Isonicotinaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 February - 23 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Isonicotinaldehyde

EC Number:

212-832-3

EC Name:

Isonicotinaldehyde

Cas Number:

872-85-5

Molecular formula:

C6H5NO

IUPAC Name:

pyridine-4-carbaldehyde

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report) : Pyridine-4-aldehyde
- Physical state : clear yellowish liquid
- Analytical purity : 98,7% (GC)
- Lot/batch No. : 1120126/001
- Storage condition of test material : Refrigerator at ca. 4°C , in the dark , under nitrogen

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCruBR

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source : Charles River UK Limited , England CT9 4LT
- Age at study initiation : appr. 8 weeks
- Weight at study initiation : 18,2 - 23,4 g
- Housing : animals were housed singly in Makrolon cages type II (22cm x 16,5cm ground area , 15cm high) , Bedding material : wood chips
- Diet : Altromin maintenance diet for rats and mice , item No. 1324forte , germ reduction by 25 kGy 60Co-gamma irradiation , ad libitum
- Water : Tap water , offered in Makrolon-bottles with stainless steel canules , ad libitum
- Acclimation period : 8 days

ENVIRONMENTAL CONDITIONS
- Temperature : average of 22,0°C (continuous monitoring and recording)
- Humidity : average of 33,1% (continuous monitoring and recording)
- Air changes (per hr) : no data
- Photoperiod (hrs dark / hrs light) : 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Group A (low dose) : 25% (v/v) solution of "Pyridine-4-aldehyde" in AOO
Group B (mid dose) : 50% (v/v) solution of "Pyridine-4-aldehyde" in AOO
Group C (high dose) : 100% (undiluted) "Pyridine-4-aldehyde"

AOO = acetone:olive oil , 4:1 (v/v)

No. of animals per dose:

5

Details on study design:

MAIN STUDY
- Criteria used to consider a positive response :
According to the guideline the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3 .
The positive control substance led to a stimulation index of 13,4 , thus demonstrating the validity of the experiment .

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was diluted with acetone:olive oil , 4:1 (v/v) or undiluted and was administered to three groups of 5 female CBA/Ca mice . Administration was performed epicutaneously to the dorsal surface of both ears , once a day on 3 consecutive days . The volume administered was 25 µl per ear .
Concentrations used (AOO = acetone:olive oil , 4:1 (v/v)) :
-Group A (low dose) : 25% (v/v) solution of "Pyridine-4-aldehyde" in AOO
-Group B (mid dose) : 50% (v/v) solution of "Pyridine-4-aldehyde" in AOO
-Group C (high dose) : 100% (undiluted) "Pyridine-4-aldehyde"
Two groups with 5 animals each served as positive and negative controls . Both control substances were administered under identical conditions as the test substance .
The following solutions served as control substances :
-Group P (positive control) : 25% (v/v) solution of hexyl cinnamic aldehyde in AOO
-Group K (negative control) : AOO

5 days after the first topical administration , each animal received 20 µCi 3H-methyl thymidine (3H-TdR) by intravenous administration . The injection solution containing 80 µCi/ml 3H-TdR was prepared by the dilution of 720 µl 3H-TdR (amersham pharmacia biotech , Kat.Nr.:TRA 310 , batch 306 , specific activity 2.0 Ci/mmol , radioactive concentration 1 mCi/ml , radiochemical purity 99,3% , thymine content 0,1%) with 8,28 ml sterile phosphate buffered saline (PBS) . Then 250 µl of the solution were intravenously administered to each animal via a tail vein .
Approximately 5 hours after 3H-TdR injection all animals were sacrificed by carbon dioxide asphyxiation and the draining auricular lymph nodes were rapidly excised . The lymph nodes of each group were pooled in PBS : A single cell suspension (SCS) of lymph node cells (LNC) was prepared by gentle mechanical disaggregation of the pooled lymph nodes through a 70 µm cell strainer . The SCS were then transferred into centrifuge tubes and LNC were pelleted by centrifugation (4°C , max. 200 g , 10 min) : Afterwards supernatants were removed by aspiration . Then the LNC were resuspended and washed twice with PBS . After the final washing the supernatants were removed leaving just a small volume (<0,5ml) and macromolecules were precipitated by incubation with 5% trichloroacetic acid (TCA) at 4°Overnight . Each precipitate was pelleted by centrifugation (4°C , max. 200g , 10 min) and resuspended in 1 ml TCA . This suspension was transferred into scintillation vials containing 10 ml scintillation cocktail (Packard Bioscience : Ultima Gold , Bestellnr. 6013329) and 3H-TdR incorporation was determined with a beta-scintillation counter (TriCarb 2200CA , PackardInstrument Co. , protocol 4 : single label 3H , dpm , AEC-quenchcurve in Ultima Gold from21 June 2002) .

Positive control substance(s):

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

Statistics:

Statistical analysis of body mass differences between the groups on Days 1 and 6 was performed by the analysis of variance followed the Scheffe test (p=0,05 , two sided)
Statistical analysis of ear thickness differences between the groups was performed by the analysis of variance followed by the Scheffe test (p=0,05 , two sided) or by the H-Test when the conditions for the analysis of variance were not met .

Results are presented as test/control ratio (Stimulation index) , calculated as : dpm test group/dpm negative control group
(dpm = disintegrations per minute , corrected by the subtraction of the mean background)
A substance is regarded as a sensitiser in the LLNA if the test substance induces a 3-fold or greater increase in 3H-TdR incorporation into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes , as indicated by the SI , together with the consideration of dose response .

Results and discussion

Positive control results:

Application of 25% hexyl cinnamic aldehyde in AOO resulted in an Stimulation index of 13,4 . This proves the sensitivity of the strain of animals used and the reliability of the experiment technique .

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The Stimulation index of the test substance groups was between 11,5 and 12,9 . There was no concentration related response . For detailed information see "Any other information on results incl. tables"

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: For detailed information see "Any other information on results incl. tables"

Any other information on results incl. tables

dpm results and calculated stimulation indices (SIs)

	dpm	SI
group K (negative control)	1818	1
group A (low dose)	23441	12,9
group B (mid dose)	20976	11,5
group C (high dose)	21516	11,8
group C (positive control)	24494	13,5

Mortality : All animals survived till the end of the study .

General observations ;

No abnormal behaviour or clinical signs were detected during the experiment in the animals . All animals of the highest test substance group (C) showed white crystalline crusts on the auricles on day 4 , they were inconspicuous the other days .

Skin reactions :

No local irritations were observed at the application sites of all animals of all test substance groups and the negative control group throughout the whole study . On days 2, 3 and 4 all animals of the positive control group had moderate erythema on the application sites indicating slight irritative skin reactions .

Body mass :

There were no statistically significant differences in mean body masses between the dosed groups and the negative control on days 1 and 6 .

Ear thickness :

Some ears in the high dosed group were thicker than the ears in the control group , but because of a high scattering , the values did not gain statistical significance . There were no differences in mean ear thickness between the other substance groups and the negative control on day 4 . The ear thickness of the positive control group was significantly different to the negative control group on day 4 .

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information

Conclusions:

The study was performed according to the OECD TG429 without deviations and according to the good laboratory practice principles, it is considered to be of highest quality (reliability Klimisch 1). The criteria of validity of the test system are fulfilled. Data from ear thickness measurements show that "Pyridine-4-aldehyde" is not a strong skin irritant . Therefore a false positive result induced by dermal irritation can be excluded .
Although no concentration related response was observed , "Pyridine-4-aldehyde" is regarded as a sensitiser in the LLNA according to the OECD-Guideline 429 , "Skin Sensitisation : Local Lymph Node Assay" , since the stimulation indices of all examined test substance concentrations were clearly greater than 3 .

Executive summary:

The Local Lymph Node Assay was performed to evaluate a possible skin sensitising potential of "Pyridine-4-aldehyde" according to the OECD-Guideline 429, "Skin Sensitisation: Local Lymph Node Assay". The test substance was used diluted with acetone:olive oil, 4:1 (v/v) or undiluted and was administered to three groups of 5 female CBA/Ca mice. Administration was performed epicutaneously to the dorsal surface of both ears, once a day on 3 consecutive days. The volume administered was 25 µL per ear.

Concentrations used (AOO= acetone:olive oil, 4:1 v/v).

Group A (low dose):	25% (v/v) solution of "Pyridine-4 -aldehyde" in AOO
Group B (mid dose):	50% (v/v) solution of "Pyridine-4-aldehyde" in AOO
Group C (high dose):	100% (undiluted) "Pyridine-4-aldehyde"

Two groups with 5 animals each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance.

The following solutions served as control substances:

- Group P (positive control):25% (v/v)solution of hexyl cinnamic aldehyde in AOO

- Group K (negative control): AOO

5 days after the first topical application, 3H-methyl thymidine was intravenously administered to all mice via a tail vein. Approximately 5 hours later all animals were sacrificed, the draining auricular lymph nodes were excised, pooled for each group, and single cell suspensions were prepared. Then the incorporation of 3H-methyl thymidine into the cells was determined (liquid scintillation counter) and compared with the negative controls. The stimulation index (SI) was calculated as the ratio of the disintegrations per minute (dpm) of the dosed group or of the positive control group to the dpm of the negative control group. Ear thickness of all animals was measured before the first and 24 hours after the last administration to get an information about the skin irritating properties of the substances administered.

All animals survived till the end of the study. Moderate erythema was noted in all animals of the positive control group on Days 2-4, indicating slight local skin irritation. This slight irritating effect was confirmed by a statistically significant difference of the ear thickness between the positive control group and the negative control group on Day 4. No skin irritating effects were observed in the test substance groups and the negative control group throughout the whole study. There was no statistically significant difference in ear thickness between the test substance groups and the negative control group on Day 4.

³H-methyl thymidine incorporation, stimulation indices :

The calculated stimulation indices (test substance/negative control ratio) were decisive for the grading of the potential of sensitisation: According to the guideline the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response. The SI of each tested test substance group was clearly above the limit of "3". There was no concentration related response. The positive control substance led to a stimulation Index of 13.4, thus demonstrating the validity of the experiment.

According to the OECD-Guideline 429, "Skin Sensitisation: Local Lymph Node Assay", "Pyridine-4-aldehyde" is regarded as a sensitiser in the Local Lymph Node Assay.