1-(3-Chloro-2-pyridinyl)-4,5-dihydro-3-[(phenylsulphonyl)oxy]-1H-pyrazole-5-carboxylic acid ethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2006 - September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Guideline for the Testing of Chemicals (2001)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

Health Effects Test Guidelines (2003)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate

EC Number:

846-153-4

Cas Number:

653592-41-7

Molecular formula:

C17H16ClN3O5S

IUPAC Name:

ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Frederick, Maryland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 11 weeks old
- Weight at study initiation: 21.3-27.4 g
- Housing: During dosing and resting phases of the study, animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. After final weighing (test Day 5) until sacrifice, animals were housed 1 group per plastic shoebox cage with bedding.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet #5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Alternating 12-hour light and dark cycles
- IN-LIFE DATES: From 26 July 2006 to 01 August 2006

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0% (vehicle control), 5%, 10%, 25% and 50%

No. of animals per dose:

5 female mice

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: A Stimulation Index (SI) greater than 3 indicates a positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
Prior to study start, a quantity of the test substance was evaluated for solubility in the vehicle. All dose preparations were formulated fresh daily. On each day of dosing (test Days 0-2), twenty-five μL of test or control substance were administered topically to the dorsum of each mouse ear.
Test Days 3-4 were days of rest followed by intravenous (tail vein) injection of 20 μCi of ³H-thymidine per mouse on test Day 5. Approximately 5 hours after the injection, animals were sacrificed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8°C overnight. On test day 6, the single cell suspensions were counted on a beta counter. The counts per minute (cpm) data were converted to disintegrations per minute (dpm).

OBSERVATIONS:
Animals were carefully observed for any clinical symptoms at least once daily throughout the study. The animal body weights were measured prior to the first treatment and at the scheduled sacrifice.

Positive control substance(s):

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

Statistics:

A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group.

When possible, an EC3 value for the stimulation index data was derived from linear interpolation of points on the dose-response curve immediately above and below the 3-fold threshold. The equation used for calculation of EC3 was:
EC3 = c + [(3-d)/(b-d)] × (a-c)
where:
a = the lowest concentration giving stimulation greater than 3
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 3
d = the actual stimulation index caused by c

Results and discussion

Positive control results:

A 25% concentration of the positive control, HCA, produced a dermal sensitisation response in mice, SI = 5.61. This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.14

Test group / Remarks:

5%

Results 2

Parameter:

SI

Value:

0.96

Test group / Remarks:

10%

Results 3

Parameter:

SI

Value:

1.66

Test group / Remarks:

25%

Results 4

Parameter:

SI

Value:

3.61

Test group / Remarks:

50%

Results 5

Parameter:

SI

Value:

5.61

Test group / Remarks:

25% positive control

Cellular proliferation data / Observations:

No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration. No clinical signs were observed in animals dosed with the test substance. Hair loss was observed in one animal from the positive control group.

Any other information on results incl. tables

Table 1 Simulation Index Data

Group	Material tested	n	Mean (dpm)	S.D. (dpm)	SI
II	0% vehicle control	5	603,7	388,29	N/A
IV	5%	5	690,1	265,15	1,14
VI	10%	5	579,9	167,47	0,96
VIII	25%	5	1000,1	176,67	1,66
X	50%	5	2177,5 (#)	1301,27	3,61
XII	25% positive control (a)	5	3386,9	960,15	5,61

a: Data were not included in the statistical analysisi of the test substance groups.

#: Statistically significant increase in dpm data from vehicle control at p < 0.01 by Jonckheere-Terpstra trend data.

 

An SI of greater than 3.0 was observed at the 50% test concentration of the test substance. The EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance was calculated to be 42%.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the experimental conditions of the study, the test item produced a dermal sensitization response in mice.

Executive summary:

The objective of this study was to evaluate the potential of the test item to produce a dermal sensitization response in mice using the local lymph node assay (LLNA) according to OECD Guideline 429.

 

Five groups of 5 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle control), 5%, 10%, 25%, or 50% test item on both ears. Dimethylsulfoxide (DMSO) was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in DMSO as a positive control. On test day 5 of the assay, mice received ³H-Thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle control group.

 

No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration. No clinical signs were observed in animals dosed with the test substance. Hair loss was observed in one animal from the positive control group.

 

A statistically significant increase in cell proliferation measurements compared to the vehicle control group was observed at the 50% test concentration. An SI of greater than 3.0 was observed at the 50% test concentration of the test item. The EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was calculated to be 42%.

Based on these data, the test item is considered a dermal sensitizer.