Ethylenediamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14:07:1999 - 31:01:2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

This study has been conducted according to the principles of Good Research Practice. The data described in this summary report have not been audited by the CTL Quality Assurance Unit.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Ethylenediamine

EC Number:

203-468-6

EC Name:

Ethylenediamine

Cas Number:

107-15-3

Molecular formula:

C2H8N2

IUPAC Name:

ethane-1,2-diamine

Details on test material:

- Name of test material (as cited in study report): Ethylendiamine
- Analytical purity: 99 %

Specific details on test material used for the study:

Source : Union Carbide Corporation (UCC)
Appearance : Transparent colorless liquid
Storage : Ambient temperature
Solvent : AOO

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

Young adult (6-12 weeks old) female BALB/c strain mice (Harlan Seralab, Bicester, Oxfordshire, UK) were used throughout these studies. Mice were housed in groups of 1, 4 or 5 in metal cages and food (SDS PCD pelleted diet; Special Diets Services Ltd, UK) and water were available ad libitum. The ambient temperature was maintained at 21 +/- 2 oC and relative humidity was 55 +/- 10% with a 12 hour light/dark cycle.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

1, 2.5, 5, 10% (w/v)
Postive control 2,4-Dinitrochlorobenzene (DNCB) was dissolved in 4:1 acetone : olive oil (AOO) prepared freshly immediately prior to dosing.

No. of animals per dose:

5

Details on study design:

Groups of mice (n = 5) received 25 µl of test material in vehicle or an equal volume of vehicle alone on the dorsum of both ears daily for 3 consecutive days. Control animals were treated with the vehicle alone (n=10 /AOO) or with 1% DNCB (n=5) dissolved in AOO. Five days after the initiation of exposure, all mice received an intravenous injection of 250 µl of phosphate buffered saline (PBS) containing 20µCi of 3H-methyl thymidine (3H-TdR specific activity 2Ci/mmol; supplied by Amersham International, UK). Five hours later mice were sacrificed and the draining auricular lymph nodes removed and pooled for each experimental group.
A single cell suspension of lymph node cells (LNC) was prepared by mechanical disaggregation through a 200-mesh stainless steel gauze. Pooled LNC were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C. Approximately 12 hours later pellets were resuspended in 1ml of TCA and transferred to 10ml of scintillation fluid. Incorporation of 3H-TdR was measured by β-scintillation counting and expressed as mean disintegrations per minute
(dpm) per node for each test group. In each case, a stimulation index (SI) relative to the concurrent vehicle-treated control was derived. Chemicals which induce an SI of three or more are considered to be positive in this assay.
The estimated concentration of chemical required to induce an SI of 3, or EC3 value, was derived by linear interpolation as described previously ( Basketter et al, 1999). The EC3 value was calculated by interpolating between two points on the SI axis, one immediately above, and the other immediately below, the SI value of 3. The vehicle-treated control value (SI = 1) cannot be used for the latter. Where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d), respectively, then the EC3 value may be calculated using the following equation: EC3 = c + [(3-d)/(b-d)] (a-c).

Positive control substance(s):

other: 2,4-Dinitrochlorobenzene (DNCB; 98%), Sigma Chemical Co (St Louis, MO, USA)

Results and discussion

Positive control results:

The historical values for proliferation induced by DNCB (1%) are stimulation indices of 24.2 +/- 3.5 (mean and SEs of 3 experiments).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.2

Test group / Remarks:

1%

Results 2

Parameter:

SI

Value:

2.6

Test group / Remarks:

2.5%

Results 3

Parameter:

SI

Value:

4.9

Test group / Remarks:

5%

Results 4

Parameter:

SI

Value:

8.5

Test group / Remarks:

10%

Cellular proliferation data / Observations:

See tables attached

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

EDA produced a positive response (EC > 3) at concentrations of 5 and 10% in AOO as measured by 3H-thymidine incorporation in lymph nodes five days after initiation of treatment. The EC3 value was calculated to be 2.9%.

Executive summary:

EDA was studied in the Local Lymph Node Assay (LLNA). Groups of young adult Balb/c mice (n=5) were administered 25 μl EDA in AOO at concentrations of 1, 2.5, 5, and 10% (w/v) on the dorsum of both ears daily for three consecutive days. Control animals were treated with the vehicle alone (n=10, water/AOO) or with 1% DNCB (n=5) dissolved in AOO. EDA ( 5 and 10%) produced a positive response as measured by 3H-thymidine incorporation in lymph nodes five days after initiation of treatment.