Registration Dossier

Administrative data

Description of key information

The key study for  oral repeated dose toxicity is an oral 90-day repeated dose toxicity study in the rat (Grosz, 2013). The resultant NOAEL (oral) is 120 mg/kg bw/day based on females. The key study for inhalation repeated dose toxicity study is a 2-week inhalation toxicity study in the rat (Dupont, 2005). The resultant NOAEL (inhalation) is > 1000 mg/m3. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
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:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
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 oral 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: 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).
- 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.
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).
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
HISTOPATHOLOGY: Yes
Other examinations:
IMMUNOTOXICOLOGY INVESTIGATION:
- T-cell dependent antibody response (TDAR) assay
- Lymphocyte subtyping
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.
Behaviour (functional findings):
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.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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.
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.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the highes dose minimal to mild 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.
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clinical signs or mortality related to the test item.

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 female. 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.


OTHER FINDINGS: IMMUNOTOXICOLOGY FINDINGS

T-CELL DEPENDENT ANTIBODY RESPONSE (TDAR) ASSAY
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.

LYMPHOCYTE SUBTYPING
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.
Dose descriptor:
NOAEL
Effect level:
480 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: 480 mg/kg bw/day is the highest tested dose
Dose descriptor:
NOEL
Effect level:
120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: based on microscopic changes in the liver and kidneys in females at 480 mg/kg bw/day
Dose descriptor:
NOEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified
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. The NOEL is 120 mg/kg/day for males and 40 mg/kg/day for females. No immunotoxicity was detected at any dose level up to and including 480 mg/kg/day.
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 according to the experimental design presented in the following table.

Table 1: Experimental design

Group designation

Dose Level (mg/kg/day)

Conc (mg/mL)

Dose volume (mL/kg bw/day)

Number of Animals

Main study

Satellite groups

Males

Females

Males

Females

Main

Recovery

Main

Recovery

Control

0

0

2.5

10

10

10

10

10

10

Low Dose

40

16

10

-

10

-

10

10

Mid Dose

120

48

10

-

10

-

10

10

High Dose

480

192

10

10

10

10

10

10

Rats assigned to Recovery groups, following the completion of 90-day treatment period were kept for a 28-Day observation period.

Satellite groups were assigned to immuntoxicology T-cell Dependent Antibody Response (TDAR) investigation.

An additional group of ten male and ten female Wistar rats served as positive control for TDAR investigation (animals were

treated with cyclophosphamide, a known immunosuppressant, via intraperitoneal injection).

The test item was formulated in propylene glycol containing 1% Tween 80 and the control group was treated with the vehicle only. The first day of the treatment was Day 0 for all animals.

During the study, observations for mortality and clinical signs were performed daily, detailed clinical examination, body weight and food consumption measurements were performed at weekly intervals.

Ophthalmoscopy and neurological assessment including a functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of treatment. A motor activity measurement was repeated for males groups during the last week the recovery.

Clinical pathology examinations (haematology, clinical chemistry and urinalysis) were conducted prior to necropsy following the end of the treatment and recovery periods (Days 90 and 118), followed by necropsy with macroscopic examination and selected organ weight measurements.

Full histopathology was performed for Groups 1 (Control) and 4 (High dose); in addition, the affected organs (kidneys and liver) from females of the Low and Mid dose and Recovery groups were also examined. Organs showing macroscopic findings from any animal of any group were also examined microscopically.

Analysis of test item formulations for concentration and homogeneity was performed four times during the treatment period, using a validated GC method. All formulations were found to be in the range of 90 to 102% of nominal concentrations and were homogeneous. No test item was detected in the control samples. Based on these results, the test item formulations were considered suitable for the study purposes.

RESULTS

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.

CONCLUSION

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, body weight, food intake, haematology, blood clotting, bone marrow cellularity, clinical chemistry or urinalysis parameters.

There were no adverse effects on organ weights or on macroscopic observations at necropsy. There were no treatment related 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 480 mg/kg/day for males and 120 mg/kg/day for females, based on microscopic changes in the liver and kidneys in females at 480 mg/kg/day. The NOEL is 120 mg/kg/day for males and 40 mg/kg/day for females. No immunotoxicity was detected at any dose level up to and including 480 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The information available is very recent and of good quality, so the overall quality of the database is considered good.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant OECD guideline study, available as unpublished report, only 2-week study but furthermore no restrictions, adequate for assessment.
Principles of method if other than guideline:
The study is conducted similarly to OECD testing guideline 403, but repeatedly during 2 weeks
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 6 weeks old on the day of arrival
- Housing: Except during exposure, animals were housed singly in stainless steel wire-mesh cages
- Diet (e.g. ad libitum): ad libitum except during exposure (PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002)
- Water (e.g. ad libitum): ad libitum except during exposure (tap water)
- Acclimation period: Rats were quarantined after arrival for 3 days prior to testing, further temporarily identified by cage identification, weighed and observed for signs of disease or injury. Then rats were assigned to groups by a computerized stratified randomization program. At study start, the animals were 8 weeks old and weighed between 241 and 286 grams.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Relative humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): approximate 12-hour light/dark cycle
IN-LIFE DATES: From: January 18, 2005 To: February 21, 2005

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Mass median aerodynamic diameter (MMAD) and other conditions: See Table 2
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus and exposure chamber volume: The exposure chamber was constructed of glass (cylindrical) with a nominal internal volume of 34 L. A polycarbonate baffle inside the chamber promoted uniform chamber distribution of the test atmosphere.
- Method of holding animals in test chamber: During exposure, animals were individually restrained in perforated stainless steel cylinders with conical nose pieces. The restrainers were inserted into a polymethylmethacrylate faceplate attached to the exposure chamber so that the nose of each animal extended into the exposure chamber.
- System of generating particulates/aerosols: Chamber atmospheres were generated by aerosolization of the test substance in air with a Spraying Systems nebulizer. The test substance was metered into the nebulizer with a Harvard Apparatus model 22 syringe infusion pump. Filtered, high-pressure air, metered into the nebulizer by a Brooks model 0154E mass flow controller, carried the resulting atmosphere into the exposure chamber. Chamber concentrations of test substance were controlled by varying the rate of the infusion pump.
- Treatment of exhaust air: Test atmospheres were exhausted through a dry-ice cold trap followed by an MSA charcoal/HEPA filter cartridge prior to discharge into the fume hood.
- Temperature, humidity, pressure in air chamber: Chamber temperature was 24°C, relative humidity ranged from 46 - 50%, airflow was 16.0 L/min and the oxygen concentration was 21.0%.

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure the atmospheric concentration of the test substance was determined by gravimetric analysis at approximately 30-minute intervals in the test chamber. Known volumes of chamber atmosphere were drawn from the sampling port through a 25 mm filter cassette containing a pre-weighed glass fiber (Type A/E) filter. The filters were weighed on a Cahn model C-31 Microbalance®. The atmospheric concentration of the test substance was calculated from the difference between the pre- and postsampling filter weights divided by the volume of chamber atmosphere sampled.- Particle size distribution: MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The atmosphere generated in this study was considered to be respirable in rats, the mass median aerodynamic diameter was 2.0 μm ± 2.0 (MMAD ± GSD), with 97% of the aerosol being less than 10 μm MMAD.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample to determine aerosol size distribution (mass median aerodynamic diameter and percent
aerosol less than 1,3, and 10 µm diameter) was taken fiom each test chamber for Groups III, V, and VII weekly with a Sierra senes 210 cyclone preseparatortcascade impactor and sierram series 110 constant flow air sampler.
Duration of treatment / exposure:
Each group of rats was exposed 6 hours/day. The starting time of each exposure was defined as the time when the generation system was turned on. The ending time of each exposure was defined as the time when the generation system is turned off. Rats were exposed to the test substance during both the time it took for the chamber to reach concentration, and the time it took for the test substance to be purged from the chamber. After each exposure, rats were returned to their cages. The exposure period was defined as the period between initiation of the first exposure and completion of the last exposure.
Frequency of treatment:
Each group of rats was over a 2-week period (weekends and holidays excluded) for a total of 9 exposures.
Remarks:
Doses / Concentrations:
0 (control), 10 ± 0.35, 300 ± 5.8, 1000 ± 19 mg/m³ (Groups I, III, V, VII, respectively)
Basis:

No. of animals per sex per dose:
10 male animals/group
Control animals:
yes
Details on study design:
At the conclusion of the 2-week exposure period, 5 rats per group were allowed to recover for 2 weeks. During the recovery period observations were continued.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
- Time scheudule: daily during 14 days

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: individual examination immediately following exposure on the days of dosing.

BODY WEIGHT: yes
- Time scheudule: twice weekly – all animals

FOOD CONSUMPTION: no
FOOD EFFICIENCY: no
WATER CONSUMPTION: no
OPHTHALMOSCOPIC EXAMINATION: no
HAEMATOLOGY: yes
- Time schedule: day 11 (1 day post-exposure); day 25 (2 weeks recovery)
- Anaesthetic: carbon dioxide anesthesia
- Animals fasted: yes (15h)
- No. of animals: all animals on day 11; 5 animals/group on day 25
- Parameters: red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, absolute reticulocyte count, platelet count, white blood cell count, differential white blood cell count, microscopic blood smear examination, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: yes
- Time schedule: day 11 (1 day post-exposure); day 25 (2 weeks recovery)
- Animals fasted: yes (15h)
- No. of animals: all animals on day 11; 5 animals/group on day 25
- Parameters: aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, cholesterol, triglycerides, glucose, total protein, albumin, globulin, calcium, inorganic phosphate, sodium, potassium, chloride
URINALYSIS: yes
- Time schedule: day 11 (1 day post-exposure); day 25 (2 weeks recovery)
- Metabolism cages used for collection: yes (15h)
- Animals fasted: yes (15h)
- Parameters: quality, color, clarity, ketone, bilirubin, blood, volume, osmolality, pH, glucose, urobilinogen, protein, microscopic urine sediment examination
NEUROBEHAVIOURAL EXAMINATION: no

Sacrifice and pathology:
- Necropsy (final body weights, organ weights, macroscopic examination): 5 animals/group on day 11 (1 day post-exposure) ; 5 animals/group on day 25 (2 weeks recovery)
- Group organ weight means and ratios (% body weight and % brain weight) were calculated.

HISTOPATHOLOGY: yes
- all organs were processed and examined.
Statistics:
See Table 1.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
other: LD50
Effect level:
> 2 000 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified

TABLE STILL TO BE ADDED APPENDIXES REQUIRED

Conclusions:
Based upon the observations, the NOAEL = 1000 mg/m3
Executive summary:

Four groups of 10 male CRI:CD(SD)IGS BR rats were exposed to aerosol atmospheres (1.9-2.4 µm) of 3G0 at 0 (control), 10+0.35,300+5.8 or 1000+19 mg/m3(mean+S.E.M.). Animals were exposed nose only, 6 hours a day for a total of 9 exposures over a 2-week period. The dosing period was followed by a recovery period in half of the animals No test substance related effects were observed in clinical observations, body weights, clinical pathology, gross necropsy observations and microscopic pathology.Based upon the observation of no significant effects following exposure to 3G0, the no observed- Adverse effect level (NOAEL) for a 2-week repeated inhalation exposure was 1000 mg/m3for male rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 000 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two key oral studies are available, one 28 -day study (Van Tuyl, 2010) and one 90 -day study (Grosz, 2013). The NOAELs originating from the 90 -day study are used further on in the risk assessment.

The first key study for repeated dose oral toxicity was performed by means of combined 28-day repeated dose toxicity study with reproduction/developmental toxicity screening test in the rat (Van Tuyl, 2010); this study was conducted according to internationally accepted OECD 422 testing guidelines and in compliance with GLP standards. Based on the results of a 5-day dose range finding study, the dose levels of 1500, 5000 and 15000 ppm in the diet were used. Males were exposed for 28 days (2 weeks prior to mating, during mating, and up to termination); females were exposed for 41-48 days (2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation). No reproductive/developmental toxicity was observed at any dose level. At 1500 and 5000 ppm, no parental toxicity was noted. At 15000 ppm, parental toxicity consisted of decreased motor activity for females, decreased body weight gain for males and females, haematological changes for females (increased prothrombin time), changes in clinical biochemistry parameters, (increased glucose and decreased sodium levels for males, increased potassium levels for both sexes), organ weight changes (increased liver and kidney weightsfor both sexes, slightly decreased thymus weights for females) and histopathological findings. Microscopic findings related to treatment were recorded in the adrenal glands (multifocal vacuolation in zona glomerulosa), kidneys (cortical hyaline droplets and corticomedullary tubular basophilia along with a single instance of granular casts), liver (diffuse midzonal/centrilobular hypertrophy), pituitary gland (adenohypophyseal multifocal hypertrophy), spleen (increased hemosiderin pigment along with a reduction in primarily erythroid hemopoietic foci), thymus (lymphoid atrophy (involution)) andthyroid glands (diffuse follicular hypertrophy/hyperplasia) for males and/or females. Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 5000 ppm was derived. The reproductive and developmental NOAEL was at least 15000 ppm. When corrected for mean test article intake the NOAEL of 5000 ppm corresponds to 314-576 mg/kg body weight/day and the NOAEL of 15000 ppm corresponds to 977-1563 mg/kg body weight/day (males and females, respectively).

The second available key study for repeated dose oral toxicity is a 90 -day repeated dose toxicity study (Grosz, 2013).

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. The NOEL is 120 mg/kg/day for males and 40 mg/kg/day for females. No immunotoxicity was detected at any dose level up to and including 480 mg/kg/day.

The key study for repeated dose inhalation toxicity was performed in a 2-week inhalation study in rats (Dupont, 2005); this study was conducted according to GLP standards. Four groups of 10 male rats were exposed to aerosol atmospheres of 3G0 (1.9-2.4 µm MMAD) at 0 (control), 10+0.35,300+5.8 or 1000+19 mg/m3. Animals were exposed nose only, 6 hours a day for a total of 9 exposures over a 2-week period. The dosing period was followed by a recovery period in half of the animals. No test substance related effects were observed in clinical observations, body weights, clinical pathology, gross necropsy observations and microscopic pathology. Based upon the observation of no significant effects following exposure to 3G0, the no observed- Adverse effect level (NOAEL) for a 2-week repeated inhalation exposure was 1000 mg/m3for male rats.

 



Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The NOAEL determined in this study is the lowest one detected. Additionally, the exposure duration was the longest in this study.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Only one study available.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Based on the results of the available oral repeated dose toxicity studies (Van Tuyl, 2010; Grosz, 2013) and the available inhalation repeated dose toxicity study (Dupont, 2005) there is no need to classify the substance in accordance to criteria listed in the CLP Regulation (EC 1272/2008).