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Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 April 1990 to 26 June 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1991

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were dosed topically with the radiolabelled test material, formulated in diethyl phthalate at 3 concentrations; 50, 5 and 0.5%. These concentrations are equivalent to dose levels of approximately 1000, 100 and 10 mg/kg. Doses were applied to the skin on gauze squares which were held in place by semi-occlusive dressing. The gauze squares and dressings were removed after 6 hours and the dose area washed with diethyl phthalate. Urine, faeces and expired air were collected for 72 hours after dosing and at termination selected tissues and the remaining carcasses were retained for analysis of total radioactivity.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-phenoxyethyl isobutyrate
EC Number:
203-127-1
EC Name:
2-phenoxyethyl isobutyrate
Cas Number:
103-60-6
Molecular formula:
C12H16O3
IUPAC Name:
2-phenoxyethyl 2-methylpropanoate
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): [14C]-2-phenoxyethyl isobutyrate and non labelled 2-phenoxyethyl isobutyrate.
Radiolabelling:
yes
Remarks:
2-[ring U 14C]-Phenoxyethyl Isobutyrate

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 9 weeks
- Weight at study initiation: 288-306 g
- Housing: individually in all-glass metabolism cages.
- Diet: SQC Rat and Mouse Maintenace Diet No. 1 ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 2°C
- Photoperiod: 12 hours dark/ 12 hours light.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: diethyl phthalate
Duration of exposure:
6 hours
Doses:
- Nominal doses: 50, 5 and 5% concentrations, equivalent to approximately 1000, 100 and 10 mg/kg respectively.
- Actual doses: Ranged from 993.58 to 1000.43 mg/kg, 98.86 to 102.95 mg/kg and from 12.08 to 12.16 mg/kg.
- Administered doses were calculated as follows: Administered doses were calculated by measuring the amount of radioactivity associated with 3 'mock doses'.
- Dose volume: 2mL.
No. of animals per group:
Four per group.
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: Appropriate amounts of radiolabelled and non labelled test material were combined in solution in ethyl acetate in a 5 mL volumetric flask. The ethyl acetate was removed under a stream of nitrogen and the volume made up to 5 mL with diethyl phthalate. Six aliquots from each dosing solution were submitted for LSC to verify the radioactive content and homogeneity of each dose solution.
> High Dose, 1000 mg/kg: 4.45 mg of radiolabelled test material and 2503.26 mg of non-labelled test material.
> Medium Dose, 100 mg/kg: 4.55 mg of radiolabelled test material and 248.91 mg of non-labelled test material.
> Low Dose, 10 mg/kg: 4.56 mg of radiolabelled test material and 25.72 mg of non-labelled test material.

TEST SITE
- Preparation of test site: The backs of the animals were shaved with animal clippers 24 hours prior to dose application. The dose solution was applied to square gauze with a silver foil back.
- Area of exposure: Approximately 4 cm x 4 cm.
- Type of cover / wrap if used: 50 mm micropore dressing held in place by non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: The dose area was swabbed with a cotton wool swab soaked in diethyl phthalate. A further cotton wool swab was used to remove excess solvent from the skin. The dressings, swabs and gloves worn were retained for analysis. Animals were then returned to the metabolism cages.
- Time after start of exposure: 6 hours.

SAMPLE COLLECTION
- Collection of blood: Blood was collected from the vena cava at termination, 72 hours. Plasma was separated from blood cells by centrifugation.
- Collection of urine: 0-6 h, 6-24 h, 24-48 h and 48-72 h.
- Collection of faeces: 0-24 h, 24-48 h and 48-72 h.
- Collection of expired air: 0-24 h, 24-48 h and 48-72 h.
- Cage wash: Collected at 24 hours and then at sacrifice, cages were rinsed with water containing detergent.
- Terminal procedure: Animals were sacrificed 72 hours post dosing by CO2 narcosis.
- Analysis of organs: Liver, kidneys, gastrointestinal tract, treated skin, non-treated skin and remaining carcass.

TOTAL REACTIVITY MEASUREMENTS
Total radioactivity was measured in all samples of urine, faeces, expired air, cage wash, skin swabs, gloves, skin dressings, plasma, liver, kidney, gastrointestinal tract, treated skin, non-treated skin and remaining carcass. All samples were analysed for 5 minutes by LSC; all biological samples were processed in duplicate wherever possible. Samples for combustion were weighed and combusted using an automatic sample oxidiser. The resultant 14CO2 was absorbed and mixed automatically with scintillation fluid.

Representative blank sample values were subtracted from sample count rates to give net dpm per sample. A limit of reliable determination of 30 dpm above the background was used.

SAMPLE PREPARATION FOR ANALYSIS:
- Clear liquid samples: Samples of urine, plasma, cage wash, dose solution etc. were made up to 1.0 mL with distilled water or compatible solvent where necessary and mixed with 10 mL of scintillator
- Skin dressings, skin swabs and gloves: Samples were soaked in known amounts of methanol for 30-60 min, and after vigorous shaking, 1 mL duplicates mixed with 10 mL of scintillator.
- Faeces: Faeces were homogenised in water and sodium carboxymethylcellulose added to stabilise the homogenates. Representative samples were taken for combustion.
- Tissues: Tissues were finely chopped and representative samples taken for combustion.
- Carcasses: Carcasses were finely minced and homogenised with water. Representative samples were taken for combustion.
- Skin: Skin samples were first soaked in a known volume of methanol overnight to remove unbound dose material and 1 mL duplicates mixed with 10 mL of scintillator. The methanol was the poured off and the skin samples solubilised with 10% sodium hydroxide/methanol/triton X 100 (80:10:10, v/v/v). The skin samples were combined with reagent in a ratio of 1:4 or 1:5 and placed in a shaking water bath (60 °C) overnight. Weighed duplicate samples were made up to 4 mL with water and mixed with 10mL of scintillator to form a thixotropic gel suitable for scintillating counting.

Results and discussion

Signs and symptoms of toxicity:
not specified
Dermal irritation:
not specified
Absorption in different matrices:
Radioactivity was quite rapidly absorbed and eliminated almost exclusively in urine, regardless of the dose concentration. The proportion of dose absorbed over the 72 hour period was similar for all 3 dose levels although there were indications that absorption was slightly more variable at the lowest dose level.

While removal of the patches and washing of the skin at 6 h post dose removed a large proportion of the dose (65-72%), a considerable amount remained associated with the skin, acting as a reservoir from which radioactivity continued to be absorbed. Of the radioactivity still associated with the skin at 72 h post dose 20-35% was bound to the skin. The small amounts of radioactivity associated with non-dosed skin can most likely be attributed to contamination of the dose area.

Less than 1% of the dose was excreted in faeces over the 72 h period. This indicates some biliary elimination, if all absorption was via the skin. However, it must be noted that all of the animals were observed grooming themselves immediately after removal of the test material, and it is therefore possible that some oral ingestion of the dose material remaining on the skin occurred. So, it is equally possible that the radioactivity observed in faeces was the result of oral ingestion of the dose.

Concentrations of radioactivity associated with the plasma and tissues investigated at 72 h post dose decreased with dose level in a linear manner. Highest concentrations, at all dose levels, were observed in the kidney and concentrations in all tissues were generally higher than those circulating in plasma. No appreciable amounts of radioactivity were observed in expired air at any dose level.

Elimination patterns for each dose level can be seen in Tables 1 to 3.
Total recovery:
- Total recovery: Recovery at dose concentrations of 50, 5 and 0.5% (1000, 100 and 10 mg/kg) were 96.22, 95.62 and 92.23% respectively.
Percutaneous absorptionopen allclose all
Dose:
1000 mg/kg
Parameter:
percentage
Absorption:
ca. 21.5 %
Remarks on result:
other: 72 h
Dose:
100 mg/kg
Parameter:
percentage
Absorption:
ca. 20.4 %
Remarks on result:
other: 72 h
Dose:
10 mg/kg
Parameter:
percentage
Absorption:
ca. 19.5 %
Remarks on result:
other: 72 h

Any other information on results incl. tables

Table 1: Recovery of Total Radioactivity - 1000 mg/kg (results expressed as % administered dose recovered)

Sample Time (h) Animal
1 2 3 4 Mean
Urine
0-6 h 2.53 2.32 3.20 3.13 2.80
0-24 h 12.36 8.78 10.15 11.33 10.66
0-48 h 18.64 14.05 16.65 17.27 16.65
0-72 h 20.76 16.13 20.58 20.49 19.49
Faeces
0-24 h 0.41 0.08 0.06 0.09 0.16
0-48 h 0.54 0.14 0.15 0.16 0.25
0-72 h 2.72 0.16 0.19 0.28 0.84
Cage wash 1.37 0.31 1.35 0.61 0.91
Expired air** 0.00 0.00 0.02 0.00 0.01
Treated skin (methanol wash) 1.15 0.51 1.57 1.32 1.14
Treated skin (residue) 0.60 0.31 0.70 0.64 0.58
Non-treated skin (methanol wash) 0.27 0.18 0.23 0.15 0.21
Non-treated skin (residue) 0.09 0.06 0.07 0.05 0.07
Kidney (72 h) 0.00 0.00 0.00 0.00 0.00
Liver (72 h) 0.01 0.01 0.01 0.01 0.01
GI tract (72 h) 0.03 0.02 0.04 0.03 0.03
Carcass (72 h) 0.86 0.72 0.60 0.69 0.72
Dressing (6 h) 51.13 66.80 59.64 59.77 59.34
Skin wash (6 h) 11.19 10.11 9.81 9.33 10.11
Gloves (6 h) 1.30 1.07 3.21 5.46 2.76
TOTAL 91.48 96.39 98.02 98.83 96.22

** Results derived from data < 10 dpm above background.

Table 2: Recovery of Total Radioactivity - 100 mg/kg (results expressed as % administered dose recovered)

Sample Time (h) Animal
1 2 3 4 Mean
Urine          
0-6 h 1.88 1.82 1.56 3.10 2.09
0-24 h 7.96 9.86 10.79 8.04 9.16
0-48 h 13.24 17.27 17.96 12.92 15.35
0-72 h 16.89 21.46 23.32 15.70 19.34
Faeces   
0-24 h 0.13 0.64 0.05 0.08 0.22
0-48 h 0.21 0.72 0.12 0.11 0.28
0-72 h 0.25 0.94 0.34 0.13 0.41
   
Cage wash 1.81 0.91 1.36 1.32 1.35
   
Expired air 0.12 0.92 0.07 0.07 0.30
   
Treated skin (methanol wash) 6.39 4.11 8.38 3.87 5.69
Treated skin (residue) 1.73 0.88 1.94 0.88 1.36
Non-treated skin (methanol wash) 0.33 0.43 0.65 0.34 0.44
Non-treated skin (residue) 0.10 0.20 0.36 0.12 0.20
 
Kidney (72 h) 0.00 0.00 0.00 0.00 0.00
   
Liver (72 h) 0.01 0.01 0.01 0.01 0.01
   
GI tract (72 h) 0.03 0.03 0.04 0.02 0.03
   
Carcass (72 h) 0.32 0.37 0.48 0.21 0.35
   
Dressing (6 h) 52.37 56.17 53.57 64.30 56.60
   
Skin wash (6 h) 7.46 2.00 4.85 8.22 5.63
   
Gloves (6 h) 3.96 3.58 4.37 3.79 3.93
   
TOTAL 91.77 92.01 99.74 98.98 95.62
   

Table 3: Recovery of Total Radioactivity - 10 mg/kg (results expressed as % administered dose recovered)

Sample/ Time (h) Animal
1 2 3 4 Mean
Urine          
0-6 h 1.36 1.33 0.73 1.42 1.21
0-24 h 6.15 10.00 6.09 10.45 8.17
0-48 h 10.97 17.20 10.90 18.74 14.45
0-72 h 13.16 21.50 14.14 23.55 18.09
Faeces   
0-24 h 0.09 0.21 0.06 0.20 0.14
0-48 h 0.28 0.36 0.12 0.34 0.28
0-72 h 0.31 0.44 0.18 0.44 0.34
   
Cage wash 0.83 3.58 1.57 1.89 1.97
   
Expired air 0.41 1.32 0.36 0.35 0.61
   
Treated skin (methanol wash) 1.25 3.61 6.05 2.99 3.48
Treated skin (residue) 0.36 1.08 1.83 1.44 1.18
Non-treated skin (methanol wash) 0.31 0.35 0.41 0.63 0.43
Non-treated skin (residue) 0.14 0.19 0.16 0.25 0.19
 
Kidney (72 h) 0.00 0.01 0.01 0.01 0.01
   
Liver (72 h) 0.00 0.01 0.01 0.01 0.01
   
GI tract (72 h) 0.02 0.04 0.03 0.03 0.03
   
Carcass (72 h) 0.20 0.53 0.52 0.50 0.44
   
Dressing (6 h) 66.84 48.25 55.97 48.84 54.98
   
Skin wash (6 h) 7.28 4.91 7.29 5.81 6.32
   
Gloves (6 h) 2.66 4.65 3.62 5.79 4.18
   
TOTAL 93.77 90.47 92.15 92.53 92.23
   

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, radioactivity was steadily absorbed through the skin and eliminated almost exclusively in urine; 3, 2 and 1% of the test material 6 h after dosing at the high, medium and low dose levels, respectively, and 19, 19 and 18% at 72 h, respectively. Less than 1% of the test material was eliminated in faeces over the 72 h period at all dose levels, and in general less than 1% of the test material was retained in the carcass at 72 h post dose. Varying amounts of dose were still associated with the dose site at 72 h post dose (approximately 2-7%) and of this material some was bound to the skin - the amount bound also varied considerably (approximately 18-37% of observed radioactivity). Small amounts of radioactivity (generally <0.5% dose) were associated with non dosed skin at this time - as the bulk of this material could be removed with solvent it would seem likely to be due to contamination from the dose site. Investigation of concentrations of radioactivity in tissues at 72 h post dose revealed a linear relationship between dose levels. In all cases, highest concentrations were observed in the kidney and concentrations in all tissues examined were generally higher than plasma concentrations. All animals were observed grooming extensively after the removal of dose patches and cleaning of the site, at 6 hours post application. Therefore there is a possibility that some oral ingestion occurred. The average rate of dermal absorption over the 72 hour observation period was determined to be between 19.5 and 21.5%.
Executive summary:

The absorption, distribution and elimination of the test material applied topically was investigated by dosing rats at concentrations of 50, 5 and 0.5% formulated in diethyl phthalate. These concentrations constituted dose levels of approximately 1000, 100 and 10 mg/kg. Doses were applied to the skin on gauze squares which were held in place by semi-occlusive dressing. The gauze squares and dressings were removed at 6 hours post dose and the dose area washed with diethyl phthalate. Urine, faeces and expired air were collected for 72 hours after dosing and at 72 hours post dose selected tissues and remaining carcasses were retained for analysis of total radioactivity.

Under the conditions of the study, radioactivity was steadily absorbed through the skin and eliminated almost exclusively in urine; 3, 2 and 1% of the dose 6 h after dosing at the High, Medium and low dose levels respectively and 19, 19 and 18% at 72 h. Less than 1% of the dose was eliminated in faeces over the 72 h period at all dose levels, and in general less than 1% of the dose was retained in the carcass at 72 h post dose. Varying amounts of dose were still associated with the dose site at 72 h post dose (approximately 2-7%) and of this material some was bound to the skin - the amount bound also varied considerably (approximately 18-37% of observed radioactivity). Small amounts of radioactivity (generally <0.5% dose) were associated with non dosed skin at this time - as the bulk of this material could be removed with solvent it would seem likely to be due to contamination from the dose site. Investigation of concentrations of radioactivity in tissues at 72 h post dose revealed a linear relationship between dose levels. In all cases, highest concentrations were observed in the kidney and concentrations in all tissues examined were generally higher than plasma concentrations. All animals were observed grooming extensively after the removal of dose patches and cleaning of the site, at 6 hours post application. Therefore there is a possibility that some oral ingestion occurred. The average rate of dermal absorption over the 72 hour observation period was determined to be between 19.5 and 21.5%.

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