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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Jul 2010 - 04 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Országos Gyógyszerészeti Intézet, National Institute of Pharmacy, Budapest, Hungary
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): ammonium iron(3+) hexakis(cyano-C) ferrate(4-)
- Physical state: solid
- Appearance: blue odourless powder
- Analytical purity: 93.6%
- Lot/batch No.: K73-4766
- Expiration date of the lot/batch: 20 Mar 2013
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J Rj
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 21.1 - 24.5 g
- Housing: Mice were housed individually in type II polypropylene/polycarbonate cages with Lignocel® Hygienic Animal Bedding and glass tunnel-tubes.
- Diet: ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance", Soest, Germany, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 20 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 22 Sep 2010
To: 28 Sep 2010

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25 and 50% (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and radioactive proliferation assay was not performed. Two animals per dose group were used.
- Compound solubility: The maximum soluble concentration was > 50% (w/v) test substance in the vehicle. For the preliminary irritation test, concentrations of 25 and 50% (w/v) of the test substance in dimethylformamide (DMF) were used. In both concentrations, the test substance precipitated on the ears of the treated animals.
- Irritation: No skin irritation and no systemic toxicity were observed during the treatment period.
- Lymph node proliferation response: not performed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Tritiated thymidine (³HTdR) incorporation determined by β-scintillation.
- Criteria used to consider a positive response:
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
1. Exposure to at least one concentration of the test substance resulted in an incorporation of ³HTdR at least 3-fold or higher than recorded in control mice, as indicated by the stimulation index.
2. Dose-response relationship, structural relationship with known skin sensitizers as well as local skin irritation must be taken into account.

TREATMENT PREPARATION AND ADMINISTRATION:
25 μl of 10, 25 and 50% (w/v) of the test substance in DMF was applied to the entire dorsal surface of both ears of each mouse. Each animal was treated once a day for 3 consecutive days. There was no treatment on Days 4 and 5. On Day 6, an injection of 250 μl phosphate buffered saline (PBS) containing 20 μCi of ³HTdR was made into the tail vein of each animal. Approximately 5 h later, the draining auricular lymph node of each ear was excised into PBS. Once removed, the nodes of mice from each test group were pooled. A single cell suspension of these pooled lymph node cells was prepared by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. Single cell suspensions were washed two-fold with PBS. After washing, cells were precipitated with 5% trichloroacetic acid (TCA) at 2 - 8 °C for 18 h. Each precipitate was transferred to a suitable sized scintillation vial with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded to a β-scintillation counter and ³HTdR incorporation was measured for up to 10 min per sample.
Positive control substance(s):
other: 25% (w/v) α-hexylcinnamaldehyde in dimethylformamide (DMF)

Results and discussion

Positive control results:
The positive control substance induced a SI value of 3.8, thus meeting the reliability criteria for the local lymph node assay (SI > 3).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: negative control: SI 1 10% (w/v) test substance: SI 1.3 25% (w/v) test substance: SI 1.2 50% (w/v) test substance: SI 1.2 positive control: SI 3.8
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: negative control: 746.5 DPM 10% (w/v) test substance: 939.5 DPM 25% (w/v) test substance: 880.5 DPM 50% (w/v) test substance: 860.5 DPM positive control: 2807 DPM

Any other information on results incl. tables

Clinical signs:

No mortality and no signs of systemic toxicity were observed in any animal during the study period. The treatment with the test substance has no influence on the body weight of the animals.

Table 1: Results of the local lymph node assay in mice.

Test group

DPM/group

corrected DPM/group

DPN

No. of nodes

SI values

Background (5% (w/v) TCA)

35.5

---

---

 

---

Negative control

782

746.5

93.3

8

1.0

10% (w/v) test substance

975

939.5

117.4

8

1.3

25% (w/v) test substance

916

880.5

110.1

8

1.2

50% (w/v) test substance

896

860.5

107.6

8

1.2

positive control

2807*

2807*

350.9

8

3.8

* In the study report, there was no difference of the positive control DPM value before and after the correction regarding the background value. 

DPM: Disintegrations per minute

DPN: DPM divided by the number of lymph nodes

SI: Stimulation index

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified