4-(1,1,3,3-tetramethylbutyl)phenol

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Objective of study:

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

The experimental details (chemicals, analytical procedures, animal husbandry and experimentation) followed those of the preceding study in Wistar rats (7.1.1 Certa et al. 1996) as closely as possible.

GLP compliance:

yes

Radiolabelling:

no

Species:

rat

Strain:

other: DA/Han rats (Harlan-Winkelmann, Borchen, Germany)

Sex:

female

Route of administration:

other: oral: gavage respectively intravenous

Vehicle:

propylene glycol

Duration and frequency of treatment / exposure:

single application

Remarks:

Doses / Concentrations:
single gavage application: 50, 200 mg OP / kg bw
single intravenous application: 5 mg OP / kg bw

No. of animals per sex per dose / concentration:

six male animals per dose and application

Control animals:

yes, concurrent vehicle

Type:

absorption

Results:

12.3% (50 mg OP/kg; gavage)

Type:

absorption

Results:

8.4% (200 mg OP/kg; gavage))

Test no.:

#1

Toxicokinetic parameters:

Cmax: 1600 ng/ml blood (immediately after 5mg OP/kg; i.v.)

Test no.:

#1

Toxicokinetic parameters:

C(time): 100 ng/ml blood (1h after 5 mg OP/kg; i.v.)

Test no.:

#1

Toxicokinetic parameters:

C(time): 1-20 ng/ml blood (48 h after 5 mg OP/kg; i.v.)

Test no.:

#1

Toxicokinetic parameters:

half-life 3rd: 36.1 h (5 mg OP/kg; i.v.)

Test no.:

#2

Toxicokinetic parameters:

Cmax: 181 ng/ml blood (90 min after 50 mg OP/kg; gavage)

Test no.:

#2

Toxicokinetic parameters:

C(time): 105 ng/ml blood (8 h after 50 mg OP/kg; gavage)

Test no.:

#2

Toxicokinetic parameters:

C(time): detection limit (32 h 105 ng/ml blood (8 h after 50 mg OP/kg; gavage)

Test no.:

#3

Toxicokinetic parameters:

Cmax: 419 ng/ml blood (8 h after 200 mg OP/kg; gavage)

Test no.:

#3

Toxicokinetic parameters:

C(time): 14 ng/ml blood (48 h after 200 mg OP/kg; gavage)

Metabolites identified:

not specified

Conclusions:

Interpretation of results (migrated information): other: gender and sex related
The present data are suggestive of major strain and gender differences in the biological disposition of OP in rats. In particular, an extensive enterohepatic circulation would be of considerable importance for the extrapolation of biological potency data obtained in vitro to relevant situations in vivo.

Executive summary:

In a toxicokinetics study p-tert-octylphenolwas administered to six female animals (DA/Han rats (Harlan-Winkelmann, Borchen, Germany)/sex/dose in single application intravenous at a dose level of 5 mg/kg bw and by gavage at dose levels of 50 and 200mg/kg bw. 

Compared to the previous data set on toxicokinetics of OP in male Wistar rats (Certa et al. 1996) the biological half-life of OP is consistently longer. The final (Gamma-phase) t1/2, upon i.v. administration of 5 mg/kg, was 310 min (5.17 h) in male Wistar rats (Certa et al. 1996), but 36.1 h in female DA/Han rats (present data). This difference is further corroborated by the data obtained after oral administration, and indicates not only gender but also strain differences.

Also in contrast to the previous data in male Wistar rats, the present data show peculiarities in the time-course of OP blood levels after oral administration of OP, which indicate extensive enterohepatic circulation of this compound.

 

This toxicokinetics publication in the female DA/Han rats (Harlan-Winkelmann) meets generally accepted scientific standards is well documented and acceptable for assessment.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

other: binding to estrogen receptor

Qualifier:

no guideline followed

Principles of method if other than guideline:

Agonist Activity:
The yeast ER and AR assays were performed as described by Routledge and Sumpter (Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 15, 241–248, 1996) and Sohoni and Sumpter (Several environmental oestrogens are also antiandrogens. J. Endocrinol. 158, 327–339, 1998).

Antagonist Activity:
To determine whether NP, OP, NPG, and OPG possessed antiestrogenic and/or antiandrogenic activity, the natural ligand (17ß-estradiol or DHT) was added to the medium of the appropriate assay at a concentration that produced a suboptimal response (5 nM for both assay systems).

GLP compliance:

no

Radiolabelling:

no

Species:

other: yeast

Remarks:

Doses / Concentrations:
Agonist Activity
Test chemicals and negative controls were serially diluted in ethanol (except Hepes which was diluted in aqueous media).
Positive control compounds were tested in the following dose ranges—17beta-estradiol (4.88 pM to 10 nM) and dihydrotestosterone (24.4 pM to 50 nM).
OPG (and all negative controls) were tested at concentrations up to 1 mM in the estrogenic and androgenic assays.
OP was only tested at concentrations up to 31 µM in all four assays.

Antagonist Activity
Positive control compounds were tested in the following dose ranges—hydroxytamoxifen (antiestrogenic assay) from 4.88 nM to 10 µM and flutamide (antiandrogenic assay) from 488 nM to 1 mM.
OP was tested in these antagonist assays at concentrations up to 31 µM.
OPG (and all negative controls) were tested up to 1 mM in the antiestrogenic assay and 156 µM in the antiandrogenic assay.

Positive control reference chemical:

estrogen: estradiol
antiestrogen: hydroxytamoxifene
androgen: DHT
antiandrogen: flutamide

OP exhibited intrinsic estrogenic activity (above 61 nM), but at a concentrations 3–4 orders of magnitude higher than 17beta- estradiol.

In contrast, at concentrations up to 1 mM, the glucuronides of OP elicited no activity in the estrogenicity assays .

The glucuronide, in common with the parent compounds, were also devoid of antiestrogenic activity.

Minimal androgenic activity, in terms of both potency and effectiveness relative to DHT, was detected for OP. However, these minimal androgenic effects were noted at concentrations >2 orders of magnitude greater than those that induced estrogen-like activity in vitro.

The glucuronide, in common with the parent compounds, failed to elicit any antiandrogenic activity in vitro.

It is recognized that OP is rapidly and extensively metabolized, largely via glucuronidation, in a number of species including rats and humans. Therefore, to understand the human relevance at anticipated levels of human exposure to these agents, of the estrogenic effects of OP in rats, it was important to investigate the estrogenic activities of OP glucuronides (OPG) relative to the parent compounds. The yeast assay systems used were shown to be appropriately sensitive to either the estrogen estradiol and the antiestrogen hydroxytamoxifene or the androgen DHT and the antiandrogen flutamide. In addition, the hormonal specificity of all positive responses reported was confirmed by the absence of activity for the three negative control agents concomitantly evaluated in all assays—Hepes buffer, sodium acetate, and niacinamide.

Conclusions:

Interpretation of results (migrated information): other: Glucuronidation of OP eliminates its estrogen-like activity in yeast harboring human ER.
Glucuronidation of OP eliminates its estrogen-like activity in yeast harboring human ER. It is likely, that the weak estrogen-like activity noted for OP 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 OP to remove the ability of these chemicals to mimic biological estrogens in humans. It is concluded that at expected exposure levels, the potential endocrine hazard posed by NP and OP to humans is likely to be negligible.

Executive summary:

This study investigates the intrinsic ability of OP, and its principal mammalian metabolite octylphenol glucuronide (OPG), to affect estrogen receptor (ER)- or androgen receptor

(AR)-mediated transcription in a yeast transcriptional activation system.

 

Glucuronidation of OP eliminates its estrogen-like activity in yeast harboring human ER. It is likely, that the weak estrogen-like activity noted for OP 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 OP to remove the ability of these chemicals to mimic biological estrogens in humans. It is concluded that at expected exposure levels, the potential endocrine hazard posed by NP and OP to humans is likely to be negligible.

 

The study is classified as acceptable because the yeast assay system was shown to be appropriately sensitive to either the estrogen estradiol and the antiestrogen hydroxytamoxifene or the androgen DHT and the antiandrogen flutamide and satisfies requirements for in vitro assay studies.

Reference 3

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.07.1995 - 27.07.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

(The study was designed according to scientifically accepted procedures and according to validated and standardized methods, developed in the testing facility.)

Principles of method if other than guideline:

The OCT concentrations in tissues were determined by a gas chromatographic method, using a mass selective detector. OCT was extracted with methyl tert.-butyl ether and p-tert.-butyl phenol (BUT) was used as internal standard. The limit of detection under the conditions of this study was determined to be in the range of 5 - 10 ng/g tissue.

GLP compliance:

yes (incl. QA statement)

Remarks:

MINISTERIUM FÜR UMWELT, RAUMORDNUNG UND LANDWIRTSCHAFT DES LANDES NORDRHEIN-WESTFALEN

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fa. Harlan-Winkelmann, D-33167 Borchen
- Age at study initiation: no data
- Weight at study initiation: 200-220 g
- Fasting period before study: No fasting period before administration
- Housing: Groups of five in Makrolon cages Type IV
- Diet: ad libitum, Ssniff R10 - laboratory standard rat diet (in pellet form), Fa. Ssniff, Spezialfutter GmbH, D-59494 Soest.
- Water: groups I - IV ad libitum, Fa. Gelsenwaser, D-45721 Haltern; group V tap water saturated with OCT ad libitum
- Acclimation period: animals were acclimated under study conditions except administration for a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3°C
- Humidity (%): 30-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light; 12/12

Route of administration:

other: : oral: gavage and drinking water (repeated application)

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Solutions of the test material in the vehicle for the gavage application were prepared once. The concentrations of the test substance were determined after preparation of the solutions, at the mid and at the end of the study.
- Storage temperature of food: room temperature, dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): used in comparable studies
- Concentration in vehicle: 50 mg OCT/ml (for 50 mg/kg dose group, gavage application); 200 mg OCT/ml (for 200 mg/kg dose group, gavage application);

For the treatment of group V, a saturated solution of OCT in tap water was prepared weekly (day 1 - 14 of application) or once (day 15 - 28 of application), respectively, by dissolving a surplus of OCT in tap water, stirring overnight, and removal of unsoluble residues by filtration.
The concentrations of these solutions were determined weekly.

Duration and frequency of treatment / exposure:

After acclimatization the animals of group I and II received gavage applications of 50 or 200 mg/kg b.w. OCT once per day for 14 consecutive days.
Other animals received drinking water satured with OCT for 14 and 28 days, respectively.

Remarks:

Doses / Concentrations:
Repeated gavage application: 50 mg/kg resp. 200 mg/kg bw
drinking application ad libitum: mean concentration = 8.39 mg/l / standard deviation = 1.182 mg/l (for average doses see Table 6 in annexed document)
One control groups received the vehicle only, another control group received pure water.

No. of animals per sex per dose / concentration:

Group I : 5 animals : 50 mg/kg bw gavage
Group II : 5 animals : 200 mg/kg bw gavage
Group III : 5 animals : vehicle control group (gavage)
Group IV : 5 animals : drinking water control group
Group V : 5 animals : about 8 mg/l drinking water (satured solution)

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

no

Details on study design:

- Rationale for animal assignment: there are not enough data in mammals. To complete a former study, the same animals (Wistar rats) were chosen.

Details on dosing and sampling:

Schedules for blood sampling in the oral gavage dose groups see: "Any other information on materials and methods incl. tables 2

Statistics:

Statistical evaluations (average, standard deviation) were performed using the PC Software Microcal Origin Vers. 3.5 (Microcal Software Inc.).

Preliminary studies:

Three preliminary studies showed low bioaccumulation and a high degree of detoxification by glucuronidation and sulfation of OCT.

Type:

distribution

Results:

After oral gavage OCT is detectable in different tissues.

Type:

distribution

Results:

The OCT drinking water group had no OCT in any tissue.

Details on distribution in tissues:

50 mg/kg dose group: OCT was detected in liver tissue and fat of 3/5 animals treated for 14 days with 50 mg/kg. The mean OCT concentrations measured were about 10 and 7 ng/g tissue for fat and liver, respectively. No OCT was detected in the remaining tissues analyzed.

200 mg/kg dose group: OCT was detected in all tissues of animals treated for 14 days with 200 mg/kg per gavage. The OCT concentration was highest in fat tissue (about 1285 ng/g tissue). In liver, kidney and muscle tissue the OCT concentrations measured were about 87, 71 or 43 ng/g tissue, respectively. Low concentrations of about 9 or 7 ng/g tissue were measured in brain and lung tissue, respectively.
For mean OCT concentration in different tissues see table 5 and figures 1 and 2 of annexed document.

Drinking water group: OCT was detectable in muscle and kidney of one single animal (#21) receiving OCT via drinking water for 14 days. No OCT was measured in any other tissue of animals receiving OCT over a period of 14 or 28 days, nor in animals of the recovery group. The reason for detecting OCT in muscle and kidney tissue, but no other tissue including fat, of one single animal after 14 day drinking water application is unknown. Since OCT is highly lipophilic the highest concentration is expected in fat tissue. Due to the fact that OCT was not detectable in fat tissue and not in animals of the 28 day group this finding is considered to be negligible.

Blood concentration: The OCT concentration in blood sampled at sacrifice of animals of the drinking water group was determined. Although the blood volume used for analysis was increased up to 5 ml, OCT was not detected in concentrations above the limit of detection, which was about 1 ng/ml blood. This results confirm the findings of the previous study BT-95/0125 where OCT was not detected in blood sampled from animals receiving OCT via drinking water for up to 28 days.

Test no.:

#1

Transfer type:

other:

Observation:

distinct transfer

Metabolites identified:

not measured

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results male rats
Concentration of up to 1285 ng OCT/g fat were detected in male rats after repeated oral gavage of 200 mg OCT/kg bw. At this dose group OCT was also detected in other tissues, but not in testes. After repeated oral gavage of 50 mg OCT/kg bw, only trace levels (7 - 10 ng/g tissue) of OCT were detected and only in fat and liver tissue of 3/5 animals.
After drinking water application (800 pg/kg bw/day) for up to 28 days OCT was not detected in any tissue or blood sample.

Executive summary:

In a toxicokinetic study p-(1,1,3,3-tetramethylbutyl)-phenol (Octylphenol PT, OCT) (98 %) was administered to 3 groups of 5 male Wistar rats by repeated gavage at dose levels of 0, 50, 200 mg/kg bw. Another group received OCT saturated water (appr. 8 mg/l) ad libitum.

At the 200 mg/kg bw group OCT was detected in any tissue tested despite testes. The maximum concentration was 1285 ng OCT/g measured in fat. After administration of 50 mg/kg bw traces of OCT (7-10 ng/g tissue) were found in fat and liver tissue. No accumulation of OCT occurs in animals of the drinking water group.

 

This toxicokinetics study in rats is classified acceptable and satisfies essential requirements for a toxicokinetics study (OECD 417) in rats. The study does not cover the total endpoint information, but is only the fourth part of a series of four supplementary studies.

Reference 4

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13.07.1995 - 13.10.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

(The study was designed according to scientifically accepted procedures and according to validated and standardized methods, developed in the testing facility.)

Principles of method if other than guideline:

Determination of the protein Content of the cytosolic fraction and of the liver homogenate by UV-spectrophotometry at 750 nm after reaction with the Folin Ciocalteus Phenol reagent.
Analysis:
OCT was determined by a gaschromatographic analysis using p-tert-butylphenol (BUT) as internal standard, subsequent to liquid/liquid extraction with methyl-tert-butyl ether (MTBE).
Phospho-adenosine-3,5-diphosphate (PAP) that was formed in the sulfotrarisferase assay was analyzed in the cytosolic preparations by HPLC, using a Spectra-Physics HPLC. A Lichrosorb RP 18-5 column (4.6 x 250 mm) with a Lichrosorb RP 18-7 precolumn was used for the analysis.

The glucuronidation rate of OCT was determined by measuring the time dependent decrease of unconjugated OCT in incubation mixtures with liver homogenate and the necessary co-factors.
The OCT sulfation rate was determined by measuring phospho-adenosine-3,5-diphosphate (PAP) that is formed from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) during the assay.
Influence of protein concentration on glucuronidation and sulfation activity has been tested.

GLP compliance:

yes (incl. QA statement)

Remarks:

MNISTERIUM FÜR UMWELT, RAUMORDNUNG UND LANDWIRTSCHAFT DES LANDES NORDRHEIN-WESTFALEN

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fa. Harlan-Winkelmann, D-33167 Borchen
- Age at study initiation: no data
- Weight at study initiation: 160-180 g
- Fasting period before study: no data
- Housing: in Makrolon cages Type IV
- Diet: ad libitum, Ssniff R10 - laboratory standard rat diet (in pellet form), Fa. Ssniff, Spezialfutter GmbH, D-59494 Soest.
- Water: ad libitum, Fa. Gelsenwaser, D-45721 Haltern
- Acclimation period: animals were acclimated for a minimum of 5 days
- number of animals: 2 for preparation of liver homogenate and 2 for preparation of cytosolic fractions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3°C
- Humidity (%): 30-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light; 12/12

Details on study design:

Microsomal and cytosolic liver fractions, as well as the liver homogenate were isolated by means of differential centrifugation

The livers of two animals were cut to pieces and homogenized in ice-cold KC1 solution (1.15 %, 6.5 ml/g liver), followed by a centrifugation at 4 °C and 9000 g for 10 min. The supernatant of this centrifugation step, the so-called "9000 g supernatant", was stored in aliquots of 1 ml each at about -80 °C until use.

For the preparation of the cytosolic liver fraction the livers of two animals were cut to pieces and homogenized in ice-cold KCI solution, followed by a centrifugation at 4 °C and 15000 g for 10 min. The resulting supernatant was subsequently centrifugated at 4 °C and 105000 g for 53 min. The supernatant of this centrifugation step, the cytosolic fraction, was stored in aliquots of 1 ml each at about -80 °C until use. The microsomes were isolated by resuspending the pellet in KCI solution and repeated centrifugation for 53 min (4 °C, 105000 g). The pellet of the last centrifugation step was homogenized with KCI solution, and aliquots of 0.5 ml each were stored at about -80 °C for possible further use.

A saturated solution of OCT in Tris-HCl buffer (pH 7.4) was prepared by dissolving a surplus of OCT for a minimum of 24 hrs at room temperature. Unsoluble residues were removed by filtration (0.2 pm filter). The concentration of OCT was determined by gaschromatographic analysis. Each determination was performed fourfold using different dilutions.

Statistics:

Statistical evaluations (average, standard deviation) were performed using either the PC Software Microcal Origin Vers. 3.5 (Microcal Software Inc.) or Quattro Pro Vers. 4.0 (Borland GmbH).

Type:

metabolism

Results:

Octylphenol is readily detoxified by UDPglucuronosyltransferases and sulfotransferases

Test no.:

#1

Toxicokinetic parameters:

other: Vmax (UDG): 11.03-11.24 nmol/min x mg microsomal protein

Test no.:

#2

Toxicokinetic parameters:

other: Km (UDG): 8.39-8.93 µmol/l

Test no.:

#3

Toxicokinetic parameters:

other: Vmax (Sulfotransferase): 10.53-11.35 nmol/min x mg cytosolic protein

Test no.:

#4

Toxicokinetic parameters:

other: Km (Sulfotranserase): 10.53-11.35 µmol/l

Metabolites identified:

not measured

Details on metabolites:

glucuronidation and sulfation was determined by measuring the decrease of unconjugated OCT

OCT is detoxified by uridine-diphospho-glucuronyltransferases and the sulfotransferases. Kinetic constants Vmax and Km based on the microsomal protein content of the liver homogenate resulted in Vmax and Km values for glucuronidation of 11.03 - 11.24 nmol/(min * mg microsomal protein) and 8.39 - 8.93 µmol/l, respectively.

Vmax and Km values for sulfation were calculated with 2.79 - 2.85 nmol/(min * mg cytosolic protein) and 10.53 - 11.35 µmol/l, respectively.

Conclusions:

Interpretation of results (migrated information): other: rat liver has a high capacity for detoxification of OCT via glucuronidation and sulfation
Vmax and Km for the glucuronidation and sulfation of OCT in vitro have been determined, using liver preparations from male Wistar rats. Glucuronidation and sulfation are generally regarded as important detoxification pathways for phenolic compounds. Rat liver has a high capacity for detoxification of OCT via glucuronidation and sulfation
In contrast, OCT concentrations decreased when active liver homogenate was present in the incubation mixture demonstrating conjugation of OCT with glucuronic acid, a reaction catalyzed by UDP-glucuronosyltransferases.
The sulfation of OCT was determined using rat liver cytosol by comparing the increases in PAP (phospho-adenosine-3,5-diphosphate) concentrations with time in the incubation mixtures in the presence and absence of OCT. Due to the instability of PAPS (3'-phosphoadenosine 5'-phosphosulfate) a slight increase in PAP was observed in the absence of OCT. However, in the presence of OCT a much higher and time dependent increase of PAP indicated reaction of OCT with the activated sulfate (PAPS) catalyzed by sulfotransferases.

In summary, the results demonstrate, that rat liver has a high capacity for detoxification of OCT via glucuronidation and sulfation.

Executive summary:

In a toxicokinetic study (BT-95/0125 -2) the metabolic parameters with regard to glucuronidation and sulfation of p-(1,1,3,3-tetramethylbutyl)-phenol (Octylphenol PT, OCT) (98 %) cytosolic fractions were assessed in vitro, using liver homogenate and cytosolic fractions from male Wistar rats. The study results demonstrate that rat liver has a high capacity for detoxification of OCT via glucuronidation and sulfation.

 

This toxicokinetics study in rat is classified acceptable and satisfies essential requirements for an in vitro studies according OECD guidelines. The study does not cover the total endpoint information, but is the third part of a series of four supplementary studies.

Reference 5

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01.03.1995 - 20.04.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

toxicokinetics

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

(The study was designed according to scientifically accepted procedures and according to validated and standardized methods, developed in the testing facility.)

Principles of method if other than guideline:

Investigation of the toxicokinetics of OCT after repeated oral (gavage) application to male Wistar rats, to assess the bioaccumulation potential.
Additionally the blood concentration of OCT after repeated application via drinking water was investigated.

GLP compliance:

yes (incl. QA statement)

Remarks:

MINISTERIUM FÜR UMWELT, RAUMORDNUNG UND LANDWIRTSCHAFT DES LANDES NORDRHEIN-WESTFALEN

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fa. Harlan-Winkelmann, D-33167 Borchen
- Age at study initiation: no data
- Weight at study initiation: 200-220 g
- Fasting period before study: No fasting period before administration
- Housing: Groups of five in Makrolon cages Type IV
- Diet: ad libitum, Ssniff R10 - laboratory standard rat diet (in pellet form), Fa. Ssniff, Spezialfutter GmbH, D-59494 Soest.
- Water: groups I - IV ad libitum, Fa. Gelsenwaser, D-45721 Haltern; group V tap water saturated with OCT ad libitum
- Acclimation period: animals were acclimated under study conditions except administration for a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3°C
- Humidity (%): 30-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light; 12/12

Route of administration:

other: oral: gavage and drinking water (repeated application)

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Solutions of the test material in the vehicle for the gavage application were prepared once. The concentrations of the test substance were determined after preparation of the solutions, at the mid and at the end of the study.
- Storage temperature of food: room temperature, dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): used in comparable studies
- Concentration in vehicle: 50 mg OCT/ml (for 50 mg/kg dose group, gavage application); 200 mg OCT/ml (for 200 mg/kg dose group, gavage application);

For the treatment of group V, a saturated solution of OCT in tap water was prepared weekly (day 1 - 14 of application) or once (day 15 - 28 of application), respectively, by dissolving a surplus of OCT in tap water, stirring overnight, and removal of unsoluble residues by filtration.
The concentrations of these solutions were determined weekly.

Duration and frequency of treatment / exposure:

After acclimatization the animals of group I and II received gavage applications of 50 or 200 mg/kg b.w. OCT once per day for 14 consecutive days.
Other animals received drinking water satured with OCT for 14 and 28 days, respectively.

Remarks:

Doses / Concentrations:
Repeated gavage application: 50 mg/kg resp. 200 mg/kg bw
drinking application ad libitum: mean concentration = 8.39 mg/l / standard deviation = 1.182 mg/l (for average doses see Table 6 in annexed document)
One control groups received the vehicle only, another control group received pure water.

No. of animals per sex per dose / concentration:

Group I : 5 males : 50 mg/kg bw gavage
Group II : 5 males : 200 mg/kg bw gavage
Group III : 5 males : vehicle control group (gavage)
Group IV : 5 animals : drinking water control group
Group V : 5 animals : about 8 mg/l drinking water (satured solution)

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

no

Details on study design:

- Rationale for animal assignment: there are not enough data in mammals. To complete a former study the same animals (Wistar rats) were chosen.

Details on dosing and sampling:

Schedules for blood sampling in the oral gavage dose groups
Group Animal dose (mg/kg) 1.Sample-Time 2.Sample-T. 3.Sample-T. 4.Sample-T. 5.Sample-T. 6.Sample-T. 7.Sample-T. 8.Sample-T.
I 01-05 50 10 min 30 min 60 min 2 h 4 h 6 h 8 h 23 h
II 06-10 200 10 min 30 min 60 min 2 h 4 h 6 h 8 h 23 h



Animals of group V (15 animals) received drinking water saturated with OCT
# of animals application period days of blood sampling during application phase recovery phase
#21 - #25 14 days days 2, 5, 9, 12 none
#26 - #29 28 days days 20 and 27 none
#30 - #31 28 days days 20 and 27 7 days
#32 - #33 28 days days 20 and 27 14 days
#34 - #35 28 days days 20 and 27 21 days
Urine and Feces were collected and stored for further determination.
Determinations of Urine and feces were not part of this study.

Tissues (liver, kidney, including adrenals, lung, testes, including epididymides, brain, stomach, caecum, colon, rectum, sceletal muscle tissue (thigh), abdominal fat) were taken, weighted and stored for further use, but not for investigation in the fram of this study.

Measurement of concentrations in blood samples. Blood was taken under light ether anaesthesia from the orbitus sinus.

Statistics:

Statistical evaluations (average, standard deviation, student's t-test) were performed using the PC Software Microcal Origin Vers. 3.5 (Microcal Software Inc.).

Type:

absorption

Results:

rapid uptake from the GI tract; detectable as early as 10 min after gavage

Type:

absorption

Results:

maximal blood level: 50-70 ng/ml in the 50 mg/kg bw group

Type:

absorption

Results:

maximal blood level: 80-100 ng/ml in the 200 mg/kg bw group

Type:

absorption

Results:

similar blood concentration profiles at day 1 and day 14: indicating that repeated oral gavage did not lead to increased blood concentrations

Type:

absorption

Results:

substance not detected in any blood sample from animals receiving OCT via saturated drinking water (0.8 mg/kg bw)

Details on absorption:

In the oral gavage groups, OCT was detected as early as 10 min after administration, indicating rapid uptake from the gastrointestinal tract. In the 50 mg/kg and the 200 mg/kg dose groups maximal blood levels of about 50 - 70 ng/ml and 80 - 100 ng/ml blood were determined, respectively. These blood levels decreased during the 24 hours after administration, but OCT was still detected at low concentrations prior to the next application. However, the blood concentration-time profile at day 14 was similar to the profile obtained at day 1, indicating that repeated oral gavage administration did not lead to increased blood concentrations.

OCT was not detected in any blood sample from animals which received OCT via drinking water as saturated solution for up to 28 days.

Test no.:

#1

Toxicokinetic parameters:

C(time): 50-70 ng/ml during first 2h after gavage (50 mg/kg bw group)

Test no.:

#2

Toxicokinetic parameters:

C(time): 80-100 ng/ml during first 6h after gavage (200 mg/kg bw group)

Metabolites identified:

not measured

The OCT blood profile after the first administration of 50 and 200 mg OCT/kg bodyweight in this study was different compared to the blood profile after single administration in the previous study (Huels BT-94/0125). This might, at least in part, be due to the fact, that the animals of the single administration study were kept fasted overnight, whereas the animals of the study with repeated administration were fed before the first application. It can be assumed, that the feeding status has a great impact on the resorption behaviour, especially for lipophilic substances.

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
Octylphenol is detectable in the blood 10 min after gavage, indicating rapid uptake from the GI tract. Repeated administration of 50 and 200 mg OCT/kg bodyweight does not increase blood concentrations with time. Administration via saturated drinking water (0,8 mg/kg bw) over a period of up to 28 days did not result in detectable levels of OCT in blood.

The results are in agreement with the hypothesis of a rapid elimination of OCT after oral ingestion, presumably via glucuronidation and sulfation in the liver. Only if these detoxification pathways are saturated excessive doses may lead to bioaccumulation. The results obtained during this study with repeated administration of OCT to male Wistar rats confirmed the results and conclusions of the study with single administration (Hüls BT-94/0125), indicating a low potential for bioaccumulation in male rats.

Executive summary:

In a toxicokinetic study (BT-95/0125) p-(1,1,3,3-tetramethylbutyl)-phenol (Octylphenol PT, OCT) (98 %) was administered to 3 groups of 5 male Wistar rats repeated by gavage at dose levels of 0, 50, 200 mg/kg bw. Although OCT residues were measured 24 h after administration, repeated administration did not increase blood concentrations.

An addition OCT was administration via saturated drinking water (appr. 8 mg OCT/l). The application did not result in detectable amounts of OCT in the blood.

 

This toxicokinetics study in rats is classified acceptable and satisfies essential requirements for a toxicokinetics study OECD 417 in rats. The study does not cover the total endpoint information, but is only the second part of a series of four supplementary studies.

Reference 6

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.12.1994 - 03.02.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

toxicokinetics

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

(The study was designed according to scientifically accepted procedures and according to validated and standardized methods, developed in the testing facility.)

Principles of method if other than guideline:

Determination of the bioavailability of p-(1,1,3,3-tetramethylbutyl)-phenol after single administration in male Wistar rats.
In addition, blood concentration profiles were obtained after oral and intravenous application to assess the potention of the substance to bioaccumulate in the rat.
The substance concentration in the blood samples was determined by a specially developed gas chromatographic method.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Bescheinigung vom Ministerium für Umwelt, Raumordnung und LAndwirtschaft des Landes Nordrhein-Westfalen

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fa. Harlan-Winkelmann, D-33167 Borchen
- Age at study initiation: no data
- Weight at study initiation: 180-200 g (up to 250 g for additional animals in groups I and III)
- Fasting period before study: Animals of the p.o. dose groups were retained from food appr. 16 h before dosing
- Housing: Groups of two (untreated) or three (dosage groups and remainder) in Makrolon cages Type IV
- Diet: ad libitum, Ssniff R10 - laboratory standard rat diet (in pellet form), Fa. Ssniff, Spezialfutter GmbH, D-59494 Soest.
- Water: ad libitum, Fa. Gelsenwaser, D-45721 Haltern
- Acclimation period: animals were acclimated under study conditions except administration for a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3°C
- Humidity (%): 30-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light; 12/12

Route of administration:

other: oral gavage and intravenous injection

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): preparation freshly on application day
- Storage temperature of food: roomtemperature, dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): used in comparable studies
- Concentration in vehicle: 5 mg OCT/ml (for i.v. dosing); 50 mg OCT/ml (for 50 mg/kg dose group, gavage application); 200 mg OCT/ml (for 200 mg/kg dose group, gavage application);

Duration and frequency of treatment / exposure:

single gavage application & single injection intravenous

Remarks:

Doses / Concentrations:
single gavage application: 50 mg/kg resp. 200 mg/kg bw
single injection intravenous: 5 mg/kg bw
control groups received the vehicle only (p.o.)

No. of animals per sex per dose / concentration:

Group I : 6 males : 5 mg/kg i.v.
Group Ic : 2 males vehicle p.o.
Group II : 6 males : 50 mg/kg p.o.
Group IIc: 2 males vehicle p.o.
Group III : 6 males : 200 mg/kg p.o.
Group IIIc: 2 males vehicle p.o.

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

no

Details on study design:

Various xenobiotics have recently been reported to show estrogenic activity, since in several in vitro- and in vivo-test systems they mimic effects of estradiol. Those substances are suspected to cause health effects, especially negative effects an reproduction and tumor incidences, in
both humans and wildlife through disruption of the endocrine system. One group of chemicals showing estrogenic activity both in vivo and in vitro are p-alkylated phenols (AP). Although the estrogenic activity of those APs is extremely low (about 4-5 magnitudes lower than the physiological estrogen estradiol-17ß), no data regarding possible bioaccumulation of APs in mammals exist. Bioaccumulation studies in aquatic animals revealed bioconcentration factors (defined as ratio of the concentration in the organism to concentration in the water) up to 300. Therefore, although the estrogenic potency of APs is very low, it is discussed that due to bioaccumulation of APs estrogenic efficient blood levels could be reached.

Because of that Wistar rats were chosen for the investigation.

Details on dosing and sampling:

Measurement: Concentration in blood samples
For blood sampling time see table 2 of annexed document
Blood was taken under light ether anaesthesia from the orbitus sinus.
Within every group subgroups of 3 rats each were formed. Within every subgroup blood samples were taken at 7 different time points after application.

Statistics:

Reproducibility:
Calculation of U (area of variation; defined as interval containing the results with a probability of P=95%
U=(100xtP/Cmean)xs
tP=student factor (p=0,05)
Cmean=average concentration
s=standard deviation

Toxicokinetic parameters:
statistical evaluation: average and standard deviation curve fitting determination of the Area under the blood Concentration Time curve (AUC) by integrating the respective curve (see eq. 1 calculation instruction of annexed document) using the PC Software Microcal Origin Vers. 3.5 (Microcal Software Inc.)

Type:

other: bioavailability

Results:

2% of 50 mg/kg (gavage)

Type:

other: bioavailability

Results:

10% of 200 mg/kg (gavage)

Details on absorption:

100 µl of blood of untreated animals was spiked with three different amounts of OTC in duplicate (50ng, 500 ng and 2 µg).
Recovery rates were estimated to taget concentration:
a) 50 ng/100 ml blood => measured concentration: appr. 25 ng/100 µl
=> recovery rate appr. 50%
b) 500 ng/100 ml blood => measured concentration: appr. 56 ng/100 µl
=> recovery rate appr. 56%
c) 2000 ng/100 ml blood => measured concentration: appr. 75 ng/100 µl
=> recovery rate appr. 75%

overall recovery rate = 60,5 % ,
standard deviation = 11,65 %

However, the results indicate, that the recovery of OCT increases with increasing OTC concentrations in the samples.

Test no.:

#1

Toxicokinetic parameters:

AUC: 0.433 µg*h/ml (group I: 5 mg/kg i.v.)

Test no.:

#2

Toxicokinetic parameters:

AUC: 0.086 µg*h/ml (group II: 50 mg/kg oral gavage)

Test no.:

#3

Toxicokinetic parameters:

AUC: 1.778 µg*h/ml (group III: 200 mg/kg oral gavage)

Test no.:

#4

Toxicokinetic parameters:

Cmax: 40 ng/ml (max blood level) within 20 min after single gavage of 50 mg/kg; 3-7 ng/ml after 4- 6h

Test no.:

#5

Toxicokinetic parameters:

Cmax: highly variable max blood level within up to 480 min after single gavage of 200 mg/kg; OCT not detectible after 32 or 48 h

Test no.:

#6

Toxicokinetic parameters:

half-life 1st: 310 min (elimination half-life by fitted to 3 compartment model)

Metabolites identified:

not measured

For further results see results in annexed document.

From area under the blood concentration-time curve the bioavailability can be calculated using the following equation:

F=(AUCoral x dose i.v.)/(dose oral x AUC i.v.)

Oral bioavailability:

F(50 mg/kg bw oral gavage) = 0,02 = 2%

F(200 mg/kg bw oral gavage) = 0,10 = 10%

=> low bioavailability

One reason might be that OCT is only incompletely resorbed from the gastrointestinal tract after oral application, due to the low solubility of OCT in aqueous media. However, the increasing bioavailability at the high dose is difficult to explain by this assumption. Another reason might be a marked liver first pass effect, i.e. extensive metabolism of OCT in the liver during the first passage through the liver. If this assumption is valid, the increased bioavailability at the high dose may be explained by saturation of elimination processes in the liver, resulting in higher OCT blood concentrations. Both explanations have to be further investigated.

The blood concentration-time curve can be mathematically described using a three-compartment model (see figure 1 in annexed document)

The best fit was achieved using the parameters:

A: 2218.91 alpha: 0.35782

B: 419.10 beta: 0.0335

C: 12.80 gamma: 0.00223

Model:

In the model used, the body is assumed to consist of 3 different compartments. These compartments are a central compartment, normally the blood, a shallow peripheral compartment (e.g. richly perfused tissues) and deep peripheral compartment (i.e. slowly perfused tissues. especially fat tissue).

The substance enters the central compartment via resorption from the gastrointestinal tract or via intravenous injection and is rapidly distributed throughout the richly perfused organs, the shallow peripheral compartment. In this phase the blood level rapidly decreases until in a second phase this decrease slows down. The second phase can be described by slow distribution of the substance into the deep peripheral compartment. The third part of the blood concentration-time curve is determined mostly by elimination processes from the body.

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
The bioaccumulation potential of OCT after injection of 5 mg/kg bw seems to be negligible. After oral application of high doses, saturating the detoxification pathways, a bioaccumulation potential of OCT cannot be excluded.

Executive summary:

In a toxicokinetic study p-(1,1,3,3 -tetramethylbutyl)-phenol (98 %) was administered to 3 groups of 6 male Wistar rats either receiving a single gavage application of 50 mg or 200 mg OCT/kg bw or a single intravenous injection of 5 mg/kg bw. Blood concentration profiles were used to assess the potential of OCT to bioaccumulate.

The elimination rate was found to be 0.00223, resulting in a half life for elimination of t1/2 = 310 min. After approximately 24 hours more than 95 % of the substance already has been eliminated. However, after application of higher doses (200 mg/kg oral gavage) OCT was detectable in blood samples of single animals 24 - 48 hours after dosing. Whether these data show a bioaccumulation potential which is relevant for doses expected for human exposure is questionable. The high dose probably resulted in saturation of elimination processes. In addition, the blood levels detected 24 - 48 hours after dosing were found to be very low, i.e. in the range of the detection limit of 1 - 5 ng/ml blood. These data indicate that the bioaccumulation potential of OCT in the blood after intravenous injection of 5 mg/kg bw seems to be negligible, whereas after oral application of high doses, saturating the detoxification pathways, a bioaccumulation potential of OCT cannot be excluded.

This toxicokinetics study in rats is classified acceptable and satisfies essential requirements for a toxicokinetics study (OECD 417) in rats. The study does not cover the total endpoint information, but is only the first part of a series of four supplementary studies.

Reference 7

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Maternal exposure to 600 mg/kg OP during late pregnancy increased the expressions of CaBP-9k mRNA and protein in maternal and neonatal uteri.

Objective of study:

other: Expression of Calbindin-D(9k) (CaBP-9k)

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effect of OP on the expression of CaBP-9k following maternal exposure during late pregnancy in maternal and fetal uteri. After administration of a high dose (600 mg/kg bw) of OP, CaBP-9k mRNA in maternal uteri was induced at day 5 of lactation. CaBP-9k mRNA was also induced after treatment with a high dose of OP (600 mg/kg bw) which is during late pregnancy transferred from the mother and exposed to the fetuses, and persisted until day 5 of lactation.

GLP compliance:

not specified

Species:

rat

Strain:

not specified

Sex:

female

Details on test animals or test system and environmental conditions:

no data

Route of administration:

other: no data

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Duration: from late pregnancy until day 5 of lactation

Remarks:

Doses / Concentrations:
600 mg/kg bw

No. of animals per sex per dose / concentration:

no data

Control animals:

not specified

Positive control reference chemical:

no data

Treatments with high doses of OP (600 mg/kg bw) reduced the expression of maternal estrogen receptor aplpha (ERalpha) mRNA and induced the reduction of neonatal ERalpha mRNA expression. Furthermore, the there was a significant decrease in the expression of CaBP-9k protein.

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Absorption and distribution of OP in maternal and neonatal uteri are extremely rapid, and OP can easily pass though the placenta during pregnancy to affect functions of neonatal reproductive tissues.

Executive summary:

The effect of OP on the expression of CaBP-9k following maternal exposure during late pregnancy in maternal and fetal uteri was examined in this study. After administration of a high dose (600 mg/kg bw) of OP, CaBP-9k mRNA in maternal uteri was induced at day 5 of lactation. CaBP-9k mRNA was also induced after treatment with a high dose of OP (600 mg/kg bw) which is during late pregnany transferred from the mother and exposed to the fetuses, and persisted until day 5 of lactation.

Treatments with high doses of OP (600 mg/kg bw) reduced the expression of maternal estrogen receptor aplpha (ERalpha) mRNA and induced the reduction of neonatal ERalpha mRNA expression. Furthermore, the there was a significant decrease in the expression of CaBP-9k protein.

To conclude, absorption and distribution of OP in maternal and neonatal uteri are extremely rapid, and OP can easily pass though the placenta during pregnancy to affect functions of neonatal reproductive tissues.

Reference 8

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

metabolism

toxicokinetics

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Principles of method if other than guideline:

Details on test methods are described in the related study reports Hüls BT-94/0125 & BT-95/0125.
Blood and tissue analysis: in vivo
Metabolism: in vitro

GLP compliance:

yes

Radiolabelling:

not specified

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkel-mann (Borchen, Germany).
- Weight at study initiation: 200—220 g
- Fasting period before study: only dose group for single administration was excluded from food for approximately 16 h prior dosing.
- Housing: Makrolon type IV cages five rats/cage
- Diet (e.g. ad libitum): Ssniff R 10 diet, Ssniff Spezialfutter GmbH, Soest, Germany
- Water (e.g. ad libitum): ad libitum tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +- 3 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: oral: gavage; oral: drinking water; intravenous

Vehicle:

propylene glycol

Duration and frequency of treatment / exposure:

Oral: gavage; single gavage and multiple gavage (14 consecutive days)
Oral: drinking water; 28 days, water consumption was measured daily
Intravenous: single intravenous injection

Remarks:

Doses / Concentrations:
Oral: gavage;
single: 0, 50, 200 mg OP/kg bw
multiple: 50, 200 mg OP/kg bw

Oral: drinking water;
concentration: approximately 8 mg/l;
mean daily dose ingested: approximately 800 µg/kg


Intravenous
5 mg OP/kg bw

No. of animals per sex per dose / concentration:

Oral: gavage;
single gavage: groups of six males
multiple gavage (14 consecutive days): groups of 5 male rats

Oral: drinking water; 5 rats per group

Intravenous: six males

Control animals:

yes, concurrent vehicle

Test no.:

#1

Toxicokinetic parameters:

Cmax: 1970 ng/ml (i.v.)

Test no.:

#1

Toxicokinetic parameters:

AUC: 0,433 µg * h / ml (i.v.)

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 310 min (i.v.)

Test no.:

#1

Toxicokinetic parameters:

other: elimination rate: 0.00223 (i.v.)

Test no.:

#2

Toxicokinetic parameters:

Cmax: 40 ng/ml (oral/gavage 50 mg/kg)

Test no.:

#2

Toxicokinetic parameters:

AUC: 0.086 µg * h / ml (oral/gavage 50 mg/kg)

Test no.:

#3

Toxicokinetic parameters:

AUC: 1.778 µg * h / ml (oral/gavage 200 mg/kg)

Metabolites identified:

yes

Details on metabolites:

OP is metabolized via glucuronidation and sulphation, the major detoxification pathways for phenolic compounds.
A Vmax of 11.24 nmol/(min * mg microsomal protein) and a Km of 8.77 lmol/l were calculated from the plots for enzyme catalysed OP glucuronidation.
For enzyme catalysed sulphation, a Vmax of 2.85 nmol/(min * mg protein) and a Km of 11.35 lmol/l were calculated.
=> from Lineweaver-Burk and Eadie-Hofstee plots

- Using the AUC values obtained from the blood profiles of the different treatment groups oral bioavailability of 2 & 10 % were calculated for the 50 and 200 mg/kg dose groups, respectively.

- The blood profiles after repeated administration do not show significant differences.

- OP could be confirmed in different tissues after repeated administration, predominantly in fat; at higher concentrations also in liver, kidney, brain, lung, and muscle, but not in testes.

Conclusions:

Interpretation of results (migrated information): other: No bioaccumulation at low doses of OP in the rat
The current study demonstrated a lack of bioaccumulation potential for low OP doses in the rat. This result was first obtained in experiments using single administration and was further corroborated by experiments with repeated OP administration. Metabolism studies in vitro provided a mechanistic explanation for this observation. OP was eliminated after a single i.v. injection with a half-life of 5 h, indicating a rather rapid removal of OP in the rat. Based on the AUC-ratios observed for animals treated with single oral (gavage) doses of 50 or 200 mg/kg and animals treated with an i.v. dose of 5 mg/kg, we calculated oral bioavailabilities of 2 and 10 %, respectively. The large variations which were observed especially with the low blood concentrations after gavage administration indicate caution in the interpretation of the data. However, the low bioavailability was tentatively attributed to a marked liver first-pass effect, i.e. extensive metabolism of OP in the liver during the first passage through the liver. The increased bioavailability after application of higher doses may consequently be explained by the saturation of elimination processes in the liver, resulting in higher OP blood concentrations over a prolonged period of time.
The analysis of OP-content in tissues indicates that fat will act as storage tissue at high doses.
Ingestion via drinking water saturated with OP over a period of up to 28 days did not result in detectable levels of OP in blood. The fact that OP was not even detected in fat from animals receiving the test compound for 28 days gives further support to the hypothesis that low oral doses of OP are efficiently removed during the first passage through the liver.
OP was demonstrated to be rapidly conjugated via glucuronidation and sulphation, in accordance with observations on the metabolism of other phenolic compounds in mammalian livers, including human liver . (see table 3 of annexed document) The Vmax and Km values listed in Table 3 have been determined using liver fractions from rats and humans. The values are of the same order of magnitude compared to the data obtained in the experiments described here. Therefore glucuronidation and sulphation are most likely also to be the predominant pathways for detoxification of OP in vivo, a conclusion supported by preliminary results from the analysis of urine and faeces obtained from the rats receiving OP via gavage or drinking water.

Executive summary:

In a toxicokinetics study in male Wistar rats p-(1,1,3,3-Tetramethylbutyl)-phenol was administered to groups of 6 respectively 5 male Wistar rats/dose as single gavage, multiple gavage and i.v. injection at dose levels of 0, 50, 200 mg/kg bw/day.

 

The publication summarizes 4 study reports conducted by Hüls (BT-94/0125 & BT-95/0125-1 to 3). The author concludes that OP does not bioaccumulate in rats when administered at low concentrations. Octylphenol was demonstrated to be rapidly conjugated via glucuronidation and sulphation. At high concentrations there is a potential of bioaccumulation, predominantly in fat tissue. It is suggested that this is due to the saturation of metabolic capacity of the detoxification pathway.

Description of key information

Short description of key information on bioaccumulation potential result: 
In a set of four consecutive studies (Hüls BT-94/0125, BT-95/0125.13-) toxicokinetic parameters of p-(1,1,3,3-tetramethylbuty1)-phenol (OCT) were determined in male Wistar rats.
Short description of key information on absorption rate: 
The dermal absorption of radioactive labelled Nonylphenol and two nonylphenol ethoxylates (NPE-4, NPE-9) was tested in an ex vivo isolated perfused porcine skin flap (IPPSF) assay and an in vitro porcine skin flow  through (PSFT) diffusion cell assay.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - dermal (%):

1

Additional information

Blood concentration profiles obtained after single oral and intravenous application were used to assess the potential of p-(1,1,3,3 -tetramethylbuty1)-phenol (OCT) to bioaccumulate in rats. The peak blood concentration was about 1970 ng/ml blood after single intravenous (i.v.) application of 5 mg/kg bw. The blood level rapidly decreased within 30 minutes after application. OCT was not detectable in the blood samples 6 - 8 hours after application. Fitting the data of blood concentration-time curves resulted in an elimination half-life (t1/2) of 310 min.

After single administration by gavage of 50 mg/kg bw a maximal blood level of about 40 ng/ml was reached within 20 min. Within 4 - 6 hours after dosing, blood levels decreased to 3 – 7 ng/ml. After oral application of 200 mg/kg, the blood concentration-time profiles of individual animals showed a high variation. The blood profiles indicated the presence of two peak blood concentrations, i.e. at 45 - 120 and 240 - 480 min after dosing, respectively, suggesting enterohepatic circulation. OCT was not detectable in blood samples of several animals 32 hours after dosing, while other animals showed low OCT concentrations in the blood 48 hours after dosing. The oral bioavailability was found to be 2 % and 10 % for the 50 mg and 200 mg dose group, respectively. This low bioavailability might be due to incomplete resorption from the gastrointestinal tract due to the low solubility of OCT in aqueous media or due to a marked first pass effect in liver or intestinal tissue.

The large variations observed in blood at low OCT-concentrations following gavage administration suggest caution in the interpretation of the data. However, the increased bioavailability after application of higher doses may be explained by saturation of elimination processes, resulting in higher OCT blood concentrations over a prolonged period of time.

The half-life of OCT elimination after single intravenous injection was 310 min: Using this value, an elimination of more than 95 % of OCT within 24 hours after intravenous application of 5 mg/kg is calculated. This calculation in addition to the blood concentration-time curves observed after single gavage application provides an indication regarding the bioaccumulation potential of OCT: At doses which are in the range of occupational and/or environmental relevant levels, the exposure to OCT should not lead to bioaccumulation. However, doses which are orders of magnitudes higher, a bioaccumulation of OCT may be expected.

In addition to the single oral and i.v. application repeated oral gavage administrations of 50 and 200 mg OCT/kg bw was investigated. And additional group of rats received OCT via drinking water saturated with 8 mg OCT/l, corresponding to a mean daily dose of 800 pg/kg over a period of 28 days. In the oral gavage groups, OCT was detected in the blood as early as 10 min after administration, indicating rapid uptake from the gastrointestinal tract. In the 50 mg/kg and 200 mg/kg dose group maximal blood levels of about 50 – 70 ng/ml and 80 – 100 ng/ml blood were determined, respectively. These blood levels decreased during the 24 hours after administration, but OCT was still detected at low concentrations prior to the next application. However, the blood concentration-time profile at day 14 was similar to the profile obtained at day 1, indicating that repeated oral gavage administration did not lead to increased blood concentrations.

OCT was not detected in blood samples from animals which received OCT via drinking water.

The results obtained during the study with repeated administration of OCT to male Wistar rats confirmed the results and conclusions of the study with single administration, that OCT has a low potential for bioaccumulation in male rats.

The same setup was used to assess OCT concentrations in tissues, obtained at sacrifice from animals receiving repeated dose oral gavage of 50, 200 mg OCT/kg bw, and ~800 pg/kg bw via drinking water, respectively. In the 50 mg/kg oral gavage group, OCT was detected in fat and liver tissue in 3 of 5 animals, with an average concentration of 10 and 7 ng/g tissue respectively. OCT has not been detected in any other tissues analyzed. In the 200 mg/kg oral gavage group, OCT was found in all tissues analyzed, but not in testes. The highest concentration was found in fat tissue (about 1285 ng/g), followed by liver, kidney, and muscle tissues with average concentrations of 87, 71, or 43 ng/g tissue. Low concentrations of about 9 or 7 ng/g tissue were found in brain and lung tissue. In the drinking water group OCT was not detected in any tissue.

The glucuronidation and sulfation of OCT has been characterized with regard to V_max and K_m in vitro, using liver preparations from male Wistar rats. Glucuronidation and sulfation are generally regarded as important detoxification pathways for phenolic compounds. It was investigated whether OCT is also metabolized via these pathways. The results demonstrate that rat liver has a high capacity for detoxification of OCT via glucuronidation and sulfation.

In a complementary in vitro study Moffat et al. (2001) showed that glucuronidation eliminates the estrogen-like activity of OCT. It is likely, therefore, that the weak estrogen-like activity noted for OCT at high doses in rats reflects the saturation of glucuronide conjugation. At concentrations present in the environment this metabolic saturation is unlikely to occur, thus enabling glucuronidation of OCT removes the ability of these chemicals to mimic biological estrogens in humans. It is therefore concluded that at the expected exposure levels, the potential endocrine hazard posed by OCT to humans is likely to be negligible.

Upmeier et al. (1999) rerun part of the toxicokinetic studies conducted in male Wistar rats using female DA/Han rates. Compared to the previous data set the biological half-life of OCT was found to be consistently longer. The final (Gamma-phase) t1/2 upon i.v. administration of 5 mg/kg was 310 min (5.17 h) in male Wistar rats (Certa et al. 1996), but 36.1 hours in female DA/Han rats. This difference is supported by data obtained after oral administration. This would indicate not only gender but also strain differences. The data show peculiarities in the time-course of OCT blood levels after oral administration of OCT, which indicate extensive enterohepatic circulation of this compound.

Conclusion:

OCT does not bioaccumulate in rats when administered at low concentrations. The substance is rapidly eliminated by a conjugated system of glucuronidation and sulfation. At high concentrations there is a potential of bioaccumulation, predominantly in fat tissue. It is suggested that this is due to the saturation of the metabolic capacity of the detoxification pathway.

Discussion on absorption rate:

The dermal absorption of nonylphenol (NP) was tested in an ex vivo perfused porcine skin flap model (IPPSF). This model has earlier been shown to be a reliable tool to predict human exposure because of its intact vasculature, as well as anatomical and physiological similarities to human skin.

NP in aqueous PEG-400 solutions is absorbed at 0.1% of the applied dose after 8h, representing the amount of systemic available compound. It has been shown earlier that this rate was not significantly altered by using different vehicles (PEG-400 vs. water) or concentrations (0.1, 1.0, or 10%). The value is therefore considered to be valid also for higher concentrations.

About 0.75% of the applied dose penetrated the stratum corneum and underlying dermis within 8 h. In a worst case scenario all of the penetrated substance could subsequently be absorbed leading to an increased systemic exposure. This estimate does not account for a loss of stratum corneum due to exfoliation. However, the overall potential systemic exposure from skin contact to NP is still considerable less than 1%. This is in accordance with previous results from anin vitroporcine skin flow through (PSFT) diffusion cell assay. Due to the structural similarity between NP and 4-tert-octylphenol it is assumed that 4-tert-octylphenol shows a comparable low absorption rate.