Registration Dossier

Administrative data

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.42 and in accordance with the Principles of Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Diisobutyl carbinol (A mixture of 2,6-Dimethyl-4-heptanol and 4,6-Dimethyl-2-heptanol isomers)
- Physical state: colourless liquid
- Analytical purity: 99.8 ± 0.02 %
- Lot/batch No.: AA0155T3YY

In vivo test system

Test animals

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: 18.0 - 23 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): 21 to 23°C
- Humidity (%): 65 to 66 %
- Air changes (per hr): 13.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Study design: in vivo (non-LLNA)

Induction
Concentration / amount:
not applicable
Challenge
Concentration / amount:
not applicable
No. of animals per dose:
not applicable
Details on study design:
not applicable
Challenge controls:
not applicable

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Diethylene glycol mono phenyl ether was diluted with AAO 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. AOO was chosen based on miscibility and previous laboratory experience.
- Irritation: Prior to the main LLNA study, concentrations of 10, 25, 50, 75% v/v Diisobutyl carbinol in AOO and 100% undiluted test substance and the vehicle control (AOO) 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 AOO 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 (AOO) or positive control (25% α-hexylcinnamaldehyde) or 5% and 25% v/v or undiluted (100%) Diisobutyl carbinol once daily for three consecutive days (Days 1, 2 and 3). No treatment was made on Days 4 and 5.
A solution of 3H-TdR (80 µCi/mL)[specific activity, 6.7 Ci/mmol; Perkin Elmer, USA] in sterile phosphate buffer saline (PBS) was freshly prepared. The prepared working solution of 3H-TdR was analyzed for radioactivity. On Day 6, a volume of 250 µL (20 µCi) of 3H-TdR in PBS was administered to each mouse via the lateral tail vein using a 1.0 mL disposable syringe fitted with a 26 SWG, 1/2 inch needle. Approximately five hours post injection of 3H-TdR, animals were euthanized by an overdose of Isoflurane anaesthesia. The auricular lymph nodes (bilateral) were excised and placed in PBS, processed and the radioactivity was counted. A single cell suspension of the lymph node cells (LNC) from each mouse was prepared by gentle mechanical disaggregation using a tissue homogeniser (Stomacher 80 MicroBiomaster, Seward Ltd, United Kingdom) for 30 seconds at medium speed using PBS (approx. 10 mL). After all the nodes had been processed, the tubes containing the suspensions of LNC were centrifuged at 200 x g for 10 minutes at 40C. The supernatant was poured into a container for collection of radiolabel waste. 10 mL of PBS was added to each tube and inverted to resuspend the pellet. The tubes were centrifuged again as described above and supernatant was poured off. After the final (second) wash, the cell pellet was suspended in 3.0 mL of 5% trichloroacetic acid (TCA) and stored overnight at 40 °C for approximately 18 hours. Clumping of LNC was avoided by ensuring that the pellet was completely resuspended in a small volume of TCA before making up to the final volume. The suspended precipitates were centrifuged at 200 x g for 10 minutes at 40 °C and the supernatant poured off into a container for collection of radiolabel waste. The pellet from each tube was reconstituted in 1 mL of 5% TCA and subsequently transferred to a scintillation vial containing 10 mL of scintillation cocktail (Aquasol-2, PerkinElmer, USA). Two additional 2 mL aliquots of distilled water were used to rinse the tubes and the rinses were added to the scintillation vial containing the pellet in TCA and cocktail. The samples were mixed using a snapping wrist action.
The radioactivity in each precipitate was measured for 5 minutes using a ß-scintillation counter (Tricarb 2900-TR, Packard Instruments, USA) as disintegrations per minute (dpm) per mouse
- 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: Diisobutyl carbinol was diluted with AOO 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

Results and discussion

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 5.55, in comparison with the vehicle-treated mice.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values for the 5, 25% v/v and undiluted (100%) Diisobutyl Carbinol groups were 1.43, 1.85 and 2.64, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The mean dpm values for the 5, 25% v/v and undiluted (100%) Diisobutyl carbionl groups were 2592.40, 3338.8 and 4772.0, respectively.

Any other information on results incl. tables

Three consecutive daily applications of 10, 25, 50 or 75% v/v Diisobutyl carbinol in acetone:olive oil (AOO) or 100% undiluted test substance or the vehicle alone (AOO) 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 AOO and undiluted test substance (100%).

Diisobutyl carbinol 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  1808.20  1.00 
G2 - Positive control  10037.20  5.55 
G3 - 5% Diisobutyl carbinol  2592.40  1.43 
G4 - 25% Diisobutyl carbinol 3338.80  1.85 
G5 - 100% Diisobutyl carbinol 4772.00  2.64 

Applicant's summary and conclusion

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 Diisobutyl carbinol. Therefore Diisobutyl carbinol 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 Diisobutyl carbinol 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 Diisobutyl carbinol in acetone:olive oil (AOO) or 100% undiluted test substance or the vehicle alone (AOO) 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 Diisobutyl carbinol in the main LLNA study. The doses selected for the main LLNA study were 5 and 25% v/v in AOO and 100% undiluted test substance.

 

Main LLNA Study: Five female CBA/Ca mice/group received vehicle (AOO) or 25% α-hexylcinnamaldehyde (HCA: positive control in AOO) or 5 or 25% v/v Diisobutyl carbinol in AOO 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.

 

Diisobutyl carbinol 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 Diisobutyl carbinol. Therefore Diisobutyl carbinol 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.