Registration Dossier

Administrative data

Description of key information

LLNA: not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany
Type of study:
mouse local lymph node assay (LLNA)
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
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)
Positive control results:
The positive control substance p-phenylendiamine induced an SI of 9.8 ± 3.8 (5 animals).
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
Key result
Parameter:
SI
Value:
1.1
Variability:
± 0.4
Test group / Remarks:
6.25%
Key result
Parameter:
SI
Value:
0.9
Variability:
± 0.3
Test group / Remarks:
12.5%
Key result
Parameter:
SI
Value:
0.7
Variability:
± 0.2
Test group / Remarks:
25%

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

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A study performed according to OECD guideline 429 and in compliance with GLP, is available for assessment of skin sensitizing properties of Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid (Lütkenhaus, 2012). Five CBA mice per dose were treated with 25 µl of 6.25%, 12.5%, and 25% test substance (diluted in DMSO) once daily for 3 consecutive days. The dosing concentrations were determined in a range-finder assay. No signs of systemic toxicity or signs of irritation at the application site were detected after treatment of the animals with up to 25% test item solution. 25% was the maximum technically applicable concentration determined in a solubility test. 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 and washed. After precipitation of macromolecules with TCA, scintillation fluid was added and 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.

The DPM were 3392±1024, 3848±1312, 2988±920, and 2268±574 for the control, 6.25%, 12.5%, and 25% dose group, respectively. The resulting stimulation indices were 1.1 ± 0.4, 0.9 ± 0.3 and 0.7 ± 0.2 for the 6.25%, 12.5%, and 25% dose group, respectively. In addition, no signs of systemic toxicity or signs of irritation at the application site were detected. In conclusion, as the stimulation index after treatment with Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid was smaller than three, the test item was not sensitising under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation of Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl) succinic acid do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.