Copper chloride

Skin sensitisation

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: GLP - Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Copper chloride

EC Number:

231-842-9

EC Name:

Copper chloride

Cas Number:

7758-89-6

Molecular formula:

Cl Cu

IUPAC Name:

λ¹-copper(1+) chloride

Details on test material:

- Name of test material (as cited in study report): Copper(I)chloride
- Test-substance No.:08/0387-2
- Physical state: solid
- Analytical purity: 98.88 %
- Lot/batch No.: 20080901
- Stability under test conditions: The stability of the test substance in the vehicle for the maximum application period was confirmed indirectly by
analysis of the homogeneity / correctness of the concentration.
- Storage condition of test material: Room temperature
- Other: The test substance was homogeneous by visual inspection

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 8 – 12 weeks
- Weight at study initiation: 17.0 g – 21.4 g
- Housing: single, Makrolon cage, type II
- Diet (e.g. ad libitum): Kliba-Labordiaet (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days before the first test-substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 30 – 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0.1%, 0.3% and 1%. The correctness of the concentration of the 0.1% test-substance preparation (2nd run) was confirmed in one sample but was too low in two samples. This variation was possibly caused by an incorrect sampling of the suspension. The deviations are considered of no influence on the evaluation of the study results.

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Concentrations: Pretest: 1% and 30%; 1st run of the study: 1%, 3% and 10%;
- Irritation and Lymph node proliferation response: The 1% test-substance preparation showed slightly increased ear weights and lymph node weights as indication of some ear irritation. Markedly increased ear weights and lymph node weights were observed after application of the 30% test-substance preparation as indication of strong ear irritation. Additionally the ears of the mice treated with the 30% preparation were sticky and hardened due to residues of test substance on the day of lymph node removal.
- Based on these results 1%, 3% and 10% test-substance preparations in propylene glycol were selected for the 1st run of the study.
- Due to increases in ear weights and lymph node parameters after application of all concentrations in the first part of the study, further concentrations of 0.1%, 0.3% and 1% were tested in a 2nd run.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Randomization: according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.
TREATMENT PREPARATION AND ADMINISTRATION:
- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- ³H-thymidine injection: about 66 to 72 hours after the last application of test substance to the ears, the mice were injected intravenously with 20 μCi of ³H-thymidine
TERMINATION AND EXAMINATION
- Termination: Animals were sacrificed on study day 5 about 5 hours after ³H-thymidine injection.
- Determination of ear weight: Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
- Preparation of cell suspension was performed and determination of cell count was carried out using a Casy®-Counter.
- Measurement of ³Hthymidine incorporation of the lymph node cells was conducted in a ß-scintillation counter.

EVALUATION OF RESULTS
• In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the ear backs is determined.
• The parameters used to characterize the response are lymph node cell count, ³H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight.
• Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test-substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
• Calculations: The stimulation indices of cell count, ³H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.

- Criteria used to consider a positive response:
• An increase SI of cell count by a factor of ≥ 1.5 and/or of ³H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.
• If biologically relevant increases in ear weights are running in parallel to the increase in cell count, ³H-thymidine incorporation and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Depending on the magnitude of lymph node response the evaluation of the sensitizing potential may be modified or additional studies might be necessary.
• If a test substance does not elicit a biological relevant increase in cell count, ³H-thymidine incorporation but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered.

Positive control substance(s):

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

Statistics:

not applicable

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Test Group 1 / Treatment: vehicle propylene glycol / Stimulation Index*: 1.00 Test Group 2 / Treatment: CuCl (1%) in propylene glycol / Stimulation Index: 4.40 Test Group 3 / Treatment: CuCl (3%) in propylene glycol / Stimulation Index: 10.70 Test Group 4 / Treatment: CuCl (10%) in propylene glycol / Stimulation Index: 24.36 Test Group 5 / Treatment: vehicle propylene glycol / Stimulation Index: 1.00 Test Group 6 / Treatment: CuCl (0.1%) in propylene glycol / Stimulation Index: 1.10 Test Group 7 / Treatment: CuCl (0.3%) in propylene glycol / Stimulation Index: 1.34 Test Group 8 / Treatment: CuCl (1%) in propylene glycol / Stimulation Index: 2.54 *test group 2 - 4 / test group 1 (vehicle control) or test group 6 - 8 / test group 5 (vehicle control) (Groups 1 - 4 were treated in the 1st run and groups 5 - 8 in the 2nd run).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Test Group 1 / Treatment: vehicle propylene glycol / ³H-thymidine incorporation: 638.6 DPM/Lymph Node Pair Test Group 2 / Treatment: CuCl (1%) in propylene glycol / ³H-thymidine incorporation: 2,806.7 DPM/Lymph Node Pair Test Group 3 / Treatment: CuCl (3%) in propylene glycol / ³H-thymidine incorporation: 6,835.5 DPM/Lymph Node Pair Test Group 4 / Treatment: CuCl (10%) in propylene glycol / ³H-thymidine incorporation: 15,552.3 DPM/Lymph Node Pair Test Group 5 / Treatment: vehicle propylene glycol / ³H-thymidine incorporation: 1,227.7 DPM/Lymph Node Pair Test Group 6 / Treatment: CuCl (0.1%) in propylene glycol / ³H-thymidine incorporation: 1,346.8 DPM/Lymph Node Pair Test Group 7 / Treatment: CuCl (0.3%) in propylene glycol / ³H-thymidine incorporation: 1,639.4 DPM/Lymph Node Pair Test Group 8 / Treatment: CuCl (1%) in propylene glycol / ³H-thymidine incorporation: 3,114.6 DPM/Lymph Node Pair (Groups 1 - 4 were treated in the 1st run and groups 5 - 8 in the 2nd run). Historical control data: Mean ³H-thymidine incorporation: 514.5 DPM/Lymph Node Pair Historical positive control study: 10 % Alpha-Hexylcinnamaldehyde, techn. 85 %: Group mean: ³H-thymidine incorporation: 3,720.2 DPM/Lymph Node Pair

Any other information on results incl. tables

Cell count, ³H-thymidine incorporation and lymph node weight:

- When applied as 1%, 3% and 10% preparations in propylene glycol, the test substance induced a biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.

- There was an increase in lymph node weights, as well.

- Concomitantly, the increase of ³H-thymidine incorporation into the cells was biologically relevant (increase above the cut off stimulation index of 3) at these concentrations, with the exception of the 1% test-substance preparation which caused an increase of ³H-thymidine incorporation at the border of this value after the 2nd run.

- When applied at the concentrations of 0.1 and 0.3 %, no relevant increases in lymph node cell counts and ³H-thymidine incorporation into the cells were observed.

- The 0.3%, 1% and 3% test-substance preparations caused some increase in ear weights as indication of ear skin irritation. On the day of lymph node removal the animals of the 3% concentration showed minimal scaling on the ears. The 10% test-substance preparation caused a strong increase in ear weights. On the day of lymph node removal incrustation, scaling and residues of test substance were observed on the ears of the 10% concentration group.

 

Because the lymph node response in the test groups treated with the 1% concentration cannot be fully attributed to the ear skin irritation observed, it is concluded that Copper (I) chloride shows a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

 

The threshold concentration for sensitization induction was > 0.3% <1%. The estimated concentration (EC) that leads to the SI of 1.5 for cell count (EC 1.5) and the estimated concentration that leads to the SI of 3.0 for ³H-thymidine incorporation (EC 3) was calculated by linear regression from the results of the 0.3% and 1% concentrations (mean of SI of test groups 2 and 8) to be 0.6% and 0.8%, respectively.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information