2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

guideline was in draft form at the time the study was conducted

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

EC Number:

260-829-0

EC Name:

2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

Cas Number:

57583-35-4

Molecular formula:

C22H44O4S2Sn

IUPAC Name:

2-ethylhexyl 2-{[({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)dimethylstannyl]sulfanyl}acetate

Details on test material:

- Name of test material (as cited in study report): Methyltin (2-ethylhexyl) mercaptoacetate
- Physical state: liquid
- Analytical purity: 80.5%
- Composition of test material, percentage of components: monomethyltin (EHTG):18.3%; dimethyltin (EHTG): 80.5%; trimethyltin (EHTG): 0.10%
- Lot/batch No.: Y10059/001/002
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Radiolabelling:

no

Test animals

Species:

other: human and rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: rat: Charles River UK Ltd., Margate, Kent, UK
- Age at study initiation: 28 +/- 2 days




IN-LIFE DATES: From: 1998-06-08 To: 1999-01-15

Administration / exposure

Type of coverage:

other: occluded and open

Vehicle:

ethanol

Duration of exposure:

24 h

Doses:

- Dose volume: 100 uL/cm2
- Rationale for dose selection: The first phase of the study was to identify the highest dose that could practically be applied to human epidermis, which was also determined to be non-damaging to human epidermis by measuring the alteration in barrier function of the epidermis, following exposures to various applications.
During the second phase of the study the absorption of tin was determined from applications of this non-damaging dose to both human and rat epidermis.

No. of animals per group:

Skin barrier damage assessment: 3 cells- human epidermis
Absorption test: 6 cells/species

Control animals:

yes

Remarks:

untreated in the skin barrier damage test

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: receptor chambers filled with receptor fluid: water or 50% ethanol in water. Samples were diluted with a diluent prepared containing a known amount of indium internal reference.


APPLICATION OF DOSE: applied undiluted to human epidermis in the skin barrier damage assessment.


VEHICLE
- Justification for use and choice of vehicle (if other than water): 50% ethanol in water; the solubility in water alone in minimal
- Amount(s) applied (volume or weight with unit): 100 uL/cm2 (24300 ug/cm2 nominal)


TEST SITE
- Preparation of test site: Extraneous tissue was removed from human whole skin samples. The skin samples were immersed in water at 60 deg C for 40-45 seconds and te epidermis teased off the dermis. Each epidermal membrane was given an identifying number and stored frozen on aluminum foil until used. Skin from rats was carefully shaved using animal clippers in the dorsal and flank region, ensuring the skin was not damaged. The clipped area was excised and any subcutaneous fat removed. The skins were soaked for approximately 20 hours in 1.5 M sodium bromide then rinsed in distilled water. The epidermis was carefully peeled from the dermis and stored frozen on aluminum foil until use.
Epidermis samples were digested in nitric acid and diluted with deionised water. Donor / wash samples were concentrated to approximately 2 mL prior to digestion in nitric acid and dilution.
- Area of exposure: cells placed in receptor chambers
- Type of cover / wrap if used: no data
- Time intervals for shavings or clipplings: one time


SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: no; skin tissue was removed


REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: Afterthe final sample of receptor fluid had been taken at the end of the exposure period, the remianing fluid in the receptor chamber was discarded and the chamber rinsed with fresh receptor fluid (5 mL) which was also discarded. The surface of the epidermis was rinsed with 50% ethanol in water (2x5 mL) and the rinsings combined.
The donor chambers were carefully removed and washed with ethanol (10 mL) and the washings retained for analysis.
The surface of the epidermis was rinsed with further 50% ethanol in water (3x5 mL) and the rinsings combined with the first rinsings (i.e. total rinse volume = 25 mL).
The epidermis was carefully removed from the receptor chamber ad placed in a glass scintillation vial.
- Time after start of exposure: at the end of the exposure period (24-hr)



SAMPLE PREPARATION
- Storage procedure: stored frozen on aluminum foil
- Preparation details: Extraneous tissue was removed from human whole skin samples. The skin samples were immersed in water at 60 deg C for 40-45 seconds and te epidermis teased off the dermis. Each epidermal membrane was given an identifying number and stored frozen on aluminum foil until used. Skin from rats was carefully shaved using animal clippers in the dorsal and flank region, ensuring the skin was not damaged. The clipped area was excised and any subcutaneous fat removed. The skins were soaked for approximately 20 hours in 1.5 M sodium bromide then rinsed in distilled water. The epidermis was carefully peeled from the dermis and stored frozen on aluminum foil until use.
Epidermis samples were digested in nitric acid and diluted with deionised water. Donor / wash samples were concentrated to approximately 2 mL prior to digestion in nitric acid and dilution.


ANALYSIS
- Method type(s) for identification: The recedptor fluid diluted samples were analysed using ICP/MS; The epidermis diluted samples were anlaysed using ICP/AES
- Validation of analytical procedure: no data
- Limits of detection and quantification: Receptor fluid:0.2 - 2 ng tin/mL for samples for human experiments and 0.4 -20 ng tin/mL for samples from rat experiments, however in calculations the LOQ was set at a value of 1 ng/mL for both species.
Epidermis samples and donor/wash samples: the limit of quantitation using the above procedure was set at 12 ug tin for both human and rat epidermis and 6 ug tin/mL for the donor / wash samples.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: extraneous human whole skin samples and skin from male rats of the Wistar-derived strain
- Type of skin: human and rat (dorsal and flank region)
- Preparative technique: Extraneous tissue was removed from human whole skin samples. The skin samples were immersed in water at 60 deg C for 40-45 seconds and te epidermis teased off the dermis. Each epidermal membrane was given an identifying number and stored frozen on aluminum foil until used. Skin from rats was carefully shaved using animal clippers in the dorsal and flank region, ensuring the skin was not damaged. The clipped area was excised and any subcutaneous fat removed. The skins were soaked for approximately 20 hours in 1.5 M sodium bromide then rinsed in distilled water. The epidermis was carefully peeled from the dermis and stored frozen on aluminum foil until use.
Epidermis samples were digested in nitric acid and diluted with deionised water. Donor / wash samples were concentrated to approximately 2 mL prior to digestion in nitric acid and dilution.
- Thickness of skin (in mm): no data
- Membrane integrity check: yes; Samples of epidermis were mounted in glass diffusion cells with an exposed area of 2.54 cm2. The cells were placed in a water bath maintained at 32 +/- 1 deg C.
The integrity of the membranes was determined by measurement of their electrical resistance across the skin membrane. Membranes with a measured resistance <10kΩ (human) or <2.5 kΩ (rat) were regarded as having a lower integrity than normal and not used for exosure to the test material.
- Storage conditions: stored frozen on aluminum foil until use
- Justification of species, anatomical site and preparative technique: In vitro techniques employing glass diffusion cells have been shown to predict percutaneous absorption of chemicals in vivo.


PRINCIPLES OF ASSAY
- Diffusion cell: donor chamber-skin-support grid-receptor chamber- sampling arm
- Receptor fluid: diluent containing a known amount of indium internal reference
- Solubility of test substance in receptor fluid: 50% ethanol in water as the solubility of the test material in water alone is minimal.
- Flow-through system: at recorde intervals, samples of receptor fluid were taken for analysis. The volume of fluid in the receptor chamber was maintained by the addition of (0.5 mL) of fresh receptor fluid to the chamber immediately after the removal of each sample.
- Test temperature: 32 +/- 1 deg C
- Occlusion: occluded and unoccluded
- Reference substance(s): no

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

no effects

Absorption in different matrices:

Human epidermis: Occluded applications: tin absorption was 0.005 ug/cm2/h during the first 10h of exposure, increased to a maximum of 0.018 µg/cm2/h during the 10-24 hour period. Unoccluded application: absorption rates during the 0-10 h and 10-24 h periods were 0.002 µg/cm2/h and 0.007 µg/cm2/h respectively. In terms of percent of applied tin, 134 x 10E-5% (0.327 µg tin/cm2) was absorbed from the occluded dose, and 44 x 10E-5 % (0.107 µg tin/cm2) was absorbed from the unoccluded dose after 24-h exposure.

Rat epidermis: Absorption was 133 times faster than human for the occluded application and 213 times faster for the unoccluded application. The occluded trial absorption rate during the first 3h was 0.069 µg/cm2/h, increasing to 2.39 µg/cm2/h between 3-24h.
From the unoccluded application the rate in the first 3h was 0.489 µg/cm2/h, increasing to 1.49 µg/cm2/h between 3-24h. In terms of percent of applied tin, 0.206% (50 µg tin/cm2) was absorbed from the occluded dose, while 0.137% (33 µg tin/cm2) was absorbed from the unoccluded dose after 24h exposure.

The amounts of tin absorbed were <0.001% (unoccluded) and 0.001% (occluded) through human epidermis and 0.14% (unoccluded) and 0.21% (occluded) through rat epidermis.

Total recovery:

- Total recovery: >80%- human; >70%- rat was obtained from the surface of the epidermis and donor chamber
- Recovery of applied dose acceptable: yes; between 80-88% from both the occluded and unoccluded applications. Of the applied tin, approximately 1.5-2% for human and 6.5-7.2% for rat was recovered from the epidermis.
- Results adjusted for incomplete recovery of the applied dose: no
- Limit of detection (LOD): no data
- Quantification of values below LOD or LOQ: where individual values were below the LOQ, the LOQ values were included in calculations to obtain mean absorption data. In such cases, ti absorption is overestimated.

Percutaneous absorption

Percutaneous absorption 1

Dose:

100 uL/cm2

Parameter:

percentage

Absorption:

0 %

Remarks on result:

other: 24h

Remarks:

135 x 10e-5; human; occluded

Percutaneous absorption 2

Dose:

100 uL/cm2

Parameter:

percentage

Absorption:

0 %

Remarks on result:

other: 24h

Remarks:

44 x 10e-5; human; unoccluded

Percutaneous absorption 3

Dose:

100 uL/cm2

Parameter:

percentage

Absorption:

0.2 %

Remarks on result:

other: 24h

Remarks:

rat; occluded

Percutaneous absorption 4

Dose:

100 uL/cm2

Parameter:

percentage

Absorption:

0.1 %

Remarks on result:

other: 24h

Remarks:

rat; unoccluded

Conversion factor human vs. animal skin:

not determined

Any other information on results incl. tables

The test substance did not damage the epidermis when applied to  human epidermis at a rate of 100 µl/cm2 and exposed under occlusion for  24 hours.

Applicant's summary and conclusion

Conclusions:

Following 24-h dermal contact, the amount of the test substance required to alter the barrier function of human epidermis was >100 uL/cm2.
At this 100 uL/cm2 dose level, the absorption of tin from the test substance through human epidermis is extremely slow, when compared with the absorption rates of other penetrants measured using this in vitro technique.
The absorption of tin from the test substance through rat epidermis significantly overestimated absorption through human epidermis.
Absorption of tin through human epidermis after 24h exposure under occlusion was 135 x 10e-5% of the applied tin dose, but was only 44 x 10e-5% when left unoccluded. Throguh rat epidermis, 0.208% of the applied tin was absorbed by 24 h from the occluded application and 0.138% from the unoccluded applications.

Executive summary:

The absorption of the test substance has been measured in vitro through human and rat epidermis. The tin species absorbed were not individually identified; absorption determinations were based on measurements of total tin in the samples analysed. The first phase of the study was to idenitify the highest dose that could practically be applied, which was also determined to be non-damaging to human epidermis. During the second phase of the study, the absorption of tin was determined from both occluded and unoccluded applications of this non-damaging dose of the test substance (100 uL.cm2) human and rat epidermis.

The test substance did not damage the skin barrier (damage ratio = 1.3), when applied to human epidermis at a rate of 100 uL/cm2 and exposed under occlusion for 24h. This dose level is regarded as an excess application and increasing the dose would not further damage the membrane.

Fromt he occluded applications to human epidermis, tin absorption was 0.005 ug/cm2/h during the first 10 h of exposure, which increased to a maximum absorption rate of 0.018 ug/cm2/h during the 10 -24 h period. From the unoccluded application, absorption rates were slower during the 0 -10 h and 10 -24 h periods (0.002 ug/cm2/h and 0.007 ug/cm2/h, respectively). In terms of percent of applied tin, 134 x 10e-5% (0.327 ug tin/cm2) was absorbed from the occluded dose, while only 44 x 10e-5% (0.107 ug tin/cm2) was absorbed from the unoccluded dose after 24 h exposure.

When compared with human epidermis, absorption of tin from the test substance through rat epidermis was 133 times faster for the occluded application and 213 times faster for the unoccluded application.

The absorption rate for tin during the first 3h of exposure was 0.069 ug/cm2/h, increasing to 2.39 ug/cm2/h between 3 -24 h. Tin absorption form the unoccluded application during the first 3 h of exposure was 0.489 ug/cm2/h, increasing to 1.49 ug/cm2/h between 3 -24 h. In erms of percent of applied tin, 0.208% (50 ug tin/cm2) was absorbed from the occluded dose, while 0.138% (33 ug tin/cm2) was absorbed from the unoccluded dose after 24 h exposure.

The overall recovery of tin from the test system after 24 h exposure was between 81 -89% from both the occulded and unoccluded applications to human and rat epidermis.

Of the recovered tin, 82 -87% (human) and 74 -78% (rat) was washed off the epidermis and donor chamber at the end of the 24 h exposure period. The amounts of tin actuallly penetrated the epidermis into the recpetor solution were relatively insignificant (<0.001% and 0.001% through human epidermis adn 0.14% and 0.21% through rat epidermis). Of the applied tin, approximately 1.5 -2% (human) and 6.5 -7.3% (rat) was recovered from the epidermis.