Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 June 2014 - 15 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 429. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 4,4,4-Trifluoro-1-(4-methylphenyl)-1,3-butanedione
- Physical state: slightly coloured crystalline powder
- Analytical purity: 99.9%
- Lot/batch No.: M13696C

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 10 weeks old
- Weight at study initiation: 21.1-22.8 g
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage type: Type II. polypropylene / polycarbonate.
- Diet (e.g. ad libitum): ad libitum, ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: al least 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1-25.0 °C
- Humidity (%): 31-78 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
(AOO)
Concentration:
25, 50 and 100 % (w/v).
No. of animals per dose:
4 animals per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test.Based on physical and chemical characteristics of the test item, 100% concentration in Acetone: Olive oil 4:1 (v/v) mixture (AOO) was achievable.
- Preliminary Irritation/Toxicity Test: 2 animals per dose were exposed to test item concentrations of 100 and 50 % (w/v) in AAO. The experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. No mortality or clinical signs of systemic toxicity were observed. No marked body weight loss (>5%) was observed. Precipitate was observed on the ears of the animals in the 100% (w/v) and 50% (w/v) dose groups. The appearance of the lymph nodes was normal in all test item treated group. Based on these results, Based on these results 100% and 50% (w/v) doses were considered to be acceptable for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
*That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in
control mice, as indicated by the stimulation index.
*The data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On day 6, each mouse received an injection of 250µl of sterile PBS (phosphate buffered saline) containing approx. 20 µCi of 3HTdR. Five hours later, the mice were euthanized and the auricular lymph nodes were extracted from the animals. A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and the samples were prepared to be examined in a β-scintillation counter.

OBSERVATIONS:
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection).

EVALUATION OF RESULTS:
Radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes and corrected with the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes). Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation index values were 1.3, 4.4 and 2.5 at concentrations of 100, 50 and 25 % (w/v), respectively. The calculated EC3 value was calculated to be 31.6% (w/v).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Test item 100 % (w/v) in AOO: 3435.0 DPM Test item 50 % (w/v) in AOO: 11155.0 DPM Test item 25 % (w/v) in AOO: 6307.0 DPM

Any other information on results incl. tables

Clinical observation:

No mortality was observed during the study. Aggressive behaviour (fighting) was observed in two animals of the 100% (w/v) dose group and for one animals of the 50% (w/v) dose group on Day 3. Slight intermittent tremors were also recorded in the 100% (w/v) dose group. Animals were repeatedly observed later on that day (at 2 hours after treatment), and the aggressive behaviour was still observed in two animals of the 100% (w/v) dose group. Test item precipitate was observed in the 100% (w/v) dose group on Days 1-3; minimal amount of test item precipitate was observed in the 50% (w/v) dose group on Days 1-2. There were no indications of any irritancy at the site of application.

Body weight:

No treatment related effects were observed on body weights. Although marked body weight loss (5%) was detected for one animal in the 50% (w/v) dose group, but this fact was considered as animal variability, not a test item related effect.

Proliferation assay:

Test Group Name

Measured DPM / group

DPM

Number
lymph nodes

DPN

Stimulation Index

Background (5 % (w/v) TCA)

35/31

-

-

-

-

(-) control (AOO)

2580

2547.0

8

318.4

1.0

Test item 25 % (w/v) in AOO

3468

3435.0

8

429.4

1.3

Test item 10 % (w/v) in AOO

11188

11155.0

8

1394.4

4.4

Test item 5 % (w/v) in AOO

6340

6307.0

8

788.4

2.5

(+) control (25 % (w/v) HCA in AOO

29693

29660.0

8

3707.5

11.6

The appearance of the lymph nodes was normal in the negative (vehicle) control group and in all test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

At 100% the aggressive behaviour indicated a systemic toxic effect in 2/4 mice, although this was not seen in the preliminary study, hence this dose level appears to be slightly above the acceptable maximum. But because the stimulation index at this dose level is not >3, it has no consequences. Since there were no confounding effects of irritation at any dose level, or significant systemic toxicity at the other concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

The resulted stimulation index observed at 50% (w/v) concentration under these exaggerated test conditions was considered to be good evidence that is a sensitizer (Figure 1). Any possible toxic effects at 100% (w/v) concentration do not influence this interpretation. The obtained data are compatible with a conventional dose response. The lower SI value observed at higher concentration of 100% (w/v) might be the limited skin penetration due to the less amount of vehicle in this 1 g/mL formulation. Furthermore, potential cytotoxicity to the immunocytes cannot be excluded in this case. The size of lymph nodes made no contradiction with the observed slight positive effect.

The calculated EC3 value was calculated 31.6% (w/v).

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item showed to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The stimulation index values were 1.3, 4.4 and 2.5 at concentrations of 100, 50 and 25 % (w/v), respectively. The calculated EC3 value was calculated to be 31.6% (w/v).
Executive summary:

The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42 (GLP study). Based on the results of the Preliminary Compatibility Test, the test item was formulated in acetone:olive oil 4:1 (v:v) mixture

(abbreviated as AOO) at a highest achievable concentration of 100 % (w/v). The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using two doses: 100 and 50 % (w/v) in AOO. The 100 % (w/v) was selected as top dose for the main test. Twenty female CBA/J Rj mice were allocated to five groups of four animals each in the main test. Three groups received the test item at 100, 50 and 25 % (w/v) concentrations, the negative control group received the vehicle (AOO) and the positive control group received 25 % (w/v) HCA (dissolved in AOO). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality was observed during the study. Aggressive behaviour and slight tremors indicating systemic toxicity was observed in 2/4 mice at 100% (w/v) (not observed in the preliminary test). No treatment related effects were observed on body weight. There were no indications of any irritancy at the site of application. Test item precipitate was observed in the 100 and 50 % (w/v). The stimulation index values were 1.3, 4.4 and 2.5 at concentrations of 100, 50 and 25 % (w/v), respectively. The calculated EC3 value was 31.6% (w/v). All validity criteria were fulfilled. In conclusion, the test item showed to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.