REAKTIV-GELB F-68 072 FW

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not specified.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP study, not conducted to any recognised guidelines, as is an investigational assessment of bioavailability. Allocated as Reliability 2 as the study is conducted by a recognised research facility, and the results are well documented.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In mice dosed orally with the test substance, the bioavailability was determined by analysis of blood and bone marrow samples taken 4 hours and 24 hours after oral dosing with 2000 mg/kg bw and in urine samples collected over 24 h after oral dosing with 2000 mg/kg bw. The expected decomposition product Reactive Yellow F68072-vinyl (formed by hydrolysis of Reactive Yellow F68072 under aqueous conditions) was quantified by HPLC with UV-detection.

GLP compliance:

no

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

EC Number:

413-090-5

Cas Number:

189574-45-6

Molecular formula:

Hill formula: C24H18LixN10NayO12S4

IUPAC Name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

See below

Test animals

Species:

mouse

Strain:

other: Not specified.

Sex:

male

Details on test animals or test system and environmental conditions:

Male B6C3F1 mice (n - 10, aged 8-10 weeks at arrival in the animal facility of the department) were purchased from Harian-Winkelmann, Borchen, Germany. On the day of treatment (July 16, 2007), mice (n = 5, body weight range 24.1 g-33.2g). The animals had free access to water and a standard diet (T.2018 Global Rodent Breeding, Harlan Teklad, Station Road, Blackthorn, Bicester, Oxon, 0X25 1TP, United Kingdom) and were kept under standard conditions (12 h day/night cycle, temperature 21 – 23 °C, humidity 45 - 55 %) before administration.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

not specified

Details on exposure:

Mice (n = 5, body weight range 24.1 g-33.2g) were orally administered Reactive Yellow F68072 at doses of 2,000 mg/kg bw in deionized water (7.5 mL/kg bw). The animals had free access to water and a standard diet (T.2Q18 Global Rodent Breeding, Harlan Teklad, Station Road, Blackthorn, Bicester, Oxon OX25 1TP, United Kingdom) and were kept under standard conditions (12 h day/night cycle, temperature 21 - 23 deg C; humidity 45 - 55 %) before administration. For blood sampling four hours after administration, mice were anaesthetized with carbon dioxide and blood samples were taken by cardiac puncture. Blood samples were kept at -20°C before sample workup. In a second experiments, mice (n = 5) were administered Reactive Yellow F68072 at doses of 2000 mg/kg bw in deionized water (7.5 mL/kg bw) and were transferred to metabolic cages to collect urine and feces immediately after administration. Animals were kept in the cages for 24 h and urine was collected at 4 deg C. After the urine collection period, animals were anaesthetized with carbon dioxide and blood samples were taken as described above.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not conducted.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Single dose

Post exposure period:

None

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

Sampling. Blood samples were collected in Monovettes (Sarsted, Germany). The blood samples were centrifuged for 15 min at 1.500 x g and the supernatants were stored at -20X until sample workup for analysis by HPLC. Volume of urine samples was determined and the samples were frozen immediately without further treatment. The samples were analyzed in August/September of 2007.

Sample preparation. To plasma samples, two volumes of water/ethanol (60.40, v:v) were added and the samples was extracted using ultrasound for 15 minutes at room temperature. The suspensions obtained wore then subjected to centrifugation (10 min at6,000 rpm) and the obtained supernatant (0.1 mL) was injected into the HPLC-system. Urine samples were also treated with two volumes of water:ethanol and subjected to centrifugation as described for blood samples. Bone marrow rinsed from femurs with phosphate butter was treated with 2 volumes of ethanol/water (60:40, v;v) subjected to sonication at room temperature for 15 minutes, centrifuged and 0.1 ml of the obtained supernatant were analysed by HPLC. Recovery of Remazol vinyl from spiked blood samples was > 90 %.

Analytical methods. The samples were separated on a HyperSK ODS3 HPLC column (4 ^ mm x 300 mm; 5///n; Maisch, Ammerbuch, Germany) using an Agilent 1090 HPLC-pump coupled to a diode array detector (Agilent, Waldbronn, Germany}. Separation was performed by gradient elution with 50 mM potassium phosphate buffer (solvent A) and methanol (solvent B) using the following conditions: linear gradient from 5 % b to 60% B within 35 min at a flow-rate of 1 ml/min. The UV-absorption of the eluate from the column was monitored at 248 and 440 nm. Calibration standards containing known concentrations of Reactive Yellow F68072-vinyl were prepared in water at pH 7 and five concentrations were used to generate calibration curves. Relative standard deviations were < 10 % on repeated injections.

Examinations

Examinations:

As detailed above.

Positive control:

None

Results and discussion

Details on results:

Concentrations of Reactive Yellow F68072-vinyl in the 24-hour urine samples was 320 µg/mL at 248 nm.

In blood samples collected 4 hours after oral administration of Reactive Yellow F68072, concentrations of Reactive Yellow F68072-vinyl were very low and close to the limit of detection. However, all samples contained a small peak co-eluting with the authentic Reactive Yellow F68072-vinyl standards at 24.9 minutes. Concentrations of Reactive Yellow F68072-vinyl in the samples collected 4 hours after substance administration was 9.53 µg/mL at 248 nm. In blood samples taken 24 hours after Reactive Yellow F68072 administration, concentrations of Reactive Yellow F68072-vinyl were below the limit of detection in all samples.

Any other information on results incl. tables

When analyzing Reactive Yellow F68072-vinyl standards, the compound gave two peaks in the HPLC separation with similar UV-spectra eluting at app. 24.9 min.

 

In urine from mice receiving 2000 mg Reactive Yellow F68072/kg bw, samples collected for 24 hours after administration contained the typical peaks for Reactive Yellow F68072-vinyl, which were characterised by recorded electronic spectra and the relative intensities of the signals recorded at 248 and 440 nm at the expected retention time of 24.9 minutes. Concentrations of Reactive Yellow F68072-vinyl in the 24-hour urine samples was 320 µg/mL at 248 nm.

In blood samples collected 4 hours after oral administration of Reactive Yellow F68072, concentrations of Reactive Yellow F68072-vinyl were very low and close to the limit of detection. However, all samples contained a small peak co-eluting with the authentic Reactive Yellow F68072-vinyl standards at 24.9 minutes. Concentrations of Reactive Yellow F68072-vinyl in the samples collected 4 hours after substance administration was 9.53 µg/mL at 248 nm. In blood samples taken 24 hours after Reactive Yellow F68072 administration, concentrations of Reactive Yellow F68072-vinyl were below the limit of detection in all samples.

 

Conclusions

The demonstration of the presence of Reactive Yellow F68072-vinyl in urine samples from mice given Reactive Yellow F68072 shows that orally administered Reactive Yellow F68072 is bioavailable. Gastrointestinal absorption of Reactive Yellow F68072 results in a translocation of the compound to blood after passing the liver and the highly water soluble Reactive Yellow F68072-vinyl is then translocated to the kidney to be excreted, in part, with urine. Absorption of Reactive Yellow F68072 from the gastrointestinal tract is also supported by the detection of Reactive Yellow F68072-vinyl in blood, although the concentrations present at the 4-hour sampling point selected for analysis were low.

Applicant's summary and conclusion

Conclusions:

The demonstration of the presence of Reactive Yellow F68072-vinyl in urine samples from mice given Reactive Yellow F68072 shows that orally administered Reactive Yellow F68072 is bioavailable. Gastrointestinal absorption of Reactive Yellow F68072 results in a translocation of the compound to blood after passing the liver and the highly water soluble Reactive Yellow F68072-vinyl is then translocated to the kidney to be excreted, in part, with urine. Absorption of Reactive Yellow F68072 from the gastrointestinal tract is also supported by the detection of Reactive Yellow F68072-vinyl in blood, although the concentrations present at the 4-hour sampling point selected for analysis were low.

Executive summary:

This study addresses the bioavailability of Reactive Yellow F68072 after oral administration in mice to support the conclusions on absence of genotoxicity in vivo based on a negative in vivo micronucleus test in mice performed by Hoechst AG. To assess bioavailability, concentrations of the decomposition product Reactive Yellow F68072-vinyl, which is formed from Reactive Yellow F68072 in aqueous solutions at pH 7, were determined in blood and urine samples from mice dosed orally with Reactive Yellow F68072 in water.

The demonstration of the presence of Reactive Yellow F68072-vinyl in urine samples from mice given Reactive Yellow F68072 shows that orally administered Reactive Yellow F68072 is bioavailable. Gastrointestinal absorption of Reactive Yellow F68072 results in a translocation of the compound to blood after passing the liver and the highly water soluble Reactive Yellow F68072-vinyl is then translocated to the kidney to be excreted, in part, with urine. Absorption of Reactive Yellow F68072 from the gastrointestinal tract is also supported by the detection of Reactive Yellow F68072-vinyl in blood, although the concentrations present at the 4-hour sampling point selected for analysis were low.