1H-Pyrazole-4-carboxylic acid, 3-(difluoromethyl)-1-methyl-, ethyl ester

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)

Rationale for reliability incl. deficiencies:

other: The study was performed according to the OECD guideline No.429 (2010) and in compliance with the GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Only the highest test substance concentration was tested. This reduced Local Lymph Node Assay (LLNA) uses fewer animals than the full standard LLNA and is considered suitable for non-toxic substances with no alerting structure for sensitizing properties.

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:

ethyl 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylate

EC Number:

619-510-5

Cas Number:

141573-95-7

Molecular formula:

C8H10F2N2O2

IUPAC Name:

ethyl 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylate

Details on test material:

- Name of test material (as cited in study report): DFMMP
- Lot/batch No.: Batch No. MKA13111
- Substance type: mono-constituent substance
- Physical state: powder
- Analytical purity: 99.0% (GC)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source : Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: 21 - 24 g
- Housing: group housed in Makrolon MII type cages.
- Diet (e.g. ad libitum): free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 - 22.1°C
- Humidity (%): 44-72%
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (non-LLNA)

Induction

Concentration / amount:

Not applicable

Challenge

Concentration / amount:

Not applicable

No. of animals per dose:

Not applicable

Details on study design:

Not applicable

Challenge controls:

Not applicable

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

Merck, Darmstadt, Germany

Concentration:

For the preliminary test the concentrations were 25 and 50% of the test item.
For the main test the concentrations were 0 and 50% of the test item.

No. of animals per dose:

For the preliminary test: 2 females/dose (no control) were treated.
For the main test: 5 females were treated for both the control and the 50% concentration (total of 10 animals).

Details on study design:

RANGE FINDING TEST
- Compound solubility: The test item was soluble in dimethyl formamide. A solution was obtained at the maximum tested concentration of 50%.
- Irritation: Measurement of the ear thickness (using a digital thickness gauge) was performed prior to dosing on Days 1, 3 and 6. Very slight erythema was observed on the dorsal surface of the ears on Day 3 for both animals at a 50% test substance concentration. No signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, a 50% test substance concentration was selected for the main study.
- Lymph node proliferation response: no measurement

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymph node cell proliferative responses were measured.
- Criteria used to consider a positive response: The results were expressed as disintegration per minute (dpm) for each animal and for each dose group. Stimulation indices (SI) were calculated according to the following formula: SI = dpm of treated group / dpm of control group. The test item was considered as a skin sensitizer when the SI for a dose group is higher than or equal to 3.
TREATMENT PREPARATION AND ADMINISTRATION
The test item was prepared in the vehicle at the chosen concentrations. All dosage form preparations were prepared within 4 hours prior to each treatment. On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The most recent check with Alpha-hexylcinnamicaldehyde showed that the calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI value calculated for the 50% test substance concentration was 1.5.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The mean DPM/animal value for the experimental group treated with a 50% test substance concentration was 319. The mean DPM/animal value for the vehicle control group was 219 DPM.

Any other information on results incl. tables

No irritation of the ears was observed in any of the animals examined. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.