Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 June 2011 to 8 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 20 July 2010 Date of signature: 29 October 2010
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ammonium perrhenate

- Substance type: white solid

- Physical state: Solid

- Analytical purity: 100%

- Lot/batch No.: 17410

- Expiration date of the lot/batch: 28 July 2011

- Stability under test conditions: Not tested. Assumed to be stable.

- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxfordshire, United Kingdom

- Age at study initiation: eight to twelve weeks old

- Weight at study initiation: 15 to 23 g

- Housing: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes

- Diet : 2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxfordshire, United Kingdom. Ad libitum.

- Water : mains tap water, ad libitum.

- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C

- Humidity (%): 30 to 70%

- Air changes (per hr): approximately fifteen changes per hour

- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

IN-LIFE DATES: From: Day 1 To: Day 6

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Preliminary test: 25% w/w
Main test: 25%, 10%, 5% w/w
No. of animals per dose:
4 animals per dose group and vehicle control in the main test.
Details on study design:
RANGE FINDING TESTS:

- Compound solubility: Dimethyl formamide was chosen as vehicle as it produced the highest concentration that was suitable for dosing (25%).

- Irritation: Local irritation was scored daily according to the scale in OECD TG 429. Ear thickness was also measured.

- Lymph node proliferation response: No assessment of lymph node proliferation was made in the preliminary screening test.

- Others: Overt signs and body weights were recorded.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
The mice were treated by daily application of 25 µl of the test item (25%, 10% and 5% w/w) in dimethyl formamide, to the dorsal surface of each ear for three consecutive days. A further group received vehicle alone.
- Name of test method: Radioactive labelling to measure lymph node cell proliferation (expressed as Stimulation Index) determined five days following the first topical application.

- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".

- Others: Any signs of toxicity or signs of ill health as well as bodyweight were recorded.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was freshly prepared as a solution in dimethyl formamide. The test item was formulated within two hours of being applied to the test system.

Rainge finding study: One mouse was treated by daily application of 25 µl of the test item at a concentration of 25% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days.

Main study: The mice were treated by daily application of 25 µl of the test item (25%, 10% and 5% w/w) in dimethyl formamide, to the dorsal surface of each ear for three consecutive days.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.

Results and discussion

Positive control results:
The Stimulation Index expressed as the mean radioactive incorporation for the group treated with α-Hexylcinnamaldehyde, tech, 85% (as a solution in dimethyl formamide at a concentration of 15% v/v) divided by the mean radioactive incorporation of the vehicle control group was 4.77. Therefore α-Hexylcinnamaldehyde, tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation index: 1.32 at 5%, 1.11 at 10% and 0.70 at 25%. See the table in 'any other information on results incl. tables'.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Disintegrations per minute: 12653.32 for vehicle control, 16664.93 at 5%, 14093.60 at 10% and 8863.04 at 25%.

Any other information on results incl. tables

Table Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
dimethyl formamide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

12653.32

1581.67

na

na

5

16664.93

2083.12

1.32

Negative

10

14093.60

1761.70

1.11

Negative

25

8863.04

1107.88

0.70

Negative

dpm = Disintegrations per minute

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test item as a solution in dimethyl formamide at concentrations of 25%, 10% or 5w/w. A further group of four animals was treated with dimethyl formamide alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in
dimethyl formamide

Stimulation Index

Result

5

1.32

Negative

10

1.11

Negative

25

0.70

Negative

The test item was considered to be a non-sensitiser under the conditions of the test.

Ammonium perrhenate does not meet the criteria for classification of skin sensitiser according to EU classification system (Council Directive 67/548/EEC and Regulation (EC) No 1272/2008).