Registration Dossier

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: This study was conducted in accordance with GLP and guidelines (OECD,USEPA,EC) and sufficient data is available for the interpretation of study results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
purity 99.65%

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
Mice were 8 weeks old at the start of the study.

Temperature: 22 ± 1°C
Humidity: 40-70%
Air Changes: 12-15 times/hour
Photoperiod: 12 hours dark/light

Animals were housed one per cage in stainless steel cages after assignment to the study. Cages had wiremesh floors that were suspended above absorbent paper and contained a feed container and a pressure activated lixit valve-type watering system. On the day of sacrifice and following the injection of 3H-thymidine, each treatment group of mice was housed in shoebox cages containing corncob bedding, food pellets, and a crock filled with water. The mice were sacrificed five hours later. The room relative humidity and temperature were maintained within a range of 40-70% and 22 ± 1°C, respectively. A 12-hour light/dark photocycle was maintained with lights on at 6:00 a.m. and off at 6:00 p.m. Room air was exchanged approximately 12-15 times/hour.

Study design: in vivo (non-LLNA)

Positive control substance(s):
yes

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 20%, and 80%
No. of animals per dose:
6
Details on study design:
PILOT IRRITATION SCREEN
Prior to the administration of a dosing solution on the ear, the thickness of each ear was measured using a digital micrometer. Individual mice received one concentration of MOD-1 (5%, 10%, 20%, 40%, or 80%) or AOO on the dorsal surface of each ear (25 l) on two consecutive days. Using an adjustable pipette with a disposable tip the test materials (25 l/ear) were spread over the dorsal surface of each ear in a manner to prevent material loss off the ear. The ears were evaluated for erythema prior to application of test material solutions. Ear thickness and erythema were determined 24 hours after the second application. The percent ear-swelling was calculated for each mouse.

MAIN STUDY
The administration of the material (25 μl/ear) was made on the dorsal surface of both ears as described above. All mice (n = six mice/group) received one of three concentrations of MOD-1 (5%, 20% or 80% v/v) or AOO once daily for three consecutive days. HCA at 30% v/v in vehicle was evaluated concurrently as a positive dermal sensitization control. On day 6, all mice received a 250 μl intravenous injection (i.v.) via the lateral tail vein containing 20 μCi of 3Hthymidine (specific activity 2Ci/mmol; Amersham code TRA310, Buckinghamshire, United Kingdom) diluted in phosphate-buffered saline (PBS). Approximately five hours later, the mice were sacrificed via CO2 asphyxiation and the auricular lymph nodes (two) located at bifurcation of the jugular veins were excised and placed in PBS. A single cell suspension of lymph node cells from one mouse was prepared by gentle mechanical disaggregation using a tissue homogenizer (Stomacher® 80 Lab System, Seward Medical Unlimited, London, United Kingdom). The cells were washed two times in PBS and were suspended in 3 ml of 5% trichloroacetic acid (TCA) for approximately 18 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 b-scintillation counter and reported as disintegration 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
1. The % ear swelling was calculated for each ear using the following equation:
% Ear swelling = (B – A)/A x 100 where:
A = mean of pretreatment measurement (mm x 10-2)
B = mean of post treatment measurement (mm x 10-2)

2. The Stimulation Index (SI) was calculated for each mouse using the following equation:
SI = (Disintegrations per minute (dpm) of individual mouse)/(Average dpm of the VH control mice)

3. EC3 Calculation:
EC3 = XL + [(3-YL)/(Yh-YL)](Xh-XL)
Where, YL = SI value below 3
XL = chemical concentration that elicits YL
Yh = SI value above 3
Xh = chemical concentration that elicits Yh

Means and standard deviation (SD) were generated for body weight data (absolute and gain), ear swelling data, and the LLNA response (dpm & SI values). These body weight and dpm data were analyzed by a one-way analysis of variance (Steele and Torrie 1960). Comparisons of treated vs. control groups were done, as necessary, by Dunnett’s t-test (Steele and Torrie 1960). The alpha level at which all tests was conducted is 0.05. The final interpretation of the biological significance of the responses was based on both statistical outcome and scientific judgement.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 0% (AOO control): 1.0 ± 0.4 5% MOD-1: 1.6 ± 1.1 20% MOD-1: 2.3 ± 0.9 80% MOD-1: 4.6 ± 2.0 30% HCA: 12.0 ± 2.3
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 0% (AOO control): 862.43 ± 356.57 5% MOD-1: 1328.4 ± 978.58 20% MOD-1: 1945.2 ± 766.52 80% MOD-1: 3989.9 ± 1730.0 30% HCA: 10308 ± 1993.7 mean±SD (n=6)

Any other information on results incl. tables

Based on the results of the screen, the maximum soluble concentration level of 80% MOD-1 was tested in the LLNA along with 5% and 20% to characterize the dose response. In the LLNA phase of the study, topical applications of 80% MOD-1 elicited no erythema on 4/6 mice, barely perceptible erythema (scores = 1) on 1/6 mice (SI = 2.3) and well defined erythema (score = 2) on 1/6 mice (SI = 7.4). Mean body weights of mice treated with MOD-1 were unaffected. Sensitivity of the LLNA was demonstrated via the positive response from the positive control, 30% HCA, which elicited proliferation (SI) that was 12- fold greater than that of vehicle control. MOD-1 demonstrated LLNA results consistent with weak dermal sensitization potential as the lymph nodes draining the area of topical application demonstrated a proliferative response just above the 3x threshold at the highest concentration tested. MOD-1 applications induced proliferation that increased dose responsively and surpassed the 3x threshold at 80% (SI = 4.6) compared to vehicle controls. The concentration of MOD-1 (that would cause a three- fold increase in proliferation (EC3) was calculated to be 38%.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
1-methoxy-2,7-octadiene demonstrated weak dermal sensitization potential as it elicited a minimal degree (5-fold) of lymphocyte proliferation in the mouse LLNA and has an EC3 value of 38%..