N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-01-20 to 1989-08-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

EC Number:

221-220-5

EC Name:

N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

Cas Number:

3033-62-3

Molecular formula:

C8H20N2O

IUPAC Name:

{2-[2-(dimethylamino)ethoxy]ethyl}dimethylamine

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)
- Molecular formula: (CH3)2NCH2CH20CH2CH2N(CH3)2
- Molecular weight: 160.26
- Substance type: Pure active substance
- Physical state: Pale yellow liquid
- Analytical purity: 49-401=97.8%; 51-487=97.5%; supplied by Sponsor.
- Radiochemical purity (if radiolabelling): 98%
- Specific activity (if radiolabelling): 1-5 mCi/mmol
- Locations of the label (if radiolabelling): uniformly labeled
- Stability under test conditions: Stable.
- Storage condition of test material: No special requirements, room temperature.
- Other: Specific Gravity: 0.8496 (20/20 deg C); Solubility: Water - complete (25 deg C); Vapor Pressure: 0.28 mmHg (20 deg C); Vapor Density: 5.5 (relative to air density = 1); Boiling Point: 189.2 deg C (760 mmHg); Flash Point: 17S deg F (open cup); Odor: Light amine smell.
- Lot/batch No.: BRRC Numbers: 49-401 and 51-487; BRRC Registry No. 49-468; unlabelled (BRRC Registry No. 49-401; No.51-487 was used in the additional rat experiments) forms for this study.

Radiolabelling:

yes

Remarks:

14-Carbon N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

Test animals

Species:

rat

Strain:

other: CDF® Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. (Indianapolis, IN)
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 215-275 g
- Fasting period before study: No, only during exposure.
- Housing: Prior to use in the study, rats were housed in plastic shoe box cages in groups of 3-4. Animals selected for the material balance study were transferred to individual Roth-type metabolism cages.
- Individual metabolism cages: Yes, standard Roth-type metabolism cage
- Diet (e.g. ad libitum): Agway Certified Rodent Chow, St. Marys, OH, ad libitum
- Water (e.g. ad libitum): Municipal drinking water (Municipal Authority of Westmoreland County, Greensburg, PA), ad libitum.
- Acclimation period: The animals were acclimated to the laboratory environment for at least 7 days prior to dosing; the animals were acclimated to the experimental environment for two days prior to test substance administration.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 deg C
- Humidity (%):40-70%
- Air changes (per hr): Air change ca. every 5 minutes
- Photoperiod (hrs dark / hrs light): 12-hour photoperiod

IN-LIFE DATES: From: 1987-02-04 To: 1987-02-06 (as documented in Protocol Amendment 2 #17 "Fourty-eight hours administration of the dose, the animals will be anesthetized with methoxyflurane."

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: Millipore water

Duration of exposure:

48 hours

Doses:

Nominal doses: 200.0 mg/kg nominal dose level for the main and additional percutaneous studies. [Target radioactivity was 10-15 microCuries/0.2 ml of the tracer to a 200 g rat]

Actual doses: 184.0, 184.0, 200.0, 198.0, 205.2 mg/kg actual dose level for the main and additional percutaneous studies for males and females.
Actual doses reported as follows:
- Percutaneous Males: 184.0 mg/kg actual dose level with 73.6 +/- 0.24 uCi/kg radioactive dose level
- Percutaneous Female Group I: 184.0 mg/kg actual dose level with 71.275 +/- 1.59 uCi/kg radioactive dose level
- Percutaneous Female Group II: 200.0 mg/kg actual dose level with 63.275 +/- 0.88 uCi/kg radioactive dose level
- Percutaneous Male Repeat Study: 198.0 mg/kg actual dose level with 169.8 +/- 9.7 uCi/kg radioactive dose level
- Percutaneous Female Repeat Study: 205.2 mg/kg actual dose level with 318.0 +/- 15.7 uCi/kg radioactive dose level

Dose volume: Target volume of 1.0 ml/kg, or 0.5 ml/kg (in the additional rat experiments)

Rationale for dose selection: The dose selected for the dermal administration was determined from kinetic information obtained via the intravenous LD50 preliminary study.

No. of animals per group:

8 rats per dose level were used.

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: Dosing solution was prepared as a water solution. Dosing solutions were analyzed for the test substnce concentration by capillary GC with a flame ionization detector after dosing.
- Method of storage: N/A

APPLICATION OF DOSE: The dose solution will be applied to the intrascapular region of the back in a 6 cm2 area in male rats aplied with a syringe.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Millipore water was used in the particular building in which the experiment took place in lieu of distilled water, this decisions was not based on specific properties of the test substance.
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): 200 mg/mL in H2O (400 mg/mL in H2O for repeat study)
- Lot/batch no. (if required): N/A
- Purity: N/A

TEST SITE
- Preparation of test site: The exposure site was lightly clipped using electric clippers while under anesthesia for the jugular cannula.
- Area of exposure: 4 (female) to 6 (male) cm2 area in the interscapular region of the back of rats
- % coverage: N/A
- Type of cover / wrap if used: (approx. 70 x 100 cm) Polyethylene film and held in place with adhesive tape and a fabric jacket to minimize oral ingestion
- Time intervals for shavings or clipplings: Only once prior to the exposure period.

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (type of restrainment not described in study report).

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: No
- Washing procedures and type of cleansing agent: no, the test substance was left in place for 24 hours.
- Time after start of exposure: N/A

SAMPLE COLLECTION
- Collection of blood: Blood samples (approx. 0.1 ml) were collected via the indwelling jugular cannulae of rats at 0 (pre-administration), 0.5, 1, 2, 4, 6, 9, 12, 18, 24, 36 and 48 hours post-dosing. In the additional rat percutaneous pharmacokinetic experiments, the following modifications were made: blood collection times were 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 30, 36 and 48 hr.
- Collection of urine and faeces: Urine and feces were collected in 12-hr intervals. In the additional rat percutaneous pharmacokinetic experiments, the following modifications were made: urine samples were collected at 6, 12, 24, 36 and 48 hr, feces were collected at 24 and 48 hr.
- Collection of expired air: In the additional rat percutaneous pharmacokinetic experiments, the following modifications were made: CO2 trapping
solutions and volatile traps were collected at 24 and 48 hr.
- Analysis of organs: Following removal of the pelts the liver, kidney, bone marrow, brain, fat (perirenal), heart, lung, muscle, spleen and tested ovaries were isolated from the carcass.

SAMPLE PREPARATION
- Storage procedure: Blood was collected in heparinized capillary tubes. Urine and feces were collected under dry ice conditions and the urine samples were frozen at approximately -80 deg C until the preliminary metabolite analysis was conducted.
- Preparation details:
- Blood: The blood collected was placed in heparinized capillary tubes and plasma prepared. An aliquot of the plasma was added to 10 ml of Aquasol-2(R) counting scintillant and counted by liquid scintillation spectrometry. Based on radioactivity levels observed, selected samples of the remaining plasma were then analyzed for metabolites and parent chemical.
- Expired air: Forty-eight hours after administration of dose, the animals were anesthetized with methoxyflurane (rats). Skin sections from each animal were removed in the following manner. The dosed area of skin, as well as the skin around the periphery of this site, were separated from each animal using blunt dissection techniques. The pelts were removed from the carcass prior to removal of organs. The dose site and peripheral skin were weighed and skin segments from two remote areas (ventral surface and sacral regions) were taken in order to examine the possibility of both radial diffusion of the test substance from the dose site and systemic distribution of test substance to the skin. The skin from both the dose site and directly surrounding it (approximately 1 cm from edge of dose site) were pulverized at liquid nitrogen temperatures in a Spex Freezer Mill and an aliquot from each sample was counted directly in a suspension of 10 ml Aquasol-2QD and 6 ml water. In the additional rat percutaneous pharmacokinetic experiments, the following modifications were made: the pelt was not removed before homogenization of the carcass.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting. Radioactivity was determined by tissue oxidation of isolated organs, and tissues, aliquota of homogenized carcass and feces (33% w/v in distilled water), and red blood cells from all collection intervals. Radioactivity recovered from the dose site and from the occlusive bandage was also quantified. Tissue oxidation was conducted in a Biological Materials Oxidizer and 14CO2 derived from combustion of these tissues was quantified by liquid scintillation spectrometry.
- Liquid scintillation counting results (cpm) converted to dpm as follows: N/A
- Validation of analytical procedure: N/A
- Limits of detection and quantification: N/A

OTHER: The pharmacokinetic description of the fate of 14C-test substance evaluated using RSTRIP to derive optimum parameter estimates for the plasma data. Mean plasma 14C concentrations (microgram Equiv/g) will be used to fit a two compartment open pharmacokinetic model in which elimination and transfer of l4C-test substance between compartments will be assumed to be first-order processes. The parameters which will be estimated include: the rate constant of absorption, ka; the rate constant of elimination, ke; and the half-lives (t1/2) of absorption and elimination.

Details on in vitro test system (if applicable):

N/A

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

Preliminary studies:
- Expired air (if applicable): 0.072% of the cutaneous dose was recovered as CO2. Recovery of 14C-labeled test substance from charcoal traps was 0.044% of the total dose administered cutaneously.

Main study- percent recovered dose (Mean +/- S.D.):
- Occlusion device + rinse + skin: Relatively large amounts of the dose were associated with the dosing site and occlusion devices in both males and females, 14.504 +/- 2.825% in males and 25.688 +/- 7.560% in females
- Skin wash: 0.392 +/- 0.106% in males and 0.192 +/- 0.131% in females
- Skin test site: 2.534 +/- 0.440 in males and 4.558 +/- 2.200% in females
- Blood: 0.034 +/- 0.015% in males and 0.025 +/- 0.010% in females
- Carcass: 1.916 +/- 0.188% in males and 2.163 +/- 0.327% in females
- Urine: 24% of the dose the recovered dose in male and female rats
- Faeces: 1.5% and 0.9% in 0-24 hr in male and female rats, respectively; 0.2% and 0.06% in 24-24 hr in male and female rats, respectively; totaling 1.752 +/- 2.511% in males and 1.006 +/- 0.900% in female rats, respectively.
- Other: Large fractions of the dose (51.8% in males and 39.3% in females) could not be accounted for in the test system.

Additional study- percent recovered dose (Mean +/- S.D.):
- Occlusion device + rinse + skin: 10.011 +/- 1.825 % in males and 12.705 +/- 0.153 % in females
- Skin wash: 0.281 +/- 0.106 % in males and 0.176 +/- 0.065 % in females
- Skin test site: 2.881 +/- 0.572 % in males and 3.127 +/- 0.288 % in females
- Blood: 0.076 +/- 0.045 % in males and 0.250 +/- 0.294 % in females
- Carcass: 11.438 +/- 1.560 % in males and 10.869 +/- 2.211% in females
- Urine: 52.189 +/- 3.407 % in males and 46.641 +/- 2.518 % in females
- Faeces: 0.506 +/- 0.342 % in males and 1.933 +/- 0.698% in females

Total recovery:

Preliminary studies:
- Total recovery: N/A
- Recovery of applied dose acceptable: N/A
- Results adjusted for incomplete recovery of the applied dose: N/A
- Limit of detection (LOD): N/A
- Quantification of values below LOD or LOQ: N/A

Main study:
- Total recovery: 48.142 +/- 5.003% in male rats and 60.749 +/- 12.042% in female rats of total recovered dose
- Recovery of applied dose acceptable: N/A
- Results adjusted for incomplete recovery of the applied dose: N/A
- Limit of detection (LOD): N/A
- Quantification of values below LOD or LOQ: N/A

Additional study:
- Total recovery: 79.435 +/- 2.769% in male rats and 75.571 +/- 0.432% in female rats of total recovered dose
- Recovery of applied dose acceptable: N/A
- Results adjusted for incomplete recovery of the applied dose: N/A
- Limit of detection (LOD): N/A
- Quantification of values below LOD or LOQ: N/A

Conversion factor human vs. animal skin:

N/A

Any other information on results incl. tables

MAIN STUDY:

Absorption rate constant= 0.02621 min^-1 for males and 0.008504 min^-1 for females

Half-life of absorption= (t1/2)= 26.45 min for males and 81.51 min for females

ADDITIONAL STUDY:

Absorption rate constant= 6.393 hr^-1 for males and 4.945 hr^-1 for females

Half-life of absorption= (t1/2)= 0.1084 hr for males and 0.1402 hr for females

Bioavailability= 82.55 % for males and 93.60% for females

Applicant's summary and conclusion

Conclusions:

The 48 hour dermal exposure to the test substance labeled with Carbon-14 resulted in rapid absorption and distribution and slow elimintaion primarily through the urine of unchanged test substance and no significant accumulation in the tissues although radioactivity was found in the liver and kidneys of rats at the time of sacrifice. Cutaneous bioavailability of the test substance was calculated as 78.2% in male rats and 50.8% in female rats.