benzovindiflupyr (ISO); N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2009 to 03 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N-[11-(dichloromethylidene)tricyclo[6.2.1.0²,⁷]undeca-2,4,6-trien-3-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

EC Number:

691-719-4

Cas Number:

1072957-71-1

Molecular formula:

C18H15Cl2F2N3O

IUPAC Name:

N-[11-(dichloromethylidene)tricyclo[6.2.1.0²,⁷]undeca-2,4,6-trien-3-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

Details on test material:

- Name of test material (as cited in study report): SYN545192 tech.
- Substance type: Technical material
- Physical state: Beige powder
- Analytical purity: 97.0%
- Methodology for characterisation: HPLC
- Reanalysis date: End February 2013
- Storage condition of test material: <30°C

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 19.4-22.1 g
- Housing: Individually in Type II polypropylene/polycarbonate cages
- Diet: Ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance" ad libitum
- Water: Municipal tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70%
- Air changes: 15-20 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 09 December 2009 To: 03 February 2010

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Assay 1: 0, 5, 10, 25% w/v
Assay 2: 0, 0.01, 0.1, 1.0% w/v

No. of animals per dose:

Assay 1: 5
Assay 2: 6

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Yes (acetone: olive oil 4:1 mixture, N,N-dimethylformamide, methyl ethyl ketone, propylene glycol and dimethyl sulfoxide were tested).
- Irritation: Yes (10 and 25% w/v preparations of SYN545192 tested in 2 mice per dose level. Conducted as for main experiment but terminated on day 6 with a body weight measurement and radioactive proliferation was not performed).
- Lymph node proliferation response: No

MAIN STUDY
- Topical application: 25 μL of the appropriate formulation topically applied the dorsal surface of each ear once a day for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
- Proliferation assay: On day 6, 250 μL of sterile phosphate buffered saline containing approximately 20 μCi of 3HTdR was injected iv into the tail vein of each mouse. Five hours after injection, mice were killed and the draining auricular lymph nodes were excised. The nodes of each animal were processed individually. Single cell suspension of lymph node cells were prepared. Lymph node cells were pelleted by centrifugation. Incorporated 3HTdR was determined by suspending the lymph node cells in 5% TCA (trichloroacetic acid) for precipitation of macromolecules. After incubation with 5% TCA at 2-8 °C overnight precipitate was recovered by centrifugation at 190 x g for 10 minutes at 4 °C, and supernatants were removed and pellets were resuspended in 1 mL of 5% TCA solution and dispersed using an ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expressed the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5% TCA.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

DPM was measured for each animal. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation and mean DPN was calculated for each treatment group (“group DPN”). Stimulation index (SI = DPN of treated group divided by DPN of the appropriate control group) for each treatment group was also calculated. A stimulation index of 3 or greater is the criteria for defining a positive result. At the Sponsor’s request the EC3 value of the test item was calculated. The calculation of the EC3 value was conducted by linear interpolation according to the equation: EC3 = c + [(3-d)/(b-d)]x(a-c) where the data points lying immediately above and below the SI value of 3 on the LLNA dose-response plot have the co-ordinates (a,b) and (c,d) respectively. Use of the individual approach to calculate SI enables the performance of a statistical analysis of the data. In the absence of any positive results, the statistical analysis of the data was not performed.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Assay 1: Stimulation index values of the test item were 0.6, 0.9 and 0.4 at concentrations of 25, 10 and 5% w/v, respectively. Assay 2: Stimulation index values of the test item were 0.8, 0.7 and 0.8 at concentrations of 1.0, 0.1 and 0.01% w/v, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Assay 1: Group DPN (DPM divided by number of nodes) were 167.7, 94.1, 147.0, 73.3, 630.2 at concentrations of 0 (negative control), 25, 10, 5 % w/v SYN545192 and 25% HCA (positive control) respectively. Assay 2: Group DPN were 116.9, 96.4, 78.4, 91.0 at concentrations of 0 (negative control), 1.0, 0.1 and 0.01% w/v SYN545192 respectively.

Any other information on results incl. tables

Assay 1: On Day 3, 1 animal from the 10% w/v treated group was noted to be moribund with markedly decreased activity and was subsequently killed due to its poor clinical condition. In addition, signs of toxicity were observed in animals at all dose levels (25, 10 and 5% w/v).

Assay 2: No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any treated groups. No test item-related clinical signs were noted.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present assay, SYN545192 tested in a suitable vehicle, was shown to have no skin sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of SYN545192 following dermal exposure when administered topically to female CBA/J Rj mice was determined using the local lymph node assay. The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, 5 hours prior to termination animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI). The following concentrations were tested: 25, 10 and 5% (w/v) in Assay 1 and 1.0, 0.1 and 0.01% (w/v) in Assay 2.

Stimulation index values of the test item were 0.6, 0.9 and 0.4 at concentrations of 25, 10 and 5% (w/v), respectively in Assay 1 and stimulation index values of the test item were 0.8, 0.7 and 0.8 at concentrations of 1.0, 0.1 and 0.01% (w/v), respectively in Assay 2.

In conclusion, under the conditions of the present assay, SYN545192 tested in a suitable vehicle, was shown to have no skin sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.