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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2006-05-03 to 2006-05-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, adopted 2002-04-24
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Peconal H- Physical state: Pink powder- Analytical purity: 98.9 % cobalt(2+) bis(4-oxopent-2-en-2-olate) dihydrate; (20.32 % total cobalt content)- Batch No.: 05022006- Expiration date of the batch: 2007-04-30- Storage condition of test material: At room temperature and under argon gasNo further information on the test material was stated.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: On the first day of the treatment period, the animals were approx. 10 weeks old.
- Weight at study initiation: On the first day of the treatment period, the animals had a mean body weight +/- standardd deviation of 21.2 +/- 1.2 g.
- Housing: The animals were housed individually in disposable crystal polystyrene cages (22.00 cm X 8.50 cm X 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet: Free access to SsniffR/M-H pelleted diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Relative humidity: 30 to 70 %
- Ventilation: approx. 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 / 12
No further information on the test material was stated.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Preliminary test: 25, 10, 5, 2.5, 1, 0.5,0.25 and 0.1 %
Main test: 0.05, 0.1, 0.25, 0.5, 1 and 25 %
No. of animals per dose:
2 females per concentration (preliminary test); 4 females per concentration (main test)
Details on study design:
RANGE FINDING TESTS:
No cutaneous reactions were noted. At the concentration ≥ 2.5 %, a significant increase in ear thickness was recorded, showing the irritant potential of the test item at these concentrations. The highest concentration retained for the main test was therefore 1 %.

CHOICE OF THE VEHICLE:
Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)), dimethylformaide was chosen among the other proposed vehicles. A solution was obtained at the maximum concentration of 25 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the Stimulation Indices (SI) for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared in the vehicle at the chosen concentration.
All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.
On days 1, 2 and 3, a dose-volume of 25µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
Lymph node cell proliferative responses were measured as described by Kimber and Dearman (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9 % NaCl containing 20 µCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
For each experimental group, a single cell suspension of auricular lymph node cell (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9 % NaCl and pellets obtained were re-suspended in exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at +5°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima Gold scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.

CLINICAL SIGNS, MORBIDITY AND MORTALITY:
The animals were observed at least once a day during the study for clinical signs, sings of morbidity or mortality.

BODY WEIGHT:
The animals were weighed individually on the first day (day 1) and on the day of sacrifice (day 6).

EAR THICKNESS MEASUREMENTS AND RECORDING OF LOCAL REACTIONS:
Ear thickness measurements and recording of local reactions were performed in order to access any possible irritant effect of the test item, as possible irritancy maybe involved in false psoitive lymphoproliferative responses.
On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice), the thickness of the left ear of each animal of group 1 to 6 was measured using a micrometer.
No measurement of ear thickness was perfomred for the animals of the positive control group.
Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (colouration, presence of residual test item, ....) was noted.
The irritation level of the test item was determined according to the table 1 (see "Any other information on materials and methods incl. tables")

No further information on details on study design (LLNA) was stated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The results were expressed as disintegration's/mn (dpm) per group and per node. Stimulation Indices (SI) were calculated according to the following fromula: SI = disintegration's/mn of treated group / disintegration's/mn of control group.

Results and discussion

Positive control results:
In the positive control group given α-hexyl cinnamic aldehyde at the concentration of 25 %, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 22.02) were noted. The study was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
3.94
Test group / Remarks:
0.05%
Parameter:
SI
Value:
6.32
Test group / Remarks:
0.1%
Parameter:
SI
Value:
17.03
Test group / Remarks:
0.25%
Parameter:
SI
Value:
24.5
Test group / Remarks:
0.5%
Parameter:
SI
Value:
25.06
Test group / Remarks:
1%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
0.05 % (w/v): 1000.64 (dpm per group); 125.08 (dpm per node) 0.1 % (w/v): 1607.04 (dpm per group); 200.88 (dpm per node) 0.25 % (w/v): 4325.89 (dpm per group); 540.74 (dpm per node) 0.5 % (w/v): 6224.47 (dpm per group); 778.06 (dpm per node) 1 % (w/v): 6367.34 (dpm per group); 795.92 (dpm per node) Vehicle group: 254.09 (dpm per group); 31.76 (dpm per node) Positive control group: 5595.62 (dpm per group); 699.45 (dpm per node)

Any other information on results incl. tables

No mortality and no clincal sings were observed during the study.

The body weight change of treated animals was similar to that of control animals.

No cutaneous reactions and no noteworthy increase in ear thickness were observed at any of the tested concentrations.

The quantity of cells obtained in each group was satisfactory and the celluarity correlated with incorporation of 3H-TdR. The cell viability was higher than 80 % in each group.

A dose related increased in the SI was noted at all the tested concentrations.

The irritation level was I.

In the absence of local irritation, the positive lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions, the test item Peconal H induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.