Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2006 - December 2006.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to internationally accepted guidelines and under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Sodium chlorate
Batch number: 0009/06
Storage: At room temperature in the dark
Expiry date: 15 February 2007
Purity: 99.66% (from CoA)
Radiolabelling:
no
Remarks:
The test substance was not radiolabelled, 3H2O was used to assess integrity of the skin membranes.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
Rat skin:
- Source: Charles River UK Ltd, Margate, Kent, UK.
- Age at study initiation: 28 - 32 days
- Weight at study initiation: 119-134g
- Fasting period before study: not applicable
- Housing: stainless-steel cages with suspended mesh floors in groups of up to a maximum of six rats per cage
- Individual metabolism cages: not applicable
- Diet (e.g. ad libitum): VRF1 diet (Special Diet Services, Witham, UK), ad libitum
- Water (e.g. ad libitum): potable water (Anglian Water), ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Human skin:
The skin samples were obtained from human donors post-mortem and were supplied by International Institute for the Advancement of Medicine and Asterand Inc. They were stored at ca -20ºC.

The provenance of each skin sample were as follows:
Skin number Sex Anatomical region Age Receipt at Huntingdon Life Sciences
H1 Female Abdominal 73 22 September 2005
H2 Male Abdominal 47 13 September 2006
H3 Female Abdominal 64 9 March 2005
H4 Female Back 47 14 December 2005

Administration / exposure

Type of coverage:
other: The skin was placed in static glass diffusion cells
Vehicle:
other: High dose: Unchanged, no vehicle. Low dose: water.
Duration of exposure:
24 hours
Doses:
- Nominal doses:
High dose (Group 1)
Solid granular sodium chlorate was supplied ready for use by the Study Sponsor and therefore no dose preparation was necessary.
Low dose (Group 2)
An aqueous dilution of the sodium chlorate at a concentration of sodium chlorate of 15 g/l was prepared. Solid granular sodium chlorate (0.1502 g) was weighed into a pre-weighed vial. Water was added to a final volume of 10 ml. The diluted dose formulation was magnetically stirred throughout the dosing procedure.
- Actual doses calculated as follows: To accurately determine the quantity of test substance administered to each cell, quality control (QC) doses (9.5 µl) of the test substance were dispensed into vials at intervals throughout the dosing procedure. The QC samples were retained for analysis using the validated analytical method.
- Actual doses: At the low dose level, the achieved dose levels of sodium chlorate applied to the skin were determined by analysis of QC samples taken before, during and after dosing the cells. The achieved doses of sodium chlorate were 145.4 µg per cell, equivalent to 153.1 µg/cm2. At the high dose level, the achieved dose levels of sodium chlorate applied to the skin were determined by direct weighing of each dose. The mean achieved doses of sodium chlorate were 4870 µg per cell (range 4680 - 5220 µg per cell), equivalent to 5130 µg/cm2. The achieved dose levels were considered acceptable to fulfil the objectives of the study.
- Dose volume: 9.5 µl
- Rationale for dose selection: The sodium chlorate was applied as the commercially available undiluted formulation (high level, 5 mg/cm2) and after dilution into water to simulate practical use (low level of 15 g/l).
No. of animals per group:
7 skins were used per dose group per human or rat, in total 14 skins per dose.
Control animals:
yes
Remarks:
one untreated control skin was taken along for the high dose and one for the low dose.
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: Solid granular sodium chlorate (0.1502 g) was weighed into a pre-weighed vial. Water was added to a final volume of 10 ml. The diluted dose formulation was magnetically stirred throughout the dosing procedure.
- Method of storage: no data

APPLICATION OF DOSE: The low dose formulation was applied to the skin membrane with a calibrated positive displacement pipette at approximately 10 µl/cm2 exposed skin area (9.5 µl of dose, unoccluded). The actual amount of sodium chlorate applied to the skin was determined from aliquots (9.5 Al) of each dose formulation (QC checks) taken at the time of dosing each group of cells. The high dose formulation was evenly applied to the skinmembrane as a granular solid at approximately 5 mg/cm2 exposed skin area (4.75 mg of dose, unoccluded). To assist skin contact and to mimic perspiration, a saline solution (0.9% w/v) was applied to the skin-membrane (9.5 µl per 0.95 cm2) prior to high dose level administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Amount(s) applied (volume or weight with unit): 9.5 µl
- Concentration (if solution): 15 mg/L
- Lot/batch no. (if required): no applicable
- Purity: not applicable

ANALYSIS

Measurement of radioactivity
Radioactivity (3H) was measured by liquid scintillation counting (LSC) using LKB-Rackbeta Spectral Rackbeta Spectral (Wallac Oy, Turku, Finland) liquid scintillation counter with automatic quench correction. Generally radioactivity in gross amounts of less than twice background (4 minute counts) was considered to be below the limit of detection.

Receptor fluid (membrane integrity test samples only)
Ultima Gold scintillator (10 ml) was directly added to the receptor fluid samples and the radioactivity measured for tritium (3H) by LSC (Appendix 3). The receptor cell chamber components were soaked in Elga water and sonicated for 30 minutes. The extraction procedure was repeated for the donor cells.

SAMPLE ANALYSIS
Measurement of the amounts of sodium chlorate in the test samples was performed using validated analytical methodology described in Appendix 2. In outline, sodium chlorate was extracted from diffusion cell components, skin, tape-strips and swab samples with water; receptor fluid samples were diluted with water. The quantification of sodium chlorate was performed using liquid chromatography with mass spectrometric detection (LC-MS).
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: from rats, details see above or from humans supplied by International Institute for the Advancement of Medicine and Asterand Inc.
- Ethical approval if human skin: no data
- Type of skin: rat and human skin frozen at -15ºC or -20ºC, respectively
- Preparative technique:
Full thickness human skin preparation
Prior to use, skin samples were thawed to room temperature. Each full thickness skin membrane was then swabbed with 70% v/v ethanol/water to remove residual fat and blood, wiped dry and re-hydrated with distilled water prior to dermatoming.
Full thickness rat skin preparation
Six rats weighing 119 - 134 g (28 - 32 days old) were used. Each rat was sacrificed by cervical dislocation. After sacrifice, the body was shaved with
electric clippers and the skin removed and stored at. <-15ºC. Prior to use, skin samples were thawed to room temperature. The skin was then prepared in the same manner as human skin.
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 of skin (in mm): dermatomed skin thickness 200 - 400 µm, measured using a digital calliper
- Membrane integrity check: tritiated water (3H2O) through each membrane prior to the application of the test formulations. Distilled water (3 ml) was added to the receptor chamber of each diffusion cell. An aliquot (250 µl) of 3H2O was applied to the surface of the skin membrane and the cells then occluded. The water in the receptor chambers was removed after 1 hour into scintillation vials and the receptor chamber replenished with 3 ml distilled water. After a further 3 hours, the receptor chamber contents were removed to scintillation vials.
- Storage conditions: rat and human skin frozen at -15ºC or -20ºC, respectively
- Justification of species, anatomical site and preparative technique: no data

PRINCIPLES OF ASSAY
- Diffusion cell: Static glass 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 following application of the test substance was 50% v/v ethanol : water. 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 32ºC by placing the diffusion cells onto a multi-plate magnetic stirrer in a water bath.
- Receptor fluid: distilled water
- Solubility od test substance in receptor fluid: The solubility of sodium chlorate in the receptor fluid 50% v/v ethanol : water was shown to be 0.288 mg/ml (range 0.273 - 0.312 mg/ml) after incubation for approximately 24 hours at 32ºC. The solubility of sodium chlorate in the receptor fluid was demonstrated to be adequate and not rate limiting to the absorption process.
- Static system: no
- Flow-through system: no, semi static. The receptor fluid (approximately 3 ml) was taken from each diffusion cell and replenished with fresh receptor fluid (3 ml) at 2, 4, 6 and 8 hours after dose application.
- Test temperature: 32ºC
- Humidity: 44.5 - 48.6%
- Occlusion: no
- Reference substance(s): none
- Other:
Administration and sampling
Following the membrane integrity test, the receptor chamber of the diffusion cell was rinsed out to remove residual 3H2O and filled with 3 ml of receptor (50% v/v ethanol : water). The diffusion cells were returned to the multi-plate magnetic stirrer in the temperature controlled water bath (maintained at 32°C). The low dose formulation was applied to the skin membrane with a calibrated positive displacement pipette at approximately 10 µl/cm2 exposed skin area (9.5 µl of dose, unoccluded). The actual amount of sodium chlorate applied to the skin was determined from aliquots (9.5 µl) of each dose formulation (QC checks) taken at the time of dosing each group of cells. The high dose formulation was evenly applied to the skin membrane
as a granular solid at approximately 5 mg/cm2 exposed skin area (4.75 mg of dose, unoccluded). To assist skin contact and to mimic perspiration, a saline solution (0.9% w/v) was applied to the skin-membrane (9.5 µl per 0.95 cm2) prior to high dose level administration.

Assessment of the skin absorption and distribution of sodium chlorate
The receptor fluid (approximately 3 ml) was taken from each diffusion cell and replenished with fresh receptor fluid (3 ml) at 2, 4, 6 and 8 hours after dose application. The receptor fluid taken at these times and that removed at the end of the experiment (24 hours) were retained for analysis. A sample of fresh 50% v/v ethanol : water was analysed as the zero-hour receptor fluid. Untreated diffusion cells containing skin membranes were also set up to assess background levels of sodium chlorate and (control cells) and to provide samples of matrices for the determination of procedural recoveries through the analytical method (procedural recovery cells).
Each skin sample was swabbed at 8 hours after dosing 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 the end of the experimental phase (24 hours after application) the skin membranes were tape-stripped using 3M Scotch ‘Magic’ tape. The initial tape strips (1 - 2) were collected into a glass vial separately and represented residual surface (non-absorbed) dose. Subsequent tape strips containing the stratum corneum were pooled as one batch and collected into sample pots. The remaining skin was retained separately. The diffusion cell components were also retained and analysed for mass balance purposes.
All samples that were not analysed immediately after collection were stored at approximately <-15ºC as soon as possible after collection.

Results and discussion

Absorption in different matrices:
See table in "Remarks on results..."
Total recovery:
See table in "Remarks on results..."

Any other information on results incl. tables

The distribution of sodium chlorate in human and rat skin is summarised below.

Dose level

 

High (5 mg/cm2)

Low (150 µg/cm2)

Skin type

Unit

Human

Rat

Human

Rat

Total non-absorbed

 

 

 

 

 

Skin surface(skin swabs + surface strips)

 

%

98.40

93.19

99.36

78.66

µg

4772.5

4451.0

144.64

114.36

Remaining on cell (Donor chamber)

 

%

0.07

0.29

0.15

0.68

µg

3.23

13.97

0.22

0.98

Total

%

98.47

93.48

99.49

79.32

Stratum corneum

%

0.22

1.20

0.81

3.67

µg

10.60

57.77

1.17

5.33

Total abosorbed

 

 

 

 

 

Receptor fluid

%

0.20

0.21

0.83

10.82

µg

9.81

10.54

1.21

15.73

skin

%

0.09

1.94

0.22

3.58

µg

4.26

93.36

0.31

5.20

Remaining on cell (receptor chamber)

 

%

0.009

0.009

<0.03

0.05

µg

0.43

0.42

<0.05

0.08

Total

%

0.30

2.17

1.05

14.42

Stratum corneum

%

0.22

1.20

0.81

3.67

µg

10.60

57.77

1.17

5.33

Total absorbable (absorbed + stratum corneum)

%

0.51

3.37

1.85

18.09

Total recovery

%

98.98

96.84

101.35

97.41

Absorption rate (µg/cm2/hr)

 

0.446

0.467

0.166

4.875

Results are expressed as mean applied dose and are calculated from the mean of the individual recoveries and not the sum of the mean components

 

Applicant's summary and conclusion

Conclusions:
Rat skin was considerably more permeable than human skin following application at the low dose level. At the high dose level, the permeability of human and rat skin was similar.
The surface swab at 8 hours was intended to reflect a simple washing regimen at the end of the working day and, as such, this material is not regarded as available for absorption. For human skin, 98.31% and 98.92% of the applied dose was recovered in the skin swabs at high and low dose levels, respectively. For rat skin, 92.98% and 77.72% of the applied dose was recovered in the skin swabs at high and low dose levels, respectively. The material recovered on the surface tape strips at 24 hours was considered to be associated with surface residues following incomplete removal at 8 hours, and/or material from the superficial stratum corneum and so was considered to be non-absorbed.
The material present in the receptor fluid, together with that located in the skin sample remaining after tape-stripping and that remaining on the receptor chamber, accounted for 0.30% and 2.17% of the high dose and 1.05% and 14.42% of the low dose for the human and rat skin respectively and this is considered to be directly absorbed.
It was evident that there was some affinity of sodium chlorate for the stratum corneum, with means of 0.22% and 1.20% of the applied dose detected in this compartment for human and rat skin at the high dose, and 0.81% and 3.67% for human and rat skin at the low dose. In vivo, material associated with the stratum corneum may be ultimately absorbed, or lost externally as a result of desquamation of the stratum corneum. In the case of sodium chlorate it was shown that material remaining in the stratum corneum should be considered as available for absorption. Thus, the total absorbable was 0.51% and 3.37% for human and rat skin for the high dose, and 1.85% and 18.09% for human and rat skin for the low dose.
The steady-state absorption rates for sodium chlorate were low, with values of 0.446 µg/cm2/hr (human) and 0.467 µg/cm2/hr (rat) at the high dose level and 0.166 µg/cm2/hr (human) and 4.875 µg/cm2/hr (rat) at the low dose level, showing that sodium chlorate does not rapidly penetrate the skin.
Executive summary:

According to OECD428 and under GLP a comparative in vitro dermal penetration study using human and rat skin was performed.The rate and extent of absorption of sodium chlorate was investigated following dermal application to excised human and rat skin at two dose concentrations. The sodium chlorate was applied as the commercially available undiluted formulation (high level, 5 mg/cm2) and after dilution into water to simulate practical use (low level of 15 g/l). Seven static diffusion cells were prepared for each skin type at each dose level. Dermatomed membranes (200 - 400 µm thickness) were maintained in the cells at approximately 32°C. The integrity of the membranes was first tested using tritiated water (3H2O). After removal of the residual 3H2O, the test formulation was applied to the unoccluded skin samples as a solution at 9.5 µl per cell (10 µl/cm2) (low dose level) and as a granular solid at approximately 5 mg/cm2 (high dose level. To assist skin contact and to mimic perspiration, a saline solution (0.9% w/v) was applied to the skin-membrane (9.5 µl per 0.95 cm2) prior to high dose administration). The skin samples were exposed to the test material for 8 hours, after which time the remaining dose was washed off the skin with a mild detergent solution. Receptor fluid was collected at 0, 2, 4, 6, 8 and 24 hours after dosing. The solubility of sodium chlorate in the receptor fluid was demonstrated to be sufficient for the study and was not rate limiting to the absorption process. At the end of the study, the skin samples were tape stripped to remove residual surface dose and the stratum corneum. The group mean distributions of sodium chlorate are summarised below:

Dose level

High (5 mg/cm2)

Low (150 µg/cm2)

 

Human

Rat

Human

Rat

Total % non-absorbed

98.47

 

93.48

99.49

79.32

Total % absorbed

0.30

 

2.17

1.05

14.42

Total % in stratum corneum

0.22

 

1.20

0.81

3.67

Total % absorbable

0.51

3.37

1.85

18.09

Absorption rate (µg/cm2/hr)

0.446

 

0.467

0.166

4.875

Total % absorbable = Total % absorbed + Total % in stratum corneum

Results are expressed as mean % applied dose and are calculated from the mean of the individual recoveries and not the sum of the mean components. Rat skin was considerably more permeable than human skin following application at the low dose level. At the high dose level, the permeability of human and rat skin was similar. The directly absorbed material (that in the receptor fluid and remaining in the skin after tape stripping) at 24 hours were 0.30% and 2.17% for the high dose level, and 1.05% and 14.42% for the low dose level, in human and rat skin respectively. There was some affinity of sodium chlorate for the stratum corneum (means of 0.22% and 1.20% of the high dose for human and rat skin respectively, and 0.81% and 3.67% of the low dose for human and rat skin respectively). In vivo, material associated with the stratum corneum may be ultimately absorbed, or lost externally as a result of desquamation of the stratum corneum, and in the case of sodium chlorate the material in this skin layer should be considered as available for absorption. Thus, the total absorbable was 0.51% and 3.37% for human and rat skin at the high dose level, and 1.85% and 18.09% for human and rat skin at the low dose level. The steady-state absorption rates for sodium chlorate applied at the high dose level were 0.446 µg/cm2/hr and 0.467 µg/cm2/hr in human and rat skin, respectively. The steady-state absorption rates for sodium chlorate applied as the aqueous diluted formulation (15 g/l) were 0.166 µg/cm2/hr and 4.875 µg/cm2/hr in human and rat skin, respectively.