2-piperidinoethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov 2018 - 28 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland Pfalz

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-piperidinoethanol

EC Number:

221-244-6

EC Name:

2-piperidinoethanol

Cas Number:

3040-44-6

Molecular formula:

C7H15NO

IUPAC Name:

2-piperidinoethanol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: B1056-070518
- Expiration date of the lot/batch: 07 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: Good solibility. Because the test-substance preparations were applied shortly after preparation, no analysis of the test substance in the vehicle was required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparation was produced on a weight per weight (w/w) basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer, the test substance was dissolved in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH, C/O Postfach 553, NL-5800 AN Venray
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 17.5 g – 20.0 g (pretest); 17.9 g – 23.7 g (main test)
- Housing: single; As group housing may increase oral exposure due to grooming of the animals and may interfere with clear observations of each individual animal, animals were single housed for the duration of the test.
- Diet: ad libitum; Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland
- Water: ad libitum; drinking water
- Acclimation period: at least 5 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 13 Nov 2018 To: 26 Nov 2018

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

Propylene glycol was used as vehicle because good solubility of the preparation was achieved.

Concentration:

1%, 5%, 10%

The highest test substance concentration that does not induce local signs of skin irritation and/or systemic toxicity was determined in a pretest (experimental conduct in accordance with GLP, but without a GLP status).

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The test-substance preparations at different concentrations were solutions in propylene glycol (PG).
- Irritation/ear thickness measurements/erythema scores: In order to determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. Two mice were treated with test-substance concentrations of 1%, 10% and 25% each on three consecutive days. At the tested concentrations, the animals did not show relevant increases in ear weights (compared to historical vehicle values) and ear thickness measurements as indication of ear skin irritation. However, one animal of the 25% concentration showed moderate crust formation, slight scaling and lesions on the ear skin on study day 5. At the tested concentrations of 10% and 25%, the animals showed moderately increased lymph node weights. Therefore, the dose levels 1%, 5% and 10% (w/w) were selected for the main study.
- Systemic toxicity: No signs of systemic toxicity were observed in the pretest.

MAIN STUDY
- Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.
- Form of application: epicutaneous
- Application volume: 25 µL per ear
- Side of application: dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Body weight determination: individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 µCi ³H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice.
- Sacrifice: on study day 5 about 5 hours after ³H-thymidine injection by cervical dislocation under Isoflurane anesthesia
- Terminal procedures:
* Determination of ear weights: Immediately after the death of each animal, 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.
* Removal and weight determination of the lymph nodes: 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 and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline (PBS) 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 phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined by using a Casy® Counter.
* Measurement of ³H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with 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 β-scintillation counter.

EVALUATION OF RESULTS
- A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of ³H-thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index (SI ≥ 3.0). The biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. lymph node cell counts, lymph node weights, ear weights). The thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.

Positive control substance(s):

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

Statistics:

For the statistical analysis of the the parameters ³H-thymidine incorporation, cell count, lymph node weight and ear weight the Wilcoxon-Test was used.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

for ³H-thymidine incorporation

Value:

1.15

Test group / Remarks:

1% test substance concentration

Results 2

Parameter:

SI

Remarks:

for ³H-thymidine incorporation

Value:

1.75

Test group / Remarks:

5% test substance concentration

Results 3

Parameter:

SI

Remarks:

for ³H-thymidine incorporation

Value:

3.21

Test group / Remarks:

10% test substance concentration

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
When applied as 10% solution in propylene glycol, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of ³H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 5% concentration was statistically significant. Concomitantly, the 10% test-substance solution induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell count. In addition, a statistically significant increase in lymph node weight was noted at the 10% concentration.
The test-substance concentrations did not cause relevant increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, statistically significant but
concentration-independent increases in ear weights were observed at the 1% and 5% concentrations.

DETAILS ON STIMULATION INDEX CALCULATION
Mean values and standard deviations of the measured parameters were calculated per test group from the individual values. The stimulation indices of ³H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.

EC3 CALCULATION
The EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI or by using the two nearest points below or above the SI.
The threshold concentration for sensitization induction was >5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for ³H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 9.3% and 7.7%, respectively.

CLINICAL OBSERVATIONS
No signs of systemic toxicity were noticed in all animals during general observation.
No local findings were observed during the observation period.

BODY WEIGHTS
No influence on the body weights was observed during the study.

Any other information on results incl. tables

Table 1: ³H-thymidine incorporation, cell count and lymph node weight: test group mean value, standard deviation and stimulation index

Test Group	Treatment	³H-thymidine incorporation [DPM/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle propylene glycol	716.5	212.7	1.00
2	1% in propylene glycol	825.7	120.9	1.15
3	5% in propylene glycol	1,256.7	733.4	1.75 #
4	10% in propylene glycol	2,302.4	968.4	3.21 ##

 

Test Group	Treatment	Cell Counts [Counts/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle propylene glycol	9,634,880	2,318,222	1.00
2	1% in propylene glycol	11,129,600	785,069	1.16
3	5% in propylene glycol	12,468,800	3,283,562	1.29
4	10% in propylene glycol	16,208,000	5,986,179	1.68 #

 

Test Group	Treatment	Lymph Node Weight [mg/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle propylene glycol	5.4	0.9	1.00
2	1% in propylene glycol	6.1	0.8	1.13
3	5% in propylene glycol	5.9	1.0	1.10
4	10% in propylene glycol	8.0	1.8	1.49 #

 

Table 2: Ear weight: test group mean value, standard deviation and stimulation index

 

Test Group	Treatment	Ear Weight [mg/animal]
		Mean	S.D.	Stimulation Index^1
1	vehicle propylene glycol	31.6	2.0	1.00
2	1% in propylene glycol	33.6	1.4	1.06 #
3	5% in propylene glycol	35.0	2.8	1.11 #
4	10% in propylene glycol	33.9	0.9	1.07

1 test group x / test group 1 (vehicle control)

# = statistically significant for the value p ≤ 0.05

## = statistically significant for the value p ≤ 0.01

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

It is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test substance was assessed using the radioactive Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 1%, 5% and 10% (w/w) preparations of the test substance in propylene glycol (PG) or with the vehicle alone. The highest concentration was selected based on the presence of lesions on the ear skin in a pretest using a 25% test substance solution. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation

or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, 20 μCi ³H-thymidine in 250 μL sterile saline were injected

into the tail vein of the mice. About 5 hours after the ³H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated

measuring ³H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter

sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation. When applied as 10% solution in propylene glycol, the test substance induced a biologically

relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of ³H-thymidine incorporation into the cells

from the auricular lymph nodes. The increase of the 5% concentration was statistically significant. Concomitantly, the 10% test-substance solution induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell count.

In addition, a statistically significant increase in lymph node weight was noted at the 10% concentration. The test-substance concentrations did not cause relevant increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, statistically significant but concentration-independent increases in ear weights were observed at the 1% and 5% concentrations.

Thus, it is concluded that the test substance exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was >5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for ³H-thymidine incorporation and the EC 1.5

(estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 9.3% and 7.7%, respectively.