Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March-April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, in accordance with guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
other: EC Regulation 2008/440/EC B.42 Skin Sensitisation: Local lymph node assay (Amendment 06 July 2012 Commission Regulation (EU) No 640/2012)
GLP compliance:
yes (incl. certificate)
Remarks:
GYEMSZI National Institute
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Test item: 4-chloro-a,a,a-trifluorotoluene (PCBTF)
CAS No.: 98-56-6
EC No.: 202-68-1
Batch No.: 11/2012
Appearance: Liquid, colourless, aromatic
Composition: 4-chloro-a,a,a-trifluorotoluene
Purity: 99.3 %
Expiry date: January 31, 2016
Solubility: 29 ppm in water
Soluble in almost all primary organic solvents
Storage: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT.
- Age at study initiation: 10-11 weeks old (at start of the preliminary test)
- Weight at study initiation: 16.1-20.0 g.The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: Grouped caging (4 animals/cage), Type II. Polypropylene / polycarbonate
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water (e.g. ad libitum): tap water from municipal supply, from a bottle ad libitum.
- Acclimation period: 21 or 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 06 March 2013 To: 14 March 2013

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
The test item was a liquid hence applicability of the 100 % concentration (the undiluted test item) was evaluated in order to test the highest test concentration possible. Further test concentrations were achieved by formulation of the test item in N,N-Dimethylformamide (DMF). Both the undiluted test item and the formulations were adequately applicable on the ears of animals.
Based on results of the preliminary irritation/toxicity test 4-chloro-a,a,a-trifluorotoluene (PCBTF) was examined in the LLNA at concentrations of 100 % as the undiluted test item and as 75 %, 50 % or 25 % (w/v) formulations in DMF according to the relevant guidelines.
No. of animals per dose:
Four groups of 4 animals each.
Details on study design:
PRELIMINARY ASSAY
The maximum dose selection was performed according to the relevant guidelines. The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used.
The test item was a liquid hence applicability of the 100 % concentration (the undiluted test item) was evaluated in order to test the highest test concentration possible. Further test concentrations (75 % and 50 % (w/v)) were achieved by formulation of the test item in DMF. Both the undiluted test item and the formulations (apparently solutions) were adequately applicable on the ears of animals.
Three groups of 2 CBA/Ca mice were treated with the undiluted test item and the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the preliminary test. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6). Both ears of each mouse were observed for erythema. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
No mortality was observed during the test. No significant, treatment related effect on the body weights was observed in any treatment group. No signs of systemic toxicity or significant local irritation (indicated by an erythema score ≥ 3 and/or an increase of ear thickness ≥ 25 %) were observed at the treatment site (ears) at the tested concentrations. Based on the preliminary test results 100 % (the undiluted test item) was used as maximum concentration in the main test in order to test the highest concentration possible. The test item was also tested at three additional, lower concentrations (75 %, 50 % and 25 %) according to the relevant guidelines.
MAIN TEST DESIGN
Animals in the treatment groups were treated with the relevant vehicles (DMF and AOO), appropriate formulations of the test item or 25 % concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test. Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicles (DMF and AOO, as negative control groups) using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. 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 was intravenously injected via the tail vein with 250 µL of sterile PBS (1 x PBS, diluted from 10x concentrate with purified water) containing 20 µCi of 3H-methyl-thymidine using a 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 sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps.
Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, 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). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and resuspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloracetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8°C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4°C and decanting the supernatants, than the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
For the measurement of incorporated radioactivity the vials were loaded into a beta-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The beta-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
EC3 calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve .
Dose-response relationship was evaluated by linear regression.

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI> 3) was noted for HCA (SI = 11.2). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation index values were 7.3, 6.9, 8.1 and 1.1 at concentrations of 100 %, 75 %, 50 % and 25 %, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPN (DPM divided by the number of pooled lymph nodes).

Any other information on results incl. tables

No significant, treatment related effect on body weights was observed during the test.

No mortality or symptoms of systemic toxicity were observed in any treatment group. No visible sign of significant irritation (indicated by an erythema score> 3) or any other local effect at the treatment site (ears) was observed in any treatment group.

Visually larger lymph nodes than the relevant controls were observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative control groups (both DMF and AOO) and in the test item treated groups.

In spite of normal visual appearance of the lymph nodes significantly increased lymphoproliferation (indicated by an SI ³ 3) compared to the relevant control (DMF) was noted for 4-chloro-a,a,a-trifluorotoluene (PCBTF) at concentrations of 100 %, 75 % and 50 %. No significantly increased lymphoproliferation was observed at concentration of 25 %.

Dose-related response was observed, although its linearity was not statistically significant (p = 0.30, r = 0.70, evaluated by linear regression using SI values).

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: Lymph nodes from all 4 animals in each dose groups were pooled for a valid experiment. Skin sensitizing effect was observed at the applied concentration of the positive control (the stimulation index was greater than 3).
Conclusions:
Under the conditions of the present Local Lymph Node Assay, 4-chloro-a,a,a-trifluorotoluene (PCBTF) tested at concentration of 100 %, as the undiluted test item and as 75 %, 50 % and 25 % (w/v) formulations in an appropriate vehicle (DMF) was shown to have sensitization potential.
Based on the EC3 value calculated using the dose-response curve 4-chloro-a,a,a-trifluorotoluene (PCBTF) was classified as a weak sensitizer in this LLNA according to the published data. Considering the Commission Regulation (EU) No 286/2011 of 10 March 2011 amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures, the classification of 4-chloro-a,a,a-trifluorotoluene (PCBTF) for skin sensitisation can be the following: Category 1, Sub-category 1B.
Executive summary:

The aim of this study was to evaluate the skin sensitization potential of 4-chloro-a,a,a-trifluorotoluene (PCBTF) following dermal exposure in the Local Lymph Node Assay. The test item was a liquid hence applicability of the 100 % concentration (the undiluted test item) was evaluated in order to test the highest test concentration possible. Further test concentrations were achieved by formulation of the test item inN,N-Dimethylformamide (DMF). Both the undiluted test item and the formulations were adequately applicable on the ears of animals. Based on results of the preliminary irritation/toxicity test 4-chloro-a,a,a-trifluorotoluene (PCBTF) was examined in the LLNA at concentrations of 100 % as the undiluted test item and as 75 %, 50 % or 25 % (w/v) formulations in DMF according to the relevant guidelines.

28 female CBA/Ca mice were allocated to 7 groups of four animals each:- four groups received4-chloro-a,a,a-trifluorotoluene (PCBTF)at four different concentrations of 100 %, 75 %, 50 % and 25 % (w/v), - the positive control group receiveda-Hexylcinnamaldehyde (HCA) at concentration of 25 % (w/v), - one control group (used as negative control for the groups treated with the test item) received the vehicle of the test item (DMF) only, - one control group (used as negative control for the group treated with the positive control substance) received the vehicle of the positive control (AOO) only. Each substance was applied on the external surface of each ear of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of3HTdR and the obtained values were used to calculate stimulation indices (SI). The positive control item (25 % HCA in AOO) induced the appropriate (SI ³ 3) stimulation over the control (SI value was 11.2), thus confirming the validity of the assay. No mortality was observed during the study. No significant, treatment related effect on body weights or any other signs of systemic toxicity were observed in any treatment group during the test. No signs of significant irritation or any other local effect were observed at the treatment site (ears) in any treatment group. Visually largerlymph nodes than the relevant controls were observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative control groups (both DMF and AOO) and in the test item treated groups. In spite of normal visual appearance of the lymph nodes significantly increased lymphoproliferation (indicated by an SI ³ 3) compared to the relevant control (DMF) was noted for 4-chloro-a,a,a-trifluorotoluene (PCBTF)at concentrations of 100 %, 75 % and 50 %. No significantly increased lymphoproliferation was observed at test item concentration of 25 %. The stimulation index values were 7.3, 6.9, 8.1 and 1.1 at concentrations of 100 %, 75 %, 50 % and 25 %, respectively. Dose-related response was observed, although its linearity was not statistically significant(p = 0.30, r = 0.70, evaluated by linear regression using SI values). 

Since the test was valid and no sign of systemic toxicity or irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay. Although

linearity of the observed dose-response proved not to be statistically significant, the increased lymphoproliferation observed at three test concentrations (100 %, 75 % and 50 %) is considered evidence that4-chloro-a,a,a-trifluorotoluene (PCBTF) has sensitization potentialaccording to evaluation criteria of therelevant guidelines. 

EC3calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve. The calculated EC3value of 4-chloro-a,a,a-trifluorotoluene (PCBTF) based on the dose-response was 31.8 % in this LLNA. Using the EC3value
4-chloro-a,a,a-trifluorotoluene (PCBTF) can be ranked among weak sensitizers (10 ≤ EC3 ≤ 100) in this LLNA according to the published data for classification of contact allergens