Registration Dossier

Administrative data

Endpoint:
dermal absorption in vivo
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study, tested with the source substance Bioban™ CS-1246 biocide.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Bioban CS-1246
IUPAC Name:
Bioban CS-1246
Constituent 2
Reference substance name:
7a-ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole
EC Number:
231-810-4
EC Name:
7a-ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole
Cas Number:
7747-35-5
IUPAC Name:
7a-ethyldihydro-1H-[1,3]oxazolo[3,4-c][1,3]oxazole
Details on test material:
- Name of test material (as cited in study report): Bioban™ CS-1246 (7a-Ethyldihydro-1H,3H,5H-oxazolo(3,4-c)oxazole)
- Molecular formula (if other than submission substance): C7H13NO2
- Molecular weight (if other than submission substance): 143.09
- Physical state: liquid (color: pale yellow)
- Analytical purity: 98.55%
- Lot No.: non-radiolabeled: TC1031LAH3
- Radiochemical purity (if radiolabelling): >98%
- Specific activity (if radiolabelling): radiolabeled 20.92 mCi/mmol
- Locations of the label (if radiolabelling): 7a-Ethyldihydro-1,7-dihydro-[1,7-14C]-oxazolo(3,4-c)oxazole
- Other: Synonyms: 1-Aza-3,7, dioxa-5-ethylbicyclo (3.3.0) octane, 7-Ethyl bicyclooxazolidine (IUPAC name), 5-Ethyl-1-aza-3,7, dioxabicyclo[3.3.0]octane
Supplier: non-radiolabeled: The Dow Chemical Company, Bedford Park, Illinois, USA;
Supplier: radiolabeled: Scynexis Europe Limited, Essex, England (Registry # DA2459)

Radiolabelling:
yes
Remarks:
7a-Ethyldihydro-1,7-dihydro-[1,7-14C]-oxazolo(3,4-c)oxazole

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: jugular cannulated rats: Taconic (Germantown, New York, USA); non-cannulated rats: Charles River Laboratories Inc. (Portage, Michigan, USA)
- Age at study initiation: 9-10 weeks
- Fasting period before study: ~ 16 h
- Housing: animals were housed singly in glass Roth-type metabolism cages, which were designed for the separation and collection of urine, faeces, CO2, and organic volatiles.
- Individual metabolism cages: yes
- Diet: Lab Diet Certified Rodent Diet #5002 (PMI Nutrition Internation, St. Louis, Missouri, USA) in pelleted form; ad libitum, except that access to feed was restricted approximately 16 h prior to the administration of radiolabeled test material with the exception of animals in group 6 (dermal group), which were not fasted prior to dosing; analyses of the feed are performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the level of selected contaminants
- Water: municipal water; ad libitum; periodically analysed for chemical parameters and biological contaminants by the municipal water department
- Acclimation period: non-cannulated animals: at least one week in stainless steel cages (including at least two days in metabolism cages); jugular vein cannulated animals (surgery performed by the supplier): for four days in metabolism cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.2 - 24.7
- Humidity (%): 38 - 78
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12/12 (on at 6:00 a.m. and off at 6:00 p.m.)

Rats in which the plasma/blood 14C time-course was determined were obtained already cannulated in the jugular vein by the supplier.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
polyethylene glycol
Remarks:
PEG-400
Duration of exposure:
6 hours
Doses:
- Dose volume: 10 µL/cm² to 12 cm² (120 µl of dose solution)
- The dermal dose solution was applied at the target radioactivity of ~ 62.5 µCi per rat (~ 250 µCi/kg).
- Rationale for dose selection: The dose of 5 mg/kg was lower than the NOEL in a 90-day repeated dose toxicity study (Stebbins et al. (2007) found the NOEL to be 10 mg/kg/day).
No. of animals per group:
4
Control animals:
yes
Details on study design:
DOSE PREPARATION
- Method of storage: Liquid scintillation spectrometer (LSS) analysis of aliquots of the dosing solution taken from various locations in the solution containers was used to confirm homogeneity of the dosing solution.
The stability of the 5 mg/kg dose solution of BIOBAN™ CS-1246 in PEG-400 was confirmed to be stable for up to 16 days (Markham and Beuthin, 2008).

APPLICATION OF DOSE:
Animals were anesthetised with isoflurane for dosing. A measured dose of 14C-BIOBAN™ CS-1246 was applied topically using a round tipped feeding needle attached to an all glass syringe to an approximately 12 cm² area. The dose solution was applied evenly to the skin a in volume of 10 µL/cm². The syringe and feeding needle used for application of the test materials was weighed before and after the dosing to determine the actual dose applied. Protective appliance: Saddle shaped ~ 1.5 mm thick Teflon frame (4cmx5cm with a 3cmx4cm cut-off opening) was positioned intrascapularly and as far anterioly as possible and attached to each animal using Permabond Industrial Grade 910 adhesive. In addition, each rat was fitted with a rodent jacket to prevent the animals from grooming the dose site.

VEHICLE
- Lot no. (if required): 036K0046 (Sigma-Aldrich Corporation, St. Louis, Missouri, USA)

TEST SITE
- Preparation of test site: Animals were anesthetised with isoflurane and the hair on the back of each rat was clipped approximately 16 h prior to dosing.
- Area of exposure: 12 cm²

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: Test animals access to the dose site was restricted by use of the rodent jacket and dosed area was semi-occluded by covering with Teflon Spectra/Mesh macroporous filter material, which was glued to the Teflon frame.

REMOVAL OF TEST SUBSTANCE
At 6 h post-dosing, the macroporous filter material covering was removed and the skin at the dosed site was washed. The site was washed five times with cotton tip applicators dipped in an aqueous solution of detergent (i.e. ~2-4% Ivory dish washing liquid) rinsed several times with gauze soaked with water and the area blotted dry with gauze squares.

SAMPLE COLLECTION/SAMPLE PREPARATION
- Collection of blood: Following washing, animals had the Teflon Spectra/Mesh covering replaced and placed in the Roth-type metabolism cages for collection of time-course blood. Blood was obtained at sacrifice via cardiac puncture and/or inferior vena cava and/or dosal aorta. The blood was centrifuged to separate plasma and plasma analysed for radioactivity.
- Collection of urine and faeces: immediately following dosing, the animals were placed in the Roth-type metabolism cages for the separation and collection of urine and faeces; following washing, animals had the Teflon Spectra/Mesh covering replaced and returned to their cages for the continuous collection of urine, faeces
All urine voided during the study was collected in dry-ice cooled traps. The urine traps were changed at 12-, 24-, and 48-h post-dosing followed by 24-h intervals for the remainder of the study. Equal volume aliquots of urine samples (per time, dose and sex) from the 0-12 h and 12-24 h collection intervals was pooled and stored at -80 °C.
Faeces were collected in dry-ice chilled containers at 24 h intervals. Faeces were collected 6 h post-dosing for the group 4. Equal volume aliquots of faecal homogenates from each animal were taken from the 0-24 h collection interval and pooled (per dose and sex) and stored at -80 °C. Inadvertently, group 1 samples were stored at -20°C
- Collection of expired air: expired volatiles: charcoal traps were used (The charcoal traps were changed at 24 h intervals.); expired CO2: following the charcoal trap, the expired air was passed through a solution of monoethanolamine: 1-methoxy-2-propanol (3:7, v/v) to trap expired CO2; the CO2 trap was changed only at the 12 h and 24 h intervals.
- Terminal procedure: 168 h post-dosing, the animals were sacrificed; the Teflon frame was removed and the components analysed for radioactivity. The skin at the dosed site was washed again and tape stripped to remove stratum corneum, which was then excised, solubilised and analysed. Following the terminal sacrifice, a final cage wash was performedl
- Analysis of organs: Tissues were collection at terminal sacrifice: adrenal, carcass (residual), kidney, skin, testis, blood (RBC and plasma), fat (perirenal), liver, spleen, thyroid, brain, GI tract (with contents), ovary, stomach (without contents), uterus
- The application-site skin was analysed separately from the remote skin.

ANALYSIS
- Method type(s) for identification: Liquid scintillation spectrometer; Metabolites were identified via HPLC with in-line radiochemical detection
- Liquid scintillation counting results (cpm) converted to dpm as follows: Counts per minutes (cpm) were corrected for quench and converted to disintegrations per minute (dpm).

OTHER:
Metabolites: pooled samples (urine and faeces) from selected intervals were analysed in duplicate via HPLC with in-line radiochemical detection or singly if fraction collection was required.

Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects

Any other information on results incl. tables

Absorption was estimated from the percent of the dermally applied radioactivity recovered in urine and rinse, final cage wash, CO2 and tissues, and faeces. A mean total of 27 ± 3% and 25 ± 5% of the dermally applied dose was systemically absorbed to males and females, respectively. The unabsorbed radioactivity of 43 ± 6% (males) and 41 ± 4% (females) was recovered from the skin wash, covering, tape strips, frame and dosed skin.

Urinary elimination of the dermally applied dose:

Over the 168 h collection period, 19% and 15% of the administered dose was recovered in urine of males and females, respectively. Slow and low urinary elimination was consistent with pattern of slow penetration of the applied dose from the dermal site of application and limited absorption of the applied dose, as expected due to cornified layer of cells on the skin surface that work as a barrier.

 

Faecal elimination of the dermally applied dose:

The 14C- BIOBAN™ CS-1246 -derived radioactivity recovered in faeces was only 4%.

 

As expected on the basis of low absorption via the dermal route of exposure, a lower percentage of administered radioactivity was associated with tissues of animals receiving a single dermal dose of 14C- BIOBAN™ CS-1246. On average, only 3% of the dermally administered radioactivity remained in tissues at terminal sacrifice (168 h post-dosing). Most (2.6 -2.7%) was recovered in the skin remote from the application site and 0.8 -1.7% was recovered from skin at the site of application.

For males, the highest concentrations of radioactivity was found in skin > liver > thyroid ≥ kidney.

For females, the highest concentrations of radioactivity was found in skin > carcass > kidney = stomach tissue.

The difference in the residual radioactivity between the dose-site skin and skin at remote from the dosed site was only 6 -fold (males) and 3 -fold (females). Such a low difference in the residual radioactivity between the dosed site skin and skin at other sites of the body was an indication that complete absorption has been achieved and any additional penetration of radioactivity from the application site was not likely to occur.

Fat and fatty tissue (e.g. brain) generally exhibited the lowest accumulation of 14C-14C-BIOBAN™ CS-1246 -derived radiaoactivity (mostly under the detection limit).

The absorbed 14C-BIOBAN™ CS-1246 was completely metabolised, affording 2-amino-2-ethyl-1,3-propanediol (AEPD) as the only metabolite above 5% of the administered dose in all urine and faecal samples analysed from all dose groups. Four minor metabolites were also observed above 0.5% of the administered dose, but not identified.

Kinetic parameters of 14C- BIOBAN™ CS-1246 in RBC and in plasma

The peak plasma concentration of  14C- BIOBAN™ CS-1246 among the dermally dosed rats occurred 2 -3 h post-application. The rate of dermal absorption was 0.14 -0.2 h-1 and elimination of absorbed radioactivity was 0.5 -1.5 h. The clearance was relatively high 6754 -8453 mL/kg/h after dermal dosing consistent with a low plasma AUC 0.7 -0.9 µg*h/g and a relatively rapid elimination of absorbed radioactivity.

The test substance derived radioactivity was also present in RBC, with Cmax concentrations ~90% of the levels seen in plasma. The rate constant of absorption of radioactivity into RBC was faster than into the plasma (0.03 -0.09 h-1) and the elimination was slower. This could be an indication of binding of radioactivity to RBC, albeit at extremly low levels (~0.01 -0.02 µg/g). The elimination of radioactivity from RBC followed a relatively rapid (α) elimination phase and a slow elimination (β) phase. The appearance of detectable radioactivity in RBC for extended period of time resulted in 8 -11 fold higher AUC and 8 -12 fold slower clearance of radioactivity from RBC than plasma.

Table 1: Kinetic parameters of 14C- BIOBAN™ CS-1246 in RBC and in plasma (mean ± SD of 4 animals)

 

RBC

Plasma

Parameter

Mean ± SD (males)

Mean ± SD (females)

Mean ± SD (males)

Mean ± SD (females)

t(max) [h]

3.99 ± 0.43

3.24 ± 0.54

3.00 ± 0.54

1.71 ± 0.55

Cmax [µg/g]

0.06 ± 0.02

0.083 ± 0.00

0.07 ± 0.02

0.09 ± 0.01

K(absorption) [1/h]

0.03 ± 0.01

0.09 ± 0.04

0.21 ± 0.07

0.14 ± 0.02

elimination [h]

-

-

1.53 ± 0.76

0.47 ± 0.20

α [h]

rapid elimination

3.39 ± 1.24

4.42 ± 1.53

-

-

t½β [h]

slow elimination

188 ± 46

109 ± 44

-

-

AUC [µg*h/g]

7.49 ± 1.83

6.84 ± 0.94

0.67 ± 0.24

0.86 ± 0.06

Cl [mL/kg/h]

723 ± 169

876 ± 154

8453 ± 2448

6754 ± 496

 

 

Applicant's summary and conclusion