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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:
supporting study
Study period:
Not reported
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: GLP status not known, published in peer reviewed literature. Method considered not to be valid following review by the European Centre for the Validation of Alternative Methods (Basketter et al, 2008)

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay 2nd round
Author:
Ehling G et al
Year:
2005
Bibliographic source:
Toxicology 212 69-79
Reference Type:
review article or handbook
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Ear-draining lymph node (LN) weight and cell counts were used to assess LN cell proliferation instead of [3H]TdR incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test item.
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
mixed xylenes
IUPAC Name:
mixed xylenes
Constituent 2
Chemical structure
Reference substance name:
Xylene
EC Number:
215-535-7
EC Name:
Xylene
Cas Number:
1330-20-7
Molecular formula:
C8H10
IUPAC Name:
xylene
Details on test material:
- Name of test material (as cited in study report): Xylene
- Analytical purity: 97.5%

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
not specified
Details on test animals and environmental conditions:
BALB/c mice from local breeder were used. The age of the animals was between 7 and 12 weeks. The animals were kept under standard conditions and had free access to food and water.

Study design: in vivo (LLNA)

Vehicle:
other: DAE433 (DAE433 = 40% dimetyhlacetamide, 30% acetone, 30% ethanol by volume
Concentration:
10, 30 and 100%
No. of animals per dose:
6
Details on study design:
Dorsal side of both ears treated with 25 µL/ear of the xylene solutions on days 1 to 3. Mice killed on day 4, 24 h after the last treatment. Four days following the last topical application (day 6), the auricular lymph nodes (LN) excised. The LN were weighed and pooled for each animal and suspended in 5 mL RPMI-1640 supplemented with 5% heat inactivated foetal calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin. Single cell suspensions were prepared under aseptic condition. Cells washed twice in standard medium then resuspended in 1 mL standard medium with 10% FCS. Cells counted (Coulter Counter) and cultured at a concentration of 1 × 10^7 cells/mL. When necessary, cell suspensions of several animals were pooled to obtain the concentration required. Cell suspensions seeded in triplicate into micro titre plates. Cells cultured with 10 mL [3H]TdR (3.7 MBq/mL) for 24 h at 37°C in a humidified atmosphere of 5% CO2 in air. [3H]TdR incorporation determined by liquid scintillation counting. [3H]TdR incorporation is expressed per animal.

To assess irritant potential, an ear punch (8 mm diameter) taken 24 h after last exposure and weighed. Auricular lymph nodes draining the ear tissue excised 24 h after the last exposure. Lymph node (LN) weights obtained from LN pairs of individual animals. For the determination of individual LN cell counts, single-cell suspensions from LN pairs of individual animals prepared by mechanical tissue disaggregation. The cells counted using automated equipment.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Measured parameters compared using non-parametric Kruskal-Wallis test as global test, and the non-parametric two-group Wilcoxon-Mann-Whitney rank test for all two-group comparisons. Because of power considerations the non-parametric Steel test was not applied for the comparison of the three concentration groups with the control group.

Results and discussion

Positive control results:
A satisfactory response was obtained with the positive control sustance (alpha-hexylcinnamaldehyde, assessed blind at all 11 laboratories) however no quantitative results were included in the publication.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Not reported
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not reported

Any other information on results incl. tables

Treatment with mixed xylene was associated with a statistically significant increase in ear-draining lymph node weight and cell count (indicative of a sensitisation response) in 7 of the 9 laboratories involved in the trial; and an increase in ear weight (indicative of irritation) in 3 of 9 laboratories.

Although not discussed in detail, one laboratory also conducted a standard LLNA protocol using mixed xylene (3H-thymidine incorporation) and obtained a "positive" result.

Overall, mixed xylene was classified as an allergen in 8 of the 9 laboratories participating in the trial.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Irritant, author concluded that may cause LN activation and acute skin reactions. Basketter and Kimber (2010) reviewed the available data for xylenes and considered the reported findings to be a false positive.
Conclusions:
Mixed xylene produced a statistically significant increase in ear-draining lymph node weight and cell count (indicative of a sensitisation response) in 7 of the 9 laboratories involved in the trial; and an increase in ear weight (indicative of irritation) in 3 of 9 laboratories. The study authors concluded that mixed xylene was an allergen.
Executive summary:

The sensitisation potential of mixed xylene was assessed in a modified local lymph node assay where increased ear-draining lymph node weight and cell counts were used in place of radioactive labelling to quantify lymph node cell proliferation. Acute skin inflammation (a potential confounder that can lead to "false positive" results) was also determined based on changes in ear tissue weight. The study was conducted at 9 independent laboratories as part of an international validation exercise under the supervision of the Swiss Agency for Therapeutic Products, Berne. Treatment with mixed xylene was associated with a statistically significant increase in ear-draining lymph node weight and cell count in 7 of the 9 laboratories, and an increase in ear tissue weight in 3 of 9 laboratories. Based on these findings, mixed xylene was classified as an allergen in a majority of laboratories participating in the trial.

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