Reaction mass of (2,4,6-trioxo-1,3,5-triazine-1,3,5(2H,4H,6H)-triyl)tri-2,1-ethanediyl triacrylate and 2-Propenoic acid, 1,1'-[[dihydro-5-(2-hydroxyethyl)-2,4,6-trioxo-1,3,5-triazine-1,3(2H,4H)-diyl]di-2,1-ethanediyl] ester

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well conducted published NTP study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Objective of study:

absorption

distribution

excretion

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Information concerning the used methods can be found at NTP homepage or under J Robert Buchanan, Leo T. Burka, Ronald L. Melnick. Purpose and Guidelines for Toxicokinetics Studies within the National Toxicology Program. Environmonmental Health Perspectives, Vol. 105, No. 5, pp. 468-471, 1997.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Trimethylolpropane Triacrylate (TMPTA)

IUPAC Name:

Trimethylolpropane Triacrylate (TMPTA)

Constituent 2

Reference substance name:

2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate

EC Number:

239-701-3

EC Name:

2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate

Cas Number:

15625-89-5

Molecular formula:

C15H20O6

IUPAC Name:

2,2-bis(prop-2-enoyloxymethyl)butyl prop-2-enoate

Details on test material:

Technical-grade unlabeled trimethylolpropane triacrylate was obtained from Aldrich Chemical Company
(Milwaukee, WI) in two lots (03115TV and 03914PV). Identity and purity analyses were conducted by the study
laboratory. Proton- and 13C-nuclear magnetic resonance and mass spectra were consistent with the structure.
Reverse phase high-performance liquid chromatography (HPLC) indicated the presence of several ultravioletabsorbing
impurities that were not identified
[14C]-Trimethylolpropane triacrylate (7.5 mCi, lot 91-350-35-02 and 5 mCi, lot R90-306-69-34) was obtained from
Chemsyn Science Laboratories (Lenexa, KS). The chemical’s identity was confirmed by coelution of
[14C]-trimethylolpropane triacrylate with unlabeled trimethylolpropane triacrylate by reverse phase HPLC. The
radiochemical purity of [14C]-trimethylolpropane triacrylate was determined to be less than 70%. After
development and application of a purification method, the average radiochemical purity of
[14C]-trimethylolpropane triacrylate was determined to be 98.8%.

Radiolabelling:

yes

Remarks:

14 C material used

Test animals

Species:

other: rats and mice

Strain:

other: male F344/N (rats) and B6C3F (mice)

Sex:

not specified

Details on test animals or test system and environmental conditions:

Young adult male F344/N rats (240-306 g) and adult B6C3F1 mice (25-35 g) were obtained from Charles River Laboratories (Raleigh, NC, and Kingston, NY). Animals were coded by ear tags and quarantined for approximately 1 week. Purina Rodent Chow No. 5002 and tap water were available ad libitum. Prior to the experiments, rats and mice were housed up to a maximum of four animals per cage in polycarbonate cages. During the excretion studies, animals were housed individually in glass metabolism chambers that allowed for separate collection of urine, feces, and exhaled CO2 beginning at least 1 day before the studies.

Administration / exposure

Route of administration:

other: dermal (occlusive) and i.V.

Vehicle:

other: acetone for dermal and a mixture of absolute ethanol, Emulphor®, and phosphate-buffered saline for i.V.

Details on exposure:

Rats received a single intravenous injection of 9.4 mg [14C]-trimethylolpropane triacrylate/kg body weight or a single dermal dose of 1.7, 15.2, or 130 mg/kg (13.3, 7.2, 10.5, and 10.5 μCi, respectively)
In a dermal-tape stripping experiment in rats, successive strips of the washed application site skin were analyzed following a single 124 mg/kg dose of [14C]-trimethylolpropane triacrylate (3.8 μCi).
In a preexposure study, rats were given a dermal dose of 151 mg/kg unlabeled trimethylolpropane triacrylate followed 24 hours later by a single dermal dose of 151 mg/kg [14C]-trimethylolpropane triacrylate (8.9 μCi).
Mice received a single dermal dose of 1.2 mg/kg [14C]-trimethylolpropane triacrylate (2.1 μCi)

Animals in dermal dosing experiments were sedated with an intramuscular dose of 60 mg/kg ketamine:xylazine (7:1) approximately 24 hours before dermal doses were applied, and the fur on the back of each animal was clipped. The clipped area was wiped with acetone, dried, and examined; animals with broken skin in the clipped area were excluded from the study. The doses were applied to 1 square inch (rats) or 0.5 square inch (mice) previously outlined with a permanent, felt-tip marker.

The intravenous injection dose was formulated in a mixture of absolute ethanol, Emulphor®, and phosphate-buffered saline by adding appropriate quantities of trimethylolpropane triacrylate. All dermal doses were prepared by dissolving appropriate quantities of trimethylolpropane triacrylate in acetone.

Duration and frequency of treatment / exposure:

single treatment for all experiments excluding preexposure study
72h duration
In a preexposure study, rats were given a dermal dose of 151 mg/kg unlabeled trimethylolpropane triacrylate followed 24 hours later by a single dermal dose of 151 mg/kg [14C]-trimethylolpropane triacrylate

Doses / concentrations

Remarks:

Doses / Concentrations:
intravenous injection:
- Nominal doses (rats) [14C]: 9.4mg/kg bw

dermal:
- Nominal doses (rats) [14C]: 1.7, 15.2 and 130 mg/kg bw
- Nominal doses (mice) [14C]: 1.2 mg/kg bw

dermal strip experiment:
- Nominal doses (rats) [14C]: 124 mg/kg bw

preexposure dermal study:
- Nominal doses (rats) [unlabeled]: 151 mg/kg bw

- Actual doses: stability was confirmed during an dermal carcinogenicity experiment; using GC, recovery rates were (with minor exception) in the range above 85% (mostly near 100).
- Actual doses calculated as follows: Nominal doses were used
- Dose volume: no data

No. of animals per sex per dose / concentration:

- 5 animals per dose group according to raw data

Control animals:

no

Positive control reference chemical:

no

Details on study design:

1. dermal, rats and mice
APPLICATION OF DOSE:

VEHICLE
All dermal doses were prepared by dissolving appropriate quantities of labeled and/or nonlabeled trimethylolpropane triacrylate in acetone.

TEST SITE
Animals in dermal dosing experiments were sedated with an intramuscular dose of 60 mg/kg ketamine:xylazine (7:1) approximately 24 hours beforedermal doses were applied, and the fur on the back of each animal was clipped. The clipped area was wiped with acetone, dried, and examined; animals with broken skin in the clipped area were excluded from the study. The doses were applied to 1 square inch (rats) or 0.5 square inch (mice)
previously outlined with a permanent, felt-tip marker. Before dosing, a self-adhering protective foam appliance Libertyville, IL). Doses were administered with either a ball-tipped gavage needle and glass syringe equipped with a Teflon®-tipped plunger or a silylated glass wiretrol (Drummond Scientific Co., Broomall, PA) to the square inch of skin exposed through the window in the foam appliance. After dosing was complete, a cloth cover was attached to the foam appliance, and a protective metal cover was secured over the appliance with either Elastoplast® or Coban® adhesive bandage (3M Medical-Surgical Division, St. Paul, MN).After dosing the mice, a halved tissue capsule (Fisher HistoPrep; Fisher Scientific Company, Pittsburgh, PA), the latticed top of which was covered with 50/50 polyester/cotton sheeting, was glued over the dosing site with Dura Quick Gel super glue (Loctite Corp., Rocky Hill, CT). Doses were administered to the shaved skin of mice with a 50 μL wiretrol.

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: (see above)

REMOVAL OF TEST SUBSTANCE
At 72 hours postdosing the dosing appliances were removed from the anesthetized animals.The dosing site skin was washed and collected for analysis of acetone-extractable and nonextractable radiolabel
- Time after start of exposure: 72h

SAMPLE COLLECTION
To measure cumulative excretion of radiolabel, collection flasks and traps were changed at 8, 24, 48, and 72 hours postdosing
- Collection of blood:Blood was withdrawn from the animals into a heparinized syringe by cardiac puncture
- Collection of urine and faeces:Urine and feces were collected separately into round-bottom flasks cooled with dry ice
- Collection of expired air:Exhaled 14CO2 was collected by passing air from the metabolism cages through two traps containing room temperature 1 N sodium hydroxide
- Terminal procedure:The animals were sacrificed by intracardiac injection of Euthanasia-6® (Sparhawk Veterinary Laboratories, Inc., Lenexa, KS) or by carbon dioxide asphyxiation. Bladder urine was removed from each animal and added to the final urine collection.
- Analysis of organs: yes

SAMPLE PREPARATION
- Storage procedure: Selected tissues were removed from the carcasses and the blood, urine, and tissues were stored at –20° C for subsequent analysis of total radiolabel.
- Preparation details:Aliquots of urine, cage rinse, skin wash, and breath trap contents were mixed with scintillation cocktail and directly assayed for 14C content by liquid scintillation spectrometry (LSS); aliquots of blood, feces, and tissues were digested in Soluene 350® (Packard Instrument Company, Meriden, CT), neutralized, decolorized with perchloric acid and hydrogen peroxide, and assayed for total radiolabel. Skin samples and the residual carcass were digested in 2 N ethanolic sodium hydroxide prior to analysis for radiolabel. The dosing appliances were cut into eight pieces, added to scintillation vials containing 2 mL methanol, and analyzed by LSS. Appropriate background samples containing the same combination of reagents as the corresponding biological samples were prepared for all sample types
For mice urine, feces, breath, cage rinse, skin wash, appliance, and tissue samples were collected, digested,and analyzed as in the definitive dermal studies in rats, except the skin samples were not extracted with acetone prior to digestion.

2. intravenous, rats
APPLICATION OF DOSE :
In the intravenous injection study, a group of five male rats was given a single bolus dose of 9.4 mg [14C]-trimethylolpropane triacrylate/kg.

SAMPLE COLLECTION
- Collection of blood:Serial blood collections were taken at 0.08, 0.5, 1, 3, 6, 24, and 48 hours from the indwelling cannula and at 72 hours by cardiac puncture to analyze for blood concentrations of radiolabel
- Collection of urine and faeces:Urine, feces, cage rinse, and exhaled 14CO2 were collected and analyzed for cumulative excretion of radiolabel as
in the definitive dermal rat studies except that collections were made at 3, 6, 24, 48, and 72 hours postdosing
- Collection of expired air: see above
- Tissue samples were collected at 72 hours postdosing and digested and analyzed for radiolabel as described for the definitive dermal studies in rats.

3. general
ANALYSIS
- Method type(s) for identification: Liquid scintillation counting

please refer to the orginal NTP Protocol (which is published) for more detailed informations

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, blood and selected tissues (Adipose, Bladder, Brain, Heart, Kidney, Liver, Lung, Muscle, Skin, Spleen, Testis, Residual carcass) , cage washes,exhaled CO2.
- Time and frequency of sampling:To measure cumulative excretion of radiolabel, collection flasks and traps were changed at 8, 24, 48, and 72 hours postdosing. At 72 hours postdosing, the cages were rinsed.

Statistics:

Common statistics

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

dermal (rat): 55.1%(1.7mg/kg), 32.7% (15.2mg/kg), 18.7% (130 mg/kg) absorption increases if preexposure to 25.4% (151mg/kg); dermal (male mice): 75.0% (1.2mg/kg)

Main ADME results 2

Type:

distribution

Results:

Tissue to blood ratio were below 1 with exepetion of kidney. Kidney:blood ratios of radiolabel (approximately 3.3-11.1) were elevated in dermal dosed rats but not in i.v. dosed. Intravenous application showed tissue:blood ratio below 0.7 for all tissues.

Main ADME results 3

Type:

excretion

Results:

i.v. injection (rats, 9.4mg/kg): 77.4% was excreted in the urine (48.6%) , feces, and exhaled CO2 (20.1%); dermal application (rats): urine (28.0% (1.7mg/kg) - 3.0% (130mg/kg), exhaled CO2 (13.1% (1.7mg/kg) - 1.4% (130mg/kg), skin (8.0-10.4%)

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Absorption of dermally administered trimethylolpropane triacrylate was inversely related to dose; a total of 18.7% of the
130 mg/kg dose was absorbed, while 32.7% of the 15.2 mg/kg and 55.1% of the 1.7 mg/kg dose were absorbed. Most of the radiolabel remaining in the animals 72 hours after dermal application was associated with the skin at the dose site. Preexposed rats absorbed 1.33 times as much [14C]-trimethylolpropane triacrylate as animals that were not preexposed.
In mice, a total of 75% of the dermally applied [14C]-trimethylolpropane triacrylate was absorbed 72 hours after a single dose of 1.2 mg/kg approximately 1.4 times as much as was absorbed by rats administered a similar dose.

Details on distribution in tissues:

Elevated kidney:blood ratios of radiolabel (approximately 3.3-11.1) were noted in rats after dermal exposures, but the kidney:blood ratio of radiolabel after intravenous bolus administration was not elevated. Tissue to blood ratio were below 1 with exepetion of kidney. It was shown that kidney:blood ratios in dermally dosed rats were not due to covalent binding of radiolabeled compounds to kidney proteins.
Intravenous application showed tissue:blood ratio below 0.7 for all tissues.

Transfer into organs

Transfer type:

other: kidney blood ratios were elevated (after dermal application). But in general less test item was found in the tissues.

Observation:

slight transfer

Remarks:

kidney

Details on excretion:

Intravenous application:
A total of 77.4% of the radiolabel was excreted in the urine, feces, and exhaled CO2. Urine and exhaled CO2 accounted for the largest percentages (approximately 48% and 20%, respectively)
Large amounts are excreted wi

Dermal application:
Most of the radiolabel remaining in the animals 72 hours after dermal application was associated with the skin at the dose site. Acetone extracted 8%, 10%, and 9%, respectively, of the dose from the site of application of rats administered single doses of 15.2, 130 or 151 mg/kg. Large amounts of TMPTA also excreted via urine 28.0%, 12.1%, 3.0% (1.7 mg/kg, 15.2 mg/kg and 130 mg/kg, rats) and exhaled 13.1%, 4.9%, 1.4%. A compareable distribution could also be seen for mice.

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

Following 72 hours of exposure, acetone extracts of the stripped, sliced skin were prepared and analyzed by HPLC; the major peak, accounting for approximately 73% of the radiolabel, was associated with trimethylolpropane triacrylate. Two more peaks accounted for 14% and 10% of the radiolabel. Parent trimethylolpropane triacrylate was therefore the major xenobiotic in the skin at the dosing site and most likely the major xenobiotic
available to the systemic circulation throughout the studies.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
TMPTA is absorpt dermal inversely related to dose. Large amounts of the dose can be found unabsorpted or in skin. Main route of excretion is via inhalation, urine and faeces. Only small amount were distributed to the tissue and carcass. No accumulation in tissue was observed. A large part of the applied dose is excreted after 72h.

Executive summary:

A study to investigate the absorption, distribution and excretion of Trimethtylolpropane triacrylate (TMPTA) was performed within the scope of an NTP toxicity profile. The study was conducted with rats and mice according to guidelines for toxicokinetics studies within the National Toxicology Program. Therefore rats and mice received different dosages of TMPTA via dermal application or tail vein injection. Excreta, blood and tissue samples were collected using different techniques.

In male F344/N rats, 18.7% of a single dermal dose of 130 mg/kg [14C]-trimethylolpropane triacrylate was absorbed and an average of 76% of the administered radiolabel was recovered unabsorbed 72 hours after dosing (Appendix L). In rats administered a 24-hour preexposure dose of nonradiolabeled trimethylolpropane triacrylate, 25% of a subsequent 151 mg/kg radiolabeled dose was absorbed and 65% was recovered unabsorbed from the dose site 72 hours after dosing. The absorption of dermally administered trimethylolpropane triacrylate was inversely related to dose; a total of 18.7% of the 130 mg/kg dose was absorbed, while 32.7% of the 15.2 mg/kg and 55.1% of the 1.7 mg/kg dose were absorbed. In mass terms, approximately five times more trimethylolpropane triacrylate was absorbed as the dose concentration increased by one order of magnitude. An average of less than 5% of the dose was recovered in the excreta of rats 72 hours after dermal application of 130 mg/kg, compared to an average of approximately 19% of the 15.2 mg/kg dose and 45% of the 1.7 mg/kg dose (Appendix L). Very little radioactivity was associated with most of the tissues 72 hours after exposure; however, the kidney had elevated tissue:blood ratios at each dose concentration. In male B6C3F1 mice, a total of 75% of dermally applied [14C]-trimethylolpropane triacrylate was absorbed 72 hours after a single 1.2 mg/kg dose. A much larger percentage of the applied radioactivity remained at the site of application in mice than in rats (31% in mice, 9% in rats). The collected tissues and residual carcass contained less than 2% of the administered dose. Approximately 21% of the dose remained unabsorbed, and approximately 42% was excreted in the urine, feces, and exhaled carbon dioxide after 72 hours. Very little radiolabel was associated with most of the tissues 72 hours after dosing; the non-application site skin, however, had an elevated tissue:blood ratio. During the 72 hours following an intravenous bolus dose of 9.4 mg/kg [14C]-trimethylolpropane triacrylate to rats, a total of 77.4% of the radiolabel was excreted in the urine, feces, and exhaled carbon dioxide, with urine and exhaled carbon dioxide accounting for the largest percentages. Total radiolabel (but not parent trimethylolpropane triacrylate) was measured in the blood, and only slight changes in radiolabel concentrations were observed in blood after 1 hour. Among the tissues collected 72 hours after dosing, the highest radiolabel concentration was in the blood, and the average total recovery of radiolabel was 90% during the 72 hours after the intravenous dose.