2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

5 September 2012 to 1 October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study was conducted on the formulated product; however, the results are expressed in terms of percentage of the acid equivalent of the active ingredient, applied as the potassium salt.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

yes

Remarks:

- see below

Principles of method if other than guideline:

In the 0.02 g a.e./L group, two skin membranes (replicates C-7 and C-8, derived from one donor) had slightly higher Kp values for tritiated water than the cut-off value. The data obtained for replicates C-7 and C-8 were comparable to the other data obtained and therefore considered appropriate to use in the calculations. This deviation is not considered to have affected the outcome and validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

soluble liquid concentrate formulation

IUPAC Name:

soluble liquid concentrate formulation

Test material form:

other: liquid mixture

Details on test material:

Non-radiolabelled active substance
- Physical state: Off-white powder
- Storage conditions of test material: Ambient conditions

Radiolabelled active substance
- Storage conditions of test material: <-18 °C

Specific details on test material used for the study:

GF-1601, which was tested at three target
oncentrations: 30 g a.e.L-1 (concentrate), 12 g a.e.L-1 (field spray dilution I) and 0.02 g a.e.L-1 (field spray dilution II). The dermal absorption of the active substance, aminopyralid (acid equivalent), was measured for each of these three test concentrations.

Radiolabelling:

yes

Remarks:

[14C]

Test animals

Species:

other: human split-thickness skin

Administration / exposure

Vehicle:

other: Formulated - equivalent to in-use product.

Duration of exposure:

8 hours of exposure (16 hours of post exposure time)

Doses:

- Nominal doses: 30 g a.e./L (concentrate), 12 g a.e./L (field spray dilution I) and 0.02 g a.e./L (field spray dilution II)
- Actual doses: 311 ± 6 µg/cm², 130 ± 3 µg/cm² and 0.19 ± 0.01 µg/cm²
- Dose volume: 10 µL/cm², resulting in approximately 6.4 µL applied over the surface (accounting for expected loss during distribution of the skin samples).
- Rationale for dose selection: 30 g a.e./L represents the maximum concentration users would be exposed to when handling the concentrated product, field spray dilution II represents the in-use concentration, and field spray dilution I was included to examine dose-response effects.

No. of animals per group:

Two skin membranes from each of 4 donors were exposed in each test group (8 skin membranes per dose level)

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions:
For the concentrate application, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 27 μL of a 95 mM KOH aqueous solution was added. The solvent and water were evaporated under nitrogen to dryness and 1.0226 g of the formulated test material was added. The test concentration was carefully vortexed in order to obtain a homogenous suspension.
For test field spray dilution I, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 27.3 μL of a 95 mM KOH aqueous solution was added. The solvent and water were evaporated under nitrogen gas to dryness after which 1.0155 g of the diluted in-use formulation was added (0.8255 g of the concentrated product was mixed with 1.2087 mL demineralised water). The test concentration was carefully vortexed in order to obtain a homogenous suspension.
For test field spray dilution II, the radiolabelled test material dissolved in methanol was added to a brown glass vial and 22.5 μL of a 3.96 mM KCl aqueous solution was added. The solvent and water were evaporated under nitrogen gas to dryness after which 0.9066 g water was added. The test concentration was carefully vortexed in order to obtain a homogenous suspension.

APPLICATION OF DOSE: The dose preparations were applied with a positive displacement pipette tip and spread evenly on the skin surface within the donor compartment (ca 10 μL/cm²) using a disposable glass rod.

SAMPLE COLLECTION
- Mass balance samples:
> Receptor fluid samples: Collected at 0-1 hour, 1-2 hours followed by 2 hour intervals up to 24 hours after application.
> Skin wash: After 8 hours of exposure, a volume of 40 µL of mild soap solution (3 % in water at 37 °C) was applied to the skin surface and removed using a cotton swab. This was repeated four times. The skin was then washed twice with 40 µL of demineralised water and cotton swab after which the skin was dried with two dry cotton swabs. The washing efficiency was evaluated and found to be sufficient (less than 1 % of the radioactivity recovered in the first three swabs was found in the fourth). After 24 hours, the application site was washed again using the same method.
> Diffusion cell: 24 hours after exposure, the diffusion cell was dismantled and both compartments were washed twice with 1.0 mL ethanol.
> Stripping: Each skin disc was tape stripped 15 times using Stripping Discs and a D-Squame® pressure device. Tape stripping was discontinued if the epidermis ruptured.
> Digestion: After stripping, the membranes were digested in 1.5 M KOH solution with 20 % ethanol for a minimum of 24 hours.

SAMPLE PREPARATION
- Preparation details: Ultima Gold™ scintillation liquid was added to samples of the receptor fluid (10 mL per sample), the diffusion cell washes (10 mL per sample), the cotton swab extracts (10 mL to a weighed aliquot of each sample), the tape strips (4 mL per sample), and to samples of the mock dosing samples (10 mL per sample). For the determination of radioactivity in digested skin preparations, 15 mL Hionic Fluor™ scintillation liquid was added to the entire digested skin membrane.
> Dose formulations: A weighed volume of water was added to aliquots of the dose formulation taken just before and directly after dosing. Scintillation liquid (Ultima Gold™) was added to weighed subsamples for analysis.
> Receptor fluid: Samples of the receptor fluid were added directly to scintillation liquid (Ultima Gold™) for analysis.
> Skin wash: The cotton swabs were pooled per skin membrane for each time point (8 and 24 hours) and extracted with ethanol for at least 24 hours at room temperature. Aliquots of the extracted cotton swabs were added directly to scintillation liquid (Ultima Gold™) for analysis.
> Donor and receptor compartments: The samples from each compartment were separately mixed with scintillation liquid (Ultima Gold™) for analysis.
> Tape stripping: Tape strips were added directly to scintillation liquid (Ultima Gold™) for overnight extraction followed by analysis.
> Skin membranes: The entire digested membrane fractions were directly added to scintillation liquid (Hionic Fluor™) for analysis.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting. The radioactivity in the samples was determined using a Canberra Packard Tricarb 3100 TR scintillation counter.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human donors. Breast skin samples were donated after surgery. All donors were female and of a similar age
- Ethical approval if human skin: All donors provided informed consent.
- Type of skin: Human skin membranes
- Preparative technique: All subcutaneous fat was removed from the skin samples upon arrival at the laboratory. Upon thawing, the tissues were cut using a Dermatome (25 mm) and measured with a digimatic micrometer. The exposed area of each membrane was 0.64 cm²
- Thickness of skin: 300 to 400 µm
- Membrane integrity check: 36 membranes were evaluated by measuring the permeability coefficient (Kp) for tritiated water. 200 µL saline containing tritiated water (17.5 kBq/mL) was applied in the donor compartment of the flow through diffusion cells. The compartments were covered with a glass slide. Samples of the receptor fluid (approximately 1.6 mL/h) were collected for up to three hours after application. The tritiated water remaining at the application site was removed with a pipette and the skin was dried with cotton swabs. 24 membranes were selected for use in the study (8 per group - two from each of the four donors for each test group).
- Storage conditions: Tissues were stored after fat removal in aluminium foil below -18 °C until use. One donor’s tissues were stored overnight at 2 - 10 °C before subcutaneous fat was removed.

PRINCIPLES OF ASSAY
- Diffusion cell: 9 mm flow-through automated diffusion cells
- Receptor fluid: Saline (0.9 % sodium chloride w/v containing 0.01 % sodium azide) supplemented with 6 % w/v polyoxy-ethylene 20-oleyl glycol (PEG)
- Solubility of test material in receptor fluid: 87 µg/mL
- Flow-through system: The receptor fluid was pumped at a speed of ca. 1.6 mL/h.
- Test temperature: 32 ± 1 °C (skin surface temperature)
- Humidity: Ambient

Results and discussion

Absorption in different matrices:

- Receptor fluid (in vitro test system): 0.21 ± 0.12 (concentrate), 0.22 ± 0.15 (field dilution I) and 0.73 ± 0.42 (field dilution II) as percentage of administered dose.
- Receptor chamber (in vitro test system): 0.01 ± 0.00 (concentrate and field dilution I) and 0.09 ± 0.03 (field dilution II) as percentage of administered dose.
- Donor chamber (in vitro test system): 0.04 ± 0.02 (concentrate), 0.03 ± 0.02 (field dilution I) and 0.10 ± 0.04 (field dilution II) as percentage of administered dose.
- Skin preparation (in vitro test system): 0.20 ± 0.10 (concentrate), 0.19 ± 0.13 (field dilution I) and 0.68 ± 0.25 (field dilution II) as percentage of dose administered.
- Stratum corneum (in vitro test system): 0.16 ± 0.07 (concentrate), 0.11 ± 0.06 (field dilution I) and 0.22 ± 0.06 (field dilution II) as percentage of dose administered.

Total recovery:

- Total recovery: 100.7 ± 1.0 (concentrate), 99.7 ± 2.7 (field dilution I) and 100.9 ± 2.6 (field dilution II) as percentage of dose administered
- Recovery of applied dose acceptable: Yes

Percutaneous absorption

Percutaneous absorption 1

Key result

Dose:

30 g a.e./L

Parameter:

percentage

Absorption:

0.41 %

Remarks on result:

other: 24 hours

Remarks:

Concentrated product (mean absorbed dose)

Percutaneous absorption 2

Key result

Dose:

12 g a.e./L

Parameter:

percentage

Absorption:

0.42 %

Remarks on result:

other: 24 hours

Remarks:

Field dilution I (mean absorbed dose)

Percutaneous absorption 3

Key result

Dose:

0.02 g a.e./L

Parameter:

percentage

Absorption:

1.5 %

Remarks on result:

other: 24 hours

Remarks:

Field dilution II (mean absorbed dose)

Any other information on results incl. tables

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (30 g a.e./L) was 0.65 µg/cm² (0.21 % of the applied dose) and the mean maximal flux was 0.13 µg/cm²/h. The lag time was 0.8 hours.

For field dilution I (12 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.28 µg/cm² (0.22 % of the applied dose) and the mean maximal flux was 0.04 µg/cm²/h. The lag time was 1.1 hours.

For field dilution II (0.02 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.0014 µg/cm² (0.73 % of the applied dose) and the mean maximal flux was 0.0001 µg/cm²/h. The lag time was 0.1 hours.

The mean absorbed dose was determined to be 0.41, 0.42 and 1.50 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

The mean potentially absorbed dose was determined to be 0.55, 0.50 and 1.68 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

Table 1: Results

	Concentrate	Field Dilution I	Field Dilution II
Total concentration (g a.e./L)	31.5	13.0	0.02
Dose (µg/cm²)	311 ± 6	130 ± 3	0.19 ± 0.1
Penetration into the receptor fluid after 24 hours (µg/cm²)	0.65	0.28	0.0014
Penetration into the receptor fluid after 24 hours (% of dose)	0.21	0.22	0.73
Maximal flux (µg/cm²/h)	0.13	0.04	0.0001
Lag time (h)	0.8	1.1	0.1
Recovery of dose (% ± SD) Receptor fluid	0.21 ± 0.12	0.22 ± 0.15	0.73 ± 0.42
Recovery of dose (% ± SD) Receptor compartment wash	0.01 ± 0.00	0.01 ± 0.00	0.09 ± 0.03
Recovery of dose (% ± SD) Skin*	0.20 ± 0.10	0.19 ± 0.13	0.68 ± 0.25
Absorbed dose (% ± SD)**	0.41 ± 0.21	0.42 ± 0.26	1.50 ± 0.45
Tape strips (1 + 2) (% ± SD)	0.03 ± 0.02	0.03 ± 0.02	0.04 ± 0.01
Tape strips (3 - last) (% ± SD)	0.14 ± 0.06	0.08 ± 0.04	0.18 ± 0.06
Stratum corneum (% ± SD)	0.16 ± 0.07	0.11 ± 0.06	0.22 ± 0.06
Potentially absorbed dose (% ± SD)***	0.55 ± 0.26	0.50 ± 0.30	1.68 ± 0.44
Skin wash t = 8 h (% ± SD)	99.9 ± 1.2	98.9 ± 2.9	98.5 ± 3.0
Skin wash t = 24 h (% ± SD)	0.21 ± 0.08	0.3 ± 0.1	0.6 ± 0.2
Donor compartment wash (% ± SD)	0.04 ± 0.02	0.03 ± 0.02	0.10 ± 0.04
Total recovery (% ± SD)	100.7 ± 1.0	99.7 ± 2.7	100.9 ± 2.6

* Epidermis + dermis (without stratum corneum)

** The amount in the receptor fluid plus the receptor compartment wash, plus the skin membrane (excluding tape strips, i.e. stratum corneum)

*** The amount in the receptor fluid plus the receptor compartment wash plus the skin membrane (except for the first 2 tape strips)

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated products) was found to be poorly absorbed through the skin.

Executive summary:

The dermal absorption of the test material was determined in a study conducted in accordance with the OECD guideline 428 and under GLP conditions.

The test material was assessed as the concentrated formulated product (31.5 g a.e./L) and as in use field dilutions (13 and 0.02 g a.e./L) using human split-thickness skin in flow through diffusion cells. The contact time was 8 hours and the post-exposure time was 16 hours.

In addition to the amount of [14C]test material in the receptor fluid, the residues remaining in/on the skin membranes and in the stratum corneum (at 24 hours) were also determined. Human skin membranes were prepared from four separate donors in duplicate (n = 8) per test concentration tested.

The mean absorption of the active substance into the receptor fluid after 24 hours for the concentrated product (31.5 g a.e./L) was 0.65 µg/cm² (0.21 % of the applied dose) and the mean maximal flux was 0.13 µg/cm²/h. The lag time was 0.8 hours. For field dilution I (13 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.28 µg/cm² (0.22 % of the applied dose) and the mean maximal flux was 0.04 µg/cm²/h. The lag time was 1.1 hours. For field dilution II (0.02 g a.e./L), the mean absorption of the active substance into the receptor fluid after 24 hours was 0.0014 µg/cm² (0.73 % of the applied dose) and the mean maximal flux was 0.0001 µg/cm²/h. The lag time was 0.1 hours.

The mean absorbed dose was determined to be 0.41, 0.42 and 1.50 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

The mean potentially absorbed dose was determined to be 0.55, 0.50 and 1.68 % of the active substance in the concentrated product, field dilution I and field dilution II, respectively.

Under the conditions of the test, the acid equivalent of the test material (dosed as the formulated products) was found to be poorly absorbed through the skin.