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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 August 2013 - 18 November 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,15-dichloro-3,5,8,11,13-pentaoxapentadecane; 1-chloro-2-[(2-chloroethoxy)methoxy]ethane; 1-chloro-2-{[2-(2-chloroethoxy)ethoxy]methoxy}ethane
EC Number:
Molecular formula:
C5H10Cl2O2 C10H20Cl2O5 C7H14Cl2O3
1,15-dichloro-3,5,8,11,13-pentaoxapentadecane; 1-chloro-2-[(2-chloroethoxy)methoxy]ethane; 1-chloro-2-{[2-(2-chloroethoxy)ethoxy]methoxy}ethane
Test material form:
other: liquid
Details on test material:
Chemical Name: Reaction mass of Bis (2-chloroethoxy)methane and 1,15-dichloro-3,5,8,11,13-pentaoxa-pentadecane and 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane
Batch/Lot Number: 030613
Purity: 100% (multi constituent substance)
Appearance: slightly brownish liquid

In vivo test system

Test animals

Details on test animals and environmental conditions:
Species and strain: CBA/J Rj mice
Route des Chènes Secs B.P. 4105
53940 LE GENEST-ST-ISLE, France
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 8 weeks old (age-matched, within one week)
Body weight range at starting: 20.3-22.0 grams
(The weight variation in animals of the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 6 days
Note: In the preliminary experiment, mice of 8 weeks of age (20.8-21.4 grams) were used after 8 days of acclimatization.

Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.6 - 24.7 °C
Relative humidity: 30 - 68 %
Ventilation: 15-20 air exchange/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (non-radioactive phase): 244/5 (preliminary experiment)
523 (main experiment)
Room/Cabinet (radioactive phase): 139 – 140

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
50-25-10-5 in AOO
No. of animals per dose:
Details on study design:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. Due to the physical characteristics of the test item (liquid), 100% (undiluted) concentration was achievable. The formulation at 50% (w/v) using Acetone : Olive oil 4:1 (v/v) mixture (abbreviated as AOO) as vehicle was suitable for the test. As AOO is one of the vehicles recommended by the relevant OECD guideline, it was selected for vehicle of the study.
The test item was weighed and formulations prepared daily on a weight : volume basis (as % (w/v) ) in the Central Dispensary Unit of CiToxLAB Hungary Ltd.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study, although the formulations were checked for visible homogeneity and physical stability.
- Irritation:
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/J Rj 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.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum achievable concentration was 100% (undiluted).
In the Preliminary Irritation / Toxicity Test, signs of systemic toxicity were observed. Increased activity, tremors, incoordination in both hind-limbs and hunched back were observed in the animals of the 100% (undiluted) and in the 50% (w/v) dose groups on Day 3. Increased respiratory rate, tremors, increased activity, incoordination was observed in one animal of the 100% (w/v) dose group on Day 4, this animal was found dead on Day 5. The observations of the preliminary experiment are summarized in Table 8 of Appendix 4. No marked body weight loss was observed in the surviving experimental animals (Table 6 of Appendix 4).
Ear thickness of the animals was measured by using a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The weights of the ear punches (2 per animal). Ear thickness data and the revealing ear punch weights were within the historical control range. Both ears of each mouse were observed for erythema. No visual signs of local irritation were observed.
The draining auricular lymph nodes of the animals were visually examined: the appearance of the lymph nodes was normal in the 100% (undiluted) dose group and larger than normal in the 50% (w/v) dose group (subjective judgement by analogy with observations of former experiments).
Based on these results, 50% (w/v) dose was considered to be acceptable as the highest examined concentration for the main test. However, since there were indications of possible systemic effects at this dose, an additional group was also treated to ensure 3 acceptable dose levels in the main study.
- Lymph node proliferation response: not performed

- Name of test method: OECD429 LLNA
- Criteria used to consider a positive response:
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.

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.

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 records were maintained.

Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

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 (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. 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.
Note: Ear punch weight determination was performed on the negative control group animals on Day 6 in the main experiment immediately after the euthanasia to collect historical control data.
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). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
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).
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)

Results and discussion

Positive control results:
Stimulation index: 7.1

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: see table
other: disintegrations per minute (DPM)
Remarks on result:
other: See table

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
The observed stimulation index values were 2.3, 1.3, 0.7 and 0.6 at concentrations of 50, 25, 10 and 5% (w/v), respectively. In conclusion, under the conditions of the present assay the test substance tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitisation potential following dermal exposure. The study was performed with vertebrate animals as no 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 and on the recommendations of the OECD Guideline [1], the test item was tested for formulation compatibility in Acetone : Olive oil 4:1 (v/v) mixture (abbreviated as AOO). The highest achievable concentration was 100% (undiluted).

The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using two doses (100% (undiluted) and 50% (w/v)) in the selected vehicle. Based on the observations recorded in the preliminary test, the 50% (w/v) concentration was selected as top dose for the main test.

In the main assay, twenty-four female CBA/J Rj mice were allocated to six groups of four animals each:

- four groups received the test substance (formulated in AOO) at 50, 25, 10 and 5% (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. 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 or signs of systemic toxicity were observed during the main study. No treatment related effects were observed on body weight. The appearance of the lymph nodes was normal in the negative (vehicle) control group and in the 25, 10 and 5% (w/v) test item treated groups. Larger than normal lymph nodes was observed in the positive control group and in the 50% (w/v) test item treated group.

The observed stimulation index values were 2.3, 1.3, 0.7 and 0.6 at concentrations of 50, 25, 10 and 5% (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 significant lymphoproliferative response (stimulation index value of 7.1) was noted for the positive control chemical, this result confirmed the validity of the assay.

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