Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, Klimisch 1

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Principles of method if other than guideline:
The study was designed to measure plasma and milk levels that would be reached as a result of oral dosing, 14C-AHTN. AHTN was administered by gavage to pregnant Charles River CD rats at 2.0 or 20 mg/kg bw as a solution in corn oil daily from day 14 of gestation up to 7 days post-parturition.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
radiolabelled AHTN
Radiolabelling:
yes
Remarks:
AHTN radio labelled with carbon-14 in the aromatic ring

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Charles river CD rats
Number: 49 (4 for pilot study, 3 controls, 22 low dose and 20 high dose).
Age: 10 - 15 weeks.
Bodyweight: ca 250 - 400 g (Appendix 2).
Supplier: Charles River UK Ltd, Margate, Kent, UK.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Duration and frequency of treatment / exposure:
Daily until sacrifice
Doses / concentrations
Remarks:
Doses / Concentrations:
Nominal daily dose of 2 mg/kg/bw and 20 mg/kg/bw using a 2 and 20 mg/ml solution in corn oil.
No. of animals per sex per dose:
18
Control animals:
no

Results and discussion

Preliminary studies:
Pilot study (milk and plasma levels)
14C-AHTN was administered as a suspension in aqueous sodium carboxymethyl cellulose (CMC) solution (1 % w/v). Suspensions were prepared and administered daily throughout the dosing period.
The radiochemical purity of the 14C-AHTN was measured by thin-layer chromatography (TLC) prior to dosing and after dose administration and was found to be >97% and stable during the dose preparation.
Four pregnant rats were administered consecutive daily oral doses of 14C-AHTN in suspension in 1 % carboxymethyl cellulose at a nominal level of 25 mg/kg beginning on Day 14 of the gestation period up until Day 3 of parturition.
At four hours after the last dose, plasma radioactivity concentrations were 14.4 - 22.5 ppm, milk radioactivity concentrations were 9.82 - 37.9 ppm AHTN.
From the results of the pilot study it was decided that the low and high dose levels used in the main study would be 2 mg/kg/day and 20 mglkg/day respectively.

Toxicokinetic / pharmacokinetic studies

Transfer into organs
Test no.:
#1
Transfer type:
blood/placenta barrier
Observation:
other: Levels of AHTN plus metabolites were barely detectable in the foetus.

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
No identification of metabolites, only quantification of changed AHTN

Any other information on results incl. tables

The highest mean levels of radiolabel were found in the 4 hr plasma samples, declining to about 35% of that level at 24 hr after dosing (see table 1).

Lower levels were seen after 7 days as opposed to 3 days indicating no significant accumulation. Plasma levels were roughly proportional to dose with levels at 20 mg/kg bw/day approximately 10 fold higher than those at 2 mg/kg bw/day.

Table 1 Analysis of total radioactivity in plasma after daily oral administration of 2 or 20 mg/kg bw/day 14C-AHTN in μg equivalents AHTN/ml plasma
Time after Time after oral Mean level after Mean level after
parturition administration oral dose of oral dose of
(days) (hours) 2 mg/kg bw/day 20 mg/kg bw/day
3 4 3.13 ± 0.40 25.1 ± 3.5
  8 1.72 ± 0.29 24.3 ± 6.4
  24 1.10 ± 0.18 9.98 ± 1.6
7 4 2.41 ± 0.46 21.0 ± 2.2
  8 2.20 ± 1.02 17.3 ± 1.9
  24 0.86 ± 0.10 6.53 ± 0.9

Levels of total residue found in the milk (table 2) were also highest at 4 hr after dosing declining 5 to 10 fold by 24 hr. Similar levels were generally seen after 7 days dosing as compared to those after 3 days dosing (except for 2 mg/kg at 4 and 8 hrs) also consistent with no significant accumulation. Additionally, the major residues in the milk were not associated with the peak assigned as AHTN based on retention time. Although not fully characterised, the AHTN peak in the milk extracts is considered authentic as the metabolites are expected to be more polar and would therefore precede AHTN on the polar column. About 66-85% and 57-81% of the radioactivity was associated with other materials (metabolites) at the low and high dose, respectively.

Table 2 Analysis of total radioactivity and unchanged AHTN in milk after daily oral administration of 2 or 20 mg/kg bw/day 14C-AHTN in μg equivalents AHTN/ml milk (ppm)
After oral dose of 2 mg/kg bw/day After oral dose of 20 mg/kg bw/day
Milk collection Time after oral Total AHTN Ratio Total AHTN Ratio
after parturition administration radiolabel Mean AHTN/total radiolabel Mean AHTN/total
(hours) Mean residue Mean residue
3 4 1.45 ± 0.43 0.35 ± 0.25 0.22 ± 0.10 25.0 ± 11.1 9.43 ± 2.57 0.41 ± 0.10
8 0.66 ± 0.12 0.10 ± 0.02 0.15 ± 0.05 10.5 ± 3.35 2.14 ± 1.39 0.19 ± 0.10
24 0.31 ± 0.07 na - 2.89 ± 0.71 na -
7 4 1.89 ± 0.57 0.63 ± 0.20 0.34 ± 0.02 18.0 ± 1.1 7.73 ± 0.71 0.43 ± 0.02
8 0.87 ± 0.21 0.14 ± 0.05 0.15 ± 0.03 8.76 ± 2.40 2.95 ± 1.65 0.32 ± 0.09
24 0.21 ± 0.06 na - 1.55 ± 0.30 na -

na = not analysed due to low radioactivity levels

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Following the last of daily oral doses of 14C-AHTN on Days 3 and 7 of parturition, at both low and high dose levels, plasma radioactivity levels steadily declined during the period 4 - 24 hours post dose. A steady decline in milk radioactivity concentrations and AHTN concentrations in milk during 4 - 24 hours after the last dose was also observed at both dose levels.

Plasma and milk total radioactivity concentrations and AHTN concentrations in milk were generally 10 times greater at the 20 mg/kg/day dose level than those observed at the 2 mg/kg/day dose level.
The milk samples taken at 24 hours contained radioactive levels which were too low for accurate determination of AHTN.

Whole-body autoradiography of pregnant rats dosed with low level 14C-AHTN and sacrificed at 4 and 24 hours after the last dose showed that radioactivity levels in the foetus were essentially not detectable.
Executive summary:

The transfer of AHTN (6-acetyl-1,1,2,4,4,7-hexamethyl tetraline) across the placenta and into milk of rats during and after pregnancy following repeated oral administration has been investigated.

 

For the main study 14C-AHTN was formulated as a solution in maize oil and was administered orally to two groups of 18 pregnant rats at low and high dose levels of 2 mg/kg/day and 20 mg/kg/day respectively.

 

Consecutive daily oral doses of 14C-AHTN began on Day 14 of the gestation period and continued up to Day 7 of parturition. Milk and blood samples from three animals at each dose level were taken at 4, 8 and 24 hours on Days 3 and 7 after parturition. 

Concentrations of radioactivity in milk and plasma have been determined by liquid scintillation counting and concentrations of AHTN in milk have been determined by an HPLC method.

 

For rats dosed with 14C-AHTN at the low level, mean plasma radioactivity levels after Day 3 of parturition declined from 3.13 to 1.10 ppm over 24 hours. After Day 7 of parturition, mean plasma radioactivity levels peaked at 2.41 ppm and declined with time.

 

For rats dosed with 14C-AHTN at the high level, mean plasma radioactivity levels after Day 3 of parturition were 25.1 ppm at 4 hours, approximately 10 times those measured in the low dose animals. Concentrations of plasma radioactivity declined with time. A similar pattern was observed in the Day 7 of parturition samples.

 

For rats dosed with 14C-AHTN at the low level, mean milk radioactivity levels after Day 3 of parturition were 1.45 ppm at 4 hours and declined at a similar rate to total plasma radioactivity levels. Concentrations of radioactivity were similar in the Day 7 of parturition samples.

 

Total radioactivity concentrations in the high level rat milk samples were approximately 10 times those measured in the low dose experiment and showed a similar pattern of decline with time.

 

Mean levels of AHTN in milk of rats dosed at the low level represented 10 - 35% of the total radioactivity in milk and declined from less than 0.7 ppm to 0.1 ppm over 8 hours.

 

Mean levels of AHTN in milk of rats dosed at high level were 9.43 ppm and 2.14 ppm at 4 and

hours respectively after Day 3 of parturition and 7.73 ppm and 2.95 ppm at 4 and 8 hours respectively after Day 7 of parturition. Those levels represented 19 - 43% of the total radioactivity in milk.

 

At both the high and low dose levels, at 24 hours after Days 3 and 7 of parturition, radioactivity levels were too low for accurate determination of AHTN.

 

Two additional animals received low level doses of 14C-AHTN for 5 days beginning on Day 14 of the gestation period. The animals were sacrificed at 4 and 24 hours after the last dose and taken for whole autoradiography. Radioactivity levels were barely detectable in the foetus at both sacrifice times.