4-fluoro-1,3-dioxolan-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Report date: 4 October 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(13 February 2003)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

483-360-5

EC Name:

-

Cas Number:

114435-02-8

Molecular formula:

C3H3FO3

IUPAC Name:

4-fluoro-1,3-dioxolan-2-one

Test material form:

other: Solid with a low melting point (i.e. liquefied solid)

Details on test material:

Name of the test susbtance: Monofluoroethylene carbonate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

(CBA/CaBkl)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B & K Universal Ltd, Hull, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: ad libitum, Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK
- Water: ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h (6:00 to 18:00)
- IN-LIFE DATES: From: 14 July 2005 To: 3 August 2005

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10, 25 and 50%

No. of animals per dose:

4 females per dose

Details on study design:

PRELIMINARY SCREENING TEST:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test material at a concentration of 50% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. Based on this information the dose-levels selected for the main test were 10%, 25% and 50% w/w in dimethyl formamide.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25 μL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

TERMINAL PROCEDURES:
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmol, Arnersharn Biosciences UK Ltd) giving a total of 20 μCi to each mouse.
Five hours following the administration of 3HTdR, all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a 10 mL centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).
After overnight incubation at 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Pol y Q TM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA).

INTERPRETATION OF RESULTS
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

Positive control substance(s):

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

Results and discussion

Positive control results:

The simulation index for the positive control group was: 2.64, 8.36 and 12.94 at concentrations of 5%, 10% and 25% w/v respectively.
α-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

5.29

Test group / Remarks:

50% w/w in dimethyl formamide

Results 2

Key result

Parameter:

SI

Value:

8.01

Test group / Remarks:

25% w/w in dimethyl formamide

Results 3

Key result

Parameter:

SI

Value:

10.29

Test group / Remarks:

10% w/w in dimethyl formamide

Cellular proliferation data / Observations:

A stimulation index of greater than 3 was recorded for the three concentrations of the test material (10%, 25% and 50% w/w in dimethyl formamide).

Any other information on results incl. tables

Table 1: Dpm, Dpm/Node and Stimulation Index (SI)

Concentration (% w/w) in dimethyl formamide	Dpm	Dpm/Node^a	Stimulation Index (SI)^b	Result
Vehicle	7856.28	982.04	N/A	N/A
10	80858.79	10107.35	10.29	Positive
25	62903.23	7862.90	8.01	Positive
50	41552.19	5194.02	5.29	Positive

a = Dpm/node obtained by dividing the Dpm value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not applicable

Mortality: There were no deaths.

Clinical observations: No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight: Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test material was considered to be a sensitiser under the conditions of the test. An inverse dose response relationship was noted in the Stimulation Index results. The reason for this is unknown but could be due to decreased bioactivity of the test material with increasing concentrations in dimethyl formamide, or due to immunosuppression at higher concentrations of test material. An EC3 value could not be calculated due to this and also because all SI values were greater than 3 which did not allow calculation (as derived by linerar interpolation).

Executive summary:

A study was performed to assess the skin sensitisation potential of Monofluoroethylene carbonate in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

- OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

- Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Following a preliminary screening test, three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test material as a solution in dimethyl formamide at concentrations of 10%, 25% or 50% w/w. A further group of four animals was treated with dimethyl formamide alone.

The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in dimethyl formamide	Stimulation index (SI)	Result
10	10.29	Positive
25	8.01	Positive
50	5.29	Positive

Monofluoroethylene carbonate was considered to be a sensitiser under the conditions of the test.

An inverse dose response relationship was noted in the Stimulation Index results. The reason for this is unknown but could be due to decreased bioactivity of the test material with increasing concentrations in dimethyl formamide, or due to immunosuppression at higher concentrations of test material.