Bis(2-ethylhexyl) phthalate

Endpoint summary

Description of key information

Absorption rates assumed:

oral exposure:

rat (repeated dose:) 75%

adults: 75%, children 100%

inhalation exposure:

adults: 75%, children 100%

dermal exposure:

adults: 5%, children 5%

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

5

Absorption rate - inhalation (%):

100

Additional information

Studies to compare the toxicokinetic behaviour of DEHP between humans, different strains of non-human primates, different strains of rats, mice, hamsters, guinea pigs, dogs, and miniature pigs have been performed in different. There are also studies that compare the toxicokinetics after different routes of exposure. In this section of toxicokinetics the whole study is described once, at the first appropriate occasion, and the next time the study is mentioned with a reference to the description.

The structures of the metabolites referred to in the text are given in Albro et al. (1983) and named according to the widely accepted nomenclature of Albro et al. (1983).

HOOC-C6H6-COO-CH2-CH-R’

│

R’’

I     2-ethyl-3-carboxypropyl phthalate (R’=-CH2COOH; R’’= -CH2CH3)

II     2-carboxyhexyl phthalate (R’=-[CH2]3CH3; R’’= -COOH)

III     2-ethyl-4-carboxybutyl phthalate (R’=-[CH2]2COOH; R’’= -CH2CH3)

IV     2-carboxymethylhexyl phthalate (R’=-[CH2]3CH3; R’’= -CH2COOH)

V     2-ethyl-5-carboxypentyl phthalate (R’= -[CH2]3COOH; R’’= -CH2CH3)

VI     2-ethyl-5-oxyhexyl phthalate (R’= -[CH2]2-CO-CH3; R’’= -CH2CH3)

VII     2-(2-hydroxyethyl) hexyl phthalate (R’= -[CH2]3CH3; R’’= -CH2CH2OH)

VIII     2-ethyl-4-hydroxyhexyl phthalate (R’= -CH2-CHOH-CH2CH3; R’’= -CH2CH3)

IX     2-ethyl-5-hydroxyhexyl phthalate (R’= -[CH2]2]-CHOH-CH3; R’’= -CH2CH3)

X     2-ethyl-6-hydroxyhexyl phthalate (R’= -[CH2]3CH2OH; R’’= -CH2CH3)

XI     2-ethyl-pentyl phthalate (R’= -[CH2]3CH3; R’’= -CH2CH3)

XII     2-ethyl-4-oxyhexyl phthalate (R’= -CH2-CO-CH2CH3; R’’= -CH2CH3)

XIV     2-carboxymethyl-4-oxyhexyl phthalate (R’= -CH2-CO-CH2CH3; R’’= -CH2COOH)

XV     2-ethyl-4-oxy-6-carboxyhexyl phthalate (R’= -CH2-CO-CH2COOH; R’’= -CH2CH3)

XVI     2-ethyl-4-hydroxy-6-carboxyhexyl phthalate (R’= -CH2-CHOH-CH2COOH; R’’= -CH2CH3)

XVII     2-(1-hydroxyethyl) hexyl phthalate (R’= -[CH2]3CH3; R’’= -CHOH-CH3)

XVIII     2-carboxymethyl-4-hydroxyhexyl phthalate (R’= -CH2-CHOH-CH2CH3; R’’= -CH2COOH)

XIX     2-(1-hydroxyethyl) -5-hydroxyhexyl phthalate (R’= -[CH2]2-CHOH-CH3; R’’= -CHOH-CH3)

XX     2-ethyl-4,6-dihydroxyhexyl phthalate (R’= -CH2-CHOH-CH2CH2OH; R’’= -CH2CH3)

XXI     2-carboxymethyl-5-hydroxyhexyl phthalate (R’= -[CH2]2-CHOH-CH3; R’’= -CH2COOH)

XXV     2-carboxymethyl-5-oxyhexyl phthalate (R’= -[CH2]2-CO-CH3; R’’= -CH2COOH)

XXVI     2-(1-oxyethyl) hexyl phthalate (R’= -[CH2]3CH3; R’’= -CO-CH3)

5.1.1.1. Oral

The metabolism and excretion of DEHP has been extensively studied in rats following oral administration. Other species in which the metabolism and excretion by the oral route have been studied include mice, guinea pigs, hamsters, non-human primates, and humans.

Humans

The primary objective of this GLP study was to determine the rate and extent of conversion of isotopically labelled DEHP into their primary and secondary metabolites in blood and urine following single oral dose administration at two different dose levels in healthy male and female subjects. The secondary objective was to collect and store biological samples for potential future chemical analysis (Douglas, 2010; Anderson et al., 2011).

The study comprised of 2 parts: Part 1 was an initial pilot investigation performed in one subject from which bioanalytical and pharmacokinetic analyses were determined and Part 2 was performed in 20 subjects (10 males and 10 females) who each took part in 2 treatment periods receiving both low (0.3 mg D⁴‑DEHP) and high (3.0 mg D⁴‑DEHP) doses of the phthalates.

Blood and urine samples were collected for the analysis of plasma and urinary concentrations ofthe primary and secondary metabolites of D⁴-DEHP (D⁴‑MEHP, D⁴‑5‑oxo‑MEHP, D⁴‑5‑OH‑MEHP and D⁴‑5‑carboxy‑MEPP). Plasma and urinary pharmacokinetic parameters were derived from these data.

For D⁴-DEHP, the primary and secondary metabolites were steadily formed following administration of single oral doses of 0.3 mg and 3.0 mg D⁴-DEHP with median t_maxvalues in plasma ranging from 2.5 to 5.0 hours postdose. The between-subject variability for AUC_0-tlastand C_maxwas high for all metabolites.

A total of 6% to 7% of the D⁴‑DEHP dose was excreted in the urine as D⁴‑MEHP, and 11% to 16% as D⁴‑5‑oxo-MEHP, D⁴‑5‑OH‑MEHP and D⁴‑5‑carboxy-MEPP, up to 48 hours postdose. The overall fraction of the 0.3 mg and 3.0 mg doses of D⁴-DEHP excreted as both the primary and secondary metabolites up to 48 hours postdose was 46% to 50%, which equates to a conversion factor for estimating the daily intake of D⁴-DEHP of 1.9 to 2.2.

Almost complete renal excretion of the metabolites assessed for D⁴-DEHP occurred over the initial 24 hours following dosing and between-subject variability (fe%) was generally low.

Overall, there appeared to be no marked gender differences in the exposure and urinary excretion of the metabolites of D⁴‑DEHP. There was also no correlation evident between renal excretion (fe%) or systemic exposure (AUC_0-tlastand C_max) and gender, body weight or age.

Single oral doses of D⁴‑DEHP were considered to be safe and well tolerated by healthy male and female subjects when administered at dose levels of 0.3 mg and 3.0 mg D⁴‑DEHP and 0.9 mg and 9.0 mg D⁴‑DINP.

The overall fraction ofD⁴-DEHP excreted in the urine is presented in Table 26:

 

Table 26. Summary of % molar elimination of D⁴-DEHP metabolites in the urine over 48 hours Postdose(taken from Anderson et al., 2011)

 

	MEHP	5oxo-MEHP	5OH-MEHP	5cx-MEPP	Total
low dose	6.94	12.53	16.33	15.9	51.7
high dose	5.67	10.00	14.86	11.97	42.51
mean	6.31	11.27	15.5	13.93	47.11
standard errors of mean	0.309	0.426	0.50	0.558	1.345

 

 

Single oral doses of D⁴‑DEHP were considered to be safe and well tolerated by healthy male and female subjects when administered at dose levels of 0.3 mg and 3.0 mg D⁴‑DEHP. The incidence of adverse events reported during the study was low, with a total of three adverse events reported by 2 subjects. All adverse events were moderate in severity and required treatment with concomitant medication. Only one adverse event of headache was considered by the investigator to be related to the study investigational product however as this occurred at the lower dose level only it is most probably unlikely to be truly substance-related. There were no serious or severe adverse events reported during the study. There were no findings considered to be of clinical importance for the clinical laboratory evaluations (serum biochemistry, haematology, or urinalysis).

Conclusions:

The primary (D⁴-MEHP) and secondary (D⁴-5-oxo-MEHP, D⁴‑5‑OH‑MEHP and D⁴‑5‑carboxy-MEPP) metabolites of D⁴-DEHP were steadily formed following administration of single oral doses of 0.3 mg and 3.0 mg D⁴-DEHP. All metabolites appeared in plasma at median t_maxvalues ranging from 2.8 to 4.0 hours postdose.

Systemic exposure (AUC_0-tlast) to D⁴-MEHP increased in a supra-proportional manner of up to 17‑fold for the 10-fold increase in dose. The dose proportionality for exposure to the secondary metabolites with increasing dose could not be assessed due to the low systemic exposure of these metabolites at the 0.3 mg dose level.

The between-subject variability for systemic exposure (assessed for AUC_0-tlastand C_max) to the primary and secondary metabolites of D⁴-DEHP was high.

A total of 6% to 7% of the D⁴‑DEHP dose was excreted in the urine as D⁴‑MEHP, and 11% to 16% as D⁴‑5‑oxo-MEHP, D⁴‑5‑OH‑MEHP and D⁴‑5‑carboxy-MEPP, up to 48 hours postdose.

The overall fraction of the 0.3 mg and 3.0 mg doses of D⁴-DEHP excreted as both the primary and secondary metabolites up to 48 hours postdose was 51.7% and 42.51% for the low and high dose, respectively.

The renal excretion of the metabolites assessed for D⁴-DEHP mainly occurred over the initial 24 hours following dosing.

Systemic exposure and urinary excretion of the primary and secondary metabolites investigated for D⁴‑DEHP was generally similar for male and female subjects.

There was no correlation evident between renal excretion (fe%) or systemic exposure (AUC_0-tlastand C_max) and gender, body weight or age.

Single oral doses of D⁴‑DEHP were considered to be safe and well tolerated by healthy male and female subjects when administered at dose levels of 0.3 mg and 3.0 mg.

These data (urinary metabolites found after application of low dose) are used to calculate external doses from measured urinary concentrations. The methodology is described in detail in chapter 9.0.

The observed urinary excretion of DEHP metabolites in the Anderson et al. (2011) study is in close agreement witht the results of another recent toxicokinetic study. Kessler et al. (2012) investigated metabolite profiles of DEHP in 4 human male volunteers, who ingested a single dose of approx. 0.645 mg/kg bw DEHP-D4.Total average amount of urinary excretion of the three metabolites MEHP-D4, 5OH-MEHP-D4 and 5 -oxo-MEHP-D4 was 29.1 and 31% after 22 and 46 h, respectively.

In another study with human volunteers (10 males/10 females) metabolites were identified and quantified after oral application of a single dose of 3 mg/kg bw. DEHP-D4 (and 9 mg/kg bw. di-iso-nonyl phthalate) (Kurata et al., 2012). MEHP-D4, 5OH-MEHP-D4 and 5 -oxo-MEHP-D4 were identified, the major part of it as glucuronides (69 to 86%). Urinary concentrations, but no cumulative excretion data are reported in this publication.

Further human studies:

Two healthy male volunteers (47 and 34 years old) received 30 mg DEHP (> 99% pure) as a single dose or 10 mg/day of DEHP for 4 days (Schmid & Schlatter, 1985). In the single dose study, urine was collected every six hours for 48 hours after dosage and metabolites were isolated and identified by GC/MS. Urinary excretion of DEHP occurred mostly within the first 24 hours and a urinary elimination half-life of about 12 hours was estimated. 11% and 15% of the administered dose was eliminated in the urine of the two volunteers, respectively. A total of 12 metabolites were detected with the major metabolites being identified and quantified as MEHP (6.4 and 12.7% of the detected metabolites, in the two volunteers, respectively) and metabolites I (1.9 and 2.1%), IV (3.7 and 1.8%), V (25.6 and 33.8%), VI (24.0 and 19.7%), VII (5.3 and 4.0%), and IX (33.0 and 25.9%). The amount of the remaining 5 metabolites was less than 1%. About 35% of the metabolites were unconjugated in both volunteers. In the repeated dose study, urine was collected in 24 hour intervals until 48 hours after the last dose. Fifteen and 25% of the administered dose was eliminated in the urine of the two volunteers, respectively. Excretion of metabolites showed strong daily fluctuations. Based on the mean excretion of MEHP (9.6%), 5-oxo-MEHP (21.8%), 5-OH-MEHP (29.4 %), and an assumed total urinary excretion of 25%, Koch et al. (2003a) have calculated conversion factors of 2.4% (MEHP), 5.5% (5-oxo-MEHP), and 7.4% (5-OH-MEHP) based on this study.

The time-course of DEHP metabolism and elimination in one human volunteer has been investigated by Koch et al. (2004). A single oral dose of 48.1 mg deuterium-labelled DEHP (0.64 mg/kg b. w.) was administered to a male volunteer (the senior author of the paper, age 61, body weight 75 kg). DEHP was spiked into butter and administered on bread. By the use of deuterium-DEHP and the most modern technique (LC-LC/MS-MS), the results are very reliable even though they only represent one individual. The urinary excretion of MEHP, 5OH-MEHP, 5oxo-MEHP was monitored for 44 hours post-dosing and the serum levels was monitored for 8 hours post-dosing. Peak concentrations in serum were found in the sample taken two hours after dosage, with MEHP as the major metabolite. The half-time of all the three measured metabolites in serum was estimated to be less than 2 hours. The excretion of DEHP metabolites in urine followed a multi-phase elimination pattern. After an absorption and distribution phase of 4 to 8 hours, the urine elimination pattern showed an initial half-time (8 to 16 hours post-dosing) of about 2 hours for all three metabolites. The second phase, beginning 14 to 18 hours post-dosing, showed a half-time of about 5 hours for MEHP but 10 hours for the secondary metabolites. The study shows that the secondary metabolites 5OH-MEHP and 5oxo-MEHP are the major metabolites of DEHP found in human urine at all time points following a single oral dose of DEHP and that the ratio between MEHP and the secondary metabolites varies over time. Thus, the ratios of MEHP to 5OH-MEPH + 5oxo-MEHP varied over time from 1 to 4.9 during the first phase (8-16 hours post dose) to 1 to 14.3 during the second phase (16 to 24 hours post dose). In the last sample, taken 44 hours after the dose, the ratio was 1 to 74. This difference in elimination half-times has to be taken into account when DEHP ingestion are calculated based on either MEHP or the secondary metabolites. After 44 h, 47% of the DEHP dose had been excreted in urine as the three measured metabolites. MEHP comprised 7.3% of the applied dose, 5OH-MEHP 24.7% and 5oxo-MEHP 14.9%. Thus, the ratio of excreted MEHP to 5OH-MEHP+5oxo-MEHP was 1 to 5.4 in this individual, which appears lower than in the two recent studies on the general population (Koch et al., 2003b; Barr et al, 2003), where the ratio of MEHP to 5OH-MEHP +5oxo-MEHP were 1 to 8.7 and 1 to 14.1, respectively.

The metabolism of di(2-ethylhexyl)phthalate (DEHP) in humans was studied after three doses of 0.35 mg (4.7 µg/kg), 2.15 mg (28.7 µg/kg) and 48.5 mg (650 µg/kg) of D4-ring-labelled DEHP were administered orally to a male volunteer (Koch et al., 2005).Two new metabolites, mono(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono[2-(carboxymethyl)hexyl]phthalate (2cx-MMHP) were monitored for 44 h in urine and for 8 h in serum for the high-dose case, in addition to the three metabolites previously analysed: mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP). For the medium- and low-dose cases, 24 h urine samples were analysed. Up to 12 h after the dose, 5OH-MEHP was the major urinary metabolite, after 12 h it was 5cx-MEPP, and after 24 h it was 2cx-MMHP. The elimination half-lives of 5cx-MEHP and 2cx-MMHP were between 15 and 24 h. After 24 h 67.0% (range: 65.8–70.5%) of the DEHP dose was excreted in urine, comprising 5OH-MEHP (23.3%), 5cx-MEPP (18.5%), 5oxo-MEHP (15.0%), MEHP (5.9%) and 2cx-MMHP (4.2%). An additional 3.8% of the DEHP dose was excreted on the second day, comprising 2cx-MMHP (1.6%), 5cx-MEPP (1.2%), 5OH-MEHP (0.6%) and 5oxo-MEHP (0.4%). In total about 75% of the administered DEHP dose was excreted in urine after two days. Therefore, in contrast to previous studies, most of the orally administered DEHP is systemically absorbed and excreted in urine. No dose dependency in metabolism and excretion was observed. The secondary metabolites of DEHP are superior biomonitoring markers compared to any other parameters, such as MEHP in urine or blood. 5OH-MEHP and 5oxo-MEHP in urine reflect short-term and 5cx-MEHP and 2cx-MMHP long-term exposure. All secondary metabolites are unsusceptible to contamination. Furthermore, there are strong hints that the secondary oxidised DEHP metabolites not DEHP or MEHP are the ultimate developmental toxicants.

Silva et al., 2003 report percentages of glucuronidation of four common phthalate monoesters, monoethyl (mEP), monobutyl (mBP), monobenzyl (mBzP), and mono-2-ethylhexyl phthalate (mEHP) in a subset of urine (mEP n=262, mBP n=283, mBzP n=328, mEHP n=119) and serum (mEP n=93, mBP n=149, mEHP n=141) samples from the general US population. The percentages of free and conjugated monoester excreted in urine differed for the various phthalates. For the more lipophilic monoesters (i.e., mBP, mBzP, and mEHP), the geometric mean of free monoester excretion ranged from 6 to 16%. The contrary was true for the most hydrophilic monoester, mEP, for which about 71% was excreted in urine as its free monoester. Furthermore, percentages of free and conjugated monoesters were similar for mEP, mBP and mEHP among serum and urine samples. Serum mBzP was largely below the method limit of detection. Interestingly, the serum mEP and mBP levels were less than 3% and 47%, respectively, of their urinary levels, whereas the level of mEHP was similar both in urine and serum.

The metabolism of DEHP in humans was investigated by identifying urinary oxidative metabolites of DEHP from individuals with urinary MEHP concentrations about 100 times higher than the median concentration in the general US population (Silva et al., 2006). In addition to the previously identified DEHP metabolites MEHP, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), and mono(2-carboxymethylhexyl) phthalate (MCMHP), we also identified for the first time in humans three additional oxidative metabolites, mono(2-ethyl-3-carboxypropyl) phthalate (MECPrP), mono(2-ethyl-4-carboxybutyl) phthalate (MECBP), and mono(2-(1-oxoethyl)hexyl) phthalate (MOEHP) based on their chromatographic behavior and mass spectrometric fragmentation patterns. Metabolites with two functional groups in the side alkyl chain were also tentatively identified as isomers of mono(2-hydroxyethyl-4-carboxybutyl) phthalate (MHECBP), mono(2-ethyl-4-oxo-5-carboxypentyl) phthalate (MEOCPP), and mono(2-ethyl-4-hydroxy-5-carboxypentyl) phthalate (MEHCPP). The presence of urinary DEHP metabolites in humans that have fewer than eight carbons in the alkyl chain was observed. These metabolites were previously identified in rodents. Although quantitative information is not available, these findings suggest that, despite potential differences among species, the oxidative metabolism of DEHP in humans and rodents results in similar urinary metabolic products.

Non-human primates

In a 65-week oral-dose toxicity study of DEHP in marmosets, which included a toxicokinetic study (Kurata, 2003), ring-labeled¹⁴C-DEHP (99.6% purity) in corn oil was given to 3 groups of marmosets. The first group was treated at 3 months of age. The second group was treated at 18 months of age. The third group was treated for 65 weeks from 3 months of age with unlabeled DEHP and studied at 18 months of age. There were 3 animals of each sex in each treatment group. Treatments were by gavage at dose levels of 100 or 2500 mg/kg bw. Blood samples were collected 1, 2, 4, 8, 12, 24, 48, 72, 120, and 168 hours after dosing. Spontaneous urine and feces were collected for radioactivity determination. At least 2 weeks after the kinetic studies, animals were dosed again and tissues collected 2 hours later for determination of radioactivity. Radioactivity determination was by liquid scintillation counting. The authors found the highest level of radiation in the kidneys after a single oral dose, and considered that high radioactivity levels in the prostate and seminal vesicles of some animals may have been due to urine contamination. Repeated dosing for 65 weeks did not appear to alter the distribution of DEHP in 18-month-old animals. The authors called particular attention to the small amount of label distributed to the testis and postulated that differences in access of DEHP metabolites to the testis may explain a lack of testicular toxicity in marmosets compared to rodents, in which large amounts of MEHP are distributed to the testis after DEHP treatment.

In a GLP study (Kurata, 2005),¹⁴C-labeled DEHP was orally administered to pregnant marmosets at a single dose of 100 mg/kg to examine the transferability of the radioactivity to foetuses. The radioactivity (0.40 µg eq/g) of the foetal blood was comparable to that (0.41 µg eq/mL) in the plasma of the pregnant animals, and the radioactivity in the foetal kidney and liver, 0.62 and 0.55 µg eq/g, respectively, was higher than that in the plasma of the pregnant animals at 24-hr post-dose. The whole-body autoradiography revealed high radioactive concentrations in the bladder urine and small intestinal contents. As for the foetal testis, the radioactivity, 0.20 µg eq/g, was lower than that in the plasma of the pregnant animals, and no specific distribution was noted in this organ.

Kessler et al.(2004) compared blood levels of DEHP and MEHP in pregnant and non-pregnant Sprague-Dawley rats and marmosets in a Good Laboratory Practice (GLP) study. Sprague–Dawley rats and marmosets were treated orally with 30 or 500 mg DEHP/kg per day, nonpregnant animals on 7 (rats) and 29 (marmosets) consecutive days, pregnant animals on gestation days 14–19 (rats) and 96–124 (marmosets). In addition, rats received a single dose of 1000 mg DEHP/kg. Blood was collected up to 48 h after dosing. Concentrations of DEHP and MEHP in blood were determined by GC/MS. In rats, normalized areas under the concentration–time curves (AUCs) of DEHP were two orders of magnitude smaller than the normalized AUCs of the first metabolite MEHP. Metabolism of MEHP was saturable. Repeated DEHP treatment and pregnancy had only little influence on the normalized AUC of MEHP. In marmosets, most of MEHP concentration-time courses oscillated. Normalized AUCs of DEHP were at least one order of magnitude smaller than those of MEHP. In pregnant marmosets, normalized AUCs of MEHP were similar to those in nonpregnant animals with the exception that at 500 mg DEHP/ kg per day, the normalized AUCs determined on gestation days 103, 117, and 124 were distinctly smaller. The maximum concentrations of MEHP in blood of marmosets were up to 7.5 times and the normalized AUCs up to 16 times lower than in rats receiving the same daily oral DEHP dose per kilogram of body weight. From this toxicokinetic comparison, DEHP can be expected to be several times less effective in the offspring of marmosets than in that of rats if the blood burden by MEHP in dams can be regarded as a dose surrogate for the MEHP burden in their fetuses.

In a study of GLP-quality, male Cynomolgus monkeys (2 animals per group) received 100 or 500 mg/kg b. w. per day of unlabelled DEHP (99.8% pure) in corn oil by gavage for 21 days (Short et al., 1987). On day 22 each monkey received a single dose of (carbonyl-¹⁴C) DEHP (radiochemical purity >97%) followed by three daily doses of unlabelled DEHP on days 23 to 25. Urine and faeces were collected at intervals on days 22 to 25 and then the animals were sacrificed. The percentages of (¹⁴C) DEHP derived radioactivity in urine, faeces, and selected tissues (blood, liver, spleen, intestines, intestinal contents, fat, brain, kidneys, adrenals, testes, urinary bladder) were determined by liquid scintillation. Urine samples collected from 0-24 hours were analysed for metabolites of DEHP by normal and reversed phase HPLC. (¹⁴C) DEHP derived radioactivity was detected in some tissues (liver and intestines) at the 500 mg/kg dose level, but represented less than 0.2% of the dose administered. The plasma concentration curves (AUC) for DEHP derived radioactivity for the first 48 hr was133 and 283 ug-hr/ml at 100 mg/kg b. w. and 387 and 545 ug-hr/ml at 500 mg/kg b. w. For the dose of 100 mg/kg b. w., the two individual monkeys excreted 20 and 55% (20 and 55 mg) in the urine and 49 and 39% (49 and 39 mg) in the faeces. For the the 500 mg/kg b. w. dose, 4 and 13% (20 and 65 mg) in the urine and 69 and 56% (345 and 280 mg) in he faeces. This was measured within 96 hours but the majority was excreted within the first 24 hours after dosing, with most of the remainder being excreted during the next 24 hours.DEHP derived radioactivity in 0-24 hour urine samples was resolved into at least 15 metabolites and identified as MEHP, phthalic acid, metabolites I, III, IV, V, VI, IX, X, XII, XIII, XIV, and unidentified fractions. Major metabolites were MEHP, phthalic acid, metabolites V, IX, X, and probably XII, however, a great variability between the two individuals in both dose groups was observed. Polar components, including possible glucuronides, made up only a small percentage of the urinary radioactivity. This study indicates a species difference in the metabolism of DEHP in rats and a nonhuman primate as two of the major metabolites identified in the urine of rats (metabolite I, the end-product of ß-oxidation of V; and metabolite VI, which is believed to be the proximate peroxisome stimulator in rodents) were minor metabolites in monkey urine.The recoveries of the amount of DEHP derived radioactivity in the urine indicated that absorption at 500 mg/kg b. w. is equivalent to that at 100 mg/kg b. w. This suggests that a dependent reduction in the absorption of DEHP from the intestinal tract of Cynomolgus monkeys (cf. Marmosets). However, the AUC is greater for 500 mg/kg b. w. than 100 mg/kg b. w. indicating that absorption is greater above 100 mg/kg b. w. than at 100 mg/kg b. w. [though one would expect proportionally higher values but AUC for a longer time than 48 hr should be conducted for 500 mg]. The difference in none recovered radioactivity in the urine at these two different dose groups may depend on dose, or a higher degree of an alternative excretion pathway (e. g. hepatobilary excretion) and/or a higher degree of retention in the body at 500 mg/kg.

A comparative species differences in the metabolism of DEHP was studied after administration of a single oral dose of 100 mg/kg b. w. (carbonyl-¹⁴C) DEHP (radiochemical purity >97%) in corn oil by gavage to three male Cynomolgus monkeys, five male Fisher 344 rats and five groups of five male B6C3F1 mice at (Short et al., 1987; Astill et al., 1986). The study was using a method equivalent to a guideline study and conducted according to GLP. Urine and faeces were collected at intervals of 12, 24, 48, 72, and 96 hours after dosing. Blood samples were taken from the femoral vein of monkeys at 2, 4, 8, 24 hours and just prior to sacrifice. All animals were killed around 96 hours after dosing for tissue collection (liver, stomach, intestines, intestine contents, gall bladder wash and bile). Concentrations of radioactivity in urine, faeces and blood were determined at the specified intervals and concentrations of radioactivity in selected tissues and other biological samples were determined by liquid scintillation at around 96 hours after dosing. Faeces samples collected from 0-48 hours were pooled for the monkeys, and urine samples collected from 0-24 hours were pooled for each species and were analysed for metabolites of DEHP by HPLC. Urinary metabolites were isolated and the major metabolites were analysed by GC/MS.All three species excreted 30-40% of the dose in the urine (rats 32.9%, mice 37.3%, monkeys 28.2%), primarily during the first 12 hours for rats and mice and during the first 24 hours for monkeys. All three species excreted around 50% of the dose in the faeces (rats 51.4%, mice 52.0%, monkeys 49.0%), primarily during the first 24 hours for rats and mice and during the first 48 hours for monkeys. The rates and extent of urinary and faecal excretion varied widely among monkeys. DEHP was detectable in some tissues in all three species. The mean concentrations detected, with the exception of monkey liver and rat intestinal contents, were less than 1 µg/g. The highest concentrations were detected in liver, intestinal contents, and fat for monkeys, rats, and mice, respectively. Total recoveries of the radioactivity administered were 79 (68-91%), 87 (82-92%) and 90% (63-102%) for monkeys, rats and mice, respectively. Radioactivity in 0-24 hour urine samples were resolved into 13, 15, and 14 components in rats, mice, and monkeys, respectively. The components in urine were identified as MEHP (not detected in rat), phthalic acid, metabolites I, II (not detected in monkey), III (not detected in rat), IV, V, VI, VII, IX, X, XII, XIII, XIV, and unidentified fractions. Major urinary components in rats were metabolites I, V, VI, and IX. Major urinary components in mice were MEHP, phthalic acid, metabolites I, VI, IX, and XIII, and in monkeys: MEHP, and metabolites V, IX, and X. In monkeys 15-26% of the radioactivity excreted may represent glucuronic acid conjugates whereas in rat glucuronides are either absent or present in negligible quantities. Radioactivity in 0-48 hour monkey faecal extracts and in 0-24 hour rat and mouse faecal extracts were resolved into 11, 10 and 10 components in rats, mice and monkeys, respectively. The faecal components were identified as DEHP, MEHP, phthalic acid, metabolites I-IV, VI, VII, IX, X, XII, XIII (not detected in monkey), and XIV (not detected in mouse). DEHP was a major faecal component in all three species and MEHP a major faecal component in rats and mice.Based on the amount of DEHP derived radioactivity recovered in the urine of Cynomolgus monkeys, rats and mice a similar degree of oral absorption of DEHP is indicated at a dose level of 100 mg/kg b. w.Two major species differences in the metabolism of DEHP in rat and mouse were observed as MEHP was a major component in mouse urine but was not detectable in rat urine; metabolite V was a major component in rat urine but a negligible component in mouse urine. Some overall similarities were observed in the metabolism of DEHP in monkeys and rats. In both species the MEHP formed by hydrolysis of DEHP was further metabolized via thew-oxidation pathway, generating metabolites X, V, and I which collectively made up 34 and 44% of the radioactivity in the urine of monkeys and rats, respectively; and via the (w-1) -oxidation pathway, generating metabolites IX and VI which collectively made up 19 and 29% of the radioactivity in the urine of monkeys and rats, respectively. However, some overall differences in metabolism were also observed between the two species. MEHP was a relatively major component of monkey urine (11%) but was not detected in rat urine. Also in monkeys 15-26% of the radioactivity excreted may represent glucuronic acid conjugates whereas in rat glucuronides are either absent or present in negligible quantities. Furthermore, MEHP was extensively converted to metabolite V in the monkey but, in contrast to the rat, further oxidation to metabolite I was negligible. Also metabolite I was a major component in mouse urine. It appears therefore that ß-oxidative metabolism of DEHP is a major pathway in rodents but not in monkeys.

The disposition of DEHP was studied in marmosets (Rhodes et al., 1983). Groups of three male marmosets received a single dose of (¹⁴C-ring labelled) DEHP (radiochemical purity 97.5%) by the oral route (100 and 2 000 mg/kg b. w.), intravenously (100 mg/kg b. w.), and intraperitoneally (1 000 mg/kg b. w.)(Rhodes et al., 1983; Rhodes et al., 1986). Urine and faeces were collected for seven days and the radioactive content determined. Tissue samples were removed 7 d after the administration.Following intravenous administration approximately 40% of the dose was excreted in urine and approximately 20% in the faeces (cumulative excretion) indicating a 2 to 1 ratio between the urinary and biliary (faecal) routes of excretion in the marmoset. Around 28% of the dose remained in the lungs with minimal levels in other tissues. A much smaller proportion of the dose was excreted following intraperitoneal administration (10% in the urine and 4% in the faeces) in a similar 2 to 1 ratio. Around 85% of the dose remained as unabsorbed¹⁴C in the peritoneal cavity with minimal amounts in the tissues (0.6%). Following oral administration of 100 mg/kg b. w. 20-40% of the dose were excreted in urine and around 25% in faeces, and following administration of 2 000 mg/kg b. w. around 4% and 84% were excreted in urine and faeces, respectively. Minimal amounts remained in the tissues (< 0.1%). This indicates that oral absorption of DEHP by marmosets is dose-limited at 2000 mg/kg b. w. compared with 100 mg/kg b. w. Dose dependent reduction in the absorption of DEHP from the intestinal tract of the marmoset (according to the authors the amount absorbed is more equivalent to that expected for a 150 to 200 mg/kg b. w. dose).

A comparative toxicokinetic study was carried out in marmosets (3 males and 3 females, 12-18 months) and Wistar derived albino rats (3 males and 3 females, Alderley Park Specific pathogen-free strain, 6-8 weeks). The animals were given (¹⁴C ring labelled) DEHP (radiochemical purity 97.9%) at doses of 2 000 mg/kg b. w. dailyby gavage for 14 days (Rhodes et al., 1986). Two samples of blood (0.5 ml) from each animal were taken during 0-8 hour period after dosing on day 1 and 14. The rats were bled via the tail vein and the marmosets via the femoral vein. Twenty-four hours after the final dose the animals were killed by inhalation of carbon dioxide/oxygen, and samples of blood (5 ml) were withdrawn from each animal via the vena cava. Excreta were collected for 24 hours after administration of the dose on days 6 and 13. Immediately after the blood samples were taken each animal was dissected and whole liver, kidneys and testes taken for radiochemical analysis. Radioactivity was measured by liquid scintillation spectrometry. The radiolabelled compounds in urine and faeces were analysed by TLC to determine the distribution between DEHP and its metabolites.The uptake of radioactivity into the blood of rats was rapid and peaked after 2-3 hours (126 and 206 µg/g in males and females, respectively) following administration on day 1, and after 6 hours (368 and 475 µg/g in males and females, respectively) on day 14. On both days, blood levels did not decline significantly during the 8-hour sampling period, but 24 hours after dosing on day 14, the levels were 66 and 158 µg/g in males and females, respectively. Blood levels in marmosets were considerably lower. They peaked 1 hour after dosing (5 and 8 µg/g in males and females, respectively) at day 1 and after 1 and 3 hours after dosing in males and females, respectively, on day 14 (13 µg/g each) and had not declined significantly after 24 hours.After dosing on day 6, male rats excreted 83% (53% in urine and 30% in faeces) and female rats excreted 63% of the dose (39% in urine, 24% in faeces). Male marmosets excreted 69 (1% in urine and 64% in faeces) and female marmosets excreted 80% of the dose (2% in urine and 75% in faeces) after the same exposure period. After dosing on day 13 male rats excreted 97% (56% in urine and 41% in faeces) and female rats excreted 96% of the dose (52% in urineand 43.6 in faeces), while male marmosets excreted 62% (1% in urine and 59% in faeces), and female marmosets excreted 75% of the dose (1% in urine and 71 in faeces). The discrepancies between the sum of urine and faeces and the total is due to cage washing. Two radiolabelled compounds were present in rat faeces (analysed by TLC), one being identified as DEHP (42% of the radioactivity from TLC), the other more polar compound (57% of the radioactivity) was not identified. In the faeces of marmosets, 98% of the recovered radioactivity was identified as DEHP. The levels of radioactivity in blood, expressed asmg equivalents of DEHP per g of blood in males and females, were 0.3 and 0.5% of the daily dose, respectively, in rats one hour after administration on day 1. The corresponding levels for marmosets were 0.02 and 0.03%, respectively. The levels of radioactivity in blood 24 hours after administration on 14thday of exposure were 0.2 and 0.5%, respectively, for male and female rats. The corresponding values for marmosets were 0.03 and 0.06%, respectively. The very high faecal elimination and the low levels of radioactivity in urea, blood and tissues in marmosets compared with rats suggests, in agreement with the single dose study by Rhodes et al., (1983) that DEHP at 2000 mg/kg b. w. was poorly absorbed, whereas the urinary elimination data for rats indicate that at least half the dose was absorbed. The study also shows that repeated administration of DEHP in both rat and marmoset did not modify the proportion of dose excreted. The tissue levels in liver and in kidney were generally higher in female rats compared to male rats (liver 216 and 286 µg/g, kidney 115 and 176 µg/g in males and females, respectively). The mean residue level in testes was lower (36mg/g) than in other tissues. Tissue levels in marmosets were considerably lower than in rats (liver 29 and 47 µg/g, kidney 15 and 35 µg/g in males and females, respectively).

Rats

Sprague-Dawley

The toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP), a major metabolite of DEHP, was investigated in Sprague-Dawley rats orally treated with a single dose of¹⁴C-DEHP (Koo, 2007). Urinary excretion of total¹⁴C-DEHP and of its metabolites was followed by liquid scintillation counting (LSC). Concentrations of DEHP and MEHP were determined 6, 24, and 48 h after treatment in rat serum and 6, 12, 24, and 48 h after treatment in urine by high-performance liquid chromatography (HPLC). After 24 h, peak concentrations of MEHP in both urine and serum were observed in animals treated with 40, 200, or 1000 mg DEHP/kg. HPLC showed that general toxicokinetic parameters, such as Tmax (h), Cmax (μg/ml), Ke (1/h), and AUC (μg-h/ml/) were greater for MEHP than DEHP in both urine and serum. In contrast, the half-lives (t1/2 [h]) of DEHP were greater than those of MEHP. The AUC ratios between DEHP and MEHP were relatively smaller in serum than in urine, suggesting the important role of urinary DEHP data for exposure assessment of DEHP.

Sjöberg et al.(1985) studied the kinetics of DEHP and MEHP in immature and mature Sprague-Dawley rats in two different studies. In one study (9-10 rats per group; 25, 40, or 60 days old on the day of dosing) were given a single dose of 1 000 mg/kg b. w. of DEHP (99% pure) in corn oil by gavage. Blood samples, 0.25 ml drawn from a jugular vein, were taken at 1, 3, 5, 7, 9, 12, 15, 24, and 30 hours after dosing. The area under the plasma concentration-time curve (AUC) and the elimination half-life was calculated. Detectable plasma concentrations of DEHP (>2 µg/ml) were found only in some of the animals 1-7 hours after dosing. MEHP was detectable in all but five plasma samples (the 24- and 30-hour sample of two 60-day old rats, and the 30-hour sample of one 60-day old rat). The maximal plasma concentration (Cmax) of MEHP generally appeared one hour after dosing, but in some 25-day old animals it was observed at 3-7 hours after dosing. No differences in Cmaxwere observed between the different age groups. Cmaxranged between 48 and 152 µg/ml, with a mean of 93 µg/ml. The mean AUC (0-30 hours) of MEHP of 25-day old rats (1213mg×hr/ml) was significantly higher than that of the 40- and 60-day old rats (611 and 555mg×hr/ml, respectively). No significant differences in the mean plasma elimination half-life of MEHP were observed when comparing the different age groups. The mean plasma elimination half-lives of MEHP were 3.9, 3.1 and 2.8 hours, respectively for 25, 40 and 60 days old rats. The binding of MEHP to plasma proteins was 98% in all dose groups. In a second experiment by Sjöberg et al. (1985), the excretion of DEHP was studied in immature and mature Sprague-Dawley rats. Two groups of 6 rats which were 25 and 60 days old, respectively, on the day of dosing were given single doses of 1000 mg/kg b. w. of (carbonyl-¹⁴C) DEHP (99% pure) in corn oil by gavage. The urine was collected daily for three days. The cumulative excretion of radioactivity was 44 and 26% in 25- and 60-day old rats, respectively, within the first 72 hours after dosing. More than 85% of the urinary radioactivity appeared within the first 24 hours. No intact DEHP or MEHP was found in the urine when analysed by TLC.

To examine the plasma concentration time profiles of MEHP and metabolites V, VI and IX after oral administration of DEHP, two separate experiments were performed by Sjöberg et al., (1986). In the first experiment, a suspension of DEHP (purity not stated) in propylene glycol was given to five 35-day old male Sprague-Dawley rats in a dose of 2.7 mmol/kg b. w. Blood samples drawn from one of the jugular veins 0.5, 1, 2, 3, 5, 7, 9, 12, 15, and 22 hours after dosing. In the second experiment, five rats were given daily doses of 2.7 mmol/kg b. w. of DEHP in propylene glycol for 7 days. After the final dose, blood samples were collected at the same time intervals as in the first experiment. The plasma concentrations of MEHP and the metabolites were determined by gas chromatography-electron impact mass spectrometry.The plasma concentrations and mean AUC’s of each of the MEHP-derived metabolites were considerably lower than those of MEHP both after single and after repeated administration. The maximal plasma concentrations (MEHP, 0.55 and 0.56 µmol/ml; metabolite IX, 0.15 and 0.09 µmol/ml; metabolite VI, 0.06 and 0.07 µmol/ml; metabolite V, 0.06 and 0.09 µmol/ml after single and repeated doses, respectively) and mean AUC’s (MEHP, 5.15 and 3.44 µmol/ml; metabolite IX, 0.84 and 0.46 µmol/ml; metabolite VI, 0.44 and 0.41 µmol/ml; metabolite V, 0.39 and 0.43 µmol/ml after single and repeated doses, respectively) did not differ significantly between animals given single or repeated doses of DEHP. The mean elimination half-life of MEHP was significantly shorter in animals given repeated doses (1.8 hours) than in those given a single dose (3 hours).

The disposition kinetics of DEHP was studied in male Sprague-Dawley rats following single or multiple administration of DEHP by various routes (peroral by gavage: 2 000 mg/kg b. w.; intra-arterial: 100 mg/kg b. w.; intraperitoneal: 4 000 mg/kg b. w.)(Pollack et al., 1985).The animals were given a single dose of 2 000 mg/kg b. w. of DEHP (purity not stated) in corn oil by gastric intubation. Blood samples were drawn over a 30-hour period. Thereafter, repetitive doses of DEHP were administered to the same animals once daily for 7 days whereafter blood samples were collected over a 48-hour period. The concentrations of DEHP and MEHP in whole blood were determined by high performance liquid chromatography (HPLC). After a single oral dose, DEHP was absorbed relatively rapidly with a peak blood concentration of DEHP observed at approximately 3 hours. Systemic bioavailability of DEHP was low, approximately 13%. Blood concentrations of MEHP were much higher than those of the parent compound after oral administration. The blood concentrations of DEHP following repeated dosing were similar to those observed after a single dose. Secondary increase in the concentration of DEHP in blood were observed following administration by all three routes. Following a single intra-arterial injection a large apparent volume of distribution and a high rate of clearance was observed for DEHP. A marked route-dependency in the formation of MEHP from DEHP was observed. Pharmacokinetic calculations revealed that approximately 80% of an oral dose of DEHP undergoes mono-de-esterification, as compared to only about 1% of the dose following either intra-arterial or intraperitoneal administration. Multiple intraperitoneal injections resulted in an apparent decrease in the rate and/or extent of DEHP absorption from the peritoneal cavity, while no significant change in the peroral absorption of DEHP was observed. The difference in the MEHP to DEHP AUC ratio between peroral and intraperitoneal routes was still evident after multiple dosing.

DEHP and MEHP were secreted into the milk of lactating Sprague-Dawley (CD) rats when given 3 oral doses of 2 000 mg/kg b. w. per day of DEHP in corn oil by gavage on days 15-17 of lactation (Dostal et al., 1987). Plasma collected 6 hours after the third dose contained virtually no DEHP but substantial amounts of MEHP (76 µg/ml). Milk collected 6 hours after the third dose contained 216 µg/ml DEHP and 25 µg/ml MEHP. A very efficient extraction mechanism for DEHP was suggested because of a high milk/plasma ratio.

Male Sprague-Dawley rats (number not stated, 250-350 g) were given two doses of 100 mg (7-¹⁴C) DEHP (purity not stated) or (7-¹⁴C) MEHP (purity not stated) in corn oil by gavage, 24 hours apart (Albro et al., 1983). Urine was collected from the time the first dose was given until 24 hours after the second dose. Metabolites were isolated and analysed by HPLC and GC, and the profiles of radioactivity of the urinary metabolites were determined. Twenty metabolites were identified in the urine of rats (Table 4.24). The metabolites identified in the urine of rats treated with either DEHP or MEHP were identical. No glucuronides or other conjugates were detected.In a second experiment of the same study, (7-¹⁴C) DEHP was given as a single dose to a rat (300 g) and the urine collected was freezed immediately. One week later the same rat was given a dose of (7-¹⁴C) DEHP identical to that above and the urine was collected. Metabolites were isolated and analysed by HPLC and GC. The profiles of radioactivity of the urinary metabolites in the two different samples were qualitatively identical indicating, according to the authors, that the presence of any of the metabolites found was not due to further metabolism by bacteria in the urine.According to the authors, previous studies of the metabolism in rats led to the suggestion that the enzymatic processes normally associated withw-, (w-1) -, a- and ß-oxidation of fatty acids could account for the known metabolites of DEHP found in the urine. Several metabolites of DEHP have been identified in the present study. Their formation requires that the initial hydroxylation process is less specific than fatty acidw- and (w-1) -oxidation are thought to be. Furthermore, it is necessary to postulate either that the aliphatic chain of MEHP can be oxidised at two sites simultaneously, or that oxidation products can be recycled for a second hydroxylation prior to excretion.

Adult male Sprague-Dawley rats (CD, 300-400 g; number not stated) were administered two doses of 200 µl (196 mg) (7-¹⁴C) DEHP (>99% pure) in corn oil by gavage, 24 hours apart (Albro et al., 1973). The urine was collected for 48 hours after the first dose was given. Metabolites in the urine were analysed by TLC and gas chromatography (GC) and characterised by infrared (IR), nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Five metabolites were identified in the urine. The metabolites identified correspond to phthalic acid and to metabolites I, V, VI, IX resulting fromw- and (w-1) -oxidation of MEHP without attack on the aromatic ring. MEHP was not detected in the urine and phthalic acid amounted to less than 3% of the urinary metabolites. Conjugates were not detected. These results indicate, according to the authors, that DEHP is firsthydrolysed to MEHP, which then undergoesw- and (w-1) -oxidation of the side chain. Alcohol intermediates may then be oxidized to the corresponding ketones. The metabolites found in the urine suggest that, in the rat, MEHP is metabolized like a fatty acid byw- and (w-1) -oxidation and then by ß-oxidation.

The elimination of DEHP was studied in rats and hamsters (Lake et al., 1984). A single dose of (carbonyl-¹⁴C) DEHP (>99% pure) was administered to 5-week-old male Sprague-Dawley rats and male DSN strain Syrian hamsters at dose levels of 100 (5 rats, 3 hamsters) or 1000 mg/kg b. w. (5 rats, 5 hamsters) in corn oil by gastric intubation. Urine and faeces were collected over a period of 96 hours and then the animals were sacrificed. Radioactivity was measured in urine, faeces, and total gut contents by liquid scintillation spectrometry. Faecal metabolites were extracted and chromatographed on thin-layer plates. In both species the bulk of the radioactivity was excreted within 24 hours. At the lower dose level, both species excreted more radioactivity in the urine (rat: 51%, hamster: 53%) than in the faeces (rat: 43%, hamster: 31%), whereas at the higher dose level, the major route of excretion was via the faeces (rat: 53%, hamster: 48%). In both species and at both dose levels, only negligible amounts of radioactivity were present at termination in either the liver, kidney, or total gut contents. Faecal radioactivity profiles were determined in 0-24 hour faeces samples. About 50% of the faecal radioactivity of rats at the higher dose level appeared to be the parent compound, the remainder comprised metabolites possibly including MEHP. In contrast, more than 95% of the faecal radioactivity of hamsters appeared to be the parent compound. Similar results were obtained with faecal extracts from rats and hamsters at the lower dose level.

Excretion and metabolism of DEHP were studied in a comparative study where DEHP (99.6% pure) was administered in the diet to adult male Sprague-Dawley rats (6 animals, 200-300 g), male beagle dogs (4 animals, approximately 1 year old, 7-10 kg), and male miniature pigs (5 animals, Hormel strain, between 4 month and 1 year, 10-25 kg) in doses of 50 mg/kg b. w. per day for 21-28 days before administration of a single dose of (carbonyl-¹⁴C) DEHP (radiochemical purity >98%) (50 mg/kg b. w.) in corn oil by gavage (Ikeda et al, 1980). Administration of the DEHP containing diets was continued until the animals were killed. Distribution and excretion of the radioactivity in urine, faeces, and various organs and tissues (liver, kidney, g. i. -tract with content, lungs, brain, fat and muscle) were analysed at various times by liquid scintillation. Excretion of radioactivity in urine and faeces during the first 24 hours was 27 and 57% (rats), 12 and 56% (dogs), and 37 and 0.1% (pigs), respectively; and after 4 days 37 and 53% (rats), 21 and 75% (dogs), and 79 and 26% (pigs), respectively. Elimination of radioactivity was rapid in rats, slightly prolonged in dogs and least rapid in pigs; excretion in all three species was virtually complete in 4 days. TLC revealed four radioactive metabolites in rat urine, three in dog urine and five in pig urine. Only a trace of unmetabolized DEHP was found in the urine of rats, dogs, or pigs. A substantial amount of radioactivity was present in the gastro-intestinal tract at day 1 in all species and a small amount remained after 4 days. In other organs there was only a small amount of radioactivity present in all samples. Of the remaining organs the highest level (about 2% of the dose) was found in the livers from rats after 4 hours. Bile samples from dogs, and to a lesser extent from pigs, accounted for a significant amount of administered¹⁴C dose. Less than 1% of the administered¹⁴C dose was secreted in the bile from bile duct cannulated rats.

Metabolism and tissue distribution of mono-2-ethylhexyl phthalate (MEHP) has been studied in male Sprague-Dawley rats (Chu et al., 1978). To study if MEHP was readily absorbed orally, the carotid arteries of 8 rats were cannulated and 4 days later 4 animals were given 69 mg (7‑¹⁴C) MEHP/kg (20mCi) in corn oil via stomach tube. Serial blood samples (0.2 ml) were collected at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 24 and 48 hours after dosing and radioactivity was determined. The blood level was highest in the first sample, after 0.5 hours, and then rapidly decreased. About five hours after dosing there was a small increase in the bloodconcentration in all animals whereafter the concentration slowly continued to decrease.Another four rats were given 35 mg (7‑¹⁴C) MEHP/kg in 5% NaHCO3via the cannula. Serial blood samples were collected at 2, 4, 6, 8, 12, 14, 16, 18 and 20 minutes after the injection and analyzed for radioactivity. Immediately after the blood samples were taken the animals were exsanguinated and tissues and organs were removed for determination of radioactivity. The rapid decrease was observed also in the rats administered i. v. The first blood sample showed that 53% of the radioactivity remained in the blood. Approximately 1/3 and 1/5 of the radioactivity was retained in the blood 10 and 20 min after administration, respectively. Twenty minutes after an i. v. dose the liver, bladder and kidney were found to possess high radioactivity, but other tissues also had some radioactivity. One of the major deposition sites for radioactivity shortly after i. v. injection was the liver.In a third experiment two groups of four rats were given 69 mg (20mCi) and 6.9 mg (2mCi) (7‑¹⁴C) MEHP/kg, respectively, in corn oil via stomach tube. The animals were exsaguinated after 24 hours and tissues and organs were removed for determination of radioactivity. Twenty-four hours after an oral dose of 69 mg (7‑¹⁴C) MEHP virually all radioactivity was removed from the body and only traces were present in the kidney, liver, heart, lung, intestine and muscle. No detectable amounts could be found in the tissues of rats 24 hours after an oral dose to 6.9 mg/kg.In a fourth study four rats were given a single oral dose of 69 mg (7‑¹⁴C) MEHP/kg (20mCi) in corn oil via stomach tube, and were kept individually in metabolism cages. Urine and faeces were collected each day for 7 days for radioactivity measurements. Only the urine was examined for metabolites as this route of excretion accounted for 81% of the dose. Excretion after 48 hours was insignificant. At least four metabolites were identified and they had previously been identified as DEHP metabolites.The bile ducts were cannulated in four rats in a fifth study by the same author. These rats were given 3.5 or 35 mg (7‑¹⁴C) MEHP/kg in 5% NaHCO3via the superficial dorsal vein of the penis. Serial bile samples were taken hourly for 8 hours and assessed for radioactivity. Within 8 hours 52% (3.5 mg/kg) and 40% (35 mg/kg) of the dose was secreted, respectively, and secretion after this time was insignificant.Thus, the present study indicate that MEHP given orally to the rat undergoesw- and (w-1) -oxidation to yield the same metabolites as does DEHP, and suggest that MEHP is the intermediary product in the DEHP metabolism. More than 80% of the radioactivity of the orally administered (7‑¹⁴C) MEHP was excreted in urine within 24 hours. Up to 52% of the radioactivity entered the intestine from the bile whereas only 8% of the dose was excreted in faeces. This would indicate that resorption of radioactivity took place in the intestine. The rise in the radioactivity in the blood after the rapid decrease could be attributed to the reabsorption of biliary secreted material. The percent radioactivity secreted in the bile was lower, 40%, at the higher exposure level (35 mg/kg) compared to 52% at the lower exposure level (3.5 mg/kg).

Calafat et al.(2005) measured MEHP in maternal urine and amniotic fluid after gavage administration of DEHP [purity not specified] in corn oil to pregnant Sprague-Dawley rats on GD 8, 10, 15, 16, and 17. [The abstract indicates administration also on GD “5/7”.] Doses were 0, 11, 33, 100, and 300 mg/kg bw (n = 2/dose group). Urine was collected approximately 6 hours after dosing and amniotic fluid was collected at necropsy on GD 18. MEHP was analyzed by HPLC-tandem MS after solid-phase extraction and enzymatic hydrolysis. There was no temporal trend in urinary MEHP levels over the collection period, and the 5 urine MEHP levels were combined for each animal. Creatinine-corrected and uncorrected urinary MEHP and uncorrected amniotic fluid MEHP were highly correlated with maternal DEHP dose (r values 0.964–0.998). [Data were presented only in graphic form. At the 300 mg/kg maternal DEHP dose level, urinary MEHP was estimated from a graph at 16.4 µg/L and amniotic fluid MEHP was estimated at 2.8 µg/L.] Maternal urinary MEHP was only 13.3% unconjugated while amniotic fluid MEHP was 88.2% unconjugated. The authors observed that the finding that MEHP was largely conjugated in urine did not agree with reports of other studies on urinary MEHP in rats. The authors also indicated that the lack of measurement of more oxidized MEHP metabolites may lead to an underestimation of exposure to DEHP and its biotransformation products.

Ono et al.(2004) evaluated the testicular distribution of DEHP in 8-week-old Sprague-Dawley rats. The rats were given a single gavage dose of DEHP 1000 mg/kg bw, radiolabeled either in the ring or the aliphatic side chains. The animals were perfusion-fixed with paraformaldehyde and glutaraldehyde under anesthesia 6 or 24 hours after DEHP administration (n = 4 animals/time point). Testis, liver, and kidney were collected and processed for light and electron microscopic autoradiography. After ring-labeled DEHP was given, light microscopy showed preferential distribution of grains to the basal portions of stage IX–I tubules at 6 hours. Grain counts were high in the kidney at 6 hours at the epithelial brush border and the abluminal cytoplasm of the proximal tubule. At 24 hours, grain counts in testis and kidney were much reduced, and hepatic grain counts were increased in a centrilobular distribution in the liver. Electron microscopic autoradiography of Stage IX–I seminiferous tubules 6 hours after ringlabeled DEHP showed grains in Sertoli cell smooth endoplasmic reticulum and mitochondria. There were also grains at cell-junctions involving neighboring Sertoli cells and Sertoli-germ cells. Fewer grains were seen in the Sertoli cell Golgi apparatus and lysomes and in spermatocyte cytoplasm. By contrast, administration of side arm-labeled DEHP resulted in few grains in the seminiferous epithelium and 6 hours and no grains in any tissue examined at 24 hours. The authors concluded that phthalic acid is transported into tissue after DEHP administration and is responsible for the testicular toxicity of both DEHP and MEHP.

Wistar

The absorption, blood concentration and excretion of DEHP were determined in pregnant and non-pregnant Wistar female rats following a single and a repeated oral administration at the dose levels of 200 mg/kg and 1000 mg/kg (Laignelet and Lhuguenot, 2000). Blood samples were taken at defined time intervals after administration for quantification of total radioactivity. Urine and faeces were collected daily and DEHP and its metabolites were extracted and then identified by GC-MS and quantified by GC.In non-pregnant rats, [¹⁴C]-DEHP was rapidly, extensively and dose-related absorbed following a single or repeated oral administration as mirrored by the blood concentration curve profile and a rapid excretion in urine and faeces. A 5-day pre-treatment did not have any significant effect on the total absorption rate but increased slightly the half-life of elimination at the high dose level. After a single or a repeated administration, [¹⁴C]-DEHP was excreted very quickly in excreta and the recovery reached or exceeded 90% of the administrated dose. [¹⁴C]-DEHP was excreted in majority as MEHP-derived metabolite essentially in urine, as MEHP mainly in faeces and DEHP almost totally in faeces. Omega-1 oxidation was the main metabolic pathway (c.a. 60-70%) of the production of MEHP-derived metabolite. The repeated administration was characterised by a decrease of the DEHP excretion and a concomitant increase of the MEHP-derived metabolites but without alteration of the w/w-1 oxidation ratio. This effect was probably related to the metabolic activation which took place after a few days of treatment.In pregnant rats, [¹⁴C]-DEHP was rapidly, extensively and dose-related absorbed following a single or repeated oral administration as mirrored by the blood concentration curve profile and a rapid excretion in urine and faeces. DEHP, MEHP and MEHP-derived metabolites were found in blood. MEHP was the main circulating compound followed by DEHP. MEHP-derived metabolites were present at low concentration. A 5-day pre-treatment increased the total absorption rate and also increased the MEHP concentrations in blood to the detriment of DEHP. The distribution of [¹⁴C]-DEHP in whole foetus was also rapid and extensive and followed by a rapid clearance parallel to the blood concentration curve in dams. The radioactivity contents in whole foetus were lower than the corresponding radioactivity concentrations in the blood of dams but did not reflect the actual concentrations in the blood of foetus. After a single or a repeated administration, [¹⁴C]-DEHP was excreted very quickly in excreta and the recovery reached or exceeded 75 and 88% of the administrated dose, respectively. [¹⁴C]-DEHP was excreted in majority as MEHP-derived metabolite essentially in urine, as MEHP mainly in faeces and DEHP almost totally in faeces. Omega-1 oxidation was the main metabolic pathway of the production of MEHP-derived metabolite.

The distribution and elimination of DEHP and MEHP after a single oral dose of 25 mmol DEHP /kg (corresponding to 9765 mg/kg b. w., purity not stated) by gastric intubation were studied in male JCL: Wistar rats (number not stated, 200 g) (Oishi and Hiraga, 1982). Samples of blood and tissues were collected at 1, 3, 6, 24, 48 and 96 hours post-intubation, and analyzed by gas-liquid chromatography and a electron capture detector. The concentration of DEHP and MEHP in blood and tissues increased to a maximum within 6-24 hours after dosing while the highest levels observed in the heart and lungs occurred within one hour. Both DEHP and MEHP were detected in brain and kidney, but the concentrations were very low. Only small amounts of MEHP was measured in the lung, and DEHP was detected in the spleen at very low levels. The concentration of DEHP in fat increased gradually until 48 hours after dosing. The concentration of DEHP in liver declined with a half-life of 1 day while that in the epididymal fat declined more slowly with a half-life of 6.5 days. At 6 hours after administration, the highest ratio of MEHP/DEHP was recorded in testes (2.1). The ratio in blood was 1.1 while the ratio in other tissues was less than one. Biological half-lives of DEHP in different tissues ranged from 8 to 156 hours in the testicular tissue and epididymal fat, respectively, and of MEHP from 23 to 68 hours in the blood and epididymal fat, respectively.

A study by Oishi (1990) reported on the distribution and elimination of DEHP after a single oral dose of DEHP (2 000 mg/kg b. w.) in male Wistar rats (35 days old). The blood was collected from the caudal vena cava under deep ether anestesia and then testes were removed at 1, 3, 6, 12 and 24 hours following DEHP administration. The concentration of MEHP in blood and in testis increased to a maximum 6 hours after administration of DEHP and then slowly decreased. For MEHP the biological half-lives in blood and testis were 7.4 and 8.0 hours, respectively, and the area under the concentration-time curve was 1497 and 436mg×h per ml or per g, respectively.

Young male Wistar rats (number not stated, 100-200 g) were treated with a single dose of (carbonyl-¹⁴C) DEHP (purity not stated) at a dose level of 2 000 mg/kg b. w. in corn oil by gastric intubation following pre-treatment with DEHP (>99% pure) for 0, 6 or 13 days (Lake et al., 1975). At the end of 4 days, when no further radioactivity was detected in the excreta, the animals were sacrificed, and the organs and tissues were removed. The radioactivity in excreta, organs, and tissues were measured by liquid scintillation spectrometry. Following a single dose of DEHP, virtually all of the administered radioactivity was excreted in the urine (52%) and faeces (48%) within 4 days, and less than 0.1% of the radioactivity remaining in organs and tissues. Similar results were observed in rats pre-treated with DEHP for 6 or for 13 days (60% of the radioactivity was recovered in urine and 40% in faeces).

In several experiments by Tanaka et al., (1975), male Wistar rats (150-250g) were given single oral doses (500 mg/kg b. w.) or a single intravenous doses (50 mg/kg bw) of (carbonyl-¹⁴C) DEHP (radiochemical purity >99%) to study distribution, metabolism and elimination. In the elimination studies there were two animals in each group, and in the distribution studies there were three animals in each. The peak blood level was observed about 6 hours after administration. The concentrations in liver and kidney reached a maximum in the first 2-6 hours. No significant retention was found in organs and tissues (brain, heart, lungs, liver, spleen, kidney, stomach, intestine, testicle, blood, muscle an adipose tissue). About 80% of the dose was excreted in the urine and faeces within 5-7 days following both oral and intravenous administration. Excretion in the urine was generally slightly greater than that in the faeces. In experiments with rats in which the bile duct was cannulated, about 5% of the dose was recovered from the bile in 24 hours after oral administration, whereas about 24% was recovered after intravenous administration. When urine and faecal extracts were analysed by thin-layer chromatography (TLC) after oral administration, four major metabolites were detected in urine. Unchanged DEHP was excreted in the faeces, but DEHP or MEHP were not detected in the urine or bile. After intravenous administration about 75% of the dose was recovered from the liver after the first hour. The radioactivity of the liver declined rapidly by about 50% within the next 2 hours and only 0.17% of the radioactivity remained on the 7thday. The intestine accumulated the next highest amount of radioactivity. The radioactivity increased as the radioactivity in the liver decreased. After intravenous application the activity in the liver and kidneys reach a maximum in the first 2-6 hours. Medium values were seen in the heart, lung and spleen. The peak blood level was observed about 6 hr after administration. The testicle and brain showed the lowest values as in the case of intravenous application.

The distribution, accumulation, and excretion of DEHP were studied in several experiments with Wistar rats (Daniel and Bratt, 1974). Following a single oral dose of (carbonyl-¹⁴C) DEHP (2.9 mg/kg b. w.; purity not stated), rats (5 adult males) excreted 42% and 57% of the administered radioactivity in the urine and faeces, respectively, within 7 days. Rats (5 adult males) fed a diet containing 1 000 ppm of DEHP 7 days prior to dosing with (carbonyl-¹⁴C) DEHP excreted 57% and 38% in the urine and faeces, respectively, within 4 days.In studies with biliary-cannulated rats, administered 2.6 mg/kg DEHP by intubation, around 10% was excreted in the bile.In rats (24 females) fed a diet containing 1 000 or 5 000 ppm of (carbonyl-¹⁴C) DEHP for 35 and 49 days, respectively, the amount of radioactivity in liver and abdominal fat rapidly attained a steady-state concentration without evidence of accumulation. When returned to a normal diet, the radioactivity in the liver declined with a half-life of 1-2 days and in fat with a half-life of 3-5 days. DEHP was extensively metabolized with 14 metabolites, including MEHP, present in urine (analysed by TLC, MS and NMR). DEHP was not detected in the urine. The principal metabolites detected correspond to phthalic acid and metabolites IV or V, VI and IX i. e. the acid, alcohol, and ketone resulting fromw- and (w-1) -oxidation of MEHP. The hexobarbital sleeping time was reduced 39 and 43% in male and females, respectively, when given five daily oral doses of DEHP. When DEHP was administered intravenuosly the hexobarbital sleeping time increased by approximately 40% in male rats compared with the corresponding controls.Rats were injected 600 mg¹⁴C-DEHP/kg as an emulsion prepared by subjecting to ultrasonication through the femoral vein. After 2, 24, 72 and 96 hours the animals were killed and the lungs, liver, spleen, blood and portions of abdominal fat were removed for radiochemical analysis. Radioactivity disappeared rapidly from the blood and about 60-70% was recovered in the liver and lung within 2 hours of dosing. After 4 days 44% was recovered from the urine, 29% from the faeces. About 1% was recovered in fat.

Lhuguenot et al.(1985) studied the metabolism of DEHP and MEHP in rats following multiple dosing. Adult male Wistar rats (180-220 g, 3 rats per group and per chemical) were administered (7-¹⁴C) DEHP (>98% pure) or (¹⁴C) MEHP (position of label not stated; highest available purity) by gastric intubation in corn oil at doses of 50 or 500 mg/kg b. w. for three consecutive days. Urine was collected for 4 days, at 24-hour intervals, metabolites were extracted, analysed by GC and detected by MS. After exposure to DEHP approximately 50 and 60% and after exposure to MEHP approximately 70 and 80% of the total daily doses were recovered in the urine at the low and high dose levels, respectively. No water-soluble conjugates were detected in the urine following administration of DEHP or MEHP. After a single dose of either compound, the main metabolites excreted were I, V, VI, IX. At the lower dose level, no or minor changes in urinary metabolite profiles were seen with time; after multiple dosing at the higher dose level, increases inw-/b-oxidation products (metabolites I and V) and decreases in (w-1) -oxidation products (metabolites VI and IX) were seen.

Fischer 344

Male Fischer 344 rats (12 animals per group) received a total of 10 daily doses of 1.8, 18, or 180 mg/kg b. w. of radiolabelled DEHP (purity not stated) in cottonseed oil by gavage. All rats received the same amount of radioactivity (1.8 mg/kg b. w. (¹⁴C) DEHP, position of label not stated) with different amounts of non-radioactive DEHP diluent (Albro et al., 1982). Urine and faeces were collected daily. Three rats from each group were sacrificed 1, 3, 10, or 12 days after receiving their first dose of DEHP. Various tissue samples and faeces were radioassayed using a tissue oxidizer; urine was radioassayed directly in liquid scintillation fluid. The profiles of the radioactive metabolites in urine were determined by HPLC. Unhydrolyzed DEHP was measured by Radio-TLC. The percentage of¹⁴C retained in the liver tended to decrease with exposure time and also with increasing dose. There was no evidence for accumulation of DEHP in the liver. Essentially the same observations applied to the testes except that testes had lower concentrations than the liver. After about 4 days, excretion (cumulative excretion of¹⁴C as a percentage of the cumulative dose) became quite independent of the dose. Up to a dose of 180 mg/kg per day there was no indication of beginning to saturate the overall elimination mechanism.In a separate experiment, rats and mice were given single oral doses of DEHP in cottonseed oil by gavage at doses ranging from 1.8 to 1 000 mg/kg bw. The animals were sacrificed 6 hours later and the livers were assayed for intact DEHP as described above. In Fisher rats, as the dose increased, a threshold was reached, at about 450 mg/kg bw, above which there was a steady increase in the amount of unhydrolysed DEHP reaching the liver. According to the authors, intact DEHP will reach the liver of rats whenever its concentration exceeds 0.43% in the diet. In contrast, an absorption threshold could not be determined in either CD-1 or C3B6F1mice for doses up to 1000 mg/kg b. w. According to the authors, this may reflect the higher level of DEHP-hydrolase in the intestines of mice than in rats. Preliminary expeiments with both Sprague-Dawley (CD) and Fischer 344 rat revealed that the maximum amount of DEHP that could be given as a single oral dose without significant excretion of unabsorbed DEHP in the faeces was 200 mg/kg bw. According to the authors, pharmacokinetic studies using¹⁴C-labelled DEHP at doses above 200 mg/kg bw would rapidly become dominated by unabsorbed DEHP and, according to the authors, could not be directly compared to rats of elimination at lower doses.

Male Fischer 344 rats (100-150 g bw, 12 animals per group) were fed diets containing 1 000, 6 000, or 12 000 ppm (estimated to correspond to 85, 550 and 1 000 mg/kg b. w. per day, respectively) of non-radiolabelled DEHP (99.8% pure) (Short et al., 1987; Astill et al., 1986). These groups were divided into three subgroups each consisting of 4 rats which received diets for 0, 6 or 20 days, followed by a diet containing a similar level of (carbonyl-¹⁴C) DEHP (radiochemical purity >97%) for 24 hours. Urine and faeces were collected at 24-hour intervals from 24-96 hours after administration of radiolabelled DEHP and then the animals were sacrificed. Four animals from each dose group were sacrificed on each of days 5, 11 and 25 and terminal blood samples were collected. Liver, lung, spleen, intestines, fat, brain, kidney, adrenals, testes and urinary bladder were removed. Urine samples collected from 0-48 hours were pooled for each of the three dose levels and three prior exposure regimens and analysed by normal and reverse phase HPLC for metabolites of DEHP. Urinary metabolites were isolated and the major metabolites were analysed and identified by GC-MS. Faeces samples were similarly pooled and analysed by normal HPLC for the metabolites of DEHP. Radioactivity was measured by liquid scintillation.At all dose levels and exposure times radioactivity was excreted primarily via the urinary route and primarily during the first 24 hours. The percentage of the dose excreted in urine increased with dose, from 53% at 1 000 ppm to 62-66% at 6 000 ppm, and 66-69% at 12 000 ppm. The faecal excretion occurred primarily during the second 24 hours. The percentage of the dose excreted in faeces decreased with dose, from 35-38% at 1 000 ppm to 26-30% at 6 000 ppm and 24-28% at 12 000 ppm. At all dose levels, prior exposure to DEHP did not affect the extent or rate of urinary or faecal excretion. Less than 1% of the administered dose remained in tissues 4 days after treatment. The radioactivity in the urine samples was resolved into 14 components and identified as phthalic acid, metabolites I, II, III, IV, V, VI, VII, IX, X, XII, XIII, XIV, and unidentified fractions. The major urinary metabolites, I and V, were followed by metabolite IX and phthalic acid; the remainders was all present in only minor quantities. DEHP and MEHP were not detected in the urine. The radioactivity in the faeces sample was partially resolved into 15 components and tentatively identified as DEHP, MEHP, phthalic acid, metabolites I-V as a pool, metabolites VI, VII, IX, X, XII, XIII, and XIV. The major faecal metabolites were MEHP, metabolites I-V, VI and IX.The urinary, and to a lesser extent the faecal, metabolite excretion patterns changed with dose and prior exposure to DEHP. The major changes in metabolism occurred between the 1 000 and 6 000 ppm dose levels and between 0 and 6 days prior exposure to DEHP. Minor changes were observed with increase in dose to 12 000 ppm. Urinary elimination of metabolites I and V were measured as a function of dose and duration of treatment. The output of metabolite 1 was relatively constant at all dietary levels on day 0, while the output of metabolite V increased with dietary level on day 0. Following prior exposure to DEHP at the 1 000 ppm dose level the urinary excretion of metabolite I doubled compared with no prior exposure, while the the urinary excretion of metabolite V remained relatively constant. Following prior exposure to DEHP at the 6 000 or 12 000 ppm dose level the urinary excretion of metabolite I increased three or four times, respectively, compared with no prior exposure. The urinary excretion of metabolite V decreased two or three times, respectively, following prior exposure to DEHP at the 6 000 or 12 000 ppm dose level compared with no prior exposure. According to the authors the increase in urinary levels of metabolite V at 6000 ppm and higher while metabolite I remained relatively constant, indicated that at high exposure levels the initial dose of DEHP exceeded the rats ability convert metabolite V to metabolite I. However, the capacity forb–oxidation appeared to increase with repeated exposure to DEHP since urinary levels of metabolite V decreased with subsequent doses of DEHP while those of metabolite I increased.The tissue distribution was examined in rats sacrificed 112-116 hours after receiving the radiolabelled DEHP. A major source of radioactivity was found in the intestinal contents. Additional sources of radioactivity included the liver, fat, kidney and adrenals. A comparison of the tissue levels of radioactivity after 0 and 20 days of pretreatment, indicated that pretreatment with DEHP for 20 days did not significantly alter the tissue distribution of¹⁴C-DEHP derived radioactivity.

To study peroxisome proliferation Fisher 344 rats (5 males / group) were fed diets containing 100, 1000, 6000, 12000, 25000 ppm DEHP, corresponding to 11, 105, 667, 1223 and 2100 mg DEHP/kg bw/d (Short et al., 1987). After an overnights fast the rats were sacrificed and their livers were examined for parameters indicative of peroxisome proliferation. Liver weight expressed as percent of body weight was significantly increased in rats that received 667 mg/kg bw/d and above. Palmitoyl-CoA oxidation and lauric acid 11- and 12-hydroxylation weight was significantly increased in rats that received 667 and 105 mg/kg bw/d, respectively, and above. Since an increased peroxisomal score was observed at dose levels that also produced significant changes in the biochemical parameters, these observations appear to be correlated.

Fischer 344 rats, CD mice, Syrian golden hamsters, and Hartley albino guinea pigs were given two doses of (carbonyl-¹⁴C) DEHP (purity not stated) in cottonseed oil by stomach tube at 24 hours intervals (Albro et al., 1982). The maximum single dose of DEHP was 180 mg/kg b. w for rats and guinea pigs, 360 mg/kg b. w for mice, and 20 mg/kg b. w for hamsters. Urine was collected for a total of 48 hours following the first dose. The urinary metabolites were analysed by HPLC and GC-MS. Rats excreted predominately metabolites having carboxyl groups on the side chain (metabolites I-V), these diacids require from three to six oxidative steps for their formation. Table 4.1.2.1.7 gives the distribution of radioactive metabolites in pools of urine from three animals, however, it should be noted that the doses were not the same between species. No components were detected in urine from hamsters or guinea pigs that were not also present in urine from rats and mice. In the hamsters the main metabolites were: di methyl phthalate (DMP), metabolite I, V, VI and IX. The main metabolites in the mouse were: DMP, MEHP, metabolites I, VI, IX, while in the guinea pigs MEHP was the dominating metabolite. MEHP accounted for 5% of the metabolites in hamsters, and 19 and 70%, respectively, in mice and guinea pigs. Rats did not excrete conjugates of DEHP metabolites. In contrast, each of the other three species excreted glucuronide conjugates. No conjugates other than glucuronides were detected in any of the species tested.

Parmar et al.(1985) studied the effects on rat pups from dams (strain not stated) given DEHP through the lactation period. Pups from 10 litters were pooled and seven pups were randomly assigned to each mother. Five mothers were given 2 000 mg/kg b. w. of DEHP (vehicle not stated) daily by oral gavage from day 1 of birth up to day 21, and five mothers served as control group and was given saline. DEHP was detected in the livers of pups from treated mothers indicating that DEHP can be transferred through the milk.

The metabolism of 2-ethylhexanol (2-EH), a metabolite of DEHP, was studied in two adult male rats administered (1-¹⁴C) ethylhexanol (purity not stated) in cottonseed oil by gavage (Albro, 1975). Carbon dioxide (from expired air), urine, and faeces were collected at hourly intervals for 28 hours after administration. Metabolites in the urine were identified by GC and MS. 2-EH was efficiently absorbed and radioactivity from 2-EH was rapidly excreted in respiratory carbondioxide (6-7%), faeces (8-9%), and urine (80-82%), with essentially complete elimination by 28 hours after administration. Other metabolites identified were 2-ethyl-5-hydroxyhexanoic acid, 2-ethyl-5-ketohexanoic acid and 2-ethyl-1,6-hexandioic acid. Only about 3% of 2-EH was excreted unchanged. Thus, these data indicate that the carbon chain of 2-EH is ultimately metabolized through oxidation pathways (w- and (w-1) -oxidation with subsequent ß-oxidation) to acetate and carbon dioxide.

Conclusion on oral absorption in rats

The two recent and highly reliable toxicokinetic studies of Laignelet and Lhuguenot (2000) with pregnant and non-pregnant Wistar rats are the most reliable data available to estimate the exposure in experimental rats after long-term exposure. It becomes clear from these data that absorption is higher after repeated exposure compared to single application. Metabolites found in urine alone summed up to 73.5% (lower amounts found in the high dose group of pregnant rats were probably due to a low recovery rate), without accounting for residues in the carcasses and amounts excreted via the bile. A nearly complete oral absorption can be assumed in rats after repeated dosing. Based on these data the oral absorption in rats with repeated dosing regimens is estimated in a conservative way at 75%.

Mice

The absorption, blood concentration and excretion of DEHP were determined in pregnant and non-pregnant CD1 female mice following a single and a repeated oral administration at the dose levels of 200 mg/kg and 1000 mg/kg (Laignelet, 2000). Blood samples were taken at defined time intervals after administration for quantification of total radioactivity. Urine and faeces were collected daily and DEHP and its metabolites were extracted and then identified by GC-MS and quantified by GC.In non-pregnant mice, [¹⁴C]-DEHP was rapidly and extensively absorbed following a single or repeated oral administration as mirrored by the blood concentration curve profile and a rapid excretion in urine and faeces. The absorption was not dose-related. It was significantly in excess of the 5-fold difference in dose levels after a single administration and significantly below after a repeated administration. At low dose, a 5-day pre-treatment increased slightly the absorption rate, but at high dose, it induced a decrease of the absorption. After a single or a repeated administration, [¹⁴C]-DEHP was excreted very quickly in urine and faeces and the total recovery reached 60 or 69% whatever the dose level, respectively. [¹⁴C]-DEHP was excreted in majority as MEHP-derived metabolite essentially in urine, as MEHP equally in urine and faeces after a single administration and mainly in urine after a repeated administration and as DEHP almost totally in faeces. Omega-1 oxidation was the main metabolic pathway of the production of MEHP-derived metabolite. However, repeated administration of DEHP induced a metabolic activation and a displacement of the oxidation pathway in favour of thew-oxidation. A large part of MEHP-derived metabolites were excreted as glucuro-conjugates in urine but this proportion decreased after a repeated administration of high dose. A low proportion of MEHP was excreted as glucuro-conjugate.In pregnant mice, [¹⁴C]-DEHP was rapidly, extensively and dose-related absorbed following a single or repeated oral administration as mirrored by the blood concentration curve profile and a rapid excretion in urine and faeces. A pre-treatment with a low dose did not have any effect on the total absorption rate but increased the MEHP concentrations in blood to the detriment of DEHP. However, a pre-treatment with a high dose induced an increase of the Cmax and paradoxically a decrease of the AUC. This effect could be related to an extensive initial absorption of non-metabolised DEHP and an increased of the excretion rate due to metabolic activation and/or entero-hepatic excretion. Only DEHP and MEHP were found in blood, no MEHP-derived metabolites were detected. MEHP was the main circulating compound. The distribution of [¹⁴C]-DEHP in whole foetus was also rapid and extensive and followed by a rapid clearance parallel to the blood concentration curve in dams. The radioactivity contents in whole foetus were lower than the corresponding radioactivity concentrations in the blood of dams but did not reflect the actual concentrations in the blood of foetus. After a single or a repeated administration, [¹⁴C]-DEHP was excreted very quickly in excreta and the recovery reached or exceeded 60% of the administrated dose. [¹⁴C]-DEHP was excreted in majority as MEHP-derived metabolite essentially in urine, as MEHP mainly in faeces and DEHP almost totally in faeces. Omega-1 oxidation was the main metabolic pathway of the production of MEHP-derived metabolite. However, repeated administration of DEHP induced a metabolic activation and a displacement of the oxidation pathway in favour of thew-oxidation. A large part of MEHP and MEHP-metabolites were excreted as glucuro-conjugates in urine but this proportion seemed to decrease after a repeated administration.

Male C57BR mice (10-12 g, three groups of one control animal and 8 exposed) were given a single oral dose of 6.72 mg (carbonyl-¹⁴C) DEHP (radiochemical purity >98%) by gavage The animals were killed after 1, 2, 4, 8, 24 hr and 3, 5, 7 days, respectively, and were examined by whole-body autoradiography (Gaunt and Butterworth, 1982). Following absorption, the radioactivity was widely distributed in organs and tissues without evidence of tissue storage. The contents of stomach and small intestine showed marked evidence of radioactivity in all mice during the first 24 h, but only a slight reaction of one animal was recorded on day 3. Radioactivity was present in the caecal contents at 1 hr, increased to a maximum at 2 hr and persisted for 1 day, but was found only in one animal on day 3. No radioactivity was detected in the colon contents or faeces after 1 hr the activity reach a maximum at 2 and 4 hrs in colon contents and faeces, respectively. The decline in radioactivity was similar to that in other parts of the gastro-intestinal tract. In the bladder there was a high level of activity between 1-24 hr and some activity in 2 of 3 mice on day 3. In the kidney activity in the parenchyma was similar to that in many tissues of the same animal, but it was more concentrated in the renal pelvis and papillae. Radioactivity in the testis was obvious only in one animal (killed after 4 hr) and was similar to the general tissue levels. In other tissues the level of radioactivity varied considerably between animals even at the same examination interval.

The distribution and tissue retention of DEHP following intravenous and oral administration was studied in mice with whole body autoradiography (Lindgren et al., 1982). In one experiment two male C57BL mice received 10 µCi of (2-ethylhexyl-1-¹⁴C) DEHP (chemically and radiochemical purity >99%) intravenously, corresponding to 9.6 mg/kg b. w. of DEHP. Another 2 male mice were given 10 µCi (carbonyl-¹⁴C) DEHP intravenously, corresponding to 3.6 mg/kg b. w of DEHP. The distribution was similar following administration of either substance. Four hours after intravenous injection, a very high activity was observed in the gall bladder, intestinal contents and urinary bladder. A high uptake was also seen in the liver, kidney, and brown fat. Some activity was observed in the white fat, myocardium, and muscles. The level in blood, bone, cartilage, testes, and nervous system was very low. Twenty-four hours after administration the activity in the gall bladder, intestinal contents and urinary bladder was still very high. The concentration in brown fat was high, but the activity in the liver and kidney was lower than after 4 hours.

In a second experiment of the same study (Lindgren et al., 1982), the effects of pre-treatment on the distribution of (¹⁴C) DEHP were studied in male mice by whole body autoradiography. Four mice were given DEHP (10 mg/kg) by oral intubation once daily for 5 consecutive days, 2 mice were given daily intraperitoneal injections of phenobarbital sodium (75 mg/kg/d) in physiological saline for 3 consecutive days, and another 2 mice were intraperitoneally treated with 3-methylcholantrene (30 mg/kg/d) for 4 consecutive days. Twenty-four hours after the last administration the animals received 10mCi DEHP in 20ml ethanol (corresponding to 9.6 mg DEHP/kg for 2-ethylhexyl-1-¹⁴C and 3.6 mg DEHP/kg forcarbonyl-¹⁴C). The mice were killed 24 hours after injection with (¹⁴C) DEHP. Following pretreatment of male mice with either DEHP, phenobarbital sodium or 3-methylcholantrene, the distribution 24 hours after the injection of either (carbonyl-¹⁴C) DEHP or (2-ethylhexyl-1-¹⁴C) DEHP was similar to that in the non-pre-treated animals, except that the concentration in the brown fat was higher in all the pre-treated animals as compared to non-pre-treated animals.

In a third experiment of the same study, Lindgren et al. (1982), six pregnant mice were each given 10mCi (¹⁴C) DEHP in soy bean oil by oral intubation. The mice were killed after 4 and 24 hours. Mice at gestation day 8 were given 7.7 mg DEHP/kg for 2-ethylhexyl-1-¹⁴C and 2.9 mg DEHP/kg forcarbonyl-¹⁴C, and mice at day 16 of gestation received 4.8 mg DEHP/kg for2-ethylhexyl-1-¹⁴C and 1.8 mg DEHP/kg for carbonyl-¹⁴C. Whole body autoradiography was performed as described previously. After the pregnant mice had been killed the uteris were removed with their embryos, except for a few fetuses from mice in the late stage of pregnancy which were removed surgically from the uterus. As for the uteris, the maternal livers and kidneys were also removed for autoradiography. At early gestation marked uptake was seen in the yolk sac. There was a high concentration in the gut of the embryo 4 hours after administration of (carbonyl-¹⁴C) DEHP on gestation day 8. On gestation day 9, 24 hours after administration of (2-ethylhexyl-1-¹⁴C) DEHP pronounced activity was observed in the neuroepithelium of the embryos. A high concentration was also seen in the uterine fluid. Except for the gut and the neuroepithelium, uptake in the embryo was low. In the mice at late gestation, a very high accumulation was seen in the yolk sac after oral administration of either (2-ethylhexyl-1-¹⁴C) DEHP or (carbonyl-¹⁴C) DEHP. The distribution in the fetuses was very similar after administration of the two¹⁴C-labelled DEHP compounds. Four hours after administration on gestation day 16 there was high activity in the renal pelvis, urinary bladder and intestinal contents. Some activity was seen in the skeleton and liver. On gestation day 17 there was little activity left in the fetuses, although a rather high activity was observed in the renal pelvis, urinary bladder and intestinal contents.

The distribution and retention of DEHP was studied in the NMRI mouse brain and liver (Eriksson and Darnerud, 1985). (7-¹⁴C) DEHP (0.7 mg/kg b. w.; purity not stated) was administered by gavage to young mice (3-20 days old). One and 7 days after treatment the amount of radioactivity in the liver and brain was measured. The amount of radioactivity in the brain was low, especially in 10- and 20 days-old mice, and retention of radioactivity in the brain was minimal. The amount of radioactivity in the liver was about 10 times that in the brain. After 24 hours the amount of radioactivity found in the livers ranged from about 27 to 2% in the order 3-, 10- and 20-day-old mice, showing significant decreases in all ages after 7 days.

Male CD mice, guinea pigs, rats, and hamsters were given two doses of (carbonyl-¹⁴C) DEHP (360 mg/kg b. w. for mice) by stomach tube at 24 hours intervals to compare the toxicokinetic behaviour of DEHP between the species (Albro et al., 1982). The study is presented in the section on oral administration to Fischer 344 rats. Rats predominately excreted metabolites having carboxyl groups on the side chain (metabolites I-V), these diacids require from three to six oxidative steps for their formation. No components were detected in urine from hamsters or guinea pigs that were not also present in urine from rats and mice. In the hamsters the main metabolites were: di-methyl phthalate (DMP), metabolite I, V, VI and IX. The main metabolites in the mouse were: DMP, MEHP, metabolites I, VI, IX, while in the guinea pigs MEHP was the dominating metabolite.MEHP accounted for 5% of the metabolites in hamsters, and 19 and 70%, respectively, in mice and guinea pigs. Rats did not excrete conjugates of DEHP metabolites. In contrast, each of the other three species excreted glucuronide conjugates. No conjugates other than glucuronides were detected in any of the species tested. Table 4.26gives the forms of excretion products in urine.

Studies to compare the toxicokinetic behaviour of DEHP between male B6C3F1 mice, rats and Cynomolgous monkeys has been performed (Short et al., 1987; Astill et al., 1986) and is presented in the section on oral administration to non-human primates.All three species excreted 30-40% of the dose in the urine (rats 32.9%, mice 37.3%, monkeys 28.2%), primarily during the first 12 hours for rats and mice and during the first 24 hours for monkeys. All three species excreted around 50% of the dose in the faeces (rats 51.4%, mice 52.0%, monkeys 49.0%), primarily during the first 24 hours for rats and mice and during the first 48 hours for monkeys. The rates and extent of urinary and faecal excretion varied widely among monkeys. DEHP was detectable in some tissues in all three species. The mean concentrations detected, with the exception of monkey liver and rat intestinal contents, were less than 1 µg/g. The highest concentrations were detected in liver, intestinal contents, and fat for monkeys, rats, and mice, respectively. Total recoveries of the radioactivity administered were 79 (68-91%), 87 (82-92%) and 90% (63-102%) for monkeys, rats and mice, respectively. Radioactivity in 0-24 hour urine samples were resolved into 13, 15, and 14 components in rats, mice, and monkeys, respectively. The components in urine were identified as MEHP (not detected in rat), phthalic acid, metabolites I, II (not detected in monkey), III (not detected in rat), IV, V, VI, VII, IX, X, XII, XIII, XIV, and unidentified fractions. Major urinary components in rats were metabolites I, V, VI, and IX. Major urinary components in mice were MEHP, phthalic acid, metabolites I, VI, IX, and XIII, and in monkeys: MEHP, and metabolites V, IX, and X. In monkeys 15-26% of the radioactivity excreted may represent glucuronic acid conjugates whereas in rat glucuronides are either absent or present in negligible quantities. Radioactivity in 0-48 hour monkey faecal extracts and in 0-24 hour rat and mouse faecal extracts were resolved into 11, 10 and 10 components in rats, mice and monkeys, respectively. The faecal components were identified as DEHP, MEHP, phthalic acid, metabolites I-IV, VI, VII, IX, X, XII, XIII (not detected in monkey), and XIV (not detected in mouse). DEHP was a major faecal component in all three species and MEHP a major faecal component in rats and mice.

In mice (strain and number not stated) given a single oral dose of 400 mg/kg b. w. of (¹⁴C) MEHP (radiochemically pure, position of label not stated) in corn oil, the major metabolites (identified by GC/MS) in urine were recovered in the form of glucuronides (Egestad and Sjöberg, 1992). Three new metabolites were isolated and characterised as conjugates of ß-glucose. Thus glucosidation has been shown to be an alternative conjugation pathway, although less important.

A single oral dose of 400 mg/kg b. w. of (carbonyl-¹⁴C) MEHP in corn oil was given to 11 male mice (strain not stated) and male guinea pigs (Dunkin Hartley, number of animals not stated) (Egestad et al., 1996). The radiolabelled DEHP was diluted with unlabelled DEHP to give a specific activity of 2.7 and 6.1mCi/mmol for the guinea pigs and mice, respectively. Urine was collected over 48 hours. Following extraction, individual metabolites were purified and separated using a combination of ion-exchange chromatography and reversed-phase HPLC. Analysis of intact conjugates, as well as nonconjugated metabolites, was performed by GC/MS. Enzymatic methods were used for further characterisation. The study confirmed glucuronidation as the major conjugation pathway for MEHP in the investigated species. The recovery of¹⁴C was 83-103% and 74-78% in guinea pigs and mice, respectively. In guinea pigs MEHP glucuronide were the dominating metabolite whereas in the mice it was an even distribution of the glucuronides of MEHP and its metabolites. In mice approximately 3% of the administered dose was found in the urine as ß-glucose conjugates. The ß-glucose conjugates were not observed in the guinea pigs.

Guinea pig

Male Hartely guinea pigs, rats, mice, and hamsters were given two doses of (carbonyl-¹⁴C) DEHP by stomach tube at 24 hours intervals to compare the toxicokinetic behaviour of DEHP between the species (Albro et al., 1982). The study is presented in the section on oral administration to Fischer 344 rats.Rats excreted predominately metabolites having carboxyl groups on the side chain (metabolites I-V), these diacids require from three to six oxidative steps for their formation. No components were detected in urine from hamsters or guinea pigs that were not also present in urine from rats and mice. In the hamsters the main metabolites were: dimethyl phthalate (DMP), metabolite I, V, VI and IX. The main metabolites in the mouse were: DMP, MEHP, metabolites I, VI, IX, while in the guinea pigs MEHP was the dominating metabolite. MEHP accounted for 5% of the metabolites in hamsters, and 19 and 70%, respectively, in mice and guinea pigs. Rats did not excrete conjugates of DEHP metabolites. In contrast, each of the other three species excreted glucuronide conjugates. No conjugates other than glucuronides were detected in any of the species tested. Table 4.26gives the forms of excretion products in urine.

A single oral dose of (carbonyl-¹⁴C) MEHP was given to male guinea pigs (Dunkin Hartely: number of animals not stated) and mice to compare the toxicokinetic behaviour of MEHP (Egestad et al., 1996). The study is presented in the section on oral administration to mice. The study confirmed glucuronidation as the major conjugation pathway for MEHP in the investigated species. The recovery of¹⁴C was 83-103% and 74-78% in guinea pigs and mice, respectively. In guinea pigs MEHP glucuronide were the dominating metabolite whereas in the mice it was an even distribution of the glucuronides of MEHP and its metabolites. In mice approximately 3% of the administered dose was found in the urine as ß-glucose conjugates. The ß-glucose conjugates were not observed in the guinea pigs.

Hamster

The elimination of DEHP was studied in rats and hamsters (Lake et al., 1984). A single dose of (carbonyl-¹⁴C) DEHP (>99% pure) was administered to 5-week-old male Sprague-Dawley rats and male DSN strain Syrian hamsters at dose levels of 100 (5 rats, 3 hamsters) or 1000 mg/kg b. w. (5 rats, 5 hamsters) in corn oil by gastric intubation. The study is presented in the section on oral administration to Sprague-Dawley rats. Urine and faeces were collected over a period of 96 hours and then the animals were sacrificed. Radioactivity was measured in urine, faeces, and total gut contents by liquid scintillation spectrometry. Faecal metabolites were extracted and chromatographed on thin-layer plates. In both species the bulk of the radioactivity was excreted within 24 hours. At the lower dose level, both species excreted more radioactivity in the urine (rat: 51%, hamster: 53%) than in the faeces (rat: 43%, hamster: 31%), whereas at the higher dose level, the major route of excretion was via the faeces (rat: 53%, hamster: 48%). In both species and at both dose levels, only negligible amounts of radioactivity were present at termination in either the liver, kidney, or total gut contents. Faecal radioactivity profiles were determined in 0-24 hour faeces samples. About 50% of the faecal radioactivity of rats at the higher dose level appeared to be the parent compound, the remainder comprised metabolites possibly including MEHP. In contrast, more than 95% of the faecal radioactivity of hamsters appeared to be the parent compound. Similar results were obtained with faecal extracts from rats and hamsters at the lower dose level.

Male guinea pigs, rats, mice, and Syrian golden hamsters were given two doses of (carbonyl-¹⁴C) DEHP by stomach tube at 24 hours intervals to compare the toxicokinetic behaviour of DEHP between the species (Albro et al., 1982). The study is presented in the section on oral administration to Fischer 344 rats. Rats excreted predominately metabolites having carboxyl groups on the side chain (metabolites I-V), these diacids require from three to six oxidative steps for their formation. No components were detected in urine from hamsters or guinea pigs that were not also present in urine from rats and mice. In the hamsters the main metabolites were: di methyl phthalate (DMP), metabolite I, V, VI and IX. The main metabolites in the mouse were: DMP, MEHP, metabolites I, VI, IX, while in the guinea pigs MEHP was the dominating nmetabolite. MEHP accounted for 5% of the metabolites in hamsters, and 19 and 70%, respectively, in mice and guinea pigs. Rats did not excrete conjugates of DEHP metabolites. In contrast, each of the other three species excreted glucuronide conjugates. No conjugates other than glucuronides were detected in any of the species tested. Table 4.26gives the forms of excretion products in urine.

Dog

A study to compare the toxicokinetic behaviour of DEHP between dog, rats and miniature pigs has been performed by Ikeda et al., 1980. The study is presented in the section on oral administration to Sprague-Dawley rats.Excretion of radioactivity in urine and faeces during the first 24 hours was 27 and 57% (rats), 12 and 56% (dogs), and 37 and 0.1% (pigs), respectively; and after 4 days 37 and 53% (rats), 21 and 75% (dogs), and 79 and 26% (pigs), respectively. Elimination of radioactivity was rapid in rats, slightly prolonged in dogs and least rapid in pigs; excretion in all three species was virtually complete in 4 days. TLC revealed four radioactive metabolites in rat urine, three in dog urine and five in pig urine. Only a trace of unmetabolized DEHP was found in the urine of rats, dogs, or pigs. A substantial amount of radioactivity was present in the gastro-intestinal tract at day 1 in all species and a small amount remained after 4 days. Bile samples from dogs, and to a lesser extent from pigs, accounted for a significant amount of administered¹⁴C dose. Less than 1% of the administered¹⁴C dose was secreted in the bile from bile duct cannulated rats.

Pigs

The kinetics of DEHP and its metabolite mono(2-ethylhexyl) phthalate (MEHP) was studied in the young male pig, an omnivore model-species for research in reproductive toxicology (Ljungvall, 2004). Eight pigs were given 1000 mg DEHP/kg bodyweight by oral gavage. The concentrations of DEHP and MEHP were then measured in the plasma and tissues of the pigs at different time points after administration. There was no consistent rise above contamination levels of concentrations of DEHP in the plasma of the pigs. However, the metabolite MEHP reached the systemic blood circulation. The half-life of MEHP in the systemic blood circulation was calculated to be 6.3 h. Absorption from the intestine was biphasic in six of the eight pigs and the mono-exponential elimination-phase started 16 h after the after the administration of DEHP. To conclude, MEHP consistently reaches the systemic circulation in the pig when DEHP is administered orally. The kinetic pattern of the parent substance on the other hand is more difficult to characterise.

A study to compare the toxicokinetic behaviour of DEHP between miniature pigs, rat and dog has been performed by Ikeda et al., 1980. The study is presented in the section on oral administration to Sprague-Dawley rats. Excretion of radioactivity in urine and faeces during the first 24 hours was 27 and 57% (rats), 12 and 56% (dogs), and 37 and 0.1% (pigs), respectively; and after 4 days 37 and 53% (rats), 21 and 75% (dogs), and 79 and 26% (pigs), respectively. Elimination of radioactivity was rapid in rats, slightly prolonged in dogs and least rapid in pigs; excretion in all three species was virtually complete in 4 days. TLC revealed four radioactive metabolites in rat urine, three in dog urine and five in pig urine. Only a trace of unmetabolized DEHP was found in the urine of rats, dogs, or pigs. A substantial amount of radioactivity was present in the gastro-intestinal tract at day 1 in all species and a small amount remained after 4 days. Bile samples from dogs, and to a lesser extent from pigs, accounted for a significant amount of administered¹⁴C dose. Less than 1% of the administered¹⁴C dose was secreted in the bile from bile duct cannulated rats.

The distribution and retention of DEHP and DBP orally administered in feed to piglets has been studied (Jarosová et al., 1999). Six piglets (33-50 kg) of which 4 received DEHP (5 g per day and head (ca. 125 mg/kg b. w. /day) for 14 days and 2 served as controls were used. After 14 days, 2 treated and the 2 controls were sacrificed. The remaining two treated-piglets were then maintained on a DEHP-free diet, and one each sacrificed on day 14 and 28, respectively, post dosing. DEHP and MEHP were determined by HPLC analysis. Whole blood and urine samples were collected before sacrifice and on from treated animals on day 7 of treatment. The concentration of DEHP was measured in the whole (wet weight) and/or fat extracted tissues/organs of muscle, renal fat, subcutaneous fat, kidneys, lungs, brain, heart, and liver. MEHP was only determined in the whole liver, whole blood and urine samples.Body and tissue/organ weights were not affected by administration of DEHP.The highest levels of DEHP were in the subcutaneous (treated cf. control: ca 19 cf.0.42 mg/kg fat) and renal fat (ca. 25 cf. 0.37 mg/kg fat), muscle (ca 25 cf. 2.4 mg/kg fat), heart (ca 12 cf. <0.2 mg/kg fat) and lungs (ca 13 cf. 0.25 mg/kg fat). The amount of DEHP in kidney (ca 2 cf. <0.2 mg/kg fat) was low. MEHP, but not DEHP, level was increased in the liver, whole blood and urine: individuals values greatly varied but an increase up to, for example, of 20-1000-fold in blood occurred. DEHP was not increased in the brain. In the 14 day post dosing (recovery) animal, the level of DEHP was decreased by around 50% in subcutaneous and renal fat, muscle, heart and lungs. MEHP returned to control levels in liver, whole blood and urine. By 28 days, DEHP was reduced to control levels in all tissues/organs except in renal fat and the lungs. Although only one animal per recovery time interval was used, these data indicate that in piglets that DEHP is retained for a considerable time post dosing.The authors further investigated the reason for the presence of DEHP in the organs (not blood) of the control animals. Analysis of the piglet feed showed that around 0.4 mg DEHP and 0.5 DBP per Kg commercial feed was present. Based on a 40 kg pig eating 2 kg of feed a day, the daily intake of DEHP is around 0.02 mg/kg b. w. Comparing the daily intake (0.8 mg DEHP) with the residue of DEHP in whole muscle of control pigs (0.10 mg/kg), a biotransfer factor (BTF: concn. in meat (mg/kg) /daily intake of DEHP (mg/d)) of 0.125 d/kg is derived.

Broiler hens

The distribution and retention of DEHP and DBP orally administered in feed to broiler hens has been studied (Jarosová et al., 1999). Eighteen broiler hens (750 g) per treated and control group were used. DEHP (100 mg per day and head (ca. 135 mg/kg b. w. /day) was administered for 14 days. Six hens each per treated and control group were sacrificed at 14 days, and on post treatment days 14 and 28. DEHP and MEHP were determined by HPLC analysis. Liver and blood samples were obtained by heart puncture to determine DEHP and MEHP. Muscle (pooled samples of breast and thigh samples), skin (thoracic area), and mesenterial fat were analysed for DEHP. Post DEHP dosing samples of blood, muscle and skin were pooled from 6 individuals.The highest levels of DEHP were in the mesenterial fat (treated cf. control: ca 31cf.0.33 mg/kg fat), skin (ca 26cf.3.8 mg/kg fat) and muscle (ca 26cf.2.5 mg/kg fat). DEHP (ca 6.3cf.0.47 mg/kg fat) and MEHP (ca 0.15cf. <0.01 mg/kg whole tissue) were detected in the liver. In whole blood, levels of MEHP varied but indicated an increase of more than 7-fold. In the 14 day post dosing (recovery) animal, the level of DEHP decreased by more than 50% in muscle, skin, adipose tissue and liver. However, by comparison with the all the control groups and the treated group, DEHP is apparently retained in the muscle, skin and adipose tissue around 30%). MEHP levels in the liver and blood were reduced to control levels by post recovery day 14.The authors further investigated the reason for the presence of DEHP in the organs (not blood) of the control animals. Analysis of the hens feed showed that around 1.0 mg DEHP and 2.0 DBP per Kg commercial feed was present.

5.1.1.2. Inhalation

There are few studies available concerning the inhalation route of exposure. Studies available for humans are not strictly toxicokinetic studies but case studies of patients and in the working environment, which are reported therafter. There is only one toxicokinetic study with rats exposed to a DEHP aerosol.

Rats

In a study performed by General Motors (1982), adult male Sprague-Dawley rats (200 g) were exposed, either once or repeatedly, by inhalation to a DEHP aerosol (98% pure).In the single exposure study, six rats were exposed to 129±26 mg/m³of (carboxyl-¹⁴C) DEHP for 6 hours (head-only chamber; radiochemical purity of the labelled compound 95%). Accurate particle size determination was not technically possibly to conduct; however, the particle size was estimated to be 0.4 - 0.5 µm. Three animals were sacrificed immediately after exposure and the remaining three after the follow-up time. The follow-up time for collection of urine and faeces samples was 72 hours before the animals were sacrificed. Samples were collected at 12- and 24-hour intervals for urine and faeces, respectively, and assayed for radioactivity. Blood samples (0.1 ml) were taken from the jugular vein 1, 3, 6, hr during exposure and 1, 3, 6, 12, 18, 24 and 36 hours post-exposure. Tissues (lung, liver, kidney, fat, adrenal, heart, spleen, thymus, testes, and brain) were removed post mortem and assayed for radioactivity. Radioactivity was determined by liquid scintillation spectrometry. Metabolites were determined by HPLC in urine samples collected between 0-12, 12-24, 24-36 hours. Data were calculated as µmole equivalents DEHP.During single dose inhalation exposure (¹⁴C) DEHP was absorbed rapidly as indicated by the amount of radioactivity in the blood. Following exposure, the decrease in radioactivity in blood was log non-linear. Immediately after exposure, approximately 1 mg (based µmole equivalents DEHP) or 75% of the body burden was recovered in the carcass and skin. 10% was recovered in the lung and ca. 2% in all the other tissues excluding the brian where no radioactivity was detected. Radioactivity associated with the lung probably represents that fraction of aerosol particles deposited in the larger airways. This fraction could be absorbed through the gastrointestinial tract following clearance from the pulmonary spaces by mucociliary ladder mechanism. It is probable that a large portion of DEHP is ingested. After 72-hours, approximately 1.5 mg (3.94 µmole equivalents DEHP) was recovered mainly in the urine and faeces. Combined, urine (52%) and faeces (40%) accounted for greater then 90% of total recovered radioactivity. Around 6% of the original body burden radioactivity was determined in tissues (low amount of radioactivity in the lung and liver, and trace amount in kidney), carcass, and skin. Baesd on this information the retention of DEHP can be derived by comparison with the calculated inhaled dose. Assuming a minute volume of 0.2 l/min and 100% inhaltion of the aerosol the inhaled dose is around 36 mg/kg b. w. /day or 7.2 mg/rat/day for a 6 hour per day inhalation exposure. Retention is thus around 21% (1.5/7.2) for DEHP with an estimated particle size of 0.4 - 0.5 µm.The rate of excretion of radioactivity in the faeces was approximately first order kinetics during 72-hour. The elimination half-life was ca. 22 hours and the elimination rate constant (Ke) 0.032 hr-1. In urine, excretion was biphasic. The initial rapid phase was ca. 10 hours (Ke = 0.069 hr-1). And was sustianed for 30 hours. The slower phase half-life was 22 hours.At least three peaks were identified by HPLC in urine. Phthalic acid (3-5% total radioactivity) was identified. The reamining radioactivity was confined to two other peaks, however, the metabolites were not identified: DEHP was not detected in the urine.Under the experimental conditions used in this study, around 1.5% of a nominal aerosol concentration of 100 mg/m³was absorbed by rat following a 6 hour exposure period. Both pulmonary and gastrointestinial tract absorption are expected to contribute to the total body burden level of detected¹⁴C-DEHP derived radioactivity.

For comparison, General Motors (1982) also studied the disposition in male Sprague-Dawley rats (200 g) (number not stated) after a single peroral (gavage) dose of (carboxyl-¹⁴C) DEHP (25 mmoles/kg bw (10 mg/kg bw): 25 mmoles/ml, 9.7mCi/ml) administered in corn oil with the results of the single exposure inhalation study (see above). The dose was selected to approximate DEHP absorbed during inhalation (2 mg/animal). Faeces were collected over 24 hours interval and urine over 12 hours interval. The animals were killed after 72 hours Tissues (lung, liver, kidney, fat, adrenal, heart, spleen, thymus, testes, and brain) were removed post mortem and assayed for radioactivity. Radioactivity was determined by liquid scintillation spectrometry. A more rapid excretion of¹⁴C- in the urine and increased body burden clearance (half-life»12 hours) was observed following oral administration compared with inhalation exposure. The percentage cumulative recovery of radioactivity in urine (54%) and faeces (43%) after 72 hours was not significantly different as compared with inhalation exposure (52 and 40%, respectively, in urine and faeces). Within 72 hours the recovery was 3.94 and 3.82mmole equivalents DEHP (ca 1.5 mg) following single oral and inhalation administration, respectively. To assess the relevance of this comparison route-specfic metabolism, biliary exretion and reabsorption, and the contribution of GI-tract absorption during and following inhalation exposure should be considered compared with unabsorbed orally administered DEHP. Urinary excretion following single oral exposure was also biphasic with half-lifes of about 10 and 22 hours, respectively. The excretion in urine and decline in body burden were more rapid following oral administration, especially initially.In the repeated exposure study General Motors (1982), 16 rats were pretreated with 100 mg/m3unlabelled DEHP for 2 weeks (6 hours per day, 5 days per week in an exposure chamber, mass median aerodynamic diameter of aerosol particles: 0.6 µm) except that the last exposure was to (carboxyl-¹⁴C) DEHP (head-only chamber).Following repeated inhalation exposure, around 90% of the radioactivity was excreted in the urine (50%) and faeces (40%), and 8% recovered in the carcass and skin. Excretion of radioactivity in urine was apparently first order with a half-life of about 25 hours. Urinary excretion seems to be initially slower compared with single inhalation exposure (see above) but was parallel to the curve from single exposure after less than 24 hours.The study indicates that following repeated inhalation exposure long-term retention does not occur and that the excretion profile is not modified by prolonged inhalation exposure (2-weeks) compared with single exposure. Hence, disposition characteristics following repeated exposure were similar to single dose exposure.

5.1.1.3. Dermal

Data are reported in section “Dermal absorption”.

5.1.1.4. Other routes

Exposure of humans to DEHP through medical treatment practices such as dialysis, respiration therapy, blood transfusions, or parenteral nutrition where the source of DEHP is the plastic materials used in the medical treatment devices or storage bags may also occur. The data are summarized in sections 7.10.5 of the IUCLID dossier and 5.10.2 of the CSR.

Non-human primates

The disposition of DEHP was studied in marmosets (Rhodes et al., 1983). Groups of three male marmosets received a single dose of (¹⁴C-ring labelled) DEHP (radiochemical purity 97.5%) by the intravenous (100 mg/kg b. w.), intraperitoneal (1 000 mg/kg b. w.) and oral (100 and 2 000 mg/kg b. w.) routes. Urine and faeces were collected for seven days and the radioactive content determined. Following intravenous administration approximately 40% of the dose was excreted in urine and approximately 20% in the faeces (cumulative excretion for 7 days) indicating a 2 to 1 ratio between the urinary and biliary (faecal) routes of excretion in the marmoset. Around 28% of the dose remained in the lungs 7 days after administration of 14C-DEHP, with minimal levels in other tissues. A much smaller proportion of the dose was excreted following intraperitoneal administration (10% in the urine and 4% in the faeces) in a similar 2 to1 ratio. Around 85% of the dose remained as unabsorbed DEHP in the peritoneal cavity with minimal amounts in the tissues (0.6%). The results for oral administration are presented above.

The Urinary excretion and metabolites of DEHP were studied in two African Green monkeys following a bolus infusion with ¹⁴C-DEHP (carbonyl-¹⁴C-DEHP-enriched plasma containing 96% DEHP and 4% MEHP) (Albro et al., 1981; Peck and Albro, 1982). To closely simulate the manner in which man is exposed to DEHP when receiving blood products, a¹⁴C-DEHP impregnated PVC plastic strip was immersed in 20-ml aliquot of plasma and stored for up to 5 months at 4°C. Serial plasma, urine and stool samples were obtained and measured. Urine metabolites were isolated and identified by GC/MS. Plasma¹⁴C concentration rapidly decreased: less than 5% by 30 min post infusion. C14-MEHP increased rapidly followed by¹⁴C-MEHP oxidation products. The levels of MEHP was greater than the oxidation products. MEHP is apparently quite stable since¹⁴C-MEHP in plasma increased to a plateau within7 minutes and then remained at a constant level for more than 30 minutes even though the total¹⁴C-plasma levels decreased in the plasma. The cumulative¹⁴C excretion in urine of three monkeys was 50% and greater than 70% by 4 and 24 hours, respectively.Urine samples from two monkeys were collected for a 5-hour post-infusion period. Approximately 80% of the urinary metabolites were excreted in the form of glucuronide conjugates (Table 4.1.2.1.8). According to the authors the glucuronide frequency is analogous to what has been reported for human leukemia patients but in clear contrast to rats where rat urinary metabolites are excreted unconjugated. Seven metabolites (MEHP, V, VI, VII, VIII, IX, X, XI) were identified (GC/MS) with MEHP (29%) and metabolite IX (38%) being the major metabolites. The remaining metabolites amounted to 7% or less.

The effects of DEHP on hepatic function and histology were evaluated in the rhesus monkey undergoing chronic transfusion (Jacobson et al., 1977). These studies demonstrates that intravenous administration of solubilized DEHP results in detectable concentrations of DEHP in biopsy material for up to 14 months following transfusion. The initial liver biopsies (100-400 mg) of all the exposed animals contained significant amount of DEHP. Plasma or PRP (platelet-rich-plasma) stored at 22°C yielded higher levels of DEHP in the livers of the transfused animals than plasma or PRP stored at 4°C. The 5 month follow up samples contained concentrations similar to that initially measured. Since the post-transfusion samples were in the lower range of sensitivity one of the animals were killed 14 month post-transfusion and the level of DEHP in different organs was analysed. Significant levels were found in the liver (0.7%), testis (0.4%), heart (0.8%) and fat (2.0%). The residual organ level excluding fat was less than 1% of the dose administered. One animal had tuberculosis and was killed 5 month post-transfusion. In this animal significant levels were found in the spleen (10%), lung (15%), fat (20%) and liver (1.7%). Based on organ weight (excluding fat) and DEHP content, the residual DEHP was 5 percent of the dose administered, according to the authors.

Rats

The disposition kinetics of DEHP was studied in male Sprague-Dawley rats following single or multiple administration of DEHP by various routes (intra-arterial: 100 mg/kg b. w.; intraperitoneal: 4 000 mg/kg b. w.; peroral: 2 000 mg/kg b. w.) (Pollack et al., 1985, the study is described in section oral; rats).

The disposition of DEHP and four of its metabolites was studied in Sprague-Dawley rats (40-days-old, 8 in each group) given single infusions of a DEHP emulsion in doses of 5, 50 or 500 mg DEHP/kg bw (Sjöberg et al., 1985). Plasma concentrations of DEHP, MEHP and metabolites V, VI and IX were followed for 24-h after the start of infusion. The kinetics of the primary metabolite MEHP was studied separately. The concentrations of DEHP in plasma were at all times higher than those of MEHP which were much higher than the concentrations of the other metabolites investigated. In animals given 500 mg/kg bw the area under the plasma concentration-time curves (AUCs) of the other investigated metabolites were at most 15% of that of MEHP. Parallell decreases in the plasma concentrations of DEHP, MEHP and metabolites V, VI and IX indicated that the elimination of DEHP was the rate limiting step in the disposition of the metabolites. This was partly supported by the observation that the clearance of MEHP was higher than that of DEHP. Nonlinear increases in the AUCs of DEHP and MEHP indicated saturation in the formation as well as the elimination of MEHP.

Transplacental transfer of DEHP has been observed following intraperitoneal administration of (carboxy-¹⁴C) DEHP on gestational day 5 or 10 in SD rats (Singh et al., 1975). The dams were killed at 24 hours interval starting on days 8 and 11 until day 20 of gestation. Radioactivity was detected in foetal tissues, amniotic fluid and placenta at all time points. The radioactivity peaked at 48 hours and declined rapidly thereafter. The concentration was less than that of maternal blood and less than 1% of the administered dose.

Mice

The distribution of (carbonyl-¹⁴C) DEHP was studied in male CD-1 mice following intravenous injection by tail vein of 1 ml DEHP-enriched plasma (2.293 µg/ml) (Waddell et al., 1977). After 1, 3, 9, 24, and 168 hours, the mice were killed and subjected to whole-body autoradiography. The radioactivity was rapidly accumulated in the kidney and liver, with with high concentrations in urine, bile, and intestinal contents. There was no evidence of retention in any tissues in the body. From the amount of radioactivity seen in the sequence of time intervals, it seems clear that the material is rapidly and completely eliminated by the kidney and the liver. The secretion by the liver, via the bile into the intestine appears to be the major route. No enterohepatic circulation of DEHP was indicated since there was a persistently high concentration in the intestinal lumen.

5.1.1.5. In vitro studies

The enzymatic hydrolysis of DEHP by different lipases was studied in tissues from the Sprague-Dawley rat (CD-strain), guinea pig, hamster and mice (Albro and Thomas, 1973). DEHP was hydrolysed to MEHP by lipases from a variety of rat tissues with pancreas, liver, and intestinal mucosa containing the bulk of the DEHP hydrolase activity. There was no difference in DEHP hydrolase activity in terms of units per mg of protein between intestinal homogenates from young adult (200g) and old (450g) rats, but there was higher activity from male than from female rats (1.7 and 0.45 units/g protein, respectively), and from fed than from fasted rats (1.7 and 0.74 units/g protein, respectively). Variation among species was not extreme, although different mouse strains showed considerable differences (e. g. 1.2 and 4.0 units/g protein for CD-1 and C57B1/6f, respectively). Out of 15 tissue preparations, only the liver alkaline lipase preparation was able to further hydrolyse MEHP to phthalic acid, at a rate of 2% at which it hydrolyzed DEHP.

The activities of liver, lung and kidney of rats of various age group and that of placenta in hydrolyzing di(2-ethylhexyl) phthalate to mono(2-ethylhexyl) phthalate have been measured (Gollamudi et al., 1985). Male and female Sprague-Dawley rats of 45 days of age, neonatal rats within 12 hours of parturition, and fetuses and placenta on day 19 of gestation were used. The liver was most active in all age groups; however, the lung and the kidney also had considarable activity. The tissues of the fetuses and the neonate had significant activity. The Km values of the enzyme were 4, 1.25 and 5.9 mM, respectively, in the neonatal, adult and old livers (Gollamudi et al., 1983).

The absorption of DEHP and MEHP was studied using an everted gut-sac preparation form the male Sprague-Dawley (300-400 g) rat small intestine (White et al., 1980). MEHP was significantly less lipophillic than DEHP, and was absorbed by the everted gut sac in a significantly greater quantity than DEHP. Esterases within the mucosal epithelium hydrolysed DEHP quantitatively to MEHP.

Lake et al.(1977) studied the hydrolysis of DEHP in hepatic and intestinal preparations from various species. Hepatic postmitochondrial supernatant preparations from Sprague-Dawley rat, olive baboon, and albino ferret were able to hydrolyse DEHP to MEHP as well as were intestinal mucosal cell preparations from rat, baboon, ferret, and man. According to the authors, these results show a species similarity in the metabolism of DEHP between man, a rodent, a nonrodent, and a nonhuman primate species. Furthermore, the results suggest that orally ingested DEHP would most probably be absorbed from the gut of the rat, baboon, ferret, and man primarily as the corresponding monoester.

Gray et al.(1982) studied the hydrolysis of DEHP in intestinal preparations from young Sprague-Dawley rats (28-42 days) and Dunkin-Hartely ham­sters. During a 16-h incubation, DEHP was hydrolysed to MEHP which was significanly different between rats (18.9±2.1%) and hamsters (4.1±1.0%).

The hydrolysis of DEHP was studied in hepatic post-mitochondrial supernatant fractions and in intestinal mucosal cell whole homogenates obtained from untreated Sprague-Dawley rats and Syrian hamsters (Lake et al., 1984). Rat hepatic DEHP hydrolase activity was more than twice as active as the enzyme present in hamster liver. Rat intestinal DEHP hydrolase activity was thrice as active as the hamster intestinal activity. According to the authors, these differences may explain the different hepatic response of rats and hamsters to DEHP.

Two groups of 6 Sprague-Dawley rats which were 25 days old and two groups of 6 Sprague-Dawley rats which were 60 days old were used to studyin vitrometabolism (Sjöberg et al., 1985). One group of each age were pre-treated with DEHP (gavage, 1000 mg/kg bw for 14 days) and the other group of each age were pre-treated with phenobarbital (intraperitoneal injection, 100 mg/kg bw for 3 days). After the animals were killed liver microsomes were prepared and concentrations of mono-(2-ethyl-5-hydroxyhexyl) phthalate was determined. The conversion of MEHP to mono-(2-ethyl-5-hydroxyhexyl) phthalate in liver microsomes from untreated 25- and 60-day-old rats were 0.37±0.07 and 0.39±0.08 nmol/mg protein/min, respectively. The rate of (w-1) hydroxylation in liver microsomes from rats pretreated with DEHP was 0.33±0.05 nmol/mg protein/min. Liver microsomes pretreated with phenobarbital showed a two fold increase in the conversion rate 0.73±0.15 nmol/mg protein/min.

In another experiment the protein binding of MEHP in plasma from 25, 40 and 60-day-old Sprague-Dawley rats at 25mg/ml was determined using the equilibrium dialysis techqnique (Sjöberg et al., 1985). The binding of MEHP to plasma proteins was 97.6±1.5, 98.0±0.2 and 97.5±0.4% in the 25, 40 and 60-day age group, respectively. The plasma protein binding was constant in the concentration interval 5-150mg/ml.

The hydrolysis rates of DEHP were measured in suspensions of contents of the Wistar rat stomach, small intestine or caecum (Rowland et al., 1977). After 16 hours, 1.0% of DEHP was hydrolysed by stomach, 22.1% by small intestine, and 6.9% by caecum contents. The hydrolysis product was identified as MEHP.

In studies reported byLhuguenot et al. (1985), the metabolism of MEHP was studied in isolated and cultured rat (Wistar derived) hepatocytes. At concentrations of 50 or 500 µM (¹⁴C) MEHP (position of label not stated, highest available purity), the substance was extensively metabolized. No water-soluble conjugates were detected. At the low concentration, recoveries of radioactivity of 71-79% were obtained. The amount of unchanged MEHP remaining in the medium increased slightly from day 1 to day 3. Metabolites I and V are final products of MEHP metabolism, no further metabolism was detected in this study. Metabolite X was transformed to metabolite V, which was further transformed to metabolite I, and metabolite IX was transformed to metabolite VI. A small increase in metabolite I and a decrease in metabolite VI were seen from day 1 to day 3. At the high concentration, recoveries of radioactivity ranged from 74 to 83%. The amount of unchanged MEHP decreased from 149 µM after 1 day to 93 µM after 3 days. A fivefold increase in metabolite I and a smaller increase in metabolite V along with time-dependent decreases in metabolites VI and IX were observed.

Ito et al.(2005) evaluated enzyme activities in tissues from rats, mice, and marmosets to assess possible specifies differences in the biotransformation of DEHP. CD-1 mice and Sprague-Dawley rats were 11 weeks old and Common marmosets were 18 months old when liver, kidney, lung, and small intestine were harvested. Tissues were stored at −85ºC until used. Tissue homogenates or microsomal fractions were assayed for lipase activity based on hydrolysis of DEHP to MEHP. UDP-glucuronyl transferase by measuring glucuronidation of MEHP, naphthol, and bisphenol A. Alcohol dehydrogenase was measured using 2-phenoxyethanol and 2-ethylhexanol as substrates, and aldehyde dehydrogenase was measured using 2- phenylpropionaldehyde and 2-ethylhexanal as substrates. Lipase activity was highest in liver, small intestine, and kidney in mice. The lowest lipase activity was found in marmosets. Marmoset hepatic lipase activity was 4–5% that of mouse activity, and small intestine lipase activity in marmosets was <1% of mouse small intestine activity. Rat lipase activities in these organs were intermediate between mouse and marmoset. Lipase activities were comparably low in rat and mouse lung and were undetectable in marmoset lung. UDP-glucuronyl transferase was detectable only in liver in the 3 species. Although activity was greater in mouse than marmoset, the difference between species was not as great as for lipase. Alcohol and aldehyde dehydrogenases were higher in marmoset than in rodents; however, the authors concluded that the possible increased ability of marmosets over rodents to convert MEHP to its ω-oxidation products was unlikely to be important given the small amount of MEHP that would be expected to be generated in marmosets from oral or intravenous exposures.

Discussion on absorption rate:

Humans

No reliable study is available.

Rats

Dermal absorption distribution, and excretion were studied in rats by Elsisi et al. (1989). Hair from a skin area (1.3 cm in diameter) on the backs of male F344 rats (number not stated) was clipped, [¹⁴C]-DEHP (> 96% radiochemically pure, uniformly labelled on the ring) was applied at a single dose of 30-40 mg/kg bw (5-8 mg/cm2) in ethanol and after evaporation the area of application was covered with a perforated cap (non-occluded). Rats were kept in metabolic cages and urine and faeces were collected every 24 hours for 7 days. On the 7th day the rats were sacrificed and samples from various organs and tissues (brain, spinal cord, lung, liver, spleen, intestine, kidney, testis, fat, muscle, and skin) were collected. The skin area of application was also removed and analysed. The radioactivity was determined using liquid scintillation spectrometry. Cumulative excretion in the urine and faeces was around 4.5%. The amount of radiolabel remaining in the body 7 days after dosing was less than 2% of the applied dose. Retention in the different organs and tissues examined was low ≥ 0.3%: muscles showed the highest amount 1.17%. Most of the unabsorbed dose (86%) remained at the skin area of application after 7 days. Dermal absorption (considered as the cumulative amount detected in excreta and tissues, excluding the dosed skin (a lower bound)) is calculated to be 6.5%.

The dermal absorption of [¹⁴C]-DEHP was evaluated in male F344 rats (Melnick et al., 1987). A single dose of 30 mg/kg bw (6 mg assuming 200 g/rat: 4.5 mg/cm2) of [14C]-DEHP (purity and position of label not stated), dissolved in ethanol, was applied to a circular area of 1.3 cm diameter (1.326 cm2) in the middle of the back of three rats. After the ethanol had evaporated, a perforated plastic cap was glued on the skin over the site of application. Urine and faeces were collected every 24 hours for 5 days and radioactivity was determined by liquid scintillation spectrometry.

Five days after dosing, recovery of 14C in the urine and faeces was around 5% with 3%, respectively. Body organs and the skin in the area of DEHP application site were also collected and analysed. The amount of radiolabel retained in the body 5 days after dosing was less than 2% of the applied dose. The highest amount was recovered in the muscle (1.2%). About 95% of the applied dose was recovered from the application site and the plastic caps, which were used to cover the application site. These results indicate that DEHP is not well absorbed through the skin of rats. Dermal absorption (considered as the cumulative amount detected in excreta and tissues, excluding the dosed skin (a lower bound)) is calculated to be 9%.

The absorption of [14C]-DEHP contained in a PVC plastic film (40% w/w (25.5mg/cm2): 0.5 mm thick) in male Fischer 344 rats was studied (Deisinger et al., 1998). Sheets of PVC film (15 cm2) were applied to the shaved backs of eight rats in two separate experiments (4 rats/experiment). In a short-term study (“study II”), the PVC was removed after 24 hours and the animals killed. In a longer term study (“study I”), after removal of the PVC film at 24 hours the animals were rewrapped with aluminium foil and bandage at the exposure site and sacrificed after a further 6 days. In both studies, [14C]-label was determined in the following: urine and faeces up to sacrifice; cage washes; washed and rinsed residue from the exposure site before sacrifice; entire clipped area including the dermal exposure site was exercised after washing and rinsing; the remainder of the body (carcass). The PVC films were also analysed for remaining [14C]-DEHP after the 24-hour exposure period. In “study I” (longer term study), the aluminium and bandage used from 24 hours to 7 days were analysed. The migration of (14C) DEHP from the film was 261 and 505.6 μg during 24 hours (0.725 and 1.4 μg/cm2/hour). Based on the materials mass balance information on the combined amount of [14C]-DEHP for urine, faeces, cage washes and carcass, and residual amount in the skin at the application site, the authors calculate that the percutaneous absorption rate (J) is around 0.24 μg/cm2/hr in both studies.

Guinea pig

Dermal absorption of a single dose radio-labelled DEHP was determined in 5 female hairless guinea pigs [Crl:LAF/HA(hr/hr)BR, 20-30 weeks old] (Ng et al., 1992). [Carbonyl-14C]-DEHP (53 μg, 13.2 µg/cm2, > 98% pure) was dissolved in 50 µl acetone and applied topically on 4 cm2 of the washed upper dorsal area. The exposed area was then covered by a protective non-occlusive pad to prevent ingestion of the compound. Animals were kept individually in glass metabolism cages. 24h after dosing the pad was removed and the dosing site cleansed with soap and water to remove unabsorbed material. Urine and faeces were collected at 6 and 12 hours on the first day and then at daily intervals for 7 days post-treatment for radioassay. Seven days post-administration the site of application was stripped with tape 10 times. Radioactivity content on the tape was determined by liquid scintillation spectrometry. To correct the dermal absorption for incomplete excretion (e.g. body retention), the excretion rate of an intramuscular dose, in 5 animals, was compared with the excretion rate of a topical dose.

Three percent (7% after correction) of the dermally administered dose was absorbed in vivo and excreted in 24 hours. Around 60% of the topical dose was not excreted in the faeces and urine after 7 days. The percent of [14C]-label recovered was 31% from skin surface wash (performed at 24 hours), 53% (21% before corrected) cumulative urine and faeces excretion up to 7 days, and 11.3% was tape strip recovery (performed at 7 days). 13% and 5% were recovered in the protective skin pad and body tissues (liver, fat, muscle, skin), respectively. The total recovery was 95% (76% before corrected). To determine the amount of DEHP that might have been have volatilized during the penetration process, DEHP was applied on a piece of skin that was kept in a petri dish at 33ºC for 7 days. Analysis of the radioactivity content revealed that 10% of the dosed material was lost. Thus volatilisation can only in part account for the loss of DEHP from the application site. Dermal absorption (considered as the cumulative amount detected in excreta and tissues, excluding the dosed skin (a lower bound)) is calculated, without consideration of correction, to be 26%.

The skin reservoir and bioavailability of dermally administered [14C]-DEHP in hairless guinea pigs was determined (Chu et al., 1996). Different amounts of [14C]-DEHP were applied to the washed dorsal region of 4 female Hartley hairless guinea pigs in four different experiments for different times and dosages: 119 µg/cm2 for 24 hours; 107 µg/cm2 for 48 hours; 442 µg/cm2 for 7 days; and, 529 µg/cm2 for 14 days. Radioactivity was measured in skin sections by autoradiography or liquid scintillation to determine the amount of radioactivity. The authors conclude that the results indicate that the amount of DEHP remaining in the skin after washing will eventually enter the systemic circulation and should be considered as part of the total dose absorbed, and that the hair follicle may play a role in percutaneous penetration.

Dermal absorption (considered as the cumulative amount detected in excreta and tissues, including skin dose) is calculated to be 9.7-18.9% from the four different experiments.

In vitro

Absorption, permeability constant and percutaneous absorption rate of [14C]-DEHP was determined in the epidermis of nonviable human skin (autopsy sample) and rats (AL/pk, Wistarderived) with a glass diffusion cell (Scott et al., 1987). 0.5 ml (14C)-labelled undiluted DEHP was applied to the skin preparations. Apparently the skin discs were 7 cm2, therefore, 70 mg/cm2 [14C]-DEHP was applied. 50% v/v aqueous ethanol was used as the receptor fluid and 50 μl samples collected at regular intervals. Diffusion cells were maintained at 30ºC. The results are presented in the following Table.

Table : Absorption rate data of DEHP. Adapted form Errata to Scot et al. (1987)

Human*                     Rat*

Permeability constant (Kp: 10-5 cm/hr)                    0.57                           2.28

Percutaneous permeability rate (J: μg/cm 22/hr)        5.59                         22.37

Lag time (hr)                                                            3.1                             3.9

* Epidermis, 50% aqueous ethanol

Absorption, permeability constant and percutaneous absorption rate of [14C]-DEHP was determined in the stratum corneum of human skin (autopsy sample) and full thickness rat skin Fischer-334 with a Franz-types diffusion cell (Barber et al., 1992). 0.3 ml [14C]-labelled undiluted DEHP was applied to 1.02 or 0.636 cm2 of skin (288-576 mg/cm2). The receptor solution was based on Dulbecco’s phosphate-buffered (pH 7.1) isotonic saline. Diffusion cells were maintained at 37 or 30ºC. The results are presented in the following Table.

Table: Absorption rate data of DEHP. Information are adapted

Human*                     Rat**

Permeability constant (Kp: 10-5 cm/hr)                    0.0105                     0.0431

Percutaneous permeability rate (J: μg/cm22/hr)         0.1                       0.42

* Stratum corneum

** Full thickness skin. Buffer

The permeability constant of [14C]-DEHP in the epidermis and dermis of male Sprague-Dawley rats was compared using buffer or aqueous ethanol with a diffusion cell (Pelling et al., 1998). Absorption was determined using both phosphate buffer saline and 50% aqueous ethanol as receptor medium. [14C]-DEHP was applied in 50 μl acetone (78.6 μg; 78.6 μg/cm2 assuming application to 1 cm2). Diffusion cells were maintained at 31.5ºC. The results are presented in the following Table. The authors comment that the rate and extent of DEHP absorption through the epidermis was greatly increased (40- and 80-fold, respectively) using 50% aqueous ethanol as receptor fluid compared with buffer. Also in comparison with Scott et al. (1987) it is noted that the Kp for the aqueous ethanol system is much higher (16-fold) in this study.

Table: Absorption rate data of DEHP. Information are adapted

Rat

Buffer                     50% aq. Ethanol

Permeability constant (Kp: 10-5 cm/hr)

Epidermis                                                      1.3                     94.6

Dermis                                                           4.76                     9.83

Percutaneous permeability rate (J: μg/cm 2/hr)*

Epidermis                                                       0.02                     0.786

* Calculated based on data detailed in the study. The area of the applied (14C)DEHP was assumed to be 1 cm2

The absorption of (14C) DEHP was determined in full thickness viable and nonviable guinea pig skin with a diffusion cell (Ng et al., 1992). The receptor solution was based on Hepes-buffered Hanks’ balanced salt solution, containing gentamicin (50 mg/l) and 4% bovine serum albumin. [14C]-DEHP was applied in 10 μl acetone (53.2, 228 and 468 μg/cm2). Diffusion cells were maintained at 37ºC. The amount of [14C] recovered in receptor fluid, on skin disc and in skin wash after 24 hours was determined. In vitro experiments were also carried out using perfusate containing an esterase inhibitor, phenylmethylsulfonyl fluoride (174 mg/l). Metabolites were identified by GC/MS.

Total recovery was between 78-90%. The results are presented in the following Table.

The authors comment that for a dose of 53.2 μg/cm2 the in vitro absorption of 47% (receptor fluid + skin disc) after 24 hours is comparable with the corrected in vivo cumulative excretion (urine + faeces) of 53% after 7 days.

To determine the amount of DEHP that may have volatised during the penetration process, DEHP was applied on a piece of skin that was kept in a petri dish at 33ºC for 7 days. Analysis of the radioactivity content revealed the 10% of the dosed material was lost. Thus volatilisation is apparently low and can only in part account for the dose of DEHP from the application site.

DEHP was metabolised to a monoester, MEHP. In the presence of the esterase inhibitor, the dose that permeated into the receptor fluid decreased from 3.36% in the control to 2.67% in 24 hours. The proportion of MEHP was also reduced from 2.36 to 1.23% in the inhibitor treated group.

Table: Percent radiolabel recovered for DEHP at 24 hr post application. Information are adapted

Dose (μg/cm2)

53.2              53.2*              228              468

Receptor fluid                                               6.1             5.0               2.4               2.5

Skin disc                                                      41              40               37              36

Receptor fluid + skin disc                             47              45                40              39

Skin wash                                                   38              41               50               40

Total recovery                                           85        86           90               78

Estimate Per Ab rate (J: μg/cm2/h)#            0.13           0.11             0.23              0.49

* Non-viable skin.

# Calculated based on % in receptor fluid and 24 hours. Information on the rate of excretion is not detailed in the study other than for at 24 hours.

The absorption of (14C) DEHP was determined in the perfused porcine skin flap (Wester et al., 1998). Isolated skin flaps were perfused in a non-recirculating chamber. 10 cm2 was dosed with (14C) DEHP (18.5 μg/cm2). After 8 hours, 0.14% was recovered in the perfusate, 14.5% was recovered in skin strips (taped 12 times), 3.8% in the remaining skin and 71% in skin surface wash, a total material balance recovery of 94%. Based on the combined amount in the perfusate and skin strips (14.6%) the percutaneuos absorption rate is 0.34 μg/cm2/hour.