5-ethyl-2-methylpyridine

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Remarks:

Toxi-Coop ZRT.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material: 5-ethyl-2-methylpyridine

In vivo test system

Test animals

Species:

mouse

Strain:

other: CRL: NMRI BR and CBA/Ca mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 - 12 weeks old
- Weight at study initiation: 16.9 – 20.7 g; The weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight.
- Housing during acclimatization period: Grouped caging in small groups, during the test: Grouped caging (5 animals/cage), cage type: Type II. Polypropylene / polycarbonate
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 21 or 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 - 70 %
- Photoperiod (hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
- From: May 16, 2012
- To: May 24, 2012

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

No., Groups , Test item concentration (% w/v ) , Positive control concentration (% w/v) , No. of animals
1, Vehicle control: AOO , - , - , 5
2, Positive control: HCA in AOO , - , 25, 5
3, LZ163 in AOO , 50, - , 5
4, LZ163 in AOO , 37.5, - , 5
5, LZ163 in AOO , 25, - , 5
6, LZ163 in AOO , 10, - , 5
7, LZ163 in AOO , 5, - , 5
AOO = Acetone:olive oil 4:1 (v/v) mixture
HCA = α-Hexylcinnamaldehyde

No. of animals per dose:

Total of 5 animals per dose and control group.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test item was a liquid and based on preliminary test results formulated for the main test (apparently solutions) in Acetone: Olive oil 4:1 (v/v) mixture (AOO).
- Irritation: The preliminary irritation/toxicity screen (conducted in two steps at a concentration range from 100 % to 10 %) indicated systemic toxic effect of the test item at high test concentrations. Based on the results 50 % was selected as the maximum applicable concentration to be used in the main test.
- Lymph node proliferation response: Proliferation values obtained reflected the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1. A minimum of four animals were used per dose group, with a minimum of three concentrations of the test substance, plus a negative control group treated only with the vehicle for the test substance and a positive control. In those cases in which individual animal data were collected and statistically analyzed, a minimum of five animals per dose group were used.
- Skin sensitizing effects were observed at the applied concentration in the positive control group (SI > 3).

TREATMENT PREPARATION AND ADMINISTRATION:
AOO was used as negative (vehicle) control both for the groups treated with the test item formulations and the positive control. The test item was administered in five different concentrations.
- In vivo Treatment: Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicle (negative control group) using a pipette, on the dorsal surface of each ear. After the treatments animals returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
- Proliferation Assay: No technically failed treatment was observed during the test. Lymph nodes from all five animals per dose groups were processed (except the 50 % dose group where 4 animals were processed).
- Injection of 3H-methyl-thymidine (3HTdR): On Day 6, the animals were intravenously injected via the tail vein with 250 μL of sterile PBS containing 20 μCi of 3HTdR using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes: Five hours (± 30 minutes) after intravenous injection the mice were humanely sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of individual mice were collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Preparation of Single Cell Suspension of Lymph Node Cells: A single cell suspension (SCS) of lymph node cells (LNCs) of each individual animal was prepared and collected in disposable tubes by a gentle mechanical disaggregation of the lymph nodes through 200 μm-mesh stainless steel gauze using the plunger of a disposable syringe. The cell strainer (stainless steel gauze) was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4°C. After centrifugation, the majority of the supernatant was aspirated off, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated and the LNCs were resuspended in 10 mL of PBS. This washing procedure was repeated twice. This procedure was repeated for lymph nodes of each individual animal.
- Determination of Incorporated 3HTdR: After the final washing step, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before resuspending the LNCs in 3 mL of 5 % trichloracetic acid (TCA) to allow for the precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed and the pellets were resuspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % TCA.
Instrument used for the measurement:
Name: Packard Tri-Carb 2300 TR Liquid Scintillation Analyzer
Serial Number: 406 117

Positive control substance(s):

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

Statistics:

The individual approach enabled statistical analysis of the data. Statistical analysis was performed by SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values. The normal distribution of data was examined by Kolmogorow-Smirnow test. Since no normal distribution was observed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. A positive result was detected and inter-group comparisons performed using the Mann-Whitney U-test. Dose-response was evaluated by linear regression using Microsoft Excel Software.

Results and discussion

Positive control results:

Statistically significant increase of the proliferation values compared to the negative control was observed in the positive control group (p < 0.01, Mann-Whitney U-test).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Visually larger lymph nodes than the control was observed in the positive control group and in the 50 % dose group. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group and in the other test item treated groups. Significant (SI ≥ 3) lymphoproliferation was noted for LZ163 at concentrations of 50 %, 37.5 % and 25 %. No significant (SI ≥ 3) lymphoproliferation was noted for LZ163 at concentrations of 10 % and 5 %. The observed stimulation index values were 4.74, 5.45, 3.27, 1.55 and 2.26 at test item concentrations of 50 %, 37.5 %, 25 %, 10 % and 5 %, respectively. Dose-related response was observed, but not statistically significant (see Statistical Analysis).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Individual DPM and DPN Values, the Mean DPM and DPN Values and the SI values for all Groups in the Main Test are shown in table 1 below. Individual DPM values (corrected with the mean background value) were statistically evaluated in the 37.5 %, 25 %, 10 % and 5 % test item treated groups and in the control groups (both positive and negative control). Statistically significant increase of the proliferation values compared to the negative control was observed in the positive control group and in the 37.5 % test item treated group (p < 0.01, Mann-Whitney U-test) and in the 25 % dose group (p < 0.05, Mann-Whitney U-test). The increase of the lymphoproliferation over the control at test item concentrations of 10 % or 5 % was not statistically significant. Results of the statistical analysis are given in Table 9 of Appendix I. Significance of the dose-response was evaluated by linear regression using the SI values calculated for evaluated test concentrations (37.5 %, 25 %, 10 % and 5 %). Although the effect was considered as dose-related, no statistical significance was observed: the r value was 0.93, the p value was 0.068.

Any other information on results incl. tables

Individual DPM and DPN Values, the Mean DPM and DPN Values and the SI values for all Groups in the Main Test are shown in table 1 below:

Table 1: Individual DPM and DPN Values, the Mean DPM and DPN Values and the SI values for all Groups in the Main Test

Test Group	Animal	Individual DPM	Individual DPM	Individual DPN	Group DPM	Group DPN	SI
Name		(Measured)	(Corrected)$	(DPM/Node)#	(Mean)	(Mean)	Values
	684	369	326.5	163.3	724.3	362	1.00
Negative (vehicle)	685	1211	1168.5	584.3
control:	700	575	532.5	266.3
AOO	705	393	350.5	175.3
	716	1286	1243.5	621.8
	683	7223	7180.5	3590.3	8824.5	4412	12.18
Positive control:	686	13383	13340.5	6670.3	**
25 % HCA	701	8891	8848.5	4424.3
in AOO	704	8301	8258.5	4129.3
	714	6537	6494.5	3247.3
	690	2824	2781.5	1390.8	3432.3	1716	4.74
LZ163	691	4648	4605.5	2302.8	NE
50 % in AOO	702	3821	3778.5	1889.3
	711	2606	2563.5	1281.8
	689	5730	5687.5	2843.8	3944.3	1972	5.45
LZ163	693	1526	1483.5	741.8	**
37.5 % in AOO	703	2508	2465.5	1232.8
	706	6666	6623.5	3311.8
	717	3504	3461.5	1730.8
	687	1892	1849.5	924.8	2371.3	1186	3.27
LZ163	694	1271	1228.5	614.3	*
25 % in AOO	718	3112	3069.5	1534.8
	719	3801	3758.5	1879.3
	720	1993	1950.5	975.3
	688	1483	1440.5	720.3	1123.5	562	1.55
LZ163	692	1681	1638.5	819.3	NS
10 % in AOO	707	829	786.5	393.3
	710	554	511.5	255.8
	712	1283	1240.5	620.3
	682	2399	2356.5	1178.3	1637.7	819	2.26
LZ163	708	804	761.5	380.8	NS
5 % in AOO	709	1109	1066.5	533.3
	713	2191	2148.5	1074.3
	715	1898	1855.5	927.8

AOO = Acetone: Olive oil 4:1 (v/v) mixture

HCA =a-Hexylcinnamaldehyde

$ = Measured DPM value – Mean background value

# = Corrected DPM value / No. of lymph nodes (per animal)

Mean background value = 42.5

No. of lymph nodes (per animal) = 2

NE= Not evaluated. The 50 % dose group was not statistically evaluated, because mortality and significant clinical symptoms were observed in this dose group during the main test.

NS= Not significant; Mann-Whitney U-test versus control (AOO)

* Significant; p < 0.05 Mann-Whitney U-test versus control (AOO)

** Significant; p < 0.01 Mann-Whitney U-test versus control (AOO)

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and also results of the preliminary irritation/toxicity tests.

The test item was a liquid and based on preliminary test results formulated for the main test (apparently solutions) in Acetone: Olive oil 4:1 (v/v) mixture (AOO). The preliminary irritation/toxicity screen (conducted in two steps at a concentration range from 100 % to 10 %) indicated systemic toxic effect of the test item at high test concentrations. Based on the results 50 % was selected as the maximum applicable concentration to be used in the main test. To ensure validity of the test should unexpected adverse effects occur, a total of five concentrations were examined in the main test. Concentrations tested were 50 %, 37.5 %, 25 %, 10 % and 5 % in vehicle (AOO).

The mortality and intensity of the clinical symptoms observed in the 50 % dose group indicated significant systemic toxic effects. Therefore even though the SI value was calculated, results were not statistically evaluated. Taking into account both the preliminary test results and main test data (body weights, clinical observations, ear thickness values and erythema scores) clinical symptoms observed in the 37.5 % and 25 % dose groups were not considered toxicologically significant. Proliferation values obtained reflected the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

Although no strong dose-response relationship was observed (as indicated by the linear regression performed using data points of the statistically evaluated test concentrations of 37.5 %, 25 %, 10 % and 5 %), the statistically significant (SI ≥ 3) proliferation observed at 37.5 % and 25 % concentrations was considered evidence that LZ163 has sensitization potential, according to evaluation criteria of the relevant guidelines.

EC3calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method. The calculated EC3value of LZ163 was 22.4 % in this LLNA. Using this EC3value according to the published data for classification of contact allergens LZ163 can be ranked among weak sensitizers (10 ≤ EC3≤ 100) in this LLNA.

Additionally EC3value based on the equation of the regression curve was also calculated: the relevant value was 18.1 in this LLNA. Using the EC3value based on the regression curve LZ163 can be ranked among the same category (weak sensitizer) as based on the actual dose-response curve.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present Local Lymph Node Assay, the test item at concentrations of 50%, 37.5%, 25%, 10% and 5 % as homogenous formulations (solutions) in a suitable vehicle (AOO) was shown to have sensitization potential (sensitizer). Based on the EC3 values calculated using dose-response and regression curve analysis the test item was considered a weak sensitizer in this LLNA.

Executive summary:

A study according to EU Method B.42, OECD Guideline 429 and EPA OPPTS 870.2600 (Skin Sensitisation: Local Lymph Node Assay) was carried out. Under the conditions of the assay the test item was tested at concentrations of 50 %, 37.5 %, 25 %, 10 % and 5 % as homogenous formulations (solutions) in a suitable vehicle (AOO). The test item was shown to shown to have sensitization potential (sensitizer).The calculated EC3-value in this LLNA was 22.4 % based on linear interpolation and 18.1 % based on regression curve analysis. Based on the EC3 values calculated using dose-response and regression curve analysis the test item was considered a weak sensitizer.