Methanone, 1,1'-(1,4-phenylene)bis[1-(4-phenoxyphenyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2013 - 05 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Deviations:

no

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:

[4-(4-phenoxybenzoyl)phenyl](4-phenoxyphenyl)methanone

EC Number:

620-097-9

Cas Number:

54299-17-1

Molecular formula:

C32H22O4

IUPAC Name:

[4-(4-phenoxybenzoyl)phenyl](4-phenoxyphenyl)methanone

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approximately 8 weeks old on the day of treatment
- Mean body weight at study initiation: the animals of the preliminary test had a mean body weight of 18.4 g (range: 18.2 g to 18.6 g) and the animals of the main test had a mean body weight of 20.4 g (range: 18.8 g to 22.5 g).
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 6 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 22 January 2014 to 03 February 2014

Study design: in vivo (LLNA)

Vehicle:

other: dimethylformamide (DMF)

Concentration:

The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
- the vehicle was selected on the basis of producing a homogeneous preparation suitable for application of the test item,
- the concentrations were selected from the concentration series 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
- the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an = 25% increase of the ear thickness.

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
- the vehicle was selected on the basis of producing a homogeneous preparation suitable for application of the test item,
- the concentrations were selected from the concentration series 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
- the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an = 25% increase of the ear thickness.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness and radioactivity levels.

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
On days 1, 2 and 3, at approximately the same time each day, a dose-volume of 25 µL of the control or dose formulation preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed.

Positive control substance(s):

other: a-hexyl cinnamaldehyde (HCA)

Results and discussion

Positive control results:

SI = 7.42

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

50 % EKKE

Value:

1.64

Results 2

Key result

Parameter:

SI

Remarks:

25% EEKE

Value:

1.15

Test group / Remarks:

EKKE

Results 3

Key result

Parameter:

SI

Remarks:

10% EKKE

Value:

2.05

Results 4

Key result

Parameter:

SI

Remarks:

5% EKKE

Value:

1.5

Results 5

Key result

Parameter:

SI

Remarks:

2.5 % EKKE

Value:

1.33

Results 6

Key result

Parameter:

SI

Value:

7.42

Test group / Remarks:

Positive control

Any other information on results incl. tables

The results are presented in the following table:

 

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	2.5	I	1.33
Test item	5	I	1.50
Test item	10	I	2.05
Test item	25	I	1.15
Test item	50	I	1.64
HCA	25	-	7.42

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).

HCA: a-hexyl cinnamaldehyde.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

EKKE, tested as a homogeneous suspension at concentrations of 2.5, 5, 10, 25 and 50% in dimethylformamide, gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.
Therefore, the test item should not be classified as a skin sensitizer according to the criteria of CLP Regulation.

Executive summary:

The potential of EKKE to induce contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with OECD Guideline No. 429 and the principles of Good Laboratory Practices. To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 5, 10, 25 or 50% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. On completion of the observation period, the animals were sacrificed then discarded without macroscopicpost-mortemexamination. In the main test, five groups of four female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 2.5, 5, 10, 25 or 50% under a dose-volume of 25 µL. One negative control group of four females received the vehicle dimethylformamide (DMF) under the same experimental conditions. Additionally, one positive control group of four females received the positive control,a-hexylcinnamaldehyde (HCA), at 25%in a mixture Acetone/Olive Oil (4/1; v/v)under the same experimental conditions.From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (³H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of³H-TdR. The results are expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

No unscheduled deaths and no clinical signs were observed during the observation period. Body weight of animals was unaffected by the test item treatment. No local reactions were observed in any animals. In all test item-treated groups, the increase in ear thickness was below 10%. The threshold positive value of 3 for the SI was reached in the positive control group (SI = 7.42). The experiment was therefore considered valid. No significant lymphoproliferation was noted with the test item at any tested concentrations as all SI values were below the cut-off value of 3.

 

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
EKKE	2.5	I	1.33
EKKE	5	I	1.50
EKKE	10	I	2.05
EKKE	25	I	1.15
EKKE	50	I	1.64
HCA	25	-	7.42

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).

 

EKKE gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties. Therefore, EKKE not be classified as a skin sensitizer according to the CLP and GHS criteria.