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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
toxicokinetics
Principles of method if other than guideline:
Evaluation of the systemic exposure and to determine the main pharmacokinetic parameters of free Hexylene glycol, and its potential metabolites, 4 Hydroxy-4-Methyl-2-Pentanone (HMP), methylisobutylketone (MIBK) and methylisobutylcarbinol (MIBC) after single oral administration to male Sprague Dawley rats
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 292 g (range: 284 g to 297 g)
- Fasting period before study: at least 14 hours before dose administration
- Housing: in threes, in suspended wire-mesh cages
- Individual metabolism cages: no
- Diet (ad libitum): pellets SSNIFF R/M-H (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): filtered tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in corn oil. Specifically, 2.9497 g of test item was mixed with 9.1998 g of vehicle. The dosage form was prepared by slowly adding the test item to the vehicle, at room temperature, under continuous stirring. The quantity of test item used for dosage form preparation was calculated, taking into consideration the molecular weight.

The dosage form suspension was continually stirred until use.
The test item dosage form was prepared on the day of treatment and was stored at room temperature and protected from light prior to use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 295 mg/ml
- Amount of vehicle (if gavage): 2 ml/kg
Duration and frequency of treatment / exposure:
Single administration
Remarks:
Doses / Concentrations:
590 mg/kg (5 mmol/kg)
No. of animals per sex per dose:
9
Control animals:
no
Details on study design:
- Dose selection rationale: The dose-level selected was consistent with a previous pharmacokinetics probe study (CIT/Study No. 20991 PAR) using methylisobutylketone and methylisobutylcarbinol, and a NOAEL of 450 mg/kg bw/day for hexylene glycol, identified in a 90-day oral subchronic toxicity study (CIT/ Study No. 15837 TCR).
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, plasma
- Time and frequency of sampling:. 0.5, 1, 1.5, 3, 4.5, 6, 9, 12 and 24 hours post gavage
- From how many animals: 3 per time point
- Method type for identification: GC-FID
- Limits of quantification: < 0.500 mg/mL

TREATMENT FOR CLEAVAGE OF CONJUGATES : no
Type:
other: Toxicokinetics
Results:
free-HG was quantifiable from 0.5 to 6 hours after gavage. The mean plasma conc. increased to a maximum of 0.833 mg/mL at 1.5 hours after administration and then decreased in an essentially monophasic manner. AUC0-t (from 0 to 6 hours) was 3.86 mg.h/mL
Type:
metabolism
Results:
The plasma concentrations of the potential metabolites HMP, MIBK and MIBC were below the limit of quantification of: 0.5 mg/mL.
Toxicokinetic parameters:
Cmax: 0.833 mg/ml
Toxicokinetic parameters:
other: Clast: 0.641 mg/ml
Toxicokinetic parameters:
Tmax: 1.5 h
Toxicokinetic parameters:
half-life 1st: 21.2 h
Toxicokinetic parameters:
AUC: 3.86 mg.h/mL
Metabolites identified:
no

Mortality, morbidity and clinical signs

No deaths, morbidity or clinical signs occurred during the study.

       

Body weight

On the day of treatment the mean body weight ± standard deviation was 292 g ± 4.4 g (range between 284 g and 297 g).

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Hexylene glycol was well tolerated following a single oral administration at 590 mg/kg to male Sprague-Dawley rats. Plasma concentrations of free Hexylene glycol were quantifiable from 0.5 to 6 hours after dose administration. The AUC0-t value was 3.86 mg.h/mL, and the Cmax value of 0.833 mg/mL was reached at 1.5 hours after gavage. No plasma metabolite (HMP, MIBK and MIBC) concentrations were identified with a limit of quantification of 0.5 mg/mL.
Executive summary:

The objective of this study was to evaluate the systemic exposure and to determine the main pharmacokinetic parameters of free hexylene glycol (HG), and its potential metabolites, 4-hydroxy-4-Methyl-2-Pentanone (HMP), methylisobutylketone (MIBK) and methylisobutylcarbinol (MIBC) after single oral administration to male Sprague-Dawley rats.

Nine Sprague-Dawley rats received 590 mg/kg bw of HG as a suspension in corn oil by the oral route (gavage) on a single occasion.

Blood samples for the determination of plasma levels of free HG, HMP, MIBK and MIBC were taken at 0.5, 1, 1.5, 3, 4.5, 6, 9, 12 and 24-hour post-gavage. For the blood sampling, the animals were divided into three sampling sets (each containing three animals) and each animal was sampled three times in total and sacrificed after its last blood sampling occasion. During the study period, the animals were observed at least twice daily for morbidity, mortality and clinical signs.

No death or morbidity occurred during the study and no clinical signs were observed. The main pharmacokinetic (PK) parameters of free HG after a single oral (gavage) administration of HG at the dose-level of 590 mg/kg to male Sprague-Dawley rats are :

PK parameters

t1/2

tmax

Cmax

Clast

tlast

AUC0-t

AUCextrapolated

Units

h

h

mg/mL

mg/mL

h

mg.h/mL

%

Results

21.2

1.5

0.833

0.641

6

3.86

84

The mean plasma concentrations increased to a maximum of 0.833 mg/mL at 1.5 hours after administration and then decreased until 6 hours after administration in an essentially monophasic manner. The terminal half-life calculated was long (higher than 6 hours), but this value should be interpreted with caution due to the high percentage of extrapolation in the AUC calculation(> 80%) and no conclusion can be drawn on the elimination behavior of the test item. The plasma concentrations of the potential metabolites, HMP, MIBK and MIBC were below the limits of quantification of 0.5 mg/mL.

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:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to same study
Objective of study:
other: Milk transfert
Principles of method if other than guideline:
Evaluation of the exposure of pups to hexylene glycol and/or its metabolites on day 1 post-partum (p.p.) via dams treated from day 6 post-coitum (p.c.) until 20 or 21 p.c. with non radiolabelled Hexylene glycol and on day 1 p.p. with [14C]-Hexylene glycol.
GLP compliance:
no
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
Strain and sanitary status: Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
Breeder: Charles River Laboratories Italia, Calco, Italy.
Age/Weight: at the beginning of the treatment period, the animals were 10-11 weeks old and had a mean body weight of 258 g (range: 227 g to 296 g). The females were primigravid.
The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France).
Mating: females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum (p.c.).
All animals had free access to SSNIFF R/M-H pelleted maintenance diet batch No. 4898480 (SSNIFF Spezialdiäten GmbH, Soest, Germany) and to bottles containing tap water (filtered with a 0.22 µm filter)
Acclimation: the animals were acclimated to the study conditions for a period of 5 days before the beginning of the treatment period (arrival of females on day 1 p.c.).

ENVIRONMENTAL CONDITIONS
The animal room conditions were set as follows:
- temperature: 22 ± 2°C,
- relative humidity: 50 ± 20%,
- light/dark cycle: 12h/12h,
- ventilation: about 12 cycles/hour of filtered, non-recycled air.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Non radiolabelled test item formulations
Based on the stability demonstrated in CiToxLAB France/Study No. 15837 TCR, the test item dose formulations were prepared and stored up to 7 days at +4°C and protected from light "prior-to-use”. After preparation, the formulation was divided into daily aliquots and stored at +4°C. On the day of dosing, the dose formulation was allowed to come at room temperature prior to delivery under light protection (amber glass beaker).
- Radiolabelled test item formulation
The isotopic dose formulation was prepared once on day 1 p.p. and delivered at room temperature and protected from light (amber glass beaker). As 2 days of parturition were anticipated, one formulation was prepared and further divided into two aliquots. The aliquot not delivered to the animal room was stored at +4°C and protected from light "prior-to-use", based on information mentioned in § Non radiolabelled test item formulations.
Duration and frequency of treatment / exposure:
Days 6 to 21 or 22 p.c. (non radiolabelled formulation) and day 1 p.p . (radiolabelled formulation)
Remarks:
Doses / Concentrations:
1000 mg/kg
No. of animals per sex per dose:
5 (control), 20 (treated
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: results of the OECD 421 study
- Chemical analysis of the dose-formulations
The GC-FID analytical method for the determination of Hexylene glycol in dose formulation samples was validated in CiToxLAB France/Study No. 36614 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.
- Determination of concentration in dose formulations
. Non radiolabelled Hexylene glycol
The dose formulation samples were assayed using a validated method.
The concentration of the test item in samples of each control and test item dose formulations prepared for the use on day 6 p.c. and on day 20 p.c. was determined.
These analyses were performed prior to administration of the dose formulations to the animals.
Acceptance criterion:
.. measured concentration = nominal concentration ± 10%.
If the results are not satisfactory, the Study Director may request the preparation of a new dose formulation or new dose formulations.
. Radiolabelled dose formulation
The High Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method for:
Total radioactivity
The total radioactivity of the radiolabelled test item solution was analyzed in triplicate by Liquid Scintillation Counting on the following occasions:
.. before dosing (and before the prepared formulation is divided into two aliquots, see § Non radiolabelled test item formulations),
.. after each dosing (two analyses at maximum, depending on the dates of complete parturition),
.. no analysis was performed from any aliquot before dosing (but samples were taken as a contingency, stored at room temperature and analyzed if requested by the Sponsor/Study Director).
. Radiochemical purity
The radiolabelled test item solution was analyzed (in duplicate) by HPLC/UV/on line radioactivity detection to determine the radiochemical purity (results expressed as %) using the developed analytical procedure according to the method provided by Quotient Bioresearch (reference chromatogram peak comparison), on the following occasions:
.. before dosing (and before the prepared formulation is divided into 2 aliquots, see § Non radiolabelled test item formulations),
.. no analysis was performed from any aliquot before or after dosing.
Acceptance criterion: >97%.
The day of dosing, three aliquots were taken from the dose formulation intended for treatment. All aliquots were analyzed by liquid scintillation counting to determine the total radioactivity.
After treatment, the same analysis of radioactivity was undertaken on the three aliquots of the dose formulations which were passed through the same administration devices as that used for the treatment. The results were expressed as Bq/g of dose formulation.
Samplings (before and after treatment) were stored at room temperature after addition of the liquid scintillation counting until analysis.
The dose formulation was analyzed (in duplicate) by HPLC/UV/on line radioactivity detection to determine the radiochemical purity (results expressed as %).
Details on dosing and sampling:
BLOOD SAMPLING OF DAMS
Venous blood (approximately 1 mL) was collected, 5 hours and 25 ± 5 minutes after dosing, from the orbital sinus of each dam just prior to sacrifice into a tube containing lithium heparin and placed on wet ice pending centrifugation. Blood was centrifuged at 3000G for 10 minutes under refrigerated conditions (set to maintain at +4ºC).
The plasma was kept on wet ice until transferred in individual tube and stored at 20°C until analysis for total amount of radioactivity (see § Radioactive biological sample analysis).

BIOLOGICAL SAMPLE ANALYSIS
The total radioactivity of all radioactive biological samples (plasma from dams and pups) was measured by Liquid Scintillation Counting (LSC) using a Tri-Carb 2910TR system (Perkin Elmer, Downers Grove IL 60515, USA).
Selected pups (four pups per litter: two males + two females) were individually solubilized by placing in an alcoholic potassium hydroxide solution before homogenization. Thereafter, an aliquot of each homogenate was combusted using a Model 307 oxidizer, Packard. Finally the total radioactivity was measured at by LSC using a Tricarb 2910 TR Liquid Scintillation system (Perkin Elmer).

Analytical apparatus and counting method for Liquid Scintillation Counting (LSC)
A Tri-Carb B2910TR counter (Perkin Elmer, Downers Grove IL 60515, USA) was used.
An external standard source (Baryum 133) was used to monitor sample quenching; the dpm values of each sample were obtained from the corresponding cpm values using a quench standard curve.
All samples were counted for up to 10 minutes.
For each sample type, the mean dpm value from the replicate analysis was used to calculate the corresponding radioactivity concentration (as Bq/g or ng-eq/g).
Statistics:
Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by the Fisher exact probability test.
Details on absorption:
Radioactivity in plasma was detected in all dams (mean value of 4381 ± 288 Bq/g) with a low interanimal variability.
Details on distribution in tissues:
Radioactivity of approximately 134 ± 59 Bq/g foetus or a recovery of approximately 0.087 ± 0.039% per pup of the radioactive dose administered was measured in 4 pups/litter. A great inter variability was observed between litters as well as high intra variability between pups from a same litter.
Transfer type:
other: blood of dams 5 hours after PND 1 administration
Observation:
distinct transfer
Remarks:
4381 ± 288 Bq/g blood
Transfer type:
other: total radioactivity in pup on PND 1
Observation:
slight transfer
Remarks:
134 ± 59 Bq/g foetus or a recovery of approximately 0.087 ± 0.039% per pup of the radioactive dose
Metabolites identified:
no
Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Radioactivity was detected in all pups sacrificed on day 1 p.p., demonstrating the transfer of test item from the plasma through the milk, although the amounts detected were low.
Executive summary:

The exposure of pups to Hexylene glycol and/or its metabolites was evaluated on day 1 post-partum (p.p.). One group of 20 time-mated female Sprague-Dawley rats received the test item,Hexylene glycol, from day 6 post-coitum (p.c.) until 20 or 21 p.c. with non-radiolabelled Hexylene glycol and on day 1 post-partum (p.p.). with [14C]-Hexylene glycol by oral route (gavage) at the dose-level of 1000 mg/kg/day. Once the parturition was completed, the radiolabelled test item was given orally to the dams, presence of milk was checked 1, 3 and 5 hours post-dosing, and blood was collected from the dams before sacrifice for determination of radioactivity in plasma. After their sacrifice on PND 1, two male and two female pups per litter were selected for determination of total radioactivity by liquid scintillation counting.

Radioactivity was detected in the plasma of dams (mean value of 4381 ± 288 Bq/g) and data were characterized by low inter animal variability. In the 64 pups analyzed (16 litters), radioactivity was also detected on day 1p.p.. Data showed that approximately 134 ± 59 Bq/g or 0.087 ± 0.039% of the radioactive dose administered was measured in 4 pups/litter. A great inter variability was observed between litters as well as high intra variability between pups from a same litter. Compared to the radioactivity in the plasma of dams (on Bq/g basis), only 3% were found in the pups.

This result confirmed the test item passage through the plasma to the milk compartment, although in limited quantities on day 1p.p..

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Species:
human
Vehicle:
unchanged (no vehicle)
Duration of exposure:
24 hours
Doses:
- Nominal doses: 8.6 mg/cm²
- Actual doses: 8.04 mg/cm² (62843 Bq)
- Dose volume: 30 µL
Dose:
8.04 mg/cm² (62843 Bq)
Parameter:
percentage
Absorption:
2.59 %
Remarks on result:
other: 24 h
Remarks:
The total absorbed dose was 2.59 ± 1.66%. The mean absorption rate varied between 0.027% and 0.030% of the applied dose/cm2/hour (corresponding to 6.75 and 7.63 µg-eq/cm2/hour) and was found globally constant between 7 and 24 hours after application

- Receptor fluid, receptor chamber, donor chamber (in vitro test system): The mean cumulative amount of (14C)-Hexylene glycol (n = 5; ± SD) recovered in the receptor fluid over the 24-hour period, including the final receptor chamber washes (mean 0.12 ± 0.08% of the radioactive dose) was low at 2.08 ± 1.56% of the radioactive dose (range from 0.84 to 4.66%).

- Skin preparation (in vitro test system): At the end of the 24-hour exposure period, the vast majority of the radioactive dose was recovered in the skin swabs and donor chamber rinses with a mean of 101.5 ± 6.2% of the applied dose, representative of the unabsorbed dose.

- Stratum corneum (in vitro test system): A low level of radioactivity was recovered in the two surface tape strips (0.04 ± 0.01% of the dose, considered as unabsorbed dose) and in the ten following stripping performed to remove the stratum corneum (0.11 ± 0.05% of the dose). The quantity of radioactivity recovered from the exposed skin with a mean of 0.41 ± 0.11% of the applied dose was low and the radioactivity recovered in the flange skin (fraction of skin not exposed as hidden by the wall of the cell) was very low at 0.16 ± 0.07% of the dose. Radioactivity from flange skin areas, although resulting from a test item diffusion from the exposed area was considered as unabsorbed dose.

- Total recovery: 93.2% to 112.9% of the applied radioactive dose.

- Recovery of applied dose acceptable: yes

- Results adjusted for incomplete recovery of the applied dose: no

- Limit of quantification (LOQ): For the different biological matrices, blank values corresponding to the mean value of radioactivity (dpm per aliquot) measured in pre-dose matrices (sample collected before dosing for receptor fluids),or in substitution matrices (ethanol/water 50/50 for rinsing of donor compartment, ethanol for rinsing of receptor compartment, alkaline solution for skin and THF for strip) were determined.

Any measurement of radioactivity (dpm) lower than two times the blank value was reported as BLQ (Below the Limit of Quantification).

Blank value (dpm/aliquot, see table below) was subtracted from each raw measurement of radioactivity (dpm/aliquot) and the value of radioactivity presented is a corrected value. Consequently, the limit of quantification (corrected value) was defined as equal to the blank value.

Conclusions:
The absorption of (14C)-Hexylene glycol through human skin was defined on the basis of mean absorption rate, percentage dose recovered in the tape strip and in the skin membrane (at 24 hour post-dosing) and percentage of the dose in the receptor fluid. The mean recovery of radioactivity was considered complete (mean of 104.3 ± 5.0%) in five of eight diffusions cells. The mean total directly absorbed radioactivity (receptor fluid + rinsing) was relatively low at 2.08 ± 1.66% and the absorbable dose (remaining radioactivity in the skin + strips) was very low at 0.55 ± 0.15%. The mean absorption rate of Hexylene glycol through human skin varied between 0.027% and 0.030% of the applied dose/cm2/hour (corresponding to 6.75 and 7.63 µg-eq/cm2/hour) and was found globally constant between 7 and 24 hours after application.
Executive summary:

The in vitro percutaneous absorption and distribution of (14C)-Hexylene glycol (14C-HG), was evaluated in human dermatomed skin (Fabreguettes, 2014). For this purpose,14C-HG was applied to excised human skin mounted in flow-through diffusion cells. This study was performed according to OECD guideline 428 and the principles of Good Laboratory Practices. Dermatomed skin samples (thickness between 0.372 and 0.452 mm) from four human donors were maintained in eight flow-through diffusion cells at 32 ± 2°C. Prior to absorption experimentation, the integrity of the skins was tested by measuring the Trans-Epidermal Water Loss (TEWL). Then,14C-HG formulation (mixture of non labelled and14C-HG) was applied to the skin samples at a dose-volume of 10 µL/cm² (i.e. 8.63 mg/cm²) and the cell was occluded with an aluminium sheet to prevent the losses by evaporation. The skin samples were exposed to the test item formulation for 24 hours, after which the remaining formulation was washed off the skin using cotton swabs dampened with an ethanol/water solution (1:1, v/v). Receptor fluid samples were collected at hourly intervals for the duration of the study (24 hours). As Hexylene glycol is described as freely miscible with water, the solubility of the test item in the receptor fluid (0.9% NaCl) was not tested and considered not to be rate limiting to the absorption process. At the end of the study, the skin was washed to remove excess of formulation, the exposed area was tape stripped with 3M scotch tape to remove stratum corneum and then the skin samples were fractionated into the exposed skin area and non-exposed area (flange skin). The total radioactivity of the skin swabs and donor chamber rinses, biological samples (exposed and flange skin areas), receptor fluid and receptor chamber rinses was measured by direct Liquid Scintillation Counting (LSC) for receptor fluids and rinses, by LSC after extraction with appropriate solvent for swabs and tape strip and by LSC after homogenization and solubilization with an ethanol/potassium hydroxide solution for exposed and flange skin samples.

The radiochemical purities of14C-HG and applied formulation were found, respectively at 92.1% and 92.2%, respectively and considered acceptable for the percutaneous absorption test. The overall radioactivity recovery of the applied dose of14C-HG was considered complete in all cells with values ranging from 93.2% to 112.9% of the applied radioactive dose. Three cells were excluded of the mean calculation: cell No.5 which had a recovery outside the range of 90-110% and an anomalous TEWL value and cells Nos. 3 and 6 presented an anomalous profile (leak/unexpected deterioration of the skin suspected). The mean cumulative amount (calculated from five cells) of (14C)-HG recovered in the receptor fluid over the 24-hour period (i.e.dose directly absorbed), including the final receptor chamber washes (mean 0.12 ± 0.08% of the radioactive dose) was relativelylow at 2.08 ± 1.56% ofthe radioactive dose. The kinetic profile of radioactivity in the receptor fluid was characterized by an increase of absorption between 0 and 7 hours to reach a level of 0.08 ± 0.07% of the applied dose/hour. Then, a mean constant absorption 0.08/0.09 ± 0.07% of the dose/hour was observed between 8h and 24h post-dose corresponding to a mean absorption rate varying from 0.027% to 0.030% of the dose/cm²/hour (equivalent to 6.75/7.63 µg-eq/cm²/hour, respectively).

At the end of the 24-hour exposure period, the vast majority of the radioactive dose was recovered in the material washed off the skin (skin swabs) and donor chamber with a mean of 101.5 ± 6.2% of the applied dose, representative of the unabsorbed dose.

A low level of radioactivity was recovered in the two surface tape strips (0.04 ± 0.01% of the dose) and in the ten following stripping performed to remove the stratum corneum (0.11 ± 0.05% of the dose). The quantity of radioactivity recovered from the exposed skin with a mean of 0.41 ± 0.11% of the applied dose was low and the radioactivity recovered in the flange skin (fraction of skin not exposed as hidden by the wall of the cell) was very low at 0.16 ± 0.07% of the dose. Radioactivity from flange skin areas, although resulting from test item diffusion from the exposed area was considered as unabsorbed dose.

The mean (± SD) distribution of radioactivity (% of dose) is summarized in the table below:

 

Donor compartment

Skin compartment

Receptor compartment

Total recovery

Rinsing + swabs

Surface tape strip

Flange Skin

Exposed skin strip

Exposed skin

Receptor fluid

Receptor rinsing

 

101.5 ± 6.2%

0.04 ± 0.01%

0.16 ± 0.07%

0.11 ± 0.05%

0.41 ± 0.11%

1.96 ± 1.48%

0.12 ± 0.08%

 

Unabsorbed dose:
101.7 ± 6.2%

Absorbable dose:
0.55 ± 0.15%

Dose directly absorbed:
2.08 ± 1.56%

104.3 ± 5.0%

Total absorbed: 2.59 ± 1.66%

 

 

In conclusion, the absorption of (14C)-HG through human skin was defined on the basis of mean absorption rate, percentage dose recovered in the tape strip and in the skin membrane (at 24-hour post-dosing) and percentage of the dose in the receptor fluid. The mean recovery of radioactivity was considered complete (mean of 104.3 ± 5.0%) in five of eight diffusions cells. The mean total directly absorbed radioactivity (receptor fluid + rinsing) was relatively low at 2.08 ± 1.56% and the absorbable dose (remaining radioactivity in the skin + strips) was very low at 0.55 ± 0.15%. The mean absorption rate of14C-HG through human skin varied between 0.027% and 0.030% of the applied dose/cm²/hour (corresponding to 6.75 and 7.63 µg-eq/cm²/hour) and was found globally constant between 7 and 24 hours after application.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
excretion
Qualifier:
no guideline followed
Principles of method if other than guideline:
Quantification of the excretion of glucuronate of Hexylene glycol in the urine of rabbits.
Species:
rabbit
Strain:
other: Chinchilla
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 2-3 kg
- Fasting period before study: no data
- Housing: no data
- Individual metabolism cages: no data
- Diet : 60g of rat cubes diet 41
- Water : 100 ml/day
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
no data
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Doses / Concentrations:
1 mmol/kg (118 mg/kg bw)
No. of animals per sex per dose:
3
Details on dosing and sampling:
Urine was analysed daily for glucuronic acid by the naphtharesorcinol method of Paul (Mead, Smith & Williams, 1958).
Type:
excretion
Results:
67 of the dose (49-93)% as glucuronides
Executive summary:

Gessner et al. (1960) administered 1 mmol/kg hexylene glycol to 3 chinchilla rabbits by stomach tube with water. Urine collected from the animals for 1-2 days after dosing showed 67% (range 49-93%) of the dose excreted as glucuronate conjugates.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
excretion
Principles of method if other than guideline:
Five male volunteers aged 50-60 were given single and repeated oral daily doses of 1 -5 g hexylene glycol using various dosing regimes
GLP compliance:
no
Radiolabelling:
no
Species:
human
Details on test animals and environmental conditions:
Five heahhy subjects between 50 and 60 years took part in the experiment.
Route of administration:
oral: feed
Vehicle:
other: aqueous solution, flavoured with black currant syrup.
Duration and frequency of treatment / exposure:
Single, 5-day or 8-11-day administrations
Remarks:
Doses / Concentrations:
1, 2 or 5 g/person
No. of animals per sex per dose:
5
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: all the urine was collected after and during the single or 5-day administration. The urine was collected each day from about 8 a. m. to 5 p. m. during the 8-11-day administration
- Method type(s) for identification: spectrophotometry (Jacobsen, 1958b)
- Limits of detection and quantification: 0.2 mg/ml urine
- Other:

TREATMENT FOR CLEAVAGE OF CONJUGATES: hydrochloric acid
Details on excretion:
After single doses of 1-2 g hexylene glycol no detectable amount could be found in the urine, indicating an excretion of less than 100 mg daily. After a single dose of 5 g, about 0.5 g was excreted during the first 24 hours, 0.2-0.3 g during the second and 0.1-0.2 g during the third period of 24 hours. After 5 g daily for several days the daily excretion increased and reached 1.3-1.4 g on the fifth day. The excretion continued after discontinuation of intake, but fell to zero during the following 10 days (fig. I).

Table 1 gives the excretion after various doses. Not included in the table are the figures from a patient who for 3 consecutive days excreted less than
100 mg hexylene glycol daily after having received 0.6 g daily for 3 months. Broadly it seems that 1/5-1/3 of ingested hexylene glycol is excreted in the urine.
The amount of hexylene glycol excreted free or conjugated (Jacobsen, 1958b) in the five experimental subjects is shown in table 2. The high variation is presumably largely, although not necessarily, due to the large error of the analytical method. However, the average of about 50% free hexylene glycol excreted is much higher than has been found in rats.
Executive summary:

Five test persons were given oral doses of a 10 % aqueous solution of hexylene glycol, either as single 5 g doses or two 2.5 g doses daily on 5 consecutive days, or 1 g daily for 8 to 11 days followed by 2 g daily for 13 to 14 days; 18% to 35% of the dose was excreted in the urine. According to the authors, half of the excreted substance was in the form of the glucuronide of hexylene glycol. Hexylene glycol was detectable in the urine for 5 to 11 days after the last dose. A half-life was not given. After administration of single doses = 2 g or daily doses = 600 mg for 90 days, with the unspecific photometric method which was used (detection limit about 100 mg in the 24-hour urine; Jacobsen 1958a) neither free hexylene glycol nor its glucuronide could be detected in the urine.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment ((No indication of test substance purity. Only urinary (and not fecal or exhaled) excretion was quantified. )
Objective of study:
excretion
Principles of method if other than guideline:
The excretion rate was determined in rats after a single dose of 400 mg hexylene glycol or after administration of 100 or200 mg hexylene glycol for 60 days or more. The urine was collected, pooled and analysed for total and free hexylene glycol.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 4-week old
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: no data
- Individual metabolism cages: yes/no
- Diet (ad libitum): hard mouse bread + 20g of fresh carrots
- Water : no data
- Acclimation period:no data

ENVIRONMENTAL CONDITIONS
no data
Route of administration:
other: Orally in milk (repeated doses) or by gavage in water (single dose)
Vehicle:
other: milk or water
Duration and frequency of treatment / exposure:
Daily for 60 days or more or single administration
Remarks:
Doses / Concentrations:
100, 200, or 400 mg/rat
No. of animals per sex per dose:
6/group
Control animals:
yes, concurrent vehicle
Details on dosing and sampling:
After administration of hexylene glycol for 60 days or more, 24 hours samples of urine were collected from 6 rats of each group. The rats chosen for excretion experiments were the ones that had consumed the 100 or 200 mg hexylene glycol offered daily for longer periods and therefore could be considered to be in a state of balance between intake and excretion of hexylene glycol. The urines from the individual rats in each group of 6 were pooled and analyzed for free and bound hexylene glycol.

In order to obtain information about the excretion rate of a single dose, 6 male rats (weighing 330-362 g) were given each exactly 400 mg hexylene glycol(i.e.one third of the LD50) in 4 ml water by stomach tube. The urine was collected, pooled, and analyzed for total and free hexylene glycol
Type:
excretion
Results:
Rats excreted 32 % to 47 % of the administered substance in the urine, 96 % as the glucuronide and 4 % as unchanged hexylene glycol.
Details on absorption:
Not measured
Details on distribution in tissues:
Not measured
Details on excretion:
When receiving 200 mg per day for 60 to 131 days, the rats excreted 40% of consumed hexylene glycol in the urine. Only 4% of the excreted quantity was in the form of free glycol, which gives a concentration of 0.40 mg/ml. See attached tables 1 and 2.
When receiving a single administration of 400 mg, the rats excreted 49% of consumed hexylene glycol in the urine. See attached table 3.
Metabolites identified:
not measured
Executive summary:

The excretion of free and bound hexylene glycol was determined in the 24 hour pooled urine of groups of 6 male rats following acute and repeated exposure to hexylene glycol. The rats receiving a single dose received 400 mg hexylene glycol in aqueous solution by stomach tube. Repeated doses were administered in milk at nominal concentrations of 100 mg/kg/day for 62-98 days or 200 mg/kg/day for 60-131 days. Rats receiving 200 mg/kg/day hexylene glycol/day excreted 40% of the amount consumed in the urine. Only 4% of the excreted quantity was in the form of free glycol. Rats receiving 100 mg/kg/day hexylene glycol excreted 51% in the urine of which about 7% was in the form of free glycol. Rat receiving a single dose of 400 mg/kg excreted 49% in the urine of which about 14% was in the form of free glycol.

Description of key information

The predominant mechanism for the metabolism of HG appears to be conjugation of the parent compound or its intermediate metabolite with glucuronide, followed by excretion in the urine. HG is absorbed through the mucosa of the gastrointestinal tract. Man excretes about 35 % of a dose of HG slowly with the urine, about half as the unchanged substance and half as the glucuronide; rodents excrete 50 % to 60 % of the dose in the urine, mainly as the glucuronide. Oral absortion rate only based on the urine excretion will be underestimated as the fecal excretion via the enterohepatic cycle of the conjugates are not taken into account. A global absorption rate of 50% seems appropriate after oral administration. Considering its physicochemical properties, the absorption of HG after inhalation exposure is expected to be of the same order than after oral administration. The absorption of (14C)-HG through human skin was relatively low at 2.59 ± 1.66% (range from 1.1 to 5.3%) of the applied dose. For the purpose of risk assessment, an absorption rate of 5 % (ca. mean + 2 x SD) will be used after dermal exposure.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
5
Absorption rate - inhalation (%):
50

Additional information

Limited data is available to describe the likely ADME properties of hexylene glycol (HG). Given its relatively high water solubility, log Pow of < 1 and molecular weight of < 500 Da., absorption might be expected following ingestion or inhalation exposure. An in vitro percutaneous absorption study with human skin was performed to evaluate the dermal absorption.

Oral route

Absorption/excretion

The excretion of free and bound HG was determined in the 24 hour pooled urine of groups of 6 male rats following acute and repeated exposure to HG (Larsen, 1958). The rats receiving a single dose received 400 mg HG in aqueous solution by stomach tube. Repeated doses were administered in milk at nominal concentrations of 100 mg/kg/day for 62-98 days or 200 mg/kg/day for 60-131 days. Rats receiving 200 mg/kg/day HG/day excreted 40% of the amount consumed in the urine. Only 4% of the excreted quantity was in the form of free glycol. Rats receiving 100 mg/kg/day HG excreted 51% in the urine of which about 7% was in the form of free glycol. Rat receiving a single dose of 400 mg/kg excreted 49% in the urine of which about 14% was in the form of free glycol.

Gessner et al, 1960 also reported excretion of glucuronate of HG in the urine of rabbits administered about 118 HG mg/kg by stomach tube. 67% of the dose was recovered from the urine in conjugated form. They were unable to characterise the conjugate.

Five test persons were given oral doses of a 10 % aqueous solution of HG, either as single 5 g doses or two 2.5 g doses daily on 5 consecutive days, or 1 g daily for 8 to 11 days followed by 2 g daily for 13 to 14 days; 18% to 35% of the dose was excreted in the urine (Jacobsen, 1958a). According to the authors, half of the excreted substance was in the form of the glucuronide of HG. HG was detectable in the urine for 5 to 11 days after the last dose. A half-life was not given. After administration of single doses = 2 g or daily doses = 600 mg for 90 days, with the unspecific photometric method (Jacobsen 1958b) which was used (detection limit about 100 mg in the 24-hour urine) neither free HG nor its glucuronide could be detected in the urine.

Toxicokinetics

The systemic exposure and the main pharmacokinetic parameters of HG, and its potential metabolites, 4-hydroxy-4-Methyl-2-Pentanone (HMP), methylisobutylketone (MIBK) and methylisobutylcarbinol (MIBC) were evaluated after a single oral administration to male Sprague-Dawley rats (Aubert, 2010). Nine Sprague-Dawley rats received 590 mg/kg bw of HG as a suspension in corn oil by the oral route (gavage) on a single occasion. Blood samples for the determination of plasma levels of free HG, HMP, MIBK and MIBC were taken at 0.5, 1, 1.5, 3, 4.5, 6, 9, 12 and 24-hour post-gavage. For the blood sampling, the animals were divided into three sampling sets (each containing three animals) and each animal was sampled three times in total and sacrificed after its last blood sampling occasion. During the study period, the animals were observed at least twice daily for morbidity, mortality and clinical signs. No death or morbidity occurred during the study and no clinical signs were observed.

The main pharmacokinetic (PK) parameters of free HG after a single oral (gavage) administration of HG at the dose-level of 590 mg/kg to male Sprague-Dawley rats are:

 

PK parameters

t1/2

tmax

Cmax

Clast

tlast

AUC0-t

AUCextrapolated

Units

h

h

mg/mL

mg/mL

h

mg.h/mL

%

Results

21.2

1.5

0.833

0.641

6

3.86

84

 

The mean plasma concentrations increased to a maximum of 0.833 mg/mL at 1.5 hours after administration and then decreased until 6 hours after administration in an essentially monophasic manner. The terminal half-life calculated was long (higher than 6 hours), but this value should be interpreted with caution due to the high percentage of extrapolation in the AUC calculation(> 80%) and no conclusion can be drawn on the elimination behaviour of the test item. The plasma concentrations of the potential metabolites, HMP, MIBK and MIBC were below the limits of quantification of 0.5 mg/mL.

Distribution

The exposure of pups to Hexylene glycol and/or its metabolites was evaluated on day 1 post-partum (p.p.) (Chevalier, 2014). One group of 20 time-mated female Sprague-Dawley rats received the test item,Hexylene glycol, from day 6 post-coitum (p.c.) until 20 or 21 p.c. with non-radiolabelled Hexylene glycol and on day 1 post-partum (p.p.). with [14C]-Hexylene glycol by oral route (gavage) at the dose-level of 1000 mg/kg/day. Once the parturition was completed, the radiolabelled test item was given orally to the dams, presence of milk was checked 1, 3 and 5 hours post-dosing, and blood was collected from the dams before sacrifice for determination of radioactivity in plasma. After their sacrifice on PND 1, two male and two female pups per litter were selected for determination of total radioactivity by liquid scintillation counting.

Radioactivity was detected in the plasma of dams (mean value of 4381 ± 288 Bq/g) and data were characterized by low inter animal variability. In the 64 pups analyzed (16 litters), radioactivity was also detected on day 1p.p.. Data showed that approximately 134 ± 59 Bq/g or 0.087 ± 0.039% of the radioactive dose administered was measured in 4 pups/litter. A great inter variability was observed between litters as well as high intra variability between pups from a same litter. Compared to the radioactivity in the plasma of dams (on Bq/g basis), only 3% were found in the pups.

This result confirmed the test item passage through the plasma to the milk compartment, although in limited quantities on day 1p.p..

Dermal route

The in vitro percutaneous absorption and distribution of (14C) -Hexylene glycol (14C-HG), was evaluated in human dermatomed skin (Fabreguettes, 2014). For this purpose,14C-HG was applied to excised human skin mounted in flow-through diffusion cells. This study was performed according to OECD guideline 428 and the principles of Good Laboratory Practices. Dermatomed skin samples (thickness between 0.372 and 0.452 mm) from four human donors were maintained in eight flow-through diffusion cells at 32 ± 2°C. Prior to absorption experimentation, the integrity of the skins was tested by measuring the Trans-Epidermal Water Loss (TEWL). Then,14C-HG formulation (mixture of non labelled and14C-HG) was applied to the skin samples at a dose-volume of 10 µL/cm² (i. e.8.63 mg/cm²) and the cell was occluded with an aluminium sheet to prevent the losses by evaporation. The skin samples were exposed to the test item formulation for 24 hours, after which the remaining formulation was washed off the skin using cotton swabs dampened with an ethanol/water solution (1:1, v/v). Receptor fluid samples were collected at hourly intervals for the duration of the study (24 hours). As HG is described as freely miscible with water, the solubility of the test item in the receptor fluid (0.9% NaCl) was not tested and considered not to be rate limiting to the absorption process. At the end of the study, the skin was washed to remove excess of formulation, the exposed area was tape stripped with 3M scotch tape to remove stratum corneum and then the skin samples were fractionated into the exposed skin area and non-exposed area (flange skin). The total radioactivity of the skin swabs and donor chamber rinses, biological samples (exposed and flange skin areas), receptor fluid and receptor chamber rinses was measured by direct Liquid Scintillation Counting (LSC) for receptor fluids and rinses, by LSC after extraction with appropriate solvent for swabs and tape strip and by LSC after homogenization and solubilization with an ethanol/potassium hydroxide solution for exposed and flange skin samples.

The radiochemical purities of14C-HG and applied formulation were found, respectively at 92.1% and 92.2%, respectively and considered acceptable for the percutaneous absorption test. The overall radioactivity recovery of the applied dose of14C-HG was considered complete in all cells with values ranging from 93.2% to 112.9% of the applied radioactive dose. Three cells were excluded of the mean calculation: cell No.5 which had a recovery outside the range of 90-110% and an anomalous TEWL value and cells Nos. 3 and 6 presented an anomalous profile (leak/unexpected deterioration of the skin suspected). The mean cumulative amount (calculated from five cells) of (14C) -HG recovered in the receptor fluid over the 24-hour period (i. e.dose directly absorbed), including the final receptor chamber washes (mean 0.12 ± 0.08% of the radioactive dose) was relatively low at 2.08 ± 1.56% of the radioactive dose. The kinetic profile of radioactivity in the receptor fluid was characterized by an increase of absorption between 0 and 7 hours to reach a level of 0.08 ± 0.07% of the applied dose/hour. Then, a mean constant absorption 0.08/0.09 ± 0.07% of the dose/hour was observed between 8h and 24h post-dose corresponding to a mean absorption rate varying from 0.027% to 0.030% of the dose/cm²/hour (equivalent to 6.75/7.63 µg-eq/cm²/hour, respectively). At the end of the 24-hour exposure period, the vast majority of the radioactive dose was recovered in the material washed off the skin (skin swabs) and donor chamber with a mean of 101.5 ± 6.2% of the applied dose, representative of the unabsorbed dose. A low level of radioactivity was recovered in the two surface tape strips (0.04 ± 0.01% of the dose) and in the ten following stripping performed to remove the stratum corneum (0.11 ± 0.05% of the dose). The quantity of radioactivity recovered from the exposed skin with a mean of 0.41 ± 0.11% of the applied dose was low and the radioactivity recovered in the flange skin (fraction of skin not exposed as hidden by the wall of the cell) was very low at 0.16 ± 0.07% of the dose. Radioactivity from flange skin areas, although resulting from test item diffusion from the exposed area was considered as unabsorbed dose.

The mean (± SD) distribution of radioactivity (% of dose) is summarized in the table below:

 

Donor compartment

Skin compartment

Receptor compartment

Total recovery

Rinsing + swabs

Surface tape strip

Flange Skin

Exposed skin strip

Exposed skin

Receptor fluid

Receptor rinsing

 

101.5 ± 6.2%

0.04 ± 0.01%

0.16 ± 0.07%

0.11 ± 0.05%

0.41 ± 0.11%

1.96 ± 1.48%

0.12 ± 0.08%

 

Unabsorbed dose:
101.7 ± 6.2%

Absorbable dose:
0.55 ± 0.15%

Dose directly absorbed:
2.08 ± 1.56%

104.3 ± 5.0%

Total absorbed: 2.59 ± 1.66%

 

 

In conclusion, the absorption of (14C) -HG through human skin was defined on the basis of mean absorption rate, percentage dose recovered in the tape strip and in the skin membrane (at 24-hour post-dosing) and percentage of the dose in the receptor fluid. The mean recovery of radioactivity was considered complete (mean of 104.3 ± 5.0%) in five of eight diffusions cells. The mean total directly absorbed radioactivity (receptor fluid + rinsing) was relatively low at 2.08 ± 1.56% (range from 0.84 to 4.66%) and the absorbable dose (remaining radioactivity in the skin + strips) was very low at 0.55 ± 0.15%. The mean absorption rate of14C-HG through human skin varied between 0.027% and 0.030% of the applied dose/cm²/hour (corresponding to 6.75 and 7.63 µg-eq/cm²/hour) and was found globally constant between 7 and 24 hours after application.