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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Already evaluated by the Competent Authorities for Biocides and Existing Substance Regulations.

Data source

Reference
Reference Type:
publication
Title:
In vitro Study of Percutaneous Absorption, Cutaneous Bioavailability and Bioequivalence of Zinc and Copper from Five Topical Formulations.
Author:
Pirot, F., Millet, J., Kalia, Y.N. & Humbert, P.
Year:
1996
Bibliographic source:
Skin Pharmacol. 9: 259-269

Materials and methods

Principles of method if other than guideline:
This was a non-regulatory study carried out to compare in vitro the percutaneous absorption and cutaneous bioavailability of zinc and copper
present in 5 topical formulations from differenct salts and vehicles. No guidelines are available to address this objective. This summary addresses
only those sections of the report that relate to copper salts.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Lot/batch: Not stated
Purity: Not stated.
Stability: Not stated
Five preparations were tested:
Emulsion A (water/oil) was formulated with 0.25% (w/w) ZnPC (Zn2+ = 0.05%), 1.5% (w/w) ZnO (Zn2+ = 1.21%)
and 0.5% (w/w) CuPC (Cu2+ = 0.1%).
Emulsion B (water/oil) was formulated with 0.25% (w/w) ZnSO4 (Zn2+ = 0.06%), 1.5% (w/w) ZnO and 0.5% (w/w) CuSO4 (Cu2+ = 0.13%).
Emulsion C (water/oil) was formulated with 0.1% (w/w) ZnSO4 (Zn2+ = 0.02%), 7% (w/w) ZnO (Zn2+ = 5.62%) and 0.2% (w/w) CuSO4 (Cu2+ = 0.05%).
Ointment D was formulated with 0.1% (w/w) ZnSO4 (Zn2+ = 0.02%), 10% (w/w) ZnO (Zn2+ = 8.03%) and 0.35% (w/w) CuSO4 (Cu2+ = 0.09%).
Ointment E was formulated with 10% (w/w) ZnO (Zn2+ = 8.03%) and 0.2% (w/w) CuSO4 (Cu2+ = 0.05%).
Radiolabelling:
no

Test animals

Species:
human
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
Samples of human abdominal skin were obtained from surgery.

Administration / exposure

Vehicle:
other: water/oil emulsions or unchanges solutions as given under test material information above.
Duration of exposure:
72 hrs
Doses:
16 mg/cm2 of each formulation was applied to the outer skin surface.
Details on study design:
Skin permeation studies; In the first experiment, percutaneous absorption and cutaneous bioavailability of zinc and copper from ZnPC and CuPC were compared with those from ZnSO4 and CuSO4, first using a similar (emulsion B) vehicle, and then in a vehicle of different composition (emulsion C). In the second experiment, the percutaneous absorption and cutaneous bioavailability of zinc and copper from ZnPC and CuPC were compared with those from ZnSO4 (and ZnO) and CuSO4 in ointments D and E.Samples were then mounted in Franz-type static diffusion cells with a 3.14 cm2 surface area. The volume of the receptor compartment was 9 ml, which was filled with 0.9% NaCl solution. The receptor fluid was magnetically stirred throughout the experiment and the temperature maintained at 33°C. 16 mg/cm2 of each formulation was applied to the outer skin surface. The receptor solution was replaced after 2, 4, 12, 24, 48 and 72 hours and kept for analysis. Three cells were used for each formulation. Receptor fluids were subjected to flame atomic absorption spectrometry (AAS) for Cu and Zn quantification at 324.7 and 213.9 nm, respectively, with an acetylene-air flame.
Details on in vitro test system (if applicable):
Skin permeation studies; Two series of experiments were done using two different sources of intact human skin. Skin samples were sliced with a dermatome to a thickness of 400 µm as assessed by high frequency (25 MHz) B-scan ultrasound imaging. All dermatomed skin samples were randomised to avoid source effect.

Results and discussion

Absorption in different matrices:
Percutaneous absorption of copper; Percutaneous copper absorption from topical application of five formulations represented less than 6% of the applied dose (range 0.66 – 5.04%), confirming the occurrence of minimal copper diffusion through human skin.

Any other information on results incl. tables

The diffusion of copper from all formulations (except emulsion B) appeared to be high for a period of up to 24 hours, as long as copper salts remained solubilised in the vehicle. After 72 hours, the amount of copper absorbed, expressed as % of applied dose) from CuPC in emulsion A was not significantly different from that of CuSO4 in emulsion B. Furthermore, there was no difference between delivery of copper from emulsion A and both ointments D and E. The results also suggested that the delivery of copper was not enhanced by replacing the sulphate counterion with the larger, more hydrophobic PC moiety (Table 1). 

Table 1. Comparisons of percutaneous absorption of Cu2+ in skin treated with 3 different emulsions (experiment 1) and 2 ointments (experiment 2); each value is the mean±SD of 3 measurements.

 

Formulations

Cu2+ absorbed (% of applied dose)

 

 

Salt

Conc. %

Total Cu2+ applied dose µg/cm2

2h

6h

24h

48h

72h

Flux for 72 h ng/cm2/h

Experiment 1

Em. A

CuPC

0.5

21±6

0.54±0.23

1.33±0.53

1.67±0.081

1.89±1.18

2.12±1.57

6±5

Em. B

CuSO4

0.5

22±4

0.52±0.08

0.52±0.08

0.52±0.08

0.66±1.17

0.66±1.17

2±4

Em. C

CuSO4

0.2

12±3

1.04±0.082

2.28±0.543

2.28±0.544

2.59±1.01

2.59±1.01

4±2

Experiment 2

Em. A

CuPC

0.5

19±5

1.26±0.52

1.67±1.15

2.90±1.05

3.60±1.59

5.04±2.86

13±8

Em. D

CuSO4

0.35

15±1

0.96±0.23

1.58±0.29

2.26±0.46

3.02±0.76

3.77±1.00

8±2

Em. E

CuSO4

0.2

12±3

0.77±0.16

1.54±0.33

2.41±0.48

3.39±0.36

3.40±0.35

6±1

 Fisher’s least significance difference test was used in all comparisons.

1p<0.05 as compared to B.

2p<0.05 as compared to A and B.

3p<0.05 as compared to A and B.

4p<0.05 as compared to B.

 

All formulations in experiment 1 increased the level of copper on average 4-fold, 2-fold and 5-fold in epidermis, dermis and whole skin respectively, as compared with skin control (Table 2). In experiment 2, a significant increase in the copper level in whole skin and epidermis was shown by skin treated with emulsion A and ointment D. No corresponding increase in copper level in dermis and in whole skin was shown from the 3 formulations or ointment E, respectively. It was considered that the increase in copper concentration in whole skin may have been due to a build-up of copper in epidermis, in effect forming a copper reservoir, followed by slow diffusion through the dermis.

 

Table 2. Cutaneous bioavailability of copper in treated skin with 3 different emulsions (experiment 1) and 2 ointments (experiment 2); each value is the mean±SD of 3 measurements, inmg/g.

  

Formulations

Epidermis

Dermis

Whole skin

Experiment 1

Em. A

473±253*

4±2 x 10–1*

10±7*

Em. B

540±334*

6±2*

11±6*

Em. C

600±310*

12±4*

21±15

Control

140±14

3±3 x 10-1

3±3 x 10-1

Experiment 2

Em. A

920±193*

10±6

33±3*

Oint. D

500±62*

18±8

25±6*

Oint. E

760±211*

16±8

11±4

Control

360±60

12±1

14±1

 * p< 0.05 as compared to control (Mann-Whitney test).

Multiple relative comparisons between treatments were made using the RII of copper (Figure 1 - attached). In experiment 1, the RII of copper in dermis and whole skin, both treated by emulsion C, was significantly higher than those calculated from emulsions A and B. RIIs of copper in epidermis, dermis and whole skin treated by emulsions A and B were not significantly different, so that higher delivery of copper from CuPC as compared to CuSO4could not be assumed. Furthermore, experiment 2 showed that RIIs of copper in epidermis and dermis treated by the 3 formulations were not significantly different. A significant RII of copper in whole skin treated by the emulsion A and the ointment D was shown as compared to skin treated by ointment E. However, RIIs of copper in whole skin treated by emulsion A and ointment D were not significantly different. As shown in experiment 2, delivery of copper in different layers of skin treated by CuPC in emulsion were not significantly higher than that from CuSO4in ointment.

Discussion:

Low copper concentrations were measured in the receptor solution, suggesting that percutaneous absorption was minimal across dermatomed skin. Conversely, topical application of formulations produced a significant increase of copper concentrations in epidermis and dermis, as compared to control data.

Bioequivalence of emulsions A and B was shown by means of the similar RII value for copper. However, no bioequivalence of copper from emulsions A and B was shown with ointments D and E. Consequently, a better cutaneous bioavailability of copper from organic salts (CuPC) as compared to mineral salts (CuSO4) was not found by using these emulsions.

Assessments of bioequivalence have been based only on dermatopharmakinetic studies in vitro, but nevertheless confirm minimal percutaneous absorption of copper through normal skin and compare the effects of the nature of the counter-ions and vehicles on cutaneous bioavailability.

Applicant's summary and conclusion

Conclusions:
Copper concentrations measured in receptor solution accounted for 0.66 – 5.04% of the applied dose, suggesting that percutaneous absorption was minimal across dermatomed skin. Conversely, topical application of formulations produced a significant increase of copper concentrations in epidermis and dermis, as compared to control data.

Bioequivalence of emulsions A and B was shown by means of the similar RII value for copper. However, no bioequivalence of copper from emulsions A and B was shown with ointments D and E. Consequently, it was not possible to demonstrate a better cutaneous bioavailability of copper from organic salts (CuPC), compared to mineral salts (CuSO4).
Executive summary:

Materials and Methods:

A study was carried out to to compare in vitro the percutaneous absorption and cutaneous bioavailability of Zn and Cu present in topical formulations containing different salts and vehicles. The study was not designed to follow an internationally accepted guideline and was not carried out in compliance with GLP.

 

The following five preparations were tested: Emulsion A (water/oil), formulated (w/w) with 0.25% Zinc 2-pyrrolidone 5-carboxylate (ZnPC), 1.5% zinc oxide (ZnO) and 0.5% copper 2-pyrrolidone 5-carboxylate (CuPC). Emulsion B (water/oil) was formulated with 0.25% zinc sulphate (ZnSO4.7H2O), 1.5% ZnO and 0.5% copper sulphate (CuSO4.5H2O). Emulsion C (water/oil) was formulated with 0.1% ZnSO4, 7% ZnO and 0.2% CuSO4. Ointment D was formulated with 0.1% ZnSO4, 10% ZnO and 0.35% CuSO4. Ointment E was formulated with 10% ZnO and 0.2% CuSO4.

 

Two series’ of experiments were done using 2 different sources of intact human skin. In the first experiment, percutaneous absorption and cutaneous bioavailability of Zn and Cu from ZnPC and CuPC were compared with ZnSO4and CuSO4, first using a similar (emulsion B) vehicle, and then in a vehicle of different composition (emulsion C). In the second experiment, percutaneous absorption and cutaneous bioavailability of Zn and Cu from ZnPC and CuPC were compared with ZnSO4(and ZnO) and CuSO4in ointments D and E.

Skin samples were sliced to a thickness of 400 µm and mounted in Franz-type static diffusion cells, which were then filled with 0.9% NaCl solution. The receptor fluid was magnetically stirred throughout the experiment and the temperature maintained at 33°C. 16 mg/cm2of each formulation was applied to the outer skin surface. The receptor solution was replaced after 2, 4, 12, 24, 48 and 72 hours and kept for analysis. Three cells were used for each formulation. Receptor fluids were subjected to flame AAS for Cu and Zn quantification.

 

After 72 hours, any remaining formulation on the skin surface was removed using cotton swabs. To ensure complete removal , the stratum corneum was stripped twice using adhesive tape. Samples of dermis and whole epidermis, were taken from treated skin by punch biopsy for assessment of Zn and Cu. A sample of whole skin was also taken. All samples were dried at 105°C, weighed, and dissolved in HNO3. The mixtures were then passed through a 0.22 µm filter and diluted 10- or 20-fold before determination of Cu and Zn using AAS.

Absorbed amounts of zinc and copper through human skin were analysed using ANOVA. When treatment effects were significant, all possible pairwise comparisons were analysed with Fisher’s protected least significant difference procedure. The chosen level of significance was p < 0.05. Mean Zn and Cu levels in treated skin were expressed as mg and µg of Zn and Cu respectively/gram dry tissue. Multiple comparisons between metal levels in treated skin to those in control skin were made using the Mann-Whitney test. The relative increase index (RII) was defined as the ratio between Zn or Cu concentration in treated skin to that in control skin (%), divided by the applied dose. RII values were compared using a Mann-Whitney test.

 

Results and Discussion:

Percutaneous Cu absorption from topical application of 5 formulations represented less than 6% of the applied dose (range 0.66 – 5.04%), confirming minimal diffusion of Cu through dermatomed human skin. Cu diffusion from all formulations (except emulsion B) appeared to be higher for up to 24 hours, as long as Cu salts remained solubilised in the vehicle. After 72 hours, the % of applied Cu absorbed from CuPC in emulsion A was not significantly different from that of CuSO4in emulsion B. Furthermore, there was no difference between delivery of Cu from emulsion A and both ointments D and E. The results suggested that the delivery of Cu was not enhanced by replacing the sulphate counter-ion with the larger, more hydrophobic PC moiety.

All formulations in experiment 1 increased the level of Cu, on average 4-, 2- and 5-fold, in epidermis, dermis and whole skin respectively, as compared with control. In experiment 2, a significant increase in the Cu level in whole skin and epidermis was shown by skin treated with emulsion A and ointment D. No corresponding increase in Cu level in dermis and in whole skin was shown from the 3 formulations or ointment E, respectively. It was considered that the increase in Cu concentration in whole skin may have been due to a build-up of Cu in epidermis, in effect forming a Cu reservoir, followed by slow diffusion through the dermis.

 

Multiple relative comparisons between treatments were made using the RII of Cu. In experiment 1, the RII of Cu in dermis and whole skin, both treated by emulsion C, was significantly higher than those calculated from emulsions A and B. RIIs of Cu in epidermis, dermis and whole skin treated by emulsions A and B were not significantly different, so that higher delivery of Cu from CuPC as compared to CuSO4could not be assumed. Furthermore, experiment 2 showed that RIIs of Cu in epidermis and dermis treated by the 3 formulations were not significantly different. A significant RII of Cu in whole skin treated by the emulsion A and the ointment D was shown as compared to skin treated by ointment E. However, RIIs of Cu in whole skin treated by emulsion A and ointment D were not significantly different. As shown in experiment 2, delivery of Cu in different layers of skin treated by CuPC in emulsion were not significantly higher than that from CuSO4in ointment.