Xylene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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). Overall, the result obtained by Ehling et al. for mixed xylenes is considered unreliable.

Data source

Reference

Reference 1

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 2

Reference Type:

publication

Title:

An evaluation of performance standards and nonradioactive endpoints for the Local Lymph Node Assay - the Report and Recommendations of ECVAM Workshop 65

Author:

Basketter, D, Cockshott, A, Corsini, E, Gerberick, GF, Idehara, K, Kimber, I, van Loveren, H, Matheson, J, Mehling, A, Omori, T, Rovida, C, Sozu, T, Takeyoshi, M and Casati, S.

Year:

2008

Bibliographic source:

ATLA 36, 243-257

Reference 3

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)

Deviations:

yes

Remarks:

Considered a non-standard, unvalidated methodology by ECVAM Workshop (e.g. based on mouse stain used, choice of vehicle, time of cell collection, magnitude of discriminating SI; determination of [3H]TdR incorporation by lymph node cells in vitro)

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

Test material information

Constituent 1

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. Lymph node weight, lymph node cell counts and topical irritation were determined on day 4, 24 h after the last treatment. 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 were counted using automated equipment. To assess irritant potential, an ear punch (8 mm diameter) was taken 24 h after last exposure and weighed.
[3H]TdR incorporation by lymph node cells was determined in vitro in other animals sacrificed 4 days following the last topical application (day 6). The auricular lymph nodes (LN) were excised, 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 from 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.

The methodology used in this paper was reviewed as part of an ECVAM Workshop on “An Evaluation of Performance Standards and Non-radioactive Endpoints for the Local Lymph Node assay”, and the discussion and recommendations reported by Basketter et al. (2008). The Workshop noted that the method used by Ehling et al. (2005) for measurement of cell numbers and ear thickness deviated from the standard method in terms of the strain of mouse used (BALB/c (inbred) or NMRI (outbred) versus CBA/Ca or CBA/J (inbred) in the Guideline); excision of lymph nodes on day 4 (rather than on day 6, as stated in Guideline); use of dimethylacetone/acetone/ethanol (4:3:3) as vehicle (versus vehicles / vehicle validation requirements of the Guideline); and a cut-off at SI = 1.4 or above (versus a cut-off of SI=3 or above in the Guideline). Together, these were concluded (by the ECVAM Workshop) to represent a MAJOR change in methodology that required further validation.

The use of [3H]TdR incorporation by lymph node cells in vitro (as opposed to incorporation of [3H]TdR following i.v. injection in vivo was not discussed but appears to represent a change versus the Guideline.

Basketter and Kimber (2010) also commented that the findings of Ehling et al. (2005) for mixed xylenes can only be considered of limited relevance since the assay used was not considered to be valid following review by ECVAM.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Measured parameters compared using non-parametric Kruskal-Wallis test as the 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 substance (alpha-hexylcinnamaldehyde, assessed blind at all 11 laboratories) however no quantitative results were included in the publication.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Not reported

Results 2

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 according to criteria applied by the study authors) in 7 of the 9 laboratories involved in the trial; and an increase in ear weight (indicative of irritation according to criteria applied by the study authors) in 3 of 9 laboratories.

Although not discussed in any detail, one laboratory also determined 3[H]TdR incorporation by lymph node cells in vitro 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:

other: ambiguous

Conclusions:

Mixed xylene produced a statistically significant increase in ear-draining lymph node weight and cell count (indicative of a sensitisation response according to criteria used by the study authors) 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, however the findings are of limited relevance because the assay was not considered valid following review by the European Centre for the Validation of Alternative methods (Basketter et al., 2008; Basketter and Kimber, 2010).

Executive summary:

The sensitisation potential of mixed xylenes was assessed in a modified local lymph node assay where increased ear-draining lymph node weight and cell counts were used to quantify lymph node cell proliferation by 9 laboratories participating in a ring-trial. [3H]TdR incorporation in vitro by lymph node cells was also assessed in a single laboratory (assay not conducted by other laboratories). Acute skin inflammation (a potential confounder that can lead to "false positive" results) was also determined based on changes in ear tissue weight. Treatment with mixed xylenes 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. An increased (but not quantified) increase in 3[H]TdR incorporation by lymph node cells was also reported by one laboratory. Based on these findings, mixed xylene was considered an allergen by a majority of laboratories participating in the trial. However the methodology used was evaluated by an ECVAM Workshop (reported by Basketter et al., 2008) which concluded the assay methods deviated from the Guideline in terms of the strain of mouse, the choice of vehicle, the time of lymph node/lymph node cell collection, and the magnitude of the SI used to differentiate positive and negative samples. Overall, the ECVAM Workshop concluded that these differences represented a major change in methodology that required further validation. The use of [3H]TdR incorporation by lymph node cells in vitro (as opposed to incorporation of [3H]TdR following i.v. injection in vivo, as required by the Guideline) was not discussed by the ECVAM Workshop but is another change relative to the Guideline. Basketter and Kimber (2010) also noted that these findings for mixed xylenes can only be considered of limited relevance given methodological deviations from the Guideline. It is concluded that this publication does not provide convincing evidence that mixed xylenes possesses a potential to induce or elicit skin sensitisation.