Hexanediamide, N1,N1,N6,N6-tetrakis(2-hydroxyethyl)-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 04, 2001 to August 09, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well described GLP compliant study conducted to recognized international test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Primid XL-552

IUPAC Name:

Primid XL-552

Details on test material:

- Name of test material: Primid XL-552
- Molecular weight: 320.39
- Physical state: Crystalline solid
- Analytical purity: 95.6%
- Lot/batch No.: 9309960/20
- Expiration date of the lot/batch: May 01, 2002
- Amount needed : approximately 1300 mg
- Amount supplied: 50g
- Solubility: water 608.3 g/l
- Melting point: 120- 125°C
- Storage: In the original container, at room temperature
- Radiochemical purity (if radiolabelling): >98%
- Molecular weight (at this specific radioactivity): 320.70
-Specific radioactivity : 10 mCi/mmol (370 MBq/mmol) or 31.18 µCi/mg (1.154MBq/mg)
- Lot no: 00BLY058
- Expiration date of radiochemical substance (if radiolabelling): The radiochemical purity was checked by RCC prior to administration. Therfore, no expiration date is needed.
- Amount supplied: 5mCi (185 MBq)
- Amount nedeed: About 2 mCi or about 78 MBq
- Storage condition of test material: at 20°C, in the dark
- Other: safety precautions: routine hygenic procedures according to the Swiss Legislation on Radiological Protection (Switzerland,ordinance of June 22, 1994)

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: HanBrl WIST: Wistar rats, outbred, SPF-quality

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology and Animal Breeding Division Wolferstrasse 4, CH-4414 Fuellinsdorf/Switzerland.
- Age at study initiation: Males 6-8 weeks, Females 7-10 weeks.
- Weight at study initiation: The body weights were determined at the beginning of acclimation ( body weight males: 140-160g, females 150-170g ) and at the day of treatment with C-PRIMID XL-552 (body weight males and females 135-172g, groups 1-8). For groups 9 and 10, the body weight prior to the radiolabelled test item administration ranged from 191-256g.
- Fasting period before study: The rats were fasted overnight prior to administration.
- Housing: In groups of 2-4 rats in Makrolon cages under conventional hygenic conditions with standard soft wooden bedding during acclimation. The accomodations during the treatments are decribed in the respective experiments.
- Individual metabolism cages: yes.
- Diet:Pelleted 3433-Kliba rat maintenance diet ad libitum (PROVIMI KLIBA AG, CH-4303 Kaiseraugust/Switzerland).
- Water: Tap water ad libitum.
- Acclimation period: At least 5 days to laboratory environment, including 1-3 days to cages with a stainless grid or to all-glass metabolism cages. The animals of groups 9 and 10 were placed into the metabolism cages immediately after the radiolabelled administration.
- Initial number: 26 males and 36 females; additionally one reserve animal of each sex for each batch.
- Actual number: 36 males and 36 females.
- Identification: Individual numbers (ear tags).
- Health status: the health status of the treated animals was checked visually at daily intervals .
ENVIRONMENTAL CONDITIONS
- Temperature: Target temperature : 19.0 -25.0 °C
- Humidity: 40.0 - 70.0 %
- Air changes: 10-15 times/hours
- Photoperiod (hrs dark / hrs light): 12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

One concentrated stock solution was prepared: The total amount of labelled test item ( 160mg) were quantitatively transferred with water into a 20 ml volumetric flask, and filled up with water. The specific radioactivity was measured by liquid scintillant counting (LSDC) . Based on a specific activity of 1.154 MBq/mg an amount of 154.86 mg C Primid XL-552 was found. Based on the target specific radioactivities of 500 kBq/mg and 50kBq/mg for the low and high dose level, two application solutions were prepared in water.

High dose application solution: an aliquot of 5.81 ml of the stock solution and 993.59 mg unlabelled Primid XL-552 was given into a 100 ml volumetric flask and filled up with water. The total amount of test item was 1035.37 mg and the new specific activity was determined to be 1.2586 µCi/mg. The specific weight of the administration solution was determined to be 0.9982 g/ml.

Low dose application solution: an aliquot of 6.46 ml of the stocks solution and 65.32 mg unlabelled Primid XL-552 was given into a 200 ml volumetric flask and filled up with water. The total amount of test item was 113.31 mg and the new specific activity was determined to be 13.2100 µCi/mg. The specific weight of the administration solution was determined to be 1.0168 g/ml. The two application solutions were stored at about -20°C.
Un-labelled test item
Based on a target dose level of 5 mg/kg and a target administration volume of 1 ml/100 g rat, 250 mg Primid XL-552 were dissolved in 500 ml purified water and aliquots of 30 ml were stored at -20°C.


HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The stability of the test item during administration was determined by its radiochemical purity using an aliquot of the administration solutions before and after the treatment as compared to the stock solution. The purity of the administration solution for group 1 was 98.7 % before and after the administration. The purity of the administration solution for group 2 and 3 was 97.3 % before and 97.5% after the administration. For the groups 6 and 8 purity measurements of the application solution were performed after the application. For group 4, the purity was 97.4% , for group 6 and 8 a purity of 98.8% was measured. These results indicated, that the test item was sufficiently stable in the administraion solution to perform the study.

Duration and frequency of treatment / exposure:

Levels of radioactivity in expired air, urine, faeces, blood plasma and organs/tissues and the identity of metabolites in plasma, urine, faeces and organs/tissues were determined based on the following experiments:
- a balance study in both sexes at two dose levels after single oral administration (groups 1-4).
Number of treatments: One treatment.
Duration of recovery: 96 hours.
- a blood and plasma level study in both sexes at two dose levels after single oral administration (groups 5-8).
Number of treatments: One treatment.
Duration of recovery: 96 hours max
- a balance study in both sexes after repeated oral administration of non-labelled test item (14x) followed by single oral administration of C-labelled test item at the low dose level (groups 9 and 10).
Number of treatments: 14 daily administrations with unlabelled test item followed bu oral administration with the C-labelled test item.
Duration of recovery: 96 hours.

Doses / concentrations

Remarks:

Doses / Concentrations:
Target dose level:
Low dose: 5 mg/kg of body weight (spec. rad. 13.5 µCi/mg or 500 kBq/mg)
High dose: 100 mg/kg of body weight (spec. rad. 1.35 µCi or 50 kBq/mg)
Target oral administration volume: 1.0 ml/100g of body weight

The oral administration of (C)-Primid XL-552 resulted in an avarage dose level of 100.5 - 101.0 mg/kg body weight for the groups 1, 4, 6 and 8 in an average dose level of 5.0-5.2 mg/kg body weight for the groups 2, 3, 5, 7, 9 and 10 .

No. of animals per sex per dose / concentration:

Balance Study including expired air after single oral administration of C-Primid XL-552 at the High Dose Level to Male Rats:

Target dose level: 100 mg/kg of body weight
(C)-Primid XL-552
Group: 1
Sex: male
Animal-no. 1-4

Balance Study after Single Oral Administration of C-Primid XL-552 at the Low Dose Level to Male Rats and the both Dose Levels to Female Rats:

Target dose levels (mg/kg)
5.0 5.0 100
Group: 2 3 4
Sex: male female female
Animal no. 5-8 9-12 13-6

Levels of radioactivity in blood and plasma after Single Oral Administration at two Dose Levels of C-Primid XL-552 to Male and Female Rats:

Target dose levels (mg/kg) Time intervals
5.0 100 5.0 100 (hours)
Group: 5 6 7 8
Sex: male male female female
Animal no. 17-20 29-32 41-44 53-56 0, 2, 8, 72
21-24 33-36 45-48 57-60 0.5, 4, 24, 96
25-28 37-40 49-52 61-64 1, 6, 48

Balance Study after Repeated Oral (14x) Administration of Unlabelled Primid XL-552 Followed by Single Oral Administration of C-Primid XL-552 at the Low Dose Level to Male and Female Rats:

Target dose level 5.0 mg/kg of body weight
Group: 9 10
Sex: male female
Animal no. 65-68 69-72

Control animals:

no

Details on study design:

- Rationale for animal assignment: Animal numbers will be given randomly.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled : expired air, urine, faeces, blood, organs/tissues, residual carcass and cage wash.
- Time and frequency of sampling: (Single Oral Administration of C-Primid XL-552)
Volatiles: 8, 24, 48, 72 and 96 hours after administration.
Urine/Faeces: 8, 24, 48, 72 and 96 hours after the administration. Faeces : 24, 48, 72 and 96 hours after administration.
Blood: after 96 hours, animals were killed by carbon dioxide. Blood was collected in the thoracic cavity after heart incision and sampled into heparinized tubes. Aliquots (0.5 - 1.0 ml) of blood were separated, the remaining blood was centrifuged and the plasma decanted.
Organs/Tissues/residual carcass: after 96 hours heart, lung, liver, stomach, thymus, spleen, intestinal tract, adrenal glands, kidney, gonads, muscle, bones, fat, thyroid gland, pancreas and residual carcass were taken.
- Time and frequency of sampling: (Single Oral Administration at Two dose levels of C-Primid XL-552 to Male and Female rats)
Blood: 0, 2 and 8 hours or
0.5, 4 and 24 hours or
1, 6 hours after administration and collected into heparinized tubes.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled : urine, faeces
- Time and frequency of sampling:
Urine were collected from 0 to 48 hours after the firts administration with C-labelled test item.
Faeces were collected from 0 to 48 hours after the firts administration with C-labelled test item.
- From how many animals:
Urine: the urine of 4 rats were pooled.
Faeces: the faeces of 4 animals were pooled.
- Method type(s) for identification :
Urine: HPLC and TLC analyses were performed directly without any further sample work-up .
Faeces: the faeces were extracted three times with acetonitrile/water (8+2, v/v). Each extraction was performed by shaking the sample for 30 min. at room temperature. Radioactivity in each extract was measured after centrifugation for 10 min. at 1500 to 2500 g .
The acetonitrile extracts were pooled and directly analysed by HPLC and TLC.

Results and discussion

Preliminary studies:

The study was performed in two steps. In the first study part, male rats were orally treated with the high dose level of C-Primid XL-552 and radioactivity was determined in expired air, urine, faeces, blood, organs/tissues, residual carcass and cage wash.
In the second part of the study, female rats were orally treated with the low and high dose levels and male rats were treated with the low dosel level. Radioactivity was determined in urine, faeces, blood, organs/tissues, residual carcass and cage wash.

Main ADME results

Main ADME results 1

Type:

excretion

Results:

After oral administration excretion of radioactivity occured rapidly, mainly via faeces, almost completely within 48 hours. No differences in the excretion patterns between male/female , low/high dose as well as single /repeated administration is observed

Main ADME results 2

Type:

other: pharmacokinetic parameters

Results:

No differences between male/female rats, low/high dose as well as single /repeated administration were found.

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:

Group 1
At 96 hours after the administration, organs/tissues with the highest residual radioactivity were : Blood (1.138 µg eq/g), plasma (0.239 µg eq/g), liver (0.130 µg eq/g) and skin (0.106 µg eq/g).
Group 2
At 96 hours after the administration, organs/tissues with the highest residual radioactivity was : liver (0.006 µg eq/g).
Group 3
At 96 hours after the administration, tissues with the highest residual radioactivity was: Intestinal tract (0.003 µg eq/g).
Group 4
At 96 hours after the administration, organs/tissues with the highest residual radioactivity were : Blood (0.029 µg eq/g), intestinal tract (0.036 µg eq/g), liver (0.070 µg eq/g) and skin (0.013 µg eq/g).
Group 9
At 96 hours after the administration, tissues with the highest residual radioactivity was: Intestinal tract (0.003 µg eq/g)
Group 10
At 96 hours after the administration, organs/tissues with the highest residual radioactivity were : Stomach (0.004 µg eq/g), intestinal tract (0.003 µg eq/g), carcass (0.003 µg eq/g).

Details on excretion:

Characterisation of residual radioactivity
Radioactivity was characterised in faeces (0-48 hours pools per group) and liver (group 1 only). It was also attemted to characterise plasma of group 1. However, due to a too low amount of radioactivity even extraction was not possible.
Faeces (o-48 hours), extraction of radioactivity
Groups 1 (male, high dose level)
Total extracted radioactivity amounted to 96.2% of the radioactivity recovered. Remaining radioactivity (non-extractable) was 3.8%.
Group 2 (male, low dose level)
Total extracted radioactivity amounted to 96.7% of the radioactivity recovered. Remaining radioactivity (non-extractable) was 3.8%.
Group 3 (female, low dose level)
Total extracted radioactivity amounted to 95.2% of the radioactivity recovered. Remaining radioactivity (non-extractable) was 4.8%.
Group 4 (female, high dose level)
Total extracted radioactivity amounted to 96.7% of the radioactivity recovered. Remaining radioactivity (non-extractable) was 3.3%.
Group 9
Total extracted radioactivity amounted to 95.3% of the radioactivity recovered. Remaining radioactivity (non-extractable) was 4.7%.
Group 10
Total extracted radioactivity amounted to 95.5% of the radioactivity recovered. Remaining radioactivity (non-extractable) was 4.5%.

Extraction of radioactivity in liver
The radioactivity was extracted from the liver tissue using three times acetonitrile. Total extracted radioactivity amounted to 38.1% . The amount of radioactivity (non-extractable) remaining in the tissue amounted to 61.9% or 0.081 µg eq/g. Due to the low radioactivity amounts in the extract, no further analyses were possible. Also, due to approximately 6200 dpm in 22g of tissue residue, no further extractions were performed.

Toxicokinetic parameters

Test no.:

#1

Toxicokinetic parameters:

AUC: levels for blood and plasma ranged from 131.2-234.1 ng*h/ml for the low dose and from 2035.1 to 4442.2 ng*h/ml for the high dose.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Metabolite patterns were determined by HPLC and TLC analyses in faeces and urine . Due to too low amounts of radioactivity, metabolite patter in organ/tissue could not be determined.
Metabolite Patterns in Faeces
Group 1 In the organic extract of the 0-48 h faeces sample at least 6 radiactive fractions were found . The parent item amounted to 76.6% of the radioactivity administered. Unknown metabolite fractions were detected in amounts ranging from 1.2 to 5.2% of the radioactivity administered.
Group 2 In the organic extract of the 0-48 h faeces sample at least 6 radiactive fractions were found . The parent item amounted to 73.8% of the radioactivity administered. Unknown metabolite fractions were detected in amounts ranging from3.0 to 5.7% of the radioactivity administered.
Group 3 In the organic extract of the 0-48 h faeces sample at least 6 radiactive fractions were found . The parent item amounted to 70.0% of the radioactivity administered. Unknown metabolite fractions were detected in amounts ranging from2.2 to 7.7% of the radioactivity administered.
Group 4 In the organic extract of the 0-48 h faeces sample at least 6 radiactive fractions were found . The parent item amounted to 78.1% of the radioactivity administered. Unknown metabolite fractions were detected in amounts ranging from 1.4 to 6.0% of the radioactivity administered.
Group 9 In the organic extract of the 0-48 h faeces sample at least 6 radiactive fractions were found . The parent item amounted to 83.2% of the radioactivity administered. Unknown metabolite fractions were detected in amounts ranging from 1.4 to 3.7% of the radioactivity administered.
Group 10 In the organic extract of the 0-48 h faeces sample at least 6 radiactive fractions were found . The parent item amounted to 71.5% of the radioactivity administered. Unknown metabolite fractions were detected in amounts ranging from 1.9 to 6.3% of the radioactivity administered.
Metabolite Patterns in Urine
Group 1 In the urine collected from 0-48 at least 4 radioactive fractions were found. The parent item was detected in an amount of 3.1%. Unknown metabolite fractions amounted from 0.2 to 0.4 of the radioactivity administered.
Group 2 In the urine collected from 0-48 at least 4 radioactive fractions were found. The parent item was detected in an amount of 3.4%. Unknown metabolite fractions amounted from 0.2 to 0.4 of the radioactivity administered.
Group 3 In the urine collected from 0-48 at least 4 radioactive fractions were found. The parent item was detected in an amount of 3.0%. Unknown metabolite fractions amounted from 0.1 to 0.3 of the radioactivity administered.
Group 4 In the urine collected from 0-48 at least 4 radioactive fractions were found. The parent item was detected in an amount of 3.0%. Unknown metabolite fractions amounted from 0.2 to 0.4 of the radioactivity administered.
Group 9 In the urine collected from 0-48 at least 3 radioactive fractions were found. The parent item was detected in an amount of 3.5%. Unknown metabolite fractions amounted from 0.2 to 0.3 of the radioactivity administered.
Group 10 In the urine collected from 0-48 at least 2 radioactive fractions were found. The parent item was detected in an amount of 2.8%. Unknown metabolite fractions amounted from 0.1%

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
Based on the results after single oral administration of (C)-Primid XL-552 to male and female rats at two dose levels as well as after repeated oral administration of unlabelled Primid followed by (C)-primid XL-552 at the low dose level to male and female rats, the conclusion are:
Excretion occured in all groups rapidly mainly via faeces. Within 48 hours, the radioactivity was almost completely excreted. At 96 hours after the administration, radioactive residues in organs/tissues were very low.
Concerning pharmacokinetic parameters, no difference between male/female rats, low/high dose as well as single/repeated administration were found.
The major amount of the radioactivity in the faeces could be extracted by using acetonitrile/purified water ( 8+2, v/v). In faeces up to 6 radioative fractions (F1-F6) were detected. F2 was identical to the parent item. Fractions F1, F3-F6 were unknown metabolite fractions.
In urine at least 4 radioactive fractions(U1-U4) were detected. U1 was identical to the parent item. Fractions U2-U4 were unknown metabolite fractions.

Executive summary:

The objective of the study was to determine the levels of radioactivity in expired air, urine , faeces, blood, plasma and organ/tisues as well as the identity of metabolities in plasma, urine, faeces and organs/tissues after oral administration of (C)-Primid XL-552 to male and female rats- The target dose levels were 100 mg/kg (high dose level, group 1,4, 6, 8) and 5 mg/kg (low dose level, group 2, 3, 5, 7, 9, 10). Based on this experiments:

- a balance study in both sexes at both dose levels after single oral administration (groups 1 -4)

- a blood and plasma level study in both sexes at both dose levels after single oral administration

- a balance study in both sexes after repeated oral administration of non-labelled test item (14x) followed by single oral administration of C-labelled test item at the low dose level (groups 9 and 10).

Excretion via faeces was measured in 24- hours intervals after administration up to 96 hours. Excretion via urine and expired air (group 1 only) was measured 8 hours and 24 hours after administration and then in 24 -hours intervals up to 96 hours. Animals were sacrified at 96 hours and radioactivity in selected organs/tissues was determined.

Based on the results after single oral administration of (C)-Primid XL-552 to male and female rats at two dose levels as well as after repeated oral administration of unlabelled Primid followed by (C)-primid XL-552 at the low dose level to male and female rats, the conclusion are:

Excretion occured in all groups rapidly mainly via faeces. Within 48 hours, the radioactivity was almost completely excreted. At 96 hours after the administration, radioactive residues in organs/tissues were very low.

Concerning pharmacokinetic parameters, no difference between male/female rats, low/high dose as well as single/repeated administration were found.

The major amount of the radioactivity in the faeces could be extracted by using acetonitrile/purified water ( 8+2, v/v). In faeces up to 6 radioative fractions (F1-F6) were detected. F2 was identical to the parent item. Fractions F1, F3-F6 were unknown metabolite fractions.

In urine at least 4 radioactive fractions(U1-U4) were detected. U1 was identical to the parent item. Fractions U2-U4 were unknown metabolite fractions.