bis(2-ethylhexyl) (4-hydroxy-3,5-dimethoxybenzyl)malonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-09-28 - 2008-04-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

30 September 1996

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

471-510-2

EC Name:

-

Cas Number:

872182-46-2

Molecular formula:

C28H46O7

IUPAC Name:

1,3-bis(2-ethylhexyl) 2-[(4-hydroxy-3,5-dimethoxyphenyl)methyl]propanedioate

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Laboratory Animal Services, Fuellinsdorf, Switzerland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks (50 days)
- Weight at study initiation: Males: 158 - 176 g (mean: 166 g)
Females: 130 - 146 g (mean 138 g)
- Fasting period before study: no
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet: ad libitum (Pelleted standard Kliba Nafag 3433 (batch no. 41/07) rat / mouse maintenance diet; Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water: ad libitum
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Identification: Acclimatization period: Cage card and tail mark. Treatment period: Cage card and individual ear tattoo
- Randomization: Computer-generated random algorithm.

DETAILS OF FOOD AND WATER QUALITY:
Pelleted standard Kliba Nafag 3433 (batch no. 41/07) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants. Results of representative analyses for contaminants are included in the report.
Community tap-water from Itingen was available ad libitum in water bottles. None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10-15
- Photoperiod: 12 hrs dark / 12 hrs light (with music during the light period)

IN-LIFE DATES: From: 2007-10-29 To: 2007-12-10 (incl. recovery)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

DOSE FORMULATION
The dose formulations were prepared weekly.
The test substance was weighed into a tared glass container on a suitable precision balance and the vehicle, PEG 300, was added to give the appropriate final concentration of the test item in the emulsion. The mixtures were prepared using a magnetic stirrer and stored at room
temperature (15 - 25 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Stability of Dose Formulations:
At least one week
Based upon the results of stability analyses

Storage of Dose Formulations:
At room temperature (20 ± 5 °C) in glass beakers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS
Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start using a HPLC method with UV detection.
Analyses were performed by the study scientist of the analytical part according to a HPLC analytical method supplied by the Sponsor and previously adapted. Details of the analytical method are documented in the raw data generated by the study scientist (and/or his/her staff). The dose formulations were delivered under ambient conditions.
The identity of the test material was confirmed by its retention time which was similar to that measured in the working standards. The mean test item content was found to be within the accepted range of ±20% of the nominal content. In addition, the homogeneous distribution of the test material in PEG 300 was demonstrated. The application formulations were considered to be stable for at least seven days when kept at room temperature.
In conclusion, the results obtained from formulation analysis confirm the correct preparation and storage of application formulations during the conduct of this study.

Duration of treatment / exposure:

Duration of Treatment: 28 days
Duration of Recovery: 14 days

Frequency of treatment:

Frequency of Administration: Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Group 1: 10 per sex (0 mg/kg bw/day)
Group 2: 5 per sex (100 mg/kg bw/day)
Group 3: 5 per sex (300 mg/kg bw/day)
Group 4: 10 per sex (1000 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

see executive summary

Positive control:

no

Examinations

Observations and examinations performed and frequency:

Viability / Mortality: Twice daily
General Clinical Observations:
- Acclimatization Period: Once daily
- Treatment Period: Twice daily on days 1-3; once daily thereafter
- Recovery Period: Once daily

Detailed Behavioral Observations:
- Acclimatization Period: Once before the first test item exposure
- Treatment Period: Once weekly (Allocation A and B)
- Recovery Period: Not performed during recovery

Food Consumption:
- Acclimatization Period: Once weekly
- Treatment Period: Once weekly
- Recovery Period: Once weekly

Body Weights:
- Acclimatization Period: Once weekly
- Treatment Period: Once weekly
- Recovery Period: Once weekly

Functional Observation Battery/Grip strength/Locomotor activity: During week 4

Clinical Laboratory Investigations
Blood and Urine Sampling: After 4 Weeks (treatment period), allocation A and B
After 6 Weeks: (recovery period), allocation B
Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube. Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.
The assays were performed under internal laboratory quality control conditions to assure reliable test results.

Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

HEMATOLOGY
The following hematology parameters were determined:
Complete Blood Cell Count:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index (low, medium, high fluorescence)
Leukocyte count, total
Differential leukocyte count:
Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes
Large unstained cells
Platelet count
Hemoglobin Derivatives
Methemoglobin
Coagulation
Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Alkaline phosphatase
Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS
Urine volume (18 hour)
Specific gravity (relative density)
Color
AppearancepH value
Nitrite
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes

Sacrifice and pathology:

Pathology

Necropsy
After 4 Weeks: 26-Nov-2007 (allocation A animals)
After 6 Weeks (Recovery): 10-Dec-2007 (allocation B animals)
All allocation A and B animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution except for eyes with optic nerve and harderian gland which were fixed in Davidson's solution or epididymides and testes which were fixed in Bouin’s solution:
Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (femur)
Brain - including section of medulla/pons,
cerebral and cerebellar cortex
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Esophagus
Eyes w/optic nerve (fixed in Davidson's
solution)
Harderian gland (fixed in Davidson's solution)
Heart
Ileum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Larynx
Lacrimal gland, exorbital
Liver
Lungs, filled w/formalin at necropsy
Lymph nodes - mesenteric, mandibular
Mammary gland area
Nasal cavity
Ovaries
Pancreas
Pharynx
Pituitary gland
Prostate gland incl. coagulating glands
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland, if possible)
Tongue
Trachea
Urinary bladder, filled w/formalin at
necropsy
Uterus
Vagina
Gross lesions


Organ Weights
The following organs were weighed before fixation and the weight recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.
Adrenal glands
Brain
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Epididymides
Thymus

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, clinical laboratory data, organ weights, and ratios:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

One male treated with 300 mg/kg/day and two males and eight females treated with 1000 mg/kg/day showed salivation on day 18 of treatment after administration. One male and four females treated with 1000 mg/kg/day still showed slight salivation after administration on day 19 of treatment. However, as salivation was observed only on days 18 and 19, this finding was considered not to be related to the test item.
One control male showed localized hair loss from day 3 of acclimatization through week 2 of treatment. A second control male had scabs from week 2 of treatment to the beginning of week 4. One male treated with 100 mg/kg/day showed scabs and a wound starting in week 2 and additional hair loss from week 3 to the end of treatment. Breathing noises were observed in one male treated with 1000 mg/kg/day on 2 days in week 2. These findings were considered to be incidental. No clinical signs were noted during recovery.

Mortality:

no mortality observed

Description (incidence):

All animals survived to scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related effects on body weights and body weight gain were noted.
The mean body weight of test item-treated females during treatment and recovery and of test item-treated males during treatment at all dose levels was comparable to that of the respective controls.
In males treated with 1000 mg/kg/day, the mean body weight during recovery was slightly higher than in controls. The mean body weight gain of test item-treated and control males at the end of treatment and
during recovery was similar. In test item-treated females at all dose levels, the mean body weight gain during treatment was slightly lower than in the respective controls except day 22 in females treated with 1000 mg/kg/day. The difference attained statistical significance on day 8 in females treated with 100 mg/kg/day and at the end of treatment in those treated with 100 or 300 mg/kg/day.
Although they attained statistical significance, the differences noted were small and did not show a dose relationship, nor were they consistent across sexes. Thus, they were considered not to be test item-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY / VIABILITY
All animals survived to scheduled necropsy.

CLINICAL SIGNS

GENERAL CAGESIDE OBSERVATIONS
One male treated with 300 mg/kg/day and two males and eight females treated with 1000 mg/kg/day showed salivation on day 18 of treatment after administration. One male and four females treated with 1000 mg/kg/day still showed slight salivation after administration on day 19 of treatment. However, as salivation was observed only on days 18 and 19, this finding was considered not to be related to the test item.
One control male showed localized hair loss from day 3 of acclimatization through week 2 of treatment. A second control male had scabs from week 2 of treatment to the beginning of week 4. One male treated with 100 mg/kg/day showed scabs and a wound starting in week 2 and additional hair loss from week 3 to the end of treatment. Breathing noises were observed in one male treated with 1000 mg/kg/day on 2 days in week 2. These findings were considered to be incidental.
No clinical signs were noted during recovery.

Functional Observational Battery
No clinical signs were noted during detailed observation in week 4

Grip Strength
No differences were noted in the mean grip strength between test item-treated animals and the respective controls.

Locomotor Activity
No differences in locomotor activity were noted between test item-treated and control females. In males treated with 300 or 1000 mg/kg/day, the locomotor activity at 20-30 minutes and in males treated with 1000 mg/kg/day the total locomotor activity (-33%) was decreased with statistical significance. As the difference in total locomotor activity showed a dose-relationship but similar differences were not present in females, this finding was considered to be only possibly test item-related and in the absence of correlating findings, e.g. in clinical signs, nonadverse.

FOOD CONSUMPTION
Absolute food consumption during treatment in males treated with 100 mg/kg/day was slightly lower and in males treated with 1000 mg/kg/day slightly higher than in the respective controls. In females, the absolute food consumption of test item-treated animals compared well to that of the control animals.
Relative food consumption in test item-treated males during treatment and recovery was comparable to that of the respective controls. In females treated with 100 or 300 mg/kg/day, the relative food consumption over treatment was very slightly lower than in controls.
During recovery, the absolute food consumption of males and females treated with 1000 mg/kg/day and the relative food consumption of females treated with 1000 mg/kg/day was slightly higher than that of the respective controls.
The observed differences were minor, without dose relationship and inconsistent across sexes. Thus, they were considered to be incidental.

BODY WEIGHT
No test item-related effects on body weights and body weight gain were noted.
The mean body weight of test item-treated females during treatment and recovery and of test item-treated males during treatment at all dose levels was comparable to that of the respective controls.

In males treated with 1000 mg/kg/day, the mean body weight during recovery was slightly higher than in controls.

The mean body weight gain of test item-treated and control males at the end of treatment and during recovery was similar. In test item-treated females at all dose levels, the mean body weight gain during treatment was slightly lower than in the respective controls except day 22 in females treated with 1000 mg/kg/day. The difference attained statistical significance on day 8 in females treated with 100 mg/kg/day and at the end of treatment in those treated with 100 or 300 mg/kg/day.

Although they attained statistical significance, the differences noted were small and did not show a dose relationship, nor were they consistent across sexes. Thus, they were considered not to be test item-related.

CLINICAL LABORATORY INVESTIGATIONS
Hematology
No test item-related changes in hematology parameters were noted.
No statistically significant changes in hematology parameters were noted in females. After 4 weeks of treatment, in males treated with 100 mg/kg/day a statistically significant but slight decrease was noted in red blood cell count (-5%), haemoglobin concentration (-7%), and hematocrit (-7%). Hemoglobin concentration distribution width in those males was increased with statistical significance (+10%). A slight increase in white blood cell count in males treated with 100 or 1000 mg/kg/day (+26% and +16%, respectively) did not attain statistical significance. In males treated with 1000 mg/kg/day, the number of monocytes (+42% relatively and +58% absolute) was increased with statistical significance. After the recovery period, males treated with 1000 mg/kg/day had a slightly decreased white blood cell count (-21%), increased neutrophils (+62% relatively), decreased lymphocytes (-11% relative and -30% absolute) and decreased basophils (-50% absolute). These changes attained statistical significance. These changes did not show a dose-relationship and the results remained within the normal range of historical data for rats of this strain and age. Changes noted after the recovery period were different from those noted after treatment. Thus, the observed changes are considered not be test item-related.

Clinical Biochemistry
No test item-related changes in clinical chemistry parameters were noted.
After 4 weeks of treatment, urea was decreased with statistical significance in females treated with 1000 mg/kg/day (-19%). This change is considered to be biologically not relevant. Cholesterol was decreased with statistical significance in females treated with 1000 mg/kg/day (-24%) but increased in females treated with 100 and 300 mg/kg/day without reaching statistical significance. An increase in phosphorus was noted in males treated with 1000 mg/kg/day and females treated with 100 mg/kg/day (both +12%, statistically significant in males only). Protein and albumin levels were decreased in females treated with 100 mg/kg/day (-5% and -7%, statistically significant) but increased in females treated with 300 mg/kg/day (+6% and +7%,
statistically significant).
Except for total protein in females treated with 300 mg/kg/day, these changes remained within the normal range of historical data for rats of this strain and age. Since they were inconsistent across dose levels and sexes, the changes noted are considered not to be test item-related. No statistically significant changes were present after the recovery period.

Urinalysis
No test item related changes in urinalysis parameters were noted. A statistically significant increase in erythrocytes in males treated with 300 or 1000 mg/kg/day remained within the normal range of historical data for rats of this strain and age and is in the absence of any correlating findings considered not to be test item-related.

PATHOLOGY
ORGAN WEIGHTS
No test item-related changes in organ weights were noted.
Slightly increased absolute and relative liver weights were recorded in males and females treated with 300 mg/kg/day. Increased absolute and relative thymus weights and decreased absolute and relative adrenal weights were noted in males treated with 1000 mg/kg/day. Males treated with 300 mg/kg/day had increased absolute and relative kidney weights. However, none of these differences showed a dose relationship, and statistical significance was not attained.
After the recovery period, an increase in absolute and relative liver weights in males treated with 1000 mg/kg/day attained statistical significance. Absolute and relative thymus weights were decreased in males treated with 1000 mg/kg/day with the difference attaining statistical significance in thymus to body weight ratio. Absolute and relative kidney and spleen weights were increased in these males and adrenal weights were decreased. Statistical significance was attained for absolute kidney weight, adrenal to body weight ratio, and kidney and spleen to brain weight ratio. In females treated with 1000 mg/kg/day, slightly increased absolute and relative thymus and spleen weights were noted with the differences not attaining statistical significance.
These few statistically significant or non-significant deviations in average organ weights at the end of the recovery period were considered to be incidental, reflecting the usual individual variability.

MACROSCOPIC / MICROSCOPIC FINDINGS
No test item-related macroscopic lesions were noted.
After 28 days of treatment, foci were found in the thymus of one control male, one male and one female treated with 300 mg/kg/day, and one female treated with 100 mg/kg/day. One control female and one female treated with 300 mg/kg/day had foci in the stomach. A watery cyst was noted in the left kidney of one male treated with 300 mg/kg/day. One male treated with 100 mg/kg/day had sores and eschars in the skin.
These isolated findings were considered to be within the range of normal background lesions, which may be seen in species of this strain and age in this type of study and were considered incidental, reflecting the usual individual variability.
After recovery, the only findings were an enlarged liver in two males treated with 1000 mg/kg/day and dilation of the uterine horns in one female treated with 1000 mg/kg/day. Since similar findings were not present at treatment end and in the absence of corresponding microscopic findings, these findings are considered to be incidental.

There were no findings, which distinguished test item-treated animals from controls.
A variety of other changes was found in this study. These commonly occur in laboratory rats of this strain and age, neither their incidences nor their distribution or morphologic characteristics gave any indication of a test item-related effect.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects up to the highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

No adverse effects have been observed in rats treated orally with the test material up to the limit dose of 1000 mg/kg bw/day for 4 weeks. Based on the results of this study, 1000 mg/kg bw/day were established as the no-observed-adverse-effect-level (NOAEL)

Executive summary:

Purpose

The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats by gavage for a period of 28 days. The reversibility of possible treatment-related changes was assessed after a treatment-free 14-day recovery period. This study should provide a rational basis for a risk assessment in man. The results of this study should indicate potential target organs and should identify chemicals with neurotoxic potential.

Study Design

In this subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, food consumption and body weights were recorded periodically during acclimatization, treatment and the recovery period. Functional observational battery, locomotor activity and grip strength were investigated during week 4.

At the end of dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

Results

Dose Formulation Analysis

The results indicate the accurate use of the test item during this study. The identity of the test item was confirmed by its retention time which was similar to that measured in the working standards. Application formulations were found to be homogenously prepared and sufficient formulation stability under storage conditions was approved. No test item was detected in the control samples.

Mortality / Viability

All animals survived to scheduled necropsy.

Clinical Signs

No test item related clinical signs were noted.

Functional Observational Battery

No clinical signs were noted during detailed observation in week 4.

Grip Strength

No differences were noted in the mean grip strength between test item-treated animals and the respective controls.

Locomotor Activity

Slightly decreased total locomotor activity in males treated with 1000 mg/kg/day (-33%) was considered to be non-adverse as similar differences were not observed in females and no correlating findings (e.g., clinical signs) at this dose level were noted.

Food Consumption

No test item-related differences in food consumption were noted.

Body Weights

No test item-related effects on body weights and body weight gain were noted.

Clinical Laboratory Investigations

Hematology

No test item-related changes in hematology parameters were noted.

Clinical Biochemistry

No test item-related changes in clinical chemistry parameters were noted.

Urinalysis

No test item related changes in urinalysis parameters were noted.

Organ Weights

No test item-related changes in organ weights were noted.

Macroscopic / Microscopic Findings

No test item-related macroscopic lesions were noted.

There were no microscopic findings, which distinguished test item-treated animals from controls.

Conclusions

Oral administration of the test item to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for 28 days resulted in no unscheduled deaths. No test item-related effects on functional observational battery, grip strength, food consumption, body weights, organ weights, or clinical laboratory parameters were noted.

No macroscopic or microscopic findings distinguished test item-treated animals from control animals.

Slightly decreased total locomotor activity in males treated with 1000 mg/kg/day (-33%) was considered to be non-adverse.

Based on the results of this study, 1000 mg/kg body weight/day of the test substance were established as the no-observed-adverse-effect-level (NOAEL).