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Description of key information

The toxicokinetic of branched p-Nonylphenol (CAS 84852-15-3) was assessed in two GLP studies in SD rats by Fennel (2001) and a toxicokinetic study in CD rats conducted by Green (2003).

Linear 4-n-Nonylphenol (CAS 104-40-5) was assessed in a 4 day in vivo metabolic balance study in Wistar rats conducted by Zalko (2003). Two organ specific studies were conducted by Daidoji (2003 and 2006) using a perfused rat liver model and everted intestine of SD rats, both in vivo. The latter study was complemented by an in vitro simulation of organ specific metabolism using microsomes from intestinal tissue.

Müller (1998) reported toxicokinetic behaviour of 4-Nonylphenol in two human volunteers. In addition the ability of p-NP, and its principal mammalian metabolite nonylphenol glucuronide (NPG), to affect human estrogen receptors in vitro, was investigated by Moffat (2001) using a yeast transcriptional activation system.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential

Additional information

Branched p-Nonylphenol (CAS 84852-15-3)

In a pilot pharmacokinetic study in male and female SD rats (Fennel 2001) single i.v. and gavage application of branched Nonylphenol was compared. Elimination half life (t1/2), bioavailability and metabolic profile were determined. The elimination half lives in plasma after i.v administration of 5 mg ring-14C labelled NP was 9.6 h (males) and 9.3 h (females); after oral administration it was 12.4 h (males) and 8.5 h (females). Cmaxin blood was 10 fold lower administered by gavage than i.v. administration in female rats and 22 fold lower in male rats. Four radioactive moieties were detected in blood after i.v. application: protein-bound NP, NP glucuronide, NP itself and an unidentified glucuronidated metabolite. Protein-bound radioactivity decreased with time and was detected at all time points during the 24 h experiment, whereas free nonylphenol was below the limit of detection within 4 h of dosing (i.v.) in female rats. The plasma protein-bound fraction was not detected after oral administration.

Liver, lung, testis, epididymis, subcutaneous fat, abdominal fat and spleen were collected from male rats at 2, 8, and 24 h following i.v. dosing. Liver contained 11.3% of the dose 2 h after application; subcutaneous fat and abdominal fat contained 0.31 and 0.2% of the dose per gram of tissue, respectively. Estimating the amount of dose contained in total body fat (~ 9%) indicates that fat is a significant reservoir for NP when administered intravenous. When administered by gavage, the peak levels in the liver were 1.8% of the dose 2 h after dosing and 2.2% after 8 h, declining to 0.8% by 24 h. Fat tissue contained approximately 0.01% of the dose per g tissue at all time points, indicating that fat is a non-significant reservoir for NP when administered orally, in contrast to i.v. application. On gavage administration to male rats, less than 0.02% of the dose was recovered in lung, spleen, testis and epididymis after 24 h. The levels of radioactivity in plasma and tissue were highly variable following gavage. This variability was particular pronounced in animals at the 50 and 100 mg/kg group, implying individual differences in uptake from the gut and/or metabolism.

The bioavailability calculated from nonylphenol-derived radioactivity in blood was 0.25 and 0.29 for female and male rats, respectively.

An associated study conducted by Fennel (2001) focussed on distribution and metabolic profile in SD rats after single gavage of 0, 5 and 200 mg/kg bw. The majority of the administered dose was excreted within seven days in faeces (81-85%) and to lesser extend in urine (12-17%). The total dose recovered in excreta amounted to approximately 90-97%, except for the high dose group (200 mg/kg bw/day), in which approximately 75% of the dose was recovered. A small amount of radioactivity was exhaled as CO2in male rats in the low dose group (amounting to 0.14% of the applied dose), suggesting a breakdown of NP into volatile metabolites. The rate of excretion of radioactivity in the urine was faster in female rats compared with males. In addition qualitative differences were observed in urinary metabolites between male and female rats. No radioactivity was detectable in the reproductive organs (testes, ovaries, epididymis, and uterus) examined at day 7 after administration. The majority of the radioactivity recovered after 7 days was in the liver (0.14%), the intestinal tissue (small: 0.08%; large: 0.1%) and the contents of the small (0.5%) and large (0.8%) intestines. There was no accumulation in abdominal and subcutaneous fat tissue in accordance with the findings of the pharmacokinetic study (Fennel 2001).

Green (2003) published a toxicokinetic study in SD rats. P-NP was administered i.v. (10 mg/kg bw) and oral (10 and 100 mg/kg bw) for up to 14 days.

75% of the radioactivity applied i.v. was being eliminated within 24 h, mainly in the faeces. After 7 days 13% of the dose applied i.v. was found in the carcass. Concentration of radioactivity in fat increased 4-5 fold over the duration of the study. The absolute amounts in fat, however, were less than 0.06% of the amount found in excreta on day 14 of the study.

Up to 64% of the dose was eliminated in bile following 10 mg/kg oral dose and up to 49% at the higher dose. Similar amounts were excreted in bile after i.v. application. From the proportion of the dose eliminated in bile and urine, an absorption rate of 65% and 80% can be concluded at 10 mg/kg, respectively. Absorption rate was calculated to be ~50% at the 100 mg/kg dose. Following absorption, NP was metabolised in the liver, with the majority of the metabolites excreted in bile, mainly as glucuronide conjugates.

Sex related difference was seen in the blood and plasma, with the maximum concentration in males being 2-3 fold higher than that in females. The ability to clear NP and its metabolites from blood was also different. In males, the half live in plasma was not affected by the increase in dose from 10 to 100 mg/kg (7 vs. 9 h), whereas in females the half-life increased approximately 4-fold with the increase in dose (3 vs. 13 h). The capacity of the female rat to metabolize and excrete NP is lower than that of males at high doses. The sex-related difference was also seen in the metabolic profiles in urine, bile and faeces. NP-glucoronide (NPG) represents the only significant metabolite in the bile at the 10 mg/kg dose; following 100 mg/kg significant amounts of NP itself were present in female but not in male bile. Similar, NP was a major component in female urine following a 100 mg/kg, but not a 10 mg/kg dose. Both findings suggest that the capacity of the liver to form glucoronide is saturated at the higher dose in females.

NP was more extensively metabolized in male rats, with a number of metabolites present in urine, bile and faecal extracts that were not seen in female rats. NPG, the major metabolite in female rats was not present in male urine, although it was present in bile.

Following repeated dosing, steady state was reached within 7 days. There was no evidence of significant accumulation into tissue compartments or of a significant change in clearance or metabolite profiles in urine. Enterohepatic circulation of metabolites does not appear to be a major feature in NP metabolism. Recirculation would be reflected in the blood concentration of radioactivity. An early peak concentration around 6 h after oral dosing may be indicative of a limited amount of enterohepatic circulation, but there is no evidence to suggest that recirculation is sustained for any length of time.

Extraction of faecal samples revealed that faeces contained mainly NP itself. With regard to the fast absorption this is an unexpected finding suggesting, that excreted NP is deconjugated by bilary and enterobacterial enzymes and being transported through the GI tract bound to diet.

Linear 4-n-nonylphenol (CAS 104-40-5)

The toxicokinetic effect of linear 4-n-nonylphenol was investigated in a 4 day in vivo metabolic balance study in Wistar rats conducted by Zalko (2003). The metabolic profile after oral administration of 10 mg/kg bw/day and 1 µg/kg bw/day was characterized in urine, faeces and bile. In addition metabolism and distribution was tested in pregnant rats up to day 20 of gestation.

4-n-nonylphenol is extensively metabolized and predominantly eliminated in urine (57% in males and 40% in females within 96 h). Ten major metabolites were characterized. Most of them were formed by ω- or β-oxidation of the 9-carbon side chain and conjugation of phenol moieties to sulfate or glucuronic acid. In rat, unlike in fish, no 5-carbon and 7-carbon side chain metabolites were detected. The main part of urinary radioactivity was associated with 1-3-carbon side chain metabolites, most or which were identified as conjugates.

Bile and faeces contain several 4-n-NP metabolites resulting from β-oxidation but the metabolites were only detected in very low amounts. Major metabolite in these samples is hydroxylated NP. Bilary hydroxyl-4-n-NP was excreted as glucuronide which is very likely deconjugated by the intestinal flora into the corresponding aglycone. Sulfo-conjugaties was notably higher in males than in females. The major metabolites are para-hydroxy benzoic acid and the corresponding sulphate. Although female rats excreted more radioactivity in feces, biliary excretion was significantly more important in males. Thus, the intestinal re-absorption of 4-n-NP residues and their possible entero-hepatic cycling show gender-related differences.

Neither the distribution pattern nor the residual levels of 4-n-nonylphenol were found to be different between the dose groups. No unexpected tissue-specific accumulation of 4-n-nonylphenol was detected, i.e. no preferential retention of 4-n-NP in fat was observed. Most tissue extractions led to the conclusion that the radioactivity present in tissues was mainly associated with volatile compounds, supporting the hypothesis of a complete breakdown of 4-n-NP.

Experiments carried out in pregnant rats exposed to 1 µg/kg bw/day from day 3 to day 19 of gestation demonstrated similar metabolic pathways. Very limited amounts, if any, of non metabolized 4-n-nonylphenol did reach fetuses, suggesting that non-significant amounts of NP cross the placenta barrier.

These conclusions are valid only for linear NP. Branched side chains will not undergo a complete breakdown of the alkyl chain.

In addition to the metabolic balance study, metabolism and excretion were determined using an in vivo perfused rat liver model (Daidoji, 2003). 4-n-nonylphenol and additional short-chain alkylphenols were injected into the portal vein of the liver of Sprague-Dawley rats at concentrations of 0.025 or 0.05 mM. Liver perfusion was carried out in a flow-through mode. Subsequent excretion into bile and vain was monitored. About 800 to 1000 nmol of injected nonylphenol could be conjugated as glucuronide within 1 h. Most of the glucuronide and free nonylphenol remained in the liver. Other alkylphenols having shorter alkyl chains were excreted smoothly into the bile as glucuronides. These results indicated that alkylphenols with shorter (including C6) alkyl chains were easily excreted into the bile as glucuronides compared to the delayed excretion of long-chained (C9) molecules like NP. In addition to the perfused liver model absorption, distribution and elimination were investigated in everted intestinal tissue of SD rats (Daidoji, 2006). NP is readily absorbed and glucuronidized within intestinal tissue of SD rats. This was confirmed by a simulation of organ specific metabolism using microsomes prepared from intestinal tissue. The intestine microsomes showed strong glucuronidation activity. Nonylphenol was glucuronidated within the intestinal wall but NP and NPG was not excreted from intestinal tissue within 10 h. Orally administered nonylphenol remained for long periods in gastrointestinal tissue as neither the parent compound nor glucuronide was excreted into the mucosal or serosal side. As though the present study confirmed that intestinal tissue possesses a strong alkylphenol elimination system using UDP glucuronosyltransferase, this system is impaired by the marginal transport of alkylphenol-glucuronide possessing long alkyl chain, such as nonylphenol.

Human data

The toxicokinetic behaviour of 4-n-nonylphenol was investigated in two human volunteers (i.v.: 14 µg/kg body weight; oral: 66 µg/kg body weight) by Müller et al. (1998). After intravenous and oral application, the elimination half-life of the parent compound from the blood was 2–3 h. Bioavailability after oral application (determined by oral and intravenous AUCs) was about 20%. After a single oral application the blood concentration did not exceed 650 pg/g blood. A distribution volume of 2800 l was calculated, suggesting migration primarily into deeper lipoid compartments after the initial 2-3 h distribution phase. The elimination half-life in blood was found to be 2-3 h. The bioavailability was calculated to be 20% based on the AUC ratio between i.v. and oral application. Less than 1% of the orally applied dose was excreted in the faeces as NP or from conjugate cleavage, indicating that the substance was quantitatively absorbed in the gastrointestinal tract. The low bioavailability was therefore probably due to extensive metabolism in the gut wall and during the first passage of the liver. This pharmacokinetics study in human volunteers is restricted by the low number of participants.

Furthermore, levels of NP in non-occupationally exposed persons were investigated by analyzing human autopsy adipose tissue samples from people aged 3 to 100. NP concentrations ranged from 19 to 85 ng/g lipids. These values were both in the range of the analytical background contamination and do not indicate a concern of bioaccumulation by non-occupational exposure.

Estrogen-like properties

The ability of p-NP, and its principal mammalian metabolite nonylphenol glucuronide (NPG), to affect human estrogen receptors (ER) or androgen receptors (AR) was investigated in vitro by Moffat (2001) using a yeast transcriptional activation system. Glucuronidation of NP was found to eliminate the estrogen-like activity of NP in yeast harbouring human ER. It is likely, that the weak estrogen-like activity noted for NP at high doses in rats reflects saturation of glucuronide conjugation. At concentrations present in the environment, this metabolic saturation is unlikely to occur, thus enabling glucuronidation of NP to remove the ability of these chemicals to mimic biological estrogens in humans.

As mentioned above Green (2003) found that oral administered NP at a dose of 100 mg/kg/d results in an increased bioavailability of NP in female rats which occurs following metabolic saturation of the glucuronidation. This suggests that adverse effects to the reproductive system in rat studies occurring at 50 mg/kg bw/d are very likely attributed to metabolic saturation.