https://ntp.niehs.nih.gov/go/tr601abs

Abstract for TR-601

Toxicology and Carcinogenesis Studies of Di(2‑ethylhexyl) Phthalate Administered in Feed to Sprague Dawley (Hsd:Sprague Dawley SD) Rats

CASRN: 117-81-7
Chemical Formula: C24H38O4
Molecular Weight: 390.6
Synonyms/Common Names: Bis(2-ethylhexyl)phthalate; dioctyl phthalate; phthalic acid di(2-ethylhexyl) ester; bis(2-ethylhexyl) 1,2-benzenedicarboxylate; 1,2-benzenedicarboxylic acid bis(2-ethylhexyl) ester
Report Date: December 2021

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Abstract

Di(2-ethylhexyl) phthalate (DEHP) is a member of the phthalate ester chemical class that occurs commonly in the environment and to which humans are widely exposed. Lifetime exposure to DEHP is likely to occur, including during the in utero and early postnatal windows of development. To date, no carcinogenicity assessments of DEHP have used a lifetime exposure paradigm that includes the perinatal period (gestation and lactation). The National Toxicology Program (NTP) tested the hypothesis that exposure during the perinatal period would alter the DEHP carcinogenic response quantitatively (more neoplasms) or qualitatively (different neoplasm types).

Two chronic carcinogenicity assessments of DEHP were conducted in which Sprague Dawley (Hsd:Sprague Dawley SD) rats were exposed to dosed feed containing 0, 300, 1,000, 3,000, or 10,000 ppm DEHP for 2 years using different exposure paradigms. In Study 1, groups of 45 F0 time-mated females were provided dosed feed beginning on gestation day (GD) 6 through lactation. On postnatal day (PND) 21, groups of 50 F1 rats per sex continued on the study and were provided dosed feed containing the same DEHP concentration as their respective dam for 2 years. In Study 2, groups of 50 rats per sex, aged 6 to 7 weeks at study start, were provided dosed feed containing DEHP for 2 years.

Perinatal and postweaning study in rats (study 1)

During the perinatal period, lower maternal mean body weight, maternal mean body weight gain, and feed consumption were observed in F0 dams exposed to 10,000 ppm DEHP relative to control animals. Also in that exposure group, litter size and pup weights on PND 1 were significantly decreased compared to the control group. Male and female pup mean body weight gains were significantly decreased in the 10,000 ppm group during lactation and resulted in significantly decreased pup body weights at weaning when compared to the control group. Pup survival was not affected following gestational and lactational DEHP exposure.

Following perinatal and 2 years of postweaning DEHP exposure, survival of exposed male and female rats to study termination was similar to that of control groups; however, there were decreases in mean body weight in the 10,000 ppm group compared to the control group.

Significant increases in the incidences of hepatocellular adenoma, hepatocellular adenoma or carcinoma (combined), pancreatic acinar adenoma, and pancreatic acinar adenoma or carcinoma (combined) were observed in the 3,000 and 10,000 ppm male rats relative to the control group. Higher incidences of hepatocellular carcinomas (10,000 ppm males) and pancreatic acinar carcinomas (3,000 ppm males) were also observed. In female rats, significant increases in the incidences of liver neoplasms occurred in the 3,000 ppm (hepatocellular adenoma and hepatocellular adenoma or carcinoma [combined]) and 10,000 ppm (hepatocellular carcinoma and hepatocellular adenoma or carcinoma [combined]) groups. Occurrences of pancreatic acinar adenomas were observed in the 3,000 and 10,000 ppm female groups, and a trend of higher incidence of uterine adenocarcinomas with increasing exposure was observed given the incidence in the 10,000 ppm group. Nonneoplastic lesions were observed in the liver (male and female), pancreas (female), testis, epididymis, kidney (male and female), heart (male only), bone marrow (male only), and pituitary gland (male only).

Postweaning-only study in rats (study 2)

Following 2 years of postweaning DEHP exposure, survival of male and female rats was commensurate with or greater than that of control animals, and lower body weights were observed in the 10,000 ppm group. Notably, the magnitude of decreased weight was smaller in the control animals in Study 2 than in the control animals in Study 1. Significant increases in the incidences of hepatocellular adenoma, carcinoma, and adenoma or carcinoma (combined) were observed in male and female rats exposed to 10,000 ppm DEHP relative to the respective control group. In male rats, significantly increased incidences of pancreatic acinar neoplasms were observed in the 3,000 (adenoma) and 10,000 ppm (adenoma and carcinomas) groups. A trend of increasing incidence of testicular interstitial cell adenoma with increasing exposure was observed in male rats given the incidence observed in the 10,000 ppm DEHP group. In female rats, significantly increased incidences of uterine adenocarcinoma and uterine adenoma, adenocarcinoma, squamous cell carcinoma, or squamous cell papilloma (combined) were observed in the 10,000 ppm group compared to the control group. Occurrences of uterine squamous cell papilloma (including multiple) were observed in the 10,000 ppm group. Nonneoplastic lesions were observed in the liver (male and female), pancreas (male and female), testis, epididymis, uterus, heart (male only), bone marrow (male), and pituitary gland (male only).

Comparative carcinogenic benchmark dose analyses

Benchmark dose (BMD) levels corresponding to a 10% increased risk of carcinogenic response (BMD10) were estimated for exposure-related carcinogenic responses that were observed in both studies. Generally, the BMDs between studies were within threefold of each other. The lowest estimated BMD10 (30.99 mg DEHP/kg body weight/day) corresponded to pancreatic acinar adenoma or carcinoma (combined) in males in the postweaning-only study (Study 2).

Genetic toxicology

DEHP was tested in a variety of genotoxicity assays in vitro and in vivo; most results were negative. In vitro, negative results were obtained in the following assays: six independent bacterial mutation assays in Salmonella typhimurium bacterial strains (TA100, TA1535, TA1537, TA97, and TA98) with and without exogenous metabolic activation systems (S9 mix; induced hamster, rat, and mouse liver S9), a single mouse lymphoma gene mutation assay (with and without induced rat liver S9 mix), and three independent chromosomal aberration assays conducted in Chinese hamster ovary (CHO) cells (with and without rat liver S9). In nine in vitro sister chromatid exchange tests conducted in CHO cells with and without S9, DEHP produced positive responses in four tests, equivocal results in three, and negative results in two.

In vivo, no increases in chromosomal aberrations were observed in bone marrow cells of female B6C3F1 mice following exposure to DEHP in dosed feed for 14 days. DEHP produced mixed results in three independent erythrocyte micronucleus assays: equivocal in female B6C3F1 mice exposed to DEHP in dosed feed for 14 days, equivocal in male TgAC (FVB/N) mice and positive in female TgAC (FVB/N) mice following exposure via dosed feed for 26 weeks, and negative in male and female TgAC (FVB/N) mice following a 26-week dermal exposure. DEHP produced negative results in two independent studies that tested for induction of sex-linked recessive lethal mutations in Drosophila melanogaster.

Conclusions

Under the conditions of the perinatal and postweaning feed study (Study 1), there was clear evidence of carcinogenic activity of di(2-ethylhexyl) phthalate (DEHP) in male Hsd:Sprague Dawley SD rats based on the increased incidences of hepatocellular adenoma or carcinoma (combined) and acinar adenoma or carcinoma (combined) neoplasms (predominately adenomas) of the pancreas. There was clear evidence of carcinogenic activity of DEHP in female Hsd:Sprague Dawley SD rats based on the increased incidence of hepatocellular adenoma or carcinoma (combined). The occurrence of pancreatic acinar adenoma or carcinoma (combined) was considered to be related to exposure. The occurrence of uterine (including cervix) adenoma, adenocarcinoma, squamous cell carcinoma, or squamous cell papilloma (combined) in female rats may have been related to exposure.

Under the conditions of the postweaning-only feed study (Study 2), there was clear evidence of carcinogenic activity of DEHP in male Hsd:Sprague Dawley SD rats based on the increased incidences of hepatocellular adenoma or carcinoma (combined) and acinar adenoma or carcinoma (combined) neoplasms (predominately adenomas) of the pancreas. The occurrence of testicular interstitial cell adenoma in male rats may have been related to exposure. There was clear evidence of carcinogenic activity of DEHP in female Hsd:Sprague Dawley SD rats based on the increased incidences of hepatocellular adenoma or carcinoma (combined) and uterine (including cervix) adenoma, adenocarcinoma, squamous cell carcinoma, or squamous cell papilloma (combined). The occurrence of pancreatic acinar adenoma or carcinoma (combined) in female rats was considered to be related to exposure.

The BMD analysis shows there was no consistent pattern indicating that perinatal and postweaning exposure was more sensitive compared to postweaning-only exposure and modeled responses were within threefold of each other. However, there was a stronger carcinogenic response in the reproductive organs (uterus and testis) in the postweaning-only exposure study compared to the perinatal and postweaning exposure study.

Perinatal and postweaning exposure to DEHP (Study 1) resulted in increased incidence of nonneoplastic lesions in the liver, kidney, heart (male), pancreas (female), pituitary gland (male), bone marrow (male), testis, and epididymis. In addition, exposure increased gross lesions within the reproductive tract of males and females.

Postweaning exposure to DEHP (Study 2) resulted in increased incidence of nonneoplastic lesions in the liver, pancreas, heart (male), pituitary gland (male), bone marrow (male), testis, epididymis, and uterus.

National Toxicology Program (NTP). 2021. NTP technical report on the toxicology and carcinogenesis studies of di(2-ethylhexyl) phthalate (CASRN 117-81-7) administered in feed to Sprague Dawley (Hsd:Sprague Dawley SD) rats. Research Triangle Park, NC: National Toxicology Program. Technical Report 601. https://doi.org/10.22427/NTP-TR-601

Studies

Summary of the Two-year Carcinogenesis and Genetic Toxicology Studies of Di(2‑ethylhexyl) Phthalate
Perinatal and Postweaning Study
(Study 1)		 Postweaning-only Study
(Study 2)
Male
Sprague Dawley Rats		 Female
Sprague Dawley Rats		 Male
Sprague Dawley Rats		 Female
Sprague Dawley Rats
Concentrations in feed 0, 300, 1,000, 3,000, or 10,000 ppm 0, 300, 1,000, 3,000, or 10,000 ppm 0, 300, 1,000, 3,000, or 10,000 ppm 0, 300, 1,000, 3,000, or 10,000 ppm
Survival rates 25/50, 33/49, 40/50, 35/50, 29/50 31/50, 32/50, 34/50, 34/50, 27/50 32/50, 35/50, 39/50, 35/50, 42/50 33/50, 34/50, 33/50, 34/50, 32/50
Body weights 10,000 ppm group 29.7% less than the control group 3,000 ppm group 9.9% less than the control group; 10,000 ppm group 31.7% less than the control group 10,000 ppm group 15.6% less than the control group 10,000 ppm group 21.9% less than the control group
Gross lesions Testis: small (2/49, 2/49, 4/50, 2/50, 45/49); enlarged (or swelling) (0/49, 0/49, 0/50, 0/50, 1/49); fluid or blood filled (0/49, 1/49, 1/50, 1/50, 1/49); right or left, abdominal, undescended (1/49, 0/49, 0/50, 0/50, 19/49); right or left, inguinal, undescended (0/49, 1/49, 1/50, 0/50, 4/49); right or left, abdominal or inguinal, undescended (1/49, 1/49, 1/50, 0/50, 23/49); right, not present (0/49, 0/49, 0/49, 0/50, 1/49); cranial suspensory ligament (0/49, 0/49, 0/50, 0/50, 5/49)

Epididymis: small (0/49, 0/49, 2/50, 0/50, 14/49); right, cauda, agenesis (0/49, 0/49, 0/49, 0/50, 2/49); right or left, caput, agenesis (0/49, 0/49, 0/50, 0/50, 4/49); right or left, cauda, agenesis (0/49, 0/49, 0/50, 0/50, 2/49); right or left, corpus, agenesis (0/49, 0/49, 0/50, 0/50, 3/49)

Levator ani/bulbocavernosus muscle: small (0/50, 0/49, 0/50, 0/50, 2/48)

Cowper’s glands: left, small (0/50, 0/49, 0/50, 0/50, 1/47); right, small (0/50, 0/49, 0/50, 0/50, 1/47)

Prostate glands: small (0/50, 0/49, 0/50, 0/50, 1/47)

Seminal vesicles/ coagulating glands: small (1/50, 0/49, 1/50, 1/50, 8/47)

Phallus: small (0/50, 0/49, 0/49, 0/50, 3/49); cleft (0/50, 0/49, 0/49, 0/50, 3/49)

Prepuce: cleft (0/50, 0/49, 0/50, 0/50, 1/49); incomplete preputial separation (0/50, 0/49, 0/50, 0/50, 7/49)

Gubernaculum: right or left, not present (0/47, 0/49, 0/49, 0/50, 18/41); ↑ right length		 Vagina: not patent (0/50, 0/50, 0/50, 0/50, 5/48)

Phallus: cleft (0/50, 0/50, 0/50, 2/50, 1/48)		 None None
Nonneoplastic effects Liver: hepatocyte, cytoplasmic alteration (0/50, 0/49, 1/50, 28/50, 37/49); hepatocyte, hypertrophy (0/50, 0/49, 0/50, 3/50, 17/49); pigment (0/50, 1/49, 5/50, 40/50, 38/49); necrosis (3/50, 4/49, 1/50, 6/50, 13/49); eosinophilic focus (4/50, 1/49, 7/50, 2/50, 11/49); basophilic focus (1/50, 1/49, 4/50, 4/50, 17/49)

Testis: germinal epithelium, degeneration (includes bilateral) (16/49, 25/49, 21/50, 21/50, 44/49); interstitial cell, hyperplasia, focal (includes bilateral) (4/49, 3/49, 6/50, 5/50, 30/49); seminiferous tubule, dysgenesis (includes bilateral) (0/49, 0/49, 0/50, 0/50, 10/49)

Epididymis: hypospermia (includes bilateral) (4/49, 5/49, 12/50, 8/50, 43/49)

Kidney: papilla, edema (0/50, 0/49, 0/50, 0/50, 39/49); papilla, hemorrhage (0/50, 1/49, 0/50, 2/50, 12/49); epithelium, papilla, hyperplasia (9/50, 4/49, 4/50, 3/50, 17/49); infarct (2/50, 10/49, 9/50, 7/50, 17/49)

Heart: valve, fibrosis (0/50, 2/49, 1/50, 3/50, 11/49); valve, thrombus (0/50, 0/49, 0/50, 0/50, 6/49)

Bone marrow: hypercellularity (21/50, 17/49, 29/50, 34/50, 36/50)

Pituitary gland: pars distalis, hypertrophy (3/50, 7/49, 5/50, 15/50, 37/49)		 Liver: hepatocyte, cytoplasmic alteration (0/49, 4/50, 7/50, 39/50, 39/48); hepatocyte, hypertrophy (0/49, 2/50, 5/50, 9/50, 34/48); pigment (0/49, 6/50, 14/50, 36/50, 40/48); necrosis (3/49, 9/50, 3/50, 7/50, 8/48); eosinophilic focus (3/49, 4/50, 4/50, 7/50, 12/48); basophilic focus (4/49, 5/50, 3/50, 2/50, 10/48); bile duct hyperplasia (9/49, 13/50, 13/50, 21/50, 8/48)

Pancreas: acinus, hyperplasia (0/49, 0/50, 0/50, 2/50, 3/48)

Kidney: papilla, edema (0/50, 0/50, 2/50, 0/50, 38/48); epithelium, papilla, hyperplasia (2/50, 1/50, 2/50, 4/50, 15/48); infarct (0/50, 3/50, 7/50, 5/50, 12/48); renal tubule, cyst (0/50, 0/50, 2/50, 0/50, 7/48); renal tubule, dilation (0/50, 0/50, 0/50, 0/50, 3/48)		 Liver: hepatocyte, cytoplasmic alteration (0/50, 1/50, 0/50, 38/50, 49/50); hepatocyte, hypertrophy (0/50, 0/50, 0/50, 2/50, 6/50); pigment (0/50, 0/50, 7/50, 45/50, 50/50); necrosis (0/50, 2/50, 4/50, 7/50, 8/50); eosinophilic focus (1/50, 0/50, 4/50, 2/50, 24/50); clear cell focus (29/50, 31/50, 33/50, 35/50, 39/50)

Pancreas: acinus, hyperplasia (7/49, 8/50, 9/50, 24/50, 26/50)

Testis: germinal epithelium, degeneration (includes bilateral) (31/50, 25/50, 21/50, 22/50, 50/50); edema (includes bilateral) (27/50, 23/50, 29/50, 24/50, 45/50); interstitial cell, hyperplasia, focal (includes bilateral) (1/50, 1/50, 0/50, 4/50, 4/50)

Epididymis: hypospermia (includes bilateral) (4/50, 4/50, 4/50, 3/50, 43/50); duct, exfoliated germ cell (includes bilateral) (2/50, 3/50, 4/50, 4/50, 36/50)

Heart: valve, fibrosis (2/50, 0/50, 0/50, 1/50, 9/50); valve, thrombus (0/50, 0/50, 0/50, 2/50, 6/50)

Liver: hepatocyte, cytoplasmic alteration (0/50, 2/50, 15/50, 38/50, 45/49); hepatocyte, hypertrophy (0/50, 0/50, 6/50, 14/50, 28/49); pigment (3/50, 0/50, 18/50, 30/50, 48/49)

Pancreas: acinus, hyperplasia (0/50, 1/50, 1/50, 1/50, 5/47)

Uterus: inflammation, chronic (2/50, 9/50, 6/50, 8/50, 8/49)
Neoplastic effects Liver: hepatocellular adenoma (0/50, 1/49, 0/50, 3/50, 8/49); hepatocellular carcinoma (1/50, 0/49, 0/50, 0/50, 3/49); hepatocellular adenoma or carcinoma (combined) (1/50, 1/49, 0/50, 3/50, 11/49)

Pancreas: acinar adenoma (10/50, 7/49, 8/50, 36/50, 22/49); acinar carcinoma (0/50, 0/49, 0/50, 3/50, 1/49); acinar adenoma or carcinoma (combined) (10/50, 7/49, 8/50, 38/50, 22/49)		 Liver: hepatocellular adenoma (1/49, 0/50, 5/50, 9/50, 5/48); hepatocellular carcinoma (0/49, 0/50, 0/50, 0/50, 8/48); hepatocellular adenoma or carcinoma (combined) (1/49, 0/50, 5/50, 9/50, 13/48)

Pancreas: acinar adenoma or carcinoma (combined) (0/49, 0/50, 0/50, 2/50, 1/48)		 Liver: hepatocellular adenoma (0/50, 2/50, 0/50, 1/50, 6/50); hepatocellular carcinoma (0/50, 0/50, 0/50, 0/50, 6/50); hepatocellular adenoma or carcinoma (combined) (0/50, 2/50, 0/50, 1/50, 12/50)

Pancreas: acinar adenoma (1/49, 4/50, 5/50, 23/50, 30/50); acinar carcinoma (0/49, 1/50, 0/50, 1/50, 5/50); acinar adenoma or carcinoma (combined) (1/49, 5/50, 5/50, 23/50, 33/50)		 Liver: hepatocellular adenoma (0/50, 0/50, 1/50, 1/50, 13/49); hepatocellular carcinoma (0/50, 0/50, 0/50, 0/50, 2/49); hepatocellular adenoma or carcinoma (combined) (0/50, 0/50, 1/50, 1/50, 14/49)

Pancreas: acinar adenoma or carcinoma (combined) (0/50, 0/50, 0/50, 1/50, 2/47)

Uterus: adenoma, adenocarcinoma, squamous cell carcinoma, or squamous cell papilloma (combined) (2/50, 4/50, 1/50, 6/50, 13/50)
Equivocal findings None Uterus: adenoma, adenocarcinoma, squamous cell carcinoma, or squamous cell papilloma (combined) (3/50, 1/50, 1/50, 3/50, 7/50) Testis: interstitial cell, adenoma (7/50, 3/50, 3/50, 6/50, 15/50) None
Level of evidence of carcinogenic activity Clear evidence Clear evidence Clear evidence Clear evidence
Genetic Toxicology
Assay Results
Bacterial gene mutations: Negative in Salmonella typhimurium strains TA100, TA1535, TA1537, TA97, and TA98, with and without S9
Mouse lymphoma L5178Y tk+/− cells: Negative with and without S9
In vitro CHO cell chromosomal aberration test: Negative with and without S9
In vitro CHO cell sister chromatid exchange test:
Without rat liver S9:
With rat liver S9:
Positive or equivocal in 7 out of 9 studies
Negative in 9 out of 9 studies
In vivo chromosome aberration test: Negative in female B6C3F1 mice exposed via dosed feed for 14 days
In vivo micronucleus test in mice:
B6C3F1 mice:
TgAC (FVB/N) mice:
TgAC (FVB/N) mice:
Equivocal in females exposed via dosed feed for 14 days
Equivocal in males and positive in females exposed via dosed feed for 26 weeks
Negative in males and females exposed dermally for 26 weeks
Drosophila melanogaster sex-linked recessive lethal test:
Adult injection:
Larval feeding:
Negative
Negative