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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Type of information:

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

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

Justification for type of information:

see Read Across Justification in section 13

Reason / purpose for cross-reference:

read-across source

Duration and frequency of treatment / exposure:

single dose

Positive control reference chemical:

-

Statistics:

-

Preliminary studies:

NA

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

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

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.

Reference 2

Endpoint:

basic toxicokinetics

Type of information:

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

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

Justification for type of information:

see Read Across Justification section 13

Reason / purpose for cross-reference:

read-across source

Statistics:

-

Preliminary studies:

NA

Details on absorption:

see kinetic study HOE 92.0568

Details on distribution in tissues:

see kinetic study HOE 92.0568

Details on excretion:

72 hours following administration of the dose, 109.1% of the administered radioactivity were recovered. Excretion mainly via feces.
108.8% excretion via feces
0.3% excretion via urine
Nearly 99% were excreted in the first 24 hours.

Metabolites identified:

yes

Details on metabolites:

No unchanged substance was found in urine or feces, indicating that rapid and total metabolisation of the substance took place in the rat. All excreted metabolites were even more polar than the substance itself.
- Urine: At least 3 metabolites were separated, but no match with one of the reference compounds was found.
- Feces: At least 7 metabolites were detected including the main metabolite. Due to the high amount of coextracted material, no clear identification could be made.

The main metabolite of the substance in urine and feces presumably lost at least one azo-linked side-chain.
No glucuronide or sulfate conjugates were excreted via feces.
Most likely, the non-extractable residues contain at least the intact naphthalene ring system.

Conclusions:

The test item was almost completely metabolised. The majority of the metabolites were excreted via faeces. As elimination occurs rapidly and quantitatively, neither intact substance nor any of its metabolites should accumulate in animal tissues.
Characterisation of the excreted metabolites gives strong evidence that the substance is metabolised by cleavage of one or both of the azo-linkages. The resulting naphthalene fragment containing one or no side-chain is thermally labile and thus could not be isolated and characterized.
These findings are fully in concordance with the results of study HOE 88.1064.

Executive summary:

Five male rats received 1,4,5,8-naphthyl-¹⁴C-labelled test substance as an aqueous solution at a nominal dose of 10 mg/kg body weight (actual dose: 9.41 mg/kg) by gastric intubation. Urine and feces from 0-24, 24-48 and 48-72 h after application were pooled and investigated using HPLC.

Elimination of the administered radioactivity was rapid, quantitative and occurred mainly via faeces.

72 h following administration of the dose 109.1% of the applied radioactivity were recovered in the excreta (108.8% via faeces and 0.3% via urine). Moreover, nearly 99% of the dose was excreted in the first 24 h following administration.

No unchanged substance was detected in urine or faeces extract, indicating that rapid and complete metabolisation of the test substance took place in the rat. Moreover, all excreted metabolites were even more polar than the test substance itself, which strongly supports the assumption that neither the substance nor any of its metabolites has a bioaccumulation potential.

In urine clear separation of at least 3 metabolites (retention times: 3.5-3.8 min; 6.4 min and 13.9-14 min) was achieved and the retention behavior gives strong evidence that each metabolite lost at least one azo-linked side-chain. Moreover it is very likely that the most polar metabolite (Rt=3.5-3.8 min) has no side-chains at all.

Following extraction with acetonitrile/water, in faeces at least 7 metabolites were detected covering retention times from 3 to 23 minutes and containing one main metabolite (Rt=13-15 min). Again, the retention behavior gives strong evidence that the main metabolite has lost at least one azo-linked side-chain. The proposed degradation pathway is given in the attached Figure 2.

The non-extractable residues in faeces accounted for approx. 53% of the applied dose, indicating that significant amounts of radioactive residues are tightly bound to that matrix. Due to rapid excretion and limited bioavailability of such bound residues, this fraction should be of no toxicological concern. Nevertheless, hydrolysis experiments were performed and revealed a broad peak (Rt=10-20 min) in addition to a main product of very polar character (Rt=3 min), which had most probably lost both side-chains. As it is very likely that cleavage of azo groups will occur during hydrolysis, it may be concluded that the non-extractable residues in faeces at least contain the intact naphthalene ring system.

Reference 3

Endpoint:

basic toxicokinetics

Type of information:

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

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

Justification for type of information:

see Read Across Justification section 13

Reason / purpose for cross-reference:

read-across source

Duration and frequency of treatment / exposure:

single dose

Positive control reference chemical:

-

Details on study design:

-

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 study HOE 88.1064

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 test substance 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 the substance to, presumably, erythrocytes.

After oral administration, excretion of radioactivity was mainly via faeces (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). Substance 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.

Reference 4

Endpoint:

basic toxicokinetics

Type of information:

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

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

Justification for type of information:

see Read Across Justification in section 13

Reason / purpose for cross-reference:

read-across source

Preliminary studies:

NA

Details on absorption:

ca. 1% (estimated from study HOE 89.0388)

Details on distribution in tissues:

Kidneys: 0.1228 µg equivalent/g
Spleen: 0.0332 µg equivalent/g
subcutaneous fat: 0.0174 µg equivalent/g
Lungs: 0.013 µg equivalent/g
All other organs: < 0.01 µg equivalent/g

In sum 0.0344% of the administered dose

Details on excretion:

Excretion mainly via feces: 95.58% of the administered dose; t1/2(I): 4.9 h, t1/2(II): 75 h
Renal excretion: 1.22% + 014% (cage washing) of the administered dose; t1/2(I): 5 h, t1/2(II): 71 h
Bi-phasic elimination
no excretion by exhalation

Test no.:

#1

Toxicokinetic parameters:

Cmax: 0.19 µg eq/g

Test no.:

#1

Toxicokinetic parameters:

Tmax: 6.8 hours

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 22.1 hours

Test no.:

#1

Toxicokinetic parameters:

AUC: 48: 5.85 µg eq*h/g

Test no.:

#1

Toxicokinetic parameters:

AUC: oo: 6.66 µg eq*h/g

Test no.:

#2

Toxicokinetic parameters:

Cmax: 0.29 µg eq/g

Test no.:

#2

Toxicokinetic parameters:

Tmax: 6.4 hours

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 18.5 hours

Test no.:

#2

Toxicokinetic parameters:

AUC: 72: 7.14 µg eq*h/g

Test no.:

#2

Toxicokinetic parameters:

AUC: oo: 8.8 µg eq*h/g

Metabolites identified:

no

Remarks:

Metabolites were identified in study DM90/001

Details on metabolites:

see metabolism study DM90/001

Conclusions:

After oral gavage, the radioactivity was predominantly eliminated via the faeces (95.58 ± 1.86%) within 7 days. The renal (including cage wash) eliminated part was 1.36 ± 0.53%. Excretion in feces and urine was biphasic. There was no tendency for the substance or its metabolites to bioaccumulate in blood or tissues.

Executive summary:

The kinetics of in the naphthalene-part ¹⁴C-labelled test substance was examined in male rats after single oral treatment at about 10 mg/kg body weight.

The slow increase of the radioactivity led to concentration maxima of 0.19 and 0.29 μg equivalent/g after on average 6.8 and 6.4 h in blood and plasma, respectively. The concentration decrease occurred with biological half times of 22.1 and 18.5 hours in blood and plasma, respectively. The comparable kinetics in blood and plasma is an indication for the fact that binding of radioactivity to formed blood components does not occur.

After oral gavage, the radioactivity was predominantly eliminated within 7 days via the faeces (95.58 ± 1.86%). The renal (including cage wash) eliminated part was 1.36 ± 0.53%. Excretion in faeces and urine was biphasic. For the first rapid phase, half times of 4.9 h (faeces) and approx. 5 h (urine) were calculated or estimated, respectively. In the slow second phase (t_½ ca. 75 h for faeces and 71 h for urine) only about 1% (faeces) and 0.1% (urine) or less were excreted.

At the end of the study, 7 days after oral administration, the highest remaining concentrations were measured in the kidneys (0.12 μg equivalent/g), followed by spleen (0.03 μg equivalent/g), subcutaneous fatty tissue (0.02 μg equivalent/g) and lung (0.01 μg equivalent/g). The remaining tissues contained less than 0.01 μg equivalent/g. Overall, 0.03% of the administered dose was found in the tissues investigated.

The recovery rate was 96.98 ± 1.45% of the administered radioactivity.

Description of key information

Based on available data, the substance has a limited oral and very low dermal bioavailability. Due to its good water solubility and low lipophilicity, there is no potential for bioaccumulation.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

30

Absorption rate - dermal (%):

5

Absorption rate - inhalation (%):

30

Additional information

The kinetics and metabolism of Reactive Black 5, as a close structural analogue to Reactive Blue 250, was investigated in studies with radiolabelled test substance. In the first part, the substance was labelled at both phenyl rings (bisphenyl-U-¹⁴C), in the second part, in the naphthalene-part.

Five male rats received ¹⁴C-labelled substance as an aqueous solution at a nominal dose of 10 mg/kg body weight by gastric intubation.

The reductive cleavage of the azo groups is the main metabolisation step in the rat. The resulting amine is excreted mainly in the faeces either directly or after N-acetylation.

Remarkable is the very high excretion rate of the metabolites via faeces. Usually, such behaviour 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 gastrointestinal tract

 

The fact that the metabolite pattern in faeces is nearly identical with that in urine gives strong evidence that biliary excretion dominates for the test item.

For assessment of the absorption rate after oral gavage, one group of 3 rats were treated intravenously with 1 mg/kg bw. The mean oral 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 C_max of 0.7 to 0.8 µg equivalent/g after 5.6 hours in plasma and 21 hours in blood. T_½ were 5 and 34 hours in plasma and about 8 days in blood, leading to the assumption of binding of Reactive Black 5 to, presumably, erythrocytes.

After oral administration, excretion of radioactivity was mainly via faeces (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). Substance levels in kidneys (0.1 µg eq/g and other tissues were lower. Totally, 0.28% of the administered dose was found in blood and tissues. The mean over-all recovery rate was 99% of the administered dose.

In the naphthalene-labelled investigation, urine and faeces from 0-24, 24-48 and 48-72 h after application were pooled and investigated using HPLC. Elimination of the administered radioactivity was rapid, quantitative, and occurred mainly via faeces. 72 h following administration of the dose 109.1% of the applied radioactivity were recovered in the excreta (108.8% via faeces and 0.3% via urine). Moreover, nearly 99% of the dose was excreted in the first 24 h following administration.

No unchanged substance was detected in urine or faeces extract, indicating that rapid and complete metabolisation of the test substance took place in the rat. Moreover, all excreted metabolites were even more polar than the test substance itself, which strongly supports the assumption that neither the substance nor any of its metabolites has a bioaccumulation potential.

In urine, clear separation of at least 3 metabolites (retention times: 3.5-3.8 min; 6.4 min and 13.9-14 min) was achieved and the retention behaviour gives strong evidence that each metabolite lost at least one azo-linked side-chain. Moreover, it is very likely that the most polar metabolite (Rt=3.5-3.8 min) has no side-chains at all.

Following extraction with acetonitrile/water, in faeces at least 7 metabolites were detected covering retention times from 3 to 23 minutes and containing one main metabolite (Rt=13-15 min). Again, the retention behaviour gives strong evidence that the main metabolite has lost at least one azo-linked side-chain. The proposed degradation pathway is given above.

The non-extractable residues in faeces accounted for approx. 53% of the applied dose, indicating that significant amounts of radioactive residues are tightly bound to that matrix. Due to rapid excretion and limited bioavailability of such bound residues, this fraction should be of no toxicological concern. Nevertheless, hydrolysis experiments were performed and revealed a broad peak (Rt=10-20 min) in addition to a main product of very polar character (Rt=3 min), which had most probably lost both side-chains. As it is very likely that cleavage of azo groups will occur during hydrolysis, it may be concluded that the non-extractable residues in faeces at least contain the intact naphthalene ring system.

The slow increase of the radioactivity led to concentration maxima of 0.19 and 0.29 µg equivalent/g after on average 6.8 and 6.4 h in blood and plasma, respectively. The concentration decrease occurred with biological half times of 22.1 and 18.5 hours in blood and plasma, respectively. The comparable kinetics in blood and plasma is an indication that binding of radioactivity to formed blood components does not occur.

After oral gavage, the radioactivity was predominantly eliminated within 7 days via the faeces (95.58 ± 1.86%). The renal (including cage wash) eliminated part was 1.36 ± 0.53%. Excretion in faeces and urine was biphasic. For the first rapid phase, half times of 4.9 h (faeces) and approx. 5 h (urine) were calculated or estimated, respectively. In the slow second phase (t½ca. 75 h for faeces and 71 h for urine) only about 1% (faeces) and 0.1% (urine) or less were excreted.

The results of the toxicokinetic studies are in agreement with the modelled information which is available for the source substances and the Substance and can therefore be transferred to the toxicokinetic behaviour for Reactive Blue 250.