Ester reaction products of 1,4-Benzenedicarboxylic acid with C11-14 iso-alcohols, C13-rich

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to sound scientific principles, similar to OECD TG 414.

Data source

Reference

Reference Type:

publication

Title:

Developmental Toxicity and Uterotrophic Studies with Di-2-Ethylhexyl Terephthalate

Author:

Faber, WD

Year:

2007

Bibliographic source:

Birth Defects Research (Part B) 80:396-405

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

In addition a uterotrophic assay was performed in rats treated by gavage on PND 19-21

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

di-2-ethylhexyl terephthalate (DEHT)

IUPAC Name:

di-2-ethylhexyl terephthalate (DEHT)

Constituent 2

Reference substance name:

Bis(2-ethylhexyl) terephthalate

EC Number:

229-176-9

EC Name:

Bis(2-ethylhexyl) terephthalate

Cas Number:

6422-86-2

Molecular formula:

C24H38O4

IUPAC Name:

bis(2-ethylhexyl) terephthalate

Details on test material:

Di-2-ethylhexyl terephthalate (DEHT) (Eastman 168 plasticizer; CAS 6422-86-2) is a clear, viscous liquid used as a general purpose plasticizer for flexible polyvinyl chloride (PVC) and polyvinyl chloride/vinyl acetate (PVC/VA) copolymers.

Gas chromatographic/mass spectral analysis was used to determine structural confirmation of the test article. Analytical results for all three studies determined that the material had a purity of 97.6–97.8%.

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)IGS BR

Details on test animals or test system and environmental conditions:

For the prenatal developmental toxicity studies, sexually mature, virgin, female rats were purchased from Charles River Laboratories (Raleigh, NC). The rats were 10 weeks old on receipt and were provided a 14-day acclimation period. The rats were housed individually in wire-mesh cages suspended above cage-board except during cohabitation for breeding. The animals were singly housed after mating.

For the uterotrophic assay, immature female rats were purchased from Charles River Laboratories. The animals were 7–8 days old on receipt and were supplied in litters of 12–14 animals with their own or another (fostering) dam. The litters were arbitrarily culled to 10 pups after receipt. The day of birth was designated PND 0. During
the 8-day acclimation period, the dams and pups were housed by litter in plastic maternity cages containing ground corncob nesting material (Bed-O’Cobs, The Andersons, Maumee, OH). After randomization, the weanlings were removed from the dam and gang-housed in plastic maternity cages (3–4 per cage) containing ground corncob bedding.

For all studies, Certified Rodent LabDiet 5002 meal (PMI Nutrition International, Richmond, IN) and water (reverse-osmosis-purified municipal water) were supplied ad lib throughout the acclimation period and during the study. The animals were maintained on a 12-hr light/dark cycle (lights on at 6 AM). Environmental conditions were set to maintain a temperature of 71.7 +/-5 °F and a relative humidity of 30–70%.

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

For the prenatal developmental toxicity studies, DEHT was blended in the basal diet at constant concentrations of 0.3, 0.6, and 1.0% (w/w) for the study in rats; the concurrent control group received basal diet only. Test diets were stored at room temperature in containers known not to contain plasticizers to eliminate the possibility of other plasticizers migrating into the feed. Test diets were offered ad lib from GD 0–20 in rats. Stability, homogeneity, and concentration verification of di-2–ethylhexyl terephthalate in the test diets was confirmed by HPLC/UV.

For the uterotrophic assay, DEHT dosing formulations were prepared in corn oil and dispensed daily (without analysis) at dosage levels of 0, 20, 200, and 2000 mg/kg/day. A 0.05 mg/mL stock formulation of EE, the positive control, was prepared by dissolving EE in small amounts
of 95% ethanol and adding corn oil to achieve the final nominal concentration. Dosing formulations were prepared daily in corn oil by diluting the stock formulation and were administered while stirring at a dosage level of 0.003 mg/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity, and concentration verification of di-2–ethylhexyl terephthalate in the test diets was confirmed by HPLC/UV.

Details on mating procedure:

After the acclimation period, each female presenting good health and meeting acceptable body weight requirements was paired with an untreated, sexually mature resident male of the same strain and source. Positive evidence of mating was confirmed by the presence of sperm in a vaginal smear. The day on which evidence of mating was identified was termed GD 0 and the animals were separated. The animals were singly housed after mating.

Duration of treatment / exposure:

Four groups of mated female Crl:CD (SD)IGS BR rats (25/group) were fed test diets containing 0, 0.3, 0.6, or 1.0% DEHT ad lib from GD 0–20.

Frequency of treatment:

rats were fed test diets containing the test substance ad lib.

No. of animals per sex per dose:

25/group

Control animals:

yes

Examinations

Maternal examinations:

All rats were observed twice daily for moribundity and mortality and detailed clinical observations were recorded for each animal from GD 0 through euthanasia. Individual body weights and food consumption were recorded daily throughout the treatment period. Mean compound consumption was calculated for the entire gestation period

All maternal rats were euthanized by carbon dioxide inhalation on GD 20. The thoracic, abdominal, and pelvic cavities were examined at necropsy. Post mortem findings were correlated with the ante mortem findings, and any abnormalities were recorded. Individual liver weights were also recorded.

Ovaries and uterine content:

The uterus and ovaries were excised, and the number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses (live or dead), early and late resorptions, and the total number of implantation sites were recorded. Placentae were also examined. Uteri with no macroscopic evidence of implantation were placed in 10% ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:

Fetal Examination for the Prenatal Developmental Toxicity Studies
Each viable fetus was examined externally, sexed, weighed, and euthanized by intrathoracic injection of sodium pentobarbital. Each viable fetus was dissected for visceral examination using a modification of a fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). Fetal kidneys were examined and those with indications of incomplete development were graded for renal papillae development
(Woo and Hoar, 1972). Renal papillae with a Woo and Hoar Grade 0 (papillae not developed) were considered to be developmental variations. No Grade 0 renal papillae were observed in any rat fetuses. Heads from approximately 50% of the fetuses in each litter were placed in Bouin’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining fetuses were examined by a mid-coronal slice. All fetal carcasses were eviscerated and fixed in 100% ethyl alcohol, macerated in potassium hydroxide, and stained with Alizarin Red S and Alcian Blue by a method similar to that described by Dawson (1926) and Inouye (1976). Findings of anatomic departures from normal were recorded as either malformations (those structural anomalies that alter general body conformity, disrupt or interfere with body function, or may be incompatible with life) or developmental variations (alterations in anatomic structure that are considered to have no significant biologic effect on animal health or body conformity, representing slight deviations from normal). Nonviable fetuses, if autolysis was minimal or absent, were examined, weighed, sexed, and the crown–rump length was measured. Crown–rump measurements and degrees of autolysis were recorded for late resorptions when present.


Weanling Observations for the Uterotrophic Assay:
Clinical observations and body weights. All rats were observed twice daily, morning and afternoon, for appearance, behavior, moribundity and mortality. Individual clinical observations were recorded daily and signs of toxicity were observed at the time of dosage administration and approximately 1 hr post-dose. Individual body weights were recorded on a daily basis
beginning the day before the initiation of dose administration.
Euthanasia and uterine weights. On PND 22, approximately 24 hr after the last dose administration, the animals were euthanized by carbon dioxide inhalation. The uteri were harvested in the same sequence and at approximately the same time as the daily dose administration. Each uterus was excised, and the wet weight was recorded. The uteri were then opened and blotted with dry filter paper to absorb the luminal fluid and reweighed subsequently to collect the blotted weight.

Statistics:

All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group. For the rat prenatal developmental toxicity studies, the litter rather than the fetus was considered to be the experimental unit for all statistical analyses. For the uterotrophic assays, the weanling was considered to be the experimental unit for statistical analyses. For the prenatal developmental toxicity studies, mean maternal body weights (absolute and net), food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, fetal body weight (separately by sex and combined), and liver weights were subjected to a one-way analysis of variance (ANOVA) to determine intergroup differences. If the nonparametric ANOVA showed statistically significant (P<0.05) intergroup variance, Dunnett’s test was used to compare the test article-treated groups to the control group. Mean litter proportions (%/litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and fetal sex distribution) and total fetal malformations and developmental variations were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA showed statistically significant (P<0.05) intergroup variance, the Mann-Whitney U-test was used to compare the test article-treated groups to the control group.

For the uterotrophic assay, mean body weights, body weight gains, uterine weights (wet and blotted), and luminal fluid weights were analyzed by a parametric ANOVA and Dunnett’s test as described for the prenatal developmental toxicity studies.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
No DEHT-related internal findings were noted in maternal rats at any dosage level during necropsies on GD 20. Although mean net body weight and net body weight gain (defined as the body weight and body weight change exclusive of the weight of the uterus and contents) in the 1.0% DEHT maternal rats were statistically significantly reduced (P<0.05 or P<0.01) when compared to the control group values, gravid uterine weights in this group were similar to the control group. This finding suggests that the systemic effects of DEHT were restricted to the maternal compartment. Mean liver weight in the 1.0% DEHT group was statistically significantly (P<0.05) increased relative to the control group value. Mean liver weights in the 0.3 and 0.6% group rats were similar to the control group value and no other test-article related effects were noted at any dosage level.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

rats

Effect level:

458 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Remarks:

rats

Effect level:

2 000 mg/kg bw/day (nominal)

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
No indication of prenatal developmental toxicity in rats was observed at any exposure concentration of DEHT on evaluation of fetal morphologic data. Fetal malformations were observed in 1(1), 1(1), 1(1), and 2(2) fetuses (litters) in the control, 0.3%, 0.6%, and 1.0% DEHT groups, respectively, and were considered to be spontaneous in origin. Although there was no difference in the overall total number of skeletal variations between groups, an increase in the number of fetuses with 14th rudimentary ribs was observed in the 1.0% group relative to the control group. When evaluated on a litter proportional basis, the increase was statistically significant (P<0.01; data not shown). In the absence of any other indications of developmental toxicity in the 1.0% group rats, however, this increase was not considered to be adverse. No other differences from the control group were observed for developmental variations at any DEHT concentration.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

rats

Effect level:

747 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: no effects observed in rats

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Results for Utertrophic Assay

Clinical signs, mortalities and body weights. All weanling females survived to the scheduled euthanasia on PND 22. No DEHT-related clinical findings were observed in the 20, 200, or 2000 mg/kg/day DEHT groups at the daily examinations or 1 hr after dosing. No differences in mean body weight were noted in treated animals when compared to the control group. Mean body weight gain in the 2000 mg/kg/day DEHT group was reduced after the first day of dosing (PND 19–20) that resulted in a 19% reduction in cumulative mean body weight gain when the entire treatment period interval (PND 19–22) was evaluated compared to the control group. No

other effects were observed in weanlings treated with DEHT at any dose level.

Uterine weights. Mean wet and blotted uterine weights were unaffected by DEHT administration at all dose levels. Differences from the vehicle control group were slight and not statistically significant. In the weanlings treated with the positive control article (EE),

mean uterine wet, blotted uterine weights, and luminal fluid were increased 3.1-, 2.9-, and 4.0-fold, respectively, compared to the vehicle control group. These differences were all significant statistically (P<=0.01).

Applicant's summary and conclusion

Conclusions:

The NOEL for developmental toxicity in rats was 747 mg/kg/day, the highest dose tested as no fetotoxic, developmental or teratogenic effects were observed.

Executive summary:

This study was conducted to evaluate the potential adverse effects of di-2-ethylhexyl

terephthalate (DEHT) exposure on in utero development in rats. In addition, a uterotrophic assay for

estrogenic activity was conducted in sexually immature rats. In the developmental toxicity studies, diet containing DEHT was fed to four groups of mated female Crl:CD(SD)IGS BR rats (25/group) from gestation day (GD) 0–20. Concentrations within the feed were 0, 0.3, 0.6, and 1.0% for the rats. Laparohysterectomies were carried out on the last day of exposure and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. The fetuses were weighed, sexed, and examined for external, visceral and skeletal malformations, and developmental variations. The dose rate from dietary DEHT exposure was 0, 226, 458, and 747 mg/kg/day in the rats for the control, low, mid, and high-exposure groups, respectively. DEHT exposure did not affect clinical observations. A slight reduction in body weight gain was noted in the high-dose level rat group; the remaining groups were unaffected. At necropsy, increased liver weights were noted in the high-dose rat group. Mean numbers of implantation sites and viable fetuses, mean fetal weights, and mean litter proportions of preimplantation loss, early resorptions, late resorptions, and fetal sex ratios were unaffected by DEHT exposures. No test article-related malformations or variations were observed at any concentration level in the rat developmental toxicity studies. In the uterotrophic assay for estrogenic activity, sexually immature female rats received oral gavage doses 20, 200, or 2000 mg DEHT/kg bw/day from postnatal day (PND) 19–21. A slight reduction in rate of body weight gain was noted on the first day of dosing in the high-dose group, but no other indications of toxicity were evident. DEHT exposure did not affect wet or blotted uterine weight parameters in any of these dose groups. The NOEL for developmental toxicity in rats was 747 mg/kg/day. The NOEL for estrogenic activity was 2000 mg/kg/day. The NOEL for maternal toxicity was 458 mg/kg/day in rats.