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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: 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:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Version / remarks:

1984

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S. EPA: Pesticide Assessment Guideline, Subdivision F; Neurotoxicity Screening Battery; NTIS, pp. 13 - 27, 1991.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EC Commission Directive 87/302/EEC of November 18, 1987; Part B: Methods for the determination of Toxicity Subchronic Oral Toxicity Test.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

EC Number:

240-539-0

EC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Cas Number:

16484-77-8

Molecular formula:

C10H11ClO3

IUPAC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Test material form:

solid

Remarks:

Slightly brown

Details on test material:

- Storage conditions: Room temperature

Test animals

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 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 except during motor activity measurements when they were housed in polycarbonate cages with wire covers.
- Diet: Ad libitum (except during motor activity measurements and for about 16 - 20 hours before sacrifice).
- Water: Ad libitum.
- Acclimation period: On the days of arrival, an 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 by a contract laboratory.

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).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral 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:

3 months

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 000 ppm

Remarks:

Females only, ca. 240 mg/kg body weight per day.

Doses / concentrations 2

Dose / conc.:

2 500 ppm

Remarks:

Males only, ca. 189 mg/kg body weight per day.

Doses / concentrations 3

Dose / conc.:

500 ppm

Remarks:

Males and females, ca. 38 mg/kg body weight/day.

Doses / concentrations 4

Dose / conc.:

75 ppm

Remarks:

Males and females, ca. 6 mg/kg body weight/day.

No. of animals per sex per dose:

15 per sex per dose, separated into 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 (subchronic toxicity and neurotoxicity), the 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 subchronic 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.

Examinations

Observations and 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
The differential blood count were evaluated visually.
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

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.
The following parameters were examined: Fur, skin colour, posture, salivation, respiration, activity, arousal level, vocalisation, tremors, convulsions, bizarre behavior, 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.

IMMUNOLOGY: No

Sacrifice and 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.

Results and discussion

Results of examinations

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.

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 substance-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 substance-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 statisitcally significant, a substance-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 substance-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 substance-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 substance-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, substance-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/tisues and locations of the central and peripheral nervous system did not reveal any substance-related neuropathological alterations.

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

Target system / organ toxicity

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

Applicant's summary and conclusion

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. The NOAEL for systemic toxicity was 75 ppm (about 6 mg/kg bw/day).

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 substance 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 bwday):

- No substance-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).