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Administrative data

Description of key information

The substance caused skin sensitisation reactions in a reliable study conducted according to Test Guideline OECD429 (Local Lymph Node Assay).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-JUL-2017 to 08-NOV-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Deviations:
yes
Remarks:
The actual temperature and relative humidity deviated from guideline range. For animal ethical reasons, mice used in the preliminary assay were 13 weeks old. These deviations were not considered to impact the results or integrity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The actual temperature and relative humidity deviated from guideline range. For animal ethical reasons, mice used in the preliminary assay were 13 weeks old. These deviations were not considered to impact the results or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BLT 03-2017
- Expiration date of the lot/batch: March 2022
- Purity test date: 10 March 2017
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, Italy
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old (age-matched, within one week) (except animals used for preliminary assay: 13 weeks old)
- Weight at study initiation: 19.9 - 21.8 g (22.3 - 24.3 g for the mice aged 13 weeks used in the premliminary assay)
- Housing: Group caging in Type II. polypropylene / polycarbonate cages / mice were provided with glass tunnel-tubes for enrichment
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.4 – 25.7°C
- Humidity (%): 30 - 77 %
- 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: 2-AUG-2017 To: 8-AUG-2017
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100%, 50%, 25% w/v in acetone/olive oil (4:1 v/v)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Based on the observation of the solubility test, the maximum available concentration was 100 % (w/v).
- Irritation: the preliminary assay was conducted in 2 mice/dose using 2 doses (100% and 50% (w/v) in acetone/olive oil.
- Systemic toxicity: no signs of systemic toxicity were observed, no mortality and no clinical signs. Marked body weight loss (> 5% reduction of bw) was seen in one animal of each group.
- Ear thickness measurements: thickness gauge (on day 1, pre-dose; day 3, 48h after the 1st dose; day 6). Additional quantification of the ear thickness was done by ear punch weight after the euthanasia of the experimental animals. Both the ear thickness value and ear punch weights were within the acceptable range.
- Erythema scores: 0 in all animals, at both test concentrations and all time-points


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
1- 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.
2- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Topical application: 25 µl of the test item or formulation
Test concentrations: 100% (undiluted), 50%, 25% w/v in acetone/olive oil (4:1 v/v)
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.

PROLIFERATION ASSAY
On Day 6, each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR. 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. The draining auricular lymph nodes were excised and pooled per test group, collected in separate Petri dishes and kept wet in a small amount of PBS 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. Following PBS washes te pooled lymph nodes were pelleted by centrifugation, resuspended in PBS, washed again.

Determination of Incorporated 3HTdR:
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.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. 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).
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)
Positive control results:
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved at a concentration of 25 % in the in the same vehicle (AOO) was used to demonstrate the appropriate performance of the assay.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 8.5) was noted for HCA in the main experiment.
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control (acetone/olive oil)
Parameter:
SI
Value:
5.5
Test group / Remarks:
100% (w/v)
Parameter:
SI
Value:
2.3
Test group / Remarks:
50% (w/v)
Parameter:
SI
Value:
1.1
Test group / Remarks:
25% (w/v)
Parameter:
SI
Value:
8.5
Test group / Remarks:
Positive control (25% (w/v) HCA)
Key result
Parameter:
EC3
Value:
ca. 60.9
Test group / Remarks:
Extrapolated value
Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
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 was calculated for each treatment group: SI = DPN value of a treated group divided by the DPN value of the negative control group.

EC3 CALCULATION
EC3 = c + [(3-d)/(b-d)] x (a-c)
In this equation, the data points lying immediately above and below the SI value of 3 on the dose-response plot have the coordinates (a,b) and (c,d), respectively.
The calculated EC3 value of 1,4-Diiodoperfluorobutane is 60.9 % (w/v).

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. No amount of test item precipitation was observed on the ears of the animals.

BODY WEIGHTS
In the main study, no marked body weight losses (≥5%) was observed on the mean body weight changes in any groups; however, the body weight loss was ≥5% for 1/4 animals in the 50 % (w/v) and the Positive Control group. These changes were considered incidental.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the present assay, 1,4-Diiodoperfluorobutane, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The EC3 value of 1,4-Diiodoperfluorobutane was calculated as 60.9 % (w/v).
Executive summary:

The skin sensitisation potential of 1,4-Diiodoperfluorobutane following dermal exposure in CBA/CaOlaHsd mice was assessed in the Local Lymph Node Assay. The study was performed according to OECD Guideline No.: 429, and in compliance with Good Laboratory Practices.

Based on the results of the Preliminary Compatibility Test, the best vehicle for the test item was Acetone:Olive Oil 4:1 (v:v) mixture (AOO) and the 100 % (w/v) formulation was chosen as the highest concentration suitable for the test.

Following the Preliminary Irritation/Toxicity Test using two doses and 2 animals/dose, 100 % (w/v) in AOO was selected as top dose for the main test.

In the main assay, twenty female mice were allocated to five groups (4 mice/group):

- three groups received 1,4-Diiodoperfluorobutane (formulated in AOO) at 100, 50, and 25 % (w/v) concentrations respectively,

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

- the positive control group received 25 % (w/v)α-Hexylcinnamaldehyde (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 assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

No mortality or signs of systemic toxicity were observed during the study. No marked bodyweight losses was observed on the mean body weight changes inany groups; however,the body weight loss was ≥5% for 1/4 animals in the50 % (w/v) and the Positive Control group.

The stimulation index (SI) values were 5.5, 2.3 and 1.1 at concentrations of 100, 50 and 25 % (w/v), respectively.

The result of the positive control substance HCA was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical 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, 1,4-Diiodoperfluorobutane, tested in a suitable vehicle, was shown to have a sensitisation potential in the Local Lymph Node Assay. The extrapolated EC3 value of 1,4-Diiodoperfluorobutane is 60.9 % (w/v).

The substance is classified as skin sensitizer Category 1 (sub-category 1B) according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Results of a sensitisation assay using Test Guideline OECD 429 showed a skin sensitising potential triggering classification in sub-category 1B, according to criteria in EC CLP Regulation 1272/2008.