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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

other: bioavailability

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

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: 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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Group 4

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.

Preliminary studies:

No

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.

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.

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.

Type:

metabolism

Results:

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

Type:

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

Results:

Males: 67.3% - Females: 30%

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.

Key result

Test no.:

#4

Toxicokinetic parameters:

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

Key result

Test no.:

#5

Toxicokinetic parameters:

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

Key result

Test no.:

#4

Toxicokinetic parameters:

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

Key result

Test no.:

#5

Toxicokinetic parameters:

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

Key result

Test no.:

#4

Toxicokinetic parameters:

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

Key result

Test no.:

#5

Toxicokinetic parameters:

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

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) 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.

- 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).

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.

Reference 2

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.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010-07-28 to 2010-09-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

toxicokinetics

Principles of method if other than guideline:

No test guideline mentioned but the procedures were well described.
- Principle of test:
This satellite study was conducted to investigate the toxicokinetic profile of the test item administered in a single oral gavage dose, or following a 27-day administration period. Blood, urine and faeces samples were sampled at regular intervals and analysed.
- Short description of test conditions: groups of animals were treated at 3 dose levels.
- Parameters analysed : Tmax, Cmax, AUC and half-life were determined in plasma. Tmax, Max elimination rate, AUC, half-life and renal clearance were determined in urine.
In addition, the plasma levels were determined 24 hours after administration on day 1 and on day 27.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water

Duration and frequency of treatment / exposure:

Satellite groups: Single oral administration
Main groups: daily oral administration for 27 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Males/Females

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Males

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Males

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Males

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Females

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Females

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Females

No. of animals per sex per dose / concentration:

3/sex in control group, 6/sex/dose in treated groups (single treatment), 6/sex/dose in treated groups (27-day treatment)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on preliminary studies
- Rationale for animal assignment : computerised stratified randomisation to give approximately equal initial mean body weights

Details on dosing and sampling:

TOXICOKINETIC
- Tissues and body fluids sampled: urine, faeces, plasma
- Time and frequency of sampling:
Blood samples collected in different animals at 0, 2, 4, 6, 8, 24, 48, 72h
Urine and feces samples were collected at 6 intervals: 0-8, 24-48, 8-24, 48-72, 72-120, 120-168 h

METABOLITE CHARACTERISATION STUDIES : No

Type:

absorption

Results:

All animals were largely exposed. Absorption was generally higher and slower in males compared to females, as indicated by the AUC values and Tmax.

Type:

distribution

Results:

Presence in plasma and urine support tissue distribution. Distribution to liver is demonstrated by increased activity of cytochromoe P450 and other effects. Urine excretion of carboxylated anion provides evidence of distribution to kidneys.

Type:

metabolism

Results:

The carboxylated anion was eliminated unchanged in urine, suggesting no metabolism.

Type:

excretion

Results:

Renal excretion. Elimination in females is higher than in males, as indicated by Cmax, AUC and the renal clearance. Most of the substance is eliminated during the first 24 hours.

Details on absorption:

There was a high variability in the absorption, as indicated in the AUC measured. The high variability in the groups might indicate problems in the absorption process.
Absorption was generally higher and slower in males compared to females, as indicated by the plasma AUC values and Tmax.

Details on distribution in tissues:

Presence in plasma and urine support tissue distribution.
Liver exposure was demonstrated by the dose-dependent increased activity of cytochrome P450 and cyanide-insensitive palmitoyl CoA oxidase in males and females, as well as the liver damages reported in section 7.5.1.

Details on excretion:

The substance was mainly eliminated via urine. Less than 1.5% was found in faeces.

Cumulative excretion over the period 0 - 168h:
The % excreted in urine was similar in both sexes. The amount of anion excreted decreased with the dose administered. Most of the carboxylated anion was excreted mainly in urine within the first 24 hours.
Males: 5 mg/kg: 52.4% - 20 mg/kg: 49.9% - 60 mg/kg: 27.9%
Females: 20 mg/kg: 56.4% - 60 mg/kg: 41.6% - 200 mg/kg: 35.6%

The renal clearance was 4.1 - 7.0 - 5.4 ml/h in males, while it was found 10-fold higher in females.

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: (plasma) Males: 4 - 2- 4h; Females: 2 - 2 - 2h

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: (plasma) Males: 15 - 57- 98 µg/ml ; Females: 6.6 - 35.6 - 116.7 µg/ml

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: (plasma) Males: 7.5 - 12.6 - 5.5 h ; Females: 2.2 - 5.8 - 10.75 h

Key result

Toxicokinetic parameters:

Tmax: (urine) 0 - 8 hrs

Remarks:

(males and females)

Key result

Toxicokinetic parameters:

Cmax: (urine) Males: 47.55 - 239.4 - 413.7 µg/mL*h ; Females: 201.2 - 450 - 1125.7 µg/mL*h

Key result

Toxicokinetic parameters:

half-life 1st: (urine) Males: 11.3 - 11 - 13 h; Females: 11.9 - 11.9 - 28.3 h

Metabolites identified:

no

Details on metabolites:

The carboxylated anion was detected unchanged in urine.

However, activity of Cytochrome P450 was increased with statistical significance in all groups with a dose-related trend in both sexes (see result tables 1 & 2 below). Following a 4-week recovery period, statistically significant increases were still observed in both sexes.

Table 1- Cytochrome P450 activity in males:

P450 activity - males	main study				recovery	recovery
group	control	5 mg/kg/d	20 mg/kg/d	60 mg/kg/d	control	60 mg/kg/d
number/group	5	5	5	5	5	5
Mean nmol/g liver	8.90	29.67	42.75	39.01	18.02	112.97
SD	2.28	5.29	14.96	20.32	4.49	4.99
group diff. at p<0.05		7.16*	18.82*	25.42*		6.94*
group diff. at p<0.01		11.93*	31.35*	42.35		10.10*
Mean nmol/g protein	0.247	0.898	0.853	0.885	0.539	2.370
SD	0.059	0.128	0.249	0.427	0.079	0.200
group diff. at p<0.05		0.176*	0.318*	0.537*		0.223*
group diff. at p<0.01		0.293*	0.530*	0.894		0.324*
Mean protein concentration mg/ml	7.195	8.854	9.985	8.818	6.622	9.571
SD	0.456	0.492	2.176	1.479	1.090	0.686
group diff. at p<0.05		0.835*	2.764*	1.924		1.332*
group diff. at p<0.01		1.390*	4.605	3.205		1.939*

Dunnett' test

Table 2 -Cytochrome P450 activity in females:

P450 activity - females	main study				recovery	recovery
group	control	20 mg/kg/d	60 mg/kg/d	200 mg/kg/d	control	200 mg/kg/d
number/group	3	5	5	4	5	4
Mean nmol/g liver	6.96	27.58	38.13	34.48	8.97	39.74
SD	1.27	10.61	6.05	14.02	0.32	2.82
group diff. at p<0.05		18.55*	18.55*	19.40*		4.53*
group diff. at p<0.01		24.60*	24.60*	25.73		7.47*
Mean nmol/g protein	0.258	0.522	0.726	0.714	0.330	0.962
SD	0.025	0.129	0.167	0.239	0.069	0.080
group diff. at p<0.05		0.318*	0.318*	0.333*		0.168*
group diff. at p<0.01		0.422*	0.422*	0.441*		0.277*
Mean protein concentration mg/ml	5.896	10.404	10.944	9.476	4.925	8.334
SD	2.427	3.602	2.932	0.748	1.201	1.324
group diff. at p<0.05		4.501*	4.501*	4.774		2.226*
group diff. at p<0.01		5.910*	5.910*	6.268		3.375*

Dunnett' test

* Plasma levels on day 1 and day 27:

Concentrations of carboxylated anion found in plasma 24 hours after dosing of day 27 was similar to those found at day 1 in males.

In females, concentrations on day 27 were higher than on day 1 in the low dose and mid-dose groups. In the high dose group the value seemed lower but it was related to an outlier value.

As no kinetics was performed at that time point, no conclusions can be drawn. Considering the half-lives determined for the substance, the time point for sampling at day 27 is probably not appropriate to assess potential accumulation.

No analysis was performed after the recovery period.

Day 1 - Plasma levels (ng/mL) in males 24 hours following first administration of day 1 (t= 24 hours)

dose groups:	control	5 mg/kg/d	20 mg/kg/d	60 mg/kg/d
individual data	BLOQ	311.5 D	429.5	605.4
individual data	BLOQ	150.7	658.1	978.2 D
individual data	BLOQ	118.9	450.6 d	618.6
mean of 3 animals	0	193.7	512.7	734.1
SD	0	103.2	126.3	211.5
CV%	0	53.30	24.64	28.82

Validation range: from LLOQ= 18.57 ng/ml to ULOQ = 928.4 ng/ml

D: diluted sample

Day 27 - Plasma levels (ng/mL) in males 24 hours following last administration of day 27 (t= 24 hours)

dose groups:	control	5 mg/kg/d	20 mg/kg/d	60 mg/kg/d
individual data	BLOQ	263.4	534.9	245.3
individual data	BLOQ	385.0	570.7	574.2
individual data	BLOQ	245.9	880.8	154.1
mean of 3 animals	0	298.1	662.1	324.5
SD	0	75.76	190.2	221.0
CV%	0	25.42	28.73	68.09

Validation range: from LLOQ= 18.57 ng/ml to ULOQ = 928.4 ng/ml

Plasma levels (ng/mL) in females 24 hours following the first administration of day 1 (t= 24 hours)

dose groups:	control	20 mg/kg/d	60 mg/kg/d	200 mg/kg/d
individual data	BLOQ	BLOQ	BLOQ	118.1
individual data	BLOQ	BLOQ	35.27	39090 D
individual data	BLOQ	BLOQ	27.99	341.6
mean of the 3 animals	0	0	21.09	13180 (230 without the outlier)
SD	0	0	18.62	22440 (158 w/o outlier value)
CV%	0	0	88.31	170.20

Validation range: from LLOQ= 18.57 ng/ml to ULOQ = 928.4 ng/ml

D: diluted sample

Plasma levels (ng/mL) in females 24 hours following last administration of day 27 (t= 24 hours)

dose groups:	control	20 mg/kg/d	60 mg/kg/d	200/100 mg/kg/d
individual data	BLOQ	160.0	281.9	431.0
individual data	BLOQ	367.6	756.0	337.6
individual data	BLOQ	222.6	358.2	472.1
mean of the 3 animals	0	250.1	465.4	413.6
SD	0	106.5	254.6	68.92
CV%	0	42.58	54.70	16.67

Validation range: from LLOQ= 18.57 ng/ml to ULOQ = 928.4 ng/ml

Conclusions:

The purpose of this study was to investigate the toxicokinetic profile of C6O4 administered at different doses. The time to reach peak or maximum C6O4 cyclic concentration (Tmax) was found to be 2 to 4 hours in the plasma from males, and 2 hours in the plasma from females and within 0 - 8 hours in the urine.
The elimination half-life was found to be 7.5, 12.6 and 5.5 hours in the plasma from males treated at 5, 20 or 60 mg/kg, respectively, and 2.2, 5.8 and 10.75 hours in the plasma from females treated at 20, 60 or 200 mg/kg, respectively. The elimination half-life for the urine was found at 11.3, 11 or 13 hours for the males treated at 5, 20, or 60 mg/kg respectively, and 11.9, 11.9, and 28.3 hours for the females treated at 20, 60, or 200 mg/kg respectively.

Reference 4

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Hepatic enzymes activity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Objective of study:

other: fatty acid oxidation activity and total cytochrome P450 activity

Principles of method if other than guideline:

Liver extracts were prepared from rats treated with cC6O4 ammonium salt for 28 days
- Peroxisome proliferation marker: a liver extract was prepared from one of two frozen samples. Extracts were assayed for each of the following endpoints: A) protein content B) cyanide-insensitive palmitoyl-CoA oxidation.
- Hepatic microsomal cytochrome P-450 enzyme content: Liver microsomal fractions were prepared from a second aliquot of frozen liver and assayed for microsomal protein content (Lowry et al (spectrophotometer) method) and total cytochrome P450 content (Omura and Sato (spectrophotometer) method).

Conclusions:

In vitro assays using liver extracts from rats treated with cC6O4 ammonium salt for 28 days showed a significant dose-dependent increase in activity of peroxisomal β-oxidation of fatty acids consistent with peroxisome proliferation.
An increased total Cytochrome P450 activity was measured in the liver of rats treated with cC6O4 ammonium salt.

Reference 5

Endpoint:

basic toxicokinetics, other

Remarks:

In vitro metabolisation study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-02-10 to 2013-07-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

metabolism

Principles of method if other than guideline:

- Principle of test: assess the metabolisation of cC6O4 in an in vitro microsomal test system.
- Short description of test conditions: the test item is incubated at 10 µM with rat liver microsomes or rat liver peroxisomes in the presence of NADPH regenerating system solution and peroxisome cofactors.
- Parameters analysed / observed: the concentration of cC6O4 is analysed to evaluate its potential transformation.

GLP compliance:

no

Radiolabelling:

no

Type:

metabolism

Results:

The concentration of the cC6O4 was not altered indicating that no formation of metabolites has occurred.

Metabolites identified:

no

Details on metabolites:

No biotransformation was observed.

1- rat liver microsomes

No decrease in the parent compound cC6O4 was observed after a 60-minute incubation with rat liver microsomes and NADPH regenerating system (Mix A) at 10 µM.

No relevant differences in cC6O4 concentration were detected after a 60 minute incubation in the presence of denatured rat liver microsomes (Mix B).

No relevant amount of cC6O4 or metabolites correlated to cC6O4 were found in the gas phase of samples at test point No. 4. The respective liquid phase was analysed and no decrease in cC6O4 concentration was observed.

In contrast there was a significant decrease in 7-ethoxycoumarin after 60 minutes of incubation with mix A.

 

Table 1 - incubation of cC6O4 with rat liver microsomes

test item	Incubation Mix	Time point No.	Time (min)	concentration %,T=0 (%) *	SD (%)
cC6O4 10 µM	A	1	0	100.00	3.20
cC6O4 10 µM	A	2	30	97.09	4.68
cC6O4 10 µM	A	3	60	97.28	4.25
cC6O4 10 µM	A	4**	60	92.24	1.54
cC6O4 10 µM	B	5	0	100.00	10.10
cC6O4 10µM	B	6	60	96.93	9.27
7-EC 100 µM	A	11	0	100.00	16.83
7-EC 100 µM	A	12	60	59.73	20.56

* Mean of 3 replicates

** sample for gas analysis

 

2- rat liver peroxisomes

When incubated with rat liver peroxisomes and Peroxisome Mix at 10 μM, no decrease in cC6O4 concentration was observed after 60 minutes.

No relevant amount of cC6O4 or metabolites correlated to cC6O4 were found in the gas phase of samples at test point No. 10. The respective liquid phase was analysed and no decrease in cC6O4 concentration was noted.

 

Table 2 - incubation of cC6O4 with rat liver peroxisomes

test item	Incubation Mix	Time point No.	Time (min)	concentration %,T=0 (%)*	SD (%)
cC6O4 10 µM	peroxisome mix	7	0.0	100.00	0.00
cC6O4 10 µM	peroxisome mix	8	30	101.40	5.50
cC6O4 10 µM	peroxisome mix	9	60	98.30	8.50
cC6O4 10 µM	peroxisome mix	10**	60	98.34	0.66

Conclusions:

Under the conditions of this study, the concentration of the cC6O4 was not altered significantly after a 60-minute incubation with rat liver microsomes or rat liver peroxisomes indicating that no formation of metabolites occurred.
The analysis of gas samples after in vitro incubation of the test item with rat liver microsome and peroxisome metabolic systems indicated the absence of cC6O4 or cC6O4 metabolites, confirming the stability of the test item under the experimental conditions.

Description of key information

Key study: toxicokinetic study (OECD Guideline 417, GLP, V2) in Sprague-Dawley rats, single dose of cC6O4 ammonium salt by the oral (20 or 200 mg/kg) or intravenous (20 mg/kg) routes.

- Peak plasma concentrations achieved within 2 to 6 hours in males and females by the oral route

- Plasma Cmax and AUC were 2-fold (200 mg/kg dose) to 7-fold (20 mg/kg dose) higher in males compared to females, resulting in a higher systemic exposure.

- Plasma concentrations dropped quickly, within 24 hours of administration.

- Urine is the primary excretion route for the carboxylated anion, with a majority of substance eliminated within 24 to 48 hours (urine Tmax: 0-8h).

- A minor fraction was found in feces (less than 1% in males, less than 2% in females).

- Plasma elimination half-lives: 5.54 - 5.95 hrs (males), and 4.24 - 7.22 hrs (females), for the doses 20 and 200 mg/kg, respectively.

- Urine elimination half-lives: 6.4 - 5.38 hrs (males), and 4.95 - 5.69 hrs (females) for the doses 20 and 200 mg/kg, respectively.

- Negligible amounts were detected in the male liver (0.02-0.04% of the dose) and kidneys (< 0.01% of the administered dose) at 48 hrs. No amount detected in female liver or kidneys.

- the volume of distribution was larger in males (49.15 ml) than in females (19.09 ml).

- Males had a higher maximum urinary elimination rate than females, however the clearance rate was higher in females.

 

 

Other supporting studies showed:

- similar results in males and females regarding the higher plasmatic concentrations in males, half-lives in plasma (4 to 13 hours in males and 2.2 to 18.7 hours females but with a larger variability of results in females), and in urine (5.38 to13h in males with no significant dose effect, and 4.95 to 28.3 hours in females, with some variability in the results at similar doses).

Based on these results, the substance is rapidly absorbed and excretion in urine occurred usually within 48 hours. There is no evidence of bioaccumulation.

The higher plasmatic exposures observed in male rats in the toxicokinetic studies are consistent with the adverse effects seen in liver at lower doses compared to females in the repeated dose toxicity studies.

 

- activation of microsome and peroxisome metabolism was clearly observed after a 4 week repeated dose administration by oral route in liver extracts. Liver toxicity, and to a minor extent kidney toxicity, were also observed.

In an in vitro toxicokinetic study (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. No significant increase was observed in the plasma concentrations in males at time point 24 hours after 27 administrations, while an increase was seen in females with results that were more variable.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

In accordance with Annex VIII (Section 8.8.1) the toxicokinetic behaviour of the substance was assessed from the relevant available information. 

The physico-chemical characteristics of the substance and results obtained from all toxicological studies of the dataset were also used to predict the toxicokinetic behaviour of cC6O4.

The toxicokinetic behaviour was investigated in Sprague-Dawley rats in a study conducted according to OECD 417 test guidelines (Key study, 2014).

In addition, the toxicokinetic properties of cC6O4 were investigated in earlier preliminary studies:

-   Preliminary Toxicokinetic Study after Single Oral (Gavage) administration at a dose level of 26.1 mg/kg b.w. (dry salt) in male Wistar Rats (non-GLP supporting study, 2008).

3 animals were used for blood sampling 15 minutes before administration (predose) and 4, 8, 12, 24, 72 and 168 hours after administration.

3 additional animals were used for urine sampling during the time intervals 0-12, 12-24, 24-72, 72-96 and 96-168 hours after administration.

Clinical signs, body weight, plasma and urine levels were determined.

 

-    Preliminary Toxicokinetic Study after single Oral (Gavage) administration at a dose level of 26.1 mg/kg b.w. (dry salt) in female Wistar rats (non-GLP supporting study, 2010).

A single group of 6 rats was allocated to the study, 3 females/time point up to 168 hours. Collection of biological samples was performed in all animals at 0, 4, 8, 12, 24, 48, 72, 168 hours for blood sampling and during the time intervals 0-12, 12-24, 24-72, 72-96 and 96-168 hours after administration for urine sampling. Clinical signs, body weight , plasma and urine levels were determined.

In addition, toxicokinetic parameters were also investigated in satellite groups of the 28 days oral toxicity study in male and female Sprague Dawley rats (GLP supporting study, 2011) after single oral (gavage) administration.

Three satellite groups were investigated; each one comprised 6 male and 6 female animals for each treatment dose:

- 5, 20 and 60 mg/kg bw/day for males;

- 20, 60 and 200 mg/kg bw/day for females.

In total 18 male and 18 female rats were investigated.

The control satellite group comprised 3 male and 3 female rats, administered once only at the same dose level of the main group (200 mg/kg bw for the high dose females). Urine and feces sampling was carried out up to 168 hours.

A proof of absorption was also carried out 24 hours after dosing of Day 27 of treatment in animals of the main groups.

 

 

Summary of results

The 28 days oral toxicity study results showed a high variability between different doses; a high variability was seen also comparing animals of the same sex at the same administered dose.

 

Key toxicokinetic results from all studies

Plasma:

rats strains, sex (study date)	Dose (mg/kg bw)	Cmax(ng/mL)	Tmax(h)	AUC0-tlast(ng*h/mL)	t1/2(h)
Wistar, males (2008) (screening)	26.1	10 400 - 16 499	4	(56 109) - 74 867	4.4
Wistar, females (2010) (screening)	26.1	2334	4	12 884	18.7
SD, males (2011)	5	15 000	4	144 000	7.5
	20	57 000	2	340 000	12.6
	60	98 000	4	724 000	5.5
SD, females (2011)	20	6 600	2	25 400	2.2
	60	35 600	2	94 200	5.8
	200	116 700	2	364 000	10.75
SD, males (2014)	20	40 123	2 - 6 - 2 - 2*	429 859	5.54
	200	342 950	4 - 2 - 4 - 2*	2 848 278	5.95
SD, females (2014)	20	6 030	2 -  4 - 24 - 6*	57 543	4.24
	200	168 150	6 - 6 - 2 - 2*	898 691	7.22

rounded values

* individual values are reported due to large variability observed

(...) less reliable value

 

 

Despite individual variability in some studies, Tmax was consistently found around 4 hours in plasma and half-lives were often found below 10 hours, and on one occasion up to 18 h.

This support a rapid absorption and distribution/elimination. The plasmatic concentrations (Cmax and AUC) were usually higher in males than in females. This higher plasmatic exposure could explain the greater sensitivity to liver toxicity observed in males in the repeated dose toxicity studies, although there was no significant levels remaining in liver or kidneys 48 hours after a single administration.

 

Urine:

	Dose (mg/kg bw)	Max rate (ng/mL)	Tmax rate (h)	AUC0-tlast (ng*h/mL)	Urinary t1/2 (h)	Clearance (mL/h)	Urinary excretion (% of dose)
Wistar, males (2008)	26.1	450 126 - 561 621	6	4 067 153 - 5 559 251	9.2 - 10.9	N.D.	82 - 91 (at 168h)
Wistar, females (2010)	26.1	96 043	6	104 585	18.3	N.D.	32.72 (at 168h)
SD, males (2011)	5	47 550	0 - 8	821 000	11.3	4.1	52.4 (at 168h)
	20	239 340	0 - 8	2 782 000	11	7.0	49.9 (at 168h)
	60	413 670	0 - 8	4 123 500	13	5.4	27.9 (at 168h)
SD, females (2011)	20	201 200	0 - 8	2 277 000	11.9	75.4	56.9 (at 168h)
	60	450 000	0 - 8	4775 000	11.9	45.3	41.6 (at 168h)
	200	1 125 700	0 - 8	12 958 000	28.3	32.3	35.6 (at 168h)
SD, males (2014)	20	441 000	0 - 8	7 372 000	6.40	14.91	104 (at 48h)
	200	2 575 000	0 - 8	39 262 000	5.38	11.60	53 (at 48h)
SD, females (2014)	20	412 000	0 - 8	4 871 000	4.95	65.81	96.1 (at 48h)
	200	1 549 000	0 - 8	7 973 000	5.69	21.99	44.02 (at 48h)

 

The urine Tmax was consistently found within 8 hours. The urine half-life varied between studies but except in a few cases it was similar in both sexes.

 

After a single oral dose male and female rats were sufficiently exposed to allow evaluation of kinetic profiles within the selected dose range, in the performed studies. The kinetic of the free acid seems to be non linear. Absorption in males was higher than in females. The value of Tmax in male and female was 4 hours with an half life of 4.4 hours in male and 18.66 hours in female. The individual time course of cC604 anion in plasma showed a high variability (%C.V of 24% to 114% in males 52.54 to 173.20% in females). The values of AUC0-tlast found in males were generally higher than in females.

 

The renal route is the main elimination route of the free acid. Maximal excretion rates (Max rate) were observed at 6 h (Tmax rate) after administration, both in males and females. The Urinary half life was 9.2 -10.9 hours in males and 18.29 hours in females. Also in urine, the individual time course of cC604 showed a high variability (%C.V of 13% to 159% in males and 14.87% to 100.20% in females).

 

The mean percentage of urinary excretion in Wistar rats appeared to be gender-dependent in that study (Males: 82-91% ; Females: 32.72%), however this was not confirmed by the other studies.

Analysis of the carboxylated anion in plasma 24 hours after the first oral administration and after 27 consecutive administrations did not show significant increase in the concentrations in males. There was more variability in females, with an increase seen between day 1 and 27.

 

In a second set of studies following oral or intravenous administration in rats (2014), the following observations were made:

 

- Absorption:

The oral absorption of cC6O4 is demonstrated in groups of rats including in a toxicokinetic study conducted similarly to the OECD Guideline 417 (GLP, V2) where cC6O4 was administered by single oral gavage to Sprague-Dawley rats at the dose-levels of 0 (purified water), 20 and 200 mg/kg. Indeed, after oral dosing at 20 and 200 mg/kg, animals of both sexes were exposed to the carboxylated anion. 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. In this study, cC6O4 was also administered by single intravenous dosing to groups of Sprague-Dawley rats (4/sex/dose) at the dose-levels of 0 (purified water), and 20 mg/kg cC6O4 in order to calculate the oral absolute bioavailability of the test item. Therefore, the estimated bioavailability (using plasma AUC from both intravenous and oral administration at 20 mg/kg) was 67% and 30% and 67% for males and females, respectively. These results based on plasma are not fully consistent with the almost complete recovery of the anion in urine (96-104%) for the dose 20 mg/kg. A high recovery in urine was also obtained in another study (supporting study, 2008) indicating the substance is well absorbed. 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. 

Available data are not sufficient to evaluate dermal and respiratory absorptions of cC6O4.

 

- Distribution:

In a repeated dose toxicity study (GLP compliant), liver toxicity, and to a minor extent kidney toxicity were observed after a 4 week repeated dose administration by oral route. In the toxicokinetic study where cC6O4 was administered by single intravenous injection to Sprague-Dawley rats, 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 indicated that the substance was more distributed in males than in females. The differences between the absolute oral bioavailability data and the urine recovery data in the study following iv or oral dosing are not well understood but could indicate a fraction of substance is distributed to tissues and/or bond to proteins prior to renal elimination, but no data are available to clarify these results.

In males the repeated exposure did not produce a significant increase in plasma levels 24 hours after administration on day 27 compared to day 1 in all 3 dose groups, but the results in females were more variable because of the very low levels observed on day 1 in low and mid-dose and a high initial concentration in the high dose.

In the toxicokinetic study where cC6O4 was administered by single oral gavage to Sprague-Dawley rats, very small amounts of carboxylated anion were found in liver and kidneys at 48h after dosing, which was considered not significant (0.02-0.04% of the dose in male liver and < 0.01% of the administered dose in male kidneys, while no amount detected in female liver or kidneys). Therefore no bioaccumulation of the substance was observed in these organs.

 

- Metabolism:

In the toxicokinetic study performed in rats, at the lower dose of 20 mg/kg the amount excreted in urine as unchanged carboxylated anion was approximately 95% indicating no metabolic transformation had occured.

In a repeated dose toxicity study (GLP compliant), activation of microsome and peroxisome metabolism was clearly observed after a 4 week repeated dose administration by oral route. In an in vitro toxicokinetic study (non GLP compliant), the possible formation of 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.

 

- Excretion:

In the toxicokinetic study where cC6O4 was administered by single oral gavage to Sprague-Dawley rats, very small amounts of carboxylated anion were found in faeces, which was not considered a significant excretion route (less than 1% in males, less than 1.5% in females). Renal excretion is the main elimination route of the carboxylated anion. Amount found in urine varied between experimental studies, but was found up to 100% of the administered dose. Most of the carboxylated anion was eliminated during the first 24-hour collection interval. In the same study, when intravenously administered, the renal route is also the main elimination route of the carboxylated anion.

In a satellite group of the 4-week repeated dose toxicity study (GLP compliant), the toxicokinetic after a single and repeated oral dose was also investigated. The renal route was found to be the main elimination route and test chemical elimination was seen to be approximately 30 to 50% in terms of carboxylated anion.