Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start: 11 Oct 2012, Experimantal Termination: 31 Jan 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

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

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The objective of this study is to determine the pharmacokinetics of diisobutyl ketone (DIBK), DIBK isomer 4,6-dimethylheptan-2-one, diisobutyl
carbinol (DIBC), DIBC isomer 4,6-dimethylheptan-2-ol, and their potential major metabolites in plasma collected from NTac:SD rats following a single oral administration. A determination of comparable pharmacokinetics parameters (such as AVC) for both ketone and alcohol and their potential
major metabolites would indicate bioequivalence of ketone and alcohol in the male NTac:SD rat.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Diisobutyl carbinol (DIBC)
- Molecular formula (if other than submission substance): C9H20O
- Molecular weight (if other than submission substance): 144.3
- Physical state: Liquid
- Analytical purity: 98%
- Composition of test material, percentage of components: Sum of 2,6-dimethyl-4-heptanol and 4,6-dimethyl-2-heptanol
- Purity test date: 2011
- Lot/batch No.: ZJ1855T3Z1

Diisobutyl Keytone (DIBK)
- Molecular formula (if other than submission substance): C9H18O
- Molecular weight (if other than submission substance): 142.2
- Physical state: Liquid
- Analytical purity: 99%
- Composition of test material, percentage of components: Sum of 2,6-dimethyl-4-heptanol and 4,6-dimethyl-2-heptanol
- Purity test date: 2009
- Lot/batch No.: XA2355T643

Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic (Germantown, New York)
- Age at study initiation: 8 to 12 weeks
- Housing: Following administration of gest material by gavages, Groups 1 and 2 for the Probe Study will be housed in the same tubs for acclimation.- Following administration of the test material by gavages, Groups 3 and 4 for the TK study will be housed singly in glass Roth-type cages.
- Diet (e.g. ad libitum): LabDiet certified Roden Diet #5002 in pellet for is provided ad libitum except that access will be restriced approximately
16-hours prior to dosing and will be returned about 4-hours post-dosing.
- Water (e.g. ad libitum): Drinking water obtained from a municipal source is provided ad libitum.
- Housing / Acclimation period: Non-cannulated rates will be acclimated to the building for at least 7-days in solid bottom tubs. Cannulated rates
will be housed individually in glass Roth type metabolism cages on arrival and acclimated for at least 5-days prior to their use in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 1°C (and a maxium permissable excursion of +/- 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

ANIMAL WELFARE
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Pans 1-4, the animal care and lise activities
required that conduct of the studies be revie\ved and approved by the Institutional Animal Care and Use Committee (IACLJC). The IACUC has
determined that the proposed Activities arc in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study are Metabolism 01, Humane Endpoints 01 and Animal ID O1.

CANNULATION
Jugular Vein (Groups 3 and 4 rats only) NTac:SD rats in which the blood/plasma time-course will he determined will be obtained already cannulated
in the jugular vein by the supplier.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose
Details on exposure:
For both the Probe and Time-course phases of this study a single 700mg/Kg dose of DIBK/DIBC will be administed as follows:

VEHICLE
- Justification: Due to the possibe effect (caused by corn oil) on the metabolite identification in plasma samples, 0.5% methylcellulose will be used as
the dose vehicle to reduce the matrix effect.
- Concentration in vehicle: A dose of approximaltely 5mmol/Kg (700mg/Kg) will be administered
- Amount of vehicle (if gavage): The final dose suspension will be administered at a target volume of ~5mL/Kg body weight, via a ball-tipped gavages needle attached to a glass syringe.


HOMOGENEITY AND STABILITY OF TEST MATERIAL:
- Homogeneity of the dose suspensions may be determined prior to dosing by taking aliquots from 2-3 locations within the container.
- Stability will not necessary as the animals will be dosed within 24 hours of preparation.
Duration and frequency of treatment / exposure:
A single dose will be administered
Doses / concentrations
Remarks:
Doses / Concentrations:
One 700mg/Kg gavages dose will be administered.
No. of animals per sex per dose:
Probe: 2 Male rats
Time-course: 4 Male rats
Vehicle Control: 1 Male
Control animals:
yes, concurrent vehicle
Positive control:
Not applicable
Details on study design:
Diisobulyl ketone (DIBK) and Diisobutyl carbinol (DIBC) are high-production volume chemicals that are widely used in the chemical industry. Due to
the catalytic large-scale production process where acetone was used as a starting material, both DIBC and DIBK contain their corresponding minor
isomers. DiBC contains about 13% of 4,6-dimethylheptan-2-ol and DIBK contains about 13% of 4,6- dimethylheptan-2-one. There are sufficient DIBK toxicity data for supporting DIBK registration (such as REACH registration). In contrast, only limited toxicity data is available for DIBC, which could
hinder the registration for DIBC. To solve the potential registration issue, a Read-cross approach has been proposed, where the ketone DIBK (or DIBK isomer 4,6-dimethylheptan-2-one) toxicity data, in conjunction with new ADME data showing bioequivalence between ketone and alcohol molecules,could be used to develop an adequate REACH registration dossier for the alcohol DIBC (or DlBC isomer 4,6-dimethylheptan-2-o1). The proposed
bioequivalence study is based on the hypothesis that this alcohol-ketone set undergo interconversion in vivo, as has been shown for the analogous compounds alcohol MIBC and ketone MIBK (Gingell et al., 2003). In that study Gingell et al. showed these two compounds are essentially
bioequivalent, with MIBC converting first to MIBK, followed by both materials being metabolized to the common HMP metabolite
(4-hydroxy-4-methyl-2-pentanone). Pharmacokinetic analysis of blood samples, following single oral doses of either MIBK or MIBC showed that the total systemic exposure of the two major metabolites (MIBK +HMP) were comparable, as measured by the sum of the plasma AUC values. Although
no mass balance of administered radiolabel and urinary metabolite profiling \vere conducted in the Gingell's study or other prior metabolism
studies with these compounds (DiVincenzo et aI., 1976; Duguay and Plaa. 1995; Granvil et al., 1994) and some differences in overall
ADME fate of these two test materials may occur. the final result from the Gingell et al. (2003) study showed that metabolic equivalency of
MIBC with MIBK support the conclusion that MIBC will have a similar toxicological profile to that of MIBK, and reduces the need for additional animal
studies (Gingell et al., 2003).

DIBK and DIBC are quite comparable in structure to the related MIBK and MIBC compounds. It would therefore be expected that DIBK and DIBC would undergo similar interconversion in mammals, and also form a common HMP-like metabolite, which would be 6-hydroxy-2,6-dimethyl-4-heptanone (HDH) (Scheme 2). In similar way, the minor isomer of DIBK (4,6-dimethylheptan-2-one) and DIBC (4,6-dimethylheptan-2-o1) will also undergo
similar interconversion in mammals and also form common metabolite 6-hydroxy-4,6-dimethylheptan-2-one. Based on this background
information, a bridging limited ADME study is proposed to confirm this hypothesis via the similar approach as reported in Gingell's publication
(Gingell et al., 2003):

Step 1: Probe Experiment
- 2 Male rates will be adminsitered either DIBC or DIBK, vial oral gavages, at a dose level of 700mg/Kg according to the previouse study on MIBC
and MIBK (Gingell et al., 2003).
- Animals weill be sacrificed at the time of common Cmax previously reported for the higher inter-conversion for both MIBK and MIBC (about 1.5
hours).
- These Cmax plasma samples will be analyzed for the two test materials (DIBK and DIBC) and their corresponding minor isomers
(4,6-dimethylheptan-2-one and 4,6-dimethyiheptan-2-(1), as well as the proposed common metaholites HDH and 6-hydroxy-4,6-
dimethylheptan-2-one (or another major common metabolite).
- If HDH (or another appropriate biomarker metabolite) levels are found comparable to, or higher than DIBK/DIBC, a standard of this metabolite will be synthesized for the subsequent quantitative TK experiment.
- This scenario will be also applied to the minor isomers of both DIBK and DlBC (4.6-dimethylheptan-2-one and 4,6-dimethylheptan-2-o1 ).

Step2: Time-course - Collecting plasma samples for TK analysis:
- Once the DlBC, 4,6-dimethylheptan-2-oL DIBK and 4,6-dimethylheptan-2-one presence in the Probe experiment is confirmed, the definitive
TK experiment will be conducted to determine kinetics of the DIBC, 4,6-dimethylheptan-2-01, DIBK 4,6- dimethylheptan-2-one, and possibly
HDH and 6-hydroxy-4,6-dimethylheptan-2-one (or other metabolite) biomarkers.
- A group of 4 male rats, with cannula implanted in the jugular vein, will be administered either DIBK or DIBC, via oral gavage, at a dose level of
700 mg/kg.
- Repetitive bloocl samples will be taken from each rat at 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 6. 12, 24, 48, 72, 96 and 120 hrs post-dosing and centrifuged
to obtain plasma.
- Chemical analysis will then be conducted on individual plasma samples for DlBC, 4,6-dimethylheptan-2-ol, DIBK 4,6-dimethylheptan-2-one and
possibly HDH and 6-hydroxy-4,6-dimethylhcptan-2-one.
- Pharmacokinetic analysis would be conducted on the TK quantitative results, to calculate absorption and eliminalion half-lives of the TK
biomarkers, as well as AUC determination of systemic bioavailabijity.
- The hopeful outcome of this study design would be a conclusion similar to that found for MlBK/MIBC, in which the DIBK/DIBC and
4,6-dimcthylheptan-2-one/4,6-dimethylheptan-2-0l would be found to interconvert in the
rat, with possible formation of comparable levels of a common metabolite.

See Table 1 for details.
Details on dosing and sampling:
Oral Dose Administration
The oral dose will be administered via a ball-tipped gavage needle attached to a glass syringe. Animals from Groups 1-4, will receive a single oral
dose. The volume of dose formulation to be administered to each animal will be calculated based on the body weighr taken on the day of dose
administration.

Statistics:
Descriptive: statistics will be used, i.e., mean ± standard deviation. All calculations in the database will be conducted using Microsoft Excel
(Microsoft Corporation, Redmond,Washington) spreadsheets and databases in full precision mode (15 digits of accuracy). Certain pharmacokinetic parameters will be estimated for blood and urine data, including AUC (area-under-the-curve), Cmax, 1/2Cmax, and elimination rate constants,
using PK Solutions (v.2.0.6., Summit Research Services, Montrose, Colorado).

Results and discussion

Preliminary studies:
Not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
DIBC and DIBK share the main major metabolites and are considered to be ‘interconvertable’. DIBC major isomer was metabolised to DIBK major isomer, to DIBK alcohol and to DIBC alcohol.
DIBC minor isomer was metabolised to DIBK minor isomer.

DIBK major isomer was converted to DIBC, DIBC alcohol and to DIBK alcohol. DIBK minor isomer can convert to DIBC minor isomer.

There is evidence that enterohepatic circulation of phase II conjugates occurs.

Bioaccessibility

Bioaccessibility testing results:
Not applicable

Any other information on results incl. tables

The objective of this study was to determine the pharmacokinetics of diisobutyl ketone (DIBK), DIBK isomer (4,6-dimethylheptan-2-one), diisobutyl carbinol (DIBC), DIBC isomer

(4,6-dimethylheptan-2-ol), and their potential major metabolites in plasma collected from NTac:SD rats following a single oral administration. A determination of comparable

pharmacokinetics parameters (such as AUC) for both ketone and alcohol and their potential major metabolites would indicate bioequivalence of these two test materials in the male

NTac:SD rat.

After single oral dose of DIBC (containing DIBC-isomer) or DIBK (containing DIBK-isomer) in male NTac:SD rat (700 mg/kg, nominal), metabolite profiling was initially conducted with plasma samples collected at the estimated time of Cmax plasma concentrations of test material and/or metabolites (1.5 hr post-dosing). In addition to the two test materials, and their isomers, two common metabolites were identified from either test material. These metabolites (DIBC alcohol and DIBK alcohol) were monohydroxylated metabolites of DIBC and DIBK, with the site of hydroxylation at the σ and σ-1 positions, respectively.

Quantitative analysis of DIBC, DIBK, DIBC-isomer, DIBK-isomer, DIBC-alcohol and DIBK alcohol was then conducted for blood samples collected at 5 min to 120 hr after a single oral dose of either DIBC or DIBK (490-635 mg/kg). DIBK, DIBK-isomer, DIBK alcohol and DIBC alcohol were found to be quantifiable from the DIBK test material while DIBC, DIBKisomer, DIBK-alcohol and DIBC alcohol were quantifiable from DIBC oral administration. The current study also showed that DIBC and DIBK, and their minor isomers are well interconverted in rats. Both DIBC and DIBK have similar metabolic pathways to form the major common metabolites (DIBC-alcohol and DIBK-alcohol). DIBC isomer was quantitatively converted to DIBK isomer, with no subsequent metabolites identified at the current dose level. Both sets of major and minor test material isomers are rapidly absorbed

following oral administration, with substantial evidence of enterohepatic recirculation of DIBK, DIBC and several of their metabolites. Quantitatively, DIBC isomer and DIBK isomer have comparable systemic bioavailability, with complete conversion of DIBC isomer to DIBK isomer, resulting in similar blood AUC values for the DIBK isomer arising from either of these minor test materials. In contrast, DIBK was shown to have higher bioavailability (AUC) than DIBC after oral administration. This higher bioavailability may be due to lower absorption of DIBC or a higher rate of Phase II conjugation of DIBC and metabolites, followed by biliary elimination. In summary, DIBK and DIBC, along with the corresponding minor isomers were interconvertible in the male rat, with comparable or lower systemic exposure of DIBC isomers than DIBK isomers.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no data
Not applicable
Executive summary:

The objective of this study was to determine the pharmacokinetics of diisobutyl ketone (DIBK), DIBK isomer (4,6-dimethylheptan-2-one), diisobutyl carbinol (DIBC), DIBC isomer (4,6-dimethylheptan-2-ol), and their potential major metabolites in plasma collected from NTac:SD rats following a single oral administration. A determination of comparable pharmacokinetics parameters (such as AUC) for both ketone and alcohol and their potential major metabolites would indicate bioequivalence of these two test materials in the male NTac:SD rat.

After single oral dose of DIBC (containing DIBC-isomer) or DIBK (containing DIBK-isomer) in male NTac:SD rat (700 mg/kg, nominal), metabolite profiling was initially conducted with plasma samples collected at the estimated time of Cmax plasma concentrations of test material and/or metabolites (1.5 hr post-dosing). In addition to the two test materials, and their isomers, two common metabolites were identified from either test material. These metabolites (DIBC alcohol and DIBK alcohol) were monohydroxylated metabolites of DIBC and DIBK, with the site of hydroxylation at the σ and σ-1 positions, respectively.

Quantitative analysis of DIBC, DIBK, DIBC-isomer, DIBK-isomer, DIBC-alcohol and DIBK alcohol was then conducted for blood samples collected at 5 min to 120 hr after a single oral dose of either DIBC or DIBK (490-635 mg/kg). DIBK, DIBK-isomer, DIBK alcohol and DIBC alcohol were found to be quantifiable from the DIBK test material while DIBC, DIBKisomer, DIBK-alcohol and DIBC alcohol were quantifiable from DIBC oral administration. The current study also showed that DIBC and DIBK, and their minor isomers are well interconverted in rats. Both DIBC and DIBK have similar metabolic pathways to form the major common metabolites (DIBC-alcohol and DIBK-alcohol). DIBC isomer was quantitatively converted to DIBK isomer, with no subsequent metabolites identified at the current dose level. Both sets of major and minor test material isomers are rapidly absorbed

following oral administration, with substantial evidence of enterohepatic recirculation of DIBK, DIBC and several of their metabolites. Quantitatively, DIBC isomer and DIBK isomer have comparable systemic bioavailability, with complete conversion of DIBC isomer to DIBK isomer, resulting in similar blood AUC values for the DIBK isomer arising from either of these minor test materials. In contrast, DIBK was shown to have higher bioavailability (AUC) than DIBC after oral administration. This higher bioavailability may be due to lower absorption of DIBC or a higher rate of Phase II conjugation of DIBC and metabolites, followed by biliary elimination. In summary, DIBK and DIBC, along with the corresponding minor isomers were interconvertible in the male rat, with comparable or lower systemic exposure of DIBC isomers than DIBK isomers.