Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 July 2013 - 03 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: COMPARATIVE PHARMACOKINETIC STUDY complies with OECD Principles of Good Laboratory Practices.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Objective of study:
absorption
metabolism
toxicokinetics
Principles of method if other than guideline:
The objective of the study was to demonstrate that the test item, Heptanal, is rapidly metabolized to Heptanoic acid. Therefore, the plasma pharmacokinetic exposure of Heptanal and Heptanoic acid was compared after a single administration to rats.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptanal
EC Number:
203-898-4
EC Name:
Heptanal
Cas Number:
111-71-7
Molecular formula:
C7H14O
IUPAC Name:
heptanal
Test material form:
other: liquid
Details on test material:
Test item
- Name: Heptanal
- Synonyms: Heptaldehyde P-PH, n-heptanal
- Name on the analytical certificate: N –HEPTANAL
- Batch number: 1305019
- CAS number: 111-71-7
- Description: colorless liquid
- Purity: 97.97%
- Storage conditions: between +2 and +8°C, protecting from light and under nitrogen and away from heat and sources of ignition
- Expiry date: 12 October 2013.

Metabolite
- Name: Heptanoic acid
- Name on the analytical certificate: n–HEPTANOIC ACID
- Batch number: 1304076
- CAS number: 111-14-8
- Description: colorless liquid
- Purity: 99.53%
- Storage conditions: at room temperature, keeping tightly closed in a dry and well-ventilated place
- Expiry date: 30 April 2015.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: approximately 14 weeks old on the day of treatment
- Mean body weight at study initiation: 412 g (range: 373 g to 470 g)
- Fasting period before study: Yes
- Housing: the animals were housed by three from the same group in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: a period of 47 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 27 August 2013 to 28 August 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:


VEHICLE
- Justification for use and choice of vehicle (if other than water): for Heptanoic acid, corn oil was used in previous study. For heptanal was chosen in order to have the vehicle for heptanoic acid
- Concentration in vehicle: 235 mg/mL of Heptanal and 211 mg/mL of Heptanoic acid
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): MKBH4894V.

HOMOGENEITY AND STABILITY OF TEST MATERIAL: no stability, the test materials were administered within 1 hour after preparation.
Duration and frequency of treatment / exposure:
The dose formulations were administered to rat sprague Dawley on a single occasion on Day 1.
Day 1 corresponds to the day of treatment.
After administration, the animals were blood collected until maximum 24 hours.

Administration
The dose formulations were administered by gavage, using a plastic syringe fitted with a metal gavage tube.
The quantity of dose formulation administered to each animal was adjusted according to the body weight recorded on the day of treatment.
A constant dosage-volume of 5 mL/kg was used.
The dose formulations were stirred continuously until filling the syringe of administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
1175 mg/kg with Heptanal.
1055 mg/kg with Heptanoic acid.
No. of animals per sex per dose / concentration:
9 males per group.
Control animals:
no
Positive control reference chemical:
none.
Details on study design:


- Dose selection rationale: the dose-levels were selected in agreement with the Sponsor, based on the results of a previous study: the 28 days repeated toxicity study (Hazleton, 1990) with the test item Heptanoic acid, in solution in corn oil administered orally (gavage) to male and female Sprague-Dawley rats at the dose-levels of 0, 875, 1750 and 3500 mg/kg/day showed that the no observable adverse effect level (NOAEL) for Heptanoic acid were concluded to be 1750 mg/kg/day.
Therefore, the dose-level of 1000 mg/kg was chosen for this study. This dose-level was considered to be sufficient for a good exposure of the test item and its metabolite.

- Rationale for animal assignment (if not random): during the acclimation period, the required number of animals (18 males) was selected according to body weight and clinical conditions, and allocated to the groups according to a computerized stratification procedure, so that the average body weight of each group was similar.
Details on dosing and sampling:
18 males Sprague Dawley were allocated into two groups of 9 rats. The first group was orally treated on a single ocasion with the heptanal and the second group with the metabolite of heptanal, heptanoic acid. Heptanal and geptanoic acid were both solubilized in corn oil as vehicle. The actual dose-levels were used for the pharmacokinetic evaluation:
-Heptanal: dose-level of 1175 mg/kg/day,
-Heptanoic acid: dose-level of 1055 mg/kg/day.
After administration, blood samples were taken at 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12 and 24 hours after administration. Three animals per group were collected at each time-point. Blood were collected on ice and centrifuged within 10 min after collection. Then, plasma samples were frozen at -80C and analyzed for determination of Heptanal and Heptanoic acid for group 1 and for determination of Heptanal and Heptanoic acid for group 2. After their respective last blood collection, the animals were sacrificed and discarded without necrospy. Mortality, morbidity and clinical signs were checked twice a day after administration.

2.4.3 Pharmacokinetic evaluation
The pharmacokinetic evaluation was carried out by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight Corporation, Mountain View, California 94040/USA) software.

For Heptanal and Heptanoic acid, the pharmacokinetic parameters were determined from the mean concentrations after averaging individual levels for each group, analyte and time-point. The Standard Deviation (SD) and Coefficient of Variation (CV) within each mean were calculated to assess inter individual variability. For samples with a concentration level below the Limit Of Quantification (LOQ), the values were considered as zero. When more than 50% of the value was below the Limit Of Quantification, the mean value was reported as BLQ.

The following pharmacokinetic parameters were calculated:
¿ Cmax (maximum concentration) was read directly from the concentration/time plots,
¿ Tmax (time to maximum concentration) was read directly from the concentration/time plots,
¿ ¿z (elimination rate constant) was determined by the log linear regression obtained from at least the last three quantifiable concentrations. When the correlation coefficient (r2) for the "goodness" of the fit of the regression line was below 0.85, the value were considered as not reliable,
¿ t1/2 (terminal half-life, in hours) was calculated as 0.693/¿z. When ¿z was considered as not reliable (r²<0.85), the t½ value was indicated as not reliable too,
¿ AUC0-t (area under the curve from 0 hours to the time-point of the last quantifiable concentration) and AUC0-8h were calculated according to the log linear trapezoidal method,
¿ AUC0-inf (area under the curve from time 0 to infinity) was calculated as the sum of AUC0-t and AUCt-inf, where AUCt-inf = Ct/¿z (the measured concentration at the last time point with quantifiable data divided by the elimination rate constant),
¿ AUC0-inf was considered less reliable when ¿z was less reliable, and when the extrapolation to infinity from the last time-point with measurable data constitutes more than 20% of the total AUC0 inf,
¿ When r² <0.85 and when AUCextra> 20%, ¿z, t1/2 and AUC0-inf were considered as not reliable.

The pharmacokinetic parameters of Heptanal and Heptanoic acid were compared by calculating ratios.
Statistics:
No statistic wer performed.

COMPUTER SYSTEMS
The major computer systems used in the study are detailed in the following table:

Software Version number Application function
CIT Pharma 2.2 Test item receipt and inventories.
CITOX D.7 Acquisition and management of in-life data (toxicology and clinical pathology).
Empower 2 Build 2154 Acquisition/management of chromatographic data (GC/FID) of Chemical analysis of the dose formulations.
Panorama E2 2.60.0000 Acquisition of temperature and humidity in study rooms, laboratories, freezers and refrigerators.
ANALYST 1.4.1
1.4.2 Acquisition/management of chromatographic data (LC/MS-MS) of Plasma levels of the test item and its metabolite.
Phoenix WinNonLin 6.3 Pharmacokinetic evaluation.


Results and discussion

Preliminary studies:
Not applicable.
Main ADME results
Type:
other: Not applicable.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Following administration of Heptanal, mean plasma concentration of heptanal were quantified from the first collected time-point (0.125h) to 12 hours after administration. Mean levels quantified from 0.125h and 2 h were just above the limit of quantification of 100 ng/ml and the mean cocnetration increased until a maximum of 572 ng/ml 8 hours after adminstration. Mean levels were below the limit of quantification (<100 ng/ml) 24 hours after administration.
Following administration of heptanal, plasma concentration of heptanoic acid were quantifed from the first collected time-point of 0.125 to 8 h. Plasma levels reached a maximum of 10554 ng/ml rapidly after administration (Tmax=0.25h). After reaching the maximum, heptanoic acid levels decreased until 8 hours (540 ng/mL) and were below the limit of quantification (<200 ng/ml) at 12 and 24 hours after adminstration.
Following adminstration if heptanoic acid, mean plasma concetrations of heptanoic acid were quantifed from the first collected time-point of 0.125 h to 12h. Mean plasma levels reached a maximum of 14400 ng/mL rapidly after admnistration (Tmax=0.25 h). After reaching the maximum, heptanoic acid levels decreased until 12 hours (468 ng/mL) and were below the limit of quantification(<200 ng/mL) at 24 hours after administration.
Details on distribution in tissues:
Not applicable.
Details on excretion:
Not applicable.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
It was demonstrated that heptanoic acid was a metabolite of heptanal. As shown in the table above, following administration of heptanal, plasma concentrations of the metabolite heptanoic acid were higher than concentration of the test item. Moreover, heptanoic acid plasma concetrations were globally similar after administration of heptanal or heptanoic acid.
Comparison of the exposure of Heptanoic acid after administration of Heptanal and Heptanoic acid was presented in the following table:

Heptanoic acid
Dose normalized PK parameters Ratios of heptanoic acid
After administration of Heptanoic acid / After administration of Heptanal
Cmax / Dose 1.52
AUC0-t / Dose 1.07
AUC0-8h / Dose 0.98
AUC0-inf / Dose 1.04*
PK: pharmacokinetic.
*: considered as less reliable as r²<0.85.

Similar dose normalized AUCs values of Heptanoic acid were observed after administration of Heptanal and Heptanoic acid: AUCs Heptanoic ratios After Administration of Heptanoic acid / After Administration of Heptanal ranged between 0.98 and 1.07. This similar exposure demonstrated that after administration, the test item, Heptanal was rapidly metabolized to Heptanoic acid.
The Heptanoic acid maximum concentration (Cmax) was 1.5-fold higher after administration of Heptanoic acid than after administration of Heptanal which could be explained by the difference of the absorption between Heptanoic acid and Heptanal.

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:
Not applicable.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Following administration of Heptanal, mean plasma concentration of Heptanal were quantified from the first collected time-point (0.125 h) to 12 hours after administration. Mean levels quantified from 0.125 h and 2 h were just above the limit of quantification of 100 ng/mL (individual values ranging between BLQ and 202 ng/mL) and then mean concentrations increased until a maximum of 572 ng/mL 8 hours after administration. Mean levels were below the limit of quantification (< 100 ng/mL) 24 hours after administration.
Following administration of Heptanal, plasma concentrations of Heptanoic acid were quantified from the first collected time-point of 0.125 h to 8 h. Plasma levels reached a maximum of 10554 ng/mL rapidly after administration (Tmax = 0.25 h). After reaching the maximum, Heptanoic acid levels decreased until 8 hours (540 ng/mL) and were below the limit of quantification (< 200 ng/mL) at 12 and 24 hours after administration.
The second peak of 7313 ng/mL reached at 1 hour was considered to be due to the inter-animal variability.
Following administration of Heptanoic acid, mean plasma concentrations of Heptanoic acid were quantified from the first collected time-point of 0.125 h to 12 h. Mean plasma levels reached a maximum of 14 400 ng/mL rapidly after administration (Tmax = 0.25 h). After reaching the maximum, Heptanoic acid levels decreased until 12 hours (468 ng/mL) and were below the limit of quantification (< 200 ng/mL) at 24 hours after administration.
Executive summary:

The objective of this study was to demonstrate that the test item, Heptanal, is rapidly metabolized to Heptanoic acid. Therefore, the plasma pharmacokinetic exposure of Heptanal and Heptanoic acid was compared after a single oral administration to rats.

Eighteen male Sprague-Dawley rats (body weight ranging from 378 g to 470 g on day 1) were allocated into two groups of 9 rats. The first group was orally treated on a single occasion with the Heptanal (batch No. 1305019) and the second group with the metabolite of the Heptanal, Heptanoic acid (batch No. 1304076). Heptanal and Heptanoic acid were both solubilised in corn oil as vehicle. Following analysis of the dose formulations, a deviation on Heptanal concentrations of +17.5% and a deviation on Heptanoïc acid of +5.3% were measured. Therefore, the actual dose-levels were used for the pharmacokinetic evaluation:

     - Heptanal: dose-level of 1175 mg/kg,

     - Heptanoic acid: dose-level of 1055 mg/kg.

 

After administration, blood samples were taken at 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12 and 24 hours after administration. Three animals per group were collected at each time-point.

Blood were collected on ice and centrifuged within 10 min after collection. Then, plasma samples were frozen at -80C and analyzed for determination of Heptanal and Heptanoic acid for group 1 and for determination of Heptanoic acid only for group 2.

After their respective last blood collection, the animals were sacrificed and discarded without necropsy.

Mortality, morbidity and clinical signs were checked twice a day after administration.

 

Results

Achieved dosage form

There was a satisfactory agreement between the theoretical and actual dose-levels calculated, as all deviations were within ± 8% of the target values.

 

Pharmacokinetics 

Pharmacokinetic parameters obtained after a single administration of Heptanal and Heptanoic acid to male Sprague-Dawley rats were presented in the following table:

 

Test item administered

Heptanal

Heptanoic acid

Dose-level (mg/kg)

1175

1055

Test item analyzed

Heptanal

Heptanoic acid

Heptanoic acid

Tmax

h

8

0.25

0.25

Cmax

ng/mL

572

10 554

14 400

AUC0-t

ng.h/mL

3 433

18 930

18 527

AUC0-8h

ng.h/mL

2 037

18 930

16 921

AUC0-inf

ng.h/mL

nc

21 957*

20 802*

AUCextra

%

nc

13.8

11.1

tlast

h

12

8

12

Cmax/ Dose

0.487

8.98

13.6

AUC0-t/ Dose

2.92

16.1

17.3

AUC0-8h/ Dose

1.73

16.1

15.7

AUC0-inf/ Dose

nc

18.7*

19.4*

nc: not calculated.

*: considered as less reliable as r²<0.85.

After administration of Heptanal or heptanoic acid, Heptanoic concentrations reached rapidly a maximum at 0.25h.

Similar dose normalized AUCsvalues of Heptanoic acid were observed after administration of Heptanal or Heptanoic acid:

AUCsHeptanoic ratios After Administration of Heptanoic acid / After Administration of Heptanal ranged between 0.98 and 1.07. This similar exposure demonstrated that after administration, the test item, Heptanal was rapidly metabolized to Heptanoic acid.

The Heptanoic acid maximum concentration (Cmax) was 1.5-fold higher after administration of Heptanoic acid than after administration of Heptanal which could be explained by the difference of the absorption between Heptanoic acid and Heptanal.

After administration of Heptanal at the dose-level of 1175 mg/kg, the exposure of the metabolite (AUCsvalues) was between 6- to 9-fold higher than the parent compound. Moreover, Cmaxvalue of Heptanoic acid was 18-fold higher than Heptanal Cmaxvalue.

These results demonstrated a rapid metabolism of the Heptanal to Heptanoic acid after administration.

 

After administration of Heptanal or Heptanoic acid, Heptanoic acid concentrations reached rapidly a maximum at 0.25 h.

 

Similar dose normalized AUCsvalues of Heptanoic acid were observed after administration of Heptanal and Heptanoic acid: AUCsHeptanoic ratios After Administration of Heptanoic acid / After Administration of Heptanal ranged between 0.98 and 1.07. This similar exposure demonstrated that after administration, the test item, Heptanal was rapidly metabolized to Heptanoic acid.


The Heptanoic acid maximum concentration (Cmax) was 1.5-fold higher after administration of Heptanoic acid than after administration of Heptanal which could be explained by the difference of absorption of the Heptanoic acid and Heptanal.

After administration of Heptanal at the dose-level of 1175 mg/kg, the exposure of the metabolite (AUCsvalues), Heptanoic acid was between 6- to 9-fold higher than the exposure of the parent compound. Moreover, Cmaxvalue of Heptanoic acid was 18-fold higher than Heptanal Cmaxvalue.

 

Conclusion

Following a single oral administration of Heptanal at the dose-level of 1175 mg/kg or Heptanoic acid at the dose-level of 1055 mg/kg to male Sprague-Dawley rats, no clinical signs were observed. Pharmacokinetic results demonstrated a rapid metabolism of Heptanal to Heptanoic acid after Heptanal administration with a Tmax at 0.25 hours for plasma heptanoic concentrations. Indeed, Heptanoic acid exposures (AUC) were similar after administration of Heptanal or Heptanoic acid. Moreover, the exposure of the metabolite Heptanoic acid was between 6- to 9-fold higher than the parent compound. After administration of Heptanal, the Heptanoïc acid maximum concentration (Cmax) of 10554 ng/mL was reached 0.25 hours after administration.

Categories Display