Registration Dossier

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:
experimental study
Adequacy of study:
key study
Study period:
From August 14, 2012 to Agusust 29, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to the OECD Guideline 429, with GLP.
Reference:
Composition 0
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
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: eight to twelve weeks.
- Weight at study initiation: 15 to 23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Vehicle:
dimethylformamide
Concentration:
25%, 10% or 5% w/w in dimethyl formamide
No. of animals per dose:
4 animals
Details on study design:
PRELIMINARY SCREENING TEST:
- Irritation: a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL at a concentration of 25% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Appendix 5. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
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".

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.
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in dimethyl formamide. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 μCi to each mouse.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
α-Hexylcinnamaldehyde, tech., 85%, as a solution in dimethyl formamide at a concentration of 15% v/v, was considered to be a sensitiser under the conditions of the test (SI = 5.74).
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 5, 10 and 25 % were 6.07, 11.36 and 11.05 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM values for the experimental groups treated with test substance concentrations 5, 10 and 25 % were 93405.21, 174784.70 and 170038.60 DPM respectively. The DPM value for the vehicle control group was 15383.92 DPM.

Table 1. Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration

(% w/w) in

dimethyl

formamide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

15383.92

1922.99

na

na

5

93405.21

11675.65

6.07

positive

10

174784.70

21848.09

11.36

positive

25

170038.60

21254.83

11.05

positive

Clinical Observations and Mortality Data:

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight:

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test.
Executive summary:

The skin sensitisation potential of the test item in the CBA/Ca strain mouse, following topical application to the dorsal surface of the ear, was determined according to the OECD Guideline 429 and the EU B.42 method (GLP study).

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/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 50μL (25μL per ear). of the test item as a solution in dimethyl formamide at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with dimethyl formamide alone.

 

The Stimulation Index values calculated for the substance concentrations 5, 10 and 25 % were 6.07, 11.36 and 11.05 respectively. As the SI of 3.0 or greater indicates a positive result, the test item was considered to be a sensitiser under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Key study: Experimental result on test item:

The possible skin sensitization potential of the test substance was carried out according to the 429 OECD Guideline and the EU Method B.42 (GLP study), by a LLNA test.

The SI values calculated for the substance concentrations 5, 10 and 25 % were 6.07, 11.36 and 11.05 respectively. These results indicate that the test substance could elicit an SI ≥ 3 therefore the product is considered to be a skin sensitizer.


Migrated from Short description of key information:
Key study: OECD Guideline 429. EU Method B.42. GLP study. The test subtance was determined to be skin sensitizer.

Justification for selection of skin sensitisation endpoint:
Only one study available. Klimisch 1. Study was performed according to the OECD Guideline 429, with GLP.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance is classified as Skin Sensitiser Category 1B according to CLP Regulation (EC3 was extrapolated from test data, resulting as 3.95).