Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Pre-test: Mice, CBA/CaOlaHsd
Main experiment: CBA/CaCrl
Rationale Recognised as the recommended test system
Source Harlan Laboratories B.V., The Netherlands
Number of animals for the pre-test 2 females
Number of animals for the main study 20 females
Number of animals per group 5 females (nulliparous and non-pregnant)
Number of test groups 3
Number of control (vehicle) groups 1
Age
Pre-test: 10 - 11 weeks (beginning of treatment)
Main study: 8 weeks (beginning of treatment)
Identification The animals were distributed into the test groups 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
Cage Type: Makrolon Type II / III, with wire mesh top
(EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG, 73494
Rosenberg, Germany)
Feed: pelleted standard diet, ad libitum
(Harlan Laboratories B.V. 5960 AD Horst / Netherlands)
Water: tap water, ad libitum
(Gemeindewerke, 64380 Rossdorf, Germany)
Environment: temperature 22 + 2°C
relative humidity 45-78%
artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
Each test group of mice was treated by topical (epidermal) application to the dorsal
surface of each ear with different test item concentrations of 5, 10, and 25% (w/w) in
propylene glycol. The application volume, 25 WL/ear/day, was spread over the entire dorsal
surface (diameter approx. 8 mm) of each ear once daily for three consecutive days. A further group of
mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
No. of animals per dose:
5 females
Details on study design:
A solubility experiment was performed according to the recommendations given by OECD
429. The highest test item concentration, which could be technically used was a 25 %
(w/w) suspension in propylene glycol. The test item formulations were prepared using a
mortar. At higher concentrations, an applicable formulation of the test item was not
achieved, neither by the use of other vehicles nor by using additional methods to formulate
the test item (e.g. vortexing, warming to 37°C).
To determine the highest non-irritant test concentration that did at the same time not
induce signs of systemic toxicity, a pre-test was performed in two animals and stated in
raw data and report. Two mice were treated by (epidermal) topical application to the dorsal
surface of each ear with test item concentrations of 10 and 25% once daily each on three
consecutive days. Prior to the first application of the test item and before sacrifice the body
weight was determined. Clinical signs were recorded at least once daily. Eventual signs of
local skin irritation were documented and a score was used to grade a possible reddening
of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3
and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247
Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were
punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm
corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an
analytical balance. Eventual ear irritation was considered to be excessive if reddening of
the ear skin of a score value S3 was observed at any observation time and/or if an
increase in ear thickness of S25% was recorded on day 3 or day 6 (for individual results
see Annex 1). At the tested concentrations the animals did not show any signs of local
skin irritation or systemic toxicity.
Thus, the test item in the main study was assayed at 5, 10, and 25% (w/w). The highest
concentration tested was the highest level that could be achieved whilst avoiding systemic
toxicity and excessive local skin irritation in the pre-experiment.

Each test group of mice was treated by topical (epidermal) application to the dorsal
surface of each ear with different test item concentrations of 5, 10, and 25% (w/w) in
propylene glycol. The application volume, 25 WL/ear/day, was spread over the entire dorsal
surface (diameter approx. 8 mm) of each ear once daily for three consecutive days. A further group of
mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124
Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL).
Five days after the first topical application (day 6) 250 WL of phosphate-buffered saline
(PBS) containing 20.1 WCi of 3HTdR (equivalent to 3HTdR 80.5 WCi/mL) were injected into
each test and control mouse via the tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanised by
intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, 30827 Garbsen,
Germany).
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per
animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells
were prepared by gentle mechanical disaggregation through stainless steel gauze (200
Wm 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, 63110 Rodgau, Germany) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a -scintillation counter (Tricarb
2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background
3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The -
scintillation counter expresses 3HTdR incorporation as the number of radioactive
disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables and
for the DPM values (group mean DPM ± standard deviation).

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by
use of a substance which is known to have skin sensitisation properties in CBA/CaOlaHsd
mice. The periodic positive control experiment was performed with -Hexyl
cinnamaldehyde in acetone:olive oil (4+1 v/v) using CBA/CaCrl mice in August 2011.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle Control Group 1,00 5 % L-Cystine 0,98 10 % L-Cystine 1,24 25 % L-Cystine 1,02
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle Control Group 472,9 5 % L-Cystine 461,9 10 % L-Cystine 584,1 25 % L-Cystine 480,9

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
In the study the test item L-Cystine suspended in propylene glycol was assessed for its
possible skin sensitising potential.
For this purpose a local lymph node assay was performed using test item concentrations
of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration
that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation
as confirmed by a pre-experiment.
The animals did not show any signs of systemic toxicity or local skin irritation during the
course of the study and no cases of mortality were observed.
In this study Stimulation Indices (S.I.) of 0.98, 1.24, 1.02 were determined with the test
item at concentrations of 5, 10, and 25% (w/w) in propylene glycol, respectively.
The test item L-Cystine was not a skin sensitiser under the test conditions of this study.
Executive summary:

In order to study a possible skin sensitising potential of L-Cystine, three groups each of five female mice were treated once daily with the test item at a concentrations of 5, 10, and 25% (w/w) in propylene glycol by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of five mice was treated with the vehicle (propylene glycol) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 0.98, 1.24, 1.02 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in propylene glycol. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

In conclusion, according to the current study under OECD 429, the test item L-Cystine is not a skin sensitiser.