Acetic acid, 2-[(5-chloro-8-quinolinyl)oxy]-

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 August 2013 - 24 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Version / remarks:

April 2004

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(5-chloroquinolin-8-yl)oxy]acetic acid

Cas Number:

88349-88-6

Molecular formula:

C11H8ClNO3

IUPAC Name:

[(5-chloroquinolin-8-yl)oxy]acetic acid

Test material form:

solid: particulate/powder

Remarks:

powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): X204558
- Molecular formula: C11H8NO3
- Molecular weight: 237.64 g.mol-1
- Substance type: Safener
- Physical state: Solid cream colored powder
- Analytical purity: 98.3%
- Purity test date: September 14, 2014
- Lot/batch No.: TSN303795
- Radiochemical purity (if radiolabelling): 100 %
- Specific activity (if radiolabelling): 38.85 mCi.mmol-1 (6.01 MBq.mg-1)
- Locations of the label (if radiolabelling): Cloquintocet-Ph-Ul-14C
- Storage condition of test material: Ambient temperature (15 – 25 ºC), Radiolabelled material: <−18 ºC

- Test Preparations:
Test preparation : GF-3015
Concentration of test substances : 45.1 % w/w X204558, 21.9 % w/w Pyroxsulam
Lot no. : ENBK-134853-074
Formulation type : Wettable granulate (WG)
Appearance : Cream colored powder
Storage conditions : Ambient temperature (15 – 25 ºC)

Test material name : GF-3015 semi blank1
Concentration of test substances : 0.0 % w/w X204558, 39.2 % w/w Pyroxsulam
Batch number : ENBK-134599-068
Appearance : Cream colored powder
Expiry date : April 9, 2015 2
Storage conditions : Ambient temperature (15 – 25 ºC)

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, the Netherlands
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 272 - 318 g
the weight variation of the animals did not exceed ± 20% of the average weight
- Fasting period before study: None
- Housing: individually housed in open Plexiglas metabolism cages
- Individual metabolism cages: yes
- Diet: ad libitum commercial rat diet (Rat & Mouse no. 3 breeding diet)
- Water: ad libitum tap-water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45% - 65%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 19 August 2013 To: 18 September 2013

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: Stock solutions of radiolabelled cloquintocet acid were in toluene/96 % ethanol (1:1, v/v) and diluted formulations were prepared with water

Duration of exposure:

The duration of exposure was 10 hours. Extended post exposure monitoring was carried out for additional 14, 62 and 158 hours to yield additional information on the bioavailability of the test substance after passing the epidermis to dermis and entering the systemic circuit.

Doses:

- Nominal doses: 451.5 g/kg, 1.05 g/L and 0.15 g/L which are equivalent to 2.26, 10.5 and 1.5 mg/cm2, respectively
- Dose volume: 50 mg (concentrate) or 100 μL (field dilutions) of dose formulation were applied to 10 cm2 of clipped skin
- Rationale for dose selection: The concentrate (451.5 g/kg) represents the maximal concentration possible when handling the undiluted formulation (e.g. during mixing and loading of the plant protection product), while field dilutions I and II (1.05 g/L and 0.15 g/L) reflect the concentrations recommended for use in the field (i.e. in-use spray dilutions)

No. of animals per group:

12 males per group (4 males per subgroup)

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions:
Concentrate (451.5 g/kg):
About 1.5 g powdered GF-3015 was weighed and transferred into a mortar. The proper amount of radiolabeled [14C]-X204558, dissolved in toluene/96% ethanol (1:1, v/v), was added together with an additional volume of 96% ethanol for adequate mixing. The solvent was evaporated under N2 gas while stirring until a dry powder remained. The dry powder was pulverized using a pestle. The homogeneity of the mixture was tested directly after preparation.
Field dilution I (1.05 g/L):
An amount of 0.5485 g of semi-blank formulation was made up to 43 mL with deionized water, resulting in a 43 times diluted blank formulation. A stock solution of 3.20 mg.g-1 nonlabelled X204558 in toluene/96% ethanol (1:1, v/v), was prepared. 2.5 MBq of [14C]-X204558 (~0.415 mg) dissolved in toluene/96% ethanol (1:1, v/v) was transferred to a brown glass vial. In addition, 0.635 mg of non-labelled X204558 dissolved in toluene/96% ethanol (1:1, v/v) was added. The solvent was evaporated under N2 gas until almost dryness and 100 μL of 43 times diluted semi-blank formulation was added. The dose preparation was kept overnight at ambient temperature protected from light while gently stirring. About 30 minutes before application 0.9 mL water was added, resulting in a 430 times diluted formulation.
Field dilution II (0.15 g/L):
An amount of 0.5485 g of semi-blank formulation was made up to 100 mL with deionized water, resulting in a 100 times diluted semi-blank formulation. From this solution, 100 μL was diluted up to 3 mL with deionized water resulting in a 3000 times diluted semi-blank formulation. 2.71 MBq of [14C]-X204558 (~0.45 mg), dissolved in toluene/96% ethanol (1:1, v/v), was transferred to a brown glass vial. The solvent was evaporated under N2 gas until almost dryness, after which 3.0 mL of 3000 times diluted semi-blank formulation was added.

Verification of the amount of radioactivity to be applied was performed before application. The concentration and homogeneity of [14C]-X204558 in all dose preparations was checked by taking aliquots just before and directly after dosing. For homogeneity, a coefficient of variation lower than 5% would be considered sufficient.

- Method of storage:
Dose preparation A (Concentrate) and the 43-times and 100-times diluted semi-blanks used to prepared the dose preparation B and C (Field dilutions I and II) were prepared the day prior to the first application and kept at ambient temperature and protected from light until application. The field dilutions were prepared fresh (within 24 h) before each application session. The radiochemical purity in the dose preparations was checked with radio-HPLC around application to the rats.

APPLICATION OF DOSE:
Concentrate: The dose preparation (~50 mg) was applied to the skin, which was moistened with physiological saline using impregnated cotton wool just before dose application. The dose preparation was evenly spread within the ‘O’-ring using a disposable plastic rod.
Field dilutions: The dose preparations were continuously stirred and visually checked for homogeneity prior to each dose administration. The dose preparations (100 μL) were applied with a syringe and spread evenly within the ‘O’-ring using a disposable plastic rod.

TEST SITE
- Preparation of test site: Hair was clipped and the area was wiped with a cotton swab wetted with acetone to remove fat and skin oils and then checked for abrasions.
- Area of exposure: 10 cm2
- % coverage: Not stated
- Type of cover / wrap if used: permeable tape
- Time intervals for shavings or clippings: At least twenty hours prior to dosing each animal had an area of around 20 cm2 of skin clipped in the shoulder and dorsal region.

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: a non-absorbing phyton ‘O’-ring with an inside area of approximately 10 cm2 was glued to the clipped skin using cyanoacrylate adhesive. The ‘O’-ring was covered by a protective device with permeable tape (cover, semi-occlusive conditions) to prevent uncontrolled loss of the test substance. The animals were then fitted with an extra strong tape around the body between the forelegs and the protective device to prevent gnawing at the ‘O’-ring and protective device.

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: the tape from the protective device was removed and retained for analysis
- Washing procedures and type of cleansing agent: The unabsorbed test substance was removed from the application site by washing 9 times with a mild lukewarm soap solution using cotton swabs. The moist skin area was dried with 1 additional cotton swab and a fresh cover was applied by fitting an extra strong tape around the body and protective device.
- Time after start of exposure: After an exposure period of 10 hours

SAMPLE COLLECTION AND PREPARATION
- Collection of blood: Blood was collected from the abdominal aorta into tubes containing lithium heparin. Duplicate sample aliquots of whole blood were weighed directly into combustible cones, air dried and stored at room temperature until combustion. Plasma samples of sacrifice blood were prepared by centrifugation of blood (10 minutes at 2000 x g). Duplicate sample aliquots of plasma were taken for analysis. The remaining plasma was transferred to vials and stored frozen (≤ -18 ºC) for possible repeat analysis. The red blood cells were discarded.
- Collection of urine and faeces: Urine was collected at ambient temperature. After each collection period, the cage was washed with approximately 5 mL demineralised water, which was collected in the same urine vial. At the end of the 24 hour collection period, the urine vials were weighed and duplicate sample aliquots were taken for analysis. The remainder or a sub-sample of the urine (ca. 20 g) was transferred to storage vials and stored frozen (≤ -18 ºC) for possible repeat analysis. On Saturdays and Sundays, the urine vials were stored in the refrigerator until processing on the Monday/working day.
Faeces were collected at ambient temperature. At the end of the 24 hour collection period, ca 3 parts of water were added to the collection vials and the samples weighed and homogenised. Duplicate sample aliquots of homogenates were directly weighed into combustible cones, air dried and stored at room temperature until combustion. A sub-sample of the homogenate (ca. 20 g) was transferred to storage vials and stored frozen (< -18 ºC) for possible repeat analysis. The remainder of the homogenate was discarded. On Saturdays and Sundays, the faeces vials were stored in the refrigerator until processing on Monday/working day.
- Cage washing: At the day of sacrifice, the cages were rinsed thoroughly with water/ethanol/triton X-100 (100/100/1; v/v/v), which was collected in
vials. After weighing, duplicate sample aliquots were taken for analysis. A sub-sample (ca. 20 g) was transferred to storage vials and kept at room temperature for possible repeat analysis. The remainder of the cage wash was discarded.
- Skin washing: Cotton swabs (n=10) used for the skin washing at 10 hours were collected sequentially in pairs in separate vials, e.g. swab 1 and 2 in vial one, swab 3 and 4 in vial 2, etc. Samples were extracted with 96% ethanol for at least 24 hours at room temperature.
- Terminal procedure: At the defined time points, the animals were killed by exsanguination after anesthesia with CO2/O2. The ‘O’-ring and protective device was carefully removed from the animal and retained for analysis. The skin at the application site was washed thoroughly to remove any remaining dose or exfoliated cells etc. on the surface to facilitate skin stripping. To this end, the skin was washed once with liquid soap and twice with aqueous washes. The moist skin area was dried with 1 additional cotton swab. Cotton swabs used were collected together in one vial. Samples were extracted with 96% ethanol for at least 24 h at room temperature.
The washed skin at the site of application was skin stripped. The individual tape strips and stripped skin were removed and retained for analysis. As control, a skin sample of a non-treated area (approximately 2 cm from the treated skin and same size as of treated skin) was also collected. The gastro-intestinal tract was removed and retained for analysis. Apart from the residual carcass, no other tissues or organs were collected separately. The residual carcass, GI-tract and skin (application and control) samples were directly digested with 1.5 M KOH in 20% ethanol and stored at room temperature until analysis.
- Skin stripping: Before skin tape stripping, any hair present on the application site was removed using an electric clipper. Any hair removed, was combined with the first tape strip. The clipper was cleaned with tape in order to remove the smaller hair particles. Skin stripping involved the application of TESA adhesive tape for 5 seconds before the tape was carefully removed against the direction of hair growth. Skin stripping was continued for maximum 20 tape strips or until a complete ‘shiny’ appearance of the viable epidermis was evident, which indicated that the stratum corneum had been removed.
Adhesion of viable epidermis to the tape strips is negligible. In the result section, it is mentioned at which tape strip the stratum corneum was completely removed. Each skin strip was collected in a separate vial and analysed individually.
- ‘O’-ring and cover The ‘O’-ring and protective device was extracted with 96% ethanol for at least 24 hours at room temperature.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting and HPLC analysis
- Liquid scintillation counting results (cpm) converted to dpm as follows: Radioactivity in all samples was determined by LSC on a Tri-Carb 3100TR liquid scintillation counter using QuantaSmartTM software. All counts were converted to DPM using tSIE/AEC (transformed Spectral Index of external standards coupled to Automatic Efficiency Correction).

Results and discussion

Signs and symptoms of toxicity:

no effects

Dermal irritation:

no effects

Absorption in different matrices:

- Non-occlusive cover + enclosure rinse: At sacrifice, the O-ring and cover contained 2.6-5.0% (concentrate), 0.25-0.55% (field dilution I) and 0.32-0.80% (field dilution II) of the applied radioactivity.
- Skin wash: The bulk of the applied radioactivity was removed by skin washing: 87.9-93.8% in the groups receiving the concentrate, 56.5-66.5% in the groups receiving the field dilution I and 55.2-62.3% in the groups receiving the field dilution II.
- Skin test site:
Skin stripping:
Concentrate: Skin stripping revealed that most of the radioactivity in the stratum corneum was concentrated in the upper layer and/or hair at 168 hours post-application. The amount of radioactivity in the skin strips (3 until the last) after 24 hours was 0.25 ± 0.14% and decrease to 0.11 ± 0.06% at 168 hours after dosing.
Field dilution I: Skin stripping revealed that most of the radioactivity in the stratum corneum was concentrated in the upper layer and/or hair at 168 hours post-application. The amount of radioactivity in the skin strips (3 until the last) after 24 hours was 7.60 ± 1.53% and decreased to 11.19 ± 2.56% at 168 hours after dosing.
Field dilution II: Skin stripping revealed that most of the radioactivity in the stratum corneum was concentrated in the upper layer and/or hair at 168 hours post-application. The amount of radioactivity in the skin strips (3 until the last) remained constant for the duration of the study (11.01 ± 2.61% at 24 hours, 12.89 ± 2.54% at 72 hours and 11.55 ± 3.65% at 168 hours after dosing), indicating that the radioactivity present in the tape strips likely will not be available for absorption.
Stripped skin:
Concentrate: After 24 hours, only 0.05 ± 0.03% of the administered dose was retained in the stripped skin. The amount remaining at 168 hours was 0.02 ± 0.01%, indicating that the absorption was essentially complete.
Field dilution I: After 24 hours, 0.76 ± 0.25% of the administered dose remained in the stripped skin. The amount remaining at 168 hours was 0.42 ± 0.22%.
Field dilution II: After 24 hours, 0.85 ± 0.14% of the administered dose remained in the stripped skin. The amount remaining at 168 hours was 0.22 ± 0.14%.
- Blood and Carcass:
Compared to the absorbed activity, the radioactivity levels in the remaining tissue were very low, indicating a fast elimination after absorption. The levels of radioactivity in plasma, blood and carcass were close to or below the limit of detection at the last time point. Throughout the study, the mean concentration was far below 1, 0.01 and 0.001 μg.g-1, for the concentrate, the field dilution I and the field dilution II, respectively.
Concentrate: The radioactivity in the residual carcass decreased from 0.37 ± 0.46% at 24 hours after application to 0.08 ± 0.03% at 168 hours after application.
Field dilution I: The radioactivity in the residual carcass slightly decreased from 0.51 ± 0.19% at 24 hours after application to 0.43 ± 0.28% at 168 hours after application.
Field dilution II: The radioactivity in the residual carcass decreased from 0.85 ± 0.29% at 24 hours after application to 0.53 ± 0.22% at 168 hours after application.
- Urine and Faeces: The absorbed radioactivity was predominantly excreted with the urine. The radioactivity in urine was a factor of 2-2.5 higher than in the faeces. The majority of the radioactivity was excreted within the first 24 hours in all the groups (concentrate, field dilution I and field dilution II).
- Cage wash + cage wipe: Presented in summary table of recovered radioactivity

Total recovery:

- Total recovery: Mean recovery in the 9 subgroups ranged from 97.5% to 86.4%. It is likely that the lower recovery found especially in the groups receiving the field dilutions, was caused by an inefficient extraction of skin wash swaps and /or protective device and not in the penetrating fractions, since the latter were measured directly.

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

24 h

Dose:

Concentrate

Parameter:

percentage

Absorption:

0.63 %

Percutaneous absorption 2

Time point:

72 h

Dose:

Concentrate

Parameter:

percentage

Absorption:

0.26 %

Percutaneous absorption 3

Time point:

168 h

Dose:

Concentrate

Parameter:

percentage

Absorption:

0.11 %

Percutaneous absorption 4

Key result

Time point:

24 h

Dose:

Field dilution I

Parameter:

percentage

Absorption:

1.38 %

Percutaneous absorption 5

Time point:

72 h

Dose:

Field dilution I

Parameter:

percentage

Absorption:

0.2 %

Percutaneous absorption 6

Time point:

168 h

Dose:

Field dilution I

Parameter:

percentage

Absorption:

0.5 %

Percutaneous absorption 7

Key result

Time point:

24 h

Dose:

Field dilution II

Parameter:

percentage

Absorption:

1.64 %

Percutaneous absorption 8

Time point:

72 h

Dose:

Field dilution II

Parameter:

percentage

Absorption:

0.52 %

Percutaneous absorption 9

Time point:

168 h

Dose:

Field dilution II

Parameter:

percentage

Absorption:

0.62 %

Any other information on results incl. tables

Summary table of recovered radioactivity

	Concentrate (451.5 g/kg) Nominal dose: 2260 μg/cm^2			Field dilution I (1.05 g/L) Nominal dose: 10.5 μg/cm^2			Field dilution II (0.15 g/L) Nominal dose: 1.5 μg/cm^2
Time group	T1 (24h)	T2 (72h)	T3 (168h)	T1 (24h)	T2 (72h)	T3 (168h)	T1 (24h)	T2 (72h)	T3 (168h)
Urine(0-24 h)	0.15 ± 0.02	0.26 ± 0.13	0.17 ± 0.03	0.58 ± 0.32	0.67 ± 0.41	0.78 ± 0.27	0.39 ± 0.15	0.93 ± 0.86	0.55 ± 0.33
(24-48 h)	-	0.05 ± 0.05	0.02 ± 0.01	-	0.02 ± 0.01	0.03 ± <0.01	-	0.02 ± <0.01	0.03 ± <0.01
(48-72 h)	-	0.02 ± 0.01	0.02 ± 0.01	-	0.01 ± <0.01	0.01 ± 0.01	-	0.01 ± <0.01	0.01 ± <0.01
(72-96 h)	-	-	0.02 ± 0.01	-	-	0.01 ± <0.01	-	-	0.01 ± <0.01
(96-120 h)	-	-	0.01 ± 0.01	-	-	0.01 ± 0.01	-	-	0.01 ± <0.01
(120-140 h)	-	-	0.01 ± 0.01	-	-	0.01 ± <0.01	-	-	0.01 ± <0.01
(144-168 h)	-	-	0.01 ± <0.01	-	-	0.01 ± 0.01	-	-	0.01 ± 0.01
Total (0-168h)	0.15 ± 0.02	0.34 ± 0.18	0.26 ± 0.07	0.58 ± 0.32	0.70 ± 0.41	0.85 ± 0.24	0.39 ± 0.15	0.96 ± 0.87	0.61 ± 0.33
Faeces(0-24 h)	0.08 ± 0.02	0.16 ± 0.12	0.11 ± 0.04	0.25 ± 0.06	0.20 ± 0.09	0.31 ± 0.08	0.18 ± 0.04	0.28 ± 0.25	0.15 ± 0.04
(24-48 h)	- 0.03 ± 0.02	0.01 ± <0.01	-	0.02 ± 0.01	0.03 ± 0.01	-	0.02 ± <0.01	0.05 ± 0.01
(48-72 h)	- 0.02 ± 0.01	0.01 ± 0.01	-	0.01 ± <0.01	0.02 ± 0.02	-	0.01 ± <0.01	0.02 ± 0.01
(72-96 h)	-	-	0.02 ± 0.01	-	-	0.02 ± 0.01	-	-	0.01 ± <0.01
(96-120 h)	-	-	0.01 ± <0.01	-	-	0.01 ± 0.01	-	-	0.01 ± <0.01
(120-140 h)	-	-	0.01 ± 0.01	-	-	0.01 ± 0.01	-	-	0.02 ± 0.01
(144-168 h)	-	-	0.01 ± <0.01	-	-	0.01 ± 0.01	-	-	0.02 ± 0.01
Total (0-168h)	0.08 ± 0.02	0.21 ± 0.14	0.17 ± 0.05	0.25 ± 0.06	0.24 ± 0.09	0.43 ± 0.04	0.18 ± 0.04	0.31 ± 0.25	0.27 ± 0.05
Cage wash	0.02 ± 0.02	0.07 ± 0.05	0.03 ± 0.02	0.01 ± 0.01	0.01 ± 0.01	0.02 ± 0.02	0.04 ± 0.02	0.02 ± <0.01	0.03 ± 0.03
Blood	<0.01 ± <0.01	<0.01 ± <0.01	<0.01 ± <0.01	<0.01 ± <0.01	<0.01 ± <0.01	<0.01 ± <0.01	<0.01 ± <0.01	<0.01 ± <0.01	<0.01 ± <0.01
Control skin	<0.01 ± <0.01	<0.01 ± <0.01	<0.01 ± <0.01	0.01 ± 0.01	<0.01 ± <0.01	0.03 ± 0.06	0.13 ± 0.19	0.02 ± 0.03	0.02 ± 0.01
GI-tract	0.01 ± <0.01	0.02 ± 0.01	0.01 ± <0.01	0.01 ± <0.01	0.01 ± <0.01	0.01 ± 0.01	0.04 ± 0.05	0.02 ± <0.01	0.04 ± 0.04
Carcass	0.37 ± 0.46	0.19 ± 0.16	0.08 ± 0.03	0.51 ± 0.19	0.17 ± 0.08	0.43 ± 0.28	0.85 ± 0.29	0.45 ± 0.21	0.53 ± 0.22
Absorbed dose	0.63 ± 0.41	0.84 ± 0.49	0.56 ± 0.13	1.38 ± 0.44	1.13 ± 0.53	1.77 ± 0.31	1.64 ± 0.41	1.79 ± 1.06	1.50 ± 0.43
Tape strips 1 + 2	0.06 ± 0.03	0.17 ± 0.03	0.15 ± 0.10	15.43 ± 6.20	14.68 ± 1.09	7.59 ± 1.18	14.59 ± 3.50	19.40 ± 6.06	15.05 ± 4.89
Tape strips 3 - last	0.25 ± 0.14	0.08 ± 0.03	0.11 ± 0.06	7.60 ± 1.53	8.05 ± 1.49	11.19 ± 2.56	11.01 ± 2.61	12.89 ± 2.54	11.55 ± 3.65
Stripped skin	0.05 ± 0.03	0.01 ± 0.01	0.02 ± 0.01	0.76 ± 0.25	0.39 ± 0.18	0.42 ± 0.22	0.85 ± 0.14	0.85 ± 0.10	0.22 ± 0.14
Total skin wash	92.35 ± 3.48	93.79 ± 2.25	87.91 ± 4.64	66.53 ± 5.49	56.52 ± 5.67	66.34 ± 1.84	63.25 ± 3.95	55.20 ± 4.69	57.32 ± 1.89
O-ring/cover	2.84 ± 2.77	2.64 ± 3.05	5.01 ± 3.07	0.25 ± 0.06	0.39 ± 0.09	0.55 ± 0.49	0.36 ± 0.03	0.32 ± 0.07	0.80 ± 0.21
Recovery	96.11 ± 0.41	97.54 ± 2.12	93.76 ± 1.83	91.96 ± 0.82	81.86 ± 4.62	87.87 ± 1.43	91.70 ± 0.69	90.44 ± 1.03	86.44 ± 2.26

Applicant's summary and conclusion

Conclusions:

Absorption is 0.63, 1.38 and 1.64% for the concentrate (451.5 g/Kg), the field dilution I (1.05 g/L) and the field dilution II (0.15 g/L), respectively.

Executive summary:

A study was carried out according to OECD test guideline 427 to examine the in vivo dermal absorption of cloquintocet acid formulated as GF-3015 (test preparation), a wettable granule (WG) formulationfor use in plant protection, and two field dilutions in rats. The test preparation was tested at three target concentrations: 451.5 g/Kg (concentrate), 1.05 g/L (field dilution I; 1:430) and 0.15 g/L (field dilution II; 1:3000). The concentrate represents the maximal concentration possible when handling the undiluted formulation (e.g., during mixing and loading), while field dilutions I and II reflect the concentrations recommended for use in the field (i.e.in-use spray dilutions). In addition to the extent of percutaneous absorption of the compound related radioactivity, the residues remaining in/on skin after washing of the application site at 10 hours were estimated, as was the kinetics of percutaneous absorption at 3 time points post application, i.e., 24, 72 and 168 hours. Furthermore, the distribution of radioactivity between the upper skin layer (i.e., in stratum corneum) and the rest of the application site was estimated.

One day before the start of the exposure, 36 male rats were housed individually in metabolic cages and an area of approximately 20 cm² of skin was clipped in the shoulder-dorsal region. The following day, the animals were weighed and dosed. Approximately 5-10 minutes before dosing, a rubber O-ring with cyanoacryl adhesive was glued on the skin. Dosing was performed by administering 100 μL of concentrate or field dilution I or II on the shaved skin in the O-ring and spreading it over the skin surface. Subsequently, the O-ring was covered with a thin polyethylene cylinder of 2 cm height with semi-permeable tape. The animals were then returned to the metabolic cages. After an exposure of 10 hours, the skin was washed at the application site. At 3 different time points (24h, 72h and 168h post application) the animals were sacrificed. Approximately 20 skin strips were collected. During the whole duration of the study, urine and faecal samples were collected at 24 hour-intervals. In order to determine the recovery, the amount of [14C]-cloquintocet acid was determined in dose formulations, liquid specimens (urine, cage wash, skin wash extract, plasma and extract of cover and ‘O’-ring), Skin strips, Faeces and blood, Application site, non-treated skin, gastro-intestinal (GI) tract and residual carcass.

Recoveries were 94-98%, 82-92% and 86-92% from concentrate, field dilution I and field dilution II, respectively. It is likely that the lower recovery found in some groups receiving the field dilutions, was caused by an inefficient extraction of skin wash swabs and or protective device and not in the penetrating fractions, since the latter were measured directly.

The total absorbed dose from the Concentrate was 0.63%, 0.84% and 0.56% after 24, 72 and 168 hours, respectively. The total absorbed dose from the Field dilution I was 1.38%, 1.13% and 1.77% after 24, 72 and 168 hours, respectively. The total absorbed dose from the Field dilution II was 1.64%, 1.79% and 1.50% after 24, 72 and 168 hours, respectively. The absorbed radioactivity was predominantly excreted with the urine. The radioactivity in urine was a factor of 2-2.5 higher than in the faeces. The majority of the radioactivity was excreted within the first 24 hours in all the groups. Moreover, there was no decline of residue from skin including stratum corneum post 24-hour in any dose level.

The absorption post-24 hours was minimal since at this time-point the plateau of absorption has already been reached. The amount in the stripped skin was very low (<1%) in all test groups. The amount in stratum corneum from different test groups was stable and did not decline post 24-hour, and is therefore not-absorbable. This was also mirrored by very low levels in the blood/plasma, confirming that the residue at the application site is not-absorbable and, therefore, not included in the absorbed dose. The stratum corneum dose will eventually be lost upwards (not inwards) via normal desquamation.

It was concluded that dermal absorption of cloquintocet acid was 0.63, 1.38 and 1.64% for the concentrate (451.5 g/Kg), the field dilution I (1.05 g/L) and the field dilution II (0.15 g/L), respectively.