3,6,9-trioxaundecamethylene bis(2-ethylhexanoate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2009- 15 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD 422 guidelines and GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-ethylenedioxydiethyl bis(2-ethylhexanoate)

EC Number:

202-319-2

EC Name:

2,2'-ethylenedioxydiethyl bis(2-ethylhexanoate)

Cas Number:

94-28-0

Molecular formula:

C22H42O6

IUPAC Name:

2,2'-ethylenedioxydiethyl bis(2-ethylhexanoate)

Details on test material:

- Name of test material (as cited in study report): 2,2’-ethylenedioxydiethyl bis (2-ethylhexanoate)
- Substance type: Clear colourless liquid
- Physical state: Liquid

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 12 weeks.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5
animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material and paper as cage-enrichment were supplied.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment under laboratory conditions.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7- 21.8°C
- Humidity (%): 30 – 100%,
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the
bulk of the diet. No correction was made for the purity of the test substance. Elix water (approximately 15% in total) was added to aid
pelleting. The pellets were dried for approximately 24 hours at 35ºC before storage. The control animals, and animals in the
acclimatization received similarly prepared pellets but without the test substance.
DIET PREPARATION
- Rate of preparation of diet (frequency): Once weekly
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany).
- Storage temperature of food: Diets were kept in the diet store room in the animal house.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on Day 8 and Day 22 (4 and 18 September 2009) during the treatment phase according to a validated method
(NOTOX Project 491294).
The concentration accuracy of diet preparations was considered acceptable if the mean concentration accuracies were 80-120% of the
target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Diet preparations were considered stable
if the relative difference before and after storage was maximally 10%.
In addition, random samples were taken from all diet preparations and stored at ≤-15ºC for possible future analysis. Any remaining
samples were discarded after approval by the sponsor or at finalization of the study report. Sampling occurred as soon as possible after
drying the pellets. If the analytical determinations were not performed on the day of preparation, the samples were stored at ≤-15ºC.
To check cleaning procedures, a diet residue test was performed once during the study.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-48
days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Ad libitum.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1500, 5000 and 15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
oral versus dermal: The rationale for the choice of an oral test versus a dermal test was based on the reliability of an oral test towards
prediction of systemic toxicity. Additionally, absorption of the test substance is more certain in an oral versus a dermal test which enables
extrapolation towards other routes of exposure.
diet versus gavage: Before start of the study trial formulations were performed at NOTOX to select an appropriate vehicle for the test
substance. Based on these trial formulations, the most appropriate vehicle was corn oil. With this information, the analytical department
tried to set up an analytical method to analyse the formulations of the test substance in the vehicle, but unfortunately no accurate
measurement could be performed. The corn oil in the GC method resulted in contamination, carry over and peak formation at the test
substance retention time. Dilution of the formulation before analysis resulted in better results for the highest dose level, but at lower
concentrations still no accurate results could be obtained. Other vehicles like ethanol or acetone might have been suitable for the
analytical method, but are not preferred in toxicity studies as these. In consultation with the Sponsor it was therefore decided to change
the route of exposure from gavage to diet before start of the study.
Rationale dose levels: Dose levels were selected based on results of the dose range finding study (NOTOX Project 491633).

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily; at least immediately after dosing. Once prior to start of treatment and at weekly intervals this was
also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or
housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females
were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION : Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food
consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

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: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes iso-flurane
- Animals fasted: yes, but water was available
- How many animals: 5 males/group (random) and all females with live pups
- Parameters examined were: white blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils,
basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean
corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated Partial thromboplastin
time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Animals fasted: yes, but water available
- How many animals: 5 males/group (random) and all females with live pups
- Parameters examined were: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total
bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested
during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention
being paid to the reproductive organs. The number of former implantation sites and corpora lutea was recorded for all paired females.

From all animals, samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate
buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Identification marks (not processed), Cervix, Clitoral gland, Epididymides*, Ovaries, Preputial gland, Prostate gland, Seminal vesicles
including coagulating gland, Testes*, Uterus, Vagina, All gross lesions.
From 5 males/group (random) and all females with live pups, samples of the following tissues and organs were collected and fixed in
addition to the abovementioned list:
Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Colon, Duodenum, Eyes (including optic nerve and Harderian
gland)*, Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, Lacrimal gland (exorbital), Larynx, Liver,
Lung (infused with formalin), Lymph nodes (mandibular, mesenteric), Nasopharynx, Oesophagus, Pancreas, Peyer's patches (jejunum,
ileum) if detectable, Pituitary gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Skeletal muscle, Skin, Spinal cord
(cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Thymus, Thyroid including parathyroid (if detectable),
Tongue, Trachea, Urinary bladder.
*Fixed in modified Davidson's solution and transferred to formalin after fixation for at least 24 hours.

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from 5 males/group (random) and all females with live pups on the
scheduled day of necropsy:
Adrenal glands, Brain, Epididymides (all males), Heart, Kidneys, Liver, Ovaries Spleen, Testes (all males), Thymus, Uterus (including
cervix), Prostate*, Seminal vesicles including coagulating glands*, Thyroid including parathyroid*
* weighed when fixed for at least 24 hours.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of 5 selected animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal
vesicles, testis, uterus, and vagina) of all animals that failed to mate, conceive, sire or deliver healthy pups.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance
estimate was applied for the comparison of the treated groups and the control groups for each sex. (Dunnett C.W., 1955)
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Miller R.G., 1981)
- The Fisher Exact-test was applied to frequency data. (Fisher R.A., 1950)

The following additional methods of statistical analysis were used:
The numbers of corpora lutea and implantation sites were transformed by using log x and x2, respectively, to obtain a normal distribution.
This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the
comparison of the treated groups and the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the l owest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test
statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations
may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet
display different test statistics values.

No statistical analysis was performed on histopathology findings.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No mortality occurred during the study period.
No clinical signs of toxicity were noted during the observation period.

BODY WEIGHT AND WEIGHT GAIN:
For high dose males, body weight was statistically significantly decreased on the last day of treatment. In addition, at 15000 ppm, body
weight gain was statistically significantly decreased for males during the complete treatment period and for females during pre-mating
and mating. A slight decrease (not statistically significant) was also observed in these females during lactation. On Day 5 of pre-mating,
almost all females of the high dose group showed a body weight loss (statistically significant).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
No statistically significant changes in food consumption before or after allowance for body weight were noted.
The mean test article intake values (mg substance/kg body weight/day) are summarized in the following table.

Mean test article intake (mg test article/kg body weight/day) based on nominal dose level:
Group 2 Group 3 Group 4
MALES
Pre-mating 105 358 1073
Mating 91 314 977
FEMALES
Pre-mating 122 397 1360
Post-coitum 120 382 1256
Lactation 161 576 1563

HAEMATOLOGY
For females treated at 15000 ppm, the percentage of neutrophils was decreased and prothrombin time was increased (statistically significant). The statistically significantly decreased values for neutrophils in 15000 ppm dosed females (20.2%) was due to a coincidental high value in the control group (27.1%) and considered unrelated to treatment as the values remained within the normal range (historical control data mean 23.0 ± 5.25%, P5 14.9%, P95 30.7%, years 2008-2010, N = 75). No toxicologically relevant changes occurred in haematological parameters of treated male rats.
Additionally, there were no changes in total white blood count, lymphocyte or any other blood cell type in both sexes. The findings on neutrophils are therefore considered coincidental.

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
• Increased glucose levels for high dose males
• Decreased sodium levels for high dose males
• Increased potassium levels for high dose males and females
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats at 1500 and 5000 ppm.

NEUROBEHAVIOUR
Females treated at 15000 ppm showed decreased motor activity at the low sensor throughout the entire 12-hour recording period
(statistically significant). For males, the variation in motor activity did not suggest a relation with treatment.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

ORGAN WEIGHTS
The following statistically significant changes in organ weights distinguished treated animals from control animals:
• Decreased terminal body weights for high dose males
• Increased liver:body weight ratios for high dose males
• Increased liver weights and liver:body weight ratios for mid and high dose females
• Increased kidney:body weight ratios for mid and high dose males and high dose females
• Slightly decreased thymus weights and thymus:body weight ratios for high dose females
The statistically significantly decreased thymus weights and thymus:body weight ratios for high dose females (0.133g and 0.059g%, respectively) were considered slight as the values remained within the normal range (historical control data for thymus weights: mean 0.190+/- 0.050g, P5 0.114g, P95 0.285g, years 2008-2010, N=78, historical control data for thymus weight-body weight ratios: mean 0.0840 +/- 0.021g%, P5 0.050g%, P95 0.116g, years 2008-2010, N=76). Additionally thymus atrophy is a common change in laboratory rodents, which might be a secondary finding to the toxicity in the same dose group (Greaves P. Histopathology of preclinical toxicity studies, 2008). It is proposed to evaluate these parameters again in the 90-day study to see whether the are consistent or not.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic findings related to treatment were recorded in the adrenal glands, kidneys, liver, pituitary gland, spleen, thymus and thyroid
glands.

These consisted of:
Adrenal glands: Multifocal vacuolation in zona glomerulosa at increased incidence in group 4 (15000 ppm) females. Recorded instances
were minimal or slight degree in two group 1 (0 ppm control), minimal in one group 2 (1500 ppm), minimal or slight in three group 3 (5000
ppm) and slight in four group 4 (15000 ppm) animals. This was not statistically significant, however there was a positive trend.
No effects on the adrenal glands were noted for males.

Kidneys: Cortical hyaline droplets were increased in incidence and severity in group 4 males. Minimal degree, two in group 1 and one in
each of groups 2 and 3 and slight or moderate degree in all five group 4. This was statistically significant in group 4 with a positive trend.
In addition a slight degree of granular casts were recorded in one group 4 animal.
Corticomedullary tubular basophilia was seen in one group 1 (minimal), three group 3 (minimal) and at minimal to moderate degree in
three group 4 animals. This was not statistically significant, however there was a positive trend.
No effects on the kidneys were noted for females.

Liver: In the liver of both sexes diffuse midzonal/centrilobular hypertrophy was recorded at minimal or slight degree in all ten animals of
group 4.
This was statistically significant in both sexes with a positive trend.

Pituitary gland: Adenohypophyseal multifocal hypertrophy was noted at minimal degree in two group 1 and 2, at minimal or slight in four
group 3 and in all five group 4 males at minimal or slight severity.
This was weakly statistically significant in group 4, however there was a positive trend.
No effects on the pituitary glands were noted for females.

Spleen: Hemosiderin pigment was slightly increased in severity in group 4 females - five instances at minimal or slight degree in group 1,
minimal in three group 2, minimal or moderate degree in four group 3 and at slight or moderate degree in all five group 4 females. This
was weakly statistically significant in group 4, however there was a positive trend.
Primarily erythroid hemopoietic foci were reduced in group 4 females - minimal to moderate in four group 1, slight or moderate in five
group 2, minimal or slight in five group 3 and none in group 4. This was statistically significant in group 4 with a positive trend.
No effects on the spleen were noted for males.
On the one hand, extramedullary haematopoiesis is normal in rats, but its degree can vary considerably under a variaty of circumstances. In rodents, many of these stimili are non-specific and occur sporadically. On the other hand, iron pigment (haemosiderin) is commonly seen in the spleen of rats, largely in the red pulp. Increased iron deposition in the spleen may indicate changes of red blood cell turnover with decreases in haemoglobin levels. (Graeves P. Histopathology of preclinical toxicity studies, 2008). There were however no haematological changes in haemoglobin, red blood cell number or haematocrit after 4 weeks of dosing. It is proposed to evaluate these parameters again in the 90-day study to see whether these findings are consistent or not.

Thymus: Lymphoid atrophy (involution) was slightly increased in incidence and severity in group 4 females - minimal in two group 1,
minimal or slight in three group 2, minimal in one group 3 and minimal or slight in four group 4. This was not statistically significant.
No effects on the thymus were noted for males.

Thyroid glands: Diffuse follicular hypertrophy/hyperplasia was slightly increased in group 4 animals of both sexes - minimal in one group
1, minimal in two group 2, slight in one group 3 and minimal or slight in three group 4 males. In females - minimal degree in three group 4
only. This was not statistically significant in males or females, however there was a positive trend in females.

The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar Han rats of this age
in this type of study and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with 2,2’-ethylenedioxydiethyl bis (2-ethylhexanoate) by dietary administration in male and female Wistar Han
rats at dose levels of 1500, 5000 and 15000 ppm revealed parental toxicity at 15000 ppm.

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 5000 ppm was derived.
When corrected for mean test article intakethe NOAEL of 5000 ppm corresponds to 314-576 mg/kg body weight/day.

Executive summary:

Chemical analysis showed that the diets were prepared accurately and homogenously, and were stable for at least 14 days at room temperature.

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 weights for 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)) and thyroid glands (diffuse follicular hypertrophy/hyperplasia) for males and/or females.

At 5000 ppm, increased liver weights (females) and kidney weights (males) were noted. In the absence of microscopic findings, these changes were not considered toxicologically significant.

No treatment-related toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, food consumption, and macroscopic examination).

CONCLUSION

In conclusion, treatment with 2,2’-ethylenedioxydiethyl bis (2-ethylhexanoate) by dietary administration in male and female Wistar Han rats at dose levels of 1500, 5000 and 15000 ppm revealed parental toxicity at 15000 ppm. Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 5000 ppm was derived. When corrected for mean test article intakethe NOAEL of 5000 ppm corresponds to 314-576 mg/kg body weight/day.