Registration Dossier

Administrative data

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-12-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with the Principles of GLP and there is sufficient information to permit a meaning ful evaluation of study results.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 14C-Dipropylene Glycol n-Propyl Ether, Dipropylene glycol N-propyl ether
- Molecular formula: C9H20O3
- Molecular weight: 176.3
- Physical state: Clear liquid
- Analytical purity: 99.8% as determined by Sigma Aldrich with gas chromatography, structure verification was performed by The Dow Chemical Company, Midland, MI.
- Impurities (identity and concentrations): not specified
- Purity test date: Radiolabeled: Moravek Biochemicals, Brea, CA (Lot# R05-089-057, Inv #863), Non-Radiolabeled: Sigma-Aldrich, Saint Louis, Missouri, 63105 (Lot # 05630TA)
- Radiochemical purity (if radiolabelling): Radiolabled: 98.4% as determined by liquid chromatography
- Specific activity (if radiolabelling): ~57 mCi/mmol
- Locations of the label (if radiolabelling):
- Synonym: DOWANOL DPnP
Radiolabelling:
yes
Remarks:
14C- Dipropylene Glycol n-Propyl Ether

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc. (Kalamazoo, Michigan)
- Age at study initiation: 5-6 months
- Weight at study initiation: 2400 to 2700 grams
- Fasting period before study:
- Housing: The animals were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least two weeks prior to the start of the study.
- Individual metabolism cages: yes. The animals were acclimated to the metabolism cages by increasing the amount of time four days prior to the initiation of the study (2, 4, 8, and 16 hours, respectively). The animals were also acclimated to lycra/spandex animal jackets by increasing amount of time three days prior to the initiation of the study (2, 4, and 6 hours, respectively). The animal jackets were placed on the animals to prevent grooming of the dose site during the 6-hour exposure period.
- Diet (e.g. ad libitum): rationed feed
- Water (e.g. ad libitum): ad libitum
- Acclimation period: two weeks prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Following administration of test material, the animals were housed individually in metabolism cages that were designed for the collection of urine, feces, CO2, and organic volatiles. The metabolism cages contained hanging feeders and water bottles containing stainless steel sipper-tubes. Air was drawn through the metabolism cages at 30 L/minute. The cages were located in rooms designed to maintain relative humidity within a range of 54-57%, and temperature at 20 ± 1°C. A combination humidity/temperature monitor was mounted inside each cage with readings recorded daily. Cage humidity ranged from 60-66% and temperature at 20 ± 1°C. A 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m. Room air was exchanged approximately 12-15 times/hour.

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
single 6-hour dermal exposure. The rabbits received a single dermal dose of ~16-18 ml DPnP/cm² applied topically to approximately 10 x 15 cm area of skin.
Doses:
The dermal dose was applied as neat (undiluted) test material. Appropriate amounts of 14Clabeled and non-radiolabeled DPnP were mixed to obtain a dose of 219 mCi/1000 mg of DPnP. Radioactivity in the dose solutions was quantified by LSS. The amount of dose solution administered was ~ 1 ml/kg body weight. The radioactivity was ~219 μCi/kg body weight.

LSS analysis of aliquots of the 14C-labeled dosing solution taken from various locations in the solution container was used to confirm the concentration of radioactivity and the homogeneity of the 14C-DPnP in the dosing solution.

The dose solution was diluted to approximately 1 mg/ml in methanol and analyzed by GC/MS in the electron impact mode. The 14C-DPnP tracer was diluted about 60-fold with unlabeled material during the dose solution preparation; therefore, only the identity of the unlabeled material could be verified. The mass spectra obtained for the isomers were similar and consistent with proposed fragmentation patterns and library spectra (data not shown). Therefore, the unlabeled material used in the dose solution was verified to be Dowanol DPnP.
No. of animals per group:
A group of four female New Zealand White rabbits were used to determine the dermal absorption of 14C-DPnP.
Control animals:
no
Details on study design:
DOSE PREPARATION
- The dermal dose was applied as neat (undiluted) test material. Appropriate amounts of 14Clabeled and non-radiolabeled DPnP were mixed to obtain a dose of 219 mCi/1000 mg of DPnP. Radioactivity in the dose solutions was quantified by LSS. The amount of dose solution administered was ~ 1 ml/kg body weight. The radioactivity was ~219 μCi/kg body weight.

APPLICATION OF DOSE: A section (10 x 15 cm) on the back of each animal was clipped free of hair the day before the start of dosing. Undiluted test material was applied over the clipped area using an all glass syringe and blunt-tipped needle. The volume of test material applied was ~1 ml/kg
body weight.

An occlusive bandage of absorbent gauze and non-absorbent cotton was placed over the dosing area. The bandage was held in place for approximately six hours using lycra/spandex jackets. Following the six-hour occlusion period, the occlusive bandage and jacket were removed and the test site was washed off with cotton gauze dipped in an aqueous solution of detergent (i.e., ~4% Ivory® dish washing liquid, Proctor and Gamble
Co, Cincinnati, Ohio) to remove any residual 14C-DPnP. The application site was then rinsed with water-dampened gauze. The jacket, occlusive bandage, and the gauze used to wipe the application site were analyzed for radioactivity via LSS.

At 6-hours post-application, the lycra/spandex jacket and occlusive bandage were removed and analyzed for radioactivity. The skin at the application-site was washed with cotton gauze dipped in ~4% aqueous solution of detergent to remove any residual 14C-DPnP and analyzed for radioactivity.

At 96-hours post-application, the animals was anesthetized with CO2/O2 and sacrificed by inhalation of CO2. The skin at the application-site was excised, solubilized, and a weighed aliquot analyzed for radioactivity by LSS.

SAMPLE COLLECTION
- Collection of blood: yes (terminal)
- Collection of urine and faeces: yes (All urine and feces voided during the study was collected together in dry-ice cooled traps at
24-hour intervals. An aqueous homogenate (~ 25% w/w) was prepared. Weighed aliquots of the excreta were oxidized and quantitated for radioactivity by LSS. Cages were rinsed with water at 24-hour intervals after the excreta jars were changed and the rinse collected. Each cage rinse was weighed, and a weighed aliquot was analyzed for radioactivity via LSS.)
- Collection of expired air: yes (Expired Volatiles - Due to the volume of air that was pulled through the cages and the inherent capacity of
the traps, it was necessary to split the flow exiting the cages to accurately determine expired volatiles and CO2. Air was drawn through the each cage from two ports, at approximately 3 and 27 liters/minute, for a total of 30 L/min. Upon exiting the cage from the 3 liters/minute portal, the air was passed through charcoal to trap expired volatiles, while the 27 liter/minute flow was diverted to an approved laboratory hood. The charcoal traps were changed at 24-hour intervals. Radioactivity trapped on the charcoal was desorbed with weighed amounts of methanol. Weighed aliquots of the solvent were analyzed for radioactivity. Total exhaled volatiles were calculated multiplying the measured radioactivity from the 3 liters/minute flow by a factor of 10 to account for radioactivity lost through the 27 liters/minute portal. Expired CO2 - Following the respective charcoal trap (described above) the expired air was passed through CO2 trap containing 850 ml of monoethanolamine:1-methoxy-2-propanol (3:7 v/v) to trap expired CO2. The total weights of the traps were recorded and weighed aliquots from each trap were analyzed for radioactivity via LSS. The CO2 traps were changed at 12-, 24-, 48-, and 72-hours post-application. Total 14C-CO2 was calculated by multiplying the measured radioactivity from the 3 liters/minute flow by a factor of 10 to account for radioactivity lost through the 27 liters/minute portal.)
- Terminal procedure: At 96-hours post-application the animals were anaesthetized with CO2/O2 and euthanasia was accomplished via inhalation of carbon dioxide. Death was confirmed by severing a major artery. Following sacrifice, the cages were washed, the contents collected, and the
weight of the sample determined. A weighed aliquot of the Final Cage Wash (FCW) was analyzed for radioactivity.
- Analysis of organs: yes (application-site skin, skin, carcass, kidney and liver).

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting/spectrometer ((Packard Tri-Carb 2900TR, Packard Bioscience Company, Meriden, Connecticut).
- Liquid scintillation counting results (cpm) converted to dpm as follows: Counts per minute (cpm) were corrected for quench and background, and was converted to disintegrations per minute (dpm). The instrument was equipped with sealed 14C-standards that were counted every 23 hours during the operation to monitor the performance of the LSS. Samples with dpm less than twice the concurrently run background (blanks) were
considered to contain insufficient radioactivity to reliably quantify
Details on in vitro test system (if applicable):
not applicable

Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects
Absorption in different matrices:
- Skin test site: 1.17 ± 0.27
- Skin, untreated site: 0.31 ± 0.14
- Blood: 0.02 ± 0.01
- Kidney: 0.01 ± 0.00
- Liver: 0.10 ± 0.04
- Carcass: not tested
- Urine + Faeces: 11.73 ± 3.12
- Cage wash + cage wipe: 0.02 ± 0.01
- Occlusive bandage, jacket and skin wash: 69.65 ± 2.73
Total recovery:
- Total recovery: 88.89 ± 1.92
- Per cent Absorbed: 18.64 ± 4.45
Conversion factor human vs. animal skin:
not applicable

Any other information on results incl. tables

The 14C-DPnP applied dermally to the rabbits was neat and contained 219 μCi/g. An average of 2320 mg 14C-DPnP and 508 μCi were applied to 150 cm² on the backs of the rabbits.

With undiluted 14C-DPnP, a mean total of 88% of applied radioactivity was recovered. Animal #03A5753 slipped partially out of the jacket during the

exposure phase of the study, and was observed grooming the exposure area. The higher amount of radioactivity, as compared to the mean of the other animals, recovered in the feces/urine (20 vs. 12%), CO2 (5 vs. 3%) and expired volatiles (3 vs. 2%), and along with the lower amount recovered on the occlusive bandage, jacket, and skin wash (61 vs. 70%) are consistent with oral ingestion of the test material. Therefore, the data are presented for this animal but are not calculated in the means and standard deviations.

At the 96-hour post-application sacrifice time interval, the mean value for absorbed dose, represented by radioactivity detected in feces/urine, CO2, charcoal trap, tissues (including application-site skin), cage rinse, and FCW, was 18.64 ± 4.45% of the applied dose. The majority of the mean absorbed radioactivity as percentage of administered dose was eliminated via the combined feces/urine (11.73 ± 3.12%) (Table 3). Animals 03A5752 and 03A5754 excreted >97% of their feces/urine total in the first 24-hours post-exposure. In contrast, animal #03A5755 voided a relatively small amount of urine during the first 24-hr post-application (but the amount of feces was equivalent with the other animals) and eliminated only 0.41% of the administered dose. During 24-48 hours post-exposure, the amount of urine voided was consistent with the other animals and 12.45% of the

administered dose was detected in the feces/urine for this animal. This would suggest that even though the feces and urine were collected together as one sample, the urine is probably the major route of elimination of derived radioactivity as compared to the feces.

A mean value of 3.15 ± 0.86 and 1.99 ± 0.51% of the administered dose was detected in the CO2 and charcoal traps, respectively. The vast majority was eliminated in the first respective time points.

A mean value of 1.56 ± .47% of the administered dose was recovered in the tissues at sacrifice. The vast majority in the tissues was in the application-site skin and skin (minus application-site skin), 1.17 ± 0.27 and 0.31 ± 0.14% of the administered dose, respectively. The blood, kidney, and liver contained 0.13% of the administered dose. The occlusive bandage, jacket, and skin wash contained the largest amount of recovered administered dose at 69.65 ± 2.73. These radioactivities are considered to be non-absorbed.

Applicant's summary and conclusion

Conclusions:
The data indicate absorption of undiluted DPnP when applied dermally to rabbits. A mean of 19% of the administered radioactivity was absorbed and detected in the feces/urine, CO2, charcoal traps, tissues, cage rinse, and FCW. The majority (12%) of the absorbed administered dose was in the combined feces/urine sample, with evidence suggesting urine is the primary route of elimination of these two. The CO2 and charcoal traps recovered 5% of the administered dose. Derived radioactivity from absorbed 14C-DPNP was quickly eliminated.
Executive summary:

The purpose of this study was to provide data on the dermal absorption of 14C-dipropylene glycol n-propyl ether (DPnP) in New Zealand White rabbits following a single 6-hour dermal exposure.

Approximately 2.4 g of the test material was topically applied to an ~150 cm2 area of skin using a glass syringe with a blunt-tipped needle. The application site was covered with an occlusive bandage of absorbent gauze and non-absorbent cotton. The bandage was held in place for approximately six hours using a lycra/spandex jacket. An average of 2320 mg (508 μCi) of DPnP was applied to three rabbits. The application site was washed with ~4% aqueous detergent solution after 6 hours of exposure. The rabbits were sacrificed at 96-hours post-application.

A total of 86-90% of the administered radioactivity applied was recovered. The majority of the applied radioactivity, 61-72%, was recovered in the 6-hour application-site wash material, jacket, and occlusive bandage. A mean of 19% of the administered radioactivity was absorbed and detected in the feces/urine, CO2, charcoal traps, tissues, cage wash, and final cage wash. The majority (12%) of the absorbed administered dose was in the combined feces/urine sample, with evidence suggesting urine is the primary route of elimination. A total of 5% of the administered dose was recovered in the CO2 and charcoal traps. Derived radioactivity from absorbed 14C-DPnP was quickly eliminated, with the majority recovered within 24 hours of application.

The data generated in this study indicate absorption of undiluted 14C-DPnP when applied dermally to rabbits.