Tetrasodium 4-amino-5-hydroxy-6-[[2-methoxy-5-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. Dec. 1987 to 21. March 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14. Dec. 1987 to 30. Sep 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

yes

Remarks:

metabolism determined in separate study

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

bisphenyl-U-14C

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 160 to 230 g
- Fasting period before study: -
- Housing: single (excretion) 2/cage (plasma levels, exhalation)
- Individual metabolism cages: yes (excretion) / no (plasma levels, exhalation)
- Diet: Altromin 1321 ad libitum
- Water: tap ad libitum
- Acclimation period: -


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26
- Humidity (%): 30 to 50
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): -


IN-LIFE DATES: From: 14. Dec To: 24. Dec. 1987
23. Sep. To: 26. Sep. 1988

Route of administration:

other: oral gavage and intravenous

Vehicle:

other: water and NaCl

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): - oral: 10 mg/kg bw
- IV: 1 mg/kg bw
- concentration (if solution): - oral: 2 mg/g in aqua bidest
- IV: 0.3 mg/g in NaCl solution

Duration and frequency of treatment / exposure:

single dose

Dose / conc.:

10 mg/kg bw (total dose)

Remarks:

p.o.: 10 mg/kg body weight (nominal) - Concentration: 2 mg/g solution

Dose / conc.:

1 mg/kg bw (total dose)

Remarks:

i.v.: 1 mg/kg body weight (nominal) - Concentration: 0.3 mg/g solution

No. of animals per sex per dose / concentration:

Exhalation: 2
Plasma levels: 5
Excretion/remaining concentration p.o.: 5
Excretion i.v.: 3

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

- Tissues and body fluids sampled: urine, faeces, blood, plasma, exhalate, tissues (spleen, stomach, small intestines, liver, kidneys, gonads, heart, lungs, skeletal muscle, subcutaneous fat, retroperitoneal fat, brain, eyes)
- Time and frequency of sampling:
- Blood sampling (tip of tail): 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 24, 32, 48, 72, 96, 120, 144, 168 h after test item administration
- Urine sampling: in polyethylen bottles 0 -2, 2 -4, 4 -8, 8 -24, 24 -48, 48 -72, 72 -96, 96 -120, 120 -144, 144 -168 h after test item administration
- Feces sampling: in glass vessels: 0 - 24, 24 -48, 48 -72, 72 -96, 96 -120, 120 -144, 144 -168 h after test item administration
- Organ/tissue sampling: directly after sacrifice - 7 days after test item administration
- Exhaled air: continuous aspiration at 0.2 m³/h
- Method type(s) for detection: Liquid scintillation counting
- Limits of detection and quantification: determination of blank value

Statistics:

-

Preliminary studies:

NA

Details on absorption:

oral: 28.6%

Details on distribution in tissues:

Blood: 0.25 µg equivalent/mL
Liver: 0.18 µg equivalent/g
Kidneys: 0.10 µg equivalent/g
All other organs: < 0.1 µg equivalent/g

In sum 0.28% of the administered dose; with mean values of 0.13% in the blood and 0.10% in the liver

Details on excretion:

Oral administration:
Excretion mainly via feces: 83.71% of the administered dose
Renal excretion: 14.79% of the administered dose
Bi-phasic elimination
no excretion by exhalation

Intravenous administration:
Main excretion renal: 51.72% - bi-phasic
Fecal excretion: 26.5%

Metabolites identified:

no

Remarks:

Metabolites were identified in the metabolism study

Details on metabolites:

see metabolism study HOE 88.1064

Conclusions:

Incomplete absorption (ca. 28.6 %) after oral administration. About 85% of the administered dose were excreted via faeces and about 15% via urine. The highest doses of radioactivity were found in blood and liver, 7 days after test item administration (about 0.28 % of the administered dose together with all other organs) . After intravenous administration, about 52% of the radioactivity were found in urine within 3 days after test item administration.

Executive summary:

The kinetics of ¹⁴C-labeled Reactive Black 5 was investigated in 5 male rats after oral gavage of 10 mg/kg body weight. For assessment of the absorption rate after oral gavage, one group of 3 rats were treated intravenously with 1 mg/kg bw.

The mean absorption rate was 28.6% - determined by comparison of the renal excretion after oral and IV administration. The slow increase of radioactivity resulted in similar Cmax of 0.7 to 0.8 µg equivalent/g after 5.6 hours in plasma and 21 hours in blood. T1/2 were 5 and 34 hours in plasma and about 8 days in blood, leading to the assumption of of binding of Reactive Black 5 to, presumably, erythrocytes.

After oral administration, excretion of radioactivity was mainly via feces (80% to 88% within 7 days); renal excretion was about 11% to 18% in a bi-phasic manner with half-times of 4 to 7 hours and 40 to 69 hours, respectively.

The highest remaining concentration after 7 days was found in blood (0.25 µg eq/mL) and liver (0.18 µg eq/g). TS levels in kidneys (0.1 µg eq/g and other tissues were lower. Totally, 0.28% of the administered dose were found in blood and tissues. The mean over-all recovery rate was 99% of the administered dose.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Objective of study:

excretion

metabolism

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

Tier 1

Deviations:

yes

Remarks:

exhalation determined in separate study

Principles of method if other than guideline:

Determination of radioactivity in the excreta of rats. Identification of metabolites in the excreta.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 4-amino-5-hydroxy-3,6-bis[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

EC Number:

241-164-5

EC Name:

Tetrasodium 4-amino-5-hydroxy-3,6-bis[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

Cas Number:

17095-24-8

Molecular formula:

C26H25N5O19S6.4Na

IUPAC Name:

Tetrasodium 4-amino-5-hydroxy-3,6-bis[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): HOE CG 0062-14C

Radiolabelling:

yes

Remarks:

bisphenyl-U-14C

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: -
- Weight at study initiation: 230 g
- Fasting period before study: -
- Housing: 2 per cage
- Individual metabolism cages: no
- Diet: Altromin 1321 ad libitum
- Water: tap ad libitum
- Acclimation period: 1 to 2 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%): 30 - 70%
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): -

IN-LIFE DATES: From: 21. Dec. 1987 To: 24 Dec. 1987

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg/kg bw
- concentration (if solution): 1.836 mg/g

Duration and frequency of treatment / exposure:

single dose

Doses / concentrations

Dose / conc.:

10 mg/kg bw (total dose)

Remarks:

10 mg/kg body weight (nominal) = 9.94 mg/kg bw actual dose

No. of animals per sex per dose / concentration:

5 males

Control animals:

no

Positive control reference chemical:

-

Details on study design:

TS was dissolved in bidistilled water (5 minutes ultrasound ) to a final concentration of 1.836 mg/g

Details on dosing and sampling:

- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: 0-24 h, 24-48 h, 48-72 h after dosing
- From how many animals: pooled from all animals
- Method type(s) for identification: Liquid scintillation counting, HPLC-UV, HPLC-14C, TSP-HPLC-MS
- Limits of detection and quantification:
- Other:


TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):
- acetylation
- enzyme cleavage with beta-glucoronidase/arylsulfatase

Statistics:

-

Results and discussion

Preliminary studies:

NA

Toxicokinetic / pharmacokinetic studies

Details on absorption:

see kinetic study HOE 89.0388

Details on distribution in tissues:

see kinetic study HOE 89.0388

Details on excretion:

More than 90% were excreted within the first 24 hours. Excretion mainly via feces.
85% excretion via feces
15% excretion via urine
no unchanged test item excreted

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Urine: 6 metabolites were separated, two main metabolites identified.
1. 8% of total radioactivity: sulfate-ester
2. 4% of total radioactivity: N-acetylate

Feces:
All extractable metabolites identified. About 17% of the radioactivity remained unextracted
1. 76% of total radioactivity: sulfate-ester amount decreased over time
2. N-acetylate increased over time

Applicant's summary and conclusion

Conclusions:

The test item was almost completely metabolized. The majority of the metabolites were excreted via feces.

Executive summary:

The proposed degradation pathway is given in the figure attached.

This means that reductive cleavage of the azo groups is the main metabolization step in the rat. The resulting amine is excreted mainly in the feces either directly or after N-acetylation.

Remarkable is the very high excretion rate of the metabolites via faeces. Usually such behavior is caused by one of the following acts:

- almost total absorption of the test substance and subsequent biliary excretion of the metabolites

- degradation of the test substance by the intestinal flora

- abiotic hydrolysis of the test substance in the gastro intestinal tract

 

The fact that the metabolite pattern in feces is nearly identical with that in urine gives strong evidence that biliary excretion dominates for the test item. However, determination of the absorption rate was done in a further study.