Registration Dossier

Administrative data

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
circa 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to guidelines but was conducted according to GLPs and the report contains sufficient data for interpretation of study results

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
14C labelled test material was applied to the skin of monkeys for 12 hours. Blood samples, urine and feces were collected for 72 hours after dosing and assayed for radioactivity,
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Nitroethane (99.5% purity, Gold Label) was purchased from Aldrich Chemicals Company and stored in the freezer until the study began. 14C-Nitroethane was synthesized by the laboratories of the Institute for Ecological Chemistry/GSF in Neuherberg, West Germany. It was received by White Sands Research Center on February 15, 1989. The following information was enclosed:total activity: 37 MBq = 1 mCi in approximately 2.2 ml etherradiochemical purity: 99%specific activity: 185 MBq/mmoldate of preparation: 6-03-86
Radiolabelling:
yes
Remarks:
14C

Test animals

Species:
monkey
Strain:
Macaca fascicularis
Sex:
female
Details on test animals and environmental conditions:
Female rhesus monkeys (Macaca fascicularis) were selected for this study because these animals are considered to be an acceptable model for man with the present test criteria.Two female rhesus monkeys (PH0041 and PH0115) were selected from the White Sands Research Center colony, examined by the study veterinarian and determined to be in good state of health.

Administration / exposure

Type of coverage:
occlusive
Vehicle:
other: In the report, ethanol (HPLC grade) is used to dilute the test material. Later, in the same paragraph, the solvent is described as 'an ethanol/ether solvent mixture' It is not clear exactly what the solvent was.
Duration of exposure:
12 hours
Doses:
Animal PH0041 got an intended dose of 300 ul (15.46 mg). However, PH0115 got only 230 ul (11.85 mg).
No. of animals per group:
2
Control animals:
no
Details on study design:
Two female rhesus monkeys (PH0041 and PH0115) were selected from the White Sands Research Center colony, examined by the study veterinarian and determined to be in good state of health. Seventy-two hours prior to application, the back of each monkey was clipped free of hair. A 20 cm2 area was demarcated by tattooing as test site. Twenty-four hours prior to dosing the test site was cleaned with isopropanol. The animals were lightly sedated with ketamine HCl (10 mg/kg IM). Catheters for blood collection were introduced into the leg vein. Animal pH0041 got an intended dose of 300 ul (15.46 mg). However, PHO115 got only 230 ul (11.85 mg) because, due to a lot of air bubbles in the syringe, some material got lost while blowing the air bubbles out of the syringe. Therefore, only 230 ul of the test solution were left over for the second animal. The test solution was evenly applied to the test site with a disposable syringe equipped with a feeding needle. A piece of plastic wrapping foil (a little bigger than 20 cmt) was taped tightly over the test site. Thereafter the animals were placed in restraint chairs. Twelve hours after dose application the animals were again slightly sedated. The patch was then removed and the test site was swabbed 3 times with a 2% solution of soap in distilled water. A fourth swab, dampened with acetone, was used as a final rinse. Seventy-two hours after dose application, the animals were sedated. The test site and an adjacent 1 cm strip were excised, The subcutaneous fat was removed from the skin. Fat and skin were weighed before freezing them. The following samples were collected:Urine (freeze-trapped, cooled with dry ice) post-dose intervals: 0-2, 2-4, 4-6, 6-8, 8-10 and 10-12 and at 12-hour intervals thereafter. After thawing, the volume of each sample was determined.Feces post-dose intervals: 0-4, 4-8, 8-12 hours and at 12-hour intervals thereafter. Before freezing, each feces sample was weighed.Blood (venipuncture): at 0.33, 0.66, 1, 2, 3, 4, 6, 8, 10 and 12 hours and at 12-hour intervals thereafter. The blood was collected into EDTA-coated tubes.AnalysesSamples were analyzed as follows:Blood: Triplicate samples (100 ul) of the whole blood were transferred to combustion cones, combusted to 14CO2 and countedby liquid scintillation methods. Urine: Triplicate samples (1 ml) of urine were transferred to liquid scintillation fluid and counted for radioactivity. The remaining urine was stored in a freezer.Feces: The frozen feces were homogenized in a blender with dry ice. At least triplicate samples were weighed into combustion cones, mixed with cellulose powder, combusted to 14CO2 and counted by liquid scintillation methods. The remaining feces were stored in a freezer.Skin: The subcutanoeus fat was minced and mixed with cellulose powder. The samples were combusted to 14CO2 and counted by liquid scintillation methods. The frozen skin (treated and untreated separately) was homogenized in a blender with dry ice. Four to six samples, depending on the accordance of the values, were weighed into combustion cones and mixed with cellulose powder. The samples were combusted to 14CO2 and counted by liquid scintillation methods. Swab Extracts: The swabs were extracted in acetone and the patch in ethanol. Triplicate samples (1 ml) of the swab acetone and the patch extracts were counted by liquid scintillation methods. Liquid Scintillation Counting: After cooling, the samples were counted twice for two minutes. When two of the three samples were within 5% of each other, these samples were considered acceptable and were used for calculations.Histopathology: A 1x1 cm section of treated and nontreated skin was examined histologically by utilizing hematoxylin and eosin staining and light microscopy.
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:
The analysis of the skin samples showed that absorption of the test material occurred only in negligible amounts.
Total recovery:
The very high loss of test material (i.e., 99.79% of the total dose) can be attributed to the very high volatility of the test material and therefore evaporation (in spite of the patch) from the test site. It should also be considered that exhaled radioactivity, i-e., nitroethane or volatile metabolites, were not trapped under the selected test conditions.
Conversion factor human vs. animal skin:
No data in report.

Any other information on results incl. tables

Neither animal showed any signs of toxicity. Feed and water consumption was low during the first 12 hours but normalized later. During the first 12 hours the urine and feces output was partly very low or even nonexistent (i.e., urine 0-2 hours, post-dose for PH0041 and PH0115 and 6-8 hours and 10-12 hours post-dose for PH0115; feces 0-4 hours post-dose for monkey PH0115 and 8-12 hours and 12-24 hours post-dose for monkey PH0041) but also normalized later. The body weight remained within 5% of the weight from day 0 (monkey PH0041 day 0 weight: 3.79 kg; monkey PH0115 day 0 weight: 4.42 kg; day 0 = dosing day; monkey PH0041 day 3 weight: 3.44 kg; monkey PH0115 day 3 weight: 4.18 kg). The histological examination of the skin samples taken from the test site did not show any signs of skin damage or irritation.

Since the applied dose of both animals did not differ drastically, average values were taken. The average excretion was determined to be 16.2 ug, 77.2% of which was excreted in the urine. Monkey PH0041 excreted 14.91 ug nitroethane (0.096% of the total dose) within 72 hours, whereas monkey PH0115 excreted 10.12 ug nitroethane (0.085% of the total dose) over the same period. An average of 65.1% of the total urine radioactivity was collected within 24 hours (PH0041: 68.9%; PH0115: 61.2%). The corresponding 48-hour figures are: average 91.4%; monkey PH0041: 90.8%; monkey PH0115: 92.0%. The main excretion was observed within 36 hours after dosing. After 36 hours, only 0.83% of the test material were excreted in the urine. The maximum concentration of nitroethane in blood was determined after 40 minutes (monkey PH0041, 41.3 ng/ml which equals 41.3 ppb) and 1 hour, respectively (monkey PH0115, 16.5 ng/ml which equals 16.5 ppb). Obviously, there is a big discrepancy in the blood values between both animals, which might be a consequence of the different dose levels. After 24 hours (PH0041) and 12 hours (PH0115), respectively, the concentration in the blood of both animals was zero.

The excretion of nitroethane in feces within 72 hours was quite different in both animals (monkey PH0041: 4.64 ug, 0.030% of the total dose and monkey PH0115: 2.76 ug, 0.023% of the total dose). The highest excretion rate of nitroethane occurred in PH0041 36 hours post-dose and in PH0115 48 hours after dosing. After 12 hours the test material remaining on the skin and not yet evaporated was wiped off with soap/water and acetone swabs. The swabs and the occlusive patches were extracted with acetone and ethanol, respectively. The analysis of the swabs showed an average recovery of 2.71 ug nitroethane (0.021% of the total dose). The average recovery in the patches was 5.40 ug which equals 0.041% of the total dose. These findings indicate that only very low amounts of the test material were found on the skin. Considering the fact that nitroethane is a high volatile compound, it has to be assumed that most of the test material evaporated from the test site although it was covered airtight with a patch during the first twelve hours.

Seventy-two hours after dosing, the test site and an adjacent 1 cm area were excised and skin and subcutaneous fat were analyzed for radioactivity. The skin contained only an average of 4.05 ug nitroethane (0.029% of the total dose; monkey PH004l: 4.87 ug = 0.032% AD and monkey PH0115: 3.23 ug = 0.026% AD). Included in these percentages are 0.024% (monkey PH0041) and < 0.001% (monkey PH0115) found on the untreated skin. These low radioactivity levels probably reflect the manual contamination during application rather than an actual migration from the test site. The subcutaneous fat did not contain any test material (i.e., under 0.001% of the applied dose). This indicates that nitroethane or more probably metabolites are absorbed only in negligible amounts in the skin and the subcutaneous fat, respectively.

The above-mentioned percentages total 0.205% (monkey PH0041) or 0.210% (monkey PH0115). The average percentage of unrecovered radioactivity was 99.792% (monkey PH0041: 99.795% and monkey PH0115: 99.790%). These values indicate that the difference in recovery between both monkeys was very little. In general, the very low recovery rate can be attributed to high evaporation from the test site. It has to be considered that large amounts of nitroethane contained for a short period in the blood were exhaled without being trapped in scintillation fluid and counted. Previous oral studies with nitroethane confirm the findings of low recovery. It also has to be assumed that the low amounts of radioactivity determined in the skin after 72 hours can be attributed to metabolites of nitroethane since the parent compound might be evaporated from the test site very fast.

The following table summarizes the results showing the amounts of nitroethane and percentages of the total dose found for both monkeys individually and the average of both animals:

      Monkey PH0041     Monkey PH0115     Average
 Sample  Percent  ug NE  Percent  ug NE  Percent  ug NE
 Urine  0.096  14.91  0.085  10.12  0.090  12.50
 Feces  0.030  4.64  0.023  2.76  0.027  3.70
 Skin  0.032  4.87  0.026  3.23  0.029  4.05
 Fat  <0.001  0.04  <0.001  0.01  <0.001  0.03
 12 -hour Swabs  0.011  1.71  0.031  3.71  0.021  2.71
 Patch  0.036  5.51  0.045  5.33  0.041  5.42
  Blood*  ----  ----  ----  ----  ----  ----
 Unrecovered radioactivity  99.795  15425.90  99.790  11825.70  99.792  13625.84
 Total  100.000  15457.62  100.000  11850.86  100.000  13654.24

*Radioactivity in blood after 72 hours. .

Applicant's summary and conclusion

Conclusions:
The analysis of the skin samples showed that absorption of the test material occurred only in negligible amounts. The very high loss of test material (i.e., 99.79% of the total dose) can be attributed to the very high volatility of the test material and therefore evaporation (in spite of the patch) from the test site. It should also be considered that exhaled radioactivity, i-e., nitroethane or volatile metabolites, were not trapped under the selected test conditions.
Executive summary:

The study was designed to provide information on the absorption of the test material when applied dermally to female adult rhesus monkeys. After shaving the backs of two female rhesus monkeys and marking a 20 cm2 area as test site by tattooing, a single dermal dose (i-e., 300 ul and 230 ul ether/ethanol solution, respectively, containing 4.9% 14C nitroethane) was applied on the intact skin by means of a disposable plastic syringe equipped with a feeding needle to guarantee a smooth application. Thereafter, the test site was covered with an occlusive plastic foil patch and taped air tight over the test site. Twelve hours after dose application the patch was removed and the skin was wiped with soap and acetone swabs to remove remaining test material. The swab and the patch were extracted with acetone and ethanol respectively. The extracts were assayed for radioactivity. Blood samples, urine and feces were collected for 72 hours after dosing and assayed for radioactivity, Seventy-two hours after dose application, the test site and an adjacent 1 cm area were excised. Skin and subcutaneous fat were assayed for radioactivity. The skin samples (treated and untreated) were also examined histologically.

Neither animal showed any signs of toxicity. Feed and water consumption was normal. Neither monkey had any urine 2 hours after dosing. Later, (i-e., between 2-8 hours after application), monkey PH0115 had only very low urine output (i.e., 100 ul). After 12 hours the output normalized. Neither animal had any feces between 8-12 hours and 0-4 hours, respectively, after dose application. The body weight remained within 5% of the starting weight, Histological examination of the skin samples did not show any signs of skin damage or irritation. All percentages given in the summary are the average (when not otherwise marked) of the two animals. The terms excretion, percentage, doses, etc. refer to the radioactivity of the corresponding samples. The excretion of the test material was determined to be 16.20 ug nitroethane (i.e., 0.117% of the total dose) within 72 hours, 77.2% of which were excreted in the urine. Forty-eight hours after dosing, 91.4% of the total urine radioactivity was excreted. The fecal samples showed 22.8% of the total excreted radioactivity. The blood levels reached the average maximum concentration of 41.3 ng nitroethane/ml blood (41.3 ppb) for animal PH0041 and 16.5 ng nitroethane/ml blood (16.5 ppb) for animal PH0115 after 40 minutes (PH0041) and 1 hour (PH0115). After 24 hours the nitroethane levels in blood in both animals were at zero. The skin excised 72 hours after dosing contained 4.05 ug nitroethane (i-e., 0.029% of the total dose). The concentration in the subcutaneous fat was determined to be much lower than 0.001% of the applied dose. A quantity of 2.71 ug nitroethane (0.021% of the total dose) was recovered 12 hours after dose application by wiping the skin with soap/water and acetone swabs, A quantity of 5.42 ug nitroethane (0.041% of the total dose) was contained in the occlusive patch. The very high loss of test material (i.e., 99.79% of the total dose) can be attributed to the very high volatility of the test material and therefore evaporation (in spite of the patch) from the test site. It should also be considered that exhaled radioactivity, i-e., nitroethane or volatile metabolites, were not trapped under the selected test conditions. The analysis of the skin samples showed that absorption of the test material occurred only in negligible amounts.