Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted according to GLP. However, data on the urinary deconjugation experiment with beta-glucuronidase/sulfatase at the high dose level were missing. This deviation did not affect the outcome of the study.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Objective of study:
distribution
excretion
metabolism
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Specific activity: 4.59 GBq/mmol; 124 mCi/mmol
Physical state: Liquid
Radiochemical purity: 96.0 - 99.8%
Solubility (20°C): Water: 0.5 +/- 0.7 µg/I; Methanol: >50 g/ml
The test material was stored below -15°C in the dark.

Non-radiolabelled test substance (supplied by Sponsor)
Purity: 98.8%
Storage: At ambient temperature in the dark.
Expiry date: 31 December 2010
Radiolabelling:
yes
Remarks:
14C- labeled phenolic ring

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
Strain as stated in the report: CD-l (albino) [Crl:CD1(ICR)]
Sex: Male
Bodyweight at dosing: 25 - 40 g
Age at dosing: 6-8 weeks
Supplier: Charles River UK Ltd., Margate, Kent, UK
Acclimatization: at least 5 days before the intended date of dosing.
Housing: During acclimatization animals were housed in groups of up to four mice in solid bottomed plastic cages.
After dosing, the animals were housed in sub-groups of 3 in glass metabolism cages (Metabowls®).
Diet ad libitum: VRFl diet manufactured by Special Diet Services
Water ad libitum: tap water

ENVIRONMENTAL CONDITIONS
Room temperature: 21 +/- 2°C.
Relative humidity: 55 - 15%.
Light/dark cycle: alternating 12 hour light/dark.
Air changes: approximately 15 per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose in 0.1% Tween 80
Details on exposure:
14C-labelled test substance was stored in acetonitrile at a concentration of 495.91 µCi/ml (1.58 mg/ml) at or below -15°C. Dose formulations were freshly prepared on the day of administration. To confirm homogeneity and concentration of radioactivity in the dose formulations (Groups 1, 2 and 4) aliquots were taken during the dosing procedure and were diluted with acetonitrile. Subsequently aliquots were taken in triplicate for radioassay. Purity checks of the dose formulations were carried out at each dosing time and all were >95%.
Duration and frequency of treatment / exposure:
single gavage.
Doses / concentrations
Remarks:
Doses / Concentrations:
0.5 and 50 mg/kg bw in 10 ml vehicle/ kg bw.
No. of animals per sex per dose:
4 subgroups of 3 males (=12 males) received 50 mg/kg bw (designated as Group2)
8 subgroups of 3 males (=24 males) splitted into two treated groups received 0.5 mg/kg bw (designated as Group 1 and 4, respectively)
Control animals:
no
Details on study design:
Rationale for the selection of the administration route and dose levels
The oral route was chosen as these ia an expected route for human exposure to the test substance. Dose levels were selected upon consultation of the Regulatory Authority (BfR) at a level where no observable effects were expected (0.5 and 50 mg/kg bw in 10 ml/kg bw vehicle, respectively).

Dose preparation and administration
14C-labelled test substance was stored in acetonitrile at a concentration of 495.91 µCi/ml (1.58 mg/ml) at or below -15°C. Dose formulations were freshly prepared on the day of administration and homogeneity and concentration of radioactivity in the dose formulations were confirmed. The dose solutions were administered orally using a graduated syringe with a rubber gavage tube. The exact amount of formulation administered was determined gravimetrically by weighing the dose syringe when loaded and again after dosing.
Administration of oral doses at 0.5 mg/kg bw (Groups 1 and 4):
A nominal portion of 141.33 - 141.48 µCi of the 14C- labeled test substance was dispensed into a scintillation vial. The solvent was evaporated under nitrogen gas at 37°C and re-suspended in vehicle (9 ml) by use of ultrasonication. Once suspended the formulation was stirred continuously.
Administration of oral dose at 50 mg/kg bw (Group 2):
Non-radiolabelled test substance (43.1 mg), was dispensed into a scintillation vial and 600 µCi 14C-labelled test substance stock solution (nominal) was added. The solution was stirred thoroughly and the solvent was evaporated under nitrogen gas at 37°C and re-suspended in vehicle by ultrasonication. Once suspended the formulation was stirred continuously. The radiochemical purity of the formulation was determined after dose administration.

Measurement of radioactivity
Radioactivity was measured by liquid scintillation counting (LSC) with automatic quench correction. Radioactivity below twice the background level was considered to be below the limit of detection.
Aliquots of liquid samples were mixed with Ultima Gold scintillator (Packard BioScience) or Monoflow 4 for measurement of radioactivity.
Solid samples were combusted in oxygen using a sample oxidizer. The combustion products were absorbed into CO2 sorbent and mixed with Permafluor E+ scintillator. The efficiency of the oxidizer was found to be above 95%. Measurements of radioactivity were corrected for oxidizer efficiency.
Details on dosing and sampling:
PHARMACOKINETIC STUDY
Excretion and tissue distribution after single oral doses (0.5 and 50 mg/kg bw; Group 1 and 2, respectively):
Following administration of radiolabeled test substance to four subgroups of three male mice, each subgroup was transferred into a glass metabolism cage.
Urine was collected into cooled receivers at time intervals of 0 - 6, 6 - 24 h, and subsequently at intervals of 24 h up to sacrifice after 168 h. Prior to dosing, pre-dose urine samples were collected for use as backgrounds.
Feces were collected separately into cooled receivers at 24 hr intervals up to 168 h. Cage washes were performed every 24 h throughout the sample collection period and retained for radioactivity measurement.
Blood samples were taken under isoflurane by cardiac puncture immediately prior to sacrifice at 168 h into heparinised tubes. Radioactivity of whole blood and plasma were measured.
At sacrifice the following tissues were removed for analysis of radioactivity:
Gastrointestinal tract with contents, Heart, Kidneys, Liver, Lungs, Muscles and Testes
Tissues were pooled for each subgroup of three mice and all samples were stored at or below -15°C prior to analysis. The residual carcass was retained. Radioactivity of tissues, carcass and feces was assessed after homogenization of the respective fraction.
Measurement of volatile radioactivity (0.5 mg/kg bw; Group 4):
Each subgroup of three mice were transferred to glass metabolism cages and 14CO2 of the expired air was trapped in a mixture of ethanolamine: 2-ethoxyethanol (1 : 3, v/v) during 24 hour intervals for up to 48 h. At 48 h the mice were sacrificed by cervical dislocation.

METABOLITE CHARACTERISATION STUDIES
Urine samples from the intervals 0 - 6 h, 6 - 24 h and 24 - 48 h from mice of group 1 and 2 (0.5 and 50 mg/kg bw, respectively) were pooled and duplicate aliquots were taken for radioassay. Urine pool concentrates were analyzed by reverse phase HPLC and normal phase TLC.
Fecal extracts from the 0 - 24 h interval were pooled (10% by weight), concentrated under nitrogen gas and duplicate aliquots were taken for radioassay. Fecal extracts were further analyzed by reverse phase HPLC and normal phase TLC.

PREPARATION OF URINE FOR MASS SPECTROMETRY ANALYSIS
To identify metabolites detected via HPLC/TLC a fresh 0 - 48 h urine pool was prepared from animals treated with 50 mg/kg bw (Group 2). Sub-samples of approximately 2 ml were freeze-dried and re-suspended in methanol. After centrifugation and further washing steps, the samples were analyzed by RP-HPLC. The major urinary metabolites were identified by mass spectrometry (ESI) coupled to a UPLC-UV-system at the Department of Mass Spectrometry for structural investigative work. Particular ions were selected for MS/MS analysis and the corresponding spectra were compared with that from the authentic test substance and the expected metabolite (Metilox acid) to confirm the structure of metabolites.

TREATMENT FOR CLEAVAGE OF CONJUGATES
Urine from sampling interval 0 - 48 h collected from groups 1 and 2, respectively was pooled and duplicate aliquots were taken for radioassay.
For the deconjugation experiments, portions of these pools were incubated with buffer and beta-glucuronidase/sulphatase at 37°C for 18h. Phenolphthalein glucuronic acid was added into a control sample to confirm beta-glucuronidase activity. Samples were analyzed by RP-HPLC and normal phase TLC.
Aliquots of the urine pools were further subjected to mild acid/base hydrolysis. Samples were acidified/basified and incubated at 37°C for 18 h. Upon neutralisation samples were analyzed by reverse phase HPLC and normal phase TLC.

Results and discussion

Preliminary studies:
No preliminary study was conducted.
Main ADME resultsopen allclose all
Type:
distribution
Results:
Upon sacrifice after 7 days, less than 0.3% radioactivity were detected in tissues at 0.5 and 50 mg/kg bw, respectively.
Type:
metabolism
Results:
A hydroxylated and a glucuronide conjugate of the hydroxylated metabolite, as well as the metabolite (14 - 18% and 0.4 - 1.1 % of dose, respectively) were detected at 0.5 and 50 mg/kg bw.
Type:
excretion
Results:
85% to 93% of applied dose were excreted within 48 hours.

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
Following administration of 0.5 mg/kg bw radio-labeled test substance radioactivity in carcass, muscle, testes and plasma was below 0.001 µg equivalents/g wet weight. Radioactivity in the other tissues ranged from 0.01 (heart, kidneys, lungs) to a maximum of 0.02 % of administered dose in whole blood.
Following administration of 50 mg/kg bw radio-labeled test substance the tissue radioactivity was highest in whole blood and liver (0.293 and 0.128 µg equivalents/g wet weight, respectively). Radioactivity detected in kidney, lungs, heart and gastro intestine tract (including content) ranged from (0.083 to0.028 µg equivalents/g). All other tissue concentrations (carcass, muscle, plasma and testes) accounted for approximately 0.015 µg equivalents/g wet weight. The radioactivity detected in organs corresponded to 0.01 to 0.02% of the ingested dose.
Following administration of 0.5 mg/kg bw radio-labeled test substance to mice the mean excretion of radioactivity in the expired air during the 0 – 48 h intervall accounted for a total of 0.07% of dose.
Details on excretion:
The overall mean recovery following administration of 0.5 mg/kg bw radio-labeled test substance to mice accounted for 99.48% of the dose (Group 1). More than 85% of the given dose was excreted within 48 hours (44% via urine and 43% via feces). Urinary and fecal excretion during the 0 - 168 h interval accounted for 47.7 % and 43.8% of the dose and 7.94% of the dose were recovered in the cage-washes at the same time interval.
Radioactivity in tissues ranged from 0.01 to a maximum of 0.02 % of administered dose.

The overall mean recovery following administration of 50 mg/kg bw radio-labeled test substance accounted for 96.92% of the dose (Group 4).Approximately 93% of dose was excreted within 48 hours (38% via urine and 55%via feces). Urinary and fecal excretion during the 0 - 168 h interval accounted for 38.8% and 56.1% of the dose and 1.94% of the dose were recovered in the cage-washes at the same time interval
Radioactivity in tissues ranged from 0.01 to a maximum of 0.02 % of administered dose.

Following administration of 0.5 mg/kg bw radio-labeled test substance to mice the mean excretion of radioactivity in the expired air during the 0 – 48 h intervall accounted for a total of 0.07% of dose

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
A total of 3 metabolites in the freshly prepared urine pools of animals treated with 50 mg/kg bw were identified via molecular weight and fragmentation pattern using a mass spectrometer operating in the negative electrospray mode (ESI LC/MS). Metabolites in the other urine and fecal fractions were correlated to these identified metabolites via their retention times.

Any other information on results incl. tables

Details on metabolites:

A total of 3 metabolites in the freshly prepared urine pools of animals treated with 50 mg/kg bw were identified via molecular weight and fragmentation pattern using a mass spectrometer operating in the negative electrospray mode (ESI LC/MS). Metabolites in the other urine and fecal fractions were correlated to these identified metabolites via their retention times.

In the feces collected 0-24 h after treatment the unchanged test substance was the major component (31.6% of applied dose). The main metabolite accounted for 7.4% while all other metabolites accounted for less than 5.6 % of dose.

Table 1: Radioactivity in selected tissues 7 days after single oral dose of radio-labeled test substance in µg equivalents/g.

Tissue

Dose (mg/kg bw)

0.5 (group 1)

50 (Group 2)

Carcass

0.000 +/- 0.000

0.005 +/- 0.005

GIT

0.001 +/- 0.000

0.028 +/- 0.012

Heart

0.001 +/- 0.000

0.071 +/- 0.014

Kidney

0.001 +/- 0.000

0.083 +/- 0.015

Liver

0.002 +/- 0.001

0.128 +/- 0.025

Lungs

0.001 +/- 0.001

0.081 +/- 0.027

Muscle

0.000 +/- 0.000

0.013 +/- 0.015

Testes

0.000 +/- 0.000

0.008 +/- 0.001

Whole blood

0.003 +/- 0.001

0.293 +/- 0.093

Plasma

0.000 +/- 0.000

0.015 +/- 0.004

GIT: Gastro intestinal tract (with contents)

Deviations from the protocol:

No data on the urinary deconjugation experiment with beta-glucuronidase/sulfatase at 50 mg/kg bw were given.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results