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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.
Justification for type of information:
Data is from experimental study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
other: Directive 96/54/EEC,Part. B.6 (30 July 1996)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Principles of method if other than guideline:
To assess the skin sensitization potential of test chemical.
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Justification for non-LLNA method:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 100%

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Age at study initiation: 9 weeks - Weight at study initiation: 19-28 grams- Housing: Animals were housed in groups of 5 of the same dose group in plastic cages (265 x 160 x 140 mm)- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 8 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22± 2 °C- Humidity (%): 55 ± 15%- Air changes (per hr): minimum 15 air changes per hour- Photoperiod (hrs dark / hrs light):12-hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0% (Vehicle Control), 0.05%, 0.25%, 1.25%
No. of animals per dose:
5 females mice per dose
Details on study design:
MORBIDITY/MORTALITY: All animals were observed twice daily.CLINICAL SIGNS: Animals were observed daily. On treatment days, animals were examined before and at least once after dosing to detect any clinical signs or reaction to treatment (especially at the treatment sites).BODY WEIGHT: All animals were weighed on days –1 and 5.EAR SWELLING MEASUREMENT: For each animal, ear thickness was measured once pretest (on day 0) and on day 5 using a constant pressure micrometer.EVALUATION OF THE CELL PROLIFERATION: Method and material: all mice received an intravenous injection via the tail vein (using an infusion pump) of 250 μL of phosphate buffered saline (PBS) containing 21.4 μCi of [3H] methyl thymidine (specific activity of 25 Ci/mmol). Five hours later the mice were sacrificed by carbon dioxide inhalation and the draining auricular lymph nodes were excised. A single cell suspension was prepared for each animal. Cells were washed twice with PBS. Cell viability and cellularity were assessed using the Trypan blue exclusion test. Cells were precipitated with ice cold 5% trichloro-acetic acid (TCA). Approximately 18 hours later, the pellets were resuspended in 1 mL of TCA and transferred into the scintillation cocktail. [3H]-methyl-thymidine incorporation was measured by liquid scintillation counting in a TRI-CARB 2700TR counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Body weights on days -1 and 5, body weight gains over days -1 to 5, ear thickness changes over days 0 to 5, cell proliferation and cellularity were analysed statistically using a SAS softare package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk’s test was used to assess the normality of the data distribution in each group. Data with homogeneous variances and normal distribution in all groups were analysed using Anova followed by Dunnett’s test. Data showing non homogeneous variances or a non normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon’s rank sum test.

Results and discussion

Positive control results:
The positive control hexyl cinnamic aldehyde was shown to be a skin sensitiser and thus validated the experimental conditions.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 2.6, 10.4, and 16.1 at 0.05, 0.25, and 1.25%, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 273.0, 1101.0, and 1710.0 at 0.05, 0.25, and 1.25%, respectively.

Any other information on results incl. tables

Table 2 : Data Summary

Treatments and Concentrations

DPM

(per node)

Stimulation Index

Cellularity index

Irritation Class

Negative Controls

(AOO)

106.0

 -

-

0.05%

273.0

2.6

1.17

1

0.25%

1101.0

10.4

2.36

1

1.25%

1710.0

16.1

2.46

1

Positive Control

(HCA 50%)

2939.5

27.8

5.10

3

Applicant's summary and conclusion

Interpretation of results:
other: sensitising
Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). The positive control hexyl cinnamic aldehyde was shown to be a skin sensitiser and thus validated the experimental conditions. Under the defined experimental conditions, the test substance induces delayed contact hypersensitivity in the murine local lymph node assay. The EC3 value for the test substance calculated on the basis of the results obtained in this experiment is equal to 0.06%.
Executive summary:

The objective of the study was to determine if the test substance is a contact skin sensitiser in CBA/J mouse as measured by cell proliferation in the draining lymph nodes after topical application on the pinnae. Morbidity/mortality checks were performed twice daily. Clinical examinations were performed daily. Individual body weights were recorded on days –1 and 5. Ear swelling measurement was performed once pretest (on day 0) and on day 5. All animals were sacrificed on day 5 for assessment of cell viability, cellularity and cell proliferation. The positive control hexyl cinnamic aldehyde induced a positive response in the local lymph node assay, as there was at least a 3-fold increase in isotope incorporation in the draining auricular lymph node relative to the vehicle. The mean stimulation index was 27.8 for the positive control at the concentration of 50%. The test substance induced a positive response in the local lymph node assay, as there was at least a 3-fold increase in isotope incorporation in the draining auricular lymph node relative to the vehicle, with a calculated EC3 value of 0.06%. The mean stimulation indices were 2.6, 10.4, and 16.1 at the concentrations of 0.05, 0.25, and 1.25%, respectively. The test substance did not increase ear thickness following topical application, whereas there was a 38.8% increase of thickness in the positive control group. In the absence of evidence of local irritation, the lymphoproliferative responses observed with the test substance were attributed to delayed contact hypersensitivity. The cellularity index was 1.17, 2.36, and 2.46 at 0.05, 0.25, and 1.25% of the test substance, respectively, and 5.10 for the positive control. The irritation class of the test substance is 1 (increase in ear thickness ≤10) at the three tested concentrations, whereas the irritation class of hexyl cinnamic aldehyde is 3 (increase in ear thickness ≥ 30%).

On the basis of observed effects, the test substance was considered as skin sensitizer.