N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

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:

1992

Report date:

1992

Materials and methods

Objective of study:

absorption

distribution

excretion

Principles of method if other than guideline:

Since the major route of human exposure is dermal, this study was designed to obtain information on the
pharmacokinetic and distribution parameters of MGK 264 following multiple dermal administrations in the rat.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

EC Number:

204-029-1

EC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Cas Number:

113-48-4

Molecular formula:

C17H25NO2

IUPAC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

impurity 1

Reference substance name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

Molecular formula:

C20H38N2O2

IUPAC Name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

impurity 2

Reference substance name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Molecular formula:

C12H19NO2

IUPAC Name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Test material form:

liquid

Details on test material:

Purity and characterisation analysis conducted on the following sample; Supplier: McLaughlin Gormley King ; Batch Number: AB9500

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: CRL:CD Br

Details on species / strain selection:

The rat is a species commonly used for pharmacokinetic
studies and is the animal of choice for this study according to
the EPA Pesticide Assessment Guidelines. In addition, a number of
other toxicological evaluations have been and/or are scheduled to be
conducted on MGK 264 utilizing this strain of rat as the test animal

Sex:

male/female

Details on test animals or test system and environmental conditions:

All experiments were performed on adult male and female Charles River CD
(CRL:CD Br) rats obtained from Charles River Breeding Laboratories, Portage,
Michigan. The rats were approximately six weeks of age when received by the BTC.
Upon arrival, the test animals were transferred to a quarantine room where they were
eartagged and maintained in individual, hanging, stainless steel cages throughout the
two week quarantine period. Soon after their arrival, the animals were examined for
general health by the staff veterinarian. The animals were weighed once each week
during the quarantine period. During the second week of quarantine, the animals
were weighed and their food consumption was monitored. Animals that showed poor
food consumption or poor weight gain during quarantine were eliminated from the
study. Morning and afternoon mortality checks were conducted each day during the
quarantine and testing periods. Prior to dosing, all animals were re-examined to
assure normal health, moved to the test room and assigned to treatment groups using
a weight stratified randomization procedure.
The animals were fasted for an eighteen hour period prior to the initial
nonradiolabeled dose and six hours post-dosing as well as an eighteen hour period
prior to the radiolabeled dosing and a six hour period post-dosing. Otherwise, the
animals received Purina Rodent Chow No. 5002 ad libitum. Water was provided ad
libitum throughout the quarantine and study periods. No known contaminants were
present in the diet or water which would affect the outcome of the study. Therefore,
no additional analyses outside those completed by the manufacturer and the local
water district were performed.
The room temperature ranged between 69°F-74°F; the relative humidity ranged
between 56%-79% during the quarantine and study periods. A 12 hour light,
12 hour dark cycle was established and the air exchange rate was at least seven per
hour.

Administration / exposure

Route of administration:

dermal

Vehicle:

other: isopropyl alcohol

Details on exposure:

The nonradiolabeled dose was applied evenly over the application area using a
precalibrated 100 μL glass wiretrol micropipette. The radiolabeled dose was also
applied evenly to the application area within the glass enclosure (backpack). All
doses were administered on a constant volume basis i.e., 100 μL of dosing solution.
The actual amount of dosing solution applied was measured by weight as a
differential between the weight of the micropipette before and after dosing. After the
radiolabeled dosing, a nonocclusive plastic cover was glued to cover the top of the
backpack. Each backpack was checked daily and reglued if necessary.

Duration and frequency of treatment / exposure:

This study consisted of two experiments conducted in rats. The first experiment involved the determination of the blood concentration of radioactivity with time, the determination of peak blood level and half-life of radioactivity in blood, following multiple dermal administration of nonradiolabeled MGK 264 for fourteen consecutive days followed by a single dermal administration of [Hexyl-l-14C] MGK 264 on day 15. The second experiment involved the euthanasia of animals at peak blood level of radioactivity, first and second half-life of radioactivity and at 168 hours following the same dosing regimen.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

12 mg/kg bw/day (nominal)

Remarks:

All male rats on both experiments.

Doses / concentrations 2

Dose / conc.:

17 mg/kg bw/day (nominal)

Remarks:

Female rats only (experiment 2 only)

No. of animals per sex per dose / concentration:

FIrst experiment - 5 male rats
Second experiment - 4 groups of 5 male rats and one group of female rats

Control animals:

no

Positive control reference chemical:

not specified.

Details on study design:

This study consisted of two experiments conducted in rats. The first experiment involved
the determination of the blood concentration of radioactivity with time, the determination of
peak blood level and half-life of radioactivity in blood for male rats. The second experiment
involved the determinations of the amount of compound remaining on the skin and in various
tissues and excreta at peak blood level, at blood half-life and second blood half-life of
radioactivity in male rats and at 168-hours in male and female rats following multiple dermal
administrations of MGK 264 for fourteen days followed by a single dermal administration
of [Hexyl-l-14C] MGK 264 (Endo/Exo Mixture).

In the first experiment, five male rats were administered a daily dose of 100 μL of an isopropyl
alcohol solution containing 5 % (w/w) of nonradiolabeled MGK 264 for fourteen consecutive days.
On day 15, each rat was administered a single dermal dose of 100 μL of 5% w/w [Hexyl-l-14C]
MGK 264 in isopropyl alcohol. Each rat received approximately 12 mg/kg body weight and between
13.9-14.5 μCi of radioactivity. The rats were fasted for approximately 18 hours prior to the first
day of dosing with the nonradiolabeled solution and prior to the administration of the radiolabeled
solution. Following the radiolabeled dose, blood samples were collected from the tail vein at
different time intervals over a 120 hour period and the radioactivity in blood was determined. Blood
radioactivity was very low at all time intervals (2 to 3 times background) suggesting that the
absorption of radioactivity was extremely slow. Two radioactive peaks were present in blood, one
at six hours post-dose administration and one at ten hours post-dose administration. The half-life of
blood radioactivity was calculated to be 28.5 hours. Since these results were very close to the peak
(12 hours) and half-life (31 hours) determined in the single dermal dose study, the decision was made
to use the same time of euthanasia from the single dermal dose, so that data from the two regimens
could be compared.
In the second experiment, four groups of five male rats and one group of five female rats were
administered multiple dermal doses of MGK 264 as described for the blood kinetics study followed by a single dermal dose of [Hexyl-l-14C] MGK 264 at approximately 12 mg/kg body weight for the
males and 17 mg/kg body weight for the females. Each rat was administered between 13. 9-14. 2 μCi
of radioactivity. Based on the blood kinetic data, one group of five males was euthanized at each
of the following time intervals; peak blood level ofradioactivity (12 hours); blood half-life (43 hours)
and second blood half-life of radioactivity (74 hours); and 168-hours post-dose administration. The
group of five females was euthanized at 168-hours post-dose. Urine and feces were collected for
all groups at predetermined time intervals until the time of euthanasia. When the rats were
euthanized, the treated skin and enclosures were removed and rinsed, the animals were then
necropsied and selected tissues and organs were harvested. Samples of urine, feces, tissues, skin
rinse and enclosure rinse were subsequently analyzed for radioactivity.

Details on dosing and sampling:

Experiment 1:
Approximately 22 hours prior to the first day of dosing with the nonradiolabeled test
article, the back and shoulders of five rats were shaved and cleansed with acetone.
The food was removed approximately 18 hours prior to the initial dose and returned
to the rats six hours after dosing. The animals were administered approximately
100 μL of the nonradiolabeled test article to the shaved area of the back using a
100 μL Wiretrol micropipette (Drummond Scientific, Broomall, PA), for fourteen
consecutive days. The nonradiolabeled MGK 264 was administered as a 5 % (w/w)
solution in isopropanol. On day 14 ( prior to the final nonradiolabeled dose, each rat
was anesthetized with Ketamine/Xylazine (7: 1 v/v) and the back and shoulders were
shaved and cleansed with acetone. Care was taken not to abrade the skin. After
shaving, a glass contoured rectangular enclosure (2.5 cm x 5 cm; maximum height,
1.3 cm) was glued onto the middle of the shaven area on each animal's back with a
cyanoacrylate glue (Super Glue). Silicone medical adhesive, Type A, was applied
as a seal around the outside of each enclosure. Each rat was administered the day 14
dose and returned to its cage. Food was removed 18 hours prior to dosing with
14C-MGK 264.On day 15, each rat was administered approximately 100 μL of the radiolabeled
dosing solution to the skin within the enclosure using a 100 μL Wiretrol micropipette
(Drummond Scientific, Broomall, PA). After dosing, a plastic cover with holes to
provide for a nonocclusive cover was glued to the top of the rectangular enclosure
to prevent disturbance of the application site and to prevent mechanical loss of the
test material. The animals were transferred to hanging, stainless steel cages and
placed in individual rat restrainers for blood sampling. Blood samples (100 μL) were
collected from the tail vein at 15, 30 and 60 minutes; 2, 3, 4, 5, 6, 8, 10, 12, 24,
36, 48, 72, 96 and 120 hours. The rats were allowed water ad libitum throughout
the study and food ad libitum six hours after dosing. After the 120-hour blood
collection, the animals were euthanized by C02 asphyxiation and discarded as
radioactive waste.

Results and discussion

Preliminary studies:

Experiment 1 results: Two radioactive peaks were present in blood, one at six hours post-dose administration and one at ten hours post-dose administration. The half-life of blood radioactivity was calculated to be 28.5 hours. As both results were close to peak (12 hours) and half-life (31 hours) determined in the single dermal dose study, same time of euthanasia from single dermal dose study will be used for experiemnt 2.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

For the animals euthanized at peak blood level of radioactivity, a majority of the administered radioactivity (71. 85 % ) was recovered in the skin rinse. For the animals euthanized at blood half-life and second blood half-life of radioactivity, the mean percentages of dosed radioactivity in the skin rinse were 49. 54 % and 3 7 .54 % , respectively. For the males and females euthanized at 168-hours, the mean percentages of dosed radioactivity in the skin rinse were 8.09% and 0.44%, respectively. The dosed radioactivity was slowly absorbed from the skin.
A comparison of the 14C-MGK 264 derived blood radioactivity following a single or multiple dose was considered. The blood levels of radioactivity peaked twice in each dosing regimen and the half-lives were similar. These data demonstrated that there was little or no difference in the blood level of radioactivity or half-life of radioactivity between the two dosing regimens.

Details on distribution in tissues:

Mean % applied radioacivity (>0.11% AR):
peak blood level of radioactivity (12 hours) - 7.3% AR in intestines; 2.2% in carcass 0.5% AR in liver
blood half-life (43 hours) - 6.6% AR in intestines; 1.7% in carcass 0.4% AR in liver
second blood half-life of radioactivity (74 hours) - 5.4% AR in intestines; 1.0% in carcass 0.3% AR in liver
168-hours post-dose administration in male rates- 1.2% AR in intestines; 0.8% in carcass 0.1% AR in liver
168-hours post-dose administration in female rates- 0.3% AR in intestines; 0.6% in carcass

Details on excretion:

Mean % applied radioacivity:
peak blood level of radioactivity (12 hours) - 71.9% AR recovered from urine/ 0.2% AR recovered from feces
blood half-life (43 hours) - 25.7% AR recovered from urine/ 11.1% AR recovered from feces
second blood half-life of radioactivity (74 hours) - 33.2% AR recovered from urine/ 16.5% AR recovered from feces
168-hours post-dose administration Male rats - 51.5% AR recovered urine/ 30.4% AR recovered from feces
168-hours post-dose administration female rats - 69.3% AR recovered urine/ 27.6% AR recovered from feces

Applicant's summary and conclusion

Conclusions:

For the animals euthanized at peak blood level of radioactivity, a majority of the administered radioactivity (71. 85 % ) was recovered in the skin rinse. For the animals euthanized at blood half-life and second blood half-life of radioactivity, the mean percentages of dosed radioactivity in the skin rinse were 49. 54 % and 3 7 .54 % , respectively. For the males and females euthanized at 168-hours, the mean percentages of dosed radioactivity in the skin rinse were 8.09% and 0.44%, respectively. The dosed radioactivity was slowly absorbed from the skin. By 168 hours, a majority of the administered radioactivity was excreted through the urine (51.48% for the males, 69.29% for the females) with a significant amount of radioactivity recovered from the feces (30.37% for the males, 27.65% for the females). This indicated that biliary excretion played an important role in the elimination of radioactivity from the body.
The radioactivity recovered from the carcass decreased with time indicating that no accumulation of radioactivity in the body occurred. In general, mean tissue radioactivity decreased with time and did not accumulate in the tissues studied. The total mean percent of administered dose recovered in the four groups ranged between 99.40% - 104.96%. A comparison of the results of the single dose and multiple dose studies indicated that, with the exception of a higher amount of radioactivity present in the skin at the different euthanasia time intervals, following the multiple dose administration, the patterns of absorption, tissue distribution and elimination between the single and multiple doses are comparable.


In conclusion, with the exception of the higher amount of radioactivity present in the skin at the different euthanasia time intervals, following multiple dose administration, the patterns of absorption, tissue distribution and elimination between the single and multiple doses are comparable.

Executive summary:

This study consisted of two experiments conducted in rats. The first experiment involved the determination of the blood concentration of radioactivity with time, the determination of peak blood level and half-life of radioactivity in blood, following multiple dermal administration of non-radiolabeled MGK 264 for fourteen consecutive days followed by a single dermal administration of [Hexyl-l-14C] MGK 264 on day 15. The second experiment involved the euthanasia of animals at peak blood level of radioactivity, first and second half-life of radioactivity and at 168 hours following the same dosing regimen. The purpose of the second study was to determine the tissue distribution, rate and route of excretion and balance of radioactivity at the time of euthanasia. Prior to the initiation of this study, an identical study was performed with the exception that a single dermal dose of 14C-MGK 264 was applied (BTC Study No. P02072). The comparison of the blood concentration of radioactivity with time and the tissue distribution and excretion between the single and multiple dermal doses are presented in this report. In the first experiment, five male rats were administered a daily dose of 100 μL of an isopropyl alcohol solution containing 5 % (w/w) of nonradiolabeled MGK 264 for fourteen consecutive days. On day 15, each rat was administered a single dermal dose of 100 μL of 5% w/w [Hexyl-l-14C] MGK 264 in isopropyl alcohol. Each rat received approximately 12 mg/kg body weight and between 13.9-14.5 μCi of radioactivity. The rats were fasted for approximately 18 hours prior to the first day of dosing with the non-radiolabeled solution and prior to the administration of the radiolabelled solution. Following the radiolabelled dose, blood samples were collected from the tail vein at different time intervals over a 120 hour period and the radioactivity in blood was determined. Blood radioactivity was very low at all time intervals (2 to 3 times background) suggesting that the absorption of radioactivity was extremely slow. Two radioactive peaks were present in blood, one at six hours post-dose administration and one at ten hours post-dose administration. The half-life of blood radioactivity was calculated to be 28.5 hours. Since these results were very close to the peak (12 hours) and half-life (31 hours) determined in the single dermal dose study, the decision was made to use the same time of euthanasia from the single dermal dose, so that data from the two regimens could be compared. In the second experiment, four groups of five male rats and one group of five female rats were administered multiple dermal doses of MGK 264 as described for the blood kinetics study followed by a single dermal dose of [Hexyl-l-14C] MGK 264 at approximately 12 mg/kg body weight for the males and 17 mg/kg body weight for the females. Each rat was administered between 13. 9-14. 2 μCi of radioactivity. Based on the blood kinetic data, one group of five males was euthanized at each of the following time intervals; peak blood level of radioactivity (12 hours); blood half-life (43 hours) and second blood half-life of radioactivity (74 hours); and 168-hours post-dose administration. The group of five females was euthanized at 168-hours post-dose. Urine and feces were collected for all groups at predetermined time intervals until the time of euthanasia. When the rats were euthanized, the treated skin and enclosures were removed and rinsed, the animals were then necropsied and selected tissues and organs were harvested. Samples of urine, feces, tissues, skin rinse and enclosure rinse were subsequently analyzed for radioactivity. For the animals euthanized at peak blood level of radioactivity, a majority of the administered radioactivity (71. 85 % ) was recovered in the skin rinse. For the animals euthanized at blood half-life and second blood half-life of radioactivity, the mean percentages of dosed radioactivity in the skin rinse were 49. 54 % and 3 7 .54 % , respectively. For the males and females euthanized at 168-hours, the mean percentages of dosed radioactivity in the skin rinse were 8.09% and 0.44%, respectively. The dosed radioactivity was slowly absorbed from the skin. By 168 hours, a majority of the administered radioactivity was excreted through the urine (51.48% for the males, 69.29% for the females) with a significant amount of radioactivity recovered from the feces (30.37% for the males, 27.65% for the females). This indicated that biliary excretion played an important role in the elimination of radioactivity from the body.

The radioactivity recovered from the carcass decreased with time indicating that no accumulation of radioactivity in the body occurred. In general, mean tissue radioactivity decreased with time and did not accumulate in the tissues studied. The total mean percent of administered dose recovered in the four groups ranged between 99.40% - 104.96%. A comparison of the results of the single dose and multiple dose studies indicated that, with the exception of a higher amount of radioactivity present in the skin at the different euthanasia time intervals, following the multiple dose administration, the patterns of absorption, tissue distribution and elimination between the single and multiple doses are comparable.