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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report date:
1983

Materials and methods

Objective of study:
absorption
distribution
excretion
Principles of method if other than guideline:
- Principle of test: Different groups of male rats were treated with 14C-labeled test material followed by analysis of excrements, blood, organs and carcass to determine absorption, distribution and excretion of the test material.

- Short description of test conditions:
A single oral dose of the radiolabeled test material was given to 4 different exposure groups. Animal killing was performed at various time (3 h, 8h, 24 h, 72 h, 168 h) after treatment in test animal group 1 for examination of whole body sections. In test animal group 2 blood samples were taken at specified times for measurements of the substance / substance metabolite concentration. Animals of group 3 were examined for the rate of excretion in feces and urine by sampling at various time. Animals of test group 4 recieved 1/20 of the substance dose of test group 3 (otherwise the objection was identical wih group 3).

- Parameters analysed / observed: Determination of radioactivity in urine, faeces, blood, organs (liver, kidneys, testicles), tissue samples (muscle, fat)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]
EC Number:
265-512-0
EC Name:
Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]
Cas Number:
65140-91-2
Molecular formula:
C17 H29 O4 P. 1/2Ca
IUPAC Name:
calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]
Details on test material:
RADIOLABELLING INFORMATION
- Radiochemical purity: > 99 %
- Specific activity: 59.388 µCi/mg
Specific details on test material used for the study:
RADIOLABELLING INFORMATION
- Radiochemical purity: > 99 %
- Specific activity: 59.388 µCi/mg

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test solution preparation with the vehicle

The solid test substance was dissolved in the vehicle;
Test solution A (for animal test groups 1 and 2) with 2605,1 µg test substance/g solution and 152,24 µCi/g;
Test solution B (for animal test group 3) with 2596,1 µg test substance/g solution and 153,41 µCi/g
Test solution C (for animal test group 4) with 150.1 µg test substance/g solution and 8,834 µCi/g


FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution
Radiolabelling:
yes
Remarks:
14C-radiolabelling

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, 8741 Sulzfeld/Germany
- Weight at study initiation: 172-182 g
- Housing: For 7 days before administration of the test substance and during the experimental phase the control and test rats were housed separately in metabolism cages.
- Individual metabolism cages: Yes
- Diet: Dr. Rupprecht Schott Nachf. GmbH + Co., Hamburg/Germany, Art.-No. 253, ca. 22 g/day (except the day before administration of test substance, only 8 g)
- Water: tap water, ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-1
- Humidity (%): 55+/-5
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: A mixture of equal parts by weight of 1,2-dihydroxypropane and tap water.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The radioactive solution as well as the non-labelled solution were prepared shortly before administration to the test animals. Calculated amounts of 14-C-labelled or non-labelled test material were weighed in the glass vessels of an Ultrasonic homogeniser and dissolved in the defined amounts of the test vehicle at 30 °C within 30 minutes.



Duration and frequency of treatment / exposure:
Single oral exposure
Doses / concentrationsopen allclose all
Dose / conc.:
10.88 mg/kg bw (total dose)
Remarks:
Test group 1 (Distribution)
Dose / conc.:
10.71 mg/kg bw (total dose)
Remarks:
Test group 2 (Absoprtion)
Dose / conc.:
10.67 mg/kg bw (total dose)
Remarks:
Test group 3 (Excretion)
Dose / conc.:
0.53 mg/kg bw (total dose)
Remarks:
Test group 4 (Excretion)
No. of animals per sex per dose / concentration:
Test group 1 (Distribution): 5 test and 2 control animals
Test group 2 (Absorption): 6 test and 2 control animals
Test group 3/4 (Excretion): 6 low dose and 6 high dose test animals, 2 control animals
Control animals:
yes
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)

- Tissues and body fluids sampled: Urine, faeces, blood, organs (liver, kidneys, testicles), tissue samples (muscle, fat)

- Time and frequency of sampling:
Tissue/organ samples after 168 h (test group 3 and 4)
Urine, faeces: Collection during the entire experimental period (test group 3 and 4)
urine: 0-3, 3-6, 6-24, 24-48, 48-72, 72-96, 96-120, 120-144, 144-168 h after treatment; feces: 0-6, 6-24, 24-48, 48-72, 72-96, 96-120, 120-144, 144-168 h after treatment
Blood after 168 h ( test group 3 and 4) and after 0.25, 0.5, 1, 2, 4, 6, 12, 24, 48, 72, 120 and 168 h (test group 2)

Results and discussion

Main ADME resultsopen allclose all
Type:
absorption
Results:
Only small amounts were absorbed from the gastrointestinal tracts into the blood, reaching a maximum within the first and second hour after administration.
Type:
distribution
Results:
Absorbed radioactivity was concentrated significantly but only temporarily in the livers, virtually completely eliminated during period of study.
Type:
excretion
Results:
Within 168 h after oral administration an average of 87.6 % and 86.3 % (high dose and low dose, respectively) of the dose was excreted, mainly via the feces (85.4 % and 84.9 %). Further amounts of radioactivity were exhaled.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The absorption of the test substance from the gastrointestinal tract of the rat has a maximum between the first and second hour after oral administration. In this period, the average equivalent concentration amounted to about 0.78 µg test substance per g blood. The subsequent decrease of the average equivalent concentration of the test substance in the blood of the rats is clearly shown by the values at 48 h and 168 h after oral administration of 0.029 and 0.005 µg, resp., test substance per g blood (for details see table 1 "Any other information on results").
Details on distribution in tissues:
Autoradiograms showed that the substance was absorbed only in small portions from the gastrointestinal tracts and was concentrated significantly only in the liver of the test rats and only temporarily. These radioactivities and the clearly lower concentrations over the remaining organs and tissues of the test animals werealmost completely eliminated during the progress of experiment.
The gastrointestinal tracts of the rats killed at 168 h contained on average about 0.01% of the dose. As a consequence of the enormous elimination of the C-labelled test substance and/or its metabolites in faeces and urine, the results show that only small amounts of radioactivity were found in the blood, the organs, tissue samples and in the residual carcass. At the time of sacrifice, the blood of the rats contained an average of less than 0.01 % of the dose and about 0.06 % and 0.1 % were present in the residual carcasses of high dose and low dose animals, respectively. The blood, organs and tissue samples taken at the time of sacrifice and the residual carcass of the rats from the high dose group contained average equivalent concentrations from 3.7 (testicles) to 29.5 ng test substance (liver) per g, whereas the blood, organs, tissue samples and the residual carcass of the rats from the low dose animals contained only average equivalent concentrations from 0.3 (blood) to 2.7 ng test substance (liver) per g (for details see table 2 and table 3 "Any other information on results").
Details on excretion:
An average of 85.4 % and 84.9 % of the given radioactivities were eliminated in the faeces by rats from the high dose and low dose, respectively, within 168 h after oral administration of the 14C-labelled test substance. In the same interval, an average of 2.2 % and 1.4 % of the radioactivity amounts orally administered in form of the 14C-labelled test substance were found in the urine of the rats. According to these results, the rats, excreted, an average of 87.6 % (high dose) and 86.3 % (low dose) within 168 h after oral administration. As pre-tests showed, the test rats exhaled further amounts of radioactivity.

Any other information on results incl. tables

Table 1: Radioactivities, the average relative and equivalent concentrations of the orally administered test substance in the blood of rats after different time points.

 

Test substance

 

Radioactivity of blood [nCi/g] at time after administration [h]

 

Radioactivity

Dose

0.25

0.5

1

2

4

6

12

24

48

72

120

168

 

[µCi]

[µCi/g bw]

 [mg/kg bw]

 

 

 

 

 

 

 

 

 

 

 

 

Average

114.12

0.6228

10.711

17.39

36.52

45.67

41.09

17.06

12.67

5.988

4.066

1.714

0.9623

0.4406

0.2786

Standard deviation

3.4

0.018

0.32

9.84

23.55

32.79

25.70

3.6

3.62

1.54

1.42

1.33

0.53

0.14

0.07

Relative standard deviation (%)

3.0

3.0

3.0

56.6

64.5

71.8

62.5

21.3

28.6

25.7

34.8

77.6

54.8

32.0

27.9

Average relative concentration

 

 

 

27.780

58.339

72.955

65.636

27.249

20.233

9.565

6.496

2.738

1.537

0.704

0.4455

Average equivalent concentration

 

 

 

0.2976

0.6249

0.7814

0.7030

0.2919

0.2167

0.1025

0.0696

0.0293

0.0165

0.0075

0.0048

Relative concentration = average measured radioactivity per g test material/administered radioactivity per g body weight at time of intubation

Equivalent concentration = Relative concentration * dose administered (mg/g bw) * 1000 [µg/g test material]

Table 2: Excretion of radioactivity (%) in urine and feces by rats given 14C-labeled test material by intubation.

 

 

 

Average excreted radioactivity amounts [%(sd)]

Group

Number of animals

Route of excretion

0-3 h

3-6 h

6-24 h

24-48 h

48-72 h

72-96 h

96-120 h

120-144 h

144-168 h

high dose

6

urine

0.31 (0.26)

0.26 (0.08)

1.41 (1.46)

0.17 (0.11)

0.04 (0.02)

0.01 (0.01)

0.01

0.01

0.01

feces

0.03 (0.03)

76.99 (5.12)

7.95 (3.65)

0.36 (0.24)

0.07 (0.01)

0.03 (<0.01)

0.01 (<0.01)

0.01

low dose

6

urine

0.17 (0.09)

0.24 (0.1)

0.72 (0.12)

0.18 (0.10)

0.04 (0.01)

0.03 (0.01)

0.03 (0.01)

0.02

<0.01

feces

 

67.95 (6.13)

16.05 (4.39)

0.74 (0.43)

0.12 (0.05)

0.04 (0.02)

0.02 (0.01)

0.02 (0.01)

 

 

Table 3. Summary of the average radioactivity amounts in %, of the average relative (P) and equivalent concentrations (C) after 168 h.

tissue radioactivity amount (%) average relative concentration (P) equivalent concentration (C) (ng/g)
group high dose low dose high dose low dose high dose low dose
urine 2.2 1.4
feces 85.39 84.94
GI tract 0.01 0.01
blood < 0.01 < 0.01 0.53 0.66 4.6 0.3
liver 0.02 0.03 3.39 6.29 29.5 2.7
kidneys < 0.01 < 0.01 1.29 2.07 11.3 0.9
testicles < 0.01 < 0.01 0.43 0.88 3.7 0.4
muscle < 0.01 < 0.01 0.63 1.16 5.5 0.5
fat < 0.01 0.01 2.38 3.95 20.7 1.7
residual carcass 0.06 0.1 0.72 1.34 6.2 0.6
recovered radioactivity 87.68 86.49

Relative concentration P = average measured radioactivity per g test material/administered radioactivity per g body weight at time of intubation

Equivalent concentration C = Relative concentration * dose administered (mg/g bw) * 1000 [µg/g test material]

Applicant's summary and conclusion

Conclusions:
No bioaccumulation potential can be concluded based on the study results. The orally given test material and its possible metabolites are retained for only a limited time in male rats and are preferentially excreted in the feces, but also via the kidneys and by respiration.
Executive summary:

Absorption, distribution and elimination of the test substance by male rats was quantitatively investigated by radiotracer techniques under GLP-conditions. Simultaneously the residues

of the test substance and/or its metabolites in blood, different organs and tissue samples and in the residual carcass of the test animals

were determined radioanalytically. For that purpose the total of 29 male Sprague-Dawley rats was divided

into control and test groups. After an adaptive phase of 7 days, each rat of the test group 1-4 was given a single oral dose of about I0.9, 10.7, 10.7 or 0.53 mg of C-labelled test substance per kg body weight. The rats of the control groups were each given an average oral dose of about 10.5 mg of non-labelled

test item per kg of body weight and produced biological material samples which were used for the determination of blanc values for

radioactivity assays of feaces, urine, organs, blood etc..

During the study period (168 h) all urine and faeces from the rats of the test groups 3/4 as well as from two rats of the control

group were collected quantitatively during certain periods of time. At the end of the test, the anaesthetized rats were killed by bleeding to death. The blood was collected separately

from each animal and as for liver, kidneys, testicles, muscle and fat samples, the residual carcass and all faeces and urine samples the radioactivity content measured. From the animals of test group 2 and the respective control group, blood samples were obtained at specific times after administration by puncture of the ophthalmic venous plexus. The blood samples were also assayed for radioactivity measurement.

From the radioactivities of the biological samples, the excretion rates in feces and urine of radioactivity and the relative or equivalent concentrations of the test item in

blood, in different organs, tissue samples and in the residual carcass were calculated. The rats of test group 1 were killed at 3, 8, 24, 72 and l68 h after oral administration of the C-labelled test substance, and two animals of the control group were killed at 8 and 72 h after oral administration of the unlabelled test substance. Sagittal whole body sections of the eye-kidney, the suprarenal gland, the thyroid gland and the median plan were made. The whole body sections, after freeze drying, were placed on photographic films in order to examine the distribution of radioactivity over the different body planes. The investigation showed that the rats from test groups 3 and 4 eliminated on average 87.6 % (85.4 % in feces and 2.2 % in urine) and 86.3 % (84.9 % in feces and 1.4 % in urine) respectively, of the oral dose of test substance. Further, not yet quantified, portions of the applied radioactivity were exhialed by the rats.

Also, at 168 h after the oral administration of the C-labelled test item only small amounts of radioactivity were found in the dead bodies of the rats from test groups 3and 4.

At the time of killing the animals, the radioactivity in the blood the organs liver/kidneys/testicles, the gastrointestinal tracts and the residual carcass of the rats from test groups 3 and 4 amounted to < 0.01, < 0.04, 0.01 and 0.06 % and 0.0l, <0.05, 0.01 and 0.10 % respectively of the dose.

The absorption of radioactivity given orally in form of the C-labelled test substance from the gastrointestinal tracts of the rats reached a maximum within the first and second hour after administration (test group 2). Radioactivity given perorally in form of C-labeled test item was absorbed in only small portions from the gastrointestinal tracts and were concentrated significantly but only temporarily in the livers - as compared with all other organs and tissues of the test rats of group 1. However, such radioactivity and that distributed clearly and evenly in lower concentrations over the residual carcass of the rats was virtually completely eliminated during period of study.