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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:
Started: december 03, 2010. Ended: december 23, 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: There are not deviations from the recommended guidelines.

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
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Lubricating oils, used, vacuum distd.
EC Number:
295-422-7
EC Name:
Lubricating oils, used, vacuum distd.
Cas Number:
92045-41-5
IUPAC Name:
Lubricating oils, used, vacuum distd.
Details on test material:
The sample used was identified as FLL-TDA.
Batch number: S/63 200810

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
The maximum concentration was selected according to the criteria specified in the International Guidelines and on the basis of the data obtained in the preliminary test.
The test item was prepared at the concentrations of 0, 5, 10, 25, 50 and 100%
No. of animals per dose:
Four females per dose were tested.
Details on study design:
Administration:
on days 1, 2 and 3, a dose-volume of 25 μL 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 anesthesia 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.

Clinical signs, morbidity and mortality:
the animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality.

Body weight:
in the main test, the animals were weighed individually on the first day of the study (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 assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.
On days 1, 2 and 3 (before each cutaneous application) and on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle control and treated groups was measured using a micrometer.
No measurement of ear thickness was performed for the animals of the positive control group. Any irritant reaction (erythema and edema) was recorded in parallel. Any other observation (coloration, presence of residual test item …) was noted.

Intravenous injection of 3H-TdR and sampling of auricular lymph nodes:
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 20 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 cells (ALNC) was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe.

Preparation of auricular lymph node cell suspensions and determination of proliferation:
cell suspensions were washed once with 15 mL of 0.9% NaCl. The pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by 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 +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.
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 as dpm per node.
Stimulation Indices (SI) were calculated according to the following formula:

SI = dpm of treated group / dpm of control group

The test item was considered as a skin sensitizer when the 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.
The EC3 value (theoretical concentration resulting in a SI value of 3) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold. The equation used for calculation of EC3 was:

EC3 = c + [(3 – d)/(b-d)] x (a – c)

Where a = the lowest concentration giving stimulation index > 3; b = the actual stimulation index
caused by a; c = the highest concentration failing to produce a stimulation index of 3; and
d = the actual stimulation index caused by c.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation undex (SI) Test item 5%: 1.02 Test item 10%: 1.34 Test item 25%: 1.36 Test item 50%: 0.93 Test item 100%: 4.31 HCA 25%: 9.28 The EC3 value (theoretical concentration resulting in a SI value of 3) is equal to 81%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Disintegrations per minute (dpm) Vehicle: 629.00 (dpm per group) and 78.63 (dpm per node) Test item 5%: 639.00 (dpm per group) and 79.88 (dpm per node) Test item 10%: 843.00 (dpm per group) and 105.38 (dpm per node) Test item 25%: 853.00 (dpm per group) and 106.63 (dpm per node) Test item 50%: 586.00 (dpm per group) and 73.25 (dpm per node) Test item 100%: 2708.00 (dpmper group) and 338.50 (dpm per node) HCA 25%: 5837.00 (dpm per group) and 729.63 (dpm per node)

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions of this study, the test item induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
According to the EC3 value obtained, the test item should be considered as a weak sensitizer; therefore, according to Regulation (EC) n. 1272/2008 and to Directive 67/548/EEC, study results indicate that the substance should be classified for skin sensitisation.