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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 06 January, 2004 to 17 November, 2005.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted according to the OECD 417 and in compliance with GLP with some acceptable deviations. However, the dermal application part suffered from design limitations, allowing for oral uptake from the skin after the 6 h exposure, and therefore invalidating the results obtained for dermal uptake.

Data source

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

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Principles of method if other than guideline:
Method: other: OECD 417
Deviation: Yes
Some deviations from study plan occurred which are not considered to have compromised the validity or integrity of the study:
- fluctuations of room temperature outside limits set
- Animals of group 7 were fed around 6 h after treatment instead of 4 h
- blood sample scheduled for D28709 (group 2) at 24 h was taken from D28704, as the earlier animal died during anesthesia prior to sampling
- Bile was collected into glass tubes instead of plastic tubes
- Animal D28763 (group 9, first treatment group - due to technical bile collection problems only second treatment group reported) was treated
according to the body weight of another animal by error.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
This is a formulation of 49.9% test substance in water

-Labelled ([ring-U-14C]BKC) and unlabelled material based on same amine raw material. Radiochemical purity of labelled material: > 99.9% (HPLC);
specific activiy: 2.15 MBq/mg test item (58.2 µCi/mg)
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
-Species: Sprague-Dawley rats
-Strain: Crl CD® (SD) IGS BR
-Source: Charles River Laboratories France, L’Arbresle, France. Caesarean Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).
-Sex: 60 males and 60 females.
-Age/weight at study initiation: Young adults approximately 7 week old; for the bile collection group, animals were around 10 week old.
-Number of animals per group: Kinetics (5 groups): 9 males and 9 females (3/group/time/sex); Excretion balance: (3 groups): 5 males and 5 females
Bile collection (1 group): 4 males and 4 females
-Control animals: Yes: For the purposes of pre-dose sample analysis, plasma, blood and tissues will be collected from at least one untreated supplementary animal/sex using the above mentioned procedures.

Administration / exposure

Route of administration:
other: Gavage and topical
Vehicle:
water
Details on exposure:
-Specific activity of test substance 2.15 MBq/mg test item (58.2 µCi/mg)
-Volume applied: Oral: 10 mL/kg; Dermal: 1.5 mL/kg bw on approximately 10% body surface area (approximately 12 µL/cm2)
-Size of test site: Dermal: approximately 10% body surface area: 25 cm2 for 200 g rat, 30 cm2 for 250 g rat.
-Exposure period: Dermal: Treated area washed after 6 hours.
-Sampling time: PK - oral: (3 animals/sex/group) post-gavage
⋅ first sampling set: 0.5, 4 and 24 hours,
⋅ second sampling: 1, 8 and 48 hours,
⋅ third sampling set: 2, 72 and 96 hours.
PK - dermal (after initiation of skin contact):
⋅ first sampling set: 3, 8 and 24 hours,
⋅ second sampling: 6, 10 and 48 hours,
⋅ third sampling set: 7, 16 and 72 hours.
MB oral and dermal:
Urine and faeces: 24 hours before radioactive treatment, and during the periods 0-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours after the radioactive gavage/dermal application.
Bile collection: 0-3, 3-6, 6-12 and 12-24 hours post gavage.

-Samples: Blood/plasma, urine, faeces, bile, exhaled air, organs, carcass, skin with substance not removable, liquid used for washing the skin, protective appliances.
Duration and frequency of treatment / exposure:
6 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
Males and females: Single and repeated oral low dose level: 50 mg/kg bw; high dose oral: 200 mg/kg bw; single dermal low dose: 1.5 mg/kg bw; high dose dermal: 15 mg/kg bw.
No. of animals per sex per dose:
Males: 64 Females: 64

Results and discussion

Main ADME results
Type:
other: Absorption, distribution and excretion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
-Percutaneous adsorption:
Dose:
Radiochemical purity (99.5%) is sufficient (>98% ; OECD guidance), and formulated material was relevant in relation to possible human exposure:
Concentrations for dermal application were 0.1 and 1% for 6 h. This is based on already avaibale information on irritancy levels of the test substance:
4 h, 5% on rabbit skin is mildly irritating; 24 h occluded patches with 0.1% concentrations in water produced a well defined erythema and very
slight oedema.
The oral high dose of 200 mg a.i./kg bw could possibly lead to some toxic effects, as the acute LD50 by gavage is between 250 and
450 mg a.i./kg bw.
Recovery:
Total recovery for oral groups were suffficient with around 100% except for males of repeated dose group resulting to 95.3% recovery.
For dermal groups, total recovery was slightly low (87.2% males and 91.0% females). However, it should be remarked that probably the total
radioactivity in the carcass samples with an avarage of about 5% was too low, as for all 4 animals for which no seperate internal organs were
measured, total carcass levels were about 10%, compared to 1 to 3% for the 6 animals for which radioactivity in individual oragns were measured.

Oral:
Most of the radioactivity were excreted via the faeces. About 3-4% left via urine, except for the males in the repeated dose groups showing a mean value of 8.31%. However, soft faeces were also observed in this group during the days of collection, possibly causing contamination of urine with faeces.
Also corresponding with high cage wash values. Elimination was quick, with 70-80% excreted within the first 24 h. No radioactivity was left in
the carcass. Bile examinations show that 4.58% (males) and 3.75% (females) of the total dose was recovered in the bile. Elimination was quick, with
25-30% already passed out in the bile in the 0-3 h period. The mean plasma and blood levels for males and females remained below quantifiable limits at all time points, except in the 50 mg/kg dose group for 0.5 to 2 h time points for plasma (161 and 251 ng -eq/g for males at 1 and 2
h respectively, and 109 ng-eq/g at 0.5 h, 212 ng-eq/g at 1 h and 192 ng-eq/g at 2 h for females), and in blood only the 1 h time point in females (173 ng-eq/g). No plasma or blood levels could be determined for the 200 mg/kg dose group or the repeated 50 mg/kg dose group.
Following single oral gavage at a nominal dose-level of 50 mg/kg bw to rats of group 1, the mean radioactivity levels were below quantifiable limits
in all tissues/organs at all time-points, except for the intestines and liver. Specifically, levels for males/females were 23.3/23.2% of the dose for
the intestines and 0.087/0.039% of the dose for the liver at the 24 h time-point. Levels decreased over time, and were all non-quantifiable by
168 h.
Following single oral gavage at a nominal dose level of 200 mg/kg bw to rats of group 2, the mean radioactivity levels were above quantifiable limits
in approximately half the analysed tissues and organs at 24 h. Specifically, high levels were present in the intestines (62.2/71.5% of the dose)
for males/females, and trace levels were present in the abdominal fat, heart, kidneys, liver, lungs, lymph nodes and or pancreas (range 0.004 to
0.24% of the dose). As previous, levels decreased over time, and were all non-quantifiable by 96 h with the exception of the intestines.
Following repeated oral gavage at a nominal dose-level of 50 mg/kg bw to rats of group 5, the mean radioactivity levels were below quantifiable
limits in all tissues/organs, except for the intestines (e.g. levels for males/females of 15.9/36.9% of the dose at 24 h) and the liver in males
(0.072% of the dose at 24 h). Levels decreased over time and were non-quantifiable by 168 h.

Dermal:
The minimal percutaneous absorption (Faeces, urine and intestines) seemed to amount to 46.4% for males, and 47.4% for females. The maximum
systemic absorption (faeces, urine, carcass and skin site) was 50.0% and 50.1% for males and females respectively. The data indicate that the skin
application site is a reservoir for absorbed radioactivity in the animals. As the dermal application site was not protected from grooming after the
6 h exposure period, test substance remained available on the skin for subsequent oral uptake from grooming.
Test substance was uniformly distributed in the stratum corneum. Cross-contamination to adjacent skin was observed. The mean plasma and blood
levels for males and females for 1.5 mg/kg dose group remained below quantifiable limits at all time points, except for the 7 and 8 h time-points
for blood (levels for males/females of 3.52/4.40 and 2.67/3.26 ng-eq/g, respectively). For the 15 mg/kg bw dose group only the 8 (levels of 70.2/68.6 ng-eq/g for males/females) and 24 (levels of 62.3/55.0 ng-eq/g for males/females) h time-points resulted in values above the quantifiable limits.
Following single dermal application at a nominal dose-level of 1.5 mg/kg to rats of group 3 and 15 mg/kg to rats of group 4, the mean
radioactivity levels were below quantifiable limits in all tissues/organs at all time-points except for the intestines, "stripped" skin fiom the
application site and adjacent site. Trace levels were found at 24 and 48 h time-points in the eyes of the 1.5 mg/kg bw dose group.

Any other information on results incl. tables

No test substance-related mortality or morbidity were observed during the study. The only death observed (Male D28768,
group 9, single oral dose at 50 mg/kg bw), was an isolated incident and not seen at the higher dose-level of 200 mg/kg bw,
was considered to be due to complications following the bile duct cannulation. In terms of clinical signs, ptyalism was
seen in 1/9 males and 1/9 females of group 5 from Day 6 (repeat oral dose of 50 mg/kg bw) and soft faeces were noted in
4/5 males of group 8 from Day 6 (also, repeat oral dose of 50 mg/kg bw). As the ptyalism was of low incidence and is often
seen in rats treated orally, this was not considered to be test substance-related. In contrast, as of high incidence, the
soft faeces were considered to be due to the repeated test substance treatment. No dermal irritation occurred.


Recovery:
Total recovery for oral groups were sufficient with around 100% except for males of repeated dose group resulting to
95.3% recovery. For dermal groups, total recovery was slightly low (87.2% males and 91.0% females). However, it should be remarked
that probably the total radioactivity in the carcass samples with an avarage of about 5% was too low, as for all 4
animals for which no separate internal organs were measured, total carcass levels were about 10%, compared to 1 to 3% for
the 6 animals for wich radioactivity in individual organs were measured.

-Oral application: 
Following single and/or repeated oral gavage at 50 and 200 mg/kg bw/day, the plasma, blood and organ radioactivity levels
were essentially non-quantifiable indicating a low oral bioavailability. The actual fraction of the oral dose
absorbed was about 8% (urine and bile fractions); this was eliminated rapidly, essentially within a 48 to 72 h
period. The vast majority of the oral dose was excreted rapidly in the faeces. At the high oral dose-level only,
quantifiable levels of radioactivity were found in some central organs at 8 h post-dosing; otherwise, the vast
majority of the dose was confined to the intestine and levels decreased over time. Only 0.62 to 8.15% of the oral
dose was eliminated in the bile in a 24 h period.

-Dermal application:
Following single dermal application at 1.5 and 15 mg/kg bw, the plasma and blood radioactivity levels were non-quantifiable
at nearly all time-points. For the 1.5 mg/kg bw group, around 2% and 43% of the dose was eliminated in the urine and
faeces, respectively, mostly within a 48 h period, suggesting that the dermal dose was highly absorbed via the
skin. However, as the test site was not protected with an Elizabethan collar during the main part of the collection
period (the collar was worn during the 6 h exposure period only), this may have been due to the animal licking the test
site. This is also supported with the finding that after oral dosing only about 4% was excreted via bile back to
intestines, and 4% excreted via urine. If similar routes of excretion are expected for dermal absorbed doses, it would
not be possible to find levels of 50% of applied doses in intestines with only 2% excreted via urine. This indicates
that about 50% of the dermal applied dose was taken up orally after all, which following the same oral kinetics
leads to the 2% excretion in urine as indeed was observed.

At 24 h post-dosing, most of the radioactivity was in the "stripped" skin (dermis/epidermis) application site
(15.02/8.74% [male/female] and 33.8/24.2% of the dose for the high and low dose groups respectively) and intestine for both
dose-levels (5.76/8.32% and 5.61/7.79% of the dose for the high and low dose groups respectively), though some radioactivity
was in the skin adjacent to the application site and minor traces were in the eyes (both most likely from
cross-contamination due to grooming). At 168 h, levels in the application site of the individual animals of the low
dose were 5.19 to 9.21% of the radioactive dose, suggesting the skin acted as a drug reservoir. In the stratum corneum
of the application site, the levels of radioactivity were of similar magnitude in the different layers at each
time-point. For all tissues/organs, the radioactivity levels essentially decreased over time.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Oral:
Following single and/or repeated oral administration at 50 and 200 mg/kg bw, the plasma, blood and organ radioactivity levels were essentially non-quantifiable indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was about 8% (urine and bile fractions); this was eliminated rapidly, essentially within a 48 to 72 h period. The vast majority of the oral dose was excreted rapidly in the faeces. At the high oral dose-level only, quantifiable levels of radioactivity (2,386 to 23,442 ng-eq/g) were found in some central organs at 8 h post-dosing; otherwise, the vast majority of the dose was confined to the intestines and levels decreased over time. Only about 4% of the oral dose was eliminated in the bile in a 24 h period of which about 30% during the first 3 h.
Dermal:
Following single dermal application at 1.5 and 15 mg/kg bw, the plasma and blood radioactivity levels were non-quantifiable at nearly all time-points. For the 1.5 mg/kg bw group, around 2% and 43% of the dose was eliminated in the urine and faeces, respectively, mostly within a 48 h period, suggesting that the dermal dose was highly absorbed via the skin. However, as the test site was not protected with an Elizabethan collar during the main part of the collection period (the collar was worn during the 6 h exposure period only), this may have been due to the animal licking the test site. This is also supported with the finding that after oral dosing only about 4% was excreted via bile back to intestine, and 4% excreted via urine. If similar routes of excretion are expected for dermal absorbed doses, it would not be possible to find levels of 50% of applied doses in intestine with only 2% excreted via urine. This indicates that about 50% of the dermal applied dose was taken up orally after all, which following the same oral kinetics leads to the 2% excretion in urine as indeed was observed.

At 24 h post-dosing, most of the radioactivity was in the "stripped" skin (dermis/epidermis) application site (15.02/8.74% [male/female]
and 33.8/24.2% of the dose for the high and low dose groups respectively) and intestines for both dose-levels (5.76/8.32% and 5.61/7.79% of the dose
for the high and low dose groups respectively), though some radioactivity was in the skin adjacent to the application site and minor traces were in the
eyes (both most likely from cross-contamination due to grooming). At 168 h, levels in the application site of the individual animals of the
low dose were 5.19 to 9.21% of the radioactive dose, suggesting the skin acted as a drug reservoir. In the stratum corneum of the application
site, the levels of radioactivity were of similar magnitude in the different layers at each time-point. For all tissues/organs, the radioactivity levels
essentially decreased over time.
Executive summary:
A guideline toxicokinetic study was conducted using radiolabelled C12-16 ADBAC. Rats were treated with single and repeated oral doses (50 or 200 mg/kg bw) as well as a single dermal dose of 1.5 or 15 mg/kg bw. Following single and/or repeated oral doses, the plasma, blood and organ radioactivity levels were essentially non-quantifiable, indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was about 8% (urine and bile fractions). This was eliminated rapidly, essentially within a 48 to 72 hour period. The majority of the oral dose was excreted in the faeces. At the high oral dose level only, quantifiable levels of radioactivity (2,386 to 23,442 ηg equivalent/g) were found in some central organs at 8 hour post-dosing; otherwise, the vast majority of the dose was confined to the intestines and levels decreased over time. Only about 4% of the oral dose was eliminated in the bile in a 24 hour period, of which 30% was eliminated during the first 3 hours. Following a single dermal application, the plasma and blood radioactivity levels were non-quantifiable at nearly all time-points. For the 1.5 mg/kg bw group, around 2 and 43% of the dose was eliminated in the urine and faeces, respectively, mostly within a 48 hour period. This apparent high absorption via the skin may have been due to indirect oral exposure via the animal licking the test site. This is also supported by the finding that, after oral dosing, only about 4% was excreted via bile back to the intestine and 4% excreted via urine. If similar routes of excretion are expected for dermally absorbed doses, it would not be possible to find levels of 50% of applied doses in intestine with only 2% excreted via urine. This indicates that about 50% of the dermally applied dose was taken in orally. Excretion in urine (2%) following dermal exposure was similar to that following oral exposure. At 24 hours post-dosing, most of the radioactivity was in the "stripped" skin (dermis/epidermis) application site (15.02/8.74% [male/female] and 33.8/24.2% of the dose for the high and low dose groups, respectively) and intestines for both dose levels (5.76/8.32% and 5.61/7.79% of the dose for the high and low dose groups, respectively), although some radioactivity was in the skin adjacent to the application site and minor traces were in the eyes (both most likely from cross-contamination due to grooming). At 168 hours post-dosing, the application site of low dose animals retained 5.19 to 9.21% of the radioactive dose. In the stratum corneum of the application site, the levels of radioactivity were of similar magnitude in the different layers at each time-point. For all tissues/organs, the radioactivity levels decreased over time.