Phenol, 4-nonyl-, branched

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment.

Data source

Reference

Reference Type:

publication

Title:

In vivo metabolic fate of the xeno-estrogen 4-n-nonylphenol in wistar rats

Author:

D. Zalko, R. Costagliola, C. Dorio, E. Rathahao, and J-P. Cravedi

Year:

2002

Bibliographic source:

Drug Metabolism and Disposition, Vol. 31, No. 02

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

1) In vivo metabolic balance study: high dose (10 mg/kg/day) and low dose (10 µg/kg/day) administration by gavage to 6 rats/sex
2) Biliary excretion: singe administration to 3 rats/sex; 1µg/kg; cannulated biliary duct
3) Metabolism in pregnant rats: 3 females; administration of 1µg/kg/day by diet up to day 20 of gestation

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

4-n-Nonylphenol

IUPAC Name:

4-n-Nonylphenol

Constituent 2

Reference substance name:

p-nonylphenol

EC Number:

203-199-4

EC Name:

p-nonylphenol

Cas Number:

104-40-5

IUPAC Name:

4-nonylphenol

Details on test material:

- Name of test material (as cited in study report): Nonylphenl (4-n-NP)
read-across: both, CAS 104-40-5 and CAS 84852-15-3 refer to Nonylphenol
- Analytical purity: no data on unlabeled NP (purchased from Riedel-de-Haen)
- Radiochemical purity (if radiolabelling): >96%
- Specific activity (if radiolabelling): 1.72 GBq/µmol and 2 MBq/µmol
- Locations of the label (if radiolabelling): Ring-2,6-3 H-labeled nonylphenol ( 3 H-4-n-NP) and ring- 14 C-labeled nonylphenol ( 14 C-4-n-NP)
- Other: radiolabeled NP was purchased from Isotopchim (Ganago-bie-Peyruis, France).
- Storage condition of test material: Prior to commencement of dosing, the 24-h stability of 3H-4-n-NP, when incorporated to the diet, had been successfully verified by HPLC following ethanol extraction (yield, 100%; only unchanged 4-n-NP detected)
- Other: Solvents used for extraction and high performance liquid chromatography (HPLC) analyses were of the highest commercial grade available from Scharlau Chemie S.A. (Barcelona, Spain) or Merck (Briare-Le-Canal, France). Ultrapure water from Milli-Q system (Millipore, Saint Quentin-en-Yvelines, France) was used for HPLC mobile phases preparation.

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks
- Weight at study initiation: mean values males: 190.3 and 189.7 g, females: 160 g both groups
- Housing: individually housed in in stainless steel metabolic cages
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): as libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

other: : oral gavage (metabolic balance, biliary excretion) and diet (metabolism in pregnant rats)

Vehicle:

corn oil

Remarks:

(gavage only)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
In vivo metabolic balance study (1) & Biliary excretion (2):
Doses were respectively adjusted to 1 µg/kg/day (group A, “low-dose”) or 10 mg/kg/day (group B, “high-dose”). The material used for intragastric gavage was washed with ethanol. The radioactivity recovered with ethanol was counted, thus allowing the calculation of the actual dose administered daily to each animal.

DIET PREPARATION
Metabolism in pregnant rats (3):
- Mixing appropriate amounts with standard diet (UAR 210; U.A.R., Villemoisson-Sur-Orge, France)

VEHICLE
In vivo metabolic balance study (1) & Biliary excretion (2):
- Amount of vehicle (if gavage): 0.5 ml

Duration and frequency of treatment / exposure:

In vivo metabolic balance study (1):
At 0, 24, 48, and 72 h, animals were gavaged with 0.8 MBq 3 H-4-n-NP fortified with the appropriate amount of unlabeled 4-n-NP prior dissolution in 0.5 ml of corn oil.

Biliary excretion (2):
Single gavage (1µg/kg)

Metabolism in pregnant rats (3):
Diet; day 3-19 of gestation (1 µg/kg/day)

Doses / concentrations

Remarks:

Doses / Concentrations:
In vivo metabolic balance study (1):
1 µg/kg/day (group A, “low-dose”)
10 mg/kg/day (group B, “high-dose”)

Biliary excretion (2):
1 µg/kg

Metabolism in pregnant rats (3):
1 µg/kg/day

No. of animals per sex per dose / concentration:

In vivo metabolic balance study (1):
3/sex/dose

Biliary excretion (2):
3/sex

Metabolism in pregnant rats (3):
3 female

Control animals:

other: Two male rats were gavaged with 1 µg/kg 14 C-4-n-NP dose. These animals were used to verify the results obtained with 3 H-4-n-NP

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: low dose was used to investigate the metabolism at a level corresponding to a possible daily oral intake for humans. The high dose was used to investigate possible modifications of 4-n-NP metabolic routes and to obtain enough material for metabolites structural identification. Additional experiments were run in pregnant rats to better understand the distribution of 4-n-NP during gestation and the possible changes occurring in the metabolic pathways of this xenobiotic.
- Rationale for animal assignment (if not random): random

Details on dosing and sampling:

Metabolic balance study:
(1) In vivo metabolic balance study:
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: daily at time points 24, 48, 72, 96 h (on day 1-4)
- Tissues and body fluids sampled: blood; brain, liver, kidney, testis (or ovaries, uteri), muscle, perirenal fat, the rest of viscera and carcasses
- Time and frequency of sampling: end of experiment (96 h after application)

(2) Biliary excretion:
- Single oral dose; biliary duct cannulated immediatetly after gavage
- Bile collection started at t = 30 min; duration: 6 h

(3) Metabolism in pregnant rat:
Diet at libitum from day 3-19 of gestation; sampling as in study (1).

Statistics:

Comparison between values was achieved using Student's t test.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The overall amount of radioactivity recovered during the 3 H-4-n-NP metabolic balance study corresponded respectively for males and females to 78.5 ±0.5% and 68.4 ±0.3% of the administered dose. In a complementary 24-h metabolic balance experiment using two male rats dosed with 1 µg/kg 14 C-4-n-NP, the total amount of radioactivity recovered was very close (73.85 ±2.1%), and again most of the radioactivity was excreted in urine and to a lesser extent in faeces.

Details on distribution in tissues:

In vivo metabolic balance study (1):
For rats receiving a 1 µg/kg dose, liver and kidney contained the highest concentrations of residues (0.20 – 0.22 ppb). These levels were only twice higher than those measured in all other tissues, as well as in carcasses. Radioactivity repartition and residual concentrations of radioactivity calculated for samples from group B rats (dosed 10 mg/kg) were about 10,000-fold higher than those recorded for group A rats (dosed 1 µg/kg). No significant difference was observed between males and females.

Residue Distribution in Pregnant Rats (3):
Pregnant rats were orally dosed with a mean 0.649 µg/kg/day 4-n-NP from day 3 to 19 of gestation. Radioactivity losses (food left by rats and radioactivity washed from cages) accounted for only 1.4% of the dose. Radioactivity levels in tissues were higher than the levels measured for the low-dose group rats in the metabolic balance study, the highest levels being measured in liver, kidney, and fat. Given the difference between the two experimental protocols, no direct comparison can be attempted between the two groups. The residual levels of 4-n-NP in tissue and fetuses of pregnant Wistar rats are summarized in table “"tissue distribution" (below). Since no significant amounts of 4-n-NP (or its metabolites) metabolites were detected in fetuses or in amniotic fluids, it is concluded that no significant amounts of 4-n-NP cross the placental barrier in Wistar rat.
Blood radioactivity was mainly associated with the plasma fraction (74.3%).

Transfer into organs

Test no.:

#1

Transfer type:

blood/placenta barrier

Observation:

no transfer detectable

Details on excretion:

In vivo metabolic balance study (1):
Over the 4-day study, most of the administered radioactivity was excreted in urine. Urinary excretion was significantly higher in male rats (57%) than in females (40%). These values were similar for groups A and B (Table 1). Fecal radioactivity excretion was found to be higher in females, for which it represented more than 20% of the dose. A significant difference between sexes was observed only for group A animals (Table 1). The cumulated amounts of radioactivity excreted in urine and faeces over the 4-day period showed no significant difference between males and females, even though the values recorded for males were slightly higher for both dosage levels.

Results of Biliary Excretion study (2):
Over the 6-h study, cumulated radioactivity excretion was higher in males (9.5%) than in females (5.9%). The difference was statistically significant at all time points with the exception of the first sample (0.5–1 h). Radiochromatographic profiles were carried out for all 1- and 6-h samples, showing a similar pattern for males and females.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Urinary Metabolites:
10 major urinary metabolites of 4-n-NP in rats were identified:
1) para-Hydroxy benzoic acid glucuronide
2) 3-(4-Hydroxyphenyl) propanol glucuronide
3) 3-(4-Hydroxy-phenyl)-2-propenoic acid glucuronide
4) 3-(4-Hydroxy-phe`nyl)-2-propionic acid glucuronide
5) para-Hydroxy benzoic acid
6) Ring-hydroxylated 3-(4-hydroxyphenyl)-2-propionic acid
7) para-Hydroxy benzoic acid sulfate
8) 3-(4-Hydroxy-phenyl)-2-propionic acid sulfate
9, 9’) 3-(4-Hydroxy-phenyl)-2-propenoic acid sulphate (Z & E isomer)

Any other information on results incl. tables

Table "tissue distribution"

Residual levels of 4-n-NP in tissues and fetuses of pregnant Wistar rats dosed orally with 0.65 µg/kg/day 4-n-NP, from day 3 to day 19 of gestation (mean of 3 animals +/- S.D.); results are expressed as nanograms of NP Eq per gram wet weight (ppb) and in percent of the total radioactivity administered to animals.

	ppb	% dose
Blood	0.12 +/- 0.02	0.03
Brain	0.08 +/- 0.02	0.004
Liver	0.34 +/- 0.41	0.15
Digestive tract	0.73 +/- 0.29	0.73
Kidney	0.26 +/- 0.05	0.26
Uterus	0.12 +/- 0.02	0.05
Placenta	0.11 +/- 0.02	0.02
Fetuses	0.13 +/- 0.06	0.01
Fat	0.21 +/- 0.06	0.03
Carcass	0.17 +/- 0.02	1.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no data
4-n-nonylphenol is extensively metabolized and eliminated predominantly in urine. There is no dose-depending difference in distribution pattern and residual levels between 1 µg/kg and 10 mg/kg groups. No tissue-specific accumulation was detected. Very limited amounts, if any, of non metabolized 4-n-nonylphenol did reach fetuses.

Executive summary:

In a 4-day metabolic balance study 4-n-nonylphenol (> 96%, ring-labelled) was administered by gavage to 3 Wistar rats/sex/dose with 1 µg/kg (“low-dose”) and 10 mg/kg (“high-dose”), respectively. Neither the distribution pattern nor the residual levels of 4-n-nonylphenol were found to be different between groups, and no unexpected tissue-specific accumulation of 4-n-nonylphenol was detected. No preferential retention of 4-n-NP in fat was observed. Most of the radioactivity was eliminated in urine (57% in males and 40% in females - regardless of the dose administered), containing hydrophilic metabolites very likely resulting from extensive beta-oxidation of the nonyl side chain and from the conjugation of the phenol to sulfate or to glucuronic acid.

Metabolite identification and radio-HPLC profiling showed that 4-n-NP is extensively metabolized in rat. About 10 major metabolites were characterized. Most of them were formed by ω or β-oxidation of the 9 -carbon side chain. In rat, unlike in fish, no 5-carbon and 7-carbon side chain metabolites were detected. 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.

It appeared that sulfo-conjugation was more pronounced in male than in female rats.

Although female rats excreted more radioactivity in feces, biliary excretion was significantly more important in males. Thus, the intestinal reabsorption of 4-n-NP residues and their possible entero-hepatic cycling could be different in male and female rats.

Experiments carried out in pregnant rats exposed to a 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.

 

This metabolic balance study in rat is classified acceptable and meets generally accepted scientific standards. It is to be stressed that the metabolic profile is valid only for unbranched NP.