Ziram

Neurotoxicity

Description of key information

Key, report number WIL-223001, acute (rat):
NOAEL (systemic toxicity): 15 mg/kg bw (males and females)
LOAEL (systemic toxicity): 300 mg/kg bw (males and females)

NOAEL (neurotoxicity): 15 mg/kg bw (males and females)
LOAEL (neurotoxicity): 300 mg/kg bw (males and females)

Key, report number WIL-223004, subchronic (rat):

NOAEL (systemic toxicity): 180 ppm (corresponding to 14 and 16 mg/kg bw/day in male and female rats, respectively)

LOAEL (systemic toxicity): 540 ppm (corresponding to 34 and 40 mg/kg bw/day in male and female rats, respectively)

NOAEL (neurotoxicity): 540 ppm (corresponding to 34 and 40 mg/kg bw/day in male and female rats, respectively)

 

Key, report number WIL-223006, developmental neurotoxicity (rat):

NOAEL (maternal systemic toxicity): 72 ppm (corresponding to 5, 8 and 12 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively)

LOAEL (maternal systemic toxicity): 207 ppm (corresponding to 13, 23 and 34 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively)

NOAEL (maternal reproductive toxicity): 540 ppm (corresponding to 27, 59 and 61 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively)

NOAEL (developmental toxicity F1 generation): 72 ppm (corresponding to 5, 8 and 12 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively in dams)

LOAEL (developmental toxicity F1 generation): 207 ppm (corresponding to 13, 23 and 34 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively in dams)

NOAEL (neurotoxicity F1 generation): 72 ppm (corresponding to 5, 8 and 12 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively in dams)

LOAEL (neurotoxicity F1 generation): 207 ppm (corresponding to 13, 23 and 34 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively in dams)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Aug 1993 - 22 Dec 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 418 (Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure)

Version / remarks:

adopted in 1995

Deviations:

yes

Remarks:

rats instead of hens, 14 days observation and not 21, methodological limitations (no biochemical, tests were performed once a week, histopathology was performed on 5 rats/sex from control and top dose, no positive control)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

humidity slightly different, administration of test substance once, food consumption, ophthalmology not examined, solely 5 animals/sex of the control and high-dose group histopathologically examined

Qualifier:

according to guideline

Guideline:

EPA OPP 81-8 (Neurotoxicity Screening Battery)

Version / remarks:

adopted in 1990

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: 43 days
- Weight at study initiation: 189 - 253 g (males), 138 - 190 g (females)
- Housing:
Weaning animals: 3 per sex/cage for 3 days following receipt.
Thereafter animals were housed individually.
- Diet: Purina® Certified Rodent Chow® #5002 in meal form, ad libitum
- Water: Municipal water from the public supply, ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 23.3
- Humidity (%): 60 - 85
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 Aug To: 22 Dec 1993

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate volume of corn oil to dose the control group animals was dispensed into a properly labeled storage container. The corn oil was stirred throughout the sampling and dosing procedures using a magnetic stir bar and plate. Each dosing admixture was prepared as follows:
A sufficient amount of test substance was weighed into a tared pre-calibrated storage container. An appropriate amount of corn oil was added to attain the required volume and test substance concentration. The container was then placed on a Polytron PT 6000 and the contents mixed for approximately 5 min. Each preparation was stirred continuously throughout the sampling and dosing procedures using a magnetic stir bar and plate. All preparations were made on the day of dosing.

VEHICLE
- Amount of vehicle: 7.5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity, stability and test article concentration analyses were conducted on test article preparations. Prior to initiation of dosing, the top, middle and bottom of the preparations were sampled for homogeneity; the middle of the preparations were sampled for stability. On the day of dosing for Replicate 1, the middle of each preparation was sampled for concentration analysis. In addition, samples from the middle of each preparation were collected on each day of dosing and stored refrigerated.

With regard to the test substance, the suspensions were homogeneous. The low-dose group mean concentrations were 2.12 mg/mL (top, 104%), 2.15 mg/mL (mid, 105%), and 2.08 mg/mL (bottom, 102%). The overall mean was 2.11 mg/mL (104% of the dose concentration). The mean concentrations for the mid-dose group were 39.4 mg/mL (top, 96.5%), 39.9 mg/mL (mid, 97.9%) and 41.1 mg/mL (bottom, 101%). The overall mean was 40.1 mg/mL (98.3% of the dose concentration). In case of the high-dose group, the location means were 85.1 mg/mL (top, 104%), 86.4 mg/mL (mid, 106%), and 83.8 mg/mL (bottom, 103%) with an overall mean concentration of 85.1 mg/mL, or 104% of the dose concentration. All groups met the requirements for homogeneity.

The suspensions were stable for 8 h at room temperature. Samples were analyzed to establish a Time Zero concentration and then analyzed after 8 h at room temperature. The Time Zero concentrations were 1.95 mg/mL (low-dose group, 95.5%), 45.4 mg/mL (mid-dose group, 111%) and 89.9 mg/mL (high-dose group, 110%). The 8 hour concentrations were 1.90 mg/mL (low-dose group, 92.9 %), 47.7 mg/mL (mid-dose group, 117 %) and 89.7 mg/mL (high-dose group, 110 %). There was no apparent loss of test material after 8 h of room temperature storage.

The suspensions used for dose administration contained the specified amounts of test substance. The mean concentrations were 2.15 mg/mL (low-dose group, 105%), 42.6 mg/mL (mid-dose group, 104%), and 87.5 mg/mL (high-dose group, 107%). Suspensions met with the requirement for concentration acceptability.

Duration of treatment / exposure:

Single oral dose

Frequency of treatment:

Once

Dose / conc.:

15 mg/kg bw (total dose)

Dose / conc.:

300 mg/kg bw (total dose)

Dose / conc.:

600 mg/kg bw (total dose)

No. of animals per sex per dose:

12 per sex for 0, 15, 300 mg/kg bw
16 per sex for 600 mg/kg bw

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked: mortality and/or moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily except on days were FOB was conducted.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded pre-study (Day -10), on Day 0, 7 and 14. Body weights were also recorded during the FOB procedures and prior to necropsy (Day 15).

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

No anticholinergic substances used

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
Observations on all animals were made pre-study, at the approximate time of "peak effect" (approximately 4-h after dose administration) and on Day 7 and 14. Testing was performed by the same technicians without knowledge of the animal group assignment. FOB was performed in a sound-proofed room equipped with a white noise generator set to operate at 70 + 10 db. with one exception; home cage observations were performed in the animal room.

Home Cage Observations: Posture, Convulsions/tremors, Feces consistency, Biting, Palpebral (eyelid) closure

Handling Observations: Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Red/Crusty deposits, Eye prominence, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone

Sensory Observations: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eye blink response, Hind limb extension, Olfactory orientation

Neuromuscular Observations: Hind limb extensor strength, Hind limb foot splay, Grip strength-hind and forelimb, Rotarod performance

Physiological Observations: Catalepsy, Body weight, Body temperature

LOCOMOTOR ACTIVITY: Yes
Observations were made on all animals during the pre-test period, at the approximate time of "peak effect" (approximately 4-h after dose administration) and on Day 7 and 14. Locomotor activity, recorded after the completion of the FOB, was measured automatically using the Digiscan 'Micro' Animal Activity System, a PC-controlled system consisting of individual test chambers, which utilize a series of infrared photo-beams surrounding a clear plastic, rectangular cage to quantify an animal's motor activity. The testing of treatment groups was done according to replicate sequence. The activity system was operated in the Stagger Start mode of operation. Data were collected in 1 min epochs and the test session was 41 min in duration for each animal. Animal placement into the activity cage in the Stagger Start mode of operation initiated the data collection process. The first epoch was often incomplete due to the placement of the animal in the activity cage. For this reason, the first minute of data was deleted. The remaining 40 min of data collection (4, 10 min subsessions) were compiled for tabular presentation.
During the Day 14 MA data collection, there was an apparent malfunction of the monitor housing 15 mg/kg bw group male no. 15378. This was discovered following the necropsy of male no. 15378.
These data were not tabulated in the group mean for this interval (this isolated incident had no impact on the scientific integrity, validity or outcome of the study).
Locomotor Activity was divided into two categories; total and ambulatory activity. Total MA was defined as a combination of fine motor skills (i.e. grooming; interruption of one or two adjacent photo-beams) and ambulatory motor activity (interruption of three or more consecutive photo-beams). Data for ambulatory and total MA were tabulated.

AUDITORY STARTLE REFLEX HABITUATION: No

LEARNING AND MEMORY TESTING: No

Sacrifice and (histo)pathology:

HISTOPATHOLOGY
Sacrifice:
- unscheduled sacrifice: A complete necropsy examination was conducted. The necropsy included an examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities including the viscera. All gross lesions were collected and preserved in 10% neutral buffered formalin. The carcasses were then discarded.
- scheduled sacrifice: Animals in each group were euthanized by CO2 after a post-exposure period of 14 days: inhalation and then perfused in situ.

Neuropathology:
Central and peripheral tissues were preserved. Brain weight (excluding olfactory bulbs) and brain dimensions (length and width) were recorded. Any observable gross changes, abnormal coloration or lesions of the brain and spinal cord were recorded. It was also recorded when no gross lesions were observed for the brain and spinal cord (not required by the protocol). Additionally, remarkable gross lesions observed internally/externally during the perfusion/dissection processes were also recorded. These deviations from the protocol had no apparent impact on the outcome of the study. Following nerve tissues were dissected and prepared for a qualitative histopathological examination (embedded in paraffin or plastic, sectioned and then stained with hematoxylin and eosin) from five animals per sex in the control group and in the high-dose group.

Central Nervous System tissues:
Brain - forebrain, center of cerebrum, midbrain, cerebellum and pons, and the medulla oblongata, Spinal cord - at cervical swellings C3 - C8 and at lumbar swellings T13 - L4, Gasserian ganglion/Trigeminal nerves Lumbardorsal root ganglion at T13 - L4, Lumbardorsal root fibers at T13 - L4, Lumbar ventral root fibers at T13 - L4, Cervical dorsal root ganglion at C3 - C8, Cervical dorsal root fibers at C3 - C8, Cervical ventral root fibers at C3 - C8, Optic Nerves, Eyes

Peripheral Nervous System tissues:
Sciatic nerves (mid-thigh region and at sciatic notch), Sural nerves, Tibial nerves, Peroneal nerves, Forelimbs, Tail

Other examinations:

no

Positive control:

none

Statistics:

Statistical tests for bw, histopathological and brain data were performed by a computer with appropriate software. All analyses were two-tailed for a significance level of 5% and 1%. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Bw and bw change data were subjected to a one-way analysis of variance (ANOVA). If significant differences were indicated by the ANOVA, Dunnett's test was used to compare the control and treated groups.
Histopathological findings of the 600 mg/kg bw group were compared to the control group data by the Kolmogorov-Smirnov test. Brain weights and dimensions were analyzed by one-way ANOVA. If significant differences were indicated by the ANOVA, Dunnett's test was used to compare the control and treated groups.
Statistical tests for the FOB and Locomotor activity data were performed using a PC installed with SAS/STATstatistical software. Each mean was presented with the S.D. and the number of animals used to calculate the mean. Animals that died on study were not included in the calculation of the means for any test period. Continuous FOB data were analyzed using a two-way repeated measure ANOVA. If significant treatment or treatment-time interactions occurred, a one-way ANOVA was conducted at each time point. If significant treatment effects were observed at a time point, Dunnett's multiple T-test was conducted to determine significant treatment differences from the control group (p<0.05). FOB parameters yielding scalar (ordinal) or descriptive data were analyzed using the repeated measures SAS CATMOD procedure. If significant treatment or treatment-time effects occurred, Fisher's Exact test or Dunnett's test were conducted to determine significant treatment differences from the control group (these tests were performed by a computer with appropriate software).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled sacrifice:
Clinical signs observed for those animals in the 600 mg/kg bw group that died on study were generally limited to 2/4 males and 5/7 females. Remarkable findings included gait alterations (rocking, lurching or swaying and/or high carriage), abnormal respiration (rales, labored respiration and/or gasping), abnormal excreta (soft stool, diarrhea, mucoid feces, decreased defecation and/or decreased urination), ptosis, hypothermia, (cool to touch, males only) and hypoactivity (females only). Other findings observed for these animals consisted of colored staining/material (tan, yellow, red and/or orange) on various body surfaces and the abdomen appearing distended.
Clinical signs observed for the 300 mg/kg bw group female that died included gait alterations (rocking, lurching or swaying and high carriage), ptosis, soft stool and tan staining on the forelimbs and around the mouth.

Scheduled sacrifice:
Treatment-related clinical signs were observed in surviving animals of the 300 and 600 mg/kg bw group. The most remarkable of these findings often were limited to or occurred most frequently in two males in the mid-dose group. Clinical signs observed for these two males only included cyanosis, hypothermia (cool to touch), enophthalmus and unkempt appearance. The aforementioned clinical signs were observed on Day 8 or later (last week of study) on one to three occasions. Other remarkable clinical signs observed for both males throughout the study included abnormal respiration, abnormal excreta, gait alterations, abdomen appearing distended, hypoactivity, ptosis and red, tan, orange and/or brown staining/material. Furthermore, signs observed in the other animals of the mid-dose and high-dose groups included gait alterations (rocking, lurching or swaying and/or high carriage), abnormal respiration (rales, labored respiration and/or gasping), abnormal excreta (soft stool, diarrhea, mucoid feces, decreased defecation and urination and small feces) and abdomen appearing distended. Overall, the occurrence of these findings was limited to the first week of study. Additionally, tan, red, orange and/or brown staining/material was observed for some males and females in the 300 and 600 mg/kg bw group generally during the first week of study.

For details, please refer to attachment 1 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Following dose administration with 600 mg/kg bw of the test substance, four males (25% mortality) and seven females (44% mortality) died. All of the males and three of the females died on Day 1. The deaths of the other females in the 600 mg/kg bw group occurred on Day 2, 4 or 5. One female (8% mortality) in the 300 mg/kg bw group died on Day 2. All other animals in the 300 and 600 mg/kg bw groups and all animals in the control and 15 mg/kg bw groups survived and were sacrificed as scheduled on Day 15.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

With regard to the body weight, transient statistically significant reductions in mean body weight gain were apparent in the 300 and 600 mg/kg bw group males and females for Day 0 to 7. The differences from the control group were 63% and 55% in the mid-dose group and 67% and 45% in the high-dose group for males and females, respectively. Even though mean bw gains in the 300 mg/kg bw group females and the 600 mg/kg bw group males and females were similar to or greater than the control group for Day 7 to 14 and 14 to 15, the mean cumulative gains were slightly lower than the control group means for Day 0 to 14 and 0 to 15. Mean body weight gain was reduced in the mid-dose group males for Day 7 to 14; this was mainly due to 2/12 of these animals (previously mentioned males with severe clinical signs). All mean cumulative gains (Day 0 to 7, 0 to 14 and 0 to 15) in the 300 mg/kg bw group males were statistically significantly lower than the control group means. The Day 7 mean body weight in the 600 mg/kg bw group males was statistically significantly lower than the control group mean. Mean body weight values were statistically significantly lower for Day 7, 14 and 15 in the 300 mg/kg bw group males. No effects on mean body weights or body weight gains were apparent in the 15 mg/kg bw group males or females.

For details, please refer to attachment 2 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Based on the FOB, differences from the control group were noticed for animals that received the test substance at dose levels of 300 and 600 mg/kg bw. Overall, responses occurred approximately 4-h following treatment on Day 0, and were transient in nature (several signs were observed at the daily clinical examinations generally during the first week of study; none of these persisted to Day 7). Specific test results included (i) altered posture, palpebral closure and feces consistency in the 300 and 600 mg/kg bw group males and females during the home cage observations on Day 0; (ii) lacrimation, salivation, changes in fur appearance, altered palpebral closure, changes in respiratory rate and/or character, red deposits around the mouth and crusty deposits around the nose and mouth in the 300 and 600 mg/kg bw group males and females during the handling observations on Day (in addition, decreased muscle tone was observed in the high-dose group males on Day 0); (iii) impaired mobility and altered gait in the 300 and 600 mg/kg bw group males and females during the open field observations on Day 0; (iv) absent tail pinch and olfactory responses and a slighter startle response in the 300 and 600 mg/kg bw group males and females, absent startle response in the 600 mg/kg bw group males and females, absent forelimb and/or hind limb extension in the 300 and 600 mg/kg bw group females during the sensory observations on Day 0; (v) decreased body temperatures in the 300 and 600 mg/kg bw group males and females during the physiological observations on Day 0, and reduced bw in the 300 and 600 mg/kg group males on Day 7. Remarkable signs observed during the Day 14 FOB evaluation were generally limited to the both named males belonging to the 300 mg/kg bw group. It should be noted that these findings were generally not observed during the Day 7 FOB evaluation. Moreover, some of these signs were not even observed at the day 0 FOB evaluation for these animals or the 600 mg/kg group males and females at any evaluation period. Regarding the low-dose group, the only possible effects on FOB parameters were transient in nature and were observed only during the Day 0 evaluation. These possible effects included altered posture for one male during the home cage observations and slight impairment of gait for two males and one female during the open field observations. Reductions in mean ambulatory and total MA counts were observed in the 300 and 600 mg/kg bw group males and females on Day 0. The values for all four subsessions, especially the first three, were remarkably reduced in the 300 and 600 mg/kg bw group males and females when compared to the control group. Reductions in mean ambulatory and total MA counts in the 600 mg/kg bw group males persisted through to Day 7; however, full recovery was apparent for these males by Day 14. Even though the mean counts were not affected in the 300 mg/kg male group on Day 14, the ambulatory and total MA counts for both named males below were reduced when compared to the control group at this time period and when compared to their Day 7 values.

For details, please refer to attachment 3 and 4 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

With regard to organ weight, no treatment-related effects on mean absolute brain weights were apparent at any dose level. The mean absolute brain weights for the 300 and 600 mg/kg bw group males were statistically significantly lower than the control group mean. However, the differences were small (6% or 7%) and no remarkable differences between these males and the control group were apparent in brain dimensions. In addition, no similar differences from the control group in mean absolute brain weights were observed for the 300 and 600 mg/kg bw group females, therefore, the slightly lower brain weights for the 300 and 600 mg/kg bw group males were not attributed to test substance administration.

For details, please refer to attachment 5 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled sacrifice:
White contents in the stomach and/or intestine were observed at 300 and 600 mg/kg bw as well as distention of the stomach and/or intestine. Two females of the 600 mg/kg bw group were emaciated (no abdominal adipose tissue was present).

Scheduled sacrifice:
No treatment-related effects on brain dimensions were noticed.

For details, please refer to attachment 6 under "Overall remarks, attachments".

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects on brain dimensions were noticed.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathologically, no treatment-related microscopic lesions were observed in any of the central or peripheral nervous system tissues examined from the 5 animals/sex in the high-dose group (600 mg/kg bw).
For details, please refer to attachment 7 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

15 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

mortality

other: behaviour (changes in FOB)

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxicity

Effect level:

15 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

neurotoxicity

Effect level:

300 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Key result

Critical effects observed:

no

Table 1         Acute neurotoxic effects
Parameter / Dose	0 mg/kg		15 mg/kg		300 mg/kg		600 mg/kg		Dose-response +/–
	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀
Clinical signs					Yes	Yes	Yes	Yes	–	–
Body weight					↓		↓		–	–
Changes in FOB					Yes	Yes	Yes	Yes	–	–

Conclusions:

The study at hand was conducted under GLP conditions and is in accordance with EPA OPP 81-8 (Neurotoxicity Screening Battery) and similar to OECD 424. The study is considered reliable and valid. The test substance was administered by the oral route (gavage) as a single dose at dose levels of 15, 300 and 600 mg/kg bw to Sprague-Dawley rats. Systemic toxicity was observed at 300 and 600 mg/kg bw in male and female rats evident by mortality, clinical signs, reduced body weight gain, and gross pathology findings. Neurotoxicity was also observed in both male and female rats at 300 and 600 mg/kg bw including impaired walk and tiptoe gait, impaired mobility and reduced body temperature. Thus, under the conditions tested the NOAEL for systemic toxicity and neurotoxicity was set to 15 mg/kg bw.

Reference 2

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Sep 1993 - 07 Oct 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

humidity slightly different, ophthalmology not performed, only 5 animals/sex of the control and high-dose group examined histopathologically

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: 50 days
- Weight at study initiation: 207-271 g (males), 151-190 g (females)
- Housing: Weanling animals: 3 per sex/cage for 3 days following receipt in suspended stainless steel wire-mesh cages. Thereafter all animals were housed individually.
- Diet: Purina® Certified Rodent Chow® #5002, ad libitum
- Water: Municipal water, ad libitum
- Acclimation period: 19 days prior to dosing initiation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23.9
- Humidity (%): 36 - 78
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Oct 1993 To: 27 Feb 1994

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
For the control group, 7.5 kg of feed was weighed out weekly and stored in a properly labelled storage bag.
For dose calculation purposes, a correction factor of 1.022 was utilized. For each dose group, the appropriate amount of test substance was weighed into a tared glass container. After weighing 1 kg of feed and placing it into a mixing bowl, the test substance was added to the bowl. A small amount of feed was added to the glass container, and then the container was scraped to ensure the transference of the entire test substance. The preparation was mixed for 5 min. The premix was transferred to a mixer and mixed for 10 minutes with enough feed to achieve a total batch of 7.5 kg. An intensifier bar was used during the first and last three min of the mixing procedure. Each test diet was prepared approximately weekly, subdivided into seven separate bags for daily administration and stored refrigerated, due to the limited stability of the test article in the diet. One bag of each test diet was withdrawn from refrigeration on a daily basis and released for administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity, stability and test substance concentration analyses were conducted on diet preparations. Samples were collected prior to study initiation (homogeneity, stability concentration analyses) and approximately weekly during the study (concentration analyses conducted on samples collected in study weeks 0, 1, 2, 3, 7 and 11).

Homogeneity
With regard to the test substance, the suspensions were homogeneous. The low-dose group mean concentrations were 62.2 ppm (top, 104%), 64.1 ppm (mid, 107%), and 63.3 (bottom, 106%). The overall mean was 63.2 ppm (105% of the dose concentration). The mean concentrations for the mid-dose group were 180 ppm (top, 100%), 181 ppm (mid, 101%) and 168 ppm (bottom, 93%). The overall mean was 177 ppm (98.1% of the dose concentration). In case of the high-dose group, the location means were 541 ppm (top, 100%), 547 ppm (mid, 101%), and 556 ppm (bottom, 103%) with an overall mean concentration of 548, or 101% of the dose concentration.

Stability
There was significant loss of test substance in Groups 2 (60 ppm) and 3 (180 ppm) (73.8% and 75.8% of target concentrations, respectively) after 16 h at room temperature (RT). The high-dose group (540 ppm) diet formulation remained within stability requirements after 16 hat RT (86.1% of target concentration). When Groups 2 and 3 were fortified to 72 and 207 ppm, respectively, the test substance concentrations decreased to the desired dosage levels [92.0% and 98.9% of the target concentrations (60 and 180 ppm), respectively] after 16 h of RT storage.

Concentration
The diet formulations used for dose administration contained the specified amounts of test substance. Mean concentrations (percent of target fortified concentration) for the six diet preparations analyzed were 69.4 ppm (Group 2, low, 96.3%), 204 ppm (Group 3, 98.5%) and 543 ppm (Group 4, 101%).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily via diet

Dose / conc.:

60 ppm

Remarks:

The concentration of test material in the low dose level was increased by 20% above nominal (72 ppm) to compensate losses during storage. In total this corresponded to 5 and 6 mg/kg bw/day in male and female rats, respectively.

Dose / conc.:

180 ppm

Remarks:

The concentration of test material in the low dose level was increased by 15% above nominal (207 ppm) to compensate losses during storage. In total this corresponded to 14 and 16 mg/kg bw/day in male and female rats, respectively.

Dose / conc.:

540 ppm

Remarks:

Corresponding to 34 and 40 mg/kg bw/day in male and female rats, respectively.

No. of animals per sex per dose:

10 (5 animals/sex were allocated for cholinesterase and brain neurotoxic esterase (NTE) determination; the remaining 5 were allocated for neuropathology evaluation at termination)

Control animals:

yes, plain diet

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality and/or moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily except on days were FOB was conducted.

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly, beginning approximately two weeks prior to treatment.

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: The mean amounts of test substance consumed (mg/kg/day) by each sex per group were calculated from the mean food consumed (g/kg/day) and the appropriate nominal concentration of the test substance in the food (ppm).
- Time schedule: Individual food consumption was measured daily.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: Yes
CHOLINESTERASE ACTIVITY (ACHE): Yes
RED BLOOD CELL (RBC): Yes

- Time schedule for examinations: Evaluation was conducted pre-study, during the 4th and 8th weeks of test substance exposure corresponding to study weeks 3 and 7, respectively) and at study termination (week 13).
- How many animals: 5 animals/sex/group
- Method of sample collection and processing:
Following euthanization and exsanguination, whole brain cholinesterase (Cholin) and neurotoxic esterase (NTE) evaluations were conducted for each of the 5 animals/sex/group in this study component. Blood samples were collected from the caudal(tail) vein for the intervals prior to study termination and from the inferior vena cava at the time of necropsy. For study weeks 3 and 7, blood evaluations followed the completion of the FOB and locomotor activity evaluation. The plasma and red blood cell cholinesterase evaluations were conducted in a sepcialised laboratory.
- Animals fasted: not stated
- Description of methodology for NTE determination: not provided

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
Observations on 10 animals/sex/group (5 animals/sex/group each from the cholinesterase/NTE group and the neuropathology group) were made pre-study (Week 2) and then during the 4th, 8th and 13th weeks of the test substance exposure period (Weeks 3, 7 and 12, respectively) prior to dose administration. Testing was performed by the same technicians, wherever possible, without knowledge of the animal group assignment. The FOB was performed in a sound-proofed room equipped with a white noise generator set to operate at 70 + 10 db with one exception; home cage observations were performed in the animal room.

Home Cage Observations
Posture, Convulsions/tremors, Feces consistency, Biting, Palpebral (eyelid) closure

Handling Observations
Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Red/Crusty deposits, Eye prominence, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone

Open Field Observations (evaluated over a 2 minute observation period)
Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behaviour, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait Score, Backing

Sensory Observations
Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eye blink response, Hind limb extension, Olfactory orientation

Neuromuscular Observations
Hind limb extensor strength, Hind limb foot splay, Grip strength-hind and forelimb, Rotarod performance

Physiological Observations
Catalepsy, Body weight, Body temperature

LOCOMOTOR ACTIVITY: Yes
Observations were made on 10 animals/sex/group (5 animals/sex/group each from the cholinesterase/NTE group and the neuropathology group) pre-study (Week -2) and during the 4th, 8th and 13th weeks of the test substance exposure period (Weeks 3, 7 and 12, respectively) prior to dose administration. Locomotor activity, recorded after the completion of the FOB, was measured automatically in a PC-controlled system. This PC-controlled system utilizes a series of infrared photo-beams surrounding a clear plastic, rectangular cage to quantify an individual animal's motor activity. The testing of treatment groups was done according to replicate sequence. Each animal was tested separately. The activity system was operated in the Stagger Start mode of operation. Data were collected in one minute epochs (print intervals) and the test session duration was 41 min. Animal placement into the activity cage in the Stagger Start mode of operation initiated the data collection process. The first epoch was often incomplete due to the placement of the animal in the activity cage. For this reason, the first minute of data was deleted for each animal. The remaining 40 minutes of data collection (divided into 10-min subsessions) were compiled for data presentation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e. grooming; interruption of one or two adjacent photo-beams) and ambulatory motor activity (interruption of three or more consecutive photo-beams).

AUDITORY STARTLE REFLEX HABITUATION: No

LEARNING AND MEMORY TESTING: No

Sacrifice and (histo)pathology:

PATHOLOGY
- NTE/Cholinesterase Animals: Brains were weighed and evaluated for brain cholinesterase and NTE levels.
- Neuropathology animals: At study termination, the 5 animals/sex/group allocated for neuropathological examination were euthanized by CO: inhalation and then perfused in situ with a buffered sodium nitrite solution followed by a solution of 1.5% glutaraldehyde-4.0% formaldehyde. The central and peripheral tissues were dissected and preserved. Brain weight (excluding olfactory bulbs) and brain dimensions (length and width) were recorded. Any observable gross changes, abnormal coloration, or lesions of the brain and spinal cord were recorded.
- Unscheduled deaths: Complete necropsy was conducted. The necropsy included an examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities including the viscera. Tissues and organs were retained for possible future histological examination only as deemed necessary by the gross findings.

HISTOPATHOLOGY
- Yes, on neuropathology animals from control and high-dosage group. The nerve tissues were embedded in plastic or paraffin, as appropriate, sectioned, then stained with hematoxylin and eosin. The following nerve tissues were sampled for a qualitative histopathological examination from animals in the control and 540 ppm groups:

Central Nervous System tissues
Brain - forebrain, center of cerebrum, midbrain, cerebellum and pons, and the medulla oblongata, Spinal cord - at cervical swellings C3 - C8 and at lumbar swellings T13 - L4, Gasserian ganglion/Trigeminal nerves Lumbardorsal root ganglion at T13 - L4, Lumbardorsal root fibers at T13 - L4, Lumbar ventral
root fibers at T13 - L4, Cervical dorsal root ganglion at C3 - C8, Cervical dorsal root fibers at C3 - C8, Cervical ventral root fibers at C3 - C8, Optic Nerves, Eyes

Peripheral Nervous System tissues
Sciatic nerves (mid-thigh region and at sciatic notch), Sural nerves, Tibial nerves, Peroneal nerves, Forelimbs (preserved for potential future examinations), Tail

Other examinations:

none

Positive control:

none

Statistics:

All statistical tests for data other than the FOB and Locomotor Activity were performed. All analyses were two-tailed (except as noted) for significance levels of 5% and 1%. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Body weights (bw), body weight changes, food consumption, cholinesterase and neurotoxic esterase values, absolute and relative brain weights and brain dimensions were analyzed by a one-way analysis of variance (ANOVA). If significant differences were indicated by the ANOVA, Dunnett's test was used to compare the control and treated groups. Histopathological findings in the treated groups were compared to the control group data by the one-tailed Kolmogorov-Smirnov test. Each mean was presented with the standard deviation and the number of animals used to calculate the mean. The animal that died on study was not included in the calculation of the means for any test period. Continuous FOB and Locomotor Activity data were analyzed using a two-way repeated measures ANOVA. If significant treatment or treatment-time interactions occurred, a one-way ANOVA was conducted at each time point. If significant treatment effects were observed at a time point, Dunnett's multiple T-test was conducted to determine significant treatment differences from the control group. FOB parameters yielding scalar (ordinal) or descriptive data were analyzed using the repeated measures SAS CATMOD procedure. If significant treatment or treatment-time interactions occurred, Fisher's Exact test or Dunnett's test were conducted to determine significant treatment differences from the control group.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One male in the 72 ppm group was hypoactive and had laboured respiration and red material around the nose on study Day 13, the day prior to death.
No apparent test substance-related clinical signs were observed for the animals that survived to the scheduled euthanization. The clinical signs observed in the treated group animals were observed at a similar incidence in the control group, were present in a non-dose-related manner, were limited to single animals in various groups, and/or were those commonly observed in laboratory rats.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in the 72 ppm group was found dead on study Day 14. All other animals in the 72 ppm group and all animals in the control, 207 and 540 ppm groups survived through to the scheduled euthanization.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

With regard to the body weight, mean body weight in the 540 ppm group males and females was lower than the control group beginning at Week 1 and continuing throughout the study period (Week 13). The differences from the control group ranged from 8% to 10% for the 540 ppm group males and from 7% to 11% for the females in this group. The mean body weight values were statistically significantly lower than the control group values at Weeks 1 through 8 for the males and at Weeks 1 through 3 and Weeks 5 through 13 for the females in the 540 ppm group. The lower mean body weight were mainly due to lower body weight gains in the 540 ppm group males and females for Week 0 to 1. In general, weekly mean body weight gains were comparable to the control group means throughout the remainder of the study, except for a statistically significantly lower body weight gain observed for the 540 ppm group females for week 9 to 10. The mean cumulative body weight gains (Weeks 0 to 1 through Weeks 0 to 13) were statistically significantly lower than the control group means in the 540 ppm group males and females. The overall (Weeks 0 to 13) differences from the control group for mean body weight gain were 18% and 32% in the 540 ppm group males and females, respectively.

No test substance-related effects were observed in the body weight data for the 72 or 207 ppm group males and females.

For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption (evaluated as g/animal/day and g/kg/day) was remarkably and statistically significantly reduced in the 540 ppm group males and females during the initial week of test substance administration (Week 0 to 1). In addition, g/animal/day means for these animals were generally slightly lower than the control group means throughout the remainder of the study period (Weeks 1 to 2 through 12 to 13). The values were statistically significant for Weeks 3 to 4, 4 to 5, 5 to 6 and 12 to 13 for the males and for Weeks 1 to 2, 2 to 3, 5 to 6, 9 to 10, 10 to 11, 11 to 12 and 12 to 13 for the females.

Food consumption in the 72 and 207 ppm group males and females was unaffected by test substance administration throughout the study. The only statistically significant difference from the control group was a slightly lower g/kg/day value in the 207 ppm group females for Week 0 to 1. However, the g/animal/day value in these females for Week 0 to 1 was similar to the control group value. Therefore, the lower g/kg/day value was attributed to a slight difference in mean body weight.

For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No remarkable differences from the control group were observed in mean plasma and red blood cell cholinesterase values at any dietary concentration during study Weeks 3, 7 and 13. No apparent effect on mean brain cholinesterase was observed at any dietary concentration. No statistically significant differences from the control group were observed in the treated groups. Mean brain neurotoxic esterase values were statistically significantly lower in the 540 ppm group males and females when compared to the control group values at Week 13. The differences from the control group were 47% and 38% for the males and females, respectively, and were attributed to the treatment with the test substance. However, the toxicological significance of the moderate inhibition of the brain NTE (Neuropathy Target Esterase) is not clear.

No remarkable differences from the control group were apparent in the 72 or 207 ppm group males and females

For details, please refer to attachment 5 under "Overall remarks, attachments".

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional Observational Battery (FOB)

Home Cage Observations
No remarkable differences were apparent between the control and treated groups when the home cage observations were evaluated during the pre-test period and during Weeks 3, 7 and 12 (4th, 8th and 13th weeks of dose administration, respectively).

Handling Observations
No remarkable differences were apparent between the control and treated groups when the handling observations were evaluated during the pre-test period and during Weeks 3, 7 and 12.

Open Field Observations
No test substance-related differences were apparent between the control and treated groups when the open field observations were evaluated during the pre-test period and during Weeks 3, 7 and 12. One female and one male in the 540 ppm group displayed a gait alteration (walks on tiptoes) at Weeks 3 and 7, respectively. In each case, the alteration was graded as slight but definite. However, these were isolated occurrences for single animals. In addition, this finding has been previously observed in females in the historical control data and no gait alterations were observed during the Week 12 open field observations or during the daily clinical examinations at any dose level. Therefore, these isolated occurrences of gait alteration in two animals were not attributed to the test substance. No other remarkable differences between the control and treated groups were observed in open field parameters.

Sensory Observations
No remarkable differences were apparent between the control and treated groups when the sensorimotor responses were evaluated during the pre-test period and during Weeks 3, 7 and 12.

Neuromuscular Observations
At the Week 7 neuromuscular evaluation, only the mean forelimb grip strength in the 540 ppm group females was statistically significantly lower than the control group mean. A similar reduction was not observed in the 540 ppm group males. In addition, mean forelimb grip strength in these females was similar to the control group means at the other evaluation periods (Weeks 3 and 12). Therefore, the difference was not attributed to treatment. No other remarkable differences were apparent between the control and treated groups when the neuromuscular observations were evaluated during the pre-test period and Weeks 3, 7 and 12.

Physiological Observations
Test substance administration had no adverse effects on catalepsy or body temperature at dietary concentrations of 72, 207 and 540 ppm. Mean body weight in the 540 ppm group males and females were reduced during Weeks 3, 7 and 12 when compared to the control group values. With the exception of the Week 12 value for the males, all of these means were statistically significant. These findings were consistent with those noted for the weekly body weight data. No effects on mean body weight were apparent in the 72 or 207 ppm group males and females when evaluated during the pre-test period and during Weeks 3, 7 and 12.

For details, please refer to attachment 3 under "Overall remarks, attachments".

Locomotor activity
Mean ambulatory and total motor activity values in the 72, 207 and 540 ppm group males and females were similar to the control group values during the pretest period and during Weeks 3, 7 and 12 (4%, 8th and 13th weeks of dose administration, respectively). Animals in all study groups during each evaluation period showed normal habituation profiles to the test chambers during the Locomotor activity sessions.

For details, please refer to attachment 4 under "Overall remarks, attachments".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Non-perfused animals
No test substance-related changes were apparent in mean absolute brain weights or mean brain weights relative to final body weights at any dietary concentration tested. All values in the treated groups were comparable to the control group values.

Perfused animals
No adverse effects were apparent on mean absolute brain weights or brain length and width measurements at dietary concentrations of 72, 207 and 540 ppm. The values in the treated groups were similar to those observed in the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One male in the 72 ppm group died on Day 14. On the day prior to death, this male was hypoactive and had labored respiration. Macroscopic examination of this animal revealed a large mass on the thymus gland. The only other gross lesions observed for this animal were enlarged lymph nodes (mediastinal and bronchial).

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No microscopic lesions were observed in any central or peripheral nervous system tissues upon histopathological examination of the 5 neuropathology animals/sex in the 540 ppm group. The only lesions observed were limited to one male and one female in the control group. Digestion chambers were noted in the sciatic nerve for both the male and the female and in the peroneal nerve for the female.
The substantia nigra was not specifically named in the 13-week neurotoxicology study report. As the substantia nigra is located in the midbrain and the midbrain was investigated in the detailed
neuropathological examinations without showing any lesions, it could be concluded that the treatment
with the test substance does no affect the substantia nigra in rats. The overall weight of the evidence dose not point towards a substance-related effect on the substantia nigra

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Please refer to "Neuropathological findings" above.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

207 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 14 and 16 mg/kg bw/day in male and female rats, respectively

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: moderate inhibition of the brain NTE (Neuropathy Target Esterase)

Remarks on result:

other: corresponding to 34 and 40 mg/kg bw/day in male and female rats, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 34 and 40 mg/kg bw/day in male and female rats, respectively

Key result

Critical effects observed:

no

Table 1         Subchronic neurotoxic effects
Parameter / Dose	0 ppm		60 ppm		180 ppm		540 ppm		Dose-response +/–
	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀
Body weight							↓	↓	−	−
Food consumption							↓	↓	−	−
Neurotoxic esterase							↓	↓	−	−

Conclusions:

The study at hand was conducted under GLP conditions and similar to OECD 424. The study is considered reliable and valid. The study assessed the potential to induce neurotoxicity related to the administration of the test substance for at least 91 consecutive days to Sprague-Dawley rats (10/sex/group) upon dietary administration of the test at a concentration of 72, 207 and 540 ppm (corresponding to 5, 14 and 34 mg/kg bw/day for males and 6, 16 and 40 mg/kg bw/day for females, respectively).
Systemic toxicity was observed at 540 ppm (corresponding to 34 and 40 mg/kg bw/day in male and female rats, respectively) evident by reduced body weight, body weight gain, food consumption and lower mean brain neurotoxic esterase values. The toxicological significance of the moderate inhibition of the brain NTE (Neuropathy Target Esterase) is not clear. Neurotoxicity was not observed in any of the treatment groups. Thus, under the conditions tested the NOAEL for systemic toxicity was set to 207 ppm (corresponding to 14 mg/kg bw/day for males and 16 mg/kg bw/day for females) and the NOAEL for neurotoxicity was set to 540 ppm (corresponding to 34 mg/kg bw/day for males and 40 mg/kg bw/day for females). Ziram did not induce behavioural and neuronal toxicity at doses producing systemic toxicity.

Reference 3

Endpoint:

neurotoxicity: oral

Remarks:

developmental

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Nov 2007 to 16 Jul 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 426 (Developmental Neurotoxicity Study)

Version / remarks:

adopted in 2007

Deviations:

yes

Remarks:

assessments of learning and memory, motor and sensory function, and habituation were not conducted in this study. These parameters were already examined during the 2-generation study (Nemec, 1996) from the same laboratory using the same dose groups.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

Version / remarks:

adopted in 1998

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: 81 days
- Weight at study initiation: 225 g - 288 g on gestation Day 0
- Fasting period before study: no
- Housing: After mating, the females were individually housed in plastic maternity cages with nesting material.
- Diet: PMI Nutrition International, LLC, Certified Rodent, LabDiet® 5002, ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 - 22.3
- Humidity (%): 38.0 - 60.4
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 November 2007 To: 16 July 2009

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002
- Storage temperature of food: refrigerated

DIETARY LEVELS
Actual target test substance concentrations were 60, 180 and 540 ppm.
Due to excessive maternal toxicity, the 540 ppm exposure level was lowered to 360 ppm beginning on lactation Day 4 and continuing throughout the remainder of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Triplicate samples were collected from the top, middle and bottom of each test substance formulation; triplicate samples were collected from the middle of the control formulation, One set of these samples from each formulation was analyzed for homogeneity. The top, middle and bottom of the second and third sets of samples were combined and analyzed for stability following 24-h room temperature storage and the other set was analyzed for stability following 10-day refrigerated storage.

Homogeneity
The assessments met the WIL Research Laboratories, LLC Standard Operating Procedures (SOP) acceptance criteria for test substance homogeneity, i.e., the variability for the mean concentration was ≤10% RSD at a concentration within the acceptable limits (85% to 115% of the fortified concentration), with the following exception. The formulation prepared at a fortified test substance concentration of 72 ppm and assessed for test substance homogeneity failed to meet the SOP requirement due to high intra-assay variability (24% RSD). However, the high RSD was the result of only 1 of 6 samples (first sample collected from the bottom strata was 45.3% of the fortified concentration whereas the second sample collected from the bottom strata was 95.0% of the fortified concentration). Therefore, the results of this analysis were considered acceptable.

Stability
Various assessments of test substance stability in formulations following storage at room temperature for 24 h met the previously stated acceptance criteria with the following exceptions. The post-storage concentration in formulations fortified at a test substance concentration of 72 ppm (target concentration 60 ppm) and prepared on 26 October 2007 and 7 November 2007 was 75.9% and 61.4% of the target concentration, respectively. Also, the formulations prepared on 26 October 2007 and following refrigerated storage for 10 days met the stated acceptance criteria with the following exception. The post-storage concentration in the formulation fortified at a test substance concentration of 540 ppm (target concentration 540 ppm) was 82.1% of the target concentration. The low concentrations of the test substance detected in the test diet relative to the target concentrations following room temperature storage were consistent with previous studies (Nemec, 1996; WIL-223003); therefore, these results were considered acceptable.

Concentrations
Exposure formulations fortified at test substance concentrations of 0, 72, 207, 360, and 540 ppm offered to animals on study were analyzed to confirm test substance concentration, and the results met the previously mentioned SOP acceptance criteria for concentration acceptability in diet admix formulations, with the following exceptions.
The formulation prepared on 7 November 2007 at a fortified concentration of 72 ppm was 80.5% of the fortified concentration. This formulation was not introduced to the animals and was successfully reformulated on 9 November 2007. The formulation prepared on 14 November 2007 at a fortified concentration of 207 ppm (target concentration of 180 ppm) was 119% of the fortified concentration.

Duration of treatment / exposure:

gestation Day 6 to lactation Day 21

Frequency of treatment:

Daily via diet

Dose / conc.:

60 ppm

Remarks:

The concentration of test material in the low dose level was increased by approximately 20% above nominal (72 ppm) to compensate losses during storage. In total, this corresponded to 5, 8 and 12 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively.

Dose / conc.:

180 ppm

Remarks:

The concentration of test material in the low dose level was increased by approximately 15% above nominal (207 ppm) to compensate losses during storage. In total, this corresponded to 13, 23 and 34 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively.

Dose / conc.:

540 ppm

Remarks:

The 540 ppm diet concentration was offered to animals through the morning of lactation Day 4. Thereafter, the 360 ppm diet concentration was offered because the magnitude of maternal toxicity observed at the 540 ppm exposure level would have precluded the objectives of the study. In total, this corresponded to 27, 59 and 61 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively.

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the 2-generation study with ziram (Nemec, 1996, IUCLID Section 7.8.1)

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor or other difficulties).

BODY WEIGHT: Yes
Individual maternal body weights were measured on gestation Says 0 and 6 - 20 (daily) and on lactation Days 1 - 21 (daily). Group mean body weights were calculated for each of these days. Group mean body weight changes were calculated for each corresponding interval of gestation and lactation, and also for gestation Days 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 20 and 6 - 20 and for lactation Days 1 - 4, 4 - 7, 7 - 11, 11 - 14, 14 - 17, 17 - 21 and 1 - 21.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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
- Time schedule: Individual maternal food consumption was recorded on gestation days 0 and 6 - 20 (daily) and on lactation Days 1 - 21 (daily).

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

None

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: No

LOCOMOTOR ACTIVITY: Yes
- Locomotor activity testing was performed in a room equipped with a white-noise generation system set to operate at 70±10 decibels (db). Locomotor activity was assessed for 20 rats/sex/group on postnatal Days 13, 17, 21 and 61.
The same animals were tested at each interval. Locomotor activity was measured automatically using the Kinder Scientific Motor Monitor System (Kinder Scientific, LLC, Poway, California).
Each animal was tested separately. Data were collected in 5-min epochs (print intervals) and the test session duration was 60 min.
Data for ambulatory and total locomotor activity were tabulated. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of 2 or more consecutive photobeams).

Sacrifice and (histo)pathology:

OFFSPRING
- Time point of sacrifice of offspring selected for brain weight or neuropathological evaluation: postnatal Days (PND) 21 or 72, 15 pups/sex/group were macroscopically examined for neuropathology

Microscopic examination
Brains from pups perfused on PND 21 and 72 were prepared for microscopic neuropathological examination (10 rats per sex). The brains were prepared for a qualitative histopathological examination by embedding in paraffin, sectioning and staining with hematoxylin and eosin. Sections from all major brain regions (including olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain [tectum, tegmentum, cerebral peduncles, central gray matter], cerebellum, pons and medulla oblongata [a full coronal section was prepared]) were examined from the first 10 rats/sex in the control and high-exposure groups on PND 21 and 72, first 10 males in the low- and mid-exposure groups on PND 21 and the first 10 females in the low- and mid-exposure groups that met the criteria for measurement. In addition to the qualitative histopathological evaluation, a simple morphometric analysis was performed. Specific measurements taken were determined by the pathologist, but included at least 2 measurements from the neocortical and hippocampalareas and 1 from the cerebellar area.
Central and peripheral nervous system tissues from the offspring perfused in situ at study termination (PND 72, Subset A) were processed for neuropathological evaluation. The following tissues were microscopically examined qualitatively for 10 rats/sex in the control and high-exposure groups:
Brain - olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalmus, midbrain (tectum, tegmentum, cerebral peduncles, central grey matter), cerebellum, pons and medulla oblangata (full coronal section)
Spinal cord - at cervical swellings C3 - C7 and at lumbar swellings T13 - L4
Trigeminal ganglion/nerves'
Lumbardorsal root ganglion at T13 - L4*
Lumbardorsal root fibers at T13 - L4*
Lumbarventralrootfibers at T13 - L4*
Cervical dorsal root ganglion at C3 - C7*
Cervical dorsal root fibers at C3 - C7*
Cervical ventral root fibers at C3 - C7*
Sciatic nerves ''+
Sural nerves ''+
Tibial nerves ''+
Peroneal nerves ''+
Optic nerves'
Eyes'
Skeletal muscle (gastrocnemius)
' = Both processed and evaluated microscopically
'' = 1 processed for microscopic evaluation
+ = Indicates that 2 sections (1 cross and 1 longitudinal) of the tissue were evaluated from the right hind leg. The tissues from the left hind leg were collected and preserved for possible future evaluation.
* = 4 - 6 tissues were collected at necropsy; 2 tissues were evaluated microscopically

The central nervous system tissues were embeddedin paraffin, and the peripheral nervous system tissues were embedded in plastic. Tissues were prepared for histopathological evaluation by sectioning and staining with hematoxylin and eosin. The histopathologic examination included a simple morphometric analysis. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during the processing or other designations as appropriate.


Morphometric analysis
Quantitative examinations of the brains from the selected PND 21 and PND 72 offspring were conducted and included the following. Specific levels analysed, designated as Levels 1, 3 and 5, were defined as follows. Level 1 was a coronal section of rostral cerebrum, including caudoputamen, Level 3 was a coronal section of mid-cerebrum (cerebral cortex, hippocampal formation, thalamus, etc.) and Level 5 was a mid-sagittal section of cerebellum and pons. Levels 1, 3 and 5 corresponded to Plates 11, 33 and 79, respectively. Measurements on Levels 1 and 3 were bilateral and were averaged for each animal. Measurements on Level 5 were not paired since the other half of these tissues were sectioned transversely to enable visualization of cerebellar nuclei. Measurements were made on homologoussections to ensure that the dimensions of the regions were comparable. In some cases, there was deformation or irregularity of the desired region. As a result, morphometric analysis could not be made for some regions in a few animals.

Other examinations:

None

Positive control:

None

Statistics:

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group by sex.

ONE-WAY ANOVA
Mean maternal and offspring body weights and body weight changes, maternal food consumption, gestation lengths, former implantation sites, unaccounted-for sites, numbers of pups born, live litter sizes, day of attainment of balanopreputial separation or vaginal patency, body weight on the day of attainment and brain weight, length, width and morphometric data

If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of pup viability and males per litter were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-exposed groups to the control group. Neuropathological data were analyzed using the Fisher’s Exact Test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F0 maternal generation:
No test substance-related clinical findings were noted during the generation at the daily examinations.

F1 litter:
With the exception of 6 pups in the 540/360 ppm group noted with a small stature (secondary to reduced body weights) on 1 - 4 incidences each, no clinical observations noted for F1 pups in this study were attributed to maternal test substance exposure.

F1 generation:
Following weaning of the F1 pups, 1 male each in the control and 540/360 ppm groups was found dead on PND 23 and PND 35 respectively. No remarkable clinical observations or internal findings were noted for these males. All other F1 animals survived to the scheduled necropsies. No test substance-related clinical findings were noted at the weekly examinations of the F1 animals. Clinical findings noted in the test substance-exposed groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group and in a manner that was not exposure-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

F0 maternal generation:
All F0 maternal animals in the control, 72, 207 and 540/360 ppm groups survived to the scheduled
necropsy.

F1 litter:
No difference was observed with regard to the number of pups (litters) that were found dead between the test groups and the control group (8(5), 11(6), 12(8) and 12(9) in the control, 72, 207 and 540/360 ppm groups, respectively). Three (3), 2(2), 0(0) and 5(4) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.

F1 generation:
Following weaning of the F1 pups, 1 male each in the control and 540/360 ppm groups was found dead on PND 23 and PND 35 respectively. Because of the mortality in the control group, the single mortality in the 540/360 ppm group was not attributed to maternal test substance exposure.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0 maternal generation:
Gestation
A test substance-related mean body weight loss was noted in the 540/360 ppm group at the onset of the test diet exposure period (gestation Days 6 - 9); the difference from the control group was statistically significant. Additionally, mean body weight gain in this group was statistically significantly lower than the control group throughout the remainder of gestation with the exception of gestation Days 15 - 18. As a result, a statistically significantly reduced mean body weight gain was noted in the 540/360 ppm group when the overall gestation exposure period (gestation Days 6 - 20) was evaluated. Due to the reduced mean body weight gains and body weight loss, mean body weights in the 540/360 ppm group were 5.3% to 14.7% lower than the control group during gestation Days 7 - 20; differences were statistically significant. In the 207 ppm group, a test substance-related, statistically significantly lower mean body weight gain was noted during gestation Days 6 - 9. Mean body weight gain in this group was similar to the control group during gestation Days 9 - 18, but was statistically ssignificantly lower during gestation Days 18 - 20 and when the overall gestation exposure period (gestation Days 6 - 20) was evaluated. As a result, mean body weights in the 207 ppm group were 2.1% to 3.8% lower than the control group during gestation Days 12 - 20; differences from the control group achieved statistical significance on gestation Days 15 and 20. The lower mean body weights were reflective of an overall gestational mean body weight gain that was 15.7% lower than the control group. Mean body weights and body weight gains in the 72 ppm group were unaffected by test substance exposure during gestation.

Lactation
Mean body weight gain in the 540/360 ppm group was slightly lower than the control group during lactation Days 1 - 4; the difference on lactation Day 2 - 3 achieved statistical significance. Following the reduction in test substance concentration in the test diet (540 ppm to 360 ppm) on lactation Day 4, mean body weight gain in this group was statistically significantly higher than the control group during lactation Days 4 - 7, and remained similar to or slightly higher (statistically significant during lactation Days 17 - 21only) compared to the control group during the remainder of lactation (lactation Days 7 - 21). As a result, mean body weight gain in the 540/360 ppm group was statistically significantly higher than the control group when the overall lactation period (lactation Days 1 - 21) was evaluated. Due to reductions in mean body weight gains during gestation, mean body weights in this group were statistically significantly lower (3.8% to 15.5%) than the control group during lactation Days 1 - 20. However, as a result of the increased mean body weight gains during lactation after the test diet concentration was reduced to 360 ppm, mean body weight in the 540/360 ppm group recovered to within 2.1% of the control group at the conclusion of the exposure period on lactation Day 21.Mean body weight changes in the 72 and 207 ppm groups were unaffected by test diet exposure during lactation.

F1 litter:
On PND 1, mean male and female body weights in the 540/360 ppm group were 11.4% and 10.6% lower, respectively compared to the control group. Mean male and female pup body weight gains in the 540/360 ppm group were statistically significantly lower than the control group during PND 1 - 4, 4 - 7 (males only) and 17 - 21. The decrease noted late in the pre-weaning period (PND 17-21) was likely the result of direct compound consumption by the pups. As a result of these decreases, in addition to the lower mean weights on PND 1, mean male and female pup body weights were 10.3% to 18.8% and 8.5% to 16.1% lower, respectively, during the pre-weaning period and were generally statistically significantly different from the control group. In the 207 ppm group, mean male and female pup body weight gains were generally similar to the control group during PND 1 - 17; no statistically significant differences were noted. During PND 17 - 21, mean male and female pup body weight gains in this group were statistically significantly lower than the control group, which was likely the result of direct compound consumption by the pups. As a result, mean male and female pup body weights in the 207 ppm group were 8.4% and 6.8% lower, respectively, as compared to the control group on PND 21; the difference from the control group for the males only achieved statistically significance. Mean pup body weights and body weight gains in the 72 ppm group males and females were unaffected by test substance exposure to the F0 maternal animals throughout the pre-weaning period. No statistically significant differences from the control group were noted.

F1 generation:
Mean weekly body weight gains in the F1 males and females were unaffected by exposure ofthe test diet to the F0 maternal animals. Differences in mean body weight gains between the control, 72, 207 and 540/360 ppm groups were slight and notstatistically significant. In the 540/360 ppm group, mean male and female body weights were 6.9% and 7.3% lower (not statistically significant), respectively, as compared to the control group on PND 28 due to the reduced mean pup body weight gains during the preweaning period. Mean male and female body weights in this group continued to be reduced throughout the post-weaning period and remained 6.9% and 3.5% lower, respectively, on PND 70 compared to the control group; differences were notstatistically significant. Mean body weights in the 72 and 207 ppm groups were similar to the control group throughout the post-weaning period.

For details, please refer to attachment 1 and 2 under "Overall remarks, attachments" and to table 1 and 2 under "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Gestation
Test substance-related lower mean food consumption was noted in the 540/360 ppm group throughout the gestation exposure period (gestation Days 6 - 20). The differences from the control group were generally statistically significant and corresponded to the reduced mean body weight gains noted in this group. In the 207 ppm group, a test substance-related reduction in mean food consumption was noted during gestation Days 6 - 9; the difference from the control group was statistically ignificant and corresponded with the reduced mean body weight gain following the onset of test diet exposure. Mean food consumption in this group was similar to the control group throughout the remainder of gestation and when the overall test diet exposure period (gestation Days 6 - 20) was evaluated. With the exception of a statistically significant increase during gestation Days 15 - 18 when evaluated as g/kg bw/day only, differences from the control group were slight and not statistically significant. Mean food consumption in the 72 ppm group was unaffected by test substance exposure during gestation.

Lactation
In the 540/360 ppm group, mean food consumption was statistically significantly (when evaluated as g/animal/day only) lower than the control group during lactation Days 1 - 4 and corresponded to a slightly lower mean body weight gain during this interval. Following the reduction of the test substance concentration in the diet (540 ppm to 360 ppm) on lactation Day 4, mean food consumption in this group was generally similar to the control group with the exception of statistically significant (when evaluated as g/animal/day only) reductions during lactation Days 9 - 10, 11 - 14 and 20 - 21; these differences were not considered test substance-related effects and did not correspond to any reductions in mean body weight gains. Mean food consumption was statistically significantly higher when evaluated on a g/kg bw/day basis during lactation Days 4 - 7; however, this difference was considered secondary to the reduced mean body weights and not a direct test substance-related effect. When the overall lactation exposure period (lactation Days 1 - 21) was evaluated, mean food consumption in the 540/360 ppm group was statistically significantly (when evaluated as g/animal/day only) lower compared to the control group. Mean food consumption in the 72 and 207 ppm groups was unaffected by test substance exposure during lactation.

For details, please refer to attachment 3 under "Overall remarks, attachments" and to table 3 and 4 under "Any other information on results incl. tables".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Developmental landmarks and behavioural testing
Balanopreputial separation
Mean ages of attainment of balanopreputial separation were unaffected by exposure of the F0 maternal animals to the test substance. The mean ages of attainment of balanopreputial separation were 46.2, 45.1 and 44.9 days in the 72, 207 and 540/360 ppm groups, respectively, when compared to 45.4 days in the control group. None of the differences from the control group were statistically significant. In the 540/360 ppm group, mean body weight (220.1 g) at the age of attainment of balanopreputial separation was statistically significantly lower (7.1%) compared to the control group (236.9 g); this difference was attributed to the test substance-related reduced mean body weights noted in the F1 generation. Mean body weights at the age of attainment in the 72 and 207 ppm groups (240.2 g and 229.8 g, respectively) were unaffected by maternal test substance exposure; differences from the control group were slight and not statistically significant.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Vaginal Patency
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by exposure of the test substance to the F0 maternal animals. The mean ages of attainment of vaginal patency were 33.4, 32.4 and 32.8 days in the 72, 207 and 540/360 ppm groups, respectively, when compared to 32.2 days in the control group. Mean body weights at the age of attainment were 111.2 g, 107.1 g and 103.6 g in the same respective groups when compared to 107.9 g in the control group. None of the differences from the control group were statistically significant.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Locomotoractivity
Mean cumulative locomotor activity counts in the 540/360 ppm group for males and females combined were 25.0% (total) and 43.9% (ambulatory) higher than the concurrent control group on PND 13 differences from the control group did not achieve statistical significance. On PND 17 and 21, males and females combined in the 540/360 ppm group showed less habituation to the test environment compared to the control group animals, resulting in higher cumulative total and ambulatory counts for these animals (30.7% to 39.8% and 40.9% to 61.9%, respectively); differences from the control group were generally statisticall significant. The monotonic dose-response relationship evaluated by the linear dose by time interaction was not statistically significant analysed on PND 13, 17 and 21. When the repeated measures analysis was conducted across testing ages (PND 13, 17 and 21), statistically significant increases in overall total and ambulatory activity were noted when the cumulative data for all testing days were combined. These increases in locomotor activity and reduced habituation to the test environment on PND 17 and 21 corresponded to reduced mean body weights (6.1% to 8.0% for animals selected for motoractivity assessment) compared to the control group and were considered, at least in part, indicative of developmental delay. In the 207 ppm group, mean total and ambulatory locomotor activity counts were unaffected by maternal test diet exposure on PND 13 and 17; differences from the control group, when evaluated with males and females combined, were slight, not statistically significant and did not occur in an exposure-related trend. However, animals in the 207 ppm group showed less habituation to the test environment on PND 21, resulting in higher mean cumulative total and ambulatory counts (46.7% and 66.2%, respectively), when pooled sexes were compared to the control group. Differences from the control group were statistically significant, corresponded to reduced mean body weights (4.9% and 5.7% for males and females selected for motor activity assessment, respectively) on PND 21. The monotonic dose-response relationship evaluated by the linear dose by time interaction was not statistically significant, analyzed on PND 13, 17 and 21. When the repeated measures analysis was conducted across testing ages (PND 13, 17 and 21), differences from the control group did not achieve statistical significance. In the 207 and 540/360 ppm groups on PND 61, locomotor activity patterns were unaffected by maternal test diet exposure. When the overall total counts were evaluated for combined sexes, statistcilaly significantly higher mean counts were noted in these groups compared to the control group. For ambulatory counts on PND 61, a statistically significant treatment by time by sex interaction was noted, resulting in ambulatory counts being statistically evaluated separately by sex. These analyses indicated statistcially significantly higher mean overall ambulatory counts for both males and females. However, all differences from the control group noted in the 207 and 540/360 ppm groups on PND 61 were not considered exposure-related due to the generally small magnitudes of change during the individual subintervals, absence of a clear exposure-related trend and the lack of any remarkable effect on the pattern of habituation. The monotonic dose-response relationship evaluated by the linear dose by time interaction was not statistically significant analysed on PND 61. Locomotor activity patterns (mean ambulatory and total locomotoractivity counts and habituation to the test environment) evaluated on PND 13, 17, 21 and 61 were unaffected by maternal test substance exposure in the 72 ppm group, differences from the control group were slight and not statistically significant, with the following exceptions. On PND 13, mean male and female combined ambulatory counts were 38.9% lower compared to the control group; however, this difference did not achieve statistical significance, did not occur in an exposure-related manner and was attributed to 2 females in the control group with very high ambulatory counts compared to all other animals in this group. On PND 21, meantotal and ambulatory locomotor activity counts in the 72 ppm group were 24.0% and 43.5% higher, respectively, as compared to the control group. However, differences from the control group did not achieve statistical significance and were primarily the result of 2 atypical females in the 72 ppm group with exceptionally high total and ambulatory counts. In addition, no remarkable differences in the pattern of habituation were noted in the 72 ppm group.As a result, differences noted on locomotor activity in the 72 ppm group were not attributed to maternal test diet exposure.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 generation:
At the PND 21 evaluation, the 540/360 ppm group males, but not females, had statistically significantly lower mean brain weight and shorter mean brain length and width. Mean brain weight for the 540/360 ppm group males was 6.4% lower and mean final body weight was 13.2% lower than the control group males. Mean brain weight relative to final body weight in the 540/360 ppm group males at PND 21 was slightly higher (not statistically significant) compared to the control group. Similarly, the 207 ppm group males had a statistically significantly lower mean final body weight (11.3%), lower (not statistically significant) mean brain weight (4.4%) and slightly higher (not statistically significant) mean brain weight relative to final body weight. Therefore, the reductions in mean brain weight and gross measurements were considered indirect effects of maternal test diet exposure and directly related to their test substance-related lower mean body weight. No test substance-related effects on mean brain weight or overall length and width were observed in the 72 ppm group.
For details, please refer to attachment 6 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F0 maternal generation:
No test substance-related internal findings were observed at any exposure level. No internal findings were noted for females that were euthanized on lactation Day 4 due to pup sex ratio criteria not being met or females that survived to the scheduled necropsy on lactation Day 21. One female that failed to deliver in the 540/360 ppm group had cystic ovaries and cervix and was noted with clear fluid in the uterus. All females that failed to deliver were nongravid. At the lactation Day 21 necropsy, no test substance-related effects were observed on the number of former implantation sites and the number of unaccounted-for sites. The differences between the control and test substance-exposed groups were slight and not statistically significant.

F1 litter:
No internal findings that could be attributed to maternal exposure to the test diet were noted at the necropsies of pups that were found dead.
No internal findings that could be attributed to maternal exposure to the test diet were noted at the necropsy of pups euthanized on PND 21.

F1 generation:
One male each in the control and 540/360 ppm groups was found dead on PND 23 and 35, respectively. No remarkable internal findings were noted for either male at necropsy.
At the PND 72 necropsies of F1 animals not selected for neuropathology, no internal gross findings related to F0 maternal test diet exposure were observed.
At the necropsies of F1 animals selected for brain weights on PND 21, no test substance-related gross findings were noted in the brain.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation:
Of the 8 measurements taken, only 1, the height of the hemisphere on Level 1, was statistically significantly smaller in the 540/360 ppm group females compared to the control group females. The other morphometric measurements were also slightly smaller in the 540/360 ppm group females, but statistical significance was not achieved. These differences were consistent with the smaller mean brain weight and shorter length in these females. However, the lower height of the hemisphere in these females was within the historical control data ranges. Therefore, none of these smaller measurements compared to the control group were considered test substance-related. Measurements in the 72 and 207 ppm females were similar to and not statistically significantly different from the control group. There were no test substance-related qualitative histological changes in the female brains and peripheral nervous system tissues at 72 and 207 ppm or male and female brains and peripheral nervous system tissues at 540/360 ppm.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Please refer to "Neuropathological findings" above.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

F0 maternal generation:
Pregnancy status
The pregnancy rates in the control, 72, 207 ppm group was 100% and in the 540/360 ppm group 92.0%, respectively. Two females in the 540/360 ppm group failed to deliver and were determined to be nongravid at necropsy.

Gestation length and parturition
No test substance-related effects were noted on mean gestation lengths or the process of parturition at any exposure level. Mean F0 gestation lengths in the test substance-exposed groups were similar to the control group value. No signs of dystocia were noted at any exposurelevel.

F1 litter data:
PND 0 litter data and postnatal survival
The mean number of pups born, live litter size, percentage of males per litter at birth and postnatal survival were unaffected by F0 maternal exposure to the test substanceat all exposure levels. Differences from the control group were slight, were not statistically significant and/or did not occur in an exposurerelated manner.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal systemic toxicity

Effect level:

72 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 5, 8 and 12 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

maternal systemic toxicity

Effect level:

207 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 13, 23 and 34 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal reproductive toxicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 27, 59 and 61 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity F1 generation

Effect level:

72 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 5, 8 and 12 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively in dams.

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity F1 generation

Effect level:

207 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 13, 23 and 34 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively in dams.

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxicity F1 generation

Effect level:

72 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: corresponding to 5, 8 and 12 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively in dams.

Key result

Dose descriptor:

LOAEL

Remarks:

neurotoxicity F1 generation

Effect level:

207 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Remarks on result:

other: corresponding to 13, 23 and 34 mg/kg bw/day during gestation, lactation Days 1 - 4 and lactation Days 4 - 21, respectively in dams.

Key result

Critical effects observed:

no

Table 1: Summary of Body Weight Changes During Gestation [g]

	0 ppm	72 ppm	207 ppm	540 / 260 ppm
DAY 0-6
MEAN	27.	30.	30.	25.
S.D.	7.4	7.2	7.8	9.2
N	25	25	25	23
DAY 6-7
MEAN	3.	0.	-7.**	-11.**
S.D.	5.5	5.8	5.4	6.2
N	25	25	25	23
DAY 7-8
MEAN	4.	6.	4.	-3.**
S.D.	4.4	4.5	3.9	4.3
N	25	25	25	23
DAY 8-9
MEAN	4.	6.	6.	-1.**
S.D.	5.0	5.4	5.4	3.4
N	25	25	25	23
DAY 9-10
MEAN	4.	4.	5.	1.**
S.D.	3.5	3.8	4.5	3.8
N	25	25	25	23
DAY 15-16
MEAN	7.	10.	11.	10.
S.D.	9.3	5.8	5.5	3.9
N	25	25	25	23
DAY 16-17
MEAN	17.	11.*	12.	13.
S.D.	9.5	8.4	3.5	5.2
N	25	25	25	23
DAY 17-18
MEAN	13.	18.*	16.	10.
S.D.	6.3	7.6	4.0	4.3
N	25	25	25	23
DAY 18-19
MEAN	17.	14.	13.*	13.**
S.D.	6.6	3.8	4.6	3.6
N	25	25	25	23
DAY 19-20
MEAN	16.	15.	13.*	10.**
S.D.	4.9	3.5	4.4	3.7
N	25	25	25	23
DAY 6-20
MEAN	115.	114.	97.**	58.**
S.D.	12.5	12.4	13.3	20.8

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

MEAN DIFFERENCES CALCULATED FROM INDIVIDUAL DIFFERENCES

NONGRAVID WEIGHT(S) NOT INCLUDED IN CALCULATION OF MEAN

 

Table 2: Summary of Body Weight Changes During Lactation [g]

	0 ppm	72 ppm	207 ppm	540 / 260 ppm
DAY 1-4
MEAN	14.	13.	8.	9.
S.D.	10.2	10.0	9.4	10.0
N	25	25	25	23
DAY 4-7-A
MEAN	8.	9.	12.	26.**
S.D.	7.8	8.1	11.2	8.1
N	22	25	25	22
DAY 7-11
MEAN	16.	13.	15.	18.
S.D.	7.9	9.0	12.5	9.6
N	22	25	25	22
DAY 11-14
MEAN	10.	10.	11.	11.
S.D.	9.0	7.3	9.4	8.1
N	22	25	25	22
DAY 14-17
MEAN	0.	7.*	7.*	7.
S.D.	8.4	9.5	10.6	8.4
N	22	25	25	22
DAY 17-21
MEAN	-8.	-6.	-4.	3.**
S.D.	10.6	13.8	8.2	9.6
N	22	25	25	22
DAY 1-21
MEAN	41.	45.	50.	74.**
S.D.	14.8	11.6	13.4	15.0

** = Significantly different from the control group at 0.01 using Dunnett's test

MEAN DIFFERENCES CALCULATED FROM INDIVIDUAL DIFFERENCES

A = The 540 ppm test diet concentration was lowered to 360 ppm on lactation day 4.

Table 3: Summary of Food Consumption During Gestation [g/animal/day]

	0 ppm	72 ppm	207 ppm	540 / 260 ppm
DAY 6-9
MEAN	20.	21.	17.**	9.**
S.D.	2.0	2.2	3.3	2.3
N	25	25	25	23
DAY 9-12
MEAN	21.	22.	20.	12.**
S.D.	2.2	1.8	2.0	3.0
N	25	25	25	23
DAY 12-15
MEAN	21.	23.*	21.	16.**
S.D.	2.2	1.9	2.4	3.0
N	25	25	25	23
DAY 15-18
MEAN	22.	24.*	23.	19.**
S.D.	3.0	2.3	2.8	2.8
N	25	25	25	23
DAY 18-20
MEAN	22.	23.	21.	18.**
S.D.	4.6	2.4	3.4	3.8
DAY 6-20
MEAN	21.	23.*	20.	14.**
S.D.	2.2	1.6	2.0	2.1
N	25	25	25	23

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

 

 

Table 4: Summary of Food Consumption During Lactation [g/animal/day]

	0 ppm	72 ppm	207 ppm	540 / 260 ppm
DAY 1-4
MEAN	35.	36.	32.	29.**
S.D.	5.6	6.0	4.1	4.2
N	25	25	25	23
DAY 4-7-A
MEAN	39.	40.	39.	38.
S.D.	5.4	5.0	4.1	3.9
N	22	25	25	22
DAY 7-11
MEAN	50.	49.	48.	46.
S.D.	5.0	6.2	4.6	3.9
N	22	25	25	22
DAY 11-14
MEAN	60.	58.	57.	54.**
S.D.	5.5	6.4	5.5	4.4
N	22	25	25	22
DAY 14-17
MEAN	62.	63.	61.	60.
S.D.	5.8	7.3	4.3	3.9
N	22	25	25	22
DAY 17-21
MEAN	66.	65.	62.	64.
S.D.	8.2	7.3	5.2	4.0
N	22	25	25	22
DAY 1-21
MEAN	53.	52.	50.	49.*
S.D.	5.2	5.5	3.5	2.5

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

A = The 540 ppm test diet concentration was lowered to 360 ppm on lactation day 4.

Conclusions:

The study at hand was conducted under GLP conditions and similar to OECD 426. The study is considered reliable and valid. The objective of this study was to determine the potential of the test substance to induce functional and/or morphological insult to the nervous system which may arise in the offspring from exposure of the mother during pregnancy and lactation (gestation Day 6 through lactation Day 21). Actual test substance consumption in the 72, 207 and 540/360 ppm F0 groups was 5, 13 and 27 mg/kg bw/day, respectively, during gestation, 8, 23 and 59 mg/kg bw/day, respectively, during lactation Days 1 - 4 and 12, 34 and 61 mg/kg bw/day, respectively, during lactation Days 4 - 21.
The NOAEL for maternal systemic toxicity of the test substance was considered to be 60 ppm (fortified concentration of 72 ppm) achieved dosage range 5 — 12 mg/kg bw/day based on reductions in mean maternal body weights, body weight gains and food consumption in the 207 and 540/360 ppm groups during gestation and/or lactation.
The NOAEL for F0 maternal reproductive toxicity (process of parturition and duration of gestation) was considered to be 540 ppm due to the absence of any test substance-related effects on reproduction while being exposed to the 540 ppm (achieved dose during gestation 27 mg/kg bw/day) test diet.
Based on the reduced mean pup body weights and body weight gains in the 207 and 540/360 ppm group males and females, the NOAEL for F1 developmental toxicity was considered to be 60 ppm (fortified concentration of 72 ppm).
Based on the increase in total and ambulatory counts and lower habituation to the test environment in the 207 and 540/360 ppm group males and females on PND 13, 17 and/or 21, and in the absence of any test substance-related effect on brain morphometry on PND 21 and 72 at any exposure level, the NOAEL for neurotoxicity of the F1 generation was considered to be 60 ppm (fortified concentration of 72 ppm achieved dosage range 5 - 12 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

12 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

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

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Neurotoxicity: oral administration

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

The acute neurotoxicity study (Report number: WIL-223001) was performed according to EPA OPP 81-8, similar to OECD 424 and in compliance with GLP. The test substance was administered by the oral route (gavage) as a single dose at dose levels of 15, 300 and 600 mg/kg bw to Sprague-Dawley rats. The NOAEL for systemic toxicity was 15 mg/kg bw, based on observed mortalities, clinical signs, reduced body weight gain, and gross pathology findings. For neurotoxicity the NOAEL was as well 15 mg/kg bw, based on impaired walk and tiptoe gait, impaired mobility and reduced body temperature.

A 13-weeks dietary neurotoxicity study (Report number: WIL-223004) was conducted similar to OECD 424 and in compliance with GLP to assess the potential neurotoxicity related to the administration of the test substance for at least 91 consecutive days to Sprague-Dawley rats. 10 rats/sex/group received the test substance via dietary administration at a concentration of 72, 207 and 540 ppm (corresponding to 5, 14 and 34 mg/kg bw/day for males and 6, 16 and 40 mg/kg bw/day for females, respectively). Systemic toxicity was observed at 540 ppm (corresponding to 34 and 40 mg/kg bw/day in male and female rats, respectively) evident by reduced body weight, body weight gain, food consumption and lower mean brain neurotoxic esterase values. The toxicological significance of the moderate inhibition of the brain NTE (Neuropathy Target Esterase) is not clear. Only one female displayed a gait alteration (walks on tiptoes) at Week 3 and one male in Week 7 in the 540 ppm dose group in the open field observations. The historical control data demonstrated that gait alterations also occur in control animals at different time points to a certain extent in either sex. As these gait alterations were observed in only one animal of each sex at only one time point and no gait alterations were observed during the Week 12 of the open field observations or during the daily clinical examinations at any dose level, these observations were isolated in occurrence and not attributed to the test substance. Therefore, neurotoxicity was not observed in any of the treatment groups. Thus, under the conditions tested the NOAEL for systemic toxicity was set to 207 ppm (corresponding to 14 mg/kg bw/day for males and 16 mg/kg bw/day for females) and the NOAEL for neurotoxicity was set to 540 ppm (corresponding to 34 mg/kg bw/day for males and 40 mg/kg bw/day for females).

A developmental neurotoxicity study (Report number: WIL-223006) was conducted according to EPA OPPTS 870.6300, similar to OECD 426 and in compliance with GLP. The potential of the test substance to induce functional and/or morphological insult to the nervous system was investigated, which may arise in the offspring from exposure of the mother during pregnancy and lactation (gestation Day 6 through lactation Day 21). Actual test substance consumption in the 72, 207 and 540/360 ppm F0 groups was 5, 13 and 27 mg/kg bw/day, respectively, during gestation, 8, 23 and 59 mg/kg bw/day, respectively, during lactation Days 1 - 4 and 12, 34 and 61 mg/kg bw/day, respectively, during lactation Days 4 - 21. The NOAEL for maternal systemic toxicity of the test substance was considered to be 60 ppm (fortified concentration of 72 ppm) achieved dosage range 5 — 12 mg/kg bw/day based on reductions in mean maternal body weights, body weight gains and food consumption in the 207 and 540/360 ppm groups during gestation and/or lactation. The NOAEL for F0 maternal reproductive toxicity (process of parturition and duration of gestation) was considered to be 540 ppm due to the absence of any test substance-related effects on reproduction while being exposed to the 540 ppm (achieved dose during gestation 27 mg/kg bw/day) test diet. Based on the reduced mean pup body weights and body weight gains in the 207 and 540/360 ppm group males and females, the NOAEL for F1 developmental toxicity was considered to be 60 ppm (fortified concentration of 72 ppm). Based on the increase in total and ambulatory counts and lower habituation to the test environment in the 207 and 540/360 ppm group males and females on postnatal Days 13, 17 and/or 21, and in the absence of any test substance-related effect on brain morphometry on postnatal Days 21 and 72 at any exposure level, the NOAEL for neurotoxicity of the F1 generation was considered to be 60 ppm (fortified concentration of 72 ppm achieved dosage range 5 - 12 mg/kg bw/day).

In addition to the above-mentioned neurotoxicity studies, a dietary two-generation reproduction and developmental neurotoxicity study was also performed with the test substance (Report number: WIL-223003). This study was designed to investigate the potential adverse effects of the test substance on reproductive capabilities in two generations of rats (F0 and F1) and the potential of the test substance to cause functional and morphological changes to the nervous system of the developing rat (F2 litters) following continuous treatment of the F0 and F1 generations. The test substance was orally administered via feed at dose level of 72, 207 and 540 ppm. The test substance did not affect reproductive performance in the 2-generation rat study at the doses studied. Parental toxicity was observed in the F0 and F1 generations, e.g. inhibition of body weight gain and reduced food intake at the highest dose level tested. At the same dose neonatal toxicity was expressed, e.g. slightly reduced pup body weights (F1 and F2). Various indicators of physical and functional development, as well as behavioral responses of the selected F2 treated pups in the developmental neurotoxicity phase were comparable to the control group values. Neuropathological examinations revealed no gross or microscopic test article-related lesions in the selected F2 pups. Based on the results of this study, a concentration of 207 ppm was considered to be the NOAEL (no observable adverse effect level) for parental systemic toxicity, 207 ppm was considered to be the NOAEL for neonatal toxicity and 540 ppm was considered to be the NOAEL for reproductive and developmental neurotoxicity. For further details, please refer to the respective study record and endpoint summary under 7.8.1 "Toxicity to reproduction".

Neurotoxicity: inhalation administration

No data available.

Neurotoxicity: dermal administration

No data available.

Conclusion:

Based on the acute and subchronic neurotoxicity studies the test substance affected behaviour and motor activity only at systemically toxic dose levels. No neuropathological changes were seen in both studies. The developmental neurotoxicity study in rats demonstrated that the test substance is not a specific developmental neurotoxicant. Taking into account all the available data and the lack of any indication for specific developmental neurotoxicity, it is concluded that the test substance is not a developmental neurotoxicant.

Justification for classification or non-classification

The test substance caused behavioral effects only at doses that had caused maternal toxicity. Therefore, the available data on neurotoxicity of the test substance do not meet the criteria for classification and labelling according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.