2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1 December 2003 to 27 February 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was conducted to assess and compare the absorption, distribution, metabolism, and elimination of [2,6-14C-ring labelled]test material and [2,6-14C-ring labelled]test material-triisopropanolamine (TIPA) following administration of single oral doses of those compounds. In this study, four male Fischer 344 rats were given a single oral dose of a solution that delivered a target dose of 50 mg 14C-test material/kg of body weight. A separate group of four male Fischer 344 rats were dosed with an equimolar amount of 14C-test material-TIPA at a target dose of 96 mg 14C-test material-TIPA/kg of body weight.

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 173 - 188 g
- Housing: Animals were housed one per cage in glass Roth-type metabolism cages. The metabolism cages were designed for the separation and collection of urine and faeces. Air was drawn through the metabolism cages at ca. 500 mL/minute
- Individual metabolism cages: yes
- Diet: ad libitum during the pre-exposure and study periods, except that at 16 hours prior to the scheduled oral administration of the dose solutions, all feed except for one pellet was withdrawn. Feed was returned to the animals at approximately 4-hour post-dosing.
- Water: municipal water, ad libitum
- Acclimation period: Animals were acclimated in metabolism cages for two days prior to dosing

CANNULATION
- Jugular Vein
The rats in which the blood plasma 14C-time-course was determined were obtained already cannulated in the jugular vein by the supplier.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 24 °C
- Humidity: 48 - 54 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The oral dose solutions were prepared as suspensions in 0.5 % aqueous methyl cellulose. Appropriate amounts of 14C-labelled and/or non-radiolabelled test material were added to deliver the target dose of 50 mg/kg body weight upon administration of ca. 5 g dose solution/kg body weight. To achieve that dose, the test material dose solution was prepared at a targeted concentration of 10.0 mg/g of dose solution (the actual analytically verified concentration was 9.9 mg/g). Similarly, appropriate amounts of 14C-labelled and/or non-radiolabelled test material-TIPA were added to deliver the target dose of 96 mg/kg body weight upon administration of ca. 5 g dose solution/kg body weight. To achieve that dose, the test material-TIPA dose solution was prepared at a targeted concentration of 19.2 mg/g of dose solution (the actual analytically verified concentration was 19.3 mg/g). Note that the 50 mg/kg dose level for test material, and the 96 mg/kg dose level for test material-TIPA were equimolar.
The amount of radioactivity administered was targeted at 125 μCi/kg for both test materials.
Due to the differences in the water solubilities of the two compounds, the physical properties of the two dose solution formulations were quite different. The triisopropanolamine salt of the test material is much more water soluble than the acid form of the test material and was completely dissolved in the aqueous-based dose solution. In contrast, much of the test material (acid) remained undissolved in the dose solution and that formulation would best be described as a suspension. Care was taken to uniformly re-suspend the test material in its dose formulation (shaking by hand) immediately prior to administering the gavage dose.

Duration and frequency of treatment / exposure:

Animals received a single oral dose of test material.

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

4 males

Control animals:

no

Details on study design:

- Dose selection rationale: The dose level selected for this “bridging” pharmacokinetic/metabolism study was equivalent to the low dose that was orally administered in a previous study that was conducted to examine the absorption, distribution, metabolism, and elimination (ADME) of 14C-test material in the male Fischer 344 rat. In the present study, rats were dosed with equimolar amounts of either 14C-test material or 14C-test material-TIPA; specifically, at target doses of 50 mg/kg of the 14C-test material, and 96 mg/kg of 14C-test material-TIPA (ae conversion factor = 0.52).
- Rationale for animal assignment: Animals were selected based on patency of the jugular vein cannulae and randomly assigned to treatment groups using a computer-driven randomisation procedure.

Details on dosing and sampling:

- Blood Collection
The rats were fitted with indwelling jugular vein cannulae and plasma [14C]-concentration-time courses were constructed. Approximately 0.1 mL of blood was collected at the following times (0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, and 120 hours post-dosing) and plasma prepared by centrifugation. The plasma was analysed for radioactivity by liquid scintillation spectroscopy (LSS).

- Urine Collection
All urine voided during the study was collected in dry- ice cooled traps. The urine traps were changed at 6, 12, 24, 36 and 48 hours post-dosing followed by 24-hour intervals for the remainder of the study (the final collection time was at 120 hours post-dosing). The cages were rinsed with water at the time the traps were changed and the rinse collected. Each urine specimen and urine/cage rinse was weighed, and a weighed aliquot of each sample was analysed for radioactivity by LSS. Equal-volume aliquots of urine samples from the 0 - 6 hour and 6 - 12 hour collection intervals were pooled and stored at –80 °C and selected urine samples from those two collection intervals underwent chemical analysis.

- Faeces Collection
Faeces were collected in dry- ice chilled containers at 24-hour intervals throughout the duration of the study. An aqueous homogenate (~ 25 % w/w) was prepared, and weighed aliquots of these homogenates were combusted and quantitated for radioactivity by LSS. In addition, equal volume aliquots of faecal homogenates from each animal were taken from the 0 - 24 hour collection interval and pooled. These pooled samples were stored at –80 °C and designated for chemical analysis.

- Identification of Radiolabelled Compounds in Urine and Faeces
Following the chromatographic radioprofiling of urine and faecal extracts, representative samples were also analysed by liquid chromatography/mass spectrometry (LC/MS) in an attempt to confirm the identity of the major radiolabelled peak as parent compound(s) and to identify those metabolites that exceeded 5 % of the administered dose in the urine and faecal specimens.

- Terminal Sacrifice
At approximately 120- hour post-dosing, the animals were anesthetized with a CO₂/O₂ mixture and sacrificed by exsanguination. Following sacrifice the Roth cages were washed and the final cage wash analysed for radioactivity.
The following tissues were collected at sacrifice: gastrointestinal (GI) tract, plasma (terminal), residual carcass, kidney, skin, liver, spleen, perirenal fat, and whole blood (terminal).
The carcass, GI tract with contents, kidneys, liver, and whole blood were collected, homogenised (~ 33 % homogenate), and a weighed aliquot oxidised and analysed for radioactivity by LSS. A portion of the blood specimen was centrifuged to obtain plasma and the plasma analysed for radioactivity by LSS. The skin was removed from the carcass and a representative skin sample was directly oxidised and analysed for radioactivity by LSS. The remaining tissues (e.g., perirenal fat and spleen) were directly oxidised without homogenisation.

- Final Cage Wash
Following the terminal sacrifice of the animals, a final cage wash was performed. The final cage wash and contents were collected and the weight of the sample was determined. A weighed aliquot of the final cage wash was analysed for radioactivity.

- Plasma
Except for the terminal blood sample (of which a portion was used for plasma), blood samples were centrifuged to obtain plasma which was analysed for radioactivity to construct the plasma 14C-concentration-time courses.

- Control Samples
Control urine and faeces were collected in dry- ice chilled traps from one male rat not dosed with either of the 14C-labelled test materials. The control excreta was collected over a period of approximately 48 hours beginning on the day that the treatment animals were dosed at the time eartag numbers were assigned. The control animal(s) were sacrificed at study termination and blood collected by the same procedure as the dosed animals. No tissues were collected from the control animals.

Statistics:

Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the database were conducted using Microsoft® Excel® spreadsheets and databases in full precision mode (15 digits of accuracy). Pharmacokinetic parameters were determined, including AUC (area-under-the-curve) and elimination rate constants, using a spreadsheet-based (Microsoft® Excel®) pharmacokinetic computer modelling application (PK Solutions™, Summit Research Services, Ashland, Ohio). Additional statistical analyses were not performed (nor warranted) since a direct comparison of the 14C plasma concentration-time curves and the pharmacokinetic parameters derived from those curves together with comparisons of the rates and extent of 14C excretion in urine and faeces were sufficient to demonstrate the bioequivalence of the test material and the TIPA salt of the test material following oral administration of those compounds.

Type:

absorption

Results:

test material was found to be readily absorbed

Type:

excretion

Results:

test material was efficiently cleared through the urine and faeces

Metabolites identified:

yes

Details on metabolites:

The pooled faecal extracts and urine produced a single radiolabelled peak that eluted at a retention time equivalent to the corresponding standard for the parent material. These peaks represented 46.28 and 50.56 % of the administered doses in urine and faeces respectively.

Dose Administered

The actual test material concentrations (as mg test material/g of dose solution) and levels of radioactivity (as µCi of radioactivity/g of dose solution) of the prepared dose solution was within 1 and 19 % of the targeted test material concentration and radioactivity level, respectively. No signs of toxicity were observed following oral administration of the test material. The mean animal body weight was 0.178 kg; the average amounts of test material administered (per kg of body weight) was therefore within 5 % of the targeted dose level of 50 mg/kg. The average amount of radioactivity administered (per kg of body weight) was within 25 % of the targeted level of 125 µCi/kg bw. The slight difference between the targeted and actual administered doses of test material had no impact on the results of this study.

 

Distribution of Radioactivity in Excreta and Tissues

The overall recovery of radioactivity obtained from excreta, tissues, and final cage wash over the 120-hour collection period ranged from 93.0 to 101.5 % of the orally administered 14C-labelled test material (overall average recovery of 97.1 ± 3.5 %). Most of the recovered radioactivity was associated with excreta (average of 96.9 % of the administered 14C-activity), which represents 99.8 % of recovered radioactivity. Of the radioactivity recovered in excreta, slightly more 14C-activity was recovered in the faeces than in the urine. Faeces contained 43.8 to 53.6 % of the administered dose (average of 50.6 ± 4.6 %). The radioactivity recovered in urine (and associated cage rinses) ranged from 39.1 to 57.6 % of the administered dose (average of 46.3 ± 7.9 %). Except for a skin sample from one animal which accounted for 0.02 % of administered radioactivity, terminal blood from one animal which contained < 0.01% of administered dose and spleens from three animals which contained < 0.01 % of administered dose, none of the tissue samples that were collected at terminal sacrifice contained 14C levels exceeding the limit of quantitation (LOQ) from the group dosed with 14C-test material. In general LOQ’s were equivalent to radioactivity levels of≤0.01 % of administered dose. The radioactivity recovered in final cage washes accounted for 0.2 % of the administered dose.

 

Concentration-Time Course of Radioactivity in Plasma

The highest concentration of radioactivity was observed in the first plasma samples (taken at 0.25 hours post-dosing), with a concentration of 26 mg equivalent test material/g plasma (defined as the Observed Cmax values). The concentration of 14C activity in plasma decreased in a bi-exponential manner over the 48-hour time period in which samples were collected. Approximately a 10- fold decrease in plasma concentrations occurred during the first two hours post-dosing. By 48- and 24-hours post-dosing, plasma radioactivity levels were non-quantifiable for the 14C-test material group.

The plasma elimination half- lives estimated for the rapid initial (a), and slower terminal (b), phases of the curve were 0.3 and 8.8 hours for the 14C-test material dosed group.

The 12-hour area-under-the-plasma-curve (AUC) was calculated as 23.0 µg eq- hour/g plasma for the 14C-test material group. As stated previously, the highest concentration of 14C-activity in plasma was measured in the 0.25- hour samples. This suggests that absorption of 14C-test material following oral administration was very rapid, with the highest concentration of radioactivity in plasma (Cmax) occurring before the first plasma samples were taken. Because of the rapid absorption, there are essentially no concentration-time values defining the absorptive phase of the plasma time curve and the pharmacokinetic parameters which would define the absorptive phase (ka, actual Cmax, actual Tmax) cannot be calculated with a high degree of certainty. However, since plasma concentrations of radioactivity decreased with each successive time point beginning with the first set of samples taken at 0.25-hour, it is reasonable to state that the actual Tmax was less than 0.25 hours.

 

Urinary Excretion of Absorbed Radioactivity

The total amount of radioactivity recovered in urine over the entire 120- hour collection period accounted for 46 % of the administered dose of 14C-test material. Radioactivity was rapidly excreted following oral administration. The highest percentage of urinary radioactivity was excreted in the 0 to 6 hour collection interval, with 38 % of the administered dose of 14C-test material recovered in that initial interval (calculated from urine and rinses, combined). The radioactivity levels recovered in that initial 0 to 6 hour interval represent 83 % of the total urinary 14C-activity collected (over the entire 120 hour collection period). An additional 4 % of the administered dose was excreted in the 6 - 12 hour collection interval. By 24 hours post-dosing, 45 % of the dose was recovered in the urine. The radioactivity excreted in urine between 24 and 120 hours post-dosing represented only 2 % of the administered dose.

Urinary elimination half- lives estimated for the rapid initial (a), and slower terminal (b), phases of the curve were 2.8 and 7.8 hours for the 14C-test material-dose group.

 

Recovery of Radioactivity in Faeces

The total amount of radioactivity recovered in faeces over the entire 120 hour collection period accounted for 51 % of the administered dose of 14C-test material. The majority of the faecal radioactivity, was eliminated during the first 24-hour collection interval following dosing, with 49 % of the administered dose being recovered in that initial 0 to 24-hour interval. The radioactivity level recovered in the 0 to 24-hour interval represent 96 % of the total faecal 14C activity collected (over the entire 120-hour collection period).

 

Identification of Radiolabelled Peaks in Urine and Faecal Extracts

HPLC analyses with in-line radioactivity monitoring (RAM) of pooled urine (0 to 6 hour and 12 to 24 hour collection intervals) and faecal extracts from pooled 0 to 24 hour collection interval from the test material dosed group revealed a single radiolabelled peak that eluted at a retention time equivalent to an authentic standard of test material. Those single radiolabelled peaks represented 46.28 and 50.56 % of the administered doses in the urine and faecal samples, respectively.

Positive electrospray ionisation (PESI)-LC/MS/MS of an authentic standard of test material produced an essentially equivalent mass spectra that was representative of a spectrum expected for the test material. Analysis of pooled 6 to 12 hour urine samples (from rats dosed with test material by PESI-LC/MS/MS confirmed the identity of the major eluting peak (Peak A) as parent test material.

Conclusions:

Under the conditions of the study, the test material was found to be readily absorbed and efficiently cleared through the urine and faeces. The overall potential for bioaccumulation is therefore low.

Executive summary:

This study was conducted to assess and compare the absorption, distribution, metabolism, and elimination of [2,6-14C-ring labelled]test material and [2,6-14C-ring labelled]test material-triisopropanolamine (TIPA) following administration of single oral doses of those compounds.

During the study, four male Fischer 344 rats were given a single oral dose of a solution that delivered a target dose of 50 mg 14C-test material/kg of body weight. A separate group of four male Fischer 344 rats were dosed with an equimolar amount of 14C-test material-TIPA at a target dose of 96 mg 14C-test material-TIPA/kg of body weight.

Both compounds were rapidly absorbed; the highest plasma concentrations of radioactivity (observed Cmax) for both compounds occurred in the first sample taken at 0.25 hours post-dosing and were 26 and 16 µg equivalent test material for the 14C-test material and 14C-test material-TIPA dose groups, respectively.

Pharmacokinetic parameters from the plasma time curves yielded the following values for 14C-test material and 14C-test material-TIPA, respectively: plasma AUC’s were 23.0 and 19.0 µg eq-hour/g of plasma; half-lives from the a phase of plasma elimination were 0.338 and 0.509 hours; and half-lives from the β phase of plasma elimination were 8.8 and 13.0 hours. The excretion of 38.3 % (for 14C-test material) and 34.6 % (for 14C-test material-TIPA) of administered radioactivity in urine within six hours of dosing confirms that the radiolabelled portion of the molecule was rapidly absorbed whether administered as test material acid or the TIPA salt of the test material. Based on the radioactivity recovered in urine through 120 hours, a minimum of 46.3 and 42.5 % of the orally administered 14C-test material and 14C-test material-TIPA was absorbed. The radioactivity associated with the two compounds was rapidly eliminated with 93.5 % (44.7 % in urine; 48.8 % in faeces), and 93.3 % (41.5 % in urine; 51.8 % in faeces) of the administered doses of 14C-test material and 14C-test material-TIPA, respectively, recovered in excreta within 24 hours post-dosing. Urinary elimination half-lives of the a phase were 2.8 hours and 2.5 hours for the 14C-test material and 14C-test material-TIPA dosed groups, respectively. Urinary elimination half-lives of the β phase were 7.8 hours and 10.7 hours for the 14C-test material and 14C-test material-TIPA dosed groups, respectively. The radiolabelled portion of both molecules was excreted essentially unchanged in urine and faeces. Except for a minor radioactive peak detected in the 0 to 6 hour pooled urine sample from the 14C-test material-TIPA dosed group (representing 0.34 % of administered dose), the only radiolabelled peak detected in analyses of urine and faecal extracts was confirmed as parent test material. The results from this study indicate that 14C-test material and 14C-test material-TIPA, when administered orally to rats, are bioequivalent in terms of absorption, distribution, metabolism, and excretion of the radiolabelled portion of the molecule(s).

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 November 2004 to 28 April 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

The absorption, distribution, metabolism, and elimination of 14C-test material orally administered to three groups of three female New Zealand White rabbits were determined. Non-pregnant rabbits (Group 1) and pregnant rabbits at gestation day 7 (GD 7; Group 2) were administered a single oral dose of 371 and 362 mg 14C-test material/kg bw, respectively. The repeated dose group (Group 3) were given daily doses of 279 mg test material/kg bw from GD 7-21, followed by 279 mg 14C-test material/kg bw on GD 22.

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Radiolabelling:

yes

Species:

rabbit

Strain:

New Zealand White

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Rabbits were non-pregnant and time-mated females
- Age at study initiation: Sexually mature adult females, 5 - 6 months old
- Weight at study initiation: 3.0 - 3.5 kg
- Housing: Animals were individually housed in stainless steel cages (floor area of 4 ft² and >14 in. tall). The cages had flattened tube grid floors suspended above catch pans with absorbent non-contact bedding. Cages contained a J-type feeder and a water bottle with a stainless steel sipper tube.
- Individual metabolism cages: no
- Diet: Upon receipt, rabbits received approximately two oz. of diet. The amount of feed increased by 1 and 2 oz. per day increments up to approximately 5 and 8 oz. for non-pregnant and pregnant rabbits, respectively, to avoid gastrointestinal disturbances during the acclimation period.
- Water: municipal water, ad libitum
- Acclimation period: at least 6 days

- Vascular Access Ports
The rabbits were surgically implanted with vascular access ports (VAP). The jugular vein was utilised for placement of the VAP and was located in the caudal area below the shoulder blades. In the case of the pregnant rabbits, the surgery was performed two weeks prior to mating. Vascular access ports (VAP) were utilised for the blood/plasma 14C concentration time-course (Groups 1-3).

- Breeding Procedure
Sexually mature virgin females were naturally mated with one buck of the same strain after a two-week recovery period from the VAP implantation. The observed day of breeding was considered day 0 of gestation.


ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 40 - 60 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % METHOCEL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Two separate oral dose suspensions were prepared in 0.5 % METHOCEL. An appropriate amount of non-radiolabelled test material added to obtain the target dose of 275 mg test material/kg body weight for administration to the Group 3 repeated dose animals from GD 7 to GD 21). The radiolabelled dose suspension administered to Groups 1, 2, and 3 (on GD 22) was prepared by adding an appropriate amount of non-radiolabeled and 14C-labelled test material to obtain the target dose of 275 mg test material/kg body weight and ca. 25 μCi/kg.

Duration and frequency of treatment / exposure:

An appropriate amount of non-radiolabelled test material was added to obtain the target dose of 275 mg test material/kg body weight for administration to the Group 3 repeated dose animals from gestation day 7 to gestation day 21). The radiolabelled dose suspension administered to Groups 1, 2, and 3 (on gestation day 22) was prepared by adding an appropriate amount of non-radiolabelled and [14C]labelled test material to obtain the target dose of 275 mg test material/kg body weight and ~25 μCi/kg.

Dose / conc.:

371 mg/kg bw/day (nominal)

Dose / conc.:

362 mg/kg bw/day (nominal)

Dose / conc.:

279 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

3 females per group

Control animals:

no

Positive control reference chemical:

Positive control samples to assess the performance of the assay were prepared with 350 μg/mL salicylic acid (in vitro plasma protein binding methods).

Details on study design:

- Rationale for animal assignment: Animals were stratified by body weight and then randomly assigned to treatment groups using a computer program

Details on dosing and sampling:

STUDY DESIGN
The study consisted of a blood/plasma 14C concentration-time course to determine peak (Cmax) blood/plasma 14C concentrations and ADME experiments to determine absorption, distribution, metabolism and elimination of 14C-test material following single or multiple doses of test material.
Rabbits from Groups 1-3 had a plasma and red blood cell (RBC) 14C concentration-time course evaluated to determine peak (Cmax) and half-peak (½ Cmax) plasma 14C concentrations. Approximately 0.2 mL blood was collected at chosen times (0.2, 0.4, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72 hours post-dosing), transferred to multiple glass micro blood collecting tubes (heparinised) (per animal/time point) and centrifuged to separate the plasma from the red blood cells (RBC).

IN VITRO PLASMA PROTEIN BINDING METHODS
For plasma protein binding, plasma from NZW rabbits (non-pregnant, GD 7, and GD 22) and non-pregnant Fischer 344 rats (for comparison purposes) were utilised to investigate in vitro plasma protein binding of the test material using an ultrafiltration technique.

SPECIMEN COLLECTION
- Urine
All urine voided during the study was collected in dry-ice cooled traps. The urine traps were changed at 12 and 24 hour post-dosing, followed by 24 hour intervals for the remainder of the study. The cages were rinsed with water at 24 hour intervals. Each urine specimen and urine/cage rinse was weighed, and a weighed aliquot of each sample was analysed for radioactivity by LSS. Equal volume aliquots of urine samples (per time and Group) from the 0 - 12 hour and 12 - 24 hour collection intervals were pooled, stored at –80 °C, and underwent chemical analyses.
- Faeces
Due to the size and construction of the rabbit metabolism cages, it was not possible to collect the faeces in dry-ice chilled containers. The faeces were at laboratory temperature prior to collection at 24 hour intervals. An aqueous homogenate (~ 25 % w/w) was prepared and weighed aliquots of these homogenates were placed in combustion cones, oxidised, and quantitated for radioactivity by LSS. In addition, equal volume aliquots of faecal homogenates from each animal was taken from the 0 - 24 hour collection interval, pooled (per group), and stored at –80 °C for chemical analysis.
- Plasma/Blood
Rabbits from Groups 1 - 3 had blood and plasma 14C concentration-time courses evaluated to determine peak (Cmax) and half-peak (½ Cmax) 14C concentrations. Approximately 0.2 mL blood was collected at chosen times (0.2, 0.4, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72 hour post-dosing) and centrifuged to separate the plasma from the red blood cells (RBC). The blood collection for Group 2 animals was altered slightly post 1 hour sampling. The plasma and RBC were analysed for radioactivity by LSS.
- Expired Volatiles and CO₂
Expired volatiles and CO₂ samples were not collected.

TERMINAL SACRIFICE
The animals (all groups) were anaesthetised with CO₂ and sacrificed at 72 hours post-dosing via cardiac puncture. Following sacrifice, the metabolism cages were washed and the final cage wash (FCW) analysed for radioactivity.
- Tissues
The tissues collected at sacrifice included: brain, blood (terminal), kidney, liver, spinal cord, perirenal fat, plasma (terminal), red blood cells, spleen, gastrointestinal (GI) tract, residual carcass, and skin.
The brain, GI tract with contents, kidney, liver, and spinal cord were collected, homogenised (~ 33 % homogenate), a weighed aliquot oxidised and analysed for radioactivity by LSS. Blood was centrifuged to obtain plasma and the plasma analysed for radioactivity by LSS. The remaining tissues were directly oxidised without homogenisation and analysed for radioactivity by LSS. Due to the high recovery of radioactivity in the excreta (average for the 3 groups >95 %), the skin and residual carcass (all groups) were not analysed.

FINAL CAGE WASH
Following the terminal sacrifice of the animals, a final cage wash was performed. The final cage wash and contents were collected and the weight of the sample was determined. A weighed aliquot of the final cage wash was analysed for radioactivity.

TERMINAL BLOOD/PLASMA/RBC
Blood was obtained at sacrifice via cardiac puncture and analysed for radioactivity. A portion of the terminal blood was centrifuged to obtain plasma and RBC, and analysed for radioactivity.

CONTROL SAMPLES
Control urine and faeces were collected (dry-ice cooled traps for the urine) from female rabbits not dosed with test material.

Statistics:

Descriptive statistics were used (i.e., mean ± standard deviation). All calculations in the database were conducted using Microsoft Excel® spreadsheets and databases in full precision mode (15 digits of accuracy). Certain pharmacokinetic parameters were estimated for both plasma/blood and urine data, including AUC (area-under-the-curve), Cmax and elimination rate constants, using a pharmacokinetic computer modelling program (PK Solutions, Carlsbad, CA). Differences in in vitro plasma protein binding between groups were used to compared using a one-way analysis of variance (Steel and Torrie, 1960).

Type:

absorption

Results:

the test material was rapidly absorbed following oral administration to rabbits

Type:

excretion

Results:

the test material was rapidly eliminated unchanged in the urine

Metabolites identified:

yes

Details on metabolites:

The pooled urine samples and pooled faeces were analysed by HPLC and yielded one radioactive peak with a retention time comparable to the standard representative of the parent. The identity of this peak was confirmed with HPLC positive ion electrospray ionisation/mass spectrometry.

Administration of Test Material

The concentration of the unlabelled test material orally dosed daily for 15 days (repeated dose) to the Group 3 animals (gestation day 7 through 21) was 64.7 mg/g. The test material was shown to be stable at this concentration range in 0.5 % METHOCEL suspension for at least to 14 days.

The Group 1 animals (non-pregnant) were inadvertently administered a single oral dose at a nominal concentration of 371 mg 14C-test material/kg. It was decided, for comparative purposes, to administer a single oral dose to the Group 2 animals (GD 7) at the same dose as Group 1. While this dose was ~45 % higher than the no-observed-effects- levels (NOEL) for maternal toxicity, as previously reported, the dose was 27 % less than the NOEL for developmental toxicity (500 mg/kg). At this point in the study, Group 3 animals (GD 7-22) had already been administered 15-daily doses of 279 mg unlabelled test material/kg. Therefore, this group was administered 279 mg 14C-test material/kg on GD 22.

Disposition of Radioactivity

At 72 hours post-dosing, tissues accounted for 0.11 - 0.15 % of the administered dose and was consistent amongst the three groups of animals.

At sacrifice, the remaining carcasses were collected. However when the recovery of the administered dose was determined to be 94 - 99 % for the three groups it was determined it was not necessary to analyse the carcass for radioactivity.

The urine/rinse accounted for 76.63, 82.52, and 85.68 % of the administered dose by 72 hours post-dosing for Groups 1, 2, and 3, respectively. The 0 - 12 hour urinary interval values were for urine only. The 12 - 24 hr interval included urine from that time interval and the 0 - 24 rinse. During the first 12 hours post-dosing, 51, 58, and 72 % of the total radioactivity recovered in the urine for Groups 1, 2, and 3, respectively. The 0 - 12 and 12 - 24 hour time intervals accounted for 94, 93, and 97 % of the total amount recovered in the respective urines.

By 72 hours post-dosing, faecal elimination was 20.17, 16.06, and 7.54 % of the administered dose for Group 1, 2, and 3, respectively. The 0 - 24 hour time interval accounted for 97, 95, and 95 % of the total amount eliminated in the faeces, respectively.

Total radioactivity recoveries were 98.15, 99.05, and 93.85 % of the administered dose for Groups 1, 2, and 3, respectively. Urine and faeces accounted for >99 % of all radioactivity recovered for the respective groups.

Distribution of Radioactivity in Tissues

The GI tract was the only tissue with detectable amounts of radioactivity for all study animals. The spleen from two of the three animals from Group 3 had detectable amounts of radioactivity. These data are indicative of the test material not accumulating in the tissues.

Chemical Analysis of Urine

The 0 - 12 and 12 - 24 hour-pooled urines for Groups 1-3 were analysed by HPLC and yielded one radioactive peak. The peak had an HPLC retention-time match with an authentic standard of test material. In addition, the pooled urine samples were subjected to HPLC positive ion electrospray ionisation/ mass spectrometry (+ESI/MS) analysis to determine the identity of the radioactive peak. Based on similar +ESI/MS spectra for the lone radioactive peak and an authentic standard of 14C-test material, the lone radioactive peak observed in the pooled urine was identified as parent test material.

Chemical Analysis of Faeces

The 0-24 hour-pooled faeces for Groups 1-3 were analysed by HPLC and yielded one radioactive peak. The peak had an HPLC retention-time match with an authentic standard of test material. In addition, each pooled faecal sample was subjected to HPLC positive ion electrospray ionisation/ mass spectrometry (+ESI/MS) analysis to determine the identity of the radioactive peak. Based on similar +ESI/MS spectra for the lone radioactive peak and an authentic standard of 14C-radiolabelled test material, the lone radioactive peak observed in the pooled faeces is identified as parent test material.

Plasma 14C-Concentration Time-Course

The test material was rapidly absorbed from the GI tract after oral dosing and detected in plasma at relatively high concentration within 10 minutes of dosing. The peak plasma concentrations (Cmax) of the test material-derived radioactivity were reached ~1 hour of dosing (Tmax) for all Groups. The decline in the plasma concentration of test material-derived radioactivity after reaching the initial peak appeared to be mono-exponential in all three groups of rabbits.

The mean Cmax of test material in the plasma of the Group 1 and Group 2 rabbits that received a single oral dose of 362-371 mg test material/kg were almost identical, 49.7 ± 15.5 vs. 50.5 ± 14.8 μg/g plasma, respectively. The mean Cmax for the Group 3 rabbits (pregnant receiving multiple doses of 279 mg test material/kg) is higher, 70.9 ± 5.3 μg/g plasma.

The mean area under the plasma concentration-time curves (AUC) were 334 ± 13, 265 ± 39, and 441 ± 55 for Groups 1, 2, and 3, respectively. As seen with Cmax, the plasma AUC for DE-750 following the repeated oral doses was not proportional to dose.

The test material was rapidly eliminated from plasma of all groups of rabbits. The mean plasma elimination half- life were 7.2 ± 2.6 hours in Group 1, to 4.4 ± 1.6 hours in Group 2 and 4.1 ± 0.7 hours in Group 3 rabbits.

Red Blood Cell 14C-Concentration Time Course

The test material was detected with RBC at relatively high concentration within 10 minutes of dosing. Peak RBC concentrations (Cmax) of the test material-derived radioactivity were reached within 1 hour of dosing for all Groups. As seen with plasma, the decline in the RBC concentration of test material-derived radioactivity after reaching the initial peak appeared to be mono-exponential in all three groups of rabbits.

The mean Cmax of test material in the RBC of the Group 1 and Group 2 rabbits that received a single oral dose of 362-371 mg test material/kg were almost identical, 37.2 ± 9.0 vs. 38.0 ± 6.1 μg/g RBC, respectively. The mean Cmax for the Group 3 rabbits (pregnant receiving multiple doses of 279 mg test material/kg) appears slightly higher, 44.9 ± 28.5 μg/g RBC.

The test material was rapidly eliminated from the RBC samples of all groups of rabbits. The RBC elimination half- life ranged from 4 to 7 hours. Similar to that observed with plasma, the half- life of elimination from RBC appears similar regardless of dose regimen or pregnancy status of the rabbits, given the distribution of the individual animal data for the 3 groups. The rapid elimination of test material-derived radioactivity from the RBC samples was supported by high clearance values, 1.1 to 1.5 litres/hour among all the groups.

Urinary Excretion

Urinary elimination of the test material-derived radioactivity was similar in all groups. The amount excreted in the urine ranged from 76 to 86 % of the administered dose for all three groups. The differences in the total mg equivalence of test material eliminated in Groups 1 and 2 urine (915 and 982 mg) vs. Group 3 (809 mg), is reflected in the dose administered. The urinary half- life of elimination ranged from 6 to 7 hours with an elimination rate constant from 0.10 to 0.12 hour^-1. There is little difference in the amount excreted in the urine or the urinary elimination rates observed with the non-pregnant rabbits (single oral dose), GD 7 rabbits (single oral dose), or the pregnant rabbits receiving repeated dose from GD 7 through GD 22.

In Vitro Plasma Protein Binding

Under the conditions used in these experiments, the plasma protein binding of the positive control compound, salicylic acid at 350 μg/mL, was in agreement with previously reported values, 60 - 80 %, indicating the assay was performing as expected.

The plasma protein concentration measured in the non-pregnant F344 rats, 85.3 ± 2 mg/mL, was slightly higher than that measured in any of the rabbit plasma samples. With the rabbits, the non-pregnant animals had plasma protein concentrations of 84.6 ± 3 mg/mL. The plasma protein concentration in the rabbits decreased during pregnancy to 77.5 ± 2 mg/mL at GD 7 and 74.9 ± 1 mg/mL at GD 22. The plasma protein binding of test material showed a concentration-dependent decrease in all plasmas. The average binding of test material in rat plasma fell by 16.8 % as the test material concentration was increased ~15- fold from 10 to 154 μg/mL. Similarly, the average binding of test material in rabbit plasma decreased by 14.8, 18.4 and 15.0 % over the same test material concentrations with non-pregnant, GD 7 and GD 22 rabbits, respectively.

The plasma obtained from rabbits demonstrated a significant, 10 - 14 % decrease in plasma protein binding among non-pregnant, GD 7 and GD 22 across the concentrations evaluated (one-way ANOVA, p = 0.05). At the lowest concentration, 10 μg/mL, 68.2 % of the test material was bound to proteins from non-pregnant rabbit plasma. At GD 7, plasma protein binding was reduced to 65.7 %. By GD 22, only 58.1 % of test material was associated with plasma protein, a >10 % reduction from the non-pregnant rabbit plasma. Similarly, at 33 μg/mL, plasma protein binding decreased from 67.5 % with the non-pregnant rabbit plasma to 62.7 % and 53.4 % with the GD 7 and GD 22 plasma, respectively. At the highest concentration, 154 μg/mL, only 53.4 % of the test material was bound to non-pregnant rabbit plasma proteins. Plasma protein binding was further reduced to 47.3% with GD 7 rabbit plasma, and, by GD 22, only 43.1 % of the test material was bound to plasma protein, an 11 % reduction from the non-pregnant rabbit plasma. This corresponds to a 25 % reduction in plasma protein binding from the lowest concentration with the non-pregnant rabbit plasma. Taken together with the in vivo test material plasma concentration data, these data imply that circulating levels of free test material are higher in pregnant rabbits, increasing the bioavailability of test material during pregnancy. Assuming that the absorption of test material during pregnancy and following repeated dosing remains constant, the increased levels of free test material may result as a consequence of the lower plasma protein binding observed in the plasma from pregnant rabbits.

Conclusions:

In conclusion, with all groups the test material was rapidly absorbed following oral administration to rabbits and rapidly eliminated unchanged in the urine.

Executive summary:

The absorption, distribution, metabolism, and elimination of 14C-test material orally administered to three groups of three female New Zealand White rabbits were determined. Non-pregnant rabbits (Group 1) and pregnant rabbits at gestation day 7 (GD 7; Group 2) were administered a single oral dose of 371 and 362 mg 14C-test material/kg bw, respectively. The repeated dose group (Group 3) were given daily doses of 279 mg test material/kg bw from GD 7 - 21, followed by 279 mg 14C-test material/kg bw on GD 22.

For all groups, the test material was quickly absorbed with peak plasma and red blood cell maximal concentrations (Cmax) both within ~1 hour of dosing. The plasma elimination rate was also rapid, with half- lives ranging from 4 to 7 hours for all three dose groups. The Cmax and AUC for the group 3 animals (279 mg/kg) were slightly higher than the other two groups, administered 362 - 371 mg/kg.

At 72 hours post-dosing, the GI tract was the only tissue with detectable amounts of radioactivity for all study animals and ranged from 3.1 to 4.6 μg-eq./g tissue. This represents 0.11 - 0.15 % of the administered dose. The spleen from two of the three animals from the repeated dose group had detectable amounts of radioactivity for an overall mean of 2.0 ± 0.9 μg-eq./g tissue (<0.005 % of the administered dose).

Orally administered test material was not metabolised by rabbits. There was one radioactive peak detected in excreta (all groups) and was positively identified as parent test material.

Urine was the principal route of elimination with 77, 83, and 86 % of the administered dose eliminated 72 hours post-dosing, for Groups 1, 2, and 3, respectively. By 12 hours post-dosing 39, 48, and 61 % of the administered dose was eliminated in the urine, respectively, with the vast majority (93 - 97 %) of the urinary total being eliminated by 24 hours post dosing. Faeces accounted for 20, 16, and 8 % of the administered dose for Groups 1, 2, and 3 at 72 hours post-dosing, with the vast majority (95 - 97 %) being eliminated in the first 24 hours. These data are consistent with the test material being more systemically available to the gestation day 22 animals. Urine and faeces accounted for >99 % of the recovered radioactivity for all three groups.

The repeated dose animals (gestation days 7 - 22) were administered 14C-test material dose ~75 % of the dose that the non-pregnant and gestation day 7 animals received. However, they had a slightly higher plasma/red blood cell Cmax, and ~25 % higher area under the curve (AUC) than the other two groups, consistent with higher bioavailability of the test material to the gestation day 22 animals. Correcting for the size of the 14C-dose administered by dividing the AUC by the dose, the relative bioavailability of the test material following the repeated dose regime was 75 - 115 % higher than the other single oral dosed groups. In vitro plasma protein binding of the test material demonstrated relative amounts of protein-bound test material decreased in order of non-pregnant rats and rabbits >GD 7 > GD 22.

No significant differences were observed between the non-pregnant and GD 7 rabbits in the absorption or the routes or rates of elimination of the test material. The test material had a greater bioavailability to the gestation day 22 animals, as shown by increased plasma levels and higher percent of urinary elimination. Some differences in plasma protein binding were observed between groups. In conclusion, with all groups the test material was rapidly absorbed following oral administration to rabbits and rapidly eliminated unchanged in the urine.

Reference 3

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 June 2002 to 12 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 99.5%

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: ca. 200 - 230 g

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % methyl cellulose

Details on exposure:

DOSE ADMINISTRATION
The dose was administered via oral gavage. Dose formulations were warmed to room temperature before dose administration and were stirred constantly on a magnetic plate to ensure homogeneity during dose administration. The dose amount was based on individual animal body weight determined within 4 hours before each dosing. The actual amount dosed was determined by the difference in dosing syringe weight before and after dosing.

Duration and frequency of treatment / exposure:

Rats were orally administered a single dose of radiolabelled test material at a targeted high dose rate of 1000 mg/kg (Single high dose- Group 1M) and at a targeted low dose rate of 50 mg/kg (Single low dose-Group 2M). An additional group of rats were orally administered daily doses of non-radiolabelled test material (Days 1 through 14) and a single dose of radiolabelled test material (Day 15) at a targeted low dose rate of 50 mg/kg/day (Multiple low dose-Group 3M).

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

4 males per group

Control animals:

no

Details on dosing and sampling:

SAMPLING
Urine and faecal samples were taken for radiochromatographic profiling using HPLC and LC-MS/MS. The elimination half-lives of [¹⁴C]test material equivalents in urine were determined using WinNonlin 4.0.1 (Pharsight Corp., Mountain View, CA).

Type:

absorption

Results:

test substance was readily absorbed

Type:

excretion

Results:

test substance was efficiently cleared through the urine (average T1/2 of 3-4 hours) and faeces

Details on absorption:

The amount of absorbed radioactivity eliminated in the urine and detected in the tissues at sacrifice was 50, 59 and 42 % of administered dose for the single low, multiple low, and single high dose groups, respectively.
The faeces contained 43, 33, and 43 % of the administered dose, for the single low, multiple low, and single high dose groups, respectively. The amount of absorbed test material in the faeces was not directly determined in this study. However, it is estimated the amount of absorbed test material eliminated in the faeces is likely to be less than 5 % of the administered dose.

Details on distribution in tissues:

[¹⁴C]test material was rapidly depleted from the body after oral dose administration. After the 7-day depletion, skin and remaining carcass contained 0.03 - 0.45 % and 0.04 - 0.26 % of the administered dose, respectively. Less than 0.01 % of the administered dose was detected in kidneys, liver and spleen.

Details on excretion:

During the 7-day collection period, the average total recovery of the administered dose was greater than 95 %, with 41.3 - 58.6 % excreted in urine and 33.0 - 43.4 % eliminated in faeces. After the 7-day depletion, less than 1 % of the administered dose remained in tissues and carcasses. During the 24 hours after the single high dose administration, 0.01 % of the administered dose was recovered in volatiles including CO2 and volatile organics.
The single high dose and the single low dose groups presented similar excretion patterns, most of the radioactivity being excreted in faeces and urine within 24 hours.
Compared to the single low dose group, the multiple dose group presented slightly higher percentage of radioactivity recovered in the urine. Similar to the other groups, most of the radioactivity in the multiple dose group was recovered during the first 24 hours.

Metabolites identified:

yes

Details on metabolites:

The parent represented more than 96 % of the chromatographed radioactivity detected via HPLC radiochromatography in the urine. The remaining 4 % was expected to be trace impurities in the labelled test material. The parent represented 100 % of the recovered radioactivity chromatographed in the faeces.

Animal Health and Observation

The animals were examined within 24 hours of receipt and deemed them healthy and free of gross abnormalities. No medications were administered and the animals remained healthy during the course of the study. No changes in behaviour or appearance were observed after dose administration.

Identity and Radiochemical Purity of the Test Material

The radiochemical purity of the test material in formulation was confirmed by HPLC before dose administration. The radiochemical purity of [¹⁴C]test material in the high and low dose formulations ranged from 97.6 to 97.9 %. The radioactivity and mass of the test material in dose formulations were determined . The specific activity was calculated for each dose level. The dose formulations were homogenous during the dose administration process. The test material was demonstrated to be stable in the 0.5 % methyl cellulose for at least 104 days at 4 °C storage conditions in the present study.

Dose Levels

The average dose levels for the groups 1M, 2M and 3M were 1174.4, 52.1 and 51.8 mg/kg bw, respectively.

Urinary Elimination Half Lives

The average α-phase urinary elimination half-lives (T1/2) of [¹⁴C]test material equivalents were 3.78, 2.85 and 3.27 hours for single high, single low, and multiple low dose groups, respectively. There were no significant differences (p = 0.37) in T1/2 of [¹⁴C]test material among three dose groups. The average β-phase urinary elimination halflives (T1/2) of [¹⁴C]test material equivalents were 10.88, 10.23 and 12.25 hours for single high, single low, and multiple low dose groups, respectively. There were no significant differences (p = 0.68) in T1/2 of [¹⁴C]test material among three dose groups.

Chromatographic Profiling and Identification of [¹⁴C]test material in Urine and Faeces

The composite urine samples (0-6 and 6-12 hours) of all three groups were used for chromatographic profiling. The 0-24 hour composite faecal samples from all three groups and the 24-48 hour composite faecal sample of the single high dose group were extracted and analysed by HPLC.

Chromatographic Profiling and LC-MS/MS Analysis of Reference Standards

A standard mix containing [¹⁴C]test material was routinely analysed to monitor the performance of HPLC system. The standard mix was also analysed on LC-MS/MS with in-line UV- and ¹⁴C-detectors. Parent compound was eluted at approximately 28 minutes.

Characterisation and Identification of Radiolabelled Components in Urine

Urine is a major contributor to the recovery of the administered [¹⁴C]test material dose in rats. Metabolic profiling was performed on pooled urine samples (0-6 and 6-12 hours), which represented greater than 90 % of the radioactivity recovered in the urine from the single and multiple low dose groups and 78 % in the urine from the high dose group. An HPLC radiochromatogram of the pre-dose urine sample, fortified with [¹⁴C]test material, was analysed to validate the HPLC method and to confirm the retention time of test material in presence of matrix.

Co-chromatography was performed using the 6-12 hour urine samples from all three dose levels fortified with [¹⁴C]test material to confirm the parent retention time. The LC-MS/MS (Product Ion Scan) analysis was performed using the 0-6 hour composite urine sample to confirm component identification. The product ion scan (m/z 207) of the 0 - 6 hour composite urine sample produced a fragmentation pattern identical to that observed during the analysis of a test material reference standard. Multiple reaction monitoring (MRM) analysis (m/z 207/161) further verified the identity of the unchanged parent in the 0 - 6 hour composite urine sample. The metabolic profiles in urine samples of rats orally dosed with [¹⁴C]test material were simple and characterised by 4 peaks. The profiles were qualitatively similar across three dose levels and collection intervals. The unchanged parent, represented the vast majority of the excreta and made up 96.0 % of the chromatographed radioactivity in all HPLC-radiochromatograms. Three unknown components were detected in urine and made up 4.0 % of the total chromatographed radioactivity. The three unknown components were also detected in similar quantities in dose formulations, suggesting that they are trace impurities. Therefore, no further characterisation was performed.

Characterisation and Identification of Radiolabelled Components in Faeces

Faeces are another major contributor to the recovery of the administered [¹⁴C]test material dose in rats. The total recovery of the administered dose in faeces was 43.43, 43.05, and 32.98 % for the single high, single low, and multiple low dose groups, respectively. Metabolic profiling was performed on the 0 - 24 hour pooled faecal samples of all three dose levels and the 24 - 48 hour pooled faecal sample from the single high dose group. These samples represented approximately 90 % of the faecal radioactivity after each treatment.

An HPLC radiochromatogram of the pre-dose faecal sample, fortified with [¹⁴C]test material, was analysed to confirm the HPLC method and the retention time of test material in the presence of matrix. The results are expressed as percentages of the chromatographed radioactivity and administered carbon-14 dose.

Co-chromatography was performed using the 0 - 24 hour faecal samples from all three dose levels fortified with [¹⁴C]test material to confirm the parent retention time. The metabolic profiles in faeces following oral administration of [¹⁴C]test material to rats were simple and characterised by a single peak. The profiles were qualitatively similar across dose level and collection interval. The unchanged parent, represented the only observed component in faeces and made up 100 % of the radioactivity in all HPLC-radiochromatograms. The LC-MS/MS (Multiple Reaction Monitoring) analysis was performed using the 0 - 24 hour composite faecal sample. The multiple reaction monitoring analysis (m/z 207/161) verified the identity of the component as the unchanged parent (test material).

Conclusions:

Under the conditions of the study, the test material was found to be readily absorbed and efficiently cleared through the urine (average T1/2 of 3-4 hours) and faeces. The overall potential for bioaccumulation is therefore low.

Executive summary:

The absorption, distribution, metabolism and excretion of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.7485, OECD 417 and B.36.

During the study, three groups of four male Fischer 344 rats were given a single oral dose of either 50 or 1000 mg [¹⁴C]test material, or a single oral dose of 50 mg [¹²C]test material /kg for 14 days followed by a single oral dose of 50 mg/kg [¹⁴C]test material.

[¹⁴C]test material was readily absorbed and efficiently cleared through the urine (average T1/2α of 3-4 hour for all groups) and faeces. The absorption and excretion patterns were similar among the three groups. An average of 74.04 - 93.13 % of the administered radioactivity was excreted during the first 24-hours post-dose administration. Urinary and faecal elimination totalled 41 - 59 and 33 - 43 % of the administered dose, respectively, with <5 % of the administered dose in faeces estimated to be absorbed test material.

Selected composite urine and faeces were analysed by HPLC. Both metabolic profiles of all groups were similar. The major peak in both matrixes was identified by mass spectrometry as parent (test) material, and accounted for 96 - 98 and 100 % of the radioactivity in the urine and faeces, respectively.

Reference 4

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

5 September 2012 to 1 October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study was conducted on the formulated product; however, the results are expressed in terms of percentage of the acid equivalent of the active ingredient, applied as the potassium salt.

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

yes

Remarks:

- see below

Principles of method if other than guideline:

In the 0.02 g a.e./L group, two skin membranes (replicates C-7 and C-8, derived from one donor) had slightly higher Kp values for tritiated water than the cut-off value. The data obtained for replicates C-7 and C-8 were comparable to the other data obtained and therefore considered appropriate to use in the calculations. This deviation is not considered to have affected the outcome and validity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GF-1601, which was tested at three target
oncentrations: 30 g a.e.L-1 (concentrate), 12 g a.e.L-1 (field spray dilution I) and 0.02 g a.e.L-1 (field spray dilution II). The dermal absorption of the active substance, aminopyralid (acid equivalent), was measured for each of these three test concentrations.

Radiolabelling:

yes

Remarks:

[14C]

Species:

other: human split-thickness skin

Vehicle:

other: Formulated - equivalent to in-use product.

Duration of exposure:

8 hours of exposure (16 hours of post exposure time)

Doses:

- Nominal doses: 30 g a.e./L (concentrate), 12 g a.e./L (field spray dilution I) and 0.02 g a.e./L (field spray dilution II)
- Actual doses: 311 ± 6 µg/cm², 130 ± 3 µg/cm² and 0.19 ± 0.01 µg/cm²
- Dose volume: 10 µL/cm², resulting in approximately 6.4 µL applied over the surface (accounting for expected loss during distribution of the skin samples).
- Rationale for dose selection: 30 g a.e./L represents the maximum concentration users would be exposed to when handling the concentrated product, field spray dilution II represents the in-use concentration, and field spray dilution I was included to examine dose-response effects.

No. of animals per group:

Two skin membranes from each of 4 donors were exposed in each test group (8 skin membranes per dose level)

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions:
For the concentrate application, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 27 μL of a 95 mM KOH aqueous solution was added. The solvent and water were evaporated under nitrogen to dryness and 1.0226 g of the formulated test material was added. The test concentration was carefully vortexed in order to obtain a homogenous suspension.
For test field spray dilution I, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 27.3 μL of a 95 mM KOH aqueous solution was added. The solvent and water were evaporated under nitrogen gas to dryness after which 1.0155 g of the diluted in-use formulation was added (0.8255 g of the concentrated product was mixed with 1.2087 mL demineralised water). The test concentration was carefully vortexed in order to obtain a homogenous suspension.
For test field spray dilution II, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 22.5 μL of a 3.96 mM KCl aqueous solution was added. The solvent and water were evaporated under nitrogen gas to dryness after which 0.9066 g water was added. The test concentration was carefully vortexed in order to obtain a homogenous suspension.

APPLICATION OF DOSE: The dose preparations were applied with a positive displacement pipette tip and spread evenly on the skin surface within the donor compartment (ca 10 μL/cm²) using a disposable glass rod.

SAMPLE COLLECTION
- Mass balance samples:
> Receptor fluid samples: Collected at 0-1 hour, 1-2 hours followed by 2 hour intervals up to 24 hours after application.
> Skin wash: After 8 hours of exposure, a volume of 40 µL of mild soap solution (3 % in water at 37 °C) was applied to the skin surface and removed using a cotton swab. This was repeated four times. The skin was then washed twice with 40 µL of demineralised water and cotton swab after which the skin was dried with two dry cotton swabs. The washing efficiency was evaluated and found to be sufficient (less than 1 % of the radioactivity recovered in the first three swabs was found in the fourth). After 24 hours, the application site was washed again using the same method.
> Diffusion cell: 24 hours after exposure, the diffusion cell was dismantled and both compartments were washed twice with 1.0 mL ethanol.
> Stripping: Each skin disc was tape stripped 15 times using Stripping Discs and a D-Squame® pressure device. Tape stripping was discontinued if the epidermis ruptured.
> Digestion: After stripping, the membranes were digested in 1.5 M KOH solution with 20 % ethanol for a minimum of 24 hours.

SAMPLE PREPARATION
- Preparation details: Ultima Gold™ scintillation liquid was added to samples of the receptor fluid (10 mL per sample), the diffusion cell washes (10 mL per sample), the cotton swab extracts (10 mL to a weighed aliquot of each sample), the tape strips (4 mL per sample), and to samples of the mock dosing samples (10 mL per sample). For the determination of radioactivity in digested skin preparations, 15 mL Hionic Fluor™ scintillation liquid was added to the entire digested skin membrane.
> Dose formulations: A weighed volume of water was added to aliquots of the dose formulation taken just before and directly after dosing. Scintillation liquid (Ultima Gold™) was added to weighed subsamples for analysis.
> Receptor fluid: Samples of the receptor fluid were added directly to scintillation liquid (Ultima Gold™) for analysis.
> Skin wash: The cotton swabs were pooled per skin membrane for each time point (8 and 24 hours) and extracted with ethanol for at least 24 hours at room temperature. Aliquots of the extracted cotton swabs were added directly to scintillation liquid (Ultima Gold™) for analysis.
> Donor and receptor compartments: The samples from each compartment were separately mixed with scintillation liquid (Ultima Gold™) for analysis.
> Tape stripping: Tape strips were added directly to scintillation liquid (Ultima Gold™) for overnight extraction followed by analysis.
> Skin membranes: The entire digested membrane fractions were directly added to scintillation liquid (Hionic Fluor™) for analysis.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting. The radioactivity in the samples was determined using a Canberra Packard Tricarb 3100 TR scintillation counter.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human donors. Breast skin samples were donated after surgery. All donors were female and of a similar age
- Ethical approval if human skin: All donors provided informed consent.
- Type of skin: Human skin membranes
- Preparative technique: All subcutaneous fat was removed from the skin samples upon arrival at the laboratory. Upon thawing, the tissues were cut using a Dermatome (25 mm) and measured with a digimatic micrometer. The exposed area of each membrane was 0.64 cm²
- Thickness of skin: 300 to 400 µm
- Membrane integrity check: 36 membranes were evaluated by measuring the permeability coefficient (Kp) for tritiated water. 200 µL saline containing tritiated water (17.5 kBq/mL) was applied in the donor compartment of the flow through diffusion cells. The compartments were covered with a glass slide. Samples of the receptor fluid (approximately 1.6 mL/h) were collected for up to three hours after application. The tritiated water remaining at the application site was removed with a pipette and the skin was dried with cotton swabs. 24 membranes were selected for use in the study (8 per group - two from each of the four donors for each test group).
- Storage conditions: Tissues were stored after fat removal in aluminium foil below -18 °C until use. One donor’s tissues were stored overnight at 2 - 10 °C before subcutaneous fat was removed.

PRINCIPLES OF ASSAY
- Diffusion cell: 9 mm flow-through automated diffusion cells
- Receptor fluid: Saline (0.9 % sodium chloride w/v containing 0.01 % sodium azide) supplemented with 6 % w/v polyoxy-ethylene 20-oleyl glycol (PEG)
- Solubility of test material in receptor fluid: 87 µg/mL
- Flow-through system: The receptor fluid was pumped at a speed of ca. 1.6 mL/h.
- Test temperature: 32 ± 1 °C (skin surface temperature)
- Humidity: Ambient

Absorption in different matrices:

- Receptor fluid (in vitro test system): 0.21 ± 0.12 (concentrate), 0.22 ± 0.15 (field dilution I) and 0.73 ± 0.42 (field dilution II) as percentage of administered dose.
- Receptor chamber (in vitro test system): 0.01 ± 0.00 (concentrate and field dilution I) and 0.09 ± 0.03 (field dilution II) as percentage of administered dose.
- Donor chamber (in vitro test system): 0.04 ± 0.02 (concentrate), 0.03 ± 0.02 (field dilution I) and 0.10 ± 0.04 (field dilution II) as percentage of administered dose.
- Skin preparation (in vitro test system): 0.20 ± 0.10 (concentrate), 0.19 ± 0.13 (field dilution I) and 0.68 ± 0.25 (field dilution II) as percentage of dose administered.
- Stratum corneum (in vitro test system): 0.16 ± 0.07 (concentrate), 0.11 ± 0.06 (field dilution I) and 0.22 ± 0.06 (field dilution II) as percentage of dose administered.

Total recovery:

- Total recovery: 100.7 ± 1.0 (concentrate), 99.7 ± 2.7 (field dilution I) and 100.9 ± 2.6 (field dilution II) as percentage of dose administered
- Recovery of applied dose acceptable: Yes

Key result

Dose:

30 g a.e./L

Parameter:

percentage

Absorption:

0.41 %

Remarks on result:

other: 24 hours

Remarks:

Concentrated product (mean absorbed dose)

Key result

Dose:

12 g a.e./L

Parameter:

percentage

Absorption:

0.42 %

Remarks on result:

other: 24 hours

Remarks:

Field dilution I (mean absorbed dose)

Key result

Dose:

0.02 g a.e./L

Parameter:

percentage

Absorption:

1.5 %

Remarks on result:

other: 24 hours

Remarks:

Field dilution II (mean absorbed dose)

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (30 g a.e./L) was 0.65 µg/cm² (0.21 % of the applied dose) and the mean maximal flux was 0.13 µg/cm²/h. The lag time was 0.8 hours.

For field dilution I (12 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.28 µg/cm² (0.22 % of the applied dose) and the mean maximal flux was 0.04 µg/cm²/h. The lag time was 1.1 hours.

For field dilution II (0.02 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.0014 µg/cm² (0.73 % of the applied dose) and the mean maximal flux was 0.0001 µg/cm²/h. The lag time was 0.1 hours.

The mean absorbed dose was determined to be 0.41, 0.42 and 1.50 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

The mean potentially absorbed dose was determined to be 0.55, 0.50 and 1.68 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

Table 1: Results

	Concentrate	Field Dilution I	Field Dilution II
Total concentration (g a.e./L)	31.5	13.0	0.02
Dose (µg/cm²)	311 ± 6	130 ± 3	0.19 ± 0.1
Penetration into the receptor fluid after 24 hours (µg/cm²)	0.65	0.28	0.0014
Penetration into the receptor fluid after 24 hours (% of dose)	0.21	0.22	0.73
Maximal flux (µg/cm²/h)	0.13	0.04	0.0001
Lag time (h)	0.8	1.1	0.1
Recovery of dose (% ± SD) Receptor fluid	0.21 ± 0.12	0.22 ± 0.15	0.73 ± 0.42
Recovery of dose (% ± SD) Receptor compartment wash	0.01 ± 0.00	0.01 ± 0.00	0.09 ± 0.03
Recovery of dose (% ± SD) Skin*	0.20 ± 0.10	0.19 ± 0.13	0.68 ± 0.25
Absorbed dose (% ± SD)**	0.41 ± 0.21	0.42 ± 0.26	1.50 ± 0.45
Tape strips (1 + 2) (% ± SD)	0.03 ± 0.02	0.03 ± 0.02	0.04 ± 0.01
Tape strips (3 - last) (% ± SD)	0.14 ± 0.06	0.08 ± 0.04	0.18 ± 0.06
Stratum corneum (% ± SD)	0.16 ± 0.07	0.11 ± 0.06	0.22 ± 0.06
Potentially absorbed dose (% ± SD)***	0.55 ± 0.26	0.50 ± 0.30	1.68 ± 0.44
Skin wash t = 8 h (% ± SD)	99.9 ± 1.2	98.9 ± 2.9	98.5 ± 3.0
Skin wash t = 24 h (% ± SD)	0.21 ± 0.08	0.3 ± 0.1	0.6 ± 0.2
Donor compartment wash (% ± SD)	0.04 ± 0.02	0.03 ± 0.02	0.10 ± 0.04
Total recovery (% ± SD)	100.7 ± 1.0	99.7 ± 2.7	100.9 ± 2.6

* Epidermis + dermis (without stratum corneum)

** The amount in the receptor fluid plus the receptor compartment wash, plus the skin membrane (excluding tape strips, i.e. stratum corneum)

*** The amount in the receptor fluid plus the receptor compartment wash plus the skin membrane (except for the first 2 tape strips)

Conclusions:

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated products) was found to be poorly absorbed through the skin.

Executive summary:

The dermal absorption of the test material was determined in a study conducted in accordance with the OECD guideline 428 and under GLP conditions.

The test material was assessed as the concentrated formulated product (31.5 g a.e./L) and as in use field dilutions (13 and 0.02 g a.e./L) using human split-thickness skin in flow through diffusion cells. The contact time was 8 hours and the post-exposure time was 16 hours.

In addition to the amount of [14C]test material in the receptor fluid, the residues remaining in/on the skin membranes and in the stratum corneum (at 24 hours) were also determined. Human skin membranes were prepared from four separate donors in duplicate (n = 8) per test concentration tested.

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (31.5 g a.e./L) was 0.65 µg/cm² (0.21 % of the applied dose) and the mean maximal flux was 0.13 µg/cm²/h. The lag time was 0.8 hours. For field dilution I (13 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.28 µg/cm² (0.22 % of the applied dose) and the mean maximal flux was 0.04 µg/cm²/h. The lag time was 1.1 hours. For field dilution II (0.02 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.0014 µg/cm² (0.73 % of the applied dose) and the mean maximal flux was 0.0001 µg/cm²/h. The lag time was 0.1 hours.

The mean absorbed dose was determined to be 0.41, 0.42 and 1.50 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

The mean potentially absorbed dose was determined to be 0.55, 0.50 and 1.68 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated products) was found to be poorly absorbed through the skin.

Reference 5

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

12 September 2012 to 19 September 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study was conducted on the formulated product; however, the results are expressed in terms of percentage of the acid equivalent of the active ingredient, applied as the olamine salt.

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: aminopyralid formulated as GF-1633 (test preparation), a Soluble Liquid Concentrate (SL) formulation, and two dilutions

Radiolabelling:

yes

Remarks:

[14C]

Species:

other: human split-thickness skin

Vehicle:

other: Formulated - equivalent to in-use product.

Duration of exposure:

8 hours of exposure (16 hours of post exposure time)

Doses:

- Nominal doses: 40.5 g a.e./L (concentrate), 12.6 g a.e./L (field spray dilution I) and 0.02 g a.e./L (field spray dilution II)
- Actual doses: 402 ± 7 µg/cm², 125 ± 2 µg/cm² and 0.20 ± 0.0 µg/cm²
- Dose volume: 10 µL/cm², resulting in approximately 6.4 µL applied over the surface (accounting for expected loss during distribution of the skin samples).
- Rationale for dose selection: 40.5 g a.e./L represents the maximum concentration users would be exposed to handling the concentrated product, field spray dilution II represents the in-use concentration, and field spray dilution I was included to examine dose-response effects.

No. of animals per group:

Two skin membranes from each of 4 donors were exposed in each test group (8 skin membranes per dose level)

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions:
For the concentrate application, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 25 μL of a 95 mM Olamine aqueous solution was added. The solvent and water were evaporated under nitrogen to dryness and 1.1658 g of the formulated test material was added. The test concentration was carefully vortexed in order to obtain a homogenous suspension.
For test field spray dilution I, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 26 μL of a 95 mM Olamine aqueous solution was added. The solvent and water were evaporated under nitrogen gas to dryness after which 970 µL of the diluted in-use formulation was added (0.7184 g of the concentrated product (~609 µL) was mixed with 1.4016 mL demineralised water). The test concentration was carefully vortexed in order to obtain a homogenous suspension.
For test field spray dilution II, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 25.2 μL of a 3.96 mM Olamine aqueous solution was added. The solvent and water were evaporated under nitrogen gas to dryness after which 975 µL of semi-blank formulated product diluted 2000 times in water was added. The test concentration was carefully vortexed in order to obtain a homogenous suspension.

APPLICATION OF DOSE: The dose preparations were applied with a positive displacement pipette tip and spread evenly on the skin surface within the donor compartment (ca 10 μL/cm²) using a disposable glass rod.

SAMPLE COLLECTION
- Mass balance samples:
> Receptor fluid samples: Collected at 0-1 hour, 1-2 hours followed by 2 hour intervals up to 24 hours after application.
> Skin wash: After 8 hours of exposure, a volume of 40 µL of mild soap solution (3 % in water at 37 °C) was applied to the skin surface and removed using a cotton swab. This was repeated four times. The skin was then washed twice with 40 µL of demineralised water and cotton swab after which the skin was dried with two dry cotton swabs. The washing efficiency was evaluated and found to be sufficient (less than 1 % of the radioactivity recovered in the first three swabs was found in the fourth). After 24 hours, the application site was washed again using the same method.
> Diffusion cell: 24 hours after exposure, the diffusion cell was dismantled and both compartments were washed twice with 1.0 mL ethanol.
> Stripping: Each skin disc was tape stripped 15 times using Stripping Discs and a D-Squame® pressure device. Tape stripping was discontinued if the epidermis ruptured.
> Digestion: After stripping, the membranes were digested in 1.5 M KOH solution with 20 % ethanol for a minimum of 24 hours.

SAMPLE PREPARATION
- Preparation details: Ultima Gold™ scintillation liquid was added to samples of the receptor fluid (10 mL per sample), the diffusion cell washes (10 mL per sample), the cotton swab extracts (10 mL to a weighed aliquot of each sample), the tape strips (4 mL per sample), and to samples of the mock dosing samples (10 mL per sample). For the determination of radioactivity in digested skin preparations, 15 mL Hionic Fluor™ scintillation liquid was added to the entire digested skin membrane.
> Dose formulations: A weighed volume of water was added to aliquots of the dose formulation taken just before and directly after dosing. Scintillation liquid (Ultima Gold™) was added to weighed subsamples for analysis.
> Receptor fluid: Samples of the receptor fluid were added directly to scintillation liquid (Ultima Gold™) for analysis.
> Skin wash: The cotton swabs were pooled per skin membrane for each time point (8 and 24 hours) and extracted with ethanol for at least 24 hours at room temperature. Aliquots of the extracted cotton swabs were added directly to scintillation liquid (Ultima Gold™) for analysis.
> Donor and receptor compartments: The samples from each compartment were separately mixed with scintillation liquid (Ultima Gold™) for analysis.
> Tape stripping: Tape strips were added directly to scintillation liquid (Ultima Gold™) for overnight extraction followed by analysis.
> Skin membranes: The entire digested membrane fractions were directly added to scintillation liquid (Hionic Fluor™) for analysis.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting. The radioactivity in the samples was determined using a Canberra Packard Tricarb 3100 TR scintillation counter.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human donors. Breast skin samples were donated after surgery. All donors were female and of a similar age
- Ethical approval if human skin: All donors provided informed consent.
- Type of skin: Human skin membranes
- Preparative technique: All subcutaneous fat was removed from the skin samples upon arrival at the laboratory. Upon thawing, the tissues were cut using a Dermatome (25 mm) and measured with a digimatic micrometer. The exposed area of each membrane was 0.64 cm²
- Thickness of skin: 300 to 400 µm
- Membrane integrity check: 36 membranes were evaluated by measuring the permeability coefficient (Kp) for tritiated water. 200 µL saline containing tritiated water (17.7 kBq/mL) was applied in the donor compartment of the flow through diffusion cells. The compartments were covered with a glass slide. Samples of the receptor fluid (approximately 1.6 mL/h) were collected for up to three hours after application. The tritiated water remaining at the application site was removed with a pipette and the skin was dried with cotton swabs. 24 membranes were selected for use in the study (8 per group - two from each of the four donors for each test group).
- Storage conditions: Tissues were stored after fat removal in aluminium foil below -18 °C until use. One donor’s tissues were stored overnight at 2 - 10 °C before subcutaneous fat was removed.

PRINCIPLES OF ASSAY
- Diffusion cell: 9 mm flow-through automated diffusion cells
- Receptor fluid: Saline (0.9 % sodium chloride w/v containing 0.01 % sodium azide) supplemented with 6 % w/v polyoxy-ethylene 20-oleyl glycol (PEG)
- Solubility of test material in receptor fluid: 87 µg/mL
- Flow-through system: The receptor fluid was pumped at a speed of ca. 1.6 mL/h.
- Test temperature: 32 ± 1 °C (skin surface temperature)
- Humidity: Ambient

Absorption in different matrices:

- Receptor fluid (in vitro test system): 0.07 ± 0.06 (concentrate), 0.10 ± 0.10 (field dilution I) and 0.46 ± 0.27 (field dilution II) as percentage of administered dose.
- Receptor chamber (in vitro test system): 0.00 ± 0.00 (concentrate), 0.01 ± 0.02 (field dilution I) and 0.05 ± 0.01 (field dilution II) as percentage of administered dose.
- Donor chamber (in vitro test system): 0.01 ± 0.01 (concentrate), 0.05 ± 0.03 (field dilution I) and 4.64 ± 6.65 (field dilution II) as percentage of administered dose.
- Skin preparation (in vitro test system): 0.04 ± 0.04 (concentrate), 0.20 ± 0.24 (field dilution I) and 0.48 ± 0.25 (field dilution II) as percentage of dose administered.
- Stratum corneum (in vitro test system): 0.012 ± 0.007 (concentrate), 0.055 ± 0.027 (field dilution I) and 0.28 ± 0.07 (field dilution II) as percentage of dose administered.

Total recovery:

- Total recovery: 95.6 ± 3.3 (concentrate), 100.1 ± 0.5 (field dilution I) and 104.9 ± 4.0 (filed dilution II) as percentage of dose administered
- Recovery of applied dose acceptable: Yes

Key result

Dose:

40.5 g a.e./L

Parameter:

percentage

Absorption:

0.12 %

Remarks on result:

other: 24 hours

Remarks:

Concentrated product (mean absorbed dose)

Key result

Dose:

12.6 g a.e./L

Parameter:

percentage

Absorption:

0.31 %

Remarks on result:

other: 24 hours

Remarks:

Field dilution I (mean absorbed dose)

Key result

Dose:

0.02 g a.e./L

Parameter:

percentage

Absorption:

0.99 %

Remarks on result:

other: 24 hours

Remarks:

Field dilution II (mean absorbed dose)

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (40.5 g a.e./L) was 0.29 µg/cm² (0.07 % of the applied dose) and the mean maximal flux was 0.035 µg/cm²/h. The lag time was 0.8 hours.

For field dilution I (12.6 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.12 µg/cm² (0.10 % of the applied dose) and the mean maximal flux was 0.006 µg/cm²/h. The lag time was 1.3 hours.

For field dilution II (0.02 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.0009 µg/cm² (0.48 % of the applied dose) and the mean maximal flux was 0.0001 µg/cm²/h. The lag time was 0.1 hour.

The mean absorbed dose was determined to be 0.12, 0.31 and 0.99 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

The mean potentially absorbed dose was determined to be 0.13, 0.36 and 1.23 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

Table 1: Results

	Concentrate	Field Dilution I	Field Dilution II
Total concentration (g a.e./L)	40.5	12.6	0.02
Dose (µg/cm²)	402 ± 7	125 ± 2	0.20 ± 0.00
Penetration into the receptor fluid after 24 hours (µg/cm²)	0.29	0.12	0.0009
Penetration into the receptor fluid after 24 hours (% of dose)	0.07	0.10	0.48
Maximal flux (µg/cm²/h)	0.035	0.006	0.0001
Lag time (h)	0.8	1.3	0.1
Recovery of dose (% ± SD) Receptor fluid	0.07 ± 0.06	0.10 ± 0.10	0.46 ± 0.27
Recovery of dose (% ± SD) Receptor compartment wash	0.00 ± 0.00	0.01 ± 0.02	0.05 ± 0.01
Recovery of dose (% ± SD) Skin*	0.04 ± 0.04	0.20 ± 0.24	0.48 ± 0.25
Absorbed dose (% ± SD)**	0.12 ± 0.09	0.31 ± 0.36	0.99 ± 0.44
Tape strips (1 + 2) (% ± SD)	0.002 ± 0.002	0.008 ± 0.003	0.04 ± 0.03
Tape strips (3 - last) (% ± SD)	0.010 ± 0.005	0.047 ± 0.027	0.24 ± 0.05
Stratum corneum (% ± SD)	0.012 ± 0.007	0.055 ± 0.027	0.28 ± 0.07
Potentially absorbed dose (% ± SD)***	0.13 ± 0.09	0.36 ± 0.36	1.23 ± 0.40
Skin wash t = 8 h (% ± SD)	95.3 ± 3.3	99.5 ± 0.9	98.0 ± 4.8
Skin wash t = 24 h (% ± SD)	0.10 ± 0.08	0.19 ± 0.11	0.31 ± 0.22
Donor compartment wash (% ± SD)	0.01 ± 0.01	0.05 ± 0.03	4.64 ± 6.65
Total recovery (% ± SD)	95.6 ± 3.3	100.1 ± 0.5	104.9 ± 4.0

* Epidermis + dermis (without stratum corneum)

** The amount in the receptor fluid plus the receptor compartment wash, plus the skin membrane (excluding tape strips, i.e. stratum corneum)

*** The amount in the receptor fluid plus the receptor compartment wash plus the skin membrane (except for the first 2 tape strips)

Conclusions:

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated products) was found to be poorly absorbed through the skin.

Executive summary:

The dermal absorption of the test material was determined in a study conducted in accordance with the OECD guideline 428 and under GLP conditions.

The test material was assessed as the concentrated formulated product (40.5 g a.e./L) and as in use field dilutions (12.6 and 0.02 g a.e./L) using human split-thickness skin in flow through diffusion cells. The contact time was 8 hours and the post-exposure time was 16 hours.

In addition to the amount of [14C]test material in the receptor fluid, the residues remaining in/on the skin membranes and in the stratum corneum (at 24 hours) were also determined. Human skin membranes were prepared from four separate donors in duplicate (n = 8) per test concentration tested.

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (40.5 g a.e./L) was 0.29 µg/cm² (0.07 % of the applied dose) and the mean maximal flux was 0.035 µg/cm²/h. The lag time was 0.8 hours. For field dilution I (12.6 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.12 µg/cm² (0.10 % of the applied dose) and the mean maximal flux was 0.006 µg/cm²/h. The lag time was 1.3 hours. For field dilution II (0.02 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.0009 µg/cm² (0.48 % of the applied dose) and the mean maximal flux was 0.0001 µg/cm²/h. The lag time was 0.1 hour.

The mean absorbed dose was determined to be 0.12, 0.31 and 0.99 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

The mean potentially absorbed dose was determined to be 0.13, 0.36 and 1.23 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated products) was found to be poorly absorbed through the skin.

Reference 6

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

10 September 2012 to 19 September 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study was conducted on the formulated product; however, the results are expressed in terms of percentage of the acid equivalent of the active ingredient, applied as the triisopropanolamine salt.

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GF-1883, which was tested at two target concentrations: 12 g a.e.L-1 (concentrate) and 0.08 g.a.e.L-1 (field spray dilution). The dermal absorption of the active substance, aminopyralid (acid equivalent), was measured for each of these two test concentrations.

Radiolabelling:

yes

Remarks:

[14C]

Species:

other: human split-thickness skin

Vehicle:

other: Formulated - equivalent to in-use product.

Duration of exposure:

8 hours of exposure (16 hours of post exposure time)

Doses:

- Nominal doses: 12.5 g a.e./L (concentrate) and 0.08 g a.e./L (field spray dilution)
- Actual doses: 128 ± 1 µg/cm² and 0.80 ± 0.02 µg/cm²
- Dose volume: 10 µL/cm², resulting in approximately 6.4 µL applied over the surface (accounting for expected loss during distribution of the skin samples).
- Rationale for dose selection: 12.5 g a.e./L represents the maximum concentration users would be exposed to handling the concentrated product, field spray dilution represents the minimum in-use concentration.

No. of animals per group:

Two skin membranes from each of 4 donors were exposed in each test group (8 skin membranes per dose level)

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions:
For the concentrate application, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 26.5 μL of a 95 mM triisopropanolamine (TIPA) aqueous solution was added. The solvent and water were evaporated under nitrogen to dryness and 1.0809 g of the formulated test material was added. The test concentration was carefully vortexed in order to obtain a homogenous suspension.
For the test field spray dilution, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 25.6 μL of a 15.8 mM TIPA aqueous solution was added. The solvent and water were evaporated under nitrogen gas to dryness after which 1 mL of the diluted in-use formulation was added (the formulated test material was diluted 150 times with water). The test concentration was carefully vortexed in order to obtain a homogenous suspension.

APPLICATION OF DOSE: The dose preparations were applied with a positive displacement pipette tip and spread evenly on the skin surface within the donor compartment (ca 10 μL/cm²) using a disposable glass rod.

SAMPLE COLLECTION
- Mass balance samples:
> Receptor fluid samples: Collected at 0-1 hour, 1-2 hours followed by 2 hour intervals up to 24 hours after application.
> Skin wash: After 8 hours of exposure, a volume of 40 µL of mild soap solution (3 % in water at 37 °C) was applied to the skin surface and removed using a cotton swab. This was repeated four times. The skin was then washed twice with 40 µL of demineralised water and cotton swab after which the skin was dried with two dry cotton swabs. The washing efficiency was evaluated and found to be sufficient (less than 5 % of the radioactivity recovered in the first three swabs was found in the fourth). After 24 hours, the application site was washed again using the same method.
> Diffusion cell: 24 hours after exposure, the diffusion cell was dismantled and both compartments were washed twice with 1.0 mL ethanol.
> Stripping: Each skin disc was tape stripped 15 times using Stripping Discs and a D-Squame® pressure device. Tape stripping was discontinued if the epidermis ruptured.

SAMPLE PREPARATION
- Preparation details: Ultima Gold™ scintillation liquid was added to samples of the receptor fluid (10 mL per sample), the diffusion cell washes (10 mL per sample), the cotton swab extracts (10 mL to a weighed aliquot of each sample), the tape strips (4 mL per sample), and to samples of the mock dosing samples (10 mL per sample). For the determination of radioactivity in digested skin preparations, 15 mL Hionic Fluor™ scintillation liquid was added to the entire digested skin membrane.
> Dose formulations: A weighed volume of water was added to aliquots of the dose formulation taken just before and directly after dosing. Scintillation liquid (Ultima Gold™) was added to weighed subsamples for analysis.
> Receptor fluid: Samples of the receptor fluid were added directly to scintillation liquid (Ultima Gold™) for analysis.
> Skin wash: The cotton swabs were pooled per skin membrane for each time point (8 and 24 hours) and extracted with ethanol for at least 24 hours at room temperature. Aliquots of the extracted cotton swabs were added directly to scintillation liquid (Ultima Gold™) for analysis.
> Donor and receptor compartments: The samples from each compartment were separately mixed with scintillation liquid (Ultima Gold™) for analysis.
> Tape stripping: Tape strips were added directly to scintillation liquid (Ultima Gold™) for overnight extraction followed by analysis.
> Skin membranes: The entire digested membrane fractions were directly added to scintillation liquid (Hionic Fluor™) for analysis.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting. The radioactivity in the samples was determined using a Canberra Packard Tricarb 3100 TR scintillation counter.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human donors. Breast skin samples were donated after surgery. All donors were female and of a similar age
- Ethical approval if human skin: All donors provided informed consent.
- Type of skin: Human skin membranes
- Preparative technique: All subcutaneous fat was removed from the skin samples upon arrival at the laboratory. Upon thawing, the tissues were cut using a Dermatome (25 mm) and measured with a digimatic micrometer. The exposed area of each membrane was 0.64 cm²
- Thickness of skin: 300 to 400 µm
- Membrane integrity check: 32 membranes were evaluated by measuring the permeability coefficient (Kp) for tritiated water. 200 µL saline containing tritiated water (20.1 kBq/mL) was applied in the donor compartment of the flow through diffusion cells. The compartments were covered with a glass slide. Samples of the receptor fluid (approximately 1.6 mL/h) were collected for up to three hours after application. The tritiated water remaining at the application site was removed with a pipette and the skin was dried with cotton swabs. 16 membranes were selected for use in the study (8 per group - two from each of the four donors for each test group).
- Storage conditions: Tissues were stored after fat removal in aluminium foil below -18 °C until use.

PRINCIPLES OF ASSAY
- Diffusion cell: 9 mm flow-through automated diffusion cells
- Receptor fluid: Saline (0.9 % sodium chloride w/v containing 0.01 % sodium azide) supplemented with 6 % w/v polyoxy-ethylene 20-oleyl glycol (PEG)
- Solubility of test material in receptor fluid: 87 µg/mL
- Flow-through system: The receptor fluid was pumped at a speed of ca. 1.6 mL/h.
- Test temperature: 32 ± 1 °C (skin surface temperature)
- Humidity: Ambient

Absorption in different matrices:

- Receptor fluid (in vitro test system): 0.07 ± 0.02 (concentrate) and 0.38 ± 0.20 (field dilution) as percentage of administered dose.
- Receptor chamber (in vitro test system): 0.00 ± 0.00 (Concentrate) and 0.01 ± 0.00 (field dilution) as percentage of administered dose.
- Donor chamber (in vitro test system): 0.08 ± 0.09 (concentrate) to 0.75 ± 1.31 (field dilution) as percentage of administered dose.
- Skin preparation (in vitro test system): 0.61 ± 0.21 (concentrate) and 1.22 ± 0.80 (field dilution) as percentage of dose administered.
- Stratum corneum (in vitro test system): 0.43 ± 0.42 (concentrate) and 0.98 ± 0.82 (field dilution) as percentage of dose administered.

Total recovery:

- Total recovery: 100.0 ± 1.4 (concentrate) and 99.4 ± 2.0 (field dilution) as percentage of dose administered
- Recovery of applied dose acceptable: Yes

Key result

Dose:

12.5 g a.e./L

Parameter:

percentage

Absorption:

0.7 %

Remarks on result:

other: 24 hours

Remarks:

Concentrated product (mean absorbed dose)

Key result

Dose:

0.08 g a.e./L

Parameter:

percentage

Absorption:

1.6 %

Remarks on result:

other: 24

Remarks:

Field dilution (mean absorbed dose)

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (12.5 g a.e./L) was 0.08 µg/cm² (0.07 % of the applied dose) and the mean maximal flux was 0.005 µg/cm²/h. The lag time was 0.0 hours.

For the field dilution (0.02 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.003 µg/cm² (0.38 % of the applied dose) and the mean maximal flux was 0.0004 µg/cm²/h. The lag time was 0.5 hours.

The mean absorbed dose was determined to be 0.7 and 1.6 % of the active substance in the concentrated product and field dilution, respectively.

The mean potentially absorbed dose was determined to be 1.0 and 2.5 % of the active substance in the concentrated product and field dilution, respectively.

Table 1: Results

	Concentrate*	Field Dilution
Total concentration (g a.e./L)	12.5	0.08
Dose (µg/cm²)	128 ± 1	0.80 ± 0.02
Penetration into the receptor fluid after 24 hours (µg/cm²)	0.08	0.003
Penetration into the receptor fluid after 24 hours (% of dose)	0.07	0.38
Maximal flux (µg/cm²/h)	0.005	0.0004
Lag time (h)	0.0	0.5
Recovery of dose (% ± SD) Receptor fluid	0.07 ± 0.02	0.38 ± 0.20
Recovery of dose (% ± SD) Receptor compartment wash	0.00 ± 0.00	0.01 ± 0.00
Recovery of dose (% ± SD) Skin**	0.61 ± 0.21	1.22 ± 0.80
Absorbed dose (% ± SD)***	0.7 ± 0.2	1.6 ± 1.0
Tape strips (1 + 2) (% ± SD)	0.06 ± 0.07	0.12 ± 0.11
Tape strips (3 - last) (% ± SD)	0.37 ± 0.35	0.86 ± 0.72
Stratum corneum (% ± SD)	0.43 ± 0.42	0.98 ± 0.82
Potentially absorbed dose (% ± SD)****	1.0 ± 0.5	2.5 ± 1.6
Skin wash t = 8 h (% ± SD)	98.20 ± 0.97	94.12 ± 2.35
Skin wash t = 24 h (% ± SD)	0.60 ± 0.33	1.90 ± 1.72
Donor compartment wash (% ± SD)	0.08 ± 0.09	0.75 ± 1.31
Total recovery (% ± SD)	100.0 ± 1.4	99.4 ± 2.0

* The results from one replicate were not included due to a high penetration of the test material in the receptor, assumed to be due to the fact that the skin was damaged during application.

** Epidermis + dermis (without stratum corneum)

*** The amount in the receptor fluid plus the receptor compartment wash, plus the skin membrane (excluding tape strips, i.e. stratum corneum)

**** The amount in the receptor fluid plus the receptor compartment wash plus the skin membrane (except for the first 2 tape strips)

Conclusions:

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated products) was found to be poorly absorbed through the skin.

Executive summary:

The dermal absorption of the test material was determined in a study conducted in accordance with the OECD guideline 428 and under GLP conditions.

The test material was assessed as the concentrated formulated product (12.5 g a.e./L) and as an in use field dilution (0.08 g a.e./L) using human split-thickness skin in flow through diffusion cells. The contact time was 8 hours and the post-exposure time was 16 hours.

In addition to the amount of [14C]test material in the receptor fluid, the residues remaining in/on the skin membranes and in the stratum corneum (at 24 hours) were also determined. Human skin membranes were prepared from four separate donors in duplicate (n = 8) per test concentration tested.

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (12.5 g a.e./L) was 0.08 µg/cm² (0.07 % of the applied dose) and the mean maximal flux was 0.005 µg/cm²/h. The lag time was 0.0 hours. For the field dilution (0.02 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.003 µg/cm² (0.38 % of the applied dose) and the mean maximal flux was 0.0004 µg/cm²/h. The lag time was 0.5 hours.

The mean absorbed dose was determined to be 0.7 and 1.6 % of the active substance in the concentrated product and field dilution, respectively.

The mean potentially absorbed dose was determined to be 1.0 and 2.5 % of the active substance in the concentrated product and field dilution, respectively.

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated products) was found to be poorly absorbed through the skin.

Description of key information

Available data show that following oral administration the substance is readily absorbed and quickly excreted, with a half-life of few hours. No differences between genders, single low and high doses, and repeated low doses were observed in rats, while an increased bioavailability was noted in pregnant rabbits compared to non-pregnant animals. In both urine and faeces the substance is excreted unchanged.  Given the relatively low MW, it was determined that less than 5 % of the dose in faeces represents absorbed material.  Based on urinary excretion, the absorption rate is estimated to be at least 45 %. 
Based on results of three in vitro dermal absorption studies with human skin, which tested the substance formulated as SL formulations, a dermal absorption rate of 1 % in humans is set for risk assessment purposes. The most precautionary approach for estimating dermal absorption has been applied (i.e. potentially absorbable dose for a diluted concentration).   
In the absence of quantitative data, inhalation absorption is set at 100 % for risk assessment purposes.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

45

Absorption rate - dermal (%):

1

Absorption rate - inhalation (%):

100

Additional information

Three studies investigating the toxicokinetics of the test material are available. In addition, three in vitro dermal absorption studies using human skin and testing the test material formulated as SL formulations are also available. The use of studies with the formulated product is considered appropriate to determine a dermal absorption rate for the substance because they include different dilutions and are potentially worst case (given the presence of potential penetration enhancers e.g. surfactants). Each study is summarised in turn below. As only the concentrations corresponding to the commercial formulation and an additional concentration at a low dilution are considered useful to determine the rate of dermal absorption of the test substance, the summaries for these studies present results obtained at these concentrations only.

 

Liu (2004)

The absorption, distribution, metabolism and excretion of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.7485, OECD 417 and B.36.

During the study, three groups of four male Fischer 344 rats were given a single oral dose of either 50 or 1000 mg [¹⁴C]test material, or a single oral dose of 50 mg [¹²C]test material /kg for 14 days followed by a single oral dose of 50 mg/kg [¹⁴C]test material.

[¹⁴C]test material was readily absorbed and efficiently cleared through the urine (average T1/2α of 3-4 hour for all groups) and faeces. The absorption and excretion patterns were similar among the three groups. An average of 74.04 - 93.13 % of the administered radioactivity was excreted during the first 24-hours post-dose administration. Urinary and faecal elimination totalled 41 - 59 and 33 – 43 % of the administered dose, respectively. Less than 5 % of the administered dose recovered in faeces was estimated to represent absorbed test material, getting into faeces via the bile.

Selected composite urine and faeces were analysed by HPLC. Both metabolic profiles of all groups were similar. The major peak in both matrixes was identified by mass spectrometry as parent (test) material, and accounted for 96 - 98 and 100 % of the radioactivity in the urine and faeces, respectively.

 

Domoradzki et al. (2004)

This study was conducted to assess and compare the absorption, distribution, metabolism, and elimination of [2,6 -14C-ring labelled]test material and [2,6-14C-ring labelled]test material-triisopropanolamine (TIPA) following administration of single oral doses of those compounds.

During the study, four male Fischer 344 rats were given a single oral dose of a solution that delivered a target dose of 50 mg 14C-test material/kg of body weight. A separate group of four male Fischer 344 rats were dosed with an equimolar amount of 14C-test material-TIPA at a target dose of 96 mg 14C-test material-TIPA/kg of body weight.

Both compounds were rapidly absorbed; the highest plasma concentrations of radioactivity for both compounds (observed Cmax) occurred in the first sample taken at 0.25 hours post-dosing and were 26 and 16 µg equivalent test material for the 14C-test material and 14C-test material-TIPA dose groups, respectively.

Pharmacokinetic parameters from the plasma time curves yielded the following values for 14C-test material and 14C-test material-TIPA, respectively: plasma AUCs were 23.0 and 19.0 µg eq-hour/g of plasma; half-lives from the a phase of plasma elimination were 0.338 and 0.509 hours; and half-lives from the β phase of plasma elimination were 8.8 and 13.0 hours. The excretion of 38.3% (for 14C-test material) and 34.6 % (for 14C-test material-TIPA) of administered radioactivity in urine within six hours of dosing confirms that the amino-dichloro-picolinate portion of the molecule was rapidly absorbed whether administered as test material acid or the TIPA salt of the test material. Based on the radioactivity recovered in urine through 120 hours, a minimum of 46.3 and 42.5 % of the orally administered 14C-test material and 14C-test material-TIPA was absorbed. The radioactivity associated with the two compounds was rapidly eliminated with 93.5% (44.7% in urine; 48.8% in faeces), and 93.3 % (41.5 % in urine; 51.8 % in faeces) of the administered doses of 14C-test material and 14C-test material-TIPA, respectively, recovered in excreta within 24 hours post-dosing. Urinary elimination half-lives of the α phase were 2.8 hours and 2.5 hours for the 14C-test material and 14C-test material-TIPA dosed groups, respectively. Urinary elimination half-lives of the β phase were 7.8 hours and 10.7 hours for the 14C-test material and 14C-test material-TIPA dosed groups, respectively. The amino-dichloropicolinate portion of both molecules was excreted essentially unchanged in urine and faeces. Except for a minor radioactive peak detected in the 0 to 6 hour pooled urine sample from the 14C-test material-TIPA dosed group (representing 0.34 % of administered dose), the only radiolabelled peak detected in analyses of urine and faecal extracts was confirmed as parent test material. The results from this study indicate that 14C-test material and 14C-test material-TIPA, when administered orally to rats, are bioequivalent in terms of absorption, distribution, metabolism, and excretion of the amino-dichloro-picolinate portion of the molecule(s).

 

Hansen et al. (2005)

The absorption, distribution, metabolism, and elimination of 14C-test material orally administered to three groups of three female New Zealand White rabbits were determined. Non-pregnant rabbits (Group 1) and pregnant rabbits at gestation day 7 (GD 7; Group 2) were administered a single oral dose of 371 and 362 mg 14C-test material/kg bw, respectively. The repeated dose group (Group 3) were given daily doses of 279 mg test material/kg bw from GD 7 - 21, followed by 279 mg 14C-test material/kg bw on GD 22.

For all groups, the test material was quickly absorbed with peak plasma and red blood cell maximal concentrations (Cmax) both within ~1 hour of dosing. The plasma elimination rate was also rapid, with half- lives ranging from 4 to 7 hours for all three dose groups. The Cmax and AUC for the group 3 animals (repeated doses at 279 mg/kg) were slightly higher than the other two groups, administered 362 - 371 mg/kg.

At 72 hours post-dosing, the GI tract was the only tissue with detectable amounts of radioactivity for all study animals and ranged from 3.1 to 4.6 μg-eq./g tissue. This represents 0.11 - 0.15 % of the administered dose. The spleen from two of the three animals from the repeated dose group had detectable amounts of radioactivity for an overall mean of 2.0 ± 0.9 μg-eq./g tissue (<0.005 % of the administered dose).

Orally administered test material was not metabolised by rabbits. There was one radioactive peak detected in excreta (all groups) and was positively identified as parent test material.

Urine was the principal route of elimination with 77, 83, and 86 % of the administered dose eliminated 72 hours post-dosing, for Groups 1, 2, and 3, respectively. By 12 hours post-dosing 39, 48, and 61 % of the administered dose was eliminated in the urine, respectively, with the vast majority (93 – 97 %) of the urinary total being eliminated by 24 hours post dosing. Faeces accounted for 20, 16, and 8 % of the administered dose for Groups 1, 2, and 3 at 72 hours post-dosing, with the vast majority (95 – 97 %) being eliminated in the first 24 hours. These data are consistent with the test material being more systemically available to the gestation day 22 animals. Urine and faeces accounted for >99 % of the recovered radioactivity for all three groups.

The repeated dose animals (gestation days 7 - 22) were administered 14C-test material dose ~75 % of the dose that the non-pregnant and gestation day 7 animals received. However, they had a slightly higher plasma/red blood cell Cmax, and ~25 % higher area under the curve (AUC) than the other two groups, consistent with higher bioavailability of the test material to the gestation day 22 animals. Correcting for the size of the 14C-dose administered by dividing the AUC by the dose, the relative bioavailability of the test material following the repeated dose regime was 75 – 115 % higher than the other single oral dosed groups. In vitro plasma protein binding of the test material demonstrated relative amounts of protein-bound test material decreased in order of non-pregnant rats and rabbits >GD 7 > GD 22.

No significant differences were observed between the non-pregnant and GD 7 rabbits in the absorption or the routes or rates of elimination of the test material. The test material had a greater bioavailability to the gestation day 22 animals, as shown by increased plasma levels and higher percent of urinary elimination. Some differences in plasma protein binding were observed between groups. In conclusion, with all groups the test material was rapidly absorbed following oral administration to rabbits and rapidly eliminated unchanged in the urine.

Reus & Maas (2013)

The dermal absorption of the acid equivalent of the test material (formulated in GF-1601 and dosed as the potassium salt of the registered substance) was determined in a study conducted in accordance with the OECD guideline 428 and under GLP conditions.

The test material was assessed as the concentrated formulated product (31.5 g a.e./L) and as in use field dilutions (13 and 0.02 g a.e./L) using human split-thickness skin in flow through diffusion cells. The contact time was 8 hours and the post-exposure time was 16 hours.

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (31.5 g a.e./L) was 0.65 µg/cm² (0.21 % of the applied dose) and the mean maximal flux was 0.13 µg/cm²/h. The mean absorbed dose was determined to be 0.41 % of the active substance in the concentrated product. The mean potentially absorbed dose was determined to be 0.55 % of the active substance in the concentrated product.

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated product) was found to be poorly absorbed through the skin.

 

Reus & Maas (2013)

The dermal absorption of the acid equivalent of the test material (formulated in GF-1633 and dosed as the olamine salt) was determined in a study conducted in accordance with the OECD guideline 428 and under GLP conditions.

The test material was assessed as the concentrated formulated product (40.5 g a.e./L) and as in use field dilutions (12.6 and 0.02 g a.e./L) using human split-thickness skin in flow through diffusion cells. The contact time was 8 hours and the post-exposure time was 16 hours.

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (40.5 g a.e./L) was 0.29 µg/cm² (0.07 % of the applied dose) and the mean maximal flux was 0.035 µg/cm²/h. The mean absorbed dose was determined to be 0.12 % of the active substance in the concentrated product. The mean potentially absorbed dose was determined to be 0.13 % of the active substance in the concentrated product.

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated product) was found to be poorly absorbed through the skin.

 

Reus & Maas (2013)

The dermal absorption of the acid equivalent of the test material (formulate din GF-1883 and dosed as the triisopropanolamine salt) was determined in a study conducted in accordance with the OECD guideline 428 and under GLP conditions.

The test material was assessed as the concentrated formulated product (12.5 g a.e./L) and as an in use field dilution (0.08 g a.e./L) using human split-thickness skin in flow through diffusion cells. The contact time was 8 hours and the post-exposure time was 16 hours.

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (12.5 g a.e./L) was 0.08 µg/cm² (0.07 % of the applied dose) and the mean maximal flux was 0.005 µg/cm²/h. The mean absorbed dose was determined to be 0.70 % of the active substance in the concentrated product. The mean potentially absorbed dose was determined to be 1.0 % of the active substance in the concentrated product.

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated product) was found to be poorly absorbed through the skin.

 

Available data show that following oral administration the substance is readily absorbed and quickly excreted, with a half-life of a few hours. No differences between genders, single low and high doses, and repeated low doses were observed in rats, while an increased bioavailability was noted in pregnant rabbits compared to non-pregnant animals. In both urine and faeces the substance is excreted unchanged. Given the relatively low MW, it was determined that less than 5 % of the dose in faeces represents absorbed material. Based on urinary excretion, the oral absorption rate is estimated to be at least 45 %.

Available in vitro dermal absorption studies, performed with SL formulations, containing up to (nominal) 40 g/L of the test substance and using human skin, showed that dilution has little effect on the penetration of the test substance through the skin. The directly absorbed dose (i.e. the amount recovered in the receptor fluid) was 0.07 -0.21 % for the commercial formulation concentrate at 31.5 -40 g/L, and 0.07, 0.10 or 0.22 % for a dilution at 12.5 -13 g/L. The absorbed dose (i.e. sum of receptor fluid + receptor compartment wash + skin remaining after tape stripping) was 0.12 -0.41 % for the concentrate, and 0.24, 0.31 or 0.7 % for the dilution. The potentially absorbed dose, inclusive of the amount recovered from the stratum corneum excluding only the first two strips accounted for 0.13 -0.55 % for the concentrate, and to 0.36, 0.55 or 1 % for the dilution. Based on these results and using the most precautionary approach, a dermal absorption rate of 1 % in humans is set for risk assessment purposes. This value is consistent with results obtained in the available repeat-dose dermal toxicity study in rats, where no systemic effects were observed at the limit dose of 1000 mg/kg/day thus indicating minimal, if any, dermal absorption, even through the more permeable rat skin.

 

In the absence of quantitative data, inhalation absorption is set at 100 % for risk assessment purposes.