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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
Objective of study:
toxicokinetics
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
Radiolabelling:
no
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
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
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.
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 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%
Metabolites identified:
yes
Details on metabolites:
MIBK: methylisobutylketone
MIBC: methylisobutylcarbinol

 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.

 

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.

Endpoint:
basic toxicokinetics in vivo
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
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Version / remarks:
This study is a pilot pharmacokinetic investigation and as such is not designed to follow specific test guidelines.
Principles of method if other than guideline:
This study was performed to obtain blood samples from rats at selected time points following a single 6-hour exposure of the test atmosphere of Diacetone Alcohol at two different concentration levels of Low (500 ppm) and High (1000 ppm) in order to determine the concentrations and the pharmacokinetics parameters of Diacetone alcohol (DAA), Methyl-isobutyl ketone (MIBK) and Methylisobutylcarbinol (MIBC) from the plasma.
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
SPF colony
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: 387g-468g
- Housing: three animals per cage
- Diet (ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice
- Water (ad libitum): tap water from municipal supply
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 25.6
- Humidity (%): 40 - 62
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose only, past flow, dynamic flow exposure unit, consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.
- Method of holding animals in test chamber: tube
- Source and rate of air: no data
- Method of conditioning air: oil-free compressor passed through a suitable filter system prior to introduction to the nebuliser
- System of generating particulates/aerosols: The test item was administered as vapours. The test item was aerosolised using a stainless steel concentric jet nebulisers (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chambers and a separator glass was installed to remove any liquid particles from the atmosphere prior entering into the inhalation tower. The rate of test item use was controlled by a syringe pump.
- Temperature, humidity in air chamber: 21.0-25.5°C, 3.0-4.5%
- Air Flow In (Inner Plenum) (L/min): 29.6-30.8
- Air Flow Out (Outer Cylinder) (L/min): 23.6-25.7
- Air change rate: no data
- Method of particle size determination: not determined since the test item was evaporated and non-condensing.
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: the concentration of the test item during the exposure was measured by gas chromatography (GC/FID). Samples for analytical determination were taken once an hour during the exposure into an impinger containing ethanol as a solvent.
- Samples taken from breathing zone: yes
Duration and frequency of treatment / exposure:
single 6-hour exposure
Dose / conc.:
500 other: ppm (target)
Dose / conc.:
506 ppm (analytical)
Remarks:
3.43 mg/L
Dose / conc.:
1 000 other: ppm (target)
Dose / conc.:
1 086 ppm (analytical)
Remarks:
7.42 mg/L
No. of animals per sex per dose:
9
Control animals:
no
Details on dosing and sampling:
TOXICOKINETIC STUDY
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: 0 (on day -1 or -3), 0.5, 1, 1.5, 3, 4.5, 6, 8, 18 and 24 hours post exposure

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: 0 (on day -1 or -3), 0.5, 1, 1.5, 3, 4.5, 6, 8, 18 and 24 hours post exposure
- From how many animals: 3
- Method type(s) for identification: Gas Chromatography coupled with MS detection after electron impact ionization (GC-MS)
- Limits of quantification: DAA < 0.5 µg/mL; MIBC < 0.1 µg/mL; MIBK < 0.2 µg/mL

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): no
Key result
Toxicokinetic parameters:
Cmax: 389 µg DAA/mL
Remarks:
500 ppm
Key result
Toxicokinetic parameters:
Tmax: 0.5 h
Remarks:
500 ppm
Key result
Toxicokinetic parameters:
half-life 1st: 2.92 h
Remarks:
500 ppm
Key result
Toxicokinetic parameters:
AUC: 2193 h*µg DAA/mL
Remarks:
0.5-24h / 500 ppm
Key result
Toxicokinetic parameters:
Cmax: 848 µg DAA/mL
Remarks:
1000 ppm
Key result
Toxicokinetic parameters:
Tmax: 0.5 h
Remarks:
1000 ppm
Key result
Toxicokinetic parameters:
half-life 1st: 4.91 h
Remarks:
1000 ppm
Key result
Toxicokinetic parameters:
AUC: 7698 h*µg DAA/mL
Remarks:
0.5-24h / 1000 ppm
Metabolites identified:
yes
Details on metabolites:
Diacetone alcohol (DAA), Methyl-isobutyl ketone (MIBK) and Methylisobutyl carbinol (MIBC)

Mean plasma concentrations of DAA, MIBK and MIBC after an inhalation exposure to DAA at 500 and 1000 ppm

 

Analytes

Exposure

Sampling time

 

Pre-dose

0.5 h

1 h

1.5 h

3 h

4.5 h

6 h

8 h

18 h

24 h

DAA

500 ppm

BLQ

389

358

346

240

247

217

100

7.13

3.11

1000 ppm

BLQ

848

804

767

684

720

569

433

110

44.3

MIBK

500 ppm

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

1000 ppm

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

MIBC

500 ppm

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

1000 ppm

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ: Below Limit of Quantification.

 

Toxicokinetic parameters of DAA, MIBC and MIBK after a single 6-hour inhalation
exposure of male Sprague-Dawley rats to 500 ppm and 1000 ppm of DAA

 

Concentration

Analytes

Number of point used to calculate lz

lz

t1/2

Tmax

Cmax

Cmax/
concentration#

AUC0.5-24h

AUC0.5-24h/
concentration#

Metabolite/
Administered compound

1/h

h

h

µg/mL

h*µg/mL

Cmax

AUC0.5-24h

500 ppm
(2.38 mg/L)

DAA

0.986

5

0.238

2.92

0.5

389

163

2193

922

na

na

MIBC

na

na

na

na

na

0

nc

na

nc

nc

nc

MIBK

na

na

na

na

na

0

nc

na

nc

nc

nc

1000 ppm
(4.75 mg/L)

DAA

1.00

5

0.141

4.91

0.5

848

178

7698

1621

na

na

MIBC

na

na

na

na

na

0

nc

na

nc

nc

nc

MIBK

na

na

na

na

na

0

nc

na

nc

nc

nc

na: not applicable.

nc: not calculated as not enough quantifiable points were available.

#:Cmaxand AUC0.5-24hwere divided by the exposure concentrations of DAA (in mg/L).

 

Conclusions:
DAA was quickly and extensively absorbed by inhalation exposure.
Executive summary:

A study was performed to obtain blood samples from rats at selected time points following a single 6-hour exposure of the test atmosphere of Diacetone Alcohol at target concentration levels of 500 and 1000 ppm in order to determine the concentrations and the pharmacokinetics parameters of Diacetone alcohol (DAA), Methyl-isobutyl ketone (MIBK) and Methyl-isobutyl carbinol (MIBC) from the plasma. The animals were exposed to the test atmosphere (in the form of a vapour) using a nose-only exposure system. Analytical concentrations of 506 ppm, 1086 ppm were achieved in the respective groups. No control animals were used in the study. The test atmosphere concentration was monitored based on a validated GC method(by trapping the vapors in an impinger containing ethanol). The results of the test atmosphere characterization were considered suitable for the study purposes. Heighteen male Sprague-Dawley rats were involved in the study, 9 animals in each treatment groups. Plasma samples were prepared from male rats following the end of the exposure at nine time-points (three animals at each sampling occasion) and the plasma level of Diacetone Alcohol, Methyl isobutyl ketone and Methyl isobutyl carbinol was determined. The analytical method consisted of a liquid/liquid extraction of the three analytes from plasma with Methyl tert-Butyl Ether (MtBE) followed by analysis of supernatant by Gas Chromatography coupled with MS detection after electron impact ionization (GC-MS). The toxicokinetic evaluation was performed using non-compartmental analysis on Phoenix WinNonlin software (Pharsight Corporation, Mountain View, California 94040/USA). DAA, MIBC and MIBK toxicokinetic parameters were determined from the mean plasma concentration collected from each animal/group, at each post-exposure time-point. A separate analysis was performed for each dose-level. The following toxicokinetic parameters were calculated t½, ¿z, AUC0.5-24h, Tmaxand Cmax.

The mean actual achieved concentration of the test item were:

Target Concentration

(ppm)* [mg/L]

Achieved Concentration

(ppm)* [mg/L]

Nominal Concentration

 mg/L

500 [2.38]

506 [2.41]

3.43

1000 [4.75]

1086 [5.16]

7.42

For the three analytes (DAA, MIBK and MIBC), the standard acceptance criteria for the calibration lines and Quality Control samples were met for a successful analysis of the study samples.

Study samples with concentrations above the calibration ranges were repeated diluted (for DAA principally and MIBK).DAA, MIBC and MIBK were not quantifiable in pre-dose samples in any group.After exposure to DAA, MIBK and MIBC were not detected and DAA concentrations increased with the dose.

The mean plasma concentrations of DAA, MIBK and MIBC after an inhalation exposure to DAA at 500 and 1000 ppm were:

 

Analytes

Exposure

Sampling time

 

Pre-dose

0.5 h

1 h

1.5 h

3 h

4.5 h

6 h

8 h

18 h

24 h

DAA

500 ppm

BLQ

389

358

346

240

247

217

100

7.13

3.11

1000 ppm

BLQ

848

804

767

684

720

569

433

110

44.3

BLQ: Below Limit of Quantification.

 

The toxicokinetic parameters of DAA after single administration (6 h inhalation) of DAA at 500 and 1000 ppm to male Sprague-Dawley rats were:

Concentration

Number of point used to calculate lz

lz

t1/2

Tmax

Cmax

Cmax/
concentration#

AUC0.5-24h

AUC0.5-24h/
concentration#

Metabolite/
Administered compound

1/h

h

h

µg/mL

h*µg/mL

Cmax

AUC0.5-24h

500 ppm
(2.38 mg/L)

0.986

5

0.238

2.92

0.5

389

163

2193

922

na

na

1000 ppm
(4.75 mg/L)

1.00

5

0.141

4.91

0.5

848

178

7698

1621

na

na

na: not applicable.

#:Cmaxand AUC0.5-24hwere divided by the exposure concentrations of DAA or MIBK (in mg/L).

After a 6-hour inhalation exposure to DAA:

·        DAA was quantifiable from 0.5 h to 24 h after the end of exposure,

·        maximum concentrations of DAA were at 0.5 h after the end of exposure,

·        the elimination rate of DAA was lower and the half-life higher at 1000 ppm than 500 ppm, indicating a saturation of the excretion pathway,

·        MIBC and MIBK were not quantifiable.

After exposure to DAA, AUC0-24hand Cmax(both normalized by exposure concentration) were relatively higher when the animals were exposed to 1000 ppm than when exposed to 500 ppm.

DAA was quickly and extensively absorbed by inhalation exposure.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
other: Federal Register, 69 FR 22402, 22402 -22441. "Test Rule; In Vitro Dermal Absorption Rate Testing of Certain Chemicals of Interest to the Occupational Safety and Health Administration"
GLP compliance:
not specified
Radiolabelling:
yes
Species:
human
Vehicle:
water
Duration of exposure:
10 and 60 min
Doses:
25 mg/cm² (313 µl/cm² of a 80 mg/ml solution)
No. of animals per group:
minimum of six replicates represented by at least three donors (for each of the three experimental scenarios)
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human cadaver
- Ethical approval if human skin: no data
- Type of skin: abdominal region
- Preparative technique: either dermatomed or heat-separated epidermal membranes (HEM)
- Thickness of skin: 200-500 µm
- Membrane integrity check: electrical resistance (impedance)
- Storage conditions: frozen (-20° C) for up to 3 months
- Justification of species, anatomical site and preparative technique:

PRINCIPLES OF ASSAY
- Diffusion cell: standard in vitro diffusion cell modeI, eilher static or flow-through
- Receptor fluid: no data
- Solubility of test substance in receptor fluid: yes
- Static system:
- Flow-through system:
- Test temperature: 32°C
- Humidity:
- Occlusion:
- Reference substance(s):
- Other: Kp = steady-state rate of absorption (in µg/ hr x cm² )/ concentration of the test substance (measured in µg/cm3) applied to the skin.
Dermal irritation:
no effects
Remarks:
No change in pre- and post-dose electrical impedance (El) values for Kp, 10 minute and 60 min experiments
Total recovery:
Kp study: 89.6%
10 min: 90.8%
60 min: 91.7%
Time point:
10 min
Dose:
25 mg/cm²
Parameter:
percentage
Absorption:
ca. 0.04 %
Remarks on result:
other: skin: 0.04%; recepto fluid < 0.001%
Key result
Time point:
60 min
Dose:
25 mg/cm²
Parameter:
percentage
Absorption:
ca. 0.15 %
Remarks on result:
other: skin: 0.11%; receptor fluid: 0.04%
Key result
Time point:
24 h
Dose:
25 mg/cm²
Parameter:
percentage
Absorption:
ca. 5.71 %
Remarks on result:
other: skin: 0.7%; receptor fluid: 5.01%
Key result
Time point:
8 h
Dose:
25 mg/cm²
Parameter:
percentage
Absorption:
ca. 1 %
Remarks on result:
other: Extrapolated from the polynomial % absorption/exposure time curve y = 1E-06x² + 0,0021x + 0,0188 (R² = 1)
Parameter:
other: Kp
Remarks:
5.77e-4 cm/h
Dose:
25 mg/cm²
Parameter:
rate
Absorption:
56.6 other: µg/cm²/h
Time point:
60 min
Dose:
25 mg/cm²
Parameter:
rate
Absorption:
37.3 other: µg/cm²/h
Executive summary:

In vitro dermal penetration rate of diacetone alcohol, as permeability coefficient (Kp) and short-term dermal absorption rates at 10 and 60 min, was determined using human cadaver skin mounted in an in vitro diffusion cell model. Total recovery, based on liquid scintillation count data for total radioactivity, was between 89.6 and 91.7% of the applied dose. Skin penetration was 0.04, 0.15 and 5.71 % of the dose after 10 min, 60 min and 24 h, respectively. Kp was 5.77e-4 cm/h. From the polynomial % absorption/exposure time curve (y = 1E-06x² + 0,0021x + 0,0188, R² = 1), a skin penetration of 1% was estimated for a 8-hour exposure.

Description of key information

The low molecular weight, log Pow value, and physical state of DAA favour its absorption via various routes of exposure (oral, dermal, and inhalation). Consistent with this prediction, are the signs of toxicity and systemic effects observed in experimental animals following acute and repeated oral and inhalation exposures. The water solubility suggests that it readily diffuses through aqueous channels and pores of various tissues and organs. Therefore, DAA is likely evenly distributed throughout the body. According to the available data, the absorption by the oral and inhalation routes is considered to be extensive and close to 100% and the dermal penetration is assumed to not exceed 1 and 5% for a 8- and 24-hour exposure, respectively.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
1
Absorption rate - inhalation (%):
100

Additional information

Pharmacokinetic profiles of DAA

Oral route

The plasma pharmacokinetic profile of DAA was evaluated following a single oral administration (gavage) to male Sprague-Dawley rats (Sabadie, 2015). Nine male Sprague-Dawley rats were allocated to the study and were treated orally (gavage) by DAA 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 alcohol 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 (attached figure).

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

lambdaz

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

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.

Inhalation route

A study was performed to obtain blood samples from rats at selected time points following a single 6-hour exposure of the test atmosphere of Diacetone Alcohol at target concentration levels of 500 and 1000 ppm in order to determine the concentrations and the pharmacokinetics parameters of Diacetone alcohol (DAA), Methyl-isobutyl ketone (MIBK) and Methyl-isobutyl carbinol (MIBC) from the plasma (Balazs, 2016). The animals were exposed to the test atmosphere (in the form of a vapour) using a nose-only exposure system. Analytical concentrations of 506 ppm, 1086 ppm were achieved in the respective groups. No control animals were used in the study. The test atmosphere concentration was monitored based on a validated GC method(by trapping the vapors in an impinger containing ethanol). The results of the test atmosphere characterization were considered suitable for the study purposes. Heighten male Sprague-Dawley rats were involved in the study, 9 animals in each treatment groups. Plasma samples were prepared from male rats following the end of the exposure at nine time-points (three animals at each sampling occasion) and the plasma level of DAA, MIBK and MIBC determined. The analytical method consisted of a liquid/liquid extraction of the three analytes from plasma with Methyl tert-Butyl Ether (MtBE) followed by analysis of supernatant by Gas Chromatography coupled with MS detection after electron impact ionization (GC-MS). The toxicokinetic evaluation was performed using non-compartmental analysis on Phoenix WinNonlin software (Pharsight Corporation, Mountain View, California 94040/USA). DAA, MIBC and MIBK toxicokinetic parameters were determined from the mean plasma concentration collected from each animal/group, at each post-exposure time-point. A separate analysis was performed for each dose-level. The following toxicokinetic parameters were calculated t½, lambdaz, AUC0.5-24h, Tmaxand Cmax.

The mean actual achieved concentration of the test item were:

Target Concentration

(ppm)* [mg/L]

Achieved Concentration

(ppm)* [mg/L]

Nominal Concentration

 mg/L

500 [2.38]

506 [2.41]

3.43

1000 [4.75]

1086 [5.16]

7.42

For the three analytes (DAA, MIBK and MIBC), the standard acceptance criteria for the calibration lines and Quality Control samples were met for a successful analysis of the study samples.

Study samples with concentrations above the calibration ranges were repeated diluted (for DAA principally and MIBK). DAA, MIBC and MIBK were not quantifiable in pre-dose samples in any group. After exposure to DAA, MIBK and MIBC were not detected and DAA concentrations increased with the dose.

The mean plasma concentrations of DAA, MIBK and MIBC after an inhalation exposure to DAA at 500 and 1000 ppm were:

 

Analytes

Exposure

Sampling time after exposure

 

Pre-dose

0.5 h

1 h

1.5 h

3 h

4.5 h

6 h

8 h

18 h

24 h

DAA

500 ppm

BLQ

389

358

346

240

247

217

100

7.13

3.11

1000 ppm

BLQ

848

804

767

684

720

569

433

110

44.3

BLQ: Below Limit of Quantification.

 

The toxicokinetic parameters of DAA after single administration (6 h inhalation) of DAA at 500 and 1000 ppm to male Sprague-Dawley rats were:

Concentration

Number of point used to calculatelz

lz

t1/2

Tmax

Cmax

Cmax/
concentration#

AUC0.5-24h

AUC0.5-24h/
concentration#

1/h

h

h

µg/mL

h*µg/mL

500 ppm
(2.38 mg/L)

0.986

5

0.238

2.92

0.5

389

163

2193

922

1000 ppm
(4.75 mg/L)

1.00

5

0.141

4.91

0.5

848

178

7698

1621

na: not applicable.

#:Cmaxand AUC0.5-24hwere divided by the exposure concentrations of DAA (in mg/L).

After a 6-hour inhalation exposure to DAA:

·        DAA was quantifiable from 0.5 h to 24 h after the end of exposure,

·        maximum concentrations of DAA were reached 0.5 h after the end of exposure,

·        the elimination rate of DAA was lower and the half-life higher at 1000 ppm than 500 ppm, indicating a saturation of the excretion pathway,

·        MIBC and MIBK were not quantifiable.

After exposure to DAA, AUC0-24hand Cmax(both normalized by exposure concentration) were relatively higher when the animals were exposed to 1000 ppm than when exposed to 500 ppm.

DAA was quickly and extensively absorbed by inhalation exposure.

Comparison oral vs. inhalation routes of exposure

A single oral administration of DAA at 580 mg/kg to rats leads to an AUC of 44.9 h*mmol/L. By inhalation, considering a ventilation rate of 0.29 m3/kg, the concentration of 500 and 1000 ppm are equivalent to external dose levels of 690 and 1377 mg/kg bw and the corresponding AUC are 18.9 and 66.4 h*mmol/L. For a similar external dose level (ca. 600 mg/kg) the internal exposure (AUC) appears to be 2-fold lower after inhalation exposure than oral administration. However, the inhalation AUC was calculated over the 24-hour period following the end of the 6-hour exposure and does not integrate the internal exposure during the inhalation exposure. Therefore, in approximation, the total AUC during and 24 hours after a 6-hour inhalation exposure should be close to the AUC after oral administration, indicating a similar level of absorption.

Dermal absorption

In vitro dermal penetration rate of diacetone alcohol, as permeability coefficient (Kp) and short-term dermal absorption rates at 10 and 60 min, was determined using human cadaver skin mounted in an in vitro diffusion cell model (Fasano and McDougal, 2008). Total recovery, based on liquid scintillation count data for total radioactivity, was between 89.6 and 91.7% of the applied dose. Skin penetration was 0.04, 0.15 and 5.71 % of the dose after 10 min, 60 min and 24 h, respectively. Kp was 5.77e-4 cm/h. From the polynomial % absorption/exposure time curve (y = 1E-06x² + 0.0021x + 0.0188, R² = 1), a skin penetration of 1% was estimated for a 8-hour exposure.