Bis(2-ethylhexyl) phthalate

Endpoint summary

Description of key information

Repeat dose effects of DEHP have been evaluated in a number of animal species by several routes of exposure. The most pronounced findings were effects on the liver (hepatomegaly, peroxisome proliferation), testes (tubular atrophy) and kidneys (increased kidney weights, mineralisation of renal papilla, tubule cell pigments and chronic progressive nephropathy). For liver and kidney toxicity (increases in serum albumin, absolute and/or relative liver and kidney weights and hepatic peroxisome proliferation) a LOAEL was established from a well-conducted 104-week rat dietary study at 146.6 mg/kg bw/d. The NOAEL was 28.9 mg/kg bw/d. Studies of DEHP in monkeys failed to elicit the liver, kidney or testicular effects seen in rodents.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

29 mg/kg bw/day

Species:

rat

Quality of whole database:

high

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

50 mg/m³

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

50 mg/m³

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

This part has been adapted from the EU Risk Assessment 2008 (p 343 -363)

Studies in animals

A few inhalation studies in experimental animals are available and the only study available following dermal exposure to DEHP is inadequate for risk assessment. A large number of studies have investigated the toxicity of DEHP following repeated oral administration to experimental animals, mainly rats. The most pronounced effects included, effects on the liver (hepatomegaly, peroxisome proliferation and replicative DNA synthesis), testes (tubular atrophy), effects on the kidneys and cardiopulmonary tissues and hypolipidemic effects (decreased plasma levels of cholesterol and triglyceride). Other, less pronounced, effects have also been observed, e.g. reduced body weight and body weight gain, and alterations in clinico-chemical parameters.

Short-term repeated dose studies (up to 28-days exposure)

Inhalation

Rats

Wistar rats (10 males and females per group in the main dose group; 2 males and females per group in satellite group I, 15 males and 2-5 females per group in satellite group II; an equal number of control rats in each group; 9 weeks old at the beginning of exposure) were exposed in head-nose inhalation systems to DEHP (99.7% pure) aerosols of respirable particle size (mass median aerodynamic diameter < 1.2 ± 2.9-9.5 μm) or air (controls) (Klimisch, 1988, 1990; Klimisch et al., 1992). Exposure duration was 6 hours per day, 5 days per week for 4 weeks at 0, 0.01, 0.05, or 1.0 mg/litre (0, 10, 50, or 1,000 mg/m3). The animals of the main dose group were sacrificed at the end of the exposure period. Before sacrifice, male rats from satellite group II had a recovery period of 2 or 6 weeks after termination of exposure. Livers of animals from satellite groups I and II were examined by light and electron microscopy. No animals died during the study. Clinical examination and blood chemistry parameters did not reveal treatment-related effects. Body weights of treated rats and controls were similar. In the highest dose group, a significant increase in relative lung weights was seen in male rats. This was accompanied by foam cell proliferation and thickening of the alveolar septi. Absolute liver weights (females) and relative liver weights (both sexes) were slightly but significantly increased but there were no corresponding histological findings. All these effects were reversible within the post-exposure observation period. No testicular toxicity was detected histologically. Electron microscopical examination of liver samples from all three concentration groups and controls at the end of exposure and after the post-exposure period did not reveal clear ultrastructural changes in hepatocytes that could be attributed to the exposure or to peroxisome proliferation. The NOAEL in this study is 50 mg/m3.

The effects of inhalation of DEHP on testes of prepubertal rats was researched (Kurahashi et al., 2005). The results showed that inhalation of DEHP by 4-wk-old male Wistar rats at doses of 5 or 25 mg/m3, 6 h per day, for 4 and 8 wk significantly increased the concentration of plasma testosterone and weight of seminal vesicles. However, the concentration of luteinizing hormone (LH), follicular stimulating hormone (FSH) and the expression of mRNAs of androgen biosynthesis enzyme, cytochrome P450 cholesterol side-chain-cleavage enzyme (P450scc), 3-beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17 alpha-hydroxylase/17, 20 lyase (CYP17 and aromatase (CYP19) did not change. Rats with precocious testes did not increase in any of the DEHP groups. It has also been found that the estimated effective dose in this study was less than those reported in previous studies, which uses oral dosing.

Peroral

Rats

Male Wistar rats (4 rats per dose group, 6 control rats) were fed a diet containing 0 or 2% DEHP (> 99.5% pure) for 3, 10, or 21 days (Mann et al., 1985). Body weights were significantly decreased after 10 and 21 days of treatment. Relative liver weights were increased in all dosed rats; electron microscopic examination showed significantly increased peroxisome proliferation, changes in mitochondria, and proliferation of the smooth endoplasmatic reticulum already after 3 days of treatment. The activities of peroxisomal enzymes (PCoA, α-GD, and catalase) were significantly increased.

In an other study comparable to a guideline study and performed according to GLP principles, Alderley Park rats (10 animals/sex) were given 2,000 mg/kg bw/day of DEHP (99.7% pure) by gavage in corn oil for 14 days (Rhodes et al., 1986). The control group was given the vehicle. The body weight gain was significantly reduced in males but not in females. Slight signs of systemic toxicity were observed in treated rats of both sexes. Plasma cholesterol and triglyceride levels were significantly reduced in male rats but not in females. Absolute and relative liver weights (both sexes) and kidney weights (females) were significantly increased. Testis weights were significantly reduced in 3 male rats and testicular tubular atrophy was observed in 4 male rats. Brain weights were unaffected. Electron microscopy of livers revealed marked peroxisome proliferation (an 8-fold increase in males and 5-6-fold increase in females). Both number and size of peroxisomes were increased. Disorganisation of rough endoplasmic reticulum, mild proliferation of smooth endoplasmic reticulum, and increased number of lysosomes and mitochondria were observed in a number of rats. A significantly increased activity in the peroxisomal enzymes α-GD and PCoA and an increased activity in catalase (males only, not significant) was observed. Peroxisome proliferation was also observed in the proximal tubule of the kidney (2-fold increase in both sexes).

In a study performed according to GLP principles, Fischer 344 rats (5 animals/sex/group) were fed 0, 0.01, 0.1, 0.6, 1.2, or 2.5% DEHP (99.9% pure; corresponding to 0, 11, 105, 667 1,224, or 2,101 mg/kg bw in males and 0, 12, 109, 643, 1,197, or 1,892 mg/kg bw/day in females) in the diet for 21 days (Barber et al., 1987). In the highest dose group, rats of both sexes lost weight during the first week of the study and body weights were significantly reduced compared to controls from day 3 of the study. Animals given 1.2% gained less weight than the controls during the first three days of treatment, although the differences were not significant. Food intake was significantly decreased in the highest dose group (both sexes) and in females from the 1.2% dose-group. Absolute and relative liver weights were significantly increased from 0.6%. Histological examination showed a reduction in cytoplasmic basophilia in livers from male rats given 0.6% and more and in female rats given 1.2% and more. Relative kidney weights were significantly increased in the highest dose group; no histological abnormalities were, however, observed. Testis weights were significantly reduced in male rats in the highest dose group and moderate to severe testicular atrophy were noted. In male rats, serum triglyceride levels were significantly increased at 0.01% but significantly reduced from 0.6%. In female rats, serum triglycerides were significantly increased from 1.2%. There was no dose-related reduction in serum cholesterol levels. Electron microscopy revealed a dose-related increase in numbers of peroxisomes from 0.1% in males and from 0.6% in females. At dietary levels of 0.6% and above, the size range of the peroxisomes was also increased and there were changes in peroxisomal morphology. PCoA showed a dose-related increase at dietary levels of 0.6% and above. LAH-11 and LAH-12 were increased in males from 0.1% and more and in females from 1.2% and more. No NOAEL can be derived from this study. The LOAEL is 0.01% in the diet (corresponding to 11 mg/kg per day in males and 12 mg/kg per day in females).

In an other study realised by Fukuhara (1977), DEHP was mixed into diet and given to Wistar male rats for 16 days. A significant and dose-dependent increase in the relative liver weight (RLW) was observed in the rats fed DEHP at dietary levels greater than 0.1%. The RLW increased progressively with the duration of the treatment, reaching its maximum in two weeks. Biochemical analysis of the principal hepatic components has shown that the increase in RLW induced by DEHP was due to an increase in the total amounts of protein, water, lipid, and nucleic acids, The increase in protein was most marked and was due mainly to the increase in non-collagen protein. The total amounts of glycogen and collagen did not change. However, most of these components were found to be unchanged or reduced in their contents per liver weight, except that of protein. The increase in RLW is caused equally by the increase in cell number and in the cell volume, the nuclear DNA content being unaffected. The content of cytochrome P-450 in microsomes increased in the rats of both sex fed a 0.5% DEHP-diet for 16 days. However, the activities per mg of microsomal protein or per g of liver of aminopyrine N-demethylase and aniline hydroxylase decreased in male rats but increased in female rats. Total activities of these enzymes increased markedly in both sex of animals. Glucose 6-phosphatase, acid phosphatase and cytochrome c oxidase were reduced significantly in their activities per liver weight but glucose 6-phosphate dehydrogenase was unchanged. The significance of the liver enlargement induced by DEHP is discussed in relation to the physiological response of liver and to possible pathological changes of liver.

In a study performed according to GLP principles, DEHP (99.9% pure) was fed to groups of 5 male Fischer 344 rats at dietary levels of 0.02, 0.05, 0.1, 0.5, 1.0, or 2.5% (24, 52, 115, 559, 1,093, or 2,496 mg/kg bw/day) for 28 days (BIBRA, 1990). The control group (10 male rats) received basic diet. A significant reduction in body weight and a reduced food intake was observed after 7 days of treatment in high-dose rats and was persistent throughout the study. Absolute liver weights were significantly increased from 0.5% while relative liver weights were significantly increased at all dose levels. Hepatic PCoA was significantly increased from 0.1% in the diet. Testis weights were significantly reduced at 2.5% in the diet; marked testicular atrophy was observed histologically. This study identifies no NOAEL; the LOAEL corresponds to the lowest dose administered, that is 0.02% in the diet (24 mg/kg/day).

Male Sprague-Dawley rats (5 animals per group) were given 25, 100, 250 or 1,000 mg/kg bw/day of DEHP (> 99% pure) by gavage in corn oil for 14 days (Lake et al., 1984). Control rats (5 animals) received the vehicle. Relative liver weights were significantly and dose-dependently increased in the three highest dose groups. A marked dose-dependent increase in the activities of PCoA and CAT was also observed.

The Mangham study (1981) was conducted on the effect of oral administration of either DEHP or dialkyl 79 phthalate (DA79P) at a dose level of 2500 mg/kg/day for 7 and 21 days in young male and female Wistar albino rats. Both DEHP and DA79P increased liver site in both sexes and reduced the relative weight of testes in male rats. Liver enlargement was accompanied by alterations in several marker enzyme activities. Both DEHP and DA79P depressed mitochondrial succinate dehydrogenase in male but not in female animals. While certain parameters of hepatic xenobiotic metabolism were elevated in female rats receiving either DEHP or DA79P, a marked inhibition of xenobiotic metabolism was observed in male rats treated with DA79P. Parallel morphological investigations revealed histological evidence of liver damage in male rats given DA79P and ultrastructural investigation revealed changes in the structure of the nuclei, mitochondria, and endoplasmic reticulum. These effects were largely absent from female animals. DEHP produced no hepatic histological changes in either sex but ultrastructural studies indicated proliferation of the smooth endoplasmic reticulum, an increase in the numbers of microbodies (peroxisomes), and mitochondrial changes. Treatment of rats with either DEHP or DA79P resulted in hepatic changes, although the effects were not necessarily common to both agents. Male animals appeared to be more susceptible than female animals. Finally, both agents caused testicular atrophy as indicated by decreased testicular weight and atrophy of seminiferous tubules in male rats treated for 21 days.

Male Fischer 344 rats (5 animals per group) were fed 0, 100, 1,000, 6,000, 12,000 or 25,000 ppm (0, 11, 105, 667, 1,223, or 2,100 mg/kg bw/day) of DEHP (99.8% pure) in the diet for 21 days (Short et al., 1987). The relative liver weight and PCoA oxidation in liver homogenates were significantly increased from 6,000 ppm. LAH-11 and LAH-12 hydroxylation were significantly increased from 1,000 ppm. Peroxisome proliferation was examined by electron microscopy and evaluated as moderate to very marked in the three highest dose groups.

In a study designed to reveal a NOAEL for peroxisome proliferation, male Wistar rats (6 animals per group) were fed 0, 60, 200, 600, 2,000 or 6,000 mg/kg of DEHP (> 98.4% pure) (0, 5, 18, 52, 182, or 549 mg/kg bw/day) in the diet for 2 or 4 weeks (Jansen, 1992). Peroxisome proliferation was evaluated by morphometric analysis (light and electron microscopy) and by measurement of the activity of peroxisomal associated enzymes (PCoA, ECoA, catalase, CAT, LAH-11, and LAH-12). There were no significant differences in body weights between control animals and treated groups. The liver weights of animals in the two highest dose groups were significantly increased in a dose-related manner compared to the control group following 2 or 4 weeks of treatment. The morphometric analysis revealed a significant increase in volume density and number of peroxisomes in animals given 200 mg/kg DEHP or more in the diet for two weeks. For all enzymes, a dose-response relationship was observed. The NOAEL for induction of LAH-11, LAH-12, and ECoA was 200 mg/kg, for PCoA 600 mg/kg, and for catalase 2,000 mg/kg. The activity of CAT (the most sensitive parameter in the study) was significantly increased in all treated groups. An overall NOAEL for peroxisome proliferation was established as 60 mg/kg DEHP (5 mg/kg bw/day) in the diet. The results for CAT were not taken into account in the establishment of the NOAEL as this enzyme is not specific for peroxisome proliferation.

In a study comparable to a guideline study, male Sprague-Dawley rats were given 10, 100, 1,000 or 2,000 mg/kg bw/day of DEHP (> 99% pure) in corn oil by gavage for 5 days beginning at an age of 6 (1-week-old), 14-16 (2-week-old), 21 (3-week-old), 42 (6-week-old) or 86 (12-week old) days (Dostal et al., 1987b). The control group was given the vehicle. After two doses of 2,000 mg/kg bw/day virtually all pups in the three youngest age groups died whereas 6- and 12-week-old rats showed significantly decreased body weights with no fatalities. Five daily doses of 1,000 mg/kg bw/day caused significant decreases in body weight gain in 1-, 2-, and 3-week-old rats. Absolute and relative liver weights were significantly increased at 100 mg/kg bw/day in all age-groups except in 1-week-old rats and in all age groups at higher dose levels. Absolute kidney weight was reduced in some cases whereas relative kidney weight was increased at doses of 1,000 mg/kg bw/day or more in 3-week-old rats or older rats. Morphological examinations revealed increased peroxisome proliferation in neonatal as well as adult rats. The activities of PCoA and CAT were increased in a dose-dependent manner in all age groups. The activities of these enzymes were similar in control rats of all ages. Plasma cholesterol concentrations were higher in suckling control rats (1- and 2-week-old) than in weanling (3-week-old) and adult controls. In DEHP-treated rats, plasma cholesterol concentrations were significantly reduced in weanling and adult rats given doses of 1,000 mg/kg bw/day or more. In suckling rats plasma cholesterol levels were increased at 1,000 mg/kg/bw/day. Plasma triglyceride levels in the control group were similar at all ages whereas significant decreases in plasma triglycerides were observed in weanling and adult rats; in suckling rats only small decreases (not significant) occurred.

The neurobehavioural effects were tested in rats by a functional observational battery (FOB) and motor activity measurements before exposure, at specified times after a single dose exposure, and during and after a 14-day repeated dose exposure (Moser et al., 1995, 2003). Female Fischer 344 rats (number not given) were administered 150, 500, 1,500 or 5,000 mg/kg bw/day of DEHP (> 99% pure) (single dose study), or 50, 150, 500 or 1,500 mg/kg bw/day of DEHP (repeated exposure, 14 days) in corn oil by gavage. The FOB included following measures: autonomic, activity, excitability, neuromuscular, sensorimotor, and physiological measures. Motor activity was measured in a maze. The FOB was performed on each rat just prior to the first dose. Thereafter, the FOB followed by motor activity assessments was conducted at 4 and 24 hours after exposure (single dose study), and on day 4 and 9 (before the daily dose) and 24 hours after the last dose. No lethality occurred. A single administration of the highest dose produced pronounced signs of general debilitation in two rats 24 hours after dosing. No changes in body weight were observed in either study. No functional domain was overall affected in either study.

Hamsters

Male DSN Syrian hamsters (5 animals per group) were given 25, 100, 250 or 1,000 mg/kg bw/day of DEHP (> 99% pure) by gavage in corn oil for 14 days (Lake et al.,1984b). Control hamsters (5 animals) received the vehicle. The relative liver weight was significantly increased in high-dose animals. No significant increase in the activities of PCoA and CAT was observed. Administration of similar doses of DEHP to rats resulted in a significant and dose-dependent (from 100 mg/kg bw/day) increase in liver weight as well as marked and dose-dependently increased activities of PCoA and CAT.

Monkeys

In a study comparable to a guideline study and performed according to GLP principles, marmosets (5 animals of each sex) were given 2,000 mg/kg bw/day of DEHP (99.7% pure) by gavage in corn oil for 14 days (Rhodes et al., 1986). The control group was given the vehicle. Body weight gains were unaffected. No effects were observed on liver and testis weights whereas the relative kidney weights were significantly reduced in females. Gross and microscopic examination of the liver, kidney, testes, and pituitary showed no changes. Electron microscopy revealed only a slight increase in peroxisomes. Plasma cholesterol and triglyceride levels were similar in treated and control animals. A significant increase in hepatic catalase activity was seen in males. In a comparative study in rats (Rhodes et al., 1986), hepatomegaly, marked peroxisome proliferation, and an increase in the activity of peroxisomal, mitochondrial and smooth endoplasmatic reticulum (microsomal) enzymes concerned with fatty acid metabolism were observed following a similar dosage regimen.

Male cynomolgus monkeys (one animal per group) were given 0, 100 or 500 mg/kg bw/day of DEHP (99.8% pure) by gavage in corn oil for 21 days (Short et al., 1987). There were no treatment related changes in relative liver weight, PCoA oxidation, CAT, or LAH-11 andLAH-12 hydroxylation. In addition, no treatment related effects were observed at light and electron microscopic examination of the livers. In contrast to these results, relative liver weight and PCoA oxidation, and LAH-11 and LAH-12 hydroxylation were significantly increased in male rats given doses from 6,000 ppm (667 mg/kg bw/day) or from 1,000 ppm (105 mg/kg bw/day), respectively, in the diet for 21 days. Peroxisome proliferation was moderate to very marked from 6,000 ppm.

The effects of DEHP as a peroxisome proliferator were evaluated in young adult male cynomolgus monkeys after 14 days of treatment, with emphasis on detecting hepatic and other effects seen in rats and mice after treatment with high doses of phthalates (Pugh et al, 2000). Monkeys weighing 2-2.9 kg were exposed to 500 mg DEHP/kg/day (dissolved in 0.5% methylcellulose) administrated in a constant volume of 10 ml/kg once a day for 14 consecutive days using an adult/pediatric nasogastric tube. All animals were observed twice a day for mortality, morbidity, and toxicological or other clinical signs, including behavioural changes, appetite, and excreta. Blood samples were collected during the second and fourth week of the pre-test period and prior to necropsy. Monkeys were sacrificed on the day following the last dose. A gross necropsy was performed including a thorough visual examination of all organs and body tissues. Organ weights were obtained for the liver, kidney, and testes/epididymis, adrenals, brain, heart, lung, spleen, and thyroid/parathyroid, and organ to body weight ratios were calculated. Sections of liver, kidney, and testes, were fixed and embedded for subsequent histopathological evaluation, assessment of replicative DNA synthesis by immunohistochemical detection, evaluation of GJIC and for assessment of peroxisomal activity. There were no overt changes in the general health or behaviour of the monkeys following14 days of dosing. Treatment with DEHP had no effect on body weights, food consumption, or relative weights of any organs assessed. There were no changes in haematological parameters, serum chemistry or in the urine analysis. No inflammation or necrosis was seen in any of the tissues examined. The test substance did not produce any toxicologically important changes in the monkeys (Pugh et al., 2000).

Other Routes

A 3-day-old neonatal rat model was used to assess DEHP toxicity following intravenous administration (Greener et al., 1987). Neonates (12 rats per group, 2 to 4 days old) were injected 30.8, 91.7, or 164.8 mg/kg bw of DEHP (purity not specified) in 4% bovine serum albumin (BSA) solution for 18 consecutive days. Control neonates were injected a solution of 4% BSA or saline, or were untreated. Neonates were examined for signs of toxicity immediately after treatment and again 1 to 3 hours later. After sacrifice, a complete necropsy was performed and selected tissues (brain, heart, lungs, liver, spleen, kidneys, injection site, eyes, stomach, duodenum, and caecum) were prepared for histopathological evaluation. Body weight gains and average weight gain per day were significantly and dose-dependently decreased from days 4 to21 of the treatment period. Absolute and relative liver weights were significantly increased in a dose-related manner. No conclusive histopathological alterations were detected in the tissues with the exception of local lesions at the injection site (subacute dermatitis), also noted in half of the BSA and saline control rats.

In the Sjoberg study (1985), six intravenous infusions of DEHP (0, 5, 50 or 500 mg/kg bw) were given to 25-day- or 40-day-old rats. In Epon-embedded testicular materials from animals given the highest dose, (dilated cisternae of endoplasmic reticulum) were observed. No age-related testicular effects were observed.

Subchronic toxicity studies (>28-days exposure <chronic exposure)

Inhalation

Rats

In the Kurahashi study (2005), the effects of inhalation of DEHP on testes of prepubertal rats was researched. The results showed that inhalation of DEHP by 4-wk-old male Wistar rats at doses of 5 or 25 mg/m3, 6 h per day, for 4 and 8 wk significantly increased the concentration of plasma testosterone and weight of seminal vesicles. However, the concentration of luteinizing hormone (LH), follicular stimulating hormone (FSH) and the expression of mRNAs of androgen biosynthesis enzyme, cytochrome P450 cholesterol side-chain-cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17 alpha-hydroxylase/17, 20 lyase (CYP17 and aromatase (CYP19) did not change. Rats with precocious testes did not increase in any of the DEHP groups. It has also been found that the estimated effective dose in this study was less than those reported in previous studies, which uses oral dosing.

Oral

Rats

Fischer 344 rats (10 animals/sex/group, five- to six-week old) were given 0, 1,600, 3,100, 6,300,12,500 or 25,000 ppm of DEHP (> 99.5% pure) in the diet for 13 weeks to determine the high and low doses for a following chronic study (NTP, 1982). One male rat fed 6,300 ppm died. Depression of mean body weight gain of male and female rats fed 25,000 ppm was 29% and55%, respectively, relative to controls. Testicular atrophy was observed in all males fed 25,000 ppm but was less pronounced in males fed 12,500 ppm (1,250 mg/kg/day). No other compound-related histopathological findings were observed.

In a study performed according to OECD guideline 408 and GLP principles, young male (105-130 g) and female (93-111 g) Sprague-Dawley rats (10 animals/sex/group) were administered 0, 5, 50, 500 or 5,000 ppm (0, 0.4, 3.7, 37.6 or 375.2 mg/kg bw in males and 0.4, 4.2, 42.2 or 419.3 mg/kg bw/day in females) of DEHP (99.6% pure) in the diet for 13 weeks after a one-week acclimatisation period (Poon et al., 1997). No clinical signs of toxicity were observed. Body weight gain and food consumption were not affected. There were slight but significant decreases in red blood cell counts and serum haemoglobin, albumin, and potassium levels in male rats fed the 5,000 ppm diet. A reduction in the cholesterol concentration was observed in female rats of the same dose group. In the 5,000 ppm dose group, the liver was enlarged in 10 male and in 5 female rats and absolute and relative liver weights and relative kidney weights were significantly increased in rats of both sexes. Microscopic examination revealed minimal to mild hepatocellular hypertrophy in the liver from all rats of both sexes, minimal focal necrosis in one male and two females. Electron microscopy of liver samples revealed an increased number of peroxisomes in rats of both sexes. Significantly decreased absolute and relative testicular weight and mild to moderate seminiferous tubule atrophy and mild to moderate Sertoli cell vacuolation in male rats (9/10) were also found at 5,000 ppm. In the thyroid, mild histological changes consisting of reduced follicle size and colloid density were detected in eight animals. In the 500 ppm dose group, the liver of one male rat was enlarged and minimal Sertoli cell vacuolation was observed in 7 male rats. The NOAEL for the testicular effects (based on Sertoli cell vacuolation in male rats) was considered to be 50 ppm DEHP in the diet (3.7 mg/kg bw/day). A NOAEL for the effects on the kidney can be considered to be 500 ppm (37.6 mg/kg bw in males).

In two separate studies with exposure duration of 9 weeks or 4 weeks, male Wistar rats were dosed with DEHP by gavage and exposed to drinking water with or without acetone (0.5% wt/v in the 9-week study, 1% wt/v in the 4-week study) (Dalgaard et al, 2000). 2-ethylhexanol is a metabolite of DEHP, which role is not fully elucidated. Both the phthalic acid and the aliphatic (ethylhexyl) part of DEHP may be toxic to the testis. The possible contribution of the aliphatic part to the testis toxicity can be examined by administering acetone concomitantly with DEHP.

In the 9-week study the doses of DEHP were 0, 125, 500 or 1,000 mg/kg bw with soya oil as a vehicle in a dose volume of 2 ml/kg bw. In the 4-week study the doses of DEHP were increased to 1,000, 5,000 and 10,000 mg/kg bw. In each study 80 male rats were used; 10 animals per group, weighing approximately 160g. In the 4-week study additionally 80 female rats, approximately 10 weeks of age, were used to investigate male fertility. The rats were housed two per cage in wire cages with a 12-hour reversed day/night cycle. They were given a standard diet and they were observed twice a day for clinical signs of toxicity. During both studies bodyweight was measured once a week. Food and water consumption per cage was registered on a weekly basis. The rats were tested in a Functional Observational Battery (FOB). On the day before sacrifice, rats were anaesthetised and blood was collected for clinical biochemical analyses. During the last week of the 4-week study all male rats from each dose group were mated with undosed females. On day 15 after mating or at the end of the allocated mating period, the female rats were sacrificed, and the uteri and ovaries removed. The uterus was opened and the number of implantations and dead or retarded foetuses was counted. All males from each group underwent a thorough autopsy. Liver, kidneys, adrenals, heart, spleen, testes, epididymis, seminal vesicles, and brain were excised and weighed. In the 9-week study the histopathology was only performed in the control group and in the group receiving the highest dose level of DEHP. Histopathological findings of the testes were graded into normal, slight/moderate atrophy, or severe atrophy. In the 4-week study immunohistochemical investigations were performed on testis vimentin.

In the 9-week study no animal died and no other clinical abnormalities were observed. No effect on body weight, food and water consumption was observed. No histopathological changes were observed in any of he investigated organs of he control and animals exposed to 1,000 mg/kg bw DEHP. The only effect of DEHP was the statistically significant increase in relative liver weight in the dose group exposed to 500 and 1,000 mg DEHP/kg bw with or without acetone in the drinking water.

In the 4-week study a statistically significant reduction in body weight and food and water consumption was observed in the middle and high dose groups. This reduction was getting more pronounced during the study and was dose-dependent for DEHP. Six animals died due to emaciation, two animals in the group dosed with 10,000 mg/kg bw DEHP and four animals dosed with the same amount of DEHP combined with 1% acetone in the drinking water. No differences were found between groups in the FOB test. Hindlimp grip strength was statistically significantly reduced in the groups receiving acetone compared to the groups not receiving acetone. Forelimb grip strength was statistically significantly reduced with increasing doses of DEHP. There was a statistically significant reduction in the level of cholesterol in animals dosed with 125mg, 500 or 1,000 mg DEHP/kg bw and in animals exposed to 250 mg DEHP/kg bw in combination with 0.5% acetone.

The number of males without recognised mating increased in a dose-related manner although the effect was only statistically significant in the group receiving 10,000 mg DEHP/kg bw plus 1%acetone (4/6). The number of pregnant females decreased with increasing DEHP dose levels (9/10→1/8). The most pronounced effect of DEHP was a statistically significant increase in relative liver weight, which was observed in treated animals. In the middle and high dose groups body weight was statistically significantly reduced, while the relative weights of kidney and brain were statistically significantly increased. Testes weight was statistically significantly reduced in the groups exposed to 5,000 and 10,000 mg DEHP/kg bw with or without acetone.

The weight of epididymals and seminal vesicles were statistically significantly reduced at the highest dose level. DEHP had no effect on the weight of spleen. The testis of two rats dosed with 5,000 mg/kg bw showed severe atrophied tubules with massive loss of spermatids and spermatocytes sloughed into the lumen. In most tubules the majority of the germinal epithelium was lined by spermatogonia and Sertoli cells or by Sertoli cells alone. 5 animals that received 5,000 mg DEHP/kg bw + acetone had some microscopic features of severe atrophy of the testes and occasionally thickening of the basal lamina of the tubule. Slight to moderate atrophy was seen in two animals: one dosed with 10,000 mg DEHP/kg bw and one dosed with 5,000 mg DEHP/kg bw + 1% acetone. All other surviving animals dosed with 10,000 mg DEHP/kg bw with or without acetone in the drinking water showed severe seminiferous tubular atrophy. A slight diffuse Leydig’s cell hyperplasia was observed in testes with severe atrophy.

Adult male Wistar rats were exposed to 0, 10, 100 mg DEHP/kg bw/d or 100 mg DEHP/kg bw/d plus vitamin E (50 mg/kg bw/d) plus vitamin C (100 mg/kg bw/d) for 30 days by gavage (Srinivasan et al., 2011). At the end of the treatment period animals were euthanized and gastrocnemius muscle was dissected and subjected to assessment of various parameters. DEHP treatment increased lipid peroxidation and formation of reactive oxygen species, insulin receptor levels and insulin signalling as well as glucose uptake and oxidation were disturbed. Oral glucose tolerance was impaired and serum testosterone and insulin levels decreased. Coadministration of antioxidative vitamins was partially protective towards DEHP effects.

Mice

B6C3F1 mice (10 animals/sex/group) were given 0, 800, 1,600, 3,100, 6,300 or 12,500 ppm of DEHP (> 99.5% pure) in the diet for 13 weeks (NTP, 1982). Seven out of 10 mice in the highest dose group died; these deaths were, however, accidental. Two female mice in the 3,100 ppm group and one female in each of the control, 6,300 and 12,500 ppm groups died. A mean bodyweight gain depression of 10% or more was observed in males fed 3,100 ppm and more and in all female dose groups except for those fed 1,600 ppm. No other compound-related effects were observed.

Monkeys

In a 13-week oral study performed according to GLP principles, marmosets (4/sex/group) were daily administered 0 (corn oil), 100, 500 or 2,500 mg/kg DEHP (purity not specified) in corn oil (Kurata et al., 1995). Males were dosed from 13 to 14 months of age and females from 12 to15 months of age. The body weight gain was significantly suppressed in males administered 2,500 mg/kg. There was a significant decrease of the absolute weight of the spleen in dosed males and also a similar trend for the relative weight; this was thought to be of little toxicological significance as no histopathological changes were found. Other organ weights, including liver, testes, and pancreas, were not different from the control weights. In the DEHP dosed groups there was a significant rise in the total and free cholesterol and phospholipid levels in administration week 4. In week13, only the total cholesterol value in the 500 mg/kg males was different from the control value. It could not be concluded that the effect was caused by the administration of DEHP. A clear rise in blood testosterone and oestradiol concentrations in all groups, including controls, were concluded to be hormonal changes accompanying sexual maturity occurring at the age of about12 months. In the 500 and 2,500 mg/kg group males, a significant increase in the average hepatic peroxisome area was observed, but there was no difference from the control group in terms of the number of peroxisomes per cell or the area density with respect to the cell area of the peroxisomes. No difference from the control group was seen concerning hepatic peroxisome enzyme activities in the DEHP dosed groups. A rising trend (not statistically significant) in the hepatic microsome protein content and an accompanying rising trend in the cytochrome P-450 content per unit liver weight were observed in all dosed males and in mid- and high-dose females. The effects on various organs of long-term oral administration of DEHP have also been studied in marmosets (Kurata et al., 1996; 1998). Marmosets (12-15 months old; 4 animals per sex and group) were given daily doses of 0, 100, 500 or 2,500 mg/kg bw of DEHP (purity not specified) by gavage in corn oil for 13 weeks. The control group was given the vehicle. A significant suppression of body weight gain was observed in high-dose males. Dose-related decreases in spleen weight were observed in all dosed males. Light and electron microscopic examination revealed no substance-related abnormality in the liver in any dosage group. A slight but significant increase in the mean hepatic peroxisome volume was observed in mid- and high-dose males, but the number of peroxisomes and their volume density was not different from those in the control group. No substance-related change of peroxisome-related enzyme activity (catalase, CAT, and PCoA) was observed in any dosage group. No effects on the testes were seen.

Chronic Toxicity studies (more than 10% of the test animals lifespan)

Inhalation

No relevant data are available.

Oral

Rats

In a study comparable to a guideline study, Sprague-Dawley rats (15 animals/sex/group) were given diets containing 0, 0.2, 1.0 or 2.0% DEHP (purity specified as conforming with British Standards Institution) (0, 143, 737 or 1,440 mg/kg/day in males and 0, 154, 797, or 1,414 mg/kg bw/day in females) for 17 weeks (Gray et al., 1977). Rats in the highest dose-group showed clinical signs of toxicity (loss of fur) from week 1 through week 17 of treatment. The bodyweights of rats in the two highest dose groups were reduced compared to controls, significantly from day 2 in both sexes given 2% DEHP and from day 6 or day 83 in males or females, respectively, given 1% DEHP. The body weights of low-dose rats were lower (not significant) than in controls. Food intake was significantly reduced only in rats given 2% DEHP. Both absolute and relative liver weights were significantly increased in all dose groups when compared to controls. The absolute weights of most other organs (including testes) in rats of the two highest dose groups were lower than those of controls while the relative weights were increased. Histological examination revealed severe seminiferous tubular atrophy and cessation of spermatogenesis, which could be related to the dietary level of DEHP. These changes were evident as early as week 2. No histological changes attributable to DEHP treatment were observed in the livers. No NOAEL can be identified from this study; the LOAEL corresponds to the lowest dose level, 0.2% in the diet (143 mg/kg/day in males and 145 mg/kg/day in females).

In a subsequent 28-day study, male weanling Sprague-Dawley rats (10 animals per group) were fed diets containing 0, 25, 100, 400, 1,600 or 6,400 ppm of MEHP (Chu et al., 1981). Major organs were excised and weighed (including testes). At 6 400 ppm a significant growth retardation was noted. Liver and heart weight were significantly increased from 1,600 ppm. Histological examination revealed, however, no treatment-related abnormalities. In the following 3- and 6-month feeding studies, weanling Sprague-Dawley rats (20 animals/sex/group) were fed diets containing 0, 1, 5, 25, 125 or 625 ppm of MEHP. After 3 months, 10 animals of each sex from each group were necropsied. The remaining animals were maintained on the same diet and sacrificed after six months. Relative organ weights were not altered in the 3-month period, but the liver weights of high-dose females were significantly increased in the 6-month period. Treatment-related lesions were found in the liver (midzonal and periportal eosinophilic cytoplasmic inclusions, and vacuolations with isolated binucleated and necrotic hepatocytes), heart (mild enlargement of myocardial nuclei and segmental deregistration of myocardial striations), and adrenals (vacuolation of the zona fasciculata).

In a study comparable to a guideline study and performed according to GLP principles, groups of male and female Alderley Park rats (20 animals/sex per treatment group, 30 animals/sex in the control group) were fed diets containing sufficient DEHP (> 99.7% pure) to ensure intakes of 0, 50, 200 or 1,000 mg/kg bw/day (Hinton, 1982; Mitchell et al., 1985a). Four rats from each treatment group and six controls and were sacrificed 3, 7, 14, 28 days, and 9 months after beginning of feeding. No clinical signs of toxicity were observed during the study. Food consumption of the treated rats was either similar to or greater than that of controls. Body weight was significantly reduced only in rats given 1,000 mg/kg bw/day for 9 months. Liver weights were increased in male rats fed 50 and 200 mg/kg bw/day for 14 days or more and in all high dose rats at all time intervals. Examination of liver sections by light microscopy showed periportal accumulation of fat and mild centrilobular loss of glycogen, both effects were dose-dependent. Electron microscopy revealed that the number of peroxisomes was significantly increased at 50 mg/kg bw/day after 14 days in males and after 9 months in females. Higher doses caused increased number of peroxisomes after 3 days in males and after 14 days in females, the increase was dose-dependent in both sexes. Dose-dependent alterations to the endoplasmatic reticulum (ER) were also observed. Smooth endoplasmatic reticulum proliferation was significantly increased at 50 mg/kg bw/day after 7 days in males and after 14 days in females. Changes in rough endoplasmatic reticulum were observed at 200 mg/kg bw/day after 3 days in males and after 28 days in females. The density of the mitochondrial matrix was increased in male rats although there was no dose dependence. Biochemical studies revealed a marked induction of the peroxisomal enzymes PCoA and α-GD; the induction was dose and time-dependent. The effects in female rats increased more slowly than in male rats but were equivalent by 28 days. The ER-associated enzymes cytochrome P-450 and LAH were dose-, but not time-dependently increased with maximal activity observed at 3 days of treatment. No effect on testis weight was observed. Alterations of the kidneys (lysosomes in the cells of the proximal tubule were enlarged) were observed at 200 and 1,000 mg/kg bw/day. In thyroids of rats fed 1,000 mg/kg bw/day for 9 months showed also alterations (basophilic deposits in the colloid and enlargement of the lysosomes).

Male Fischer 344 rats (5-10 animals per group) were fed 1.2% DEHP (purity not specified) in the diet for 1, 2, 4, 8, 18, 39, 77, 151 or 365 days (Conway et al., 1989). Livers were examined biochemically. Catalase activity was increased (25%) after 8 days and remained at this level up to 365 days. Glutathione peroxidase activity showed a 50% and 80% decrease after 8 or 365 days of treatment, respectively. Lipofuscin, which was contained within lysosomes, was increased 3-fold after 39 days and remained at this level up to 365 days of treatment. The activities of the lysosomal enzymes α-fucosidase, ß-galactosidase and N-acetylglucosaminidase were increased50-100% for 39-365 days of treatment.

In a more recent long-term study, comparable to a guideline study and conducted according to the principles of GLP (David et al., 2000, 2001), F-344 rats were administered DEHP at dietary concentrations of 0, 100, 500, 2,500 or 12,500 ppm (0, 5.8, 28.9, 146.6 or 789.0 mg/kg/day, respectively, for males, and 0, 7.3, 36.1, 181.7 or 938.5 mg/kg/day, respectively, for females), 70 males and females/group, for at least 104 weeks. An additional group was administered 12,500 ppm DEHP for 78 weeks, followed by a recovery period of 26 weeks. There were no treatment-related effects at 100 and 500 ppm. At 2,500 ppm the mean serum albumin concentration and mean liver weights were significantly increased. At Week 79 and at study termination also absolute and relative kidney weights were increased in both sexes at 2,500 ppm. At the highest dose level, there was a decreased survival, increased incidence of clinical abnormalities, and decreased body weight gain in both sexes. A diffuse hepatomegaly and histopathological hepatic changes were demonstrated as were effects on the kidneys, including increased absolute and relative kidney weights (both sexes), increased incidence and severity of mineralisation of the renal papilla in males, increased incidence and/or severity of tubule cell pigment in both sexes, and increased severity of chronic progressive nephropathy in the males. In the males, also absolute and relative testis weights were significantly decreased at 12,500 ppm, with associated increased incidence of bilateral aspermatogenesis and decreased incidence of interstitial cell neoplasms. In the pituitary, an increased number of castration cells were observed in 30/60 males compared to 1/60 of the control males. There was no indication in rats killed at study termination that DEHP-related changes in the kidney, testis, and pituitary were reversible upon cessation of DEHP-exposure. The NOAEL for systemic non-neoplastic effects, including the effects on the kidney is considered to be 500 ppm DEHP in the diet (corresponding to 28.9 mg/kg bw/day in the males and 36.1 mg/kg/day in the females) based on increased absolute and relative kidney weight in both sexes at the next higher dose level (LOAEL: 2,500 ppm corresponding to 146.6 mg/kg bw/day in the males and 181,7 mg/kg bw/day in the females).

In a two years-study, Fischer 344 rats (50 animals/sex/group) were given 0, 6,000 or 12,000 ppm of DEHP (> 99.5% pure) in the diet. (NTP, 1982) Mean daily ingestions of DEHP were calculated to 322 and 674 mg/kg bw/day for low- and high-dose male rats, respectively, and to 394 and 774 mg/kg bw/day for low- and high-dose female rats, respectively. The survival rate was unaffected. At the end of the study, mean body weights of dosed male rats and high-dose female rats were marginally to moderately lower than those of the corresponding controls. Food consumption was slightly reduced in rats of either sex. In high-dose males, the incidence of hypertrophy of the anterior pituitary was significantly increased (45%); and in the testis, degeneration of the seminiferous tubules occurred in 90% of the animals.

Male Sprague-Dawley rats (a total of 520 animals) were fed 0, 0.02, 0.2 or 2% DEHP (0, 7, 70 or 700 mg/kg bw/day; > 99% pure) in the diet for 102 weeks (Ganning et al., 1987, 1990). The body weights were significantly reduced in the highest dose group reaching 20% lower values after 25 weeks of treatment. Significantly reduced body weights were also observed in the mid dose group but to a lesser extent (around 10%). Electron microscopy revealed characteristic changes in hepatocytes. After one week of treatment with 2% DEHP, peroxisome proliferation (varying size) was observed and was persistent throughout the study. The number of mitochondria was increased whereas no changes were observed in rough and smooth endoplasmatic reticulum. After feeding with 0.2% DEHP for 16 months, an increased number of peroxisomes and mitochondria and well-developed endoplasmic reticulum were observed. The number of peroxisomes and mitochondria was not significantly increased after feeding of 0.02%DEHP when compared with the control group. The activity of PCoA was significantly increased in all dose-groups with a doubling of the activity in the lowest dose group after 2 years of treatment. This long-term treatment with DEHP had complex effects on catalase activity with decreasing activity during the initial phase, at 10 weeks the activity exceeded that of the control, but returned to normal at the end of the study. The activity of urate oxidase decreased throughout the study. No hyperplastic nodules or primary liver carcinoma or other tumours were observed. In all dose-groups, DEHP exerted a pronounced effect on the function of the testes after prolonged treatment, consisting of inhibition of spermatogenesis and general tubular atrophy. No NOAEL can be derived from this study; the LOAEL is 0.02% in the diet (7 mg/kg bw/day), the lowest dose administered. In a following study investigating the reversibility of the observed effects, rats were fed a diet containing 2% DEHP for 1 year and then the basal diet for 3 weeks. PCoA and catalase activities decreased after cessation of treatment and reached control levels after 2 weeks.

Mice

In a study comparable to a guideline study and performed according the principles of GLP (David et al., 2000, 2001) B6C3F1 mice (70-85 of each sex/dose group) were administered DEHP daily in the diet at concentrations of 0, 100, 500, 1,500 and 6,000 ppm (0, 19.2, 98.5, 292.2 or 1,266.1 mg/kg/day, respectively, for males, and 0, 23.8, 116.8, 354.2 or 1,458.2 mg/kg/day, respectively, for females), for 104 weeks. One additional group (55 males and females/group) were administered 6,000 ppm DEHP for 78 weeks, followed by a 26-week recovery period. At 1,500 ppm, there was a significant decrease in kidney weight in males and an increased incidence and/or severity of chronic progressive nephropathy in both sexes. The testicular weight was also significantly decreased, with an increased incidence and severity of bilateral hypospermia and an associated increased incidence of immature/abnormal sperm forms and hypospermia in the epididymis. At the highest dose level, there was a statistically significant decrease in survival in males, treatment-related clinical signs and a significantly reduced body weight gain for both males and females. In both males and females, the kidney weight indices were significantly decreased at study termination. In the recovery group, some treatment-related findings were reversible or did not progress after cessation of DEHP exposure such as effects in the liver, including induction of neoplasms. In contrast to the liver, the effects of DEHP on the kidney and testis were not reversible following cessation of exposure. The NOAEL in this study is considered to be 500 ppm (due to effects on the kidney at the next higher dose level) corresponding to 98.5 mg/kg in males and 116.8 mg/kg in females.

In a study performed according to GLP principles, B6C3F1 mice (50 animals/sex/group) were given 0, 3,000, or 6,000 ppm of DEHP (> 99.5% pure) in the diet for 103 weeks (NTP, 1982). Mean daily ingestion of DEHP was calculated to 672 and 1,325 mg/kg bw for low- and high-dose males, respectively, and to 799 and 1,821 mg/kg bw for low- and high-dose females, respectively. The survival rate was unaffected in male mice but several female mice in the lower dose group died after 75-90 weeks of treatment. These deaths were not attributed to DEHP administration. Mean body weights of female mice were marginally to moderately lower than those of the corresponding controls at the end of the study. A significantly higher incidence of chronic inflammation of the kidney was observed in high-dose males. Bilateral seminiferous tubular degeneration and testicular atrophy were observed in 14% of the male mice in the high dose group. This lesion was also found in one control male mouse and in two low-dose males. Non-neoplastic lesions were not observed in female mice.

Monkeys

In a study realised according to guidelines and GLP (Kurata 2003), DEHP was administered by oral gavage at doses of 0, 100, 500, and 2500 mg/kg for 65 weeks to juvenile common marmosets (about 3 months old) of both sexes, and its toxicity was assessed. An extensive and intensive investigation focused on testicular morphology, and function was realised. Treatment-related change in the body weight was not evident. Treatment-related changes were not observed in general except for adaptive liver changes. During the treatment period, all males experienced a surge in testosterone, and the testosterone levels in all treated groups were similar to that of the control group. For the testis, electron microscopic examination was additionally applied, however, this revealed no treatment-related abnormalities. Histochemical examination after 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining did not reveal any alteration in steroid synthesis. Consumption of peroxide scavenger like GSH, GST, or GSH-Px in the testis was not noticed, which suggests that the peroxysomal enzyme may not be operating in this organ. For functional examination, sperm count was conducted to show no treatment related effect in numerical changes. The liver weight and its body weight ratio were not affected. P450 content tended to increase with dose-dependent manner in general, and that was considered to be adaptive change to the DEHP-exposure. However, with regard to specific CYP, CYP3A and 2E these are related to testosterone-6-beta-hydroxylation and lauric acid gamma-1 hydroxylation, respectively, and CYP4A related to lauric acid hydroxylation, no marked increases were noticed. That showed induction of non-PP dependent oxidation and proved absorption of the test substance. In the toxicological study, despite the high dose of 2500 mg/kg/day and adoptive liver change proving absorption, no testicular change was morphologically or functionally noticed in the extensive examinations. In conclusion, when DEHP was administered orally to juvenile marmoset at dosage levels of 100, 500, and 2500 mg/kg/day for 65 weeks, the testicular effect that is well known in rodents were not observed despite extensive examination.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

reliable study

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

According to the criteria edicted in REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 16 December 2008, no classification is warranted for specific target organ toxicity-repeated exposure.