5-Brom-2-[(3,4,5-trifluorphenoxy)-difluormethyl]-1,3-difluorbenzene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 18 - September 08, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

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

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:

5-[(4-bromo-2,6-difluorophenyl)difluoromethoxy]-1,2,3-trifluorobenzene

EC Number:

610-623-5

Cas Number:

511540-64-0

Molecular formula:

C13 H4 Br F7 O

IUPAC Name:

5-[(4-bromo-2,6-difluorophenyl)difluoromethoxy]-1,2,3-trifluorobenzene

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst
- Age at study initiation: Pre-test: 7-8 weeks
- Weight at study initiation: 20.1-20.5 g
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Winkelmann GmbH, Borchen, Germany), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3 °C
- Humidity (%): 30 - 82 %
- Photoperiod (hrs dark / hrs light): 12/12

HUSBANDRY
Cage type: Makrolon, Type I with wire mesh top )EHRET GmbH, Emmendingen, Germany)
Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, Borchen, Germany)

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

12.5, 25, and 50 % (w/w)

No. of animals per dose:

4

Details on study design:

PREPARATION OF TEST MATERIAL:
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle was quantitatively added. The test concentrations were prepared serially. Homogeneity of the test material was maintained during treatment with the magnetic stirrer. Preparations were made freshly before each dosing occation.

RANGE FINDING TESTS:
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a pretest (non GLP) was performed in 2 mice. The data showed that the highest concentration which could be technically used was a 50 % solution. The treatment of the mice with 6.25, 12.5, 25 and 50 % test item in Acetone/Olive oil (4+1) did not show any signs of severe local irritation or systemic toxicity.

MAIN STUDY
Each test group of mice was treated by topical application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The application volume of 25 µl was spread over the entire dorsal surface of each ear lobe onca daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (controls).
5 days after the first topical application, all mice were administered with 250 µl of 78.6µCi/mL 3HTdR (corresponding to 19.65 µL 3HdTR per animal) by intravenous injection via tail vein.
Approx. 5 days after treatment with 3HTdR all mice were euthanized . The draining lymph nodes were excised and pooled per group. Single cell suspensions were prepared and incubated at ca. 4 °C for at least 18 h for precipitation of macromolecules. The precipitates were then resuspended in 5 % Trichloroacetic acid and transferred to glass scintillation vials with scintillation liquid (Ultima gold) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter

In the first main experiment 3/4 animals treated with the high dose died and the one remaining did not show any signs of sensitisation. In order to confirm this data the high dose test group was repeated. In the repeated experiment the treated animals were observed daily for clinical signs of toxicity.

INTERPRETATION OF RAW DATA:
A test item is considered a sensitizer in the LLNA if the following criteria are fulfilled:
- Exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3x greater than that recorded in controls, as indicated by SI.
The data are compatible with a conventional dose-response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS:
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality/Viability: once daily from experimental start to necropsy.
Body weights: prior to first application and prior to treatment with 3HTdR
Clinical signs: at 1-2 h after each application, especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated

Results and discussion

Positive control results:

Conc. SI
0%: 1.00
5% 2.04
10% 2.88
25% 3.56

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.72

Test group / Remarks:

Test Group 12.5 %

Results 2

Key result

Parameter:

SI

Value:

1.15

Test group / Remarks:

Test Group 25 %

Results 3

Key result

Parameter:

SI

Value:

0.09

Test group / Remarks:

Test Group 50 % (first main experiment)

Remarks on result:

other:

Remarks:

(1 remaining animal)

Results 4

Key result

Parameter:

SI

Value:

0.99

Test group / Remarks:

Test Group: 50 % (repetition of main experiment)

Results 5

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Control Group

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The EC3 value could not be calculated since none of the tested concentrations induced an SI >3.

VIABILITY/MORTALITY
First main experiment:
3 animals died after the 3rd application at the high dose (50 %)
Repetition of the main experiment:
1 animal was euthanized an the day after the 3rd application.

CLINICAL OBSERVATIONS
First main experiment:
The animals did not show any clinical signs at the low and mid dose during the course of the study. The the high dose, all 4 animals showed redness at the ears after the 2nd application.
Repetition of the main experiment:
The animals treated with the high dose showed the following signs after the last application: reduction of the spontaneous activity, eyelid closure and ruffled fur. On the day after the last application one animal was moribund and was therefore euthanised.

BODY WEIGHTS
The body weight range was within the range commonly recrded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Conclusions:

The test material was not a skin sensitiser under the test conditions of this study.

Executive summary:

Objective

The purpose of this Local Lymph Node Assay was to assess the skin sensitizing potential of the test material when administered to the dorsum of both ears of mice.

This study should provide a rational basis for risk assessment to the sensitizing potential of the test item in man.

Study Design

For this purpose a local lymph node assay was performed using test item concentrations of 12.5, 25, and 50%. The highest concentration tested was the highest concentration that could technically be achieved.

Results

First main experiment:

The animals did not show any clinical signs at the low and mid dose during the course of the study. The the high dose, all 4 animals showed redness at the ears after the 2nd application.

Repetition of the main experiment:

The animals treated with the high dose showed the following signs after the last application: reduction of the spontaneous activity, eyelid closure and ruffled fur. On the day after the last application one animal was moribund and was therefore euthanised.

In this study Stimulation Indices (S.I.) of 0.72, 1.15, and 0.99 were determined with the test item at concentrations of 10, 25, and 50% in Acetone/Olice oil (4 +1), respectively.

Conclusion

The test material was not a skin sensitiser under the test conditions of this study.