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

Principles of method if other than guideline:
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, a single dermal dose (i.e., 300 ul ether/ethanol solution containing 5.5% 14C nitromethane) was applied on the intact skin. 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 (i.e., plasma and erythrocytes separately), 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.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Nitromethane (99+% purity, Gold Label) was purchased from Aldrich Chemicals Company and stored in the freezer until the study began. 14C-Nitromethane 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: 74 MBq = 2 mCi in approximately 2.2 ml ether
radiochemical purity: 99%
specific activity: 185 MBq/mmol
date of preparation: 9-30-88

After opening the seal the content of the vial was transferred to a more suitable vial with a sealed cap, labeled with a White Sands Research Center number (i.e., 104) and stored in a freezer. The test material was prepared as follows: a total volume of 750 ul solution was prepared, i.e., two doses of 300 ul per each monkey plus 150 ul excess. A quantity of 40 mg commercial nitromethane (35.2 ul) and 64.8 ul ethanol (HPLC grade) were placed in a vial which was cooled on ice. A quantity of 6.78 mg of the radiolabeled nitromethane was added to yield a 5% solution of nitromethane in an ethanol/ether solvent mixture. A quantity of 5 ul of the test solution was diluted to 10 ml with ethanol. Two samples (10 ul each) of the diluted solution were counted in the scintillation counter twice for 2 minutes. A final concentration of 47.1 mg nitromethane, i.e., 40 mg "cold" nitromethane plus 7.1 mg 14C-nitromethane resulted, yielding a test solution containing 5.5% nitromethane. The vial was appropriately labeled. The doses
were drawn into a disposable plastic syringe provided with a feeding needle for easier and smoother application on the skin.
Radiolabelling:
yes

Test animals

Species:
monkey
Strain:
Macaca fascicularis
Sex:
female
Details on test animals and environmental conditions:
Two female rhesus monkeys (pH0113 and PH0114) 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.

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.

Administration / exposure

Type of coverage:
occlusive
Vehicle:
other: report states vehicle was an ethanol/ether mixture. However no mention of ether is made anywhere else in the report.
Duration of exposure:
12 hours for exposure to the test material
Doses:
300 ul of a 5.5% solution of nitromethane in ethanol was applied to the back of each monkey.
No. of animals per group:
2
Control animals:
no
Details on study design:
Two female rhesus monkeys (PH0113 and PH0114) 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. Three hundred ul of the material (18.84 mg dose for each monkey) were applied evenly to the test site with a disposable syringe equipped with a feeding needle. A piece of plastic wrapping foil (a little bigger than 20 cm2) 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 and an adjacent 1 cm area around the test site were swabbed 3 times with a 2% solution of soap in distilled water. A fourth swab, dampened with acetone, was used as a final rinse. Thereafter the animals were transferred to metabolism cages. 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.

Analysis
Samples were analyzed as follows:
Blood: Whole blood was centrifuged at 1200 rpm for approximately 5 minutes and the plasma separated from the red blood cells. Triplicate samples (100 ul) of the plasma were transferred to liquid scintillation fluid and counted for radioactivity. Triplicate samples of erythrocytes were weighed into combustion cones, combusted to 14C02 and counted by 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. Triplicate samples were weighed into combustion cones, mixed with cellulose powder, combusted to 14C02 and counted by liquid scintillation methods. The remaining feces were stored in a freezer.

Skin: The subcutanoeus fat was minced, divided into two samples and mixed with cellulose powder. The samples were combusted toCO2 and counted by 1iquid scintillation methods. The frozen skin (treated and untreated separately) was homogenized in a blender with dry ice. Three to seven samples, depending on the accordance of the values, were weighed into combustion and mixed with cellulose powder. The samples were combusted to 14C02 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 1x:1 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 terms excretion, percent and total dose refer to the radioactivity and not to the weights and volumes of the corresponding samples; the numbers pH0113 and pH0114 refer to the WSRC animal numbers of the study animals.

The average excretion was determined to be 15.39 ug, 74.3% of which was excreted in the urine. Monkey PH0113 excreted 18.54 ug nitromethane (0.099% of the total dose) within 72 hours, whereas monkey PH0114 excreted 12.23 ug nitromethane (0.065% of the total dose) over the same period. An average of 76.8% of the total urine radioactivity was collected within 24 hours (PH0113: 79.2%; PH0114: 74.3%). The corresponding 48-hour
figures are: average 90.3%; monkey PH0113: 90.8%; monkey PH0114: 89.8%. The main excretion was observed within 24 hours after dosing. Monkey PH0114 shows in the beginning much lower excretion values than monkey PH0113. This could be due to the fact that PH0114 urinated less than pH0113 until the 12-hour post-dose interval. But it could also be possible that the metabolism of monkey pH0114 proceeded slower. After 24 hours, only low ug amounts of the test material were excreted. The course of the blood plasma levels and erythrocyte levels looked very similar. The maximum concentration of nitromethane in plasma was determined after 1 hour (monkey PH0113, 48.4 ng/ml which equals 48.4 ppb) respectively 2 hours (monkey PH0114, 44.2 ng/ml which equals 44.2 ppb). After 72 hours the concentration in plasma of both animals was 5.3 ng/1 ml plasma which equals 5.3 ppb, The correspondence between the more or less constant low urine values after 36 hours and the plasma values is evident. It can be assumed that as long as there are little amounts in the blood, low amounts will also be excreted in urine even after 72 hours. Monkey PH0113 had its maximum nitromethane concentration (44.3 ng/g which equals 44.3 ppb) in the erythrocytes already 20 minutes after dose application, whereas monkey pH0114 had the maximum nitromethane concentration in the erythrocytes after 2 hours (40.8 ng/g which equals 40.8 ppb). At 60 hours post-dose, the nitromethane level in the erythrocytes of both animals went against zero.

The excretion of nitromethane in feces within 72 hours was quite different in both animals (monkey PH0113: 5.31 ug, 0.027% of the total dose and monkey PH0114: 2.58 ug, 0.013% of the total dose). The highest excretion rate of nitromethane occurred in both animals 24 hours post-dose. Monkey PH0114 did not have any feces during the first four hours after dosing, and even during the next hours and days respectively, the excretion of feces was much lower compared to monkey PH0113. After 12 hours the test material remaining on the skin and not yet evaporated was wiped off with soap/water and acetone swabs, respectively. The swabs and the occlusive patches were extracted with acetone and ethanol, respectively. The analysis of the swabs showed an average recovery of 2-02 ug nitromethane (0.021% of the total dose). The average recovery in the patches was 0.76 ug which equals 0.004% 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 nitromethane 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 3.49 ug nitromethane (0.018% of the total dose; monkey PH0113: 3.87 ug = 0.020% AD and monkey PH0114: 3.10 ug = 0.016% AD). Included in these percentages are 0.001% (monkey PH0113) and <0.001% (monkey PH0114) respectively, 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., radioactivity in the samples was equal to background radioactivity). This indicates that nitromethane or more probably metabolites are absorbed only in negligible amounts in the skin; nothing at all migrated through the skin in the subcutaneous fat.

Total recovery:
The above-mentioned percentages total 0.138% (monkey PH0113) or 0.108% (monkey PH0114). The average percentage of unrecovered radioactivity was 99.877% (18.82 mg nitromethane) of the total dose (monkey PH0113: 99.862% = 18.81 mg; monkey PH0114: 99.892% = 18.82 mg). 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 nitromethane contained for a short period in the blood were exhaled without being trapped in scintillation fluid and counted. Previous oral studies with nitromethane 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 nitromethane since the parent compound might be evaporated from the test site very fast.
Conversion factor human vs. animal skin:
Not applicable.

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 when the animals were back in the metabolism cages. During the first 12 hours the urine and feces output was partly very low or even nonexistent (i.e., urine 2-4 hours, 4-6 hours, 6-8 hours post-dose for animal PH0113 and PH0114 and feces 0-4 hours post-dose for monkey PH0114 and 8-12 hours post-dose for animal PH0113) but also normalized after the animals were removed from the restraint chairs and returned to their cages. The body weight remained within 5% of the weight from day 0 (monkey PH0113 day 0 weight: 3.94 kg; monkey PH0114 day 0 weight: 4.84 kg; day 0 = dosing day; monkey PH0113 day 3 weight: 3.90 kg; monkey PH0114 day 3 weight: 4.78 kg). The histological examination of the skin samples taken from the test site did not show any signs of skin damage or irritation.

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

      Monkey PH0113     Monkey PH0114     Average
 Sample  Percent  ug NM  Percent  ug NM  Percent  ug NM
 Urine  0.072  13.23  0.052  9.65  0.062  11.44
 Feces  0.027  5.31  0.013  2.58  0.020  3.95
 Skin  0.020  3.87  0.016  3.10  0.018  3.49
 Fat  --  --  --  --  --  --
 12 hr swabs  0.010  1.90  0.011  2.14  0.011  2.02
 patch  0.004  0.71  0.004  0.80  0.004  0.76

 plasma1*

(72 hrs)

 0.005  0.96  0.011  2.10  0.008  1.53
 Erythrocytes (72 hrs)  --  --  --  --  --  --
 Unrecovered radioactivity  99.862  18814.02  99.896  18820.42  99.877  18817.22
 Total  100.000  18840.00  100.000  18840.00  100.000  18840.00

*The plasma volume was calculated as follows:

A monkey has an average of 80 ml blood/kg of body weight. Taking the final weight of 4.78 kg for PH0114 and 3.90 kg for

PH0113, 382.4 ml of blood and 312.0 ml of blood result at the end of the study, respectively. With the hematocrit of 43.9% (PH0113) and 36.1% (PH0114), the distribution between plasma and red cells results as follows:

PH0113: Total Volume: 312.0 ml

Plasma : 175.0 ml

Erythrocytes: 137.0 g

PH0114: Total Volume: 382.4 ml

Plasma: 244.4 ml

Erythrocytes: 138.0 g

Applicant's summary and conclusion

Conclusions:
Analysis of the skin samples demonstrated absorption of the test material occurred only in negligible amounts.
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 ether/ethanol solution containing 5.5% 14C nitromethane) 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 (i.e., plasma and erythrocytes separately), 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, In both animals the urine output between

2-8 hours after dermal application was very low. Thereafter, the output normalized. One animal (PH0114) had no feces at all between

0-4 hours after application. Animal PH0113 had very few feces output between 8-12 hours after application. Animal PH0114 in

general had less feces than PH0113. 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 15.39 ug nitromethane (i.e., 0.082% of the total dose) within

72 hours, 74.3% of which were excreted in the urine. Forty-eight hours after dosing, 90.4% of the total urine radioactivity was

excreted. The fecal samples showed 25.7% of the total excreted radioactivity. The blood plasma levels reached the average maximum concentration of 37.8 ng nitromethane/l ml blood plasma (37.8 ppb) for animal PH0114 and 40.3 ng nitromethane/1 ml blood plasma (40.3 ppb) for animal PH0113 after 40 minutes to 2 hours (PH0114) and 20 minutes to 6 hours (PH0113). After 72 hours the plasma levels in both animals were only 5.3 ppb. In monkey PH0113 the nitromethane levels in the erythrocytes reached a maximum of 44.3 ng/g erythrocytes after 20 minutes. In monkey PH0114 the maximum concentration was 40.8 ng/g erythrocytes after 2 hours. Forty-eight hours after dosing there were no detectable amounts of nitromethane in the erythrocytes of both animals. The skin excised 72 hours after dosing contained 3.49 ug nitromethane (i.e., 0.018% of the total dose). This equals a concentration of 789.8 ppb, whereas the concentration in the subcutaneous fat was determined to be zero. A quantity of 2.02 ug nitromethane (0.011% of the total dose) was recovered 12 hours after dose application by wiping the skin with soap/water and acetone swabs. A quantity of 0.76 ug nitromethane (0.004% of the total dose) was contained in the occlusive patch. The very high loss of test material (18.84 mg nitromethane, i.e., 99.88% 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 as nitromethane 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.