3,5-dimethylpyridine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Conducted in a reliable industry laboratory according to OECD guideline method under GLP; however, preliminary screening assay showed no irritation (substance is corrosive) making this test system suspect.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dimethylpyridine

EC Number:

209-708-6

EC Name:

3,5-dimethylpyridine

Cas Number:

591-22-0

Molecular formula:

C7H9N

IUPAC Name:

3,5-dimethylpyridine

Test material form:

other: liquid

Details on test material:

3,5-Lutidine (3,5-dimethylpyridine), purity 99.0%; 0.16% water by Karl Fisher titration, stucture characterization by infrared spectrum. Purchased from Sigma-Aldrich, St. Louis, MS., (Product # L4206, Lot # 09427TA)

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

Because the recommended strain of mouse (CBA/Ca) was not readily available from commercial supplies, female BALB/c mice were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier. Female BALB /c mice have been shown to be equivalent with CBA mice when simultaneously evaluating the LLNA response towards known chemical sensitizers (Woolhiser et al., 2000).
The animals were 8-10 weeks of age at the beginning of the study.
Animals were housed one per cage in suspended, stainless steel cages fitted with wire mesh floors, suspended above catch pans.
Diet: LabDiet (R) Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) in pellet form, ad libitum.
Water was provided ad libitum from municipal source (St. Louis, MO). This was periodically analyzed for chemical parameters and biological contaminants byt he municpal water department. In addition, specific analyses for chemical contaminants were condicted at periodic intervals by an independent testing facility.
Acclimatized for at least 1 week prior to testing.
Temperature: 22 ± 1 degrees C
Humidity 40-70%
Photoperiod: 12 h.
Room air change: 12-15 times/h.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Irritation Screening study: 0.4, 2, 10, 20 or 40%.
Main Study: 0, 2, 10 and 50%

No. of animals per dose:

6 per group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: soluble
- Irritation: yes, checked in a screening test. The test material (25 µl/ear) was spread over the dorsal surface of each ear in a manner to prevent test material loss off the ear using an adjustable pipettor with a disposable tip. Ear thickness, measured using a digital micrometer, was determined 24 hours after the second application and the percent ear-swelling was calculated for each mouse

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Asses
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
The initial and terminal body weights were obtained and recorded.
Administration of the test material (25 µl/ear) was made to the dorsal surface of both ears of the mice. All mice received one of three concentrations of the test substance (2, 10 or 50% v/v) or AOO vehicle once daily for three consecutive days. The positive control Hexyl cinnamic acid, HCA) at 30% v/v was run concurrently.
Twice each day a cage-side examination was conducted by animal care personnel, and to the extent possible the following parameters were evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behavior, morbidity, mortality, and the availability of feed and water. The ears were evaluated for erythema.
On day 6, all mice received a 250 µl intravenous injection (iv) via the lateral tail vein containing 20 µCi of 3H-thymidine (specific activity 2Ci/mmol; Amersham code TRA310, Buckinghampshire, UK) diluted in phosphate-buffered saline (PBS). Approximately five hours later, the mice were sacrificed via CO2 asphyxiation and the auricular lymph nodes (two) located at bifurcation of the jugular veins were excised and placed in PBS. A single cell suspension of lymph node cells from one mouse was prepared by gentle mechanical disaggregation using a tissue homogenizer (Stomacher® 80 Lab System, Seward Medical Unlimited, London, U.K.). The cells were washed two times in PBS and were suspended in 3 ml of 5% trichloroacetic acid (TCA) for approximately 18 hours. The suspended precipitates were centrifuged (200 x g for 10 minutes) and the supernatant removed. The pellet from each mouse was reconstituted in 1 ml of 5% TCA and subsequently transferred to a scintillation vial containing 10 ml of Aquasol-2 scintillation cocktail (Packard Instrument Company, Meridan, Connecticut), along with pellet rinses.
The radioactivity in each precipitate was measured using a scintillation counter and reported as disintegrations per minute (dpm) per mouse.

Positive control substance(s):

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

Statistics:

A mean dpm value + SD (standard deviation) was calculated for each experimental group. The Stimulation Index (SI) was calculated using the absolute dpm value for each mouse as the numerator and the mean dpm value from the vehicle control mice as the denominator, and the mean SI + SD was calculated for each experimental group. Any chemical that produces a SI of > 3 in the LLNA should be considered “positive” for contact sensitization. The EC3 value is determined by extrapolating between two values above and below the SI value of 3.
Means and standard deviation (SD) were generated for body weight data (absolute and gain), ear swelling data, erythema, and the LLNA response (dpm & SI values).
The body weight and dpm data were analyzed by a one-way analysis of variance (Steele and Torrie 1960). Comparisons of treated vs. control groups were done, as necessary, by Dunnett’s t-test (Steele and Torrie 1960). The alpha level at which all tests were conducted was 0.05. The final interpretation of the biological significance of the response was based on both statistical and scientific judgement.
The initial and terminal body weights were obtained and recorded. Twice each day a cage-side examination was conducted by animal care personnel, and to the extent possible the following parameters were evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behavior, morbidity, mortality, and the availability of feed and water. The ears were evaluated for erythema by

Results and discussion

Positive control results:

Conduct of the LLNA was confirmed via a positive response using 30% HCA, which elicited proliferation that was 11-fold (SI) greater than that of vehicle controls.

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Proliferative lymphocyte responses (SI) of approximately 4.5 and 5.0, occurred in lymph nodes draining the site of topical application of the 10% and 50% concentrations, respectively. The result at 2% was not statistically different from the vehicle control. These results are consistent with a slight dermal sensitization potential. The effective concentration resulting in a stimulation index of 3 (EC3), was calculated for this substance to be 6.4%. According to the ECETOC categorization scheme, an EC3 greater than 2% is consistent with weak sensitization potential.

Any other information on results incl. tables

Table 2: Summary of Body Weight Data, Lymph Node Proliferation (DPM and SI) and Erythema Scores for BALB/c Mice Treated with 3,5 -Lutidine

Treatment Group	Body Weight (g) (g Gained)	DPM	SI^&	Erythema Score è , &
Vehicle (AOO)	18.2 + 1.1
(n=6)	(0.3 + 0.3)	563 + 471	1.0 + 0.8	0
30% HCA	18.3 + 0.5
(n=6)	(0.3 + 0.7)	6196* + 3313	11.0 + 5.9	0
2% 3,5-lutidine	18.6 + 0.6
(n=5)	(0.4 + 0.5)	703 + 531	1.2 + 0.9	0
10% 3,5-lutidine	18.8 + 0.9
(n=6)	(0.6 + 0.4)	2540 + 1137	4.5 + 2.0	0
50% 3,5-lutidine	18.4 + 0.8
(n=6)	(0 + 0.2)	2811 + 856	5.0 + 1.5	0

Mice were treated topically with vehicle, 30% HCA or 3,5-lutidine (2, 10 or 50% v/v) for three consecutivedays. Mice were injected intravenously with³H-thymidine and draining auricular lymph nodes were evaluated for³H-thymidine incorporation into proliferating lymphocytes. Values represent means and standard deviationson day 6 of the LLNA.

*Represents statistically significant difference from vehicle control at p < 0.05 via Dunnett’s test.

^&Indicates no statistical comparison of means.

è Erythema scores represent the mean from each group on day 6 of the LLNA.

AOO = 4:1 acetone:olive oil; HCA = alpha-hexylcinnamaldehyde; g = grams; DPM = disintegrations perminute; SI = stimulation index.

Applicant's summary and conclusion

Interpretation of results:

other: Inconclusive

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

3,5-Lutidine was tested in female BALB/c mice in a local lymph node assay (LLNA) and found to be weakly sensitizing, with a SI of 5 after topical exposure of 50% in vehicle. However, this substance was found not to be irritating in a preliminary screening assay measuring mouse ear swelling after topical exposure. The weak response and the absence of irritation lead to questioning the validity of the findings.