2-Pyridinecarboxylic acid, 4-(acetylamino)-3,6-dichloro-, methyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2013 to 12 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

not specified

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 9 to 12 weeks
- Weight at study initiation: 20.1 to 23.8 g on day 1 of the test
- Housing: Animals were housed up to six per cage in filter tubs containing corncob bedding, food pellets and a water bottle. On the day the animals were euthanised (following the injection of 3H-thymidine) each treatment group of mice was housed in shoebox cages containing corncob bedding, food pellets, and a crock filled with water.
- Diet (e.g. ad libitum): Certified rodent diet in pelleted form was provided ad libitum.
- Water (e.g. ad libitum): Municipal water was provided ad libitum.
- Acclimation period: At least one week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): 12 to 15 times per hour (on average)
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle (light from 06:00 to 18:00)

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

Selected based upon maximum solubility of the test material while maintaining a solution suitable for application.

Concentration:

5, 25 and 75 % of the test material

No. of animals per dose:

5 female animals per dose

Details on study design:

RANGE FINDING TEST
A pre-screen test was performed in order to define the maximum dose level for use in the LLNA. The ears of six mice (one female/concentration) were topically treated for three consecutive days with one of six concentrations (0, 5, 10, 25, 50, or 75 %) of the test material. The initial concentration of the test material was based upon solubility and viscosity.
All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to dosing on Day 1 and prior to termination on Day 6. Both ears of each mouse were observed for erythema prior to dosing on days 1, 2 and 3 and on day 6 prior to termination and scored.
Ear thickness measurements were taken for each ear using a digital micrometer prior to dosing on Day 1 and 3 and on Day 6 prior to termination. Excessive local skin irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25 % on Day 3 or 6 relative to the measurement on Day 1. The highest dose selected for the main LLNA study was the next lower dose in the pre-screen concentration series that does not induce systemic toxicity and/or excessive local skin irritation.
Erythema was absent, and body weights and ear thickness were unaffected at all dose levels.

MAIN STUDY
The application of 25 µL of the test material was made on the dorsal surface of both ears as described above. Ears were inspected prior to application of the test material solutions, and erythema was evaluated on days 1, 2, 3, and 6. All mice were weighed on days 1 and 6.
On day 6, all mice received a 250 μL intravenous injection (i.v.) via the lateral tail vein containing 20 μCi of 3H-thymidine (specific activity 2 Ci/mmol) diluted in phosphate-buffered saline (PBS). Approximately five hours post administration, the mice were euthanised via CO₂ asphyxiation and both auricular lymph nodes located at the bifurcation of the jugular veins were excised and placed in PBS. A single cell suspension of the auricular lymph nodes from one mouse was prepared by gentle mechanical disaggregation using a tissue homogeniser. The cells were washed two times and were suspended in 3 mL of 5 % trichloroacetic acid (TCA) for approximately 18 to 70 hours. The suspended precipitates were centrifuged (200 x gravity 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.
Two additional 2 mL aliquots of water were used to rinse the tubes and the rinses were added to the scintillation vials containing the 1 mL of pellet in TCA and cocktail. The radioactivity in each precipitate was measured using a β-scintillation counter and reported as disintegrations per minute (dpm) per mouse. 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; the mean SI ± SD was calculated for each experimental group. Any test material that produces an SI of ≥3 in the LLNA should be considered “positive” for contact sensitisation.

CLINICAL OBSERVATIONS
The initial and terminal body weights were obtained and recorded. A cage-side examination was conducted twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary.
Significant abnormalities that could be observed included, but are not limited to: decreased/increased activity, repetitive behaviour, vocalisation, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency, and faecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. The ears were evaluated for erythema prior to application of test material solutions and 3H-thymidine injections and scored as follows:
- No visual effect: 0
- Slight erythema (barely perceptible): 1
- Well-defined erythema: 2
- Moderate to severe erythema: 3
- Eschar: 4

Positive control substance(s):

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

Statistics:

The % ear swelling is calculated for each ear using the following equation:
% Ear swelling = [(B - A) / A] x 100
where:
A = ear thickness measurement on Day 1 (mm x 10-²)
B = ear thickness measurement on Day 3 or 6 (mm x 10-²)

The Stimulation Index (SI) was calculated for each mouse using the following equation:
SI = Disintegration per minute (dpm) of individual mouse / Average dpm of the VH control mice

EC₃ Calculation:
EC₃ = XL + [(3 - YL) / (Yh - YL)](Xh - XL)
where:
YL = SI value below 3
XL = chemical concentration that elicits YL
Yh = SI value above 3
Xh = chemical concentration that elicits Yh

Means and standard deviation (SD) were generated for body weight data (absolute and gain) and the LLNA response (dpm and SI values). Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969).
These body weight and dpm data were analysed by a one-way analysis of variance (Steele and Torrie, 1960). When differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a 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 responses was based on both statistical outcome and scientific judgment.

Results and discussion

Positive control results:

A positive response was elicited from the positive control, achieving a stimulation index (SI) of 8.4 in comparison to vehicle-treated mice. See Table 1.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The test material at concentrations of 5, 25 and 75 % elicited proliferative responses with stimulation indices (SI) of 1.3, 1.7, and 2.0, respectively, in comparison to vehicle-treated mice. See Table 1. Therefore, the test material did not demonstrate dermal sensitisation potential in the mouse LLNA as the lymph nodes draining the area of topical application did not have a proliferative response greater than the 3-fold threshold.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 1.

Any other information on results incl. tables

Table 1: Mean Disintegrations Per Minute (dpm) and Stimulation Indices (SI)

Dose (%)		dpm	SI
0 (Vehicle)	Mean SD N	659.00 308.18 5	1.0 0.5 5
5	Mean SD N	851.20 230.97 5	1.3 0.4 5
25	Mean SD N	1152.4 575.82 5	1.7 0.9 5
75	Mean SD N	1299.0 690.36 5	2.0 1.1 5
25 HCA (Positive Control)	Mean SD N	5506.4* 1363.1 5	8.4 2.1 5

*Statistically different form the control by Dunnett’s test, alpha = 0.05

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, topical application of the test material at 5, 25 and 75 % in DMSO did not elicit a stimulation index (SI) that met the 3-fold threshold, thus indicating a lack of dermal sensitisation potential in the mouse LLNA. As such the test material requires no classification in accordance with EU criteria.

Executive summary:

The potential of the test material to cause contact sensitisation was investigated in a mouse LLNA study conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and USA EPA OPPTS 870.2600 under GLP conditions.

A screening study was conducted in which three daily topical applications of 0, 5, 10, 25, 50 or 75 % of the test material in dimethyl sulphoxide were given to one animal at each dose level. Erythema was absent and body weights and ear thickness were unaffected. Therefore the results from this study were used to determine the dosing concentrations in the definitive LLNA test.

In the main study, on Days 1 to 3 five female mice/group received 5, 25 or 75 % of the test material in dimethyl sulphoxide or the vehicle alone (DMSO). One group of five mice received 25 % α-hexylcinnamaldehyde in DMSO and served as the positive control. On day 6, uptake of 3H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours post administration.

Erythema was absent and body weights were unaffected in all dose groups. The test material at concentrations of 5, 25 and 75 % elicited proliferative responses with stimulation indices (SI) of 1.3, 1.7, and 2.0, respectively, in comparison to vehicle-treated mice.

Proper conduct of the LLNA was confirmed via a positive response using 25 % α-hexylcinnamaldehyde (a moderate contact sensitiser) which elicited proliferation with a stimulation index of 8.4 in comparison to vehicle-treated mice.

Under the conditions of this study, topical application of the test material at 5, 25 and 75 % in DMSO did not elicit a stimulation index (SI) that met the 3-fold threshold, thus indicating a lack of dermal sensitisation potential in the mouse LLNA. As such the test material requires no classification in accordance with EU criteria.