Registration Dossier

Administrative data

Description of key information

LLNA in mice: according to OECD 429, conducted by DOW, 2008 - not sensitizing.

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:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan (Indianapolis, Indiana)
- Age at study initiation: 9-12 weeks
- Weight at study initiation:21-24 gm
- Housing:Six/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 25% and 75% of DEGHE
No. of animals per dose:
Six
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:Stimulation indices

On day 6, all mice received a 250 μl intravenous injection (i.v.) via the lateral tail vein containing 20 μCi of 3H-thymidine (specific activity 2Ci/mmol; Amersham code TRA310, Buckinghamshire, United Kingdom) diluted in phosphate-buffered saline (PBS). Approximately five hours post administration, the mice were euthanized via CO2 asphyxiation and both auricular lymph nodes located at the bifurcation of the jugular veins were excised and placed in PBS. A single cell suspension of the auricular lymph nodes from one mouse was prepared by gentle mechanical disaggregation using a tissue homogenizer (Stomacher 80 Lab System, Seward Ltd., London, United Kingdom). The cells were washed two times and were suspended in 3 ml of 5% trichloroacetic acid (TCA) for approximately 18-70 hours. The suspended precipitates were centrifuged (200 x g for 10 minutes) and the supernatant removed. The pellet from each mouse was reconstituted in 1 ml of 5% TCA and subsequently transferred to a scintillation vial containing 10 ml of Aquasol-2 scintillation cocktail (Packard Instrument Company, Meridan, Connecticut). Two additional 2 ml aliquots of water were used to rinse the tubes and the rinses were added to the scintillation vials containing the 1 ml of pellet in TCA and cocktail. The radioactivity in each precipitate was measured using a β-scintillation counter and reported as disintegrations per minute (dpm) per mouse. A mean dpm value + SD (standard deviation) was calculated for each experimental group. The SI was calculated using the absolute dpm value for each mouse as the numerator and the mean dpm value from the vehicle control mice as the denominator; the mean SI + SD was calculated for each experimental group. Any test material that produces a SI of > 3 in the LLNA should be considered “positive” for contact sensitization
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
ANOVA is used and whencomparison of treated vs. control groups was done Dunnett’s t-test was used.
Positive control results:
In the repeat study, six female mice/group received 5%, 25% or 75% of the test substance vehicle (AOO) or HCA. Proper conduct of the LLNA was confirmed via a positive response using 30% HCA, which elicited proliferation that was 5.6 in comparison to vehicle treated mice, with 5 of 6 mice having an SI of 4.0 or greater
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: At 5% 1554.8 At 25% 3033.7 At 75% 1355.5
Key result
Parameter:
SI
Value:
0.4
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
75%

Table 1: Disintegration Per Minute(DPM) and Stimulation indices (SI) of Animals Treated with Vehicle (AOO),30 % HCA or 5%,25% and 75% DEGHE REPEAT STUDY

Fig.1: Chemical Structure of the test substance

Interpretation of results:
GHS criteria not met
Conclusions:
Mice treated with 5%, 25% or 75% test substance elicited proliferative responses with stimulation indices (SI) that were respectively 0.4, 1.0, and 0.7 in comparison to vehicle-treated mice. The test substance did not demonstrate dermal sensitization potential in the mouse LLNA as the lymph nodes draining the area of topical application did not demonstrate a 3-fold proliferation when compared to vehicle-treated mice.
Executive summary:

The study is a GLP study and conducted under the Skin Sensitization: LLNA; OPPTS 870.2600, OECD 429 guideline.

The Local Lymph Node Assay (LLNA) was conducted to assess the potential of the test substance to cause contact sensitization by measuring lymphocyte proliferative responses from auricular lymph nodes following topical application of the test material to the mouse ear.

Screening Study: Three daily topical applications of 1%, 5%, 25%, 50%, 75% or 100% test substance were given to one animal at each dose level. Erythema was absent in all dose groups. Body weights were unaffected in mice treated with 1%, 5%, 25%, 50% and 75%, while the mouse dosed with 100% test substance lost 1.1 grams of body weight. Results from this study were used to determine the dosing concentrations for the test substance in the LLNA.

LLNA: Six female mice/group received 5%, 25% or 75% of the test substance vehicle (acetone in olive oil (AOO)) or 30% α-hexylcinnamaldehyde (HCA; positive control) on days 1-3. On day 6, uptake of 3H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours post administration. Proper conduct of the LLNA was not confirmed as 4/6 mice dosed with the positive control 30% HCA, a moderate contact sensitizer, elicited stimulation indices that were 1.7, 1.5, 1.6, and 1.2 which fail to meet the required 3-fold threshold for a positive response. Due to these results, the assay needed to be repeated.

In the repeat study, six female mice/group received 5%, 25% or 75% of the test substance, vehicle (AOO) or HCA. Proper conduct of the LLNA was confirmed via a positive response using 30% HCA, which elicited proliferation that was 5.6 in comparison to vehicle treated mice.

Erythema was absent and body weights were unaffected in all dose groups.

Mice treated with 5%, 25% or 75% test susbstance elicited proliferative responses with stimulation indices (SI) that were respectively 0.4, 1.0, and 0.7 in comparison to vehicletreated mice. The test substance did not demonstrate dermal sensitization potential in the mouse LLNA as the lymph nodes draining the area of topical application did not demonstrate a 3-fold proliferation when compared to vehicle-treated mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The Local Lymph Node Assay (LLNA) was conducted to assess the potential of diethylene glycol monohexyl ether (DEGHE) to cause contact sensitization by measuring lymphocyte proliferative responses from auricular lymph nodes following topical application of the test material to the mouse ear. Mice treated with 5%, 25% or 75% DEGHE elicited proliferative responses with stimulation indices (SI) that were respectively 0.4, 1.0, and 0.7 in comparison to vehicle-treated mice.DEGHE did not demonstrate dermal sensitization potential in the mouse LLNA as the lymph nodes draining the area of topical application did not demonstrate a 3-fold proliferation when compared to vehicle-treated mice.



Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the test substance does not have to be classified as sensitising according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.