clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Neurotoxicity

Description of key information

Oral neurotoxicity studies

WoE, M-027750-03-1, EPA-FIFRA, rat, acute,
NOAEL (systemic, neurotoxicity): 60 mg/kg bw (males), 102 mg/kg bw (females)
LOAEL (systemic, neurotoxicity): 102 mg/kg bw (males), 213 mg/kg bw (females)

 

WoE, M-021561-02-1, Japanese Guidelines on Agricultural Chemicals, mice, rat and guinea-pig, males, pharmaceutical study,

NOAEL (mice, neurotoxicity): 25 mg/kg -bw

LOAEL (mice, neurotoxicity): 50 mg/kg bw

NOAEL (rat, neurotoxicity): 100 mg/kg bw 

LOAEL (rat, neurotoxicity): 300 mg/kg bw 

 

Key, M-027986-01-1, EPA-FIFRA, rat, 90d,
NOAEL (systemic): 150 ppm (corresponding to 60.0 mg/kg bw/day for males and 71.0 mg/kg bw/day  for females)
LOAEL (systemic): 3000 ppm (corresponding to 177.0 mg/kg bw/day for males and 200.1 mg/kg bw/day  for females)
NOAEL (neurotoxicity): 3000 ppm (corresponding to 177.0 mg/kg bw/day for males and 200.1 mg/kg bw/day for females, highest dose tested)

 

Key, M-027178-02-1, EPA-FIFRA, rat, developmental,
NOAEL (maternal, systemic): 500 ppm (corresponding to 42.9 and 90.0 mg/kg bw/day during gestation and lactation, respectively)
LOAEL (maternal, systemic): 1750 ppm (corresponding to 142.0 and 299.0 mg/kg bw/day during gestation and lactation, respectively) 
NOAEL (developmental): 500 ppm (males, 42.9 and 90.0 mg/kg bw/day during gestation and lactation, respectively), 150 ppm (females, 12.9 and 27.3 mg/kg bw/day during gestation and lactation, respectively)
LOAEL (developmental): 1750 ppm (males, corresponding to 142.0 and 299.0 mg/kg bw/day during gestation and lactation, respectively), 500 ppm (females, 42.9 and 90.0 mg/kg bw/day during gestation and lactation, respectively)
NOAEL (developmental neurotoxicity): 1750 ppm (both sexes, 142 and 299 mg/kg bw/day during gestation and lactation, respectively, highest dose tested)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity, other

Remarks:

Pharmacological Studies (considering neurotoxicity)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 Oct 1999 - 26 Nov 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

according to Japanese guidelines and GLP standards

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Agricultural Chemicals (Ministry of Agriculture, Forestry and Fisheries, 59-Nohsan-4200)

Version / remarks:

adopted 1985, revision of part 1997

Deviations:

yes

Remarks:

Effects on general physical condition and behavior in the mouse were not only observed up to 6h after administration, but up to the following day.

GLP compliance:

yes (incl. QA statement)

Remarks:

Japanese GLP Standards for Toxicity Studies on Agricultural Chemicals (Ministry of Agriculture, Forestry and Fisheries, 11-Nohsan-No.6283,1999)

Limit test:

no

Species:

other: rat, mouse and guinea pigs

Strain:

other: CD (SD) strain rats (SPF), CD-1 (ICR) strain mice (SPF) and Hartley strain guinea pigs (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (rats and mice), Japan Sic Co. Ltd. (guinea pigs)
- Age at study initiation: 4, 4.5, 5, or 6 weeks (rats), 4 or 4.5 weeks (mice) and 6 weeks (guniea pigs)
- Housing: Mice were housed in autoclaved polycarbonate cages (220WX325DX130H mm; Tokiwa Kagaku Kikai Ltd.). Rats were housed in autoclaved hanging-type stainless steel cages (246W X360DX180H mm; Clea Japan Inc.). Guinea pigs were housed in autoclaved hanging-type aluminum cages (320WX480DX330H mm, Tokiwa Kagaku Kikai Ltd.). The number of animals per cage were as follows: mice, 4 or 5; rats, 1 or 2; guineapigs, 2 or 3.
- Diet: pelleted diet ad libitum (mice and rats: MF; guinea pigs: LRC4; Oriental Yeast Co. Ltd.), ad libitum
- Water: filtered tap water (5 µm filter and irradiated by UV ray), ad libitum
- Acclimation period: 5 to 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23
- Humidity (%): 45 - 71
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 6 Oct 1999 To: 26 Nov 2000

Route of administration:

oral: gavage

Vehicle:

other: arabic gum dissolved in water for injection (5%), DMSO in in vitro experiments

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: in vivo experiments: the following concentrations of dosing suspensions were used 0.625,1.25, 2.5, 3, 5, 7.5,10, 20, 22.5, 28, 30, 39, 40, 55, 77,100,175, 228, 296, 300, 385, and 500 mg/mL: in vitro experiment: the concentrations were 10"3, 10"2, and 10'1 mol/L. All administration preparations were prepared on the day of use.

VEHICLE
- Concentration in vehicle: 5%
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

single exposure (in vivo), 5 min (4 preparations, in vitro)

Frequency of treatment:

once (in vivo), once (in vitro)

Dose / conc.:

12.5 mg/kg bw/day

Remarks:

Observations of physical condition and behavior (Irwin's method) (mice)

Dose / conc.:

25 mg/kg bw/day

Remarks:

Observations of physical condition and behavior (Irwin's method) (mice)

Dose / conc.:

50 mg/kg bw/day

Remarks:

Observations of physical condition and behavior (Irwin's method) (mice)

Dose / conc.:

100 mg/kg bw/day

Remarks:

Observations of physical condition and behavior (Irwin's method) (mice)

Dose / conc.:

200 mg/kg bw/day

Remarks:

Observations of physical condition and behavior (Irwin's method) (mice)

Dose / conc.:

400 mg/kg bw/day

Remarks:

Observations of physical condition and behavior (Irwin's method) (mice)

Dose / conc.:

25 mg/kg bw/day

Remarks:

Anesthetic effect (mice), Synergjstic effect on convulsions(pentylenetetrazol)(mice), Effect on intestinal transport (mice), Effect on muscle strength (mice)

Dose / conc.:

75 mg/kg bw/day

Remarks:

Anesthetic effect (mice), Synergjstic effect on convulsions(pentylenetetrazol)(mice), Effect on intestinal transport (mice), Effect on muscle strength (mice)

Dose / conc.:

225 mg/kg bw/day

Remarks:

Anesthetic effect (mice), Synergjstic effect on convulsions(pentylenetetrazol)(mice), Effect on intestinal transport (mice), Effect on muscle strength (mice)

Dose / conc.:

6.25 mg/kg bw/day

Remarks:

Synergistic effect on convulsions(electroshock)(mice)

Dose / conc.:

12.5 mg/kg bw/day

Remarks:

Synergistic effect on convulsions(electroshock)(mice):

Dose / conc.:

25 mg/kg bw/day

Remarks:

Synergistic effect on convulsions(electroshock)(mice):

Dose / conc.:

75 mg/kg bw/day

Remarks:

Synergistic effect on convulsions(electroshock)(mice):

Dose / conc.:

225 mg/kg bw/day

Remarks:

Synergistic effect on convulsions(electroshock)(mice):

Dose / conc.:

30 mg/kg bw/day

Remarks:

Effect of body temperature (rats)

Dose / conc.:

100 mg/kg bw/day

Remarks:

Effect of body temperature (rats), Effects on blood pressure and heart rate (unanesthetized rats)

Dose / conc.:

300 mg/kg bw/day

Remarks:

Effect of body temperature (rats), Effects on blood pressure and heart rate (unanesthetized rats), Effect on blood coagulation (rats)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Effect of body temperature (rats), Effects on blood pressure and heart rate (unanesthetized rats), Effect on blood coagulation (rats)

Dose / conc.:

3 000 mg/kg bw/day

Remarks:

Effect of body temperature (rats), Effects on blood pressure and heart rate (unanesthetized rats), Effect on blood coagulation (rats)

Dose / conc.:

0 other: mol/L

Remarks:

Effects on agonists induced contraction inisolated ileum (guinea pigs)

Dose / conc.:

0 other: mol/L

Remarks:

Effects on agonists induced contraction inisolated ileum (guinea pigs)

Dose / conc.:

0 other: mol/L

Remarks:

Effects on agonists induced contraction inisolated ileum (guinea pigs)

No. of animals per sex per dose:

Observations of physical condition and behavior (Irwin's method) (mice): 3
Anesthetic effect (mice): 8
Synergjstic effect on convulsions (electroshock) (mice): 10
Synergjstic effect on convulsions (pentylenetetrazol) (mice): 10
Effect of body temperature (rats): 6
Effects on blood pressure and heart rate (unanesthetized rats): 4
Effects on agonists induced contraction inisolated ileum (guinea pigs): 4 preparations (in vitro)
Effect on intestinal transport (mice): 8
Effect on muscle strength (mice): 8
Effect on blood coagulation (rats): 6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose finding experiments were conducted in mice and rats.
Mice weighing 28.0 to 33.0 g (5 weeks of age) were allocated 3 per group over 5 dose groups for a total of 15 animals (200, 280, 390, 550, and 770 mg/kg bw dose groups). Before administration, the animals were checked to confirm lack of abnormalities in their general physical condition, after which they were administered the appropriate dose p.o.; they were then observed at 0.5,1,2,4, 6, 8, and 12 hours after administration, and then at 1,2,3,4,5,6, and 7 days after administration, to monitor appearance of acute toxic signs or mortalities.
Administration of the test substance at 200 mg/kg bw produced decreased spontaneous locomotor activity as well as prone posture and deep respiration. In addition to these acute lexicological signs, staggering gait at 280 mg/kg bw and tremor at 390 mg/kg bw were also seen. With administration of 550 mg/kg bw, in addition to the signs seen following administration of 390 mg/kg bw, decreased body temperature was seen, with 1 out of 3 animals being found dead at 4 hours after administration. With 770 mg/kg bw, the additional signs of gait disturbance, dosed eyes, cyanosis were observed, and 1 dead animal was found at 1 hour after administration and another at 1 day after administration. From day 2 through day 7 after administration, no toxicity signs or mortalities were observed.
From these results, the dose of 400 mg/kg bw, as a dose near 390 mg/kg bw which was the highest dose tested with no fatalities, was selected as the highest dose level of test substance in the following observation of the effects on general physical condition and behavior, and using the common ratio of 2, the 3 lower dose levels were set at 200,100, and 50 mg/kg bw.


Rats weighing 160.7 to 174.2 g (5 weeks of age) were allocated 3 per group over 5 groups for a total of 15 animals (1750, 2280, 2960, 3850, and bw 5000 mg/kg dose groups). Before administration, the animals were checked to confirm lack of abnormalities in their general physical condition, after which they were administered the appropriate dose p.o.; they were then observed at 0.5, 1, 2, 4, 6, 8, and 12 hours after administration, and then at 1, 2, 3, 4, 5, 6, and 7 days after administration, to monitor appearance of acute toxic signs or mortalities.
Administration at 1750 mg/kg bw resulted in decreases in spontaneous locomotor activity and body temperature as well as closed eyes. In addition to these acute toxicity signs, 2280 mg/kg bw produced tremor and deep respiration, while at 2960 mg/kg bw, prone posture, staggering gait, abnormal fur were noted. With administration of 3850 mg/kg bw, in addition to the signs seen at 2960 mg/kg bw, hunchback position was observed, and 4 days after administration 1 out of 3 animals was found dead. Administration of 5000 mg/kg bw produced the signs seen at 3850 mg/kg bw as well as cyanosis and convulsion, and 1 dead animal was found at 8 hours after administration, and another at 6 days after administration.
From these results, the dose of 3000 mg/kg bw, as a dose near 2960 mg/kg bw which was the highest dose tested with no fatalities, was selected as the high dose level in the following experiments with rats, and using the common ratio of 3, the lower 2 dose levels were set at 1000 mg/kg bw and 300
mg/kg bw.

Observations and clinical examinations performed and frequency:

MICE

CLINICAL OBSERVATIONS: Yes
- Time schedule: before, and at 0.5, 1, 3, 6 hours and 1 day after p.o. administration
- Animals: control, 50, 100, 200 and 400 mg/kg bw, 3 per group. As effects were observed at 50 mg/kg bw, an additional group (25 mg/kg bw) was tested.
- Procedure: General physical condition and behavior were observed based on twin's multiple observation method. The observation items were alertness, passivity, stereotypy, grooming, vocalization, restlessness, irritability, reactivity, spontaneous locomotor activity, touch response, pain response, startle response, Straub tail, tremors, twitches, convulsions, body position, staggering gait, abnormal gait, somersault, limb tone, grip strength, body tone, abdominal tone, pinna reflex, corneal reflex, ipsilateral flexor reflex, pupil size, palpebral opening, exophthalmos, urination, salivation, lacrimation, writhing, piloerection, body temperature, skin color, respiration, diarrhea, and death, and for all items excepting death, the severity of each sign was expressed on a 5-level scale of "no change", "slight", "moderate", "marked", and "extreme".

BODY WEIGHT: Yes
- Time schedule for examinations: at administration.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

OTHER:
ANESTHETIC EFFECT
- Animals: control, 25, 75 and 225 mg/kg bw, 8 per group
- Procedure: At 1 hour after p.o. administration of the test substance, hexobarbital at 80 mg/kg bw was intraperitoneally injected to the animals. The sleeping animals were placed on a heating pad warmed to approximately 37 °C, and the time between the disappearance and recovery of the righting reflex was measured as the sleeping time.

SUBTHRESHOLD ELECTROSHOCK
- Animals: control, 25, 75 and 225 mg/kg bw, 10 per group. As an effect was seen at 25 mg/kg bw, two additional groups with 6.25 and 12.5 mg/kg bw were tested.
- Procedure: At 1 hour after p.o. administration of the test substance, electrodes were attached to the cornea, and a stimulator (MK-800, Muromachi Kikai Co. Ltd.) was used to apply electroshock stimulation consisting of the maximum subthreshold current of 8.0 mA (pulse duration: 5 msec; stimulation interval: 10 msec; stimulation period: 0.6 sec); appearance of tonic flexor and tonic extensor convulsions was monitored immediately after applying the current.

SUBTHRESHOLD STIMULATION BY PENTYLENETETRAZOL
- Animals: control, 25, 75 and 225 mg/kg bw, 10 per group.
- Procedure: At 1 hour after p.o. administration of the test substance, pentylenetetrazol at 55 mg/kg bw was subcutaneously injected to the animals; appearance of clonic and tonic extensor convulsions was monitored for 30 minutes.

INTESTINAL TRANSPORT:
- Animals: control, 25, 75 and 225 mg/kg bw, 8 per group.
- Procedure: At 1 hour after p.o. administration of the test substance, a 5 % charcoal suspension in 5% arabic gum solution at a volume of 0.2 mL/animal was orally given to the animals. The animals were sacrificed by cervical dislocation and the gastric tube was isolated 30 minutes after the charcoal administration. The length from the pylorus to the farthest point reached by the charcoal as well as the total length of the small intestine was measured.
The intestinal transport was calculated as the transfer rate of charcoal, according to the following equation.
Transfer rate (%)
= (the length from pylorus to the farthest point reached by charcoal / total intestine length) X100


MUSCLE STRENGTH
- Animals: control, 25, 75 and 225 mg/kg bw, 8 per group.
- Procedure: On a horizontally stretched wire, mice were placed so that their front paws were gripping the wire, and those animals able to grasp the wire with their hind paws within 5 seconds were chosen for this experiment. At 1, 3, and 6 hours after p.o. administration of the test substance, the mice were again checked on the horizontal wire as in the preadministration trial, and were observed for 10 seconds after their front paws were grasping the wire to see whether they would successfully grasp with their hind paws also.


RATS
BODY WEIGHT: Yes
- Time schedule for examinations: at administration.

FOOD CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

OTHER:
NORMAL BODY TEMPERATURE
- Time schedule: before, and at 0.5, 1, 3, and 6 hours after p.o. administration
- Animals: control, 300, 1000 and 3000 mg/kg bw, 6 per group. As body temperature reducing effect was seen with administration of the test substance at 300 mg/kg bw, an additional group (100 mg/kg bw) was tested.
- Procedure: The thermosensor of a digital electronic thermometer (TD-300, Shibaura Electronics Co. Ltd.) was inserted into the rectum of each animal.

BLOOD PRESSURE AND HEART RATE IN UNANESTHEZIED RATS
- Time schedule: before, and at 0.5, 1, 3, and 6 hours after p.o. administration
- Animals: control, 300, 1000 and 300 mg/kg bw, 4 per group. As heart rate increasing effect was noted with administration of the test substance at 300 mg/kg be, an additional group (100 mg/kg bw) was tested.
- Procedure: The rat's tail was inserted into the cuff of a non-intrusive automatic blood pressure meter (BP-98A, Softron Inc.) and the systolic and mean blood pressures were determined by plethysmography method. In addition, heart rate was determined based on the pulse wave.

BLOOD COAGULATION:
- Time schedule for collection of blood: at 1 hour after p.o. administration of the test substance
- Animals: control, 300, 1000, and 3000 mg/kg bw, 6 per group.
- Anaesthesia used: Yes (ether)
- Parameters measured: activated thromboplastin time

GUINEA PIGS
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weighed at treatment

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

OTHER: Effect on agonist-induced contraction in isolated guinea pig ileum
- Animals: 6 guinea-pigs (6 - 8 weeks of age)
- Procedure: The guinea pigs were sacrificed by exsanguination under ether anesthesia, and the ileum was isolated and cut into 2- to 3-cm strips as preparations. Each preparation was affixed to a Magnus bath containing 20 mL of Krebs solution at approximately 32 °C), and was subjected to a static tensive load of approximately 0.5 g. The composition of the Krebs solution was: NaCI 6.92 g/L, KC1 0.35 g/L, MgSO4-7 H2O 0.29 g/L, CaCl2-2 H2O 0.37 g/L, NaHCO3 2.10 g/L, KH2PO4 0.16 g/L, glucose 2.10 g/L; this was aerated by a gas mixture (95% O2 + 5% CO2. Contraction in the preparation was recorded on a recorder (8K23, NEC San-ei, Inc.) measuring through an isotonic transducer (45347S, NEC San-ei, Inc.). As contractile agonists, acetylcholine 10^-6 mol/L, histamine 3 X10^-6 mol/L, and barium 10^-3 mol/L were used. Once the response of the preparation stabilized, DMSO, then the test substance at 10^-6, 10^-5, and 10^-4 mol/L (each being the final concentration) were applied in succession. Each of the treatments with DMSO or the test substance was applied for 5 minutes, and before and after treatment by each of the concentrations of the test substance, the previously specified concentrations of acetylcholine, histamine, and barium were applied, and the interactions between the test substance and each contractile agonist were observed. In each case, 4 preparations per group were used.

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: No

LOCOMOTOR ACTIVITY: No

AUDITORY STARTLE REFLEX HABITUATION: No

LEARNING AND MEMORY TESTING: No

Tests performed are described under "Observations and examinations performed and frequency".

Sacrifice and (histo)pathology:

All animals were sacrificed after the procedures decribed above.

- Brain weight: No
- Length and width of brain: No
- Other: No

Positive control:

No

Statistics:

Quantitative data were expressed as the mean ± S.D. Statistical analyses among multiple groups were performed using Bartlett's test for homogeneity of variance. When the variance was homogenous, the data were analyzed by One-Way analysis of variance. When the differences among the groups were significant, the data were analyzed by Dunnett's mean multiple comparison test. When the variance was not homogenous, the data were then analyzed by Kruskal-Wallis's test. When differences among the groups were significant, the data were analyzed by Dunnett's mean multiple comparison test. Statistical analyses between two groups were performed using the F test for homogeneity of variance. When the variance was homogenous, the data were analyzed by Student's t test. When the variance was not homogenous, the data were analyzed by Aspin-Welch's t test. Incidences were analyzed by the chi-square test. Differences of p<0.05 in comparison with the control were considered to be statistically significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

MICE
Please refer to the section "Behaviour".

RATS
NORMAL BODY TEMPERATURE
- 30 and 100 mg/kg bw: No difference from the control group was seen in the rat body temperature values.
- 300 mg/kg bw: Decrease of 0.8 °C in comparison to the control at 1 hour after administration.
- 1000 mg/kg bw: The body temperature decreased with time, so that at 6 hours after administration it was 4.1 °C lower than the control group, representing an extreme decrease.
- 3000 mg/kg bw: The body temperature decreased with time, so that at 6 hours after administration it was 6.4 °C lower than the control group, representing an extreme decrease.

For details, please refer to Attachment 1.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, treatment-related

Description (incidence):

Mice
- 12.5, 50, 100 and 200 mg/kg bw: No mortalities were observed.
- 225 mg/kg bw/day: 2/8 animals (anesthetic effects) died following administration of hexobarbital.
- 400 mg/kg bw: 1/3 animals was found dead 3 h after administration of the test substance.

Details can be found in the respective sections.

Body weight and weight changes:

not examined

Description (incidence and severity):

not applicable

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

no effects observed

Description (incidence and severity):

Prothrombin time and activated thromboplastin time in rats showed no change after administration of the test substance at 300, 1000, and 3000 mg/kg bw.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not specified

Description (incidence and severity):

No ED-related parameters were investigated in the study.

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Mice
EFFECT ON GENERAL PHYSICAL CONDITION AND BEHAVIOUR
- 12.5 and 25 mg/kg bw: No effects were observed.
- 50 mg/kg bw: The test substance caused decreased spontaneous locomotor activity in all 3 animals, and tremor and deep respiration in 1 animal. All of these signs were slight and disappeared by 3 hours after administration.They were judged to be reversible changes.
- 100 mg/kg bw: decreases in grooming and reactivity, spontaneous locomotor activity, body tone, abdominal muscle tone, and body temperature were observed along with tremor, prone posture, staggering gait, mydriasis, and deep respiration. Except that the decrease in spontaneous locomotor activity and the mydriasis were slight to moderate, all the signs were graded as slight and all had disappeared by 6 hours after administration. The effects were djudged to be reversible changes.
- 200 mg/kg bw: In addition to the signs seen at 100 mg/kg bw, decreases in touch response and grip strength were also seen. In all 3 animals of the 200 mg/kg bw dose group, almost all of the signs were graded as slight to moderate, and by 6 hours after administration all changes had disappeared.
- 400 mg/kg bw: in addition to the signs seen at 200 mg/kg, decreases in comeal reflex and pinna reflex, suppression of ipsilateral flexor reflex, reduced limb tone, Straub tail, and cyanosis were observed. Further, at 3 hours after administration, 1 out of 3 of the administered animals was found dead. In the surviving animals, the changes due to test substance administration ranged in severity from slight to marked, and 1 day after administration, the signs of decreased spontaneous locomotor activity as well as tremor and staggering gait were still observable, although the severity was slight.

For details, please refer to Attachment 2.

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

not examined

Description (incidence and severity):

not applicable

Neuropathological findings:

not specified

Description (incidence and severity):

Please refer to the respective sections.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

effects observed, treatment-related

Description (incidence and severity):

MICE
ANESTHETIC EFFECTS
- 25 and 75 mg/kg bw: No effects were observed.
- 225 mg/kg bw: The sleeping time was statistically significantly prolonged in comparison with the control, to the extent of approximately 1.6-fold. 2/8 animals died following administration of hexobarbital.

For details, please refer to Attachment 3.

SUBTHRESHOLD ELECTROSHOC
- 6.25 mg/kg bw: tonic flexor convulsion and tonic extensor convulsion in 3/10 animals (not statistically significantly different compared to the control group).
- 12.5 mg/kg bw: tonic flexor convulsion and tonic extensor convulsion in 2/10 animals (not statistically significantly different compared to the control group).
- 25 mg/kg bw: Tonic flexor convulsion and tonic extensor convulsion in 8/10 animals (statistically significantly different compared to the control group).
- 75 mg/kg bw: Tonic flexor convulsion and tonic extensor convulsion in 10/10 animals (statistically significantly different compared to the control group).
- 225 mg/kg bw: Tonic flexor convulsion and tonic extensor convulsion in 10/10 animals (statistically significantly different compared to the control group).

For details, please refer to Attachment 4.

SUBTHRESHOLD STIMULATION BY PENTYLENETETRAZOL
No adverse effects were observed. In the control and 25 mg/kg bw groups, subthreshold administration of pentylenetetrazol induced clonic convulsion in 2/10 mice, but this result was not statistically significant. In the 75 and 225 mg/kg bw groups, no incidences of clonic nor tonic extensor convulsions were observed.

For details, please refer to Attachment 5.

GASTROINTESTINAL SYSTEM
- 25 mg/kg bw: No effect was observed.
- 75 mg/kg bw: Intestinal transport of charcoal was suppressed by 40.2% compared to the control group. The difference was statistically significant.
- 225 mg/kg bw: Intestinal transport of charcoal was suppressed by 52.0% compared to the control group. The difference was statistically significant.

For details, please refer to Attachment 6.

MUSCLE STRENGTH
- 25 and 75 mg/kg bw: No effect on muscle strength was noted.
- 225 mg/kg bw: suppression of muscle strengths was observed 1 and 3 hours after administration in 4/8 animals, and in 2/8 animals 6 hours after administration. In no case the value was statistically significant. However, this result is consonant with the observation of slight suppression of the skeletal muscular system up to 3 hours after administration of 200 mg/kg bw as seen regarding grip strength and body tone (see effect on general physical condition and behavior in mice). Thus, the test substance was adjudged to exert slight suppressive effect on the skeletal muscular system.

For details, please refer to Attachment 7.

RATS
BLOOD PRESSURE AND HEART RATE IN UNANESTHETIZED RATS
- 100 mg/kg bw: No effect on blood pressure or heart rate was observed.
- 300 mg/kg bw: Increase of heart rate by 49 beats/minute compared to the control group at 0.5 hours after administration, but systolic and mean blood pressures showed no influence through 6 hours after administration.
- 1000 mg/kg bw: The heart rate showed no statistically significant change, but systolic blood pressure in comparison with the control was 16 mmHg lower 1 hour after administration, and mean blood pressure also showed statistically significantly lowered values 1 and 6 hours after administration.
- 3000 mg/kg bw: The heart rate was increased 49 beats/minute compared to the control at 0.5 hours after administration, and mean blood pressure was lowered 12 mmHg at 6 hours after administration.

As the changes were not dose dependent and slight (a maximum heart rate increase of 12%, and maximum systolic and mean blood pressures decreases of 15% in comparison with the control values), the test substance was not judged to produce major effect on the circulatory system.

For details, please refer to Attachment 8.

GUINEA PIGS
AGONIST-INDUCED CONTRACTION IN ISOLATED GUINEA PIG ILEUM
- 10^-6 mol/L: No effect on contractile response to acetylcholine, histamine, and barium in the isolated ileum preparations was observed compared to the control.
- 10^-5 mol/L: No effect on contractile response to acetylcholine, histamine, and barium in the isolated ileum preparations was observed compared to the control.
- 10^-4 mol/L: The contractile response to barium in isolated guinea
pig ileum was 12.2% suppressed in comparison with the control, but the contractile response to acetylcholine and histamine were not affected. While the change in the barium response was statistically significant in comparison with the control values, the extent of suppressive effect was with 12.2% only slight. Therefore, this was not considered to be a noteworthy effect.

For details, please refer to Attachment 9.

Key result

Dose descriptor:

NOAEL

Remarks:

mice, neurotoxicity

Effect level:

25 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects regarding neurotoxicity were observed at this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

mice, neurotoxicity

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

Remarks on result:

other: at higher doses: clinical signs included hypothermia, mydriasis, abnormal gait/position and decreased muscle tone, indicating a nicotinoic effect on the CNS

Key result

Dose descriptor:

NOAEL

Remarks:

rats, neurotoxicity

Effect level:

100 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects were observed at this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

rats, neurotoxicity

Effect level:

300 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

Dose descriptor:

NOAEL

Remarks:

guinea pigs, in vitro

Effect level:

0 other: mol/L

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects were observed at this dose level.

Dose descriptor:

LOAEL

Remarks:

guinea pigs, in vitro

Effect level:

0 other: mol/L

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Effects on agonists induced contraction in isolated ileum.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw (total dose)

System:

central nervous system

Organ:

other: decreased spontaneous locomotor activity, tremor, and deep respiration.

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The present study was conducted in compliance with Japanese GLP standards and according to Japanese Guidelines on Agricultural Chemicals (Ministry of Agriculture, Forestry and Fisheries, 59-Nohsan-4200).

In mice, the test substance induced convulsion with subthreshold electroshock at doses of 25 mg/kg bw and greater (mice), exerted suppressive effect on the central nervous system at 50 mg/kg bw and greater (mice and rats) and on the gastrointestinal system at 75 mg/kg bw and greater (mice), as well as exerting slight suppressive effect on the skeletal muscular system at 225 mg/kg bw (mice). However, the test substance was not judged to have particularly great effect on the circulatory system in rats or on contractile response to various agonists in isolated guinea pig ileum preparations, nor did it exhibit effect on the blood system in rats. In conclusion, the NOAEL for neurotoxicity in mice was set at 25 mg/kg bw and for rats at 100 mg/kg bw.

Reference 2

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 - 21 Nov 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

adopted 2997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1991

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SASCO, Inc. Portage, Michigan, USA
- Age at study initiation: 9 weeks
- Weight at study initiation: 149 - 191 g (males), 112 - 138 g (females)
- Fasting period before study: overnight
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 25.6
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

0.5% methylcellulose/0.4%Tween 80 in deionized water

Remarks on MMAD:

Not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test substance was solubilized in the vehicle and administered via gavage in the respective concentration

VEHICLE
- Justification for use and choice of vehicle: 0.5% methylcellulose/0.4% Tween 80 in deionized water
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was analyzed using nuclear magnetic resonance and mass spectroscopy for concentration of the active substance. In between measurements, the substance was kept in the freezer for approximatly 8 months, to verify its stability under these conditions.

To assess the concentration and stability of the test substance in the vehicle, liquid chromatography was used with samples at nominal concentrations of 5 and 50 mg/mLthat bracketed the range of test concentrations used in the study. Homogeneity was verified for the 5 mg/mL and the 50 mg/mL mixtures by analysis. Each dosing mixture was also analysed to measure the concentration of the test item.. Based on these results, the analytically-confirmed doses were 0, 102, 213 and 373 mg/kg bw for both sexes.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single treatment

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

actual dose ingested: 102 mg/kg bw

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

actual dose ingested: 213 mg/kg bw

Dose / conc.:

400 mg/kg bw (total dose)

Remarks:

actual dose ingested: 373 mg/kg bw

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were based on a dose finding experiment. Groups of 5 rats per sex and dose received a single oral dose of the test substance at 250, 500 or 1000 mg/kg bw and were examined 0.5, 1, 2, 3, 4 and 24 h after administration. Thereafter, they were examined once daily. At 250 mg/kg bw, no effects were observed in any sex. Treatment with 500 mg/kg produced clear signs of toxicity starting approximately three hours after treatment and evident as decreased activity and tremors in males and females and one female of this group died two days after treatment. At 1000 mg/kg bw, all females and 2 males died. Clinical signs at all dose levels became apparent at 3 h, with additional effects at 4 h following treatment. Surviving animals showed signs of toxicity for up to 72 h after treatment.
Based on these results, the doses for this study were set as 100, 200 andd 400 mg/kg bw for both sexes to induce no, intermediate and severe toxicity. Also, the beginning of the FOB was set to 4 h after treatment.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: 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 center 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 following parameters were analyzed: 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 tested, rats were transferred individually in figure-eight mazes to test motoractivity.
- Minimization of bias: Inter-observer reliability has been established
- Same technicians used throughout testing: Yes, if possible, with a second person performing the measurements
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: once during the week prior to initiating the exposure and again 4 h, 7 and 14 days after administration
- Environmental conditions: uniformity of light intensity (100+70 lux)
- Noise level: Broad-spectrum background noise (approximately 70dB(A)), generated by a white noise generator (Model S81-02, Coulbourn Instuments)

MOTOR AND 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 4 h, 7 and 14 days after administration
- Type of equipment used: figure-eight mazes, each maze had eight infrared emitter/ detector pairs (three in each of the figure-eight alleys and one in each of the blind alleys) to measure activity. 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. Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and (histo)pathology:

- Time point of sacrifice: Day 14
- Number of animals sacrificed: all surviving animals (6 with perfusion, see below, the rest without perfusion)
- Parameters measured: complete gross necropsy with examination of all organs (including the brain), body cavities, cut surfaces, external orifices and surfaces.
- Brain weight: yes
- Length and width of brain: yes

PERFUSION
- Procedures for perfusion: animals were deeply anaesthetized 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: Coronal sections from eight levels of the brain (olfactory bulbs, cerebral cortex, caudateputamen/globus pallidus, hippocampus, thalamus, hypothalamus, midbrain including tectum, tegmentum, and cerebral peduncles, cerebellum and medulla oblan), sections from four levels of the spinal cord (cervical, thoracic and lumbar, cauda equine)
- 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, gasserian ganglion, eyes, optic nerves, gastrocnemius muscle
- Thickness: 2 - 3 μm
- Number of sections: not reported

Toluidine Blue staining
- Type of staining: Toluidine Blue
- Embedding media: Epoxy resin
- Tissues: Peripheral nerve tissue, sciatic nerve (cross section)
- 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 Computer Systems or SAS. Statistical significance was set at p<0.05 or for Barlett's Test at p<0.001.

Continuous data was analyzed 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 analyzed using a Repeated-Measures 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 analyzed in the same manner. Categorical data collected in the FOB were analyzed in a similar manner, using General Linear Modeling (GLM) and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively.

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, treatment-related

Description (incidence and severity):

- 400 mg/kg bw: tremors, decreased activity and ataxia (both sexes), signs of red nasal stain and an oral stain (1 female), urine stain in females

Other signs were considered incidental (urine stain for one control and one low-dose female)

Summarized results can be found in Attachment 1 in the attached background material.

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:

no effects observed

Description (incidence and severity):

No differences occurred between control and treatment groups.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

FOB
Males
- 100 mg/kg bw: lower levels of activity during open field observation compared to controls
- 200 mg/kg bw: decreased activity compared to controls, pin-point pupils, uncoordinated righting response and decreased body temperature
- 400 mg/kg bw: decreased activity compared to controls, pin-point pupils, uncoordinated righting response, decreased body temperature, tremor and gait incoordination

Females
- 200 mg/kg bw: decreased activity compared to controls, pin-point pupils, uncoordinated righting response and decreased body temperature
- 400 mg/kg bw: decreased activity compared to controls, pin-point pupils, uncoordinated righting response, decreased body temperature, tremor and gait incoordination

Not all of these signs were statistically significant but biological significance was determined based on a combination of statistical significance and/or consistency with respect to other dose levels and occurrence in both sexes.

Summarized data can be found in Attachment 3 in the attached background material.

Motor and locomotor activity
Established variability: +/- 20%
On the day of treatment, motor- and locomotor activity of both sexes was reduced compared to controls:
- 100 mg/kg bw: males (23-37%), during first two intervals
- 200 mg/kg bw: males (59-62%), females (43-45%), during first two or three intervals
- 400 mg/kg bw: males (81-88%), females (72-83%), during first two or three intervals

Summarized data can be found in Attachment 2 in the attached background material.

Habituation was not affected by treatment.

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 differences were observed between control and treatment groups

Gross pathological findings:

no effects observed

Description (incidence and severity):

No differences were observed between control and treatment groups

Neuropathological findings:

no effects observed

Description (incidence and severity):

No differences were observed between control and treatment groups

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No differences were observed between control and treatment groups

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No differences were observed between control and treatment groups

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

clinical signs

Remarks on result:

other: corresponding to 102 mg/kg bw (actual dose ingested)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 102 mg/kg bw (actual dose ingested)

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

clinical signs

Remarks on result:

other: corresponding to 213 mg/kg bw (actual dose ingested)

Critical effects observed:

not specified

Conclusions:

The present study was conducted in compliance with GLP and according to EPA-FIFRA, Addendum 10, EPA 540/09-91-123, PB 91-154617. Under the test conditions used, the NOAEL for female rats was set at 100 mg/kg bw. For males, no NOAEL could be established because evidence of toxicity was observed also at 100 mg/kg bw, the lowest dose tested. Therefore, a follow up study was conducted. Toxicity was evident as decreased activity (FOB and motor activity) and higher incidences of clinical signs including hypothermia. Signs of toxicity were most apparent within the first hours following treatment, with complete recovery within seven days.

Reference 3

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 May - 18 May 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

adopted 1997

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 1991

Deviations:

yes

Remarks:

one sex, no organ weights, no pathology

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc, Raleigh, North Carolina, USA
- Age at study initiation: 9 weeks
- Weight at study initiation: 158 - 193 g
- Fasting period before study: overnight
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

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

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

0.5% methylcellulose/0.4%Tween 80 in deionized water

Remarks on MMAD:

not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test substance was solubilized in the vehicle and administered via gavage in the respective concentration

VEHICLE
0.5%methylcellulose/0.4%Tween 80 in deionized water
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was analyzed using nuclear magnetic resonance and mass spectroscopy for concentration of the active substance.
To assess the concentration and stability of the test substance in the vehicle, liquid chromatography was used with samples at nominal concentrations of 5 and 50 mg/mL. Homogeneity was accepted if the coefficient of variation (C.V.) was <6%. Each dosing suspension was also analyzed to measure the concentration of the test item in it, receiving actual ingested doses of 18, 36 and 60 mg/kg bw.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single exposure

Dose / conc.:

20 mg/kg bw (total dose)

Remarks:

actual dose received: 18 mg/kg bw

Dose / conc.:

40 mg/kg bw (total dose)

Remarks:

actual dose received: 36 mg/kg bw

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

actual dose received: 60 mg/kg bw

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were based on two previously performed studies in which no NOAEL could be established for male rats. In the first study, 0, 100, 200 and 400 mg/kg bw of the test substance were administered to male and female rats. For females, the NOAEL was established at 100 mg/kg bw but for males, effects were observed at all dose levels, evident by decreased activity in the FOB and motor and locomotor activity. In the second study, males only received 60 and 80 mg/kg bw of the test substance. No clinical signs, mortality or effects in the FOB occurred, but motor and locomotor activity were decreased. However, this decrease was neither statistically significant nor dose-dependent. Therefore, no NOAEL could be established. Based on the results of the two previous studies doses of 20, 40 and 60 mg/kg bw.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment, as component of the FOB conducted during pretreatment and on Day 0 prior to treatment. Body weight was not measured after treatment in this study since a NOAEL for effects on body weight was established in the initial study.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

No

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 center 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 following parameters were analyzed: 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 tested, rats were transferred individually in figure-eight mazes to test motoractivity.
- Minimization of bias: Inter-observer reliability has been established
- Same technicians used throughout testing: Yes, if possible, with a second person performing the measurements
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: once during the week prior to initiating the exposure and again 4 h after administration
- Environmental conditions: uniformity of light intensity (100+70 lux)
- Noise level: Broad-spectrum background noise (approximately 70dB(A)), generated by a white noise generator (Model S81-02, Coulbourn Instuments)

MOTOR AND 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 4 h after administration
- Type of equipment used: figure-eight mazes, each maze had eight infrared emitter/ detector pairs (three in each of the figure-eight alleys and one in each of the blind alleys) to measure activity. 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. Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and (histo)pathology:

Animals were not assigned to gross pathological examination because the NOAEL for this was already established in the first study conducted.

Other examinations:

No

Positive control:

No

Statistics:

In general, statistical evaluations were performed using software from SAS. Statistical signifance was set at p<0.05.

Continuous data was analyzed 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 analyzed using a Repeated-Measures 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 analyzed in the same manner. Categorical data collected in the FOB were analyzed in a similar manner, using General Linear Modeling (GLM) and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively.

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, non-treatment-related

Description (incidence and severity):

Occasional clinical signs occurred in all dose groups. These were perinal stain (1 control, 2 20 mg/kg bw, transient sign associated with the vehicle), red lacrimal stain (1 60 mg/kg bw), abnormally small eye (1 control)

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:

not examined

Description (incidence and severity):

not applicable

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FOB
Occasional findings occurred but sporadically and without any dose-relationship. Additionally to the signs described under clincial signs, these were altered pupil size and pupil response (1 control), slightly uncoordinated righting response (3 controls, 1 40 mg/kg bw).

Motor and locomotor activity
Variability was set to +/- 20%. This was exceeded (-21%) in the 20 mg/kg bw group before treatment but this was considered acceptable.
No difference in motor and locomotor activity was found.

Summarized data can be found in Attachment 5 in the attached background material.

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

not examined

Description (incidence and severity):

not applicable

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: highest dose tested

Critical effects observed:

no

Conclusions:

The study was conducted under GLP and similar to the EPA-FIFRA, Addendum 10, EPA 540/09-91-123, PB 91-154617 . Under the conditions of the assay, the NOAEL was set to 60 mg/kg bw, the highest dose tested. All signs that occurred during the study were considered incidental and not treatment-related.

Reference 4

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 Mar -2 Apr 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 1991

Deviations:

yes

Remarks:

Pathology and organ weights were not assessed. Body weight was only recorded before and one day after treatment. Only neurobehavior was evaluated.

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SASCO, Inc. Portage, Michigan, USA
- Age at study initiation: 9 weeks
- Weight at study initiation: 172 - 216 g
- Fasting period before study: overnight
- Housing: individually housed in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

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

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

0.5% methylcellulose/0.4%Tween 80 in deionized water

Remarks on MMAD:

not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test substance was solubilized in the vehicle and administered via gavage in the respective concentration

VEHICLE
0.5% methylcellulose/0.4% Tween 80 in deionized water
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was analyzed using nuclear magnetic resonance and mass spectroscopy for concentration of the active substance.
To assess the concentration and stability of the test substance in the vehicle, liquid chromatography was used with samples at nominal concentrations of 5 and 50 mg/mL. Homogeneity was accepted if the coefficient of variation (C.V.) was < 6%. Each dosing suspension was also analyzed to measure the concentration of the test item in it, receiving actual ingested doses of 63 and 76 mg/kg bw.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single exposure

Dose / conc.:

60 mg/kg bw (total dose)

Remarks:

actual dose ingested: 62 mg/kg bw

Dose / conc.:

80 mg/kg bw (total dose)

Remarks:

actual dose ingested: 76 mg/kg bw

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were based on a previously performed study in which 0, 100, 200 and 400 mg/kg bw of the test substance were administered to male and female rats. For females, the NOAEL was established at 100 mg/kg bw but for males, effects were observed at all dose levels, evident by decreased activity in the FOB and motor and locomotor activity. To determine a NOAEL for males, the current study was designed with males receiving 60 and 80 mg/kg bw of the test substance.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: 7 days before treatment, prior to administration of the test substance and 4 h after treatment

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

No

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 center 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 following parameters were analyzed: 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 tested, rats were transferred individually in figure-eight mazes to test motoractivity.
- Minimization of bias: Inter-observer reliability has been established
- Same technicians used throughout testing: Yes, if possible, with a second person performing the measurements
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: once during the week prior to initiating the exposure and again 4 h after administration
- Environmental conditions: uniformity of light intensity (100+70 lux)
- Noise level: Broad-spectrum background noise (approximately 70dB(A)), generated by a white noise generator (Model S81-02, Coulbourn Instuments)

MOTOR AND 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 4 h after administration
- Type of equipment used: figure-eight mazes, each maze had eight infrared emitter/ detector pairs (three in each of the figure-eight alleys and one in each of the blind alleys) to measure activity. 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. Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and (histo)pathology:

Animals were not assigned to gross pathological examination because the NOAEL for this was already established in the first study conducted.

Other examinations:

No

Positive control:

No

Statistics:

In general, statistical evaluations were performed using software from SAS. Statistical signifance was set at p<0.05.

Continuous data was analyzed 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 analyzed using a Repeated-Measures 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 analyzed in the same manner. Categorical data collected in the FOB were analyzed in a similar manner, using General Linear Modeling (GLM) and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively.

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:

no effects observed

Description (incidence and severity):

No differences were found between the control and treatment groups.

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:

not examined

Description (incidence and severity):

not applicable

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):

no effects observed

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

FOB
No differences were found between control and treatment groups.

Motor and locomotor activity
Variability was +/- 20%.
Overall, there were no differences between control and treatment groups but males in both substance groups showed lower motor and locomotor session activity at Day 0 (-40 % at 60 mg/kg bw and -24 to -26% at 80 mg/kg bw), that occurred especially in the first intervalls of the test. Even though these results were above the variability threshold, there was no statistical significance and no dose-relationship, so a treatment-related effect is questionable.

Summarized data can be found in Attachment 4 in the attached background material.

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

not examined

Description (incidence and severity):

not applicable

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

LOAEL

Effect level:

60 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

Remarks on result:

other: It is not clear whether the differences from control represent a border-line, compound-related effect, or were incidental and unrelated to treatment. Therefore, the second follow-up study was performed.

Conclusions:

The study was conducted under GLP and similar to EPA-FIFRA, Addendum 10, EPA 540/09-91-123, PB 91-154617. In both treatment groups decrased activity in the motor and locomotor activity were found. However, the reduction was more severe in the lower dose group and no statistical significance could be established. Also, no dose gave altered results in the FOB. Therefore, it is not clear, whether the effect seen in the motor and locomotor activity is treatment-related or incidental and an additional study is needed. Under the conditions of the present study, the LOAEL for effects on motor and locomotor activity in male rats was 60 mg/kg bw.

Reference 5

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Mar - 11 Jun 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Version / remarks:

adopted 1998

Deviations:

no

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 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sasco, Inc., Portage, Michigan, USA
- Age at study initiation: 9 weeks
- Weight at study initiation: 185.3 - 191.2 g (males, mean of all groups), 126.0 - 127.0 g (females, mean of all groups)
- Housing: individually in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Remarks on MMAD:

Not applicable

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: not reported but rations were changed every week. At changing the batch of feed, any uneaten feed of the previous week was collected and disposed of by incineration.
- Mixing appropriate amounts with: Purina Mills Rodent Lab Chow 5001-4 in "etts" form
- Storage temperature of food: not reported

VEHICLE
Corn oil was used as a vehicle at 1% (w/w) in the diet. A small amount of acetone served as a solvent in the diet preparation process and was allowed to evaporate.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was analyzed using nuclear magnetic resonance and mass spectroscopy for concentration of the active substance. In between measurements, the substance was kept in the freezer for approximatly 8 months, to verify its stability under this conditions.

To assess the concentration and stability of the test substance in the feed, liquid chromatography was used. For stability testing, 50 and 5000 ppm were analyzed. The concentration of the test article in the feed was measured during weeks 1, 2, 3, 7, 11 and 14 of the study. The analytical analysis of the test item determined concentrations of 135, 890 and 2677 ppm in the feed. Stability and homogeneity were confirmed.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

150 ppm

Remarks:

corresponding to an actual ingested dose of 9.2 and 10.6 mg/kg bw/day for males and females, respectively.

Dose / conc.:

1 000 ppm

Remarks:

corresponding to an actual ingested dose of 60.0 and 71.0 mg/kg bw/day for males and females, respectively.

Dose / conc.:

3 000 ppm

Remarks:

corresponding to an actual ingested dose of 177.0 and 200.1 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A three-week range-finding study was conducted in the same test strain. Rats received 100, 250, 1250, 2500 and 3750 ppm of the test substance in the diet. At term, male rats that received 2500 and 3750 ppm of the test substance had had 7% and 11% lower body weights than controls, respectively. Body weight in females was only reduced at 370 ppm (-8%). Accordingly, food consumption was reduced in animals of the higher dose groups, at 1250 (11-12%), 2500 (21-25%) and 3750 (27-34%) ppm. Apart from that, no effects were observed and the dietary concentrations for the main subchronic neurotoxicity study were set to 0, 150, 1000 and 3000 ppm. 150 ppm was expected to produce no evidence of toxicity, 1000 ppm an intermediate level of toxicity and the 3000 ppm dietary level was selected for the high dose, based on evidence that this dose level satisfies criteria of approximating a MTD (10% reduction in body weight, relative to controls).

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on holidays and weekends) with the exception of one day at the end of week 4

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, perfused animals were also weighed on the day of sacrifice for terminal body weight measurement.

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

WATER CONSUMPTION AND COMPOUND INTAKE: 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: Pupil size and pupil response, pupillary reflex (using a penlight or transilluminator), mydriasis, examination of eyelids, conjunctiva, cornea, aqueous humor, and lens (with slit lamp microscope), vitreous humor, retina, choroid, and optic disc (with indirect ophthalmoscope and a condensing lens)

Specific biochemical examinations:

Not applicable

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, with a second person performing the measurements (e.g., grip strength and foot splay)
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: once during the week prior to initiating the exposure and again during weeks 4, 8 and 13
- Noise level: Broad-spectrum background noise (approximately 70dB(A)), generated by a white noise generator (Model S82-24, Coulbourn Instuments)

MOTOR AND 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 had eight infrared emitter/ detector pairs (three in each of the figure-eight alleys and one in each of the blind alleys) to measure activity. 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. Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and (histo)pathology:

- Time point of sacrifice: week 13
- Number of animals sacrificed: all surviving animals (6 per sex and group with perfusion, see below, the rest without perfusion) by CO2 asphyxiation
- Parameters measured: complete gross necropsy with examination of all organs (including the brain), body cavities, cut surfaces, external orifices and surfaces.
- Brain weight: yes
- Length and width of brain: yes

PERFUSION
- Procedures for perfusion: animals were deeply anaesthetized 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, both forelimbs, and physical identifier (tail)

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: Coronal sections from eight levels of the brain (olfactory bulbs (level 1), cerebral cortex (Levels 2 to 6), caudateputamen/globus pallidus (Levels 2 to 5), hippocampus (Levels 5 to 6), thalamus (Levels 4 to 5), hypothalamus (Levels 4 to 5, midbrain including tectum, tegmentum, and cerebral peduncles (Level 6), cerebellum (Levels 7 to 8) and medulla oblan (Levels 7 to 8), cross- and longitudinal-sections from three levels of the spinal cord (cervical, thoracic and lumbar), cauda equina (longitudinal section only)
- Thickness: approx. 5 µm
- Number of sections: 3 sections collected from each level

Modified Lee's staining
- Type of staining: Modified Lee's
- Embedding media: glycol methacrylate (GMA)
- tissues: dorsal root ganglia (including dorsal and ventral root fibers), gasserian ganglion, eyes, optic nerves, gastrocnemius muscle
- Thickness: 2 - 3 μm
- Number of sections: not reported

Toluidine Blue staining
- Type of staining: Toluidine Blue
- Embedding media: Epoxy resin
- tissues: Peripheral nerve tissue
- 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: paraffin
- 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 Computer Systems or SAS. Statistical significance was set at p<0.05 or for Barlett's Test at p<0.001.

Continuous data was analyzed 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 analyzed using a Repeated-Measures 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 analyzed in the same manner. Categorical data collected in the FOB were analyzed in a similar manner, using General Linear Modeling (GLM) and Categorical Modeling (CATMOD) Procedures, with post-hoc comparisons using Dunnett's test and an Analysis of Contrasts, respectively.

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):

Males
- 3000 ppm: the male that was found dead (see below) exhibited decreased activity, ataxia and convulsions prior to death

Females
- Controls: urine stain (1), clear lacrimation (1), red lacrimal stain (1)
- 3000 ppm: clear lacrimation

These findings were considered incidental and non-treatment related.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 3000 ppm: one male was found dead on Day 4, this was considered incidental since no other deaths occurred

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 3000 ppm: reduced body weight for males and females, significantly different from controls from week 11-13 for males and week 9-13 for females (range: 4-5% reduced body weight compared to controls)

Summarized Data can be found in Attachment 1 in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males
- 3000 ppm: reduced (-17%) for the first week of treatment and significantly different from control for weeks 1-5 and weeks 8-13

Females:
- 3000 ppm: reduced (-20%) in week 1 and significantly reduced compared to controls thereafter

Summarized data can be found in Attachment 2 of the attached background material.

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):

Only occasional findings were reported that were not considered treatment related because they were incidental. They were occasional corneal opacity and retinal degeneration that occurred in the control (1 animal) and high dose (1 animal) group .

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Only occasional effects occurred and were observed throughout all groups (including the control). These effects were dilated pupils under room light condition (various test occasions, including prior to treatment), pupils did not appear to constrict in response to penlight under subdued light conditions (prior to treatment), red crusty zone on the ventral neck on week 4 was found on one male (3000 ppm) but was assigned to be an injury, and slightly uncoordinated aerial righting response.

Motor and locomotor activity during the test sessions was not affected by treatment in males or females at any dietary level. Based on a comparison of the pretreatment values for motor and locomotor activity among the four test groups, a measure of inherent variability was derived. As a general rule, differences of less than 20% were considered within the range of normal variability in the testing laboratoty and for the used rat strain. Few incidences exceeded the variability established in the pre-treatment observation (>20% of control) but this was considered incidental since no dose-dependency or any other pattern could be established.

For interval motor and locomotor activity, various minimal differences in activity for both sexes in various dietary groups (both increases and decreases) were found but are considered to be incidental and not related to treatment. In the same manner, habituation was not affected in any testing condition.

Summarized data of motor and locomotor activity can be found in Attachment 3 in the attached background material.

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- 3000 ppm: statistically significant increase in relative brain weight in males (+9.7%) and females (+6.9%) compared to controls but this was considered due to the decreased body weight described above. Absolute brain weight did not differ from controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no differences in gross lessions observed in control and treated animals.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No differences were found between control and treated animals.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No differences were found between control and treated animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No differences were found between control and treated animals.

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

ca. 1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 60.0 mg/kg bw/day in males and 71.0 mg/kg bw/day in females

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 177.0 and 200.1 mg/kg bw/day in males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxicity

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 177.0 and 200.1 mg/kg bw/day in males and females, respectively.

Key result

Critical effects observed:

no

Conclusions:

The present study was conducted in compliance with GLP and according to the guideline EPA OPPTS 870.6200. Under the test conditions used, the NOAEL for male and female rats regarding overall toxicity was set at 1000 ppm which was corresponding to an actual ingested dose level of 71.0 mg/kg bw/day for the females and 60.0 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
200.1 mg/kg bw/day for the females and 177.0 mg/kg bw/day for the males.

Reference 6

Endpoint:

neurotoxicity, other

Remarks:

Developmental neurotoxicity screening study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Apr - 6 Aug 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 1996

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Proposal for a New Guideline 426 (1998)

Version / remarks:

proposed 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 426 (Developmental Neurotoxicity Study)

Version / remarks:

adopted 2007

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: at least 60 days
- Weight at study initiation: 207 - 254 g
- Fasting period before study: not applicable
- Housing: F0 generation - individually in stainless-steel, wire-bottomed cages, during cohabitation in groups of two, from day 20 of gestation in nesting boxes, F1 generation: after weaning individually in stainless steel, wire-bottomed cages
- Diet: Purina Mills Rodent Lab Chow® #5001-4 in "etts" Form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

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

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks on MMAD:

not applicable

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The preparations are prepared by the sponsor and shiped to the testing facility as needed. The rations available to the rats will be changed weekly.
- Mixing appropriate amounts with (Type of food): Purina Mills Rodent Lab Chow® #5001-4 in "etts" Form
- Storage temperature of food: Freezer (-20°C), stored at room temperature during each week of use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance itself and the concentration of active ingredient in the diet, the homogeneity of the diet admixtures and the stability of the test substance in the diet were analyzed. Homogenity was verified and the mean test substance concentrations in the diet above 90% in all concentrations tested. The test substance was stable in rodent ration for a minimum of seven days at room temperature and refrigerated for 28 days.

Duration of treatment / exposure:

F0 generation: From beginning of gestation (Gestation Day 0, GD0) and continuing through Gestation Day 24 (for rats that did not deliver a litter) or until the end of lactation (LD22)
F1 generation: animals received the diet without test substance. However, the F1 pups may have been exposed to the test substance during maternal gestation, during lactation or via feed from approx. Gestation Day 12 through 22.

Frequency of treatment:

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

Dose / conc.:

150 ppm

Remarks:

corresponding to actual ingested doses of 12.9 mg/kg bw/day and 27.3 mg/kg bw/day during gestation and lactation, respectively

Dose / conc.:

500 ppm

Remarks:

corresponding to actual ingested doses of 42.9 mg/kg bw/day and 90.0 mg/kg bw/day during gestation and lactation, respectively

Dose / conc.:

1 750 ppm

Remarks:

corresponding to actual ingested doses of 142.0 mg/kg bw/day and 299.0 mg/kg bw/day during gestation and lactation, respectively

No. of animals per sex per dose:

25 (dams)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Doses were selected based on results from previous studies. In a developmental toxicity study, maternal body weight gain and feed consumption were reduced at 125 mg/kg bw/day (corresponding to 1900-2000 ppm) without any evidence of developmental toxicity. In a two-generation reproductive study, maternal body weight change and male and female pup body weights were reduced at 2500 ppm, while no effects were observed at 500 ppm. Preliminary data from the Subchronic Neurotoxicity Screening Battery as done by the sponsor found that parental body weight (male/female) and feed consumption were reduced at 3000 ppm of the test substance, while no effects were seen at 1000 ppm.
Based on these results, the concentrations of the test substance in the diet for the current study were set as 150, 500 and 1750 ppm.

Observations and clinical examinations performed and frequency:

P generation - Maternal animals
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- parameters checked: autonomic dysfunction, abnormal postures, abnormal movements or abnormal behavior patterns and unusual appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly, maternal behavior on Days 1, 5, 8, 14 and 22 postpartum.

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly (preexposure), daily from beginning of gestation

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No


F1 Generation

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during preweaning and postweaning

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: preweaning: daily, postweaning: weekly, Subsets 2 and 3 when they were taken out for behavioral testing, subset 4 was evaluated outside the home cage for signs of autonomic dysfunction, abnormal postures, abnormal movements or abnormal behavior patterns and unusual appearance. For explanations of subsets, please refer to "Any other information on materials and methods inl. tables".

BODY WEIGHT: Yes
- Time schedule for examinations: preweaning: Day 1 (birth), 5, 8, 12, 14, 18 and 22 postpartum, postweaning: weekly

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

None

Neurobehavioural examinations performed and frequency:

P Generation - Maternal Animals
FUNCTIONAL OBSERVATIONAL BATTERY: No

LOCOMOTOR ACTIVITY: No


F1
MOTOR ACTIVITY: Yes
- Number of animals: 40 offspring per sex and dose (1 male and female per litter), Subset 3
- Days of testing: Postnatal Days 14, 18, 22, 61 (± 2 days)
- Type of equipment used: passive infrared sensor mounted outside a stainless steel, wire-bottomed cage, during preweaning sessions, each pup was tested on Plexiglass® flooring.
- Length of session: 1 hour
- Parameters measured: number of movements and time spent in movement tabulated at each five-minute interval
- Same offspring evaluated at each preweaning time point: yes

AUDITORY STARTLE REFLEX HABITUATION: Yes
- Number of animals: 40 offspring per sex and dose (1 male and female per litter), Subset 3
- Days of testing: Postnatal Days 23 and 61 (±2 days) postpartum
- Same offspring evaluated at each preweaning time point: yes
- Exact age: 23 and 61 (±2 days)
- Type of equipment used: rats were placed in a small cage above a plattform with a force transducer in its base. A microcomputer sampled the output of the force transducer and controlled the test session.
- Environmental conditions: not reported
- Number of trials performed: 50
- Length (msec) and intensity (dB) of sound: 30 msec, 120 dB
- Length of interval between trials: 10 s

LEARNING AND MEMORY TESTING: Yes
Passive Avoidance Test
(1) Overall testing design
- Number of animals: 40 offspring per sex and dose (1 male and female per litter), Subset 2
- Days of testing: Beginning on Days 23 to 25 postpartum
- Evaluation of both short and long term recall: yes
(2) Equipment used
- Environmental conditions: Testing took place in two-compartment chamber with hinged Plexiglas® lids, in which one compartment was fitted with a bright Plexiglas floor while the other was fitted with a grid floor. A sliding door separated the two compartments and a current could be delivered to the dark compartment through the grid floor. The latency to enter the dark compartment or the maximum 60-second interval was recorded for each trial.
(3) Testing and training procedures
- Procedure: At the beginning of the trial, the rat was set in the light department. The light compartment was illuminated from the start of each trial until either the animal crossed to the dark compartment or the trial ended. When the animal crossed to the dark compartment, the door shut and a mild electronic pulse (1 mA, 1 s) was delivered through the grid floor.
- Number of trials per day: 15
- Number of days of testing: 2 (one week interval)
- Changes across days: none
- Inter-trial intervals: 30 seconds in a holding cage
- Definition of errors: enter in the dark compartment
- Inter-trial intervals: between trials, animals were kept in the holding cage
- End of procedure: the procedure was terminated when the rat remained in the "bright" compartment for 60 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.
- Latency (in seconds) to enter the dark compartment in the first trial of the first session as a measure of activity and exploration tendency
- Number of trials to criterion in the second test session (long term retention)
- Latency (in seconds) to enter the dark compartment in the first trial of the second session (long-term retention)

Watermaze test
Water Maze
(1) Overall testing design
- Number of animals: 40 offspring per sex and dose (1 male and female per litter), Subset 2
- Days of testing: Beginning on Days 59 to 63 postpartum
- Evaluation of both short and long term recall: yes
(2) Equipment used
- Type of equipment: 16-gauge stainless steel, modified M-maze, with water of 22 cm depths
- 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 consecutive 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
- Number of days of testing: 2 (one week interval)
- Inter-trial intervals: 15 seconds
- 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 or no goal reached in 60 seconds
(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 - Maternal animals
Special cases: dams that did not deliver litter were sacrificed on Day 25 of presumed gestation and examined for gross lesions, dams with no surviving pups were sacrificed on the day on that the last pup was found dead, gross necropsy of thoracic, abdominal and pelvic viscera were performed and the number of implantation sites recorded.
- Time point of sacrifice: dams that were chosen for further observation were sacrificed on Day 22 postpartum
- Number of animals sacrificed: all
- Parameters measured: gross necropsy and histological examination of all gross lesions

All animals were sacrifixed by CO2 asphyxiation.


F1 generation
- found dead: pups that were found on Day 1 postpartum were evaluated for vital status at birth, the lung will be evaluated and pups with gross lesions were preserved in Bouin's solution. Pups found dead or sacrificed for humane reasons on Day 2 - 22 postpartum were observed for gross lesions and cause of death. Pups with gross lesions were preserved in Bouin's solution (death on Days 2 to 5 postpartum) or in neutral buffered 10% formalin (death on Days 6 - 22 postpartum)
- Standardization of litter: on Day 5 postpartum, litter size was adjusted using CO2 asphyxiation
- if not stated otherwise, rats chosen for continuous evaluation were sacrificed after completion of the evaluation.

Subset 1
- Subset 1: sacrificed on PND (postnatal day) 12, brain weight determination and neurohistological evaluation were done, gross lesions were evaluated.
- Brain weight: yes
- Length and width of brain: yes
- Histopathology:
- Tissues evaluated: six coronal sections, olfactory bulbs
- Type of staining: hematoxylin and eosin
- Methodology of preparation of sections:
- Thickness: 7 µm
- Embedding media: paraffin embedding
- Number of sections: 8 sections for each brain (6 coronal sections plus olfatory bulbs)
- Number of animals evaluated from each sex and treatment group: 10/sex/dosage group from control and high-dose group. If differences were observed, further dosage groups would have been tested.

- Subset 5:
sacrificed on PND 22 and examined for gross lesions

- Subset 4:
- selected for neurohistological evaluation: sacrificed by sodium pentobarbital formalin
- Brain weight: yes
- Length and width of brain: yes
- Procedures for perfusion: in situ with neutral buffered 10% formalin
- tissues examined: head (without calvaria) immersed in neutral buffered 10% formalin, vertebral column was dissected to expose the spinal cord, hindlimbs were removed and dissected to expose the peripheral nerves. All these tissues were immersed in neutral buffered 10% formalin. After weighing and meassuring, the brains were further processed for histological examination
- Number of animals perfused: 10/sex and dose

Histology for Subset 4
- Tissues evaluated: eleven coronal sections (including olfactory bulbs), gasserian ganglia and associated trigeminal nerves, spinal nerve roots and ganglia, dorsal and ventral nerve roots (including the dorsal root ganglia), spinal cord (cervical and lumbar swellings and mid-thoracic cord), cervical cord, sciatic nerve, tibial nerve, common peroneal (fibular) and the sural nerve
- Number of sections: 11
- Number of animals evaluated from each sex and treatment group: 10/sex/dosage group from control and high-dose group. If differences were observed, further dosage groups would have been tested.

- Type of staining: Hematoxylin and eosin, luxol fast blue/cresyl violet and the Bielschowsky's technique
- tissues evaluated: anterior and posterior half of the brain, longitudinal sections of the dorsal root ganglia and spinal nerve roots, cross and longitudinal sections of the spinal cord
- Methodology of preparation of sections:
- Thickness: 5 µm
- Embedding media: paraffin

- Type of staining: Hematoxylin and eosin, toluidine blue and the Bielschowsky's technique
- tissues evaluated: Cross and longitudinal sections of the sciatic nerve and cross section of the tibial nerve, longitudinal sections of the common peroneal (fibular), tibial and sural nerves
- Thickness: 2 µm
- Embedding media: Glycol methacrylate (GMA)

Other examinations:

Female rats will be evaluated for:
- Adverse Clinical Signs Observed During Parturition.
- Duration of Gestation (Day 0 of presumed gestation to the time the first pup is
observed).
- Litter Size (defined as all pups delivered).
- Pup Viability at Birth.

F1 Generation
Sexual maturation: preputial separation (males) from Day 39 postpartum, vaginal patency (females) from Day 28 postpartum

Physical Landmarks
Number of pups that met the criterion were noted for each day
Surface righting reflex (ability to right in 5 seconds): From Day 1 postpartum (Subsets 1 through 5)
Pinna Unfolding: From Day 2 postpartum (Subsets 1 through 5)
Eye Opening: From Day 12 postpartum (Subsets 2 through 5)
Acoustic Startle Response: From Day 13 postpartum (Subsets 2 through 5).
Pupil Constriction: Once on Day 21 postpartum (Subsets 2 through 5).

Positive control:

No

Statistics:

Significance was set to p < 0.05 with the exception of the Bartlett's test, were significance was defined as p < 0.001.

For parametric data such as body weight, feed consumption, latency and errors per trial scores in behavioral tests and percent mortality per litter, a Bartlett's test was used to assess homogeneity of the variance. If this was significant (meaning variances were not homogeneous), a nonparametric test was performed (see below). If variances were homogeneous, data were compared using the Analysis of Variance Test (ANOVA) followed by Dunnett's Test if differences were found. Nonparametric data like litter size, numer of trials to criterion etc, were analyzed with the Kruskal-Wallis Test, when 75% or fewer of the scores in all the groups were tied, folllowed by a Dunn's Test if differences were found. When more than 75% of the scores in any dosage group were tied, Fisher's Exact Test was used to compare the proportion of ties in the dosage groups.

For data from the motor activity and auditory startle habituation tests, with measurements recorded at intervals (Blocks) throughout each test session, the Analysis of Variance with Repeated Measures was used for statistical evaluation. If this test is significant, the difference could come from the dosage groups or from an interaction between blocks and dosage (differences in the patterns of dosage group values across the measurement periods) which had an effect on the following test. If the dosage effect was significant, a Dunnett's test was performed. If the Dosage x Block interaction was significant, an ANOVA was performed to evaluate data at each measurement period, and a significant result (p<0.05) was followed by a comparison of the dosage groups using Dunnett's test.

Clinical observation incidence data, as well as other proportion data, were analyzed as contingency tables using the Variance Test for Homogeneity of the Binomial Distribution.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

P generation - maternal animals
Occasional observations of clincial signs were made. However, they occurred sporadically and through all dose groups and were therefore considered incidental. Among these were scabs, localized alopecia on the limbs, altered feces and abrasions.

F1 generation
Pups (Day 1 to Day 22 postpartum)
Occasional observations of clincial signs were made. However, they occurred sporadically and through all dose groups and were therefore considered incidental. Among these were for example abnormal curvature of the lumbar and/or lower thoracic region of the back, black or missing tip of tail, scabs and abrasions.

Adult animals (Day 22 until termination)

Occasional observations of clincial signs were made. However, they occurred sporadically and through all dose groups and were therefore considered incidental. Among these were for example scabs, localized alopecia on the limbs, chromodacryorrhea, swollen snout and abrasions.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, treatment-related

Description (incidence):

P generation - maternal animals
No mortality observed

F1 generation
Pups (Day 1 to Day 22 postpartum)
In the 150 and 500 ppm dosage group, significantly more pubs were found dead or
presumed cannibalized on Lactation Days 9 to 12 compared to controls but this finding was not dose-dependent and the dead pups came predominantly from two litters in the low dosage and one litter in the intermediate exposure group.

Postweaning
- 1750 ppm: 2 males (Days 26 and 27 postpartum), 3 females (Days 25 and 26 postpartum), deaths were presumably linked to a failure to thrive postweaning due to reduced body weight gains and weight losses
A non-treatment related death occurred in one female of the 150 ppm dosage group on Day 44 postpartum (no dose-dependency)

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

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

P generation - maternal animals
Gestation period
- 1750 ppm: significantly reduced body weight gains throughout gestation compared to controls (91% of control)

Lactation
- 1750 ppm: significantly reduced body weight (p<0.05 or p<0.01) on Lactation Days 1, 2, 3, 6, 7, 13 to 15 and 19 but comparable to controls at the end of lactation (Lactation Days 20 to 22), body weight changes were significantly reduced on Lactation Days 4 to 7 and significantly increased compared to controls on Lactation Days 14 to 22.

Effects that were significantly different compared to controls but not considered treatment related were a reduction in body weight gain in the 500 ppm group from Gestation Days 0 to 3, an increase in body weight gain on Lactation Days 14 to 22 at 500 ppm and a reduction in body weight gain on Lactation Days 4 to 7 at 150 ppm. They were concidered incidental because they were were transient fluctuations and/or not dose-dependent.

Summarized data can be found in Attachment 2 in the attached background material.

F1 generation
- 500 ppm: reduced average body weight gains compared to controls for the entire preweaning period in females and at several intervals within this period (Days 5 to 22 postpartum and Days 12 to 14 and 14 to 18 postpartum), reduced body weight on Days 14, 18 and 22 postpartum in females
- 1750 ppm: reduced average body weight gains compared to controls for the entire preweaning period in males and at several intervals within this period (Days 5 to 22, 12 to 14, 14 to 18 and18 to 22 postpartum), reduced average body weight in male pups compared to controls on Days 12, 14, 18, 22, 23, 30, 37 and 72 postpartum, reduced body weight gain and reduced average body weight gains for the entire preweaning period in females (Days 5 to 22 postpartum) and on and on Days 12 to 14, 14 to 18, 18 to 22, and 65 to 72 postpartum, reduced body weights on Days 14, 18, 22, 23 and 30 postpartum in females compared to controls.

Incidental fluctuation of body weight and body weight gain were observed at statistical significance compared to controls for males at 500 ppm (significantly reduced body weight gain on Days 14 to 18 postpartum) and 150 ppm (increased body weight gain on Days 5 to 65 and 23 to 65 postpartum, increased body weight on Days 51, 58 and 65 postpartum), but these changes were transient and not dose-dependent.

Summarized data can be found in Attachment 3 (attached background material).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

P generation - maternal animals
- 1750 ppm: reduced absolute (g/day) and relative (g/kg bw/day) feed consumption in several intervals of both gestation lactation period compared to controls

Summarized data can be found in Attachment 4 within the attached background material.

F1 generation
No biological significant differences were found. Occasional findings include reduced absolute feed consumption in 1750 ppm males on Days 51 to 58 and 65 to 72 postpartum and increased absolute feed consumption in 150 ppm females on Days 30 to 37 postpartum (both transient and not dose-dependent) compared to controls.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

F1 generation

Passive avoidance test (subset 2)
No differences were found between control and treatment groups.

Watermaze performance (subset 2)
No differences were found between control and treatment groups.

Motor activity
-1750 ppm: reduced average number of movements or the time spent in movement as evaluated in a 1.5 hour session (18 five-minute blocks) on Day 22 postpartum compared to controls.
On Day 62 postpartum, male rats moved significantly less in regard of movement numbers in Block 1-4 (1750 ppm) and more in regard of time spent in movement in Block 5 (500 ppm) compared to controls, female rats depicted a fewer number of movements in Block 4 (1750 ppm) and less time in movement in Block 6 (150 ppm). These changes were not considered treatment-related due to lack of dose-dependency and persistancy.

Summarized data can be found in Attachment 5 in the attached background material.


Auditory startle
-1750 ppm: average magnitude of response to the auditory startle and each individual block response magnitude were significantly reduced compared to controls on Day 23 postpartum (females)
A non-treatment related reduction of response magnitude was found for females (500 ppm) in Block 2 on Day 23 postpartum compared to controls, but this was transient and not dose-dependent.

Summarized data can be found in Attachment 6 within the attached background material.

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):

Subset 1
No differences in terminal body weight or brain/body weight ratio were observed between all groups sacrificed on Day 12 postpartum.

Subset 4
No differences in terminal body weight or brain/body weight ratio were observed between all groups sacrificed on Days 83 to 87 postpartum.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

P generation - maternal animals
No treatment related effects occurred, the only observation was a slight dilation of the pelvis of the left kidney in one control dam.

F1 generation
Pups
No treatment related effects occurred. All deviations (no milk in the stomach, white subcutaneous substance on the back in stillborn or dead pups) were regarded as incidental because there was no statistical significance or the observation was not present in more than 1 of 3 pups.

F1 generation
Subset 1
No differences occurred between treatment and control groups. One male rat in each of the 500 ppm and 1750 ppm maternal exposure groups had brown liver lobes, corresponding with mild, multifocal extramedullary hematopoiesis and moderate (500 ppm) or mild (1750 ppm) dark pigmented Kupffer cells at histopathological examination. However, this was considered incidental.

Subset 2
No biological significant differences were found between control and treatment groups. Observations in the rats included moderate (one male, 150 ppm) and marked (one female, 1750 ppm) dilation of the pelvis of the kidney, tail bent with tip missing (one male, 1750 ppm) and an missing left ear (female, 1750 ppm). Histolopathological examination confirmed these findings on a cellular level.

Subset 3
No biological significant differences were found between control and treatment groups. Observations in the rats included slide to marked dilation of the pelvis of the kidney (males: all treatment groups, females: control), multiple white areas on the spleen (males, 1750 ppm). Histolopathological examination confirmed these findings on a cellular level.

Subset 4
No biological significant differences were found between control and treatment groups. Observations in the rats included slide to marked dilation of the pelvis of the kidney (males: all treatment groups), small, purple and flaccid left testis and small and flaccid left epididymis (males, 1750 ppm).

All findings were considered non-treatment related since no dose-dependency was established, findings occurred sporadically or were commonly seen in this strain of rat.

Neuropathological findings:

no effects observed

Description (incidence and severity):

The neurohistological observations in Subset 1 and 4 (12 and 83-87 days postpartum, respectively) did not reveal any biologically significant differences between control and treatment groups. No statistically significant differences were apparent between treated and control male groups in any of the morphometric measurements on Day 12 postpartum (subset 1), but there were statistically significant increases in the thickness of the dentate gyrus and the height of the cerebellum, and decreased thickness of the external germinal layer of the cerebellum in females treated at 1750 ppm. However, the values of the 1750 ppm group were within the historical control data and the values of control animals were at the lower limit of historical control data. The slight differences in the size of these structures at 1750 ppm were considered to reflect slightly more advanced brain development; especially the external germinal layer becomes thinner and eventually disappears when the cerebellum enlarges. No morphometric changes were apparent in the brains of females treated at 500 ppm.
The diagonal width of the caudate putamen and the thickness of the dentate gyrus of the hippocampus were significantly shorter than controls in Days 83 – 87 postpartum of females in the 1750 ppm group (subset 4). However, the differences were not considered to be biologically significant. These differences were approximately 5%, and both the diagonal width of the caudate putamen and the thickness of the dentate gyrus of the hippocampus in the females of the 1750 ppm were within the historical control ranges. The values in females in the 500 ppm group and males at 1750 ppm were comparable to the controls. All other brain morphometric parameters were comparable to the controls. There were no treatment-related histomorphological effects in central or peripheral nerve tissues in Days 83 – 87 postpartum of both males and females at 1750 ppm..

Summarized data can be found in Attachment 7 in the attached background mateiral.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

F1 generation
Histological examinations only confirmed the gross lesions seen described above as dilated cells or inflammed sports.
Subset 1: mild, multifocal extramedullary hematopoiesis and moderate (500 ppm) or mild (1750 ppm) dark pigmented Kupffer cells.
Subset 2: The 150 ppm male rat had moderate dilation of the renal pelvis and a cyst in the cortex, in the male with the missing tip (1750 ppm), moderate necrosis was found. The female with the dilatation of the pelvis had a marked dilation of the renal pelvis and revealed a cyst in the medulla and minimal, chronic focal inflammation. In the female with the missing ear mild, chronic inflammation at the base of the ear was observed.
Subset 3: Evaluation of kidneys from the male and female rats confirmed the slight (mild), moderate or marked dilation of the renal pelvis. In the spleen of the male rat in the 1750 ppm exposure group minimal, chronic focal inflammation of the capsule was observed.
Subset 4: In testis and epididymis, diffuse, moderate testicular degeneration and minimal, focal mononuclear cell infiltration with mild, necrotic germ cells was observed, respectively.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Other effects:

no effects observed

Description (incidence and severity):

Reproductive Parameters (natural delivery observations, number of dams delivering, duration of gestation, averages for implantation sites per delivered litter, gestation index, number of dams with stillborn or dying pups)
No effects occured.

Reflex and physical development of pups (surface righting, pinna unfolding, eye opening, acoustic startle or pupil constriction)
No differences were observed between the control and treatment groups. The only effect was a transient reduction in the surface righting reflex on Day 3 postpartum in all groups receiving the test substance compared to controls but no treatment-related effect was noted since the values for the animals of the 150 and 500 ppm groups were within the historical control range. The value of the animals of the 1750 ppm group was only slightly lower than the lower limit of the historical control data and transient, so that it was not considered treatment-related.

Sexual maturation of pups
No differences occurred between control and treatment groups.

Details on results:

Historical control data was available in the original study report and was also provided in an additional review (Inawaka,2014) that re-evaluated the results based on the OECD Guideline for the Testing of Chemicals, Proposal for a New Guideline 426 (1998).

Key result

Dose descriptor:

NOAEL

Remarks:

maternal systemic

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects at this dose level

Remarks on result:

other: corresponding to 42.9 (gestation) and 90.0 (lactation) mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

maternal systemic

Effect level:

1 750 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 142 (gestation) and 299 (lactation) mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

developmental neurotoxicity

Effect level:

>= 1 750 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level.

Remarks on result:

other: corresponding to 142 (gestation) and 299 (lactation) mg/kg bw/day (highest dose tested)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental (offspring)

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

other: No adverse effects observed at this concentration.

Remarks on result:

other: corresponding to 42.9 (gestation) and 90.0 (lactation) mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

developmental (offspring)

Effect level:

1 750 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponding to 142 (gestation) and 299 (lactation) mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

developmental offspring

Effect level:

150 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

other: No effects adverse effects observed at this dose level.

Remarks on result:

other: Developmental NOAEL for female offspring, corresponding to 12.9 (gestation) and 27.3 (lactation) mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

developmental (offspring)

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponding to 42.9 (gestation) and 90.0 (lactation) mg/kg bw/day

Key result

Critical effects observed:

no

Conclusions:

The study was performed under GLP and according to the proposal for the OECD Guideline f426 (1998), since no EU guideline was available when the study was carried out. It also fulfills the requirements of the U.S. EPA guideline; Developmental Neurotoxicity Study (OPPTS 870.6300, 1996). Under the conditions of the test, the test item caused maternal toxicity at 1750 ppm, seen as reduced body weight and food consumption. Male offspring were effected at 1750 ppm in development, evident as reduced body weight and consequently mortality. Female pups showed reduced body weights and weight gain already in the 500 ppm dose group. There was no evidence of significant developental neurotoxicity in the offspring from dams exposed to the test substance up to dose levels exhibiting maternal toxicity. Reductions in motor activity on Day 22 post partum and in the auditory startle response on Day 23 post partum in F1 rats of the high dose group were not seen at later time points. As these findings were only observed in the presence of maternal and developmental toxicity characterised by reduced body weights and weight gains and in the absence of adverse histomorphological and morphometric changes in the brain, the NOAEL for developmental neurobehavioral effects was established at 1750 ppm.
Taken together, the NOAEL for maternal toxicity and developmental toxicity for male rats was established as 500 ppm (42.9 and 90.0 mg/kg bw/day during gestation and lactation, respectively), the developmental NOAEL for females was set as 150 ppm (12.9 and 27.3 mg/kg bw/day during gestation and lactation, respectively) and the NOAEL for developmental neurotoxicity was set at 1750 ppm for both sexes (142 and 299 mg/kg bw/day during gestation and lactation, respectively).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

142 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The quality of the database is good. The NOAEL was derived from a developmental neurotoxitiy study.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

To characterize the neurotoxic potential of the test substance, three acute, one subchronic and one developmental neurotoxicity in vivo studies on the test substance are available. In the following, all studies will be described and evaluated.

Acute neurotoxicity studies

To assess acute neurotoxicity, three studies were conducted in accordance to guideline EPA-FIFRA, Addendum 10, EPA 540/09-91-123, PB 91-154617 and GLP. All studies run under the number M-027750-03-1. A Weight of Evidence appproach was used to set the NOAEL for acute neurotoxicity.

 

Firstly, overnight fasted rats were fed with the test material via gavage and different parameters for neurotoxicity were assessed.  Groups of 12 male and female rats received 0, 100, 200 or 400 mg/kg bw of the test material in a single dose. Homogeneity and stability of the test material in the vehicle were analytically verified and the analytical concentrations of the doses were 102, 213 and 373 mg/kg bw. The doses were based on a dose range finding experiment (97-912-OE), where groups of 5 rats per sex and dose received a single oral dose of the test substance at 250, 500 or 1000 mg/kg bw and were examined 0.5, 1, 2, 3, 4 and 24 h after administration. Thereafter, they were examined once daily. At 250 mg/kg bw, no effects were observed in any sex. Treatment with 500 mg/kg bw produced clear signs of toxicity starting approximately three hours after treatment and evident as  decreased activity and tremors in males and females and one female of this group died two days after treatment. At 1000 mg/kg bw, all females and 2 males died. Clinical signs at all dose levels became apparent at 3 h, with additional effects at 4 h following treatment. Surviving animals showed signs of toxicity for up to 72 h after treatment.

In the main study, clinical signs were recorded daily and body weight was measured weekly. All animals underwent a functional observation battery (FOB) one week prior to treatment and 4 h, 7 days and 14 days after administration. Furthermore, motor and locomotor activity was tested in the figure-eight maze. 14 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.

No mortality occurred during the study and body weight did not change between treatment and control animals. In the high dose group, tremors, decreased activity and ataxia (in both sexes), signs of red nasal stain and an oral stain (in 1 female) and urine stain in females were observed.

Neurobehavioral effects were evident as decreased motor and locomotor activity in the figure-eight maze and FOB for males at all dose groups and for females at 200 and 400 mg/kg bw. 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. A NOAEL could be established for females at 100 mg/kg bw but no NOAEL was set for males, since administration of the lowest dose resulted in neurobehavioral changes. Therefore, an additional study was conducted with lower concentrations and males only.  

 

In the second study, the same test substance was used and groups of 12 rats were treated with the substance at doses of 0, 60 or 80 mg/kg bw. Apart from the dose levels, the study was performed as the one previously described and rats were observed for two days after treatment. However, gross pathology, body weight changes and histopathology were not assessed because no effects had been found regarding these parameters in the previous study. Here, no clinical signs occurred and all animals survived the course of the study. In the FOB, no differences were found between control and treatment groups but lower motor and locomotor activity was found in males of both treatment groups on the treatment day after administration of the test substance. However, the reduction was more severe in the lower dose group, no statistical significance could be established and all effects were reversible. Therefore, it is not clear, whether the effect seen in the motor and locomotor activity is treatment-related or incidental and an additional study was needed.

 

In this third study, the substance was administered to male rats at 0, 20, 40 and 60 mg/kg bw. The same parameters were evaluated as described in the second study. No differences were found between control and treatment groups, neither in mortality (no mortalities occurred), nor in clinical signs or behavior. Therefore, the NOAEL for males was established to be 60 mg/kg bw.

 

Pharmacological study

The pharmacological effects of the test substance were investigated in mice, rats, and guinea pigs (M-021561-02-1) in accordance to guideline Japanese Guidelines on Agricultural Chemicals (Ministry of Agriculture, Forestry and Fisheries, 59-Nohsan-4200,1985, revision of part 1997) and GLP. A Weight of Evidence appproach was used.

 

In this study, only male animals were investigated. Animals were grouped according to the investigated effect in groups of various sizes and were treated with the test substance in doses of 6.25 - 400 mg/kg bw for mice and 30 - 3000 mg/kg bw in rats. The administration levels used in the in vivo experiment were decided based upon the results of a dose-finding experiment in mice and rats; the route of administration was per os. The concentration levels of the test substance for application in the in vitro experiment as final concentrations were set at 10^-6,10^-5, and 10^-4 mol/L. The in vitro experiment was conducted using isolated guinea pig ileum. 

Regarding the general physical condition and behavior in mice, administration of the test substance at 50 mg/kg bw resulted in decreased spontaneous locomotor activity, tremor, and deep respiration. At 100 mg/kg bw, decreases in grooming, reactivity, body tone, abdominal muscle tone, and body temperature as well as prone posture, staggering gait, and mydriasis were observed in addition to the signs noted at 50 mg/kg bw. Most of these signs were slight, and all had disappeared at 6 h after administration.

With administration of the test substance at 200 mg/kg bw, decreased touch response and grip strength were seen in addition to the signs found after 100 mg/kg bw administration. The signs found with administration of the test substance at 200 mg/kg bw were also found to have disappeared at 6 h after administration. With administration of the test substance at 400 mg/kg bw, in addition to the signs noted following administration at 200 mg/kg bw, decreases in corneal reflex, pinna reflex, and ipsilateral flexor reflex, reduced limb tone, Straub tail, and cyanosis were seen, with 1/3 animals being found dead at 3 h after administration.

In the surviving animals, the signs produced by administration of the test substance were slight to marked, and even at 1 day after administration, the signs of decreased spontaneous locomotor activity, tremor, and staggering gait, while slight, were still observable. However, administration of the test substance at 12.5 and 25 mg/kg bw exhibited no effects on general physical condition and behavior in mice.

Hexobarbital-induced sleeping time in mice was significantly prolonged by administration of the test substance at 225 mg/kg bw, although no effect was seen with administration at 25 and 75 mg/kg bw.

Following administration of the test substance at 25, 75, and 225 mg/kg bw in mice, application of subthreshold electroshock stimulation produced significant convulsion-inducing effect, while with administration at 6.25 and 12.5 mg/kg bw, no convulsions were induced. With subthreshold pentylenetetrazol administration, no convulsions were induced in the the test substance 25, 75, and 225 mg/kg bw administration groups.

With respect to body temperature in rats, at 1 h after administration of the test substance at 300 mg/kg bw, and at 0.5 to 6 h after administration at 1000 and 3000 mg/kg bw, significantly decreased the temperature was recorded, while with administration at 30 and 100 mg/kg bw, no effect was noted.

With administration of 300 mg/kg bw, the heart rate in unanesthetized rats showed significant increase at 0.5 h after administration, systolic blood pressure and mean blood pressure were not affected. With administration of 1000 mg/kg bw, the heart rate showed no significant change, but systolic blood pressure was decreased at 1 hour after administration, and mean blood pressure was decreased at 1 and 6 h after administration. Administration of 3000 mg/kg bw produced increased heart rate at 0.5 h after administration, and decreased mean blood pressure at 6 h after administration. However, with administration of 100 mg/kg bw in unanesthetized rats, no effect on blood pressure or heart rate was found.

With application of 10^-4 mol/L, isolated guinea pig ileum showed suppression of bariuminduced contractile response, while the contractile responses with acetylcholine and histamine were not affected. Test substance applications at 10^-6 and 10^-5 mol/L showed no effect on contractile responses by acetylcholine, histamine, or barium in isolated guinea pig ileum.

Regarding charcoal intestinal transport in mice, the test substance administered at 75 and 225 mg/kg bw produced suppressive effect, but produced no effect at 25 mg/kg bw.

Regarding effect on muscle strength in mice, administration of 225 mg/kg bw produced suppression in 4/8 animals at 1 and 3 h after administration, and in 2/8 animals at 6 h after administration. These suppressed values were in no case significant, but rather only slight in severity. The test substance administered at 25 and 75 mg/kg bw showed no effect on muscle strength in mice.

Regarding blood coagulation time in rats, administration of the test substance at 300,1000, and 3000 mg/kg bw produced no effect on prothrombin time or on activated partial thromboplastin time.

In conclusion, the test substance induced convulsion with subthreshold electroshock at doses of 25 mg/kg bw and greater, exerted suppressive effect on the central nervous system at 50 mg/kg bw and greater and on the gastrointestinal system at 75 mg/kg bw and greater, as well as exerting slight suppressive effect on the skeletal muscular system at 225 mg/kg bw. However, the test substance was not judged to have particularly great effect on the circulatory system or on contractile response to various agonists in isolated guinea pig ileum preparations, nor did it exhibit effect on the blood system.

Taken together, effects on the nervous system were evident as decrease in spontaneous locomotor activity and tremor starting at a dose of 50 mg/kg bw in mice, at 100 mg/kg bw, decreasd reactivity was also observed, a decrease in grip strength was observed at doses of 200 mg/kg bw and higher. At 400 mg/kg bw, inhibition of pinna reflex was also noted. Based on the severity of the effects and the doses in which they were observed, a classification as STOT SE 1 is warranted.

 

Subchronic neurotoxicity study

The subchronic neurotoxicity 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 and in compliance with GLP (M-027986-01-1). The study was suitable to be a key study. 

Subchronic neurotoxicity was investigated in Fischer 344 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 150, 1000 and 3000 ppm (nominal concentration). Liquid chromatography investigations of the feed rations gave analytical concentrations of 135, 890 and 2677 ppm test substance in the diet. Ingested doses were 9.3, 60.03 and 177.0 mg/kg bw/day for males and 10.6, 71.0 and 200.1 mg/kg bw/day for females. Homogeneity and stability of the test material in the vehicle as well as concentrations of the administered test material were analytically verified. Cage sides were observed daily and clinical signs, body weight and food consumption were measured weekly. Ophthalmoscopy examinations were performed pre-exposure and pre-terminal (Week 12). 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.

One male of the high dose died prematurely on Day 4 and showed clinical signs of ill-being before (decreased activity, ataxia and convulsions). This death was considered incidental since no other death occurred. In addition, no severe clinical signs were found in the other animals. Males and females of the high dose group showed reduced significant body weight from Week 11-13 for males and Week 9-13 for females (range: 4-5% reduced body weight compared to controls). Correspondingly, food consumption was also reduced to -17 (males) or -20% (females) in Week 1 and altered to controls thereafter (for females throughout the study, for males in Weeks 1-5 and Weeks 8-13). Moreover, due to the lower body weight, relative brain weight was increased in males (+9.7%) and females (+6.9%) that received 3000 ppm of the test substance, but absolute brain weight was not altered and this was hence determined as a secondary effect. Motor and locomotor activity were unaltered in treatment groups and only minor differences were found regarding the behavior in the FOB. These effects occurred throughout all treatment groups without any pattern. No treatment-related ophthalmological changes were found in the treatment groups. Gross necropsy and histology did not show any differences between control and treatment groups. Based on these results, the NOAEL for general systemic toxicity is 1000 ppm (60.0 and 70.1 mg/kg bw/day for males and females, respectively) and the NOAEL for neurotoxicity 3000 ppm (177 and 200.1 mg/kg bw/day for males and females, respectively), the highest dose tested.

 

Developmental neurotoxicity

The developmental screening study (M-027178-02-1) was performed according to GLP and the guideline EPA OPTS 870.6300, and to the draft version of the OECD guideline 426. The study was suitable to be a key study. 

 

Maternal rats were fed with the test material at dietary concentrations of 150, 500 and 1750 ppm from the start of gestation (gestation Day 0) until the end of lactation (lactation Day 22). Homogeneity and concentration of dosing solutions were analytically verified by liquid chromatography. Stability of the test substance in the diet was demonstrated. Ingested doses were 12.9, 42.9 and 142 mg/kg bw/day during gestation and 27.3, 90.0 and 299.0 mg/kg bw/day during lactation. The offspring did not receive the test substance directly. Doses were selected based on results from previous studies. In a developmental toxicity study, maternal body weight gain and feed consumption was reduced at 125 mg/kg bw/day (corresponding to 1900-2000 ppm) without any evidence of developmental toxicity. In a two-generation reproductive study, maternal body weight change and male and female pup body weights were reduced at 2500 ppm, while no effects were observed at 500 ppm. Preliminary data from the Subchronic Neurotoxicity Screening Battery as done by the sponsor found that parental body weight (male/female) and feed consumption was reduced at 3000 ppm of the test substance, while no effects were seen at 1000 ppm. Based on these results, the dose levels for the current study were set as 150, 500 and 1750 ppm.

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. On Day 12 postpartum, one male and one female pup were randomly allocated to each of 5 subsets in each group for continuation on study. The subsets were investigated as follows: A subset of pups underwent motor activity testing on Days 14, 18, 22 and 62 postpartum and acoustic startle habituation at on Days 23 and 63 postpartum. Another subset was tested for passive avoidance conditioning (Days 23-24 postpartum) and in the water maze (Days 62-63 and 69-70 postpartum). Thereby, long and short term recall were tested. Dams were sacrificed after the gestation period, offspring was sacrificed on Days 5 (to adjust litters), 12, 22 (used to adjust litter sizes at DPs 12-22), 72-77 (after behavioral testing) and 83-87 postpartum. 10 animals/sex/dose level that were sacrificed on Day 12 postpartum 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. 10 animals/sex/dose level that were sacrificed on Days 83-87 postpartum were further investigated regarding gross pathology and brain measurements. Micropathology was performed with control and high dose animals. If lesions were found, all dose groups underwent micropathologic evaluation.

Effects in the maternal generation were reduced food consumption (absolute and relative) in the highest dose group in several intervals of both gestation and lactation period. Correspondingly, body weight of dams was also reduced throughout gestation and in several intervals of lactation in the 1750 ppm group. Body weight and body weight gain was also reduced in the female offspring from 500 ppm and in males at 1750 ppm throughout the preweaning period. Additionally, 2 males and 3 female pups died prematurely (on Days 25, 26 and 27 postpartum) and these deaths were presumably linked to a failure to thrive postweaning due to reduced body weight gains and weight loss.

There was no difference in the passive avoidance testing or in the performance in the water maze. On Day 22 postpartum, motor activity was reduced in the high dose group in males and females but not on the other days tested. Auditory startle habituation was also reduced in females of the high dose group on Day 23 but not on Day 63 postpartum. This was considered treatment related but was seen as a secondary effect to delayed development evident as reduced body weight and premature deaths in this dose group. Other effects seen in this study include occasional findings of clinical signs like lesions or scabs. These effects were evenly distributed through all treatment groups and were therefore considered incidental. Regarding brain morphology, shorter dentate gyrus and caudate putamen in females on Days 83-87 postpartum (both ca -5%) were recorded. However, these differences were considered to be within the biological variance. Moreover, no corresponding effect was observed in the male offspring and on Day 12 postpartum, dentate gyrus was slightly longer than controls. Gross necropsy and histology did not reveal any differences between the control and the high dose group. No differences were found regarding natural delivery, litter observations, reflex and physical development or sexual maturation of pups.

 

Thus, there was no evidence of significant developmental neurotoxicity in the offspring from dams exposed to the test substance up to dose levels exhibiting maternal toxicity. Reductions in motor activity on day 22 post partum and in the auditory startle response on Day 23 post partum in F1 rats of the high dose group were not seen at later time points. As these findings were only observed in the presence of maternal and developmental toxicity characterised by reduced body weights and weight gains and in the absence of adverse histomorphological and morphometric changes in the brain, the NOAEL for developmental neurobehavioural effects was established at 1750 ppm, the highest dose tested.

The NOAEL for the test material for maternal toxicity and developmental toxicity for male rats was established as 500 ppm (42.9 and 90.0 mg/kg bw/day during gestation and lactation, respectively). The developmental NOAEL for females was set as 150 ppm (12.9 and 27.3 mg/kg bw/day during gestation and lactation, respectively) and the NOAEL for developmental neurotoxicity was set at 1750 ppm for both sexes (142 and 299 mg/kg bw/day during gestation and lactation, respectively).

 

Conclusion on neurotoxicity:

In the acute neurotoxicity study, neurobehavioral findings evident as decreased motor and locomotor activity were observed in males and females at sublethal doses, which were fully reversible resulting in a NOAEL of 60 and 100 mg/kg bw for males and females, respectively. In the pharmacological study, reductions in spontaneous locomotor activity, tremors and deep respiration were observed at 50 mg/kg bw. At 25 mg/kg bw and above, both tonic flexor and tonic extensor convulsions were induced in the presence of sub-threshold electroshock application. Based on these results, a NOAEL for central nervous effects of clothianidin was considered to be 50 mg/kg bw.  

According to the Guidance on the Application of the CLP Criteria substances that warrant classification as STOT SE are those that, based on evidence from studies in experimental animals, can be presumed to have the potential to impair organ function and produce significant toxicity in humans following single exposure. Thus, following a weight of evidence assessment and the opinion of RAC, classification as STOT SE 1, H370 (nervous system) is warranted.

In the subchronic neurotoxicity study, reduced food consumption was seen at 177.0 and 200.1 mg/kg bw/day for males and females, respectively. However, no neurotoxic effects occurred up to 177.0-200.1 mg/kg bw/day, for males and females, which was the highest dose tested. In the developmental neurotoxicity study, dams showed reduced food consumption and body weight (gain) at 1750 ppm (142 and 299 mg/kg bw/day during gestation and lactation, respectively). For female pups, first effects of systemic toxicity were evident at 500 ppm (42.9 and 90.0 mg/kg bw/day during gestation and lactation, respectively) as reduced body weight (gain). At 1750 ppm, both female and male pups were impaired in body weight and showed partially reduced neurobehavioral activity. However, these effects were not seen at later time points and occurred in the absence of morphological or morphometric changes of the brain. Therefore, they were attributed to systemic toxicity seen at this dose level, so that the NOAEL for developmental neurotoxicity was set to 1750 ppm.

 

References not included in IUC:

RAC opinion, adopted 16 Sept, 2021: Committee for Risk Assessment - RAC - Opinion proposing harmonised classification and labelling at EU level of clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine, EC Number: 433-460-1, CAS Number: 210880-92-5

 

 

 

Justification for classification or non-classification

The available data on neurotoxicity of the test substance meet the criteria for classification and labelling as STOT SE 1, H370 (nervous system) according to Regulation (EC) 1272/2008. The concentrations supporting STOT SE 1 are always above 30 mg/bw kg and therefore no SCL is needed. No narcotic effects or respiratory tract irritation were reported in the single dose toxicity studies and therefore classification as STOT SE 3 is not supported.