[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/Jun/05 - 28/Jun/05

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

EC Number:

609-858-6

Cas Number:

406-78-0

Molecular formula:

C4H3F7O

IUPAC Name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

Test material form:

liquid

Specific details on test material used for the study:

Name: HFE-347pc-f
Source: Asahi Glass
Colour: Colourless
Physical state: Liquid
Batch reference number: 50312080
CTL test substance reference number: Y12880/001
Purity (%w/w): 99.9%
Storage conditions: Ambient temperature in the dark
Stability: 27th September 2005

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

The mice used for the study were young adults (8-12 weeks of age). A maximum of 4 mice were housed per cage, in cages suitable for this strain and weight range. Environmental enrichment provided included tents, bases and nestlets. The animal room was designed to have the following environmental conditions (temperature and relative humidity were recorded daily):

Temperature - 22 ± 3°C
Relative humidity - 30-70%
Air - A minimum of 15 changes per hour
Light cycle - Artificial, giving 12 hours light and 12 hours dark

Diet (RM1) and mains water, supplied by an automatic system, were available ad libitum. Each batch of diet is routinely analysed for composition and contaminants and water is periodically analysed for contaminants. No contaminants were found in the diet or water at levels considered likely to interfere with the purpose or outcome of the study. Animals were housed under the above experimental conditions for at least 5 days prior to the start of dosing. Animals were individually identified, within each cage, by a clipped area on the side. The first animal in each cage had a clipped area on the left side, the second on the right side, the third on both flanks and the fourth was not clipped.

Study design: in vivo (LLNA)

Vehicle:

other: Acetone

Concentration:

25μl of a 25, 50 or 100% w/v of the test substance in acetone

No. of animals per dose:

4

Details on study design:

Groups of 4 female mice were used for this study. Approx. 25ul of 25, 50 or 100% w/v preparation of the test substance in acetone was applied, using a variable volume micro-pipette, to the dorsal surface of each year. A vehicle control group was similarly treated, but using acetone alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected via the tail vein with approx. 250ul of phosphate buffered saline (PBS), containing 20uCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine. 5 hours later, the animals were humanely killed by inhalation of halothane vapour, followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and placed, together with the nodes of the other animals in the group, in a container of PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed 3x by centrifugation with approx. 10ml of PBS. Approx. 3ml of 5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approx. 1ml of TCA. The lymph node suspensions were transferred to scintillation vials and 10ml of scintillant (Optiphase) was added prior to β-scintillation coutning using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

Clinical observations were made at least once daily, during the study, for signs of systemic toxicity. The bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of 3H-methyl thymidine.

A positive control study was also carried out, in which the sensitisation potential of hexylcinnamaldehyde was assessed using the method outlined above. Approx. 25ul of a 5%, 10% or 25% w/v preparation of hexylcinnamaldehyde in acetone olive oil (4:1) was applied. A vehicle control group was also treated, in which acetone in olive oil alone (4:1) was applied.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

25%w/v

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

50%w/v

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 3

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

100%w/v

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Any other information on results incl. tables

The results are expressed as disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test: control ratio, known as the stimulation index (SI), for each concentration. The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3 -fold or greater increase in isotope incorporation, relative to the vehicle control group. Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a skin sensitiser.

The application of the test substance at concentrations of 25, 50 and 100% w/v in acetone resulted in an isotope incorporation which was less than 3 -fold at all concentrations (see table below). Consequently, the test substance is designated as unlikely to be a skin sensitiser under the conditions of the test.

Concentration of test substance (% w/v)	Number of lymph nodes assayed	Disintegrations per minute (dpm)	dpm per lymph node	Test: control ratio (SI)
0 (vehicle only)	8	3352	419	N/A
25	8	3748	469	1.1
50	8	3500	438	1.0
100	8	2813	352	0.8

In the positive control test, the application of hexylcinnamaldehyde at concentrations of 5%, 10% and 25% in acetone in olive oil (4:1) resulted in a >3 -fold increase in isotope concentration at the 10 and 25% w/v concentrations (see table below). Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

Concentration of hexylcinnamaldehyde (% w/v)	Number of lymph nodes assayed	Disintegrations per minute (dpm)	dpm per lymph node	Test: control ratio (SI)
0 (vehicle only)	8	1636	205	N/A
5	8	3793	474	2.3
10	8	6821	853	4.2
25	8	24807	3101	15.1

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether is unlikely to be a skin sensitiser under the conditions of the test.

Executive summary:

The purpose of this study was to assess the skin sensitisation potential of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether, using the Local Lymph Node Assay. The assay determines the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application, by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. The test substance was applied as 20, 50 or 100% w/v preparations in acetone. A vehicle control group was similarly treated using acetone alone. The test substance did not have the capacity to cause skin sensitisation when applied as 25 or 50%w/v preparations in acetone or, when applied neat. In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 10% and 25%w/v preparations in acetone in olive oil (4:1), confirming the validity of the protocol used for this study. In conclusion, therefore, 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether is unlikely to be a skin sensitiser under the conditions of the test.