Ethanol, 2-amino-, reaction products with ammonia, by-products from

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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]ethanol

Constituent 2

Reference substance name:

Cyclohexyldimethylamine

EC Number:

202-715-5

EC Name:

Cyclohexyldimethylamine

Cas Number:

98-94-2

Molecular formula:

C8H17N

IUPAC Name:

N,N-dimethylcyclohexanamine

Details on test material:

- Name of test material (as cited in study report): Cyclohexanamine, N ,N -dimethyl-

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Animals:
- Origin: Harlan Winkelmann GmbH, Gartenstr. 27, 33178 Borchen, Germany
- Sex: Female
- Identification of the animals: The single housed animals were identified by cage cards
- Age at the beginning of the study: 6 to 10 weeks
- Reasons for the selection: The test method was developed in mice.

Housing conditions:
- Air conditions: The animals were housed in fully air-conditioned rooms in which a central air-conditioning system ensured a temperature in the range of 20 - 24 °C and a relative humidity in the range of 30 - 70 %.

- Illumination period: 12 h light (6.00 a.m. - 6.00 p.m.) , 12 h darkness (6.00 p.m. - 6.00 a.m.)
- Type of cage: Makrolon type I
- No. of animals per cage: 1
- Watering: Tap water ad libitum

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

1) 3 %
2) 10 %
3) 30 %
4) vehicle acetone

No. of animals per dose:

6

Details on study design:

The study comprised three treatment groups and a vehicle control group. Each group consisted of 6 mice. A check for dead or moribund animals was made twice each workday and once on Saturdays, Sundays and on public holidays.
No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.

- 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
- Test substance application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously (i.v.) 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.

- Immediately after the death of each animal ear thickness was measured with a suitable gauge (Oditest®-device (measurement steps 0.01 mm), Kroeplin, Germany). Thereafter a 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. These measurements serve for detecting a potential inflammatory ear swelling.

- 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.

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 E1.5 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

Parameter:

SI

Value:

14.7

Test group / Remarks:

20% test substance

Results 2

Parameter:

SI

Value:

6.3

Test group / Remarks:

10% test substance

Results 3

Parameter:

SI

Value:

2.8

Test group / Remarks:

5% test substance

Results 4

Parameter:

SI

Value:

2.2

Test group / Remarks:

2,5% test substance

Cellular proliferation data / Observations:

- Cell counts, 3H-thymidine incorporation and lymph node weights

When applied as 30 % preparation in acetone, the test substance induced a statistically significant and biologically relevant (increase to 1.5 fold or above of control value = stimulation index (SI) 1.5) increase in cellularity of the auricular lymph nodes. Concomitantly, the increase of 3H-thymidine incorporation into the cells was statistically significant and biologically relevant (increase above the cut off stimulation index of 3). The 10 % and 3 % test substance preparation caused small but statistically significant increases in cellularity of the auricular lymph nodes and 3H-thymidine incorporation into the lymph node cells, which failed to reach the cut off stimulation indices and thus lie below the threshold of immunologic relevance. The lymph node weights were statistically significantly increased in all substance treated groups.

- Ear weights and ear thickness

Statistically significant increases in ear weights and ear thickness were observed in mice treated with the 30 % test substance preparation, which was accompanied by incrustation of the ear skin observed before the third application and on the day of lymph node removal. The 10% test substance preparation caused a statistically significant increase in ear weights, but not in ear thickness. The magnitude of ear skin irritation observed in the test group treated with a 30 % preparation might be considered relevant for evaluating the sensitizing potential of the test substance. As the stimulation indices at this concentration, especially that for cellularity, are well beyond the cut off criteria, an immunologic background of the lymph node response cannot be excluded.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In a dermal sensitisation study with AEEA, with groups of 6 female CBA/Ca mice each were treated with 3 %, 10 % and 30 % w/w preparations of the test substance in acetone/olive oil or with the vehicle alone using the Local Lymph Node Assay.AEEA shows a skin sensitizing effect under the test conditions chosen. The threshold concentration for sensitization induction was between 10 % and 30 %. The EC 3 for ³H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 15.2 %.

Executive summary:

Aminoethylethanolamine (AEEA) exhibited clear signs of irritation of the ear skin at a concentration of 30%. It had been reported to be skin-sensitizing in a guinea pig maximization test after intradermal induction with 5%, epidermal induction with 50% in distilled water, and challenge with 25% in distilled water, showing a response in 40% of the animals (Leung and Auletta, 1997). According to the categorization proposed in the ECETOC Technical Report No. 87, this makes it a weak sensitizer. On the other hand, an unpublished report (CTL, 2001) about an LLNA with 2.5%, 5%, 10% and 20% AEEA in acetone/olive oil (4:1) presents stimulation indices (SIs) of 2.2, 2.8, 6.3 and 14.7, resulting in an EC3 value of 5.3%, i.e. ‘‘moderate”. This is a clearly lower EC3 than that observed in the present study (13–15% depending on endpoint evaluated and method of calculation), but the AEEA study described in the unpublished report, which investigated a number of ethyleneamines, showed the lowest vehicle control value of [3H]-thymidine incorporation (range 197–386 dpm/lymph node). If the SIs had been calculated using the upper end of the vehicle control values, an EC3 of about 10% would have resulted. In conclusion, AEEA is a weak skin sensitizer and skin irritant.