Registration Dossier

Administrative data

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 days
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable with restrictions; the study was conducted according to GLPs and good scientific methodology, but no analytical data were collected to verify the stability, homogeneity, or concentration of the test substance in the vehicle.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study evaluated the ability of di (2-ethylhexyl) terephthalate to demonstrate or mimic biological activities consistent with agonism of natural estrogens. Di (2-ethylhexyl) terephthalate in Mazola® corn oil was administered by oral gavage to groups of 10 immature female rats once daily from postnatal days (PND) 19 through 21. Dosage levels were 0 (solvent control, corn oil only), 20, 200 and 2000 mg/kg bw/day, administered in a dosage volume of 10 mL/kg. A positive control group composed of 10 immature females received 0.003 mg/kg bw/day of 17α-ethinyl estradiol (EE) on a comparable regimen, again in a volume of 10 mL corn oil/kg. Animals were observed for clinical signs and were weighed daily beginning on PND 18. Weight gains and final weights were calculated. Twenty-four hours after the last dose administration, animals were euthanized, but no necropsies were conducted. The uteri were harvested and wet and blotted uterine weights were recorded. The luminal fluid weight was calculated by subtracting the blotted uterine weight from the wet uterine weight. The uteri, along with the vagina and ovaries, were preserved in 10% neutral buffered formalin for possible further microscopic evaluation.
GLP compliance:
yes
Type of method:
in vivo
Endpoint addressed:
not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
-Test substance (as cited in report): Di-2-ethylhexyl terephthalate (DOTP)
-Lot Number: 0022268
-Purity: 97.77%
-Physical description: Clear, colorless liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Test animals:
-Strain: Crl:CD®(SD)IGS BR
-Source: Charles River Laboratories, Portage, Michigan
-Sex: female
-Age at receipt: 7-8 days
-Age at study initiation: 19 days
-Acclimation period: 11-12 days
-Weight at study initiation: 37.9-50.4 g
-Housing: All rats were housed by litter with their own or a fostering dam in plastic maternity cages containing ground corn cob nesting material. Following randomization, the animals were weaned and gang-housed in plastic maternity cages.
-Diet: PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002, ad libitum
-Water: Reverse osmosis water, ad libitum
-Method of animal identification: Pups were tail tattooed on postnatal day 15
-Method of animal distribution: Animals judged to be in good health and meeting body weight requirements were randomly assigned to the study using a computer program.

Environmental Conditions:
-Temperature: 71 ± 5 °F
-Humidity: 30 - 70% relative humidity
-Photoperiod: 12 hour light/dark photoperiod
-Air Exchanges: 10 fresh air exchanges per hour

In-Life Study Dates:
-Study Initiation Date: 22 October 2001
-Experimental Start Date: 25 October 2001
-Experimental Completion Date: 9 November 2001

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Animals received single doses of the test substance by gavage at 19, 20, and 21 days of age.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
On the first day of dosing, samples from the top, middle, and bottom of each di (2-ethylhexyl) terephthalate test solution, the 17 α-ethinyl estradiol solution, and the control solution were collected prior to dosing, and samples from the middle of each solution were collected immediately after dosing. In addition, on subsequent days of dosing, samples from the middle of each solution were collected prior to dosing. All samples were stored frozen at approximately -20 °C for possible future analysis for homogeneity, stability, and concentration.

Di (2-ethylhexyl) terephthalate dosing solutions were prepared daily. The 17α-ethinyl estradiol was dissolved in 95% ethanol and diluted with corn oil to prepare a stock solution. The stock solution was diluted with corn oil daily to prepare the dosing solution.
Duration of treatment / exposure:
single daily oral gavage dose
Frequency of treatment:
daily for three days (19, 20, and 21 days of age)
Doses / concentrations
Remarks:
Doses / Concentrations:
2000, 200, 20, or 0 mg/kg bw
Basis:
no data
No. of animals per sex per dose:
10 females per dose
Control animals:
yes

Examinations

Examinations:
Clinical Observations: Animals were observed twice daily for appearance, behavior, moribundity and mortality. The animals were also observed at the time of dosing and one hour after dosing.

Body Weights: Body weights were measured daily.

Euthanasia: Approximately 24 hours after the last dose, all surviving animals were euthanized with carbon dioxide.

Gross Necropsy: Full necropsies were not performed. The uterus (with luminal fluid) from each animal was harvested, trimmed, and weighed. Then the uterus was opened, blotted, and re-weighed. The uterus, vagina, ovaries, and tail (with tattoo) were fixed in 10% buffered neutral formalin for possible histopathology.
Positive control:
Positive Control Test Substance:

-Test substance: 17α-Ethinyl Estradiol
-Lot number: 12201EO
-Purity: 98%
-Physical description: Fine, white powder

Results and discussion

Details on results:
NOAEL: 2000 mg/kg bw/day

Mortality: No mortality was observed during the study.

Clinical Abnormalities: There were no treatment-related clinical abnormalities noted during the study.

Body Weights: All mean body weights were comparable among groups throughout the study. Mean body weight gains were significantly (p ≤0.01) lower than controls between PND 19-20 (1.2 g vs. 3.2 g) and PND 19-22 (11.2 g vs. 13.8 g) for the high-dose group and were also lower than controls (p ≤ 0.01) between age Days 20-21 (4.2 g vs. 5.7 g) for the positive control group. No differences in body weight gains were seen in any other group at any time during the study.

Organ Weights: Mean wet and blotted uterine weights were significantly higher (p ≤ 0.01) for the positive control group: wet weights were .1338 g vs. .0438 g; blotted weights were .1099 g vs. .0378 g, and luminal fluid weights were .0238 g vs. .0060 g.. No other differences between groups were noted at any time during the study.

Applicant's summary and conclusion

Conclusions:
Under the conditions used in this assay, di (2-ethylhexyl) terephthalate did not demonstrate or mimic biological activities consistent with agonism of natural estrogens at dose levels up to 2000 mg/kg bw/day.

Based on the results of this study, di (2-ethylhexyl) terephthalate is not expected to cause adverse effects in developing females and is not classified for “Developmental or Reproductive Toxicity” according to GHS.
Executive summary:

In a uterotrophic assay, di (2-ethylhexyl) terephthalate was administered by oral gavage to groups of 10 immature female Sprague-Dawley rats once daily from postnatal days 19 through 21, at dose levels of 0, 20, 200 and 2000 mg/kg bw/day in corn oil (dose volume 10 mL/kg). A positive control group composed of 10 immature females received 0.003 mg/kg bw/day of 17α-ethinyl estradiol (EE) in corn oil on a comparable regimen. No test substance-related clinical observations or effects on mean body weights were noted, although in the 2000 mg/kg bw/day group, there were reductions in mean body weight gain between postnatal days 19-20 and 19-22. No test substance related differences in mean uterine weights (wet or blotted) or luminal fluid weights were observed, while uterine weights in the positive control (EE) group were increased approximately three- to four-fold over control group values. Under the conditions of the study, di (2-ethylhexyl) terephthalate did not demonstrate or mimic biological activities consistent with agonism of natural estrogens at dose levels of up to 2000 mg/kg bw/day.