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Administrative data

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed and reported GLP study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J strain, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
Number of animals: 20 females (nulliparous and non-pregnant), five females per group.
Age and body weight: Young adult animals (approx. 11 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Identification: Tail mark with marker pen.

Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were recorded in the raw data.

Accommodation
Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures.

Vehicle:
other: 1% Pluronic © L92 in Elix water
Concentration:
10, 25 and 50%
No. of animals per dose:
5
Details on study design:
On days 1, 2, and 3, the dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.

On day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No
Positive control results:
The SI values calculated for the positive test substance HCA concentrations 5, 10 and 25% were 1.7, 1.7 and 4.7 respectively. An EC3 value of 16.5% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.9, 16.0, 11.9, 16.9, 14.4 and 12.8%. Based on these results it was concluded that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Remarks on result:
other: See Table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table below

Table: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

group

TS1

(%)

animal

Size nodes2

DPM3/ animal

mean

DPM ±4

mean

SI ± SEM

left

right

 

 

 

 

 

 

 

 

1

0

1

n

n

456

513

±

95

1.0

±

0.3

 

 

2

n

n

334

 

 

3

n

n

880

 

 

4

n

n

458

 

 

5

n

n

435

 

 

 

 

 

 

 

 

 

 

 

 

2

10

6

n

n

306

466

±

79

0.9

±

0.2

 

 

7

n

n

747

 

 

8

n

n

356

 

 

9

n

n

518

 

 

10

n

n

403

 

 

 

 

 

 

 

 

 

 

 

 

3

25

11

n

n

430

571

±

87

1.1

±

0.3

 

 

12

n

n

748

 

 

13

n

n

752

 

 

14

n

n

317

 

 

15

n

n

607

 

 

 

 

 

 

 

 

 

 

 

 

4

50

16

n

n

234

612

±

161

1.2

±

0.4

 

 

17

n

n

558

 

 

18

n

n

438

 

 

19

n

n

632

 

 

20

n

n

1196

 

 

 

 

 

 

 

 

1. TS = test substance (% w/w).

2. Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

3. PM= Disintegrations per minute

4. SEM = Standard Error of the Mean

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
EDTA-CuNa2 did not show sensitizing properties in the mouse LLNA
Executive summary:

The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2010); EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"; EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test substance concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (1% watery pluronic L92). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating theof the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation of the ears was observed in any of the animals examined. White/grey test substance remnant were present on the dorsal surface of the ears of all animals at 10, 25 and 50% on Days 1-3, and at 50% also on Day 4 for one animal, which did not hamper scoring of skin reactions.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 466, 571 and 612 DPM respectively. The mean DPM/animal value for the vehicle control group was 513 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 0.9, 1.1 and 1.2 respectively. Since there was no indication that the test substance elicited an SI3 when tested up to 50%, EDTA-CuNa2was considered to be a non skin sensitizer.

The six-month reliability check withAlpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. 

Based on these results, EDTA-CuNa2 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and theRegulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

 

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

Migrated from Short description of key information:

EDTA-CuNa2 did not show skin sensitizing properties in the murine LLNA; the same is expected for EDTA-CuK2.

Justification for selection of skin sensitisation endpoint:

Well performed and reported mouse LLNA study available with EDTA-CuNa2 (see also read across doucment in section 13).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Based on the knowledge that so far all known respiratory sensitizers have also tested positive in a skin sensitization test, it is not expected that, based on the outcome with EDTA-CuNa2, EDTA-CuK2 would show respiratory sensitizing properties.

Justification for classification or non-classification

Based on the results of the LLNA, EDTA-CuNa2 does not need classification for skin sensitization; the same is expected for EDTA-CuK2.