Bis(2-ethylhexyl) terephthalate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliable without restriction; study conducted according to GLPs and to Redbook 2000 Guideline IV.C.9.b., OPPTS 870.3700, ICH Guideline Section 4.1.3, and OECD Guideline 414.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Redbook 2000 Guideline IV.C.9.b

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline Section 4.1.3

Deviations:

yes

Remarks:

One deviation was noted but it did not impact the quality or integrity of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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:

1,4-bis(2-ethylhexyl) benzene-1,4-dicarboxylate

Constituent 2

Reference substance name:

Reference substance 001

Cas Number:

6422-86-2

Details on test material:

Test Substance:
-Test substance: bis (2-ethylhexyl) terephthalate (Eastman™ 168 Plasticizer)
-Lot number: TD-4016856
-Purity (at study start): 99.79%
-Purity (at study termination): 97.6%
-Homogeneity and Stability: Analytical analysis determined that the test substance in the vehicle, standard rat chow, was homogeneous and stable for up to 15 days.

Test animals

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

Test animals:
-Source: Charles River Laboratories, Portage, Michigan
-Sex: female
-Condition at receipt: sexually mature female virgins
-Age at receipt: approximately 80 days old
-Age at study initiation: approximately 12 weeks
-Acclimation period: approximately 6 days
-Housing: Animals after the first 6 days of acclimation were single housed in stainless-steel cages. Cage paper was changed at least three times a week during the study.
-Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, with or without test material, ad libitum
-Water: Reverse osmosis-purified drinking water was supplied via an automatic watering system, ad libitum
-Method of animal identification: Each mouse was uniquely identified by a tail tattoo

Environmental Conditions:
-Temperature: 70.4 to 70.6 °F (21.3 - 21.5 °C)
-Humidity: 39.7 - 46% relative humidity
-Photoperiod: 12 hours light/12 hours dark
-Air Exchanges: approximately 10 per hour

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Certified Rodent LabDiet 5002 (PMI Nutrition International, LLC)

Details on exposure:

The control and test diets (1000, 3000 and 7000 ppm) were offered ad libitum and supplied weekly from gestation Days 0-18 to randomly selected groups of 25 pregnant females.

Study Schedule:
-Experimental start date (animal receipt): January 25, 2005
-Experimental start date: February 4, 2005
-Test diet administration: February 4-25, 2005
-Last laparohysterectomy: February 25, 2005
-Experimental termination/completion date: April 18, 2005.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Animals were fed PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, with or without test substance. For test diets, an appropriate amount of test substance and diet for each group was weighed, placed into a Hobart mixing bowl, and mixed for 5 minutes. The premix was transferred to a V-blender and mixed for 10 minutes with enough basal diet to form batches of diets containing 1000, 3000 and 7000 ppm. Dose levels were selected based on a previous range-finding study in which doses of 500 to 10000 ppm were well tolerated in pregnant mice.

The test diets were prepared biweekly, placed in labeled high density polyethylene (HDPE) plastic drums with no liners, and stored at room temperature. Diets thus prepared were stable for 15 days, as shown by previous analyses with the test substance. The control diet was weighed biweekly into a plastic bag and placed in a HDPE plastic storage drum with no liner.

Prior to use, samples (approximately 100 g each) of each diet (including the control) were taken from the top, middle and bottom strata for homogeneity/ concentration analyses. A GC/FID area % purity determination was also performed. Samples of at least 10 g were collected prior to and after treatment and were analyzed for structure of the test substance. All prepared diets were homogeneous (samples from all strata were within 100-102% of target concentrations) and contained the amount of test substance desired (ranged from 96-106% of target concentrations of 1000, 3000 and 7000 ppm). The substance used in the study was at least 97.6% pure at the end of the study (compared to a purity of 99.79% on the Certificate of Analysis). A GC-MS analysis showed the substance to be authentic di (2-ethylhexyl) terephthalate. No contaminants were present in the feed or water at concentrations sufficient to affect the outcome of the study.

Details on mating procedure:

Females in good health that weighed at least 20 g were paired for mating in the home cage of a resident, sexually mature Crl:CD1(ICR) male from the same source. Each mating pair was examined daily. Following positive evidence of mating (vaginal copulatory plug), the females were returned to their individual cages. The day on which evidence of mating was identified was designated as Gestation Day 0. Actual body weight values ranged from 25.8 to 34.2 g on Gestation Day 0.

Duration of treatment / exposure:

Gestation Days 0-18

Frequency of treatment:

continuous

Duration of test:

18 days

No. of animals per sex per dose:

25 pregnant females per dose

Control animals:

yes, concurrent no treatment

Examinations

Maternal examinations:

-Clinical observations: All mice were observed twice daily for moribundity and mortality. Individual, detailed clinical observations were recorded daily from Gestation Days 0-18. All significant findings were recorded.
-Body Weight: All animals were weighed daily from Gestation Days 0-18
-Food Consumption: All animals were weighed daily from Gestation Days 0-18
-Mean Compound Comsumption: Mean compound consumption (in mg/kg/day) for each group was determined by dividing the concentration of test material in the diet (in mg/kg) by the g/kg/day food consumption value for each interval.

Ovaries and uterine content:

All surviving mice were euthanized on Gestation Day 18 by CO2 inhalation. The thoracic, abdominal and pelvic cavities were opened and examined, and any abnormalities were recorded. Liver weights were recorded. The uterus and ovaries were exposed and excised. The numbers of corpora lutea on each ovary were recorded. All implantation sites (including resorptions) were numbered in consecutive order (beginning with the left distal to the left proximal uterine horn), noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues exhibiting gross changes were retained in 10% neutral-buffered formalin for possible future examination. Uteri with no evidence of implantation were placed in 10% ammonium sulfide solution for detection of early implantation loss.

One female in the high dose group delivered on Gestation Day 18. This animal was euthanized and necropsied. The number and location of implantation sites, corpora lutea and viable pups were recorded and included in the mean fetal data. Viable pups from this female were euthanized and examined viscerally and skeletally. A control female was euthanized on Gestation Day 6 in extremis. The uterus had no macroscopic evidence of implantation and was placed in 10% ammonium sulfide. Any grossly affected tissues from this animal were retained in 10% neutral-buffered formalin and subsequently examined.

Fetal examinations:

At necropsy each fetus was weighed, sexed, examined macroscopically for any external findings, and euthanized by an intrathoracic injection of sodium pentobarbital (if necessary). The external examination included (but was not limited to) an examination of the eyes, palate and external orifices. The crown-rump length, weight, and sex of nonviable fetuses (not autolyzed) were determined. Crown-rump measurements and degrees of autolysis were recorded for late resorptions (if present). Each viable fetus was examined viscerally (included examination of the heart and major blood vessels). Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one half of the fetuses in each litter were placed in Bouin's fixative and subsequently examined using the Wilson sectioning technique. The heads from the rest of the animals were examined by a mid-coronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation, each fetus was macerated in KOH and stained with Alizarin Red S and Alcian Blue. External, visceral and skeletal findings were recorded as developmental variations (changes representing slight deviations from normal that were considered to have no effect on animal health or body conformity) or malformations (anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Fetal data were presented according to the numbers of fetuses and litters available for examination in each group, and the number of affected fetuses per litter on a proportional basis.

Statistics:

Analyses were conducted using two-tailed tests (except where noted) at significance levels of p < 0.05 and 0.01, comparing each test group to the control group. Data were presented as mean ± SD. Mean maternal body weight (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, fetal body weights (separately and by sex combined) and liver weights were analyzed using a one-way analysis of variance (ANOVA). If the ANOVA revealed a significant intergroup variance, Dunnett's test was used to compare test groups to the control group. Mean litter proportions of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post implantation loss and fetal sex distribution) and total malformations and developmental variations (external, visceral, skeletal and combined) and of each malformation or variation were analyzed using the Kruskal- Wallis nonparametric ANOVA test to determine intergroup variances. If the variance was significant, the Mann- Whitney U-test was used to compare test to control data.

Indices:

Intrauterine data were summarized using two methods of calculation. An example of each method of calculation follows:

1. Group Mean Litter Basis:
No. Dead Fetuses, Postimplantation Loss/Litter = [Resorptions (Early/Late)/Group] / [No. Gravid Females/Group]

2. Proportional Litter Basis:
Summation Per Group (%) = [∑ Postimplantation Loss/Litter (%)] / [No. Litters/Group]

Where:
Postimplantation Loss/Litter (%) = [No. Dead Fetuses, Resorptions (Early/Late)/Litter] / [No. Implantation Sites/Litter] x 100



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 and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = [∑ Viable Fetuses Affected/Litter (%)] / [No. Litters/Group]

Where:
Viable Fetuses Affected/Litter (%) = [No. Viable Fetuses Affected/Litter] / [No. Viable Fetuses/Litter] x 100

Historical control data:

Historical Control data from the testing laboratory was provided as part of the study report consisting of 22 developmental toxicity/teratogenicity studies.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal data with dose level:
Clinical observations and survival: All animals survived to study termination, with the exception of one high dose animal that was euthanized after delivery on Gestation Day 18 and one control animal that was euthanized in extremis on Gestation Day 6. This animal had facial lacerations and was hypoactive, cool to the touch, and exhibited tremors. At necropsy, this animal was found to have a fractured nasal bone and was nongravid. Clinical findings were limited to single animals and/or did not occur in a dose-related manner.

Body weight: Maternal body weight, body weight gain, net body weight, net body weight gains and gravid uterine weights were not affected by treatment with any dose. The only significant change was reduced body weight gain during Gestation Days 12-13 in the 3000 ppm group (2.0 ± 0.63 g in treated vs. 2.5 ± 0.43 g in control), which was not seen at 7000 ppm.
Food consumption: There was no effect of test material on food consumption.

Gross pathology, incidence and severity: There were no internal findings in the high dose animal that delivered on Gestation Day 18. All females were internally normal, with the exception of one female in the high dose group that had a white area on the liver. The numbers of females that were nongravid in the control, 1000, 3000 and 7000 ppm groups were 3, 4, 4 and 1, respectively.

Organ weight changes: Mean absolute liver weights of animals in the 3000 (3.0053 ± 0.35957) and 7000 (3.2516 ± 0.29470) ppm groups were 8.4% (p < 0.05) and 17.3% (p < 0.01) higher than control.

Reproductive parameters: The numbers of nongravid females in the control, 1000, 3000 and 7000 ppm groups were 3/25, 4/25, 4/25 and 1/25, respectively (no significant difference). Preimplantation loss, number of implantation sites and numbers of corpora lutea were not affected by any dose of test material. The only significant change was a higher mean litter proportion of preimplantation loss in the 3000 ppm group (6.7 ± 8.18% in treated vs. 3.0 ± 9.21% in control). Since this was not observed in the 7000 ppm group (preimplantation loss was 2.7 ± 5.87%), it was not considered to be related to test material.

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

1 000 ppm

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal data with dose level:
Litter size, weight and sex ratio: There was no effect of treatment on live litter size, fetal body weight or sex ratios.

Total numbers of malformations/variations: The numbers of fetuses (litters) available for examination were 265(21), 276(21), 246(21) and 308(24) in the control, 1000, 3000 and 7000 ppm groups, respectively. Malformations (all of which were considered to be spontaneous) were observed in 4(3), 1(1), 8(4) and 3(3) fetuses (litters) in the respective groups. There was no effect of test material on the fetal incidence or mean litter percent with malformations.

Grossly visible abnormalities: No external developmental variations were reported in any group. External malformations occurred in the control and 3000 ppm groups only. One fetus in the control group had a meningocele. Four fetuses from one litter in the 3000 ppm group had tarsal flexure and cleft palate, and one fetus each in two different litters from this group had tarsal flexure or cleft palate. Although the litter proportions of these malformations in this group (2.0% and 2.2%, respectively) were higher than study (0% for both malformations) and historical controls (0.6% and 0.7%, respectively), they were not observed in the 7000 ppm group and were primarily clustered in one litter. Therefore, they were not considered to be related to treatment.

Visceral malformations and variations: The only visceral malformation noted was an absent kidney and ureter in one fetus in the 3000 ppm group. Visceral variations were noted in single fetuses in the 3000 (retroesophageal right subclavian artery; no brachiocephalic trunk) and 7000 ppm (hemorrhagic iris) groups. None of these changes were considered to be related to administration of test material.

Skeletal malformations and variations: Skeletal malformations were noted in three controls (fused sternebrae in two animals and severely malaligned sternebrae in another), one low dose (fused and/or malpositioned costal cartilage), one mid dose (severely malaligned sternebrae) and three high dose fetuses (severely malaligned sternebrae, fused ribs and fused and/or malpositioned costal cartilage). There were no differences in the incidences of these malformations in treated animals vs. controls when evaluated on a percent litter basis. Variations were noted in all groups (including controls) and consisted of 14th rudimentary rib(s), 14th full rib(s), 7th sternebra, accessory skull bones, malaligned sternebrae (slight or moderate), and 7th cervical rib(s). Vertebral centra unossified, extra site of ossification anterior to sternebra No. 1, extra site of ossification anterior to cervical arch No. 2, 25 presacral vertebrae and 27 presacral vertebrae were found in one, two or three fetuses in the 1000, 3000 or 7000 ppm groups. There were no significant differences in the incidences of any skeletal variations between treated and control animals when evaluated by incidence or on a percent litter basis and there was no dose-response relationship.

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

7 000 ppm

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a teratology study in which groups of pregnant CD-1 mice were exposed to 0, 1000, 3000, and 7000 ppm di (2-ethylhexyl) terephthalate ad libitum via the diet from gestation days 0-18, the only evidence of maternal toxicity was higher mean absolute liver weights noted at the two highest concentrations. Intrauterine growth and survival was unaffected at all dose levels and there was no evidence of teratogenicity or fetotoxicity, even at maternally toxic doses. The no-observed-effect-level (NOEL) for maternal toxicity was 1000 ppm (197 mg/kg bw/day) and the NOEL for developmental toxicity was 7000 ppm (1382 mg/kg bw/day) under conditions of the study.

Based on the results of this study, di (2-ethylhexyl) terephthalate is not classified for “Developmental or Reproductive Toxicity” according to GHS.

Executive summary:

In a teratological evaluation, groups of 25 pregnant CD-1 mice were exposed to di (2-ethylhexyl) terephthalate via the diet at concentrations of 0, 1000 (197 mg/kg bw/day), 3000 (592 mg/kg bw/day) or 7000 (1382 mg/kg bw/day) ppm from gestation days 0-18. Dams were sacrificed on Gestation Day 18 and fetuses were individually weighed and examined for external, skeletal and visceral malformations and variations. The two highest concentrations were maternally toxic, as manifested by higher mean absolute liver weights. There was no evidence of teratogenicity or fetotoxicity at any dose concentration. Under conditions of the study, the NOEL for maternal toxicity was 1000 ppm (197 mg/kg bw/day) and the NOEL for developmental toxicity was 7000 ppm (1382 mg/kg bw/day).