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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Already evaluated by the Competent Authorities for Biocides and Existing Substance Regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
yes
Principles of method if other than guideline:
OECD Test Guideline 428 (adopted 12 April 2004) indicates that the amount of test substance should be measured in the receptor fluid, skin preparations, skin surface washings and apparatus rinse. In this study copper content was measured in the receptor fluid only. However, the objectives of this study (to determine the extent of absorption and to compare the absorption of the test substance) are met.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Cu2+ as Cuprous Oxide
IUPAC Name:
Cu2+ as Cuprous Oxide
Constituent 2
Reference substance name:
Cu2+ as Cuprous thiocyanate
IUPAC Name:
Cu2+ as Cuprous thiocyanate
Constituent 3
Reference substance name:
Cu2+ as Copper powder
IUPAC Name:
Cu2+ as Copper powder
Constituent 4
Reference substance name:
Cu 2+ as Cupric sulphate
IUPAC Name:
Cu 2+ as Cupric sulphate
Details on test material:
For the purpose of this experiment a composite sample was prepared from three different suppliers of cuprous oxide (Red Copp 97N Premium, Cuprous oxide paint grade red, and Cuprous oxide red).Lot/Batch Number:Cuprous oxide (Red Copp 97N Premium): 39140Cuprous oxide (Cuprous Oxide, Paint Grade, Red Micromilled): Product No. 230902Cuprous oxide (Cuprous oxide, Red): Lot 30159Cuprous thiocyanate: Batch ref. 02.4.2Copper powder (Copper Flake Powder): NACupric sulphate: NADescription:Cuprous oxide (Red Copp 97N Premium): Red powderCuprous oxide (Cuprous Oxide, Paint Grade, Red Micromilled): Red powderCuprous oxide (Cuprous oxide, Red): Red powderCuprous thiocyanate: Near white powderCopper powder (Copper Flake Powder): Copper coloured powderCupric sulphate: NAPurity:Cuprous oxide (Red Copp 97N Premium): Total Copper: 88 %. Cu2O: 97.5 %.Cuprous oxide (Cuprous Oxide, Paint Grade, Red Micromilled): Total Copper: 87 %. Cu2O: 98 %.Cuprous oxide (Cuprous oxide, Red): Total Copper: 87.5 %. Cu2O: 98.1 %.Cuprous thiocyanate: Total CuSCN: 99.51 %. Total Copper: 51.99 %.Copper powder (Copper Flake Powder): Total Copper > 96 %. Copper oxide < 3 %.Cupric sulphate: NAStability:Cuprous oxide (Red Copp 97N Premium): Expiry date - 23 April 2004.Cuprous oxide (Cuprous Oxide, Paint Grade, Red Micromilled): NACuprous oxide (Cuprous oxide, Red): NACuprous thiocyanate: NACopper powder (Copper Flake Powder): NACupric sulphate: NA
Radiolabelling:
no

Test animals

Species:
human
Sex:
male/female

Administration / exposure

Doses:
Trial dose preparations were used to ascertain the optimum slurry density that could be applied to the skin, giving a homogeneous formulation as well as maintaining equivalent levels of copper in each dose.
A single application of the test formulations using a finite volume (10 µl) was applied. The achieved doses with respect to the copper content in the cells were as follows:

Cuprous oxide: 997.8µg
Cuprous thiocyanate: 693.1µg
Copper powder: 925.7µg
Cupric sulphate: 894.9µg
Details on study design:
Assessment of skin absorption:
Aliquots of the receptor fluid (2.5 ml) were taken from each diffusion cell at the end of the experiment (either 6 or 24 hours), and analysed for copper content by atomic absorption spectrophotometry. Untreated control diffusion cells containing un-dosed skin membranes were also set up to assess background levels of copper.
Details on in vitro test system (if applicable):
Full thickness human skin:
The skin samples were supplied by University College London tissue bank. They were received at Huntingdon Life Sciences, Eye, Suffolk and stored at -20°C.
The specifications associated with each skin sample were as shown in Table 1.

Prior to use, the skin samples were thawed to room temperature. The resulting full thickness skin membrane was then swabbed with 70% ethanol/water to remove residual fat and blood, and rehydrated with a thin film of water ready for dermatoming.

Dermatomed skin:
The full thickness skin sample was pinned out on a dermatome board (cork board with raised rubber cutting surface) and a mini-dermatome used to cut slices of skin which contained epidermis and some dermis (thickness measured using a digital calliper to be approximately 300 µm).

Membrane integrity:
The integrity of the skin membranes was determined by measurement of the rate of penetration of tritiated water (3H20). Skin membranes with a Kp value of greater than 3.5 x 10-3 cm/hr were considered damaged and were therefore not used in the calculation of absorption of the test substance. Membranes that had absorption rates for test substance which were clearly distinct from the remaining absorption rates for that group (i.e. contamination was suspected) were also excluded.

Group assignment:
For each of the test substances there were three sampling regimes. The first terminated at 6 hours after application of the test substance, and the second was terminated at 24 hours after application of test material. In each case the skin surface was swabbed (skin was swabbed using 1% Tween 80 in distilled water on three cotton wool buds. A dry cotton wool bud was then used to remove residual 1% Tween 80 solution) at 6 hours after application of the test substance to remove unabsorbed material (representing a wash procedure at the end of a working day). The third sampling regime terminated at 24 hours post-application but no swab was performed. For each regime, the receptor fluid was sampled at termination only
Five static diffusion cells were prepared for each test formulation at each sampling regime.

Diffusion cell design and set up:
Static diffusion cells providing an exposure area of approximately 0.95 cm2 were used. Skin samples were cut from the dermatomed slice and placed onto the receptor chamber of the diffusion cell. The donor chamber was then fixed in place. The receptor fluid used during the membrane integrity test was distilled water. The receptor fluid used following application of the test formulations was 10 mM phosphate buffered saline, supplemented with 5% w/v bovine serum albumin, adjusted to pH 7.4. The receptor chamber was filled so that the underside of the skin membrane was in full contact with the receptor fluid. A stirrer bar was inserted into the receptor chamber and the receptor fluid continuously stirred and maintained at 32°C by placing the diffusion cells onto a multi-plate magnetic stirrer in a water bath.

Administration and sampling:
After the receptor chamber of the diffusion cell was rinsed out to remove residual 3H20 following the membrane integrity test, the receptor chamber was filled with 3 ml of receptor fluid. The diffusion cells were returned to the multi-plate magnetic stirrer in the temperature controlled water bath (maintained at 32°C).
The test formulation (10 µ1) was applied to the skin-membrane and the cell was left non-occluded.
The actual amount of test formulation applied was determined from quality control (QC) checks taken before, during and after dosing each group of cells.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
See Table 2 for the copper content found in the receptor fluid for each test substance and each sampling regime.

Any other information on results incl. tables

Copper content in receptor fluid samples following a single topical application of test material to dermatomed human skin.

Results are expressed both as percent applied dose and as ng copper.

Table 2.

Test Material

Cuprous oxide composite

Cuprous thiocyanate

Copper powder

Cupric sulphate

Dose level (mg/cm2)

1.050

0.730

0.974

0.942

Group

1

2

3

4

5

6

7

8

9

10

11

12

Termination time (h)

6

24

24

6

24

24

6

24

24

6

24

24

Swabbed at 6 h (y/n)

y

y

n

y

y

n

y

y

n

y

y

n

Amount absorbed (%)

0.098

0.157

0.193

0.128

0.126

0.147

0.086

0.038

0.046

0.097

0.136

0.038

Amount absorbed (ng)

977.2

1564.6

1926.0

887.8

871.3

1019.6

800.0

352.9

427.9

868.4

1216.5

337.6

Rate of absorption (ng/cm2/hr)

171.4

68.62

84.47

155.8

38.22

44.72

140.4

15.48

18.77

152.4

53.36

14.81

Absorption ratio*

1.15

0.92

0.28

1.00

* Absorption ratio given with respect to cupric sulphate, using the amount absorbed at 24 hours following a swab at 6 hours post-dosing.

Applicant's summary and conclusion

Conclusions:
The level and rate of penetration of copper resulting from the exposure of human skin to each of the test materials were compared against those for cupric sulphate. The results for cuprous oxide composite, cuprous thiocyanate and cupric sulphate are all similar with only the copper powder showing lower penetration. If all the dermal penetration values for copper (6 hour exposure, 24 hour sampling) are pooled, then the mean value is 0.1106% with a standard deviation of 0.067 (CV 60.94%).
Executive summary:

Materials and Methods

The comparative in vitro dermal penetration of copper through human skin was examined following the application of four different copper containing formulations.  Cuprous oxide composite (approximately 1.05 mg/cm2), cuprous thiocyanate (approximately 0.73 mg/cm2), copper powder (approximately 0.97 mg/cm2) and cupric sulphate (approximately 0.94 mg/cm2), were applied at similar dose levels with respect to copper content.

Five static diffusion cells were prepared for each test formulation at each sampling regime.  Dermatomed membranes (approximately 300 µm thickness) were maintained in the cells at approximately 32°C.  The integrity of the membranes was first tested using tritiated water (3H20), and any cells showing a permeability coefficient for water of greater than 3.5 x 10-3 cm/hr were considered damaged and therefore not reported.  After removal of the residual 3H20, the test formulation was applied to the unoccluded skin samples at a rate of l0µl/cm2.

For each of the test substances there were three sampling regimes.  The first terminated at 6 hours after application of the test substance, and the second was terminated at 24 hours after application of test material.  In each case the skin surface was swabbed at 6 hours after application of the test substance to remove unabsorbed material (representing a wash procedure at the end of a working day).  The third sampling regime terminated at 24 hours post-application but no swab was performed.  For each regime, the receptor fluid was sampled at termination only.

 

Results and Discussion

Comparison of the amount of copper absorbed through human skin following the application of each test material formulation is best assessed using the most occupationally relevant sampling regime (swab at 6 hours, receptor fluid collected at 24 hours post application).

The results may be summarised as follows:

 

Product name

% applied dose

Ratio

Cuprous oxide composite

0.157

1.15

Cuprous thiocyanate

0.126

0.92

Copper powder

0.038

0.28

Cupric sulphate

0.136

1.00

 

The absorption rate (ng/cm2/h) would normally be calculated from the steepest part of the absorption profile, which is indicative of steady-state absorption.  Since the cells that were sampled at 6 hours gave the highest rate of absorption (and are therefore the closest to steady-state absorption), the absorption rate has been measured using these values. 

The results may be summarised as follows:

 

Product name

ng/cm2/h

Cuprous oxide composite

171.4

Cuprous thiocyanate

155.8

Copper powder

140.4

Cupric sulphate

152.4

 

The level and rate of penetration of copper resulting from the exposure of human skin to each of the test materials were compared against those for cupric sulphate (see the ratios for % dose applied in the table above).  There was very little penetration of copper through the skin from any of the test formulations.  The absorption of copper from the cuprous oxide composite, cuprous thiocyanate and cupric sulphate formulations was similar with between 0.126 and 0.157% of the dose being absorbed over the 24 hour period.  The level of absorption was lower from the copper powder formulation (0.038%).  If all the dermal penetration values for copper (6 hour exposure, 24 hour sampling) are pooled, then the mean value is 0.1106% with a standard deviation of 0.067 (CV 60.94%).  It should however be noted that there was some variation in the data between cells.  This was in part due to the fact that the level of absorption was extremely low for all of the formulations and the values obtained were very close to the LOQ level, and in part due to the natural variation in absorption between individual skin donors.

It is clear from the results that none of the copper compounds tested readily penetrate human skin when applied as aqueous formulations.