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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
Study limited to the toxicokinetic part
Principles of method if other than guideline:
Evaluation of the plasma pharmacokinetic profile of Diacetone alcohol (DAA), and its metabolites (Methyl isobutyl carbinol, MIBC and Methyl isobutyl ketone, MIBK) following single oral administration (gavage) to male Sprague-Dawley rats
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: 7 weeks old
- Weight at study initiation: 205 g to 231 g
- Fasting period before study:
- Housing: by three in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted maintenance diet, ad libitum
- Water: tap water filtered with a 0.22 µm filter, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was administered as a solution in the vehicle.
The test item, 5.81 g, was weighed and mixed with the 18.25 g of vehicle and then homogenized using a magnetic stirrer.
Dose formulations were analysed by Gas Chromatography with FID detection (GC-FID).
Duration and frequency of treatment / exposure:
Single adminbistration
Doses / concentrationsopen allclose all
Remarks:
5 mmol/kg bw
Dose / conc.:
580 mg/kg bw (total dose)
No. of animals per sex per dose:
9
Control animals:
no
Details on study design:
- Dose selection rationale: The dose-level was selected to be consistent with similar studies (CiToxLAB France/Study No. 20991 PAR) performed with methyl isobutyl carbinol (MIBC) and methyl isobutyl ketone (MIBK) in order to be able to compare the results.
- Morbidity and mortality
Each animal was checked for mortality and morbidity once a day, including weekends and public holidays.
- Clinical signs
Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.
- Body weight
The body weight of each animal was recorded once before the beginning of the treatment and on the day of treatment.
Details on dosing and sampling:
Blood samples were sampled from animals at:
- 0.25, 0.5, 1, 2, 3, 6, 9, 12 and 24 hours post-dosing.

Animals were divided into three sub-groups for blood sampling (each composed by three animals) and each animal was sampled three times. See detailed sampling scheme here below:
- first set of 3 animals: 0.25, 2 and 9 hours post-dosing,
- second set of 3 animals: 0.5, 3 and 12 hours post-dosing,
- third set of 3 animals: 1, 6 and 24 hours post-dosing.

Venous blood (approximately 0.8 mL) was collected from the orbital sinus of each animal, under light isoflurane anesthesia, into a tube containing lithium heparin and placed on wet ice pending centrifugation. Blood was centrifuged at 3000 g for 10 minutes under refrigerated conditions (set to maintain +4°C).
Two aliquots of plasma (at least 200 µL) were kept on wet ice before being transferred in labeled tubes and stored at 20°C until bioanalysis.
The concentration of Diacetone alcohol and its metabolite (Methyl isobutyl carbinol, MIBC and Methyl isobutyl ketone, MIBK) were determined in plasma using GC-MS method.
Statistics:
The pharmacokinetic evaluation was carried out by extravascular non-compartmental analysis using WinNonlin (Pharsight Corporation, Mountain View, California 94040/USA) software.

Diacetone alcohol, DAA, pharmacokinetic parameters were determined from the mean concentration of each time-point. The Standard Deviation (SD) and Coefficient of Variation (CV) were also calculated to assess inter-individual variability. For samples with a concentration level below the Limit Of Quantification (LOQ), the values were considered as zero. As MIBK and MIBC levels in plasma were below the Limit Of Quantification (< 0.000861 mmol/L) at all time-points, the pharmacokinetic parameters could not be determined for both metabolites.

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 collected after Cmax. The correlation coefficient (r2) for the "goodness" of the fit of the regression line should be 0.85 or higher for the value to be considered reliable,
- t½ (terminal half-life, in hours) was calculated as 0.693/¿z,
- AUC0-t (area under the curve from 0 hour to the time-point of the last quantifiable concentration) and AUC0-12h (area under the curve from 0 hour to 12 hours) was calculated according to the linear up log down method, using nominal sampling times,
- AUC0-8 (area under the curve from time 0 to infinity) was calculated as the sum of AUC0-t and AUCt-8, where AUCt-8 = Ct/¿z (the measured concentration at the last time point with quantifiable data divided by the elimination rate constant).

N.B.: as no pre-dose samples have been collected, the pre-dose concentrations on Day 1 were considered to be equal to zero.

Results and discussion

Main ADME resultsopen allclose all
Type:
other: Toxicokinetics
Results:
DAA was quantifiable in plasma from 0.25h to 24h. An initial plasma concentration peak at 4.40 mmol/L was reached after 1 hour post-dosing but Cmax was observed (at 4.82 mmol/L) after 6 hours post-dosing, indicating a prolonged absorption phase.
Type:
other: Toxicokinetics
Results:
MIBK and MIBC levels in plasma were below the Limit Of Quantification (< 0.000861 mmol/L) at all time-points.

Toxicokinetic / pharmacokinetic studies

Toxicokinetic parametersopen allclose all
Toxicokinetic parameters:
other: lambaz: 0.297 1/h
Toxicokinetic parameters:
other: T1/2: 2.3 h
Toxicokinetic parameters:
Tmax: 6 h
Toxicokinetic parameters:
Cmax: 4.82 mmol/L
Toxicokinetic parameters:
other: AUC0-t: 44.9 h*mmol/L
Toxicokinetic parameters:
other: AUC0-12h: 39.9 h*mmol/L
Toxicokinetic parameters:
other: AUC0-8: 45.0 h*mmol/L
Toxicokinetic parameters:
other: AUCextrapolated: 0.3%

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
MIBK: methylisobutylketone
MIBC: methylisobutylcarbinol

Any other information on results incl. tables

 CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS

The concentration in the administered dose formulation was found at -0.8% (2.48 mmol/L) of the nominal concentration (2.5 mmol/L) that is within the acceptable range of ± 10% of the nominal concentration.

ACHIEVED DOSAGE

There was good agreement between the nominal and actual calculated dose-level, as all deviations were within ± 3%, specifically deviations ranged from -0.83 to 2.53%.

CLINICAL EXAMINATION

Mortality, morbidity and clinical signs

No mortality, morbidity and clinical signs were observed during the study.

Body weight

The body weight of the animals was not affected during the study.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Following a single oral (gavage) administration of Diacetone alcohol DAA at the dose-level of 5 mmol/kg to male Sprague-Dawley rats, the test item was well tolerated. The test item Diacetone alcohol was quantifiable in plasma from 0.25h, reached an absolute maximum at 6 hours post-dosing and then decreased slowly until the last time-point at 24 hours where the concentration is closed to zero. The terminal half-life was of 2.3 hours for DAA. Methyl isobutyl carbinal (MIBC) and Methyl isobutyl ketone (MIBK) plasma levels were below the lower Limit Of Quantification at all time-points, suggesting that the test item, DAA, may not be significantly metabolized in MIBC and MIBK in rat or that those eventual metabolites are not significantly circulating in rat plasma.
Executive summary:

The plasma pharmacokinetic profile of Diacetone alcohol (DAA), and its potential metabolites (Methyl isobutyl carbinol, MIBC and Methyl isobutyl ketone, MIBK) were evaluated following a single oral administration (gavage) to male Sprague-Dawley rats.

Nine male Sprague-Dawley rats were allocated to the study and were treated orally (gavage) by the test item at the dose-level of 5 mmol/kg (580 mg/kg bw). A constant dose-volume of 2 mL/kg was used. Blood samples were collected in three animals per time-point before administration and in nine time-points over 24 hours. Each animal was sampled three times. The concentration of DAA, MIBC and MIBK were determined in plasma using GC-MS method. Mortality, morbidity and clinical signs were checked at least once a day. The body weight of each animal was recorded once before the beginning of the treatment period, then on the day of treatment.

No mortality, morbidity and clinical signs were observed during the study.

DAA was quantifiable in plasma from the first to the last time-points (from 0.25 h to 24 h). An initial plasma concentration peak at 4.40 mmol/L was reached after 1 hour post-dosing but absolute DAA maximum plasma concentration was observed (at 4.82 mmol/L) after 6 hours post-dosing, indicating a prolonged absorption phase. MIBK and MIBC levels in plasma were below the Limit Of Quantification (< 0.000861 mmol/L) at all time-points. Pharmacokinetic parameters of DAA following single oral administration of DAA at 5 mmol/kg to male rats are presented in the table below:

 

PK parameters

Units

Value

¿z

1/h

0.297

t1/2

h

2.3

Tmax

h

6

Cmax

mmol/L

4.82

AUC0-t

h*mmol/L

44.9

AUC0-12h

h*mmol/L

39.9

AUC0-8

h*mmol/L

45.0

 

Therefore, DAA appeared well tolerated and absorbed following a single oral (gavage) administration at the dose-level of 5 mmol/kg to male Sprague-Dawley rats. DAA was not significantly metabolized in MIBC and MIBK or that those eventual metabolites were not significantly circulating in rat plasma.