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Description of key information

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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted accroding to OECD TG 429, US EPA OPPTS 870.2600, EU Method B.24 and in accordance with the Principles of Good Laboratory Practice (GLP).
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Remarks:
The homogeneity, concentration and stability of the test substance or positive control substance in the vehicle was not analysed
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
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 Laboratories B.V. The Netherlands
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 19.0 - 21.3 grams
- Housing: individually housed
- Diet (e.g. ad libitum): ad libitum standard pelletted feed (Ssniff mice pellet feed – maintenance, manufactured by Ssniff Spezialdiäten GmbH., Ferdinand-Gabriel-Weg 16, D-59494 SÖest, Germany)
- Water: ad libitum deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier (manufactured by Eureka Forbes Ltd., Mumbai 400 001, India)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23°C
- Humidity (%): 57 to 66 %
- Air changes (per hr): 13.0 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Concentration / amount:
not applicable
Concentration / amount:
not applicable
No. of animals per dose:
not applicable
Details on study design:
not applicable
Challenge controls:
not applicable
Vehicle:
dimethyl sulphoxide
Concentration:
Diethylene glycol mono phenyl ether was diluted with DMSO to obtain concentrations of 5 and 25% v/v for the main LLNA study.
No. of animals per dose:
5 female mice/group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The undiluted test substance (100%) was suitable for application. Solubility / miscibility test was performed using AOO, DMF, MEK, PG and DMSO at concentrations 90, 75, 60, 50, 40, 25 and 10% v/v. At concentrations of 90% and below, the test substance in all the tested vehicles was pipettable and suitable for application to the dorsal surface of the mouse ear for conduct of the LLNA. DMSO was chosen based on miscibility and previous laboratory experience.
- Irritation: Concentrations tested for the irritation screen were selected based on information of dermal irritancy potential in rabbits and maximum miscibility/solubility in an appropriate LLNA vehicle while maintaining a solution suitable for application. Toxicity data regarding irritation potential were also taken into consideration. Prior to the main LLNA study, concentrations of 10, 25, 50, 75% v/v Diethylene glycol mono phenyl ether in DMSO and 100% undiluted test substance and the vehicle control (DMSO) were evaluated to determine the highest achievable concentration that avoids overt systemic toxicity and excessive local irritation.
- Lymph node proliferation response: Both ears of six female mice (one mouse/concentration) were topically treated for three consecutive days (Days 1, 2 and 3) with one of the above-listed concentrations of the test substance or DMSO alone. No treatment was made on Days 4 and 5. The test substance was administered using an adjustable micropipette with a disposable tip. All mice received 25 μL of one concentration of the test substance, spread over the dorsal surface of each ear in a manner to prevent test substance loss (50 μL total/mouse). Similarly, the vehicle alone was applied to the ears of one animal. Prior to each application, both the ears were evaluated for erythema for scoring of skin irritation. Ears were also evaluated on Day 6. All mice were weighed on Days 1 and 6. Animals were observed daily for clinical signs of toxicity. Ear thickness was measured using a micrometer (Digimatic micrometer, Mitutoyo, Japan) prior to dosing on Days 1 and 3 and prior to euthanasia on Day 6. Additionally, on Day 6, ear thickness was determined by ear punch weight, after animals were euthanized. Erythema scores, ear thickness and body weight data following test substance applications were compared to the response of the animals treated with vehicle alone.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT - The application of the test substance (25 μL/ear) was made on the dorsum of both ears as described above. Five female mice/group received vehicle (DMSO) or positive control (25% α-hexylcinnamaldehyde) or 5% and 25% v/v or undiluted (100%) Diethylene glycol mono phenyl ether once daily for three consecutive days (Days 1, 2 and 3). No treatment was made on Days 4 and 5.
- Criteria used to consider a positive response: Any test substance that produced a SI > 3 in the LLNA was normally considered “positive” for dermal sensitization potential. While a SI > 3 was originally developed empirically, a robust statistical evaluation indicated that it was an acceptable practical value for hazard identification. Furthermore, by determining EC3 values (estimated concentration resulting in a 3-fold SI), one can compare relative sensitization potency of chemicals and/or formulations. While a test substance that produces a SI of > 3 in the LLNA should be considered “positive” for contact sensitization, recent opinions have suggested circumstances in which the LLNA result and sensitization potential should be further considered in the context of additional scientific judgment. Therefore, the final interpretation of the biological significance of the responses was based on both statistical outcome and scientific judgment.

TREATMENT PREPARATION AND ADMINISTRATION: Diethylene glycol mono phenyl ether was diluted with DMSO to obtain concentrations of 5 and 25% v/v for the main LLNA study. Test substance solutions were prepared daily just prior to dosing. Preparation of the dosing materials was documented in the study file. The concentration of the dosing solution was not verified analytically.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Standard statistical methods were employed
Positive control results:
Proper conduct of the LLNA was demonstrated via the response from the positive control, 25% HCA in DMSO, which elicited a stimulation index (SI) of 5.29, in comparison with the vehicle-treated mice.
Parameter:
SI
Remarks on result:
other: The SI values for the 5, 25% v/v and undiluted (100%) Diethylene glycol mono phenyl ether groups were 1.37, 1.66 and 1.71, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The dpm values for the 5, 25% v/v and undiluted (100%) Diethylene glycol mono phenyl ether groups were 1922.8, 2316.8 and 2388.8, respectively.

Three consecutive daily applications of 10, 25, 50 or 75% v/v Diethylene glycol mono phenyl ether in dimethyl sulphoxide (DMSO) or 100% undiluted test substance or the vehicle alone (DMSO) were topically applied to one animal at each dose level. There were no clinical signs or effects on body weight that would suggest excessive toxicity. In addition, there were no effects on ear erythema, ear thickness or ear punch weights that would suggest excessive irritation. Based on the results from this screening study, concentrations selected for the main LLNA study were 5, 25% v/v test substance in DMSO and undiluted test substance (100%).

Diethylene glycol mono phenyl ether did not elicit erythema in any of the mice at any of the dose levels and there was no significant effect of body weight gains.

A table summarizing the results of the LLNA is presented below:

Group  Mean DPM  Stimulation Index 
G1 - Vehicle control  1399.4  1.00 
G2 - Positive control  7399.2  5.29 
G3 - 5% Di-EPh  1922.8  1.37 
G4 - 25% Di-EPh  2316.8  1.66 
G5 - 100% Di-EPh  2388.8  1.71 
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, a stimulation index (SI) of greater than 3.0 was not observed in mice treated with Diethylene glycol mono phenyl ether. Therefore Diethylene glycol mono phenyl ether was considered negative for dermal sensitization potential in the LLNA.
Executive summary:

The Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of Diethylene glycol mono phenyl ether to cause contact sensitization by measuring lymphocyte proliferative response in the auricular lymph nodes following topical application of the test substance to the female CBA/Ca mouse ear.

 

Screening Study: Three consecutive daily applications of 10, 25, 50, 75% v/v Diethylene glycol mono phenyl ether in dimethyl sulfoxide (DMSO) or 100% undiluted test substance or the vehicle alone (DMSO) were topically applied to one animal at each dose level. There were no clinical signs or effects on body weight. In addition, there were no effects on ear erythema, ear thickness or ear punch weights. Results from this screening study were used to determine the dosing concentrations of Diethylene glycol mono phenyl ether in the main LLNA study. The doses selected for the main LLNA study were 5 and 25% v/v in DMSO and 100% undiluted test substance.

 

Main LLNA Study: Five female CBA/Ca mice/group received vehicle (dimethyl sulfoxide [DMSO]) or 25% α-hexylcinnamaldehyde (HCA: positive control in DMSO) or 5 or 25% v/v Diethylene glycol mono phenyl ether in DMSO or undiluted test substance (100%) on Days 1 to 3. Three days after the last application (on Day 6), the mice were given a 20 μCi intravenous injection of 3H-methyl thymidine. Approximately five hours after the injection, mice were euthanized and the auricular lymph nodes draining the site of test substance application were removed for assessment of 3Hmethyl thymidine incorporation.

 

Diethylene glycol mono phenyl ether did not elicit erythema in any of the mice at any of the dose levels and there was no significant effect on body weight gains.

 

Under the conditions of this study, a stimulation index (SI) of greater than 3.0 was not observed in mice treated with Diethylene glycol mono phenyl ether. Therefore Diethylene glycol mono phenyl ether was considered negative for dermal sensitization potential in the LLNA. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.

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

In a well conducted mouse local lymph node assay, Di-EPh exposure did not cause the stimulation index to exceed 3 at any dose level. As such, this substance is not considered to be a skin sensitiser.


Migrated from Short description of key information:
A Local Lymph Node Assay on Di-EPh is available

Justification for selection of skin sensitisation endpoint:
Reliability 1 OECD guideline study

Respiratory sensitisation

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

In a well conducted mouse local lymph node assay Di-EPh was not a skin sensitiser. In the three available genotoxicity assays there is no evidence of genotoxicity or clastogenicity. Given this apparent lack of reactivity it is highly unlikely that this substance would trigger an immune response following inhalation. In addition, the vapour pressure of this substance is sufficiently low that the inhalation route would not typically be a relevant route for exposure.


Migrated from Short description of key information:
No specific study is available, but an assessment of sensitising potential is made using the skin sensitising data and genotoxicity data

Justification for classification or non-classification

This substance is not a sensitiser and therefore no classification is necessary.