2-(2-aminoethylamino)ethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 January 2006 - 25 January 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

24 April 2002.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

16 June 2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-aminoethylamino)ethanol

EC Number:

203-867-5

EC Name:

2-(2-aminoethylamino)ethanol

Cas Number:

111-41-1

Molecular formula:

C4H12N2O

IUPAC Name:

2-[(2-aminoethyl)amino]ethan-1-ol

Test material form:

not specified

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstr. 27, 33178 Borchen, Germany
- Age at study initiation: Ca. 7 weeks
- Weight at study initiation: 17.1 g - 20.7 g
- Type of cage: The mouse were single housed in Makrolon type I cages.
- Housing conditions: the animals were housed in fully air-conditioned rooms with a central air-conditioning system.
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8 days .

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30- 70
- Photoperiod : 12/12
- IN-LIFE DATES: From:10 January 2006 To: 16 January 2006

Study design: in vivo (LLNA)

Vehicle:

other: Acetone

Concentration:

3%, 10% and 30% w/w in actone

No. of animals per dose:

6

Details on study design:

PRE- SCREEN TEST:
No Pre-screen test was performed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Three groups of 6 animals were treated with one test item concentration per group. One group of 6
animals was treated with the vehicle.
Group 1 was treated with the vehicle to rule out any influence of the vehicle.
Group 2,3,4 were treated with 3%, 10% and 30% w/w concentation, respectively.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test substance preparations were produced on a weight by weight (w/w) basis shortly, before the application by stirring with a magnetic stirrer. The test substance was applied as a solution for all applications.
- Induction:Test item (25 μL) was applied in the dorsal part of both ears of each animal, for 3
consecutive days to the same application site.
- ³H-thymidine injection: On day 5 the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μl of sterile saline into a tail vein. The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.
- Ear thickness: Immediately after the death of each animal ear thickness was measured with a
suitable gauge (Oditest®-device (measurement steps 0.01 mm), Kroeplin, Germany).
- Detecting potential inflammatory ear swelling: circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal.
- Lymph node weight: Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each
animal.
- Preparation of cell suspension: the pooled lymph nodes of each animal were stored in phosphate
buffered saline in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 6 mL of phosphate-buffered physiological saline.
- Determination of Cell count: an aliquot of each suspension was further diluted with Casy®ton in a
ratio 1:500. The cell count was determined using a Casy®-Counter.
- Measurement of 3H-thymidine incorporation of the lymph node cells:The remaining cell suspensions were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic
acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ßscintillation counter.

ANALYSIS
- Mean values and standard deviations of the measured parameters were calculated for the test
and control groups from the individual values. The stimulation indices of cell count, 3H-thymidine incorporation, lymph node weight, ear weight and ear thickness measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control
group.

INTERPRETATION:
The increase (stimulation index; SI) of cell count by a factor of >1.5 and/or of 3H-thymidine
incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.
The estimated concentrations (EC) leading to the respective SI values were calculated by linear regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.
If a test substance does not elicit a statistical significant increase in cell count, 3H-thymidine incorporation and/or lymph node weight but shows a clear concentration related increase in response, further investigating of the sensitization potential at higher concentrations should be considered.

Positive control substance(s):

other:

Statistics:

WILCOXON - Test: Cell counts, 3H-thymidine incorporation, lymph node weight, ear weights as well asear thickness.

Results and discussion

Positive control results:

The Murine Local Lymph Node Assay is able to show the skin sensitizing effect of Alpha-Hexylcinnamaldehyde, techn. 85% under the test conditions chosen. The threshold concentration for sensitization induction was between 5% and 10% under the test conditions chosen. The EC 1.5 for cell count and the EC 3 for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 6.9% and 10.5%, respectively.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

3% in acetone

Results 2

Key result

Parameter:

SI

Value:

1.72

Test group / Remarks:

10% in acetone

Results 3

Key result

Parameter:

SI

Value:

6.6

Test group / Remarks:

30% in acetone

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation indices of cell count, 3H-thymidine incorporation, lymph node weight, ear weight and ear thickness measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
- Stimulation index Cell count: 1.22, 1.30 and 2.23 for 3%, 10% and 30%, repectively
- Stimulation index 3H-thymidine incorporation: 2.0, 1.72 and 6.60 for 3%, 10% and 30%, repectively
- Stimulation index lymph node weight: 1.18, 1.16 and 1.74 for 3%, 10% and 30%, repectively
- Stimulation index ear weigh: 1.03, 1.07 and 1.67 for 3%, 10% and 30%, repectively
- Stimulation index ear thickness: 1.00, 1.02 and 1.16 for 3%, 10% and 30%, repectively

EC3 CALCULATION :
The EC3 for 3H-thymidine incorporation was calculated to be 15.2.

CLINICAL OBSERVATIONS:
Incrustation of the ear skin was observed before the third application and on the day of lymph node removal in mice treated with 30% test substance preparation.
No signs of systemic toxicity were noticed.

BODY WEIGHTS
The expected body weight gain was generally observed in the course of the study.

Any other information on results incl. tables

- Animals were checked for dead or moribundity twice daily

- Body weights of the individual animals were determined on study day 0 prior to the first

application and on day 5 prior to the sacrifice of the animals.

- The stability of the test substance in acetone over a time period of 4 hours was confirmed by

analysis. Additionally the stability was determined indirectly by the concentration control

analysis.

- Homogeneity analyses: The test substance preparation was visually homogeneous (solution).

- The correctness of the concentration of the test substance preparations (30%, 10%, 3%) for the fist application was confirmed by analysis.

- The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay under the test conditions chosen is an appropriate model for testing for skin sensitization.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results of a Local Lymph node Assay performed according to OECD/EC guidelines and GLP principles, N-(2-Hydroxyethyl)-ethylendiamin was found to have skin sensitizing properties.

Executive summary:

An LLNA study was performed following OECD/EC guidelines and GLP principles. Three experimental groups of 6 females were treated with test item concentrations of 3%, 10% and 30%. One control group of six females was treated with the vehicle (Acetone).

The SI values calculated for the test item concentrations 3%, 10% and 30% were 2.0, 1.72 and 6.6, respectively. Incrustation of the ear skin was observed before the third application and on the day of lymph node removal in the mice treated with the 30% test substance preparation. No signs of systemic toxicity were noticed.

It is concluded that N-(2-Hydroxyethyl)-ethylendiamin 99% shows a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

The EC 3 for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 15.2%.

Based on these results, N-(2-Hydroxyethyl)-ethylendiamin 99% is classified category 1B for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).