Chlorothalonil

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2013 to 28 Jun 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 28. The Conduct of Skin Absorption Studies

Version / remarks:

2004

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Note No. 156. Dermal Absorption.

Version / remarks:

2011

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EFSA Guidance on Dermal Absorption.

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Chlorothalonil

EC Number:

217-588-1

EC Name:

Chlorothalonil

Cas Number:

1897-45-6

Molecular formula:

C8Cl4N2

IUPAC Name:

tetrachlorobenzene-1,3-dicarbonitrile

Radiolabelling:

yes

Administration / exposure

Vehicle:

water

Duration of exposure:

24 hours

Doses:

- Nominal doses: concentrate formulation: 400 g/L, three different spray dilutions: 10 g/L, 1.875 g/L and 0.667 g/L (equivalent to 1/40, 1/213.3 or 1/600 w/v)
respectively.
- Dose volume: 25.4 µL
- Rationale for dose selection: The application rates and exposure conditions used in this study were designed to simulate predicted normal human exposure to the test material

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: See "Any other information on materials and methods incl tables" section

APPLICATION OF DOSE: The test substance formulations were applied to the skin surface by volume (target =10 μL/cm2) using a suitable positive displacement pipette. The weight of the dose applied was recorded and used for calculations. The concentrations were expressed as g/L using the specific gravity of 1.22 for the formulation concentrate and 1.00 for the dilutions.

TEST SITE
- Area of exposure: 2.54 cm2
- Time intervals for sampling: Samples (0.5 mL) of receptor fluid were taken from the receptor chambers of the static cell system at pre-treatment and at 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hours after application using an autosampler. The receptor fluid in the chambers was stirred continuously and the receptor volume was maintained by the replacement of a volume of fresh receptor fluid, equal to the sample volume, after each sample had been taken.

ANALYSIS
- Method type for identification: Liquid scintillation counting

OTHER:
- Measurement of mass balance: After the final receptor fluid sample had been taken at 24 hours, the remaining fluid in the receptor chamber was discarded. To remove residual receptor fluid from under the surface of the skin, the receptor chamber was again refilled with fresh receptor fluid (approximately 5 mL), which was afterwards discarded.
The donor chamber was carefully removed and the underside (surface contact with the membrane) wiped with at least a single sponge pre-wetted with 3% Teepol L® in water which was added to the wash sponges (below). The donor chambers were washed with acetone and the sample of the washing taken for analysis by LSC.
The epidermal surface of the skin was decontaminated by gently swabbing the application site with natural sponges pre-wetted with 3% Teepol L® in water, and with further sponges pre-wetted with water. This continued until the decontamination appeared complete or until it was apparent that radiolabel may be being extracted from the epidermis (this was following assessment of radioactivity levels on the skin surface and/or on the sponges with a Geiger counter during the procedure). The sponges were digested in Soluene 350® and a sample taken for analysis by LSC. The surface of the skin was allowed to dry naturally.
- To assess penetration through human stratum corneum, successive layers of the stratum corneum were removed by the repeated application of adhesive tape to a maximum of five strips. The total number of tape strips was recorded. The 5 strips were extracted individually for approximately 20 hours in acetone. The extracts were sequentially numbered and analysed by LSC.
- The remaining skin was carefully removed from the receptor chamber and were digested separately in Soluene 350®. The digest was made up to a recorded volume and a sample taken for analysis by LSC

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: human
- Preparative technique: Human skin samples were obtained from a tissue bank. Skin membranes were cut from the samples at a thickness setting of 400 µm using an electric dermatome.
- Thickness of skin (in mm): 0.4
- Membrane integrity check: Skin integrity was determined by measurement of the electrical resistance across the skin membrane. Membranes with a measured resistance of <10 kΩ (Davies et al, 2004) were regarded as having a lower integrity than normal and not used for exposure to the test materials, due to the possibility of compromised barrier function.
- Storage conditions: -20ºC, on aluminium foil until required for use

PRINCIPLES OF ASSAY
- Diffusion cell: static glass diffusion cell with an exposed membrane area of 2.54 cm2 and a receptor fluid volume of approximately 4.5 mL
- Receptor fluid: 50% ethanol in water
- Solubility od test substance in receptor fluid: 0.02376 mg/mL of the test substance was found to be fully soluble in 50% ethanol water, which is equivalent to 20433% of the total amount of formulation concentrate actually absorbed per cell in a nominal 4.5mL of receptor fluid being absorbed.
- Test temperature: 32ºC ± 1ºC
- Other: discs of approximately 3.3 cm diameter of prepared skin samples were mounted, dermal side down, in such diffusion cells held together with individually numbered clamps and placed in a water bath

Results and discussion

Absorption in different matrices:

400 g test substance/L formulation concentrate
The mean absorption rate of the test substance from the formulation concentrate through human dermatomed skin was 0.008 µg/cm²/h over 24 hours. The amounts of the test substance absorbed at 6, 8, 12 and 24 hours were 0.049, 0.072, 0.096 and 0.206 µg/cm2, respectively. These respective amounts, expressed as percentages of the applied dose, equated to 0.001, 0.002, 0.003 and 0.006%.

10 g test substance/L Aqueous spray dilution 1 (1/40 w/v)
The mean absorption rate of the test substance from the 10 g/L aqueous dilution through human dermatomed skin was 0.005 µg/cm²/h during the 24 hour exposure period. The amounts of test substance absorbed at 6, 8, 12 and 24 hours were 0.025, 0.038, 0.058 and 0.120 µg/cm2, respectively. These respective amounts, expressed as percentages of the applied dose, equated to 0.025, 0.037, 0.057 and 0.119%.

1.875 g test substance/L Aqueous spray dilution 2 (1/213.3 w/v)
The mean absorption rate of the test substance from the 1.875 g/L aqueous dilution through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour exposure period. The amounts of test substance absorbed at 6, 8, 12 and 24 hours were 0.019, 0.026, 0.034 and 0.056 µg/cm2, respectively. These respective amounts, expressed as percentages of the applied dose, equated to 0.100, 0.131, 0.174 and 0.285%.

0.667 g test substance/L Aqueous spray dilution 3 (1/600 w/v)
The mean absorption rate of Test substance from the 0.667 g/L aqueous dilution through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour exposure period. The amounts of test substance absorbed at 6, 8, 12 and 24 hours were 0.013, 0.018, 0.027 and 0.054 µg/cm2, respectively. These respective amounts, expressed as percentages of the applied dose, equated to 0.189, 0.261, 0.390 and 0.765%.

Total recovery:

400 g test substance/L formulation concentrate
A mean of 105% of the applied test substance was washed off the skin after 6 hours, with a further 0.238% removed at 24 hours. Small proportions were recovered from the remaining study compartments. A mean of 0.039% of the dose applied was recovered from the donor chamber, tape strips 1-2 (0.009%), tape strips 3-5 (0.007%) and 0.401% was found in the remaining skin. The mean total recovery was 105% of the dose applied.

10 g test substance/L Aqueous spray dilution 1 (1/40 w/v)
A mean of 90.6% of the applied test substance was washed off the skin after 6 hours, with a further 4.39% removed at 24 hours. Small proportions were recovered from the remaining study compartments. A mean of 0.299% of the dose applied was recovered from the donor chamber, tape strips 1-2 (0.181%), tape strips 3-5 (0.165%) and 7.89% was found in the remaining skin. The mean total recovery was 104% of the dose applied.

1.875 g test substance/L Aqueous spray dilution 2 (1/213.3 w/v)
A mean of 99.7% of the applied test substance was washed off the skin after 6 hours, with a further 1.61% removed at 24 hours. Small proportions were recovered from the remaining study compartments. A mean of 0.076% of the dose applied was recovered from the donor chamber, tape strips 1-2 (0.119%), tape strips 3-5 (0.182%) and 6.31% was found in the remaining skin. The mean total recovery was 108% of the dose applied.

0.667 g test substance/L Aqueous spray dilution 3 (1/600 w/v)
A mean of 85.1% of the applied test substance was washed off the skin after 6 hours, with a further 4.69% removed at 24 hours. Small proportions were recovered from the remaining study compartments. A mean of 0.322% of the dose applied was recovered from the donor chamber, tape strips 1-2 (0.145%), tape strips 3-5 (0.412%) and 10.8% was found in the remaining skin. The mean total recovery was 102% of the dose applied

Percutaneous absorption

Key result

Time point:

24 h

Dose:

0.667 g/L

Parameter:

rate

Absorption:

12.12 %

Remarks on result:

other: This value is derived from: reception fluid + exposed skin + skin strips

Any other information on results incl. tables

Table 1. Distribution of the test substance in the Test System (% administered dose)

Formula	400 g/L		10 g/L		1.875 g/L		0.667 g/L
Samples (test compartment)	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Sum strips 1-2	0.009	0.207	0.181	3.7	0.119	5.17	0.145	4.46
Sum strips 3-5	0.007		0.165		0.182		0.412
Remaining skin	0.401		7.89		6.31		10.8
Receptor fluid	0.006		0.119		0.293		0.765
sum absorption	0.423		8.355		6.904		12.122

Applicant's summary and conclusion

Conclusions:

The results obtained in this study demonstrate that the absorption of the test substance through human dermatomed skin following the application of the test substance/Azoxystrobin is slow and the vast majority of the test substance can be washed off the skin by normal decontamination procedures. These data predict that the dermal absorption of the test substance from potential exposure to this formulation concentrate and its aqueous spray strength dilutions would be generally low and will not exceed 12.12%.

Executive summary:

The absorption and distribution of test substance from a test substance/Azoxystrobin suspension concentrate (SC) formulation was measured in vitro through human dermatomed skin conforming to the OECD 428 testing guideline. The doses were applied to the dermatomed skin as the test substance/Azoxystrobin formulation concentrate containing 400 g test substance/L and three aqueous spray strength dilutions: nominally containing 10 g, 1.875 g and 0.667 g test substance/L: equivalent to 1/40, 1/213.3 and 1/600 w/v, respectively. The formulation and the aqueous spray strength dilutions were applied at rates of 10 µL/cm2 and left unoccluded for an
exposure period of six hours and a total run time of 24 hours. The absorption process was followed by taking samples of the receptor fluid (50% ethanol in water) at recorded intervals throughout the exposure period. At the end of the experiment, the distribution of test substance in the test system was assessed, which included a tape stripping technique to determine its distribution in the stratum corneum and in the remaining skin.
The formulation concentrate was included to assess exposure to mixer/loaders. The spray strength dilutions used (10 g, 1.875 g and 0.667 g test substance/L) represented typical in use concentrations. These applications were designed to simulate potential human dermal exposure to the formulation during normal use.
The absorption and distribution were followed using [14C]-test substance which was incorporated into the doses prior to application. The samples were analysed by liquid scintillation counting (LSC).
The surface of the dermatomed skin was decontaminated after a six hour exposure period to investigate the amount of test substance absorbed by the end of a typical ‘working day’ period. After decontamination, the absorption of test substance was monitored for the remainder of the 24 hour observation period.

Absorption of test substance from the formulation suspension concentrate (400 g test substance/L) through human dermatomed skin was at a mean absorption rate of 0.008 µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours following a six hour wash was 0.206 µg/cm² (0.006% of the dose applied). The vast majority of the applied test substance (mean 105%) was washed off the skin at six hours, and an additional 0.238% was removed in the skin wash after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.039%), tape strips 1-2 (0.009%), tape strips 3-5 (0.007%) and 0.401% was found in the remaining skin. The mean total recovery was 105% of the dose applied.
The absorption rate of test substance from the 10 g/L aqueous dilution 1 through human dermatomed skin was 0.005µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours, following a six hour wash, was 0.120µg/cm² (0.119% of the dose applied). The majority of the applied test substance (mean 90.6%) was washed off the skin at six hours, and an additional 4.39% was removed in the skin wash after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.299%), tape strips
1-2 (0.181%), tape strips 3-5 (0.165%) and 7.89% was found in the remaining skin. The mean total recovery was 104% of the dose applied.

The absorption rate of test substance from the 1.875 g/L aqueous dilution 2 through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour experimental period. The amount
absorbed into the receptor fluid at 24 hours following a six hour wash was 0.056µg/cm² (0.285% of the dose applied). The proportion of the applied test substance that was washed off the skin at 6 hours was 99.7% with an additional 1.61% being removed by skin washing after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.076%), tape strips
1-2 (0.119%), tape strips 3-5 (0.182%) and 6.31% was found in the remaining skin. The mean total recovery was 108% of the dose applied.

The absorption rate of test substance from the 0.667 g/L aqueous dilution 3 through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours following a six hour wash was 0.054µg/cm² (0.765% of the dose applied). The proportion of the applied test substance that was washed off the skin at 6 hours was 85.1% with an additional 4.69% being removed by skin washing after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.322%), tape strips
1-2 (0.145%), tape strips 3-5 (0.412%) and 10.8% was found in the remaining skin. The mean total recovery was 102% of the dose applied.

The results obtained in this study demonstrate that the absorption of the test substance through human dermatomed skin following the application of the test substance/Azoxystrobin is slow and the vast majority of the test substance can be washed off the skin by normal decontamination procedures. These data predict that the dermal absorption of the test substance from potential exposure to this formulation concentrate and its aqueous spray strength dilutions would be generally low and will not exceed 12.12%.