Copper glycinate sulfate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Detection of contact sensitivity of metal salts using the murine local lymph node assay

Author:

Yoshiaki Ikarashi, Toshie Tsuchiya and Akitada Nakamura

Year:

1992

Bibliographic source:

Toxicology Letters, 62 (1992) 53-61

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Copper (II) sufate pentahydrate

Cas Number:

7758-99-8

Molecular formula:

CuSO4 · 5H2O

IUPAC Name:

Copper (II) sufate pentahydrate

Specific details on test material used for the study:

Source: Wako Pure Chemical Industries, Ltd., Osaka, Japan.

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6-8 weeks

Study design: in vivo (LLNA)

Vehicle:

other: ethanol (20 %)

Concentration:

10%

No. of animals per dose:

3

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The mice (n = 3) received 25 µL of test chemical solution, or vehicle alone, on the dorsum of each ear. The application was repeated for three consecutive days. In some experiments, the dorsal surface of each ear was gently abraded by lightly dragging a 19-g needle across the dorsal surface of each ear five times (without causing bleeding) just prior to the application of test chemicals. Four days following the initial application, mice were killed and the draining lymph nodes (auricular and axillary) were excised and pooled per animal. A single cell suspension of LNC was prepared by mechanical disaggregation through sterile 200-mesh steel gauge. Lymphocyte suspensions were washed twice in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 culture medium supplemented with 10% fetal calf serum (FCS), 25 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), 100 µg/ml penicillin and 100 U/ml streptomycin. The cell concentration was adjusted to give 5 E+06 cells/mL.. Lymphocyte suspensions were seeded into 96-well microtiter plates at a concentration of 1 E+06 cells/well (5 wells per animal), and cultured with 0.5 µCi [3H]methyl thymidine ([3H]TdR) for 18 h at 37°C in a humidified atmosphere of 5% CO2 in air.
Culture was terminated by automatic cell harvesting. The incorporation of [3H]TdR was measured using a liquid scintillation counter and expressed as mean counts per min (cpm) ± standard deviation per node of three animals for each test group. Increases in [3H]TdR incorporation relative to vehicle-treated controls were calculated for each test group, and expressed as stimulation indices (SI).

Positive control substance(s):

other:

Results and discussion

Positive control results:

The SI values observed with CoCl2 and K2Cr2O7 were 4.3 and 3.1, respectively.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.67

Test group / Remarks:

10 % copper sulfate pentahydrate in 20 % ethanol solution

Results 2

Parameter:

SI

Value:

4.33

Test group / Remarks:

positive control CoCl2

Results 3

Parameter:

SI

Value:

0

Test group / Remarks:

negative control, 20 % ethanol solution

Results 4

Parameter:

SI

Value:

3.16

Test group / Remarks:

positive control K2Cr2O7

Cellular proliferation data / Observations:

In the present study only the results from one treatment group were reported.

Any other information on results incl. tables

THE LOCAL LYMPH NODE ASSAY PERFORMED WITH FIVE METAL SALTS

Chemical	[3H]TdR incorporation (mean cpm±SD (x 10^-3)	SI
20% EtOH	1.52± 0.67	-
10% FeSO4	2.03±0.63	1.32
10% MnCl2	1.26±0.08	0.82
10% ZnSO4	2.14±0.77	1.41
10% CuSO4	4.08±1.20	2.67
10% NiSO4	3.56±0.25	2.32

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In the present study conducted by Ikarashi et al. (1992), copper sulfate pentahydrate is not considered to be a sensitizer in the LLNA.

Executive summary:

In the present study conducted by Ikarashi et al. (1992), copper sulfate pentahydrate was dissolved in 20% ethanol and applied to the dorsal surface of the ear on three consecutive days. After four days after initial application the mice were killed and the draining lymph nodes were dissected and incubated for 18 hours at 37°C in humidified air with [³H]TdR. After the incubation period the [³H]TdR incorporation was detected and the SI was determined.

Incubation with 10% CuSO₄ in 20% ethanol resulted in a SI value of 2.67. Thus, under the conditions of the present test, the substance was not considered a sensitizer.