Cobalt(2+) hydrogen citrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Feb 2012 - 06 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study (The LLNA is not recommended for certain metals)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium fuer Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Tricobalt dicitrate

EC Number:

212-751-3

EC Name:

Tricobalt dicitrate

Cas Number:

866-81-9

IUPAC Name:

tricobalt dicitrate

Details on test material:

- Name of test material (as cited in study report): Cobalt Citrate
- Physical state: solid
- Analytical purity: 99%
- Purity test date:
- Batch No.: 10142709
- Storage: room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Additional information on strain: CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, The Nederlands
- Age at study initiation: 8-9 weeks (pre-test: 10-12 weeks)
- Mean weight at study initiation: 19.7 g (pre-test: 21.1 g)
- Housing: 3/cage in Makrolon Type II (pre-test)/Type III (main test) with wire mesh top (Ehret GmbH, Emmendingen, Germany)
- Diet: pelleted standard diet (Harlan Laboratories V.V., Horst, Netherlands); ad libitum
- Water: tap water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 32 - 65 (acclimation phase); 45-65 (pre-test and main study)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0.5, 1 and 2.5% (w/v)

No. of animals per dose:

4 (pre-test: 2)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given in the OECD guideline 429. The highest concentration which could be technically used was a 25% suspension in AOO. Grinding of the test substance was necessary.
- Irritation: To determine the highest non-irritant test concentration, that did not induce systemic signs of toxicity, a pre-test was performed in 2 animals. The animals were treated with 10 and 25% of the test substance once daily on 3 consecutive days. Besides recording of body weight, signs of toxicity and grading of erythema, ear thickness was measured before sacrifice on day 6. Excessive local erythema were seeen; therefore, a second pre-test was performed using test item concentrations of 2.5 and 5%. At both concentrations an increase in ear thickness was observed that exceeded the threshold value for excessive local skin irritation. Both animals showed erythema of the ear skin on day 4 and 5 which decreased to score 1 on day 6. At 5%, an increase in ear weight was observed that distinctly exceeded the threshold value for excessive local skin irritation. Thus, in the main study the test item was assayed at 0.5, 1, and 2.5%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: 1) Exposure to at least one concentration of the test item resulted in an icorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation index (SI). 2) The data are compatible with a conventional dose response, although allowance must be made for either local toxicty or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- Site: topical application at the dorsum of each ear once daily each on three consecutive days with 3 different concentrations of the test substance in AOO
- Application volume: 25 µL per application
- Control: same treatment with vehicle only
5 days after the first application, the mice were intravenously injected with radio-labelled thymidine (³H-methyl thymidine; ³HTdR) in PBS (250 µL). Approx. 5 hours after injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured in a beta-scintillation counter. ³HTdR incorporation was expressedas 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.

Results and discussion

Positive control results:

The EC3 value of the positive controls (test performed in December 2011) was 14.4%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: - 0.5% : 3.73 - 1%: 4.18 - 2.5%: 6.07 Since the SI values for all treatment groups were > 3, no EC3 value was determined. A clear dose-response was observed.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: - Vehicle control: 1886 - 0.5%: 6976 - 1%: 7819 - 2.5%: 11340

Any other information on results incl. tables

Table 1: LLNA – Details on results

Test item concentration (%)	Group	Measurement DPM	Calculation			Result
			DPM-BG a)	No. of lymph nodes	DPM per lymph node b)	S.I.
-	BG I	19	-	-	-	-
-	BG II	20	-	-	-	-
0	1	1886	1867	8	233.3	1.00
0.5	2	6976	6957	8	869.6	3.73
1	3	7819	7800	8	974.9	4.18
2.5	4	11340	11321	8	1415.1	6.07

BG = background (5% trichloroacetic acid)

a) calculated with the mean value of BG I and II

b) DPM/node was determined by dividing the measured values by the number of lymph nodes pooled.

The animals did not show any signs of systemic toxicity during the study. On day 2, the animals of all test groups showed erythema of the ear skin (score = 1) and from day 3 to day 6 the observed erythema was score = 2 in all test groups.

The body weights and body weight gains were within the normal range.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: Skin sens 1A, H317
DSD: Xi, R43