Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 April 2012 to 08 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted similarly to OECD Guideline 417 with some deviations: age at study initiation was not mentioned in the study report

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 April 2012 to 08 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted similarly to OECD Guideline 417 with some deviations: age at study initiation was not mentioned in the study report

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

distribution

excretion

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

age at study initiation was not mentioned in the study report; Artificial light in the animal room was not for 12 hours on Day 1 for the 8h bleeding. All the above deviations are not considered to have compromised the purpose or conduct of the study

Qualifier:

according to guideline

Guideline:

other: Note for Guidance on Toxicokinetics: “A Guidance for Assessing of Systemic Exposure in Toxicology Studies”, CPMP/ICH/384/95.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Radiolabelling:

no

Remarks:

From Sponsor communication, an ADME study with radiolabelled compound was not feasible due to the chemical characteristics of the test item.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
For STEP1 of the study: 60 Hsd: Sprague Dawley SD rats ordered from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy and supplied by Harlan Nederland, Kreuzelweg, 53, Horst NL-5960 AD Horst, The Nederlands.
For STEP2 of the study: 12 Sprague Dawley: Crl:CD(SD) rats ordered to and supplied by Charles River Italia S.p.A., Calco, Lecco, Italy for Step 2 of the study.
- Age at study initiation: no data
- Weight at study initiation:
STEP1: 239.5 to 253.3 g for males and 195.1 to 198.0 for females.
STEP2: 250 ± 30 g
- Fasting period before study: no
- Housing: The animals were housed in a limited access rodent facility. The animals were housed 5 of one sex to a cage, in polisulphone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags; nesting material was changed at least twice a week.
- Individual metabolism cages: yes, only group 6 (animals of Groups 1 and 2 were maintained in the same condition)
- Animals of the preliminary screening were housed 2/cage.
- Diet: 4 RF 18 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) laboratory rodent diet, ad libitum
- Water: drinking water supplied via water bottles, ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: approximately 15 to 20 per hour
- Photoperiod: 12 h dark / 12 h light, with the exception of Day 1 for the 8 h bleeding

IN-LIFE DATES: From: 2 May 2012 To: 13 December 2012

Route of administration:

intravenous

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A sufficient amount of the test item was diluted with sterile water to give the required concentrations of 2 and 20 mg of ammonium salt/mL. Concentrations were calculated and expressed in terms of dry ammonium salt.

VEHICLE
- Concentration in vehicle: 2 and 20 mg of ammonium salt/mL
- Amount of vehicle: 1 mL/kg

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
- The analytical method was validated in RTC Study nos. 82340, 82350 and 89720 in the range from 0.03 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in RTC’s SOPs for solutions (r > 0.98; accuracy 97-103%; precision CV < 5%).
- The proposed formulation procedure for the test item was checked in the range from 2 to 20 mg/mL by chemical analysis (concentration) during the pre-treatment period to confirm that the method was acceptable.
- Acceptance criterion: Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (95-105%).
- Results: Results of all analyses were within the limits of acceptance.
- Samples of the formulations, prepared on Day 1, were analysed to check the concentrations which were within the limits of acceptance stated in RTC’s SOPs for the concentration of solutions (95-105%)

Duration and frequency of treatment / exposure:

Single intravenous administration

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (STEP 1)

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Group 2 (STEP 1) and 6 (STEP 2)

No. of animals per sex per dose / concentration:

4 in control group and 8 at 20 mg/kg bw

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

Not applicable

Details on study design:

- Dose selection rationale: a preliminary toxicity screening was carried out to confirm that the dose level of 20 mg/kg was suitable for the intravenous dosing. Briefly, two males and two females were dosed by the intravenous route and observed for 48 hours for clinical signs (including body weight recorded on Day 2). On Day 3 animals were sacrificed (no necropsy was performed).
- Rationale for animal assignment (if not random): The rats were allocated to groups (including animals of the preliminary screening) by computerised stratified randomization to give approximately equal initial group mean body weights.
- Intravenous bolus injection into the tail vein with an approximate speed of 1 mL/minute
- The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled:
Urine samples
Animals of group 6 were individually caged in a metabolism cage for the indicated time period. At the end of each collection interval, urine was collected. A cage wash was performed with approximately 10 mL of water rinsing the whole cage (all intervals for Group 6). Urine samples collected during the interval and the correspective volume of water used for the cage wash were pooled for each single animal, mixed, and the total volume was measured. Urine samples were frozen and stored at -20°C, pending analysis. Only urine samples were analysed from animals of Group 6.

Plasma samples
Approximately 0.3 mL blood samples were collected from the tail vein of animals at each sampling time. Samples were transferred into tubes containing heparin anticoagulant, centrifuged and the plasma frozen in aliquots (2 aliquots of 50 μL plus residues were preserved) at –20°C pending analysis.

- Time and frequency of sampling:
Plasma samples
In groups treated with the test item, blood samples were withdrawn from all animals in the
group at 6 time points. In control groups only 1 bleeding was performed.
Group 1: at 5 minutes after dosing
Groups 2 and 6: at 5 minutes, 30 minutes and 2, 6, 24, 48 hours after dosing.

Urine samples
Only Group 6: at 0-8, 8-24 and 24-48 hours post-dose

- Method type(s) for identification:
Plasma and urine samples
Analysed according to a validated method (under RTC Study no. 82330) to determine cC6O4 carboxilated anion. In addition, an extension of validation was required and performed in the present study to analyse urine samples with high concentration of cC6O4 (effect of dilution up to 5000 fold). For plasma and urine, samples coming from animals of Group 6 (Step 2) analysis was carried out using a new validated method (RTC Study no. 94340) with an higher concentration range.

- Other:
All the toxicokinetic parameters were estimated or calculated by the Kinetica™, version
4.4.1, PK/PD Analysis (Thermo Electron Corporation Informatics, Philadelphia - USA) software. Means and/or medians, standard deviations and coefficient of variations were obtained using a Microsoft Excel worksheet.

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.

Preliminary studies:

A preliminary screening was carried out since no data on intravenous toxicity were available. No mortality occurred and no clinical signs were recorded in two males and two females treated at 20 mg/kg.

Type:

absorption

Results:

Not relevant for iv dosing.

Type:

distribution

Results:

The values of clearance and volume of distribution suggested that the test chemical is fairly distributed and eliminated from the body through the kidneys.

Type:

metabolism

Results:

No metabolism seem to take place based on the high % of compound excreted unchanged and absence of phase I metabolites.

Type:

excretion

Results:

Renal route is the main elimination route of the carboxylated anion when administered intraveinously. The renal clearance is slower in males than in females.

Details on absorption:

- Mean group parameters per sex and dose level are summarised in Table 7.1.1/2.
- The test chemical concentration-time profiles following single intravenous administration declined rapidly to zero (6 hours). For this reason, for calculation purpose, the half lives were calculated considering the last three points (6 to 48 hours) in both sexes. For the same reason, a single value (animal no. 90010013 time 48 hours) was excluded to allow a consistent calculation of the slope, even if the animal was not considered an outlier.
- At the last time point observed (48 h), the mean plasma concentrations were slightly higher than the lower limit of quantitation in males (approximately 4 to 20 fold, Steps 1 and 2) while only one female (no. 90010013, Step 1) showed a value slightly over the limit of quantitation.
Variability was limited to males at 24 and 48 hours. Plasma levels were not measurable in the females of Step 2 (20 mg/kg) at 48 hour post-dose.
- The values of slope and half-life were similar in the two sexes.
- The AUC and MRT values indicated that the exposure is higher and the elimination is lower in males than in females (approximately 3 and 6 times in terms of AUC and MRT respectively).
- The values of clearance and volume of distribution suggested that the test chemical is fairly distributed and eliminated from the body. Furthermore the value of the Vss indicated that the substance was more distributed in males than in females, in agreement with other kinetic parameters.

Details on distribution in tissues:

Not examined in specific tissues following iv administration. Only the plasma concentration was measured:
The single iv dose of 20 mg/kg resulted in a plasma concentration at the earliest assessed sampling (at ca. 5 min) of ca. 220000-237300 ng/ml (males and females) (the 2nd group of males had a lower concentration at that time point, 141300 ng/ml). However, the plasma concentration then dropped more rapidly in females than in males at the subsequent time points. The overall plasmatic AUC which provides an indication of the internal dose was found to be lower in females (200 - 431 ng*h/ml for group 2 and 6, respectively) compared to males (641 - 745 g*h/ml for group 2 and 6, respectively) , despite similar urinary excretion half-life and recovery.

Details on excretion:

- Urinary excretion: Intra-group variability was moderate in the males (CV% of the total percentage excreted of 38.8%), where one of the 4 animals (no. 90010046) was excluded from the calculation, as no carboxylated anion was found in the sample collected at 0-8 hour (which likely consisted of the cage wash only) (Table 7.1.1/3). The percentage of carboxylated anion found in the urine of males was approximately 94.56 ± 36.66. Low intra-group variability was observed in the females (CV% of the total percentage excreted of 11.0%), with a percentage of carboxylated anion in the urine of approximately 91.29±10.03 (Table 7.1.1/3).
- The elimination of the carboxylated anion seemed similar in both sexes in terms of urinary half-lives and elimination rate (Tmax-rate 0-8 hours in both sexes).
- The renal clearance was similar to the plasmatic one in the males, while no comparison was possible for females (plasmatic clearance not calculable).

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: Males: 6.80h; Females: 6.20h

Remarks:

Group 2 (plasma)

Key result

Test no.:

#6

Toxicokinetic parameters:

half-life 1st: Males: 4.42h; Females: 4.85h

Remarks:

group 6 (plasma)

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: Males: 641094 ng*h/mL; Females: 200212 ng*h/mL

Remarks:

Group 2 (plasma)

Key result

Test no.:

#6

Toxicokinetic parameters:

AUC: Males: 745952 ng*h/mL; Females: 431499 ng*h/mL

Remarks:

Group 6 (plasma)

Key result

Test no.:

#6

Toxicokinetic parameters:

Tmax: Males/Females: 0-8h

Remarks:

Group 6 (urine)

Key result

Test no.:

#6

Toxicokinetic parameters:

half-life 1st: Males: 4.4h ; Females:5.83h

Remarks:

Group 6 (urine)

Key result

Test no.:

#6

Toxicokinetic parameters:

other: AUC Urine 0-48h: Males: 6731322 ng*h/ml ; Females: 5718673 ng*h/ml

Remarks:

Group 6 (urine)

Key result

Test no.:

#6

Toxicokinetic parameters:

other: Cumulative excretion (0-48h): Males: 5035 µg ; Females: 4953.5 µg

Remarks:

Group 6 (urine)

Metabolites identified:

no

Details on metabolites:

In RTC Study no. 89980EXT (non GLP compliant), the formation of possible Phase I metabolites was investigated in vitro after incubation of the test item with microsomal or peroxisomal extracts. No Phase I metabolites were formed. Phase II metabolism was not investigated.

Bioaccessibility (or Bioavailability) testing results:

See TK. oral, SS, V1, RTC 2014

- No mortality occurred and no clinical signs were recorded in any animal of the study.

- No significant body weight changes were noted during the study.

- No significant variation of terminal body weight, absolute and relative organ weight was recorded in any animal of the study

Conclusions:

No bioaccumulation potential based on study results
Under the test conditions, the systemic exposure of the male and female rats to cC6O4 appeared to decline in a probable biphasic manner, after a single intravenous dosing at 20 mg/kg. While the earliest measure in plasa (at 5 min post-injection) showed similar concentrations in males and females, the plasma AUC 0-48h was lower in females compared to males. The amount excreted in urine was 95% of the administered dose after 48hours indicating no significant accumulation of the product.

Executive summary:

In a toxicokinetic study conducted similarly to the OECD Guideline 417 and in compliance with GLP, cC6O4 was administered by single intravenous dosing to groups of Sprague-Dawley rats (4/sex/dose) at the dose-levels of 0 (purified water; group 1), and 20 mg/kg (group 2). An additional group of rats (4/sex/dose) (Group 6) was subsequently dosed intravenously at the same dose level in order to obtain plasma, urine and faecal sample for further evaluation of cC6O4 (carboxylated anion) excretion.

A preliminary screening was carried out since no data on intravenous toxicity were available.

No mortality occurred and no clinical signs were recorded in two males and two females treated at 20 mg/kg.

Each animal of Groups 2 and 6 was bled at 5 minutes, 30 minutes and 2, 6, 24 and 48 hours after dosing, while Group 1 animals were bled only at 5 minutes after dosing. Analytical activities were carried out in all blood samples and in excreta in order to evaluate the presence of the unchanged carboxylated anion.

Maximum plasma concentrations of test substance (Cmax) and their time of occurence (Tmax) were determined.

 

After intravenous dosing at 20 mg/kg, the plasma concentrations of the carboxylated anion declined in a probable biphasic manner. The area under the plasma concentration-time curve (AUC) was approximately from 2- (group 6) to 3- (Group 2) times higher in males than in females. The elimination process was quite similar in the two sexes (excretion in urine of approximately 95% of the administered dose). The values of clearance and volume of distribution suggested that the test chemical is fairly distributed and eliminated from the body. Furthermore the value of the volume of distribution at steady state (Vss) indicated that the substance was more distributed in males than in females in agreement with the other kinetic parameters.

On the other hand, the values of half-lives indicated that the elimination process is similar in the two sexes, with plasma T1/2 of 4.42h (males) and 4.85h (females), and urine T1/2 of 4.44h (males) and 5.83h (females). The percentages of the administered dose recovered in urine of Group 6 animals were approximately 94.56 ± 36.66 and 91.29 ± 10.03 in males and females, respectively, suggesting an almost total excretion through the urinary route. The renal route is therefore the main elimination route of the carboxylated anion.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Objective of study:

absorption

distribution

excretion

toxicokinetics

other: bioavailability

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

age at study initiation was not mentioned in the study report; Artificial light in the animal room was not for 12 hours on Day 1 for the 8h bleeding. All the above deviations are not considered to have compromised the purpose or conduct of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Note for Guidance on Toxicokinetics: “A Guidance for Assessing of Systemic Exposure in Toxicology Studies”, CPMP/ICH/384/95.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

EC Number:

682-238-0

Cas Number:

1190931-27-1

Molecular formula:

C6H4F9NO6

IUPAC Name:

ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): cC6O4 (alternative names: C6O4 cyclic, cC6O4 Ammonium salt aqueous solution)
- MW dry salt : 357 .1
- MW carboxilated anion : 339.1
- Substance type: carboxilated anion
- Physical state: colourless liquid
- Analytical purity (in terms of dry salt): 37.6% (water solution)
- Composition of test material, percentage of components: Anti/(anti+sin) = 52.6% w; cC904NH4 = 0.4% w; Structural H = 20 ppm (as H); other fluorinated organic compound = 400 ppm (as F); H by total organic contaminations < 10 ppm (as H).
- Purity test date: 2012-01-18
- Lot/batch No.: SPI-011-NH4-CA
- Expiration date of the lot/batch: 31 December 2020
- Stability under test conditions: In RTC Study nos. 82340 and 82350, a 24 hour stability at room temperature was verified in the range from 0.03 to 20 mg/mL. Concentrations, after the defined period of storage, were still acceptable according to RTC’s SOPs (95-105%).
- Storage condition of test material: ambient condition
- Other: RTC reference number: 13167

Radiolabelling:

no

Remarks:

From Sponsor communication, an ADME study with radiolabelled compound was not feasible due to the chemical characteristics of the test item.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 60 Hsd: Sprague Dawley SD rats ordered from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy and supplied by Harlan Nederland, Kreuzelweg, 53, Horst NL-5960 AD Horst, The Nederlands.
- Age at study initiation: no data
- Weight at study initiation: 239.5 to 253.3 g for males and 195.1 to 198.0 for females.
- Fasting period before study: no
- Housing: The animals were housed in a limited access rodent facility. The animals were housed 5 of one sex to a cage, in polisulphone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags; nesting material was changed at least twice a week.
- Individual metabolism cages: yes
- Diet: 4 RF 18 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) laboratory rodent diet, ad libitum
- Water: drinking water supplied via water bottles, ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: approximately 15 to 20 per hour
- Photoperiod: 12 h dark / 12 h light, with the exception of Day 1 for the 8 h bleeding

IN-LIFE DATES: From: 2 May 2012 To: 13 December 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A sufficient amount of the test item was diluted with sterile water to give the required concentrations of 2 and 20 mg of ammonium salt/mL. Concentrations were calculated and expressed in terms of dry ammonium salt.

VEHICLE
- Concentration in vehicle: 2 and 20 mg of ammonium salt/mL
- Amount of vehicle (if gavage): 10 mL/ kg bw

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
- The analytical method was validated in RTC Study nos. 82340, 82350 and 89720 in the range from 0.03 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in RTC’s SOPs for solutions (r > 0.98; accuracy 97-103%; precision CV < 5%).
- The proposed formulation procedure for the test item was checked in the range from 2 to 20 mg/mL by chemical analysis (concentration) during the pre-treatment period to confirm that the method was acceptable.
- Acceptance criterion: Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (95-105%).
- Results: Results of all analyses were within the limits of acceptance.
- Samples of the formulations, prepared on Day 1, were analysed to check the concentrations which were within the limits of acceptance stated in RTC’s SOPs for the concentration of solutions (95-105%).

Duration and frequency of treatment / exposure:

Single oral administration

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Group 4

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 5

No. of animals per sex per dose / concentration:

4

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

Not applicable

Details on study design:

- Dose selection rationale: the dose levels were defined in agreement with the Sponsor based on information from other studies.
- Rationale for animal assignment: The rats were allocated to groups by computerised stratified randomization to give approximately equal initial group mean body weights.
- The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
- The toxicokinetic of cC6O4 was investigated, when given by a single oral or intravenous administration to the Sprague Dawley rat, in order to calculate bioavailability.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled:
Urine and faeces samples
Animals were individually caged in a metabolism cage for the indicated time period. At the end of each collection interval, faeces and urine were separately collected. A cage wash was performed with approximately 10 mL of water rinsing the cage urine container (0-8 h interval and 0-24 h interval for Group 3) or the whole cage (8-24 h and 24-48 h interval for Groups 4 and 5). Urine samples collected during the interval and the correspective volume of water used for the cage wash were pooled for each single animal, mixed, and the total volume was measured. Faeces were also weighed. Urine and faeces samples were frozen and stored at -20°C, pending analysis.

Plasma samples
Approximately 0.3 mL blood samples were collected from the tail vein of animals at each sampling time. Samples were transferred into tubes containing heparin anticoagulant, centrifuged and the plasma frozen in aliquots (2 aliquots of 50 μL plus residues were preserved) at –20°C pending analysis.

Tissue samples
- All animals were sacrificed by carbon dioxide inhalation approximately 48 hours after dosing, at the end of the last bleeding procedure. No necropsy was performed on any animal.
-Based on the results of a 4 week study (RTC Study no. 82340 ), the following ten tissues were collected just after sacrifice from animals treated with oral gavage (24 animals): liver, kidney, thymus, fat, spleen, lungs, whole blood, stomach, ileum, caecum.
Whole blood was stored in 3 aliquots of approximately 200 μL each. The other organs were collected, weighed and preserved in a single aliquot. Tissue samples were frozen and stored at -20°C. They will be destroyed shortly after finalisation of the report.

- Time and frequency of sampling:
Plasma samples
In groups treated with the test item, blood samples were withdrawn from all animals in the group at 6 time points. In control groups only 1 bleeding was performed.
Group 3: at 2 hours after dosing
Groups 4 and 5: at 2, 4, 6, 8, 24, 48 hours after dosing.

Urine and faeces samples
Group 3: 0-24 hour post-dose
Groups 4 and 5: at 0-8, 8-24 and 24-48 hour post-dose.

Tissue samples: At termination

- Method type(s) for identification:
Plasma, urine and faeces samples
Analysed according to a validated method (under RTC Study no. 82330) to determine cC6O4 carboxilated anion. In addition, an extension of validation was required and performed in the present study to analyse urine samples with high concentration of cC6O4 (effect of dilution up to 5000 fold).

Tissue samples (liver and kidneys)
Analysed according to a validated method (under RTC Study no. 91780) to determine cC6O4 carboxylated anion.

- Other:
F : Bioavailability determined from plasma kinetics of the oral and intravenous groups, as follows: F = ( AUCoral/ AUCiv ) x ( Doseiv / Doseoral)
AUC : area under the plasma concentration-time curve from time zero to infinity. [ng•h/mL]
iv: intravenous

All the toxicokinetic parameters were estimated or calculated by the Kinetica™, version 4.4.1, PK/PD Analysis (Thermo Electron Corporation Informatics, Philadelphia - USA) software. Means and/or medians, standard deviations and coefficient of variations were obtained using a Microsoft Excel worksheet.

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.

Results and discussion

Preliminary studies:

No

Main ADME results

Main ADME results 1

Type:

absorption

Results:

All animals were largely exposed to the test material. The exposure in terms of AUC was approx. 7 and 3 x higher in males than in females at low and high dose respectively. The bioavailable fraction for oral dosing was ca 67% in males, ca 30% in females.

Main ADME results 2

Type:

distribution

Results:

A very small amount of carboxylated anion was found in the liver and kidney of high dosed animals and low dosed males.

Main ADME results 3

Type:

excretion

Results:

Renal route is the main elimination route of the carboxylated anion which was found essentially in the urine. Very small amounts of carboxilated anion were found in faeces, liver and kidneys, which was considered not significant.

Main ADME results 4

Type:

metabolism

Results:

No metabolism seem to take place based on the high % of compound excreted unchanged and absence of phase I metabolites.

Main ADME results 5

Type:

other: Bioavailability determined at the dose 20 mg/kg (AUCpo/AUCiv)

Results:

Males: 67.3% - Females: 30%

Toxicokinetic / pharmacokinetic studies

Details on absorption:

- The test chemical concentration-time profiles indicated that all animals were largely exposed to the carboxylated anion both at 20 and 200 mg/kg.
- Mean group parameters per sex and dose level are summarised in Table 7.1.1/2.
- High variability, particularly in females where the Tmax ranged from 2 to 24 hours.
- The Cmax and AUC values indicated that the exposure was higher in males than in females (approximately 7 and 3 times respectively at 20 and 200 mg/kg).

Details on distribution in tissues:

LIVER:
- A very small amount of carboxylated anion was found in the liver of high dosed animals and low dosed males. Only 1 low dosed female showed detectable amounts of carboxylated anion.
- The amount found in the whole organ ranged from below the limit of quantitation to 0.01% of the administered dose in females and from 0.01 to 0.06% of the administered dose in males.
- Results (percentages found in the whole tissue) are summarised in Table 7.1.1/4
- No carboxylated anion was found in control animals (Group 3).

KIDNEYS:
- A very small amount of carboxylated anion was found in the kidney of high dosed animals and low dosed males. Only 2 low dosed females showed detectable amounts of carboxylated anion.
- The amount found in the whole organ ranged from below the limit of quantitation to 0.01% of the administered dose in females and from slightly over the limit of quantitation to 0.01% of the administered dose in males.
- Results (percentages found in the whole tissue) are summarised in Table 7.1.1/5
- No carboxylated anion was found in control animals (Group 3).

Details on excretion:

- Urinary excretion is the main elimination route of the parent compound (unchanged carboxilated anion) in animals orally administered with the test item at both doses. Most of the carboxylated anion was eliminated during the first 24-hour collection interval. At the high dose, the amount found in urine was approximately 53.20 ± 11.89 and 44.02 ± 9.09 % of administered dose in males and females respectively (Table 7.1.1/3). In the low dose group, elimination by the renal route may also represent a main elimination route (the percentages found in urine were 104.37 ± 42.64 and 96.10 ± 12.64% in males and females respectively) (Table 7.1.1/3). However considering the high variability, these data are not always in agreement with the bioavailability data.
- A minimal amount of carboxylated anion was found in faeces of animals orally treated with the test item at both doses, while in control animals no carboxylated anion was found. The biliary route is not the route of elimination of the carboxylated anion. In terms of percentages of the administered dose, the amount found ranged from 0.06 to 3.36% in females and from 0.11 to 1.17% in males.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: Males: 5.54h; Females: 4.24h

Toxicokinetic parameters 2

Key result

Test no.:

#5

Toxicokinetic parameters:

half-life 1st: Males: 5.95h; Females: 7.22h

Toxicokinetic parameters 3

Key result

Test no.:

#4

Toxicokinetic parameters:

AUC: Males:431360 ng*h/mL; Females: 60232 ng*h/mL

Toxicokinetic parameters 4

Key result

Test no.:

#5

Toxicokinetic parameters:

AUC: Males:2867150 ng*h/mL; Females: 902331 ng*h/mL

Toxicokinetic parameters 5

Key result

Test no.:

#4

Toxicokinetic parameters:

Cmax: Males:40123 ng/mL; Females: 6030 ng/mL

Toxicokinetic parameters 6

Key result

Test no.:

#5

Toxicokinetic parameters:

Cmax: Males:342950 ng/mL; Females: 168150 ng/mL

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

In the satellite study RTC Study no. 89980EXT (non GLP compliant), the formation of possible Phase I metabolites was investigated in vitro after incubation of the test item with microsomal or peroxisomal extracts. No Phase I metabolites were formed. Phase II metabolism was not investigated.

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

- The Cmax and AUC values indicated that the exposure was higher in males than in females (approximately 7 and 3 times respectively at 20 and 200 mg/kg). Therefore, the calculation of bioavailability at 200 mg/kg cannot give reliable results. The oral kinetic of the substance seemed to be non-linear. As no data at 200 mg/kg are available after intravenous administration the lack of linearity in the increase of exposure cannot be explained.
- The bioavailability (F) of carboxylated anion following oral administration of 20 mg/kg was 67.28% and 30.08% in males and females respectively. The value of approximately 45% calculated at 200 mg/kg was not considered reliable for the reasons indicated above.

Any other information on results incl. tables

- No mortality occurred and no clinical signs were recorded in any animal of the study.

- No significant body weight changes were noted during the study.

- No significant variation of terminal body weight, absolute and relative organ weight was recorded in any animal of the study. A slight, dose-related, statistically significant, increase in liver weight only in male groups was evident in animals orally treated with the test item at 20 and 200 mg/kg (Groups 4 and 5).

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the systemic exposure of the male and female rats to cC6O4 appeared to be characterized by a nonlinear dose dependent kinetics over the range of 20 to 200 mg/kg/day, after a single oral administration. The test substance disappeared from plasma within 24 hours (plasma half-life was found 5.54 h and 4.24 h in males and females treated at 20 mg/kg, respectively, and 5.95 h and 7.22 hr in males and females treated at 200 mg/kg, respectively) and was excreted via urine. The Tmax excretion in urine was within 8 hours.
The results indicate a rapid elimination of the unchanged substance via urine.

Executive summary:

In a toxicokinetic study conducted similarly to the OECD Guideline 417 and in compliance with GLP, cC6O4 was administered by single oral gavage to groups of Sprague-Dawley rats (4/sex/dose) at the dose-levels of 0 (purified water; group 3), 20 and 200 mg/kg (groups 4 and 5)

Urine and faeces were individually collected to evaluate excretion. Excreta of Groups 4 and 5 animals were collected at 0-8, 8-24 and 24-48 hour post-dose, while in Group 3 animals collection occurred at 0-24 hours. After sacrifice (approximately 48 hours after dosing), the following tissues were weighed and collected for analytical purpose from Groups 3, 4 and 5: liver, kidney, thymus, fat, spleen, lungs, whole blood, stomach, ileum, caecum.

Analytical activities were carried out in all blood samples, in excreta, in liver and kidney tissue in order to evaluate the presence of the unchanged carboxylated anion.

Maximum plasma concentrations of test substance (Cmax) and their time of occurence (Tmax) were determined.

 

After oral dosing at 20 and 200 mg/kg, animals of both sexes were exposed to the carboxylated anion. The calculated parameters indicated that the exposure in terms of AUC (area under the plasma concentration-time curve) was approximately 7 and 3 times higher in males than in females at low and high dose respectively.

The volume of distribution was larger in males than in females.

Very small amounts of carboxylated anion, considered not significant, were found in faeces, liver and kidneys, at the 48 hour time point. The renal route is the main elimination route of the carboxylated anion which was found essentially in the urine. Most of the carboxylated anion was eliminated during the first 24-hour collection interval. At the high dose, the amount found in urine was approximately 53.20 ± 11.89 and 44.02 ± 9.09% of administered dose in males and females respectively. In the low dose group, elimination by the renal route may also represent a main elimination route (the percentages found in urine were 104.37 ± 42.64 and 96.10 ± 12.64% in males and females respectively). However considering the high variability, these data are not always in agreement with the bioavailability data.

Since no data are available at 200 mg/kg for the intravenous route and the oral uptake at 200 mg/kg seems to be not linear, bioavailability at 200 mg/kg cannot be estimated. Therefore, further considerations on the bioavailability of this dose can only be considered as speculative. However, it seems that the amount found into urine after oral administration, was only part of the amount administered. At the lower dose of 20 mg/kg the amount excreted in urine was approximately 95% although the estimated bioavailability (both intravenous and oral administration at 20 mg/kg) was between 30 and 67 for females and males, respectively. The differences between the absolute oral bioavailability data and the urine data may be explained by the metabolic degradation of the test chemical.