Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 August 2005 and 23 December 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050 (Repeated Dose 28-day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): EA-3098
- Substance type: White powder
- Physical state: solid
- Purity test date: Not indicated: treated as 100% pure
- Lot/batch No.: P-32681
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: Males: 191 - 194 mg (mean); Females: 155 - 161 mg (mean)
- Housing: Group housing of 5 animals per sex in Macrolon plastic cages (MIV type, height 18 cm; durig overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilised sawdust as bedding material. No cage-enrichment was provided during overnight activity monitoring.
- Diet (e.g. ad libitum): ad libitum, pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany).
- Water (e.g. ad libitum): ad libitum, tap water.
- Acclimation period: At least 5 days prior to study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 +/- 3.0 deg C (actual range: 21.5 - 22.8 deg C).
- Humidity (%): 30 - 70 % (actual range 38 -84 %). Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
- Air changes (per hr): Approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial flourescent light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.



VEHICLE
- Vehicle: Ethylene glycol.
- Justification for use and choice of vehicle (if other than water): Based on trial formulations at NOTOX.
- Concentration in vehicle: Specific gravity 1.036
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared for use on day 13 were analysed to check for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours was also fetermined (highest and lowest concentration). The analytical method used was based on the results of a separate project for the development and validation of the analytical method.

Test substance formulations in propylene glycol were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 84-122% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type, taking into account the variation in the analytical method.

See Appendix 4: Analytical report, in attached background material for full details.
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days per week.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (vehicle control)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
5 males and 5 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study.

TREATMENT:
Method: Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.

Frequency: Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Dose volume: 5 ml/kg bodyweight. Actual dose volumes were calculated weekly according to the latest body weight.
Positive control:
Not applicable.

Examinations

Observations and examinations performed and frequency:
Mortality/Viability: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was performedoutside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.

BODY WEIGHT: Yes
On days 1, 8, 15, 22 and 28

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was checked weekly.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Water consumption: Subjective appraisel was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

FUNCTIONAL OBSERVATIONS:
During week 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, (Pearson Technical Services, Debenham, Stowmarket, England).

Clinical Laboratory Investigations:
HAEMATOLOGY:
Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post mortem examination, between 7.00 and 10.30 am. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected in tubes prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (1.0 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). The following parameters were determined:

Haematology: white blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets.

Clotting Potential: Prothrombin time, activated partial thromboplastin time.

Clinical Biochemistry: Alanine aminotransferase, Aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.
Sacrifice and pathology:
Necropsy:
All animals surviving to the end of the observation period were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and adedscriptions of all macroscopic abnormaltieis recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4 % formaldehyde solution.

Identification marks: not processed, Adrenal glands, aorta, brain (cerebellum, mid-brain, cortex), caecum, cervix, (clitoral gland), colon, duodenum, epididymides, (eyes with optic nerve [if detectable] and harderian gland), (female mammary gland area), (femur including joint), heart, ileum, jejunum, kidneys, (larynx), (lacrimal gland, exorbital), liver, lung, infused with formalin, lymph nodes - mandibular, mesenteric, (Nasopharynx), oesophagus, ovaries, pancreas, peyers patches [jejunum, ileum] if detectable, pituitary gland, (preputial gland), prostate gland, rectum, (salivary glands - mandibular, sublingual), sciatic nerve, (seminal vesicles), skeletal muscle), (skin), spinal cord - cervical, midthoracic, lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid [if detectable], (tongue), trachea, urinary bladder, uterus, vagina, all gross lesions.

Tissues mentioned within brackets were not examined microscopically as there were no signs of toxicity or target organ involvement.

Organ Weights:
The following organ weights (and terminal body weight) were recorded from all animals on the scheduled day of necropsy:

Adrenal glands, brain, epididymes, heart, kidneys, liver, spleen, testes, thymus.

Histopathology:
The following slides were examined by a pathologist:

-all tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group.
- all gross lesions
Other examinations:
None.
Statistics:
Interpretation
The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one-t-test) based on a pooled varience estimate was applied for the comparison of the treated groups for each sex.
-The Stee-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
-The exact Fisher-test was applied to frequency data.
all tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistical analysis was performed on histopathology findings. Group means were calculated for continuous data and medians were calculated for descrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variences. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Mortality:
no mortality observed
Description (incidence):
Please see details on results below for further information.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Please see details on results below for further information.
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
No mortality occurred during the study period.

CLINICAL SIGNS:

There were no clinical signs of toxicity noted during the observation period.

Incidentally alopecia, scabs and a wound were seen on different parts of the body. These findings are commonly noted in rats of this age and strain and housed and treated under the conditions in this study. Therefore, they were considered of no toxicological significance.

Difficulties in breathing (males) were noted in 1/5 males of the high dose group. As this clinical sign was limited to one observation it was considered of no toxicological significance.

FUNCTIONAL OBSERVATIONS:
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

BODY WEIGHTS:
Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

FOOD CONSUMPTION:
Food consumption before or after allowance for body weight remained in the same range as controls over the 4-week study period.

Clinical Laboratory Investigations:

HAEMATOLOGY:
No toxicologically relevant changes occurred in haemotological parameters of treated rats.
Minor statistically significant differences in red blood cell counts arising in males between controls and animals receiving 150 mg/kg/day were within normal range for this type of study and were considered to have arisen by chance and not to represent a change of toxicological significance.

CLINICAL CHEMISTRY
In males dosed at 150 mg/kg/day decreased albumin, total protein and calcium levels were observed. There are no macroscopic or microscopic observations that correlate with decreased protein synthesis or adverse protein loss from the animals. In addition, in the absence of a dose relationship these findings were considered to have accurred by chance and are toxicologically irrelevant.
The decreased calcium levels correlates well with the reduced albumin levels, which is a calcium-binding protein. The observation for calcium is therefore considered to be false positive and is toxicologically irrelevant.
Decreased bilirubin (females, 50 mg/kg/day) and increased chloride (males, 150 mg/kg/day) values compared to controls were considered to have arisen by chance in the absence of a treatment-related distribution or corroborative findings in macroscopy or microscopy or, with respoect to chloride, concurrent increased sodium levels. These findings are therefore considered toxicologically irrelevant.

PATHOLOGY:
MACROSCOPIC EXAMINATION:
Necropsy did not reveal any toxcologically relevant alterations.

Incidental findings among control and treated animals included mandibular lymph node enlargement, pelvic dilation, foci in the thymus and alopecia. These findings are occasionally seen among rats used in these types of studies. In the absence of correlated microscopic findings and a treatment-related distribution they were considered changes of no toxicological significance.

Watery fluid in the uterus, found in several control and treated females, is related to a stage in the oestrous cycle and is a normal finding.

ORGAN WEIGHTS:
Both decreased relative testes weight in males at 150 mg/kg/day, and relative kidney weight in females at 1000 mg/kg/day were statistically significantly different from their respective controls. These changes were of a slight nature and well within the historical control range for rats of this age and strain. Furthermore, a clear dose-response relationship was absent and/or no corroborative findings were noted at the microscopic examinations. Therefore, no toxicological significance was ascribed to these changes.

MICROSCOPIC EXAMINATION:
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and stain and occurred at similar incidences and severity in both control and treated rats.


ANALYSIS OF DOSE PREPARATIONS:
Test substance formulations in propylene glycol were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 84-122% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type, taking into account the variation in the analytical method.


















Effect levels

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 toxicologically relevant effects were found at any dose level.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

DISCUSSION:

Wistar rats were treated with EA-3098 for 28 consecutive days by oral gavage at dose levels up to 1000 mg/kg/day.

There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination that were considered to be an effect of treatment.

From the results obtained a definitive No Observed Adverse Effect Level (NOAEL) for EA-3098 of 1000 mg/kg/day was established.

Applicant's summary and conclusion

Conclusions:
From the results obtained a definitive No Observed Effect Level (NOAEL) for EA-3098 of 1000 mg/kg/day was established.
Executive summary:

Method

Repeated dose 28 -day oral toxicity study with EA-3098 by daily gavage in the rat.

The study was based on the following guidelines:

- EC Directive 96/54/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996

- OECD 407, Repeated Dose 28 - day Oral Toxicity Study in Rodents, 1995

- OPPTS 870.3050, Repeated dose 28 - day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712 -C-00 -366, 2000.

Based on the results of a 5 -day range finding study, the dose levels for the 28 - day toxicity study were selected to be 0, 50, 150, and 1000 mg/kg/day.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated: clinical signs (daily); functional observation tests; body weight and food consumption (weekly); clinical pathology and macrosopy at termination; organ weights and histopathology on a selection of tissues.

Results

Accuracy, homogeneity and stability over 5 hours at room temperature of formulations of test substance in propylene glycol were demonstrated by analyses.

No treatment related findings were noted.

Conclusion

From the results presented in the report a definitive No Observed Adverse Effect Level (NOAEL) for EA-3098 of 1000 mg/kg/day was established.