N-(2-nitrophenyl)phosphoric triamide

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-10-07 - 2014-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

477-690-9

EC Name:

-

Cas Number:

874819-71-3

Molecular formula:

Hill formula: C6H9N4O3P CAS formula: C6H9N4O3P

IUPAC Name:

N-(diaminophosphoryl)-2-nitroaniline

Test material form:

solid: crystalline

Details on test material:

Reference substance: 2-NPT
Chemical description (IUPAC): N-(2-nitrophenyl)phosphoric triamide
CAS number: 874819-71-3
EC number: 477-690-9
Molecular formula: C6H9N4O3P
Molecular weight: 216.1

Radiolabelling:

no

Test animals

Species:

other: human skin

Strain:

other: not applicable

Administration / exposure

Type of coverage:

other: not applicable, in vitro

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: human skin from surgical operations
- Ethical approval if human skin: yes
- Type of skin: abdomen
- Preparative technique: Dermatomization, tape stripping and cryo-sectioning
- Thickness of skin (in mm): mean thickness of around 500 ± 100 µm
- Membrane integrity check: yes, with caffeine
- Storage conditions: cooled to 4 °C
- Justification of species, anatomical site and preparative technique:

PRINCIPLES OF ASSAY
- Diffusion cell: Franz cell
- Receptor fluid: PBS pH 7.4
- Solubility od test substance in receptor fluid: yes as determined
- Static system: yes
- Flow-through system: no
- Test temperature: 32 °C ± 1 °C

Results and discussion

Total recovery:

All calculations and evaluations were performed with the measured amounts of test substances in the test items. 166.55 µg•cm-2 2-NPT was applied (corresponds to 0.03 % (w/v)). The recoveries of 2-NPT after extraction development was 100.03 % for 2-NPT if the applied amount is set to 100 %. These values were within the set specifications (recovery of 90 % to 110 %). The development of an extraction method for skin was successful.
 

Percutaneous absorption

Key result

Time point:

24 h

Dose:

0.06%

Parameter:

percentage

Absorption:

0.26 %

Any other information on results incl. tables

 Solubility

To analyze the solubility of 2-NPT and to ensure sink conditions during the resorption experiments, one concentration (ca. 587µg·mL^-1for 2-NPT) in the preselected acceptor medium was prepared and diluted to the linear range for measurement.The solution was stored for 4 hours at room temperature on a stirrer. Samples were taken in triplicate at time points 0 h, 1 h and 4 h.The following table shows the results of the solubility measurements of 2-NPT in the selected acceptor medium PBS pH 7.4.

Solubility of 2-NPT in PBS pH 7.4

Storage time [h]	0	1	4
Storage temperature	RT	RT	RT
n1 [µg∙mL^-1]	570.79	586.11	606.75
n2 [µg∙mL^-1]	568.57	591.09	618.46
n3 [µg∙mL^-1]	568.82	596.51	620.05
Mean [µg∙mL^-1]	569.4	591.2	615.1
SD [µg∙mL^-1]	1.0	4.2	5.9
RSD [%]	0.2	0.7	1.0
Theoretical c for t=0 [µg∙mL^-1]	586.53
Appearance	Clear yellow solution with few particles	Clear yellow solution with few particles	Clear yellow solution with few particles
Dissolved [µg∙mL^-1]*	569.4 ± 1.0	591.2 ± 4.2	615.1 ± 5.9

The measured solubilities ensure sink conditions in the resorption experiments with PBS pH 7.4 as acceptor medium.

Skin quality control experiment (MEA)

In an earlier study the permeability of caffeine from an aqueous solution across full-thickness skin, dermatomized skin, heat-separated epidermis and isolated SC had been studied at Across Barriers to establish quality assurance benchmarks for skin integrity [Bock et al., 2002]. Subsequent quality control studies have been performed on a range of human skin samples.

The table below provides an overview of caffeine permeability through dermatomized skin specimens measured at Across Barriers and includes the P_appvalues determined in the present study.

Comparison of apparent permeability coefficients for caffeine through different dermatomized skin specimens with intact SC. Skin No.617-01-0714 wasused in the present study.

Skin number	Mean Papp[cm∙s^-1] (n=3)	RSD [%]
061-01-0701	9.38E-08	29
153-01-0104	8.41E-08	28
059-01-0601	8.24E-08	39
060-01-0601	5.63E-08	6
062-01-0701	5.34E-08	18
155-01-0204	5.03E-08	27
209-01-0605	3.82E-08	7
157-01-0304	3.23E-08	27
057-01-0601	3.21E-08	16
150-01-1203	2.54E-08	56
058-01-0601	2.00E-08	7
308-01-1107	1.28E-08	25
239-01-0707	1.04E-08	3
617-01-0714*	6.14E-08	15

*Evaluated as n=2 because one Franz cell was declared as outlier due to a significant higher transport most likely derived from a damaged skin biopsy.

 

The skin tightness is comparable with skins used in the past. The permeation coefficient for caffeine applied as infinite dose is in the same magnitude compared with previous data.

Mass balance of 2-NPT after transport through human skin over a time period of 24 h:

	2-NPT
Applied amount [µg·cm^-2]	166.55
Amount remaining on skin surface [µg·cm^-2]	165.36
Amount in stratum corneum [µg·cm^-2]	0.20
Amount in epidermis/dermis [µg·cm^-2]	0.18
Amount resorbed [µg·cm^-2]	0.68
Sum of absorbed & not absorbed [µg·cm^-2]	166.60
Recovery from applied amount [%]	100.03

All calculations and evaluations were performed with the measured amounts of test substances in the test items. 166.55 µg·cm^-2 2-NPT were applied (corresponds to 0.03 % (w/v)). The recovery of 2-NPT after extraction development was 100.03 % for 2-NPT if the applied amount is set to 100 %. These values were within the set specifications (recovery of 90 % to 110 %). The development of an extraction method for skin was successful.

Penetration of 2-NPT

see attached figure

Transport of 2-NPT through human skin

Mass balance reported as µg·cm^-2and recovery reported as % for 2-NPT after transport through human skin over a time period of 24 h.

Parameter	2-NPT
Amount remaining on skin surface [µg·cm^-2] (Not absorbed: residual test items in donor chamber + first two tape strips)	360.47 ± 9.67
Amount in stratum corneum [µg·cm^-2] (All tape strips except for the first two strips)	0.66 ± 0.14
Amount in epidermis/dermis [µg·cm^-2] (All slices of the skin layers)	0.26 ± 0.07
Absorbed into skin [µg·cm^-2] (Sum of stratum corneum + epidermis/dermis)	0.92 ± 0.19
Amount resorbed [µg·cm^-2] (Found in acceptor medium)	0.00
Sum [µg·cm^-2] (Absorbed + resorbed + not absorbed)	361.39 ± 9.88
Applied amount [µg·cm^-2]	349.32
Recovery of sum from applied amount [%] (Absorbed + resorbed + not absorbed)	103

2-NPT could not be detected in the acceptor compartment during and after 24 h of incubation time.

The maximum absorbed quantity into the skin was 0.92 µg^.cm^-2 for 2–NPT after 24 hours contact with the skin. This corresponds to 0.26 % of the applied dose 2-NPT.

For 2-NPT no permeation coefficient could be calculated because no transport into the acceptor compartment during and after 24 h incubation took place.

With respect to the LLOQ of the analytical method (0.0527 µg·mL^-1) and the applied amount of 2-NPT in the donor (658.32 µg) the theoretical permeation coefficient is lower than 6 E-9 cm·s^-1. Therefore the maximum absorbed amount of 2-NPT is the amount of substance found in the stratum corneum and epidermis/dermis.

Calculations of the theoretical permeation coefficient for 2-NPT

Applied concentration of 2-NPT in the donor [µg·mL^-1]	653.23
Donor compartment volume [mL]	1
Applied mass API in the donor [µg]	653.23
Acceptor compartment volume [mL]	ca. 12
Concentration of API in the acceptor after 24 h with an estimated Pappof 6 E-9 cm·s^-1[µg·mL^-1]	0.05

Applicant's summary and conclusion

Conclusions:

The study was performed as a risk assessment for the test substance 2-NPT. It was the objective to find out, which amounts of the substance are capable of permeating through and penetrating into the human skin. In practice, the maximum possible exposure concentrations is 0.03 % (w/v) for 2-NPT, that can contaminate human skin. These concentration is based on the finished mixtures of the substance that will be marketed.
During the study these expected concentrations in a product were doubled and the duration of incubation set to 24 h to get a measurable transport of the substance through the skin. Even with the doubled concentrations (0.06 % for 2-NPT) and a duration of 24 h , no transport through the skin could be detected. Therefore, only a limit value < 6E-9 cm∙s-1 could be estimated. The maximum absorbed quantity into the skin was 0.92 µg.cm-2 for 2–NPT after 24 hours contact with the skin. This corresponds to 0.26 % of the applied dose 2-NPT.
Based on these results, it can be stated that there will be a negligible transport of 2-NPT into or through human skin, especially as the real life exposure levels are half of the used ones. Furthermore it can be assumed that the contact time will be much shorter, because the skin will be cleaned faster.
 

Executive summary:

In the present study, an in vitro examination of the dermal absorption of N-(2-Nitrophenyl) phosphoric triamide (2-NPT) was performed according to OECD-Guideline 428. The test item was applied to human skin as follows, as no interferences with analytics were determined:

1 mL of 0.06 % 2-NPT (w/v) in PBS pH 7.4

The amount of 2-NPT which can be absorbed into the human organism through the skin was determined using human excised skin as an in vitro model for dermal absorption. The resorbed amounts of the test substance were quantified over a time period of 24 hours by analyzing samples from the acceptor compartment of Franz diffusion cells.

After the resorption experiment the amounts of 2-NPT taken up into the skin were determined. For this penetration study the stratum corneum was stripped by the so-called “tape stripping technology”. The strips were collected in two samples. The deeper skin layers were sliced in parallel sections using a cryo microtome. The skin slices were collected in one sample.

 

The used skin was qualified by performing a resorption study with caffeine over a period of 24 hours. Resorbed caffeine was quantified by a method developed and validated at Across Barriers. The resorption data were compared with historical data gained by the testing facility.

The analytical method for 2-NPT was obtained from sponsor and adapted. For the quantitative analysis of the test substances the following validation parameters were measured: the system suitability, the linearity and the LLOQ.

During the study the expected concentration of 0.03% in a product was doubled and the duration of incubation set to 24 h to get a measurable transport of the substance through the skin. For 2-NPT, no transport through the skin could be detected. Therefore, only a limit value < 6E-9 cm∙s^-1could be estimated. The maximum absorbed quantity into the skin was 0.92 µg.cm^-2for 2–NPT after 24 hours contact with the skin. This corresponds to 0.26 % of the applied dose 2-NPT.

Based on these results, it can be stated that there will be a negligible transport of 2-NPT into or through human skin, especially as the real life exposure levels are half of the used ones. Furthermore it can be assumed that the contact time will be much shorter, because the skin will be cleaned faster.