2,2-Difluoroethyl Acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 JULY 2013 to 16 SEPT 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

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:

2,2-difluoroethyl acetate

EC Number:

801-773-4

Cas Number:

1550-44-3

Molecular formula:

C4H6F2O2

IUPAC Name:

2,2-difluoroethyl acetate

Test material form:

liquid

Remarks:

clear and colorless.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/JHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Frederick, Maryland, U.S.A.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: mice were 10 weeks old at study start (day 1).
- Weight at study initiation: 23.0 to 23.9 grams.
- Housing: solid-bottom cages with appropriate bedding and nestlets toys as enrichment. During quarantine, animals were housed in groups of 5 or fewer. After assignment to test groups, during the dosing and resting phases of the study, and until sacrifice, animals were housed in groups of 5 or less per solid bottom cage with appropriate bedding. Animals were single housed for approximately 2 hours following each application of the vehicle, test substance, or positive control to allow additional time for drying and/or absorption. Following the 2-hour single-housing period, animals were returned to their group housing status.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum.
- Water: tap water ad libitum.
- Acclimation period: 8 days of quarantine. The mice were released from quarantine based on normal observations from body weight gain and clinical signs in all individuals.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-26ºC.
- Humidity: 30-70%.
- Photoperiod: 12-hour light/dark cycles.

IN-LIFE DATES: from 17 July 2013 to 22 July 2013.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

(MEK)

Concentration:

Tested concentrations: 0 (vehicle), 5, 25, 50, 100 % of test material.
The test material is a liquid and did not appear to have severe skin-irritating capability (pH ~5). Consequently the 100% concentration was chosen as the high dose based on solubility testing. For subsequent concentrations, the test substance was prepared in MEK.

No. of animals per dose:

5 animals per dose group.

Details on study design:

PRE-SCREEN TEST
- Compound solubility: Prior to study start, a quantity of the test substance was evaluated for solubility in the vehicle. The control and test substance concentrations and method of preparation were based on solubility information.

ANIMAL ASSIGNMENT
Mice, selected based on adequate body weight gain and freedom from any ear abnormalities (e.g., torn, scratched) or clinical signs of disease or injury, were distributed into study groups. Prior to study start, each animal was assigned to a group using a randomly generated, computer-based algorithm such that individual pretest body weights did not vary more than 20% of the group mean.

TREATMENT PREPARATION
- Test substance treatment: The test substance was prepared in the MEK vehicle at different concentrations. The 100% concentration corresponded to the neat test substance. Dose preparations were not analyzed for homogeneity or accuracy of concentration. The dose preparation procedures were believed to provide homogeneous mixtures at the targeted concentrations. In the absence of visible change in color or physical state, all dose preparations were assumed to be stable throughout the study.
- Positive control treatment: A 25% hexylcinnamaldehyde (HCA) solution in the MEK vehicle was blended using a vortex mixer and stored in a vial protected from light until dosing was completed.
All dose preparations were formulated fresh daily.

MAIN STUDY
25 μL of vehicle, test substance preparation, or positive control were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 1-3). Test days 4-5 were days of rest followed by intravenous injection of 20 μCi of ³H-thymidine in PBS per mouse on test day 6.
Approximately 5 hours after the injection, animals were sacrificed by isoflurane anaesthesia followed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8°C overnight. On test day 7, the single cell suspensions were counted on a beta counter and reported as disintegrations per minute (dpm).

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE
A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle group. The decision process in regard to a positive response included an SI of greater than or equal to 3.0 together with consideration of dose response and, where appropriate, statistical significance.
If possible, an EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the SI data was derived from linear interpolation of points on the dose-response curve immediately above and below the 3-fold threshold. The equation used for calculation of EC3 was:
EC3 = c + [(3 - d)/(b - d)] × (a - c)
where:
a = the lowest concentration giving stimulation greater than 3
b = the actual SI caused by a
c = the highest concentration giving stimulation lower than 3
d = the actual SI caused by c

Positive control substance(s):

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

Statistics:

Significance was judged at p < 0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances.
Methods:
- Test for lack of trend then either sequential application of the Jonckheere-Terpstra trend test or preliminary test for pairwise comparison.
- OR Levene's test for homogeneity and Shapiro-Wilk test for normality, then either one-way analysis of variance followed by Dunnett's test or Kruskal-Wallis test followed by Dunn's test.

Results and discussion

Positive control results:

A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice (see Table 1 in "Any other information on results" below). Therefore, the LLNA test system was valid for this study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

14.64

Test group / Remarks:

Positive control (25% HCA)

Results 2

Key result

Parameter:

SI

Value:

1.17

Test group / Remarks:

Test material at 100%

Results 3

Key result

Parameter:

SI

Value:

1.08

Test group / Remarks:

Test material at 50%

Results 4

Key result

Parameter:

SI

Value:

0.93

Test group / Remarks:

Test material at 25%

Results 5

Key result

Parameter:

SI

Value:

1.21

Test group / Remarks:

Test material at 5%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
See the cellular proliferation data expressed in dpm in Table 1 in "Any other information on results" below.
No statistically significant increases in cell proliferation measurements compared to the vehicle group were observed at any test concentration.

DETAILS ON STIMULATION INDEX CALCULATION and EC3 CALCULATION:
See the detailed results in Table 1 in "Any other information on results" below.
SIs of less than 3.0 were observed at all test concentrations of the test material. Therefore, the EC3 value for the test substance under the conditions of this study could not be calculated.

CLINICAL OBSERVATIONS:
No clinical signs of toxicity were observed in the study.

BODY WEIGHTS:
No test substance-related changes in body weights were observed at any test concentration. Body weight losses of <1-3% of initial body weight were not considered test substance related, as they were observed in single animals or did not exhibit a dose response.

Any other information on results incl. tables

Table 1 : Stimulation Index data

 

Group	Test Material concentration	n	Mean (dpm)	S.D. (dpm)	SI
1	0% (= vehicle)	5	435.60	220.92	N/A
2	5%	5	525.20	126.55	1.21
3	25%	5	403.00	128.25	0.93
4	50%	5	469.00	219.15	1.08
5	100%	5	510.00	215.88	1.17
6	Positive control (25% HCA)	5	6378.00	1313.96	14.64

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, 2,2-Difluoroethyl acetate did not produce a dermal sensitization response in mice.

Executive summary:

The skin sensitization potential of 2,2-Difluoroethyl acetate was investigated in a study performed according to OECD test guideline 429 (Local Lymph Node Assay, LLNA) under GLP compliance.

Six groups of five female CBA/JHsd mice were treated for three consecutive days with four concentrations of 2,2-Difluoroethyl acetate: 5%, 25%, 50% and 100% in vehicle (methyl ethyl ketone, MEK), with the vehicle alone and with a positive control substance (25% hexylcinnamaldehyde, HCA, solution in MEK). Administration of dosing formulations occurred on days 1, 2 and 3 by administrating topically 25 μl on the dorsum of each mouse ear. On day 6, all animals received a tail vein injection of 20 μCi of ³H-thymidine in PBS. Approximately 5 hours after the injection, animals were sacrificed, draining auricular lymph nodes were removed, and single cell suspensions were prepared. On day 7, the ³H-thymidine incorporation in the draining auricular lymph nodes was determined by counting on a beta counter the disintegrations per minute (dpm) of single cell suspensions. A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle group. A SI >=3 (with consideration of dose response and, where appropriate, statistical significance) is regarded as a positive response.

No clinical signs of toxicity were observed in the study. In the positive control group, a stimulation index of 14.64 was calculated which demonstrated the sensitivity and validity of the system. No statistically significant increases in cell proliferation measurements compared to the vehicle group were observed at any 2,2-Difluoroethyl acetate concentration. SIs of less than 3.0 were observed at all test concentrations (1.21, 0.93, 1.08 and 1.17 at 5%, 25%, 50% and 100% 2,2-Difluoroethyl acetate in vehicle, respectively). Therefore, the EC₃ value (the estimated concentration required to induce a threshold positive response, i.e. SI = 3) under the conditions of this study was not calculable.

Under the conditions of this study, the stimulation indices are <3 for all tested concentrations. Therefore, 2,2-Difluoroethyl acetate is not a dermal sensitizer in mice.