1H-Pyrazole-5-carboxylic acid, 3-bromo-1-(3-chloro-2-pyridinyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-11-28 to 2006-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD guideline 429 and GLP.

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)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at time of study conduct.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at time of study conduct.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

481-150-8

EC Name:

-

Cas Number:

500011-86-9

Molecular formula:

C9H5BrClN3O2

IUPAC Name:

3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid

Constituent 2

Reference substance name:

DBC80

IUPAC Name:

DBC80

Constituent 3

Reference substance name:

1H-Pyrazole-5-Carboxylic Acid, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-

IUPAC Name:

1H-Pyrazole-5-Carboxylic Acid, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-

Details on test material:

- Name of test material (as cited in study report): IN-DBC80
- Physical state: Tan solid
- Analytical purity: 96.7 %
- Purity test date: 2005-07-22
- Lot/batch No.: IN-DBC80-008
- Expiration date of the lot/batch: 2008-07-22
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study. No evidence of instablility, such as a change in color or physical state, was observed.
- Storage condition of test material: At room temperature (ambient < 25 degrees Celcius).

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- female (nulliparous and non-pregnant) CBA/JHsd
- Source: Harlan Sprague-Dawley (Frederick, Maryland, USA)
- Age at study initiation: About 5 weeks on the day of arrival. At study start (test day 0), mice were approximately 9 weeks old.
- Weight at study initiation: Test day 0- 18.7-25.3 grams
- Housing: All mice were housed 2-3 per cage in stainless steel, wire-mesh cages suspended above cage boards during quarantine. Prior to assignment to groups and during the dosing and resting phases of the study, each mouse was 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 appropriate bedding.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: Minimum of 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 degrees Celcius.
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark.

IN-LIFE DATES: From: 2005-11-01 To: N/A

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 5, 25, 50 and 100 %

No. of animals per dose:

5

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT- Prior to study start, mice were selected based upon adequate body weight gain and freedom from any ear abnormalities (e.g. torn, scratched), as determined in the pretest period, and were assigned to a group using a randomly generated, computer-based algorithm such that individual pretest body weights did not vary more than 20 % of the group mean.
- Name of test method: Mouse local lymph node assay (pooled and individual data for DPM calculations reported).
- Criteria used to consider a positive response: Statistically significant increases in cell proliferation in the test substance groups compared to the vehicle control group and/or stimulation indexes of greater than or equal to 3.0 indicated a positive response.


TREATMENT PREPARATION AND ADMINISTRATION: The test substance was prepared in the vehicle, including the 100 % concentration, which was prepared at 1 g/mL. All dose preparations were formulated fresh daily.
Twenty-five uL of the test substance were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 0-2). Test days 3-4 were days of rest followed by intravenous injection of 20 uCi 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 degrees Celcius 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). A stimulation index (SI) was derived from each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group.

Positive control substance(s):

other: hexylcinnamaldehyde (25 %); prepared in 4:1 mixture of acetone:olive oil

Statistics:

Significance was judged at p<0.05 except for dpm data which were judged significant at <0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances.
Body weight and body weight gains were first analyzed by Levene's test for homgeneity and the Shapiro-Wilk test for normality. One-way analysis of variance followed by a Dunnett's test was performed if the preliminary test was not significant. The Kruskal-Wallis test followed by the Dunn's test was used in the preliminary test was significant.

The Cochran Armitage test was used to analyze trends of Clinical observations.

The Lymph node dpm data was first analyzed by Levene's test for homgeneity and the Shapiro-Wilk test for normality. One-way analysis of variance followed by a Dunnett's test was performed if the preliminary test was not significant. The Kruskal-Wallis test followed by the Dunn's test was used if the preliminary test was significant.
Or, a test for a lack of trend was performed on the preliminary data. If this result was not significant a sequential application of the Jonckheere-Terpstra trend test was applied. If a significance was identified a preliminary test for a pairwise comparison was performed.

Results and discussion

Positive control results:

The positive control produced a dermal sensitization response in the mice.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Stimulation indexes of less than 3.0 were observed at all concentrations of the test substance. Therefore, the EC3 value (the estimated concentrationrequired to induce a threshold positive response, i.e., stimulation index=3) for the test substance under the conditions of this was not calculable.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Overall remarks, attachments section

Any other information on results incl. tables

No statistically significant differences in mean body weights or 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.

Pooled Data

Group	n	Mean (dpm)	Std..Dev. (dpm)	SI
Vehicle Control	5	626.55	170.13	N/A
5%	5	803.75	437.78	1.28
25%	5	1078.75	283.62	1.72
50%	5	747.55	334.13	1.19
100%	5	708.15	247.17	1.13
25 % Positive Control	5	4236.75	1407.11	6.19
Positive Control Vehicle	5	684.75	238.71	N/A

SI = Stimulation Index

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Based on the results of the study, the test substance was not deemed a dermal sensitizer.