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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-02-19 to 2008-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 417 with the following deviation: Only one dose was used, but the guideline recommends using at least two dose levels in the case of single dose administration.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Objective of study:
toxicokinetics
other: absorption, distribution, metabolism, excretion
Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
See rationale for reliability above.
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
481-170-7
EC Name:
-
Cas Number:
502453-61-4
Molecular formula:
Hill formula: C23H30BrN3O2 CAS formula: C23H30N3O2.Br
IUPAC Name:
dimethyl(3-{[4-(methylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}propyl)propylazanium bromide
Constituent 2
Reference substance name:
Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide
IUPAC Name:
Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide
Details on test material:
- Name of test material (as cited in study report): B119 HC Blue 16
- Molecular formula (if other than submission substance): C23H30N3O2.Br
- Molecular weight (if other than submission substance): non-radiolabelled (460.42); radiolabelled (461.1)
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: active
- Physical state: dark blue powder
- Radiochemical purity (if radiolabelling): 99.7% (HPLC)
- Specific activity (if radiolabelling): 777 MBq/mmol (by mass spectrometry), 1.78 MBq/mg (by gravimetric analysis)
- Locations of the label (if radiolabelling): on two carbonyl groups
- Expiration date of radiochemical substance (if radiolabelling): 2007-06-28
Radiolabelling:
yes
Remarks:
Carbon 14 on two carbonyl groups

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 9-12 weeks old
- Weight at study initiation: at least 200 grams
- Fasting period before study: Approximately 18 h prior to and 4 h after dose administration.
- Housing: ADME group (stainless steel metabolism cages, 18.5x19x20 cm [LxWxH]); TK group (Macrolon cages, type MII, height 13.5 cm)
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days under laboratory conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 - 22.0 deg. C
- Humidity (%): 34 - 60%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark /12 hrs light


IN-LIFE DATES: From: 2007-02-22 To: 2007-02-26

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Milli-Q water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: A stock solution of [carbonyls-14C]HC Blue 16 was prepared in Milli-Q water, containing 4.7448 MBq/mL (=2.737 mg/mL). Homogeneity of the resulting stock solution was checked by liquid scintillation counting of triplicate 10 uL aliquots; radiochemical purity of the stock solution was 98.06%. To prepare the final dose solution, 8.4422 g of the stock solution and 376.7 mg of unlabelled test substance were placed into an empty glass container. Subsequently, 11.1673 g Milli-Q water were added, and the formulation was mixed, vortexed and stirred. The resulting formulation was a clear, dark blue solution. The nominal concentration and specific gravity were 20 mg/ml in milli-Q water with a specific activity of 0.10 MBq/mg.


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: Nominal concentration of test substance in final dosing solution = 20 mg/mL.
- Amount of vehicle (if gavage): See preparation of dosing solutions above.
- Lot/batch no. (if required): N/A
- Purity: N/A


HOMOGENEITY AND STABILITY OF TEST MATERIAL: Homogeneity of the resulting stock solution was checked by liquid scintillation counting of triplicate 10 uL aliquots. Before and after dose administration, the homogeneity and radioactivity concentration of the dose formulation was verified by radioanalysis. For this purpose, weighed aliquots of 25 uL were taken from the top, middle and bottom of the mixture and analyzed by liquid scintillation counting using Ultima Gold scintillation cocktail. To confirm the stability of the radiolabelled substance, an aliquot of the treatment solution was analyzed on each treatment day before (time = 0) and after (time = 4 hours) dosing using HPLC.

OTHER: Dose volume for oral gavage dosing was 5 mL/kg bw. Actual dose volumes were based on the latest individual animal body weights.
Duration and frequency of treatment / exposure:
A single oral gavage dose was used for both the ADME and TK groups. Duration of observations/sampling for the ADME group was 96 hours, and for the TK group was 48 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
The oral dose was 100 mg/kg bw (range 84.9 - 98.9 mg/kg bw). Each oral dose of the test substance contained approximately 10 MBq/kg bw of radioactivity.
No. of animals per sex per dose / concentration:
4 female rats were used for the ADME group.
6 female rats were used for the TK group (3 rats were used for each blood sampling time point; rats 19, 20, 21 were sampled at odd-numbered sampling intervals and rats 22, 23, 24 were sampled at even-numbered sampling intervals).
Control animals:
not specified
Positive control reference chemical:
N/A
Details on study design:
- Dose selection rationale: The dose level was chosen based on available data from a 28-day oral study in rats. The NOAEL in this 28-day study was 100 mg/kg bw/day.
- Rationale for animal assignment (if not random): N/A
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): ADME (urine, faeces, cage washes, blood, plasma, heart, lung, spleen, gonads, abdominal fat, muscle, adrenals, bladder, thymus, thyroid, liver, kidney, brain, bone, GI tract, residual carcass); TK (blood, plasma)
- Time and frequency of sampling: ADME (urine and faeces were collected pre-dose, 0-8 h, 8-24 h, 24-48 h, 48-72 h, 72 -96h; a single cage wash was collected at study termination; blood, plasma and all tissues/organs were collected at necropsy). TK (blood/plasma sampling times were 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours).
- Other: 1. For the TK group, 3 rats were used for each blood sampling time point; the first three rats in this group were sampled at odd-numbered sampling intervals and the latter three were sampled at even-numbered sampling intervals. 2. For the ADME group, the urine and faeces collection assembly of the metabolism cage was cooled using dry ice; urine and faeces were freeze-trapped to avoid atmospheric oxidation, evaporation and bacterial degradation. Urine and faeces samples were stored at <= 75 deg. C prior to analysis. At study termination, the interiors of the metabolism cages were rinsed with methanol/water (50/50); the cage rinse was weighed and determined directly.


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): ADME group (urine and faeces). TK group (plasma).
- Time and frequency of sampling: ADME group (urine [8-24 h and 24-48 h], faeces [8-24 h and 24-48 h]). TK group (plasma from all animals at each time point was pooled).
- From how many animals: ADME (4 animals, samples pooled). TK (3 animals/time point, samples pooled).
- Method type(s) for identification: liquid chromatography-photodiode array-radioactivity-mass spectrometry (LC-PDA-RAD-MS)
- Limits of detection and quantification: N/A
- Other: No significant radioactivity was measured in the plasma samples; the test substance equivalent concentrations were < 3 mg/mL. Therefore, the plasma samples were not analyzed by LC-RAD-PDA-MS.


TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): N/A
Statistics:
N/A

Results and discussion

Preliminary studies:
A non-GLP pilot study of the test substance was conducted. Following a single oral administration of 14C-HC Blue 16 (100 mg/kg bw), it was concluded that the minimum oral absorption (excluding possible biliary excretion) was low, and that the majority of the administered dose was excreted via the faeces within 24 hours after dosing.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The oral absorption, calculated as fractional absorption from the urine data, was 5.1%. When calculated with plasma data, the oral absorption was 0.8% based on all 6 animals dosed intravenously. However, two of these intravenously dosed animals showed plasma concentrations that were over 10-fold higher than the plasma concentrations of the other four intravenously dosed animals. When these two animals were excluded from the analysis, the oral absorption was 10.9%.
Details on distribution in tissues:
At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was approximately 0.3% of the administered dose, indicating no major accumulation of radioactivity. The residual concentration in blood was 0.033 mg/kg. In liver (0.446 mg/kg), carcass (0.267 mg/kg), kidney (0.235 mg/kg) and brain (0.103 mg/kg), concentration equivalents of approximately 3-14 times the concentration in blood were observed.
Transfer into organs
Transfer type:
other: N/A
Details on excretion:
The urinary excretion of radioactivity in the ADME group showed that the excretion via urine was a minor route of elimination for [carbonyls-14C]HC Blue 16 after oral administration. Urinary excretion accounted for 0.90% after oral dosing. The highest rate of urinary excretion was observed during the first 24 hours; thereafter, a decreasing excretion rate was noted with increasing time intervals.

The faecal excretion of radioactivity in the ADME group showed that excretion via faeces was the major route of excretion after oral administration. Faecal excretion accounted for 87.0% after oral dosing. The bulk of radioactivity was excreted in the 8-24 hour interval.

The total excretion of radioactivity in the ADME group showed that 87.9% of the administered dose was excreted during the study period. After oral administration, excretion was fast and almost complete within 24 hours post-dose.
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: approximately 22.1 hours
Test no.:
#1
Toxicokinetic parameters:
AUC: AUC(last) was approximately 4.41 hr*mg/kg
Test no.:
#1
Toxicokinetic parameters:
AUC: AUC(last) was approximately 0.0441 hr*mg/kg/mg*kg
Test no.:
#1
Toxicokinetic parameters:
AUC: AUC(infinity) was approximately 5.66 hr*mg/kg
Test no.:
#1
Toxicokinetic parameters:
AUC: AUC(infinity) was approximately 0.0625 hr*mg/kg/mg*kg
Test no.:
#1
Toxicokinetic parameters:
Cmax: 0.309 mg/kg
Test no.:
#1
Toxicokinetic parameters:
Cmax: 0.00341 mg/kg/mg*kg
Test no.:
#1
Toxicokinetic parameters:
Tmax: 0.25 hr

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
In the faeces pool of the ADME group, two main peaks were observed at a retention time of 8.5 (12.5% of total radioactivity) and 20.1 (83.1% of total radioactivity). The peak at 20.4/20.1 minutes was identified as parent compound. The peak at 8.5 indicated reduction of a carbonyl group. Additionally, three other metabolites not observed in the radioactivity chromatogram were observed in the MS chromatogram. These three metabolites indicated demethylation and hydroxylation as metabolic pathways for the test substance.

In plasma samples, no significant radioactivity could be detected and therefore no metabolite analysis was performed on these samples. The urine samples contained only one peak corresponding to the parent compound; these samples were not subjected to further metabolite screening.

Any other information on results incl. tables

Possible metabolites detected in extracted faeces samples of rats dosed orally with [carbonyls-14C]HC Blue 16

RAD R.t. in min (% of total radioactivity in chromatogram)

m/z

MS R.t. (min)

Supposed metabolic reaction

Presence in*

8.5-8.9

(12.5%)

382

8.4

Reduction of carbonyl group

r

13.9

(N/A)

382

13.8

Demethylation and hydroxylation

m

17.3

(N/A)

366

17.0

Demethylation

m

17.7

(N/A)

396

17.5

Hydroxylation

m

20.1-20.4

(83.1%)

380

20.3-20.4

Parent compound

r

R.t. = Retention time.  * r  = present in radioactivity chromatogram of sample;  m = present in MS chromatogram, but no peak observed in the radioactivity chromatogram. 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
It was concluded that B119 HC Blue 16 administered orally has only low absorption (only between 0.8-10.9%); if absorbed, it was readily distributed into all organs, metabolized to some extent and excreted mainly via the feces (via bile). Following oral exposure only one possible metabolite was detected and it suggested reduction of a carbonyl group.
Executive summary:

Absorption, distribution, metabolism and excretion of HC Blue 16 was studied in the Wistar rat.  A single group of 4 females was used for the Absorption, Distribution, Metabolism and Excretion (ADME) investigation and a single group of 6 females for the toxicokinetics (TK) investigation.  All rats were treated via oral gavage with a single dose of 100 mg/kg bw.  

In the mass-balance group, urine and faeces were collected in 0-8, 8-24, 24-48, 48-72 and 72-96 hour intervals. Animals were euthanized 96 hours after dose administration, and several tissues and organs were collected.  Total radioactivity in urine, faeces, tissues and organs was determined.  Selected urine and faeces samples were pooled per group and the metabolite profile in these pooled samples was investigated.  In the toxicokinetic group, blood was sampled from three rats alternately at the following time points: 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours after dosing. Total radioactivity and B119 HC Blue 16 equivalent concentrations were determined.

No mortality was observed after oral dosing.  After oral administration of the test substance, red brown urine was observed in the ADME group and green/blue faeces were observed in the ADME and TK groups; these observations were related to the color of the test substance.  In addition, three animals in the TK group showed piloerection. 

Oral absorption was calculated in two ways: with the urine data and with the plasma data. With the urine data, the fractional absorption was calculated by dividing the percentage of radioactivity recovered in the urine after oral administration by the percentage of radioactivity recovered in the urine after intravenous administration. With the plasma data, absorption was calculated by dividing the dose-normalized area under the curve (AUC) after oral administration by the AUC after intravenous administration. Calculation of the fractional oral absorption from the urine data assumed that the ratio of urinary excreted radioactivity to systemically available radioactivity was constant for both oral and intravenous routes.

The average oral absorption was low: 5.1% when calculated from the urine data, and 0.8% and 10.9% when calculated with the plasma data.  The oral absorption was 0.8% based upon comparison with 6 animals dosed intravenously.  However, two of these intravenously dosed animals showed plasma concentrations that were over 10-fold higher than the plasma concentrations of the other four intravenously dosed animals.  When these two animals were excluded from the analysis, the oral absorption was 10.9%.  Animals were all dosed with the same formulation and by the same person.  Therefore, no apparent reason could be given for this large variability in concentrations between the animals. 

From the plasma data, it was evident that oral absorption was fast, with a Tmax value of 0.25 hour. Plasma concentrations were below the limit of quantitation at study termination. The terminal half-life was approximately 22.1 hours.

Faeces was the most important route of excretion of B119 HC Blue 16.  Faeces excretion accounted for 87% of the administered dose after oral dosing, indicating that absorbed B119 HC Blue 16 was excreted mainly via the feces and implying an important role for biliary excretion of the test substance.  The highest rate of faeces excretion was during the first 24 hours, with similar excretion rates afterwards.  Excretion via urine was only a minor route of excretion after oral administration.  Urinary excretion accounted for 0.9% of the administered dose after oral dosing. 

At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was low, indicating no major accumulation of radioactivity after 96 hours.  The average total recovery of radioactivity in the ADME group was 88.21% of the administered dose.  

In plasma samples, no significant radioactivity could be detected and therefore no metabolite analysis was performed on these samples.  The urine samples contained only one peak corresponding to the parent compound; these samples were not subjected to further metabolite screening.  In the faeces extracts, one possible metabolite was detected in the radioactivity chromatogram; this metabolite indicated that reduction of a carbonyl group was a metabolic pathway for B119 HC Blue 16.  Additionally, three other metabolites not observed in the radioactivity chromatogram were observed in the MS chromatogram.

It was concluded that HC Blue 16 administered orally had low absorption; if absorbed it was readily distributed into all organs, metabolized to some extent  and excreted via the faeces (via bile).  Following oral exposure, only one possible metabolite was detected, which suggested reduction of a carbonyl group as a metabolic pathway for B119 HC Blue 16.

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