Pyridine-2-carbaldehyde oxime

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-28 - 2016-08-13 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the testing of chemicals 429. Skin Sensitisation: Local Lymph Node Assay (Adopted: 22 July 2010).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

by Slovak National Accreditation Service

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Pyridine-2-carbaldehyde oxime

EC Number:

212-849-6

EC Name:

Pyridine-2-carbaldehyde oxime

Cas Number:

873-69-8

Molecular formula:

C6H6N2O

IUPAC Name:

pyridine-2-carbaldehyde oxime

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: AnLab Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9-10 weeks at first dose
- Weight at study initiation: 18.87 - 22.44 g
- Housing: The animals were housed individually in polypropylene cages suspended on stainless steel racks, in a room equipped with central air-conditioning.
- Diet (e.g. ad libitum): A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time.
- Water (e.g. ad libitum): The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodical monitored (including microbiological control) and recorded.
- Acclimation period: The animals were acclimated in identical conditions as during the experiment for 6 days prior to the start of treatment. Bedding was Lignocel S3/4, Lufa - ITL GmbH, Germany. The acclimation was according to standard operation procedures. The sanitation
was performed according to standard operation procedures.
- Indication of any skin lesions: none stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 -60 %
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle
- IN-LIFE DATES: From: 21.7.2016 To: 13.8.2016

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

for test item; vehicle fro positive control was Acetone/Olive Oil, 4:1, v/v

Concentration:

100% (pre-screen test)
100%, 50%, 25% (main test)

No. of animals per dose:

5 females – control (vehicle)
5 females – positive control
15 females – test item (5 per dose)
4 females - pre-screen test

Details on study design:

PRE-SCREEN TESTS: The pre-screen test was conducted under the same conditions as the main LLNA study, except there was no assessment of lymph node proliferation. The test item was tested at a concentration of 100%. All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6).
- Compound solubility: The vehicle (DMSO) was selected from the recommended vehicles according to OECD 429. The test item was insoluble in the vehicle, therefore a homogeneous suspension was obtained.
- Irritation: Both ears of each mouse were assessed for any sign of irritation.
- Ear thickness measurements: Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6.
- Erythema scores: Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.
Erythema Scores
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4


MAIN STUDY
Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear 25μL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5:
No treatment.
Day 6:
The body weight of each animal were recorded. 250μL of phosphate-buffered saline (PBS) containing 2 μCi (7.4 x 10E4 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.
Five hours later, the animals were humanely killed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach) and weighted.

Preparation of cell suspensions
Cell suspension of lymph node cells from pooled treatment groups were prepared by gentle mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged (SOPA-00156- BIO) by 600g at 4°C for 10 min. Suspension of cells were precipitated with 5% trichloroacetic acid (TCA) at 4°C for 18-20h. Pellets were centrifuged by 2000g at 4°C for 5 min., re-suspended in 1 ml TCA and transferred to gamma counting tubes for 125I-counting.

Determination of cellular proliferation (incorporated radioactivity)
Incorporation of 125I-iododeoxyuridine was measured by 125I-counting on Automatic Gamma Counter Wizard2 (Perkin Elmer) as counts per minute (CPM). The incorporation is expressed as disintegrations per minute (DPM)/treatment group. DPM were calculated according to formula:
DPM= CPM / absolute detector efficiency (Absolute detector efficiency for Gamma Counter Wizard2 2470 = 66.73%)

Clinical Observation
Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of the systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal. For Clinical observation score, see below.

Body Weight
The animal body weights were determined at the start of the test and at the scheduled day of euthanasia of the animals.

Calculation of results
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled radioactive incorporation for each treatment group by the radioactive incorporation of the pooled vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on dose-response curve, immediately above and below of SI value, according to the equation: EC3=c+[(d-3)/(b-d)]x(a-c)
a – lower concentration, b – SI of lower concentration, c – higher concentration
d – SI of higher concentration
If all points are below the stimulation index of three, no EC3 value can be stated.


ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: random
- Criteria used to consider a positive response: A substance is regarded as sensitizer in the LLNA test when SI≥3.

TREATMENT PREPARATION AND ADMINISTRATION:
Dose Preparation
The required amount of the test item (according to the concentration) was mixed in vehicle shortly before the administration (i.e. 100% concentration was obtained by mixing of 1g of test item with 1mL of vehicle). The dose preparation data are listed in the raw data. The preparations were made freshly before each dosing occasion.
Dose Administration
The test item was administrated in volume of 25 µL to the dorsum of each ear. The alpha-Hexyl cinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume.

Positive control substance(s):

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

Statistics:

For calculation of mean and S.D. values of body weights MS Excel was used.

Results and discussion

Positive control results:

SI = 4.98

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.63

Test group / Remarks:

pyridine-2-carbaldehyde oxime 100%

Results 2

Parameter:

SI

Value:

2.16

Test group / Remarks:

pyridine-2-carbaldehyde oxime 50%

Results 3

Parameter:

SI

Value:

2.04

Test group / Remarks:

pyridine-2-carbaldehyde oxime 25%

Results 4

Parameter:

SI

Value:

4.98

Test group / Remarks:

Positive Control

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Lymph Node Proliferation
In comparison with the control group, an increase of the pooled lymph node weights was recorded at all used concentrations. The pooled lymph node weights of treated groups were 0.0483g for 25% concentration, 0.0507g for 50% concentration and 0.0555g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0421g and 0.0809g, respectively. The DPM values for the three treated groups were 1603 (25%), 1697 (50%) and 2065 (100%), respectively. The SI values for the three treated groups were 2.04 (25%), 2.16 (50%) and 2.63 (100%), respectively.

EC3 CALCULATION
The EC3 value could not be calculated, as all measured points were below the stimulation index of three.

CLINICAL OBSERVATIONS: Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

BODY WEIGHTS: The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the sensitizing potential of pyridine-2-carbaldehyde oxime in mice. All animals survived throughout the test period without showing any clinical signs of toxicity or local irritation. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance a sensitizer.
Pyridine-2-carbaldehyde oxime was so identified as a non-sensitizing agent in the Local Lymph Node Assay. Hence, no classification as skin sensitizer is triggered.

Executive summary:

The sensitization potential of pyridine-2-carbaldehyde oxime was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the contact sensitization potential of chemicals.

Based on the recommendations of the OECD Guideline, the test item was formulated in Dimethyl sulfoxide (DMSO). The positive control (alpha-Hexylcinnamaldehyde) (25%) was formulated in acetone:olive oil 4:1 (v/v).

The Pre-screen test was performed using a dose of 100 % (w/v). Based on the observations recorded in the preliminary test, the concentration of 100 % was selected as top dose for the main test.

Five female mice (CBA/Ca) were topically exposed (dorsum of both ears) to the test item at concentrations of 25%, 50% and 100%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10E-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (25%, 50% and 100% w/v) the animals did not show visible clinical symptoms or local irritation or systemic toxicity.

In this study Stimulation Indices of 2.04, 2.16, and 2.63 were determined with the test item at concentrations of 25%, 50%, and 100% in dimethylsulfoxide, respectively. The EC3 value could not be calculated, as all measured points were below the Stimulation Index of three.

From the results of this study, test item pyridine-2-carbaldehyde oxime is not considered a skin sensitizer in the murine local lymph node assay.