3-[(2-hydroxyethyl)amino]propanol

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:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH (Harlan CCR), Rossdorf, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-[(2-hydroxyethyl)amino]propanol

EC Number:

242-978-3

EC Name:

3-[(2-hydroxyethyl)amino]propanol

Cas Number:

19344-29-7

Molecular formula:

C5H13NO2

IUPAC Name:

3-[(2-hydroxyethyl)amino]propan-1-ol

Details on test material:

- Name of test material (as cited in study report): 3-(2-Hydroxyethylamino)-1-propanol
- Physical state: Liquid, colorless, clear
- Analytical purity: 99.2 g/100 g via H-NMR spectroscopy and 98.9 g/100 g via potentiometric titration
- Lot/batch No.: 74568175L0
- Expiry Date: 29 January 2016
- Storage Conditions: At room temperature
- Homogeneity: The test item appeared to be homogeneous

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Horst / The Netherlands
- Age at study initiation: 1st pre-test: 8 - 9 weeks; 2nd pre-test: 10 -11 weeks; Main study: 8 - 9 weeks
- Weight at study initiation: Main study 21.2 ± 0.8
- Housing: Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): approx. 45 - 65%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Main test: 1, 2 and 5 %

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50% solution in DMF. Vortexing and warming to 37°C was used to formulate the test item.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
From day 3 to day 5 the animal treated with 50% test item concentration showed an erythema of the ear skin (score 1). Additionally on day 6 eschar formation (score 4) was observed. The animal treated with 25% test item concentration showed slight eschar formation (score 4) from day 3 to day 6 as well as visible ear swelling from day 4 to day 6. Moreover, increase in ear weights (compared to historical vehicle values) for the animal treated with 25% test item concentration was 79.96% on day 6.
Therefore, a second pre-test was performed using test item concentrations of 5 and 10%. From day 2 to day 6 erythema of the ear skin was observed for the animal treated with 10% test item concentration (score 1 on day 2, score 2 from day 3 until day 5, score 4 on day 6). Additional substance residual (day 5) and scabby ear skin (day 6) was observed. No signs of relevant irritation were observed in the animal treated with 5% test item concentration, except for a slight erythema (score 1) from day 3 until day 6.

MAIN STUDY
- Topical application: Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 1, 2, and 5% in DMF. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
- Administration of 3H-methyl-thymidine: Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.5 μCi of 3H-methyl thymidine (equivalent to 81.8 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Determination of incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
- Determination of Lymph Node Weight and Cell Count: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY® DT, Schärfe System). The values obtained were taken down manually.
- Determination of Ear Weights: After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
- Interpretation of raw data: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: 1) Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index. 2) The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Furthermore, an index was calculated for the lymph node weight and –cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 (Ehling et al., 2005) was reported for a positive response and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1 (Ulrich et al., 2001). However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.

Observations:
- Mortality / Viability: At least once daily from experimental start to necropsy.
- Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
- Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
- Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.
- Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
- Lymph node cell count: The lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter.
- Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and DMF was added. The different test item concentrations were prepared individually. The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations validated statistical program R Script DecisionTree_2.Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw). An outlier was identified in the Grubb’s, but not in the Dean-Dixon-Test and was therefore not excluced from calculations. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2015 and a EC3 of 10.8% was determined.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: In this study Stimulation Indices (S.I.) of 1.11, 1.59 and 1.98 were determined with the test item at concentrations of 1, 2, and 5% in DMF, respectively. The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Mean DPM per animal (2 lymph nodes)* was 982.7± 312.6, 1091.9 ± 245.1, 1559.5 ± 698.7, and 1944.5 ± 1047.2 for the vehicle control group, 1%, 2%, and 5% test group, respectively. * Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

Any other information on results incl. tables

- Viability / Mortality: No deaths occurred during the study period.

- Clinical Signs: Neither signs of systemic toxicity nor local skin effects were observed during the study period.

- Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

- Lymph Node Weights and Cell Counts: The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or –cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response (Ehling et al., 2005). The indices determined for the lymph node cell count did not reach or exceed this threshold.

- Ear Weights: The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information