diethyl pyridine-2,4-dicarboxylate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-10-09 to 2009-06-16

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

R0037857A

IUPAC Name:

R0037857A

Constituent 2

Reference substance name:

Mexoryl SBU

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

Mexoryl SBU

Constituent 3

Reference substance name:

diethyl pyridine-2,4-dicarboxylate

EC Number:

680-341-5

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

diethyl pyridine-2,4-dicarboxylate

Test material form:

other: Liquid (partly or fully solidified according to the temperature)

Radiolabelling:

yes

Test animals

Species:

other: rat and human

Strain:

other: Rat Wistar and human skin

Sex:

male

Details on test animals or test system and environmental conditions:

Skin membranes1 Human dermatomed skin preparationHuman skin samples were obtained from a tissue bank and stored frozen at approximately -20 °C until required for use. Skin membranes were cut from the samples at a thickness setting of 400 μm using an electric dermatome.2 Rat full thickness skin preparationClipped dorsal skin from euthanised male rats aged 42-56 days of the Wistar Crl:(WI)BR strain supplied by Charles River UK Ltd, Margate, Kent, UK. were used.Each membrane was given an identifying number and stored frozen, at approximately -20°C, on aluminium foil until required for use.The thickness of the rat skin membranes used on the study was measured with digital callipers and ranged from 1.01 mm to 1.92 mm. Assembly of diffusion cellsThe type of static glass diffusion cell used in this study has an exposed membrane area of 2.54 cm2 and a volume of approximately 4.5 mL. Discs of approximately 3.3 cm diameter of prepared skin membrane were mounted, dermal side down, in diffusion cells held together with individually numbered clamps and placed in a water bath maintained at 32 °C ± 1 °C

Administration / exposure

Type of coverage:

open

Vehicle:

other: denatured alcohol

Duration of exposure:

24h

Doses:

One dose was prepared, conforming to instructions supplied by the client, as close to the time of application as was practicable. The preparation was analysed to confirm its suitability for use in the study .The radioactivity content of the dosing preparation was determined by taking 10 μL aliquots, in triplicate, into 25 mL volumetric flasks. The volumetric flasks were made up to volume with denatured alcohol before taking 100 μL subsamples, in triplicate for analysis by LSC. The dose preparation was analysed by LSC to determine homogeneity and the specific activity of radiolabel dosed.The radiochemical purity of the [14C]-radiolabelled R0037857A and the radiolabelled content of the R0037857A in the dose preparation was established by HPLC both prior to application and following the 24 hour exposure. The stability of the radiolabelled R0037857A in the dose preparation was also established over an interval longer than the intended period of use in this study by HPLC.[14C]-R0037857A (TS00043/001) in denatured alcohol, equivalent to an activity of 17.6 MBq (1.99 mg R0037857A) was placed into a glass vial. The weight of denatured alcohol present in the vial was adjusted to 1139 mg following reduction of the volume using nitrogen gas and the small addition (70 mg) of further denatured alcohol. Unlabelled R0037857A (TS00043/002), actual weight 97.7 mg, was added and the preparation mixed thoroughly. Once the unlabelled material had dissolved, 761 mg of water was added. The preparation was mixed thoroughly by hand shaking and continuously stirred with a magnetic stirrer.

Control animals:

yes

Remarks:

Two control skin membranes were also included and remained untreated for the duration of the study

Details on study design:

Measurement of membrane integrityMembrane integrity was determined by measurement of the electrical resistance across the skin membrane. Membranes with a measured resistance of <10 kΩ (human) or <2.5 kΩ (rat) were regarded as having a lower integrity than normal and not used for exposure to the test materials

Details on in vitro test system (if applicable):

Cells were selected such that each application was represented by eight intact membranes from at least three different donors/animals. Two control skin membranes were also included and remained untreated for the duration of the study.The receptor chambers of the cells, containing small magnetic stirrer bars, were filled with a recorded volume of phosphate buffered saline (without CaCl2 and MgCl2). The cells were not placed in a water bath at this stage. This receptor fluid ensured that the test substance could freely partition into the receptor fluid from the skin membrane and never reached a concentration that would limit its diffusion.A pre-treatment sample (500 μL) was taken from each receptor chamber for analysis by LSC. An equal volume of fresh receptor fluid was added to each receptor chamber to replace the volume removed.The dose was applied to the skin and the weight of the applied dose recorded. Immediately after dosing, an activated carbon filter was placed over the donor chamber and the cells were placed in a water bath maintained at a temperature of 32°C ± 1°C. The cells remained unoccluded for the duration of the 24 hour exposure period.Sampling of receptor fluid: Samples of receptor fluid (500 μL) were taken using an autosampler 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours after application for analysis by LSC.The volume of fluid in the receptor chamber was maintained by the replacement of a volume of receptor fluid, equal to the sample volume immediately after each sample was taken.Measurement of mass balance: After the final receptor fluid sample had been taken, the remaining fluid in the receptor chamber was discarded and the chamber rinsed with fresh receptor fluid which was also discarded.The carbon filters were removed and immediately placed into a vial containing ethanol. A subsample of the extract was analysed by LSC.The epidermal surface of the human and rat skin was rinsed using the following procedure:1) 300 μL/cm2 aliquot of 3.5% sodium lauryl sulphate aqueous solution. The same aliquot was aspired and repelled three times on the skin.2) 300 μL/cm2 aliquot of 3.5% sodium lauryl sulphate aqueous solution. The same aliquot was aspired and repelled three times on the skin.3) 300 μL/cm2 aliquot of water. The same aliquot was aspired and repelled three times on the skin.4) 300 μL/cm2 aliquot of water. The same aliquot was aspired and repelled three times on the skin.5) Skin drying with natural spongesThe SLS and water skin wash samples plus the sponges used for skin drying, were placed in a 25 mL volumetric flask. A 20 mL volume of ethanol was added and the samples left to extract for approximately 5 days. The ethanol extracts were decanted into further 25 mL volumetric flasks that were made up to volume with ethanol. Subsamples of the extracts were taken for LSC analysis. The volumetric flasks containing the sponges were made up to volume with Soluene 350® and the sponges allowed to digest fully before taking subsamples for LSC analysis.The donor chamber was carefully removed and washed with ethanol. Subsamples were taken for analysis by LSC.The surface of the skin was allowed to dry naturally.To assess penetration through the human and rat stratum corneum, successive layers of the skin surface were removed by the repeated application of adhesive tape (e.g. Scotch 3M Magic Tape, 1.9cm wide), to a maximum of 21 strips. A strip of adhesive tape was pressed onto the skin surface and then carefully peeled off to remove the stratum corneum. The adhesive strips were soaked in ethanol to extract any test material. The first 5 strips were extracted individually and the remaining strips were combined in groups of 4 and each group extracted. The extracts were sequentially numbered and analysed by LSC. In some cases, it was not possible to take the full 21 tape strips as the skin began to tear, therefore, tape stripping was discontinued. The last tape strip for this diffusion cell was digested/extracted with the remaining epidermis, so as not to underestimate residues in the remaining epidermis/dermis compartment.The human and rat flange skin (skin that was held between the donor and receptor chamber) was cut away from the epidermis/dermis, digested with Soluene 350® and analysed by LSC.Human skin only – The epidermis on the remaining skin disc was separated from the dermis by heat separation. The skin was placed dermis side down, on cling film and a second piece of cling film was used to cover the epidermis side. A 200 g weight was heated to 65 °C in a water bath and placed onto the epidermal surface with moderate pressure for approximately 90 seconds. The epidermis was peeled away from the dermis using forceps. The remaining dermis was digested with Soluene 350® and analysed by LSC.Rat skin only – The heat separation of the epidermis from the dermis was not performed for rat skin owing to its fragility. The remaining skin disk (epidermis/dermis) was digested with Soluene 350® and analysed by LSCData evaluation:Results of the analysis of the samples of receptor fluid collected in the study were expressed as amounts of R0037857A in the receptor solution in terms of μg/cm². The amounts penetrated, rates of penetration (μg/cm²/h) and ‘percentage of dose penetrated’ were determined using the calculation:Equation 1:Receptor volume (mL) = VSample replacement volume (mL) = vConcentrations (μg/mL) at sampling times (h) t1, t2, t3 etc. are c1, c2, c3 etc.The total amount of penetrant having passed through the epidermal membrane into the receptor fluid, corrected for sample volume removed (c1v, c2v, c3v etc.) at sampling times t1, t2, t3 etc. are:t1 = c1Vt2 = c2V + c1vt3 = c3V + c1v + c2vt4 = c4V + c1v + c2v + c3vetc.The amount of test penetrant at each time point is divided by the area of epidermal membrane (2.54cm²) and the results plotted as amount of penetrant penetrated (μg/cm²) versus time (h). The slope of this penetration profile between given time points gives the average rate of penetration of the penetrant per cm² of the skin (μg/cm²/h) during that period.Equation 2% Penetrated = (Mean amount penetrated (μg/cm²) /Mean amount applied (μg/cm²)) x 100.The results of the mass balance and distribution determinations were expressed in terms of amount penetrated and ‘percentage of applied dose’.Tables and appendices presented in the report are computer generated. The group mean and individual data are rounded up appropriately for inclusion in the report. As a consequence, calculation of group mean data from the individual data may yield a minor variation in the value reported.Definition of systemically available dose: The systemically available dose is considered to be the R0037857A detected in the sum of the (remaining) epidermis, dermis and receptor fluid. Material removed from the surface of the epidermis by the washing procedure is regarded as not penetrated.

Results and discussion

Absorption in different matrices:

Human dermatomed skin: The penetration of R0037857A through human skin was linear throughout the entire 24 hour exposure period. Between 0-24 hours, the penetration rate was 5.64 μg/cm²/h.The amounts that penetrated through human skin at 6, 8 and 10 hours were 33.6, 43.7 and 54.5 μg/cm², respectively. These respective amounts expressed as percentages of the applied dose were 6.73, 8.74 and 10.9 %. The amount penetrated over the entire 24 hour exposure period was 140 μg/cm² corresponding to 27.9 % of the applied dose.No dosed cells were rejected, all eight individual mass balances being within the range of 90-110%.Mean recovery of the applied test material was 92.3 ± 1.4 %.The greatest proportion of the applied dose was removed by washing the surface of the skin 24 hours after application and amounted to 54.8 % of the applied dose. The amount trapped in the carbon filter was very low (0.091%).The amount of the dose present in the outer layers of the stratum corneum was 0.257 % of the applied dose (1.28 μg/cm²) with 0.558 % of the dose (2.79 μg.cm²) present in the remaining epidermal tissue.The amount recovered from dermis was 4.04 % (20.2 μg/cm²) of the applied dose with 1.98 % of the dose being present in the flange.The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 27.9 %. This percentage equated to 140 μg/cm2.The total non-absorbed dose (carbon filter, donor chamber, skin wash, stratum corneum and flange skin) represented 59.8% (299 μg/cm²).The total systemically available value (remaining epidermis, dermis and receptor fluid) was 32.5 ± 7.3 % of the applied dose corresponding to 162 ± 36.3 μg/cm².Rat full thickness skin: After a short lag phase of about 2 hours, the R0037857A penetration rate was 2.01 μg/cm²/h between 2-4 hours after which penetration through rat skin increased to 4.16 μg/cm²/h between 4-12 hours. The fastest rate of R0037857A penetration occurred between 12-24 hours (5.34 μg/cm²/h). Between 0-24 hours, the penetration rate was 4.41 μg/cm²/h.The amounts that penetrated through rat skin at 6, 8 and 10 hours were 11.1, 19.1 and 28.1 μg/cm², respectively. These respective amounts expressed as percentages of the applied dose were 2.23, 3.82 and 5.62 %. The amount penetrated over the entire 24 hour exposure period was 102 μg/cm² corresponding to 20.4 % of the applied dose.No dosed cells were rejected, all eight individual mass balances being within the range of 90-110%.Mean recovery of the applied test material was 101 ± 3.3 %.The greatest proportion of the applied dose was removed by washing the surface of the skin 24 hours after application and amounted to 62.6 % of the applied dose.The amount of the dose present in the outer layers of the stratum corneum was 2.09 % of the applied dose (10.4 μg/cm²) with 12.0 % of the dose present in the epidermal (plus dermal) tissue (60.2 μg/cm²).The proportion of the dose recovered from the flange was 3.57 % of the applied dose.The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 20.4 %. This percentage equated to 102 μg/cm².The total non-absorbed dose (carbon filter, donor chamber, skin wash, stratum corneum and flange skin) represented 68.6% (342.9 μg/cm²).The total systemically available value (epidermis plus dermis and receptor fluid) was 32.4 ± 7.6 % of the applied dose corresponding to 162 ± 38 μg/cm².

Percutaneous absorption

Percutaneous absorption 1

Dose:

162 μg/cm²

Parameter:

percentage

Absorption:

ca. 32.5 %

Remarks on result:

other: 24h

Remarks:

Human

Percutaneous absorption 2

Dose:

162 μg/cm²

Parameter:

percentage

Absorption:

ca. 32.4 %

Remarks on result:

other: 24h

Remarks:

Rat

Any other information on results incl. tables

Human dermatomed skin

 

Test Compartmentn = 8	μg R0037857A per cm²		% of applied dose
	Mean	SD	Mean	SD
Carbon filter	0.454	0.101	0.091	0.020
Donor chamber	12.9	8.39	2.59	1.68
Skin wash	274	40.5	54.8	8.10
Flange	9.91	11.0	1.98	2.20
Stratum corneum	1.28	1.23	0.257	0.246
Remaining epidermis	2.79	1.38	0.558	0.276
Dermis	20.2	7.39	4.04	1.48
Receptor fluid	140	30.7	27.9	6.14
Total non-absorbed	299	32	59.8	6.6
Systemically available	162	36.3	32.5	7.26
Total recovered	461	7.03	92.3	1.41

 

Rat full-thickness skin

Test Compartmentn = 8	μg R0037857A per cm²		% of applied dose
	Mean	SD	Mean	SD
Carbon Filter	0.241	0.039	0.048	0.008
Donor chamber	1.61	1.30	0.322	0.259
Skin wash	313	55.1	62.6	11.0
Flange	17.9	10.7	3.57	2.13
Stratum corneum	10.4	4.29	2.09	0.858
Epidermis (+ dermis)	60.2	17.8	12.0	3.55
Receptor fluid	102	39.6	20.4	7.93
Total non-absorbed	343	45	68.6	9.0
Systemically available	162	38.1	32.4	7.62
Total recovered	505	16.4	101	3.28

 

Systemically available = Sum of (remaining) epidermis, dermis and receptor fluid.

Total non-absorbed = Sum of carbon filter, donor chamber, skin wash, flange andstratum corneum.

Skin wash = amount in skin wash plus sponge digests.

Stratum corneum= amount in tape strips.

Remaining epidermis = epidermal tissue remaining after tape stripping.

Applicant's summary and conclusion

Conclusions:

The results obtained in this study indicate that R0037857A penetrated through human and rat skin at a slow rate from a 5 %R0037857A test preparation. The extent of penetration through human and rat skin amounted to 27.9 % (140 μg/cm2) and 20.4 % (102 μg/cm2), respectively after 24 hours.For both human and rat skin, a large proportion of the applied dose remained on the skin surface after a 24 hour exposure interval and was readily removed by a gentle skin washing procedure. Low proportions of the dose were associated with the stratum corneum and remaining epidermal membranes.The total systemically available value (epidermis plus dermis and receptor fluid) was similar for human and rat skin and represented 32.5 % and 32.4 % of the applied dose, respectively (corresponding to 162 μg/cm² and 162 μg/cm², respectively).

Executive summary:

The purpose of this study was to determine the in vitro penetration of R0037857A through human dermatomed skin and rat full thickness skin over a 24 hour exposure period to aid the quantitative assessment of the risk arising from skin contact with a formulation containing a nominal 5 % R0037857A. The mass balance and distribution of R0037857A within the test system following the 24 hour exposure was also determined.

The study was conducted according the OECD principles of Good Laboratory Practice and was performed following the OECD and SCCP guidelines and guidance documents for skin absorption studies.

Prior to dosing, the membrane integrity was checked by measurement of electrical resistance. The doses were applied to the 8 skin membranes of each skin model at a rate of 10 mg/cm2 corresponding to 0.5 mg/cm2 of R0037857A. The applications were left unoccluded, however the donor chambers were covered with activated carbon filters to capture any volatile components. The exposure period was 24 hours following which, the skin surface was washed with 3.5% sodium lauryl sulphate aqueous solution (2 x 300μL/cm²), followed by water (2 x 300μL/cm²) and then dried with natural sponges.

The application rates and exposure conditions used in this study were designed to simulate predicted normal human exposure to the test material.

The penetration process was monitored using [14C]-radiolabelled R0037857A, which was incorporated into the formulation, prior to application. The receptor fluid was phosphate buffered saline (PBS). The distribution of R0037857A within the test system was investigated and a 24 hour penetration profile was determined by collecting receptor fluid samples in hourly samples from 0 to 4 hours and then 2 hourly samples from 6 to 24 hours. The samples were analysed by liquid scintillation counting (LSC).

Human dermatomed skin: The penetration of R0037857A through human skin was linear throughout the entire 24 hour exposure period. Between 0-24 hours, the penetration rate was 5.64 μg/cm2/h.

The amounts that penetrated through human skin at 6, 8 and 10 hours were 33.6, 43.7 and 54.5 μg/cm2, respectively. These respective amounts expressed as percentages of the applied dose were 6.73, 8.74 and 10.9 %. The amount penetrated over the entire 24 hour exposure period was 140 μg/cm2, corresponding to 27.9 % of the applied dose.

Rat full thickness skin: After a short lag phase of about 2 hours, the R0037857A penetration rate was 2.01 μg/cm2/h between 2-4 hours after which penetration through rat skin increased to 4.16 μg/cm2/h between 4-12 hours. The fastest rate of R0037857A penetration occurred between 12-24 hours (5.34 μg/cm2/h). Between 0-24 hours, the penetration rate was 4.41 μg/cm2/h.

The amounts that penetrated through rat skin at 6, 8 and 10 hours were 11.1, 19.1 and 28.1 μg/cm2, respectively. These respective amounts expressed as percentages of the applied dose were 2.23, 3.82 and 5.62 %. The amount penetrated over the entire 24 hour exposure period was 102 μg/cm2, corresponding to 20.4 % of the applied dose.

Human dermatomed skin: No dosed cells were rejected. Mean recovery of the applied test material was 92.3 ± 1.4 %.

The greatest proportion of the applied dose was removed by washing the surface of the skin 24 hours after application and amounted 54.8 % of the applied dose. The amount trapped in the carbon filter was very low (0.091%).

The amount of the dose present in the outer layers of the stratum corneum was 0.257 % of the applied dose (1.28 μg/cm²) with 0.558 % of the dose (2.79 μg.cm²) present in the remaining epidermal tissue.

The amount recovered from dermis was 4.04 % of the applied dose (20.2 μg/cm²).

The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 27.9 %. This percentage equated to 140 μg/cm2. The total non-absorbed dose (carbon filter, donor chamber, skin wash, stratum corneum and flange skin) represented 59.8% (299 μg/cm²). The total systemically available value (remaining epidermis, dermis and receptor fluid) was 32.5 ± 7.3 % of the applied dose corresponding to 162 ± 36.3 μg/cm².

Rat full thickness skin: No dosed cells were rejected. Mean recovery of the applied test material was 101 ± 3.3 %.

The greatest proportion of the applied dose was removed by washing the surface of the skin 24 hours after application and amounted to 62.6 % of the applied dose.

The amount of the dose present in the outer layers of the stratum corneum was 2.09 % of the applied dose (10.4 μg/cm²) with 12.0 % of the dose present in the epidermal (plus dermal) tissue (60.2 μg/cm²).

The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 20.4 %. This percentage equated to 102 μg/cm². The total non-absorbed dose (carbon filter, donor chamber, skin wash, stratum corneum and flange skin) represented 68.6% (342.9 μg/cm²). The total systemically available value (epidermis plus dermis and receptor fluid) was 32.4 ± 7.6 % of the applied dose corresponding to 162 ± 38 μg/cm².