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Reference
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.
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
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
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.
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.


Preliminary studies:
Not applicable.
Type:
other: Not applicable.
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.
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) testing results:
Not applicable.
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.

Description of key information

In 2013, A comparative pharmacokinetic study after singly oral administation of heptanal or heptanoic acid to rat was performed by Jaillet. The objective of this study was to demonstrate whether Heptanal, is rapidly metabolized to heptanoic acid so that read across of toxicological data with heptanoic acid is justified to be used for Heptanal toxicological assessment.  In this context, the plasma pharmacokinetic exposure of heptanal and heptanoic acid was compared after a
single oral administration to rats.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
100
Absorption rate - inhalation (%):
100

Additional information

General information on aldehydes from the litterature:

Aldehydes (Brabec, 1993), are absorbed through the gastrointestinal tract and are rapidly eliminated from the blood. Plasma half-lives are normally difficult to measure since many low molecular weight aldehydes, and carboxylic acid (e.g., propionic acid) are endogenous in humans (Lington and Bevan, 1994). Prior to absorption, simple aliphatic aldehydes may undergo oxidation to yield the corresponding carboxylic acid. The primary metabolism of linear saturated aliphatic aldehydes and acids is a fundamental part of cell biochemistry. Aldehydes are successively oxidized to their corresponding carboxylic acids. The acid as the coenzyme A (CoA) ester then participates in the fatty acid pathway and the tricarboxylic acid cycle. To a minor extent, aldehydes also may be reduced to alcohols or conjugated with labile sulfhydryl containing substances, such as glutathione (Brabec, 1993). Medium chain carboxylic acids are condensed with acetyl CoA to form fatty acids (e.g., C10 to C18) or undergo omega-oxidation to form diacids that are further metabolized by beta-oxidation in the fatty acid cycle (Katz and Guest, 1994). A combination of high capacity dehydrogenase {alcohol (ADH) and aldehyde (ALD) dehydrogenase} and oxidase enzymes rapidly oxidise linear saturated aldehydes to the corresponding carboxylic acids. Then, the carboxylic acid resulting from oxidation of the aldehyde (heptanic acid for heptanal) enters cellular fatty acid metabolism (Voet and Voet, 1990). Degradation products will either be exhaled as CO2 or re-used within the intermediary metabolism.

Heptanal physicochemical properties

The low molecular weight (114.8 g/mol), log Pow (2.8), and water solubility along with the physical state (liquid) of Heptanal should favour its absorption via various routes of exposure (oral, dermal, and inhalation). Consistent with this prediction, pharmacokinetic analysis of Heptanal demonstrated that the compound was rapidly absorbed into the systemic circulation of rats following oral administration where it was quantified at the fisrt collected time point (0.125 h) and a maximum plasma concentrations of the metabolite heptanoic acid occurring also at the first ime point of 0.25 hours post-dosing (Jaillet, 2013).

Heptanal gastrointestinal absorption

The absorption of Heptanal was evaluated in a toxicokinetic study in male Sprague-Dawley rats. Heptanal was administered orally at a single dose of 1175 mg/kg/day body weight of Heptanal in corn oil, (Jaillet, 2013). Heptanal was rapidly absorbed into the systemic circulation following oral exposure where it was quatified at the first time point of 0.25 hours post-dosing to 12 hours after administration. The mean concentration increased until a maximum of 572 ng/ml 8 hours after administration for heptanal.

Heptanal metabolism

The metabolism of Heptanal was evaluated in a toxicokinetic study in rats (Jaillet, 2013). Rats were administered orally a single dose of 1175 mg/kg/body weight of Heptanal in corn oil (Jaillet, 2013). Pharmacokinetic results demonstrated a rapid metabolism of Heptanal to Heptanoic acid after Heptanal administration. Indeed, Heptanoic acid exposures 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. 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.

Elimination

After administration of a single dose-level of 1175 mg/kg bw of Heptanal in corn oil, by gavage to male Sprague-Dawley rats (Jaillet, 2013). 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 the quantification of 100 ng/ml and the mean cocentration 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. The metabolite, heptanoic acid 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 administration.

Hall, B.L. and Oser, B.L. (1965) Recent Progress in the Consideration of Flavoring Ingredients under the Food Additive Amendment.3. GRAF Substances. Food Technology, 19, 151-197.

 

 

K. Bauer und D. Garbe: Common fragrance and flavour materials. Preparation, properties and uses. 219 Seiten. 450 Einzelkomponenten. VCH Verlagsgesellschaft mbH. Weinheim 1985. Preis: 112,– DM

 

 

Katz GV, Guest D (1994). Aliphatic Carboxylic Acids. In: Clayton GD, Clayton FE, eds. Toxicology. 4th Edition. New York: John Wiley & Sons: 3523-3671. (Patty's Industrial Hygiene and Toxicology, Vol II, Part E).

 

 

Dawson, A.M., Holdsworth, C.D., Webb, J., 1964. Absorption of short chain fatty acids in man. Proc. Soc. Exp. Biol. Med. 117, 97- 100.

 

 

Von Oettingen (1960). The aliphatic acids and their esters: Toxicity and potential dangers. Arch Ind Health 21(1):28-32.

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