Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

An Extended One Generation Reproductive Toxicity Study in rats according to OECD Test Guidance 443 (basic study design incl. Cohorts 1A, 1B with extension to F2 generation) was conducted with the registered substance at dietary target concentrations of 0, 83, 250, 750 mg/kg bw/day. Based on the results, the parental NOAEL for both F0-generation and F1-generation was 250 mg/kg/day. The dose of 750 mg/kg/day was considered to be adverse based on the combination of the observed lower food efficiency (males and females of both generations), together with effects on kidneys (F0- and F1-males only) and liver (males and females of both generations) which manifested themselves by higher organ weights of kidneys and/or liver, increased incidence and severity of hyaline droplets accumulation and tubular basophilia in the kidneys, and changes in biochemical chemistry parameters specific for kidney and/or liver. The Reproduction NOAEL for both F0-generation and F1-generation is at least 750 mg/kg/day. The Developmental NOAEL for both F1-generations and F2-generation is at least 750 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

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
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
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
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
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
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
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
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.
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.
Reproductive effects observed:
not specified
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.

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental start date was 01 Feb 2018 and the experimental completion date was 02 Jul 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
June 2018
Qualifier:
according to
Guideline:
other: OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151
Version / remarks:
July 2013
Principles of method if other than guideline:
To reduce variability among the litters, on postnatal day 4 eight pups from each litter of equal sex distribution (if possible) were selected for F1 and F2 Generation. This is lower than specified by OECD 443 (as nealry as possible to 5 of both sexes); the strain used does not allow to reach 5 pups of each sex in the litters.
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:
- Premating exposure duration for parental (P0) animals: 10 weeks prior to mating
- Basis for dose level selection: Dietary concentrations in this study were adjusted regularly to maintain the target test item intake of 0, 83, 250, 750 mg/kg bw/day, based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test) with dietary exposure of 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) in rats, Test Facility Study No. 491295.
- exclusion of extension of Cohort 1B
- Termination time for F2: on PND 21-23
- exclusion of developmental neurotoxicity Cohorts 2A and 2B
- exclusion of developmental immunotoxicity Cohort 3
- Route of administration: oral: feed
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental/neurological/immunological historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
The test item was administered to the appropriate animals by inclusion in the diet ad libitum.

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Chemical Company Batch 9490701
- Expiration date of the lot/batch: Not provided (Stability of test item under storage conditions at least 6 months. Dates of analysis: 30 March 2018 (t=0) and 04 Oct 2018 (T=6 months).
- Purity test date: Not provided (Ship date 13 NOV 2017)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: Stability in pelleted diet (SM R/M-Z ): Stability for at least 17 days at room temperature and at least 17 days in the freezer (≤ -15°C) is confirmed over the concentration range 500 ppm to 15000 ppm (Test Facility Study No. 20134673).
Stability of test item under storage conditions at least 6 months. Dates of analysis: 30 March 2018 (t=0) and 04 Oct 2018 (T=6 months).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used to prepare pelleted diets. The test item was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. Diets were prepared freshly for use at room temperature for a maximum of 17 days. Diets were kept in the freezer (≤ - 15°C) until use for a maximum of 17 days, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 17 days (stability was confirmed under Test Facility Study No. 20134673, i.e. Analytical Method Development and Validation Study) for supplementing food during the respective food consumption measurement interval.

OTHER SPECIFICS:
- other information: Manufacturing date 02 November 2017
Species:
rat
Strain:
Wistar
Remarks:
Han
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental/neurological/immunological historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: On 31 Jan 2018, male and female Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation:
(P) At initiation of administration, animals were 6 weeks old;
(F1) x wks
- Weight at study initiation:
(P) At initiation of administration, animals weighed between 130 and 169 g (males) and between 105 and 140 g (females).
(F1) Males: 36-65 g; Females: 36-61 g
- Fasting period before study: no
- Housing: On arrival, prior to mating and during the post-weaning period (Cohorts 1A, 1B and 1C), animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination (Cohort Surplus and Spares) or weaning (on PND 21; Cohorts 1A, 1B and 1C).
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labelled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): Prepared diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to similarly prepared pellets without the test item (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: The F0-animals were allowed to acclimate to the Test Facility toxicology accommodation for 5 days before the commencement of administration.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target temperatures of 18 to 24°C; actual daily mean temperature during the study period was 21 to 23°C
- Humidity (%): relative target humidity of 40 to 70%; actual daily mean relative humidity of 34 to 65%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures.

IN-LIFE DATES: From: 05 Feb 2018 To: 24 Oct 2018

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
Trial preparations were performed to establish a suitable diet preparation procedure. These trials were performed as part of the Analytical Method Development and Validation Study (Test Facility Study No. 20134673) and these diets were not used for administration. Raw data of these trials are retained by the Test Facility.
- Rate of preparation of diet (frequency):
Diets were prepared freshly for use at room temperature for a maximum of 17 days.
- Mixing appropriate amounts with (Type of food):
Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used to prepare pelleted diets. The test item was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test item.
The amount of test item incorporated in the diet was adjusted to approximate a constant intake in terms of mg test item per kg body weight.
-F0-Generation
Study Weeks 1 and 2: When preparing the test diets for use in the first two weeks of the study, the previous intake of the test diet by males and females was unknown, as it was not measured during the acclimatization period. Therefore, concentration of test item in the test diet were determined using a fixed factor based on historical control data for relative food consumption. In Week 1 the amount of test item incorporated in the diet was 830, 2500 and 7500 ppm to achieve a target test item intake of approximately 83, 250 and 750 mg/kg bw/day (both sexes). In Week 2, the amount of test item incorporated in the diet was increased by 5% as compared to Week 1 (both sexes), based on historical food consumption data for rats of the same age.
From study Week 3 onwards until start of mating: The latest group mean body weights and group mean food consumption (treating males and females separately) were used to predict expected food consumption and body weight and to calculate the amount of test substance to be included in the diet to achieve the required intake.
From start of the mating period onwards until end of gestation: Females were given diets with a fixed concentration of test item. Selected dietary concentrations were based on test article intake until Week 9 of the study.
As long as a male was cohabited with a female for mating, he had access to the same diet as the female. Once mating had occurred, each male was separated from his female and placed back into his home cage. From that moment onwards, he had access to the ‘male diet’. Dietary concentration of this ‘male diet’ were adjusted weekly based on the latest available data on test article intake for males and projected body weight and food consumption changes over the next week.
During lactation:
Dietary concentrations used during the lactation period were calculated based on available historical control data from the Test Facility. As a starting point the dietary concentrations administered during the post-coitum period were used (i.e. Gr. 2: 1204 ppm; Gr. 3: 3589 ppm; Gr. 4: 10251 ppm). These concentrations were lowered by a factor of 1.41x (lactation Days 1-4), 1.83x (lactation Days 4-7) and 2.26x (lactation Days 7-21) to correct for the higher food intake during lactation as compared to the relative food intake during gestation Days 7-11.
After weaning: F0-females continued on the same diet used for lactation Days 7-21 up to and including the day prior to scheduled necropsy.
-F1-Generation (Cohorts 1A, 1B, 1C)
Similar to the F0-generation, also for F1-animals in Cohorts 1A, 1B and 1C dietary concentrations were adjusted on a regular basis based on latest data on test article intake and projected body weight and food consumption changes over the next week to achieve a target test item intake of approximately 83, 250 and 750 mg/kg/day bw (both sexes). However, when preparing the diets for use in the first three weeks after start of weaning on 29 May 2018 (PND 21), insufficient data on test article intake was available. Therefore, the following approach was used:
As a starting point, dietary concentrations were taken which were administered to the F0-generation during the first week of the study (i.e. Gr. 2: 830 ppm; Gr. 3: 2500 ppm; Gr. 4: 7500 ppm). It should be noted that F0-animals were 5-6 weeks old at start of treatment, while animals of the F1-generation were treated with the test diets from weaning on PND 21 onwards. At this younger age relative food consumption (g/kg body weight/day) was expected to be higher. To correct for this the dietary concentrations for F1-animals in the first week of treatment was lowered by a factor of 1.21x. This factor was obtained by extrapolating historical control data for male Wistar Han rats from the Test Facility. In the second week, slightly higher concentrations were applied. The same dietary concentrations were used for both sexes although relative food consumption was expected to be slightly lower in F1-females than in F1-males at start of treatment (PND 21).
In the third week after weaning, i.e. when first F1-animals reached the age of 5 weeks, dietary concentrations for Groups 2 and 3 were increased to the same concentrations as were used for 5-6 weeks old F0-animals at start of the study. For Group 4, a slightly lower concentration was used based on data from the F0-generation.
To select dietary concentrations from Week 4 onwards, dietary concentrations administered to F0-animals at comparable age were taken as a starting point, but also actual test article intake in the F0-generation and F1-generation were taken into consideration.
From start of the mating period onwards until end of gestation, females of Cohort 1B were given diets with a fixed concentration of test item. Selected dietary concentrations were based on test article intake until Week 12 of the pre-mating period.
As long as a Cohort 1B male was cohabited with a Cohort 1B female for mating, he had access to the same diet as the female. Once mating had occurred, each male was separated from his female and placed back into his home cage. From that moment onwards, he had access to the ‘male diet’. Dietary concentration of this ‘male diet’ was adjusted weekly based on the latest available data on test article intake for males and projected body weight and food consumption changes over the next week.
Dietary concentrations to be used for Cohort 1B females during the lactation period were calculated based on available historical control data from the Test Facility. As a starting point the dietary concentrations administered during the post-coitum period were used (i.e. Gr. 2: 1110 ppm; Gr. 3: 3193 ppm; Gr. 4: 9297 ppm). These concentrations were lowered by a factor of 1.41x (lactation Days 1-4), 1.83x (lactation Days 4-7) and 2.26x (lactation Days 7-21) to correct for the higher food intake relative to body weight during lactation as compared to the relative food intake during gestation Days 7-11.
-F1-Generation (Cohort Surplus and pups of spare litters)
As pups of Cohort Surplus and pups of Spare Litters remained with their mothers until scheduled necropsy, they had access to the same dietary concentrations used for lactation Days 7-21 (see also information above under F0-Generation – after weaning).
- Storage temperature of food:
Diets were kept in the freezer (≤ - 15°C) until use for a maximum of 17 days, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 17 days (stability was confirmed under Test Facility Study No. 20134673, i.e. Analytical Method Development and Validation Study) for supplementing food during the respective food consumption measurement interval.

Details of the preparation and dispensing of the test item have been retained in the Study Records.
Details on mating procedure:
- M/F ratio per cage: After 10 weeks of treatment (pre-mating period), animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating.
- Length of cohabitation: Once mating had occurred, the males and females were separated.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
Detection of mating was not confirmed in first instance for Female No. 171. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
- A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (type III, height 18 cm)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Six samples (approximately 5 g) were taken at middle position from diet preparations for Groups 1 and 3 (concentration analysis only) and duplicate samples (approximately 5 g) were taken at top, middle, and bottom position from diet preparations for Groups 2 and 4 (concentration and homogeneity analysis). Sampling occurred as soon as possible after drying the diet pellets. If the analytical determinations were not performed on the day of preparation, diet samples were stored at ≤ - 15ºC for a maximum of 17 days.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Duplicate sets of samples (approximately 5 g) for each sampling time point were taken on the same day samples were collected for analyses and retained at the Test Facility (≤ - 15ºC) as backup samples. After acceptance of the analytical results, backup samples were discarded.
Duration of treatment / exposure:
The test item was administered to the appropriate animals by inclusion in the diet ad libitum.
F0-males were treated for 12 weeks, including 10 weeks prior to mating (with the objective of covering at least one spermatogenic cycle) and during the mating period, up to and including the day before scheduled necropsy. F0-females were treated for a minimum of 16 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy.
During lactation (up to PND 21), pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, spilled diet from the food hopper or when they commenced eating for themselves.
From weaning onwards (PND 21), F1-animals were treated up to and including the day before scheduled necropsy (Cohort 1A) or until the day of necropsy (Cohorts 1C, Surplus). The Spare animals (not assigned to one of the cohorts) were not treated.
The F1-animals of Cohort 1B were treated from weaning onwards, including at least 10 weeks prior to their mating period, the duration of pregnancy and at least 21 days after delivery up to and including the day of scheduled necropsy (i.e. for a minimum of 15 weeks).
Frequency of treatment:
continuous in the feed
Details on study schedule:
- F1 parental animals not mated until at least 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study:
F0: at least 15-16 weeks; F1 Cohort 1B: at least 13weeks.
Dose / conc.:
83 mg/kg bw/day
Remarks:
target test item intake
Dose / conc.:
250 mg/kg bw/day
Remarks:
target test item intake
Dose / conc.:
750 mg/kg bw/day
Remarks:
target test item intake
No. of animals per sex per dose:
F0: 25
F1 Cohort 1A: 20
F1 Cohort 1B: 20
F1 Cohort C: 20
F1 Surplus: 10 (Not treated)
Control animals:
other: The control animals received similarly prepared pellets but without the test item.
Details on study design:
- Dose selection rationale:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with dietary exposure of 2,2’-Ethylenedioxydiethyl bis(2-Ethylhexanoate) in rats (Test Facility Study No. 491295, data on file at Sponsor site) and in an attempt to produce graded responses to the test item.
In that repro-screening study, four groups of ten male and ten female Wistar Han rats were exposed by dietary administration to the test substance at 1500, 5000 and 15000 ppm. 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.
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, and 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 1500 and 5000 ppm, no parental toxicity was noted. The parental NOAEL of 5000 ppm corresponded to 314-576 mg/kg bw/day. No reproductive/developmental toxicity was observed at any dose level. As such a reproductive and developmental NOAEL of 15000 ppm was established, corresponding to 977-1563 mg/kg bw/day.
Based on these results, and taking into account the higher susceptibility to toxic insults of very young (non-adult) animals and the longer duration of exposure in the current study, intended test item intake values of 83, 250, 750 mg/kg bw/day, respectively, are selected for the current study. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Rationale for animal assignment (if not random):
F0-animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected to reduce variability among the litters. Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable. The non-selected pups were culled on PND 4.
On PND 21, F1-pups from available litters per group were selected and assigned according to the following schedule. Litters were selected and approved by the Study Director in the study files. The selected female numbers were specified in the report.
- Fasting period before blood sampling for clinical biochemistry:
The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
- Other:
The oral route of administration via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Animals showing pain, distress or discomfort which was considered not transient in nature or was judged likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.
Clinical observations were performed at least once daily, beginning on the day of first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy (see deviation in Appendix 14).
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
- Cage side observations: Appendix 2: 2.1 (mortality data); 2.2 (clinical signs).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations:
Animals were weighed individually on the first day of treatment (prior to administration), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
A terminal weight was recorded on the day of scheduled necropsy.
Appendix 2: 2.3 Body weights; 2.4 Body weight gain

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured 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 during lactation on PND 1, 4, 7, 14 and 21.
Appendix 2: 2.5 Food consumption (g/animal/day); 2.6 Relative Food consumption ( g/kg bw/day)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The actual test substance intake for each period (pre-mating, post-mating, post-coitum and lactation) was estimated based on body weight and food consumption values during the same interval and expressed as: mg test item/kg body weight/day.
Note: No correction was made for the purity of the test item when calculating concentrations of the preparations.
Appendix 2: 2.7 Test item intake (mg substance/kg bw/day)

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect of treatment on water consumption was suspected.


OTHER: GENERAL REPRODUCTION DATA F0-GENERATION
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.



Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken (except for female 124).
Sperm parameters (parental animals):
Parameters examined in male parental generations:
For all surviving P (F0) males, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right side) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (left) was removed, placed in labelled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
As for animal no. 19 (Group 1), abnormalities were noted in the left epididymis, the left side organ(s) was fixed in modified Davidson's solution, and the right side organ was used for evaluation of sperm numbers.
Litter observations:
STANDARDISATION OF LITTERS (CULLING – F1 AND F2-GENERATION)
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done.
- If yes, maximum of 4/sex/litter as nearly as possible.
Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
Non-selected F1-animals from litters used for cohort assignment were sacrificed on PND 22 and spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by oral administration of intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). Animals were externally examined, with particular attention to the external reproductive genitals to examined signs of altered development, and sex was determined (both externally and internally, if possible). Descriptions of all external abnormalities were recorded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
MORTALITY/MORIBUNDITY CHECKS – F1 AND F2-GENERATION
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Pups showing pain, distress or discomfort which was considered not transient in nature or was judged likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.
CLINICAL OBSERVATIONS – F1 AND F2-GENERATION
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.
BODY WEIGHTS – F1 AND F2-GENERATION
Live pups were weighed individually on PND 1, 4, 7, and weekly thereafter until all pups reached PND 21.
For animals of Cohort Surplus, Cohort 1A and F2-animals of Cohort 1B (10 selected litters/group; one male and one female), a terminal weight was recorded on the day of scheduled necropsy.
SEX – F1 AND F2-GENERATION
Sex was externally determined for all pups on PND 1 and 4.
ANOGENITAL DISTANCE – F1 AND F2-GENERATION
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
AREOLA/NIPPLE RETENTION– F1 AND F2-GENERATION
All male pups in each litter were examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
TERMINAL PROCEDURES – F1 AND F2-GENERATION UNTIL WEANING (PND 21)
-Unscheduled Deaths – F1 and F2-Generation
Pups that were sacrificed in extremis, younger than 7 days, were euthanized by decapitation.
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology.
All pups that were killed in extremis or found dead until 29 May 2018 were preserved in 10% buffered formalin. From 29 May 2018 onwards, preterm sacrificed pups were not preserved by default. Only external abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. Descriptions of all external abnormalities were recorded.
The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, developmental defects and/or cause of death were evaluated.
-Culled Pups (PND 4) – F1 and F2-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood was collected, if possible.
Sex was determined both externally and internally (if possible). Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.
-Scheduled Euthanasia – F2-Generation
Scheduled necropsy of the F2-animals of Cohort 1B was conducted on PND 21-23. These animals were sacrificed using Euthasol®20% by intraperitoneal (ip) injection. The animals were not fasted overnight.
From 10 selected litters/group, terminal body weight was determined for one male and one female pup. Subsequently, these pups were deeply anaesthetized using isoflurane and subsequently exsanguinated. The animals were subjected to a limited examination, with special focus on macroscopically visible abnormalities in the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table were weighed and collected. The selected litters were documented in the study files by the study director in advance.
All remaining pups were sacrificed using Euthasol®20% by intraperitoneal (ip) injection. Also these pups were subjected to a limited gross pathological examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Postmortem examinations (parental animals):
SACRIFICE F0 GENERATION
- Male animals: All surviving animals after successful mating and a minimum of 12 weeks of treatment.
- Maternal animals: All surviving animals
Females which delivered: LD 23-25
Females which failed to deliver:
With evidence of mating: Post-coitum Days 25-27.
Without evidence of mating: 24 days after the last day of the mating period
All animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available ad libitum.
SACRIFICE F1 GENERATION COHORT 1B
- Male animals: All surviving animals following completion of the mating period.
- Maternal animals: All surviving animals
Females which delivered: LD 21-23
Females which failed to deliver:
With evidence of mating: Post-coitum Days 25-26.
Without evidence of mating: 24 days after the last day of the mating period
The F1-animals of Cohort 1B were not fasted overnight before necropsy. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated.

GROSS NECROPSY F0 GENERATION
- Gross necropsy consisted of external and internal examinations.
All animals were subjected to a full post mortem examination, with special focus on macroscopically visible abnormalities in the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
GROSS NECROPSY F1 GENERATION COHORT 1B
- Gross necropsy consisted of external and internal examinations
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea were recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights were weighed at necropsy for all sceduled euthanasia F0 and F1 animals. Organ weights were not recorded for male no. 5 euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated..
Histopathology for F0 was done on the full list of tissues for control and high dosed animals and on gross lesions, target tissues and reproductive tissues for the low and mid dosed animals. A full list was also done for unscheduled deaths.
Histopathology for F1 was done on the full list of tissues for control and high dosed animals of Cohort 1A and on gross lesions and target tissues for the low and mid dosed animals. Reproductive organs of group 1 nad 4 of Cholrt 1B were processed to block stage. Reproductive organs of infertile animals in Cohort 1B were also prepared in this group.

Postmortem examinations (offspring):
SACRIFICE
- Non-selected F1-animals from litters used for cohort assignment were sacrificed on PND 22 and spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24.
Scheduled necropsy of the F2-animals of Cohort 1B was conducted on PND 21-23.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
SCHEDULED EUTHANASIA – F2-GENERATION
From 10 selected litters/group, terminal body weight was determined for one male and one female pup. Subsequently, these pups were deeply anaesthetized using isoflurane and subsequently exsanguinated. The animals were subjected to a limited examination, with special focus on macroscopically visible abnormalities in the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation table were weighed and collected. The selected litters were documented in the study files by the study director in advance.
All remaining pups were sacrificed using Euthasol®20% by intraperitoneal (ip) injection. Also these pups were subjected to a limited gross pathological examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
TERMINAL PROCEDURES – F1-GENERATION FROM WEANING ONWARDS
No F1-animals died after weaning.
Non-selected F1-animals from litters used for cohort assignment were sacrificed on PND 22 and spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by oral administration of intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). Animals were externally examined, with particular attention to the external reproductive genitals to examined signs of altered development, and sex was determined (both externally and internally, if possible). Descriptions of all external abnormalities were recorded.
For all animals, necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available for consultation during the necropsy. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated. The organs identified for weighing and representative samples of the tissues mentioned in Appendix 12 were weighed and collected.

GROSS NECROPSY
- Gross necropsy of 10 selected litters/group (F2 Generation) consisted of limited examination, with special focus on macroscopically visible abnormalities in the reproductive organs.
All remaining pups (F2 Generation) were subjected to a limited gross pathological examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Non-selected F1-animals from litters used for cohort assignment and spare F1-animals which were not assigned to one of the Cohorts were externally examined, with particular attention to the external reproductive genitals to examined signs of altered development, and sex was determined (both externally and internally, if possible).

HISTOPATHOLOGY / ORGAN WEIGTHS
See under "Any other information on materials and methods incl. tables".
Table 3. Tissue Collection and Preservation for Cohort 1A
Table 4. Tissue Collection and Preservation for Cohort 1C
Table 5. Tissue Collection and Preservation for the F1-Generation (Cohort Surplus) and 10 selected F2-pups/sex/group.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data Collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
PARAMETRIC
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
NON-PARAMETRIC
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
INCIDENCE
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females and F1-females (Cohort 1B), the remaining group values were calculated from the total number in each group.
Mating index (%): (Number of females mated/ Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females/ Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1/ Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100

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

Viability index (%): (Number of live offspring on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100

Weaning index (%): (Number of live offspring on Day 21 after littering/ Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted in males and females up to 750 mg/kg/day during daily detailed clinical observations or during weekly arena observations.
Incidental findings that were noted in all groups, including controls, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be signs of toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was related to treatment with the test item.
One control male (No. 5) was euthanized in extremis one day before scheduled necropsy, after 12 weeks of treatment, due to a large superficial wound on his tail (taken from study daybook) which was considered to have been caused by an accident. No further observations were made for this animal.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of males at 750 mg/kg/day were slightly, but statistically significantly lower than controls from Week 5 of the pre-mating period onwards, lasting until the end of the mating period. Mean body weight gain was 0.90x and 0.91x of controls at the end of the pre-mating and mating period, respectively, resulting in a terminal body weight of 0.92x of controls.
Body weight (gain) in males up to 250 mg/kg/day and females up to 750 mg/kg/day remained in the same range as for the concurrent control group.
The statistically significantly lower body weights of females at 250 mg/kg/day, when compared to controls, at the end of the post-coitum period were considered to be unrelated to treatment as the differences were only minimal and no trend was apparent regarding dose and duration of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In both males and females, higher absolute and relative food consumption was observed in all treated groups from the pre-mating period onwards, until the end of the treatment period (not always statistically significant) with changes compared to the concurrent control group generally increasing with increasing dose. During the last two weeks of the lactation period, recovery to normal values was seen for females of all treated groups.
Mean test item intake over the different study periods with food consumption measurementis Mean of means, weighed for number of measurement intervals per period, remained within the nominal dietary inclusion level ± 11% for males and females treated up to 750 mg/kg/day.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
In both sexes there was a general trend towards higher food intake (absolute and relative to body weight) in all treated groups compared to the concurrent control group from start of treatment onwards. The delayed growth of high dose males in the presence of higher food intake is indicative for a lower food efficiency, i.e. more food has to be ingested to reach the same growth in the animal’s mass. This effect was less pronounced in females, who had body weight gains in the same range as concurrent controls throughout the whole treatment period. Importantly, food efficiency in lactating females recovered to normal values during the last two weeks of treatment (lactation Days 7-21; all treated groups).
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect of treatment on water consumption was suspected.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters of treated rats were considered not to have been affected by treatment.
Minor statistically significant differences in neutrophil count and mean corpuscular haemoglobin (MCH), arising between controls and females at 750 mg/kg/day were not biologically relevant as the magnitude of change was only minimal and/or values remained within the range of available internal control data.
Coagulation parameters of treated rats were not affected by treatment.
The statistically significantly longer activated partial thromboplastin time (APTT) of both males and females at 250 mg/kg/day was unrelated to treatment as mean values remained within the range of available internal control data , and the change was not dose-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes in clinical biochemistry parameters were observed only in females at 250 and 750 mg/kg.
The following statistically significant changes were noted in treated lactating females. Relative differences in mean values as compared to the control group are indicated between parentheses.
• Slightly increased mean plasma activity of alanine aminotransferase (ALAT) at 250 and 750 mg/kg/day (1.42x and 1.45x control level, respectively). Mean ALAT values remained within the normal range. However, at the individual level there was an increased number of females with higher activity of ALAT compared to the concurrent controls.
• Higher (3.3x) mean plasma concentration of bile acids at 750 mg/kg/day. Group mean value (102.4 µmol/L) was outside the range of available internal control data. The observed change in mean value could be attributed mainly to four high dose females (nos. 183, 187, 192 and 198) with values of 221.3, 362.0, 282.0 and 132.0 µmol/L (7.1x, 11.5x, 9.0x and 4.2x group mean control level), respectively. Also at the lower dose levels of 83 and 250 mg/kg/day incidentally higher values for bile acids were noted, but at a lower severity (Gr. 2 female no. 145: 209.9 µmol/L; Gr. 3 female nos. 168 and 170: 152.7 and 181.3 µmol/L, respectively).
It should be noted that not always the same set of parameters was affected in each female. For example, some females with increased bile acids showed normal values of ALAT, and vice versa. In addition, incidentally higher levels of ASAT and ALP, and/or higher liver organ weights (relative to body weight) were observed. As clearly more females were affected at 750 mg/kg/day than at 250 mg/kg, liver changes observed at the high dose were considered to be adverse.
Any remaining statistically significant changes in clinical biochemistry parameters observed (higher sodium in males and females of Groups 2, 3 and/or 4, higher chloride in males of Groups 2 and 3, and higher glucose in females of Group 3) were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses:
In lactating females at 750 mg/kg/day, serum levels of T4 were statistically significantly higher (1.36x) than those in controls. Except for one female (no. 183: 8.00 µg/dL), all individual values remained within the normal range . Serum levels of thyroid stimulating hormone (TSH) in F0-females were not affected by treatment.
Serum levels of both TSH and T4 in F0-males were unaffected by treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinary parameters of treated rats were considered not to have been affected by treatment.
The statistically significant difference in epithelial cells in males treated at 750 mg/kg/day was considered to be of no toxicological relevance, as values remained within the control range.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with 2,2’-Ethylenedioxydiethyl bis(2-Ethylhexanoate) were noted in the thyroid gland and kidneys of males.
Thyroid gland, an increased incidence and mean severity of follicular cell hypertrophy was present in males treated at 750 mg/kg/day.
Kidneys: an increased incidence and mean severity of hyaline droplet accumulation was present in males treated at 750 mg/kg/day.
Kidneys: an increased incidence and mean severity of tubular basophilia was present in males treated at 750 mg/kg/day.
One 750 mg/kg/day male showed minimal hepatocellular hypertrophy, which can occasionally be observed in control males as well. At the noted low incidence (1/25) and severity (minimal) this was considered not to be test item-related.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment up to 750 mg/kg/day.
All females had regular cycles of 4 to 5 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm motility, concentration and morphology were unaffected by treatment up to 750 mg/kg/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were 1/25 couples of the control group, 4/25 couples of the 83 mg/kg/day group, 2/25 couples of the 250 mg/kg/day group and 1/25 couples of the 750 mg/kg/day group without offspring. No histopathologically identified abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproduction Data – F0-Generation
• Sperm Analysis – F0-Generation
Sperm motility, concentration and morphology were unaffected by treatment up to 750 mg/kg/day.
• Estrous Cycle – F0-Generation
Length and regularity of the estrous cycle were not affected by treatment up to 750 mg/kg/day.
All females had regular cycles of 4 to 5 days.
• Mating Index – F0-Generation
Mating index was unaffected by treatment. The mating indices were 100% for the control and 750 mg/kg/day groups, and 92% for the 83 and 250 mg/kg/day groups.
Two females at 83 mg/kg/day (nos. 141 and 143) and two females at 250 mg/kg/day (nos. 163 and 175) were not mated. In the absence of a dose-relationship, this was considered unrelated to treatment.
• Precoital Time – F0-Generation
Precoital time was considered not to be affected by treatment.
All females showed evidence of mating within 4 days, except for 1, 2, 4, 1 females in the control, 83, 250 and 750 mg/kg/day group, respectively, for which mating took 12-14 days. In the absence of a dose-relationship, this was considered to be unrelated to treatment.
• Number of Implantation Sites – F0-Generation
Number of implantation sites was considered not to be affected by treatment.
One female at 83 mg/kg/day (no. 142), had a single implantation site only. As this occurred in absence of a dose response relationship, this was considered to be unrelated to treatment.
• Fertility Index – F0-Generation
Fertility index was considered not to be affected by treatment. The fertility indices were 96% for controls and females treated at 83 and 750 mg/kg/day, and 100% for females at 250 mg/kg/day.
The number of non-pregnant females versus mated females was 1/25, 1/23 and 1/25 in the control, 83 and 750 mg/kg/day groups, respectively. At 250 mg/kg/day all mated females were pregnant. As these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive toxicity, this was considered not to be related to treatment.
Developmental Data – F0-Generation
• Gestation Index and Duration – F0-Generation
Gestation index and duration of gestation were considered not to be affected by treatment.
All pregnant females had live offspring, except for one female at 83 mg/kg/day (no. 142) that had one implantation site only. The gestation index was 100% for the control, 250 and 750 mg/kg/day groups, and 95% for the 83 mg/kg/day group.
The failed pregnancy of female no. 142, without related histopathology changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.
• Parturition/Maternal Care – F0-Generation
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
• Post-Implantation Survival Index – F0-generation
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 95, 92 and 90% for the control, 83, 250 and 750 mg/kg/day groups, respectively.
For female no. 176 (750 mg/kg/day), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal remodelling of these areas of former implantations during the 21-23 days of lactation. No toxicological relevance was attached to this finding in the current study.
• Litter Size – F0-generation
Litter size was considered unaffected by treatment.
Mean litter sizes were 11.7, 12.3, 10.5 and 10.7 living pups/litter for the control, 83, 250 and 750 mg/kg/day groups, respectively.
• Sex Ratio – F1-pups
Sex ratio was considered not to be affected by treatment.
• Live Birth Index – F0-generation
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
Live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 100% for the control, 83 and 750 mg/kg/day groups, and 99% for the 250 mg/kg/day group.
One pup of the control group (litter no. 119) and two pups at 250 mg/kg/day (litter nos. 159 and 167) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
• Viability Index – F0-generation
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered not to be affected by treatment.
Viability indices were 99, 96, 99 and 98% for the control, 83, 250 and 750 mg/kg/day groups, respectively.
One pup of the control group (litter no. 107) was sacrificed in extremis on PND 1, based on observed paleness and absence of milk in the stomach, and two pups at 83 mg/kg/day (litter No. 149) were sacrificed in extremis on PND 4, based on lean appearance, dehydration and absence of milk in the stomach.
In addition, one pup of the control group (litter no. 119), 6 pups at 83 mg/kg/day (two in litter no. 137, one in litter no. 139 and three in litter no. 149), one pup at 250 mg/kg/day (litter no. 160) and 5 pups at 750 mg/kg/day (two in litter no. 181 and one in litter nos. 183, 193 and 200 each) were found dead or missing between PND 2 and 4. Pups missing were most likely cannibalised.
No toxicological relevance was attributed to these dead/missing pups as the mortality incidence did not show a dose-related trend and values remained well within the normal range of biological variation.
• Weaning Index – F0-generation
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.
One pup in the 83 mg/kg/day group (litter no. 145) was found missing on lactation Day 9 (most likely cannibalised), resulting in a weaning index of 99% for this low dose group compared to 100% for all remaining groups. In the absence of a dose-related trend, no toxicological relevance was attributed to this finding.
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: Higher serum levels of total T4 were observed in lactating females of the F0-generation at 750 mg/kg/day. Except for one F0-female, all individual values remained within the normal range.
Remarks:
Possible adversity of this effect could not be assessed within this type of study and was therefore not taken into account when determining the parental NOAEL.
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
F1-generation Data (Cohort 1A, 1B and 1C) from Weaning onwards: No treatment-related clinical signs were noted in males and females up to 750 mg/kg/day during daily detailed clinical observations or during weekly arena observations.
Incidental findings that were noted in all groups, including controls, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were no signs of toxicological relevance
Mortality:
no mortality observed
Description (incidence):
F1-generation Data (Cohort 1A, 1B and 1C) from Weaning onwards: No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1-generation Data (Cohort 1A, 1B and 1C) from Weaning onwards: A trend towards lower body weights and body weight gains was observed in males at 750 mg/kg/day. Differences in body weight compared to the concurrent control group were relatively slight, reaching statistical significance on Day 64 only (absolute value: 0.95x of control; Cohorts 1A and 1B combined). Mean terminal body weight of Cohort 1A males, treated for 11-12 weeks, was 0.94 of control (reaching statistical significance). In high dose males of Cohort 1B, mean body weight gain was 0.96x of controls at the end of both the premating and mating period, resulting in a mean terminal body weight of 0.95x of controls after 16 weeks of treatment (not statistically significant).
Body weight (gain) in males up to 250 mg/kg/day and females up to 750 mg/kg/day remained approximately in the same range as for the concurrent control group. Any statistically significant changes in body weight gain in these groups were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1-generation Data (Cohort 1A, 1B and 1C) from Weaning onwards: In both males and females, higher absolute and relative food consumption was observed in all treated groups from the second week of the treatment period onwards (not always statistically significant) with changes compared to the concurrent control group, generally increasing with increasing dose. In treated F1-females of Cohort 1B, relative food intake recovered to normal values during the period of lactation.
Mean test article intake of means, weighed for number of measurement intervals per period, remained within the nominal dietary inclusion level ± 6% for males and females treated up to 750 mg/kg/day.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Similar to the F0-generation, lower food efficiency was observed in all treated groups (both sexes). Changes were most pronounced in males at 750 mg/kg/day which tended to have lower body weights and body weight gains when compared to concurrent controls, despite a general trend towards higher food intake from the second week of treatment onwards. Mean terminal body weight of Cohort 1A males, treated for 11-12 weeks at the high dose, was 0.94 of control (statistically significant). In high dose males of Cohort 1B, mean body weight gain was 0.96x of controls at the end of both the pre-mating and mating period, resulting in a mean terminal body weight of 0.95x of controls after 16 weeks of treatment (not statistically significant). Also in males up to 250 mg/kg/day and females up to 750 mg/kg/day, a trend towards higher absolute and relative food consumption was observed from start of the treatment period onwards (not always dose-related/statistically significant), but body weight/body weight gain remained within concurrent control values. Similar to the F0-generation, food efficiency in treated F1-females of Cohort 1B recovered to normal values during the course of the lactation period.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect of treatment on water consumption was suspected
Haematological findings:
no effects observed
Description (incidence and severity):
F1-generation Data (Cohort 1A): Haematological parameters of treated rats were considered not to have been affected by treatment. Coagulation parameters of treated rats were considered not to have been affected by treatment.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1-generation Data (Cohort 1A): The following statistically significant changes were noted in males and females treated at 750 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
• Increased (1.96x) mean bile acid level in females at 750 mg/kg/day. Group mean value of 22.4 µmol/L remained within the normal range. The observed change in mean value could be attributed mainly to two high dose females (nos. 702 and 704) with values of 63.9 and 43.6 µmol/L (5.61x and 3.82x group mean control levels), respectively.
• Slightly decreased (0.95x) mean total protein levels in males at 750 mg/kg/day. Group mean value of 61.0 g/L was below the available internal control range .
• Slightly increased (1.16x) mean urea levels in males at 750 mg/kg/day. Group mean value of 5.9 mmol/L remained within the normal range16.
The observed changes in mean values for total protein and urea could be attributed mainly to a single high dose male (no. 418) with values of 54.2 g/L and 8.2 mmol/L (0.84x and 1.61x when compared to the group mean control levels), respectively. In the same high dose male (no. 418) relatively high values of aspartate aminotransferase (ASAT) activity (250.3 U/L; 3.01x group mean control level) and glucose (15.34 mmol/L, 1.91x; group mean control level) were recorded.
Any other statistically significant changes in clinical biochemistry parameters (i.e. decreased mean value of inorganic phosphates in Group 3 males, and increased mean values of ALP and chloride in Group 2 females) were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses: In females at 250 and 750 mg/kg/day, serum levels of T4 were statistically significantly higher (1.37x and 1.54x of control, respectively). Individual values of both T4 and TSH remained within the available (limited) range of internal control data , even for the two females at 250 mg/kg/day (nos. 621 and 640) and one female at 750 mg/kg/day (no. 706) which had a TSH value that was 3.97x, 8.48x and 7.44x the mean control group level, respectively.
Serum levels of thyroid stimulating hormone (TSH) and T4 in males were not affected by treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
F1-generation Data (Cohort 1A): Urinary parameters of treated rats were considered to be unaffected by treatment.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on splenic lymphocyte subpopulations observed.
Slightly higher T-cell and slightly lower B-cell splenic subpopulations were observed for males at 750 mg/kg/day and a slightly higher T-cytotoxic cell splenic subpopulation was observed for females at 750 mg/kg/day. Differences compared to the concurrent control group were relatively slight (< 10%), reaching no statistical significance. Therefore, they were considered to represent biological variability and thus not treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1-generation Data (Cohort 1A, 1B and 1C) from Weaning onwards: Test item-related higher liver weights were noted in 750 mg/kg/day group males and females of Cohort 1A (absolute and/or relative to body weights) and test item-related higher kidney weights (relative to body weights) were noted in 750 mg/kg/day group males in Cohort 1A. The statistically significant differences in mean adrenal gland and thyroid gland weights of 250 mg/kg/day group males (relative to bodyweight) compared to their concurrent controls were in absence of a dose response regarded to be unrelated to treatment with the test item.
For the Cohort 1B males at 750 mg/kg/day, seminal vesicle weight (absolute and relative to body weight) was statistically significantly increased (+14% and +20% compared to controls, respectively). This change was unrelated to treatment, because values remained within the available control range, no correlating findings were observed at the microscopic level, and seminal vesicle weights in the F0-generation were unaffected by treatment up to 750 mg/kg/day.
Any other differences, including those that reached statistical significance, were considered not to be 2,2’-Ethylenedioxydiethyl bis(2-Ethylhexanoate)-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1-generation Data (Cohort 1A, 1B and 1C) from Weaning onwards: There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
F1-generation Data (Cohort 1A, 1B and 1C) from Weaning onwards: Test item-related microscopic findings after treatment with 2,2’-Ethylenedioxydiethyl bis(2-Ethylhexanoate) were noted in the kidneys of the 750 mg/kg/day group F1-males (Cohort 1A).
Kidneys, an increased incidence and mean severity of hyaline droplet accumulation was present in males treated 750 mg/kg/day (up to slight).
Kidneys, an increased incidence and mean severity of tubular basophilia was present in males treated at 750 mg/kg/day (up to slight).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
F1-Generation – Cohort 1A: Length and regularity of the estrous cycle were not affected by treatment up to 750 mg/kg/day. Most females had a regular cycle of 4-5 days between PND 75 and 88.
No cycle classification could be performed for 1/20 control female (no. 498), 1/20 female at 83 mg/kg/day (no. 561), 2/20 females at 250 mg/kg/day (nos. 626 and 638) and 1/20 female at 750 mg/kg/day (no. 697). In addition, one female at 83 mg/kg/day (female no. 564) had an irregular cycle. Given their incidental nature and absence of a dose-related incidence, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
F1-Generation – Cohort 1A: Sperm motility, concentration and morphology were considered to be unaffected by treatment up to 750 mg/kg/day.
Total sperm count in the epididymis was slightly higher in treated males, reaching statistical significance at 250 mg/kg/day only. As mean values remained within the available control range, and in the absence of a dose-relationship, this finding was considered to be of no toxicological relevance.
Reproductive performance:
no effects observed
Description (incidence and severity):
F1-Generation – Cohort 1B: There were 1/20 couples of the control group, 2/20 couples of the 83 mg/kg/day group, 1/20 couples of the 250 mg/kg/day group and 2/20 couples of the 750 mg/kg/day group without offspring. No histopathologically identified abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
F1-Generation – Cohort 1A: There were no test item-related effects on the ovarian follicle counts (primordial and primary oocytes) and corpora lutea counts in the 750 mg/kg/day group F1-females (Cohort 1A) when compared to control group females. Any variation between group mean counts represented biological variability.
Reproduction Data – F1-Generation Cohort 1B
• Mating Index – F1-Generation Cohort 1B
Mating index was unaffected by treatment. The mating indices were 95% for the control and 750 mg/kg/day groups, and 100% for each the 83 and 250 mg/kg/day groups.
One control female (no. 514) and one female at 750 mg/kg/day (no. 729) did not mate. In the absence of a dose-relationship, these single occurrences were considered to be unrelated to treatment.
• Precoital Time – F1-Generation Cohort 1B
Precoital time was unaffected by treatment up to 750 mg/kg/day.
All females showed evidence of mating within 4 days, except for one female each in the control and 83 mg/kg/day group, and two females in the 250 mg/kg/day group for which mating took 12-14 days. In the absence of a dose-relationship, these exceptions were considered to be unrelated to treatment.
• Number of Implantation Sites – F1-Generation Cohort 1B
Number of implantation sites was unaffected by treatment up to 750 mg/kg/day.
• Fertility Index – F1-Generation Cohort 1B
Fertility index was considered not to be affected by treatment. The fertility indices were 100%, 90%, 95% and 95% for controls and females treated at 83, 250 and 750 mg/kg/day, respectively.
The number of non-pregnant females versus mated females was 2/20, 1/20 and 1/19 in the 83, 250 and 750 mg/kg/day groups, respectively. As these cases of non-pregnancy remained within the normal biological variation, showed no dose-related incidence across the dose groups, and in the absence of any reproductive toxicity, these were considered not to be related to treatment.
Developmental Data – F1-Generation Cohort 1B
• Gestation Index and Duration – F1-Generation Cohort 1B
Gestation index and duration of gestation were unaffected by treatment up to 750 mg/kg/day. All pregnant females had live offspring, resulting in a gestation index of 100% for all groups.
• Parturation/Maternal Care – F1-Generation Cohort 1B
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
• Post-Implantation Survival Index – F1-Generation Cohort 1B
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 93, 97, 93 and 89% for the control, 83, 250 and 750 mg/kg/day groups, respectively. The slightly lower survival index at 750 mg/kg/day was caused by one female (no. 713) that had 14 implantation sites, but only 4 pups. As all other values, and the mean group value at this high dose level remained within the control group range, this single occurrence was considered to be unrelated to treatment.
For control female no. 515, the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal remodelling of these areas of former implantations during the 21-23 days of lactation. No toxicological relevance was attached to this finding in the current study.
• Litter Size – F1-Generation Cohort 1B
Litter size was considered not to be affected by treatment.
Mean litter sizes were 11.3, 10.9, 11.5 and 11.2 living pups/litter for the control, 83, 250 and 750 mg/kg/day groups, respectively.
• Live Birth Index – F1-Generation Cohort 1B
The number of live offspring on Day 1 after littering compared to the total number of offspring born was unaffected by treatment up to 750 mg/kg/day.
Live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 98, 100, 99 and 100% for the control, 83, 250 and 750 mg/kg/day groups, respectively.
Four pups of the control group (all in litter no. 505) and three pups at 250 mg/kg/day (one in litter nos. 654, 657 and 658 each) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
• Viability Index – F1-Generation Cohort 1B
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was unaffected by treatment up to 750 mg/kg/day.
Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) were 100, 98, 99 and 100% for the control, 83, 250 and 750 mg/kg/day groups, respectively.
Three pups at 83 mg/kg/day (one in litter nos. 571, 572 and 581 each), two pups at 250 mg/kg/day (both in litter no. 644) and one pup at 750 mg/kg/day (litter no. 728) were found missing on PND 2 or 3. These pups were most likely cannibalised. No toxicological relevance was attributed to these missing pups as the mortality incidence did not show a dose-related trend and values remained well within the normal range of biological variation.
• Weaning Index – F1-Generation Cohort 1B
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.
One pup in the 750 mg/kg/day group (litter no. 725) was found dead on lactation Day 8, resulting in a lactation (weaning) index of 99% for this high dose group compared to 100% for all remaining groups. No signs were noted for this pup prior to its death. As the weaning index remained within the normal range of biological variation, this single occurrence was considered to be unrelated to treatment.
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: Higher total T4 serum levels were observed in F1 females of Cohort 1A (not lactating) at 250 and 750 mg/kg/day. All F1 individual values remained within the normal range.
Remarks:
Possible adversity of this effect could not be assessed within this type of study and was therefore not taken into account when determining the parental NOAEL.
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
Developmental
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical Signs during Lactation – F1-pups: No clinical signs occurred among pups that were considered to be related to treatment.
For some pups of the control, 83 and 250 mg/kg/day groups that were found dead/missing, paleness and/or absence of milk in the stomach was noted. At the incidence observed and in the absence of a dose-related trend, no toxicological relevance was attributed to these signs.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Mortality during Lactation – F1-pups: For some pups of the control, 83 and 250 mg/kg/day groups that were found dead/missing, paleness and/or absence of milk in the stomach was noted. At the incidence observed and in the absence of a dose-related trend, no toxicological relevance was attributed to these signs.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body Weights during Lactation – F1-pups: Body weights of pups were unaffected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
During lactation, food consumption could not be examined. From week 3 onwards, after weaning, cfr. P1 genereration.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical Biochemistry (T4 and TSH levels) on PND 4 and 22 – F1-pups: At 750 mg/kg/day, serum T4 levels were slightly, but statistically significantly higher (mean: 1.38 µg/dL; 1.22x of controls) in pups culled at PND 4. As values remained well within the range considered normal for pups of this age , this slight difference was considered to be of no toxicological relevance.
Serum thyroid stimulating hormone (TSH) and T4 levels of male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment.
In male pups of this Cohort Surplus, serum T4 and TSH levels were slightly increased after treatment, reaching statistical significance for TSH at 83 mg/kg/day only. In the absence of a dose-relationship, and as mean values remained within the available normal range , these differences were considered to be unrelated to treatment.

Sexual maturation:
no effects observed
Description (incidence and severity):
Sexual maturation F1-generation Data (Cohort 1A, 1B and 1C) from Weaning onwards: Sexual maturation (both sexes) was considered to be unaffected by treatment up to 750 mg/kg/day.
Time to vaginal patency (vaginal opening) in females and balanopreputial separation (prepuce opening) in males were slightly, but statistically significantly, delayed at 750 mg/kg/day. Vaginal opening occurred on average on PND 32 (versus PND 31 in controls) and balanopreputial separation occurred on PND 42 (versus PND 41 in controls). As for both sexes individual values generally remained within the concurrent control range and group mean values remained within the available control range , these slight differences were of no toxicological relevance. Mean body weight on the day of vaginal opening was statistically significantly higher at 83 mg/kg/day without a dose-related trend at higher doses. The slightly delayed time of balanopreputial separation observed in high dose males was considered to be an indirect effect of the slightly delayed growth as reflected in slightly lower body weights and body weight gains; body weight at attainment of sexual maturity was similar between all groups. As such, this was not considered to reflect male developmental toxicity in the high dose group.
The time between acquisition of vaginal patency and the first estrus was unaffected by treatment. Time until first estrus was on average 4 days for the control, 83, 250 and 750 mg/kg/day groups, respectively. First estrus was observed on average on PND 35, 35, 36 and 36, respectively. As values generally remained within the available control range , these slight differences were considered to be of no toxicological relevance.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital Distance – F1-pups: Anogenital distance (absolute and normalized for body weight) in male and female pups not affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areola/Nipple Retention – F1-pups: Treatment up to 750 mg/kg/day had no effect on areola/nipple retention. No nipples were found on male pups at PND 13.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross Pathology until Weaning – F1-pups: No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Histopathological findings:
not examined
Description (incidence and severity):
Histopathology of the Cohort 1A animals was conducted at the end of the premating/mating/gestation/lactation dosing period. Cohort 1B animals were processed to block stage for evaluation if needed.
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not fasted overnight before necropsy and no terminal body weight was recorded.
The animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not fasted overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
Dose descriptor:
NOAEL
Remarks:
Reproduction
Generation:
F1
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical Signs – F2-Pups: No clinical signs occurred among pups that were considered to be related to treatment.
For some pups of the 250 mg/kg/day group that were found dead at first litter check, absence of milk in the stomach was noted. In addition, for one pup of the 83 mg/kg/day group, a lean appearance was noted between PND 19 and 21. At the incidence observed and in the absence of a dose-related trend, no toxicological relevance was attributed to these signs.
The nature and incidence of these and other clinical signs (alopecia, scabs/spots on different parts of the body, swollen tattoo mark and/or discoloration of parts of the body) remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Mortality– F2-Pups: For some pups of the 250 mg/kg/day group that were found dead at first litter check, absence of milk in the stomach was noted.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body Weights - F2-Pups: Body weights of pups were considered not to be affected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
During lactation, food consumption could not be observed.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital Distance – F2-Pups: Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
The anogenital distance (absolute and normalized for body weight) was slightly higher (1.08x and 1.09x of control, respectively; reaching statistical significance for the normalized distance only) for male pups of the 750 mg/kg/day group. This was mainly caused by litter no. 712, for which the mean value was based on one male pup only, having a relatively high anogenital distance. As all values remained within the range of the control group, this single occurrence was considered to be not treatment-related.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areola/Nipple Retention – F2-Pups: Treatment up to 750 mg/kg/day had no effect on areola/nipple retention. No nipples were found on male pups at PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights - F2-Pups: Weights (absolute and relative to body weight) of the brain, thymus and spleen were not affected by treatment up to 750 mg/kg/day in male and female pups at PND 21-23.
Gross pathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Sex Ratio – F2-Pups: Sex ratio was considered not to be affected by treatment.
Dose descriptor:
NOAEL
Remarks:
Reproduction
Generation:
F2
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F2
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Reproductive effects observed:
no

Study 20134673 was performed in order to validate an analytical method for the quantitative analysis of 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) in diet including trial diet analysis to confirm the accuracy, homogeneity and stability. An additional study was performed in order to establish stability of the test item.

 

Method Validation

The analytical method was validated for the following parameters:

-Specificity: The chromatogram of the QC sample showed one major test item peak and several matrix related peaks. The area of the major test item peak was used as response in the calculations. A response at the retention time of the test item was detected in the chromatogram of the blank QC sample. The contribution of the response to the LOQ level (see 'Limit of quantification’) was 12%. Since this value was > 5%, the specificity requirements were not met and it was decided to analyze extra blank QC samples during trial diet analysis.

-Calibration curve: There was a linear relationship between response and test item concentration in the range of 0.0500 – 1.00 mg/L (in end solution). Since the coefficient of correlation (r) was > 0.99 and the back calculated accuracies of the remaining data points were in the range 85-115% the calibration line was accepted.

-Accuracy and repeatability: 98 -100%. Since the mean accuracy at each concentration level fell in the criterion 80-120% and the coefficient of variation was ≤ 10% the analytical method was accepted for the analysis of the test item in blank powder diet in the target concentration range of 500 – 15000 ppm.

-Limit of quantification (LOQ): 500 ppm in blank powder diet.

-Stability of the analytical system and end solutions: 9.1 -28%. Since the coefficient of variation at concentration levels 0.0500 and 0.100 mg/L were > 20% the samples were measured with bracketing.

-Stability of stock solutions: CV 3.2%. Since the value was≤ 10% the stock solutions were stable when stored at room temperature for at least 9 days.

Diets Analysis

Trial pellet diets were prepared at 500 ppm (500 mg/kg) and 15000 ppm (15000 mg/kg).

The concentrations analyzed in the trial diets were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

The diets were homogeneous (i.e. coefficient of variation ≤ 10%).

Diets were stable when stored at room temperature under normal laboratory light conditions in open containers and in a freezer (≤ -15°C) for at least 17 days.

Storage stability of the test item

After 6 months of storage in the refrigerator (2-8°C), analysis of the test item yielded a relative difference of ≤ 10%. The test item was found to be stable when stored in the refrigerator (2-8°C) for at least 6 months.

Conclusions:
Parental NOAEL: F0-generation and F1-generation: 250 mg/kg/day.
Reproduction NOAEL: F0-generation and F1-generation at least 750 mg/kg/day.
Developmental NOAEL: F1-generations and F2-generation at least 750 mg/kg/day.
Executive summary:

The objective of this study was to provide an evaluation of the pre- and postnatal effects of 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring.

In addition, the study provided and/or confirmed information about the effects of 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

Dietary concentrations in this study were adjusted regularly to maintain the target test item intake of 0, 83, 250, 750 mg/kg bw/day, based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test) with dietary exposure of 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) in rats, Test Facility Study No. 491295.

The study design was as follows:

Text Table 1: Experimental Design F0-animals

Group No.

Test Item Identification

Target Test Item Intake (mg/kg bw/day)a

Number of F0-Animals

Males

Females

1

-

0 (control)

25

25

2

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

83

25

25

3

250

25

25

4

750

25

25

 

Text Table 2: Experimental Design F1-animals

Group

Test Item Id.

Target Test Item Intake (mg/kg bw/day)a

Cohort

Number of F1-Animals

M

F

1

-

0 (control)

1A

20

20

1B

20

20

1C

20

20

Surplusb

10

10

 

Group

Test Item Id.

Target Test Item Intake (mg/kg bw/day)a

Cohort

Number of F1-Animals

M

F

2

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

83

1A

20

20

1B

20

20

1C

20

20

Surplusb

10

10

 

Group

Test Item Id.

Target Test Item Intake (mg/kg bw/day)a

Cohort

Number of F1-Animals

M

F

3

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

250

1A

20

20

1B

20

20

1C

20

20

Surplusb

10

10

 

Group

Test Item Id.

Target Test Item Intake (mg/kg bw/day)a

Cohort

Number of F1-Animals

M

F

4

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

750

1A

20

20

1B

20

20

1C

20

20

Surplusb

10

10

Id.= identification.

a The test item has a purity of 98.43% (see section 8); dose calculations were not corrected for purity.

b The F1-animals of Cohort Surplus were not treated.

 

Chemical analyses of dietary preparations were conducted at four occasions during the study to assess accuracy and homogeneity.

For the F0-generation, the following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs and arena observations, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations.

For the F1-generation, the following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs and arena observations, body weight, food consumption, vaginal patency (Cohorts 1A, 1B and 1C), balanopreputial separation (Cohorts 1A, 1B and 1C), stage of estrus determination (on day of necropsy - Cohorts 1A and 1B), estrous cycle determination (only Cohort 1A), clinical pathology including measurement of thyroid hormones and urinalysis (only Cohort 1A), gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis (only Cohort 1A), organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined for the F1- generation (Cohort 1B): mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones (only Cohort Surplus and Cohort 1A)).

For the F2-generation the following parameters and end points were evaluated in this study: gross necropsy findings and organ weights.

Dietary analyses confirmed that formulations of test item in diets were prepared accurately and homogenously.

F0-Generation (F0-generation is also known as P0-generation)

Body weights and body weight gain of males at 750 mg/kg/day were slightly, but statistically significantly lower than controls from Week 5 of the pre-mating period onwards, lasting until the end of the mating period. Mean body weight gain was 0.90x and 0.91x of controls at the end of the pre-mating and mating period, respectively, resulting in a terminal body weight of 0.92x of controls. Body weight (gain) in males up to 250 mg/kg/day and females up to 750 mg/kg/day were comparable to concurrent control values. It should be noted that in both sexes there was a general trend towards higher food intake (absolute and relative to body weight) in all treated groups compared to the concurrent control group from start of treatment onwards. The delayed growth of high dose males in the presence of higher food intake is indicative for a lower food efficiency, i.e. more food has to be ingested to reach the same growth in the animal’s mass. This effect was less pronounced in females, who had body weight gains in the same range as concurrent controls throughout the whole treatment period. Importantly, food efficiency in lactating females recovered to normal values during the last two weeks of treatment (lactation Days 7-21; all treated groups).

At the organ level, toxicologically relevant changes were observed in kidneys, liver and thyroid gland of males at 750 mg/kg/day and in liver of females at 750 mg/kg/day.

Kidneys:

In the kidneys of males treated at 750 mg/kg/day, an increased incidence and mean severity of hyaline droplets accumulation and tubular basophilia was noted, next to a higher kidney weight (relative to body weight: 1.19x of control). The observed hyaline droplets resemble alpha 2u-globulin, a normal protein present in male rats including controls and absent in female rats and humans. However, it should be noted that no specific staining was applied to prove the presence of alpha 2u-globulin. These morphological changes occurred in the absence of any sign for impaired kidney function; all clinical biochemistry/urinalysis parameters examined remained within the normal range of biological variation.

Liver:

The higher liver weights observed inmalesat 750 mg/kg/day (relative to body weight: 1.17x of control) occurred in the absence of correlating microscopic changes and clinical biochemistry parameters were unaffected.

Also in the liver of females treated up to 750 mg/kg/day no test item-related morphological changes were noted (i.e. liver organ weights remained within the normal range, and macroscopic/microscopic examination of the livers revealed no findings). However, various clinical biochemistry parameters specific for liver were affected at 250 and/or 750 mg/kg/day. Statistically significant changes consisted of slightly increased mean plasma activity of ALAT at 250 and 750 mg/kg/day (1.42x and 1.45x of control, respectively) and higher mean plasma concentration of bile acids at 750 mg/kg/day (3.3x of control). The observed changes in mean values could be attributed mainly to a few females within each group. Furthermore, not always the same set of parameters was affected in each female. For example, some females with increased bile acids showed normal values of ALAT, and vice versa. In addition, incidentally higher levels of ASAT and ALP, and/or higher liver organ weights (relative to body weight) were observed. As clearly more females were affected at 750 mg/kg/day than at 250 mg/kg/day, liver changes observed at the high dose were considered to be adverse.

Thyroid gland:

The increased incidence and mean severity of follicular cell hypertrophy of the thyroid gland observed in males at 750 mg/kg/day was within the range of background pathology encountered in rats of this age and strain.

No treatment-related or toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/viability, clinical appearance, haematology, and coagulation parameters, and macroscopic examination).

Reproduction results – F0-generation

No reproductive toxicity was observed up to the highest dose level tested (750 mg/kg/day).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. sperm motility, concentration and morphology, mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Developmental results – F1generation (pre-weaning)

No developmental toxicity was observed up to the highest dose level tested (750 mg/kg/day).

In pups of the F0-generation (i.e. F1-pups), no treatment-related or toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration of gestation, parturition, live birth and viability indices, sex ratio, litter size, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), macroscopic examination and brain and spleen weight.

F1-Generation (F1-generation is also known as P1-generation) (post-weaning)

Similar to the F0-generation, lower food efficiency was observed in all treated groups (both sexes). Changes were most pronounced in males at 750 mg/kg/day which tended to have lower body weights and body weight gains when compared to concurrent controls, despite a general trend towards higher food intake from the second week of treatment onwards. Mean terminal body weight of Cohort 1A males, treated for 11-12 weeks at the high dose, was 0.94 of control (statistically significant). In high dose males of Cohort 1B, mean body weight gain was 0.96x of controls at the end of both the pre-mating and mating period, resulting in a mean terminal body weight of 0.95x of controls after 16 weeks of treatment (not statistically significant). Also in males up to 250 mg/kg/day and females up to 750 mg/kg/day, a trend towards higher absolute and relative food consumption was observed from start of the treatment period onwards (not always dose-related/statistically significant), but body weight/body weight gain remained within concurrent control values. Similar to the F0-generation, food efficiency in treated F1-females of Cohort 1B recovered to normal values during the course of the lactation period.

Exposing the F1-generation of Wistar (Han) rats to 2,2’-Ethylenedioxydiethyl bis(2-Ethylhexanoate) in utero, through nursing during lactation, and via the diet, following weaning was associated in Cohort 1A with treatment-related morphological changes in the kidneys (males) and liver (both sexes) in the high dose group.

Kidneys:

Similar to the previous generation, an increased incidence and mean severity of hyaline droplets accumulation and tubular basophilia were noted in males at 750 mg/kg/day, next to a higher kidney weight (relative to body weight: 1.11x of control). In addition, high dose males had a slightly lower mean level of total protein (0.95x of control) and slightly higher mean urea level (1.16x of control). Both changes in mean values could be attributed mainly to a single high dose male with values for total protein and urea that were 0.84x and 1.61x group mean control level, respectively.

Liver:

The higher liver weights observed in males and females at 750 mg/kg/day (relative to body weight: 1.09x of control in both sexes) occurred in the absence of correlating microscopic changes, and clinical biochemistry parameters were affected in only a few high dose animals.

The statistically significantly higher mean bile acid level (1.96x of control) in females at 750 mg/kg/day could be attributed mainly to two high dose females with values of 5.61x and 3.82x of control, respectively. Elevated values of ASAT and glucose (3.01x and 1.91x of control, respectively) were noted in a single high dose male (the same animal as with relatively high values of total protein and urea).

In lactating F0-females at 750 mg/kg/day and non-lactating F1-females (Cohort 1A) at 250 and 750 mg/kg/day, mean serum levels of T4 were statistically significantly increased compared to the concurrent control mean (1.36x, 1.37x and 1.54x, respectively). It should be noted that except for one F0-female, all individual levels of both T4 and TSH remained within the available (limited) range of internal control data, and no correlating hypertrophy was seen either in the thyroid gland or liver of high dose females. Possible adversity of the higher T4 level seen in females of both generations could not be assessed within this type of study and was therefore not taken into account when determining the parental NOAEL.

No treatment-related effects were recorded for any of the developmental parameters investigated in this study including balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, and sperm motility, concentration and morphology. No test item-related shifts in splenic lymphocyte subpopulations were observed. Histopathologically, no test-item related effects were noted at stage-dependent qualitative evaluation of spermatogenesis, ovarian follicle and corpora lutea counts of females of Cohort 1A and morphology of reproductive organs. Mating and fertility indices, precoital time, number of implantations, gestation index and duration of gestation, parturition, live birth and viability indices, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), macroscopic examination and organ weights of brain, thymus and spleen were unaffected by treatment up to 750 mg/kg/day.

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1A, 1B, 1C, and a second generation), the following no-observed-adverse-effect level (NOAEL) of 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) were established for both F0-generation and F1-generation:

Parental NOAEL:

F0-generation and F1-generation: 250 mg/kg/day.

The higher dose of 750 mg/kg/day was considered to be adverse based on the combination of the observed lower food efficiency (males and females of both generations), together with effects on kidneys (F0- and F1-males only) and liver (males and females of both generations) which manifested themselves by higher organ weights of kidneys and/or liver, increased incidence and severity of hyaline droplets accumulation and tubular basophilia in the kidneys, and changes in biochemical chemistry parameters specific for kidney and/or liver.

Note: In this study, higher serum levels of total T4 were observed in lactating females of the F0-generation at 750 mg/kg/day, and females of Cohort 1A (F1-generation; not lactating) at 250 and 750 mg/kg/day. Except for one F0-female, all individual values remained within the normal range. Possible adversity of this effect could not be assessed within this type of study and was therefore not taken into account when determining the parental NOAEL.

Reproduction NOAEL: F0-generation and F1-generation at least 750 mg/kg/day.

Developmental NOAEL: F1-generations and F2-generation at least 750 mg/kg/day.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
high
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicityscreening test of 2,2’-ethylenedioxydiethyl bis (2-ethylhexanoate) in rats by dietaryadministration (Van Tuyl, 2011). 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).

No reproductive/developmental toxicity was observed at 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).

According to amende REACH 1907/2006 legislation Annex X Column I, an Extended One-Generation Reproductive Toxicity Study is required (B.56 of the Commission Regulation on test methods as specified in Article 13(3) or OECD 443), basic test design (cohorts 1A and 1B without extension to include a F2 generation), one species, most appropriate route of administration, having regard to the likely route of human exposure, unless already provided as part of Annex IX requirements.

The basic test design will be followed with 10 weeks premating oral dosing in males and 2 weeks in females. Based on the available information, an extension of the reproduction toxicity Cohort 1B to F2 will be performed if internal triggers (described in OECD Guidance 117, 2011) for a second generation are hit during the study. However there is no need to include the developmental neurotoxicity and immunotoxicity cohorts. More information on conditions/triggers for the EOGRTS as described in REACH Amendment (EU) 2015/282 and REACH Guidance R.7a (July 2015) is annexed.


In short: 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 (corresponding to 977-1563 mg/kg bw).

Furthermore, an Extended One Generation Reproductive Toxicity Study (EOGRTS) according to OECD Test Guidance 443 was conducted with 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) to evaluate pre- and postnatal effects on development and systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats (basic study design incl. Cohorts 1A, 1B with extension to F2 generation). In addition, the study provided information on the integrity and performance of the adult male and female reproductive systems. Dietary concentrations in this study were adjusted regularly to maintain the target test item intake of 0, 83, 250, 750 mg/kg bw/day. Chemical analyses of dietary preparations during the study to confirmed accuracy and homogeneity (Peter, 2019).

F0-Generation (also known as P0-generation). 

Body weights and body weight gain of males at 750 mg/kg/day were slightly, but statistically significantly lower than controls from Week 5 of the pre-mating period onwards, lasting until the end of the mating period. In both sexes there was a general trend towards higher food intake (absolute and relative to body weight) in all treated groups compared to the concurrent control group from start of treatment onwards. The delayed growth of high dose males in the presence of higher food intake is indicative for a lower food efficiency, which was less pronounced in females. At the organ level, toxicologically relevant changes were observed in kidneys, liver and thyroid gland of males at 750 mg/kg/day and in liver of females at 750 mg/kg/day. In the kidneys of males treated at 750 mg/kg/day, an increased incidence and mean severity of hyaline droplets accumulation and tubular basophilia was noted, next to a higher kidney weight. Higher liver weights observed in males at 750 mg/kg/day occurred in the absence of correlating microscopic changes and clinical biochemistry parameters were unaffected. In the liver of females treated up to 750 mg/kg/day no test item-related morphological changes were noted, however, various clinical biochemistry parameters specific for liver were affected at 250 and/or 750 mg/kg/day (slightly increased mean plasma activity of ALAT at 250 and 750 mg/kg/day and higher mean plasma concentration of bile acids at 750 mg/kg/day). An increased incidence and mean severity of follicular cell hypertrophy of the thyroid gland observed in males at 750 mg/kg/day was within the range of background pathology encountered in rats of this age and strain.No reproductive toxicity was observed up to the highest dose level tested (750 mg/kg/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. sperm motility, concentration and morphology, mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).No developmental toxicity was observed up to the highest dose level tested (750 mg/kg/day). In pups of the F0-generation (i.e. F1-pups), no treatment-related or toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration of gestation, parturition, live birth and viability indices, sex ratio, litter size, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), macroscopic examination and brain and spleen weight.

F1-Generation (post-weaningalso known as P1-generation)

Similar to the F0-generation, lower food efficiency was observed in all treated groups (both sexes). Changes were most pronounced in males at 750 mg/kg/day which tended to have lower body weights and body weight gains when compared to concurrent controls, despite a general trend towards higher food intake from the second week of treatment onwards. Also in males up to 250 mg/kg/day and females up to 750 mg/kg/day, a trend towards higher absolute and relative food consumption was observed from start of the treatment period onwards, but body weight/body weight gain remained within concurrent control values. Exposing the F1-generation in utero, through nursing during lactation, and via the diet, following weaning was associated in Cohort 1A with treatment-related morphological changes in the kidneys (males) and liver (both sexes) in the high dose group. Similar to the previous generation, an increased incidence and mean severity of hyaline droplets accumulation and tubular basophilia were noted inuat 750 mg/kg/day, next to a higher kidney weight. In addition, high dose males had a slightly lower mean level of total protein and slightly higher mean urea level. Higher liver weights observed in males and females at 750 mg/kg/day in the absence of correlating microscopic changes, and clinical biochemistry parameters were affected in only a few high dose animals (higher mean bile acid level in females at 750 mg/kg/day could be attributed mainly to two high dose females; elevated values of ASAT and glucose in a single high dose male).

In lactating F0-females at 750 mg/kg/day and non-lactating F1-females (Cohort 1A) at 250 and 750 mg/kg/day, mean serum levels of T4 were statistically significantly increased compared to the concurrent control mean, however it should be noted that except for one F0-female, all individual levels of both T4 and TSHremained within the available range of internal control data,and no correlating hypertrophy was seen either in the thyroid gland or liver of high dose females. Possible adversity of the higher T4 level seen in females of both generations could not be assessed within this type of study and was therefore not taken into account when determining the parental NOAEL.

No treatment-related effects were recorded for any of the developmental parameters investigated in this study including balanopreputial separation, vaginal patency, first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, and sperm motility, concentration and morphology. No test item-related shifts in splenic lymphocyte subpopulations were observed. Histopathologically, no test-item related effects were noted at stage-dependent qualitative evaluation of spermatogenesis, ovarian follicle and corpora lutea counts of females of Cohort 1A and morphology of reproductive organs. Mating and fertility indices, precoital time, number of implantations, gestation index and duration of gestation, parturition, live birth and viability indices, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), macroscopic examination and organ weights of brain, thymus and spleen were unaffected by treatment up to 750 mg/kg/day.

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1A, 1B, 1C, and a second generation), the parental no-observed-adverse-effect level (NOAEL) for both F0-generation and F1-generation is 250 mg/kg/day. The dose of 750 mg/kg/day was considered to be adverse based on the combination of the observed lower food efficiency (males and females of both generations), together with effects on kidneys (F0- and F1-males only) and liver (males and females of both generations) which manifested themselves by higher organ weights of kidneys and/or liver, increased incidence and severity of hyaline droplets accumulation and tubular basophilia in the kidneys, and changes in biochemical chemistry parameters specific for kidney and/or liver. The Reproduction NOAEL for both F0-generation and F1-generation is at least 750 mg/kg/day. The Developmental NOAEL for both F1-generations and F2-generation is at least 750 mg/kg/day.

Effects on developmental toxicity

Description of key information
2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) administered to pregnant Hannover Wistar rats by oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day, daily from gestation days GD 5 to 19, was associated with the following effects:  At 1000 mg/kg bw/day maternal toxicity was observed as evidenced by with decreased body weight gain versus control groups and transient decreased food consumption (GD 5-14). This was associated with foetal toxicity evident as a reduction in mean foetal body weight (>6%), and increased incidence of skeletal variations consisting of incomplete ossification of skull and delayed ossification of metatarsals and of sternal bodies. There were two visceral malformations (dilated malpositioned oesophagus and transpositioned heart and great arteries) and three skeletal malformations (split thoracal XI vertebra, absence of sacral II & caudal vertebrae and unilateral short, bent femur). One of the skeletally malformed foetus (malformation of sacral/caudal vertebrae) was also externally malformed (absence of anus and tail). The findings were considered to be secondary to maternal toxicity, evidenced in the repeated dose toxicity studies. 
No maternal or developmental toxicity was observed at 300 mg/kg bw/day. One viscerally malformed foetus (unilateral ectopy of adrenal gland) was found and was considered incidental. No maternal or developmental toxicity was observed at 100 mg/kg bw/day.
The NOAEL for maternal toxicity and embryo-/foetotoxicity was determined to be 300 mg/kg bw/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-2014
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Hannover Wistar rats (CRLHan)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, at least 11 weeks old
- Weight at treatment initiation: Not exceeding ± 20% of the mean weight, and was in the range of 205-262 g
- Housing: Standard laboratory conditions; group housing, up to 3 animals of the same group per cage. Type II and/or III polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). The bedding material was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Diet (e.g. ad libitum): ssniff® SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, D-59494 Soest Germany) ad libitum.
- Water (e.g. ad libitum): tap water as for human consumption, in water bottles, ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 – 24.0°C
- Humidity (%): 32 - 64 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 29 October 2013 (first mating, when the females were approximately 11 weeks old)
To: 03 December 2013 (last necropsy)
Route of administration:
oral: gavage
Vehicle:
other: propylene glycol containing 1% Polysorbate 80 (Tween 80)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (propylene glycol containing 1% Polysorbate 80) at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd.
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 (400, 120 and 40 mg/mL) of the test item for each dose level (1000, 300 and 100 mg/kg bw/day, respectively) and was stirred until a homogenous dosing form was obtained.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Based on the results of the 90-day study (CiToxLAB study code: 12/072-101P) propylene glycol containing 1% Polysorbate 80 (Tween 80) was selected as vehicle. Vehicle of this composition proved to compose stable suspension of the test item, considered suitable for the study purposes.
- Lot/batch no. Propylene glycol (1,2-Propanediol): SZBD1000V (Sigma-Aldrich); Polysorbate 80 (Tween 80): BCBJ7603V (Fluka)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations (concentration and homogeneity) using a validated GC method, was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd.
Top, middle and bottom samples were taken in duplicate from the test item formulations (Low, Mid and High doses) twice during the study (during the first and last weeks of treatment; November 4 and 25, 2013, respectively). Similarly, one sample (duplicate) was taken on each occasion from the Group 1 (control) solution, for concentration measurements.
All formulations were found to be in the range of 93 to 100% of nominal concentration. All formulations were shown to be homogeneous. No test item was detected in the control samples
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male: 1-3 females
- Length of cohabitation: approximately 2 hours in the morning, until at least 24 sperm positive females/group were attained
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (GD0)
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
from Gestation Day (GD) 5 to GD19
Frequency of treatment:
once daily
Duration of test:
necropsy on GD20
No. of animals per sex per dose:
107 female animals, plus an appropriate number of spares: 26-28 mated female animals/group, 4 groups (one control and 3 test item-treated groups); 22-25 pregnant female animals/group (with implantation sites at necropsy); 60 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from CiToxLAB Hungary Ltd. spare colony were used
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set by the Sponsor in consultation with the Study Director based on available data from the previous studies, including the results of a pilot developmental study in the rat (CiToxLAB study code 13/220-105PE), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the pilot study (Study code 13/220-105PE) treatment at 1000 mg/kg bw was associated with decreased maternal body weight gain (by approximately 10% and 7% for cumulative body weight gain and corrected gain values, respectively,) and the mean weight of foetuses was lower by approximately 5% when compared to control mean.

According to the Sponsor’s information, 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 Test Guideline 422 by dietary administration, was available (Study No. 491295). In this study a parental No Observed Adverse Effect Level (NOAEL) was found to be of 5000 ppm test item concentration in rodent diet (Mid dose level), equivalent to 314-576 mg/kg body weight/day test item intake. Target organs at 15000 ppm (equivalent to 977-1563 mg/kg body weight/day) were adrenal glands, kidneys, liver, pituitary gland, spleen, thymus and thyroid glands. There were no reproductive/developmental effects up to the highest dose.

The oral route is a possible route of exposure to the test item in humans and is considered suitable to provide the systemic exposure required on this developmental toxicology study.
- Rationale for animal assignment (if not random):
The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side clinical observations were made at least daily after the treatment as practical.
- Cage side observations:
The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical sign were noted during the study.
On GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals at the onset of treatment (GD5) then at least weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food was measured with a precision of ±1 g on GD 0, 3, 5, 8, 11, 14, 17 and 20. Food consumption was calculated for each interval, including overall values for GD5-20 and GD0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes.
The ovaries and uterus were removed and the pregnancy status ascertained.
The uterus including the cervix was weighed (accuracy ±1 g) and examined for early and late embryonic or foetal deaths and for the number of live foetuses.

The corrected body weight was calculated (body weight on GD20 minus weight of the gravid uterus).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of live/dead fetuses. The placentae were examined macroscopically.
Fetal examinations:
Fetal examinations
- Sex distribution
- Fetal body weigths
- External examinations: Yes: all live foetuses per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter. The heads were examined by Wilson's free-hand razor blade method.

Statistics:
The statistical evaluation of all numerical data was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method (Bartlett, ANOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, Chi2). The homogeneity 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 (ANOVA) was made. If the obtained result was significant Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons were further compared using Kruskal-Wallis, and Mann-Whitney U-tests.
The following data were excluded from statistical analysis:
-sperm positive but non pregnant females (total exclusion) [No. 141, 201 and 188 (control), 142 (100 mg/kg) 104, 132, 143, 129, 156 and 183 (300 mg/kg) and 112 (1000 mg/kg)]
-female no. 1513 (control) exclusion of body weight of GD20 due to incorrect data

No exclusion was possible in the food consumption data for all females, as it was calculated based on cage.

The limit for growth retarded foetuses was calculated from the average body weight of the vehicle control foetuses. A foetus was considered as growth retarded if the deviation from the mean control values was greater than minus two fold standard deviation of all control foetuses. In the present study, this cut-off value was set at 2.894 g.
Indices:
The number and percent of pre and post-implantation losses were calculated.
Historical control data:
Historical data of Hannover Wistar rats were appended to the report.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Lower body weight gain (GD5-20) and transient decreased in food consumption was observed in dams at 1000 mg/kg bw/day.

Details on maternal toxic effects:
There was no unscheduled mortality during the study.
There were no toxicologically significant or test-item related clinical signs noted following administration of test item.
Lower body weight gain (GD5-20) was observed in dams at 1000 mg/kg bw/day, overall mean value by 2.4 %, and corrected net body weight gain (-gravid uterus weight) by 9.9%, , when compared to control mean. This was mainly attributed to lower body weight gain compared to control mean on GD5-8 (-32.6%) and GD8-11 (-13.2%). Transient decrease in food consumption was also noted at the beginning of the treatment period (approximately 6-10% from GD5-14, with highest value and statistical significance on GD5-8). This maternal toxicity may probably correlate with liver and kidney toxicity, which was revealed in 90-day gavage study with this test item (ref.6) and toxicity in target organs seen in the 28-day repeated dose study with the reproductive/developmental screening test at 15000 ppm in the feed (equivalent to 977-1563 mg/kg body weight/day) i.e. adrenal glands, kidneys, liver, pituitary gland, spleen, thymus and thyroid glands.
No relevant effect on body weight (gain) or food consumption was noted at 100 and 300 mg/kg bw/day.
No test item related macroscopic observations were made at necropsy on GD20.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: The weight of foetuses at 1000 mg/kg bw/day was decreased. Skeletal examination revealed an increased incidence of minor changes (variations) and sporadic skeletal malformations at 1000 mg/kg bw/day.

Details on embryotoxic / teratogenic effects:
There were no toxicologically significant differences in intrauterine mortality between test item treated and control females.

The mean number of corpora lutea and implantation sites was comparable with the controls in all treated groups.
The mean number of viable foetuses was comparable with the control mean.
There were no toxicological significant differences in the placenta at evaluation of the treated groups compared to controls.

The weight of foetuses at 1000 mg/kg bw/day was decreased, by approximately 6%, evaluated by both litter mean as well as male and female mean values separately, when compared to the controls. The differences attained statistical significance.
At 300 mg/kg bw/day the mean litter foetal weights were comparable to control.
At 100 mg/kg bw/day the mean litter foetal weights were slightly decreased (by 5-6%) but this observation is equivocal in correlation with test item administration in the conditions of this study.

External examination revealed one malformed foetus at 1000 mg/kg bw/day in the form of absence of anus and tail. This malformation was accompanied by absence of sacral II and caudal vertebrae observed during the skeletal examination.
In control group two malformed foetuses (omphalocele) were found, confirmed by visceral examination, both in one litter. There were no external variations in control and dosed groups.
There were two foetal visceral malformation observed at 1000 mg/kg bw/day: dilated mal-positioned (across trachea) oesophagus and transpositioned heart and great arteries.
In one foetus at 300 mg/kg bw/day unilateral ectopy of adrenal gland was observed, however based on the isolated occurrence, this observation was considered incidental and not related to treatment.
Three skeletally malformed foetuses (split thoracal (Th.XI) vertebra, absence of sacral II&caudal vertebrae and unilateral short, bent femur) were found in three litters in the 1000 mg/kg bw/day treated group.
In addition, at skeletal examination of the foetuses, an increased incidence of minor changes (variations) was noted at 1000 mg/kg bw/day and consisted of incomplete ossification of skull, delayed ossification of metatarsals and of sternal bodies.
Incomplete ossification of skull was observed in approximately 15% of foetuses and the severity of finding was also increased.
The incidence of delayed ossification of sternal bodies and of un-ossified metatarsals were slightly increased (observed in approximately 9% and 7% of foetuses, respectively) and occurred also more frequently in foetuses of low body weight.
These variations were regarded as evidence of the test item toxicity-related delay in foetal development which occurred at dose level associated with established maternal toxicity.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) administered to pregnant Hannover Wistar rats by oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day, daily from gestation days GD 5 to 19, was associated with the following effects:
At 1000 mg/kg bw/day maternal toxicity was observed as evidenced by with decreased body weight gain versus control groups (2.4% and 9.9% for absolute and net mean values, respectively, with peaks of 32.6% and 13.2% on GD5-8 and GD8-11, respectively) transient decreased food consumption (GD 5-14). This was associated with foetal toxicity evident as a reduction in mean foetal body weight (>6%), and increased incidence of skeletal variations consisting of incomplete ossification of skull and delayed ossification of metatarsals and of sternal bodies. There were two visceral malformations (dilated malpositioned oesophagus and transpositioned heart and great arteries) and three skeletal malformations (split thoracal XI vertebra, absence of sacral II & caudal vertebrae and unilateral short, bent femur). One of the skeletally malformed foetus (malformation of sacral/caudal vertebrae) was also externally malformed (absence of anus and tail).
No maternal or developmental toxicity was observed at 300 mg/kg bw/day. One viscerally malformed foetus (unilateral ectopy of adrenal gland) was found and was considered incidental. No maternal or developmental toxicity was observed at 100 mg/kg bw/day.
The NOAEL for maternal toxicity embryo-/foetotoxicity was determined to be 300 mg/kg bw/day.
Executive summary:

Test item, 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) administered to pregnant Hannover Wistar rats was administered by oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day, daily from gestation days GD 5 to 19, was associated with the following effects:

At 1000 mg/kg bw/day maternal toxicity was observed as evidenced by decreased body weight gain versus control groups (2.4% and 9.9% for absolute and net mean values, respectively with peaks of 32.6% and 13.2% on GD5-8 and GD8-11, respectively), transient decreased food consumption (GD 5-14). This was associated with foetal toxicity evident as a reduction in mean foetal body weight (>6%), and increased incidence of skeletal variationsconsisting of incomplete ossification of skull and delayed ossification of metatarsals and of sternal bodies.There were two visceral malformations (dilated malpositioned oesophagus andtranspositioned heart and great arteries)and three skeletal malformations (split thoracal XI vertebra, absence of sacral II & caudal vertebrae and unilateral short, bent femur). One of the skeletally malformed foetus (malformation ofsacral/caudal vertebrae) was also externally malformed (absence of anus and tail).

 

Delayed or incomplete ossification of developing fetal bones are common skeletal alterations in developmental toxicity studies. It refers to a decrease in the amount of mineralized bone compared with that expected for a given developmental age. The reasons for these findings are that fetuses are evaluated near the end of gestation at a time when ossification in certain regions of the skeleton is proceeding quite rapidly. But also, fetal ossification is highly dependent on maternal physiologic factors, including utero-placental blood flow, nutritional status, etc., so it is not unexpected that fetal ossification rates can be altered by maternal toxicity. The relevance of these findings is limited for following reasons. First, human skeletal development takes place postnatally so these findings are not applicable to human situation. Secondly, studies in rats have demonstrated that delayed ossifications do not persist postnatally (so they are reversible). (Carney and Kimmel, 2007)

 

In addition to the changes in body weight gain and food consumption seen in the current study, target organs supporting maternal toxicity were identified during repeated dose toxicity testing with the current substance. Especially at the dose level of 1000 mg/kg bw, target organs were evident within one month dosing:

- In a 28 day dietary toxicity and reproductive toxicity study in Wistar Han rats (Van Tuyl, 2010), following changes were seen at 15000 ppm (corresponding to mean of 977 mg/kg bw/day in males and 1256 mg/kg bw/day in females during gestation): increased serum potassium levels in both sexes, increased (relative) kidney weights and increased (absolute and relative) liver weights in both sexes. Microscopic changes were observed in the adrenal glands (multifocal vacuolation in zona glomerulosa), liver (diffuse midzonal/centrilobular hypertrophy),thyroid glands (diffuse follicular hypertrophy/hyperplasia).

- In a 90-day oral gavage toxicity study in Wistar rats (Grósz, 2013), the highest dose of 480 mg/kg bw/day showed higher absolute and relative weights of kidneys and livers in females only. Microscopic changes in the kidney comprised minimal epithelial degeneration of cortical tubules (cytoplasmic vacuolation, cellular sloughing) associated with the presence of homogenous eosinophilic content (casts) in tubular lumen. Following a 28-day treatment-free period, signs of the recovery were detected in the kidney. Microscopic 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; this was fully reversible during the 28-day recovery period. In a 14-day DRF by oral gavage study in Wistar rats, higher serum potassium, calcium and phosphorus concentrations were observed in females at 1000 mg/kg bw/day. Slightly higher thyroids weights were found in both sexes at 1000 mg/kg bw/day and in females dosed at 300 mg/kg bw/day.

 

No maternal or developmental toxicity nor teratogenicity was observed at 300 mg/kg bw/day.One viscerally malformed foetus (unilateral ectopy of adrenal gland)was found and was considered incidental. No maternal or developmental toxicity was observed at 100 mg/kg bw/day. Based on the above results, the NOAEL for maternal toxicity and for embryo-/foetotoxicity was determined to be 300 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
high
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) was tested in an OECD TG 414 study in rats (Grosz, 2014b) and preceding dose range finding toxicity study (Grosz, 2014a). When daily dosed by gavage to pregnant Hannover Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day from gestation days GD 5 to 19, following effects were observed. At 1000 mg/kg bw/day maternal toxicity was observed as evidenced by decreased body weight gain versus control groups (2.4% and 9.9% for absolute and net mean values, respectively with peaks of 32.6% and 13.2% on GD5-8 and GD8-11, respectively), and transient decreased food consumption (GD 5-14). This was associated with foetal toxicity evident as a reduction in mean foetal body weight (>6%), and increased incidence of skeletal variations consisting of incomplete ossification of skull and delayed ossification of metatarsals and sternal bodies.There were two visceral malformations (dilated malpositioned oesophagus andtranspositioned heart and great arteries) and three skeletal malformations (split thoracal XI vertebra, absence of sacral II & caudal vertebrae and unilateral short, bent femur). One of the skeletally malformed foetus (malformation ofsacral/caudal vertebrae) was also externally malformed (absence of anus and tail).

 

Delayed or incomplete ossification of developing fetal bones are common skeletal alterations in developmental toxicity studies. It refers to a decrease in the amount of mineralized bone compared with that expected for a given developmental age. The reasons for these findings are that fetuses are evaluated near the end of gestation at a time when ossification in certain regions of the skeleton is proceeding quite rapidly. But also, fetal ossification is highly dependent on maternal physiologic factors, including utero-placental blood flow, nutritional status, etc., so it is not unexpected that fetal ossification rates can be altered by maternal toxicity. The relevance of these findings is limited for following reasons. First, human skeletal development takes place postnatally so these findings are not applicable to human situation. Secondly, studies in rats have demonstrated that delayed ossifications do not persist postnatally (so they are reversible).

Reference: Carney, E.W.and Kimmel, C.A. (2007) Interpretation of Skeletal Variations for Human Risk Assessment: Delayed Ossification and Wavy Ribs. Birth Defects Research (Part B) 80:473–496

 

In addition to the changes in body weight gain and food consumption seen in the current study, target organs supporting maternal toxicity were identified during repeated dose toxicity testing with the current substance. Especially at the dose level of 1000 mg/kg bw, target organs were evident within one month dosing:

- In the 28 day dietary toxicity and reproductive toxicity study in Wistar Han rats (Van Tuyl, 2010), following changes were seen at 15000 ppm (corresponding to mean of 977 mg/kg bw/day in males and 1256 mg/kg bw/day in females during gestation): increased serum potassium levels in both sexes, increased (relative) kidney weights and increased (absolute and relative) liver weights in both sexes. Microscopic changes were observed in the adrenal glands (multifocal vacuolation in zona glomerulosa), liver (diffuse midzonal/centrilobular hypertrophy),thyroid glands (diffuse follicular hypertrophy/hyperplasia).

- In the 90-day oral gavage toxicity study in Wistar rats (Grósz, 2013), the highest dose of 480 mg/kg bw/day showed higher absolute and relative weights of kidneys and livers in females only. Microscopic changes in the kidney comprised minimal epithelial degeneration of cortical tubules (cytoplasmic vacuolation, cellular sloughing) associated with the presence of homogenous eosinophilic content (casts) in tubular lumen. Following a 28-day treatment-free period, signs of the recovery were detected in the kidney. Microscopic 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; this was fully reversible during the 28-day recovery period.In the 14-day DRF by oral gavage study in Wistar rats, higher serum potassium, calcium and phosphorus concentrations were observed in females at 1000 mg/kg bw/day. Slightly higher thyroids weights were found in both sexes at 1000 mg/kg bw/day and in females dosed at 300 mg/kg bw/day.

 

No maternal or developmental toxicity nor teratogenicity was observed at 300 mg/kg bw/day in the OECD 414 study.One viscerally malformed foetus (unilateral ectopy of adrenal gland)was found and was considered incidental. No maternal or developmental toxicity was observed at 100 mg/kg bw/day. Based on the above results, the NOAEL for maternal toxicity and for embryo-/foetotoxicity was determined to be 300 mg/kg bw/day.

 

According to REACH 1907/2006 legislation Annex X Column I, pre-natal developmental toxicity is studied according to OECD TG 414 in one species at the most appropriate route of administration, having regard to the likely route of human exposure. According to Annex IX Column 2, a decision on the need to perform a study at the >100 t/y level or the next, thus at the > 1000 t/y level, on a second species is based on the outcome of the first test and all other relevant available data. According to OECD TG 414, the preferred non-rodent species is the rabbit. A waiver for a second species however is proposed based on expected gastro-intestinal intolerance in rabbits, which may prevent to test higher dosages and volumes. A second species teratogenicity study is not expected to lead to better assessment of developmental toxicity potential.

In the current dossier, the results from different studies on rats (OECD422; OECD408 and OECD414) have been provided concluding that there was no need to classify the substance for reproductive or developmental effects. Specifically for this substance, rat was considered the most appropriate species for the reasons stipulated above. In addition, a test proposal for a two-generation reproductive toxicity study (OECD416) is present in the current dossier, and this will bring additional data to be considered when deciding to require a 2nd species OECD 414 or not.


Justification for selection of Effect on developmental toxicity: via oral route:
Key study

Justification for classification or non-classification

Based on the results of the available oral repeated dose toxicity studies (Van Tuyl, 2011; Grosz, 2013) and the available prenatal developmental toxicity study in rats (Grosz, 2014a and b) and the available EOGRTs study (Peter and Meijer, 2019) there is no need to classify the substance in accordance to criteria listed in the CLP Regulation (EC 1272/2008).