mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Neurotoxicity

Description of key information

Acute Dose Oral Route: Mellert et al. (1995)

Under the conditions of this study, the NOAEL was therefore slightly below 175 mg/kg body weight in both sexes. It was considered that the test material did not cause permanent damage to the nervous system when administered as single gavage to Wistar rats.

Sub-Chronic Repeated Dose Oral Route: Mellert et al. (1995)

Under the conditions of this study the no effect level for neurotoxicity was above 3 000 ppm in females and above 2 500 ppm in males.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 1994 to 23 August 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPP 82-1

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: All animals were 42 days old at the start of the test material administration (i.e. day 0 for each subset).
- Weight at study initiation: At the start of the administration period (day 0) the groups had a mean body weight of: Male animals: 176 (156 - 201) g; female animals: 145 (114 - 158) g.
- Housing: The rats were housed singly in type DK III stainless steel wire cages. The motor activity measurements were conducted in polycarbonate cages with wire covers
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: On the days of arrival, a 7-day acclimatisation period started, in which the animals were accustomed to housing and diet.

DETAILS OF FOOD AND WATER QUALITY:
The food used in the, study was assayed for chemical and microbiological contaminants.
The drinking water is regularly assayed for chemical contaminants as well as for the presence of microorganisms.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light):12 hours (12 hours light from 06.00 - 18.00 h, 12 hours dark from 18.00 - 6.00 h).

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet:
The test material was weighed out and thorougly mixed with a small amount of food in a beaker. Subsequently a premix was prepared in a BOSCH household mixer by adding an appropriate amount of food and mixing for about 3 min. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a GEBR. LODIGE laboratory mixer. The mixtures were prepared in intervals for which the stability of the test material in the diet was guaranteed. The stability of the test material in the diet was proven with a comparable batch before the start of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration control analyses were performed with samples taken at the start of the administration period and after 8 weeks of administration.

The stability of the test material in the diet was proven for a period of 33 days at room temperature.
The homogeneous distribution of the test material in the diet was proven in a previous study with the test material using the same mixing procedure and comparable concentrations in the diet.
Analytical checks of the concentrations confirmed that the concentrations were within an acceptable range (between 93.8 % and 110.3 % of the target concentrations.

Duration of treatment / exposure:

Three months

Frequency of treatment:

Daily

Dose / conc.:

3 000 ppm

Remarks:

Females only, ca. 240 mg/kg bw/day

Dose / conc.:

2 500 ppm

Remarks:

Males only, ca. 189 mg/kg bw/day

Dose / conc.:

500 ppm

Remarks:

Males and females, ca. 38 mg/kg bw/day

Dose / conc.:

75 ppm

Remarks:

Males and females, ca. 6 mg/kg bw/day

No. of animals per sex per dose:

15 per sex per dose, separated in to subgroups of 5 per sex per dose.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses were chosen on the basis of previous studies.
- Rationale for animal assignment: The animals were randomly assigned to the groups based upon body weight, separated by sex prior to the first functional observational battery. The randomisation list was drawn up by a computer.
- Rationale for selecting satellite groups: As this study was conducted as a combined study (sub-chronic toxicity and neurotoxicity), following subgroups consisting of 5 males and 5 females per dose were used:
Section A: Used for functional observational battery, motor activity measurement, perfusion fixation and neuropathology.
Section B: Used for functional observational battery, motor activity measurement, clinical chemistry, haematology, urinalyses, immersion fixation and histopathology according to guidelines for subchronic toxicity.
Section C: Used for clinical chemistry, haematology, urinalyses, immersion fixation and histopathology according to guidelines for sub-chronic toxicity.
In order to balance the groups for functional observational batteries and motor activity measurements, the study was conducted with 4 subsets (Section A males, Section B males, Section A females, Section B females). Within each subset, animals of control, low, mid and high dose group were tested again in randomised order, whereas randomisation was not based upon body weight. The randomisation list was drawn up by a computer.
a) All animals were examined on the same day after start of dosing. Time of testing was therefore identical for all animals;
b) For each examination animals from all test groups (including control) could be used;
c) The examinations for all subsets could be performed on the same time of the day, thus diurnal effects could be neglected.

Observations and clinical examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The general state of health concerning mortality or moribund state of the animals was checked twice a day (Monday to Friday) and once a day (Saturdays, Sundays and public holidays). The animals were additionally examined in detail and palpated once a week.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight was determined before the first neurofunctional test in order to randomise the animals. During the conduct of the study, the body weight was determined on day 0 (start of administration period) and thereafter in weekly intervals. Body weight was also determined on the days, when functional observational batteries were carried out. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as the body weight change.

FOOD CONSUMPTION: Yes
- The food consumption of the animals was determined weekly. The values were calculated as grams per animal per day.

- Intake of the test substance: The mean daily intake of the test material (group means) was calculated based upon individual values for body weight and food consumption using the equation below:

Substance intake for day x = (FCx x C) / BWx

Where:
BWx = Body weight on day x (g)
FCx = mean daily food consuption on day x (g)
C = ppm in the diet (mg/kg)

FOOD EFFICIENCY:
Yes
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption using the equation below:

Food efficiency for day x = ((BWx - BWy) / FCy to x ) x 100

Where:
BWx = Body weight on day x (g)
BWy = Body weight on day y (last weighing date before day x) (g)
FCy to x = Mean food consumption from dau y to day x; calculated as mean daily food consumption on day x multiplied with the number of days from day y to day x (g)

WATER CONSUMPTION: Yes
- Time schedule for examinations: In the early phase of the study, water consumption was checked daily by visual inspection of the water bottles for any overt changes in volume. As water consumption was obviously higher in high dose animals, the water consumption was determined quantitatively by weighing the water bottles once a week from day 21 onwards (representative values over 4 days in males and 3 days in females). The values were calculated as grams per animal per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start and at the end of the administration period.
- Dose groups that were examined: The eyes of each 10 males and 10 females of the control and high dose groups were examined for pathological changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, FRG).

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: The following examinations were carried out in 10 animals per test group and sex of sections B and C.
- Parameters checked: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (H1E model, by Bayer, Munich, FRG): leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets
Clotting analyses: The clotting analyses were carried out using a ball coagulometer (KC 10 A model, by Amelung, Lemgo, FRG) and the results transferred to the computer. The following parameter was determined: prothrombin time (Hepato Quick's test)

CLINICAL CHEMISTRY: Yes
- Animals fasted: No
- How many animals: The following examinations were carried out in 10 animals per test group and sex of sections B and C.
- Parameters checked: An automatic analyser (Hitachi 737; by Boehringer, Mannheim, FRG) was used to examine the clinicochemical parameters.
Chloride was measured with a chloride analyser (model 925; by Ciba-Corning, Fernwald, FRG).
The following parameters were determined:
1. Enzymes: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-ϒ-glutamyltransferase
2. Blood chemistry: sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes. For urinalysis the individual animals were transferred to metabolism cages and urine was collected overnight.
- Animals fasted: Yes, food and water was withdrawn.
- Parameters checked: With the exception of volume, colour, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semi-quantitatively using test strips (Combur-9-test RL, by Boehringer, Mannheim, FRG) and a reflection photometer (Urotron RL9 model by Boehringer, Mannheim, FRG) .
The specific gravity was determined using an urine refractometer. The sediment was evaluated microscopically.
The following examinations were carried out: volume, colour, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

IMMUNOLOGY: No

Neurobehavioural examinations performed and frequency:

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At days -7, 22, 50 and 85 functional observational batteries and measurements of the motor activity were carried out.
- Battery of functions tested: A functional observational battery (FOB) was carried out in all animals of Section A and B on days -7, 22, 50 and 85, starting each time at about 10:00 a.m.
The FOB consisted of 4 parts, starting with passive observations, without disturbing the animals, followed by removal from the home cage and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests as well as quantitative measurements were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. The findings were ranked according to the degreee of severity, if applicable. In order to ensure that the observer was unaware of the treatment groups the tattoo numbers of the animals were covered with adhesive labels outside the cage, and examinations were carried out in randomised order.
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behaviour of the animals.
Attention was paid to: Posture, tremors, convulsions, behaviour, defecation, urination, general observations (all other abnormal findings).

- Open field observations:
The animals were transferred to a standard arena (62 x 62 cm with sides of 25 cm high) and observed for at least 2 minutes.
Following parameters were examined: Fur, skin colour, posture, salivation, respiration, activity, arousal level, vocalisation, tremors, convulsions, bizarre behaviour, impairment of gait, lacrimation, exophthalmus, number of rearings within 2 minutes.
Thereafter, the animals were removed from the standard arena and subjected to following sensorimotor or reflex tests: hyperesthesia, abdominal tension, palpebral closure, winking reflex, pupil size, pupillary reflex, pinna reflex, audition ("startle response"), olfaction, pain perception ("tail pinch"), coordination of movements ("righting response"), vision ("visual placing response").

Then quantitative parameters were determined: Grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, motor activity measurement.
Motor activity was measured on the same day as FOB was performed. The measurement was performed using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in polycarbonate cages with absorbent material. The cages were cleaned prior to each use. The animals were put into the cages in a randomised order. The measurements started at about 2.00 p.m. and the number of beam interrupts were counted over 18 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack. Measurements ended exactly 1.5 hours therafter. During the measurements the animals received no food and no water.

Sacrifice and (histo)pathology:

At the end of the administration period 5 animals per dose and sex (Section A) were sacrificed by perfusion fixation and subjected to neuropathological examinations. The remaining 10 animals per dose and sex (Section B and C) were sacrificed by decapitation under CO2 anesthesia and subjected to gross pathological assessment, followed by histopathological examinations.
All animals were sacrificed after a fasting period (withdrawal of food) for about 16 - 20 hours.

Statistics:

See below.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One mid dose male animal showed skin lesion from day 21 to day 56 and one low dose female animal showed tooth anomaly from day 49 onwards till the end of the study. Both findings were assessed as being incidental and not related to test material administration.
No other abnormal clinical symptoms were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animal died during the conduct of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In high dose males and females, body weights were impaired statistically significant on most of the days. The values on day 91 were about 18 % below controls in males and about 14 % below controls in females.
Corresponding body weight change values were about 28 % and 29 % below controls, respectively.
No test material-related effects were seen in mid and low dose males and females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In high dose males and females food consumption was reduced with statistical significance on several days. The impairment was about 4 % - 19 % in males and about 6 % - 19 % in females.
In mid and low dose groups no statistically or biologically relevant deviations were seen.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency was impaired statistically significant in high dose males on days 14, 21, 35, 49 and 56 and in high dose females on day 35. This was assessed as being test material-related.

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

effects observed, treatment-related

Description (incidence and severity):

In high dose males and females water consumption was increased with statistical significance on all measurement days (except day 56, males). The increase was about 13 % - 55 % in males and about 47 % - 87 % in females when compared to controls.
Water consumption was also increased in mid dose females, the values being about 4 % - 20 % above controls. Although not statistically significant, a test material-related effect cannot be excluded.
In mid dose males and in low dose males and females no statistically or biologically relevant deviations were seen.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No substance related effects were obtained. All findings were spontaneous in nature and within the biological variation in this strain of rats.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After a treatment period of 3 months high dose males and females had statistically significantly lower red blood cell counts, haemoglobin concentrations and haematocrit values than controls.
Slightly but statistically significantly decreased erythrocytes, haemoglobin and haematocrit values were also found in mid dose males.
No significant changes in the morphology of the red blood cells were seen in the differential blood count of treated animals of either sex.
Haematology examinations revealed no changes in white blood cell counts and in the differential white blood cell count of the treated animals.

Clotting analyses
There are no treatment-related changes in the clotting parameters measured.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Enzymes
In the sera of high dose animals statistically significantly increased alkaline phosphatase activities were measured. Furthermore, alanine aminotransferase activities were statistically significantly increased in high dose females.

Blood chemistry
Statistically significantly decreased calcium, total protein, globulin, triglyceride and cholesterol concentrations were detected in the sera of high dose males and females.
In mid dose males triglyceride and cholesterol levels were also decreased. Moreover, statistically significantly increased urea concentrations were found in high dose animals of either sex and increased creatinine levels were measured in high dose males. In high dose females serum chloride was slightly lowered.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males of the high dose group excreted statistically significantly increased amounts of transitional epithelial cells. No other test material-induced changes were noted in the urinalyses of the treated males and females.
There is one additional statistically significant difference in the results of urinalysis (males; low dose group; urinary crystals) which is considered to be of no toxicological concern, because this deviation is incidental, inconsistent when compared with the other sex, and lacks dose-response relationship.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home cage observations:
One mid dose male animal showed skin lesion on day 50. This was assessed as being incidental. No other abnormalities were obtained in any of the test groups when observing the animals in their home cages.

Open field observations:
One mid dose male animal showed skin lesion on day 50 and one low dose female animal showed tooth anomaly on days 50 and 85. Both findings were incidental.

Sensorimotor tests/Reflexes:
No test material-related effects were obtained.

Quantitative observations:
In high dose females rearing was decreased statistically significant on day 85. However, as there was no dose-response relationship (the values of mid dose females were even increased) and as only females were affected, this was assessed as being incidental and not test material-related.
In low dose males, grip strength of hindlimbs was slightly, but statistically significantly decreased on day 85. However, as there was no dose-response relationship and as only males were affected, this minor deviation was assessed as being incidental and not test material-related.

Motor activity measurement
Regarding the overall motor activity (summation of all intervals) no statistically significant deviation was seen in any of the treatment groups.
Comparing the single intervals with the control group, some few statistically significant deviations of single intervals (either increased or decreased values) occurred in various groups on different test days. However as these deviations from controls occurred only sporadically and without dose-response relationship, they were assessed as being incidental and not treatment-related.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In high dose males and females, substance-related findings were seen in the liver (increase in weight in both sexes, alterations of hepatocytes with cytoplasmic eosinophilia and granular cytoplasm in both sexes, centrolobular hypertrophy of hepatocytes in males).
Additionally, test material-related findings were seen also in the adrenal cortex of 8 males and 10 females (presence of fine lipid vacuoles; lipid storage).

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopy and histopathology of selected organs/tissues and locations of the central and peripheral nervous system did not reveal any test material-related neuropathological alterations.

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

> 240 mg/kg diet

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No neurotoxicological effects observed.

Dose descriptor:

NOAEL

Effect level:

> 189 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No neurotoxicological effects observed.

Critical effects observed:

yes

Lowest effective dose / conc.:

38 mg/kg diet

System:

hepatobiliary

Organ:

adrenal glands

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the conditions of this study the no effect level for neurotoxicity was above 3 000 ppm in females and above 2 500 ppm in males.

Executive summary:

The sub-chronic toxicity of the test material following repeated dose administration of the test material via the oral route was assessed according to OECD Test Guideline 408 and in compliance with GLP.

The test material was administered to groups of 15 male and 15 female Wistar rats for 3 months at dietary concentrations of 0 ppm, 75 ppm, 500 ppm, 2 500 ppm (males only) and 3 000 ppm (females only). Based upon the results of a range finding study, different high concentrations were selected for males and females.

Food consumption and body weight were determined at least once a week. Water consumption was determined once a week from day 21 onwards. A check of the general state of health of the rats was made at least daily. Furthermore, the animals were thoroughly examined and palpated once a week. In addition to these general clinical examinations, functional observational batteries and measurements of motor activity were carried out in 10 animals per group and sex before the start of the administration period, and in the 4th, 8th and 13th week of the administration period. Before the commencement and at the end of the administration ophthalmological examinations were carried out in 10 males and 10 females of control and high dose groups. Haematological and clinic-chemical examinations as well as urinalyses were carried out at the end of the administration period in 10 animals per sex and dose. Five animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations. All other animals were subjected to gross-pathological assessment, followed by histopathological examinations.

The following findings were obtained and assessed as being related to the test material administration:

3 000 ppm (females; about 240 mg/kg bodw/day) and 2 500 ppm (males; about 189 mg/kg bw/day):

- Reduced food consumption in both sexes.

- Increased water consumption in both sexes.

- Decreased body weights, resulting in reduced values of about 18 % (males) and 14 % (females) on day 91.

- Decreased body weight change, resulting in reduced values of about 28 % (males) and 29 % (females) on day 91.

- Reduced food efficiency in males and females.

- Decrease in red blood cells, haemoglobin, haematocrit, calcium, total protein, globulins, triglycerides and cholesterol in both sexes.

- Decrease in chloride in females increase in alkaline phosphatase and urea in both sexes.

- Increase in serum creatinine and transitional epithelial cells in the urine of males.

- Increase in alanine aminotransferase in females.

- Increase of absolute and relative liver weights in both sexes.

- Alterations of hepatocytes with cytoplasmic eosinophilia and granular cytoplasm in both sexes.

- Centrolobular hypertrophy of hepatocytes in males (2/10).

- Presence of fine lipid vacuoles (lipid storage) in the adrenal cortex of 8/10 males and 10/10 females.

500 ppm (about 38 mg/kg bw/day):

- Increased water consumption in females.

- Decrease in red blood cells, haemoglobin, haematocrit, triglycerides and cholesterol in males.

75 ppm (about 6 mg/kg bw/day):

- No test material-related findings.

In conclusion, the oral administration of the test material led to signs of toxicity at dietary concentrations of 3 000 ppm (females), 2 500 ppm (males) and 500 ppm (both sexes). Target organs were liver and adrenals. Toxicity was further characterised by an anaemic process and slight functional impairment of kidneys. Lipids were also lowered in either sex. No signs of neurotoxicity were detected. 

Under the conditions of this study the no effect level for neurotoxicity was therefore above 3 000 ppm in females and above 2 500 ppm in males. The NOAEL for systemic toxicity was 75 ppm (about 6 mg/kg bw/day).

Reference 2

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 1994 to 26 May 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-8 (Neurotoxicity Screening Battery)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Chbb: THOM (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: All animals were 42 days old on the day of test material administration.
- Weight at study initiation: At the day of the administration the groups had a mean body weight of male animals: 193 (178 - 210) g and female animals: 150 (135 - 163) g.
- Fasting period before study: Food and drinking water were available ad libitum throughout the study (except during motor activity measurement).
- Housing: Rats were housed singly in stainless steel wire cages (floor area about 800 cm^2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding). The motor activity measurements were conducted in polycarbonate cages with wire covers (floor area about 800 cm^2) and small amounts of absorbent material.
- Diet: Ad libitum except during motor activity measurements.
- Water: Ad libitum except during motor activity measurements.
- Acclimation period: On the day of arrival, an acclimatisation period started of at least 11 days, in which the animals were accustomed to housing and diet.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: 30 to 70 % (relative)
- Photoperiod: The day/night rhythm was 12 hours (12 hours of light from 06.00 - 18.00 h, 12 hours of dark from 18.00 - 6.00 h).

IN-LIFE DATES:
From: 25 April 1994
To: 26 May 1994

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % aqueous

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was administered as a suspension in 0.5 % CMC solution. Test material was weighed out in a calibrated beaker depending on the dose group, then 0.5 % CMC solution was filled up to the desired volume. The suspensions were mixed thoroughly for about one minute using a high speed sonicator (Ultra-Turrax, Janke & Kunkel GmbH, Staufen, FRG). During the administration the doses were kept homogeneous by using a magnetic stirrer. The test material preparations were prepared prior to administration.

VEHICLE
- Vehicle: 0.5 % aqueous carboxymethyl cellulose preparation: “Tylose 30.000” (HOECHST, Frankfurt/Main, FRG) in doubly distilled water.
- Concentration in vehicle: 1.75, 3.50 and 7.00 g/100 mL for the 175, 350 and 750 mg/kg bw doses, respectively.
- Administration volume: 10 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Investigations to characterise the test material in respect of purity and homogeneity were carried out before the start of the study. The stability for the duration of the study was ensured. The correctness of the concentrations was checked in samples drawn from each concentration.
The homogeneity of the test material preparation was checked before the start of the study. The stability of the test material in the vehicle was proven.
The test material and the test material preparations were analysed using GC and reversed phase HPLC with UV-detection. The analyses yielded concentrations between 91.2 and 97.2 % of the theoretical contents.
The analyses confirm the theoretical concentrations of the samples.
It was confirmed before the start of the study that the test material is stable in 0.5 % CMC-solution for a period of 24 hours at room temperature. As dosing was completed within this time, the stability in the vehicle was ensured.
The check of the homogeneity in the vehicle confirmed that the test material was homogeneously distributed in the vehicle. Concentration control analyses confirmed that the concentrations were within an acceptable range.

Duration of treatment / exposure:

A single oral dose followed by a two week observation period.

Frequency of treatment:

Once

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were chosen on the basis of previous investigations.
In an acute toxicity study groups of ten fasted Wistar rats (five males and five females) were given a single dose by gavage of the test material in 0.5 % aqueous carboxymethylcellulose at dose levels of 2 150, 1 470, 1 000 and 681 mg/kg body weight. All animals surviving treatment were killed and examined macroscopically after an observation period of 14 days. Mortalities occurred within 2 days at 2 150 mg/kg body weight (all animals), 1 470 mg/kg body weight (4 males/5 females), and 1 000 mg/kg body weight (5 females). Macroscopic examination of these animals revealed general congestion, haemorrhagic ulcerations in the glandular stomach, slightly atonic intestinum, and remarkably filled urinary bladders. Principal clinical signs of reaction to treatment at dose levels ≥1 000 mg/kg body weight were dyspnoea, lethargy, abnormal body position, atonia, paresis, tremor, spastic gait, piloerection, exsiccosis, and/or bad clinical condition. Recovery of surviving rats, as judged by external appearance and behaviour, was complete by day 5.
In a first study designed to determine peak effect time, the test material was administered to each 5 non-fasted Wistar rats at dose levels of 1 000 mg/kg body weight (males) or 800 mg/kg body weight (females). As 3 males and 1 female died within 2 days, the remaining animals were sacrificed at that time; these animals showed severe clinical symptoms such as convulsions, lethargy, paresis, and tremors from 2 hours after dosing by gavage until necropsy. As these dose levels proved to be too high, a second peak finding study was carried out.
In the second study designed to determine peak effect time, doses of 800 mg/kg body weight (males) and 700 mg/kg body weight (females) were used. 1 male animal died 3 days after administration. Clinical symptoms such as ataxia, tremors, half-closure of eyelids, and urine-smeared anogenital region were observed in all animals from 2 hours after administration onwards until necropsy on day 3, without any substantial change in the intensity of the effects.
Based upon the findings above, following doses were selected: 700 mg/kg body weight as high dose which is close to the LD0 value, 350 mg/kg body weight as intermediate dose and 175 mg/kg body weight as low dose.
- Rationale for animal assignment: The animals were randomly assigned to the groups based upon body weight, separated by sex, prior to the first functional observational battery. The randomisation list was drawn up by a computer.

EXPERIMENTAL PROCEDURE
In order to balance the groups, the study was conducted with 4 subsets (Section A males, Section B males, Section A females, Section B females). Within each subset, animals of control, low, mid and high dose group were tested again in randomised order. The randomisation list was drawn up by a computer. Thus, it was ensured, that:
a) All animals were examined within the same time after dosing. Time of testing was therefore identical for all animals;
b) For each examination day animals from all test groups (including controls) could be used;
c) The examinations for all subsets could be performed on the same time of the day, thus diurnal effects could be neglected.

The test material was administered at about 08.00 a.m.; Functional Observational Batteries were carried out from about 10:00 a.m. onwards; measurements of motor activity were carried out from about 02:00 p.m. onwards.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The general state of health concerning mortality or moribund state of the animals was checked twice a day (Monday to Friday) and once a day (Saturdays, Sundays and public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined in detail and palpated once at the time of body weight determination (i.e. prior to treatment on day 0).

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was determined before the first functional observational battery in order to randomise the animals. During the conduct of the study, the body weight was determined on day -7, day 0 (day of test material administration), on day 7 and on day 14. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY
A functional observational battery (FOB) was carried out in all animals on day -7, on day 0 (between 2 and 8 hours after gavage; time of peak effect), on day 7 and on day 14.
The FOB consisted of 4 parts, starting with passive observations, without disturbing the animals, followed by removal from the home cage and open field examinations in a standard arena. Thereafter, sensorimotor tests and reflex tests as well as quantitative measurements were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. The findings were ranked according to the degree of severity, if applicable.
In order to ensure that the observer was unaware of the treatment groups the tattoo numbers of the animals were covered with adhesive labels outside the cage, and examinations were carried out in randomised order.

> Home cage observations
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behaviour of the animals. Attention was paid to:
- Posture
- Tremors
- Convulsions
- Behaviour
- Defecation
- Urination
- General observations (all other abnormal findings)

> Open field observations
The animals were transferred to a standard arena (62 x 62 cm with sides of 25 cm high) and observed for at least 2 minutes. The following parameters were examined:
- Fur
- Skin colour
- Posture
- Salivation
- Respiration
- Activity
- Arousal level
- Vocalisation
- Tremors
- Convulsions
- Bizarre behaviour
- Impairment of gait
- Lacrimation
- Exophthalmus
- Number of rearings within 2 minutes

> Sensorimotor and reflex tests
After the open filed observations, the animals were removed from the standard arena and subjected to the following sensorimotor or reflex tests:
- Hyperesthesia
- Abdominal tension
- Palpebral closure
- Winking reflex
- Pupil size
- Pupillary reflex
- Pinna reflex
- Audition ("startle response")
- Olfaction
- Pain perception ("tail pinch")
- Coordination of movements ("righting response")
- Vision ("visual placing response")
Then quantitative parameters were determined:
- Grip strength of forelimbs
- Grip strength of hindlimbs
- Landing foot-splay test

MOTOR ACTIVITY MEASUREMENT
Motor activity was measured on the same day as FOB was performed. The measurement was performed using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage.
During the measurement the animals were kept in Polycarbonate cages with bedding. The cages were cleaned prior to each use. The animals were put into the cages in a randomised order. The measurements started at about 2.00 p.m. and the number of beam interrupts were counted over 18 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack. Measurements ended exactly 1.5 hours thereafter.

Sacrifice and (histo)pathology:

SACRIFICE: All animals not selected for perfusion fixation were sacrificed under CO2-anaesthesia without any further examinations.

NEUROPATHOLOGY (ANIMALS SELECTED FOR PERFUSION FIXATION)
The five surviving animals per sex and dose that showed the most distinct neurological symptoms were deeply anaesthetised (Nembutal, about 4 mL/kg body weight) at the end of the study and sacrificed by perfusion fixation. If no neuroclinic symptoms were seen in one of the test or control groups, the first five animals of the respective group were fixed by perfusion.
Soerensen's phosphate buffer served as rinsing solution and the fixation solution according to Karnovsky served as fixative.
The sacrificed animals were necropsied and the visible organs assessed by gross pathology as thoroughly as possible for perfused animals. The organ samples listed below were carefully removed.

> Plastic embedding (epoxy resin), sectioning, staining and storage
The following PNS organ samples were processed histotechnically from all perfused animals per group and sex:
- Dorsal root ganglia (C3-C6, L1-L4)
- Dorsal root fibres (C3-C6, L1-L4)
- Ventral root fibres (C3-C6, L1-L4)
- Proximal sciatic nerve
- Tibial nerve (at knee)
- Sural nerve (at knee)

For the control and high dose group, plastic embedding in epoxy resin, semithin sectioning and staining with toluidine took place. For the low and mid dose groups, storage of fixed specimen in buffer solution took place.
The semithin sections were examined light-microscopically and assessed

> Paraffin embedding (paraplast), sectioning, staining (haematoxylin-eosin (H&E)) and preservation
The following organ sections were processed histotechnically for all perfused animals in the control and high dose group:
Brain: (cross sections)
- Olfactory bulb
- Frontal lobe
- Parietal lobe with diencephalon (thalamus/hypothalamus)
- Midbrain with occipital and temporal lobe
- Pons
- Cerebellum
- Medulla oblongata with Nucleus gracilis and cuneatus
The brain was preserved in toto for all perfused animals in the low and mid dose groups in 4 % formaldehyde.

The following organ sections were processed histotechnically for all perfused animals (paraffin embedding for control and high dose group, preservation in 4 % formaldehyde for low and mid dose groups):
Spinal cord: (cross and oblique sections)
- Cervical swelling (C3-C6)
- Lumbar swelling (L1-L4)
Others:
- Gasserian ganglia with nerve
- Eyes with optic nerve
- Olfactory epithelium (nasal cavity [IV])
- Pituitary gland
- Gastrocnemius muscle
- Anterior tibial muscle

- All macroscopic findings (all affected animals) were also embedded in paraffin, sectioned and stained.

The H&E-stained paraplast slides were examined light-microscopically and assessed.

Statistics:

For the statistical evaluation of the study, means and standard deviations were calculated for various parameters for the animals in each test group and printed out in the form of tables.
For the statistical significance of the quantitative parameters of functional observational battery, and motor activity, a non-parametric one-way analysis of variance was done via the Kruskal-Walis-h-test. If the resulting p-value was equal or less than 0.05 a pairwise comparison of each dose group with the control group was carried out. This comparison was done using the Mann-Whitney-U-test for the hypothesis of equal medians. If the results of this test were significant, p-markers (* for p < 0.05, ** for p < 0.02, *** for p < 0.002) were printed together with the group mean in the summary tables.
For body weight data a parametric one-way analysis of variance was done via the F-test (ANOVA) if the resulting p-value was equal or less than 0.05, a comparison of each dose group with the control group was carried out. These comparisons were performed simultaneously via Dunnett's test for the hypothesis of equal means. If the results of this test were significant, labels (* for p < 0.05, ** for p < 0.01) were printed together with the group means in-the summary tables.
Both tests were performed two-sided.

Clinical signs:

no effects observed

Description (incidence and severity):

During the observations for mortality or moribund state of the animals, no abnormalities were detected. The examinations of the animals during the weekly body weight determinations also revealed no abnormal signs.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

During the conduct of the study, no animal died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight change was statistically significantly lower in high dose males on day 7 (about 17 % below control). This was assessed as being a material-related effect.
No statistically or biologically significant deviations were seen on other days or in other test groups.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

HOME CAGE OBSERVATIONS
On day 0, half closure of eyelids was seen in 7 high dose males, 3 mid dose males, 3 high dose females and 1 mid dose female. Abnormal posture (abdominal (prone) position, rank 2) was observed in 4 high dose females and 2 mid dose females. All findings were assessed as being related to treatment.
No abnormalities were obtained in all other groups or on other test days.

OPEN FIELD OBSERVATIONS
- The following abnormal signs were observed on day 0:
In the highest dose group, abnormal posture (abdominal (prone) position, rank 2) was observed in 2 males and 3 females. Impaired activity in the open field (hypoactivity, rank 2) was seen in 5 males and 8 females. Impairment of gait was seen in 4 males and 6 females: Slight ataxia in 4 males and 5 females; severe ataxia with spreading of fore- and hindlimbs in 1 female. One male and 3 females showed hypothermia (rats were cold to touch). One female showed a decreased arousal level (rank 2). As only one animal was affected, this might be incidental. A reddish area in the bulbus was seen in 2 females; the origin of this latter finding is difficult to explain, but a test material-relationship seems to be unlikely.
In the mid dose group, abnormal posture (abdominal (prone) position, rank 2) was observed in 2 males and 1 female. The activity in the open field was impaired in 8 males and 2 females (hypoactivity, rank 2). Impairment of gait (slight ataxia, rank 2) was seen in 4 males and 1 female.
In the low dose group, impaired activity in the open field (hypoactivity, rank 2) was seen in 1 female. Impairment of gait (slight ataxia, rank 2) was seen in 1 male. A test material-relationship cannot be ruled out, as the same findings occurred also at higher incidences in the higher dose groups.

- Day 7 observations:
1 mid dose male animal showed impaired activity (hypoactivity, rank 2). As only one mid dose animal was affected, this was assessed as being incidental.

- Day 14 observations:
No abnormal signs were detected in any of the treated animals.

One male animal of the control group showed vocalisation during handling on days 7 and 14.

SENSORIMOTOR TESTS/REFLEXES
On day 0, half closure of eyelids was seen in 2 high dose males and 1 high dose female during testing pupillary reflexes.
No abnormalities were obtained in all other groups or on other test days.

QUANTITATIVE OBSERVATIONS (Rearing Grip strength, Landing foot-splay test)
In high and mid dose females, the number of rearings was statistically significantly lower on day 0. This was related to the test material administration.
The values of the landing foot-splay test were statistically significantly increased in high and mid dose males on day 0. This was assessed as being test material related. By contrast, the statistically significantly reduced values of low dose males on day 7 were assessed as being incidental, as there was no dose-response relationship. Moreover, an impairment of gait usually results in increased values of landing foot-splay test, but not in a decrease.
Grip strength was not affected statistically significant by treatment.

MOTOR ACTIVITY MEASUREMENT
Regarding the overall motor activity, the values were statistically significantly decreased in high and mid dose males and high dose females on day 0. No statistically significant deviations were seen in the other dose groups or during the examinations on day -7, day 7 and day 14.
Comparing the single intervals with the control group, some statistically significant impairments were seen in high, mid and low dose males and high and mid dose females on day 0. This was assessed as being test material related. Increased values with statistical significance were seen in male animals of groups 2 and 3 on day -7. This was related to the unusual low activity of control animals during the first few intervals. As this occurred prior to treatment, a test material-related effect can be excluded. Some few statistically significant deviations of single intervals (increased values) occurred also on other test days. However, as they occurred only sporadically and without dose-response relationship, this was assessed as being incidental and not treatment-related.

A clear impairment of motor activity on day 0 was seen in high and mid dose males and in high dose females; a slight impairment was seen also in low dose males and in mid dose females.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross lesions noted.
No test material-related findings were obtained in the central or peripheral nervous system.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No test material-related findings were obtained in the central or peripheral nervous system.

The light-microscopic investigation did not reveal any test material-dependent, neuropathological changes in the organs examined. All findings recorded are seen to be incidental or spontaneous in character.

- High dose animals (700 mg/kg)
Macroscopy and histopathology of selected organs/ tissues and localisations of the central and peripheral nervous system did not reveal any test material-dependent and neuropathological alterations.

- Medium dose animals (350 mg/kg) and low dose animals (175 mg/kg)
There were no gross lesions recorded and no microscopic examinations performed. From the neuromorphological point of view, there is no acute neurotoxicity noted at the dose levels used.

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Clear test material-related and dose-dependent clinical signs were seen in males and females at 700 and 350 mg/kg body weight. At 175 mg/kg body weight, single animals still showed impairment of gait or activity, and motor activity was slightly reduced in males. Although the findings at the low dose level were limited to single animals, a test material relationship cannot be ruled out. The "no observed adverse effect level" (NOAEL) was therefore slightly below 175 mg/kg body weight in both sexes.
All clinical signs were reversible by day 7 post exposure (except impaired body weight change). Moreover, comprehensive neuropathological examinations of the central and peripheral nervous system did not reveal any test material-related effects. The signs recorded in the present study therefore were pharmacological effects of the test material at near-lethal dose levels and were due to acute systemic toxicity, not to selective neurotoxicity.
Thus it was considered that the test material did not cause permanent damage to the nervous system when administered as single gavage to Wistar rats.

Dose descriptor:

NOAEL

Effect level:

< 175 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Critical effects observed:

no

Conclusions:

Under the conditions of this study, the NOAEL was therefore slightly below 175 mg/kg body weight in both sexes. It was considered that the test material did not cause permanent damage to the nervous system when administered as single gavage to Wistar rats.

Executive summary:

The potential of the test material to cause neurotoxicity when administered in a single dose was investigated in accordance with the standardised guideline US EPA OPP 81-8 under GLP conditions.

The test material was given to groups of 10 male and 10 female Wistar rats as a single oral administration by gavage at dose levels of 0, 175, 350 and 700 mg/kg body weight. As vehicle, 0.5 % aqueous carboxymethyl cellulose preparation was used and the administration volume was 10 mL/kg body weight.

The animals were observed up to 2 weeks after dosing. Body weight was determined weekly. The general state of health of the rats was checked at least daily and the animals were additionally examined and palpated once a week. Functional observational batteries and motor activity measurements were carried out before test material administration, between 2 and 8 hours after test material administration (covering the time of peak effect), as well as 7 and 14 days after dosing. Tissues from 5 animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations.

Body weight change was impaired in high dose males on day 7. This indicates a systemic toxic effect of the test material administration. Comparing the values of day 7 with the values on day 14, a recovery was observed.

Abnormal clinical signs were seen dose-dependently in high and mid-dose animals during the functional observational battery performed on day 0 (day of test material administration). Typical signs of treatment were abdominal (prone) position, impaired activity in the open field, impairment of gait, half-closure of eyelids, hypothermia (rats were cold to touch), decreased number of rearings, increased values of landing foot-splay test and/or impairment of motor activity.

In single animals of the low dose group, impaired activity in the open field or impairment of gait was seen. A material-relationship cannot be ruled out, as the same findings occurred also at higher incidences in the higher dose groups. Moreover, motor activity was also slightly impaired in low dose males.

On day 7 recovery from all clinical symptoms was observed. The impaired activity observed in one mid dose male on day 7 was assessed as being incidental, as only one animal was affected and as there was no dose-response relationship.

Macroscopy and histopathology of the central and peripheral nervous system did not reveal any test material-induced neuropathological alterations.

In conclusion, the findings below were obtained on the day of treatment (except body weight change) and assessed as being test material-related:

 - 700 mg/kg body weight: Decreased body weight change on day 7 in males, half-closure of eyelids in the home cage (7 males, 3 females) and in the open field (2 males, 1 female), abdominal (prone) position in the home cage (4 females) and in the open field (2 males, 3 females), impaired activity in the open field (5 males, 8 females), impairment of gait (ataxia) (4 males, 6 females), hypothermia (1 male, 3 females), decreased number of rearings in females, increased foot-splay values in males and decreased motor activity (males and females).

- 350 mg/kg body weight: Half-closure of eyelid in the home cage (3 males, 1 female), abdominal (prone) position in the home cage (2 females) or in the open field (2 males and 1 female), impaired activity in the open field (8 males, 2 females), impairment of gait (ataxia) (4 males, 1 female), decreased number of rearings in females, increased foot-splay values in males and decreased motor activity (males, and less pronounced also in females).

- 175 mg/kg body weight: Impaired activity in the open field (1 female), impairment of gait (ataxia) (1 male) and slight impairment of motor activity (males).

Clear test material-related and dose-dependent clinical signs were seen in males and females at 700 and 350 mg/kg body weight. At 175 mg/kg body weight, single animals still showed impairment of gait or activity, and motor activity was slightly reduced in males. Although the findings at the low dose level were limited to single animals, a test material relationship cannot be ruled out. The "no observed adverse effect level" (NOAEL) was therefore slightly below 175 mg/kg body weight in both sexes.

All clinical signs were reversible by day 7 post exposure (except impaired body weight change). Moreover, comprehensive neuropathological examinations of the central and peripheral nervous system did not reveal any test material-related effects. The signs recorded in the present study therefore were pharmacological effects of the test material at near-lethal dose levels and were due to acute systemic toxicity, not to selective neurotoxicity.

Thus it was considered that the test material did not cause permanent damage to the nervous system when administered as single gavage to Wistar rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

189 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

Acute Dose Oral Route: Mellert et al. (1995)

The potential of the test material to cause neurotoxicity when administered in a single dose was investigated in accordance with the standardised guideline US EPA OPP 81-8 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was given to groups of 10 male and 10 female Wistar rats as a single oral administration by gavage at dose levels of 0, 175, 350 and 700 mg/kg body weight. As vehicle, 0.5 % aqueous carboxymethyl cellulose preparation was used and the administration volume was 10 mL/kg body weight.

The animals were observed up to 2 weeks after dosing. Body weight was determined weekly. The general state of health of the rats was checked at least daily and the animals were additionally examined and palpated once a week. Functional observational batteries and motor activity measurements were carried out before test material administration, between 2 and 8 hours after test material administration (covering the time of peak effect), as well as 7 and 14 days after dosing. Tissues from 5 animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations.

Body weight change was impaired in high dose males on day 7. This indicates a systemic toxic effect of the test material administration. Comparing the values of day 7 with the values on day 14, a recovery was observed.

Abnormal clinical signs were seen dose-dependently in high and mid-dose animals during the functional observational battery performed on day 0 (day of test material administration). Typical signs of treatment were abdominal (prone) position, impaired activity in the open field, impairment of gait, half-closure of eyelids, hypothermia (rats were cold to touch), decreased number of rearings, increased values of landing foot-splay test and/or impairment of motor activity.

In single animals of the low dose group, impaired activity in the open field or impairment of gait was seen. A material-relationship cannot be ruled out, as the same findings occurred also at higher incidences in the higher dose groups. Moreover, motor activity was also slightly impaired in low dose males.

On day 7 recovery from all clinical symptoms was observed. The impaired activity observed in one mid dose male on day 7 was assessed as being incidental, as only one animal was affected and as there was no dose-response relationship.

Macroscopy and histopathology of the central and peripheral nervous system did not reveal any test material-induced neuropathological alterations.

In conclusion, the findings below were obtained on the day of treatment (except body weight change) and assessed as being test material-related:

 - 700 mg/kg body weight: Decreased body weight change on day 7 in males, half-closure of eyelids in the home cage (7 males, 3 females) and in the open field (2 males, 1 female), abdominal (prone) position in the home cage (4 females) and in the open field (2 males, 3 females), impaired activity in the open field (5 males, 8 females), impairment of gait (ataxia) (4 males, 6 females), hypothermia (1 male, 3 females), decreased number of rearings in females, increased foot-splay values in males and decreased motor activity (males and females).

- 350 mg/kg body weight: Half-closure of eyelid in the home cage (3 males, 1 female), abdominal (prone) position in the home cage (2 females) or in the open field (2 males and 1 female), impaired activity in the open field (8 males, 2 females), impairment of gait (ataxia) (4 males, 1 female), decreased number of rearings in females, increased foot-splay values in males and decreased motor activity (males, and less pronounced also in females).

- 175 mg/kg body weight: Impaired activity in the open field (1 female), impairment of gait (ataxia) (1 male) and slight impairment of motor activity (males).

Clear test material-related and dose-dependent clinical signs were seen in males and females at 700 and 350 mg/kg body weight. At 175 mg/kg body weight, single animals still showed impairment of gait or activity, and motor activity was slightly reduced in males. Although the findings at the low dose level were limited to single animals, a test material relationship cannot be ruled out. The "no observed adverse effect level" (NOAEL) was therefore slightly below 175 mg/kg body weight in both sexes.

All clinical signs were reversible by day 7 post exposure (except impaired body weight change). Moreover, comprehensive neuropathological examinations of the central and peripheral nervous system did not reveal any test material-related effects. The signs recorded in the present study therefore were pharmacological effects of the test material at near-lethal dose levels and were due to acute systemic toxicity, not to selective neurotoxicity.

Thus it was considered that the test material did not cause permanent damage to the nervous system when administered as single gavage to Wistar rats.

Sub-Chronic Repeated Dose Oral Route: Mellert et al. (1995)

The sub-chronic toxicity of the test material following repeated dose administration of the test material via the oral route was assessed according to OECD Test Guideline 408 and in compliance with GLP.The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was administered to groups of 15 male and 15 female Wistar rats for 3 months at dietary concentrations of 0 ppm, 75 ppm, 500 ppm, 2 500 ppm (males only) and 3 000 ppm (females only). Based upon the results of a range finding study, different high concentrations were selected for males and females.

Food consumption and body weight were determined at least once a week. Water consumption was determined once a week from day 21 onwards. A check of the general state of health of the rats was made at least daily. Furthermore, the animals were thoroughly examined and palpated once a week. In addition to these general clinical examinations, functional observational batteries and measurements of motor activity were carried out in 10 animals per group and sex before the start of the administration period, and in the 4th, 8th and 13th week of the administration period. Before the commencement and at the end of the administration ophthalmological examinations were carried out in 10 males and 10 females of control and high dose groups. Haematological and clinico-chemical examinations as well as urinalyses were carried out at the end of the administration period in 10 animals per sex and dose. Five animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations. All other animals were subjected to gross-pathological assessment, followed by histopathological examinations.

The following findings were obtained and assessed as being related to the test material administration:

3 000 ppm (females; about 240 mg/kg bw/day) and 2 500 ppm (males; about 189 mg/kg bw/day):

- Reduced food consumption in both sexes.

- Increased water consumption in both sexes.

- Decreased body weights, resulting in reduced values of about 18 % (males) and 14 % (females) on day 91.

- Decreased body weight change, resulting in reduced values of about 28 % (males) and 29 % (females) on day 91.

- Reduced food efficiency in males and females.

- Decrease in red blood cells, haemoglobin, haematocrit, calcium, total protein, globulins, triglycerides and cholesterol in both sexes.

- Decrease in chloride in females increase in alkaline phosphatase and urea in both sexes.

- Increase in serum creatinine and transitional epithelial cells in the urine of males.

- Increase in alanine aminotransferase in females.

- Increase of absolute and relative liver weights in both sexes alterations of hepatocytes with cytoplasmic eosinophilia and granular cytoplasm in both sexes.

- Centrolobular hypertrophy of hepatocytes in males (2/10).

- Presence of fine lipid vacuoles (lipid storage) in the adrenal cortex of 8/10 males and 10/10 females.

500 ppm (about 38 mg/kg bw/day):

- Increased water consumption in females.

- Decrease in red blood cells, haemoglobin, haematocrit, triglycerides and cholesterol in males.

75 ppm (about 6 mg/kg bw/day):

- No test material-related findings.

In conclusion, the oral administration of the test material led to signs of toxicity at dietary concentrations of 3 000 ppm (females), 2 500 ppm (males) and 500 ppm (both sexes). Target organs were liver and adrenals. Toxicity was further characterised by an anaemic process and slight functional impairment of kidneys. Lipids were also lowered in either sex. No signs of neurotoxicity were detected. 

Under the conditions of this study the no effect level for neurotoxicity was therefore above 3 000 ppm in females and above 2 500 ppm in males. The NOAEL for systemic toxicity was 75 ppm (about 6 mg/kg bw/day).

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to specific target organ toxicity following acute and sub-chronic neurotoxicity testing.