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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
absorption
distribution
excretion
metabolism
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Qualifier:
according to
Guideline:
other: FIFRA Guideline No. 85-1
Qualifier:
according to
Guideline:
other: EEC Directive 87/302/EEC Toxicokinetics
GLP compliance:
yes
Specific details on test material used for the study:
Triisopropanolamine salt of 2,4-Dichlorophenoxyacetic acid (2,4-D TIPA salt)

A liquid sample of 2,4-D-TIPA salt was obtained from DowElanco, Midland, MI. Assay of this sample by nuclear magnetic resonance (NMR) indicated the following composition (w/Ww%): 22.0 +/- 0.9% H20, 40.3 +/- 1.5% 2,4-D and 37.1 +/- 1.5% TIPA. The molar ratio of amine to acid was 1.06 +/- 0.5. The test material was also characterized by high performance liquid chromatography (HPLC) and found to be 38.7 +/- 0.1% 2,4-D (acid equivalents) or 72.2 +/-0.2% 2,4-D TIPA salt at the 95% confidence level. Reanalysis of the sample indicated no significant change in the purity.

Triisopropanolamine-1-14C (14C-TIPA) with a specific activity of 31.9mCi/mmol, was obtained from DowElanco, Midland, MI. The 14C-TIPA was diluted with hexane/benzene (1:1; v/v) prior to being analyzed for radiochemical purity. The radiochemical purity of 14C-TIPA was determined to be 97.5 +/- 1.1%.
Radiolabelling:
yes
Remarks:
14C labeled
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Male Fischer 344 rats (10 weeks old) were purchased from the Charles River Breeding Laboratories (Kingston, NY) and weighed between 170 and 183 grams when dosed. Care and husbandry of these animals was in accordance with the Standard Operating Procedures of the Laboratory (Fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC)). Upon arrival at the laboratory, the rats were examined by a veterinarian and found to be in good general health. The rats were then acclimated to the laboratory for at least one week prior to use. The rooms in which the animals were housed had a 12-hr photocycle, and were designed to maintain adequate temperature and relative humidity for rats. Prior to the start of the experiment, rats were selected from those available using a computer driven randomization procedure and were individually identified by numbered metal ear tags. The animals were acclimated overnight to glass Roth-type metabolism cages. On the next day, they were anesthetized with methoxyflurane and an indwelling jugular vein cannula implanted (Harms and Ojeda, 1974). The rats were then allowed approximately 1 day to recover from surgery prior to administration of the oral dose. Certified rodent chow #5002 (Purina Mills Inc., St. Louis, MO) and municipal drinking water were provided ad libitum, except that food was withdrawn from these animals approximately 17 hr prior to dosing and returned approximately 4 hr post-dosing. Feed and water were analyzed in accordance with the Standard Operating Procedures of The Toxicology Research Laboratory.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Rats were housed in Roth-type metabolism cages designed for the separate collection of urine, feces, 14CO2 and expired organic 14C. Air was drawn through the cages at approximately 500 ml/min. The air, upon exiting the cage, was passed through a trap containing about 10 grams of charcoal to capture expired organic 14C and then through a trap containing about 150 ml of 3:7 (V/V) monoethanolamine: 1-methoxy-2-propanol to capture expired 14CO2. The charcoal trap was changed at 12 hours post-dosing and analyzed for radioactivity. There was insufficient radioactivity in the 0-12 hr charcoal trap to quantify (i.e., < twice background), thus, collection of the charcoal traps was discontinued. The 14CO2 traps were changed at 12, 24, 36, 48 and 72 hours post-dosing. Following collection, the weight of the 14CO2 trap solution was determined, and a weighed aliquot of the trap solution was mixed with liquid scintillation fluid (12% SPECTRAFLUOR: 22% 1-methoxy-2-propanol: 66% toluene) and analyzed for radioactivity.

Blood samples of about 0.2 ml each were drawn from the jugular cannula at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 18, 24, 48 and 72 hr post-dosing using a syringe. Following collection of each blood sample, samples were centrifuged to obtain plasma. The plasma was subsequently weighed, mixed with Aquasol liquid scintillation fluid and analyzed for radioactivity.

Urine was collected in dry-ice chilled containers that were changed at 6, 12, 24, 48 and 72 hr post-dosing. To minimize carry over between specimens, the cage was rinsed with distilled water following collection of each urine specimen. The weight of each urine and cage rinse specimen was determined, and weighed aliquots of each specimen were mixed with Aquasol liquid scintillation fluid (NEN Research Products, Boston, MA) and analyzed for radioactivity. The urine and cage rinse radioactivity was combined for each collection interval and expressed as radioactivity excreted in the urine. In addition, pooled urine samples were prepared from the 0-6 and 6-12 hr collection intervals by mixing the 0.5 ml of urine from each rat. These pooled urine samples were stored frozen (-80C) until analyzed for 2,4-D and for 14C-TIPA and metabolites.

Feces were collected at 24-hr intervals in dry-ice chilled containers. The feces were weighed and an aqueous homogenate (~33% w/w) prepared. Weighed aliquots (~95 mg) of these homogenates were placed in scintillation vials, mixed with 1.5 ml Soluene-350 (Packard Instruments, Downers Grove, IL) and incubated at 40C for 1-2 hr. Then, the sample was decolorized following the addition of 0.75 ml isopropanol and 0.3 ml of 30% hydrogen peroxide, and incubated at 40C for an additional 1-2 hr. Finally, the sample was mixed with 15 ml Hionic-Fluor liquid scintillation fluid (Packard Instruments, Downers Grove, IL). Glacial acetic acid was added to 100 ul increments to minimize photo-chemiluminescence.

Animals were euthanatized with CO2 and exsanguinated 72 hr post-dosing. The following tissues were collected and analyzed for radioactivity: liver, kidneys, perirenal fat, skin and remaining carcass. Weighed aliquots of skin and aqueous homogenates (~33% w/w) of the carcass and other tissues were solubilized and radioactivity quantified in a manner similar to that described for the feces except the samples did not require decolorization.
Duration and frequency of treatment / exposure:
Single oral gavage administration
Dose / conc.:
10.7 mg/kg bw/day
Remarks:
2,4-D-TIPA (0.045 mmol/kg bw), equivalent to 10 mg TIPA/kg bw
No. of animals per sex per dose:
4 males
Control animals:
no
Positive control:
no
Details on study design:
Rats were housed in Roth-type metabolism cages designed for the separate collection of urine, feces, 14CO2 and expired organic 14C. Air was drawn through the cages at approximately 500 ml/min. The air, upon exiting the cage, was passed through a trap containing about 10 grams of charcoal to capture expired organic 14C and then through a trap containing about 150 ml of 3:7 (V/V) monoethanolamine: 1-methoxy-2-propanol to capture expired 14CO2. The charcoal trap was changed at 12 hours post-dosing and analyzed for radioactivity. There was insufficient radioactivity in the 0-12 hr charcoal trap to quantify (i.e., < twice background), thus, collection of the charcoal traps was discontinued. The 14CO2 traps were changed at 12, 24, 36, 48 and 72 hours post-dosing. Following collection, the weight of the 14CO2 trap solution was determined, and a weighed aliquot of the trap solution was mixed with liquid scintillation fluid (12% SPECTRAFLUOR: 22% 1-methoxy-2-propanol: 66% toluene) and analyzed for radioactivity.

Blood samples of about 0.2 ml each were drawn from the jugular cannula at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 18, 24, 48 and 72 hr post-dosing using a syringe. Following collection of each blood sample, samples were centrifuged to obtain plasma. The plasma was subsequently weighed, mixed with Aquasol liquid scintillation fluid and analyzed for radioactivity.

Urine was collected in dry-ice chilled containers that were changed at 6, 12, 24, 48 and 72 hr post-dosing. To minimize carry over between specimens, the cage was rinsed with distilled water following collection of each urine specimen. The weight of each urine and cage rinse specimen was determined, and weighed aliquots of each specimen were mixed with Aquasol liquid scintillation fluid (NEN Research Products, Boston, MA) and analyzed for radioactivity. The urine and cage rinse radioactivity was combined for each collection interval and expressed as radioactivity excreted in the urine. In addition, pooled urine samples were prepared from the 0-6 and 6-12 hr collection intervals by mixing the 0.5 ml of urine from each rat. These pooled urine samples were stored frozen (-80C) until analyzed for 2,4-D and for 14C-TIPA and metabolites.

Feces were collected at 24-hr intervals in dry-ice chilled containers. The feces were weighed and an aqueous homogenate (~33% w/w) prepared. Weighed aliquots (~95 mg) of these homogenates were placed in scintillation vials, mixed with 1.5 ml Soluene-350 (Packard Instruments, Downers Grove, IL) and incubated at 40C for 1-2 hr. Then, the sample was decolorized following the addition of 0.75 ml isopropanol and 0.3 ml of 30% hydrogen peroxide, and incubated at 40C for an additional 1-2 hr. Finally, the sample was mixed with 15 ml Hionic-Fluor liquid scintillation fluid (Packard Instruments, Downers Grove, IL). Glacial acetic acid was added to 100 ul increments to minimize photo-chemiluminescence.

Animals were euthanatized with CO2 and exsanguinated 72 hr post-dosing. The following tissues were collected and analyzed for radioactivity: liver, kidneys, perirenal fat, skin and remaining carcass. Weighed aliquots of skin and aqueous homogenates (~33% w/w) of the carcass and other tissues were solubilized and radioactivity quantified in a manner similar to that described for the feces except the samples did not require decolorization.

14C Analysis: Radioactivity was quantified with a Beckman LS 3801 liquid scintillation counter (Beckman Instruments, Fullerton, CA). Counts per minute were corrected for background and quench, and converted to disintegrations per minute (dpm). At least one sealed standard was counted with each group of samples to monitor the performance of the liquid scintillation counter.

Mass Spectrometry Analysis of Urinary 14C-TIPA: Aliquots of the 0-6 and 6-12 hr pooled urine samples were made basic with 5N NaOH and extracted with methylene chloride. The extracts were then blown to dryness with nitrogen, derivatized with TFAI and analyzed using GC/MS.

Gas Chromatography of Urinary Metabolites of 14C-TIPA: The TFAI derivatized methylene chloride extracts were also analyzed by GC with a Radiomatic FLO-ONE/BETA Model GCR radioactivity-GC-detector.
Details on dosing and sampling:
The dose solution was prepared by adding a measured volume of 14C-TIPA to a 5 ml volumetric flask and removing the hexane/benzene solvent by evaporation. Then, a measured volume of the non-radiolabeled 2,4-D-TIPA was added and the volumetric flask was diluted to volume with distilled water. Targeted concentrations of TIPA and radioactivity in this solution were 5.35 mg TIPA and 60 uCi of 14C per ml. Administration of this dose solution at the rate of 2 ml/kg body weight resulted in a targeted dose of 10.7 mg TIPA and 20-30uCi of 14C per animal. Radioactivity in the dose solution was quantified using a liquid scintillation counter. The concentration of TIPA in the dose solution was calculated to be 5.55 mg/ml based on the amount of 2,4-D (5.18 mg TIPA/ml; molar ratio of 2,4-D to non-radiolabeled TIPA was 0.92) and 14C-TIPA (0.37 mg/ml) in the dose solution.

The rats were weighed and based on their body weight a measured volume of the dose solution was administered by gavage using a glass syringe and stainless steel feeding needle (Popper & Sons, Inc., New Hyde Park, NY). The quantity of dose solution actually administed was determined by weighing the syringe prior to and following dosing.
Statistics:
Descriptive statistics were calculated (mean +/- S.D.) where appropriate. The plasma 14C-concentration time data were described by a polyexponential equation using the method of residuals (Gibaldi and Perrier, 1975).
Preliminary studies:
No preliminary studies were conducted since information was available from previous studies
Details on absorption:
The highest concentration of radioactivity (4.48 +/- 1.19 ug eq/g plasma) was found in the initial sample collected 0.25 hr post-dosing. The concentration of 14C in the plasma then decreased in what appeared to be a triexponential manner. A 5-fold decrease in plasma radioactivity occurred during the first 4 hr post-dosing. After 24 hr, the plasma 14C concentration was only 3% of peak level, and by 72 hr the plasma contained only 1.3% of the peak concentration.

Half-lives estimated for the rapid initial, middle and terminal phases were 0.76, 3.6 and 38.5 hr. The area under the plasma 14C-concentration time curve (AUC), volume of distribution (Vd) and whole body (CLT) and renal clearance (CLr) were calculated as 24.03 hr ug equiv 14C-TIPA/g plasma, 1.8 l/kg body weight and 7.77 and 6.51 ml/min/kg body weight, respectively.
Details on distribution in tissues:
Less than 1% of the administered dose remained in the carcass and tissues 72 hr post-dosing. Concentrations of radioactivity were low in all tissues with the liver containing the greatest concentration, approximately 0.02% of the dose/g wet weight. The remaining tissues and carcass contained less than 0.01% of the dose/g wet weight. However, due to its greater mass, the majority of the radioactivity was in the carcass.
Details on excretion:
Most of the dose (64-70%) was excreted in the urine during the 0-6 hr collection interval. An additional 9-14% of the radioactivity was excreted in the 6-12 hr interval. By 24 hr post-dosing, 82.8% of the dose was recovered in the urine and only 1% of the dose was excreted in the urine between 24 and 72 hr post-dosing. The excretion of radioactivity in the urine was even faster than predicted by the plasma 14C-data. Normally the fraction of the dose excreted during an interval will be proportional to the AUC for the material in the plasma for that interval versus the AUC for the material in the plasma from time 0 to infinity. In this case, 98.8% of the radioactivity in the urine was excreted within 24 hr. However, the AUC for radioactivity in the plasma for the 0 to 24 hr interval represented only 68.9% of AUC for the 0 to infinity interval (i.e., 16.56 versus 24.03 hr µg equiv 14C-TIPA/g plasma). Thus, the potential for TIPA to accumulate is even less than predicted by the plasma 14C-data.

As with the urine, most (84.6%) of the fecal radioactivity was eliminated during the first 24-hr post-dosing.

The amount of radioactivity in the 0-12 hr collection interval (3-4% of the dose) represented over 86% of the 14CO2 excreted during the entire 72 hr post-dosing interval.

GC/MS analysis of the pooled urine specimens indicated that virtually all radioactivity in the urine represented unchanged 14C-TIPA. Mass spectra and extracted ion chromatograms for the derivatized urine extract and TIPA standard were identical. Only a single peak, with the same retention time as derivatized TIPA standard, was observed in the GC/MS/Radiogas chromatogram of the derivatized urine extract. Additionally, 95 and 80% of the radioactivity in the 0-6 and 6-12 hr pooled urine sample, respectively, could be accounted for by the amount of TIPA found in these samples by GC/MS.
Toxicokinetic parameters:
half-life 1st: 0.76 hr in plasma
Toxicokinetic parameters:
half-life 2nd: 3.6 hr in plasma
Toxicokinetic parameters:
half-life 3rd: 38.5 hr in plasma
Toxicokinetic parameters:
AUC: 24.03 hr ug equiv 14C-TIPA/g plasma
Metabolites identified:
yes
Details on metabolites:
GC/MS analysis of the pooled urine specimens indicated that virtually all radioactivity in the urine represented unchanged 14C-TIPA. Mass spectra and extracted ion chromatograms for the derivatised urine extract and TIPA standard were identical. Only a single peak, with the same retention time as derivatised TIPA standard, was observed in the GC/MS/Radiogas chromatogram of the derivatised urine extract. Additionally, 95 and 80% of the radioactivity in the 0-6 and 6-12 hr pooled urine sample, respectively, could be accounted for by the amount of TIPA found in these samples by GC/MS.

- No signs of toxicity were noted during the in-life phase of the study. The doses delivered were within 7-12% of the targeted dose levels. Between 94% and 96% of the administered radioactivity was recovered in the urine, feces, 14CO2, tissues / carcass, and final cage wash.

- The principle route of excretion was urine, which contained 81-85% of the total dose. Feces contained 4-7%, 3-5% was eliminated as 14CO2, <2% was recovered in the tissues/carcass and final cage wash. The amount of 14C in the traps for volatile organics was negligible.

- Highest concentration of radioactivity was found in the initial blood sample 0.25 hours post-dose. The concentration decreased in a triexponential manner. A 5-fold decrease in radioactivity was accomplished in the first 4-hour post-dosing.

- Half-lives for the rapid initial, middle, and terminal phases were 0.76, 3.6, and 38.5 hours, respectively. Most of the dose of radioactivity (64-70%) was excreted in the urine 0-6 hours post-dosing. An additional 9-14% was excreted in the 6-12 hour interval. By 24 hours post-dosing, 82.8% of the dose was excreted. As with urine, 84.6% of the fecal radioactivity was eliminated during the first 24 hours.

Conclusions:
- Orally-administered TIPA was rapidly and extensively absorbed by the rat. Based on the amount of radioactivity in urine and cage wash, a minimum of 83.8% of the orally-administered TIPA was absorbed.
- GC/MS analysis of pooled urine indicated that virtually all radioactivity in the urine represented unchanged 14C-TIPA, and was rapidly excreted.
- Based on its rapid elimination, TIPA is not assumed to accumulate in the rat upon daily administration. The data suggest that the excretion of accompanying 2,4-D was not affected by co-administration with TIPA.
Executive summary:

This study examined the metabolism and excretion of triisopropanolamine-1-14C (14C-TIPA) in male rats when administered concomitantly with 2,4 -dichlorophenoxyacetic acid (2,4 -D), and was conducted to support re-registration of products containing the TIPA salt of 2,4 -D (2,4 -D TIPA).

Four male Fischer 344 rats were given a single oral dose of a solution providing targeted doses of 10 mg 2,4 -D/kg and 10.7 mg 14C-TIPA/kg of body weight.

The concentration of radioactivity in the plasma peaked 0.25 hr post-dosing at 4.48 +/- 1.19 ug eq/g plasma and then decreased in a tri-exponential manner. Between 94 and 96% of the administered radioactivity was recovered in the urine, feces, expired 14CO2, tissues/carcass and final cage wash. The major route of excretion was the urine with approximately 80% of the dose excreted by this route in the first 24 hr post-dosing and 81 to 85% excreted by 72 hr post-dosing. The feces accounted for only 4 to 7 % of the dose. Expired 14CO2 accounted for 3 to 5% and the final cage wash ~1% of the dose. Less than 1% of the administered radioactivity remained in the tissues and carcass when these rats were sacrificed 72 hr post-dosing. Essentially all radioactivity excreted in the urine represented unchanged 14C-TIPA based on GC/MS and GC/MS/radiogas analysis of the urine excreted 0 -12 hr post-dosing. Additionally, the urinary excretion of 2,4 -D during the 0 -12 hr post-dosing interval (70.5% of the dose) was nearly identical to that excreted during this interval following oral administration of a 1 mg 2,4 -D/kg dose (69.3 +/- 13.1%).

These data demonstrate that orally administered 14C-TIPA was rapidly absorbed and rapidly excreted primarily in the urine as unchanged TIPA. Due to its rapid elimination, 14C-TIPA should not accumulate in the rat upon daily administration.

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP
Reason / purpose:
reference to same study
Objective of study:
toxicokinetics
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
only 1 dose level examined
GLP compliance:
yes
Radiolabelling:
yes
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals and environmental conditions:
Female CDF Fischer 344 rats were purchased from Charles River Breeding Laboratories, Kingston, NY, and weighed between 148 and 162 g when used. The rat is the preferred species for pharmacokinetic studies, and this strain was selected because it is available from a reliable commercial source. Female rats were used because Bronauah et al (1983) found their skin to be more permeable to polar chemicals than the skin of male rats.

Upon arrival at the laboratory, these animals were examined by a veterinarian and judged to be in good general health. The animals were given at least 7 days to acclimate to the laboratory prior to being placed on study, and were uniquely identified with numbered metal ear tags. While being acclimated to the laboratory, the rats were housed in pairs in stainless steel cages with wire-mesh floors. Deotized cage board was placed under the cages to minimize odor and aid in maintaining a clean environment. The cages were washed and the cage board changed in accordance with good laboratory practices. Throughout both the acclimation period and study, the animals were housed in rooms designed to control temperature (22 +/- 2°C) and relative humidity (40 to 60%), and to provide a 12 hour photoperiod and about 12 air changes per hour. Food (Purina Certified Rodent Chow #5002, Ralston Purina Co., St. Louis, MO) and municipal water were available ad libitum.

Three days prior to being dosed, the rats were transferred to all glass metabolism cages designed for the separate collection of urine, feces, and expired volatiles . Two days prior to being dosed, they were anesthetized with methoxyflurane and an indwelling cannula implanted in the right jugular vein using a modification of the procedure described by Harms and Ojeda (1974). Six animals were cannulated for both the intravenous (iv) and dermal segments of this study to insure that there would be at least 4 rats with patent cannulae when each dose was administered. Following surgery, these rats were returned to the metabolism cages for the final 36 hours of the acclimation period.
Route of administration:
other: intravenous or dermal
Vehicle:
other: intravenous dose vehicle was water; dermal dose vehicle was acetone
Details on exposure:
The iv dose solution was prepared by dissolving a weighed amount of unlabeled DIPA in distilled water and adjusting the pH to 7.3 with 12 N HCl. Sufficient 14C-DIPA was then added to produce a specific activity of 1.54 pCi/mg of DIPA. This solution was administered via the jugular cannula at a rate of 1 ml/kg of body weight and flushed into the rat with 0.5 ml of normal saline. Administration of these volumes resulted in each rat receiving about 5 pCi of radioactivity and a dose of 19.0 mg/kg of body weight. The dermal dose solution was prepared by dissolving sufficient unlabeled and 14C-labeled DIPA in acetone to provide a solution containing 18.9 mg DIPA/ml with a specific activity of 6.32 uCi/mg of DIPA. This dose solution was applied at a rate of 1 ml/kg to a shaved 1 cm2 area on the back (intrascapular region) of the rat with a Hamilton@ syringe. To apply this volume it was necessary to allow the acetone to evaporate and treat the same 1 cm2 of skin several times. A template cut from a Teflon sheet was used to define the 1 cm2 area to which the dose was applied. After the entire dose was applied this template was removed and analyzed for radioactivity. A 4 cm2 piece of Teflon was then used to cover the dose site and was fixed in place with adhesive tape. To further restrict access to the dose site the rats were placed in lycra-Spandex jackets for the duration of the study (i.e., 48 hr). Administration of these volumes resulted in each rat receiving about 19 uCi of radioactivity and a dermal dose of 19.5 mg/kg of body weight.
Duration and frequency of treatment / exposure:
single iv administration
dermal administration was 48 hours
Dose / conc.:
19 mg/kg bw/day
Remarks:
iv administration
Dose / conc.:
19.5 mg/kg bw/day
Remarks:
dermal administration
No. of animals per sex per dose:
Four
Control animals:
no
Positive control:
no data
Details on study design:
see details on exposure
Details on dosing and sampling:
Sample Collection and Analysis.
Blood specimens, of about 0.2 ml each, were collected via the jugular cannuala 10, 20, and 40 min, and 1, 2, 4, 6, 12, 18, and 24 hours following the iv dose, and at 0.5, 1, 2, 4, 6, 12, 24, 36, and 48 hr following the dermal dose. The blood specimens were collected in heparinized capillary tubes and spun to separate the plasma from the red blood cells (RBC). Urine was collected in traps immersed in dry ice. The urine traps were changed at 6, 12, 24, 36 (dermal dose only), and 48 hours post-dosing. To reduce carry over between samples, the cages and urine traps were rinsed with water when the urine was collected 6 and 12 hr after the iv dose and each time urine was collected following the dermal dose. Feces were collected at 12 hr intervals. To collect volatile organics and 14C02, air was drawn through the metabolism cage at a rate of about 500 ml/min. The air exiting the cage was then passed through a trap filled with charcoal (to trap volatile organics) and a trap filled with about 170 ml of a 3:7 (v/v) mixture of monoethanolamine and propylene glycol monomethyl ether to trap expired 14C02. The charcoal and 14C02 traps were changed at 12 hr intervals. The rats were anesthetized with C02 and killed by exsanguination 48 hr post dosing. The animals given the dermal dose were skinned, and the liver, kidney, and a sample of perirenal fat collected for analysis. Weighed aliquots of the plasma, urine, cage rinse, and 14C02 traps were mixed with ACS liquid scintillation fluid (Amersham, Arlington Heights, IL) and analyzed for radioactivity. The charcoal traps were divided into 10 fractions prior to being mixed with ACS liquid scintillation fluid and analyzed for radioactivity. Aqueous homogenates were prepared of the feces, carcass, liver, and kidneys from the dermally dosed rats. Weighed aliquots of these homogenates, and of the perirenal fat, skin (2 sites), and RBC samples from the dermally dosed rats were oxidized in a Biological Materials Oxidizer (R. J. Harvey Corp., Hillsdale, NJ). The 14C02 released on combustion of these samples was trapped in monoethanolamine and quantified in a liquid scintillation spectrometer. Radioactivity was quantified with either a LS9000 (Peckman Instrument Corporation, Irvine, CA) or a Mark 11 (Searle Analytical, Inc., Elk Grove Village, IL ) liquid scintillation counter. Counts per min were corrected for background and quench and converted to ug equivalents using the specific activity of 14C-DIPA in the dosing solution. The radiotracer, dose solutions, and selected urine specimens were analyzed by LC. The plasma did not contain sufficient radioactivity to justify LC analysis. The LC system consisted of a Water Associates Rodel M6000A pump, a Rheodyne Model 7125 injector, an Altex Model 156 refractive index detector (RI) and a Packard Model A7130 radioactivity monitor (RAM). The two detectors were connected in series with the RI detector immediately following the LC column. Two LC colurnn/eluent combinations were used. The radiotracer, dose solutions, and urine and plasma specimens from the iv segment were analyzed using a 5 mm (id) x 250 mm Partisil PXS 10125 SLX column (Whatman, Inc.) and a mobile phase of 70:30 (v/v) water/acetonitrile containing 0.005 M ammonium acetate and adjusted to a pH of 4.5 with acetic acid. The flow was maintained at 1 ml/min and the retention time measured at the RI detector was 13.8 min for MIPA and 15.2 min for DIPA. The high ionic strength of the urine specimens caused a reduction in the retention times which varied with the injection volume. To eliminate this problem, urines from the dermal dose segment were chromatographed using a 4.6 mm (id) x 300 mm Chromegabonds RP-SCX (10 um particles) column (E.S. Industries) and a mobile phase of 90: 10 (v/v) water:acetonitrile containing 0.05 M ammonium acetate and having a pH of 6.7. The flow was maintained at 1 ml/min and the retention times measured at the RI detector were 18.3 and 24.0 min for MIPA and DIPA, respectively. For all samples, a 4 mm ( i d ) x 26 mm precolumn packed with pellicular cation exchanger (Whatman, Inc.) was used to prolong column life. The urine samples were filtered through a 25 mm diameter, 0.45 um pore size nylon membrane syringe filter (MSI, Inc.) prior to being injected into the LC system (recovery of 14C from this filtration was 105 + 3.9%). The plasma was injected into the LC system without any sample cleanup. Recovery of radioactivity averaged 96.7% (SD = 6.3) for the Whatman Partisil column and 94.1% (SD +/- 4.4) for the E.S. Industries Chromegabond column.
Statistics:
Mean plasma concentrations and urinary excretion rates (i.e., mg DIPA in urine specimen/collection interval in hr) observed following intravenous and dermal administration of 14C-DIPA simultaneously to a two compartment open pharmacokinetic model in which the absorption, elimination and transfer between the rapid and slow exchange compartments were assumed to be first order processes. The differential equations which define this model are given below:

dAO/dt = -AO * Ka
dA1/dt = AO * ka - A1 *( ke + k12) + A2 * k21
dA2/dt = A1 * k12 -A2 * k21
dA3/dt = A1 * ke
A, Al, A2, and A3 represent the amounts of 14C-DIPA on the skin, in the rapid and slow exchange compartments, and excreted in the urine, respect
ively. ka and ke represent first order rate constants describing the dermal absorption and urinary excretion o f 14C-DIPA, and k12 and, k21 are first-order rate constants describing the transfer between the rapid and slow exchange compartments. Concentrations of 14C-DIPA in the plasma (Cp), the renal clearance of I4C-DIPA (Clr), and the dispositional rate constants a and B were calculated as follows:

Cp = A1/(V1)
Clr = k * V1 * BWT * 60 min/hr
a,B = 0.5 * ((k12 + k21 +ke) +/-((k12 +k21 +k2)2 -4 * k21 *ke)1/2
VI represents the apparent volume of the rapid exchange compartment, and BWT the average body weight of the rats. Optimum estimates for the
model parameters were obtained using DACSL (Agin and Blau, 1982).
Preliminary studies:
Intravenous Dose
During the first 12 hr after iv administration, the concentration of radioactivity in the plasma decreased rapidly in a biexponential manner that was well described by a two compartment pharmacokinetic model. The half-life for the rapid initial a phase was 0.7 hr and that of the slower terminal B phase was 6.3 hr. Concentrations of radioactivity in the plasma specimens collected more than 12-hr post-dosing were at or below the limit of quantitation (i.e., 0.5 to 1.5 times background), and were not used when the model parameters were estimated. An average of 96.8% (range 93.2 to 102.8%) of the intravenously administered radioactivity was excreted in the urine.

Most (71.3%) of the dose was eliminated during the first 6 hr and by 12 hr 93.1% of the dose had been excreted in the urine. Analysis of the urine by LC indicated that over 99% of the radioactivity in the urine was unchanged 14C-DIPA.
Details on absorption:
An average of 69.2% (SD = 17.0%) of the dermal dose was recovered. Most of the administered radioactivity was recovered from the bandage (25.7%) and the skin (27.3%). The low recovery from one of the four dermally dosed rats (i.e., 47.8% for rat 85A-9094) was due to the small amount of radioactivity recovered from its Teflon patch; 7.6% of the dose was recovered from this rat's patch versus 26-32% from the patch from each of the other animals. Of the radioactivity recovered from the skin, most (87%) was at or adjacent to the site where the dose was applied.

Following the dermal dose, the highest concentration of radioactivity in the plasma (1.1 ug/g) was observed 0.5 hr after the dose was applied to the skin. The pharmacokinetic model as developed could not predict the spike in plasma 14C-concentrations observed 0.5 hr postdosing, and underpredicted the concentration of radioactivity in the plasma 24, 36, and 48 hr post-dosing. Concentrations of radioactivity in the red blood cells were similar to those found in the plasma
Details on distribution in tissues:
1.4% of the dose was found in the liver, kidneys and carcass.
Details on excretion:
14.8% was found in the excreta.

Consistent with the intravenous data, urine was the only major route for the excretion of 14C-DIPA. Based on the urinary excretion of 14C-DIPA between 6 and 48 hr post-dosing, little radioactivity appeared to have been eliminated during the first 3 to 4 hr after the dermal dose was administered. After this 3 to 4-hr delay, radioactivity was excreted into the urine at a constant rate which was well described by the pharmacokinetic model. Analysis of the urine by LC indicated that over 99% of the radioactivity in the urine was unchanged 14C-DIPA.
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 0.39 h
Test no.:
#2
Toxicokinetic parameters:
Cmax: 11 µg/ml
Test no.:
#3
Toxicokinetic parameters:
AUC: 13.52 µg h ml-1
Metabolites identified:
yes
Details on metabolites:
There were no metabolites identified in urine samples

Following intravenous administration, 14C-DIPA was rapidly cleared from the plasma and excreted in the urine in a biexponential manner. The half-life for the rapid initial phase was 0.7 hr and that of the slower terminal phase was 6.3 hr. Within 48 hr, 96.8% if the intravenous dose was excreted unchanged in the urine. When the dermal animals were sacrificed at 48 hr post-dosing, 22.5% of the dose was found on the skin at the dose site, and another 25.7% was recovered from the bandage. In addition, 1.4% of the dose was found in the liver, kidney, and carcass, and 14.8% was found in the excreta. These data indicate that in 48 hr only 16.2% of the 14C-DIPA applied to the back of rats was absorbed and represents an absorption rate of less than 0.3% of the dose per hour. Because DIPA was slowly absorbed through the skin and rapidly eliminated via the urine, these data suggest that toxicologically significant concentrations of DIPA are unlikely to occur as a result of dermal exposure to this chemical in cosmetics.

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Because DIPA was slowly absorbed through the skin and rapidly eliminated via the urine, these data suggest that toxicologically significant concentrations of DIPA are unlikely to occur as a result of dermal exposure to this chemical in cosmetics.
Executive summary:

Diisopropanolamine (DIPA) is a component of many cosmetic formulations. The purpose of this study was to obtain data on the percutaneous absorption and subsequent elimination of DIPA which could be used in support of the continued use of DIPA in cosmetics. In this study, groups of 4 female Fischer 341 rats were given a single intravenous (iv) or dermal dose o f 19 mg of 14C-labeled DIPA/kg of body weight. Following i v administration, 14C-DIPA was rapidly cleared from the plasma and excreted into the urine in a biexponential manner. The half-life for the rapid initial phase was 0.7 hr and that of the slower terminal phase was 6.3 hr. Within 48 hr, 96.8% of the iv dose was excreted unchanged in the urine. The dermal dose was applied to the back of the rat and covered with an occulsive bandage for 48 hr. When these animals were sacrificed 48 hr post-dosing, 22.5% of the dose was found on the skin at the dose site, and another 25.7% was recovered from the bandage. In addition, 1.4% of the dose was found in the liver, kidney, and carcass, and 14.8% was found in the excreta. These data indicate that in 48 hr only 16.2% of the 14C-DIPA applied t o the back of these rats was absorbed, and this represents an absorption rate of less than 0.3% of the dose per hour. Because DIPA was slowly absorbed through skin and rapidly eliminated via the urine, these data suggest that toxicologically significant concentrations of DIPA are unlikely to occur as a result of dermal exposure to this chemical in cosmetics.

Description of key information

MIPA can be absorbed after oral exposure, which is experimentally determined for the structurally similar substance TIPA to be 90%. Supported by marginal findings in acute and repeated dose toxicity studies an oral absorption of 90% for MIPA is suggested.

MIPA has a low volatility and therefore inhalation is assumed to be limited, however, in the absence of reliable studies an absorption of 100% is determined.

Accumulation in specific organs/tissues is suggested to be low, due to the physico-chemical properties of MIPA and missing observations in the toxicological studies.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
90
Absorption rate - inhalation (%):
100

Additional information

There are no specific studies available in which the toxicokinetic properties (absorption, metabolism, distribution, elimination) of (MIPA) 1-aminopropan-2-ol, CAS 78-96-6 have been investigated. The expected toxicokinetic behaviour is derived from physico-chemical properties of MIPA and a toxicokinetic study (according to OECD 417) with a structural similar substance, TIPA (1,1',1''-nitrilotripropan-2-ol, CAS 122-20-3. The structural similarity and similar properties and/or activities between the chemicals are discussed within the justification for the adaptation of the standard information in this dossier.

Physico-chemical properties

MIPA is an amine with an isopropanol substituent and the molecular formula C3H9NO. Its reactivity is mainly triggered by its basicity, i.e. a dissociation constant (pKa) of 9.62 at 20 °C. MIPA has a molecular weight of 75.11 g/mol, melts at 1°C and boils at 160°C, measured at 1,013 hPa. The calculated vapour pressure is 63 Pa at 25°C, the octanol-water partition coefficient (log Pow) is ‑0.93 at 23°C, and the substance is miscible in water.

Experimental study from structurally similar TIPA: metabolism and excretion in vivo (1992, RL1 [reliability])

Triisopropanolamine-1-14C (14C-TIPA) was administered concomitantly with 2,4‑dichlorophenoxyacetic acid (2,4 -D) orally as a single dose to four male Fischer 344 rats, according to OECD 417. A single oral dose, equivalent to 10.7 mg 14C-TIPA/kg bw, was administered, followed by blood sampling at intervals up to 72 hours post-dose. Radioactivity peaked in the plasma at 0.25 hrs post-dosing and declined tri-exponentially. The urine was the major route of excretion with about 80% of the dose excreted in 24 hours and 81 to 85% of the dose excreted in 72 hours, primarily unmetabolized. The faces and expired air accounted for only 4% to 7% and 3% to 5%, respectively. After 72 hours, 94% to 96% was recovered with only less than 1% remaining in the tissues or carcass.

Oral absorption

The substance is tested highly soluble and has a hydrophilic character, due to its ionisable isopropanol group. An absorption by passive diffusion is suggested. MIPA is a small molecule and will be taken up easily through aqueous pores or via bulk passages of water through epithelial barriers. The hydrolysis of MIPA is not expected.

A systemic low acute toxicity was observed for MIPA in an acute oral toxicity study, comparable to OECD TG 401 (1965, RL2). Rats were administered 194-6208 mg/kg bw by gavage followed by a 14-days observation period. Clinical signs included restlessness, stagger, creep, slight abdominal position, anemia, mouth discharge, compulsive chewing, dyspnoea, apathy, tonic-clonic convulsions, and overnight exitus from doses 2425 mg/kg bw and beyond. The LD50 was 2813 mg/kg bw.

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats with a 65.4% solution of the hydrochloride salt of MIPA, dose levels of 67, 202 and 673 mg/kg bw/d expressed as organic MIPA were administered (2008, RL1). Only slightly reduced hemoglobin and hematocrit values were observed in the high dose male rats after 38 days of exposure, indicating a mild anemic process. Neither toxicity effects were observed in female rats after 45 days of exposure at all dose levels nor in the F1 pups and no developmental and fertility effects were observed. Based on the mild effects in the high dose male rats, the NOAEL for general, systemic toxicity of the test substance was set at the mid dose level (i.e. 202 mg/kg bw/d expressed as organic MIPA).

In the experimental toxicokinetic study withstructurally similar TIPA it was concluded that orally administered TIPA was rapidly absorbed and therefore, an oral absorption percentage of 90% is assumed for TIPA based on the study results (1992, RL1).

Due to the limited systemic effects observed in animals and the knowledge from the structurally similar substance TIPA, the oral absorption is assumed to be 90%.


 

Respiratory absorption

Due to its relatively low volatility (0.63 hPa at 25°C), inhalation appears to be marginal for MIPA.

Multiple studies equivalent to OECD 403 in rat and mice are available (1981, RL2 and 1991, RL2) and the endpoint is assessed in a weight of evidence approach from the available toxicological data. The acute inhalation exposure study in rats for 6 hours at a concentration of 1126 ppm (3460 mg/m3) failed to cause any deaths in rats (LC50 was not determined) and clinical signs were not observed. In the report about respiratory irritation mice were exposed to aerosol concentrations of 230-1005 mg/m3 MIPA (4 mice/concentration) for 3 hours without the finding of a dead animal.

The absorption by inhalation is assumed to be 100% for MIPA in a conservative approach.

Dermal absorption

There are no specific studies available in which the dermal toxicokinetic properties have been investigated. MIPA is experimentally tested to be corrosive to the skin and this may enhance its absorption through the skin barrier.

Distribution

There are no specific studies available in which the toxicokinetic distribution have been investigated. Based on its physico-chemical properties (low molecular weight, highly miscible in water and hydrophilicity) a wide distribution over the body has to be assumed. Besides, no target organs were observed in the toxicological studies.

Accumulative potential

Accumulation in specific organs/tissues is suggested to be low, due to the physico-chemical properties of MIPA and missing observations in the toxicological studies.

Summary

There is experimental evidence that MIPA can be absorbed after oral exposure. An oral absorption of 90% was experimentally determined in the structurally similar substance TIPA and the suggestion is supported by marginal findings in acute and repeated dose toxicity studies. The absorption by the inhalative route was assessed in conservative approaches. MIPA has a low volatility and therefore inhalation is assumed to be limited, however, in the absence of reliable studies an absorption of 100% is determined.