Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Mar - 30 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Version / remarks:

adopted in 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-(3,4-dimethyl-1H-pyrazol-1-yl)butanedioic acid; 2-(4,5-dimethyl-1H-pyrazol-1-yl)butanedioic acid

EC Number:

940-877-5

Cas Number:

2241455-89-8

Molecular formula:

C9H12N2O4

IUPAC Name:

2-(3,4-dimethyl-1H-pyrazol-1-yl)butanedioic acid; 2-(4,5-dimethyl-1H-pyrazol-1-yl)butanedioic acid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsD

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 18-23 g
- Fasting period before study: no
- Housing: single housing in IVC cages, type II L
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot No. 1114), ad libitum
- Water: tap water (sulphur acidified to a pH of 2.8), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55±10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

6.25%, 12.5%, and 25% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
2 animals were treated by topical application with the test item on 3 consecutive days at a concentration of 25% (suspended in DMSO) to the entire dorsal surface of the ear. Immediately before the first application, 48 h thereafter and shortly before sacrificing the thickness of both ears of all animals was measured. No signs of systemic toxicity or signs of irritation at the application site were detected. The measurement of ear thickness revealed no difference between control and treated animals and no change of ear thickness was observed at the different time points. All animals showed the expected body weight development throughout the duration of the preliminary test. The maximum technically applicable concentration in the vehicle was 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation (determined by beta-scintillation).
- Criteria used to consider a positive response: A substance will be regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine-incorporation into lymph node cells of the test group animals relative to that recorded for the lymph nodes of control group animals (SI ≥ 3).

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.

Five days after the first topical application all mice were dosed with 250 µL 3H-methyl thymidine (corresponding to 20 µCi) by intravenous injection (tail vein).
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected with phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approximately 1 mL 5% TCA at 4°C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
The 3H-methyl thymidine–incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: p-phenylendiamine (CAS 106-50-3, 1% on three consecutive days; reliability check in February 2012)

Results and discussion

Positive control results:

The positive control substance p-phenylendiamine induced an SI of 9.8 ± 3.8 (5 animals).

In vivo (LLNA)

Results

Results 1

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Values are given as mean ± SD Control: 3392 ± 1024 6.25%: 3848 ± 1312 12.5%: 2988 ± 920 25%: 2268 ± 574

Results 2

Key result

Parameter:

SI

Value:

1.1

Variability:

± 0.4

Test group / Remarks:

6.25%

Results 3

Key result

Parameter:

SI

Value:

0.9

Variability:

± 0.3

Test group / Remarks:

12.5%

Results 4

Key result

Parameter:

SI

Value:

0.7

Variability:

± 0.2

Test group / Remarks:

25%

Any other information on results incl. tables

No effects on the body weight development were observed.

Table 1: DPM and stimulation index of the LLNA.

Concentration	Animal No.	DPM	Stimulation index
6.25%	1	2990
	2	4336
	3	4269
	4	1891
	5	5754
	Mean ± SD	3848 ± 1312	1.1 ± 0.4
12.5%	6	2260
	7	4387
	8	1953
	9	2617
	10	3726
	Mean ± SD	2988 ± 920	0.9 ± 0.3
25%	11	2916
	12	2278
	13	1563
	14	2897
	15	1688
	Mean ± SD	2268 ± 574	0.7 ± 0.2
Negative control	16	2654
	17	2543
	18	2557
	19	4188
	20	5019
	Mean ± SD	3392 ± 1024	1.0
Background (scintillation fluid and trichloroacetic acid)		14
		14
		19
		32
		12
	Mean ± SD	18 ± 7	0.0

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified