Registration Dossier

Administrative data

Description of key information

Daily oral administration in rats at 40, 120 and 480 mg/kg/day did not result in relevant observations (clinical, ophthalmological or neurotoxicological), body weight and food intake, or haematology and urinalysis changes. Some slight and reversible non-adverse serum changes were noted in males and females at the highest dose. There were no effects on any immunotoxicology endpoints including bone marrow cellularity, T-cell Dependent Antibody Response (TDAR) and Lymphocyte subtyping up to 480 mg/kg/day. At 480 mg/kg/day minimal to mild changes were found in the liver and kidneys of females only: livers with periportal hepatocellular hypertrophy and mixed cell infiltrate and proliferation of the Kupffer cells  (correlating with slightly higher organ weights, fully reversible during 28-day recovery period) and kidneys with minimal bilateral focal/multifocal degeneration of the epithelium of cortical tubules (cytoplasmic vacuolation, cellular sloughing) associated with the presence of eosinophilic casts in tubular lumen. Signs of recovery were also observed in the cortical tubuli. Some sporadic histopathological changes in liver and kidney were observed in females dosed at 120 mg/kg however these were not adverse. The NOAEL was 480mg/kg/day for males and 120mg/kg/day for females, based on microscopic changes in the liver and kidneys. No immunotoxicity was detected at any dose level up to and including 480 mg/kg/day, therefore this dose was considered as NOAEL for immunotoxicity in males and females. 

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
immunotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study according to international guideline.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.7800
Principles of method if other than guideline:
The immunotoxicology was integrated in the OECD 408 study in rats.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: young adult rats, approx. 7 weeks at the onset of treatment
- Weight at study initiation: main study: males: 251-287g, females: 157-198g; satellite: males: 246-295g, females: 162-211g
- Housing: Type III polycarbonate
- Diet: ad libitum, ssniff® SM R/M "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest)
- Water: ad libitum, tap water from municipal supply, as for human consumption
- Acclimation period: ≥ 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0-23.4°C
- Humidity (%): 35-68 %
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: propylene glycol containing 1% polysorbate 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of the test item was weighed with accuracy of 10 mg into calibrated mixing vessels. The required amount of polysorbate (Tween 80) was added to achieve 1% polysorbate content in dose solutions (e.g. 100 µl polysorbate was added, if final volume was 10 mL). Dosing solution was made by adding the required amount of propylene glycol to achieve the final volume and desired concentrations (192, 48 and 16 mg/mL) of the test item for each dose level (480, 120 and 40 mg/kg bw/day, respectively) and was stirred until a homogenous dosing form was obtained.

VEHICLE
- Justification for use and choice of vehicle: test item was stable in propylene glycol containing 1% Polysorbate 80 in the concentration range of 10 mg/mL- 250 mg/mL for 5.5 hours at room temperature and for up to three days when stored refrigerated at 2-8ºC
- Concentration in vehicle: 16, 48 and 192 mg/mL
- Amount of vehicle (if gavage): 2.5 mL/kg bw/day
- Lot/batch no. (if required): propylene glycol: SZBC0820V (99.7%) and SZBC2900V (≥ 99.5%), polysorbate 80: BCBH5882V and BCBJ7603V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed using a validated GC method, in the Analytical Laboratory of CiToxLAB Hungary Ltd. Duplicate top, middle and bottom samples were taken from test item formulations on four occasion (during the first, second, eighth and the last weeks of treatment). Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 40, 120, 480 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
other: other: positive control treated with cyclophosphamide
Details on study design:
- Dose selection rationale:
The dose levels were selected by the Sponsor in consultation with the Study Director, based on the available data, including the results of a dose range finding study performed by the Sponsor with the test item at CiToxLAB Hungary Ltd., where doses of 300, 600 and 1000 mg/kg bw/day were administered to Wistar rats for 14 consecutive days. In this study the treatment was well tolerated and no signs of overt toxicity were observed, however haematological changes (increased % of lymphocytes versus decreased % of neutrophils in males) and serum biochemistry changes (increased calcium, phosphate, potassium in females) and slightly higher thyroids weights were found in both sexes at 1000 mg/kg bw/day.
According to the Sponsor’s information, results from a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of 2,2’-ethylenedioxydiethyl bis (2-ethylhexanoate) in rats, performed according to OECD 422 by dietary administration, are available. In this study a parental NOAEL was found to be of 5000 ppm test item concentration in rodent diet (Mid dose level), equivalent to 314-576 mg/kg bw/day test item intake. Target organs at 15000 ppm (equivalent to 977-1563 mg/kg bw/day) were adrenal glands, kidneys, liver, pituitary gland, spleen, thymus and thyroid glands.
For this 90-day repeated dose toxicity study in rats, dose levels of 40 – 120 – 480 mg/kg bw/day were selected. The high dose is based on the findings at the highest dose in the 14-day DRF study (CiToxLab P12/072-101PE, 2012) and the NOAEL of 314-576 mg/kg bw/day in the OECD 422 study (NOTOX Project 491295, 2010).

- Post-exposure recovery period in satellite groups:
Rats assigned to Recovery groups (10 male and 10 female in control and High dose groups), following the completion of 90-day treatment period were kept for a 28-Day observation period and then euthanized. See Table 1 wiht animal assignment in Main study

- Rationale for selecting satellite groups:
Fifty additional male and 50 female Wistar rats (ten male and ten female rats in each group) were assigned to satellite groups for immuntoxicology T-cell Dependent Antibody Response (TDAR) investigation according to the designed presented in Table 2 (satellite groups).

- Rationale for animal assignment: During the acclimation period, the animals were assigned to their respective dose groups by stratified allocation based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight. SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.
Observations and clinical examinations performed and frequency:
Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Morbidity and mortality twice daily. General clinical observations were made at least once a day at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations outside the home cages were made on all animals at randomisation (Day -1 or -2) and once a week thereafter, before treatment.
- Observations were performed on the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (hunchback posture, etc), gait, posture and response to handling and to environmental stimulation. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded with a precision of 1 g at randomisation, on Day 0, prior to start of treatment, then weekly, including on Day 89 (last treatment day) and Day 117 (last day of recovery), and prior to necropsy (fasted, on Days 90 or 118).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopy examination was conducted in all animals before initiation of the treatment and in the Control Group 1 and High dose Group 4 animals, during Week 13. No treatment related alterations are found at Week 13 (Day 85), therefore no examination of the lower dose groups and recovery groups was necessary.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment or recovery periods, prior to scheduled necropsy on Day 90 or 118
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: red blood cell count, haemoglobin concentration, haematocrit (%), mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) haemoglobin, red cell (erythrycyte) distribution width (%), platelet (thrombocyte) count, mean platelet (thrombocyte) volume, reticulocyte count (absolute and %), white blood cell (leukocyte) count, neutrophil (absolute and %), lymphocyte (absolute and %), monocyte (absolute and %), eosinophil (absolute and %), basophil (absolute and %), large unstained cells (absolute and %)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment or recovery periods, prior to scheduled necropsy on Day 90 or 118
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: glucose, total bilirubin concentration, urea concentration, cholesterol concentration, triglycerides, creatinine concentration, phosphorus concentration, sodium concentration, potassium concentration, calcium concentration, chloride concentration, total protein concentration, albumin concentration, Alb/glob ratio, aspartate aminotransferase activity, alanine aminotransferase activity, alkaline phosphatase, gamma glutamyltransferase activity, bile acids

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: leukocyte, nitrite, pH, protein, glucose, urobilinogen, bilirubin, ketones, blood/erythrocytes, specific gravity, sediment, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the treatment period, during Week 13 (on Day 84 am), each animal in the main and recovery groups was subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsy and macroscopic examination were performed on all Main study animals, at the end of treatment or recovery periods, on Days 90 or 118 (after the sample collection for clinical pathology evaluation).
The animals were euthanized by exsanguination under pentobarbital anaesthesia.
Necropsy and macroscopic examination were performed also on one animal (1003, control group) which was found dead on Day 76.
After exsanguination the external appearance of each rat was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size.
The following organs were trimmed of fat and weighed in surviving Main study animals (Main and Recovery Groups):
- With precision of at least 0.01g: Brain, Seminal vesicles with coagulating glands, Epididymides, Heart, Spleen, Kidneys, Testes, Liver, Thymus, Prostate, Uterus including cervix.
- With precision of at least 0.001g: Adrenal glands, Ovaries, Thyroids with parathyroids

ORGAN WEIGHTS: Yes
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated. In one High dose male (4007), significant difference in size was noted between right and left testis and epididymis, therefore their weights were measured individually.

HISTOPATHOLOGY: Yes
- Retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed in Groups 1 (Control) and 4 (High dose). In addition, any organs or tissues with macroscopic abnormalities were subjected to histological examination from all groups.
As test item-related microscopic findings were identified in the kidneys and livers of females, the affected organs from females of the Low and Mid dose level and from Recovery groups were also examined microscopically.
Cell viabilities:
SPLEEN: Yes
- Method: Histopathology
- Dose groups: all Main control and high dosed study animals
- No. of animals: 10/sex/group

THYMUS: Yes
- Method: Histopathology
- Dose groups: all Main control and high dosed study animals
- No. of animals: 10/sex/group

BONE MARROW: YES
- Method:
a. Histopathology sternum and marrow
Dose groups: all Main study animals
No. of animals: 10/sex/group
b. Bone marrow smears
Bone marrow smears from the femur of each animal were prepared at necropsy, fixed and stained with May-Grünwald and Giemsa stain. Control and High Dose group slides from Day 90 were evaluated.
- Dose groups: control and high dose
- No. of animals: 10/sex/group
- Physiological ranges for specific populations of bone marrow cells in rats (Valli, V.A., McGrath, J.P., Chu, I., Handbook of Toxicologic Pahology) are following:
myeloblast: 0.1-1.9 %
promyelocyte 1.1-2.9 %
myelocyte 3.5-5.3 %
metamyelocyte 5.4-8.2 %
band form segmented neutropbil granulocyte 12.1-17.5%
segmented neutrophil granulocyte 8.3-15.2%
premature and mature eosinophil granulocyte 1.8-3.6%
basophil granulocyte 0.4-0.5%
lymphocyte 15.4-22.3%
monocyte: 0.1-1.8%
proerythroblast 1.2-2.5 %
macroblast 19-22.3%
normoblast 11.2-15.2%
Average M: E ratio: 1.23
According to Schalm's Veterinary Hematology the age of the rats at the time of sampling was influencing the M:E ratio, that was varying between 0.62-2.7:1. In adult rats there was found a ratio of 1.44-1.88:1.
Humoral immunity examinations:
TDAR assay : T-cell Dependent Antibody Response assay
- Method:
Satellite Groups were involved for all 3 dose levels, negative control and positive control.
All animals were treated with a single intravenous injection (tail vein) of sheep red blood cells (SRBCs, 2x10^8 cells/rat) on Day 84. Blood was taken from the animals 6 days later (on Day 90), serum was prepared and analysed in a commercial ELISA kit for anti-SRBC IgM levels (Life Diagnostics, Inc., PA, USA; Catalogue Number: 4200-2).

For SRBC preparation, blood of healthy Merino sheep was used. Sterile sheep blood was stored 2-8°C according to the manufacturer’s instruction. SRBCs were prepared fresh on the day of use at CiToxLAB Hungary Ltd. at a concentration of 4x10^8 cells/mL in sterile PBS, and were administered at a dose level/volume of 0.5 mL SRBCs preparation/rat

The positive control substance (CP) was administered to animals assigned to Positive Control satellite group (5S), formulated in phosphate-buffered saline (PBS). Animals of the positive control group received the positive control substance at a dose volume of 5 mL/kg/day (dose level: 25 mg/kg bw/day) by intraperitoneal injection for 6 consecutive days (from Day 84 to Day 89) prior to the scheduled blood sampling time point.

The Satellite animals were sampled on Day 90. Prior to euthanasia, blood was collected from each animal by heart puncture under pentobarbital anaesthesia to obtain serum for ELISA measurements (up to 2.4 mL blood as practical, in tubes with no anticoagulant). After sample collections, satellite animals were euthanized by exsanguination under anaesthesia. One set of blood samples (with no anticoagulant) was transferred for ELISA measurements. After serum preparation, those samples were aliquoted and stored at -80±10ºC until analysis.

The anti–SRBC antibody contents of the samples were determined according to the method validation using commercially available ELISA kits for rat anti-SRBC IgM levels (Life Diagnostics, Inc., PA, USA, Catalogue No: 4200-2). Kits were stored unopened at 2-8ºC until use according to the manufacturer’s instruction.
The kit used detergent solubilised SRBC ghosts for solid phase immobilization and horseradish peroxidase (HRP) conjugated anti-rat IgM antibodies for detection. Calibrators, blank, quality control samples and test serum samples were diluted and incubated in the wells of the microtiter plates for 45 minutes at room temperature. Then, the wells were subsequently washed and HRP conjugate was added and incubated for 45 minutes at room temperature. Anti-SRBC molecules were thus sandwiched between the immobilized SRBC antigen and the detection antibody conjugate. The wells were washed to remove unbound HRP-labelled antibodies. TMB reagent was added into the wells and incubated for 20 minutes at room temperature. Colour development was stopped by the addition of stop solution, changing the colour from blue to yellow. Optical density at 450 nm was measured by an ELISA reader. The concentration of anti-SRBC IgM was proportional to the measured optical density (absorbance) of the test sample.
Six calibrators (nominal concentration of 100, 50, 25, 12.5, 6.25 and 3.125 u/mL, respectively), a blank sample and at least three quality control samples were used to create a standard curve for each ELISA run.
An ELISA run was considered as acceptable, if:
-six non-zero calibrators were presented,
-at least three quality controls were presented,
-at least 67% of QC samples were within 15% of their respective nominal value (or 20% at the LLOQ level).
Results were calculated by using Ascent Software and Microsoft Excel®. For the calculation of the results, a standard curve was plotted using the concentrations and average absorbances of the reference standard samples. Using the average absorbance value for each test sample, the corresponding concentration of anti-SRBC IgM was determined from the standard curve. Derived concentrations were multiplied by the dilution factors to get the actual concentrations of anti-SRBC IgM in the tested serum samples. If the measured absorbance of a sample fell outside the standard curve, sample was diluted appropriately and re-tested.

Any samples not employed in the primary analysis (back-up set 2 of sera, if any are stored frozen (approximately - 70 ºC) at CiToxLAB Hungary Ltd., until it is determined by the analyst and Study Director that it will not be required for confirmatory analysis, prior to finalization of the study report. These samples will then be discarded and their disposition will be recorded in the raw data.

- Dose groups: vehicle control, low dose , mid dose , high dose, positive control (5 groups)
- No. of animals: 10/sex/group

Other functional activity assays:
ENUMERATION TOTAL B CELLS, TOTAL T CELLS AND T CELL SUBPOPULATIONS: Lymphocyte subtyping
- Method:
Lymphocyte subtyping was performed from Main study animals.
For lymphocyte typing, blood was collected from Main Group animals on Day 90. Blood samples were collected from each animal by heart puncture under pentobarbital anaesthesia into tubes containing K3-EDTA (1.6 mg/mL blood) as anticoagulant, as described in section: 4.5 Clinical Pathology. Special attention was paid to avoid microbial contamination during blood preparation.
No blood sample was collected from the male no. 1003 (Control group) due to loss of the animal on Day 76.

Blood stabilization for lymphocyte typing
After collection on EDTA tubes, whole blood and TransFix® were mixed in the appropriate dilution (150µL Transfix® /1 mL blood). Samples were gently mixed (not vortexed) by inversion and aliquoted (2x200µL samples) and kept at +4°C until shipment. The total number of samples was 158.

Lymphocyte subtyping by flow-cytometry was carried out by Principal Investigator.
The tubes were shipped to the Test Site by a courier (at +4°C) once, the day following the completion of the sampling on Day 90.

Immunofluorescent staining
Blood samples were resuspended by inversion just before staining. One aliquot of 200 μL was used for lymphocyte subset determination.
The following parameters were determined for each animal/sex from all dose groups at the end of the treatment period. As no positive finding was identified, therefore the recovery groups were not examined.

Cell surface markers, antibodies and antibody labels are summarized in Table 3 below.

After a 20-minute incubation period (at room temperature) of the blood with the corresponding antibodies, red blood cells were lysed using TQ-Prep™ workstation (Beckman Coulter, France). Flow count™ fluorospheres were added to each sample before analysis on the flow cytometer for direct determination of lymphocyte subset absolute counts according to CiToxLAB France SOP.
Analysis of all samples was performed within stability frame (9 days from blood collection).

Flow cytometry
Acquisition was performed on a Navios flow cytometer, according to a validated method and data were analysed using the Navios v1 software (Beckman Coulter, France).
The lymphocyte population was gated using the following strategy: for each antibody combination a “Total cells” gate containing all nucleated cells was set on a size (Forward Scatter) vs. structure (Side Scatter) histogram in order to exclude debris. The events in this gate were then displayed on a bi parametric histogram set on CD3+ vs size:
• One bi parametric histogram gated on CD3 positive cells (Total T cells) was displayed and set on CD4+ vs CD8+ cells. Percentages of T helper and T cytotoxic cells were calculated from the total number of T cells.
• One bi parametric histogram gated on CD3 negative cells was displayed and set on CD45RA+ vs CD161a+ cells. Percentages of B and NK cells were calculated from the total number of CD3- cells.

Results are expressed in terms of percentage of gated cells and in absolute lymphocyte subset counts.
Results expressed in terms of absolute count by the flow cytometer were divided by 0.87 by Citox Software in order to correct the result by the dilution factor due to the addition of TransFix® solution.

- Dose groups: vehicle control, low dose, mid dose, high dose, vehicle control recovery, high dose recovery
- No. of animals: 10/ sex/group
Positive control:
An additional group of age matched positive control animals for TDAR was treated with cyclophosphamide via intraperitoneal injection for six consecutive days. (Cyclophosphamide monohydrate, a known immunosuppressant, was administered at 25 mg/kg body weight/day).
Statistics:
Data were collected using the software PROVANTIS v.7, or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the software PROVANTIS v.7, Microsoft Office Word and/or Excel, as appropriate. Results of ELISA measurements were calculated using Ascent Software and Microsoft Excel®.

The statistical evaluation of the numerical data was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest).

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of abnormal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. T-test was used in case of two groups’ comparison (Recovery groups).
The mean and standard deviations values were calculated.
No statistical analysis was carried out on food consumption data, due to small number of elements (2 cages in Low and Mid groups during treatment period and all groups during recovery period).

Statistical evaluation of results of lymphocyte subtyping measurements was performed by Principal Investigator using Citox software (version D.7).
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or mortality related to the test item.
Mortality:
no mortality observed
Description (incidence):
There were no clinical signs or mortality related to the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item related effects on food consumption. The average food consumption was comparable to the control in all treated groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination in the Control and High dose Group 4 animals, during Week 13 (Day 85), therefore no examination of the lower dose groups and recovery groups was necessary.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse effects at any dose level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse effects at any dose level.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse effects at any dose level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings considered related to test item administration under the conditions of this study.
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clinical signs or mortality related to the test item.
One male (No. 1003) died in the Control Group on Day 76, however the death of this animal was accidental (gavage accident).

BODY WEIGHT AND WEIGHT GAIN
The mean body weight and body weight gain of males and females in all experimental groups were comparable to the control throughout the treatment period. Minor variations were noted on occasion attaining statistical significance (e.g. slightly lower body weight gain values in Mid dose males on Week 12 or higher values in High dose females on Weeks 5 and 10) and were regarded as incidental and with no toxicological significance.
During the recovery period, the body weight and body weight gain values were comparable to the control in both males and females. A minor difference limited to one occasion was recorded for males on Week 17 (p <0.05) which was considered incidental.

OPHTHALMOSCOPIC EXAMINATION
No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination in the Control and High dose Group 4 animals, during Week 13 (Day 85), therefore no examination of the lower dose groups and recovery groups was necessary.

HAEMATOLOGY
There were no test item related adverse effects at any dose level.

CLINICAL CHEMISTRY
Clinical chemistry evaluation did not reveal any obvious toxicity for test item following the 90-day treatment or recovery periods.

URINALYSIS
Clinical chemistry evaluation did not reveal any obvious toxicity for test item following the 90-day treatment or recovery periods.

NEUROBEHAVIOUR
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.

ORGAN WEIGHTS
The statistically significant changes after 90 days, including the increased relative liver weights in both sexes and kidney weights in females were considered to be test item related, however they were of low magnitude and were reversible.
There were no biologically significant differences among groups in the weights of other organs measured, compared with control, although statistically significant variations were noted (i.e higher spleen and thymus weight, when adjusted for body weight and higher spleen weight when adjusted for brain weight in Mid dose females on Day 90, higher brain related thyroid/parathyroids weights in Mid dose males on Day 90). The values were within physiological range and were not considered to reflect an adverse effect.

GROSS PATHOLOGY
There were no macroscopic findings considered related to test item administration under the conditions of this study.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related microscopic findings were noted in the liver and kidney in the females at dose levels of 120 and 480 mg/kg/day.
At 480 mg/kg/day, hepatic changes were characterized as periportal hepatocellular hypertrophy, mixed cell infiltrate and proliferation of the Kupffer cells. These alterations correlated with minimal organ weight changes. Minimal hypertrophy was observed in 3 of 10 High dose females. Mixed cell infiltrate diffusely distributed in the sinusoidal space, was noted with minimal to mild degree in 4 of 10 High dose female rats. In addition, proliferated Kupffer cells (enlarged size/number) were associated with infiltrate in 1 of 4 altered females. The hepatic changes observed in the High dose were regarded to be adverse.

At 120 mg/kg/day, minimal hepatic changes were observed in 2 of 10 females, consisted of minimal periportal hepatocellular hypertrophy and were regarded as adaptive response. In 3 of 10 females minimal mixed cell infiltration was also observed. In two females this minimal infiltration was noted additionally to hepatocellular hypertrophy and in one female it was the only hepatic observation. Mixed cell infiltration may spontaneously occur in rats (ref. 4); therefore the causative role of the treatment is equivocal.

Renal changes were recorded in the cortical tubules. The degeneration of tubule epithelium (cytoplasmic vacuolation, cellular sloughing) associated with the presence of homogenous eosinophilic content (casts) filled tubular lumen was seen in 1 of 10 Mid dose and 2 of 10 High dose females. The degenerative changes altered tubular epithelium with minimal severity. Bilateral focal/multifocal distribution was present in High dose animals while unilateral/focal localization appeared in the Mid dose female. There was no evidence of the peritubular inflammatory response.
The renal changes at 480 mg/kg/day were regarded as adverse. Taking into account unilateral occurrence and minimal focal degree at 120 mg/kg/day, the finding was not regarded to be adverse.

Although minimal unilateral focal homogenous eosinophilic casts in the lumen of the cortical tubules were observed in 3/10 Low dose females, their microscopic feature was similar to minimal unilateral focal homogenous eosinophilic casts present in 1 of 10 Control female in recovery period. In addition, there was no damage to tubular epithelium or inflammatory reaction. Therefore, the casts seen in the Low dose group were considered to be without relationship to the treatment.

The spleen and thymus were examined closely in accordance with the Study Plan to clarify any potential immunotoxicity. Minimal extramedullary haematopoiesis in the spleen was present in 2/9 Control and 7 of 10 High dose males and 5 of 10 Control and 8 of 10 High dose females. There was no meaningful difference in these splenic observations between controls and treated rats, and they were considered to be a common background. In the thymus, minimal decrease of size/cellularity of the cortex/medulla in 1 of 10 female (4502) and 1 of 10 male (4003; associated with minimal prominent epithelial cords and tubules) from the High dose groups, were seen.

Decreased size/cellularity of cortical/medullary compartments was not associated with an increase of severity grade of lymphocyte apoptosis comparing to controls or inflammatory reaction. No notable reduction in thymic weight was detected in these two High dose animals. Therefore, it seems that thymic changes were not ascribed to the administration of the test item, but rather reflected individual stress conditions in these rats. It is concluded that there was no evidence for any immunotoxic effects in these tissues following a 90 day exposure of up to 480 mg/kg/d of the test item.

Following a 28-day treatment-free period, the recovery process was completed in the liver, however, effects of treatment were evident in the kidney.
In kidneys, mild bilateral or minimal unilateral multifocal tubular degeneration /regeneration with the presence of luminal homogenous eosinophilic casts was observed in 2 of 10 High Dose females. The tubules also had evidence of a regenerative process accompanied with increased tubule cells basophilia, transition of cell shape from flattened to cuboidal and/or mitosis.

In one of this female (4511), bilateral minimal glomerulonephritis as spontaneous change cannot be excluded since other inflammatory disorder, mild interstitial pneumonia affected all lobes of lungs was also observed in this animal, in addition to mild bile duct hyperplasia was observed, however, being a common background observation in the liver, was not considered to be test item related.

Other changes were without meaningful incidence and/or severity represented incidental or background observations without relationship to the treatment.

BONE MARROW SMEARS: results
In Control group, males and females, normal hematopoiesis was noted in all animals. In this Group, Mean M:E ratio for males was 0.99 and for females it was 0.95.
In the High dose group all male and female animals showed normal haematopoiesis.In males, slight increase in proerythroblast and normoblast count and a slight decrease in myelocyte, metamyelocyte and stab form neutrophil granulocyte count could he observed, within physiological ranges. The mean M:E ratio (of 0.91) was 8.1 % lower than in Control Group.Infemales there wasn't any significant difference in cell counts, all were within physiological ranges. The mean M:E ratio of 0.96 was very similar to the Control Group (0.95).
CONCLUSION
All slight changes in the M:E ratio or specific bone marrow cell populations in males and females were within the normal physiologic range. No pathological cells or pathological alteration of normal cell distribution were observed.

CELL VIABILITIES histopathology bone marrow
The bone marrow cellularity of the High dose males and females did not differ significantly from the control on Day 90. In males, there was statistical significance for decreased metamyelocyte and Myeloid to Erythroid ratio (M/E), compared to the control mean, but without biological significance.
The hematopoiesis in the test item treated animals was not disturbed, the number of the myeloblasts and promyelocytes as well as the granulocytes were comparable to the control.
No examination of the lower dose groups and recovery groups was necessary.

HUMORAL IMMUNITY EXAMINATIONS: T-CELL DEPENDENT ANTIBODY RESPONSE (TDAR) ASSAY (See Table 4 for group results - Individual data attached in Phase report)
No statistically or biologically significant changes were detected in the anti-SRBC IgM level of the test item treated dose groups of male animals. Decreased anti-SRBC IgM levels compared to the negative control group were detected in the mid and low dose group of the female animals. However, no dose response was observed, and the observed values were statistically not different from negative control values of the male animals.

Treatment with the positive control substance (cyclophosphamide) gave the anticipated strong response: the decrease in the rat anti-SRBC IgM level of the positive control animals compared to the negative control animals was statistically significant in male and female animals as well, indicating the impaired functional immune response.

OTHER FUNCTIONAL ACTIVITY ASSAYS: LYMPHOCYTE SUBTYPING (See Table 5 for group results - Individual data attached in Phase report)
There were no test item effects on lymphocyte analysis that could be attributed to the treatment at any of the dose level up to and including 480 mg/kg/day.
For males a trend to decrease in absolute count was observed for all parameters (total T cells, helper and cytotoxic T cells, B cells and NK cells) in groups treated at 120 and 480 mg/kg/day (Mid and High dose). However these changes were not dose-related and not statistically significant.
No significant changes were observed in the percentage of each lymphocyte population.

Cell viabilities:
no effects observed
Description (incidence and severity):
histopathology/smear bone marrow; histopathology spleen/thymus
Humoral immunity examinations:
no effects observed
Description (incidence and severity):
See details on results
Specific cell-mediated immunity:
not examined
Non-specific cell-mediated immunity:
not examined
Other functional activity assays:
no effects observed
Description (incidence and severity):
There were no test item effects on lymphocyte analysis that could be attributed to the treatment at any of the dose level up to and including 480 mg/kg/day.
Other findings:
effects observed, treatment-related
Description (incidence and severity):
At the highes dose histopathological changes were found in the liver and kidneys of females only. At the mid dose minimal changes were found in the liver (minimal periportal hepatocellular hypertrophy) of few females and were regarded as adaptive.
Dose descriptor:
NOAEL
Effect level:
480 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Systemic toxicity
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Systemic toxicity (microscopic changes in the liver and kidneys in females at 480 mg/kg bw/day)
Dose descriptor:
NOAEL
Effect level:
480 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Immunotoxicology

Table 4: Mean anti SRBC IgM values of the experimental groups (Day 90)

 

 

Groups/Dose (mg/kg/day)

 

 

 

Control

 

 

Low dose

40

 

Mid dose

120

 

High dose

480

 

 

Positive

control

 

 

Males (n)

 

10

 

10

 

10

 

10

 

10

 

 

Mean SRBC

IgM concentration (u/mL)

 

9971.7

 

11207.5

 

4299.1

 

5915.5

 

732.1**

 

DN

 

SD

 

13411.1

 

13557

 

3115.9

 

4239.2

 

515.8

 

 

Females (n)

 

10

 

10

 

10

 

10

 

10

 

 

Mean SRBC

IgM concentration (u/mL)

 

24329.6

 

11013.8

 

7860.2

 

16573.6

 

1227.9**

 

DN

 

SD

 

17642.1

 

10270.4

 

6250.5

 

14020.7

 

824.5

 

** = p<0.01; DN: Duncan’s Multiple Range Test, comparison to negative male control group

Table 5: Mean lymphocyte subpopulation counts of males (Day 90). In brackets percentage from control group are presented.

 

 

Cell population

 

Mean concentrations (cells/µL) and changes from control group

 

Control

(n=9)

 

Low dose

(n=10)

 

Mid dose

(n=10)

 

High dose

(n=10)

 

Total T cells

 

Mean ± SD

 

1449 ± 629

 

1468 ± 639

(+1.3%)

 

1041 ± 452

(-28.2%)

 

1169 ± 408

(-19.3%)

 

Helper T cells

 

Mean ± SD

 

1022 ± 452

 

1003 ± 404

(-1.9%)

 

737 ± 345

(-27.9%)

 

815 ± 288

(-20.3%)

 

Cytotoxic T cells

 

Mean ± SD

 

316 ± 150

 

350 ± 205

(+10.8%)

 

230 ± 89

(-27.2%)

 

274 ± 130

(-13.3%)

 

B cells

 

Mean ± SD

 

1183 ± 787

 

1103 ± 478

(-6.8%)

 

1037 ± 743

(-12.3%)

 

921 ± 446

(-22.1%)

 

NK cells

 

Mean ± SD

 

200 ± 109

 

224 ± 90

(+12%)

 

147 ± 77

(-26.5%)

 

147 ± 60

(-26.5%)

 

Conclusions:
A daily oral (gavage) administration of 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) to Wistar rats for 90 consecutive days at 40, 120 and 480 mg/kg/day was associated with the following findings. There were no toxicologically significant effects on clinical signs, ophthalmology or neurotoxicology endpoints. There were no effects on body weight, food intake, bone marrow cellularity, or haematology or urinalysis parameters. For clinical chemistry no adverse effect was noted, however, slight and reversible changes were noted in males (increased serum cholesterol and bile acids) and in females (decreased triglycerides). There were no adverse effects on organ weights or on macroscopic observations at necropsy. There were no effects on any immunotoxicology endpoints including lymphocyte subpopulation analysis or T-cell Dependent Antibody Response (TDAR) assay. At 480 mg/kg/day minimal to mild changes were found in the liver and kidneys of females only. Changes in the liver consisted of periportal hepatocellular hypertrophy and mixed cell infiltrate and proliferation of the Kupffer cells, correlated with slightly higher organ weight and were fully reversible during 28-day recovery period. The renal changes consisted of the minimal bilateral focal/multifocal degeneration of the epithelium of cortical tubules (cytoplasmic vacuolation, cellular sloughing) associated with the presence of eosinophilic casts in tubular lumen. Signs of recovery were observed in the cortical tubuli. A decreased performance was noticed during motor activity measurements in males dosed at 480 mg/kg/day, however the effect was reversible and not considered to be adverse. No other adverse effects were noted in males. At 120 mg/kg/day minimal changes were found in the liver (minimal periportal hepatocellular hypertrophy) of few females and were regarded as adaptive. In one female, unilateral focal degeneration of tubule epithelium with eosinophilic casts in tubular lumen was seen in kidney, but taking into account unilateral occurrence and minimal focal degree, the finding was not regarded to be adverse. No effects were observed for males. Treatment at 40mg/kg/day was not associated with any adverse effects or histopathological changes. In conclusion, the NOAEL of 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) administered by oral gavage to Wistar rats for 90 consecutive days is considered to be 480mg/kg/day for males and 120mg/kg/day for females, based on microscopic changes in the liver and kidneys in females at 480 mg/kg/day. No immunotoxicity was detected at any dose level up to and including 480 mg/kg/day, therefore this dose was considered as NOAEL for immunotoxicity in males and females.
Executive summary:

The objective of this study was to obtain information on the toxicity of 2,2’- ethylenedioxydiethyl bis(2-ethylhexanoate) when administered daily for 90 days by oral gavage to the Wistar rat at 3 dose levels. Doses of 40, 120, and 480 mg/kg /day were administered to male and female Wistar rats in the main group (toxicology, including lymphocyte subtyping at day 90 and recovery groups for 28 days) and satellite groups (TDAR assay). An additional group of ten male and ten female Wistar rats served as positive control group (cyclophosphamide) for TDAR investigation. The test item was formulated in propylene glycol containing 1% Tween 80 and the control group was treated with the vehicle only.No mortality or clinical signs considered related to test item was observed during the study. There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups. No test item related effects were observed in the landing foot splay test or grip strength measurements. In motor activity measurements, the total travelled distance was slightly lower (by approximately 22%), in males at 480 mg/kg/day. The average velocity and total time moving fast were also slightly lower, than control mean. As the patterns of activity were comparable with those of the control and the individual values were within the physiological range, therefore the effect was not regarded as adverse. Following the 28-day recovery period, all parameters in the motor activity test examined in males at 480 mg/kg/day, were comparable with controls. The oestrus cycle of the females was undisturbed by the treatment with the test item. No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination. There were no test item related effects on body weight and body weight gain or food consumption. There were no test item related adverse effects in haematology, blood clotting, bone marrow cellularity, clinical chemistry or urinalysis parameters at any dose level. There were no macroscopic findings considered related to test item administration. For organ weight, minimal differences were measured on Day 90 in females for liver and kidneys at 480 mg/kg/day and for liver at 120 mg/kg/day. The absolute and relative weights of liver were higher than control means by approximately 8-10% (Mid dose) and 8-12% (High dose). The changes were not considered adverse. Microscopically a minimal periportal hepatocellular hypertrophy and minimal to mild periportal mixed cell infiltrate were seen in the liver in 2-3 of 10 Mid Dose and 3-4 of 10 High Dose females, occasionally accompanied by proliferation of the Kupffer cells (1 of 10 High Dose females). The overall microscopic picture in the High dose was classified as adverse, while findings in the Mid dose rather as adaptive change. High dose females had also slightly higher weights of kidneys (no more than 11%). Microscopically in the kidneys, minimal degeneration of epithelium of cortical tubule (cytoplasmic vacuolation, cellular sloughing) associated with the presence of homogenous eosinophilic content (casts) filled tubular lumen was seen in 2 of 10 High dose females. Although minimal focal degeneration and eosinophilic content was observed in right kidney in 1 of 10 Mid dose female, but taking into account unilateral occurrence and minimal focal degree, the finding was not regarded as adverse. Following a 28 -day treatment-free period, complete recovery was observed in the liver and signs of the recovery were detected in the kidney. In kidneys, mild bilateral or minimal unilateral multifocal tubular degeneration/regeneration with the presence of luminal homogenous eosinophilic casts was observed in 2 of 10 High Dose females. The tubules also underwent regenerative process accompanied with increased tubule cells basophilia, transition of cell shape from flattened to cuboidal and/or mitosis. During Immuntoxicology investigation, in a T-cell Dependent Antibody Response (TDAR) assay, no changes were detected in males. Decreased anti-SRBC IgM levels compared to the negative control group were detected in the mid and low dose group of the females. However, no dose response was observed, and the observed values were statistically not different from negative control values. There were no test item effects on lymphocyte subpopulation analysis that could be attributed to the treatment at any of the dose level up to and including 480 mg/kg/day. Minor differences between groups were observed which were considered to represent normal variation and of no toxicological or biological relevance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
480 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High (Klimisch 1 study conducted under GLP)

Effect on immunotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A subchronic toxicity study in Wistar rats was performed to obtain information on the toxicity of 2,2’- ethylenedioxydiethyl bis(2-ethylhexanoate), including immunotoxicity parameters (Grosz, 2013a). The test item was administered daily for 90 days by oral gavage to the Wistar rat at 3 dose levels. Doses of 40, 120, and 480 mg/kg /day were administered to male and female Wistar rats in the main group (toxicology, including lymphocyte subtyping at day 90 and recovery groups for 28 days) and satellite groups (TDAR assay). An additional group of ten male and ten female Wistar rats served as positive control group (cyclophosphamide) for TDAR investigation. The test item was formulated in propylene glycol containing 1% Tween 80 and the control group was treated with the vehicle only.No mortality or clinical signs considered related to test item was observed during the study. There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups. No test item related effects were observed in the landing foot splay test or grip strength measurements. In motor activity measurements, the total travelled distance was slightly lower (by approximately 22%), in males at 480 mg/kg/day. The average velocity and total time moving fast were also slightly lower, than control mean. As the patterns of activity were comparable with those of the control and the individual values were within the physiological range, therefore the effect was not regarded as adverse. Following the 28-day recovery period, all parameters in the motor activity test examined in males at 480 mg/kg/day, were comparable with controls.The oestrus cycle of the females was undisturbed by the treatment with the test item. No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.There were no test item related effects on body weight and body weight gain or food consumption. There were no test item related adverse effects in haematology, blood clotting, bone marrow cellularity, clinical chemistry or urinalysis parameters at any dose level.There were no macroscopic findings considered related to test item administration.For organ weight, minimal differences were measured on Day 90 in females for liver and kidneys at 480 mg/kg/day and for liver at 120 mg/kg/day. The absolute and relative weights of liver were higher than control means by approximately 8-10% (Mid dose) and 8-12% (High dose). The changes were not considered adverse.Microscopically a minimal periportal hepatocellular hypertrophy and minimal to mild periportal mixed cell infiltrate were seen in the liver in 2-3 of 10 Mid Dose and 3-4 of 10 High Dose females, occasionally accompanied by proliferation of the Kupffer cells (1 of 10 High Dose females). The overall microscopic picture in the High dose was classified as adverse, while findings in the Mid dose rather as adaptive change.High dose females had also slightly higher weights of kidneys (no more than 11%). Microscopically in the kidneys, minimal degeneration of epithelium of cortical tubule (cytoplasmic vacuolation, cellular sloughing) associated with the presence of homogenous eosinophilic content (casts) filled tubular lumen was seen in 2 of 10 High dose females. Although minimal focal degeneration and eosinophilic content was observed in right kidney in 1 of 10 Mid dose female, but taking into account unilateral occurrence and minimal focal degree, the finding was not regarded as adverse.Following a 28 -day treatment-free period, complete recovery was observed in the liver and signs of the recovery were detected in the kidney. In kidneys, mild bilateral or minimal unilateral multifocal tubular degeneration/regeneration with the presence of luminal homogenous eosinophilic casts was observed in 2 of 10 High Dose females. The tubules also underwent regenerative process accompanied with increased tubule cells basophilia, transition of cell shape from flattened to cuboidal and/or mitosis.During Immuntoxicology investigation, in a T-cell Dependent Antibody Response (TDAR) assay, no changes were detected in males. Decreased anti-SRBC IgM levels compared to the negative control group were detected in the mid and low dose group of the females. However, no dose response was observed, and the observed values were statistically not different from negative control values.There were no test item effects on lymphocyte subpopulation analysis that could be attributed to the treatment at any of the dose level up to and including 480 mg/kg/day. Minor differences between groups were observed which were considered to represent normal variation and of no toxicological or biological relevance.


Justification for selection of effect on immunotoxicity via oral route endpoint:
Key study including immunotoxicity parameters.

Justification for classification or non-classification

Based on the results of the subchronic toxicity and immunotoxicology studies (Grosz, 2013a), there is no need to classify the substance in accordance to criteria listed in the CLP Regulation (EC 1272/2008).