfusion: animals were deeply anesthetized with an intraperitoneal dose of sodium pentobarbital (approximately 50 mg/kg bw). Perfusion was performed via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by in situ fixation using Universal fixative (4% (w/v) glutaraldehyde and 4% (w/v) EM-grade formaldehyde) in phosphate buffer
- Number of animals perfused: 6 of each dose/sex
- Tissues evaluated: entire brain and spinal cord, both eyes (with optic nerves) and selected (bilateral) peripheral nerves (sciatic, tibial and sural), the gasserian ganglion, gastrocnemius muscle and physical identifier

HISTOLOGY
- Number of animals evaluated from each sex and treatment group: 6 of control and high dose group

H&E staining:
- Type of staining: Hematoxylin and Eosin (H&E)
- Embedding media: paraffin
- tissues: olfactory region of the brain, forebrain, midbrain, pons, medulla oblongata and cerebellum, cross- and longitudinal-sections from three levels of the spinal cord (cervical, thoracic and lumbar)
- Thickness: not reported
- Number of sections: 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), cauda equina, gasserian ganglion, eyes, optic nerves, gastrocnemius muscle and additional tissue from the hippocampus and cerebellar cortex
- Thickness: 2 - 3 µm
- Number of sections: not reported

Toluidine Blue staining
- Type of staining: Toluidine Blue
- Embedding media: Epon
- tissues: Peripheral nerve (sciatic, tibial and sural) tissues, sciatic nerve (longitudinally)
- Thickness: 1 µm
- Number of sections: 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: not reported
- tissues: Additional sections from each region of the brain and spinal cord

Other examinations:

none

Positive control:

no

Statistics:

In general, statistical evaluations were performed using software from either INSTEM Computer Systems or SAS. Statsistical significance was set at p<0.05 or for Barlet's Test at p<0.001.

Continuous data was analysed using an Analysis of Variance (ANOVA). If a significant F-Test was determined, a Dunnett's Test was performed. For the FOB, continuous data were first analysed using a Repeated-Measurers ANOVA, followed by a one-way ANOVA if there was a significant interaction between dose group and test week. To identify groups that were different from the control group, a Dunnett's test was performed. For motoractivity, the session activity was analysed in the same manner. Categorical data collected in the FOB were analysed 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.
For interval data of the motoractivity test, a two-way Repeated-Measures ANOVA, using both test interval and test occasion as the repeated measures was performed, followed by a Repeated Measures ANOVA to determine on which weeks there was a significant treatment by interval interaction. Thereafter, the data was subjected to a one-way ANOVA followed by Dunnett's test to determine which groups differed from the control.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The number in brakets give the number of animals depicting the described symptom
- 150 mg/kg bw: 1 male showed tremors and red nasal stain
- 300 mg/kg bw: males showed tremors (9), red nasal stain (7) incoordinated gait (8), decreased activity (7), a cool-to-touch body (4), urine stain (2), females showed tremors (6), incoordinated gait (5), decreased activity (5), increased reactivity (2), red nasal stain (3) and a cool-to-touch body (4).
Clinical signs of toxicity were generally observed on day 0 and resolved in all surviving males and females within one to five days following treatment.
Some additional findings were reported, which however were not considered to be treatment-related. For females, these included urine stain, red lacrimal stain and a partially closed left eyelid in 1 control animal and red lacrimal stain in 1 high dose animal. In males, one animal of 150 mg/kg bw dose had a broken teeth, and therefore oral stain, red lacrimal stain and labored breathing. In 1 control animal, alopecia on front paws was observed. 1 middle dose and 1 high dose animal showed a perianal stain, which was comparable to control animal data from other studies.

Details can be found in Table 1 in Attachment 1.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicabel

Mortality:

mortality observed, treatment-related

Description (incidence):

- 4/18 males in the high dose group (2 in main study group, 2 in satellite group), and 10/18 females in the high dose group (8 in main group, 2 in satellite group) died. All deaths occurred on the day of administration or the day after.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Surviving high-dose males had a slightly lower body weight than control animals (4%). However, this was not considered biologically significant.

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 applicabel

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 300 mg/kg bw: increased hemoglobin concentration, increased hematocrit, mature neutrophilia and a lymphopenia (females)

Summarized data on hematology can be found in Table HE1-SUM, HE2-SUM and HE3-SUM in Attachment 2.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 150 mg/kg bw: decreased serum triglycerides (males and females)
- 300 mg/kg bw: decreased serum triglycerides, reduction in serum ALT activity (males and females), decreased serum potassium and cholesterol (females)

Summarized data on Clinical Chemistry can be found in Attachment 3 (Table CC1-SUM). Historical control data can be found in Attachment 6.

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Observational Battery (FOB)
- 150 mg/kg bw: increased incidence of sitting or lying in a normal position (rather than standing) in the home cage, tremors and nasal stain (males, day 0), tremors (females, day 0)
- 300 mg/kg bw: incoordinated gait, tremors, increased reactivity, decreased activity in the home cage, an increased incidence of sitting or lying in a normal position (rather than standing) in the home cage, evidence of decreased activity during open field observation (i.e., sitting or lying in a normal position with either minimal head or body movement or only some exploratory movement and a decreased incidence of rearing), decreased response to touch and tail pinch stimuli, impaired righting response, decreased grip strength and decreased core body temperature (all symptoms for males and females), nasal stain (males), decreased response to an auditory stimulus and urine stain (females).

All treatment-related effects were considered to be caused by acute cholinergic toxicity. Signs of toxicity were reversible in surviving animals by the next test occasion (7 days after treatment). 4 females were not tested on the day of treatment because they were physically unable to perform the test at that time.

Observations that were not considered treatment related were
- Controls: perianal stain, alopecia (males), palpebral closure, lacrimal stain (females), various vocalizations upon removal from the home cage (males), slightly impaired righting responses (males and females), slightly impaired righting responses (males and females, several days)
- 20 mg/kg bw: ease of removal (day 7), approach response (pretreatment and day 7), only for females examined
- 50 mg/kg bw: slight response (rather than no response) to tail pinch (females, occurred already prior to treatment), lower incidence of vocalization when removed from the home cage (males, , occurred already prior to treatment), no reaction to touch and tail pinch (1 male, single occurrence), decreased incidences of rearing on non-treatment days (females), slightly impaired righting responses (males and females, several days)
- 150 mg/kg bw: lacrimation, nasal stain and labored breathing (1 male, due to the broken teath described above), lower incidence of vocalization when removed from the home cage (males, , occurred already prior to treatment), decreased incidences of rearing on non-treatment days (males and females)

Motor and Locomotor Activity:
In general, differences of less than 20% were considered to be within the range of normal variability in the testing laboratory for groups of 10-12 rats/sex/dose level. Statistics for the high dose were done with the four females and 10 males that survived and were tested on all four occasions.
- 50 mg/kg bw: decrease in session motor and locomotor activity (females, 25 - 27%) on day 0, no effects were observed on day 7
- 150 mg/kg bw: decrease in session motor and locomotor activity (males, 21-25%, and females, 46-48%) on day 0, no effects were observed on day 7 (males and females)
- 300 mg/kg bw: decrease in session motor and locomotor activity (males, 73-78%, and females, 81-89%) on day 0, no effects were observed on day 7 (females), males recovered almost completely at day 7 but still showed slightly lower activity on day 14

Results on 10 minute intervals:
- 50 mg/kg bw: decrease in interval motor and locomotor activity; females: on day 0 for the first seven or eight intervals, no effects were observed on day 7
- 150 mg/kg bw: decrease in interval motor and locomotor activity; males: on day 0 during the first two to four intervals; females: on day 0 for the first seven or eight intervals, no effects were observed on day 7 (males and females)
- 300 mg/kg bw: severely reduced activity during the first interval and minimal levels of motor and locomotor activity throughout the remainder of the test session (males and females), no effects were observed on day 7 (females), slightly reduced activity during the early intervals of the test session (males, day 7 and 14)

Observations that were not treatment related:
- Control animals: substantially lower levels of motor and locomotor activity on day 0, relative to all other test days (contributed to overnight fasting)
- 50 mg/kg bw: minimally (5-9%) lower levels of motor and locomotor activity (males, within the normal variability),

Data can be found in Table 3-9 of the main study and Table 2 - 7 of the supplementary study (Attachment 4 and 5, respectively)

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):

Mean absolute and relative organ weights were determined, no significant differences to controls were found.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Because of early deaths, only 4 high-dose females were available for necropsy at terminal sacrifice, 15 days following treatment.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Description (incidence and severity):

Not applicable

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

clinical biochemistry

clinical signs

haematology

Conclusions:

The present guideline study was done in compliance with GLP. Under the conditions of the test, the NOAEL for overall toxicity was set at 42 mg/kg bw for males and females; the NOAEL for neurotoxicity was 307 mg/kg bw for both sexes.

Reference 3

Endpoint:

neurotoxicity, other

Remarks:

Developmental Neurotoxicity Screening Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jul - Oct 1999

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:

yes

Remarks:

the period of exposure was extended to cover the entire period of gestation and lactation. i.e., from gestation day GD 0 to lactation day LD 21 rather than from GD 6 to LD 10.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:W(HAN)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 15 weeks (males) and 12 weeks (females) at the time of co-housing. Males served only as breeders
- Fasting period before study: not applicable
- Housing: For co-housing one male and one female in the males cage, apart from that, animals were kept individually in individually in suspended stainless-steel cages. During gestation and lactation, individual dams and their litters were housed in plastic cages with corn cob bedding.
- Diet: Purina Mills Rodent Lab Chow 5001-4 in ‘etts’ form, ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: From 12 Jul to 15 Oct 1999

Route of administration:

oral: feed

Vehicle:

corn oil

Mass median aerodynamic diameter (MMAD):

other: not applicable

Remarks on MMAD:

not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Corn oil was used as the vehicle for the test substance; acetone served as a solvent in the diet preparation process and was allowed to evaporate prior to treatment.

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: Purina Mills Rodent Lab Chow 5001-4 in ‘etts’ form
- Storage temperature of food: stored in the freezer (-20 °C until use)

VEHICLE
- Vehicle used: corn oil
- Concentration in diet: 1% by weight of the diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogenity of the test material in the diet has been verified over the range of dietary concentrations. Concentration of the test material in the rations were measured three times during the study. Concentrations were tested by liquid chromatography (LC).

The analytical concentration in the diet is 95.5, 227 and 691 ppm.

Duration of treatment / exposure:

From beginning of gestation (Gestation Day 0, GD0) until the end of lactation (LD21), after that, animals received the diet without test substance

Frequency of treatment:

daily, 7 days/week, the rations were changed weekly and the residual feed was discarded

Dose / conc.:

100 ppm (nominal)

Remarks:

corresponding to 8.0-8.3 mg/kg bw/day ingested dose during gestation (GD 0-21) and 12.8-19.5 mg/kg bw/day ingested dose during lactation (LD 0-21)

Dose / conc.:

250 ppm (nominal)

Remarks:

corresponding to 19.4-19.7 mg/kg bw/day ingested dose during gestation (GD 0-21) and 30.0-45.4 mg/kg bw/day ingested dose during lactation (LD 0-21)

Dose / conc.:

700 ppm (nominal)

Remarks:

corresponding to 54.7-58.4 mg/kg bw/day ingested dose during gestation (GD 0-21) and 80.4-155.0 mg/kg bw/day ingested dose during lactation (LD 0-21)

No. of animals per sex per dose:

25-30 P males, 25-20 P females
ca 80 F1 males and 80 F1 females

Control animals:

yes

Details on study design:

- Dose selection rationale: the doses were based on a two-generation reproduction study in Wistar rats, where rats were fed with 0, 100, 250 and 700 ppm test substance. Toxicity was observed at 700 ppm only, and manifested as reduced body weight gain and food consumption in parental animals and the F1-generation males and females. The overall NOEL for this study was 250 ppm for males and females. There were no compound related clinical signs or effects on reproduction or pup viability at any dietary level nor were there compound-related gross or microscopic lesions at any level of exposure. Therefore, the following dose levels were selected for the present study: 100, 250 and 750 ppm.. 750 ppm should cause toxicity and can be seen as the maximum-tolerated dose (MTD). The 100 ppm dietary level was selected to produce no evidence of toxicity and the 250 ppm dietary concentration was selected as an intermediate dose to assist in establishing compound related effects and deriving a NOAEL.

Observations and clinical examinations performed and frequency:

1. Parental Generation

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly (for gestation: GD 0-6, 6-13, 13-20, for lactation: LD 0-7, 7-14 and 14-21)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: weekly (for gestation: GD 0-6, 6-13, 13-20, for lactation: LD 0-7, 7-14 and 14-21), especially in the third week after birth, pups may have eaten some of the feed
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

2. F1 Generation

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the preweaning period and once weekly after weaning

BODY WEIGHT: Yes
- Time schedule for examinations: Postnatal day (PND) 0, 4, 11, 17, and 21, and once weekly thereafter, also at the time of vaginal patency and preputial separation

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: weekly from the week of PND 28, when they were placed into single housing, until termination
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: approximately 50-60 days of age
- Dose groups that were examined: minimum of 10/sex/dietary level; representing 20 litters per level (all of them were selected for perfusion at study termination
- parameters examined: pupillary reflex (with a penlight or transilluminator), pupillary dilatation, eyelid, conjunctiva, cornea, aqueous humor, and lens (with a slit lamp microscope either), mydriasis, after that vitreous humor, retina, choroid, and optic disc (with an indirect ophthalmoscope equipped with a condensing lens)

Specific biochemical examinations:

CLINICAL BIOCHEMISTRY: No

HEMATOLOGY: No

Neurobehavioural examinations performed and frequency:

1. P Generation
DETAILED OBSERVATIONAL BATTERY (Abbreviated FOB): Yes
- Number of animals: min 10 dams/dietary level
- Time schedule: GD 6, 13 and 20, LD 4, 11 and 21
- Description of procedures: observations in the home cage, during handling, and outside the home cage in an open field, including lacrimation, salivation, piloerection, exophthalmia, urination, defecation, pupillary function, palpebral closure, convulsions, tremors, abnormal movements, unusual behaviors, posture and gait abnormalities
- Minimization of bias: the laboratory maintains evidence of inter-observer reliability (agreement) for individuals who were involved with performing these observations
- Same technicians used throughout testing: No
- Technicians were blind to treatment status of animals: Yes
- Environmental conditions: not reported
- Noise level: not reported
- Duration of observation period for open field observations: not reported

2. F1 Generation
Additional information for developmental neurotoxicity study:
- Number of offspring/sex/group examined on postnatal days (unless given in animal assignment table) outside the home cage: 16/sex/dietary level, representing at least 20 litters/level
- Description of procedures used for each age at which offspring were examined: same as for parents but neonates (i.e., PND 4 and 11) were not evaluated in the open field
- Same offspring evaluated at each time point: yes
- Same parameters assessed in the maternal abbreviated FOB examined for offspring: yes

Developmental Landmarks and Behavior:
Developmental landmarks
- Number of animals: 20 offspring per sex and dose (1 male and female per litter)
- Time schedule: daily
- parameters checked: surface righting (from PND 4), auditory startle response (from PND 10) and eye opening (from PND 11)
Sexual Maturation and Pupil Constriction
- Number of animals: all pups
- Time schedule: daily
- parameters checked: vaginal patency (females, from PND 29),preputial separation (males, from PND 38), pupil constriction (only on PND 21)

MOTOR ACTIVITY
- Number of animals: 16 offspring per sex and dose (1 male and/or female per litter)
- Days of testing: PND 13, 17, 21 and 60 (+2 days)
- Type of equipment used: figure-eight mazes, 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. A Columbus Instruments (Columbus, OH) Universal Maze Monitoring System and a personal computer were used for automated data collection.
- Length of session, number and length of subsessions: 60 min and during each ten-minute interval
- Parameters measured: Motor activity was measured as the number of beam interruptions that occurred during the test session, Locomotor activity was measured by eliminating consecutive counts for a given beam, Habituation was evaluated as a decrement in activity over consecutive intervals of the test session.

ACOUSTIC STARTLE HABITUATION: Yes
- Number of animals: 16 offspring per sex and dose (1 male and/or female per litter)
- Days of testing: PND 22, 38 (+2 days) and 60 (+2 days)
- Same offspring evaluated at each preweaning time point: yes
- Exact age: not reported
- Type of equipment used: personal computer to control the operation of an integrated startle response test system (Coulbourn Instruments, Allentown, PA) and for automated data collection.
- Environmental conditions: The enclosure was ventilated, lined with sound-attenuating and vibration-absorbing material, and had two speakers
- Number of trials performed: 50-trial test session, blocks of 10 trials
- Length (msec) and intensity (dB) of sound: eliciting stimulus (S2) - a 50-msec burst (0 msec rise/fall) of broadspectrum "white" noise [approximately 120 dB(lin)]
- Length of interval between trials: not reported
- Other procedural details: testing strategy was referred to as in P. H. Ruppert, K. F. Dean and L. W. Reiter, "Development of behavioral toxicity following acute postnatal exposure of rat pups to trimethyltin”, Neurobehavioral Toxicology and Teratology, 5, pp. 421-429, 1983.

LEARNING AND MEMORY TESTING: Yes
Passive Avoidance Conditioning
(1) Overall testing design
- Number of animals: 16 offspring per sex and dose (1 male and/or female per litter)
- Days of testing: PND 24 and PND 31
- Evaluation of both short and long term recall: yes
(2) Equipment used
- Type of equipment (including manufacturer, if available): testing was performed with an integrated system of equipment and computer programs from Coulbourn Instruments (Allentown, PA) for equipment control and data collection (automated).
- Environmental conditions: Testing took place in individual isolation cubicles with single shuttle cages made out of plexiglas and a stainless- steel rectangular chamber fitted with front-loading access. The floor of the cage consisted of a grid of stainless-steel bars. Each shuttle cage (10 x 10 cm) was lined with foam insulation to attenuate sound in the chamber and had a fan with a baffled air intake and exhaust system for ventilation. In each cage, were two compartments of equal size, separated by a wall that supported a centrally-located sliding (guillotine-type) door. In one compartment, the walls were black, the other compartment was illuminated. The light compartment was illuminated from the start of each trial until either the animal crossed to the dark compartment or the trial ended. The movement of the animal was detected by a phtocell system. A Coulbourn solid-state scanning shock generator delivered a brief (0.5 sec) pulse of mild (0.5 mA) distributed shock to the grid floor when the animal crossed to the dark
compartment.
(3) Testing and training procedures
- Number of trials per day: 15
- Number of days of testing: 2 (see above)
- procedure: The rats were placed individually in cages facing the light side. After 20 seconds, illumination started (beginning of the trial), the door to the dark compartment was opened. When the rat crossed into the dark compartment, the door automatically closed, the shock was delivered, and the light switched off (end of trial). The trial was also ended when the rat did not cross to the dark side after 180 sec
- Inter-trial intervals: between trials, animals was kept in the holding cage
- end of procedure: the procedure was terminated when the rat remained in the "bright" compartment for 180 sec on two consecutive trials or until 15 trials had elapsed, whichever occurred first.
(4) Performance measures
- Number of errors or trials to criterion: Number of trials-to-criterion. If the rats did not stay in the bright compartment during the 15 trials, they were assigned a value of 15 for the trials-to-criterion variable
Animals that failed to meet the criterion on Trial 1 or failed to crossduring the first two trials during acquisition were not assigned to trial 2 (one week later). All other animals retook the test after 7 days.


Water Maze
(1) Overall testing design
- Number of animals: 16 offspring per sex and dose (1 male and/or female per litter)
- Days of testing: PND 60 (+2) and 7 days later
- Evaluation of both short and long term recall: yes
(2) Equipment used
- Type of equipment: Water Maze (M-Maze), constructed of opaque plexiglas. Corridors were ca. 12.5 cm wide and the walls were ca. 40 cm high with 19 cm of water
- Environmental conditions: water in the M-maze was maintained at 22 +1 °C
(3) Testing and training procedures
- Number of trials per day: 15
- Number of days of testing: 1 training day (PND 60 (+2)), 1 test day (seven days later)
- procedure: Rat was placed between lateral arms, on the first trial, the arm that the rat chose first was designated to be the wrong goal for the consequitive 15 trials. In this way the rat had to enter both arms in the first trial to being provided access to the exit ramp to escape the water. Rats that failed to make a correct goal choice within 60 seconds in any given trial were guided to the correct goal with the exit ramp and then removed from the water.
- Changes across days: no
- Inter-trial intervals: 15 (+5) seconds, the animal was returned to a transport cage in intervals
- Stimulus parameters: none
- Time or latency to reach goal: five consecutive error-less trials or 15 trials in total
- Definition of errors: wrong goal choice
(4) Performance measures at first test occasion (aquisition)
- Parameters measured: trials-to-criterion, average number of errors (incorrect turns in the maze) for each trial (both as overall learning performance), latency (in seconds) to reach the correct goal on trial 2 (short term retention)
(5) Performance measures at second test occasion (retention)
- Parameters measured: trials-to-criterion, average number of errors (incorrect turns in the maze) for each trial (both as overall learning performance), latency (in seconds) to reach the correct goal on trial 1 (long term retention)

Sacrifice and (histo)pathology:

P Generation
Males: following co-habitation, males were sacrificed by CO2 asphyxiation and discarded
unless an alternative use was found
Females: dams were sacrificed by CO2 asphyxiation on LD 21, after the weaning of their respective litters. If female animals were found to be sperm positive and/or with an internal vaginal plug, but did not deliver, they were sacrificed after gestation day 24 (GD 24).
The parent generation did not undergo gross pathological investigation at necropsy.

F1 Generation

1) PND 4:
The litters were adjusted to approximately 4 males and 4 females. Pups that were not selected for further testing were sacrificed by decapitation and discarded without necropsy.

2) PND 11:
- Number of animals: 10/sex/dietary level
- procedure for sacrifice: euthanized by intraperitoneal injection of Fatal Plus
- Parameters measured: gross necropsy examination (all animals)
- procedure: after slicing the calvaria and exposing the brain, the head was immersed in buffered 10% formalin and fixated for 24 h or longer. Then, the brain with olfactory bulbs was removed by careful dissection and weighed.
- Brain weight: yes
- Length and width of brain: Anterior-to-posterior (AP) length of cerebrum and anterior-to-posterior (AP) length of the cerebellum were measured.
- Type of staining: hematoxylin and eosin (H & E), luxol fast blue/cresyl violet and Sevier-Munger stains
- Methodology of preparation of sections: standard procedures
- Thickness: 5 µm
- Embedding media: paraffin embedding
- Number of sections: eight coronal sections

Histology
Animals: Control and high dose animals
Analysis: gross examination and extensive histopathologic evaluation
If no treatment-related lesions were found: no further analysis
If treatment-related lesions were found: semi-quantitative analysis with sections from all dose groups including frontal cortex thickness (Level 4), parietal cortex thickness (Level 4), caudate putamen and underlying globus pallidus horizontal width (Level 4; maximum cross-sectional width), corpus callosum thickness (Level 4), hippocampal gyrus thickness (Level 5), cerebellum height (Level 7), external germinal layer (cerebellum) thickness (Level 7)

Detail on tissues examined can be found in Table 1 in the attached background material (pathology report).

3) PND 75 (+5)
- Number of animals sacrificed: approximately 20/sex/dietary level (10 for perfusion and collection of neural tissue and 10 for determination of fresh brain weight and gross necropsy examination)
- Parameters measured: necropsy examination
- neuropathology and fixed brain weight: 10 animals/sex/dietary level were selected for perfusion and collection of neural tissues
- Brain weight: fixed and fresh
- Length and width of brain: Anterior-to-posterior (AP) length of cerebrum and anterior-to-posterior (AP) length of the cerebellum were measured
- Procedures for perfusion: animals were deeply anesthetized with an intraperitoneal dose of sodium pentobarbital (approximately 50 mg/kg bw). Perfusion was performed via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by in situ fixation using 10% buffered formalin.
- Number of animals perfused: 10 of each dose/sex
- Tissues evaluated: entire brain and spinal cord, both eyes (with optic nerves) and selected (bilateral) peripheral nerves (sciatic, tibial and sural), the gasserian ganglion, gastrocnemius muscle, both forelimbs and physical identifier
- Embedding media: 10% buffered formalin

Histology
- Number of animals evaluated from each sex and treatment group: all perfused animals
H&E staining:

- Type of staining: Hematoxylin and Eosin (H&E)
- Embedding media: paraffin
- Tissues: eight coronal sections of the brain, sections from three levels of the spinal cord (cervical, thoracic and lumbar), the cauda equina, eyes, optic nerves and gastrocnemius muscle
- Thickness: not reported
- Number of sections: 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 and gasserian ganglia, Peripheral nerve tissues (sciatic, tibial and sural nerves, sectioned longitudinally)
- Thickness: 2 - 3 μm
- Number of sections: not reported

Histopathology
Animals: Control and high dose animals
Analysis: gross examination and extensive histopathologic evaluation
If no treatment-related lesions were found: no further analysis
If treatment-related lesions were found: semi-quantitative analysis with sections from all dose groups including frontal cortex thickness (Level 4), parietal cortex thickness (Level 4), caudate putamen and underlying globus pallidus horizontal width (Level 4; maximum cross-sectional width), corpus callosum thickness (Level 4), hippocampal gyrus thickness (Level 5), cerebellum height (Level 7), external germinal layer (cerebellum) thickness (Level 7)

Other examinations:

Delivery and Culling
Number of animals: all dams
Time schedule: Daily

Ovaries and uterine content: no

Positive control:

no

Statistics:

All statistical evaluation was done using software from either INSTEM Computer
Systems, SAS, or TASC. Statsistical signifance was set at p<0.05 or for Barlet's Test at p<0.001.

To determine the level of variance, Bartlett’s test was performed. If unequal variances occurred, nonparametric statistical procedures were used, namely Kruskal-Wallis ANOVA followed by the Mann-Whitney U test for between-group comparisons. If variance was equal, continuous data was analysed using an Analysis of Variance (ANOVA). If a significant F-Test was determined, a Dunnett's Test was performed.

Session activity of the motoractivity test was compared using an ANOVA followed by a Dunnett's test, if differences occurred. For interval data of the motoractivity test, a two-way Repeated-Measures ANOVA, using both test interval and test occasion as the repeated measures was performed, followed by a Repeated Measures ANOVA to determine on which weeks there was a significant treatment by interval interaction. Then, this data was subjected to an ANOVA followed by Dunnett's test to determine which groups differed from the control.

The acoustic startle response amplitude data (peak amplitude) was compared using an ANOVA followed by a Dunnett's test, if differences occured. The response amplitude data for each block of ten trials (five blocks/test session) were subjected to a Repeated-Measures ANOVA, using test block as the repeated measure and a Dunnett's test was performed if differences occurred.

Passive avoidance data were analysed with the Wilcoxon Test for time to failure (i.e., time to cross). Number of trials-to-criterion was analysed using Kruskal-Wallis and Wilcoxon tests for the acquisition phase and Fisher’s Exact Test for retention.

Details on statistics for the water maze test and histopathology can be found in "Any other information on materials and methods including tables"

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

MATERNAL GENERATION
Effects during gestation that were not treatment related
- Control: hair thinning (4/30), light brown stool (1/30)
- 100 ppm: hair thinning (1/30)
- 250 ppm: hair thinning (3/29), wounds, cuts, scratches or lactation (1/29)
- 700 ppm: hair thinning (3/30), wounds, cuts, scratches or lactation (1/30), red vaginal discharge (1/30)

Effects during lactation:
Effects during lactation that were not treatment related
- Control: hair thinning (4/24), dehydration (1/24)
- 100 ppm: hair thinning (3/29)
- 250 ppm: hair thinning (3/27)
- 700 ppm: hair thinning (1/27), dehydration (2/27)

F1 GENERATION
Effects on days 0-21 (postnatal) that were not treatment related:
- Control: wound/cut (2/28), bruising (1/28), unopened eye (1/28), red nasal discharge (1/30)
- 100 ppm: wound/cut (1/30), bruising (3/30)
- 250 ppm: wound/cut (2/30), bruising (1/30), weak (1/39), cold to touch (1/30), non-use of limb (1/30)
- 700 ppm: bruising (2/28), weak (1/28), dehydrated (1/28)

Post Weaning
Only effects that were not treatment related were observed, these effects were
Males
- Control: lesions or scabs on right shoulder (3/63), broken tail tip (1/63), exophthalmus on left eye (1/63), exophthalmus on right eye (1/63)
- 100 ppm: lesions or scabs on both shoulders (1/69), lesions or scabs on left shoulder (3/69), lesions or scabs on dorsal thorax (1/69), lesions or scabs on right side of the neck (1/69), incisors in both upper teeth (1/69), dark red discharge in right eye (1/69), dark red discharge in nose (1/69), exophthalmus on left eye (2/69), general opacity in left eye (1/69)
- 250 ppm: lesions or scabs on both shoulders (4/58), lesions or scabs on right shoulder (2/58)
- 700 ppm: lesions or scabs on left shoulder (1/65), lesions or scabs on right shoulder (1/65), maloccluded teeth (1/65), dark red discharge in left eye (1/65), exophthalmus on right eye (1/65)

Females
- Control: vibratory shake of the head and shoulders (1/63), both eyes fully closed (1/63), exophthalmus on right eye (1/63), alopecia on both forelimbs (1/63), alopecia on right forelimbs (1/63)
- 100 ppm: lesions or scabs on both shoulders (1/69), vibratory shake of the head and shoulders (1/69), exophthalmus on right eye (1/69)
- 250 ppm: lesions or scabs on left shoulder (1/60), vibratory shake of the head and shoulders (3/60), alopecia on ventral thorax (1/60), broken tail tip (1/60)
- 700 ppm: lesions or scabs on nose (1/64), vibratory shake of the head and shoulders (1/64)

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

not applicable

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MATERNAL GENERATION
Body weight and body weight gain were not affected by treatment during gestation and lactation at any dietary level.

F1
PND 0-21
Body weight measurements at birth (PND 0) were comparable to control for all dose levels.
At PND 4, pups in the high dose group had reduced body weight (11%). This was continued on PND 11 (13%) and PND 21 (13%). Body weight gain was also reduced males and females in the high-dose group but not at lower concentrations of the test substance. From birth to PND 4, animals that received 700 ppm of the test substance gained 22% less weight than controls. This was also the case for all other periods measured (PND 4-11 was 15% lower, PND 4-17 13% lower and PND 4-21 11% lower).

Data is summarized in Table 12 and 13 (Attachment 1 and 2).

Post weaning
In coherence with PND 0-21, body weight and body weight gain was reduced for both sexes in the high dose group but not in any other group. The following notes are only for the high dose groups
Males: body weight was statistically lower than controls for all seven measurements (starting with 11% less with partial recovery to 4% less than control at study termination)
Females: body weight was significantly lower than controls for each of the first three measured weeks (average 8% at first time point, recovery to control levels at week 4)

Data is summarized in Table 15 in Attachment 3.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MATERNAL GENERATION
- 700 ppm: During the third week of gestation, food consumption was reduced by 9% (not statistically significant but in line with following results). During lactation, food consumption was reduced by 14% in the first week

Data is summarized in Table 4 and 7 in Attachment 4 and 5.

F1 GENERATION
Post weaning
There were no differences in food consumption that were ascribed to treatment at any dietary level.

Data is summarized in Table 16 in Attachment 6.

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):

Occasional effects that were observed were minimal to slight corneal opacity in both eyes and slight diffuse degeneration of the retina. These effects were not severe and occurred in all groups.

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

MATERNAL GENERATION
Abbreviated FOB
For the dams, occasional effects were reported, which however rather were incidental than treatment-related. These consisted of lesions (e.g., scabs on ears and localized areas of alopecia) and vaginal discharge on occasion in various dose groups. On LD 21, the number of urine pools was statistically significantly increased for animals that received 250 ppm (1.3±0.5 vs 0.5±0.5 in controls). Since this was neither seen in higher dosed animal nor on other days, the finding was considered incidental.

F1 GENERATION
Abbreviated FOB
Occasional effects were observed that were rather were incidental than treatment-related. These consisted of lesions (e.g., apparent bite marks or a cut on one forelimb) and red lacrimal stain on occasion in various dose groups, animals with broken teeth or malocclusion, and pups with eye lesions, noted as exophthalmia, pupil size (dilated) and pupil unresponsive to penlight. On PND 21, the number of urine pools was slightly but statistically significantly decreased for femals of the 250 ppm group (0.3±0.5 vs 0.9±0.7 in controls). Since this was neither seen in higher dosed animal nor on other days, the finding was considered incidental.

Motor and Locomotor activity
1) Session activity
As expected, motor and locomotor activity changed with age of pups. In youngest animals (PND 13), activity was very low due to relatively undeveloped ambulatory skills and sensory function (e.g., eyelids closed). The activity increased with age.

Treatment related effects were observed but were not statistically significant. These effects were
- 700 ppm: reduced motor activity (31-38% on PND 17 for males and females, 37% on PND 21 for females), reduced locomotor activity (37-39% on PND 17 for males and females, 26% on PND 21 for females), no effects were seen after PND 21.

These effects may be attributed the test substance that may have been ingested during the last days of weaning. The fact that these effects were reversibel supports this hypothesis.

Data is summarized in Table 19 and 20 in (Attachment 7) as well as in Table S1 (Attachment 8) in the attached background material (F1).

2) Interval activity
- 700 ppm: reduced motor and locomotor activity during two or more intervals (PND 17 and 21 for males and females), the only statistically significant reduction was seen in interval 1 on PND 17 for females.

Data is summarized in Table 21 and 22 in (Attachment 7).

Acoustic startle
In general startle response amplitude increased with age. Treatment-related differences were not observed in any dose group. There were two cases where the startle response amplitude was statistically different from control (significant increase in amplitude on PND 60, females receiving 100 ppm (for the overall session and for blocks 2-5) and for 250 ppm females (block 5 only). This observation was attributed to the lower activity of the controls on this test occasion.

Passive avoidance
There were no differences observed in any dose group.

Water maze
There were no compound-related differences for males or females at any dietary level. Occasional effects were observed in males that were not considered treatment related, since they were not severe and not seen in any dose related manner or in females. Specifically these were
-100 ppm: modestly greater number of errors in first aquisition trial (0.9, males)
-700 ppm: longer trial duration in first tral on first test occasion (males), modestly greater number of errors in first aquisition trial (1.2, males)

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):

Absolute and relative organ weights were determined, no differences between control and treated animals were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Maternal generation
Following sacrifice after lactation, the dams were not subjected to gross pathology. No differences between control and treatment groups were observed.

F1 Generation

PND 11
There were no compound-related necropsy findings in offspring found dead or sacrificed
on PND 11 or at study termination.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 Generation
A slight but significant decrease in caudate-putamen width was noted at study termination in females of the high dose group (-1.9%, p = 0.03). This was not considered treatment related since it did not occur in males of the high dose (there, the caudate-putamen widths was wider than in controls) and compared to PD 11, the decrease was smaller. In case of neurotoxicity, the difference would have increased. Also, controls showed a very high caudate-putanum widths compared to previous studies in this laboratory.

Summarized data of the caudate-putamen width and other brain measurements can be found in Tables BM1-SUM and BM2-SUM in the attached background material (Attachment 9).

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No obvious histopathological findings were reported.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

no effects observed

Description (incidence and severity):

REPRODUCTION
There were no effects of treatment on reproduction parameters, including gestation length. It is noted, for example, that the fertility index was 93.3% for both the control and 700 ppm dietary groups.

Summarized data can be found in Table 1 in the attached background material.

LITTER DATA
There were no compound-related effects on the number of litters, litter size, number of stillborn pups, the live birth index, or the viability index.

Summarized data can be found in Table 9 in the attached background material.

DEVELOPMENTAL LANDMARKS AND PUPIL CONSTRUCTION
The occurrence of developmental landmarks including preputial separation and vaginal patency were comparable between pups of all dose groups and the control. Pupil constriction in response to a pen light was apparent in all pups on PND 21.

Key result

Dose descriptor:

NOAEL

Effect level:

250 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: NOAEL for dams (female).

Key result

Dose descriptor:

NOAEL

Effect level:

250 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: NOAEL for F1 generation (male/female)

Key result

Dose descriptor:

LOAEL

Effect level:

700 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: LOAEL for dams (female)

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

Remarks on result:

other: LOAEL for F1 generation (male/female)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

700 ppm (nominal)

System:

other:

Conclusions:

The present developmental neurotoxicity guideline study was conducted in compliance with GLP. Under the test conditions used, the overall NOAEL for the dams was set at 250 ppm, which was the mid-dose tested, based on decreased food consumption. With respect to the offspring, the overall NOAEL was also set at 250 ppm for both, males and females, due to decreased body weight and reduced activity; examining the neural tissue revealed no abnormalities.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

32.4 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

With respect to neurotoxicity, an acute, a subchronic as well as a development 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

Please refer to the information provided in the endpoint summary.

Additional information

Neurotoxicity: oral administration
Data on neurotoxicity are available after acute exposure and subchronic exposure 90-days to characterize the potency of the test substance to induce neurotoxicity after short-term and sub-chronic exposure. Moreover, a developmental neurotoxicity study is available to assess the toxicity to offspring. 

 

The acute neurotoxicity study (M-028815-02-1) was conducted in accordance to guideline EPA-FIFRA, Addendum 10, EPA 540/09-91-123, PB 91-154617.

To determine the acute neurotoxicity of the test material rats were fed with the test material via gavage and different parameters for neurotoxicity were assessed.  Groups of 18 male and female rats received 0, 42, 152 or 307 mg/kg bw of the test material in a single dose (6 rats of each dose were satellite animals). An additional group of 12 females received a dose of 20 mg/kg bw in a supplementary study. Homogeneity and stability of the test material in the vehicle were analytically verified by gas chromatography. Clinical signs were recorded daily and body weight was measured weekly. 24 h after administration of the test substance, blood was taken for hematology and clinical chemistry investigations. All animals underwent a functional observation battery (FOB) one week prior to treatment and 90 min, 7 days and 14 days after administration. Furthermore, motor and locomotor activity was tested in the figure-eight maze. 15 days after treatment, all surviving animals were sacrificed and all organs, body cavities cut surfaces, external orifices and surfaces were examined. 6 animals per sex and dose group were perfused and histologically examined.

One male in the 151 mg/kg bw dose was excluded from the study since it was treated 3 h instead of 90 min before neurobehavioral observation and received a lower volume of the dosing solution than scheduled.  Mortality was observed in the highest dose group in both sexes (4/18 males and 10/18 females died on the day of administration or the following day), accompanied by decreased activity of the surviving animals in the neurobehavioral observations. Moreover, animals showed various clinical signs of toxicity such as incoordinated gate, tremors or a cool-to-touch body. Surviving males in the high dose group had slightly reduced body weight than control animals. Females that received the high dose had increased hemoglobin concentration, increased hematocrit, mature neutrophilia and a lymphopenia as well as decreased serum potassium and cholesterol. Males and females of the high dose group showed decreased serum triglycerides and a reduction in serum ALT activity. In sub-lethal doses, clinical signs (one male showed tremors and a red nasal stain) and neurobehavioral effects like decreased motor and locomotor activity in the figure-eight maze for males at 151 mg/kg bw and for females at 42 and 151 mg/kg bw are ascribed to acute cholinergic toxicity as previously described for the test material. All clinical signs and neurobehavioral changes were reversible within 7 days. Gross necropsy and histology did not show any differences between control and treatment groups. No effects were observed at 42 mg/kg bw for males and 20 mg/kg bw for female rats. Thus, the NOAEL for overall toxicity was set at 42 mg/kg bw for males and 20 mg/kg bw for females; the NOAEL for neurotoxicity was 307 mg/kg bw for both sexes.

 

The subchronic neurotoxicity study (M-027944-01-1) was performed according to the guideline US-EPA-FIFRA, Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Guideline Addendum 10, Neurotoxicity; NTIS, EPA 540/09-91-123, PB 91-154617.

Subchronic neurotoxicity was investigated in rats. Therefore, three treatment groups were fed with the test material via the diet over 13 weeks (90 days). The amount of the test material in the diet was 140, 963 and 3027 ppm, corresponding to ingested doses of 9.3, 63.3 and 196 mg/kg bw/day for females and 10.5, 69.3 and 213 mg/kg bw/day for males. Homogeneity and stability of the test material in the vehicle as well as concentrations of the administered test material were analytically verified by gas chromatography. Cage sides were observed daily and clinical signs, body weight and food consumption were measured weekly. Ophthalmoscopic examinations were performed pre-exposure and pre-terminal (Week 12). In Week 4 and 13, blood was taken from 6 non-fasted animals for hematology and clinical chemistry analysis. All animals underwent a functional observation battery (FOB) in the week prior to treatment and during Weeks 4, 8 and 13. Furthermore, motor and locomotor activity was tested in the figure-eight maze. In Week 13, all surviving animals were sacrificed and all organs, body cavities cut surfaces, external orifices and surfaces were examined. 6 animals per sex and dose group were perfused and histologically examined.

No mortality was observed until the day of sacrifice but clear evidence of toxicity were noted as changes in body weight, food consumption, and clinical chemistry and in the functional observational battery (FOB) in the middle and high dose. Males had a reduced mean body weight by 8%, females by 4% in the middle and 15% (males) and 7% (females) in the high dose. Reduced food consumption throughout the study was also observed in both sexes receiving the middle and high dose. Regarding clinical chemistry, males and females in the middle and high dose showed a reduction in creatine kinase activity. In females of the middle dose, lower triglyceride levels and reduced LDH activity was oberverd. In the high dose, lower triglyceride levels (males, females), reduced lactate dehydrogenase (LDH) activity (males, females), decreased phosphate levels (males, females), reduced total protein (females) and reduced albumin (females) levels were found. Only males receiving the high test material dose showed altered behavior in the FOB as slightly uncoordinated righting response (Week 13) and lower forelimb grip strength (Week 8). All other observations regarding neurological behavior were considered incidental due to the lack of statistical significance or because they occurred evenly throughout dose groups. Gross necropsy and histology did not show any differences between control and treatment groups. The NOAEL for male and female rats regarding overall toxicity was set at 150 ppm which was the lowest dose tested, corresponding to an actual ingested dose level of 9.3 mg/kg bw/day for the females and 10.5 mg/kg bw/day for the males. Since no obvious treatment-related effects indicative of neurotoxicity were seen up to the highest dose tested of 3000 ppm, this dose was set as NOAEL for male and female rats with respect to neurotoxicity, corresponding to an actual ingested dose level of 196 mg/kg bw/day for the females and 213 mg/kg bw/day for the males.

 

The developmental neurotoxicity study (M-084646-01-1) was performed according to the guideline EPA OOPTS 870.6300.

 

Maternal rats were fed with the test material at dietary concentrations of 100, 200 and 700 ppm from the start of gestation (GD 0) until the end of lactation (LD 21). Homogeneity and concentration of dosing solutions were analytically verified by gas chromatography. Stability of the test substance in the diet was demonstrated. The offspring did not receive the test substance via the diet. Clinical signs were recorded daily and body weight and food consumption were measured weekly. Detailed physical examination of maternal animals was performed daily for dams and pubs until the end of weaning, after that, pups were examed weekly. Ophthalmological examinations were performed in offspring at 50-60 days of age. Maternal animals and pubs underwent an abbreviated functional observation battery (FOB) (dams weekly during gestation and lactation, pubs were not examined in the open field). Furthermore, motor and locomotor activity was tested in the offspring at postnatal day (PND) 13, 17, 21 and 60-62 in the figure-eight maze. Other tests for neurobehavior were acoustic startle habituation, passive avoidance conditioning and the water maze. Thereby, long and short term recall were tested. Dams were sacrificed after the gestation period, offspring was sacrificed on PND 4 (to adjust litters), PND 11 or PND 75 (+5). 10 animals/sex/dose level that were sacrificed at PND 11 were further investigated regarding gross pathology and brain measurements. Control and high dose animals were histologically investigated. If lesions were found, all dose groups underwent micropathologic evaluation. 20 animals/sex/dose level that were sacrificed at PND 75 were further investigated regarding gross pathology and brain measurements. 10 were perfused while the other 10 were not (measurement of fresh brain weight and gross necropsy). Histopathology was performed with control and high dose animals. If lesions were found, all dose groups underwent histopathologic evaluation.

Effects in the maternal generation were reduced food consumption in the highest dose group in the last week of gestation and the first week of lactation. In the high dose group, offsprings showed reduced body weighed from postnatal day (PND) 4 to 21 and reduced activity in the figure-eight maze at PND 17 (males and females) and PND 21 (females only). This was considered treatment related but was assigned to test substance intake by feed that was ingested during the last days of weaning, since the effect did not occur when the test substance was removed from the feed. Moreover, neurotoxic effects tend to increase activity in the figure-eight maze rather than decreasing it, the reduced activity seen here cannot be attributed to neurotoxicity of the substance. The EFSA stated concerns regarding the results of the study, so that an additional statement (M-488888-01-1) was written by the study sponsor in which effects were evaluated in detail. A reduced decrease in caudate-putamen width was noted at study termination in females of the high dose group (-1.9%, p = 0.03). This was not considered treatment related since it did not occur in males of the high dose (there, the caudate-putamen widths was wider than in controls) and compared to PND 11, the decrease was smaller (-1.9% at termination vs -5.5% on PND 11). In case of neurotoxicity, the difference would have increased. Also, controls showed a very high caudate-putanum widths compared to previous studies in this laboratory. The corpus callosum thickness of females in the high dose group was 27.6% smaller than in controls on PND 11. This was not statistical significant and decreased to a reduction of 7.1% on PND 75 (termination). However, both the data from this study as well as other studies from the laboratory showed a high variability in this parameter so that it should not be used to determine developmental neurotoxicity. Other effects seen in this study include occasional findings of clinical signs like lesions or scabs, minimal to slight corneal opacity in one or both eyes or thinner hair (maternal animals). These effects were evenly distributed through all treatment groups and were therefore onsidered incidentally. Gross necropsy and histology did not reveal any differences between the control and the high dose group. Based on these results, the overall NOEL for the test substance was set at 250 ppm for maternal animals. With respect to the offspring, the overall NOAEL was also set at 250 ppm for both, males and females, due to decreased body weight and reduced activity; examining the neural tissue revealed no abnormalities. 250 ppm was reported as equivalent to 19.4 - 19.7 mg/kg bw/day during gestation, and to 30 - 45.4 mg/kg bw/day during lactation. Thus, a mean value of 32.4 mg/kg bw/day over the whole exposure time can be derived as NOAEL with respect to both, dams and offspring. The statement can be provided upon request.

 

References not included in IUC:

Sheets, L. 2014: Imidacloprid Developmental Neurotoxicity Study: - Interpretation of Findings of Concern to the European Food Safety Authority (EFSA), Report no: US0426, Owner company: Bayer AG, Monheim, Germany, Study number: M-488888-01-1, Report date: Jun 20, 2014

Justification for classification or non-classification

No classification is required since neurotoxicity occurred only at concentrations that caused systemic toxicity.