Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
- Analytical purity: 98.68%
- Lot/batch No.: 9X010
- Expiration date of the lot/batch: 15 June 2010 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals 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

IN-LIFE DATES: 26 August 2009- 15 October 2009

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DIET:
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.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 13 days was allowed for mating.
- Proof of pregnancy: sperm in vaginal smear, staging of the oestrus cycle and/or or intravaginal copulatory plug referred to as day 0 of
pregnancy.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no
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
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1500, 5000, 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

Parental animals: Observations and examinations:
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
Oestrous cyclicity (parental animals):
not determined (not required)
Sperm parameters (parental animals):
Parameters examined in all male parental animals:
testis weight, epididymis weight.
in addition, for 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural
abnormalities.

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals on lactation days 5-7 .
- Females which failed to deliver: Post-coitum Day 27 (evidence of mating) or 22 days after the last day of the mating period (without
evidence of mating).

GROSS NECROPSY
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.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If
possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for
possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
no
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.
Reproductive indices:
Percentage mating males: Number of males mated/Number of males paired x 100
Percentage mating females Number of females mated/Number of females paired x 100
Fertility index males Number of males generating a pregnancy/Number of males paired x 100
Fertility index females Number of pregnant females/Number of females paired x 100
Conception rate Number of pregnant females/Number of females mated x 100
Gestation index Number of females bearing live pups/Number of pregnant females x 100
Duration of gestation Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x
100
Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x
100
Viability index: Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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

BODY WEIGHT AND WEIGHT GAIN (parental animals):
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/parental animals)):
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

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Percentage mating, fertility index, conception rate, precoital time, and number of corpora lutea and implantation sites were unaffected by
treatment.
There were a total of 16 animals which failed to mate, conceive or deliver healthy offspring which were distributed amongst all dose
groups. The number of pregnant animals was 7 in the control group, 9 in the low dose group and 8 in the mid and high dose group.
There was no relation to treatment as the lowest number of pregnant animals was in the control group.

ORGAN WEIGHTS (parental animals)
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 (parental animals)
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

HISTOPATHOLOGY (parental animals)
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 (P0)

Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No effect. Three pups of the mid dose group were found to be missing on Days 3 or 4 of Lactation, and were most probably cannibalised.
These pups were observed and weighed up to these days and no cause was established. In addition, at the incidence observed no
relationship with treatment could be established for these deaths.

CLINICAL SIGNS (OFFSPRING)
No effect. Incidental clinical symptoms of pups consisted of small size, pale appearance and a wound or scabs on the head. No
relationship with treatment was established for these observations and they were considered to be of no toxicological significance.

BODY WEIGHT (OFFSPRING)
No effect.

GROSS PATHOLOGY (OFFSPRING)
No effect. Incidental macroscopic findings of pups included small size, absence of milk in the stomach, and a wound on the head. No
relationship with treatment was established for these findings and they were considered to be of no toxicological significance.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity was observed at any dose level.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
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. No reproduction and developmental toxicity was observed
for treatment up to 15000 ppm.

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.
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of 2,2’-ethylenedioxydiethyl bis (2-ethylhexanoate) in rats by dietary administration.

Parental results:

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).

Reproductive/Developmental results:

No reproductive/developmental toxicity was observedat any dose level.

At the control group, out of ten paired females only seven were pregnant. The guideline mentions that eight pregnant females per group normally is the minimum acceptable number of pregnant females per group. However, seven for the control group was considered sufficient for meaningful evaluation of the potential of the test substance to affect reproduction and development as historical control data was available.

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. No reproduction and developmental toxicity was observed for treatment up to 15000 ppm. Based on these results, a parental NOAEL of 5000 ppm (corresponding to 314 -576 mg/kg bw) was derived. The reproductive and developmental NOAEL was at least 15000 ppm (corresponding to 977 -1563 mg/kg bw).

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.