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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-28 - 2012-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, reliable without restrictions
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010, July 22
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
- Source: Harlan Laboratories B.V., Postbus 6174, Nl-5960 AD Horst/ The Netherlands
- Age at study initiation: 8 -9 weeks
- Weight at study initiation: 18.7 - 24.0 g
- Housing: The animals were kept singly in MAKROLON cages (type III) with a basal surface of approx. 39 cm X 23 cm and a height of approx. 15 cm. - Bedding: Granulated soft wood bedding
- Diet (ad libitum): 2018C Teklad Global 18 % protein rodent diet
- Water (ad libitum): Tap water

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C (maximum range)
- Relative humidity: 20.7 - 91 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: ethanol : water (3+7,v/v)
Concentration:
0 % w/w, 10 % w/w, 25 % w/w, and 50 % w/w
No. of animals per dose:
4 female mice (nulliparous and non-pregnant)
Details on study design:
RANGE FINDING TESTS:
A preliminary experiment was carried out in 2 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Two concentrations of 25 and 50% of l-cysteine hydrochlorid monohydrate in ethanol : water (3+7,v/v) were examined.
Results:
In the preliminary experiment, concentrations of 25% and 50%, employing 1 animal per concentration, were examined. No pronounced irritating properties were observed in this preliminary experiment at concentrations of 25% or 50%, no differences in ear weight and ear thickness were noted.

MAIN STUDY:
The test item was suspended in a mixture of ethanol : water (3+7,v/v).
The test item suspension was administered to the dorsum of animal's ears at an application volume of 25 μL/ear.
The concentrations were chosen based on a preliminary dose range finding.

The experimental schedule of the assay was as follows:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0, 10, 25 and 50 % (w/w). The application volume, 25 µl/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. Five days after the first topical application 250 µl of phosphate-bufffered saline containing 19.7 µCi of 3H-methyl thymidine was injected into each mouse via tail vein.
Approximately 5 hours after the intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated withe trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl-thymidine measured in a beta-scintillation counter.
The clinical signs were recorded at least once daily, especially the treatment sites were observed carefully.
Day 1:
The weight of each animal was individually identified and recorded. The ear thickness was measured.
Open application of 25 μL of the appropriate dilution of the test item or the vehicle alone were applied to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated. On day 3 the ear thickness was measured.
Day 5: 48 hours after the last application the animals were sacrificed.
Ear swelling measurements (immediately before sacrificing the mice) were carried out.
Punch biopsies were prepared and immediately weighed on an analytical balance.

ANALYSIS OF RESULTS:
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in a 3-fold or greater increase in incorporation of 3H-methyl thymidine compared with concurrent controls, as indicated by the Stimulation Index. The estimated concentration of test item required to produce a Stimulation Index of 3 is referred as the EC3 value.
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.
Positive control results:
The positive control was performed in October 2012. The expected increases in the Stimulation Index were observed (S.I. = 5.7 in 25% (w/v) Positive control item concentration). Therefore, the study could be regarded as valid.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Threshold value of the stimulation index (SI) of lymph node cell count weight were calculated by dividing the value per group of the test item treated animals by the vehicle treated ones. Values above 3 are considered positive (these values were fixed empirically during the inter-laboratory validation of this method). 10 % w/w: SI: 2.0 25 % w/w: SI: 2.3 50 % w/w: SI: 2.3
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 0 % 95.6 10% 195.8 25 % 219.4 50 % 234.7

In the main study treatment with l-cysteine hydrochloride monohydrate at concentrations of 10%, 25% or 50% did not cause symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed.

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
Under the present test conditions, L-Cysteine hydrochloride monohydrate at concentrations of 10%, 25% or 50% (w/w) in ethanol : water (3+7,v/v)
did not reveal sensitising properties in the local lymph node assay.
According to Regulation (EC) No.: 1272/2008 and subsequent regulations, the test item is not classified as skin sensitiser.
Also, according to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is not classified as skin sensitiser.
Executive summary:

L-Cysteine hydrochloride monohydrate was suspended in a mixture of ethanol : water (3+7,v/v). The vehicle was selected on the basis of maximising contact of the test item with the skin. The test item suspension was administered to the dorsum of the animal's ears. The concentrations were chosen based on a preliminary dose range finding. The experimental schedule of the assay was as follows: Each test group of 4 female mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0, 10, 25 and 50 % (w/w). The application volume, 25 µl/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. Five days after the first topical application 250 µl of phosphate-buffered saline containing 19.7 µCi of 3H-methyl thymidine was injected into each mouse via tail vein. Approximately 5 hours after the intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated withe trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl-thymidine measured in a beta-scintillation counter.

The clinical signs were recorded at least once daily, especially the treatment sites were observed carefully.

Day 1: The weight of each animal was individually identified and recorded. The ear thickness was measured. Open application of 25 μL of the appropriate dilution of the test item or the vehicle alone were applied to the dorsum of each ear.

Days 2 and 3: The application procedure carried out on day 1 was repeated. On day 3 the ear thickness was measured.

Day 5: 48 hours after the last application the animals were sacrificed. Ear swelling measurements (immediately before sacrificing the mice) were carried out. Punch biopsies were prepared and immediately weighed on an analytical balance.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in a 3-fold or greater increase in incorporation of 3H-methyl thymidine compared with concurrent controls, as indicated by the Stimulation Index.

Results: No deaths occurred during the study periods. No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Stimulation Indices of 2.0, 2.3 and 2.3 were determined with test item concentrations of 10, 25 and 50 %. The threshold value for a sentisizer of 3 is not reached. L-Cysteine hydrochloride monohydrate is classed as not sensitizing.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

L-Cysteine hydrochloride monohydrate was suspended in a mixture of ethanol : water (3+7,v/v). The test item suspension was administered to the dorsum of the animal's ears. Each test group of 4 female mice was treated by (epidermal) topical open application to the dorsal surface of each ear with test item concentrations of 0, 10, 25 and 50 % (w/w) for three consecutive days. The test was performed according to the guideline: Injection of 3H-methyl thymidine in phosphate-buffered saline, followed by sacrifice, excising the draining auricular lymph nodes, preparation of cell suspensions of lymph node cells and determining the proliferative capacity of poolde lymph node cells by incorporation of 3H-methyl-thymidine measured in a beta-scintillation counter.

The clinical signs were recorded at least once daily, especially the treatment sites were observed carefully. Weight recorded and ear thickness was measured on certain days.

Results: No deaths occurred during the study period. No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Stimulation Indices of 2.0, 2.3 and 2.3 were determined with test item concentrations of 10, 25 and 50 %. As the threshold value for a sensitizer of 3 is not reached, l-cysteine hydrochloride monohydrate is classed as not sensitizing.


Migrated from Short description of key information:
L-Cysteine hydrochloride monohydrate at concentrations of 10%, 25% or 50% (w/w) in ethanol : water (3+7,v/v) did not reveal sensitising properties in the local lymph node assay with mice.
In accordance with article 2(7)(b) and Annex V 6., REACH the hydrate free form of L-Cysteine hydrochloride is registered. L-Cysteine hydrochloride monohydrate (CAS 7048-04-6, hydrate part of the molecule = 10.25 %) is the manufactured and imported substance and was used for testing.
It is generally accepted that water and especially water of crystallisation has no toxicologically relevant effects. Therefore, the effect levels of L-Cysteine hydrochloride monohydrate can be used to calculate the corresponding dosages of L-Cysteine hydrochloride (anhydrous (free of water of crystallisation)). Thus, the conclusion on hazard assessment and classification holds true for the anhydrous form as well.

Justification for selection of skin sensitisation endpoint:
fully reliable guideline study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

L-Cysteine hydrochloride monohydrate at concentrations of 10%, 25% or 50% (w/w) in ethanol : water (3+7,v/v) did not reveal sensitising properties in the local lymph node assay.

According to Regulation (EC) No.: 1272/2008 and subsequent regulations, the L-Cysteine hydrochloride monohydrate and its anhydrous form are not classified as skin sensitiser.

Also, according to the criteria specified by Directive 67/548/EEC and subsequent regulations, the substance is not classified as skin sensitiser.