Registration Dossier

Administrative data

Description of key information

There is a reliable 28-day oral subacute study available on the substance itself and a reliable oral combined one generation reproduction/subchronic toxicity study available on a close structural analogue. Furthermore, a 90-day subchronic toxicity study (OECD 408) is available on a second close structural analogue to provide additional read-across data to further support the repeated dose toxicity endpoint.

Repeated dose 28 -day oral toxicity study (on substance EC 434-430-9):

Male and female Wistar rats were treated with EA-3098 for 28 consecutive days by oral gavage at dose levels up to 1000 mg/kg bw/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 NOAEL for EA-3098 of 1000 mg/kg bw/day was established.

Combined one generation/sub-chronic toxicity study (on structural analogue EA 2854, EC 432 -430 -3)

Male and female Wistar rats were treated with EA 2854 for 108 consecutive days by oral gavage at dose levels up to 1000 mg/kg bw/day. There were no toxicologically significant effects observed on mortality, clinical observations, behavioural assessments, functional performance, sensory reactivity, bodyweight, food consumption, water consumption, ophthalmoscopy, urinalysis, haematology, blood chemistry, organ weights, macroscopic or microscopic pathology at dose levels up to 1000 mg/kg bw/day. From the results obtained a definitive NOAEL of 1000 mg/kg bw/day was established for systemic toxicity.

Repeated dose 90-day oral toxicity study (on structural analogue EW-84, EC 204-613-6)

In this study, administration of the test substance, for 90 consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 1000 mg/kg bw/day did not result in any adverse treatment-related effects. There were no toxicologically significant effects detected in the haematological or blood chemistry parameters examined. No treatment-related findings were reported at post mortem macroscopic observations and histopathological examination. No toxicologically significant effects were detected in the organ weights measured. The NOAEL was considered to be >1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 (Repeated Dose 28-day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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.
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.
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.
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.
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.


















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.
Critical effects observed:
no

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.

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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 December 2017 - 19 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted 21 September 1998
Deviations:
yes
Remarks:
All females on Day 50 received a higher dose amount than required due to technician error. Dose amounts were correct for remainder of study. There were no adverse effects in any female on this day and no treatment related findings overall on the study.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Physical State/Appearance: Off white powder
Purity: 100%
Batch Number: S/99/17
Storage Conditions: Stored in ambient temperature in darkness, used/formulated in light
Expiry Date: 30 March 2021
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
The animals were acclimatized for eight days during which time their health status was assessed. At the start of treatment the males weighed 197 to 232g, the females weighed 146 to 175g, and were approximately six weeks old. The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. The animals were allowed free access to food and water. A pelleted diet was used. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the substance formulations were taken on four occasions and analyzed for concentration. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Low
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Intermediate
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Clinical Observations: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Body Weight: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption: Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

Water Consumption: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Functional Observations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

Functional Performance Tests: Motor Acivity, Forelimb/Hindlimb Grip Strength, Sensory Reactivity, Ophthalmoscopic Examination.

In-Life Sampling and Analysis included:
Haematology:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)
Sacrifice and pathology:
Necropsy: On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights:
Adrenals
Ovaries
Brain
Spleen
Epididymides
Testes
Heart
Thymus
Kidneys
Uterus
Liver

Histopathology:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint) (Retained only and not processed)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Caecum
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides (preserved in Modified Davidson’s fluid)
Skin
Esophagus
Spinal cord (cervical, mid-thoracic
Eyes
and lumbar)
Gross lesions (where applicable)
Spleen
Heart
Stomach
Ileum (including Peyer’s patches)
Testes (preserved in Modified Davidson’s fluid)
Jejunum
Thymus
Kidneys
Thyroid/Parathyroid
Liver
Tongue (Retained only and not processed)
Lungs (with bronchi)
Trachea
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary glands
Uterus (with cervix)
Muscle (skeletal) (Retained only and not processed)
Vagina

Pathology: Microscopic examination was conducted by the Study Pathologist.

Statistics:
Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidences of increased salivation were evident in five males treated with 1000 mg/kg bw/day throughout the majority of the treatment period and in one male treated with 100 mg/kg bw/day on two occasions. An observation of this nature is commonly observed following the oral administration of an unpalatable substance formulation and is considered not to represent an adverse effect of treatment.

Noisy respiration was also evident in three males treated with 1000 mg/kg bw/day between Days 52 and 90, in one female treated with 1000 mg/kg bw/day on two occasions, in one male and two females treated with 100 mg/kg bw/day on one occasion and in one male and one female treated with 10 mg/kg bw/day on one occasion only. At these low frequencies and distributions, this is considered to indicate possible difficulties in dosing particular animals on isolated occasions and not indicative of systemic toxicity.

Incidental observations, showing no dose related response and considered to be unrelated to test item administration included one control male had noisy respiration on Day 43, one male treated at 100 mg/kg bw/day with an open wound on the left shoulder between Days 9 and 11, which then became a scab between Days 12 and 39 and a male treated with 10 mg/kg bw/day was observed to have misaligned posture, holding its head tilted to the left from Day 13 to termination.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female was found dead on Day 75. At necropsy this female had black colored contents in the cecum, colon, duodenum, ileum, jejunum and stomach and a pale liver with dark patches. There were no changes at histopathology to account for the death or the macroscopic changes, however, lymphoid necrosis/atrophy/depletion was present along with cortical hypertrophy in the adrenal glands which indicate poor clinical condition/stress.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males treated with 1000 and 100 mg/kg bw/day showed statistically significantly higher (p<0.05-0.01) body weight gains during Week 2, with males treated at 1000 mg/kg bw/day also showing statistically significantly higher body weight gains during Weeks 4 (p<0.01), 5 (p<0.05) and 7 (p<0.01). An increase in body weight gain is considered not to be an adverse effect of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Total leukocyte count was statistically significantly lower (p<0.05) in all treated males. With the exception of one individual male treated at 100 mg/kg bw/day, all individual values for treated males were within the historical control range. Mean corpuscular hemoglobin concentration and lymphocytes for males treated with 1000 mg/kg bw/day were statistically significantly lower (p<0.05) compared to controls. For mean corpuscular hemoglobin concentration, four of the individual control values exceeded the historical control range, whereas all of the individual values for males treated with 1000 mg/kg bw/day were within the historical control range. For lymphocytes, although all individual control values were within the historical control range, only one individual value for males at 1000 mg/kg bw/day was below this range. As the majority of values were within the historical control normal ranges, and in the absence of any histopathological correlates, these intergroup differences were considered to be of no toxicological significance.

Eosinophils were statistically significantly lower (p<0.05) than controls for males treated with 10 mg/kg bw/day. All individual values were within the historical control range and in the absence of any similar findings in males treated with 100 or 1000 mg/kg bw/day, the intergroup difference was considered to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males treated at 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in calcium concentration. All of the individual values were within the historical control range and in the absence of any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance. Bilirubin levels were also statistically significantly higher (p<0.05) than controls in males treated with 100 and 1000 mg/kg bw/day. However, two individual control values were below the historical control range and only one individual value for males at 1000 mg/kg bw/day was above the historical control range. All values for males treated with 100 mg/kg bw/day were within the historical control range. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Occasional incidences of noisy respiration were evident in one male treated with 1000 mg/kg bw/day during the final two weeks of treatment, but this is likely to be due to slight difficulties in dosing this particular animal on these occasions and is not indicative of true systemic toxicity.

The incidental clinical finding of mis-aligned posture was evident in one male treated with 10 mg/kg bw/day from Week 2 assessments onwards.

Motor activity assessment did not indicate any effect of treatment for either sex at 10, 100 or 1000 mg/kg bw/day. There was no effect of treatment at 10, 100 or 1000 mg/kg bw/day on forelimb or hindlimb grip strength.

There were no treatment-related changes in sensory reactivity. Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 10, 100 or 1000 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males treated with 100 and 10 mg/kg bw/day showed statistically significant increases (p<0.05) in absolute and body weight relative kidney weights in comparison with controls. The majority of individual values were within historical control ranges and in the absence of any similar findings in males treated with 1000 mg/kg bw/day or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Both absolute and body weight relative liver weights were statistically significantly higher (p<0.05) for males treated with 100 and 1000 mg/kg bw/day compared to controls. However, the majority of these individual values were within historical control normal ranges, a true dose related response for relative weights was not evident and there were no histopathological correlates. The intergroup differences were therefore considered not to be of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Red or dark coloration of the lungs was observed for one female at 10 mg/kg bw/day, three males and one female treated with 100 mg/kg bw/day and two males treated with 1000 mg/kg bw/day. Such findings are common in this type of study and were considered to be unrelated to treatment with the substance.

Increased pelvic space in both kidneys was observed in one control female and mottled kidneys was observed in one control male. In the absence of treatment, these were considered to be incidental findings. One female treated with 100 mg/kg bw/day had a dark liver and one female treated with 10 mg/kg bw/day had a dark patch on the liver. In the absence of a similar effect at 1000 mg/kg bw/day or any associated histopathological correlates, these findings were considered incidental and unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Centrilobular hypertrophy was noted in the liver of one male treated with 1000 mg/kg bw/day, however, due to the low incidence and minimal severity after ninety days of treatment this is considered not to be related to the administration of the substance. No changes were noted which could account for the weight increase noted in the liver of males and all are considered to be incidental.
Histopathological findings: neoplastic:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Conclusions:
The oral (gavage) administration of the substance, for ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 1000 mg/kg bw/day did not result in any adverse treatment-related effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be >1000 mg/kg bw/day.
Executive summary:

Male and female Wistar rats (10/sex/dose) were administered the substance (dissolved in Arachis oil BP) by oral gavage at doses of 0 (vehicle only), 10, 100 or 1000 mg/kg bw/day once daily for 90 consecutive days. The study was conducted according to OECD Guideline 408 (Subchronic Oral Toxicity - Rodent: 90 Day Study) and in accordance with GLP. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. No mortality occurred and no treatment-related clinical signs were observed during the study. Incidences of increased salivation were evident in males treated with 1000 mg/kg bw/day throughout the majority of the treatment period and in males treated with 100 mg/kg bw/day albeit to a lesser extent. Noisy respiration was also evident in three males treated with 1000 mg/kg bw/day between Days 52 and 90 and in one female treated with 1000 mg/kg bw/day, one male and two females treated with 100 mg/kg bw/day and one male and one female treated with 10 mg/kg bw/day on one occasion only. There were no toxicologically significant changes in the behavioral parameters measured. There were no treatment-related changes in functional performance. There were no treatment-related changes in sensory reactivity. No adverse effect on body weight development was evident in treated animals when compared to controls. No effect on food consumption or food efficiency was evident in treated animals when compared to controls. Visual inspection of water bottles did not reveal any intergroup differences. Ophthalmoscopic examination of animals of both sexes from the control and surviving 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences. There were no toxicologically significant effects detected in the hematological or blood chemistry parameters examined. No treatment-related findings were reported at post mortem macroscopic observations and histopathological examination. No toxicologically significant effects were detected in the organ weights measured. In conclusion, no treatment-related changes, which could be considered adverse, were observed in male and female rats following dosing with the substance, when administered by oral gavage for 90 consecutive days at the dosages of 10, 100 and 1000 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for this study was >1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on a reliable 28-day oral subacute study available on the substance itself and a reliable oral combined one generation reproduction/subchronic toxicity study available on a close structural analogue as well as a 90-day subchronic toxicity study is available on a second close structural analogue, no effects were observed in repeated dose toxicity studies. Therefore, no classification required under Directive 67/548/EEC or Regulation EC 1272/2008 for repeated dose toxicity.