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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-03-24 to 2009-07-21
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline-conform study under GLP without deviations

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Temperature animal room: approx. 19 °C. This deviation does not affect the validity of the study.
GLP compliance:
yes (incl. QA statement)
GLP statement and certificate attached to full study report.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Purity: 99.5%
Batch No: 14.01.2008

In vivo test system

Test animals

other: CBA/CaOlaHsd
Details on test animals and environmental conditions:
Source: Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst / The Netherlands
Number of animals for pre-test: 4
Number of animals for main study: 16
Number of animals per group: 4 (nulliparous and non-pregnant)
Number of test groups: 3
Number of control groups: 1
Age: 8-12 weeks
Identification: The animals were distributed into test group at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Housing: group housing
Cage Type: Makrolon Type II, with wire mesh top
Bedding: granulated soft wood bedding
Diet: pelleted standard diet, ad libitum
Water: tap water, ad libitum
temp: 22 +/- 2°C
rel. humidity: 45-65%
artificial light 6 a.m. - 6 p.m.

Study design: in vivo (LLNA)

propylene glycol
0, 2.5, 5 and 10% (w/v), application volume 25 µl
No. of animals per dose:
Details on study design:
Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal
surface of each ear lobe (left and right) with different test item concentrations of 2.5, 5,
and 10% (w/v) in propylene glycol. The application volume, 25 Xl, was spread over the
entire dorsal surface (diameter approx. 8 mm) of each ear lobe once daily for three consecutive days. A
further group of mice was treated with an equivalent volume of the relevant vehicle alone
(control animals).

Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare product
code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 Xl of
80.8 XCi/ml 3HTdR (corresponds to 20.2 XCi 3HTdR per mouse) by intravenous injection
via a tail vein.

Determination of Incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by
intraperitoneal injection of Pentobarbital-Natrium (Release(R), WDT, D-30827 Garbsen).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group).
Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were
prepared by gentle mechanical disaggregation through stainless steel gauze (200 Xm
mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the
lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and
incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred
to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS)
GmbH, D-63110 Rodgau) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter (Tricarb
2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR
levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-
scintillation counter expresses 3HTdR incorporation as the number of radioactive
disintegrations per minute (DPM).

Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test
concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared
with concurrent controls, as indicated by the Stimulation Index (S.I.).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: test item conc. 2.5 % (w/v): 1.83 test item conc. 5 % (w/v): 2.07 test item conc. 10 % (w/v): 2.65
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per lymph node: control group: 255.4 test item conc. 2.5 % (w/v): 467.6 test item conc. 5 % (w/v): 528.6 test item conc. 10 % (w/v): 677.7

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information
The test item m-Xylidine was not a skin sensitiser under the described conditions.
Executive summary:

In the study the test item m-Xylidine dissolved in propylene glycol was assessed for its possible contact allergenic potential. For this purpose a LLNA was performed using the test item concentrations of 2.5, 5, and 10%. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. A relevant increase on ear weights (punches) was not observed and a statistically significance of the ear weight was not determined. In this study Stimulation Indices (S.I.) of 1.83, 2.07, and 2.65 were determined with the test item at concentrations of 2.5, 5, and 10% in propylene glycol, respectively. The test item m-Xylidine was not a skin sensitiser.