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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007 - 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed OECD study with GLP
Composition 0
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Details on test animals and environmental conditions:
- Strain: CBA/CaOlaHsd
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 18.8 - 22.5 g
- Housing: single
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation: at least 5 days under test conditions after health examination

- Temperature (°C): 22+- 3
- Humidity (%): 30 - 78
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

0% (control), 5, 10, 25 %
No. of animals per dose:
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
α-Hexylcinnamaldehyde; Vehicle: acetone:olive oil (4+1)
EC3 value: 6.7 %
Remarks on result:
other: see above
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Test item Calculation Result concentration Group Measurement number of DPM per % (w/v) DPM DPM-BG lymph nodes lymph node S.I. --- BG I 24 --- --- --- --- --- BG II 24 --- --- --- --- 0 1 2268 2244 8 280.5 1.00 5 2 9323 9299 8 1162.4 4.14 10 3 11902 11878 8 1484.8 5.29 25 4 5064 5040 8 630.0 2.25 BG = Background 1 = Control Group The EC3 value could not be calculated due to the odd dose response.
Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
The test item Carbethoxypyrazolone TTR was found to be a skin sensitiser under the test conditions of this study.
Executive summary:

A local lymph node assay (LLNA, OECD 429) was performed in order to study a possible skin sensitising potential of Carbethoxypyrazolone TTR. Three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/v) in DMF by topical application to the dorsum of each ear for three consecutive days.

The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (DMF) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were

subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 4.14, 5.29, and 2.25 were determined with the test item at concentrations of 5, 10, and 25% in DMF. The EC3 value could not be calculated.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:
Migrated from Short description of key information:
Two LLNA are available:
a screening test with only the highest applicable concentration (25%)(1099600) and a complete assay with three concentrations (1117102). In both studies the stimulation index for the highest concentration (25%) is below 3. In the complete test stimulation indices greater than 3 could be observed at concentration of 5 and 10 %..
It cannot be ruled out that the measured S.I. for 25% is an effect bases n the solubility of the test item

Justification for selection of skin sensitisation endpoint:
Complete LLNAwith several concentrations

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Although the stimulation index for the highest concentration (25%) is below 3 in both assays the results of the S.I. s for 5 and 10% concentration have to be taken into account for the classification of this substance.