Copper dioleate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 November- 13 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

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

Test material information

Constituent 1

Reference substance name:

Copper dioleate

EC Number:

214-307-4

EC Name:

Copper dioleate

Cas Number:

1120-44-1

Molecular formula:

C18H34O2.1/2Cu

IUPAC Name:

copper(2+) bis((9Z)-octadec-9-enoate)

Test material form:

solid

Remarks:

paste

Details on test material:

Solid green paste. Batch number 21092015

Specific details on test material used for the study:

Test Item: Copper dioleate
Lot Number: 21092015
EINECS-No: 214-307-4
CAS No: 1120-44-1
Molecular Formula: C18H34O2.1/2Cu
Molecular Weight: 626.45 g/mol
Composition: 99-100%
Appearance: Green, solid/paste
Purity: >99%, IR
Homogeneity: Homogeneous
Production Date: Not stated
Expiry Date: 21.9.2018
Storage: Room temperature: (20 ± 5oC)
Test Item Receipt: According to SPPA-00147-BIO, Test and Reference Items

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5% etc. according to OECD Guideline 429. The vehicle (Acetone:Olive Oil 4:1 (v/v)) was selected from the recommended vehicles according to OECD 429. The test item was insoluble in the vehicle, therefore a homogeneous suspension was obtained.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Species Mice CBA/Ca
Source: AnLab Prague, Czech Republic
Number and Sex of Animals: 31 females
Age at First Dose: 8-12 weeks, female animals were non-pregnant and nulliparous were used
Animal Health: The health condition of animals was examined by a veterinarian before initiation of the study
Acclimation: The animals were acclimated in identical conditions as during the experiment for 6 days prior to the start of treatment. The acclimation was according to standard operation procedures.

Housing Condition: The animals were housed in polypropylene cages (5 animals /cage) suspended on stainless steel racks, in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 3°C, relative humidity was within the range of 50 - 60 %. The light regimen was set to a 12-hour light / 12-hour dark cycle. The sanitation was performed according to standard operation procedures.

Die:t A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time. The
certificate of analysis is included in the raw data.

Water: The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is
periodical monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.

Bedding: Lignocel S3/4, Lufa - ITL GmbH, Germany Animals Identification Each animal was marked with permanent pen on the tail. Each cage
was affixed with a cage card containing pertinent animal and study information.

Justification for the Choice of Species: The CBA/Ca mice are the standard experimental rodent of choice and recommended by OECD Guideline.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5, 10, 25%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
The pre-screen test was conducted under the same conditions as the main LLNA study, except there was no assessment of lymph node proliferation. The undiluted test item was tested. Test procedure of Pre-screen test. All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any sign of irritation. Erythema scores were given from 0 to 4. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.

MAIN STUDY
Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear 25μL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6: The body weight of each animal were recorded. 250μL of phosphate-buffered saline (PBS) containing 2 μCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein. Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach) and weighted.

CHALLENGE EXPOSURE
Based on the observations recorded in the preliminary tests, the concentration of 25%, 10% and 5% was selected for the main test.
Clinical Observations
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.
Body Weights
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the study.

Positive control substance(s):

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

Results and discussion

Positive control results:

Date: 09/2016
DPM - positive control: 4355
Stimulation Index: 5.60


Date: 07/2016
DPM - positive control: 7601
Stimulation Index: 7.49

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

It was determined with the test item at concentration of 5 % in acetone:olive oil 4:1.

Value:

2.87

Test group / Remarks:

5 females – control (vehicle) 5 females – positive control 15 females – test item 6 females - pre-screen test

Remarks on result:

other: animals did not show visible clinical symptoms or local irritation or systemic toxicity.

Results 2

Key result

Parameter:

SI

Remarks:

It was determined with the test item at concentration of 10 % in acetone:olive oil 4:1.

Value:

9.37

Test group / Remarks:

5 females – control (vehicle) 5 females – positive control 15 females – test item 6 females - pre-screen test

Remarks on result:

other: animals did not show visible clinical symptoms or local irritation or systemic toxicity.

Results 3

Key result

Parameter:

SI

Remarks:

It was determined with the test item at concentration of 25 % in acetone:olive oil 4:1.

Value:

12.94

Test group / Remarks:

5 females – control (vehicle) 5 females – positive control 15 females – test item 6 females - pre-screen test

Remarks on result:

other: animals did not show visible clinical symptoms or local irritation or systemic toxicity.

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
In comparison with the control group, an increase of the pooled lymph node weight was recorded at all used concentrations. The pooled lymph node weights of treated groups were 0.0692g for 5% concentration, 0.1067g for 10% concentration and 0.1442g for 25% concentration of tested item. The lymph node weight of control group and positive control group were 0.0440g and 0.0812g, respectively. The DPM values for the three treated groups were 2875 (5%), 9389 (10%) and 12968 (25%), respectively. The SI values for the three treated groups were 2.87 (5%), 9.37 (10%) and 12.94 (25%), respectively. The EC3 value is calculated to be 5.10%.

Lymph node weight (g)
Control 0.0440
Positive Control 0.0812
Copper dioleate 5% 0.0692
Copper dioleate 10% 0.1067
Copper dioleate 25% 0.1442

Number of lymph nodes
Control 10
Positive Control 10
Copper dioleate 5% 10
Copper dioleate 10% 10
Copper dioleate 25% 10

DPM
Control 1002
Positive Control 6189
Copper dioleate 5% 2875
Copper dioleate 10% 9389
Copper dioleate 25% 12968

SI
Control -
Positive Control 6.18
Copper dioleate 5% 2.87
Copper dioleate 10% 9.37
Copper dioleate 25% 12.94

EC3 5.10


DETAILS ON STIMULATION INDEX CALCULATION
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled radioactive incorporation for each treatment group by the radioactive incorporation of the pooled vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test
when SI≥3.

EC3 CALCULATION
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on doseresponse curve, immediately above and below of SI value, according to the equation: EC3=c+[(d- 3)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated.

CLINICAL OBSERVATIONS:
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

BODY WEIGHTS
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the study.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The skin sensitizing potential of Copper dioleate was assessed using the murine local lymph node assay. Based on the results of this study, Copper dioleate is considered a skin sensitizer under the condition of this study.

Executive summary:

The sensitization potential of Copper dioleate was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the contact sensitization potential of chemicals. Based on the recommendations of the OECD Guideline, the test item was dissolved in acetone:olive oil 4:1 (v/v). The positive control (a-Hexylcinnamaldehyde, 25%) was formulated in the same vehicle.

The Pre-screen test was performed using a doses of 100 %, 50% and 25% (w/v). Based on the observations recorded in the pre-screen tests, the concentration of 25 % was selected as top dose for the main test. Five female mice (CBA/Ca) were topically exposed (dorsum of both ears) to the test item at concentrations of 5%, 10% and 25%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5M

fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (5%, 10% and 25% w/v) the animals did not show visible clinical symptoms or local irritation or systemic toxicity. In this study Stimulation Indices (SI) of 2.87, 9.37 and 12.94 were determined with the test item at concentration of 5, 10, and 25% in acetone:olive oil 4:1, respectively. The calculated concentration

eliciting SI of 3 (EC3) was 5.10%. The test item Copper dioleate is considered a skin sensitizer under the test condition of this study.