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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-2014
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate)
- Substance type: Industrial chemical
- Physical state: Colourless liquid
- Analytical purity: >97%
- Lot/batch No.: 2X010
- Expiration date of the lot/batch: 16 April 2014
- Stability under test conditions: Stable
- Storage condition of test material: Refrigerator (2-8°C)
- Other: Manufacturer: Solutia UK Limited, Corporation Road Newport, South Wales, Gwent NP19 4XF, United Kingdom

Test animals

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)

Administration / exposure

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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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.