Nitrapyrin

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 1996 to 25 March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7600 (Dermal Penetration)

Version / remarks:

1996 draft

Deviations:

no

Principles of method if other than guideline:

This study was conducted to provide data on the dermal absorption of the test material in male Fischer 344 rats.
For rats were exposed dermally for 24 hours to 10 mg 14C-test material/10 cm2 skin surface area in the vehicle dipropylene glycol monomethyl ether (Group I) and sacrificed after 24 hours. An additional group of four rats was exposed to 10 mg 14C-test material/10 cm2 skin surface area in the same vehicle for 24 hours, followed by a skin wash at 24 hours (Group II). The dermal dose site was then re-occluded until the animals were sacrificed at 72 hr post-dosing.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Nitrapyrin

EC Number:

217-682-2

EC Name:

Nitrapyrin

Cas Number:

1929-82-4

Molecular formula:

C6H3Cl4N

IUPAC Name:

2-chloro-6-(trichloromethyl)pyridine

Test material form:

solid

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 198-223 g
- Housing: Rats were individually housed post-dosing in glass Roth-type metabolism cages for the purpose of separating and collecting urine, faeces and final cage wash. Rats were acclimated to the glass Roth-type metabolism cages for 2 days and to the bandage for 1 day prior to the administration of the 14C-test material.
- Diet: Ad libitum
- Water: Municipal drinking water ad libitum.
- Acclimation period: At least one week.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 to 23 °C; adequate environmental conditions concerning temperature for this species were maintained.
- Humidity: 44 to 48 % relative; adequate environmental conditions concerning relative humidity for this species were maintained.
- Photoperiod: 12 hour photocycle.

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: dipropylene glycol monoethyl ether

Duration of exposure:

24 hours

Doses:

- Nominal doses: 10 mg 14C-test material/10cm2.
- Dose volume: 10 µL/cm2
- Rationale for dose selection: A 1 mg/cm2 dose level was selected since it is the recommended maximum practical dose outlined in the draft EPA Health Effects Test Guidelines, 1996.

No. of animals per group:

4 animals per group

Control animals:

no

Details on study design:

STUDY DESIGN AND RATIONALE
A single dermal dose of 1 mg 14C-test material/cm2 skin surface area was applied topically to a 10 cm2 area of skin to four male rats and the dosed area was occluded. Rats were individually housed post-dosing in glass Roth-type metabolism cages for the purpose of separating and collecting urine, faeces and final cage wash. The rats were euthanatised 24 hours post-dosing and the dose site was washed with an aqueous detergent solution. Absorbed dose was calculated based on the radioactivity found in excreta, blood, liver, kidney, and remaining carcass.
An additional group of four male rats was given a single dermal dose of 1 mg 14C-test material/cm2 skin surface area to determine the amount of test material on the skin that was absorbed post-washing. At 24 hours post-dosing the occlusion was removed from the dose site, the dosed skin was washed and the washed site was re-occluded. The animals were returned to the Roth-type metabolism cages and urine, faeces and final cage wash was collected. The animals were sacrificed at 72 hours post-dosing and the blood, liver, kidney, skin and dosed skin were collected. The dermal absorption was determined as described above.

DERMAL OCCLUSIVE DEVICE
The dermal occlusive devices were constructed from skin barrier, film and bandage. The devices tested were found to be effective in holding the test material on the skin without leaking and prevented the rats from removing, spreading or ingesting the test material via grooming. The area of skin to which the test material was applied was ~5 x 2 cm, i.e., 10 cm2.

PREPARATION OF ANIMALS FOR DOSING
Animals were anaesthetised with methoxyflurane and the hair on the back of each rat was clipped approximately 24 hours prior to dosing. Access to the dermal application site was restricted by use of a protective appliance created from wafers of skin barrier and bandage. The skin barrier frames were attached 24 hours prior to dosing. The barrier frame had an open window of approximately 10 cm2 and was positioned intrascapularly and as far anteriorly as possible.

PREPARATION AND ADMINISTRATION OF DOSE SOLUTION
The radiotracer was mixed with the dipropylene glycol monoethyl ether vehicle. Appropriate amounts of 14C-labelled test material were added to obtain a target dose of 1 mg/cm2 skin surface area. The dose solution was applied evenly to the skin in a volume of 10 µL/cm2 skin surface area. Aliquots of the dosing solutions containing 14C-test material were analysed to determine radioactivity and concentration.
Animals were anaesthetised with methoxyflurane prior to topical application of the test material. The 14C-dose solution was applied topically using a blunted stainless steel cannula attached to an all-glass syringe to approximately a 10 cm2 area. The test material was spread evenly over the area. The syringe was weighed before and after dosing to quantitate the amount of the dosing solution applied.
The dose site was occluded to prevent loss of the compound via grooming. Access to the dose site was restricted by use of the appliances described above. The dosed area was occluded with film which was attached to the skin barrier appliance with surgical adhesive. The dosed site was then covered with the bandage. Following dosing with radiolabelled material, rats were returned to the all glass Roth-type metabolism cages and air was drawn through the cages at approximately 500 mL/min.

SPECIMENS
Faeces were collected in dry-ice chilled containers at 24 hour intervals. An aqueous homogenate (~25 % w/w) was prepared and weighed aliquots of these homogenates were placed in scintillation vials, solubilised and quantitated for radioactivity.
Following the administration of the radiolabelled dose, all urine voided was collected in dry-ice cooled traps at 24 hour intervals. Each urine specimen and urine/cage rinse was weighed, and a weighed aliquot was analysed for radioactivity.

GROUP I
The animals were anaesthetised with methoxyflurane 24 hours post-dosing and sacrificed by exsanguination via cardiac puncture. The bandage and the film were removed and the bandage components were analysed for radioactivity. The skin at the dosed site was washed (5 X) with cotton buds dipped in a 2-4 % aqueous solution of liquid detergent, rinsed with gauze soaked in water and the area blotted dry with gauze squares. The skin barrier appliance was removed and analysed for radioactivity. The skin at the dosed site was excised, solubilised, weighed and analysed for radioactivity. The entire remaining skin, blood, kidneys, liver and remaining carcass were collected, solubilised and analysed for radioactivity. Urine and faeces were also collected at 24 hours.

GROUP II
The animals were dosed as described for Group 1. After a 24 hour exposure the dose site was washed and the site re-occluded. These animals were sacrificed at 72 hours post-dosing and the same samples were collected and analysed as described for Group I animals. Urine and faeces were collected at 24 hour intervals.

14C-ANALYSIS
Radioactivity was quantified in a liquid scintillation spectrometer (Beckman LS 3801 or LS 1801). Counts per minute (cpm) were corrected for quench and background and converted to disintegrations per minute (dpm). At least one sealed 14C-standard was counted with each group of samples to monitor the performance of the liquid scintillation counter. Samples with dpms less than twice the concurrently run background (blanks) were considered to contain insufficient radioactivity to reliably quantify.

CALCULATIONS
All calculations were performed in Excel v4.0, full precision. The percent of the dose absorbed was calculated as the sum of the radioactivity found in the excreta and tissues, excluding the dosed skin site and the materials used for the occlusion of the dose site.

CHEMICAL ANALYSIS
The concentration of the test material in the dose solution was determined as follows. Two 10 µL aliquots from the dosing solution were transferred using a Hamilton syringe into tared 25 mL volumetric flasks and weighed to verify the aliquot transfer. The aliquots were diluted to volume with acetonitrile:Milli-Q water (70:30) and analysed via high performance liquid chromatography with UV detection. Analytical standards were prepared and analysed with samples to define the detector response.
HPLC analysis conditions for dose solution concentration determination:
Pump: Spectra Physics SP8800-010
Flow Rate: 2 mL/minute
Mobile Phase: 70:30, Acetonitrile: Milli-Q water
Autosampler: Anspec AN728
Injection: 50 µL, 2 injections/vial
Column: YMC ODS-AQ 25 cm x 4.6 mm, 5 µm
Detector: LDC SpectroMonitor 3200 UV detector @ 220 nm, Sensitivity @ 0.005 AUFS

Results and discussion

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

DISTRIBUTION OF RADIOACTIVITY
The average percentage of the applied dose which was dermally absorbed was 24.6 % for Group I and 34.6 % for Group II. The calculation of the absorbed dose included the radioactivity recovered in the excreta and tissues excluding the dosed site. The majority of the administered dose was associated with the dosed site: Group I: 65.5 %; Group II: 48.6 %. The dosed site included the frame, the film covering, the bandage, the skin washes, the dosed skin, the final cage wash and the remote skin. The average percentage of the administered dose associated with the dosed skin after washing at 24 hours post-dosing for Group I was 17.8 %, which was considered available for further absorption. This decreased to 4.12 % for Group II indicating that approximately 14 % of the dose remaining in the dosed skin was further absorbed within 72 hours.
The average percentage of the dose removed by washing with detergent and water at 24 hours post-dosing was approximately 4-5 % for Group I and II. For Group II animals the average percentage of the dose removed by washing at 72 hours post-dosing was less than 0.15 %. The percentage of administered dose associated with the materials used to occlude the dose site (i.e. bandage, covering and frame) was approximately 38 % for Group I and II animals. The average percentage of the dose associated with the blood, carcass, liver and kidneys was low, approximately 4.5 and 0.81 %, for Group I and Group II animals respectively.

EXCRETION OF RADIOACTIVITY
The average amounts of the administered dose eliminated via the urine by 24 hr post-dosing was 17.9 % for Group I and 15.9 % for Group II. The total amount of radioactivity excreted in the urine (including the cage/urine rinse) of Group II animals for the 0-72 hour interval was 30.6 %.
For Group I and Group II, 0.81 and 0.83 % of the dose respectively was excreted via the faeces by 24 hours post-dosing. The total amount of radioactivity excreted in the faeces of Group II animals by 72 hours post-dosing was 3.3 %.

Total recovery:

The mean total recoveries were 90.1 % for Group I and 83.2 % for Group II.
Based on data obtained in Groups I and II, a total absorption of 25 % for Group I and 35 % for Group II was estimated.

Any other information on results incl. tables

Administration of Test Material

The dose solution was 91.2 % of the targeted concentration and the actual radioactivity was 344.5 µCi/g of dose solution. The differences between the actual and targeted doses are not anticipated to significantly impact the results of this study.

The mean number of mg of 14C-test material administered to male rats ranged from 14.3-15.6 mg. In addition, the average amount of radioactivity administered to the animals ranged from 54-59 µCi/rat.

 

Discussion

The total recovery of radioactivity that had been administered to the animals was approximately 90 and 83 % for Group I (24 hour exposure, wash and sacrifice at 24 hours post-dosing) and Group II (24 hour exposure, wash and sacrifice at 72 hours post-dosing), respectively.

At the 24 hr sacrifice (Group 1), about 25 % of the administered dose was absorbed based on radioactivity found in urine, faeces, carcass and tissues of male Fischer 344 rats. Approximately 65 % of the dose was not absorbed by 24 hours post-dosing based on the amount of radioactivity associated with the skin at the dosed site, the bandage components and the skin washes. The percentage of the administered dose associated with the dose skin site was 18 % and represents test material bioavailable for absorption.

At the 72 hour sacrifice (Group II), approximately 35 % of the radioactivity was absorbed based on the radioactivity found in the urine, faeces, carcass and tissues. These animals had been exposed for 24 hours, followed by a skin wash at 24 hours. The animals were then re-bandaged and sacrificed at 72 hours post-dosing. The percentage of the dose still remaining on the skin after the 24 hour wash was estimated from Group I animals to be approximately 18 %. Therefore, the animals were still exposed to the test material for another 48 hours before sacrifice at 72 hours post-dosing. The percentage absorbed, approximately 35 %, included the radioactivity found in urine (31 %) and faeces (3 %) collected over the 72 hour period and the radioactivity found in blood, carcass and tissues (1 %).

In summary, an average of about 25 % of the dose was absorbed in male rats following a single dermal 24 hour exposure of 10 mg/10cm2 in the vehicle dipropylene glycol monomethyl ether. The absorption is based on the amount of radioactivity recovered in the urine, faeces, tissues and carcass after the 24 hour exposure. Approximately 18 % of the dose was associated with the skin dose site after washing and was available for further absorption. Of the 18 % of the dose remaining on the skin, an additional 14 % of that amount was absorbed in animals that were sacrificed at 72 hours post-dosing. In total approximately 35 % of the applied dose was absorbed in the 72 hour period (Group II), with 25 % absorbed in the first 24 hour (Group I).

Table 1: Percent of Administered Dose Recovered (Group I)

Sample	Average	SD
Bandage	7.97	7.50
Covering	2.74	0.74
Dosed Skin	17.83	3.79
Frame	27.28	3.91
Remote Skin	1.63	0.02
Skin Wash	4.87	1.67
Final Cage Wash	3.17	0.90
% Unabsorbed	65.49	3.49
Blood	0.14	0.06
Carcass	3.59	1.53
Faeces	0.86	0.21
Kidneys	0.17	0.05
Liver	0.62	0.21
Urine*	19.19	4.88
% Absorbed	24.58	6.43
Total Recovery	90.07	4.03

*Value includes sum of urine plus urine rinse

 

Table 2: Percent of Administered Dose Recovered (Group II)

Sample	Average	SD
24 hour Bandage	7.90	9.74
72 hour Bandage	0.86	0.40
Bandage Sum	8.77	9.77
24 hour Covering	3.99	2.98
72 hour Covering	0.69	0.80
Covering Sum	4.68	2.67
Dosed Skin	4.12	2.08
Frame	24.28	5.32
Remote Skin	0.57	0.26
24 hour Skin Wash	3.52	0.58
72 hour Skin Wash	0.13	0.06
Skin Wash Sum	3.64	0.61
Final Cage Wash	2.50	1.93
% Unabsorbed	48.56	8.40
Blood	0.02	0.01
Carcass	0.48	0.16
Faeces	3.25	0.86
Kidneys	0.06	0.02
Liver	0.25	0.09
Urine*	30.55	9.57
% Absorbed	34.61	10.64
Total Recovery	83.17	5.36

*Value includes sum of urine plus urine rinse

 

Table 3: Interval Excretion for Group I Rats Expressed as Percent of Administered Dose

Sample	Time (hours)	Average	SD
Faeces	24	0.81	0.20
Urine*	24	17.90	4.75
Total		18.71	4.93

*Value includes sum of urine plus urine rinse

 

Table 4: Interval Excretion for Group II Rats Expressed as Percent of Administered Dose

Sample	Time (hours)	Average	SD
Faeces	24	0.83	0.30
	48	1.72	0.64
	72	0.71	0.23
Faeces Sum		3.25	0.86
Urine*	24	15.93	5.05
	48	10.66	3.79
	72	3.96	2.16
Urine Sum		30.55	9.57
Total		33.81	10.41

*Value includes sum of urine plus urine rinse

Applicant's summary and conclusion

Conclusions:

An average of about 25 % of the dose was absorbed following a single dermal application to rats based on the amount of radioactivity recovered in the urine, faeces, tissues and carcass.

Executive summary:

This study was conducted under GLP conditions to provide data on the dermal absorption of the test material in male Fischer 344 rats.

Male Fischer 344 rats were exposed dermally for 24 hours to 10 mg 14C-test material/10 cm2 skin surface area in the vehicle dipropylene glycol monomethyl ether (Group I). Approximately 25 % of the administered dose was absorbed based on the radioactivity found in urine, faeces, carcass and tissues at the 24 hour sacrifice. The percentage of the administered dose detected in the dosed site, the bandage components and the skin washes was approximately 65 %. The average total recovery of radioactivity that had been administered to the animals was approximately 90 %.

Male rats were exposed to 10 mg 14C-test material/10 cm2 skin surface area in the same vehicle for 24 hours, followed by a skin wash at 24 hours (Group II). The dermal dose site was then re-occluded until the animals were sacrificed at 72 hours post-dosing. The percentage of the dose still remaining on the skin after the 24 hour wash and available for further absorption was approximately 18 %. The average percentage absorbed was approximately 35 % based on the radioactivity found in urine (31 %) and faeces (3 %) collected over the 72 hour period and in carcass/tissues (1 %) at sacrifice. The percentage of the administered dose detected at the dosed site, the bandage components and the skin washes was 49 %. The average total recovery of radioactivity that had been administered to the animals was approximately 83 %.

In summary, following a single dermal exposure for 24 hours to 10 mg of 14C-test material/10 cm2 skin surface area an average of about 25 % of the dose was absorbed following a single dermal application to rats based on the amount of radioactivity recovered in the urine, faeces, tissues and carcass. Approximately 18 % remained on the skin after washing and could be available for further absorption. Of the 18 % of the dose remaining on the skin, approximately 14 % of that amount was absorbed in the animals that were sacrificed at 72 hours post-dosing. In total approximately 35 % of the applied dose was absorbed in the 72 hour period (Group II), with 25 % absorbed in the first 24 hours (Group I).