Oligomerisation products of ethylene oxide with reaction products of rape oil and ethanolamine

Endpoint summary

Description of key information

The administration of the substance by oral gavage for a period of ninety consecutive days resulted no treatment related effects up to highest dose level of 200 mg/kg/day. Therefore, the No Observed Effect Level and the No Observed Adverse Effect Level (NOEL and NOAEL) was considered to be 200 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-Feb-2014 to ...

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted according to GLP in compliance with internationally accepted test guidelines. The substance is considered to be a Multiconstituent substance and the exact purity was unknown. This information was therefore excluded from the Statement of Compliance

Guideline:

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

Deviations:

no

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system
- Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 15 males and 15 females each (Groups 1 and 4) and 10 males and 10 females each (Groups 2 and 3)
- Age (at Delivery): Ca. 7 weeks
- Body Weight Range (at Acclimatization): males 210 to 245 g (mean 229 g) and females 124 to 153 g (mean 139 g)
- Identification: cage card and tail mark (later ear tattoo) during acclimatization and cage card and individual ear tattoo during treatment
- Randomization: Randomly allocated to groups by body weight
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). During the recovery period, the relative humidity occasionally exceeded this range, and was considered not to have any influence on the study. Therefore, these data are not reported but are retained at Harlan Laboratories Ltd. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of two, three or four in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batches 53/13 and 10/14) rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

DOSE FORMULATIONS

The test item was prepared as 100%. The purity was not corrected.

As the test item may have had an affinity for glass, the beakers for each dose level were pre-saturated with the test item before the first application and rinsed with vehicle only.

The dose formulations were prepared daily. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories study D82390), the stability of the test item formulations was considered to require daily preparation.

Product LE 097 was weighed into a glass beaker on a tared Mettler balance. The mixtures were stirred using a magnetic stirrer and used at room temperature (20 ± 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily.
- Dose Levels: 0 mg/kg/day (Group 1), 12.5 mg/kg/day (Group 2), 50 mg/kg/day (Group 3) and 200 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, Harlan Laboratories study D82390.
- Dose Volume: 5 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (Group 1), 2.5 mg/mL/day (Group 2), 10 mg/mL/day (Group 3) and 40 mg/mL/day (Group 4)
- Duration of Acclimatization Phase: 7 days
- Duration of Treatment Phase: 91/92 days
- Duration of Recovery Phase: 28 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor.

After experimental start, at week 2, week 3, week 7 and week 13 duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour).

The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Zelgliweg 1, 4452 Itingen / Switzerland) at room temperature and directly analyzed.

The test item was used as analytical standard.

Dose formulation samples (primary samples or duplicates) were discarded upon written confirmation by the study director after acceptance of the draft report.

RESULTS

The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peaks areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by power regression analysis. The coefficients of determination (R2) were higher than 0.99.

The Product LE 097 peaks were assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no peaks appeared at the retention time of Product LE 097 and, therefore, the absence of the test item in the vehicle control samples (PEG 300) was confirmed.

The Product LE 097 concentrations in the dose formulations ranged from 83.3% to 116.1% with reference to the nominal and were within the accepted range of ±20%, except for sample of dose 2 prepared on 25-Feb-2014.

The homogeneous distribution of Product LE 097 in the preparations was approved because single results found did not deviate more than 4.4% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In addition, the test item was found to be stable in application formulations when kept four hours at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

In conclusion, the results indicate the accurate preparation and storage of the test item Product LE 097 in vehicle during this study.

Duration of treatment / exposure:

91/92 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 12.5, 50 and 200 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1: 15 males and 15 females
Group 2: 10 males and 10 females
Group 3: 10 males and 10 females
Group 4: 15 males and 15 females

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this oral toxicity study was to assess the cumulative toxicity of Product LE 097 when administered daily to rats by gavage for a period of at least 90 days. The reversibility of test item-related changes was assessed after a treatment-free 28-day recovery period.

This study should provide a rational basis for toxicological risk assessment in man.


In this subchronic toxicity study, Product LE 097 was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 12.5, 50 and 200 mg/kg body weight/day for a period of 91/92 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 10 animals per sex which were sacrificed after 91/92 days of treatment. Additional 5 rats per sex and group were used at 0 and 200 mg/kg. These animals were treated for 91/92 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, ophthalmoscopy, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13.
At the end of the 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.

Positive control:

Not required

Observations and examinations performed and frequency:

VIABILITY / MORTALITY

Observations for viability / mortality were recorded twice daily.

DAILY OBSERVATIONS

The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 – 92/93 (twice daily during days 1 - 3) during the treatment period and once daily during days 1 to 28 of the recovery period.

WEEKLY BEHAVIORAL OBSERVATIONS

The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter.

More details on the investigations are detailed in the section “Any other information on materials and methods incl. results” below.

FUNCTIONAL OBSERVATIONAL BATTERY (SCREEN)

During week 13, relevant parameters from a modified Irwin screen test were evaluated in all animals. More details on the investigations are detailed in the section “Any other information on materials and methods incl. results” below.


- Grip Strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

- Locomotor Activity: Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the thirteenth treatment week for a 30-minute period and the total activity of this time period was recorded.

Low beams count was reported in 6-minute intervals as well as the total activity of the measuring period.

FOOD CONSUMPTION

The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

BODY WEIGHTS

Body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

OPTHALMOSCOPY

Ophthalmoscopic examinations were recorded once during acclimatization, and once during week 13 in animals of the control and high dose groups.

CLINICAL LABORATORY INVESTIGATIONS

Blood and Urine Sampling after Treatment: 27/28-May-2014 (allocation A and B)
Blood and Urine Sampling after Recovery: 24-Jun-2014 (allocation B)

Blood samples were drawn by sublingual venipuncture 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.

Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.

In the summary and individual tables the names of some parameters have been abbreviated.

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

The following hematology parameters were determined:

1. 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

2. Coagulation

- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Phospholipids
- Aspartate aminotransferase
- Alanine aminotransferase
- Lactate dehydrogenase
- Alkaline phosphatase
- Bile acids
- Gamma-glutamyl-transferase
- Creatine kinase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

The following urine parameters were determined:

1. Physical Examination

- Urine volume (18 hour)
- Specific gravity (relative density)
- Color
- Appearance

2. Chemical Examination

- pH value
- Nitrite
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Erythrocytes
- Leukocytes

Sacrifice and pathology:

NECROPSY

Sacrifice after Treatment: 27/28-May-2014 (allocation A)
Sacrifice after Recovery: 24-Jun-2014 (allocation B)

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 unless indicated otherwise.

- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (sternum)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in modified Davidson's solution)
- Esophagus
- Eyes w/optic nerve (fixed in Davidson's solution)
- Harderian gland (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum
- Kidneys
- Larynx
- Lacrimal gland, exorbital
- Liver
- Lungs, filled w/formalin at necropsy
- Lymph nodes – mesenteric and mandibular
- Mammary gland area
- Nasal cavity
- Ovaries
- Pancreas
- Pharynx
- Pituitary gland
- Prostate gland and seminal vesicles incl. coagulating glands
- Rectum
- Salivary glands - mandibular, sublingual
- Sciatic nerve
- Skeletal muscle
- Skin
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in modified Davidson's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Tongue
- Trachea
- Ureters
- Urinary bladder, filled w/formalin at necropsy
- Uterus (incl. oviducts, cervix and vagina)
- All gross lesions

ORGAN WEIGHTS

The organs from allocation A and B animals listed below were weighed before fixation and recorded on the scheduled dates of necropsy.

- Adrenal glands
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Epididymides (fixed in modified Davidson's solution)
- Heart including auricles
- Kidneys
- Liver
- Ovaries
- Spleen
- Testes (fixed in modified Davidson's solution)
- Thymus
- Uterus (incl. oviducts, cervix and vagina)

Relative organ weights were calculated on the basis of the body weight and brain weight.

The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.

HISTOTECHNIQUE

All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. Organ and tissue samples taken from animals which died spontaneously or which were killed in extremis were evaluated similarly to those organs taken from animals of the high-dose group.

A description of all abnormalities was included. Attempts were made to correlate gross observations with microscopic findings.

A peer review was performed by W. Henderson. The findings of the study pathologist and the peer-reviewing pathologist compared favorably. The peer review report is retained in the raw data.

Statistics:

The following statistical methods were used to analyze body weight, ophthalmoscopy, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

- The Dunnett-test (many to one t-test) based on a pooled variance estimate was 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 could not be assumed to follow a normal distribution.
- Fisher's exact-test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

1. IN-LIFE DATA

VIABILITY/MORTALITY

All animals survived the scheduled treatment or recovery periods.

DAILY OBSERVATIONS

No test item-related clinical signs were noted in any animal during daily observations.

A small number of typical background findings (kinked tail apex, breathing noises, reddish nasal discharge, scarring) were noted but were transient and/or infrequent, and were therefore considered to be of no toxicological relevance.

WEEKLY BEHAVIORAL OBSERVATIONS

No test item-related clinical signs were noted in any animal during weekly detailed/behavioral observations (treatment weeks 1 - 12 and recovery).

A small number of typical background findings similar to those seen during daily observations and were also transient and/or infrequent, and were therefore considered to be of no toxicological relevance.

FUNCTIONAL OBSERVATIONAL BATTERY (SCREEN)

During treatment week 13, there were no test item-related changes of toxicological relevance nor were there any changes of possible neurological relevance.

Typical background findings were noted with low incidence and were therefore considered to be of no toxicological relevance.

- Grip Strength: During the functional observational battery performed during week 13, the test item-treated males had similar fore- and hindlimb grip strength values as the controls. In females, however, the mean hindlimb grip strength was significantly lower (p>0.01) when compared to the control females. The forelimb grip strength values of all treated females compared favorably with the controls. Therefore the difference seen in the hindlimb grip strength was considered to be an incidental finding.

- Locomotor Activity: During the functional observational battery performed during week 13, there were no test item-related differences to the control values.

There were transient differences (reduced during 24 - 30 minutes in males treated with 200 mg/kg/day, p<0.05 and increased during 0 - 6 minutes, 6 - 12 minutes, and 12 - 18 minutes in females treated with 12.5 mg/kg/day) but these occurred in a single measurement interval in males and were dose unrelated in females, and therefore considered to be not related to the treatment with the test item.

FOOD CONSUMPTION

Test item-treated males and females showed no differences in the mean daily food consumption when compared with the respective control groups.

BODY WEIGHTS

During the treatment and recovery periods, the mean body weights of the males and females of all dose levels compared favorably with those of the respective controls.

OPHTHALMOSCOPIC EXAMINATION

There were no ophthalmoscopic findings at any dose level.

A small number of typical background and/or juvenile findings (uni- or bilateral persistent pupillary membrane or hyaloid vessel, corneal opacities) were recorded but were considered to be without toxicological relevance.


2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

There were no test item-related changes in the hematology parameters of male or female rats after treatment period and no late effects after the recovery period.

All differences of statistical significance were either isolated changes without concomitant changes in related parameters, which remained within the ranges of the historical control data, or which were related to incidentally increased control group values, and therefore considered to be without toxicological significance.

CLINICAL BIOCHEMISTRY

There were no test item-related changes in the clinical biochemistry parameters of male or female rats after treatment period and no late effects after the recovery period.

After 13 weeks of treatment, males and females treated with the test item at 50 or 200 mg/kg/day had significantly elevated sodium levels (p<0.01) when compared with the controls that remained within the ranges of the historical control values. All other values compared favorably with the respective control values.

Significantly decreased bile acid levels were noted in females treated with 200 mg/kg/day; reductions in bile acids are generally recognized as toxicologically irrelevant. The mean globulin level of these females was significantly elevated (p<0.05) when compared to the controls but remained within the range of typical variation

At 50 mg/kg/day, the mean potassium level was significantly reduced (p<0.05) in males. Females were unaffected. The mean globulin level of these females was significantly elevated (p<0.01) but was unrelated to dose and remained within the range of the historical control data.

At 12.5 mg/kg/day, the mean glucose level was significantly increased (p<0.05) in males only and the creatine kinase levels were significantly lower (p<0.05). In the absence of any dose-response relationship, these differences were considered to be toxicologically irrelevant.

After the recovery period, the mean activity of alanine aminotransferase was significantly decreased in the males previously treated with 200 mg/kg/day; reductions of activity for this (and other) liver parameters is generally recognized as toxicologically irrelevant. The mean globulin level of the females was significantly higher (p<0.05) than the controls but remained within the range of the historical control values.

URINALYSIS

There were no test item-related changes in the urinalysis parameters of male or female rats after treatment period and no late effects after the recovery period.

After 17 weeks, differences in the mean urine volume and mean relative density of males treated previously with 200 mg/kg/day suggested that individual outlying samples were diluted with water from the drinking bottles. The differences in the mean values remained within the ranges of the respective historical control data and were therefore considered to be without toxicological significance. Females were unaffected.

3. PATHOLOGY

ORGAN WEIGHTS

After 13 weeks treatment, absolute and relative spleen weights of males treated with 200 mg/kg/day were significantly reduced (p<0.05). This finding was not detected in the females at this dose level, but significantly reduced mean absolute kidney weights (p<0.05) were recorded. Since no microscopic findings were recorded for any of those animals, these findings were considered to be incidental.

Females treated at 12.5, 50 and 200 mg/kg/day had significantly reduced absolute and relative heart weights when compared with the controls. Since no microscopic findings were recorded for any of those females, these findings were considered to be incidental.

After the 4-week recovery period, there were no differences of toxicological relevance between control rats and rats previously treated with the test item at 200 mg/kg/day.

MACROSCOPIC FINDINGS

There were no test item-related macroscopic differences when compared with the controls.

Reddish discoloration of lymph nodes (either mandibular or renal) was seen in two males treated with 50 mg/kg/day and in two males treated with 200 mg/kg/day. These were considered to be typical post-mortem agonal changes of no toxicological relevance.

All females were without macroscopic findings.

MICROSCOPIC FINDINGS

There were no treatment-related microscopic findings.

Specifically, there were no changes of toxicological relevance in any reproductive organ or lymphatic tissue of either sex, and the distribution of estrus cycles of all females was similar across all groups.

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant findings noted during the course of this study. Therefore, the NOEL is set at the highest dose tested.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant findings noted during the course of this study. Therefore, the NOEL is set at the highest dose tested.

Critical effects observed:

not specified

Conclusions:

Oral administration of Product LE 097 to Wistar rats at doses of 12.5, 50 and 200 mg/kg/day, for 91/92 days resulted in no effects on mortality, food consumption, body weight development, clinical signs at daily or weekly observations (during weeks 1 - 12) or at functional observational battery (at week 13). The mean fore- and hindlimb grip strength values were not affected and no relevant differences in the mean locomotor activity were noted. No ophthalmoscopic changes of toxicological relevance were seen. Hematology, clinical biochemistry and urinalysis parameters showed only minor differences without toxicological relevance.

At necropsy, there were no macroscopic changes of note. Specifically, there were no changes of toxicological relevance in any reproductive organ or any lymphatic tissues of either sex. Follicular cell hypertrophy, noted in the high-dose males (treated with 1000 mg/kg/day) of a previous 28-day oral toxicity gavage study, was not evident in any animal in this 90-day study.

A statistically significant decrease in spleen weight in males at 200 mg/kg/day and a statistically significant decrease in heart weight in females at 12.5 mg/kg/day were without correlating microscopic findings, and were therefore considered to be incidental findings.

Based on the results of this study, 200 mg/kg body weight/day of Product LE 097 was established as the no-observed-effect-level (NOEL) and as the no-observed-adverse-effect-level (NOAEL).

Executive summary:

GENERAL

 

In this subchronic toxicity study, Product LE 097 was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 12.5, 50 and 200 mg/kg body weight/day for a period of 91/92 days. A control group was treated similarly with the vehicle, PEG300, only.

 

The groups comprised 10 animals per sex which were sacrificed after 91/92 days of treatment. Additional 5 rats per sex and group were used at 0 and 200 mg/kg. These animals were treated for 91/92 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed.

 

Clinical signs, outside cage observation, ophthalmoscopy, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13.

 

At the end of the 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.

 

MORTALITY / VIABILITY

 

All animals survived the scheduled treatment or recovery periods.

 

CLINICAL SIGNS (DAILY AND WEEKLY)

 

No test item-related clinical signs were noted in any animal during daily observations. No test item-related clinical signs were noted in any animal during weekly detailed observations (treatment weeks 1 - 12 and recovery).

 

FUNCTIONAL OBSERVATIONAL BATTERY

 

During treatment week 13, there were no test item-related changes of toxicological or neurological relevance.

 

- Grip Strength: During the functional observational battery performed during week 13, there were no test item-related differences to the control values.

- Locomotor Activity: During the functional observational battery performed during week 13, there were no test item-related differences to the control values.

 

FOOD CONSUMPTION

 

Test item-related males and females showed no differences in the mean daily food consumption when compared with the respective control groups.

 

BODY WEIGHTS

 

During the treatment and recovery periods, the mean body weights of the males and females of all dose levels compared favorably with those of the respective controls.

 

OPHTHALMOSCOPIC EXAMINATIONS

 

There were no ophthalmoscopic findings at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS

 

- Hematology: There were no test item-related changes in the hematology parameters of male or female rats after treatment period and no late effects after the recovery period.

- Clinical Biochemistry: There were no test item-related changes in the clinical biochemistry parameters of male or female rats after treatment period and no late effects after the recovery period.

- Urinalysis: There were no test item-related changes in the urinalysis parameters of male or female rats after treatment period and no late effects after the recovery period.

 

ORGAN WEIGHTS

 

No test item-related differences in absolute and relative organ weights were noted between the groups.

 

MACROSCOPIC / MICROSCOPIC FINDINGS

 

There were no test item-related macroscopic or microscopic differences in any organ when compared with the controls.

 

CONCLUSION

 

Based on the results of this study, 200 mg/kg body weight/day of Product LE 097 was established as the no-observed-effect-level (NOEL) and as the no-observed-adverse-effect-level (NOAEL).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The repeated dose (oral) toxicity endpoint was covered by subacute and subchronic GLP studies conducted according to internationally accepted test guidelines. The both studies are reliable without restrictions, therefore for the endpoint assessment, the NOAEL of the subchronic study was considered.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- Repeated dose toxicity, oral (gavage)

The sub-acute toxicity (28-day) of LE097, given daily by oral administration via gavage, was investigated in Sprague-Dawley rats according to OECD TG 407. Animals were treated with dose levels of 15, 150 or 1000 mg LE097/kg bw/day. The treatment-related effects were observed at dose of 1000 mg/kg bw/day in the both sexes of animals. On the basis of treatment-related minimal irritation changes in the forestomach of two males at 150 mg/kg bw/day level, the NOAEL for male was established at 15 mg/kg bw/day, however, the NOAEL for female was considered to be 150 mg/kg bw/day.

The sub-chronic toxicity (90-day) of LE097, given daily by oral administration via gavage, was investigated in Wistar rats according to OECD TG 408. Animals were treated with dose levels of 12.5, 50 and 200 mg/kg bw/day. No adverse effects were observed in this study and NOAEL was above 200 mg/kg bw/day.

As subchronic study overrules the findings of sub-acute study, in which the treatment-related effects observed only in two males may be regarded as non-adverse, the NOAEL for the repeated dose toxicity was taken for the further LE097 assessment from the subchronic study.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable subchronic GLP study according to internationally accepted test guidelines

Justification for classification or non-classification

-Repeated dose toxicity, oral:

Based on the above stated assessment of the specific target organ toxicity potential after repeated oral exposure of LE097, the compound does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.