Methyl 3-pyridyl ketone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-18 - 2015-12-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-documented GLP OECD guideline study without deviations on the registered substance itself.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

OECD: Guideline for the Testing of Chemicals; Section 4: Health Effects: 442B Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA. Adopted: 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Fürstenwall 25, 40219 Düsseldorf, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-pyridyl ketone

EC Number:

206-496-7

EC Name:

Methyl 3-pyridyl ketone

Cas Number:

350-03-8

Molecular formula:

C7H7NO

IUPAC Name:

1-(pyridin-3-yl)ethan-1-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 3-Acetylpyridine
- Substance type: pure substance
- Storage condition of test material: Room temperature (20±5°C)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, 5961 NM Horst, NL
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17-22 g
- Housing: OP3 (SPF-barrier), In groups of 4 animals in open Eurostandard type III macrolon cages, with Lignocel hygienic animal bedding (J. Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg), heat-treated, Specified pathogen free (SPF) housing with permanent health monitoring by room specific sentinel animals (bedding sentinels) in accordance with FELASA recommendations, with Wooden gnawing blocks, size medium 10 x 2 x 2 cm, debarked, aspen wood, NGM E-022 (ABEDD LAB & VET Service GmbH), heat-treated
- Diet (e.g. ad libitum): Maintenance diet rat/mouse, pellets, No. 1324 TPF (Altromin GmbH & Co. KG, 32791 Lage), ad libitum
- Water (e.g. ad libitum): Sterilized community tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): artificial lighting, 12 h light/12 h dark

IN-LIFE DATES: From: 2015-11-11 To: 2015-11-24

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

2.5%, 5%, 10%

No. of animals per dose:

4 females / dose group

Details on study design:

RANGE FINDING TESTS:
In a pre-study, 3-Acetylpyridine was applied on test animals in doses of 50%, 25% and 10%. 24h after the first application the animals that received the 50% and 25% doses were found dead in their cages. The LD50 for oral intake of 3-Acetylpyridine was specified in a range of 51-57 mg/kg bodyweight in rat. The 50% test item dose equals a test item content of 1250 mg/kg bodyweight. Therefore the test animals died most probably due to test item intoxication. The animal, which received the 10% dose, showed no clinical signs indicating systemic intoxication and showed no skin reaction indicating an excessive local skin irritation. Therefore the 10% suspension was selected as high dose for this study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA: BrdU-ELISA
- Criteria used to consider a positive response: Evaluation criteria according to OECD 442B
For assay validity the following system suitability criteria should be fulfilled:
1) ODVC MEAN: 0,1 ≤ ODVC ≤ 0,2
2) SIVC AVERAGE ≅!1,0
3) SIPC ≥ 1,6 over SIVC*
Classification of SI values is as follows:
SI indicates a positive result when:
1,6 ≤ SI ≤ 1,9 = borderline positive**
2,0 ≤ SI = positive
*No excessive skin irritation or systemic toxicity should be observed in parallel.
**Additional information (such as dose-response relationship, evidence of systemic toxicity or excessive irritation) should be considered to confirm that such results are positives.

TREATMENT PREPARATION AND ADMINISTRATION:
Application
25 μL of the extracted test substance, the vehicle alone or the positive control (PC), were applied to the dorsum of each ear (50 μL per animal) using a tipped pipette. Animals were fixed until evaporation of acetone. In brief, the complete experimental schedule of the assay was as follows:
• Day 1: Individually identify and record the weight of each animal and any clinical observation. Apply 25 μL of the extracted test substance, the vehicle alone or the PC, to the dorsum of each ear.
• Day 2: Repeat the application procedure carried out on day 1.
• Day 3: Repeat the application procedure carried out on day 1.
• Day 4: No treatment.
• Day 5: Inject 0.5 mL (5 mg/mouse) of BrdU2 (10 mg/mL) solution intraperitoneally.
• Day 6: Record the weight of each animal and any clinical observation. To further monitor the local skin response, score an occurring ear erythema approximately 24 h after BrdU injection and humanely kill the animals. Punch both ears and weigh ear punch-pair. Excise the draining auricular lymph nodes from each mouse ear and process separately in phosphate buffered saline for each animal.

Positive control substance(s):

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

Statistics:

Calculation of results
For each experimental group, a stimulation index (SI) was calculated by dividing the mean BrdU labeling index for each individual animal by the mean BrdU labeling index for thevehicle group (the average SI for the vehicle group is 1).
The BrdU labeling index is defined as:
BrdU labelling index = (OD370nm – OD blank370nm) – (OD492nm – OD blank492nm)

Results and discussion

Positive control results:

Group SI = 4.9 (Medium of 3 replicates, 4.5±0.7, 6.0 ±0.6, 4.2±1.2)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: S.I. of test group (2.5 %): 1.1±0.4 S.I. of test group (5 %): 1.0±0.3 S.I. of test group (10 %): 1.1±0.3

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: not applicable

Any other information on results incl. tables

Results

1 Health monitoring

1.1 Fatalities

There were no fatalities or moribund animals.

 

1.2 General clinical signs and behavior

No abnormalities concerning clinical signs and behavior were observed in the animals of the positive control group, all dose groups or vehicle group, respectively. No signs of general toxicity were observed.

 

1.3 Body weight

The mean weight at the start of the study (d1) was 20,9 g; the minimum weight was 19,2 g (8% difference to the mean weight), the maximal weight was 22,7 g (8% difference to the mean weight). Therefore the stratification and randomization procedures were sufficient. During the in life phase, the mean body weights and the body weight gains of all experimental animal groups were within normal range for healthy mice of this strain and age.

 

1.4 Erythema

Approximately 24 hours (24 h) after BrdU injection (day 6), both ears of each mouse were observed for erythema. The animals of the positive control group showed a very slight erythema. In contrast the animals of the dose groups or vehicle group did not show any signs of erythema on day 6.

 

1.5 Ear punch weight

Ear thickness was determined through ear punch weight at necropsy (day 6).

 

Ear punch weight [mg]. Asterisks denote significance levels (t-test, two tailed, unpaired): *: 0.05 ≥ p > 0.01; **: 0.01 ≥ p > 0.001; ***: p ≤ 0.001 (n.s.: not significant).

Group	Pos.-Control, 25% HCA		3-AP, 10%		3-AP, 5%		3-AP, 2.5%		Vehicle
Animal No.	Box	Ear punch weight [mg]	Box	Ear punch weight [mg]	Box	Ear punch weight [mg]	Box	Ear punch weight [mg]	Box	Ear punch weight [mg]
1	E 990	12.7	E 991	9.4	E 992	8.4	E 993	9.9	E 994	9.4
2		11.6		9.5		9.3		9.8		9.0
3		12.3		9.7		10.2		10.5		8.8
4		12.1		10.4		10.3		10.3		9.3
Mean		12.2		9.8		9.6		10.1		9,1
SD		±0.5		±0.5		±0.9		±0.3		±0.3
ttest vs. Veh. p=		0.000		0.056		0.396		0.004
Significance		***		n.s.		n.s		**

3-AP: 3-Acetylpyridine

 

Using the two tailed unpaired Student’s t-test, a significantly increased ear punch weight was found for the animals of the positive control (PC) group (p= 0,00003) and for the animals of the low dose group (p=0,004), when compared to the vehicle group. The animals of the PC group showed also a very slight erythema and a stimulation of the LCN’s. The slight erythema and the stimulation of the LCN`s were regarded as signs of sensitization. No such correlates were found for the animals of the low dose group in this regard. Therefore the increase of ear thickness in the low dose group is most probably not due to a sensitizing reaction. An increase of ear thickness due to a skin irritating effect in the absence of erythema or eschar is highly unlikely. No significant difference in ear punch weight was found for the animals of the high (P= 0,609) and medium (P= 0,396) dose groups, when compared to the vehicle group, respectively.

 

 

2 Determination of cellular proliferation (BrdU ELISA)

The content of BrdU, incorporated into the DNA of proliferating lymphnodes, was measured by ELISA, using a commercial kit. Using this non- radioactive assay, results were calculated appropriately for those method as described in the OECD series 442B. In brief, a stimulation index of 2,0 or higher clearly identifies a test item as a sensitizer, whereas an SI between 1,6 and 1,9 is considered as a borderline positive response where additional information (such as dose-response relationship, evidence of systemic toxicity or excessive irritation) should be used to confirm that such a result is true positive.

 

2.1 Validity of the assay

The mean absorbance value of the vehicle groups (ODVC MEAN) at a sample volume of 10,0 mL was 0,108 (0,118; 0,092 and 0,114 for the three test plates) and therefore within the assay’s acceptance range of ODVC MEAN 0,1 to 0,2. The positive control groups (PC) at 10 mL yielded SI’s ≥ 2,0, which are considered as true positive results, therefore, the assay is considered to be valid.

 

2.2 Proliferation of LNCs

In order to assess the sensitizing potential of 3-Acetylpyridine in high, medium and low dose, respectively, a sample volume of 10 mL at an incubation time of 30 minutes was chosen for calculation of the results

 

 

High dose group: Proliferation of LNCs in terms of BrdU incorporation.Individual BrdU labeling indices, group means of BrdU labeling indices and Stimulation Indices (SI) ± SD are given for the 10 mL sample volume.

Group	BrdU labeling indices/ animal				Group mean SD	Group SI SD
pos-Contr., 25% HCA	E 990/0	E 990/1	E 990/2	E 990/3	0.531 ± 0.055	4.5 ± 0.7
	0.648	0.492	0.474	0.509
High dose, 3-Acetylpyridine	E 991/0	E 991/1	E 991/2	E 991/3	0.132 ± 0.035	1.1 ± 0.3
	0.099	0.180	0.132	0.116
Vehicle, Acetone: olive oil (4:1 v/v)	E 994/0	E 994/1	E 994/2	E 994/3	0.118 ± 0.034	1.0 ± 0.3
	0.160	0.123	0.110	0.078

 

 

Medium dose group: Proliferation of LNCs in terms of BrdU incorporation.Individual BrdU labeling indices, group means of BrdU labeling indices and Stimulation Indices (SI) ± SD are given for the 10 mL sample volume.

Group	BrdU labeling indices/ animal				Group mean SD	Group SI SD
pos-Contr., 25% HCA	E 990/0	E 990/1	E 990/2	E 990/3	0.556 ± 0.055	6.0 ± 0.6
	0.613	0.539	0.488	0.585
Medium dose, 3-Acetylpyridine	E 992/0	E 992/1	E 992/2	E 992/3	0.097 ± 0.026	1.0 ± 0.3
	0.130	0.097	0.066	0.093
Vehicle, Acetone: olive oil (4:1 v/v)	E 994/0	E 994/1	E 994/2	E 994/3	0.092 ± 0.030	1.0 ± 0.3
	0.131	0.101	0.070	0.068

 

 

Low dose group: Proliferation of LNCs in terms of BrdU incorporation.Individual BrdU labeling indices, group means of BrdU labeling indices and Stimulation Indices (SI) ± SD are given for the 10 mL sample volume.

Group	BrdU labeling indices/ animal				Group mean SD	Group SI SD
pos-Contr., 25% HCA	E 990/0	E 990/1	E 990/2	E 990/3	0.480 ± 0.137	4.2 ± 1.2
	0.460	0.358	0.427	0.675
Low dose, 3-Acetylpyridine	E 993/0	E 993/1	E 993/2	E 993/3	0.125 ± 0.047	1.1 ± 0.4
	0.065	0.116	0.144	0.175
Vehicle, Acetone: olive oil (4:1 v/v)	E 994/0	E 994/1	E 994/2	E 994/3	0.114 ± 0.031	1.0 ± 0.3
	0.091	0.098	0.109	0.160

 

 

In the Local Lymph Node Assay, the application of 3-Acetylpyridine in the high, medium and low dose led to the following stimulation indexes (SI’s):

 

Summary of SI’s of experimental dose groups.

Groups	% 3-AP	SI
High dose	10.0	1.1 ± 0.3
Medium dose	5.0	1.0 ± 0.3
Low dose	2.5	1.1 ± 0.4

 

In conjunction with the absence of evidence of systemic toxicity or irritation the proliferative responses of the LNCs in the high, medium and low dose groups were regarded to be negligible in terms of sensitization.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 442B 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 3-Acetylpyridine in mice. 3-Acetylpyridine did not induce signs of general toxicity. No significantly increased ear thickness, in terms of ear punch weight, was detected. Together with the negligible proliferative responses of the LNCs, 3-Acetylpyridine was identified as a non-sensitizing agent in the Local Lymph Node Assay. Hence, no classification as skin sensitizer is triggered.

Executive summary:

A dermal sensitization study was carried out according to guideline OECD 442B (Guideline for the Testing of Chemicals; Section 4: Health Effects: 442B Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA) in CBA/Ca mice using 25 % α-Hexylcinnamaldehyde (HCA) as positive control and acetone: olive oil (4:1 v/v) as vehicle. The test item 3-Acetylpyridine was diluted in vehicle solution to receive a 10% suspension (high dose). In a preliminary screen test the 10% test item suspension did not show any signs of systemic intoxication or excessive skin irritation. Therefore the 10% test item suspension was found to be the feasible high dose for this LLNA. In this study the high dose and two subsequent dilutions of 5% and 2,5%, medium and low dose respectively, of the test item were applied to the dorsal side of the ears.

Animals were monitored daily. Determination of cellular proliferation was performed using the BrdU method in reference to the OECD guideline 442B. Additionally, ear thickness was determined through ear punch weight on day 6 of the study.

3-Acetylpyridine did not induce signs of general toxicity. No significantly increased ear thickness, in terms of ear punch weight, was detected. Together with the negligible proliferative responses of the LNCs, 3-Acetylpyridine was identified as a non-sensitizing agent in the Local Lymph Node Assay.

The study was classified as acceptable, and no classification of 3-Acetylpyridine as skin sensitizer is triggered.