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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 - 31 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node 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:
Sex: Female, nulliparous and non-pregnant.

Species/Strain: Mouse, CBA/J

Age/Body weight: Preliminary Animals: Young adult (12 weeks)
Test and Control Animals: Young adult (9-12 weeks)/19.0-23.4 g at experimental start.

Source: Received from Harlan, Indianapolis, IN on July 20, 2010 (Preliminary Irritation Groups) and August 17, 2010 (Test and Control Groups).

Housing: The animals were individually housed in plastic solid bottom cages with bedding during the dosing and resting phases of the study. After final weighing until sacrifice, animals were housed in their respective dose groups in plastic cages with bedding. Bedding in the plastic, solid bottom cages was changed at least once per week. All caging conformed to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 1996).

Animal Room Temperature and Relative Humidity Ranges: 19-22ºC and 60-67%, respectively.

Animal Room Air Changes/Hour: 12. Airflow measurements are evaluated regularly and the records are kept on file at Eurofins PSL.

Photoperiod: 12-hour light/dark cycle

Acclimation Period: 8 or 29 days

Food: Purina Rodent Chow #5001 ad libitum

Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system.

Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Eurofins PSL.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10 and 25 % (w/v)
No. of animals per dose:
Preliminary Irritation (5 groups): 2
Test (3 groups): 5
Vehicle (Negative) Control: 5
Positive Control: 5
Details on study design:
Preliminary Toxicity Testing
Four test substance concentrations and the vehicle control were used. Test substance concentrations of 2.5%, 5%, 10% and 25% were tested to determine the highest achievable level that avoids overt systemic toxicity and excessive local irritation. The top dose of 25% was selected based on maximum solubility/compatibility of the test substance with the vehicle while maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw data). Each group consisted of two mice. The ears of each mouse were scored for erythema and edema prior to dosing on Days 1, 2, 3, and prior to termination on Day 6.

Twenty-five μL of the appropriate dilution of the test substance concentration or the vehicle alone was applied to the dorsum of both ears of each mouse for three consecutive days (Days 1, 2 and 3). Application was done using an appropriate size micropipette to accurately deliver 25 μL. The dose was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette. No treatment was made on Days 4 and 5. On Day 6, each site was evaluated for local irritation (erythema & edema).

Animals were observed daily for signs of toxicity. The Study Director and Sponsor used this data in conjunction with any pre-existing data to select the maximum concentration to be tested. Test substance concentrations of 5%, 10% and 25% w/w mixtures in acetone/olive oil (AOO; 4:1 v/v)
were selected for testing.

Selection of Animals/Dose Levels
Prior to dosing, the animals were weighed and the ears were checked for any abnormalities or clinical signs of diseases or injury. Twenty-five healthy naive female mice without pre-existing ear irritation were selected and distributed (5 mice per group) into the following test groups:
Group # ...Purpose...................................Concentration
1 ..............Vehicle................................................0%
2.............. Test Substance..................................5%
3.............. Test Substance................................10%
4...............Test Substance................................25%
5.............. Positive Control Substance...........25% HCA

Concentrations were selected based on toxicity, solubility, irritancy, and viscosity.

Sample Preparation
Dilutions of the test substance were prepared as w/w mixtures in acetone/olive oil (AOO; 4:1 v/v). Concentrations of 5%, 10% and 25% were selected for the main test based on results of the preliminary screening test. A single concentration of a 25% w/w mixture of HCA in AOO (4:1 v/v) was prepared as a positive control. All dosage preparations were freshly prepared on the day of administration.

Test Substance Application
Beginning on Day 1, a volume of 25 μL of the vehicle, the appropriate test substance concentration or the positive control substance was applied to the dorsum of both ears of each mouse once per day for three consecutive days (Days 1, 2, and 3) using a micropipette. During application, the material was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette.

Dermal Scoring
Prior to each application (Days 1, 2 and 3), the ears were evaluated for erythema and edema according to the modified Draize scoring system (Draize et al., 1944). All ears were also evaluated on Day 6.

Thymidine, [Methyl-3H]- Injections
On Day 6 of the study (three days after the final topical application) 250 μL of sterile phosphate buffered saline (PBS, Lot #: 039K8200) containing 20 μCi of Thymidine, [Methyl-3H]- (Lot #201008) was injected intravenously via the tail vein of each mouse.

Lymph node assessment
Approximately five hours after the injection, the draining auricular lymph nodes from all animals were excised. The lymph nodes were pooled for each individual mouse. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently massaging the lymph nodes between the frosted ends of two microscope slides over a collection vessel. The slides were then rinsed briefly with PBS into the vessel. The contents of the vessel were transferred to a centrifuge tube and washed with an excess of PBS and centrifuged for approximately 10 minutes at 1750 rpm, with a g-force of 283.15 grams. This process was carried out twice. In both cases, the supernatant was decanted and discarded following each centrifugation. After the second wash, approximately 5 mL of 5% trichloroacetic acid (TCA, Lot #: 086862) was then added to the sediment and the tube was vortexed briefly. The DNA was then precipitated in the TCA at approximately 4°C overnight (approximately 18 hours).

Following the overnight precipitation of the DNA, the tubes were centrifuged again for approximately 10 minutes and the supernatant was discarded. The resulting precipitate was resuspended using 1 mL of TCA and transferred to 10 mL of scintillation fluid. Incorporation of Thymidine, [Methyl-3H]- was measured by Β-scintillation counting and expressed as disintegrations per minute, minus background dpm.

Clinical Observations
All test and preliminary screening mice were observed for signs of mortality, gross toxicity, and/or behavioral changes daily. All test mice were euthanized via overdose of inhaled Isoflurane anesthetic on Day 6.

Body Weights
All test mice were weighed on Day 1 (prior to the first application) and prior to sacrifice on test Day 6.

Reference:
Draize, J.H., Woodward, G., and Calvery, H.O. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 1944; 82:377-390.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was performed on the dpm values. Significance was judged at p <0.05. The treated groups and vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett’s t-test for multiple comparisons. Where variances are considered significantly different by Bartlett’s test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn’s test) (INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using Grubbs test (1969).

Results and discussion

Positive control results:
Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer, which elicited a proliferative response with a SI value of 3.66 relative to vehicle controls.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Treatment of mice with 5%, 10% and 25% of the test material resulted in stimulation index values of 1.19, 0.63 and 0.76, respectively, relative to vehicle control mice.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Treatment of mice with 0, 5, 10 or 25% of the test material or 25% HCA resulted in disintegrations/minute values of 962.76 +/- 393.91, 1148.78 +/- 539.78, 606.54 +/- 158.78, 732.28 +/- 228.46 or 3525.30 +/-2269.29, respectively.

Any other information on results incl. tables

A preliminary screening study was conducted with concentrations of 2.5%, 5%, 10% and 25%. The top dose of 25% was selected based on maximum solubility/compatibility of the test substance with the vehicle while maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw data). On Days 1, 2, 3 and 6, each dose site was evaluated for local irritation. Very slight erythema was noted in mice dosed at all concentrations. Based on the results of the screening assay, 5%, 10% and 25% were evaluated in the main study. All mice appeared active and healthy, and there were no consistent treatment-related effects on body weight over the course of the study. All treatment groups displayed very slight erythema on Days 3 and 6. Very slight to well-defined erythema was noted in the positive control group. Treatment of mice with 5%, 10% and 25% of the test material resulted in stimulation index values of 1.19, 0.63 and 0.76, respectively, relative to vehicle control mice. As a stimulation index (SI) of greater than 3.0 was not observed, the test material was considered negative for dermal sensitization potential. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer, which elicited a proliferative response with a SI value of 3.66 relative to vehicle controls.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified