Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15th to 30th of October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without deviations and test procedure according to GLP standards

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: Twenty-four female CBA/J [SPF] mice
- Source: from Charles River Japan, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: 20.7-22.5. None of the animals showed any abnormalities.
- Housing: The animals were individually housed in wire mesh metal cages (W 10.0 × D 19.6 × H 13.0 cm).
- Diet (e.g. ad libitum): Animals had free access to commercial pellet diet, CRF-1 (lot No. 120403, Oriental Yeast). The analytical report on contaminants in the diet was obtained from the manufacturer and the Study Director confirmed that the contaminant levels were within the acceptable limits (draft) of the Japan Experimental Animal Feed Association (analytical report code: AR-12-JP-001938-01).
- Water (e.g. ad libitum): Animals were allowed free access to tap water with an automatic water supply system. The quality of drinking water was examined by EcoPro Research Co., Ltd. in October 2012, according to the specifications of the Water Works Law. The Study Director confirmed that the analytical results met the Tap Water Quality Standard (certificate No. 126014-3). In addition, examinations for bacteria (common bacteria and Escherichia coli) in the water were conducted by the BSRC in September and November 2012 when the delegated quality analysis was not conducted. No bacteria were detected in the drinking water (test certificate No. GT12-09, GT12-11).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.9 to 23.1
- Humidity (%): 51.9 to 61.7 %RH
- Air changes (per hr): 12 times or more per hour
- differential air pressure: +30 Pa or more (with the door closed)
- Photoperiod (hrs dark / hrs light): 12 hours (lights on 7 a.m., off 7 p.m.)

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
92.7%, 50% and 25% (three concentration in total)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
A pre-screening test [Exp. No. E412 (474-111), non-GLP] was conducted before this main study to determine the concentrations for dosing. In the pre-screening test, three concentrations of 23.18, 46.35 and 92.70% (92.70% solution was an undiluted solution; these were concentrations of active ingredient) were selected using AOO as a solvent, and 25 μL each of the dose formulations were applied on the dorsal skin of both auricles of each animal, 2 mice for each concentration, once a day for 3 consecutive days. The general conditions including the application site condition were observed from 1 to 3 hours after the application on each day of application. As a result, no animals showed any abnormalities. The body weights and ear thickness were measured before the initial application and on Day 6. As a result, none of the animals showed any changes exceeding the criteria.

MAIN STUDY:
From the results mentioned above, for the main study, 92.70% was selected as the high concentration because it was expected not to induce any toxic general condition, 25% or more increase of the ear thickness, dermal erythema with score of 3 (Table Erythema Scores) or more on the auricles nor more than 5% of body weight loss, and two lower concentrations of 50 and 25% (three concentrations in total).

PREPARATION OF DOSE FORMULATIONS:
The undiluted solution of the test substance was used for 92.70% dose formulation. 0.54 g of the test substance [including 0.5 g of the active ingredient using a conversion factor of 1.08 (purity: 92.70%)] was weighed with an electronic balance (AT460DR, Mettler Toledo), and dissolved in and diluted to 1 mL with acetone and olive oil mixture (AOO, v/v=4:1, acetone: lot No. TLQ4136, Wako Pure Chemical Industries, olive oil: lot No. V2B8025, Nacalai Tesque) to make the concentration of 50%. 0.5 mL of this solution was diluted to 1mL with AOO to make the concentration of 25%.
0.25 g of the positive control substance was weighed with an electronic balance and dissolved in and diluted to 1mL with AOO to make the concentration of 25%.
These formulations were prepared just before use.

RATIONALE FOR SELECTION OF ADMINISTRATION ROUTE AND METHOD OF ADMINISTRATION:
The test substance was applied to the auricles in accordance with the common administration route for LLNA.
Twenty-five microliters each of a dose formulation were applied on the dorsal skin of auricles of each animal once a day using a MICROMAN (Model M100, Gilson).

ADMINISTRATION PERIOD AND SCHEDULE:
The administration period was 3 consecutive days.

LLNA
protocol Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
T T T - - 3H C
T: Administration of dose solutions
3H: Administration of 20 μCi [methyl-3H] thymidine (3HTdR)
Local lymph nodes were collected and minced at 5 hours after administration of 3HTdR, and then the pooled lymph node cells (LNC) were treated with 5% TCA overnight (approximately 18 hours).
C: Counting of 3HTdR incorporation into pooled LNC

PREPARATION OF 3HTdR SOLUTION:
The [methyl-3H] thymidine (3HTdR, lot No. #238-025-002-A-20120306-THN, code No. MT-6032, Moravek Biochemicals; specific radioactivity: 2.0 Ci/mmol, concentration: 1.0 mCi/mL) was diluted with phosphate buffered saline (PBS, lot No. 1006377, GIBCO, pH 7.2) to a concentration of 80 µCi/mL just before use.

CONFIRMATION OF 3HTdR SOLUTION:
Eighty microliters of 3HTdR solution were diluted to 200 mL with water for injection, and 1 mL of this solution was mixed with 10 mL of liquid scintillator (Clear-sol II, lot No. L1R1124, Nacalai Tesque). Amounts of radioactivity were counted with the liquid scintillation counter (LSC-6100B, Aloka). Because the actual amount of radioactivity of the 3HTdR solution was within ±20% of the calculated value, it was confirmed that the 3HTdR solution was appropriately prepared (actual measurement value: 1,279 Bq, calculated value: 1,184 Bq).

INCORPORATION OF 3HTdR
On Day 6, all animals in the same dosage group were transferred to polycarbonate cages and transported to the RI laboratory.
Twenty µCi of 3HTdR (250 µL of 80 µCi/mL 3HTdR solution) were administered intravenously to each mouse via the tail vein with disposable syringes and 27G needles.

PREPARATION OF SINGLE CELLS SUSPENSIONS
At approximately 5 hours after 3HTdR injection, all mice were sacrificed by dislocation of cervical vertebra under isoflurane anesthetizing. The auricular lymph nodes were immediately removed, and the pooled lymph nodes for each animal were weighed with an electronic balance (AB104, Mettler Toledo), then these tissues were collected into 1.0 mL of a phosphate buffered saline (PBS) solution for each experimental group (4 animals/group). A single cell suspension (SCS) of pooled lymph node cells (LNC) was prepared and collected into the base of a culture dish by gentle mechanical disaggregating of pooled lymph nodes through a nylon mesh (Cell Strainer, 70 μm mesh, BD Falcon) using the plunger of a 2.5 mL disposable syringe. The cell strainer was washed with 4-5 mL of PBS and the rinse was added into the base of the culture dish, and the SCS was transferred into a 10 mL graduated round-bottomed centrifuge tube. A final volume of 10 mL of SCS was made with rinsing the culture dish with PBS. This procedure was repeated. Pooled LNC was pelleted by centrifugation at 1,500 rpm (approx. 438 × g, RCF) for 10 min. After centrifugation, each supernatant was removed by aspiration, leaving 1-2 mL of supernatant above each pellet. Each pellet was gently agitated and mixed with PBS to make a 10-mL LNC suspension. The washing procedure was repeated twice.

OBSERVATIONS, MEASUREMENTS AND EXAMINATIONS:

DETERMINATION OF INCORPORATED 3HTdR
After the final wash, each supernatant was removed leaving just a small volume (≤ 0.5 mL) of supernatant above each pellet. Each pellet was gently agitated before re-suspending the LNC in 3 mL of 5% trichloroacetic acid (TCA: lot No. PKL0552, Wako Pure Chemical Industries). After incubation with 5% TCA at 4C for about 18 hours, each precipitate was recovered by centrifugation at 1,500 rpm for 10 minutes, removing each supernatant and re-suspending in 1 mL of 5% TCA. Each precipitate was transferred to a 20 mL-scintillation vial with 10 mL of liquid scintillator and thoroughly mixed. The vials were loaded into a liquid scintillation counter, and after approximately 30 minutes, 3HTdR incorporation was measured. The 3HTdR incorporation was expressed as the number of radioactive disintegrations per minute (DPM). Similarly, background 3HTdR level was also measured with 1 mL of 5% TCA.

CALCULATION OF RESULTS
Results were expressed as the stimulation index (SI). The SI was derived by dividing the mean DPM/mouse within each test substance group and positive control group by the mean DPM/mouse for the vehicle control group. A substance was regarded as a sensitizer in the LLNA if the SI of any concentration of the test substance and positive control substance were 3 or greater. When the test substance was regarded as a sensitizer, EC3 value was estimated via linear interpolation or log-linear extrapolation if possible ). The allergenic potency class of the test substance was assigned depending on the EC3 value using the GHS categorization scheme.

Hazard category and sub-categories for skin sensitizers:
Category 1: Skin sensitizer
Sub-category 1A: Substance showing a high potency in animals can be presumed to have the potential to produce significant sensitization in humans. EC3 value ≤2%
Sub-category 1B: Substance showing a low to moderate potency in animals can be presumed to have the potential to produce sensitization in humans. EC3 value >2%
Globally Harmonized System of Classification and Labelling of Chemicals (GHS), 4th revised edition, United Nations, 2011.

OBSERVATION OF GENERAL CONDITION:
General conditions of mice were observed once a day from the first administration to the anatomy. All signs were recorded.

BODY WEIGHT:
The mice were weighed with an electronic balance (PG802-S, Mettler Toledo) before the first administration and before transportation to the RI facility.
Observation of auricles
Both auricles of each mouse were observed for erythema and scored using the next table on every application day and before transportation to the RI facility.

Table Erythema Scores
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

EAR THICKNESS MEASUREMENT:
Thickness of the right ear of each animal was measured three times continuously with a thickness gauge (Mitutoyo) before the first administration and before transportation to the RI facility.

STATISTICAL ANALYSIS
No statistical analyses were used.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistical analyses were used

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The Stimulation Index (SI) for the 25, 50 and 92.70% 3,6-dimethylheptan-2-ol groups were calculated to be 2.4, 2.9 and 3.1, respectively. See Table 1 attached.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per lymph nodes for each individual animal and the stimulation index are given in Table 1 attached.

Any other information on results incl. tables

RESULTS

The DPM/animal of the vehicle control group and 3 test substance groups (25, 50 and 92.70%) were 760.6, 1792.1, 2239.0 and 2358.2, respectively. The SI for the 25, 50 and 92.70% 3,6-dimethylheptan-2-ol groups were calculated to be 2.4, 2.9 and 3.1, respectively. Therefore, 92.70% 3,6-dimethylheptan-2-ol was judged as positive, and the EC3 value of 3,6-dimethylheptan-2-ol was calculated as 52.4% via linear interpolation. On the other hand, DPM/animal of the positive control group was 4029.4, and the SI of this group was calculated as 5.3 (see Table 1 attached).

No animals showed abnormal clinical signs due to test substance administration during the observation period (see Table 2 attached).

No animals showed erythema on the auricles during the observation period (see Table 3 and Appendix 1 attached).

There were no body weight increases or decreases due to test substance or positive control administration during the sensitization period. The weights of lymph nodes in the positive control group were higher than that in the vehicle control group (see Table 4 and Appendix 2).

There were no evident increases in the ear thickness due to test substance or positive control administration during the sensitization period (see Table 5 and Appendix 3).

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
Test item was judged to be a sensitizer under the conditions of this study, and was categorized in sub-category 1B of GHS classification for skin sensitization.
Executive summary:

A mouse local lymph node assay (LLNA) was conducted to investigate the potential of 3,6-dimethylheptan-2-ol to induce allergic contact dermatitis. Twenty-four female CBA/J [SPF] mice were purchased from Charles River Japan, Inc., and 20 mice were used for this study. There were 5 dose groups, each consisting of 4 mice, and the animals in each group were exposed to 3,6-dimethylheptan-2-ol at 1 of the 3 concentration levels, positive control substance or their vehicle (acetone and olive oil mixture: v/v=4:1).

 

The results are summarized as follows.

The stimulation indices (SI) for the 25, 50 and 92.70% 3,6-dimethylheptan-2-ol groups were calculated to be 2.4, 2.9 and 3.1, respectively. These results were judged to be positive. On the other hand, the SI for the 25%α-Hexylcinnamaldehyde (HCA, positive control) was calculated to be 5.3, which was judged to be positive.

No animals showed abnormal clinical signs due to test substance administration during the observation period. No animals showed erythema on the auricles during the observation period. There was no evident increase in the ear thickness due to test substance administration during the sensitizing period. There were no body weight increases or decreases due to test substance administration during the sensitization period. The weights of lymph nodes in the positive control group were higher than that in the vehicle control group.

 

In conclusion, 3,6-dimethylheptan-2-ol was judged to be a sensitizer under the conditions of this study, and was categorized in sub-category 1B of GHS classification for skin sensitization.