pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jul 2011 to 28 Jul 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD (Guidance Document No. 28). The Conduct of Skin Absorption studies

Version / remarks:

2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: European Commission Guidance Document on Dermal Absorption

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Dermal Technology Laboratory Ltd. Med IC4, Keele University Science and Business Park, Keele, Staffordshire, ST5 5NL, United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one

EC Number:

602-927-1

Cas Number:

123312-89-0

Molecular formula:

C10H11N5O

IUPAC Name:

6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one

Radiolabelling:

yes

Administration / exposure

Type of coverage:

open

Vehicle:

water

Duration of exposure:

24 hours

Doses:

- Test solutions: 1/2000 w/w aqueous spray strength dilution containing a nominal 0.25 g test substance/kg, a 1/500 w/w aqueous spray strength dilution containing a nominal 1 g test substance/kg and a formulation concentrate containing a nominal 500 g test substance/kg
- Nominal doses: 2.59, 10.5 and 1626 µg test substance per cm2
- Dose volume: 25.4 µL

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Type of skin: Human skin, abdomen, female, age 51 to 91 years
- Preparative technique: The skin samples were immersed in water at 60ºC for 40-45 seconds and the epidermis teased away from the dermis. Each membrane was given an identifying number and stored frozen, at approximately -20ºC, on aluminium foil until required for use.
- Membrane integrity check: yes, range 10.19 to 24.94 kΩ
- Storage conditions: stored for 50 to 56 weeks and one sample 192 weeks

PRINCIPLES OF ASSAY
Cells were selected such that each application was represented by six intact membranes from at least four different donors. The receptor chambers of the cells containing small magnetic stirrer bars were filled with a recorded volume of receptor fluid (50% v/v ethanol/water) and placed in a water bath maintained at a temperature of 32ºC ± 1ºC.
A pre-treatment sample (0.1 mL (aqueous dilutions) or 0.5 mL (slurry)) was taken from each receptor chamber for analysis by LSC. The volume reduction in the receptor chamber was immediately compensated by fresh receptor fluid.
The test substance formulations were applied to the skin surface by volume using a suitable positive displacement pipette. The dose was applied drop-wise over the skin to maximise coverage. The weight of dose applied was recorded.
Skin absorption for each application was followed for 24 hours. The exposed skin area was left unoccluded.
Samples (0.1 mL (aqueous dilutions) or 0.5 mL (slurry)) of receptor fluid were taken from the receptor chambers of this static cell system 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. The samples of receptor fluid were analysed by LSC.
After the final receptor fluid sample had been taken at the end of the exposure period, the remaining fluid in the receptor chamber was discarded. 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 in contact with the membrane) wiped with one natural sponge pre-wetted with a dilute soap solution (3% Teepol in water) which was added to the wash sponges (below). The donor chambers were washed with methanol 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 in water, decontamination was assessed to be complete using a Geiger counter to measure the radioactivity on the skin/sponges, and with a further two sponges pre-wetted with water (one cell received three sponges) .The sponges were digested in a solubilising agent (Soluene 350) and a sample taken for analysis by LSC.
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 5 strips.
The surface of the skin was allowed to dry naturally. Strips of adhesive tape were sequentially pressed onto the skin surface and then carefully peeled off to remove layers of the stratum corneum. The adhesive strips were immersed individually in methanol to extract any test material. The extracts were sequentially numbered and analysed by LSC. The total number of tape strips was recorded.
The remaining epidermis was removed from the receptor chamber, digested in Soluene 350 and the whole digest analysed by LSC.

Results and discussion

Absorption in different matrices:

The mean data for distribution in the test system are presented in Table 1 to 3 in terms of amount and percentage of the applied dose.
The absorption rates for each individual cell are given in Table 4.
Key result in 'percutaneous absorption', based on 1/2000 dilution of the formulation concentrate. Absorption = absorption in stratum corneum (tape strips 3-5) + remaining epidermis + absorbed = 2.425%
Undiluted concentrate: amounts of test substance absorbed at 8, 12 and 24 hours were 0.160, 0.234 and 0.410 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.010, 0.014 and 0.025%. Absorption flow was 0.015 µg/cm2/h during the 24 hour exposure period.
1/500 dilution: amounts of the substance absorbed at 8, 12 and 24 hours were 0.017, 0.025 and 0.051 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.157, 0.236 and 0.484%. Absorption flow was 0.002 µg/cm2/h during the 24 hour exposure period.
1/2000 dilution: amounts of the substance absorbed at 8, 12 and 24 hours were 0.012, 0.014 and 0.021 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.459, 0.557 and 0.801%. Absorption flow was 0.001 µg/cm2/h during the 24 hour exposure period.

Total recovery:

- Total recovery: Table 1 to 3 in ‘any other information on results incl. tables’.
- Recovery of applied dose acceptable: Yes
- Limit of detection (LOD): Table 5 to 7 in ‘any other information on results incl. tables’.
- Undiluted concentrate: Total recovery 108%, of which 107% was washed off at 24 hours.
- 1/500 dilution: Total recovery was 105%, of which 103% was washed off at 24 hours.
- 1/2000 dilution: Total recovery was 106%, of which 103% was washed off at 24 hours.

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

24 h

Dose:

1/2000 dilution of formulation concentrate: 2.59 µg/cm2

Parameter:

percentage

Absorption:

2.451 %

Percutaneous absorption 2

Time point:

24 h

Dose:

1/500 dilution of formulation concentrate: 10.5 µg/cm2

Parameter:

percentage

Absorption:

1.2 %

Percutaneous absorption 3

Time point:

24 h

Dose:

33.3% w/w concentrate: 1626 µg/cm2

Parameter:

percentage

Absorption:

0.051 %

Any other information on results incl. tables

Table 1. 33.3% w/w aqueous slurry of the formulation concentrate: 1626 µg test substance per cm²

Test Compartment	µg test substance per cm²		% of applied dose
n=6	Mean	SEM	Mean	SEM
Donor chamber	0.185*	0.081	0.011*	0.005
Skin wash	1748	7.08	107	0.435
Stratum corneum(tape strips 1 & 2)	0.120*	0.031	0.007*	0.002
Stratum corneum(tape strips 3 – 5)	0.096*	0.046	0.006*	0.003
Remaining epidermis	0.321	0.074	0.020	0.005
Absorbed	0.410	0.064	0.025	0.004
Total recovered	1749	7.14	108	0.439

* < Limit of Quantitation Values

 

Table 2. 1/500 w/w aqueous dilution of the formulation concentrate: 10.5 µg test substance per cm²

Test Compartment	µg test substance per cm²		% of applied dose
n=6	Mean	SEM	Mean	SEM
Donor chamber	0.045	0.017	0.432	0.157
Skin wash	10.9	0.479	103	4.56
Stratum corneum(tape strips 1 & 2)	0.008	0.003	0.074	0.025
Stratum corneum(tape strips 3 – 5)	0.008	0.002	0.079	0.017
Remaining epidermis	0.066	0.013	0.630	0.126
Absorbed	0.051	0.019	0.484	0.177
Total recovered	11.1	0.463	105	4.41

* < Limit of Quantitation Values

 

Table 3. 1/2000 w/w aqueous dilution of the formulation concentrate: 2.59 µg test substance per cm²

Test Compartment	µg test substance per cm²		% of applied dose
n=6	Mean	SEM	Mean	SEM
Donor chamber	0.014	0.013	0.540	0.491
Skin wash	2.67	0.057	103	2.19
Stratum corneum(tape strips 1 & 2)	0.008	0.004	0.301	0.140
Stratum corneum(tape strips 3 – 5)	0.012	0.007	0.454	0.266
Remaining epidermis	0.030	0.022	1.17	0.861
Absorbed	0.021	0.006	0.801	0.218
Total recovered	2.75	0.043	106	1.65

* < Limit of Quantitation Values

Table 4. Mean absorption rates

Application of Test Materials and Actual Concentration of Dose Preparation	Mean Absorption Rates		Mean Amount and Percentage of Dose Absorbed
	Time period (h)	Absorption rate (µg/cm^2/h±SEM)	Time (h)	Amount (µg/cm^2)	Percentage Absorbed (%)
33.3% w/w aqueous slurry
(163 g/kg)	0-8	0.016 ± 0.004	6	0.136	0.008
10 µLcm^2(1626 µg/cm^2)	8-12	*0.024 ± 0.008	8	0.160	0.010
Unoccluded	12-24	0.014 ±0.004	12	0.234	0.014
Duration of exposure: 24h	0-24	0.015 ± 0.003	24	0.410	0.025
n = 6
1/500 w/w aqueous dilution
(1.05 g/kg)	0-8	0.002 ± 0.001	6	0.013	0.125
10 µL/cm^2(10.5 µg/cm^2)	8-12	0.002 ± 0.001	8	0.017	0.157
Unoccluded	12-24	0.002 ± 0.001	12	0.025	0.236
Duration of exposure: 24h	0-24	0.002 ± 0.001	24	0.051	0.484
n = 6
1/2000 w/w aqueous dilution
(0.259 g/kg)	0-8	0.001 ± 0.0003	6	0.010	0.403
10 µL/cm^2(2.59 µg/cm^2)	8-12	0.001 ± 0.0001	8	0.012	0.459
Unoccluded	12-24	0.001 ± 0.0001	12	0.014	0.557
Duration of exposure: 24h	0-24	0.001 ± 0.0001	24	0.021	0.801
n = 5

Table 6. Mean limit of quantitation values for 33.3% w/w Aqueous slurry of the formulation concentrate: 1626 µg test substance per cm2

Study compartment	DPM Value	µg/cm^2/h	µg/cm^2	% of applied dose
Absorption rate (0-8 hours)	-	0.015	-	0.001
Absorption rate (8-12 hours)	-	0.031	-	0.002
Absorption rate (12-24 hours)	-	0.010	-	0.0006
Absorption rate (0-24 hours)	-	0.005	-	0.0003
Receptor fluid	39.6	-	0.124	0.008
Donor chamber	16	-	0.204	0.013
Skin wash	17	-	0.542	0.033
Stratum corneum	18	-	0.574	0.035
Remaining epidermal membranes	24	-	0.008	0.0005

 

Table 6. Mean limit of quantitation values for 1/500 w/w Aqueous dilution of the formulation concentrate: 10.5 µg test substance per cm2

Study compartment	DPM Value	µg/cm^2/h	µg/cm^2	% of applied dose
Absorption rate (0-8 hours)	-	0.0007	-	0.006
Absorption rate (8-12 hours)	-	0.001	-	0.012
Absorption rate (12-24 hours)	-	0.0004	-	0.004
Absorption rate (0-24 hours)	-	0.0002	-	0.002
Receptor fluid	33.3	-	0.005	0.050
Donor chamber	28	-	0.004	0.037
Skin wash	20	-	0.007	0.066
Stratum corneum	25	-	0.009	0.083
Remaining epidermal membranes	19	-	0.001	0.010

 

Table 7. Mean limit of quantitation values for 1/2000 w/w Aqueous dilution of the formulation concentrate: 2.59 µg test substance per cm2

Study compartment	DPM Value	µg/cm^2/h	µg/cm^2	% of applied dose
Absorption rate (0-8 hours)	-	0.0007	-	0.026
Absorption rate (8-12 hours)	-	0.001	-	0.051
Absorption rate (12-24 hours)	-	0.0004	-	0.017
Absorption rate (0-24 hours)	-	0.0002	-	0.009
Receptor fluid	34.8	-	0.005	0.206
Donor chamber	26	-	0.004	0.140
Skin wash	17	-	0.006	0.229
Stratum corneum	32	-	0.011	0.432
Remaining epidermal membranes	18	-	0.001	0.039

 

Applicant's summary and conclusion

Conclusions:

In this GLP compliant OECD 428 study, the penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w), 0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absoption of 2.425% was derived. This value is used as reference value for risk assessment, since it is considered to be conservative.

Executive summary:

The in vitro dermal absorption of the test substance concentrate formulation through human epidermal membranes was measured in an OECD 428 study under GLP. [14C]-test substance doses were applied as a nominal 33.3% w/w aqueous slurry of the formulation concentrate (500 g/kg) and as 1/500 w/w (nominally, 1 g/kg) and 1/2000 w/w (nominally, 0.25 g/kg) aqueous dilutions of the formulation concentrate. The doses were applied at 10 μL/cm²

and left unoccluded for an exposure period of 24 hours.The absorption process was followed by taking samples of the receptor fluid (50% v/v ethanol/water) at recorded intervals throughout the exposure period. The distribution of the test substance within the test system and a 24 hour absorption profile were determined, using liquid scintillation counting (LSC).

For the 33.3% w/w aqueous slurry of the formulation concentrate the mean recovery was 108% of the dose applied. The vast majority of the applied test substance (mean 107%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.015 µg/cm²/h, accounting for a 24-hour accumulating dose of 0.827 µg/cm² (0.051% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction. For the 1/500 w/w aqueous dilution of the formulation concentrate the mean recovery was 105% of the dose applied.The vast majority of the applied test substance (mean 103%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.002 µg/cm²/h, accounting for a 24-hour accumulating dose of 0.125 µg/cm² (1.193% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction. For the 1/2000 w/w aqueous dilution of the formulation concentrate the mean recovery was 106% of the dose applied.The vast majority of the applied test substance (mean 103%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.001 µg/cm²/h, accounting for a 24-hour accumulating dose of 0.063 µg/cm² (2.425% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction.

In conclusion, the penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w), 0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absoption of 2.425% was derived. The latter value is used as reference value for risk assessment, since it is considered to be conservative.