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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:
18 - 31 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
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)
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.
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).
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.
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.

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.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD guideline 429 and GLP (Pénzes, 2011). In a preliminary test, suitable treatment concentrations for the main study were determined in two female CRL: NMRI BR mice. No skin irritation was observed up to 25% (w/v) concentration in dimethylformamide (DMF), which corresponded to the maximum solubility of the test substance. In the main assay, 4 female CBA/Ca mice per test group received the test substance at concentrations of 5, 10 and 25% (w/v) in DMF and the positive control substance α-hexylcinnamaldehyde at a concentration of 25% (w/v) in acetone/olive oil (4:1, v/v) (AOO) for three consecutive days. Ear thickness was measured at Day 1, 3 and 6. The formation of erythema was monitored during the whole study period. On Day 6, the cell proliferation of lymph nodes was determined by incorporation of ³H-methyl thymidine, as measured by a β-scintillation counter. The group disintegration per minute (DPM) values for the cell suspensions of pooled lymph nodes from each test group were 5416, 5734 and 9322 at concentrations of 5, 10 and 25% (w/v) of the test substance in DMF, respectively. No significant changes in DPM values compared to the controls (group DPM = 6350) were observed after treatment with the test substance, resulting in SI values were 0.9, 0.9 and 1.5 after treatment with 5, 10 and 25% (w/v) of the test substance, respectively. No mortality, no systemic toxicity, no effects on body weights and no local cutaneous reactions occurred during the study. The positive control substance yielded the expected results (SI = 8.1). Under the above mentioned conditions, the test substance was shown to have no sensitisation potential in the LLNA.

A second LLNA assay with the test material was conducted according to OECD 429 and GLP with female CBA mice to determine the potential of the test substance to produce sensitisation after repeated topical applications (Durando, 2011). Three concentrations (5, 10 and 25% (w/v)) of the test substance in acetone/olive oil (AOO; 4:1 v/v) or the vehicle alone were topically applied to 5 mice per group for three consecutive days. Three days after the last application, the mice were given a 20 μCi i.v. injection of Thymidine, [Methyl-3H]-. Five hours later, the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in disintegrations per minute per mouse (DPM/mouse). Each animal’s ears were also evaluated for erythema and edema prior to each application and again on Day 6, prior to the i.v. injection. A positive control group (five female mice) was maintained under the same environmental conditions and treated with a 25% w/w mixture of alpha-hexyl cinnamaldehyde Technical (HCA) in AOO (4:1 v/v) in the same manner as the test animals. The group disintegration per minute (DPM) values for the cell suspensions of pooled lymph nodes from each test group were 1148.78, 606.54 and 732.28 at concentrations of 5, 10 and 25% (w/v) of the test substance in DMF, respectively. No significant changes in DPM values compared to the controls (group DPM = 962.76) were observed after treatment with the test substance, resulting in SI values were 1.19, 0.63 and 0.76 after treatment with 5, 10 and 25% (w/v) of the test substance, respectively.The positive control (25% HCA) showed the expected results and thus confirmed the sensitivity of the assay. Based on the results of this study, the test substance is considered to lack dermal sensitization potential in the LLNA at concentrations of less than or equal to 25%.


Migrated from Short description of key information:
Skin sensitisation (OECD 429): not sensitising

Respiratory sensitisation

Endpoint conclusion
Additional information:

This information is not available.

Justification for classification or non-classification

The available data on skin sensitisation of the substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.