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

Endpoint summary

Description of key information

Ca 28.6% of the dye is found to be orally absorbed, when the structural analogue Reactive Black 5 is labelled at the parabase-structures. The 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. No unchanged Reactive Black 5 was detected in urine or feces extract, indicating that rapid and complete metabolization 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 Reactive Black 5 nor any of its metabolites has a bioaccumulation potential.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

28.6

Additional information

The kinetics and metabolism of the structural analogue Reactive Black 5 was investigated in studies with radiolabeled substance. In the first part, the TS was labeled at both phenyl rings (bisphenyl-U-¹⁴C), in the second part, in the naphthalene-part.

Five male rats received ¹⁴C-labelled HOE CG 0062 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 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 feces. 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.

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 C_max of 0.7 to 0.8 µg equivalent/g after 5.6 hours in plasma and 21 hours in blood. T_1/2 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 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 was found in blood and tissues. The mean over-all recovery rate was 99% of the administered dose.

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

No unchanged HOE CG 0062 was detected in urine or feces extract, indicating that rapid and complete metabolization 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 HOE CG 0062 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 feces 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 above.

The non-extractable residues in feces 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 feces 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 a hint 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 feces (95.58 ± 1.86%). The renal (including cage wash) eliminated part was 1.36 ± 0.53%. Excretion in feces and urine was biphasic. For the first rapid phase, half times of 4.9 h (feces) and approx. 5 h (urine) were calculated or estimated, respectively. In the slow second phase (t½ca. 75 h for feces and 71 h for urine) only about 1% (feces) and 0.1% (urine) or less were excreted.