1H-Imidazolium, 3-ethyl-1-methyl-, C11-13-branched alkylbenzenesulfonates

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 429.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

see box below

Principles of method if other than guideline:

During the acclimatisation period of the preliminary test, variations from the target humidity ranges were observed (Room number: 244/2; RH: 35 – 87%). These variations were considered to have no impact on the animal health, as certified by the Clinical Veterinarian, or on the outcome of the study and interpretation of the results due to their low magnitude.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1H-Imidazolium, 3-ethyl-1-methyl-, C9-14-branched alkylbenzenesulfonates

IUPAC Name:

1H-Imidazolium, 3-ethyl-1-methyl-, C9-14-branched alkylbenzenesulfonates

Constituent 2

Reference substance name:

EMI-DBS

IUPAC Name:

EMI-DBS

Constituent 3

Reference substance name:

1065642-34-3

Cas Number:

1065642-34-3

IUPAC Name:

1065642-34-3

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Details on test material:

Substance name: EMI-DBS
Description of the test item: Yellow paste
Storage conditions: Room temperature (15 - 25°C)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj mice

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
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/treatment group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 9-10 weeks old
Body weight range at starting: 19.7 – 21.9 grams (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight)
Acclimatization time: 13 days
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: 22 ± 3°C
Relative humidity: 30-70%
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): 244/6
Room/Cabinet (radioactive phase): 139 - 140

Food and feeding: Animals received ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 683 5949 Expiry Date: January 2012) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.

Water supply: Animals received tap water from the municipal supply from 500 ml bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at CiToxLAB Hungary Ltd.

Bedding: Lignocel(R) Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co. KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study.

Identification: A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd..’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software according to the actual body weights, verifying the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

In the main assay, twenty four female CBA/J Rj mice were allocated to six groups of four animals each four groups received the appropriate formulation of EMI-DBS at concentrations of 10, 5, 2.5 and 1 (w/v) %.

No. of animals per dose:

4

Details on study design:

Dose Selection and Justification of Dose Selection
A Preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using five doses, at test item concentrations of 50, 25, 10, 5 and 2.5 (w/v) %, respectively. This preliminary experiment was conducted in a similar experimental manner to the main study and was terminated on Day 6 with a body weight measurement, a radioactive proliferation assay was not performed. An assessment of ear swelling was made by measuring thickness on Days 1, 3 and 6, and the weight of an ear punch on Day 6.
During the Preliminary Irritation/Toxicity Test no mortality was observed. No treatment related effect on body weights was observed in the treated groups. Erythema grade 3 was observed at 25 and 50% (this is unacceptable for a main study). Ear thickness was measured by using a thickness gauge on Days 1, 3 and 6 and the weight of an ear punch on Day 6, these two measurements did not give a consistent indication of adverse effects. The observations recorded in this preliminary test suggest that the maximum of 10 (w/v) % formulation, the application of the material and the local effects on the animal are acceptable for a valid LLNA.

Topical application
During the assay, each mouse was topically dosed on the dorsal surface of each ear with 25 μl of the appropriate formulation applied using a pipette. 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

Injection of Tritiated Thymidine (3HTdR)
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 25G1" hypodermic needle with 1 ml sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
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 were pooled and collected in separate Petri dishes containing a small amount (1-2 ml) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
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.

Determination of Incorporated 3HTdR
After the final wash, supernatant were removed leaving a small volume (<0.5 ml) of supernatant above each pellet. Each pellet was gently agitated before suspending the LNCs in 3 ml of 5% TCA (trichloroacetic acid) for precipitation of macromolecules. After incubation with 5% TCA at 2-8 °C overnight (approximately 18 hours) precipitate was recovered by centrifugation at 190xg for 10 minutes, supernatants were removed and pellets were suspended in 1 ml of 5% TCA and dispersed using ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 ml of scintillation liquid and thoroughly mixed. The vials were loaded to a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample.
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 ml aliquots of 5% TCA.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Background (5 w/v)% TCA) = N/A Negative control: 1.0 EMI-DBS (10% in vehicle): 9.7 EMI-DBS (5% in vehicle): 11.4 EMI-DBS (2.5% in vehicle): 9.6 EMI-DBS (1% in vehicle): 3.7 Positive control (25% HCA in AOO): 18.8

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Background (5 w/v)% TCA) = 41.5 Negative control: 1241 EMI-DBS (10% in vehicle): 11730 EMI-DBS (5% in vehicle): 13768 EMI-DBS (2.5% in vehicle): 11534 EMI-DBS (1% in vehicle): 4474 Positive control (25% HCA in AOO): 22556

Any other information on results incl. tables

CLINICAL OBSERVATION

No mortality, systemic toxicity was observed during the study. On Day 2 and 3 erythema (score 1 or 2) was observed in 10 and 5 (w/v) % treated animals.

PROLIFERATION ASSAY

The appearance of the lymph nodes was normal in the negative control group and the 1 (w/v) % treated group. Larger than normal lymph nodes was observed in the 10, 5 and 2.5 (w/v) % test item treated groups and in the positive control group.

INTERPRETATION OF OBSERVATIONS

The test item was a yellow paste, which was dissolved 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. A significant lymphoproliferative response (SI≥ 3) was noted for EMI-DBS at concentrations of 10, 5, 2.5 and 1 (w/v) %. Stimulation index values of the test item were 9.7, 11.4, 9.6 and 3.7 at treatment concentrations of 10, 5, 2.5 and 1 (w/v) %, respectively. The stimulation index values were compatible with a biological dose- related response showing a clear positive response

RELIABILITY OF THE TEST

The positive control group animals were treated with 25 (w/v) % HCA solution in a relevant vehicle (AOO) concurrent to the test item groups. The positive control substance was chosen according to the OECD guideline.

No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lymphoproliferative response (stimulation index value of 18.8) was noted for α-Hexylcinnamaldehyde in this experiment. The results of the positive control group demonstrated the appropriate performance of the assay.

See the attached background material for more information on the results of the LLNA.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, under the conditions of the present assay EMI-DBS (Batch No.: GE0031758) tested in a suitable vehicle, was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 429. Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test item was dissolved in acetone/olive oil (4:1 v/v) (abbreviation: AOO). The test item is soluble in this solvent at 50 (w/v) %. This is the maximum achievable concentration, therefore it was chosen as vehicle for the test. The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using five doses, at test item concentrations of 50, 25, 10, 5 and 2.5 (w/v) % in the selected vehicle. The observations recorded in the preliminary test suggest that the maximum of 10 (w/v) % formulation, the application of the material and the local effects on the animal are acceptable for a valid LLNA. In the main assay, twenty four female CBA/J Rj mice were allocated to six groups of four animals each:

- four groups received the appropriate formulation of EMI-DBS at concentrations of 10, 5, 2.5 and 1 (w/v) %,

- the negative control group received AOO,

- the positive control group received 25 % α-Hexylcinnamaldehyde (HCA) in AOO*.

*: To minimise animal use, the positive control group was part of a concurrent study with the same solvent.

The solutions of the test item 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, systemic toxicity was observed during the study. On Day 2 and 3 erythema (score 1 or 2) was observed in 10 and 5 (w/v) % treated animals. No treatment related effects were observed on animal body weights in any other treated groups. Stimulation index values of the test item were 9.7, 11.4, 9.6 and 3.7 at treatment concentrations of 10, 5, 2.5 and 1 (w/v) %, respectively. α-Hexylcinnamaldehyde (25 (w/v) % dissolved in AOO) was used as a positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 18.8) was noted for the positive control chemical and this result confirmed the validity of the assay. In conclusion, under the conditions of the present assay EMI-DBS (Batch No.: GE0031758) tested in a suitable vehicle, was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay.