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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:
12SEP2012 to 01OCT2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Physical appearance: yellow crystals
- Storage conditions: at room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 20- 25g
- Housing: Animals were group housed in labeled makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 12SEP2012 to 01OCT2012

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0, 10, 25, 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Two test substance concentrations were tested; a 25% and 50% concentration. The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels. In order to obtain homogeneity, the formulations were heated in a water bath with a maximum temperature of 69ºC for a maximum of 25 minutes. The formulations were allowed to cool down to a temperature of maximally 40ºC prior to dosing.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing).

Necropsy: No necropsy was performed according to protocol.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.

Results and discussion

Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.2
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
2.7
Test group / Remarks:
50%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 560, 285 and 697 DPM respectively. The mean DPM/animal value for the vehicle control group was 254 DPM.

Any other information on results incl. tables

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White test substance remnants were present on the dorsal surface of the ears of both animals at 50%, on Days 1-3, which did not hamper scoring of the skin reactions.

Other results - main study:

No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals at 50%, on Days 1-3, which did not hamper scoring of the skin reactions.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal at 10, 25 and 50% which were larger in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to Regulation (EC) 1272/2008
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 50%.
Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines and GLP principles. The test substances was tested up to 50%. No mortality occurred, no clinical signs of systemic toxicity were observed and body weights and body weight gain of experimental animals remained in the same range as controls over the study period. No skin irritation was seen in any of the animals, most auricular lymph nodes were considered normal in size and no macroscpic abnormalities were noted. The SI values calculated for the substance concentrations 10, 25 and 50% were 2.2, 1.1 and 2.7 respectively.

Based on these data, sodium feroocyanide is not regarded as a skin sensitizer.