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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:
11 Feb 2020 - 11 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene dibenzoate
EC Number:
202-338-6
EC Name:
Ethylene dibenzoate
Cas Number:
94-49-5
Molecular formula:
C16H14O4
IUPAC Name:
2-(benzoyloxy)ethyl benzoate
Test material form:
solid: particulate/powder
Details on test material:
Appearance: white to off white powder or flakes
Storage conditions: at room temperature protected from light

Test animals

Species:
rat
Strain:
other: Wistar Han
Details on test animals or test system and environmental conditions:
The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 181 and 258 g
- Fasting period before study: no
- Housing: individually in Makrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and equipped with water bottles.
- Diet (ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (ad libitum): Municipal tap water
- Acclimation period: at least 5 days
- For psychological/environmental enrichment and nesting material, animals were provided with paper and aspen wooden sticks, except when interrupted by study procedures/activities.
- It was considered that there were no known contaminants in the feed or in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 46 to 52
- Air changes (per hr): Ten or greater, 100% fresh air, no air recirculation
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 16 Feb 2020 to 05 Mar 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Polyethylene Glycol 400
Remarks:
Specific gravity: 1.125
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a suspension. They were heated to a maximum temperature of 60 ± 5°C for at least 30 minutes to obtain visual homogeneity. Formulations were filled out in daily portions and stored in the refrigerator until use. On the morning of administration, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 5 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
The dose volume for each animal was based on the most recent body weight measurement. The dosing formulations were stirred continuously during dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected in week 1 of dosing. The acuracy of preparation (concentration) was analysed for all groups, homogeneity was checked for groups 2 and 4. The homogeneity results obtained from the top, middle and bottom for the preparations of Groups 2 and 4 were averaged and utilized as the concentration results.
Analyses were performed using a validated method. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was lower or equal to 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 516416) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
Females were already mated upon arrival.
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum, inclusive
Frequency of treatment:
Once daily
Duration of test:
Animals were euthanized by an oxygen/carbon dioxide procedure on Day 21 post-coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
- Dose selection rationale: Dose levels were selected based on the results obtained in a Combined 90-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of Ethylene Glycol Dibenzoate by Oral Gavage in Rats (Test Facility Study No. 516410), and in an attempt to produce graded responses to the test item. In this study, dose levels of 100, 300 and 1000 mg/kg bw/day were administered by daily oral gavage. A parental NOAEL of 300 mg/kg bw/day was established based on decreased Thyroxine (T4) levels (for females in combination with increased incidence of follicular cell hypertrophy in the thyroid) at 1000 mg/kg bw/day. The NOAEL for reproductive toxicity was established at 1000 mg/kg bw/day, and the NOAEL for developmental toxicity was established at 300 mg/kg bw/day, based on a skewed sex ratio towards females, a reduced live birth index, increased postnatal loss and decreased body weights at 1000 mg/kg bw/day. Since the dosing period in this study (93-98 days for females that delivered) extensively exceeds the dosing period in this prenatal and developmental toxicity study (14 days), it was considered that 1000 mg/kg bw/day could be used as highest dose level.

- Rationale for animal assignment (if not random): n.a.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: between 7:00 and 9:00 a.m.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Yes
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21.
- All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
- The thyroid gland and uterus were weighed at necropsy for all animals. Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) was calculated.
- In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
- The thyroid gland from all animals were examined histopathologically.
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 and distribution of live and dead fetuses, sex of each fetus based on the anogenital distance
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected: 0.1 mL (target volume)
- Other: Blood samples were processed for serum, and serum was analyzed for the thyroid hormones Triiodothyronine (T3), Thyroxine (T4), and Thyroid-Stimulating Hormone (TSH).
TSH and T4 analysis with the IMMULITE® 1000 method, T3 using LC-MS according to the bioanalytical method validated in Test Facility Study No. 20213516.
Fetal examinations:
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: yes. The AGD was normalized to the cube root of the fetal body weight.
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 (or %CV or SE when deemed appropriate) 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.

Parametric:
Datasets with at least 3 groups (control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.
Indices:
- Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.
- Corrected Body Weight Gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
- Relative Food Consumption: Calculated against the body weight for scheduled intervals.
- Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum.

For each group, the following calculations were performed:
Preimplantation loss (%): ((number of corpora lutea - number of implantation sites) x 100) / number of corpora lutea

Postimplantation loss (%): ((number of implantation sites - number of live fetuses) x 100) / number of implantation sites

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%): (number of viable fetuses affected/litter x 100) / number of viable fetuses/litter
Historical control data:
Historical control data on fetal examination were provided.
Test species: Rat (Crl:WI(Han) (ourbred, SPF Quality)),
Study range: 2015-2019
No. of studies: 60
Total No. of dams in the control group: 1321
Total No. of fetuses/Litters examined externally: 13756

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations did not reveal any alterations that were considered to have arisen as a result of dosing with the test item.
Salivation (slight) was seen after dosing in 5/22 females treated at 1000 mg/kg bw/day, mainly in the second half of the dosing period. This was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in body weight and body weight gain were noted following dosing up to 1000 mg/kg bw/day.
Mean body weights and body weight gains of treated animals remained in the same range as controls over the dosing period. However, after correction for gravid uterus weight mean body weight gain was slightly, but statistically significantly lower at 1000 mg/kg bw/day when compared to the concurrent control group (24.7 gram vs 30.4 gram; 0.81x of control). As the mean value of the high dose group remained well within the available historical control range , no toxicological relevance was attached to this finding.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was comparable to the control level following dosing up to 1000 mg/kg bw/day.
The slightly, but statistically significantly lower absolute food consumption observed in females treated at 100 mg/kg bw/day from Days 15-18 post-coitum and in females at 1000 mg/kg bw/day from Days 18-21 post-coitum was considered unrelated to dosing with the test item since no clear dose-related trend could be established.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Note: At 300 mg/kg bw/day, one female was not gravid and was therefore excluded from the data tables.
A trend towards slightly lower serum levels of thyroid stimulating hormone (TSH), total triiodothyronine (T3) and total thyroxine (T4) was observed from 100 mg/kg bw/day onwards, as is indicated in the table below. Changes compared to the concurrent control group were relatively small and did not always reached statistical significance. These changes were considered test item-related, but not adverse since all mean values remained within the available historical control range See table on thyroid hormone analysis in "Any other information on results incl. tables".
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Note: At 300 mg/kg bw/day, one female was not gravid and was therefore excluded from the data tables.
There were no treatment-related effects attributed to administration of the test item.
Higher thyroid gland weights (absolute and relative to body weight) were noted in the 100, 300 and 1000 mg/kg bw/day group females, compared to the concurrent control group (not statistically significant at 300 mg/kg bw/day). This was regarded to be unrelated to dosing with the test item, based on absence of a clear dose-response. Moreover, all means remained within the range considered normal for rats of this age and strain, and no macroscopic or microscopic correlate was present.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of dosing with the test item.
Incidental findings that were noted among control and/or treated animals included a control female with two missing toes, one female at 300 mg/kg bw/day with agenesis of thyroid gland and another female at this mid dose with many dark-red foci on the thyroid gland. These findings are occasionally seen among rats used in these types of study. At the isolated incidence and in absence of a dose-related trend they were considered unrelated to dosing with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid glands.
All 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:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Litter incidence of pre implantation loss, as well as litter incidence of post-implantation loss in the control and test item groups were similar. All values remained within the normal range of biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Mean numbers of early and late resorptions in the control and test item groups were similar. All values remained within the normal range of biological variation.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Numbers of pregnant females and mean numbers of corpora lutea in the control and test item groups were similar. All values remained within the normal range of biological variation.

At 300 mg/kg bw/day, one female was not pregnant, i.e. she had no corpora lutea and no implantation sites (confirmed by Salewski staining; taken from raw data). This isolated case of non-pregnancy was unrelated to dosing with the test item as it occurred at the mid dose only.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: The for uterus corrected maternal body weight gain decrease of 19% when compared to concurrent controls was not considered adverse.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male and female fetal body weights were significantly reduced at 1000 mg/kg bw/day (relative difference to concurrent controls: -6% for both, males and females). Values were below the lower limit of the historical control range.
Fetal weights (male, female and combined) in the 100 and 300 mg/kg bw/day dose groups were comparable to the concurrent control group and remained within the historical control range of the Test Facility.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
In the 1000 mg/kg bw/day group, mean percentage of viable fetuses per litter was statistically significantly increased when compared to the concurrent control group. Mean incidences were 90.2, 93.4, 95.3 and 98.8% in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. This was accompanied by a decreased mean litter incidence of early resorptions, total resorptions and post-implantation loss in the high dose group. Mean values were just below the available range of historical control data. However, in view of the direction of change these observations were considered unlikely to be related to dosing with the test item.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no changes in litter size observed that were considered to be related to dosing with the test item. No information on litter weight is available.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant effects on fetal anogenital distance (both sexes) noted after dosing up to 1000 mg/kg bw/day.
The statistically significantly higher mean value of corrected anogenital distance observed in male pups of the 1000 mg/kg bw/day group when compared to the concurrent controls was considered due to biological variation and does not signify a biological relevant change. The lower body weight of pups in this dose group aided to the relative increase in corrected anogenital distance. Mean value (1.54 mm) of the high dose group (1000 mg/kg bw/day) was identical to that of the low dose group (100 mg/kg bw/day), and all individual levels in high dose males (ranging from 1.27-1.67 mm) remained below the highest value (1.71 mm) measured in concurrent control males.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in external morphology noted that could be attributed to administration of the test item.
Four fetuses were externally malformed, two each in the 100 and 300 mg/kg bw/day groups. At 300 mg/kg bw/day, two littermates were affected. One of these 300 mg/kg bw/day fetuses had exencephaly and a small lower jaw, and at visceral examination, it was noted that also both eyes were absent. Skeletal examination substantiated these malformations and additionally revealed a vertebral and skull anomaly. The littermate at 300 mg/kg bw/day had a cleft palate and one missing eye bulge that both were confirmed at skeletal examination, whereby also a vertebral and rib anomaly was observed.
One of the two affected fetuses at 100 mg/kg bw/day had besides a small lower jaw and protruding tongue with matching skeletal jaw findings also a skull anomaly. The other fetus had an omphalocele.
These malformations were considered to be spontaneous in origin due to random occurrences and the lack of a dose response. Moreover, all findings except protruding tongue had been observed in historical control fetuses before.
No external variations were seen in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in skeletal morphology noted that were considered to be a direct effect of exposure to the test item.
Besides the underlying skeletal malformations in the externally malformed fetuses at 100 and 300 mg/kg bw/day and their vertebral, rib and/or skull anomalies described in a paragraph above, a vertebral anomaly occurred in one fetus at 1000 mg/kg bw/day. All these findings are previously observed in historical control fetuses, and at the single or low incidences they occurred, they were considered chance findings.
At 1000 mg/kg bw/day, two fetuses had bent limb bones which also occurred in a dead fetus at 100 mg/kg bw/day and control fetus. The remaining malformations in this study, sternoschisis and vertebral centra anomaly occurred singly in a 300 mg/kg bw/day fetus and a control fetus, respectively. The incidence and group distribution of these findings does not indicate a relationship to treatment with the test item, and as all were listed in historical control data, they were considered chance findings.
Skeletal variations occurred at a mean litter incidence of 87.3%, 82.1%, 69.9% and 91.0% in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The incidence at 300 mg/kg bw/day was statistically significantly decreased compared to the control value, which was caused mainly by significant lower incidences of reduced ossification of the skull and bent ribs. Mean litter incidences for these respective variations were 28.1%, 22.4%, 7.8%, 35.1% and 35.1%, 15.0%, 13.5%, 39.8% in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The cause of less fetuses having reduced ossification of the skull and bent ribs at 300 mg/kg bw/day is not known, but considered not related to dosing with the test item due to absence of a dose related trend. Moreover, it was noted that the 300 mg/kg bw/day incidences for these two variations were within the historical control data range, while both the control and high dose incidences were above the maximum value.
Among the variations, the incidence of unossified metacarpals and/or metatarsals was increased at 1000 mg/kg bw/day. Incidences were 1.6%, 0.9%, 2.8% and 11.0% per litter in the control 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at 1000 mg/kg bw/day was not statistically significant and the value remained within the historical control data range (0.0% - 17.6% per litter). However, this parameter is a major indicator of delayed skeletal ossification, and all six affected male fetuses and 6/9 affected female fetuses had weights below the male or female group mean weights. Moreover, as group mean fetal weights at 1000 mg/kg bw/day were statistically significantly lower compared to the control value (4.9 versus 5.3 grams, respectively), it was considered that the higher incidence of unossified metacarpals and/or metatarsals at 1000 mg/kg bw/day was secondary to the fetal weight effect and not a direct effect of the test item.
The other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within or near the range of available historical control data. Therefore, they were not considered related to dosing with the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in visceral morphology noted that could be attributed to administration of the test item.
Besides the externally malformed fetus without eyes at 300 mg/kg bw/day described in the previous section, two cases of situs inversus were noted. These occurred in a 300 mg/kg bw/day fetus and a control fetus which did not indicate a relationship to dosing with the test item.
The visceral variations that were noted occurred infrequently, in the absence of a dose-related incidence trend, in control fetuses exclusively and/or at frequencies that were within the range of available historical control data.

Effect levels (fetuses)

Key result
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

Key result
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
No changes in external, skeletal or visceral morphology noted that could be attributed to administration of the test item.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Dose formulation analysis

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks. The maximal contribution to the Group 2 sample was as low as 0.0028%, and therefore considered negligible.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

 

Thyroid Hormone analysis

Mean Percent Differences in Thyroid Hormone Levels from Control Group

 

Dose level (mg/kg/day)

100

300

1000

TSH

-4

-15

-19

Total T3

-8

-15*

-24**

Total T4

-10

-13

-18*

*: P<0.05, **: P<0.01

 

Historical Control Data

Historical control data on maternal developmental effects such as pregnacy, corpora lutea, implanation loss, and on fetal parameters such as viable fetuses, sex, weight, AGD, malformations and variations can be found in appendix 3 of the study report.

Applicant's summary and conclusion

Conclusions:
Maternal NOAEL:   1000 mg/kg bw/day (the for uterus corrected maternal body weight gain decrease of 19% when compared to concurrent controls was not considered adverse).
Developmental NOAEL: 300 mg/kg bw/day (based on the 6% lower fetal body weights observed at 1000 mg/kg bw/day when compared to concurrent controls).
Executive summary:

The objectives of this study were to determine the potential of Ethylene Glycol Dibenzoate (EGDB) to induce developmental toxicity in the rat after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested.

Time-mated female Wistar Han rats (22 females/dose level) were treated with Ethylene Glycol Dibenzoate by daily oral gavage from Day 6 to Day 20 post-coitum, inclusive, at 0, 100, 300, 1000 mg/kg bw/day. The study is performed in accordance with OECD 414 (June 2018), EU Method B.31 (2008) and EPA OPPTS 870.3700 (1998).

Formulation analyses confirmed that the formulations were prepared accurately and homogenously.

 

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

A trend towards slightly lower serum levels of thyroid stimulating hormone (TSH), and total triiodothyronine (T3) and thyroxine (T4) was observed from 100 mg/kg bw/day onwards. Changes compared to the concurrent control group were relatively small and did not always reach statistical significance. At the highest dose of 1000 mg/kg bw/day, mean values for TSH, T3 and T4 reached respectively 0.81x (not statistically significant), 0.76x and 0.82x of control. These changes were considered possibly test item-related, but not adverse since all mean values remained within the range of historical control data. This study is not suited to evaluate the possible mechanism how the observed effects on the hormones could be brought about, but it is noteworthy that the same direction and magnitude of the observed changes in the TSH level and the T3 and T4 levels, is against the normal physiological feedback mechanism for these hormones. (Attached graph shows that the small differences between averages of the groups for each of the hormones are insignificant compared to the individual variability.)

No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, body weight, food consumption, gross pathology, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss). Although there was a trend of lower body weight gain after correction for gravid uterus weight, which was 19% lower for the 1000 mg/kg bw/day group compared to control (See attached graph), this was not considered to be adverse, based on available historical control.

 

No developmental toxicity was observed in the 100 and 300 mg/kg bw/day groups.

At 1000 mg/kg bw/day, mean male and female fetal body weights were statistically significant reduced (relative difference to concurrent controls: -6% for both sexes). As fetal body weights have a small variation in general and mean values at 1000 mg/kg bw/day were below the lower limit of the historical control range, these findings were considered adverse.

There was an increased litter incidence of unossified metacarpals and/or metatarsals observed. Incidences were 1.6%, 0.9%, 2.8% and 11.0% per litter in the control 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at 1000 mg/kg bw/day was not statistically significant and the value remained within the historical control data range (0.0% - 17.6% per litter).

Both the lower pup BW, and the increased litter incidence of unossified metacarpals and/or metatarsals were minimal and hardly significant, as was the 19% lower body weight gain after correction for gravid uterus weight in the dams all at 1000 mg/kg bw. Together it is suggestive for a possible secondary effect of delayed fetal growth, and not a direct effect of the test item.

Remarkable, but likely just to be fortuitous, are the a highly statistically significant increase in number of viable fetuses observed at 1000 mg/kg, as well as decreased early and total resorptions, and a decreased post-implantation loss. Otherwise, no test item-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, anogenital distance, external, visceral and skeletal malformations, and external and visceral developmental variations).

Based on the results in this prenatal developmental toxicity study with Ethylene Glycol Dibenzoate the maternal NOAEL is 1000 mg/kg bw/day and the developmental NOAEL is 300 mg/kg bw/day (based on the 6% lower fetal body weights observed at 1000 mg/kg bw/day when compared to concurrent controls).