2-(3-Chloro-2-pyridinyl)-5-oxo-3-pyrazolidinecarboxylic 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:

May 2006 - July 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 2-(3-chloropyridin-2-yl)-5-oxopyrazolidine-3-carboxylate

EC Number:

610-490-3

Cas Number:

500011-88-1

Molecular formula:

C11H12ClN3O3

IUPAC Name:

ethyl 2-(3-chloropyridin-2-yl)-5-oxopyrazolidine-3-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 10 weeks old
- Weight at study initiation: 20.6 - 25.4 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: Approximately 2 weeks

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 17 May 2006 to 23 May 2006

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0% (vehicle control), 6.25%, 12.5%, 25% and 50%

No. of animals per dose:

5 female mice

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test substance was evaluated for solubility in dimethyl sulphoxide. Due to the solubility of the test substance, doses greater than 50% were not evaluated.
- Irritation: No irritation was observed
- Systemic toxicity: No clinical signs or toxicity were observed.

MAIN STUDY
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), 25 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:
Cage-site examinations to detect moribund or dead mice and abnormal behaviour and appearance among mice were conducted 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 = 8.16. 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:

1.1

Test group / Remarks:

6.25%

Results 2

Parameter:

SI

Value:

1.36

Test group / Remarks:

12.5%

Results 3

Parameter:

SI

Value:

1.67

Test group / Remarks:

25%

Results 4

Parameter:

SI

Value:

1.24

Test group / Remarks:

50%

Results 5

Parameter:

SI

Value:

8.16

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 of toxicity were observed in the study.

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	1202,00	371,17	N/A
IV	6,25%	5	1323,00	645,45	1,10
VI	12,5%	5	1639,20	609,79	1,36
VIII	25%	5	2010,20	492,36	1,67
X	50%	5	1484,60	328,63	1,24
XII	25% positive control (a)	5	6588,80	1597,86	8,16
XIV	0% positive control vehicle (a)	5	807,20	342,41	N/A

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the study, the test item did not produce a dermal sensitization response in mice and is not considered to be a dermal sensitizer.

Executive summary:

The objective of this study was to evaluate the potential of the test substance 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), 6.25%, 12.5%, 25%, or 50% test item on both ears. Dimethylsulfoxide was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in 4:1 acetone:olive oil (AOO) as a positive control and one group of 5 female mice was dosed for 3 consecutive days with AOO as a positive control vehicle.

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.

A rangefinding experiment was conducted to evaluate the toxicity potential of the test substance. Twenty-five μL of test substance were administered to 5 female mice, topically to the dorsum of each ear for 3 consecutive days, at concentrations of 0% (vehicle control), 6.25%, 12.5%, 25%, or 50% test substance. No clinical signs of toxicity were observed. All animals survived to the scheduled sacrifice.

 

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 of toxicity were observed in the study. No statistically significant  ncreases in cell proliferation measurements compared to the vehicle control group were observed at any test concentration. Stimulation indexes of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., stimulation index = 3) for the test substance under the conditions of this study was not calculable.

A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice, SI = 8.16. Therefore, the LLNA test system was valid for this study.

 

Under the conditions of this study, the test substance did not produce a dermal sensitization response in mice and is not considered to be a dermal sensitizer.