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Administrative data

Description of key information

Skin sensitisation (OECD 429), mouse: Skin sensitising

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 2012-06-07 to 2012-09-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted under GLP in accordance with OECD TG 429.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Change of supplier and/or catalogue number for trichloroacetic acid and phosphate buffered saline.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories BV, Maasheseweg 87c, 5804 AB Venray / Netherlands
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 18.9-23.4 g
- Housing: group housing
- Diet (e.g. ad libitum): Teklad Global Rodent 2914C – Rezeptur 3255 GLP Batch 73/11 (Provimi Kliba AG, 4303 Kaiseraugst/Switzerland), available ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen/Switzerland, available ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 20 and 40 % (w/v) in vehicle
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration which can be technically used was a 75% (w/w) solution in acetone:olive oil (4:1 v/v). As the test item is a well pipettable liquid, it was also tested neat (100%).
- Irritation: At the tested 50 and 100 % concentrations, the animals did not show any signs of irritation.
- Lymph node proliferation response: is expressed as the number of radioactive disintegrations per minute per lymph node (dpm/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of the test groups relative to that recorded for the control group (Stimulation Index S.I.). Before dpm/lymph node values were determined, the mean scintillation-background dpm was subtracted from test and control raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Topical application / Administration of 3HTdR / Determination of incorporated 3HTdR
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the S.I.
- Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: Test item preparation was made freshly before each dosing occasion, no more than 4 hours prior to application to the ears.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For the body weight mean values and standard deviations were calculated.
Parameter:
SI
Value:
1.5
Test group / Remarks:
10% (w/v) test item
Parameter:
SI
Value:
1.9
Test group / Remarks:
20% (w/v) test item
Parameter:
SI
Value:
6.1
Test group / Remarks:
40% (w/v) test item

The following results were obtained for (ethoxymethoxy)cyclododecane using acetone:olive oil (4:1 v/v) as vehicle:

Test item concentration % (w/v)

Groups

Measurement dpm

Calculation

Result SI

dpm-BG

Number of lymph nodes isolated

dpm / lymph nodea)

--

BG I

2

--

--

--

--

--

BG II

4

--

--

--

--

--

Group 1

1295

1292

8

162

--

10

Group 2

1909

1906

8

238

1.5

20

Group 3

2510

2507

8

313

1.9

40

Group4

7923

7920

8

990

6.1

BG = Background (1 mL 5 % trichloroacetic acid) in duplicates

SI = Stimulation Index (Ratio = dpm/lymph node test item treated group / dpm/lymph node Group 1)

a) = Since the lymph nodes of the animals of a dose group were pooled, dmp/lymph node was determined by dividing the measured value by the number of lymph nodes pooled.

Calculation of the EC3 Value

 

Test item concentration

% (w/v)

SI

TG 2

20 (a)

1.9 (b)

TG 3

40 (c)

6.1 (d)

EC3 = (a-c) [(3-d)/(b-d)] + c = 25.1 %

EC3 = Estimated concentration for a SI of 3

a, b, c, d = Co-ordinates of the two pairs of data lying immediately above and below the SI value of 3 on the LLNA dose response plot

Interpretation of results:
other: EU CLP criteria
Remarks:
Skin Sensitising (Category 1B)
Conclusions:
The test item shows an allergenic potential when tested up to the concentration of 40%.
Executive summary:

In order to study the possible contact allergenic potential of the substance, three groups of four female mice each were treated daily with the test item at concentrations of 10%, 20% and 40% (w/v) in acetone:olive oil (4:1 v/v) by topical application to the dorsum of each ear lobe for three consecutive days. A control group of four female mice was treated with the vehicle acetone:olive oil (4:1 v/v) only.

Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter.

All treated animals survived the scheduled study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.

The results obtained [Stimulation Index (S.I.)] are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

 

Test item concentration % (w/v)

SI

Group 2

10

1.5

Group 3

20

1.9*

Group 4

40

6.1*

EC3: 25.1 %

A dose-response relationship was observed. A calculation of the EC3 value was performed using the indicated concentrations (*).

In conclusion, the test item was found to be a skin sensitizer and an EC3 value of 25.1% (w/v) was derived.

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

One fully reliable study (2012a) in vivo is available that shows skin sensitisation potential of the substance. The test item was found to be a skin sensitizer and an EC3 value of 25.1% (w/v) was derived.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Study not required according to Annex VII-X of Regulation (EC) No. 1907/2006.

Justification for classification or non-classification

-skin sensitisation:

Referring to Table 3.4.4 of the 2nd Amendment to Regulation (EC) No. 1272/2008 (CLP, as implementation of UN-GHS in the EU), a substance needs to be classified as Category 1B "Warning - H317: May cause an allergic skin reaction", if a local lymph node assay reveals an EC3 value > 2%, which applies to the test item.