2-[[3-(dimethylamino)propyl]methylamino]ethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2013 until 26 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 22 July 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No. 440/2008

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-[[3-(dimethylamino)propyl]methylamino]ethanol

EC Number:

279-903-9

EC Name:

2-[[3-(dimethylamino)propyl]methylamino]ethanol

Cas Number:

82136-26-3

Molecular formula:

C8H20N2O

IUPAC Name:

2-{[3-(dimethylamino)propyl](methyl)amino}ethan-1-ol

impurity 1

Reference substance name:

3-aminopropyldimethylamine

EC Number:

203-680-9

EC Name:

3-aminopropyldimethylamine

Cas Number:

109-55-7

Molecular formula:

C5H14N2

IUPAC Name:

N,N-dimethylpropane-1,3-diamine

impurity 2

Reference substance name:

unknown impurities

Molecular formula:

not applicable

IUPAC Name:

unknown impurities

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

liquid

Details on test material:

- Expiry date: 09 July 2018

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant.
After an acclimatization period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card.
At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were group housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
Free access to mains tap water and food (Teklad Global Rodent diet) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25 °C and 30 to 70%, respectively.
The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

- Preliminary screening test: test item at concentrations of 50%, 25% and 10% v/v in acetone/olive oil 4:1.
- Main test: test item at concentrations of 10%, 5% or 2.5% v/v in acetone/olive oil 4:1.

No. of animals per dose:

- Preliminary test: one mouse per test item concentration.
- Main test: four mice per test item concentration.

Details on study design:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Please see above in "Details on test animals and environmental conditions".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test item was freshly prepared as a solution in acetone/olive oil 4:1. Test concentrations were determined based on the results of the preliminary test (detailed in section "Any other information on results").
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.

H-Methyl Thymidine Administration:
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 pCi/mL, specific activity 2.0 Ci/mmoL) giving a total of 20 pCi to each mouse.

Observations:
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures:
Termination: Five hours following the administration of ³HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells, and centrifuged for 10 minutes (1400 rpm). The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm for ten minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. HTdR incorporation was measured by (3-scintillation counting. The samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not applicable

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

8.27

Test group / Remarks:

2.5 (% v/v) in acetone/olive oil 4:1

Remarks on result:

other: Positive

Results 2

Key result

Parameter:

SI

Value:

15.25

Test group / Remarks:

5 (% v/v) in acetone/olive oil 4:1

Remarks on result:

other: Positive

Results 3

Key result

Parameter:

SI

Value:

15.81

Test group / Remarks:

10 (% v/v) in acetone/olive oil 4:1

Remarks on result:

other: Positive

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION (main test)
Concentration (% v/v) in acetone/olive oil 4:1 dpm dpm/Node Stimulation Index Result

Vehicle 16713.79 2089.22 na na
2.5 138247.10 17280.89 8.27 Positive
5 254853.80 31856.73 15.25 Positive
10 264206.20 33025.78 15.81 Positive
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group

CLINICAL OBSERVATIONS and SIGNS OF TOXICITY (main test):
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Very slight redness on the ears was noted on Day 2 (post dose) in animals treated at a concentration of 10% v/v in acetone/olive oil 4:1.

BODY WEIGHTS (main test):
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Results of the preliminary screening test:

Mice treated at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 showed a greater than 25% increase in mean ear thickness on Day 6. Very slight erythema was noted on Days 4 to 6 and on Days 2 to 4 in these mice treated at 50% and 25% respectively.

No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted at a concentration of 10% v/v in acetone/olive oil 4:1. Very slight erythema was noted on Days 2 and 3.

Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% v/v in acetone/olive oil 4:1.

Applicant's summary and conclusion

Interpretation of results:

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

Remarks:

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 is regarded as a sensitizer 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-sensitizer". The results were interpreted according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Conclusions:

The test item was considered to be a sensitizer under the conditions of the test.

Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% v/v, 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 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 10%, 5% or 2.5% v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone.

The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group. SI values of all three dose groups (2.5, 5, 10 (% v/v) in acetone/olive oil 4:1) indicated a positive result (SI: 8.27, 15.25, 15.81 respectively).   

The test item was considered to be a sensitizer under the conditions of the test. Thus, the test item was classified as a skin sensitizer (Category 1B) according to the CLP Regulation (EC) No. 1272/2008 for the Classification, Labelling and Packaging of Substances and Mixtures.