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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

Data source

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

Materials and methods

Test guideline
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
Chemical structure
Reference substance name:
L-cysteine
EC Number:
200-158-2
EC Name:
L-cysteine
Cas Number:
52-90-4
Molecular formula:
C3H7NO2S
IUPAC Name:
L-cysteine
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: L-Cysteine
Chemical Name: L-( +)-Cysteine
CAS No.: 52-90-4
Batch No.: 12030506
Physical State: solid
Colour: white
Molecular Weight: 121.1
pH: 4.5-6
Storage Conditions: 2-8°C, protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsD mice
Sex:
female
Details on test animals and environmental conditions:
Source: Harlan Winkelmann, 33178 Borchen, Germany
Sex: female (nulliparous and non-pregnant)
Age at the beginning of the study: 8 - 9 weeks
Number of animals: 5 mice I group; 3 mice I prescreen test


The animals were derived from a controlled full-barrier maintained breeding system
(SPF).

Full barrier in an air-conditioned room
Temperature: 22 ± 3°C
Relative humidity: 55± 10%
Artificial light, sequence being 12 hours light, 12 hours dark
Air change: at least 10 x I hour
Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test: lot
no. 1114, main study: lot no. 1335) Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular inter-vals) The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone
cages on Altromin saw fibre bedding (prescreen test: lot no. 110811, main study: Jot
no. 300512). Adequate acclimatisation period (at least five days) under laboratory conditions.

Study design: in vivo (LLNA)

Vehicle:
other: carboxymethylcellulose
Concentration:
3.125%, 6.25% and 12.5% (w/v)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The maximum technically applicable concentration of the test item was found to be 12.5% in 2% CMC in aqua ad inject
- Irritation: Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in any animal.
- Lymph node proliferation response: There was no assessment of lymph node proliferation

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted. EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index
of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated. A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine – incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).


TREATMENT PREPARATION AND ADMINISTRATION:
Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.
Administration 3H-methyl thymidine
Five days after the first topical application all mice were dosed with 20 1-1Ci 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.
Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining "auricular lymph nodes" were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS.
This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was
added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
Determination oflncomorated 3H-methyl thymidine
The 3H-methyl thymidine- incorporation was measured in a fi-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The positive control worked as expected.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
None of the three tested concentrations of the test item reached the stimulation index of3. The stimulation index at a concentration of 3.125% was 0.9 The stimulation index at a concentration of 6.25% was 1.2 The stimulation index at a concentration of 12.5% was 1.0.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 1052.8 (SD 196.1) with 3.125% L-Cysteine in CMC, 1328.0 (SD 265.0) with 6.25% L-Cysteine in CMC, 1175.8 (SD 137.7) with 12.% L-Cysteine in CMC, 1131.0 (SD 301.6) with negative control, 9473.0 (SD 711.9)with positive control substance

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.
Consequently, according to OECD 429 the test item L-Cysteine as described in this report is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.
Executive summary:

A skin sensitization study according to OECD Guideline 429 was performed with L-Cysteine (purity 99.6%) and female mice. Following a pretest on solubility and irritating properties of the test substance the concentrations for the main test were set to be 3.125%, 6.25% and 12.5% (w/v), carboxymethylcellulose in aqua ad injectionem served as vehicle. 25% alpha-Hexylcinnamaldehyde in 2% CMC in aqua ad injectionem served as positive control. Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. Five days after the first topical application all mice were dosed with 20 µlCi3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.

Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining "auricular lymph nodes" were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS.

This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight. The 3H-methyl thymidine- incorporation was measured in a fi-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal. The Disintegrations Per Minute (DPR) were 1052.8 (SD 196.1) with 3.125% L-Cysteine in CMC, 1328.0 (SD 265.0) with 6.25% L-Cysteine in CMC, 1175.8 (SD 137.7) with 12.% L-Cysteine in CMC, 1131.0 (SD 301.6) with negative control, 9473.0 (SD 711.9)with positive control substance. The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. Consequently, according to OECD 429 the test item L-Cysteine as described in this report is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.