Registration Dossier
Registration Dossier
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EC number: 240-539-0 | CAS number: 16484-77-8
Endpoint summary
Administrative data
Link to relevant study record(s)
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 14 January 2003 to 10 February 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Objective of study:
- absorption
- excretion
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 417 (Toxicokinetics)
- Version / remarks:
- 1984
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability of the radiolabelled test material in the vehicle (0.5 % aqueous Carboxymethylcellulose supplemented with 1 % Cremophor) was confirmed in all experiments. The stability of non-radiolabelled test material in feed at ambient temperature for at least 14 days was demonstrated previously. The homogeneity and achieved concentrations were verified analytically in all experiments. - Radiolabelling:
- yes
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- CrlGlxBrlHan:WI
- Details on species / strain selection:
- Recognised by international guidelines as the recommended test system. Study results will be used in relation to already available data from the same test system.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: About 7 weeks at the day of first administration of unlabelled material.
- Housing: During acclimatisation and prior to the experiment in Macrolon Cages Type Ill (2 animals per cage); during pre-treatment and plasma kinetics experiments in steel wire mesh cages (1 animal per cage).
- Diet: Ad libitum prior to and during experiments.
- Water: Tap water ad libitum.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 % relative humidity
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.).
- Route of administration:
- other: Oral gavage and diet
- Vehicle:
- other: diet (diet administration), 0.5 % Carboxymethylcellulose/ 1 % Cremophor in double-distilled water (gavage administration)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Material for oral administration via gavage: In order to achieve the required concentration and specific activity, the respective amount of non-radiolabelled test material was added to the respective amount of the stock solution of radiolabelled test material in acetone and the organic solvent was evaporated to dryness, 0.5 % Carboxymethylcellulose/ 1 % Cremophor in double-distilled water was added and filled up to the final volume. Prior to administration the preparation was sonicated and stirred to produce a homogeneous suspension. Samples were taken to check the amount of radioactivity in the preparation and to demonstrate the stability, homogeneity and the achieved concentration of the test material in the preparation.
- About 1 mL of the test material preparation per 100 g bw was administered to rats by gavage.
DIET PREPARATION
- Rate of preparation of diet: Material for oral administration via feed:
For each concentration, the test material was weighed out and thoroughly mixed with a small amount of food in a beaker. Subsequently a premix was prepared in a household mixer by adding an appropriate amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a laboratory mixer.
On day 15, mixed feed was substituted by control feed at 6:00 a.m. for 24 hours in order to avoid interference of unlabelled test material in feed with the radiolabelled test material given via gavage. - Duration and frequency of treatment / exposure:
- 14 consecutive days plus gavage on Day 15.
- Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Remarks:
- Experiment 1
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- Remarks:
- Experiment 2
- Dose / conc.:
- 40 mg/kg bw/day (nominal)
- Remarks:
- Experiment 3
- Dose / conc.:
- 80 mg/kg bw/day (nominal)
- Remarks:
- Experiment 4
- Dose / conc.:
- 120 mg/kg bw/day (nominal)
- Remarks:
- Experiment 5
- Dose / conc.:
- 180 mg/kg bw/day (nominal)
- Remarks:
- Experiment 6
- No. of animals per sex per dose / concentration:
- 4 males per experiment
- Control animals:
- no
- Details on study design:
- - Dose selection rationale: In order to clarify whether changes in toxicokinetic parameters occurred in the intended dose range which was selected using the results from previously performed studies of the subacute, sub-chronic and chronic toxicity of the test material, plasma kinetics were investigated after oral administration at the following six dose levels:
Dose 1: 10 mg/kg bw radiolabeled gavage dose, corresponding to 125 ppm in feed for feeding period.
Dose 2: 20 mg/kg bw radiolabeled gavage dose, corresponding to 250 ppm in feed for feeding period.
Dose 3: 40 mg/kg bw radiolabeled gavage dose, corresponding to 500 ppm in feed for feeding period.
Dose 4: 80 mg/kg bw radiolabeled gavage dose, corresponding to 1 000 ppm in feed for feeding period.
Dose 5: 120 mg/kg bw radiolabeled gavage dose, corresponding to 1 500 ppm in feed for feeding period.
Dose 6: 180 mg/kg bw radiolabeled gavage dose, corresponding to 2 200 ppm in feed for feeding period.
The dietary concentrations in the present study were designed to reflect those which achieve a similar mean daily intake in mg/kg bw (i.e. 10, 20... 180 mg/kg bw/day) in a sub-chronic toxicity study. Thus, the achieved dose was expected to exceed target for the early phase of this 14 day study but approach it more closely towards the end. The single dose of radiolabelled material was at the specific level. - Details on dosing and sampling:
- Blood/Plasma level
- Animals were treated with non-radiolabelled test material via feed for 14 days. On day 15, mixed feed with the test material was substituted by control feed at 6:00 a.m. for 24 hours and animals received a single administration of 14C-labelled test material by gavage at 9.00 am. Blood samples (100 - 200 μL) were taken from the retroorbital sinus at the following times after administration: 1; 2; 4; 6; 8; 12; 24; 48 hours.
- Plasma samples were checked for total radioactivity.
- In all groups body weight and feed consumption were determined weekly. Feed consumption was calculated using the mean body weight of the respective feeding period. Clinical signs were recorded daily at workdays during acclimatisation and application period.
TOXICOKINETIC / PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: Blood samples
- Time and frequency of sampling: Taken from the retroorbital sinus at the following times after administration: 1; 2; 4; 6; 8; 12; 24; 48 hours.
HPLC ANALYSIS OF TEST SUBSTANCE PREPARATIONS
Feed mixtures:
Feed samples were preincubated with double-distilled water and than extracted twice with acetonitrile. Aliquots of these extracts were used for HPLC analysis. The homogeneity and achieved concentration of the test material in feed was checked via HPLC under the following conditions:
Column: Nucleosil 100, 5 C18, 250 x 4 mm
Eluent:
60 % Acetonitrile + 0.5 M Sulfuric acid (1 000 mL + 5 mL)
40 % Double-distilled water+ 0.5 M Sulfuric acid (1 000 mL+ 5 mL)
Flow: 1 mL/min
Detection: UV-Extinction at 230 nm
Radioactive Preparations
The homogeneity and achieved concentration of the test material in 0.5 % Carboxymethylcellulose in double-distilled water as well as the radiochemical purity of 14C-test material were checked via HPLC (HP 1050 series) under the following conditions:
Column: Nucleosil 120, 7C18, 250 x 4 mm
Eluent: Acetonitrile / double-distilled water / 0.5 M Sulfuric acid (500/500/5 (v/v/v))
Flow: 1.2 mL/min
Detection: UV-Extinction at 230 nm HPLC Radioactivity Monitor LB 509 (Cell: VG 150 U4D)
Preparation of samples and measurement of radioactivity
Aliquots of plasma samples were mixed with scintillation cocktail (Hionic-Fluor, Packard) and counted for 10 minutes in a liquid scintillation counter (LSC; Wallac type 1409) and the disintegration rate corrected by the respective background. - Statistics:
- Analysis of kinetic data was performed based on the group mean values using the PC program system TOPFIT Version 2.0 (3).
- Details on absorption:
- After radioactive dosing at the dose levels of 10 and 20 mg/kg bw on day 15, plasma levels reached a maximum plateau after the first hour. The time point of maximum plasma concentration (T max) increased with increasing dose; i.e. maximum plasma concentrations were observed at 4 h, 2 h, 6 h, and 6 h after dosing at 40, 80, 120, and 180 mg/kg bw, respectively. The shift of T max with dose and the under-proportional increase of Cmax at 120 and 180 mg/kg bw might be due to a delay of gastrointestinal absorption.
After having reached peak levels, plasma concentrations declined continuously. Irrespective of the dose level, plasma concentration time curves showed three different phases: Phase 1 can be characterised as a mixed absorption/excretion phase and ranges - depending on the dose - from 0 to 8 - 12 h after dosing, whereas phase 2 represents a pure excretory phase (about 12 - 24 h). Phase 3 is a mixed absorption/excretion phase, since animals are dosed with non-radiolabelled test material via feed, again (24 hours and beyond).
The AUC0∞ increased with increasing dose. The increase in the AUC0∞ was not linear with dose. A slight over-proportional increase of the AUC0∞ was already detectable at 40 mg/kg bw being more pronounced at 80 mg/kg bw. Whereas the dose increased 4- and 8-fold relative to the low dose, the AUC0∞ increased by factors of 4.5 and 11.3. At 120 and 180 mg/kg bw, the over-proportional increase in the AUC0∞ was even much more pronounced. Whereas the overall increase in dose from the lowest to the two highest dose levels was 12- and 18-fold, the respective AUC0∞ increased by factors of 18 and 23. When comparing the AUC during the main excretory phase (AUC12-24) which is a more sensitive parameter for the excretory capacity, the over-proportional increase in AUC12-24 at 40 mg/kg bw and above, and thus, saturation of excretion, becomes even more evident. These data clearly demonstrated saturation of excretion, indicated by the over-proportional increase of the AUC with increasing dose, which already occurred at test material doses of 40 mg/kg bw. This was also reflected in the plasma concentration/time curves.
When considering half-lives during the main excretory phase (phase 2), values remained more or less unchanged at doses of 10 and 20 mg/kg bw. At doses of 40 mg/kg bw and above, half-lives continuously increased with increasing dose which is also reflected in a change of the time course of plasma concentrations. Although being a rather insensitive indicator for excretory saturation phenomena, data of initial half-lives in plasma further supported the conclusions drawn from the AUG-data.
Another parameter of interest is the plasma concentration 24 hours after dosing which can be interpreted to represent carryover of systemically available test substance to the subsequent application day.
The data clearly show that up to 20 mg/kg bw, carryover increased virtually linearly with dose. At 40 mg/kg bw, a clearly over-proportional plasma concentration was measured indicating that this dose level approximates to the point at which saturation is achieved.
Due to saturation of excretion, the carryover to the next application day increased dramatically at doses of 80 mg/kg bw and above leading to a markedly increased body burden of the test material when 'entering' the subsequent treatment day. The kinetics indicate that this effect would be progressive, leading to a very rapid build-up of body burden at high doses. Clearly, such doses cannot be sustained over a prolonged period and successful sub-chronic and chronic studies would require a high dose below 40 mg/kg/day. - Test no.:
- #1
- Toxicokinetic parameters:
- AUC: AUG values of 14C-test material in rats increased non-linearly with increasing dose. A marked over-proportional increase in the AUG and thus, saturation of excretion was demonstrated at doses above 20 mg/kg bw.
- Metabolites identified:
- not measured
- Conclusions:
- Under the conditions of the study the results demonstrate saturation of excretion capacity of the test material in rats at a dose of 40 mg/kg bw and higher in a 14 day treatment period. Long term administration of doses of 40 mg/kg/day or higher are likely to result in progressive toxicity which may compromise the health and even the survival of the rats.
- Executive summary:
The plasma kinetics of 14C-test material were assessed in male Wistar rats according to OECD Test Guideline 417 and in compliance with GLP.
In order to check the potential for saturation of excretion groups of 4 male rats were fed non-radiolabelled test material for 14 days at dietary concentrations of 125, 250, 500, 1 000, 1 500, and 2 200 ppm and the daily equivalent by a single gavage dose of radiolabelled test material at day 15. Blood was sampled at various time points up to 48 hours post dosing and the plasma concentrations of radioactivity were determined. The plasma concentration versus time curves were used to calculate the AUC0∞ and plasma half-lives.
After radioactive dosing at the dose levels of 10 and 20 mg/kg bw on day 15, plasma levels reached a maximum plateau after the first hour. The time point of maximum plasma concentration (Tmax) increased with increasing dose; i.e. maximum plasma concentrations were observed at 4 h, 2 h, 6 h, and 6 h after dosing at 40, 80, 120, and 180 mg/kg bw, respectively. The shift of Tmax with dose and the under-proportinal increase of Cmax at 120 and 180 mg/kg bw might be due to a delay of gastrointestinal absorption.
After having reached peak levels, plasma concentrations declined continuously. Irrespective of the dose level, plasma concentration versus time curves showed three different phases: phase 1 can be characterised as a mixed absorption/excretion phase and ranges - depending on the dose - from 0 to 8 - 12 h after dosing, whereas phase 2 represents a pure excretory phase (about 12 - 24 h). Phase 3 is a mixed absorption/excretion phase, since animals are dosed with non-radiolabelled test material via feed, again after day 15.
The AUC0∞ increased with increasing dose. The increase in the AUC0∞ was not linear with dose. A slight over-proportional increase of the AUC0∞ was already detectable at 40 mg/kg bw being more pronounced at 80 mg/kg bw. Whereas the dose increased 4- and 8-fold relative to the low dose, the AUC0∞ increased by factors of 4.5 and 11.3. At 120 and 180 mg/kg bw, the over-proportional increase in the AUC0∞ was even more pronounced. Whereas the overall increase in dose from the lowest to the two highest dose levels was 12- and 18-fold, the respective AUC0∞ increased by factors of 18 and 23.
When comparing the AUC during the main excretory phase (AUC12-24) which is a more sensitive parameter for the excretory capacity, the over-proportional increase in AUC at 40 mg/kg bw and above, and thus, saturation of excretion, becomes even more evident.
These data clearly demonstrated saturation of excretion, indicated by the over-proportional increase of the AUC with increasing dose, which already occurred at test material doses of 40 mg/kg bw.
Although being a rather insensitive indicator for excretory saturation phenomena, data of initial half-lives in plasma further supported the conclusions drawn from the AUC-data.
When comparing plasma concentrations at 24 hours after dosing, which can be interpreted to represent carryover of systemically available test material to the subsequent treatment day, the data clearly show that up to 20 mg/kg bw, carryover increased virtually linearly with dose. At 40 mg/kg bw, a clearly over-proportional plasma concentration was measured. Due to saturation of excretion, the carryover to the next treatment day increased dramatically at doses of 80 mg/kg bw and more leading to a markedly increased body burden of the test material when 'entering' the subsequent treatment day.
Under the conditions of the study the results demonstrate saturation of excretion capacity of the test material in rats at a dose of 40 mg/kg bw and higher in a 14 day treatment period. Long term administration of doses of 40 mg/kg/day or higher are likely to result in progressive toxicity which may compromise the health and even the survival of the rats.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 09 December 2003 to 18 February 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Objective of study:
- absorption
- excretion
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 417 (Toxicokinetics)
- Version / remarks:
- 1984
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test material in the solvent/dispersant/vehicle/test medium: The stability of the radiolabelled test material in the vehicle (0.5 % aqueous carboxymethylcellulose) was confirmed in all experiments. The stability of non-radiolabelled test material in feed at ambient temperature for at least 33 days was demonstrated previously.
- The homogeneity and achieved concentrations were verified analytically in all experiments. - Radiolabelling:
- yes
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- CrlGlxBrlHan:WI
- Details on species / strain selection:
- Recognised by international guidelines as a recommended test system. Study results will be used in relation to already available data from the same test system.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: About 8 weeks at the day of first administration of unlabelled material
- Housing: During acclimatisation and prior to the experiment in Macrolon Cages Type Ill (2 animals per cage); during pre-treatment and plasma kinetics experiments in steel wire mesh cages (1 animal per cage).
- Diet: Ad libitum prior to and during experiments.
- Water: Tap water ad libitum.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 % relative humidity
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.).
- Route of administration:
- other: Diet and gavage
- Vehicle:
- other: diet (diet administration), 0.5 % carboxymethylcellulose in double-distilled water (gavage administration)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Material for oral administration via gavage:
In order to achieve the required concentration and specific activity, the respective amount of non-radiolabelled material was added to the respective amount of the stock solution of radiolabelled material in acetone and the organic solvent was evaporated to dryness; 0.5 % carboxymethylcellulose in double-distilled water was added to the residue and filled up to the final volume. Prior to administration the preparation was sonicated and stirred to produce a homogeneous suspension. Samples were taken to check the amount of radioactivity in the preparation and to demonstrate the stability, homogeneity and the achieved concentration of the test substance in the preparation.
DIET PREPARATION
- Mixing appropriate amounts with: For each concentration, the test material was weighed out and thoroughly mixed with a small amount of food in a beaker. Subsequently a premix was prepared in a household mixer by adding an appropriate amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a laboratory mixer. - Duration and frequency of treatment / exposure:
- 60 consecutive days
- Dose / conc.:
- 15 mg/kg bw/day (nominal)
- Remarks:
- Experiment 1
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- Experiment 2
- Dose / conc.:
- 45 mg/kg bw/day (nominal)
- Remarks:
- Experiment 3
- Dose / conc.:
- 60 mg/kg bw/day (nominal)
- Remarks:
- Experiment 4
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Remarks:
- Experiment 5
- No. of animals per sex per dose / concentration:
- 4 males per experiment
- Control animals:
- no
- Details on study design:
- - Dose selection rationale:
Dose selection for this study was based on information gathered in a previous kinetic study. Plasma kinetics were investigated after oral administration at the following five dose levels:
Dose 1: 15 mg/kg bw radiolabelled dose (gavage) corresponding to 190 ppm in feed.
Dose 2: 30 mg/kg bw radiolabelled dose (gavage) corresponding to 380 ppm in feed.
Dose 3: 45 mg/kg bw radiolabelled dose (gavage) corresponding to 570 ppm in feed.
Dose 4: 60 mg/kg bw radiolabelled dose (gavage) corresponding to 760 ppm in feed.
Dose 5: 75 mg/kg bw radiolabelled dose (gavage) corresponding to 950 ppm in feed.
The dietary concentrations in the present study were designed to reflect those which achieve a similar mean daily intake in mg/kg bw in a subchronic toxicity study (i.e. 15, 30, 45, 60, and 75 mg/kg bw/day). Thus, the achieved dose was expected to exceed target for the early phase of this 60 day study but approach the target more closely towards the end of the study. The single dose of radiolabelled material was at the specified level.
- Rationale for animal assignment: The animals were selected based on health status and to provide a narrow range of body weights; animals were assigned to the groups randomly, i.e. without conscious bias. - Details on dosing and sampling:
- Blood/Plasma level
- Animals were treated with non-radiolabelled test material via feed for 60 days. On day 61, mixed feed with the test material was substituted by control feed at 6:00 a.m. for 24 hours and animals received a single administration of 14C-labelled test material by gavage at about 9.00 a.m. Blood samples (100 - 200 μL) were taken from the retroorbital sinus at the following times after administration: 1; 2; 4; 6; 8; 12; 24; 48 hours.
- Plasma samples were checked for total radioactivity.
- Administration via feed: Feed was available ad libitum. On day 61, mixed feed was substituted by control feed at 6:00 a.m. for 24 hours in order to avoid interference of non-labelled test material in feed with the radiolabelled test material given via gavage.
- Administration via gavage: About 1 mL of the test material preparation per 100 g bw was administered to rats by gavage.
- In all groups, body weight and feed consumption were determined weekly. Feed consumption was calculated using the mean body weight of the respective feeding period. Clinical signs were recorded daily on workdays (Monday- Friday) during acclimatisation and application period.
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Blood
- Time and frequency of sampling: Samples were taken from the retroorbital sinus at the following times after administration: 1; 2; 4; 6; 8; 12; 24; 48 hours.
- Method type(s) for identification: HPLC
HPLC Analysis of Feed Mixtures
Feed samples were preincubated with double-distilled water and than extracted twice with acetonitrile. Aliquots of these extracts were used for HPLC analysis. The homogeneity and achieved concentration of the test material in feed was checked via HPLC under the following conditions:
Column: Nucleosil 100, 5 C18, 250 mm x 4 mm
Eluent:
50 % Acetonitrile + 0.5 M Sulfuric acid (1 000 mL + 5 mL)
50 % Double-distilled water+ 0.5 M Sulfuric acid (1 000 mL+ 5 mL)
Flow: 1 mL/min
Detection: UV-Extinction at 230 nm
HPLC Analysis of Radioactive Preparations
The homogeneity and achieved concentration of the test matrial in 0.5 % carboxymethylcellulose in double-distilled water as well as the radiochemical purity of 14C-test material were checked via HPLC (HP 1050 series) under the following conditions:
Column: Nucleosil 120, 7C18, 250 mm x 4 mm
Eluent: Acetonitrile / double-distilled water / 0.5 M Sulfuric acid (500/500/5 (v/v/v))
Flow: 1.2 mL/min
Detection: UV-Extinction at 230 nm HPLC Radioactivity Monitor LB 509 (Cell: VG 150 U4D)
- Preparation of samples and measurement of radioactivity: Aliquots of plasma samples were mixed with scintillation cocktail (Hionic-Fluor, Packard) and counted for 10 minutes in a liquid scintillation counter (LSC; Wallac type 1409) and the disintegration rate corrected by the respective background. - Statistics:
- Analysis of kinetic data was performed based on the group mean values using the PC program system TOPFIT Version 2.0.
- Details on absorption:
- The AUC versus dose plot shows the AUC12-24 versus dose plot at the dose levels of 15 - 75 mg/kg bw in comparison to the expected AUC12-24 values calculated by linear extrapolation of the AUC12-24 of the 15 mg/kg bw group.
After radioactive dosing at dose levels of 15, 30, 45, 60 and 75 mg/kg bw on day 61, plasma levels reached a maximum concentration within the first hour except for the group dosed at 30 mg/kg bw, which reached the maximum plasma concentration (Tmax) within 4 hours. After having reached peak levels, plasma concentrations declined continuously. Irrespective of the dose level, plasma concentration/ time curves showed three different phases: Phase 1 can be characterised as a mixed absorption/excretion phase and ranges from 0 to about 10 - 12 h after dosing, whereas phase 2 represents a pure excretory phase (about 10/12 - 24 h post-dosing). Phase 3 is a further mixed absorption/excretion phase, since animals were dosed again with non-radiolabelled test material via feed (24 hours and beyond).
The AUC0-∞ increased with increasing dose. The increase in the AUC0-∞ was not linear with dose. A slight over proportional increase of the AUC0-∞ was already detectable at 30 and 45 mg/kg bw. Whereas the dose increased 2- and 3-fold relative to the low dose, the AUC0-∞ increased by factors of 2.1 and 3.4. At 75 mg/kg bw, the over proportional increase in the AUC0-∞ was more pronounced. Whereas the overall increase in dose from the lowest to the highest dose level was 5-fold, the AUC0-∞ increased by a factor of 6.0.
The effect is more transparent when comparing the AUC12-24 values. This reflects a condition where rats were in a purely excretory phase at all dose levels. This should be more representative of the position with dietary uptake, where there is likely to be a relatively constant level in the blood, reflecting the feeding habits of the rat: small meals are taken at frequent intervals. It can be seen from the AUC12-24-data, that excretion rates reflected by increased AUC values were impaired at 30 mg/kg bw and above.
These data demonstrated saturation of excretion, indicated by the over proportional increase of the AUC with increasing dose, which occurred at test material doses of 30 mg/kg bw and above.
When considering half-lives during the mixed absorption/excretion phase (phase 1), a clear increase in plasma half-life was evident at 30 mg/kg bw and above. During the main excretory phase (phase 2), which is more representative as this reflects pure excretion, values remained more or less unchanged at doses of 15 and 30 mg/kg bw. At doses of 45 mg/kg bw and above, half-lives were about 1 hour longer as compared to the two low doses. Although being a rather insensitive indicator for excretory saturation phenomena, data of half-lives in plasma further supported the conclusions drawn from the AUC-data. Another parameter of interest is the plasma concentration 24 hours after dosing which can be interpreted to represent carryover of systemically available test material to the subsequent application day.
The data also clearly reflect the saturation of excretion of test material with increasing dose. Already at 30 mg/kg bw and even more pronounced at 45 mg/kg bw, an over-proportional plasma concentration was measured indicating that in this dose range saturation is achieved. Due to saturation of excretion, the carryover to the next application day increased much more at 75 mg/kg bw leading to a markedly increased body burden of test material when 'entering' the subsequent treatment day. The kinetics indicate that this effect would be progressive, leading to a build-up of body burden at high doses. Doses above the threshold of renal excretion may lead to unacceptably high toxicity. This is seen in this study as reduced body weight gain, evident at doses of 45 mg/kg bw/day and above. - Test no.:
- #1
- Toxicokinetic parameters:
- AUC: AUC values of 14C-test material in rats increased non-linearly with increasing dose. A marked overproportional increase in the AUC; saturation of excretion was demonstrated at doses above 15 mg/kg bw.
- Metabolites identified:
- not measured
- Conclusions:
- Under the conditions of the study the results demonstrate saturation of excretion capacity of the test material in rats at a dose of 30 mg/kg bw and higher in a 60 day treatment period. Therefore, long term administration of doses of 30 mg/kg/day or higher are likely to result in progressive toxicity, which may compromise the health and even the survival of the rats.
- Executive summary:
The plasma kinetics of 14C-test material in male Wistar rats was assessed according to OEC Test Guideline 417 and in compliance with GLP.
In order to determine the potential for saturation of excretion over an extended period groups of 4 male rats were fed non-radiolabelled test material for 60 days at dietary concentrations of 190, 380, 570, 760 and 950 ppm and the daily equivalent by a single gavage dose of radiolabelled test material at day 61.
Blood was sampled at various time points up to 48 hours post dosing and the plasma concentrations of radioactivity were determined. Plasma concentration versus time curves were used to calculate the AUC0-∞ and plasma half-lives.
After radioactive dosing at dose levels of 15, 30, 45, 60 and 75 mg/kg bw on day 61, plasma levels reached a maximum concentration within the first hour except for the group dosed at 30 mg/kg bw, which reached the maximum plasma concentration (Tmax) within 4 hours. After having reached peak levels, plasma concentrations declined continuously.
Irrespective of the dose level, plasma concentration/ time curves showed three different phases: Phase 1 can be characterised as a mixed absorption/excretion phase and ranges from 0 to about 10 - 12 h after dosing, whereas phase 2 represents a pure excretory phase (about 10/12 - 24 h post-dosing). Phase 3 is a further mixed absorption/excretion phase, since animals were dosed again with non-radiolabelled test material via feed (24 hours and beyond).
The AUC0-∞ increased with increasing dose. The increase in the AUC0-∞ was not linear with increasing dose. A slight over proportional increase of the AUC0-∞ was already detectable at 30 and 45 mg/kg bw. Whereas the dose increased 2- and 3-fold relative to the low dose, the AUC0-∞ increased by factors of 2.1 and 3.4. At 75 mg/kg bw, the over proportional increase in the AUC0-∞ was more pronounced. Whereas the overall increase in dose from the lowest to the highest dose level was 5-fold, the AUC0-∞ increased by a factor of 6.0. When comparing the AUC during the main excretory phase (AUC12-24), which is a more sensitive parameter for the excretory capacity, the over proportional increase in AUC12-24 clearly commenced at 30 mg/kg bw. At this dose a two-fold rise in the dose level gave rise to a 2.7-fold rise in AUC. Saturation of excretion therefore was evident at 30 mg/kg bw and above.
Although half-life in plasma is a rather insensitive indicator for excretory saturation phenomena, these data further supported the conclusions drawn from the AUC-data. Plasma concentrations at 24 hours after dosing were also compared. These levels can be interpreted to represent carryover of systemically available test material to the subsequent application day. The data clearly show that at 30 mg/kg bw and above, an over-proportional increase in plasma concentration was measured indicating that this dose level approximates to the point at which saturation of excretion is achieved. This effect was even more pronounced at 45 mg/kg bw. The kinetics indicate that this effect would be progressive, leading to a respective build-up of body burden at high doses. It follows that doses above approximately 30 mg/kg bw/day when administered in sub-chronic or chronic studies may result in excessive toxicity to the animals.
Under the conditions of the study the results demonstrate saturation of excretion capacity of the test material in rats at a dose of 30 mg/kg bw and higher in a 60 day treatment period. Therefore, long term administration of doses of 30 mg/kg/day or higher are likely to result in progressive toxicity, which may compromise the health and even the survival of the rats.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- other: Expert review of study findings
- Adequacy of study:
- supporting study
- Study period:
- Not applicable - expert review
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- secondary literature
- Reason / purpose for cross-reference:
- reference to same study
- Objective of study:
- other: determination of rate constant for the ADME processes
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Expert report to identify the most appropriate value of the rate constant for the ADME processes for use in body-burden calculations.
Specifically, in a study of ADME processes in the rat (Lappin, 1997), the losses of test material from the rat were studied. Two doses were evaluated: 5 mg/kg bw and 100 mg/kg bw. Inspection of the graphs depicting the decline of radioactivity over time, shows that at the lower dose, the decline of the test material is largely linear, certainly up to 24 hours, after which there is a tailing off in the rate of decline, probably due to diffusion of test material into body liquids or co-measurement of metabolites. At the higher dose, there is a delay in Tmax before the fast decline of the test material most likely due to extended absorption often seen in higher dose profiles. The terminal elimination half-lives of the low dose group were estimated by Lappin (1997) as 6.4 h (male) and 4.2 h (female). At the higher doses, the values are reported as 7.9 h (male) and 7.8 h (female). This report investigates this calculation, and investigates whether it is appropriate to use the same half-life for both the low and high dose animals. - GLP compliance:
- no
- Remarks:
- Not required for expert report.
- Species:
- rat
- Test no.:
- #1
- Toxicokinetic parameters:
- half-life 1st: 5.4 h
- Remarks:
- (male)
- Test no.:
- #1
- Toxicokinetic parameters:
- half-life 1st: 4.5 h
- Remarks:
- (female)
- Test no.:
- #1
- Toxicokinetic parameters:
- other: rate constant (k) = 0.0022/min
- Remarks:
- (male)
- Test no.:
- #1
- Toxicokinetic parameters:
- other: rate constant (k) = 0.0025/min
- Remarks:
- (female)
- Conclusions:
- The results clearly show an excellent visual fit and low parameter errors obtained in the Michaelis-Menten model using a fixed value of the terminal elimination half-life at time points up to 48 h after dosing.
This evidence suggests that a suitable value for the half-lives are 5.4 h (male) and 4.5 h (female) similar to those published by Lappin (1997). Good fits would not be possible if this value of the terminal elimination half-time was inappropriate. These half-lives correspond to rate constants as follows:
Male k = 0.0022/min
Female k = 0.0025/min
These values of half-times and rate constants are suitable for application in, for example, body burden modelling for mammalian risk assessment. - Executive summary:
In a study of ADME processes in the rat (Lappin, 1997), the losses of test material from the rat were studied. Two doses were evaluated: 5 mg/kg bw and 100 mg/kg bw. Inspection of the graphs depicting the decline of radioactivity over time, shows that at the lower dose, the decline of the test material is largely linear, certainly up to 24 hours, after which there is a tailing off in the rate of decline, probably due to diffusion of test material into body liquids or co-measurement of metabolites. At the higher dose, there is a delay in Tmax before the fast decline of the test material most likely due to extended absorption often seen in higher dose profiles. The terminal elimination half-lives of the low dose group were estimated by Lappin (1997) as 6.4 h (male) and 4.2 h (female). These data were considered in order to identify the most appropriate value of the rate constant for the ADME processes for use in body-burden calculations.
In order to generate meaningful kinetic parameters for use in a body-burden calculation, the data must be handled in a way that is suitable to minimise the errors associated with fitting multivariate curves to few data points. In this study, a 2-step approach was employed.
1) The decline shown within the low-dose (5 mg/kg) groups was analysed using a non-compartmental analysis to generate an estimate of the terminal rate constant and its associated t1/2 value (ln2/λz). This was performed using the data from the arithmetic mean of the experimental results (mean of 5 animals in each batch) covering the period 9 - 24 h. The later time points were excluded in order to minimise interference in the data set from diffusion of the test material from bodily fluids back into the blood, and the effects of metabolism of the test material to other radioactive metabolites. The earlier time points were excluded in order to ensure a conservative measure of the t1/2.
2) The concentration-time data within the high dose (100 mg/kg) groups, where possible, were subjected to numerical analysis with PCModfit (Ver. 4.0) using iteratively re-weighted non-linear least squares analysis utilising a weighting factor of 1/c^2. Consideration of the number of data points and the shape of the profiles by visual assessment of the ln(concentration) vs. time plots, the model chosen was a 1-compartment oral function, for both genders, with a Michaelis-Menten and 1st order (λz) elimination phases. The terminal rate constant was fixed at the value generated from the first step. This provided a validation that the terminal rate constant was appropriate, even at the higher dose. Again, this was performed using all the animals in each batch, covering the period 0 - 48 h.
The findings of this evaluation clearly show an excellent visual fit and low parameter errors obtained in the Michaelis-Menten model using a fixed value of the terminal elimination half-life at time points up to 48 h after dosing.
This evidence suggests that a suitable value for the half-lives are 5.4 h (male) and 4.5 h (female) similar to those published by Lappin (1997). Good fits would not be possible if this value of the terminal elimination half-time was inappropriate. These half-lives correspond to rate constants as follows:
Male k = 0.0022/min
Female k = 0.0025/min
These values of half-times and rate constants are suitable for application in, for example, body burden modelling for mammalian risk assessment.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 15 March 1994 to 16 August 1996
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Objective of study:
- absorption
- distribution
- excretion
- metabolism
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The absorption, distribution, metabolism and excretion of (14C)-test material was investigated after single and multiple oral administrations to the rat (nominally 5 mg/kg and 100 mg/kg body weight).
Samples were analysed by liquid scintillation counting directly or following solubilisation or combustion.
HPLC and TLC were used to profile radiolabelled metabolites in pooled urine and faeces samples from dose groups A, B and C.
Pooled urine and faeces from male rats collected up to 48 h were analysed by LC-MS (groups A and C) and NMR (group C, urine only). The spectra obtained were compared to those of an authentic standard of the test material.
An additional experiment was carried out using LC-MS (MRM) to confirm the presence and concentration of a metabolite in female urine (groups A and C). - GLP compliance:
- yes
- Radiolabelling:
- yes
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Crl:(WI)BR strain
- Details on species / strain selection:
- The rat was chosen as it is a readily available rodent species acceptable to the regulatory authorities. There is a considerable amount of published material available for assessing the significance to man of data generated from this species.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Approximately six to eight weeks of age on arrival.
- Weight at study initiation: Between 150 and 200 g body weight on arrival. The weight range of the animals at the time of dosing was 185 to 261 g and 163 to 208 g for males and females respectively.
- Housing: The rats were housed in groups of up to five per cage according to sex, in wire floor polypropylene cages suspended over polypropylene dirt trays containing soft white wood sawdust.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: The animals were acclimatised in an experimental room for approximately one week, during which time their health status was reassessed and suitability for experimental purposes confirmed.
- Nulliparous and non pregnant: The females were nulliparous and non-pregnant.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature range was 19 to 29 °C.
- Humidity (%): Relative humidity was 57 to 68 %.
- Air changes (per hr): Experimental rooms were air conditioned and designed to provide a minimum of 10 air changes/h.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 h light (0600 to 1800 h) and 12 h darkness.
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 1 % w/v
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- The test material was prepared as a suspension in 1 % w/v carboxymethyl cellulose to provide a nominal dose volume of 5 mL/kg body weight.
- (14C)-test material was administered orally, by gavage, corresponding to a nominal radioactive dose of 15 μCi (555 kBq) per animal. - Duration and frequency of treatment / exposure:
- Groups A and C - F: Single dose
Group B: Repeat dose administration for 15 days. - Dose / conc.:
- 5 mg/kg bw/day
- Remarks:
- Group A designated low dose single administration.
- Dose / conc.:
- 5 mg/kg bw/day
- Remarks:
- Group B designated low dose repeated administration
- Dose / conc.:
- 100 mg/kg bw/day
- Remarks:
- Group C designated high dose single administration
- Dose / conc.:
- 5 mg/kg bw/day
- Remarks:
- Group D designated low dose single administration
- Dose / conc.:
- 100 mg/kg bw/day
- Remarks:
- Group E designated high dose single administration
- Dose / conc.:
- 5 mg/kg bw/day
- Remarks:
- Group F designated low dose single administration
- No. of animals per sex per dose / concentration:
- 5 per sex per dose were required for groups A - E. Additional animals were dosed to ensure data from 5 animals were available at study completion. Six animals of each sex were dosed.
For the repeat dose administration non-radiolabelled test material was administered for 14 days with a radiolabelled dose on day 15.
12 per sex per dose were used for Group F. - Control animals:
- no
- Details on study design:
- - Dose selection rationale: Not specified.
- Details on dosing and sampling:
- Excretion study:
Following the administration of (14C)-test material, the rats were placed in individual all-glass metabolism cages suitable for the separate collection of urine and faeces (all groups) and expired air (pilot group only). The collection vessels were surrounded by solid carbon dioxide.
A pilot study was performed with two male animals from group C in order to achieve a material balance, to determine the rate of elimination of dose in excreta and to monitor expired air for (14C)-carbon dioxide.
Expired air was trapped into a suitable medium. The traps on the group C pilot animals contained levels of radioactivity below the limit of detection and therefore expired air was not collected from the remaining animals from group C or the animals in groups A and B.
On achieving a material balance the remaining animals from group C and the animals in groups A and B were dosed. Additional animals (one of each sex) were dosed in group B to ensure valid data from five animals would be available in the event of problems being experienced during the practical phase. No practical problems were experienced and therefore data from the original five animals is reported.
After each collection of excreta, cage debris was removed and cages rinsed with water. At the end of the collection period, cages were rinsed thoroughly with water and then methanol.
Blood was collected into heparinised tubes and an aliquot was centrifuged to prepare plasma.
Radioactivity was determined in urine, faeces, expired air traps, (pilot study only) cage debris, cage washings, tissues and residual carcases.
- Tissues and body fluids sampled: Bone, brain, blood, fat (abdominal), gonads, heart, kidney, adrenals, thyroid, stomach (minus contents), liver, lung, muscle (quadriceps), plasma, spleen, uterus, residual carcass, skin, stomach contents.
- Time and frequency of sampling: At 168 h post-dose animals were exsanguinated under halothane anaesthesia and the tissues above were removed or sampled:
Expired air was collected from the two animals in the pilot study at the following times: 0 to 12, 12 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144 and 144 to 168 h post-dose.
Urine and faeces were collected at the following time intervals:
Urine: 0 to 6, 6 to 12, 12 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144 and 144 to 168 h post-dose.
Faeces: 0 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144 and 144 to 168 h post-dose.
Pharmacokinetic study
Following administration of (14C)-test material animals from dose groups D and E were replaced in wire floor cages. The blood was collected into microhaematocrit tubes and centrifuged to prepare plasma.
At 168 h post-dose, animals were killed by CO2 asphyxiation and discarded. Radioactivity was determined in plasma.
- Tissues and body fluids sampled: Blood was sampled from a lateral caudal vein
- Time and frequency of sampling: Pre-dose, 1.5, 3, 6, 9, 12, 24, 48, 72, 120 and 168 h post-dose.
Tissue distribution study
Following administration of (14C)-test material, 12 male and 12 female animals from group F were returned to wire floor cages.
- Tissues and body fluids sampled: The tissues were removed or sampled and assayed for radioactivity.
- Time and frequency of sampling: After dosing, four animals of each sex were exsanguinated under halothane anaesthesia at 0.5, 3 and 6 h post-dose. All samples were stored at <-15 °C.
Morbidity and mortality
All animals were observed for possible overt pharmacological or toxic signs throughout the working day as necessary.
Body weights
Individual body weights were recorded within 48 h of arrival at the Test Facility, on the day of dosing and at necropsy.
METABOLITE CHARACTERISATION STUDIES
Extraction and identification of radiolabelled metabolites in urine and faeces
Urine and faeces collected from dose groups A, B and C were examined by various chromatographic (TLC and HPLC) and spectrometric (LC-MS) techniques to profile and identify 14C-labelled metabolites.
- Urine
Composite pools of urine from samples containing > 90 % of the urinary radioactivity were formed according to time point and dose group. Sub-samples of the neat urine pools were taken for TLC, HPLC and LC-MS.
- Faeces
Composite pools of faeces homogenates were formed from samples containing > 90 % of the faecal radioactivity according to time point and dose group. Portions of each faecal pool were extracted sequentially with methanol. The organic extracts were combined and excess solvent removed. The residue was reconstituted in HPLC mobile phase prior to analysis by TLC, HPLC and LC-MS.
Characterisation of radiolabelled metabolites in urine and faeces.
To further characterise the nature of radiolabelled metabolites in urine, samples were analysed following chemical and enzyme hydrolysis by HPLC and TLC to assess their stability.
- Enzyme hydrolysis
Aliquots of unprocessed urine were added to vials and ammonium acetate buffer (pH 5) and either β-glucuronidase H-1, β-glucuronidase B-1 or aryl sulphatase (Patella vulgata extract) admixed. The samples were incubated overnight at 37 °C. Aliquots of the extract were analysed by HPLC and TLC.
Control samples were also prepared using phenolphthalein glucuronide and p-nitrophenol sulphate solutions. Liberation of phenolphthalein after incubation provided proof that the glucuronidase enzyme preparations were active, was confirmed by the production of a pink colour upon admixing a few drops of sodium hydroxide. Similarly a yellow colour due to the release of p-nitrophenol indicated that the sulphatase enzyme was active.
- Chemical hydrolysis
Aliquots of the pooled urine samples were mixed with 2.5M HCl or 5M NaOH and incubated at 95 °C for 3 h. Following cooling to room temperature the incubates were extracted with ethyl acetate. The organic extracts were combined and excess solvent removed prior to analysis by HPLC and TLC.
Radiolabelled metabolite characterisation by high performance liquid chromatography and thin layer chromatography:
The nature of radiolabelled metabolites in unprocessed urine and organic extracts prepared from faeces were investigated by HPLC and TLC. Non-radiolabelled test material was analysed similarly.
- HPLC conditions
For metabolite profiling of urine and faeces.
- TLC conditions
For metabolite profiling of urine and faeces:
Plates: Merck silica gel 60 F254
Solvent system: Chloroform: cyclohexane: acetic acid (80:20:10 v/v/v)
Development: 15 cm
Detection: UV (254 nm) and radiolabelled regions of interest were detected using a Lablogic RITA linear analyser and by autoradiography
Data were acquired and processed using commercially available software (Labchrom, Lablogic, Sheffield, UK).
- Metabolite identification by LC-MS
Unprocessed urine and organic extracts prepared from faeces from dose groups A, B and C were investigated by LC-MS.
The LC-MS conditions used for analysis were as follows:
LC-MS conditions: VG Quattro quadrupole mass spectrometer with electrospray LCMS Interface, coupled to a Jasco ternary gradient HPLC, a Hewlett Packard UV detector and a LabLogic β-ram radiodetector
Column: YMC ODS-AQ (250 X 4.6 mm i.d)
Mobile phase:
1 % aqueous Ammonium acetate (A)
1 % Ammonium acetate in methanol (B)
Gradient:
Initial: 60 % A, 40 % B
15 mins: 5 % A, 95 % B
30 mins: 5 % A, 95 % B
32 mins: 60 % A, 40 % B
Flow rate: 1 mL/min (ca. 20: 1 v/v split into radiochemical detector:mass spectrometer)
Detection: Radiodetection and UV
- Mass spectrometer conditions:
Source temperature: 150 °C
Scan range: m/z 70 to 400 and 70 to 170
Ionisation: Negative ion electrospray (ESP-)
- Identification of the major urinary metabolite using 1H and 13C Nuclear Magnetic Resonance Spectroscopy (NMR)
A preparative HPLC system was used to isolate the radio labelled metabolite at a retention time of approximately 10 minutes.
The isolated metabolite and standard test material were analysed using 1H and 13C NMR. Proton NMR spectra were obtained at 500.13 MHz and carbon spectra at 125 MHz on a Bruker AMX 500 instrument.
A sample of the purified metabolite was despatched, packed in solid carbon dioxide to the laboratory for analysis.
- β-glucuronidase deconjugation of urine pools for use in MRM analysis
Urine pools (low and high dose female, 0 - 24 h) were incubated with β-glucuronidase (pre-coated vials obtained from Sigma, UK) at 37 °C for 1 h. A solution of phenolphthalein glucuronide was also incubated in a similar manner as a positive control. Addition of base to the control at the end of the incubation period to release a pink colour confirmed that the enzyme was active.
- Confirmation of Carboxy-test material in rat urine by LC-MS (MRM)
Untreated and β-glucuronidase treated urine pools (low and high dose female, 0 - 24 h) were analysed by high performance liquid chromatography-mass spectrometry (LC-MS) using Multiple Reaction Monitoring (MRM) to confirm the presence of Carboxy-test material. Results were compared to those obtained from urine spiked with standard Carboxy-test material (purity of 96. 9 %).
The LC-MS conditions used for analysis were as follows:
LC-MS conditions: VG Quattro quadrupole mass spectrometer with electrospray LCMS interface, coupled to a Jasco ternary gradient HPLC, a Hewlett Packard UV detector and a LabLogic β-ram radiodetector
Column: Spherisorb ODS2 (250 X 4.6 mm i.d)
Mobile phase:
0.1 % aqueous formic acid (A)
0.1 % formic acid in methanol (B)
Gradient
Initial: 60 % A, 40 % B
15 mins: 5 % A, 95 % B
25 mins: 5 % A, 95 % B
27 mins: 60 % A, 40 % B
Flow rate: 1 mL/min (ca. 20: 1 v/v split into radiochemical detector: mass spectrometer)
Detection Radiodetection and UV (230 nm)
- Mass spectrometer conditions:
Source temperature: 150 °C
MRM transition: m/z 243 to 171
Ionisation: Negative ion electrospray (ESP-)
- Sample preparation
Volumes and weights of all biological samples were measured where appropriate.
Portions of urine, cage washings and plasma were added directly to scintillant and assayed by liquid scintillation counting.
Faeces, liver, stomach, stomach contents and cage debris were homogenised in an appropriate volume of deionised water.
The remaining tissues (except adrenals, ovaries and thyroids which were analysed as whole tissues) were macerated with scissors. Residual carcasses were digested under reflux using an appropriate volume of 40 % (w/v) potassium hydroxide in methanol.
- Analysis of samples for radioactivity
Solubilisation
A suitable volume of Soluene®-350 solubilising agent (Canberra Packard, Pangboume, Berkshire, UK) was added to tissue samples. After an appropriate period of incubation, liquid scintillant was added and the samples allowed to dark adapt prior to liquid scintillation counting.
- Combustion
Samples of blood, bone and faecal and debris homogenates were added to ashless floc in Combusto Cones™ and combusted in oxygen using a Packard Sample Oxidiser. The combusted products were absorbed in Carbo-Sorb®, mixed with Permafluor® E+, and the radioactivity determined by liquid scintillation counting.
Standards of 14C-Spec-Chec™ were combusted at the beginning of each day and at regular intervals throughout the day to check the carry-over between samples and to determine the efficiency of the combustion process. Combustion and trapping efficiencies were found to be in excess of 96 %. Consequently all data are reported uncorrected.
- Determination of radioactivity
Radioassays were performed at a minimum in duplicate.
Radioactivity was measured for 2 or 10 min or 0.2 sigma % using Beckman (Beckman Instruments, High Wycombe, Buckinghamshire, UK) or Packard Tri-Carb liquid scintillation counters (Canberra Packard, Pangbourne, Berkshire, UK) with the facilities for computing quench-corrected disintegrations per minute (dpm).
Efficiency correlation curves were prepared for organic, aqueous and combusted sample types and were routinely checked by the use of (14C)-n-hexadecane standards. The spectrometer was recalibrated when a deviation of greater than 3 % was observed when counting quality control standards.
DATA ANALYSIS
- Data collection: Liquid scintillation counts and weighing data were either entered manually or captured on-line using a validated data acquisition system.
- Limit of detection: The limit of detection for the analysis of each sample type was taken as twice the background disintegration rate obtained from the measurement of blank samples of the same type.
Calculations performed on experimental data
Original total weight of sample = Tg
Total weight after processing (i.e. homogenisation or freeze-drying etc.) (NB. For samples not subjected to processing before radioactivity analysis, H = T) = Hg
Weight of aliquot of sample assayed = Wg
Radioactivity (dpm - background value) in aliquot of sample analysed = R dpm
Concentration of radioactivity in sample = C = R / W dpm / g
Specific radioactivity of test material = S mCi / g
Weight of test material administered to each animal = D mg
Radioactive dose administered to each animal = S x D µCi
Calculation of μg equivalents of (14C)-test material in biological samples
Homogenised tissues: μg equivalents of test material/g of sample
((C x H) / (2.22 x 10^3 x S x T)) - K
Macerated or intact tissues: μg equivalents of test material/ g of sample
(C / (2/.22 x 10^3 x S)) – K
Calculation of percentage of radioactive dose in biological samples
Percent of radioactive dose/sample:
((K x T) / 10 x D)) - P
Pharmacokinetic Data
Non-model dependent pharmacokinetic evaluations of plasma data were performed with the aid of TopFit V 1.1 installed on an IBM or IBM compatible personal computer.
The input consisted of: Sample time (h); sample concentration; units of concentration; dose administered and units of dose administered. Non-detected values were not used in half-life calculations.
The output consisted of: The input (as above); time point range used for half-life determination; elimination rate constant (ke1); terminal half-life of elimination (t,h); AUC values (0-t, t-∞ and 0-∞ AUC (0-∞ per unit dose; dose administered; maximum concentration (Cmax) and time (tmax) to reach Cmax.
Plasma half-lives of total radioactivity following administration of test material were calculated by regression analysis of the linear portion of the individual concentration/time curves following Cmax when plotted semi-logarithmically.
The area under the plasma concentration time curves (AUC) were calculated using the trapezoidal rule. - Type:
- excretion
- Results:
- Urine excretion Group A: 95.3 % (male) and 92.3 % (female) recovered within 24 h (single administration).
- Type:
- excretion
- Results:
- Faecal excretion (Group A): 8.2 % (males) and 3.6 % (females) mostly within 24 h after dosing (single administration).
- Type:
- excretion
- Results:
- Urinary excretion Group B: 89.0 % (male) and 86.5 % (female) recovered in the urine and cage wash within 24 h (repeated dose group).
- Type:
- excretion
- Results:
- Faecal excretion Group B): 5.3 % (male) and 4.9 % (females) (repeated dose group).
- Type:
- distribution
- Results:
- Group A: Low levels were detected in carcass, skin, fat, liver (male only) and kidney of both sexes ranging from 0.010 to 0.167 μg equiv/g.
- Type:
- distribution
- Results:
- Group B: In male animals, carcass, skin, plasma, fat, liver, kidney and adrenals contained less than 0.1 μg equiv/g. In female animals, carcass, skin, plasma, fat, spleen, kidney, ovaries, uterus and adrenals contained less than 0.2 μg equiv/g.
- Type:
- excretion
- Results:
- Urinary excretion Group C: 48 h to eliminate 85.4 % (male) and 76.8 % (female) of the administered dose in the urine and cage wash.
- Type:
- excretion
- Results:
- Faecal elimination Group C: 12.5 % (male) and 9.1 % (female).
- Type:
- distribution
- Results:
- Group C: Skin (male 7.401 μg equiv/g, female 7.328 μg equiv/g), fat (male 23 .03 μg equiv/g, female 20.38 μg equiv/g), ovaries (5.854 μg equiv/g) and adrenals (male 6.698 μg equiv/g, female 5.441 μg equiv/g) contained levels above 5 μg equiv/g.
- Details on absorption:
- Pharmacokinetic study, Group D, 5 mg/kg and Group E, 100 mg/kg:
Following a single oral administration of 14C-test material at a nominal dose level of 5 mg/kg, plasma levels of radioactivity rose rapidly reaching mean maximum concentrations of 27.77 μg equiv/g and 31.54 μg equiv/g within 1.8 h, for male and female animals respectively. Thereafter, levels decreased rapidly up to 48 h and then more slowly, levels becoming undetectable at 120 h after dosing. The terminal elimination half-lives were determined as 6.354 h (male) and 4.234 h (female). The corresponding areas under the concentration/time curves (AUC) were 252.0 μg equiv.h/g (male) and 182.7 μg equiv.h/g (female).
Following administration of (14C)-test material at the higher dose level (100 mg/kg), plasma levels of radioactivity again rose rapidly, maximum levels (384.0 μg equiv/g male, 394.1 μg equiv/g female) being attained within 4.2 h. Levels remained comparable to Cmax until 24 h post-dose, thereafter decreasing to non-detectable levels within 120 h. Terminal elimination half-lives were estimated at 7.886 h (male) and 7.787 h (female). AUC values were determined as 8 449 μg equiv.h/g and 7 884 μg equiv.h/g for male and female animals respectively. - Details on distribution in tissues:
- Tissue distribution study, Group F, single oral administration, 5 mg/kg:
Radioactivity was detected in all tissues sampled in male and female animals at 30 minutes, 3 h, and 6 h after administration of (14C)-test material at a nominal dose level of 5 mg/kg.
For male animals, the highest levels of radioactivity (excluding tissues associated with the GI tract) were detected in thyroid (30.17 μg equiv/g), kidney (23.62 μg equiv/g), blood (18.86 μg equiv/g), plasma (16.60 μg equiv/g), adrenals (9.477 μg equiv/g), liver (9.444 μg equiv/g), heart (7.983 μg equiv/g) and lung (7.717 μg equiv/g) at 30 minutes after dosing. All other tissues sampled contained levels less than 5 μg equiv/g.
Tissue concentrations at the subsequent sampling times remained comparable to levels at 30 minutes. In the majority of tissues, concentrations had begun to decrease at 3 h after dosing apart from plasma, fat, gonads and adrenals, where maximum concentrations were detected at 3 h. Levels had slightly decreased in all tissues by 6 h after dosing.
For female animals, the highest levels of radioactivity (excluding tissues associated with the GI tract) were detected in thyroid (42.16 μg equiv/g), kidney (19.08 μg equiv/g), plasma (24.37 μg equiv/g), blood (19.66 μg equiv/g), gonads (11.16 μg equiv/g), uterus (9.451 μg equiv/g), adrenals (9.298 μg equiv/g), lung (8.801 μg equiv/g), heart (8.468 μg equiv/g) and liver (8.436 μg equiv/g) at 30 minutes after dosing. All other tissues sampled contained levels less than 5 μg equiv/g.
At 3 h after dosing, tissue levels of radioactivity, excluding GI Tract and contents, had slightly elevated in the majority of tissues sampled. Exceptions were blood, brain, muscle, lung, liver, and thyroid where maximum concentration occurred at 30 minutes. However, levels had not decreased to a great extent in these tissues.
At 6 h after dosing, levels had decreased in all tissues sampled compared to 3 h levels but not by a substantial degree. - Details on excretion:
- Excretion balance study, Group A, single oral administration, 5 mg/kg:
Following a single oral administration of (14C)-test material at a nominal dose level of 5 mg/kg, elimination of the radioactivity was rapid and complete. The majority of excreted radioactivity was detected in the urine. A high level of radioactivity was also recovered in the cage washings (24.1 % male, and 19.5 % female) which was most likely due to urinary contamination of the metabowl. If the values for urine and cage wash are combined, 95.3 % and 92.3 % of the total administered radioactivity was recovered within 24 h for male and female animals respectively. Faecal excretion accounted for 8.2 % in male animals and 3.6 % in females, the major proportion being excreted 24 h after dosing. At 168 h, most of the tissues contained levels of radioactivity below the limit of detection. Low levels were detected in carcass, skin, fat, liver (male only) and kidney of both sexes ranging from 0.010 to 0.167 μg equiv/g.
Excretion balance study, Group B, multiple oral administration, 5 mg/kg:
As with the single oral administration, elimination of radioactivity was rapid and complete. Urinary excretion accounted for the majority of recovered radioactivity, a combined mean of 89.0 % and 86.5 % being recovered in the urine and cage wash within 24 h for male and female animals respectively. Elimination in the faeces accounted for 5.3 % in male animals and 4.9 % in females. At 168 h, most of the tissues contained levels of radioactivity below the limit of detection. In male animals, carcass, skin, plasma, fat, liver, kidney and adrenals contained less than 0.1 μg equiv/g. In female animals, carcass, skin, plasma, fat, spleen, kidney, ovaries, uterus and adrenals contained less than 0.2 μg equiv/g.
Excretion balance study, Group C, single oral administration, 100 mg/kg:
Following administration of (14C)-test material at the high dose level (100 mg/kg) the total mean recovered radioactivity was 104.9 % and 92.1 % in male and female animals respectively. The major route of elimination was in urine. However, the time course was extended compared with the low dose studies, taking 48 h to eliminate 85.4 % (male) and 76.8 % (female) of the administered dose in the urine and cage wash. Faecal elimination of radioactivity accounted for 12.5 % and 9.1 % in male and female animals respectively. At 168 h the majority of tissues contained detectable levels of radioactivity of which skin (male 7.401 μg equiv/g, female 7.328 μg equiv/g), fat (male 23.03 μg equiv/g, female 20.38 μg equiv/g), ovaries (5.854 μg equiv/g) and adrenals (male 6.698 μg equiv/g, female 5.441 μg equiv/g) contained levels above 5 μg equiv/g. No radioactivity was recovered as (14C)-carbon dioxide. - Test no.:
- #1
- Toxicokinetic parameters:
- half-life 1st: 6.354 h (male) and 4.4234 h (female)
- Test no.:
- #1
- Toxicokinetic parameters:
- AUC: 252.0 μg equiv.h/g (male) and 182.7 μg equiv.h/g (female).
- Remarks:
- Nominal dose level of 5 mg/kg
- Test no.:
- #1
- Toxicokinetic parameters:
- AUC: 8 449 μg equiv.h/g(male) and 7 884 μg equiv.h/g (female)
- Remarks:
- Dose level of 100 mg/kg
- Metabolites identified:
- yes
- Details on metabolites:
- Metabolite Profiling:
- Urine: Urine samples were pooled at the 0 to 6, 6 to 12, 12 to 24 and 24 to 48 h intervals according to dose group. Collectively, these pools represented approximately 70% of the administered dose and were analysed using HPLC and TLC.
Radiochromatograms obtained by TLC compared favourably with the HPLC profiles of these samples. The major peak co-eluted with a standard solution of the test material under these conditions and accounted for between 38.63 % and 65.58 % of the administered radioactivity excreted between 0 and 48 h.
The chromatograms also showed one major metabolite accounting for up to 32.6 % of the dose (Group B male). There were at least six minor components although all of them were less than 2 % of the administered radioactivity, most of them considerably less.
All samples from both male and female rats were qualitatively similar. However, in the 5 mg/kg single dose study, male rats metabolised (14C)-test material to a greater extent than females. After correction for the radioactivity recovered in cage wash, at 48 h approximately 66 % of the radioactivity administered was excreted in urine as parent in males compared to approximately 83 % in females. In the repeat dose study, a similar sex difference was observed but metabolism of the test material was approximately 10 to 20 % higher in both sexes. A similar result was obtained for the high dose group. These values were calculated using the percent total from TLC traces.
Using representative urine samples, enzymic digests were performed using p-glucuronidase H-1 (Helix pomatia), B-1 (Bovine liver), or sulphatase (Patella vulgata) gave identical profiles to those obtained in the control incubation, up to eight peaks being displayed in each chromatograph and sharing a similar profile.
A portion of urine from the same pool was incubated under acidic conditions using 2.5 M HCl. In comparison with the chromatogram obtained from untreated urine, the chromatography profile is similar after hydrolysis with 2.5 M HCl with the exception that a number of lipophilic components were observed. These were produced due to degradation of (14C)-test material under strong acid conditions as evidenced by the fact that they appeared in samples which prior to hydrolysis, only contained parent compound.
Treatment with 5 M alkaline did not appear to affect the major components of the chromatogram to any great extent.
- Faeces: Faeces samples were pooled at the 0 to 24 and 24 to 48 h intervals according to dose group. They were analysed using HPLC, TLC and LC-MS.
The major peak in the 0 to 24 h samples, which accounted for approximately 60 % of the radioactivity, co-eluted with a standard solution of the test materal and the chromatograms were similar to those obtained for urine. The increased metabolism of the test material comparing males and females and comparing single and repeat doses also mirrored that observed for urine.
Characterisation and identification of radiolabelled metabolites in urine and faeces using LC-MS
The test material was confirmed as the major component in both urine and faeces, giving an HPLC peak which eluted at a similar retention time and had a similar mass spectrum to an authentic standard of the test material. The mass spectrum showed two ions, a pseudomolecular ion [M-H]- at m/z 213 and a fragment at m/z 141 indicating loss of the propionic acid moiety. Between 42.68 % and 55.52 % of the dose was excreted as the test material in urine and faeces for males and between 55.35 % and 68.93 % in females.
The mass spectrum of the radio labelled HPLC peak at 8 minutes, showed a [M-H]^- at m/z 229 and a fragment at m/z 157. This result would indicate that the metabolite was hydroxylated test material but the exact position of hydroxylation could not be determined by mass spectroscopy. Between 23.38 % and 34.22 % of the dose was excreted as hydroxylated test material in urine and faeces for males and between 4.18 % and 7.34 % in females. No interpretable spectra were obtained for the minor urinary metabolites.
Identification of the major urinary metabolite of (14C)-test material using 1H and 13C-NMR:
Comparison of the NMR spectra obtained for the test material and the purified metabolite confirmed hydroxylation of the 2-methyl moiety of the test material.
The most notable feature of the proton spectrum for the metabolite was the loss of the methyl signal at ca. 2.3 ppm and the appearance of a pair of doublets at ca. 4.7 ppm. This signal was consistent with two non-equivalent protons most likely produced by restricted rotation of a hydroxy-methyl group due to hydrogen bonding between the hydroxyl group and the propionic acid moiety. The carbon-13 spectrum was also consistent with the formation of an hydroxymethyl moiety evidenced by the shift in the methyl carbon from ca. 16.5 to ca. 60.0 ppm. The metabolite was therefore identified as Hydroxymethyl-test material.
Confirmation of Carboxy-test material in rat urine by LC-MS (MRM):
Untreated and β-glucuronidase treated urine pools (low and high dose female, 0 - 24 h) were analysed by high performance liquid chromatography-mass spectrometry (LC-MS) using Multiple Reaction Monitoring (MRM) to confirm the presence of Carboxy-test material. The presence of Carboxy-test material was confirmed in urine pools from both dose groups (HPLC retention time ca. 3.02 min). A semi-quantitative method involving urine spiked with known amounts of the authentic standard estimated levels to be 0.05 % and 0.07 % for low and high dose levels respectively. - Conclusions:
- Under the conditions of the study, the test material was absorbed rapidly and extensively at both dose levels. The distribution of radioactivity was determined at the low dose level. Levels of radioactivity were similar at all time points. The highest levels of radioactivity (greater than 5 μg equiv/g) were detected in heart, lung, liver, kidney, thyroid, adrenals, ovaries and uterus. All other tissues sampled contained levels less than 5 μg equiv/g.
The only significant components in urine and faeces were parent and Hydroxymethyl test material. None of the administered radioactivity was detected in expired air (100 mg/kg) and there was little accumulation in any of the tissue samples excised after the 168 h period or in the carcass. Apart from an increase in the amount of Hydroxymethyl test material produced in males, there was no significant sex difference observed in any of the parameters investigated in this study. - Executive summary:
The absorption, distribution, metabolism and excretion of (14C)-test material was investigated after single and multiple oral administrations to the rat (nominally 5 mg/kg and 100 mg/kg body weight).
(14C)-test material (radiochemical purity 99.5 %) was formulated in 1 % carboxymethyl cellulose and a single oral dose (15 μCi/animal) was administered to the following groups of Wistar Crl: (Wl)BR strain rats:
Dose group A: Excretion balance study at 5 mg/kg in 5 males and 5 females for 168 h.
Dose group B: Excretion balance study at 5 mg/kg in 5 males and 5 females for 168 h. Non-radiolabelled test material was administered daily as a single oral dose for 14 days. Twenty-four hours after receiving the last dose, a single oral dose of (14C)-test material was administered.
Dose group C: Excretion balance study at 100 mg/kg in 5 males and 5 females for 168 h.
Dose group D: Pharmacokinetic study at 5 mg/kg in 5 males and 5 females for 168 h.
Dose group E: Pharmacokinetic study at 100 mg/kg in 5 males and 5 females for 168 h.
Dose group F: Tissue distribution study at 5 mg/kg in 12 males and 12 females (3 sub-groups of 4 animals for each sex) for 0.5, 5 and 6 h.
Samples were analysed by liquid scintillation counting directly or following solubilisation or combustion.
HPLC and TLC were used to profile radiolabelled metabolites in pooled urine and faeces samples from dose groups A, B and C.
Pooled urine and faeces from male rats collected up to 48 h were analysed by LC-MS (groups A and C) and NMR (group C, urine only). The spectra obtained were compared to those of an authentic standard of the test material.
An additional experiment was carried out using LC-MS (MRM) to confirm the presence and concentration of Carboxy-test material in female urine (groups A and C). Carboxy-test material is a known plant metabolite.
Excretion balance studies (Dose groups A, B and C) - mean data, n = 5
The excretion and tissue retention of radioactivity was determined after single and multiple oral doses of (14C)-tets material at nominal dose levels of 5 and 100 mg/kg body weight. Radioactivity was rapidly absorbed and excreted predominately in urine. For dose groups A and B up to 98.3 % of the urinary radioactivity was recovered within 24 h. Repeat administration of the test material had no apparent effect on the rate or route of excretion.
For dose group C up to 96.4 % of the urinary radioactivity was recovered within 48 h. There was no accumulation of radioactivity in any of the tissues excised after the 168 h study period. In the pilot study using two animals, expired air was collected for 168 h after dosing. No radioactivity was recovered as (14C)-carbon dioxide.
Pharmacokinetics studies (Dose groups D and E) - mean data, n = 5
In the high dose group there was an increase in the time taken to reach maximum concentrations in the plasma levels. This was mirrored in the elimination in urine with significant levels of radioactivity being present up to 48 h in the high dose groups.
Tissue distribution study (Dose group F) - mean data, n=4
The distribution of radioactivity (expressed as μg equiv./g) after a single oral dose of (14C)-test material at a nominal dose level of 5 mg/kg body weight was determined at various time points.
Levels of radioactivity for the above tissues were similar at all time points. For both sexes, the highest levels of radioactivity were detected in heart, lung, liver, kidney, thyroid and adrenals at between 0.5 and 3 h after dosing. For female animals, high levels of radioactivity were also detected in ovaries and uterus at 3 h after dosing. All other tissues sampled contained levels less than 5 μg equiv/g.
Characterisation of metabolites
The metabolite profiles of urine and faeces samples from both male and female rats showed that (14C)-test material was excreted predominately as parent (42.68 % to 68.93 % of the administered dose). Hydroxymethyl test material was also present (4.18 % to 34.22 % of the administered dose). There were at least six additional minor components but none of these metabolites accounted for more than 2 % of the administered radioactivity. There was a quantitative sex difference in metabolism with males excreting a greater proportion of the dose as Hydroxymethyl test material (23.38 % to 34. 22 %) compared to females (4 .18 % to 7. 34 %). There was no evidence that metabolism was affected by repeat administration of the test material. The presence of Carboxy-test material was confirmed in urine which accounted for up to 0.07 % of the administered dose.
Under the conditions of the study, the test material was absorbed rapidly and extensively at both dose levels. The distribution of radioactivity was determined at the low dose level. Levels of radioactivity were similar at all time points. The highest levels of radioactivity (greater than 5 μg equiv/g) were detected in heart, lung, liver, kidney, thyroid, adrenals, ovaries and uterus. All other tissues sampled contained levels less than 5 μg equiv/g.
The only significant components in urine and faeces were parent and Hydroxymethyl test material. None of the administered radioactivity was detected in expired air (100 mg/kg) and there was little accumulation in any of the tissue samples excised after the 168 h period or in the carcass. Apart from an increase in the amount of Hydroxymethyl test material produced in males, there was no significant sex difference observed in any of the parameters investigated in this study.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- other: Expert evaluation of study findings
- Adequacy of study:
- supporting study
- Study period:
- not applicable
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- secondary literature
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Objective of study:
- absorption
- excretion
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- This evaluation summarises and assesses the results of the investigations on the plasma kinetics of 14C-test material in male rats after single and repeated oral administration (14- or 60-days pretreatment).
- GLP compliance:
- no
- Remarks:
- Summary of data
- Species:
- rat
- Type:
- absorption
- Results:
- Not measured
- Observation:
- other: Not examined
- Test no.:
- #1
- Toxicokinetic parameters:
- AUC: Not specified
- Metabolites identified:
- not measured
- Conclusions:
- Due to saturation of excretion capacity, the plasma kinetics of 14C-test material in rats was is non-linear with dose. After treatment for 14 days, kinetic parameters changed progressively with dose at 40 mg/kg bw and above. After treatment for 60 days, deviation of kinetic parameters from linearity was evident at 30 mg/kg bw. Thus, there is evidence for saturation of excretion of the test material starting at dose levels of approximately 30 - 40 mg/kg bw. Long-term administration of dose above this threshold are likely to lead to progressive toxicity over time which will compromise the health and survival of the rats.
- Executive summary:
This evaluation summarises and assesses the results of the investigations on the plasma kinetics of 14C-test material in male rats after single and repeated oral administration (14- or 60-days pretreatment).
Due to saturation of excretion capacity, the plasma kinetics of 14C-test material in rats was is non-linear with dose. After treatment for 14 days, kinetic parameters changed progressively with dose at 40 mg/kg bw and above. After treatment for 60 days, deviation of kinetic parameters from linearity was evident at 30 mg/kg bw. Thus, there is evidence for saturation of excretion of the test material starting at dose levels of approximately 30 - 40 mg/kg bw. Long-term administration of dose above this threshold are likely to lead to progressive toxicity over time which will compromise the health and survival of the rats.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 August 2000 to 26 January 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Objective of study:
- distribution
- metabolism
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 860.1300
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Radiolabelling:
- yes
- Remarks:
- Goat
- Species:
- other: Goat
- Strain:
- other: British Saanen
- Details on species / strain selection:
- The test material is an established broad leaf herbicide. During and following its use residues may result in, or on, plant material destined to be fed to food producing animals. It is necessary, therefore, as part of the safety evaluation, to investigate the extent to which these residues may be transferred to animal tissues and milk, which may be destined for human consumption and also to establish the nature of any transferred residues. This study was designed to investigate the disposition and metabolic fate of [14C]-test material in the lactating goat following multiple oral administration.
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 78.8 kg and 82.2 kg on arrival.
- Housing: On the day prior to the start of the study, the animals were transferred to metabolism crates suitable for the separate collection of urine and faeces.
- Diet: The animals were offered hay ad libitum and the amount consumed each day measured. In addition, the animals were fed protein concentrate rations at an approximate rate of 1 kg per day (500 g at each time of milking).
- Water: Ad libitum mains tap water.
- Acclimation period: The goats were acclimatised to the experimental unit for a period of 7 days prior to dosing. The animals were milked twice daily at ca. 0830 h and ca. 1630 h. The animals were weighed during the acclimatisation period, the last occasion being 24 h prior to the first dose administration
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 34 - 78 %
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle.
- Route of administration:
- oral: gavage
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Low Dose Formulation (5 ppm):
The dose solution was prepared by weighing 6.90 mg of [14C]-test material and 89.32 mg of non-radiolabelled test material into a 5 mL capacity volumetric flask which was made up to volume with acetone. Sub-samples (3 x 10 μL) of this dose solution were transferred to 10 mL volumetric flasks and made up to volume with acetone. Duplicate aliquots (100 μL) were taken from each flask for LSC analysis to determine the concentration of the test material in the dose solution. The specific activity of the [14C]-test material in the dose solution was determined as 9.751 μC.mg^-1. Aliquots of the dose solution (volume calculated from concentration of dose solution) were then dispensed into 14 capsules (272 μL per capsule) and the solvent allowed to evaporate in a flow of air. A further 2 aliquots were taken from the dose solution (one at the beginning of capsule dispensing and one at the end), and made up to 20 mL with acetone. Duplicate samples from these aliquots (25 μL) were subsequently analysed by LSC to determine the amount of radioactivity applied to each capsule.
- High Dose Formulation (50 ppm):
The dose solution was prepared by weighing 73.14 mg of [14C]-test material and 953 mg of non-radiolabelled test material into a 5 mL capacity volumetric flask, which was made up to volume with acetone. Sub-samples (3 x 10 μL) of this dose solution were transferred to 10 mL volumetric flasks and made up to volume with acetone. Duplicate aliquots (25 μL) were taken from each flask for LSC analysis to determine the concentration of the test material in the dose solution. The specific activity of the [14C]-test material in the dose solution (volume calculated from concentration of dose solution) was determined as 9.357 μC.mg^-1. Aliquots of the dose solution were then dispensed into 15 capsules (255 μL per capsule) and the solvent allowed to evaporate in a flow of air. A further 2 aliquots were taken from the dose solution (one at the beginning of capsule dispensing and one at the end) and made up to 100 mL with acetone. Duplicate samples from these aliquots (25 μL) were subsequently analysed by LSC to determine the amount of radioactivity applied to each capsule.
All the capsules were capped and stored at ca +4 °C until dosing.
A [14C]-test material radiochemical purity check was also carried out on the pre-dose formulation for confirmation prior to dosing. A radiochemical purity value of 97.0 % was determined by TLC analysis using the same method that was used for the dose stability checks. - Duration and frequency of treatment / exposure:
- The daily dose for each goat was administered over 7 consecutive days in 2 equal portions, one after morning milking (ca. 0830 h) and one following afternoon milking (ca. 1630 h). Goat 1 was dosed at the low dose level and Goat 2 at the high dose level.
- Dose / conc.:
- 5 ppm
- Remarks:
- Low dose level - 100 µCi per day
- Dose / conc.:
- 50 ppm
- Remarks:
- High dose level - 1000 µCi per day
- No. of animals per sex per dose / concentration:
- Two females
- Control animals:
- no
- Details on dosing and sampling:
- The appearance and behaviour of the animals were monitored daily.
Urine and faeces were collected during the day prior to the first dose administration and at intervals of 24 h following administration of the first daily dose until sacrifice. The cages were rinsed with water at each collection time and the rinses retained.
Milk samples were collected from each animal in the morning prior to administration of the first dose and then twice daily throughout the study period, immediately prior to the morning (ca. 0830 h) and afternoon (ca. 1630 h) doses. The final milk collection was made immediately prior to sacrifice.
The weights of urine, faeces, cage wash and milk samples were recorded and total radioactivity measured.
Approximately 23 h after administration of the final dose, the goats were stunned using a captive bolt, pithed and exsanguinated by severance of the major neck vessels. The following biological fluids and tissues were removed and assayed for total radioactivity:
Whole Blood, Omental fat, Plasma, Renal fat, Kidneys, Skeletal muscle (maximum amounts of hind and fore quarter), Liver, Gastrointestinal Tract, Gastrointestinal Tract Contents.
Total radioactivity was measured in all samples collected except for the gastrointestinal tract and contents, which were collected as a precautionary measure.
Haematology and clinical chemistry parameters were measured immediately prior to sacrifice to back up daily animal observations. Although there was some degree of variation between the goats, the haematology and clinical chemistry parameters were within the normal range of values expected for goats.
Storage of Biological Samples
Biological samples were analysed on the day of collection wherever possible, prior to storage at ca. -20 °C. Where immediate analysis was not possible, biological samples were stored at ca. -20 °C prior to and following analysis. The longest frozen storage interval, of 2 - 3 months, was for the tissue samples (from initial extraction to HPLC analysis). Since metabolite profiles for the kidney methanol/water and enzyme hydrolysis extracts were similar, this indicates that the [14C]-residues in the tissues are stable over the longest storage interval.
DETERMINATION OF RADIOACTIVITY
Liquid Scintillation Counting (LSC)
All samples prepared in scintillant were counted for 5 min using a Packard 1600 TR Liquid Scintillation Analyser (Canberra Packard Limited) with automatic quench correction by external standard method. All biological samples were processed in duplicate. Scintillation vials were allowed to heat and light stabilise prior to analysis. Representative blank sample values were subtracted from sample count rates to give net d.p.m. per sample. A limit of quantification of 30 d.p.m. above background has been instituted in these laboratories. If results arose from data less than 30 d.p.m. above background, the fact is so noted in the results section of the report. 30 d.p.m. is typical of the background count obtained throughout the study.
Combustion Analysis
Samples for combustion were weighed into Combustocones® (Packard Instruments Company Limited) and combusted using a Model 306 or 307 Tri-Carb Automatic Sample Oxidiser (Canberra Packard Limited). The resultant 14CO2 was absorbed in Carbo-Sorb® and mixed automatically with Permafluor®E+ scintillation fluid. The efficiency of the combustion process was routinely checked several times throughout each production run by combusting quality control standards (Spec-ChecTM-14C). Combustion efficiency was shown to be greater than 97 % throughout the experimental period.
Preparation of Samples for LSC
Urine and cage wash
Duplicate samples of urine (ca. 0.9 g) and cage wash (ca. 0.9 g) were mixed with 10 mL Quickszint 1® (Zinsser) scintillation fluid prior to scintillation counting.
Plasma
Whole blood was centrifuged at ca. 3 500 rpm for 10 min and plasma and blood cells separated. Duplicate aliquots of plasma (ca. 0.5 g) were mixed with 10 mL Quickszint 1® (Zinsser) scintillation fluid prior to LSC.
Whole Blood
Duplicate samples of whole blood (ca. 0.3 g) were combusted as described previously.
Milk
Duplicate ca. 4 g aliquots were mixed with 10 mL Quickszint scintillation fluid to form a gel, then analysed by LSC.
Faeces
Faeces samples were homogenised in 3 mL/g of acetronitrile:water (50:50, v/v) using a Silverson overhead homogeniser. Samples of each homogenate (2 x ca. 0.3 g) were taken for combustion as described previously.
Liver and Kidney
Liver and kidney samples were allowed to thaw prior to homogenising in a Waring Blender. Duplicate homogenate samples (ca. 0.3 g) were taken for combustion as described previously.
Muscle
Muscle samples were initially processed frozen using a Hobart mincer then homogenised with dry ice using a Hobart blender to produce a fine powder. Samples of each homogenate (2 x ca. 0.3 g) were taken for combustion as described previously.
Omental and Renal Fat
Omental and renal fat samples were chopped into small pieces while partially frozen and then homogenised with dry ice using a Hobart blender to produce a fine powder. Duplicate homogenate samples (ca. 0.5 g) were taken for combustion as described previously.
EXTRACTION OF TISSUES, MILK, FAECES, AND URINE
Based on the residue levels, samples from Goat 2 were selected for extraction and chromatographic analysis. General procedures for the extraction and processing of samples prior to chromatographic analysis were as follows.
Each sample (except urine and milk) was extracted with solvent (3 mL/g) by maceration using an overhead Silverson macerator. Following each extraction, the solvent extract was separated from the post-extracted solid (PES) by centrifugation (3 000 rpm; 10 min) and decanting. Sub-samples of the PES were submitted for combustion analysis. The volume of individual and/or combined extracts was measured and aliquots analysed by liquid scintillation counting (LSC). Total combined extracts were concentrated under nitrogen. The final volume of concentrated extracts was measured and aliquots analysed by LSC. Details of additional procedures for different sample types are as follows:
Liver and kidney
Liver and kidney were processed using the same methods, except that the kidney enzyme aqueous extract was further processed for HPLC analysis following methanol precipitation, concentration and centrifugation. A number of stages were involved in the extraction process:
Approximately 30 g of liver and kidney were extracted by maceration on 2 occasions with methanol (3 mL/g) followed by one occasion with 3 mL/g of methanol:water (9:1, v/v). Between extractions, each sample was centrifuged (3 000 rpm; 10 min) and the supernatant removed by decanting. The supernatants were combined, the total volume was measured and aliquots removed for LSC. Supernatants were concentrated under nitrogen following hexane partitioning (3 x supernatant volume) to remove fat. Total volumes were measured and aliquots removed for LSC. The hexane extracts were not processed further, since all the radioactivity was quantitatively accounted for in each methanol layer following partitioning.
The PES from each liver and kidney sample was further extracted by mixing with approximately 2 g protease enzyme (Type 1: Crude, from bovine pancreas) and ca. 25 mL of 0.1 M potassium phosphate buffer (pH 7.4) and then homogenised using a Silverson overhead homogeniser. The sample was agitated at 36 °C for ca. 48 h in a water bath then centrifuged (10 000 r.p.m., 30 min). Duplicate aliquots of the supernatant were taken for LSC.
The PES remaining after protease extraction was then further extracted by mixing with approximately 2 to 3 g of pepsin (from porcine stomach mucosa) and then homogenising with 0.1 M hydrochloric acid (50 or 75 mL) using a Silverson overhead homogeniser. The sample was agitated at 37 °C for ca. 48 h in a water bath then centrifuged (4 000 rpm, 30 min). Duplicate aliquots of the supernatant were taken for LSC.
The activity of the pepsin enzyme was confirmed by hydrolysis of bovine haemoglobin to trichloroacetic acid soluble peptides (37 °C, pH 2) using u.v. spectrophotometry at 280 nm. The activity of the protease enzyme was confirmed by hydrolysis of N-t-BOC-L-Glutamic acid Phenyl Ester Substrate Solution (prepared by dissolving N-t-BOC-L-Glutamic acid Phenyl Ester in 1,4- dioxane) to N-t-BOC-L-Glutamic acid and phenol (37 °C, pH 7.8) using u.v. spectrophotometry at 270 nm.
The PES remaining after pepsin extraction was subjected to base hydrolysis in 3N sodium hydroxide and methanol (1:4:1, weight PES: ml base: mL methanol) at approximately 58 °C overnight in a shaking water bath. Following hydrolysis, the sample was adjusted to pH 2 using HCl and then filtered. The volume of filtrate was measured and aliquots removed for LSC. The remaining PES was further hydrolysed as above except that the 3N sodium hydroxide: methanol ratio was 1:1, the temperature of hydrolysis was increased to 70 °C and the time extended to 2 days. The sample was centrifuged (3 000 rpm, 15 min), the volume of supernatant measured and aliquots removed for LSC.
Milk
The 56 h and 152 h milk samples were extracted using the same methods. An approximately equal volume of acetonitrile was added to 50 mL of milk and the sample mixed by shaking and vortexing. The sample was centrifuged (4 000 rpm, 10 min) and aliquots of the supernatant were removed for liquid scintillation counting (LSC). Following removal of the acetonitrile by rotary evaporation at 30 °C, the aqueous phase was partitioned against diethylether (1 x 150 mL) followed by hexane (2 x 150 mL) to remove fatty material. The organic phases were not processed further, since all the radioactivity was quantitatively accounted for in the aqueous layer following partitioning. Aliquots of the aqueous phase were removed for LSC. The aqueous sample was concentrated under nitrogen and aliquots of the concentrated sample removed for LSC. The concentrated aqueous extract was centrifuged at 4 000 rpm for 10 min and the pellet back washed with acetonitrile (2 x 2 mL) by vortex mixing followed by centrifugation (4 000 rpm, 10 min) to recover radioactivity losses.
The supernatants from the back washes were combined with the supernatants of the concentrated aqueous extract and subsequently concentrated under nitrogen and centrifuged (4 000 rpm, 10 min). Duplicate aliquots were analysed by LSC.
Extraction efficiencies were calculated by comparing the levels of radioactivity remaining at key stages of processing with those determined following direct LSC of the milk samples prior to processing.
Faeces
For Goats 1 and 2 (48 h and 168 h) faeces samples, approximately 20 g of sample was extracted on 3 occasions with methanol (3 mL/g) by maceration as detailed previously. The extracts were combined and aliquots removed for LSC.
Goat 1 faeces (48 h and 168 h) combined methanol extracts were partitioned with hexane (3 x equal volume) to remove fatty material prior to concentration of the extracts. The Goat 1 (hexane partitioned methanol extracts) and Goat 2 (methanol extracts) were evaporated under nitrogen to a smaller volume and a sub-sample (1 mL) was centrifuged at 10 000 rpm for 5 min. Duplicate aliquots of the sub-sample supernatant were taken for LSC prior to HPLC analysis.
Urine
Sub-samples (1 mL) of 48 h and 168 h urine from Goat 2 were centrifuged at 10 000 rpm for 5 min (Jouan A14 centrifuge) to remove particulate material prior to chromatography. Urine samples (40 mL) from Goat 1 (48 h and 168 h) were concentrated under nitrogen to a lower volume (17 mL). In each case, duplicate aliquots of the supernatant were taken for LSC prior to HPLC analysis.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HPLC was performed using the following equipment and conditions:
HPLC Model:
HP 1100 Series Modules Liquid Chromatograph
HP 1100 Series Variable Wavelength Detector
HP 1100 Series Vacuum Degasser
HP 1100 Series Quaternary Pump
HP 1100 Series Thermostatted Autosampler
HP 1100 Series Thermostatted Column Compartment
Radiodetector Model: Radiomatic™ Flo-one®\Beta, Flow Scintillation Analyser (Model 150TR)
Fraction Collector: Gilson Model 202 Fraction Collector and fractions collected every minute
Conditions:
Column: YMC ODS-AQ (250 x 4.6 mm, 5 μm)
Guard Column: Phenomenex Security Guard (10 x 4.6 mm, 5 μm)
Mobile Phase A: 1 % Ammonium acetate in water
Mobile Phase B: 1 % Ammonium acetate in methanol
Gradient:
0 min: 90 % A, 10 % B
40 min: 10 % A, 90 % B
50 min: 10 % A, 90 % B
55 min: 90 % A, 10 % B
Flow Rate: 1.0 mL/min
Column Temperature: Ambient
u.v. Detector Wavelength: 254 nm
Scintillant: Ultima-Flo™M
Sample Analysis
Non-radiolabelled test material analytical reference standard was prepared in methanol:water (1:1, v/v) at a concentration of ca. 1 mg/mL. Sub-samples of sample extracts and urine were mixed with non-radiolabelled test material for accurate comparison of retention times.
For the urine samples, analysed with on line radiodetection, data were captured using the Multichrom system and quantification carried out by peak integration. The sample extracts were analysed using fraction collection/LSC as a means of radiodetection.
Fractions containing less than background levels + 30 d.p.m. of radioactivity were not included in further calculations. Background levels of radioactivity were calculated as the mean of the values for the first two fractions in each analytical run (i.e. void volume of the HPLC column).
During HPLC analysis, the recovery of radioactivity from the HPLC column was checked by quantifying the radioactivity eluted from the system and comparing this with the level injected. Column recoveries were determined for one of each sample type.
HPLC column recoveries for Goat 2 liver, kidney enzyme hydrolysis extract, 152 h milk, 48 h faeces, 168 h faeces, 48 h urine and 168 h urine were 110.0 %, 112.6 %, 155.7 %, 96.5 %, 97.5 %, 101.6 % and 96.8 %, respectively, demonstrating acceptable recovery of injected radioactivity. In some samples the column recoveries are high due to the low levels of radioactivity injected.
LIQUID CHROMATOGRAPHY – MASS SPECTROSCOPY (LC-MS)
HPLC Model: HP 1090 Series Liquid Chromatograph
Mass Spectrometer Model: Finnigan MAT TSQ 7000 Mass Spectrometer
Radiodetector Model: Packard 150TR Radiochemical Detector
Data Handling: Xcalibur Software Version 1.1
HPLC Spectrometer Conditions
Guard Column: Phenomenex Security Guard (10 mm x 4.6 mm, 5 μm)
Column: YMC ODS-AQ (250 mm x 4.6 mm, 5 μm)
Mobile Phase A: 0.5 % Ammonium acetate solution
Mobile Phase B: 0.5% Ammonium acetate in acetonitrile
Gradient:
0 min: 90 % A, 10 % B
40 min: 10 % A, 90 % B
50 min: 10 % A, 90 % B
55 min: 90 % A, 10 % B
Flow rate: 1 mL/min
Split: ca. 100 - 200 μL/min to mass spectrometer
Injection Volume: 5 - 100 μL
Mass Spectrometer Conditions
The following types of mass spectrometer techniques were employed for the analysis of samples:
A) Full Scan Analysis
Ionisation Mode: Negative ion electrospray ionisation
Spray Voltage: 4.5 kV
Capillary Temperature: 210 °C
Sheath Gas: 70 psi
Auxiliary Gas: 5 units
APICID Offset: 10 V
Scanning Mode: 50 – 500, scan rate 1 sec
B) Product Ion Scan Analysis
Collision Gas: Argon
Collision Gas Pressure: ca. 2 mTorr
Collision Offset: 15 V
Parent Ion: m/z 213
Product Ion Range: m/z 50 - 220
Scan Time: 1 sec
C) Multiple Reaction Monitoring
Collision Gas: Argon
Collision Gas Pressure: ca. 2 mTorr
Collision Offset: 15 V
Transition 1
Parent Ion: m/z 213
Product Ion: m/z 141
Scan Time: 0.5 sec
Transition 2
Parent Ion: m/z 215
Product Ion: m/z 143
Scan Time: 0.5 sec
CALCULATIONS
Total radioactivity data were collected and calculated using the validated Debra 5.2a computerised data acquisition system.
The following principles were used in the calculation of data within this report:
Total radioactivity data
Specific activity of [14C]-test material in the dose solutions:
Low Dose Level = 9.751 μCi/mg
High Dose Level = 9.357 μCi/mg
Calculated daily dose (mg) = ((mean total d.p.m. in a dose capsule / 2.22x 10^6 (d.p.m./µCi)) / Specific activity) x 2
Total dose administered (d.p.m.) = Mean total d.p.m. in a dose capsule x 14 doses
Total dose administered (mg) = (Total d.p.m. administered / 2.22 x 10^6 (d.p.m./µCi)) / Specific activity (µCi/mg)
Calculated dose level (p.p.m.) = Calculated daily dose (mg/day) / Mean food consumption on study (kg/day)
Total d.p.m. in samples = Mean d.p.m/g x Total Sample Weight (g)
μg equiv/g = Mean d.p.m.6^-1 in sample / specific activity (d.p.m/µg)
% Administered dose in samplle = (Total d.p.m. in sample / Total d.p.m. in administered dose) x 100
Chromatography data
Total radioactive residue (TRR) = Total d.p.m. in extract + P.E.S.
%Initial extraction efficiency (%TRR) = (Total d.p.m in extract / Total d.p.m. in extract + P.E.S) x 100
% Final extraction efficiency (% TRR) = % initial extraction efficiency (% TPR) x (Overall procedural recovery / 100)
Extraction efficiency (ppm) = (Total d.p.m. in extract / Total d.p.m. in extract + P.E.S) x ppm in sample.
% Final extraction efficiency (ppm) = %Extraction efficiency (ppm) x (Overall procedural recovery / 100)
Metabolite (%TPR) = % total peaks / 100) x % final extraction efficiency (TPR).
Metabolite (ppm) = (% total peaks / 100) x % final extraction efficiency (ppm)
Limit of detection (LOD) = Background (d.p.m.)
Limit of quantification (LOQ) = Background (d.p.m.) + 30 d.p.m. - Details on distribution in tissues:
- Nominal 5 ppm Dose Level, Goat 1
The highest residue levels in tissues at the time of killing (23 h post last dose) were recorded in the kidney, which accounted for only 0.007 μg equiv/g (0.002 % dose).
Levels in the liver, renal fat and omental fat did not exceed 0.001 μg equiv/g and concentrations in hind and fore muscle were below the limit of reliable determination.
Concentrations of total radioactivity in the whole blood and plasma were also low at 0.005 and 0.004 μg equiv/g, respectively. The amount of total radioactivity recorded in tissues represented <0.01 % of the administered dose. By the end of the study period the total recovery of radioactivity in all samples accounted for 97.3 % of the administered dose.
Nominal 50 ppm Dose Level, Goat 2
The highest concentration of total radioactivity found in tissues was in kidney at 0.097 μg equiv/g (0.002 % of the administered dose). The concentration of total radioactivity in liver was 0.031 μg equiv/g and this accounted for 0.005 % of the dose.
Levels of radioactivity in the omental and renal fat were 0.003 μg equiv/g and concentrations in the hind muscle and fore muscle were very low at 0.001 μg equiv/g.
Concentrations of total radioactivity in the whole blood and plasma were 0.029 and 0.035 μg equiv/g, respectively.
The amount of total radioactivity recorded in tissues represented < 0.01 % of the administered dose. The total recovery of radioactivity in all samples at the end of the study period accounted for 95.9 % of the administered dose.
Goat 2 (High Dose) Liver and Kidneys
Liver and kidney samples from Goat 2 were processed using a sequential extraction approach. Each sample was initially extracted with methanol, followed by methanol/water (9:1, v/v). Aqueous methanol extraction was followed by protease enzyme extraction and then by pepsin enzyme extraction of the post-extracted solid (PES). Each remaining PES was then base hydrolysed with 3M NaOH in methanol, initially at 58 °C for 20 h and then at 70 °C for 48 h in a shaking water bath. The combined methanol/water extracts were partitioned with hexane and then concentrated and centrifuged prior to HPLC analysis. Goat 2 kidney pepsin enzyme aqueous extracts were further processed for HPLC analysis by methanol precipitation followed by concentration and centrifugation of the supernatant.
Initial extraction efficiencies with methanol followed by methanol/water (9:1, v/v) were highest in the liver, with the combined initial extracts representing 58.0 % TRR (0.018 ppm). Kidney combined initial extracts represented 47.7 % TRR (0.046 ppm). Following hexane partitioning to remove co-extracted fat and subsequent concentration and centrifugation of the methanol/water extracts, minor losses of radioactivity were observed such that the final liver and kidney concentrated aqueous/methanol extracts were 54.7 % TRR (0.017 ppm) and 44.3 % TRR (0.043 ppm), respectively.
Protease enzyme hydrolysis recovered a further 5.7 % TRR (0.002 ppm) and 3.5 % TRR (0.003 ppm) from the liver and kidney PES samples, respectively. Following pepsin enzyme hydrolysis a further 20.1 % TRR (0.006 ppm) and 20.2 % TRR (0.020 ppm) was recovered from the liver and kidney PES samples, respectively. Losses of radioactivity from Goat 2 kidney pepsin enzyme processed aqueous extracts were low, with 16.6 % TRR (0.016 ppm) recovered. Both the liver and kidney PES remaining following the enzyme hydrolysis experiments were further hydrolysed with 3M NaOH in methanol as described previously. Levels of 2.7 % (0.001 ppm) and 13.5 % TRR (0.004 ppm) were released following incubation of the liver PES at 58 °C for 20 h and 70 °C for 48 h, respectively. Corresponding levels for the kidney PES were 2.4 % (0.002 ppm) and 13.0 % TRR (0.013 ppm).
Goat 2 (High Dose) Liver
HPLC analysis of the liver aqueous/methanol extracts, containing 0.017 ppm (54.7 % TRR), showed the presence of 1 polar unknown component at a retention time of 5 min.
Goat 2 (High Dose) Kidney
HPLC analysis of the kidney aqueous/methanol extracts, containing 0.043 ppm 44.3 % TRR), showed the presence of 3 components. The most abundant of these represented 0.030 ppm (31.1 % TRR) and was identified as test material by cochromatography. The remaining 2 components were unidentified and represented 0.003 ppm and 0.010 ppm (2.7 % TRR and 10.5 % TRR). The kidney pepsin enzyme hydrolysis extract, containing 0.016 ppm (16.6 % TRR), showed the presence of 1 component corresponding to test material by co-chromatography.
Goat 2 (High Dose) Kidney
The reconstructed ion chromatogram of the ion at m/z 213 contained a peak which corresponded to the small peak in the radiochromatogram with a retention time of 25.21 min. The low intensity of the signal resulted in a spectrum containing numerous background ions. However, the spectrum contained the ion at m/z 213, its chlorine atom associated isotope peak at m/z 215 and the fragment ion at m/z 141. The low amount of material in this sample prevented the generation of a product ion spectrum. However, the mass spectrometer was programmed to monitor the transition from 213 => 141 and 215 => 143, as these transitions were characteristic of the test material. The retention times, the full scan spectrum and the reconstructed ion chromatograms of the transitions from m/z 13 =>141 and m/z 215 => 143 were consistent with those observed for authentic test material. Therefore, this component was characterised as the test material. - Details on excretion:
- Nominal 5 ppm Dose Level, Goat 1
Following 14 twice daily oral administrations of [14C]-test material at a nominal dose level of 5 ppm of daily food consumption per day, the major route of excretion was via urine, with 81.0 % of the administered dose accounted for by the end of the study period (23 h post last dose). Excretion via faeces over the same time period accounted for 11.0 % of the dose.
Radio-HPLC analysis of urine and faeces indicated that the test material accounted for a mean of ca. 96.4 % of the radioactivity present in urine and a mean of ca. 90.3 % of the radioactivity present in faeces. This indicates that at least 88.0 % of an oral dose of [14C]-test material was excreted as parent molecule at the low dose level.
Residues in milk were low at all timepoints, with levels never rising above 0.001 μg equiv/g. Residues in milk in total accounted for 0.019 % of the administered dose.
Nominal 50 ppm Dose Level, Goat 2
Following 14 twice daily oral administrations of [14C]-test material at a nominal dose level of 50 ppm of daily food consumption per day, the main route of excretion was via urine with 64.5 % of the dose being recovered. Excretion via faeces over the same time period accounted for 24.9 % of the dose.
Radio-HPLC analysis of urine and faeces indicated that test material accounted for a mean of ca. 94.1 % of the radioactivity present in urine and a mean of ca. 92.5 % of the radioactivity present in faeces. This indicates that at least 83.7 % of an oral dose of [14C]-Test material was excreted as parent molecule at the high dose level.
Levels of total radioactivity in milk throughout the study period were low, with a maximum concentration of 0.013 μg equiv/g at 128 and 152 h declining to 0.007 μg.equiv/g at 175 h (8 h post last dose). The total recovery of radioactivity in milk at 175 h post dose accounted for only 0.019 % of the administered dose.
Goat 1 (Low Dose) Faeces and Urine
At the 5 ppm dose level (Goat 1), faeces (48 h and 168 h post dose) were extracted with methanol and the concentrated extracts analysed by HPLC following hexane partitioning and concentration to a lower volume and centrifugation. Two urine samples from Goat 1 (48 h and 168 h) were analysed by HPLC following concentration under nitrogen to a lower volume.
Extraction of faeces (48 h and 168 h) with methanol recovered 95.1 % TRR and 94.7 % TRR. Following hexane partitioning, to remove co-extracted fat, and subsequent concentration and centrifugation of the methanol extracts, some losses of radioactivity were observed such that the final faeces concentrated extracts collected were 95.1 % TRR (48 h) and 87.3 % TRR (168 h), respectively.
No procedural losses were incurred following concentration and centrifugation of urine samples (48 h and 168 h) prior to HPLC analysis.
Goat 2 (High Dose) Faeces and Urine
At the 50 ppm dose level (Goat 2), faeces (48 h and 168 h post dose) was extracted with methanol and the concentrated extracts analysed by HPLC, following concentration to a lower volume and centrifugation. Two urine samples from Goat 2 (48 h and 168 h) were analysed by HPLC following centrifugation to remove particulate matter.
Extraction of faeces (48 h and 168 h) with methanol recovered 97.0 % TRR and 96.7 % TRR, respectively. Following concentration and centrifugation of the methanol extracts, minor losses of radioactivity were observed such that the final faeces concentrated extracts collected at 48 h and 168 h were 97.0 % TRR and 94.6 % TRR, respectively.
There were minor procedural losses following centrifugation of urine samples (48 h and 168 h) prior to HPLC analysis, the samples analysed representing 99.8 % TRR (18.22 % dose) and 97.2 % TRR (9.22 % dose), respectively.
Goat 2 (High Dose) Milk
At the 50 ppm dose level (Goat 2), milk (48 - 56 h and 144 - 152 h post dose) was extracted with acetonitrile, partitioned with diethyl ether and hexane, then concentrated under nitrogen prior to analysis by HPLC. Extraction with acetonitrile recovered 89.8 % TRR and 86.6 % TRR from the 56 h and 152 h milk samples, respectively. Following partitioning with hexane and diethyl ether to remove co-extracted fat and subsequent concentration and centrifugation of the extracts, high losses of radioactivity were observed in both extracts such that the final values were 30.8 % TRR and 29.6 % TRR, respectively. The concentrations of radioactivity in the milk samples were low (0.011 μg.equiv/g and 0.013 μg.equiv/g). It is highly likely that this, as well as the low levels of radioactivity in the concentrated initial extracts, accounts for the low procedural recoveries. Since no or minor procedural losses occurred following the hexane partitioning, no fat soluble metabolites would have been present in the hexane extracts.
Goat 1 (Low Dose) Urine (48 h)
HPLC analysis of the 48 h urine sample, which contained 10.66 % of the total administered radioactivity (100 % TRR), showed the presence of 4 components. The most abundant of these represented 10.33 % of the total administered radioactivity (96.9 % TRR) and was identified as test material by co-chromatography. The remaining 3 minor components were unidentified and represented 0.04 % to 0.16 % of the total administered radioactivity (0.3 % to 1.5 % TRR).
Goat 1 (Low Dose) Urine (168 h)
HPLC analysis of the 168 h urine sample, which contained 13.70 % of the total administered radioactivity (100 % TRR), showed the presence of 4 components. The most abundant of these represented 13.14 % of the total administered radioactivity (95.9 % TRR) and was identified as test material by co-chromatography. The remaining 3 minor components were unidentified and represented 0.15 % to 0.21 % of the total administered radioactivity (1.1 % to 1.5 % TRR).
Goat 1 (Low Dose) Faeces (48 h)
HPLC analysis of the 48 h faeces extracts, which contained 2.23 % of the total administered radioactivity (95.1 % TRR), showed the presence of 2 components. The most abundant of these represented 2.19 % of the total administered radioactivity (93.3 % TRR) and was identified as test material by co-chromatography. The remaining minor component was unidentified and represented 0.04 % of the total administered radioactivity (1.8 % TRR).
Goat 1 (Low Dose) Faeces (168 h)
HPLC analysis of the 168 h faeces extracts, which contained 1.14 % of the total administered radioactivity (87.3 % TRR), showed the presence of 1 component which was identified as Test material by co-chromatography.
Goat 2 (High Dose) Urine (48 h)
HPLC analysis of the 48 h urine sample, which contained 18.20 % of the total administered radioactivity (99.8 % TRR), showed the presence of 4 components. The most abundant of these represented 17.42 % of the total administered radioactivity (95.4 % TRR) and was identified as Test material by co-chromatography. The remaining 3 minor components were unidentified and represented 0.24 % to 0.28 % of the total administered radioactivity (1.3 % to 1.6 % TRR).
Goat 2 (High Dose) Urine (168 h)
HPLC analysis of the 48 h urine sample, which contained 9.22 % of the total administered radioactivity (97.2 % TRR), showed the presence of 7 components. The most abundant of these represented 8.79 % of the total administered radioactivity (92.7 % TRR) and was identified as Test material by co-chromatography. The remaining 6 minor components were unidentified and represented 0.02 % to 0.13 % of the total administered radioactivity (0.2 % to 1.3 % TRR).
Goat 2 (High Dose) Faeces (48 h)
HPLC analysis of the 48 h faeces extracts, which contained 2.46 % of the total administered radioactivity (97.0 % TRR), showed the presence of 3 components. The most abundant of these represented 2.38 % of the total administered radioactivity (93.9 % TRR) and was identified as test material by co-chromatography. The remaining 2 minor components were unidentified and represented 0.02 % and 0.06 % of the total administered radioactivity (0.9 % and 2.2 % TRR).
Goat 2 (High Dose) Faeces (168 h)
HPLC analysis of the 168 h faeces extracts, which contained 3.80 % of the total administered radioactivity (94.6 % TRR), showed the presence of 3 components. The most abundant of these represented 3.66 % of the total administered radioactivity (91.1 % TRR) and was identified as test material by co-chromatography. The remaining 2 minor components were unidentified and represented 0.05 % and 0.09 % of the total administered radioactivity (1.3 % and 2.2 % TRR).
Goat 2 (High Dose) Milk (56 h)
Due to the very low residue levels in the 56 h milk final concentrated extract (30.8 % TRR, 0.003 ppm), no radiolabelled residues were observed as the levels of radioactivity were below the limit of quantification.
Goat 2 (High Dose) Milk (152 h)
HPLC analysis of the 152 h milk final concentrated extract, which represented a residue level of 0.004 ppm (29.6 % TRR), showed the presence of a single unidentified component. There was evidence for the presence of test material at a retention time of 29 min, but at levels less than the limit of quantification.
Confirmation of Test material in Urine, Faeces and Kidney by Mass Spectroscopy
The analysis of test material by negative ion electrospray ionisation liquid chromatography-mass spectrometry (ESI-LC-MS) revealed a peak with a retention time of 25.18 min. The spectrum of this peak contained the ion at m/z 213, which corresponded to the deprotonated molecular ion ([M-H]-). The spectrum also contained an ion at m/z 141.
The product ion spectrum of the deprotonated molecular ion ([M-H]-) at m/z 213, fragmented to form the product ion at m/z 141.
This multiple reaction monitoring approach permitted a specific and sensitive method for the characterisation of the test material at low concentrations.
Sample Analysis
Goat 2 (High Dose) Urine (168 h)
The reconstructed ion chromatogram of the ion at m/z 213 contained a peak which corresponded to the peak in the radiochromatogram with a retention time of 24.80 min. The spectrum contained the ion at m/z 213 and its chlorine atom associated isotope peak at m/z 215. The fragment ions at m/z 141 and 143 were also present in the spectrum. The product ion spectrum of the ion at m/z 213, fragmented to form the ion at m/z 141. The retention time of both full scan spectrum and product ion spectrum for this component were consistent with those observed for authentic test material. Therefore, this component was characterised as the test material.
Goat 2 (High Dose) Faeces (168 h)
The reconstructed ion chromatogram of the ion at m/z 213 contained a peak which corresponded to the peak in the radiochromatogram with a retention time of 24.98 min. The spectrum contained the ion at m/z 213 and its chlorine atom associated isotope peak at m/z 215. The fragment ions at m/z 141 and 143 were also present in the spectrum. The product ion spectrum of the ion at m/z 213, fragmented to form the ion at m/z 141. The retention time, full scan spectrum and product ion spectrum of this component were consistent with those observed for authentic test material. Therefore, this component was characterised as the test material. - Metabolites identified:
- not measured
- Conclusions:
- Under the conditions of the study the test material is rapidly excreted by ruminants, mainly as the unchanged compound. Low doses are excreted mainly via the urine, indicating a high level of absorption. The higher dose showed an increased proportion excreted in the faeces, possibly indicating a lower absorption efficiency. Excretion via the milk was minimal. There was no evidence for accumulation in tissues; levels were negligible in most tissues within 23 hours of the final dose.
- Executive summary:
The metabolism and residue profile of the test material was assessed according to EPA Pesticide Assessment Guidelines, Subdivision 0, Series 171-4: Nature of the Residue in Livestock (Residue Test Guidelines, OPPTS 860.1300, Nature of the Residue - Plants, Livestock, US Environment Protection Agency, August 1996), and FAO Guidelines as Recommended by EU Commission Directive 96/68/EC Annex 1, Section 6.2, (21 October 1996) and in compliance with GLP.
This report describes a study in which the metabolism and residue profile of the test material, an established broad leaf herbicide, was investigated in the lactating goat. The test substance was uniformly radiolabelled with carbon-14 in the aromatic ring.
Two lactating goats each received a twice-daily oral administration of [14C]-test material in gelatin capsules over a period of 7 consecutive days. Goats 1 and 2 received doses at nominal levels of 5 ppm and 50 ppm of daily food consumption per day, respectively. Milk was collected twice daily immediately prior to each dosing. Faeces and urine were collected during the day prior to the first dose and at 24 h intervals after the first dose. The animals were killed at ca.23 h after the last dose and selected tissues collected. All biological samples were assayed for total radioactivity by liquid scintillation counting (LSC), either directly or following sample combustion.
The major route of excretion at both dose levels was via urine, with 81.0 % (Goat 1, low dose) and 64.5 % (Goat 2, high dose) of the administered dose recovered.
Excretion in faeces accounted for 11.0 % and 24.9 % of the administered dose for the low dose and high dose levels, respectively. Radio-HPLC analysis of urine and faeces from Goat 2 ndicated that the test material accounted for a mean of ca. 94.1 % of the radioactivity present in urine and a mean of ca.92.5 % of the radioactivity present in faeces. This indicates that ca.83.7 % of an oral dose of [14C]-test material was excreted as parent molecule at the high dose level. Radio-HPLC analysis of urine and faeces from Goat 1 indicated that the test material accounted for a mean of ca. 96.4 % of the radioactivity present in urine and a mean of ca. 90.3 % of the radioactivity present in faeces. This indicates that ca. 88.0 % of an oral dose of [14C]-test material was excreted as parent molecule at the low dose level.
Concentrations of radioactivity in milk were low at both dose levels. At the 50 ppm dose level a maximum concentration of 0.013 μg equiv/g at 128 and 152 h post dose declined to 0.007 μg equiv/g at 176 h post dose. Levels did not rise above 0.001 μg equiv/g at the low dose level. For both dose levels, the total levels of residues in milk accounted for 0.02 % of the administered dose.
The highest tissue residue levels were found in the kidney where the concentrations were 0.007 μg equiv/g (low dose) and 0.097 μg equiv/g (high dose). Residue levels in the liver were 0.001 μg equiv/g (low dose) and 0.031 μg equiv/g (high dose). The overall distribution of total radioactivity within the tissues was broadly similar for both dose levels, with very low residue levels found in the renal fat, omental fat, hind muscle and fore muscle. Concentrations of total radioactivity in the whole blood and plasma were also low with levels of only 0.005 μg equiv/g and 0.004 μg equiv/g, respectively, recorded for the low dose goat. Corresponding levels for the high dose goat were 0.029 μg equiv/g and 0.035 μg equiv/g.
The amount of total radioactivity recorded in tissues of Goat 1 and Goat 2 represented <0.01 % of the administered dose.
The overall recovery of the total administered radioactivity was high for both Goat 1 (97.3 %) and Goat 2 (95.9 %).
Sub-samples of Goat 1 urine (48 h and 168 h) were concentrated under nitrogen to a lower volume. Sub-samples of Goat 2 urine (48 h and 168 h) were centrifuged only.
Faeces samples (48 h and 168 h) from Goat 1 and Goat 2 were extracted with methanol. The radioactivity in the combined methanol extracts was then quantified by liquid scintillation counting (LSC). For faeces and tissues, radioactivity remaining in the post-extracted solids (PES) was quantified by combustion analysis and LSC. Goat 1 faeces combined methanol extracts were then partitioned with hexane. For each sample, the solvent extracts were combined and concentrated under nitrogen to a lower volume.
Sub-samples of milk (56 h and 152 h) were processed using acetonitrile precipitation to remove protein followed by diethylether and hexane partitioning to remove fats. The aqueous phase was concentrated, combined with acetonitrile washes of the protein pellet and concentrated further.
No procedural losses occurred following processing of Goat 1& (48 h and 168 h) urine samples. Procedural recovery values of 99.8 % and 97.2 % for the 48 h and 168 h urine samples from Goat 2, respectively, indicated quantitative recovery of residues.
Faeces final extraction efficiency values of 95.1 % TRR (Total Radioactive Residue) (48 h) and 87.3 % TRR (168 h) were obtained for Goat 1, with values of 97.0 % TRR and 94.6 % TRR observed for Goat 2 48 h and 168 h faeces samples, respectively.
Final extraction efficiencies of 29.6 % and 30.8 % TRR were observed for the 56 h and 152 h milk samples, respectively. The low values for milk were due to the low initial residue levels of 0.011 μg equiv/g to 0.013 μg equiv/g and attributed to the low levels of radioactivity in the concentrated initial extracts. No or minor procedural losses occurred following hexane partitioning. HPLC analysis of Goat 2 milk (56 h and 156 h) showed the presence of the test material by co-chromatography, but at levels close to or less than the limit of quantification.
Liver and kidney samples from Goat 1 and Goat 2 were processed using a sequential extraction approach due to low initial extraction efficiencies using solvent alone. Aqueous methanol extraction was followed with protease enzyme extraction, pepsin enzyme extraction and finally by caustic methanol extraction. Initial extraction efficiencies with methanol, followed by methanol/water (9:1, v/v), were highest in the liver with the combined initial extracts representing 58.0 % TRR (0.018 ppm). Kidney combined initial extracts represented 47.7 % TRR (0.046 ppm). Minor losses of radioactivity were observed following processing, such that the final liver and kidney concentrated aqueous/methanol extracts represented 54.7 % TRR (0.017 ppm) and 44.3 % TRR (0.043 ppm), respectively.
Residues remaining in post extracted solids from both the liver and kidney samples were further extracted by protease enzyme hydrolysis, recovering 5.7 % TRR (0.002 ppm) and 3.5 % TRR (0.003 ppm), respectively. Following pepsin enzyme hydrolysis a further 20.1 % TRR (0.006 ppm) and 20.2 % TRR (0.020 ppm) was recovered from the liver and kidneys, respectively. Similar levels of radioactivity were released from the liver and kidney PES samples following caustic extraction, where up to 2.7 % (0.002 ppm) and 20.5 % TRR (0.013 ppm) was released following incubations at 58 °C for 20 h and 70 °C for 48 h, respectively.
Processed urine from both goats was analysed directly by reverse phase HPLC with on-line radiodetection with peak integration. Concentrated and processed initial extracts of faeces, liver, kidneys and milk were analysed by reverse phase HPLC with fraction collection/LSC. Further extracts were not processed as the small quantity of radioactivity present precluded meaningful attempts to identify the species present.
Radio-HPLC analysis of urine samples from both the low and high level dosed goats indicated that the test material was the major residue accounting for 96.9 % TRR (10.33 % dose, this refers to the cumulative dose over the 7 days of the study) and 95.9 % TRR (13.14 % dose) in Goat 1&, 48 h and 168 h, respectively. Totals of 95.4 % TRR (17.42 % dose) and 92.7 % TRR (8.79 % dose) were recovered in the 48 h and 168 h urine samples, respectively, from Goat 2&. Three additional minor components were detected in Goat 1& urine, accounting for up to 0.16 % dose (48 h) and 0.21 % dose (168 h). In Goat 2& urine six additional minor components were detected, accounting for up to 0.28 % dose in the 48 h sample and 0.13 % dose in the 168 h sample. These minor components were not further identified due to the small amount of radioactivity present.
In the faeces samples, the test material was also the major component detected at both dose levels. Parent accounted for 2.38 % dose (93.9 % TRR) in the 48 h sample and 3.66 % dose (91.1 % TRR) in the 168 h sample, at the high dose level. Minor (unidentified) components accounted for up to 0.06 % dose in the 48 h sample and 0.09 % dose (2.2 % TRR) in the 168 h sample. At the low dose level parent accounted for 2.19 % dose (93.3 % TRR) in the 48 h sample and 1.14 % dose (87.3 % TRR) in the 168 h sample. A single minor unknown component, representing 0.04 % dose (1.8 % TRR) was detected in the 48 h sample.
Due to the very low residue levels (0.003 ppm, 30.8 % TRR) in the 56 h milk extracts from Goat 2, no radiolabelled residues were detected during HPLC analysis. Analysis of the 152 h milk sample indicated the presence of a single unidentified component (29.6 % TRR, 0.004 ppm). There was evidence for the presence of the test material in this sample, but at levels less than the limit of quantification. Confirmation of the test material in Goat 2 urine (168 h), faeces (168 h) and kidney was carried out using ESI-LC-MS. The reconstructed ion chromatograms for the urine, faeces and kidney were consistent, with each containing the ion at m/z 213 and its chlorine atom associated isotope peak at m/z 215, corresponding to the peak in the radiochromatograms of each sample analysis. The fragment ions at m/z 141 and 143 were also present. The retention time, full scan spectrum and product ion spectrum of this component were consistent with those observed for authentic the test material in each sample type, thereby confirming the component as the test material.
HPLC analysis of the liver aqueous/methanol extracts, containing 0.017 ppm (54.7 % TRR), showed the presence of 1 polar unknown component at a retention time of 5 min. HPLC analysis of the kidney aqueous/methanol extracts, containing 0.043 ppm (44.3 % TRR), showed the presence of 3 components. The most abundant of these represented 0.030 ppm (31.1 % TRR) and was identified as the test material by co-chromatography. The remaining 2 unidentified polar components represented 0.003 ppm and 0.010 ppm (2.7 % TRR and 10.5 % TRR). The kidney pepsin enzyme hydrolysis extract contained 1 component at 0.016 ppm (16.6 % TRR), corresponding to the test material by chromatography.
It is concluded that under the conditions of the study the test material is rapidly excreted by ruminants, mainly as the unchanged compound. Low doses are excreted mainly via the urine, indicating a high level of absorption. The higher dose showed an increased proportion excreted in the faeces, possibly indicating a lower absorption efficiency. Excretion via the milk was minimal. There was no evidence for accumulation in tissues; levels were negligible in most tissues within 23 hours of the final dose.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 August 2012 to 28 May 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Objective of study:
- distribution
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 860.1480
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Test Guideline 505
- Version / remarks:
- 2007
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Radiolabelling:
- no
- Species:
- cattle
- Strain:
- other: Friesian/Holstein dairy cows
- Details on species / strain selection:
- Regulatory guidelines indicate dairy cattle are the preferred ruminant species for use in livestock feeding studies.
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 3 - 12 years
- Weight at study initiation: 552 - 822 kg
- Housing: Cows used in this study were housed in individual pens measuring approximately 3.75 m x 3.45 m. Animals within each treatment group were housed adjacent to one another with nose-to-nose contact possible. Where cows from different treatment groups were housed next to each other, contact was prevented by a barrier.
- Diet: The cows were offered grass silage fed to appetite throughout the study. Assurances were obtained from the supplier of the silage that no treatments had been made to the crop during the current harvest year. The silage was chopped prior to feeding to reduce the amount dragged by the cows onto the feeding areas and provide a better assessment of feed intake. Silage remaining uneaten from the previous day was weighed and discarded and the weight of fresh silage offered was recorded. Samples of approximately 300 g of the silage offered were taken three times each week and stored at 5 ± 3 °C. Once each week, the three samples were mixed to form a composite sample. Approximately 400 g of this was used for a weekly dry matter analysis.
In addition, all animals were offered 8 kg of a pelleted compound feed, split into two feeds. Each week, a representative sample of the compound feed was taken for dry matter determination.
- Water: Water troughs provided the cows with ad libitum access to water.
- Acclimation period: at least 7 days
ENVIRONMENTAL CONDITIONS
- Ventillation: Ventilation of the buildings was supported by extractor fans.
- Photoperiod (hrs dark / hrs light): Animals were housed under natural light with artificial lighting where necessary.
- Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared by dissolving the appropriate amount of the test material in acetone. The prepared dosing solutions were transferred to colour-coded glass bottles and were stored in a refrigerator at 4 - 5 °C.
Samples of each week’s dosing solutions were analysed to confirm the concentrations of the test material. In addition, samples of the first week’s dosing solutions were re-analysed after storage at ca. 4 °C for approximately 7 and 14 days to confirm stability.
For each cow, the pelleted compound feed was placed into an appropriately colour-coded feeding bucket (green, blue, yellow or red for Groups A, B, C and D respectively). The dosing solution (40 mL) was added to the compound feed via a pipette.
DIET PREPARATION
- Storage temperature of food: In all cases, the compound feed was offered to the cows for consumption immediately after the addition of the dosing solution.
The intake of the compound feed was checked by visual examination of the feed buckets and any incomplete uptake was recorded. Incomplete uptake was noted on only three occasions during the study: Cow 9 refused 0.54 kg on the morning of 11th September 2012 (study Day 0) and 0.06 kg on the morning of 12th September 2012. Cow 12 refused 1.72 kg on the morning of 11 September 2012. Both cows were in the highest dosing group and were subsequently offered a small amount of molasses in their feed to overcome any palatability problems. This very low level of compound feed refusal gives confidence in the complete consumption of the test material. - Duration and frequency of treatment / exposure:
- The test material was administered to the cows for 28 (1x and 10x dose groups) or 29 (3x dose group) consecutive days. The treatment doses were administered to the cows twice daily at their individual feed stations.
- Dose / conc.:
- 0 mg/kg diet
- Remarks:
- (On a dry matter basis), Group A
- Dose / conc.:
- 194 mg/kg diet
- Remarks:
- (On a dry matter basis), Group B
- Dose / conc.:
- 582 mg/kg diet
- Remarks:
- (On a dry matter basis), Group C
- Dose / conc.:
- 1 940 mg/kg diet
- Remarks:
- (On a dry matter basis), Group D
- No. of animals per sex per dose / concentration:
- Group A: Two
Group B: Three
Group C: Three
Group D: Six - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The three doses used in the livestock feeding study were calculated from the maximum residue level of 38.8 mg/kg found in a Northern European residue trial. The test material was approved for one application of 1.5 kg a.s/ha. The dose selection used in the livestock feeding study was based on the maximum residue at day 7 from trials conforming to this GAP, however data from the other trials was used as confirmatory supporting data. Of the 11 trials following the single application GAP the mean and maximum 7 days after treatment (DAT) residue were 13.4 and 38.8 mg/kg respectively. Two trials where 2 x 1.5 kg were applied have day 7 data available. In one trial the day 7 residue was 12.3 mg/kg (almost exactly on the mean of the preceding data set). In the other trial the residue on day 7 after treatment (DAT) was 118 mg/kg. However, this result is deemed unreliable because the residue levels found were erratic (they increased markedly between days 0 and 3) and were considered as an outlier result in comparison to the rest of the dataset. Excluding this result, the maximum and mean residue 7 DAT is 38.8 mg/kg and 13.4 mg/kg respectively. The maximum residue level of 38.8 mg/kg obtained from the GLP trials is a wet weight measure. Assuming a theoretical feed intake per cow per day of 100 kg wet weight per cow per day the maximum residue would be 3880 mg wet weight/cow. To calculate the feeding level in terms of dry weight/dry matter (DM) an intake of 20 kg DM/day it can be assumed.
- Rationale for animal assignment: The cows were introduced to the study accommodation on 25th August 2012. On 31st August 2012, prior to Day 0, one cow became ill and had to be removed from the study. A replacement animal was moved into the accommodation on 4th September 2012 and the planned start of dosing was postponed to 11th September 2012, to allow the new cow to acclimatise. The individual milk yields were measured daily and the cows were allocated to their treatment groups on the basis of milk yield.
The cows were ranked in increasing order of recent milk yield. Two blocks of four cows and one block of six cows were formed from the cows ranked by milk yield data, such that within block variation in milk yield was minimised. Within each block, cows were allocated to each treatment group at random. One cow was allocated to each treatment in blocks one and two. In block three, one cow was allocated to each of treatments B and C and four cows were allocated to treatment D. Three of the cows allocated to treatment D were designated as the depuration animals. - Details on dosing and sampling:
- Individual body weights were obtained prior to dosing and on two further occasions during the study.
Milk Production
The cows were milked twice per day (morning and evening) in a four-abreast milking parlour, equipped with individual graduated collection jars. Animals were milked in order of increasing dose level, with control animals always being milked first. The milk yield was recorded on each occasion for each cow. Between milking sessions, the entire milking plant was cleaned with an appropriate dairy detergent / disinfectant and rinsed thoroughly.
Study Schedule
At the end of the dosing period, the cows were sacrificed for tissue collection between 15 and 21 hours after administration of the final dose, with the exception of three depuration cows from the 10x dose group. At the end of the dosing period, the depuration cows were transferred to the control diet and were sacrificed 3, 5 or 10 days after withdrawal of the test item from their diet.
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Whole Milk, Cream and Skimmed Milk Specimens:
On selected days of the study, a combined proportional whole milk sample for each cow was constructed from the evening and morning samples, on the basis of milk yields recorded at the corresponding milkings. For example, the proportional sample for Day 10 consisted of the morning sample for Day 10 and the evening sample for Day 9. A combined sample, of at least 200 mL, was constructed within 24 hours of collection of all contributing samples. For example, the proportional milk from a cow that produced a yield of 15 kg in the evening and 8 kg the following morning would be constructed from the evening and morning milk samples in a ratio of 15:8. After thorough mixing, 4 replicate sub-samples (R1 to R4) of approximately 50 mL were transferred to screw-cap HDPE bottles and transferred to a freezer set to maintain a temperature of less than -15 °C. Milk samples were placed in a refrigerator set to 5 ± 3 °C immediately after collection. Fridges were monitored and no significant deviations from these values were reported.
Processing of milk samples was carried out in order of increasing treatment, with control samples processed first.
On Day 22, an additional proportional sample of approximately 1 L was retained from the combined evening and morning milk yield for each cow and stored at approximately 5 °C overnight. After overnight storage, a combined proportional sample was constructed for each cow from the AM and PM samples, on the basis of milk yields recorded at the corresponding milkings. They were then separated into cream and skimmed milk samples by centrifugation for approximately 15 minutes at 3 000 rpm. Four replicate samples (R1 to R4) of approximately 50 mL skimmed milk and two replicate samples (R1 and R2) of approximately 20 g of cream were taken and transferred to a freezer set to maintain a sample temperature of less than -15 °C.
- Necropsy and Tissue Specimens: Cows were euthanised. Slaughter was effected by use of a captive bolt and a pithing rod while the cow was confined in a killing crate, followed by exsanguination. All cows were sacrificed within 15 to 21 hours of administration of the final dose, with the exception of the cows used to collect depuration data.
Tissue samples were collected from each cow immediately after slaughter. For all tissues, two replicate specimens (R1 and R2) of approximately 1 kg (or as available) were taken. Skeletal muscle specimens comprised approximately equal pieces of hind leg or flank, loin and diaphragm muscle. Fat samples consisted of approximately equal pieces of subcutaneous fat, mesenteric fat and perirenal fat. Liver samples were taken from at least 6 subsamples from different areas of the organ. Kidney samples were taken from at least three subsamples of each kidney. The weight of each tissue sample was recorded. All tissue samples were double wrapped in polythene bags. Immediately upon collection, the samples were stored in a polystyrene box containing ice packs to reduce the temperature and then transferred to a freezer set to maintain a temperature of less than -15 °C within approximately three hours of collection.
All samples remained frozen during shipment and were stored in temperature-monitored freezers at approximately ≤-18 °C on arrival.
Complete samples of muscle, fat, liver and kidney were homogenised in a Robot Coupe processor. Dry ice was used for fat specimens. After appropriate mixing of each sample, samples were transferred to HDPE plastic containers. No preparation was required for milk, skimmed milk or cream samples.
Analytical Methodology
Residues of the test material, were determined using a method that had been validated for the determination of test material, test material 2- ethylhexyl ester, test material glycine conjugate, HMCPP, CCPP and PCOC in whole milk, skimmed milk, cream, muscle, liver, kidney and fat. A summary of the method follows.
Residues were extracted from the sample by heating overnight with a methanolic sodium hydroxide solution. Following addition of sulphuric acid, monochloroacetic acid solution and acetonitrile, the extract was shaken with QuECHERS salts and then shaken with hexane. An aliquot of the acetonitrile layer was mixed with magnesium sulphate and aluminium oxide and, following centrifugation, an aliquot was evaporated to near dryness and reconstituted in a mixture of water, formic acid and methanol. A mixture of internal standards was added to the final extract prior to analysis by LC-MS/MS.
The method is designed to measure total test material residues, including its esters and conjugates. The method was validated as part of this study for the analysis of residues of test material, test material ethylhexyl ester, test material glycine conjugate, HMCPP, CCPP and PCOC. The limit of quantitation (LOQ) of test material, HMCPP, CCPP and PCOC in all matrices is 0.01 mg/kg.
Study specimens were analysed in batches, each batch containing at least one control specimen, one control specimen fortified at the limit of quantification and at least one control specimen fortified at a higher level. Recovery data were generated to cover the range of residues found in the specimens from the study.
Where extract solutions needed to be stored during the extraction process this was done in a cold room set at 4 °C. The procedural recoveries demonstrate the stability of the analyte during this storage.
Dosing solutions were diluted in acetone, followed by a mixture of water:methanol:formic acid (60:40:0.2, v/v/v/) prior to analysis by LC-MS/MS.
Method Validation
The analytical method was validated for the measurement of residues of test material, test material ethylhexyl ester, test material glycine conjugate, HMCPP, CCPP and PCOC in whole milk, skimmed milk, cream, muscle, liver, kidney and fat. For each specimen type, two control specimens, five specimens fortified at 0.01 mg/kg (proposed LOQ of the method) and five specimens fortified at 0.10 mg/kg were analysed. Validation was considered to be acceptable if the mean recovery for each matrix was in the range 70 to 120 %, with a relative standard deviation of less than 20 %.
In addition, selected specimen extracts were reanalysed after storage at approximately 4 °C, in order to assess the storage stability of the analytes in the final extracts. The limit of detection for each analyte in each matrix was estimated by comparing the response of the lowest calibration standard with the height of the baseline noise of a control specimen extract at the same retention time. The limit of detection is defined as the residue concentration (mg/kg) that would produce an instrument response equivalent to three times the baseline noise. - Details on distribution in tissues:
- Residue results are not corrected for procedural recoveries. Procedural recoveries of the main residue, the test material, were generally in the range 90 % to 110 %, so any correction of residues was considered to be unnecessary.
Residues of the test material, HMCPP, CCPP and PCOC in milk and tissues were measured using an analytical method based on LC-MS/MS. This method is designed to measure residues of test material that include esters and conjugates. The limit of quantitation (LOQ) for each of the analytes in milk, skimmed milk, cream, muscle, liver, kidney and fat is 0.01 mg/kg. The test material residue also includes any esters or conjugates.
Residues of the test material were found in all matrices from cows in the 1x, 3x and 10x dosing groups. Residues in whole milk reached a plateau after 1 to 3 days of dosing and remained stable throughout the dosing period. The residues in the 10x dosing group declined to less than the LOQ after 2 days of withdrawal of the test material from the diet. Residues of the test material did not partition selectively into skimmed milk and cream. Residues of the test material in muscle, liver, kidney and fat in the 10x dosing group showed a decline after withdrawal of the test material from the diet.
Residues of PCOC were found in cream (but not skimmed milk), liver and fat specimens from cows in the 10x dosing group only and in kidney specimens from cows in the 1x, 3x, and 10x dosing groups. Residues of PCOC in the 10x dosing group declined to less than the LOQ in liver after 3 days of withdrawal of the test material from the diet and in kidney and fat after 5 days of withdrawal of the test material from the diet.
No residues of HMCPP or CCPP were found in any of the specimens in any treatment group. Regression analysis for the test material in whole milk, skimmed milk and cream demonstrated a linear relationship between the dose level and the resulting residue concentration. A nonlinear relationship between the dose level and residue concentration was found for the test material in all other matrices and for PCOC in kidney. - Conclusions:
- Under the conditions of the study, no residues of HMCPP or CCPP were found in any of the specimens in any treatment group.
Regression analysis for test material in whole milk, skimmed milk and cream demonstrated a linear relationship between the dose level and the resulting residue concentration. A non-linear relationship between the dose level and residue concentration was found for test material in all other matrices and for PCOC in kidney. - Executive summary:
An examination of the residues in milk, muscle, liver, kidney and fat of lactating dairy cattle was undertaken according to OECD Test Guideline 505 and in compliance with GLP.
During the study, lactating Friesian/Holstein dairy cows were dosed orally for 28 or 29 consecutive days via a compound feed containing the test material. The test material was added to the compound feed as a solution in acetone. The daily compound feed ration was administered as a split feed on two occasions each day (morning and afternoon feeding). The animals were divided into 4 separate treatment groups. One treatment group of 2 cows was an untreated control group, which was dosed by adding acetone only to the compound feed. The remaining groups were treated with test material, targeted at a nominal dose based on the maximum 7-day residue in pasture grass, equivalent to a concentration in the animals’ diet (on a dry matter (DM) basis) of:
194 mg/kg test material (1x, 3 cows)
582 mg/kg test material (3x, 3 cows)
1940 mg/kg test material (10x, 6 cows)
The animals were dosed for 28 or 29 consecutive days. Three of the cows in the 10 x treatment group were used to generate depuration data. At the end of the dosing period, they were transferred to the control diet to measure the decline in residues following withdrawal of the test material from the diet. Milk specimens from each animal were collected twice daily and combined as one pooled sample. On Day 22, milk was also separated into cream and skimmed milk. Animals were sacrificed between 15 and 21 hours of final dosing (except for the three cows used to generate depuration data, which were sacrificed 3, 5 and 10 days after administration of the final dose) and specimens of muscle, liver, kidney and fat were taken.
All animals were observed at least twice daily for general health. No adverse treatment-related effects were observed on body weight, feed consumption or milk production. Additionally, no treatment-related behavioural reactions or systemic signs of toxicity were noted. Gross necropsies showed no effects that appeared to be treatment-related.
Residues of the test material and the potential metabolites 2-(2-hydroxymethyl-4-chlorophenoxy) propionic acid (HMCPP), 2-(2-carboxy-4-chlorophenoxy)propionic acid (CCPP) and 4-chloro-2- methyl phenol (PCOC) in milk and tissues were measured using an analytical method based on LC-MS/MS. This method is designed to measure residues of test material including its esters and conjugates. The limit of quantitation (LOQ) for each of the analytes in milk, skimmed milk, cream, muscle, liver, kidney and fat is 0.01 mg/kg.
The analytical method was validated as part of this study.
Residues of test material were found in all matrices from cows in the 1x, 3x and 10x dosing groups. Residues in whole milk reached a plateau after 5 days of dosing and remained stable throughout the dosing period. The residues in the 10x dosing group declined to less than the LOQ after 2 days of withdrawal of test material from the diet. Residues of test material did not partition selectively into skimmed milk or cream. Residues of test material in muscle, liver, kidney and fat in the 10x dosing group showed a decline after withdrawal of test material from the diet.
Residues of PCOC were found in cream (but not skimmed milk), liver and fat specimens from cows in the 10x dosing group only and in kidney specimens from cows in the 1x, 3x, and 10x dosing groups. Residues of PCOC in the 10x dosing group declined to less than the LOQ in liver after 3 days of withdrawal of test material from the diet and in kidney and fat after 5 days of withdrawal of test material from the diet.
Under the conditions of the study, no residues of HMCPP or CCPP were found in any of the specimens in any treatment group.
Regression analysis for test material in whole milk, skimmed milk and cream demonstrated a linear relationship between the dose level and the resulting residue concentration. A non-linear relationship between the dose level and residue concentration was found for test material in all other matrices and for PCOC in kidney.
- Endpoint:
- basic toxicokinetics in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 August 2015 to 3 December 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Objective of study:
- metabolism
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 417 (Toxicokinetics)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Radiolabelling:
- yes
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: [14C]test material dosing solution was prepared at a target concentration of 1 000 μM by dissolving [14C]test material in acetonitrile. Marker substrate dosing solutions (both Testosterone and 7-Ethoxycoumarin) were prepared by mixing 14C-labelled marker substrate and unlabelled marker substrate in acetonitrile. Target concentrations of [14C]Testosterone and [14C]7-Ethoxycoumarin dosing solutions were 25 mM and 50 mM, respectively. Solutions were stored at -20 °C ± 5 °C.
In the microsomal assays, a 5 μL aliquot of [14C]test material or marker substrate dosing solution was added to the incubation medium which final volume was 0.5 mL. Final target concentration of [14C]test material in microsomal assays was 10 μM. Final target concentration of Testosterone and 7-Ethoxycoumarin in microsomal assays was 250 μM and 500 μM, respectively. Final concentration of acetonitrile in microsomal assays was 1 %.
14C-Labelled Marker Substrates:
- 14C-Testosterone
Specific activity: 53 mCi/mmol
Radiochemical purity: 99 % (Radio-HPLC)
- [3-14C]7-Ethoxycoumarin
Specific activity: 56 mCi/mmol
Radiochemical purity: > 97 % (Radio-HPLC) - Dose / conc.:
- 1 000 other: µM
- Control animals:
- yes
- Positive control reference chemical:
- Testosterone 6β-Hydroxylation in Human Liver Microsomes
Testosterone 6β-hydroxylation, which is a marker substrate reaction for human CYP3A4/5 activity, was used as positive control to check the metabolic competences of HLM.
HLM were preincubated with [14C]Testosterone in 100 mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2) for 3 to 5 minutes in a shaking water bath at 37 °C. Incubations were initiated by the addition of NADPH-regenerating system. The samples were then incubated for 20 minutes in a shaking water bath at 37 °C.
7-Ethoxycoumarin O-Dealkylation in Animal Liver Microsomes
7-Ethoxycoumarin O-dealkylation, which is a marker substrate reaction for multiple cytochrome P450 activities, was used as positive controls to check the metabolic competences of rat, mouse, and dog and rabbit liver microsomes.
Animal liver microsomes were preincubated with [14C]7-Ethoxycoumarin in 100 mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2) for 3 to 5 minutes in a shaking water bath at 37 °C. Incubations were initiated by the addition of NADPH-regenerating system. The samples were then incubated for 20 minutes in a shaking water bath at 37 °C.
Positive controls were performed in duplicate. - Details on dosing and sampling:
- In Vitro Test Systems
Human Liver Microsomes (HLM)
The study was conducted with a pool of mixed gender human liver microsomes (100 male and 100 female) purchased from Xenotech.
Rat Liver Microsomes (RLM)
The study was conducted with a pool of mixed gender rat liver microsomes prepared from separated pools of male (191 animals) and female (200 animals) Sprague Dawley rat liver microsomes purchased from Xenotech.
Mouse Liver Microsomes (MLM)
The study was conducted with a pool of mixed gender CD1 mouse liver microsomes prepared from separated pools of male (1120 animals) and female (400 animals) CD1 Mouse liver microsomes purchased from Xenotech (Lenexa KS, US).
Dog Liver Microsomes (DLM)
The study was conducted with a pool of mixed gender dog liver microsomes prepared from separated pools of male (8 animals) and female (12 animals) Beagle dog liver microsomes purchased from Xenotech.
Rabbit Liver Microsomes (RbLM)
The study was conducted with a pool of mixed gender rabbit liver microsomes prepared from separated pools of male (8 animals) and female (3 animals) New Zealand rabbit liver microsomes purchased from Xenotech.
Preparation of Microsomal Suspension
After thawing, human, rat, mouse, dog and rabbit liver microsomes (20 mg protein/mL) were diluted at a ratio of 1:1 with a solution containing cryoprotectant substance (sucrose) to a concentration of 10 mg protein/mL. For rat, mouse, dog and rabbit, mixed gender liver microsomes were prepared by mixing male and female diluted microsomes at a ratio of 1:1, maintaining the concentration at 10 mg protein/mL. In the microsomal assays, an aliquot of 25 μL of diluted microsomal suspension (10 mg protein/mL) was added to the incubation medium (final volume of 0.5 mL) in order to obtain a final microsomal protein concentration of 0.5 mg/mL.
Incubation Buffer
Incubations of liver microsomes with [14C]test material and marker substrates were performed in a 100 mM potassium phosphate buffer system (pH 7.4; 3 mM MgCl2). Potassium phosphate buffer was used to maintain the pH of the incubation medium at 7.4. MgCl2 was added to the buffer system to reduce the rate of unwanted lipid peroxidation and to obtain a functional NADPH-regenerating system.
NADPH-Regenerating System
The NADPH-regenerating system consisted of a mixture of 8.3 mM NADP, 41.7 mM G6P and 8.3 Unit/mL G6PDH in 100 mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2). In the microsomal assays, an aliquot of 60 μL of this mixture was added to the incubation medium (final volume of 0.5 mL) in order to obtain the desired final concentrations of 1 mM NADP, 5 mM G6P and 1 Unit/mL G6PDH. NADPH-regenerating system was prepared daily, and only fresh solutions were used. Once the solution was prepared, it was stored on wet ice for immediate use.
Microsomal Assays
Metabolic Profiling of [14C]Test Material
In vitro metabolic profiling of [14C]test material was performed for 1 concentration of [14C]test material (10 μM), 1 microsomal protein concentration (0.5 mg/mL) and 3 incubation time points (30, 60 and 120 minutes).
Microsomes were preincubated with [14C]test material in 100 mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2) for 3 to 5 minutes in a shaking water bath at 37 °C. Incubations were initiated by the addition of NADPH-regenerating system. The samples were then incubated for 30, 60 and 120 minutes in a shaking water bath at 37 °C. Incubations were performed in triplicate.
Negative Controls
Control incubations without microsomes were performed to check the chemical stability of [14C]test material under incubation conditions. [14C]test material was preincubated in 100
mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2) for 3 to 5 minutes in a shaking water bath at 37°C. Microsomal suspension was replaced by an equal volume of 100 mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2). Incubations were initiated after addition of NADPH-regenerating system. Samples were then incubated for 120 minutes in a shaking water bath at 37 °C.
Control incubations without cofactor (NADPH-regenerating system) were performed to validate NADPH-dependent metabolism. Microsomes were preincubated with [14C]test material in 100 mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2) for 3 to 5 minutes in a shaking water bath at 37 °C. Incubations were initiated after addition of 100 mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2) instead of NADPH-regenerating system. Samples were then incubated for 120 minutes in a shaking water bath at 37 °C.
Negative controls were performed in duplicate.
Time Zero Controls ([14C]test material and marker substrates)
Time zero controls were performed for human and animal liver microsomes incubated with [14C]test material and marker substrates. Time zero controls were performed in duplicate. Microsomes, 100 mM potassium phosphate buffer (pH 7.4; 3 mM MgCl2), NADPH regenerating system and [14C]test material or marker substrates were added to an equal volume of ice-cold solvent in order to stop the reaction immediately.
Stop Reaction
Incubations with [14C]test material were terminated by cooling on ice and addition of an equal volume (0.5 mL) of ice-cold acetonitrile containing 1 % trifluoroacetic acid for microsomal protein precipitation. Incubations with marker substrates were terminated by cooling on ice and addition of an equal volume (0.5 mL) of ice-cold acetonitrile. All the samples were then vortexed and centrifuged at 10,000g for 10 minutes at 4 °C. Supernatant and microsomal pellet were stored at -20 °C ± 5 °C until analysis.
Sample Analysis
Quantification of radioactivity:
Radioactivity concentration in [14C]test material and marker substrate dosing solutions was measured by liquid scintillation counting (LSC) of 5 μL aliquots. After termination of incubations by solvent protein precipitation and centrifugation, the radioactivity present in the resulting supernatant was measured by LSC of 10 μL aliquots in duplicate.
Radioactivity remaining in the microsomal pellet was measured after solubilisation of the pellet. Solubilisation was achieved by incubating the pellet in 1 mL of Solvable (Perkin Elmer) at room temperature for at least 4 hours. The LSC measurement was performed on the whole solubilised pellet.
Aliquots of liquid samples were added directly to IRGA-SAFE Plus scintillation cocktail Perkin Elmer). LSC was performed with Packard liquid scintillation counters equipped with DPM and luminescence options and the measurements were automatically corrected for background and using quench curves. Measurements were performed by counting samples for a time interval allowing a counting error below 5 %, typically 1 or 5 minutes.
Chromatographic Analysis:
Radio-HPLC was used to determine the purity of [14C]test material and marker substrates dosing solutions.
After termination of incubations by solvent protein precipitation and centrifugation, all the resulting supernatants were analysed by radio-HPLC. Supernatants from microsomal incubations with [14C]test material were injected directly. Supernatants from microsomal incubations with marker substrate were diluted with Milli-Q water so that the concentration of acetonitrile in the injected sample was reduced to 20 %.
[14C]test material and its metabolites were analysed by co-chromatography with unlabelled test material and reference material available, namely PCOC. Radio-HPLC peaks corresponding to 6β-hydroxytestosterone and 7-hydroxycoumarin (marker substrate metabolites used for determination of positive control enzymatic activities) were attributed by co-chromatography with the corresponding reference material.
The analytical systems used are described below.
Instruments
HPLC 3 and HPLC 4
HPLC gradient pump: Waters 2695 Separations Module
UV-detector: Waters 2487 Dual Wavelength Absorbance Detector
14C-detector: Perkin Elmer Radiomatic Flo-One beta Series A500
HPLC 5
HPLC gradient pump: Agilent 1260 Quaternary pump VL 400 bar
UV-detector: Agilent 1260 DAD
14C-detector: Berthold Technologies LB509
HPLC 6
HPLC gradient pump: Agilent 1260 Quaternary pump VL 400 bar
UV-detector: Agilent 1260 DAD
14C-detector: Lablogic Systems B-RAM 5C Radio Isotope Detection System
Method for Testosterone 6β-hydroxylation:
Column: 250 mm × 4.6 mm, 5 μm, Phenomenex Luna C18
UV-detection: 254 nm
Mobile Phase:
Solvent A: ammonium acetate buffer (20 mM; pH3.5)
Solvent B: acetonitrile
Column temperature: Ambient
Injected volume: 350 μL
Flow: 1 mL/min
14C-Detection: 1 mL eluent was continuously mixed with 2 ml Flo-Scint A (Perkin Elmer)
Gradient:
0 min: 80 % solvent A, 20 % solvent B
35 min: 60 % solvent A, 40 % solvent B
50 min: 40 % solvent A, 60 % solvent B
55 min: 0 % solvent A, 100 % solvent B
55.1 min: 80 % solvent A, 20 % solvent B
65 min: 80 % solvent A, 20 % solvent B
Method for 7-Ethoxycoumarin O-dealkylation:
Column: 250 mm × 4.6 mm, 5 μm, Phenomenex Luna C18
UV-detection: 300 nm
Mobile Phase:
Solvent A: 0.05 % orthophosphoric acid in water
Solvent B: acetonitrile
Column temperature: Ambient
Injected volume: 350 μL
Flow: 1 mL/min
14C-Detection: 1 mL eluent was continuously mixed with 2 ml Flo-Scint A (Perkin Elmer)
Gradient:
0 min: 75 % solvent A, 25 % solvent B
3 min: 75 % solvent A, 25 % solvent B
8 min: 35 % solvent A, 65 % solvent B
15 min: 35 % solvent A, 65 % solvent B
15.1 min: 75 % solvent A, 25 % solvent B
25 min: 75 % solvent A, 25 % solvent B
Method for [14C] test material metabolic profiling:
Column: 250 mm × 4.6 mm, 5 μm, Phenomenex Luna C18
UV-detection: 230 nm
Mobile Phase:
Solvent A: 0.1% trifluoroacteic acid in water
Solvent B: 0.1 % trifluoroacteic acid in acetonitrile
Column temperature: Ambient
Injected volume: 100 - 200 μL
Flow: 1 mL/min
14C-Detection: 1 mL eluent was continuously mixed with 2 ml Flo-Scint A
(Perkin Elmer, Shelton, CT, USA)
Gradient:
0 min: 95 % solvent A, 5 % solvent B
3 min: 95 % solvent A, 5 % solvent B
33 min: 0 % solvent A, 100 % solvent B
40 min: 0% solvent A, 100 % solvent B
40.1 min: 95 % solvent A, 5 % solvent B
50 min: 95 % solvent A, 5 % solvent B
Data Analysis:
Radioactivity Recovery
The radioactivity recovery of each sample incubated with [14C]test material and marker substrate was recorded. Radioactivity recovery after incubation was determined by the sum of radioactivity measured in the supernatant and the microsomal pellet. Radioactivity recovery was considered valid if ranging between 90 - 110 % of [14C]test material or marker substrate amount applied to the in vitro system.
Radio-HPLC Quantification:
Radio-HPLC quantification was done by integrating the area under the radio-chromatographic peaks. Quantification was done by using ROI´s (regions of interest), i.e. background regions between radio-chromatographic peaks were not considered in the quantification.
Positive Control Enzymatic Activities:
The rates of Testosterone 6β-hydroxylation and 7-Ethoxycoumarin O-dealkylation were calculated by quantifying 6β-hydroxytestosterone and 7-hydroxycoumarin produced in human and animal liver microsomal incubates, respectively. Testosterone 6β-hydroxylation and 7-Ethoxycoumarin O-dealkylation activities were calculated in units of picomoles of metabolite formed per minute per milligram of microsomal protein (pmol/min/mg).
Metabolic competences of human liver microsomes were considered acceptable if measured Testosterone 6β-hydroxylation reached, at least, 80 % of the rate given by the supplier for each lot of microsomes.
Metabolic competences of animal liver microsomes were considered acceptable if measured 7-Ethoxycoumarin O-dealkylation reached, at least, 80 % of the mean of the rates given by the supplier for each lot of male and female liver microsomes.
[14C]test material Metabolism Rate and Metabolic Profiling:
[14C]test material metabolic profiles were analysed qualitatively and quantitatively. Peak names (P1 to P9) were attributed according to their relative retention time. Relative retention time was calculated by dividing the retention time of the peak by the retention time of the parent [14C]test material. Quantification of parent compound and metabolites was performed for each radiochromatogram and results were expressed as percentage of total radio-chromatogram and as percentage of total radioactivity recovered in the sample (% TRS).
Calculations:
Numerical values were rounded to a smaller degree of precision (number of digits) to increase readability and to indicate the approximate precision of the reported results.
Minor differences in the results obtained with such “rounded” values in comparison to those obtained with higher precision values are well within the limits of the experimental accuracy and are therefore of no practical concern. - Type:
- metabolism
- Results:
- Metabolism rate of [14C]test material was extremely low in both human and animal liver microsomes.
- Metabolites identified:
- no
- Details on metabolites:
- [14C]Test Material Metabolism in Human and Animal Liver Microsomes
- Radioactivity Recovery: The recovery ranged between 91.6 % and 107.6 % in HLM incubates. The recovery ranged from 100.7 % to 108.3 % in RLM, 101.0 % to 06.2 % in MLM, 102.0 % to 106.7 % in DLM and 102.6 % to 108.3 % in RbLM incubates. In all incubates, more than 90 % of the recovered radioactivity was located in the supernatant, therefore no further extraction of the microsomal pellet was performed.
[14C]Test Material Metabolism
A total of 9 different HPLC peaks (P1 to P9) were observed among human and animal liver microsome incubates. Peak P7 corresponded to [14C]test material.
In time zero controls, no significant degradation of [14C]test material was observed, the percentage of parent compound (98.1 % TRS in HLM, 97.2 % TRS in RLM, 96.8 % TRS in MLM, 97.9 % TRS in DLM and 97.6 % TRS in RbLM) being close to the radio-purity determined in [14C]test material dosing solution (97.7 % to 98.5 %).
In negative controls without microsomes, remaining parent compound accounted for 97.9 % to 98.6 % TRS after 120 minutes of incubation, showing that [14C]test material did not degrade in the incubation medium. In negative controls without NADPH-GS remaining parent compound accounted for 96.9 % to 98.1 % TRS after 120 minutes of incubation, indicating that no NADPH-independent metabolism occurred.
In time zero and negative controls, radio-HPLC peaks P1, P3, P4, P5, P6, P8 and P9 were observed. All these peaks accounted for less than 1 % TRS. Peaks P3, P4, P6, P8 and P9 were already present at similar levels in [14C]test material dosing solution.
In human and animal liver microsomes, the percentage of remaining parent compound decreased very slightly with incubation time. After 120 minutes of incubation, remaining parent compound accounted for 96.1 %, 96.7 %, 95.4 %, 97.6 % and 96.6 % TRS in HLM, RLM, MLM, DLM and RbLM incubates, respectively; showing that metabolism of [14C]test material was extremely low in both human and animal liver microsomes.
After 120 minutes of incubation, the largest peak observed was P3. P3 was already observed as an impurity in [14C]test material dosing solution, time zero and negative controls. However, this peak increased with incubation time in all the species and after 120 minutes of incubation it accounted for 1.2 %, 1.3 %, 2.7 %, 1.0 % and 2.1 % TRS in HLM, RLM, MLM, DLM and RbLM incubates, respectively. All the other peaks observed remained below 1 % TRS, as observed in time zero and negative controls.
P3 did not correspond to the putative metabolite 4-chloro-2-methylphenol (PCOC). - Conclusions:
- Under the conditions of the study metabolic profiles and kinetics observed in human and animal liver microsomes incubated with 10 μM [14C]test material were similar qualitatively and quantitatively. Metabolism rate of [14C]test material was extremely low in both human and animal liver microsomes. Only one metabolic product increased slightly with incubation time, namely P3.
- Executive summary:
The metabolism of the test material was assessed according to OECD Test Guideline 417 and in compliance with GLP.
The purpose of this study was to compare Phase I NADPH-dependent metabolism of [14C]test material in human liver microsomes (HLM) and animal liver microsomes, namely rat liver microsomes (RLM), mouse liver microsomes (MLM), dog liver microsomes (DLM) and rabbit liver microsomes (RbLM).
In vitro metabolic profiling of [14C]test material was carried out by incubating human and animal liver microsomes with 10 μM [14C]test material (nominal concentration) in 100 mM potassium phosphate buffer (pH 7.4) containing 3 mM MgCl2 and NADPH-regenerating system for 30, 60 and 120 minutes at 37 °C. After termination of incubations by solvent protein precipitation and centrifugation, the radioactivity present in the resulting supernatant was measured by liquid scintillation counting. Radioactivity remaining in the microsomal pellet was measured after solubilisation of the pellet. Metabolite profiling was conducted by analysing the supernatant by radio-HPLC.
Marker substrate reactions, namely Testosterone 6β-hydroxylation and 7-Ethoxycoumarin O-dealkylation, were used as positive controls to check the metabolic competences of human and animal liver microsomes, respectively. Positive control enzymatic activities showed that both human and animal liver microsomes used in the present study possessed metabolic competences in agreement with the acceptance criteria (80 % of the rate given by the supplier).
The radioactivity recovery ranged between 91.6 % and 107.6 % in HLM, from 100.7 % to 108.3 % in RLM, 101.0 % to 106.2 % in MLM, 102.0 % to 106.7 % in DLM and 102.6 % to 108.3 % in RbLM incubates. In all incubates, more than 90 % of the recovered radioactivity was located in the supernatant, therefore no further extraction of the microsomal pellet was performed.
A total of 9 different HPLC peaks were observed in human and animal liver microsome incubates.
In time zero and negative controls, no significant degradation of [14C]test material was observed. Only radio-HPLC peaks accounting for less than 1 % of total radioactivity recovered in the sample (TRS) were observed. Most of these peaks were already present at similar levels in [14C]test material dosing solution. Therefore, [14C]test material did not degrade in the incubation medium and no NADPH-independent metabolism occurred.
In human and animal liver microsomes, the percentage of remaining parent compound decreased very slightly with incubation time. After 120 minutes of incubation, remaining parent compound accounted for 96.1 %, 96.7 %, 95.4 %, 97.6 % and 96.6 % TRS in HLM, RLM, MLM, DLM and RbLM incubates, respectively; showing that metabolism of [14C]test material was extremely low in both human and animal liver microsomes.
The largest metabolic peak observed was P3. P3 was already observed as an impurity in [14C]test material dosing solution, time zero and negative controls. However, this peak increased slightly with incubation time in all the species. After 120 minutes of incubation it accounted for 1.2 %, 1.3 %, 2.7 %, 1.0 % and 2.1 % TRS in HLM, RLM, MLM, DLM and RbLM incubates, respectively. P3 did not correspond to the putative metabolite 4-chloro-2-methylphenol (PCOC) as shown by the co-chromatographic analysis. All the other peaks observed remained below 1 % TRS, as observed in time zero and negative controls.
Under the conditions of the study metabolic profiles and kinetics observed in human and animal liver microsomes incubated with 10 μM [14C]test material were similar qualitatively and quantitatively. Metabolism rate of [14C]test material was extremely low in both human and animal liver microsomes. Only one metabolic product increased slightly with incubation time, namely P3.
Referenceopen allclose all
Stability, Homogeneity and Concentration Control Analyses of the Test Material Preparations
The stability of non-radiolabelled test material in feed at ambient temperature for at least 14 days was demonstrated previously. Considering the low standard deviation in the homogeneity analysis, it was concluded that the test material was distributed homogeneously in feed. The mean values of the test material in feed were found to be in the range of 100.0 - 104.6 % of the nominal concentrations, demonstrating the target concentrations were achieved.
0.5 % Aqueous Carboxymethylcellulose preparations of 14C-test material:
The analytical investigations demonstrated the homogeneity and that the target concentrations of radiolabelled test material in the vehicle (0.5 % aqueous Carboxymethylcellulose supplemented with 1 % Cremophor) were achieved.
Body Weight Changes, Test Material Intake and Clinical Observations
As compared to the two lowest dose groups, body weight development and thus, body weight changes were slightly decreased in groups dosed with 500 and 1 000 ppm and markedly in groups fed with 1 500 and 2 200 ppm.
In the first week of feeding, achieved doses showed the expected excess over target doses. However, in the second feeding week, i.e. the week before radioactive dosing, feed doses were closer to target at all dose levels.
No test material related clinical findings were observed.
The AUC0∞ and AUC12-24-Values Calculated from the Plasma Concentration Versus Time Curves at the Respective Dose Level
Nominal Dose (mg/kg bw) |
AUC0-∞ [μg Eqꞏh/g] |
Increase in AUC0-∞ Relative to Previous Dose
|
Overall increase in AUC0-∞ Relative to Low Dose |
AUC12-24 [μg Eqꞏh/g] |
Increase in AUC12-24 Relative to Previous Dose |
Overall increase in AUC12-24 Relative to Low Dose |
|
10 |
- |
318 |
- |
- |
42 |
- |
- |
20 (2 X*) |
(2 X**) |
662 |
2.1 X |
2.1 X |
121 |
2.9 X |
2.9 X |
40 (2 X*) |
(4 X**) |
1425 |
2.15 X |
4.5 X |
273 |
2.4 X |
6.5 X |
80 (2 X*) |
(8 X**) |
3584 |
2.5 X |
11.3 X |
1129 |
4.1 X |
26.9 X |
120 (1.5 X*) |
(12 X**) |
5771 |
1.6 X |
18 X |
1983 |
1.8 X |
47.2 X |
180 (1.5 X*) |
(18 X**) |
7276 |
1.3 X |
23 X |
2610 |
1.3 X |
62.1 X |
* Increase relative to previous dose.
** Increase relative to low dose.
Plasma Half-Lives
Nominal Dose [mg/kg bw] |
10 |
20 |
40 |
80 |
120 |
180 |
Half-Life in Plasma [h] |
||||||
Initial phase (phase 1) |
5.96 |
6.93 |
6.52 |
19.81 |
88.99 |
45.25 |
Intermediate phase (phase 2) |
3.40 |
3.41 |
3.63 |
5.78 |
6.55 |
7.31 |
Terminal phase (phase 3) |
12.00 |
11.18 |
8.36 |
- |
- |
- |
Plasma Concentrations and their Increase with Dose
Nominal Dose (mg/kg bw) |
Plasma Conc. After 24 h [μg Eq/g] |
Increase Relative to Previous Dose |
Overall Increase Relative to Low Dose |
|
10 |
- |
0.68 |
- |
- |
20 (2 X*) |
(2 X**) |
1.55 |
2.3 X |
2.3 X |
40 (2 X*) |
(4 X**) |
4.32 |
2.8 X |
6.4 X |
80 (2 X*) |
(8 X**) |
26.66 |
6.2 X |
39.2 X |
120 (1.5 X*) |
(12 X**) |
82.52 |
3.1 X |
121 X |
180 (1.5 X*) |
(18 X**) |
135.85 |
1.6 X |
200 X |
* Increase relative to previous dose.
** Increase relative to low dose.
Stability, Homogeneity and Concentration Control Analyses of the Test Material Preparations
Feed:
The stability of non-radiolabelled test material in feed at ambient temperature for at least 33 days was demonstrated previously. Considering the low standard deviation in the homogeneity analysis, it was concluded that the test material was distributed homogeneously in feed. The mean values of the test material in feed were found to be in the range of 92.5 - 102.9 % of the nominal concentrations, demonstrating the target concentrations were achieved.
0.5 % Aqueous Carboxymethylcellulose preparations of 14C-test material:
The analytical investigations demonstrated the homogeneity and that the target concentrations of radiolabelled test material in the vehicle (0.5 % aqueous Carboxymethylcellulose) were achieved.
Body Weight Changes, Test Material Intake and Clinical Observations
As compared to the two lowest dose groups, body weight development and thus, body weight changes were slightly decreased in groups dosed with 570, 760, and 950 ppm.
During the first month of feeding, achieved doses showed the expected excess over target doses. However, in the second feeding month, i.e. the month before radioactive dosing, feed doses were closer to target at all dose levels.
No test material-related clinical findings were observed.
The AUC0-∞- and AUC12-24-Values Calculated from the Plasma Concentration versus Time Curves at the Respective Dose Levels
Nominal Dose (mg/kg bw) |
AUC0-∞ [μg Eqꞏh/g] |
Increase in AUC0-∞ Relative to Previous Dose |
Overall Increase in AUC-0-∞ Relative to Low Dose |
AUC12-24 [μg Eqꞏh/g] |
Increase in AUC12-24 Relative to Previous Dose |
Overall Increase in AUC12-24 Relative to Low Dose |
|
15 |
- |
680 |
- |
- |
156 |
- |
- |
30 (2 X*) |
(2 X**) |
1432 |
2.1 X |
2.1 X |
414 |
2.7 X |
2.7 X |
45 (1.5 X*) |
(3 X**) |
2300 |
1.6 X |
3.4 X |
657 |
1.6 X |
4.2 X |
60 (1.33 X*) |
(4 X**) |
2930 |
1.3 X |
4.3 X |
899 |
1.4 X |
5.8 X |
75 (1.25 X*) |
(5 X**) |
4052 |
1.4 X |
6.0 X |
1190 |
1.3 X |
7.6 X |
* Increase relative to previous dose.
** Increase relative to low dose.
Plasma Half-Lives
Nominal Dose (mg/kg bw) |
15 |
30 |
45 |
60 |
75 |
Half-life in Plasma (h) |
|||||
Initial phase (Phase 1) |
10.32 |
14.93 |
14.39 |
14.09 |
12.16 |
Intermediate phase (Phase 2) |
2.53 |
2.67 |
3.89 |
3.40 |
3.75 |
Terminal phase (Phase 3) |
10.75 |
8.76 |
6.60 |
7.03 |
6.31 |
Plasma Concentrations and their Increase with Dose
Nominal Dose (mg/kg bw) |
Plasma Conc. After 24 h [μg Eq/g] |
Increase Relative to Previous Dose |
Overall Increase Relative to Low Dose |
|
15 |
- |
0.94 |
- |
- |
30 (2 X*) |
(2 X**) |
2.94 |
3.1 X |
3.1 X |
45 (1.5 X*) |
(3 X**) |
11.57 |
3.9 X |
12.3 X |
60 (1.33 X*) |
(4 X**) |
11.93 |
1.0 X |
12.7 X |
75 (1.25 X*) |
(5 X**) |
19.51 |
1.6 X |
20.8 X |
* Increase relative to previous dose.
** Increase relative to low dose.
Modelling the Low-Dose Elimination
For the purpose of the modelling, the column of mean values was used from 9 h to 24 h. The rate constant in the upper pane (males) is 0.128/h^-1 (equivalent to a t1/2 of 5.43 d); the rate constant in the lower pane (females) is 0.153/h^-1 (equivalent to a t1/2 of 4.54 d). In both cases, the error is ≤10 %, and the line gives a good visual fit to the data, which indicates that the values are a statistically reliable description of the decline over this time period.
Data Used for Half-Life Determination (Mean Data Set 9 – 24 h)
| 5 mg/kg | µg Equivalents/g of (14C)-Test Material | |||||||
| Male | Time (h) | 401M | 402M | 403M | 404M | 405M | Mean | SD |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 1.5 | 28.34 | 24.1 | 27.2 | 28.71 | 29.45 | 27.56 | 2.097 | |
| 3 | 26.31 | 25.13 | 25.19 | 26.58 | 28.9 | 26.42 | 1.53 | |
| 6 | 15.27 | 20.9 | 21.64 | 15.89 | 22.13 | 19.17 | 3.307 | |
| 9 | 4.17 | 10.31 | 13.95 | 7.768 | 9.901 | 9.219 | 3.592 | |
| 12 | 1.503 | 4.553 | 9.439 | 4.379 | 6.507 | 5.276 | 29.33 | |
| 24 | 0.402 | 0.981 | 1.419 | 1.091 | 2.55 | 1.288 | 0.795 | |
| 5 mg/kg | µg Equivalents/g of (14C)-Test Material | |||||||
| Female | Time (h) | 406F | 407F | 408F | 409F | 410F | Mean | SD |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 1.5 | 29.07 | 33.32 | 31.29 | 31.89 | 28 | 30.72 | 2.156 | |
| 3 | 29.22 | 34.94 | 32.47 | 30.63 | 29.2 | 31.29 | 2.441 | |
| 6 | 8.308 | 18.36 | 9.848 | 6.495 | 14.54 | 11.51 | 4.856 | |
| 9 | 2.26 | 5.637 | 4.552 | 1.441 | 3.895 | 3.557 | 1.702 | |
| 12 | 1.096 | 1.881 | 2.487 | 0.99 | 2.371 | 1.765 | 0.698 | |
| 24 | 0.176 | 0.283 | 0.606 | 0.138 | 0.473 | 0.335 | 0.20 | |
Modelling the High-Dose Elimination
For the purpose of the modelling, the column of mean values was used from 9 h to 24 h. The entire database of all animals was used for the time period 0 - 48 h.
Data Used in the Modelling of the Michaelis-Menten Decline of Test Material in Rats Dosed at 100 mg/kg
100 mg/kg |
µg Equivalents/g of (14C)-Test Material |
|||||
Male |
Time (h) |
501M |
502M |
503M |
504M |
505M |
0 |
0 |
0 |
0 |
0 |
0 |
|
1.5 |
273.8 |
328 |
359.2 |
359.6 |
345.2 |
|
3 |
366.5 |
376.8 |
373.9 |
387.1 |
384.1 |
|
6 |
386.2 |
388.6 |
370 |
384.1 |
355.2 |
|
9 |
340.5 |
315.2 |
325.3 |
319.4 |
332.3 |
|
12 |
300.6 |
286.9 |
287.1 |
280.3 |
282.5 |
|
24 |
159 |
139.4 |
181.1 |
116.6 |
130 |
|
48 |
8.889 |
6.648 |
9.611 |
4.108 |
6.303 |
|
100 mg/kg |
µg Equivalents/g of (14C)-Test Material |
|||||
Female |
Time (h) |
506F |
507F |
508F |
509F |
510F |
0 |
0 |
0 |
0 |
0 |
0 |
|
1.5 |
365.6 |
314.5 |
285.8 |
311.8 |
295.8 |
|
3 |
394.2 |
370.2 |
370.3 |
389.8 |
416.7 |
|
6 |
386.7 |
382.4 |
387.6 |
371.5 |
370.5 |
|
9 |
302.3 |
337.4 |
318.4 |
294.1 |
299.9 |
|
12 |
240.2 |
295.8 |
284.3 |
229.1 |
252.7 |
|
24 |
142.4 |
183.1 |
127.1 |
61.05 |
97.85 |
|
48 |
9.847 |
12.65 |
3.158 |
3.31 |
|
|
Kinetic Parameters Generated from the 2-Stage Fitting of the Terminal Half-Time (t1/2) and the Michaelis-Menten Model
Parameter |
V (mL/kg) |
ka (/h) |
t1/2 (h) |
Vm (µg equiv/mL/h) |
Km (µg equiv/mL) |
Male 100 mg/kg |
223.22 |
0.992 |
5.43 |
3687.0 |
86.8 |
%Error |
5 |
17 |
(fixed value) |
7 |
16 |
Female 100 mg/kg |
214.06 |
0.757 |
4.54 |
4886.8 |
159.7 |
%Error |
11 |
31 |
(fixed value) |
15 |
26 |
The results clearly show an excellent visual fit and low parameter errors obtained in the Michaelis-Menten model using a fixed value of the terminal elimination half-life at time points up to 48 h after dosing.
This evidence suggests that a suitable value for the half-lives are 5.4 h (male) and 4.5 h (female) similar to those published by Lappin (1997). Good fits would not be possible if this value of the terminal elimination half-time was inappropriate. These half-lives correspond to rate constants as follows:
Male k = 0.0022/min^-1
Female k = 0.0025/min^-1
These values of half-times and rate constants are suitable for application in, for example, body burden modelling for mammalian risk assessment.
Animal Observations
During the course of the study no overt pharmacological or toxic signs were observed in any of the animals.
Results of Excretion Balance Study (Dose Groups A, B and C)
Sample |
Group A (5 mg/kg) |
Group B* (5 mg/kg) |
Group C (100 mg/kg) |
|||
Male |
Female |
Male |
Female |
Male |
Female |
|
Urine |
76.01 |
74.53 |
79.19 |
67.22 |
77.88 |
60.97 |
Faeces |
8.23 |
3.56 |
5.27 |
4.90 |
12.53 |
9.09 |
Cage wash |
24.07 |
19.50 |
15.13 |
23.94 |
11.53 |
18.76 |
Cage debris |
0.60 |
0.44 |
0.64 |
0.97 |
ND |
NS |
Final wash |
0.02 |
0.01 |
0.03 |
0.03 |
0.03 |
0.05 |
Tissues |
0.39 |
0.59 |
0.26 |
0.91 |
2.93 |
3.19 |
Total |
109.30 |
98.63 |
100.50 |
97.98 |
104.90 |
92.06 |
* Non-radiolabelled test material was administered daily as a single oral dose for 14 days. Twenty-four hours after receiving the last dose, a single oral dose of (14C)-test material was administered.
Results of Pharmacokinetics Studies (Dose Groups D and E)
Plasma |
Group D (5 mg/kg) |
Group E (100 mg/kg) |
||
Male |
Female |
Male |
Female |
|
Cmax (µg equiv./g) |
27.77 |
31.54 |
384.0 |
394.1 |
Tmax (h) |
1.8 |
2.7 |
4.2 |
4.2 |
T½elim (h) |
6.354 |
4.234 |
7.886 |
7.787 |
AUC (0-t) (µg equiv./g) |
250.7 |
181.6 |
8425 |
7852 |
AUC (0-∞) (µg equiv./g) |
252.0 |
182.7 |
8449 |
7884 |
Results of Tissue Distribution Study (Dose Group F)
Tissue |
0.5 h Post Dose |
3 h Post Dose |
6 h Post Dose |
|||
Male |
Female |
Male |
Female |
Male |
Female |
|
Plasma |
16.60 |
24.37 |
27.46 |
25.25 |
20.09 |
22.23 |
Blood |
18.86 |
19.66 |
16.49 |
17.08 |
13.41 |
14.72 |
Heart |
7.98 |
8.49 |
7.31 |
9.11 |
6.59 |
6.85 |
Lung |
7.72 |
8.80 |
6.85 |
8.30 |
6.88 |
7.13 |
Liver |
9.44 |
8.44 |
8.80 |
7.37 |
7.69 |
6.54 |
Kidney |
23.62 |
19.08 |
23.43 |
20.23 |
13.79 |
15.86 |
Thyroid |
30.17 |
42.16 |
23.02 |
19.77 |
12.26 |
32.01 |
Gonads |
1.75 |
11.16 |
3.51 |
11.58 |
3.36 |
9.36 |
Uterus |
NA |
9.45 |
NA |
12.10 |
NA |
6.92 |
Adrenals |
9.48 |
9.30 |
10.74 |
10.78 |
9.76 |
7.75 |
NA - Not applicable
Additional Analytical Data
Standard Test Material
A sample of the test material was prepared to show that all expected interactions could be observed under the conditions used.
1H NMR Analysis
The proton spectrum showed three aromatic signals of equal intensity with splitting characteristic of a 2-4 tri-substituted benzene ring. The aromatic protons show no coupling to any other protons. There is a single peak of intensity at 2.25 ppm, characteristic of an aromatic methyl group.
There is a quartet of intensity at 4.76 ppm and a doublet of intensity at 1.62 ppm with the same coupling constant, characteristic of a methyne and methyl group, the shift of the methyne hydrogen is indicative of its attached carbon being connected to an electronegative atom such as oxygen.
13C & DEPT-135 NMR Analysis
The carbon and DEPT-135 (Distortionless Enhancement by Polarisation Transfer) spectra confirm the presence of three protonated aromatic carbons. They also show three non-protonated aromatic carbons, one of which is connected to an electronegative atom such as oxygen. A signal at 175 ppm, the chemical shift of which is indicative of an acid carbonyl. A protonated aliphatic carbon connected to an electronegative atom such as oxygen. Two other protonated aliphatic carbon signals. The 1H and 13C spectra are consistent with the proposed structure. There are however several isolated spin systems and to confirm their arrangement, 2-Dimensional nOe and long-range C/H correlations were performed.
2D NOESY NMR Analysis
The 2-D NOESY (Nuclear Overhauser Effect SpectroscopY) Spectrum shows through space interaction between methyl protons and aromatic proton, between methyne proton and aromatic proton, between methyl protons and methyne proton and between aromatic proton and aromatic proton.
2D-HMBC NMR Analysis
The 2-D HMBC (Hetronuclear Multiple Bond Coherence) Spectrum shows long range (2JcH & 3JcH) coupling between methyl protons and carbonyl, between methyne proton and carbonyl, between methyl protons and methyne carbon, between methyne proton and methyl carbon, between methyne proton and aromatic carbon, between methyl protons and aromatic carbon, between methyl protons and aromatic carbon and between aromatic proton and methyl carbon. These results confirm the structure of the test material. The nOe experiments additionally show that steric effects result in the proprionic acid methyl and acid pointing away from the aromatic ring bringing the methyne proton close to the aromatic ring.
1H NMR Analysis
The proton spectrum at 300 K shows three aromatic signals of equal intensity with splittings characteristic of a 2-4 tri-substituted benzene ring. The aromatic protons show no coupling to any other protons. There is multiplet of intensity at 4.72 ppm, the chemical shift and intensity being consistent with a methylene group attached to an electronegative atom such as oxygen. There is a multiplet which is partially obscured by the HOD signal from the solvent and a doublet of intensity at 1.62 ppm characteristic of a methyl group attached to a methyne.
The proton spectrum at 320K which causes a change in the chemical shift of the HOD signal, shows that the previously obscured multiplet at 4.8 ppm is a quartet with the same coupling constant as the methyl at 1.62 ppm and is characteristic of a methyne attached to the methyl and to an electronegative atom such as oxygen.
13C & DEPT-135 NMR Analysis
The carbon and DEPT-135 spectra confirm the presence of three protonated aromatic carbons. They also show:
Three non-protonated aromatic carbons, one of which is connected to an electronegative atom such as oxygen.
A signal at 175 ppm, the chemical shift, of which is indicative of an acid carbonyl.
A protonated aliphatic carbon connected to an electronegative atom such as oxygen.
A doubly protonated aliphatic carbon connected to an electronegative atom such as oxygen.
A further protonated aliphatic carbon signal.
The 1H and 13C spectra are consistent with the proposed structure.
There are, once again, several isolated spin systems and to confirm their arrangement, 2-Dimensional COSY, nOe and long-range C/H correlations were performed.
2D-COSY NMR Analysis
The 2-D COSY (COrelation SpectroscopY) Spectrum shows through bond couplings between methylene protons and aromatic proton, between methyl protons and methyne proton, between aromatic proton and aromatic proton and between aromatic proton and aromatic proton.
2D-NOESY NMR Analysis
The 2-D NOESY Spectrum shows through space interaction between methyne proton and aromatic proton, between methyl protons and methyne proton and between aromatic proton and aromatic proton.
2D-HMBC NMR Analysis
The 2-D HMBC Spectrum shows 2 JcH & 3JcH coupling between methyl protons and carbonyl, between methyne proton and carbonyl, between methyl protons and. methyne carbon, between methyne proton and methyl carbon, between methyne proton and aromatic carbon, between methylene protons and aromatic carbon, between methylene protons and aromatic carbon, between methylene protons and aromatic carbon and between aromatic proton and methylene carbon.
These results confirm the structure of the sample. The splitting of the methylene signal is characteristic of a pair of geminal hydrogens with different chemical shifts and likely to arise from a -CH2OH function with restricted rotation, it is proposed that this is due to hydrogen bonding between the methylene-OH and the acid carbonyl.
Conclusion
After extensive spectroscopic analysis using both 1 & 2 Dimensional 1H and 13C NMR techniques the structure has been assigned as the 2-hydroxymethyl analogue of the test material.
No work to determine the stereochemistry of the sample was carried out during the course of this study.
The evaluation of the plasmakinetics of 14C-test material is based on the following kinetic parameters which are relevant to address the issue:
• AUC0-∞ and AUC12-24h
• plasma half-life
• plasma concentration at 24 hours post radio-labelled dosing.
In general, plasma concentration vs. time curves showed three different phases:
Phase 1 can be characterised as a mixed absorption/excretion phase and ranges from 0 to about 8 - 12 h after dosing being dependent on dose and pre-treatment duration.
Phase 2 represents a pure excretory phase (about 8 or 12 - 24 h post-dosing).
Phase 3 is a further mixed absorption/excretion phase in pre-treated animals, since animals were dosed again with non-radiolabelled test material via feed (24 hours and beyond).
AUC0-∞ and AUC12-24H
For the evaluation of plasmakinetics, the area under the curve ("AUC'') is the most relevant parameter since the AUC is a surrogate for the body burden with the test material. Usually, the AUC0-∞ is taken for such purposes which in case of the test material is a mixed phase parameter covering all three phases of the plasma kinetics. If, however, saturation of excretion is expected, this can be best worked out using the AUC during the pure excretory phase (Phase 2), i.e. AUC12-24h. This reflects a condition where rats are in a purely excretory phase at all dose levels. This should be more representative of the position with dietary uptake, where there is likely to be a relatively constant level in the blood, reflecting the feeding habits of the rat: small meals are taken at frequent intervals. For this reason, saturation effects become more evident when comparing the AUC12-24 values and the plasma half-life during this phase.
After single oral application of 14C-test material at dose levels of 5 and 100 mg/kg bw, AUC0-∞ increased by a factor of 32 whereas dose only increased by a factor of 20. As expected, the increase in AUC was more pronounced using the AUC12-24h resulting in a 65-fold increase. Therefore, even after single oral application, saturation of excretion is evident in a dose range between 5 and 100 mg/kg bw.
After dietary feeding for 14 days at concentrations of 125, 250, 500, 1 000, 1 500, and 2 200 ppm and the daily equivalent by a single gavage dose of radio-labelled test material at day 15 (i.e. 10, 20, 40, 80, 120, and 180 mg/kg bw), the AUC0-∞ increased with increasing dose. The increase in the AUC0-∞ was not linear with dose. A slight over-proportional increase of the AUC0-∞ was already detectable at 40 mg/kg bw being more pronounced at 80 mg/kg bw. Whereas the dose increased 4- and 8-fold relative to the low dose, the AUC0-∞ increased by factors of 4.5 and 11.3. At 120 and 180 mg/kg bw, the over-proportional increase in the AUC0-∞ was much more pronounced. Whereas the overall increase in dose from the lowest to the two highest dose levels was 12- and 18-fold, the respective AUC0-∞ increased by factors of 18 and 23. When comparing the AUC during the main excretory phase (AUC12-24) which is a more sensitive parameter for the excretory capacity, the over-proportional increase in AUC12-24h at 40 mg/kg bw and above, and thus, saturation of excretion, becomes even more evident.
After dietary feeding for 60 days at concentrations of 190, 380, 570, 760, and 950 ppm and radioactive dosing at dose levels of 15, 30, 45, 60 and 75 mg/kg bw on day 61, the increase in the AUC0-∞ was not linear with dose. A slight over-proportional increase of the AUC0-∞ was already detectable at 30 and 45 mg/kg bw. Whereas the dose increased 2- and 3-fold relative to the low dose, the AUC0-∞ increased by factors of 2.1 and 3.4. At 75 mg/kg bw, the over-proportional increase in the AUC0-∞ was more pronounced. Whereas the overall increase in dose from the lowest to the highest dose level was 5-fold, the AUC0-∞ increased by a factor of 6.0. Again, saturation of excretion became more obvious when using the AUC12-24h data. Excretion rates reflected by over-proportionally increased AUC12-24h values were clearly impaired at 30 mg/kg bw and above.
Thus, the experiments with single oral administration indicated that saturation of excretion occurred somewhere between 5 and 100 mg/kg bw. After 14 days pre-treatment, an over-proportional increase in the AUC and, thus, impairment of excretion was evident at 40 mg/kg bw. As compared to experiments with 14-day pretreatment, the AUC0-∞ and AUC12-24h values were clearly increased, i.e. body burdens were higher after 60 days treatment. Consequently, impairment of excretion already occurred at 30 mg/kg bw. These data clearly indicate that there is a progression of saturation of excretion with prolongation of treatment. When plotting the AUC vs. dose data after 14 and 60-day pretreatment in one graph, this progression is reflected by a "left-shift" of the curve with prolonged treatment leading to a shift of the breakpoint for saturation of excretion to lower doses. This is further confirmed by the fact that once saturation of excretion occurred, the slope of the respective part of the AUC vs. dose curve was independent of the treatment duration.
In summary, these data demonstrated saturation of excretion, indicated by the over-proportional increase of the AUC with increasing dose, which occurred at test material doses of 30 mg/kg bw and above. The AUC0-∞ and, in particular, the AUC12- 24h data give clear evidence that saturation of excretion of the test material progressed with increasing treatment duration, i.e. the longer the animals are treated, the lower the dose required for impairment of excretion. Such a progression of kinetic effects may result in progression of toxicity with prolonged treatment.
Plasma Half-Life
In addition to the AUC, plasma half-life is another parameter indicative for saturation phenomena. Saturation of excretion will lead to a longer residence time in plasma and, thus, to a higher half-life. However, due to the fact that even slight variabilities in plasma concentrations at single timepoints can have large consequences on the plasma half-life, plasma half-life is a somewhat less reliable indicator for excretory saturation phenomena and is therefore more of supportive nature. When considering plasma half-life for the evaluation of saturation phenomenon, the half-life during the pure excretory phase (phase 2) is the most appropriate one.
After single oral dosing with 5 mg/kg bw, the half-life of the test material during the pure excretory phase (phase 2) was 3.22 h. Plasma half-life during this phase increased to 5.52 h after dosing with 100 mg/kg bw giving clear evidence for saturation of excretion at this dose.
After pre-treatment for 14 days, half-lives during the main excretory phase (phase 2) remained more or less unchanged at doses of 10 and 20 mg/kg bw. At 40 mg/kg bw, a slightly higher half-life was observed becoming more pronounced at doses of 80 mg/kg bw and above.
When rats were pre-dosed for 60 days, phase 2 plasma half-lives remained more or less unchanged at doses of 15 and 30 mg/kg bw. However, a clear increase in phase 2 plasma half-life was evident at 45 mg/kg bw and above with half-lives being about 1 hour longer as compared to the two low doses.
These data on the half-lives in plasma further supported the conclusions drawn from the AUC-data in a two-fold way: they confirm that saturation of excretion occurred. The fact that saturation appeared to occur at higher doses as compared to the AUC data, simply due to the insensitivity of this parameter as explained earlier. Half-life data also reflect the progression of the effect with prolonged dosing: whereas phase 2 half-life was only slightly increased after 14 days pretreatment with about 40 mg/kg bw, a very clear increase could be observed at 45 mg/kg bw after 60 days pretreatment.
Plasma Concentration at 24 h Post-Radio-labelling Dosing
Another parameter of interest is the plasma concentration 24 hours after dosing which can be interpreted to represent carryover of systemically available test material to the subsequent application day. This carryover is important insofar, as it increases the body burden with the test material on the subsequent application day.
After single oral application of 14C-test material at dose levels of 5 and 100 mg/kg bw, the plasma concentration at 24 h post radio-labelled dosing, increased by a factor of 113 whereas dose only increased by a factor of 20.
After 14 days pretreatment, 24 h plasma concentrations clearly showed that up to 20 mg/kg bw, carryover increased virtually linearly with dose. At 40 mg/kg bw, a clearly over-proportional plasma concentration was measured indicating that this dose level approximates to the point at which saturation is achieved. Due to saturation of excretion, the carryover to the next application day increased dramatically at doses of 80 mg/kg bw and above leading to a markedly increased body burden of the test material when 'entering' the subsequent treatment day. The kinetics indicate that this effect would be progressive, leading to a very rapid build-up of body burden at high doses.
After 60 days of pretreatment, the data also clearly reflect the saturation of excretion of the test material with increasing dose. Already at 30 mg/kg bw and even more pronounced at 45 mg/kg bw, a over-proportional plasma concentration at 24 h was measured indicating that saturation is achieved in this dose range. Due to saturation of excretion, the carryover to the next application day increased much more at 75 mg/kg bw leading to a markedly increased body burden of the test material when 'entering' the subsequent treatment day.
These data on the 24 h plasma concentrations further confirm the conclusions drawn from the other kinetic parameters evaluated: They clearly indicate that the saturation effect is progressive with dose leading to a build-up of body burden at high doses. The data clearly demonstrate that carryover of the test material to the subsequent application day is over-proportionally increased at 30 mg/kg bw after both 14 and 60 days pretreatment.
When comparing the absolute AUC values of the 14- and 60-day studies, it is of interest to note that there were consistently higher AUC values in the 60-day study. For example, a 40 mg/kg dose after 14-days yielded an AUC value of 1 425, a similar value (1432) was obtained in the 60 day study at a dose of 30 mg/kg. After 60-days feeding with 75 mg/kg an AUC value of 4 052 was obtained, which is in between a dose of 80 mg/kg (AUC: 3584) and 120 mg/kg (AUC: 5 771) in the 14-day study. These data thus demonstrate a gradual build-up of body burden over time.
Based on the results of the reviewed data, long term administration of doses above a level of 30 - 40 mg/kg/day or higher are likely to result in progressive toxicity, which may compromise the health and survival of the rats.
Summary of the Distribution, Excretion and Recovery of Administered Radioactivity.
Summary of Cumulative† Total Radioactivity Data for Goats Expressed as μg equiv/g and % Total Dose Administered |
||||
Sample |
5 ppm Goat 1 |
50 ppm Goat 2 |
||
μg equiv/g |
% Dose |
μg equiv/g |
% Dose |
|
Urine |
NA |
80.96 |
NA |
64.53 |
Faeces |
NA |
10.97 |
NA |
24.86 |
Cage wash |
NA |
5.31 |
NA |
6.52 |
Milk |
NA |
0.02 |
NA |
0.02 |
Omental fat |
*0.001 |
NA |
*0.003 |
NA |
Renal fat |
*0.001 |
NA |
0.003 |
NA |
Kidney |
0.007 |
<0.01 |
0.097 |
<0.01 |
Liver |
*0.001 |
<0.01 |
0.031 |
<0.01 |
Muscle hind |
<0.001 |
NA |
*0.001 |
NA |
Muscle fore |
<0.001 |
NA |
*0.001 |
NA |
Whole blood |
0.005 |
NA |
0.029 |
NA |
Plasma |
0.004 |
NA |
0.035 |
NA |
| Total | NA | 97.3 | 0.035 | 95.9 |
NA: Not applicable
†: Urine, faeces, cage wash and milk only.
*: Results calculated from data less than 30 dpm above background.
Mean Residues of the Test Material in Milk
Day of Dosing |
Mean Residue (mg/kg) |
|||
Group A 0x |
Group B 1x |
Group C 3x |
Group D 10x |
|
-1 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
1 |
< 0.01 |
0.012 |
0.047 |
0.116 |
3 |
< 0.01 |
0.015 |
0.046 |
0.154 |
5 |
< 0.01 |
0.014 |
0.034 |
0.109 |
7 |
< 0.01 |
0.014 |
0.036 |
0.120 |
10 |
< 0.01 |
0.016 |
0.034 |
0.108 |
14 |
< 0.01 |
0.013 |
0.029 |
0.099 |
18 |
< 0.01 |
0.017 |
0.038 |
0.110 |
20 |
< 0.01 |
0.014 |
0.044 |
0.108 |
22 |
< 0.01 |
0.015 |
0.037 |
0.109 |
24 |
< 0.01 |
0.013 |
0.047 |
0.100 |
28 |
- |
0.015 |
- |
0.152 |
29 |
< 0.01 |
- |
0.049 |
- |
Depuration Period |
||||
29 |
- |
- |
- |
0.031 |
30 |
- |
- |
- |
< 0.01 |
31 |
- |
- |
- |
< 0.01 |
33 |
- |
- |
- |
< 0.01 |
35 |
- |
- |
- |
< 0.01 |
38 |
- |
- |
- |
< 0.01 |
Mean Residues of the Test Material in Skimmed Milk and Cream
Matrix |
Mean Residue (mg/kg) |
|||
Group A 0x |
Group B 1x |
Group C 3x |
Group D 10x |
|
Skimmed milk |
< 0.01 |
0.013 |
0.036 |
0.111 |
Cream |
< 0.01 |
0.015 |
0.040 |
0.133 |
Mean Residues of HMCPP in Skimmed Milk and Cream
Matrix |
Mean Residue (mg/kg) |
|||
Group A 0x |
Group B 1x |
Group C 3x |
Group D 10x |
|
Skimmed milk |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
Cream |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
Mean Residues of CCPP in Skimmed Milk and Cream
Matrix |
Mean Residue (mg/kg) |
|||
Group A 0x |
Group B 1x |
Group C 3x |
Group D 10x |
|
Skimmed milk |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
Cream |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
Mean Residues of PCOC in Skimmed Milk and Cream
Matrix |
Mean Residue (mg/kg) |
|||
Group A 0x |
Group B 1x |
Group C 3x |
Group D 10x |
|
Skimmed milk |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
Cream |
< 0.01 |
< 0.01 |
< 0.01 |
0.024 |
Mean Residues of the Test Material in Muscle, Liver, Kidney and Fat
Dose Group |
Dose |
Days of Depuration |
Mean Residue (mg/kg) |
|||
Muscle |
Liver |
Kidney |
Fat |
|||
A |
0x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
B |
1x |
- |
0.084 |
0.196 |
0.999 |
0.192 |
C |
3x |
- |
0.142 |
0.404 |
2.201 |
0.255 |
D |
10x |
- |
0.317 |
0.773 |
6.226 |
0.451 |
D |
10x |
3 |
0.063 |
0.215 |
0.215 |
0.183 |
D |
10x |
5 |
0.044 |
0.220 |
0.182 |
0.104 |
D |
10x |
10 |
0.034 |
0.160 |
0.182 |
0.128 |
Mean Residues of HMCPP in Muscle, Liver, Kidney and Fat
Dose Group |
Dose |
Days of Depuration |
Mean Residue (mg/kg) |
|||
Muscle |
Liver |
Kidney |
Fat |
|||
A |
0x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
B |
1x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
C |
3x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
3 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
5 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
10 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
Mean Residues of CCPP in Muscle, Liver, Kidney and Fat
Dose Group |
Dose |
Days of Depuration |
Mean Residue (mg/kg) |
|||
Muscle |
Liver |
Kidney |
Fat |
|||
A |
0x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
B |
1x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
C |
3x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
3 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
5 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
10 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
Mean Residues of PCOC in Muscle, Liver, Kidney and Fat
Dose Group |
Dose |
Days of Depuration |
Mean Residue (mg/kg) |
|||
Muscle |
Liver |
Kidney |
Fat |
|||
A |
0x |
- |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
B |
1x |
- |
< 0.01 |
< 0.01 |
0.016 |
< 0.01 |
C |
3x |
- |
< 0.01 |
< 0.01 |
0.029 |
< 0.01 |
D |
10x |
- |
< 0.01 |
0.016 |
0.059 |
0.012 |
D |
10x |
3 |
< 0.01 |
< 0.01 |
0.023 |
0.017 |
D |
10x |
5 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
D |
10x |
10 |
< 0.01 |
< 0.01 |
< 0.01 |
< 0.01 |
Bodyweights
- Treatment did not affect bodyweights.
Daily Observations and Necropsy
- There were no clinical observations made during the study which were attributed to treatment.
No treatment-related effects were noted post-mortem in any of the cows at any dose level.
Actual Dose Levels
Cow Number |
Nominal Dose (mg/kg Dry Matter Intake) |
Nominal Dose (mg/kg bodyweight) |
Nominal Dose (g/animal/day) |
Actual Concentration (mg/kg Dry Matter Intake)* |
Actual Daily Dose (mg/kg bodyweight)** |
1 |
0 |
0 |
0 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
Average |
0 |
0 |
0 |
0 |
0 |
3 |
194 |
7 |
3.88 |
180 |
5.1 |
4 |
194 |
7 |
3.88 |
187 |
6.9 |
5 |
194 |
7 |
3.88 |
176 |
5.3 |
Average |
194 |
7 |
3.88 |
181 |
5.8 |
6 |
581 |
21 |
11.6 |
662 |
17.0 |
7 |
581 |
21 |
11.6 |
572 |
18.3 |
8 |
581 |
21 |
11.6 |
536 |
17.7 |
Average |
581 |
21 |
11.6 |
590 |
17.7 |
9 |
1940 |
70 |
38.8 |
2201 |
52.2 |
10 |
1940 |
70 |
38.8 |
1829 |
47.5 |
11 |
1940 |
70 |
38.8 |
1884 |
57.7 |
12 |
1940 |
70 |
38.8 |
1998 |
62.3 |
13 |
1940 |
70 |
38.8 |
1835 |
62.3 |
14 |
1940 |
70 |
38.8 |
1779 |
52.0 |
Average |
1940 |
70 |
38.8 |
1921 |
55.7 |
* based on average feed intake during the dosing period.
** based on average bodyweight during the dosing period.
Dosing Solution Concentrations
Dose Group |
Target Concentration (mg/mL) |
Test Material Measured Concentration (mg/mL) |
|||
Week 1 |
Week 2 |
Week 3 |
Week 4 |
||
B |
48.5 |
55.8 |
56.5 |
64.0 |
60.6 |
C |
145.5 |
160.2 |
158.9 |
193.4 |
191.2 |
D |
485.0 |
546.5 |
567.9 |
553.7 |
580.7 |
Stability of Dosing Solutions
Dose Group |
Target Concentration (mg/mL) |
Test Material Measured Concentration (mg/mL) |
||
Storage Week 0 |
Storage Week 1 |
Storage Week 2 |
||
B |
48.5 |
55.8 |
61.7 |
61.9 |
C |
145.5 |
160.2 |
183.3 |
189.5 |
D |
485.0 |
546.5 |
566.0 |
532.4 |
Note: The results listed here were generated for confirmatory purposes only. Target concentration values are used in calculating actual doses throughout this report.
The results demonstrate adequate stability of the test material in the dosing solutions during storage.
Validation Results
The recovery results demonstrate successful validation of the analytical method for the determination of the test material, HMCPP, CCPP and PCOC in bovine whole milk, skimmed milk, cream, muscle, liver, kidney and fat. In all cases, mean recovery values were within the 70 % to 120 % range, with RSD values below 20 %.
Some individual values were slightly outside this range, mostly for the test material ethyl hexyl ester, but this does not affect the validity of the results.
The results for storage stability of extracts over a period of 8 to 23 days generally show good stability, with the exception of PCOC in some matrices. The high recovery values found in some stored extracts are believed to be caused by changes to the specimen matrix during storage. It is therefore advisable to analyse extracts promptly after they have been produced. The extracts of specimens in this study were analysed within 7 days of extraction.
Evaluation of the Stability of Residues of Test Material, H-Test Material, CCPP and PCOC in Bovine Whole Milk, Skimmed Milk, Cream, Muscle, Liver, Kidney and Fat When Stored Under Frozen Storage Conditions
Mean procedural recoveries were in the range 89 % to 111 % for test material, 91 % to 107 % for H-test material, 94 % to 111 % for CCPP and 76 % to 98 % for PCOC.
The mean stored recoveries of test material (corrected for the mean procedural recovery) after approximately 9 months of storage were 98 % in whole milk, 92 % in skimmed milk, 101 % in cream, 100 % in muscle, 100 % in liver, 94 % in kidney and 110 % in fat.
The mean stored recoveries of H-test material (corrected for the mean procedural recovery) after approximately 9 months of storage were 105 % in whole milk, 99 % in skimmed milk, 99 % in cream, 112 % in muscle, 98 % in liver, 101 % in kidney and 116 % in fat.
The mean stored recoveries of CCPP (corrected for the mean procedural recovery) after approximately 9 months of storage were 112 % in whole milk, 93 % in skimmed milk, 98 % in cream, 113 % in muscle, 96 % in liver, 99 % in kidney and 133 % in fat.
The mean stored recoveries of PCOC (corrected for the mean procedural recovery) after approximately 9 months of storage were 110 % in whole milk, 114 % in skimmed milk, 106 % in cream, 64 % in muscle, 59 % in liver, 47 % in kidney and 40 % in fat.
Overall, the results show that no significant degradation of test material, H-test material or CCPP residues was observed in whole milk, skimmed milk, cream, muscle, liver, kidney or fat after approximately 9 months of frozen storage. No significant degradation of PCOC residues was observed in whole milk, skimmed milk or cream after approximately 9 months of frozen storage. Residues of PCOC degraded to a mean value of 64 % in muscle, 59 % in liver, 47 % in kidney and 40 % in fat after approximately 9 months of frozen storage.
Radiochemical Purity of [14C]Test Material and Marker Substrates Dosing Solutions
Radiochemical purity of the [14C]test material dosing solution was determined prior use. It was 97.7 % at the beginning of the study and 98.5 % at the end of the study, showing that [14C]test material did not degrade in the dosing solution. Radiochemical purity of marker substrate dosing solutions was determined prior use. Radiochemical purity of [14C]Testosterone dosing solution was 99.6 %. Radiochemical purity of [14C]7-Ethoxycoumarin dosing solution used in rat, mouse and rabbit assays was 99.7 %. Radiochemical purity of [14C]7-Ethoxycoumarin dosing solution used in dog assays was 100 %.
Concentration of [14C]Test Material and Marker Substrates in Microsomal Assays
Radioactivity concentration in [14C]test material and marker substrate dosing solutions was measured prior to each test performed. Actual concentration of [14C]test material and marker substrates in microsomal assays was calculated according to those measurements.
Concentration of [14C]Test Material and Marker Substrates in Microsomal Assays
In Vitro Test System |
[14C]Test Material Concentration |
Testosterone Concentration |
7-Ethoxycoumarin Concentration |
Target (µM) Actual (µM) |
Target (µM) Actual (µM) |
Target (µM) Actual (µM) |
|
Human liver microsomes |
10.0 |
250 |
n.p. |
10.1 |
256 |
n.p. |
|
Rat liver microsomes |
10.0 |
n.p. |
500 |
10.3 |
n.p. |
498 |
|
Mouse liver microsomes |
10.0 |
n.p. |
500 |
10.3 |
n.p. |
511 |
|
Dog liver microsomes |
10.0 |
n.p. |
500 |
10.5 |
n.p. |
512 |
|
Rabbit liver microsomes |
10.0 |
n.p. |
500 |
10.2 |
n.p. |
584 |
n.p.: not performed
Positive Control Enzymatic Activities
Radioactivity Recovery
The mean radioactivity recovery was 100.6 % in HLM incubated for 20 minutes with Testosterone. In animal liver microsomes incubated with 7-Ethoxycoumarin for 20 minutes, the mean radioactivity recovery was 104.4 % in RLM, 105.7 % in MLM, 103.9 % in DLM and 106.2 % in RbLM. For human and animal liver microsomal incubates, more than 98 % of the recovered radioactivity was located in the supernatant.
Enzymatic Activities
Testosterone 6β-hydroxylation rates observed in HLM were higher than the rate reported by the supplier for the corresponding lot of microsomes. 7-Ethoxycoumarin O-dealkylation rates observed in rat, mouse, dog and rabbit liver microsomes were ≥ 80 % of the mean of the rates given by the supplier for each lot of male and female liver microsomes. Positive control enzymatic activities showed that both human and animal liver microsomes used in the present study possessed metabolic competences in agreement with the acceptance criteria.
Description of key information
Key value for chemical safety assessment
- Bioaccumulation potential:
- no bioaccumulation potential
- Absorption rate - oral (%):
- 100
- Absorption rate - dermal (%):
- 100
- Absorption rate - inhalation (%):
- 100
Additional information
INTRODUCTION
In accordance with the section 8.8.1 of Annex VIII in Regulation (EC) No 1272/2008, the toxicokinetic profile of MCPP-P (Mecoprop-P; IUPAC name of (R)-2-(4-chloro-o-tolyloxy)propionic acid) was derived from all available information collated in the dossier. The ADME studies are available in the rat via the oral route for MCPP-P (Beimborn and Leibold, 2003, 2004; Lappin, 1997) and for the racemate (Mellert, 1993; Reinert, 1977). Additionally, there is an in vitro comparative metabolism assay using liver microsomes of the rat, mouse, dog, rabbit and human (Thibaut, 2016). Furthermore, the physicochemical properties of the substance and the results of mammalian toxicity studies, performed either for MCPP-P or the racemate Mecoprop, were used to supplement the toxicokinetic properties of the substance. A potential difference associated with stereoisomerism is not systematic and/or does not necessarily impact the general behaviour of MCPP-P and the racemate, in particular for the properties relevant for toxicological assessment and their toxicokinetic behaviour are considered to be very similar, supported by the similar physico-chemical properties between MCPP-P and the racemate.
The conclusions drawn in the EFSA peer-review (EFSA, 2017) were also taken into consideration for the assessment.
PHYSICOCHEMICAL PROPERTIES
MCPP-P is a white solid with a melting point of 93.5 °C. The boiling temperature was not determinable since the test material appears to decompose at temperatures above its melting point prior to boiling. The molecular weight of the substance is 214.65 g/mol. The substance is soluble in water (the water solubility ranging from 860 to 880 mg/L at 20 °C for MCPP-P; 699 – 734 mg/L for the racemate) with poor lipophilicity as indicated by a low log Pow value of -0.19 at 20 °C (pH 7) for MCPP-P and -0.43 for the racemate. Due to its moderate molecular weight and high water solubility, the substance is expected to be absorbed via the oral route. Its low lipophilicity property would limit dermal penetration. The substance has low vapor pressure values (0.0014 Pa at 25 °C for MCPP-P and 0.0016 at 25 °C for the racemate).This suggests that the substance is poorly available as a vapour. Furthermore, the registered substance is not used in a granular form and therefore inhalation is not considered to be the most significant route of exposure.
ABSORPTION:
MCPP-P is rapidly and extensively absorbed after oral administration (EFSA, 2017). In rats, the absorption of radiolabelled MCPP-P after oral administration was shown to be rapid and almost complete; oral absorption rates in rats were found to be between 90 to 100 % in males and 80 to 95 % in females based on urinary excretion within 168 hours following single administration (Lappin, 1997;cited in EFSA, 2017). Signs of absorption were observed in the available 4-, 13- and 104-week studies with rats (Mellert et al., 2003a; Mellert et al, 1995; Milburn, 2008), 4-week study with mice (Mellert et al., 2003b), and 52-week study with dogs (Bachmann et al., 1997) for MCPP-P, as well as in the 4- and 13-week study with rats (Kirsch et al., 1986;Reinert, 1977), 13- and 78-week study with mice (Mellert, 1993, 1996, 1999) for the racemate.
Following a single oral dose of 5 mg/kg bw, the Tmax for total radioactivity in plasma was 1.8/2.7 hours (males/females), the Cmax was 27.8/31.5 μg eq/g (males/females), and the Area Under Curve (AUC) was 252.0/182.7 hr·μg equivalents/g (males and females) with the elimination half-life of 6.4/4.2 hours (males/females). At 100 mg/kg bw, the Tmax for total radioactivity in plasma was 4.2 hours (both males and females), the Cmax was 384.0/394.1 μg eq/g (males/females), and the AUC was 8449/7884 hr·μg eq/g (males/females), with the elimination half-life of 7.9/7.8 hours (males/females) (Lappin, 1997). Following oral dosing in rats, it was found that the increase in Cmax with increasing dose was less than dose proportional, while the increase in AUC was greater than dose proportional. For a 20-fold increase in dose from 5 to 100 mg/kg, the mean Cmax increased 13-fold, and mean AUC increased 38-fold. The analysis of the results on the plasma kinetics of radiolabeled test materials in rats concluded that there is evidence for saturation of excretion of the substance starting at dose levels of approximately 30 – 40 mg/kg bw. This would suggest that an increasing body burden at high dose levels more apparent in long-term studies.
There are no signs of dermal absorption in the available acute dermal studies in rats, rabbits and guinea pigs (Allan et al., 1993; Cummins, 1990). This is supported by apoor lipophilicity with a low log Pow value of -0.19 at 20 °C.
Given its low vapour pressure, the substance is not considered to be available as a vapour. The results of the acute inhalation study in rats (snout-only exposure) did not show any signs of absorption (Cracknell, 1990).
Distribution
MCPP-P is mainly distributed in the thyroid, kidney, blood and plasma (EFSA, 2017). The high water solubility and relatively small molecular weight of the test material will allow it to disperse into the water compartment of blood for distribution. Due to the low log octanol/water partition coefficient value,the substanceis unlikely to be accumulated in body fat. In the EFSA review, it was concluded that there is no evidence of accumulation (EFSA, 2017).
Metabolism
Hydroxylation is the main metabolic step of MCPP-P (EFSA, 2017). Hydroxylated MCPP-P, and to a lesser extent, carboxyl-MCPP-P were found as urine metabolites in rats, with male rats showing a greater degree of metabolism than female rats (Lappin, 1997).
There is no convincing evidence to suggest the hepatic metabolism enhances the activity of the substance. The metabolite hydroxymethyl-MCPP-P (HMCPP) showed lower toxicity than the parent in the acute oral toxicity and 28-day repeated dose studies. Both MCPP-P and HMCPP was found to be non-genotoxic (EFSA, 2017).
Thein vitro comparative metabolism assay showed a limited metabolism of MCPP-P in human, rat, mouse, dog and rabbit liver microsomes (Thibaut, 2016). The metabolic profiles and kinetics observed in human and animal liver microsomes incubated with radiolabeled MCPP-P were similar qualitatively and quantitatively. Based on this assay, mouse and rats were considered more relevant to humans (EFSA, 2017).
Excretion
Following oral administration in rats,MCPP-P is rapidly eliminated, mostly with urine accounting for up to 90 % of the dose, with faecal excretion of up to 12 %. A half-life is less than 8 hours (Lappin, 1997; Garratt and Allen, 2014). MCPP-P is largely excreted as unchanged parent in urine. Hydroxylated MCPP-P and carboxy-MCPP-P were found at low levels in rat urines. No metabolites were found in faeces. No radioactivity was found in the expired air in the rats (Lappin, 1997).
References
Allan, S.A. et al. (1993) Twenty-one-day dermal toxicity study in the rabbit with MCPP-p acid. Report No. JEL 50/921436. Unpublished report.
Bachmann, S. et al. (1997) Mecoprop P: Chronic oral toxicity study in Beagle dogs Administration in the diet for 12 months. Report No. 33D0002/91166. Unpublished report.
Cracknell, S. (1990) Mecoprop-P: Acute inhalation toxicity study in the rat. Report No. 90/AMS022/0278. Unpublished report.
Cummins, H.A. (1990) Mecoprop-P: Acute percutaneous toxicity study in the rat. Unpublished report.
Garratt, J. & Allen, G. (2014) Derivation of the ADME rate constant of mecoprop p in rats, suitable for body burden calculations. Report No. E2014-15. Unpublished report.
EFSA, European Food Safety Authority (2017) Peer review of the pesticide risk assessment of the active substance mecoprop-P. EFSA Journal 2017;15(5):4832.
Kirsch, P. et al. (1986) Report on the comparative study of the toxicity of the racemate and D-form of Mecoprop in rats after 7-week administration in the diet. Report No. 30S0047/8330. Unpublished report.
Lappin, G. (1997) (C14) Mecoprop-P: Adsorption Distribution, Metabolism and Excretion in the Rat. Report No. 149/3-1007. Unpublished report.
Mellert, W. et al. (2003a) Mecoprop-P: Peroxisome proliferation study in Wistar rats Administration in the diet over 4 weeks. Report No. 48C0002/91188. Unpublished report.
Mellert, W. et al. (2003b) Mecoprop-P: Mecoprop-p Peroxisome proliferation study in 86C3F1 mice Administration in the diet over 4 weeks. Report No. 49C0002/91189. Unpublished report.
Mellert et al., W (1993) Report on the study of the oral toxicity of Mecoprop-p acid in B6C3F1 mice, administration in diet for 3 months. Report No. 35C0002/91002. Unpublished report.
Mellert et al., W (1995) Mecoprop-P: Subchronic oral dietary toxicity and neurotoxicity study in Wistar rats. Report No. 50C0002/91157. Unpublished report.
Mellert et al., W (1996) Mecoprop-P: Carcinogenicity study in B6C3F1/CrlBR mice. Administration in the diet for 18 months. Report No. 76S0002/91102. Unpublished report.
Mellert et al., W (1999) Mecoprop-P: Carcinogenicity study in B6C3F1/CrlBR mice, Administration in the diet for 18 months (Supplementary study). Report No. 99/10101. Unpublished report.
Milburn, G.M. (2008) Mecoprop-P: Dietary two year carcinogenicity study in the rat. Report No. PR1324-REG. Unpublished report.
Reinert, H. (1977) Mecoprop, 3-month oral toxicity study in the rat (racemate, D-isomer). Report No. IFREB-R 807266.Unpublished report.
Thibaut, R. (2016) [14C]Mecoprop-P: In Vitro comparative metabolism in human, rat, mouse, dog and rabbit liver microsomes. Report No. 20140215.Unpublished report.
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