Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-06 to 2010-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD guideline and in accordance with GLP.

Data source

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

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 lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK.
- Age at study initiation: 8-12 wk
- Weight at study initiation: 16.9-22.9 g
- Housing: 1/polycarbonate cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 2010-01-06 To: 2010-01-18

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25%, 50%, 100% TS
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: clear solution formed with 50% test substance in vehicle
- Irritation: no irritation reported in main study
- Lymph node proliferation response: tested on 1 animal using neat test substance.

MAIN STUDY
- Name of test method: LLNA (individual animal approach)
- Criteria used to consider a positive response: The test substance is regarded as a sensitizer if at least one concentration of the chemical results in a three-fold greater increase in 3HTdR incorporation compared to control values

25 ul applied daily to dorsal surface of both ears on 3 consecutive days.

Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline containing 3H-methyl Thymidine1 (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to each mouse.

Five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised for each experimental animal. 1.0 mL of phosphate buffered saline was added to the lymph nodes for each animal. The animals were then discarded and no further investigations were carried out. The following procedures were carried out with the lymph nodes from these animals only.

A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The LNC were then washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash. After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Analysis of variance was carried out on the data. If Bartlett’s test for homogeneity of variance was significant at the 1% level, the data were logarithmically transformed prior to analysis in order to stabilise the variances (Bartlett 1937). Comparisons were made between the control and Ethylthioethanol treated groups using Dunnett’s test (Dunnett 1955, 1964). To investigate the nature of the dose response curve the linear contrast was isolated.

Results and discussion

Positive control results:
ratio (dmp/node) HCA (25%):control = 6

(Tested at this facility in October 2009 with the same strain of mouse.)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: test/control ratio: 25% TS = 1.2 50% TS = 1.4 100% TS = 0.8
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM/node: 0% TS = 337.60 25% TS = 389.95 50% TS = 468.80 100% TS = 262.60

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
A reliable local lymph node assay, conducted according to a standard method and GLP, failed to identify any sensitization potential in the test substance when applied at up to 100% to the ears of mice.