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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 17th to August 24th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
September 22nd, 2015
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Isopropyl trifluoroacetate
EC Number:
206-922-1
EC Name:
Isopropyl trifluoroacetate
Cas Number:
400-38-4
Molecular formula:
C5H7F3O2
IUPAC Name:
isopropyl trifluoroacetate
Test material form:
liquid
Details on test material:
Name: Isopropyl trifluoroacetate

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo - San Pietro al Natisone (UD) - Zona Industriale Azzida, 57, 33049 Italy
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF at arrival
- Age at study initiation: 9 weeks old (age-matched, within one week)
- Weight at study initiation: 19.1 – 21.4 g
- Housing: Group caging (Type II. polypropylene / polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 25.6°C
- Humidity (%): 29 - 80 %
- 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.
- IN-LIFE DATES: From: June 1st, 2016 To: June 7th, 2016

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50 and 100%
No. of animals per dose:
4 animals per dose
Details on study design:
PRE-SCREEN TESTS:
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100% (undiluted) and 50% (w/v) in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
Based on the observation of the solubility test, the maximum available concentration was 100% (undiluted).
No mortality or signs of systemic toxicity were observed. No test item precipitate was observed on the ears of the animals. No marked body weight loss (>5% reduction of body weight) was detected in the experimental animals.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. The ear punch values and ear punch weights were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 100% (undiluted) dose is selected as top dose for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Desintegration per minutes (DPM) was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
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. A stimulation index of 3 or greater is an indication of a positive result.

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, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide and the draining auricular lymph nodes were excised. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL).
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules at 2-8°C overnight.
After a last centrifugation, pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
According to the OECD guideline 429, a statistical analysis of the data is not mandatory.

Results and discussion

Positive control results:
The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 8.2) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: DPN
Value:
776.3
Test group / Remarks:
Negative control
Parameter:
other: DPN
Value:
274.4
Test group / Remarks:
100%
Parameter:
other: DPN
Value:
489.3
Test group / Remarks:
50 (w/v)%
Parameter:
other: DPN
Value:
665.7
Test group / Remarks:
25 (w/v)%
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control
Key result
Parameter:
SI
Value:
0.4
Test group / Remarks:
100%
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
50 (w/v)%
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
25 (w/v)%
Cellular proliferation data / Observations:
PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group and in all the test item treated dose groups.
Larger than normal lymph nodes were observed in the positive control group.
The stimulation index values were 0.4, 0.6 and 0.9 at concentrations of 100% (undiluted), 50% (w/v) and 25 % (w/v), respectively.

EAR THICKNESS MEASUREMENTS
Similarly to the preliminary experiment, ear thickness of the animals was measured using by a thickness gauge and by ear punch weight determination. The ear thickness values and revealing ear punch weights were within the historical control range. There was no indication of
local irritation by visual assessment.
Increased ear thickness values (indicating excessive local irritation) were detected in one animal (right ear) in the positive control group on Day 3, but the biopsy weights were within the acceptable range. Therefore, this fact was considered not to adversely affect the results or integrity of the study.

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the main study. There were no indications of any irritancy at the site of application on the experimental animals.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the body weight changes of experimental animals. No marked body weight loss (≥5%) was observed on the mean body weight changes.

INTERPRETATION OF OBSERVATIONS
The test item was liquid, which was formulated in AOO. Since there were no confounding effects of irritation or systemic toxicity at the applied 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 resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that Isopropyl trifluoroacetate is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion. Based on the observed results, the test item does not need classification according to the GHS or CLP

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present assay, Isopropyl trifluoroacetate, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The objective of the study was to determine the skin sensitisation potential of Isopropyl trifluoroacetate following dermal exposure. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

Based on the results of the preliminary compatibility test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100% (undiluted).

The preliminary irritation/toxicity test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100% (undiluted) and 50% (w/v) in AOO. Based on the observations recorded in the preliminary test, the 100% (undiluted) was selected as top dose for the main test.

In the main assay, twenty female CBA/CaOlHsd mice were allocated to five groups of four animals each:

- three groups received Isopropyl trifluoroacetate at 100% (undiluted); or diluted in AOO at 50% (w/v), 25% (w/v) concentrations,

- the negative control group received the vehicle (AOO),

- 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. 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 or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application on the experimental animals. No marked body weight loss (≥ 5%) was observed on the mean body weight changes.

The simulation index values were 0.4, 0.6 and 0.9 at concentrations of 100% (undiluted), 50% (w/v) and 25% (w/v), respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, Isopropyl trifluoroacetate, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

The following classification/labelling is triggeered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: none.

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