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Description of key information

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:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD testing guideline compliant study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
other: mice
Strain:
other: CBA/ CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Species/strain: Healthy CBAICaO1aI-Isd mice
Source: Harlan Winkelmann GmbH, D-33 178 Borchcn
Sex: female
The female animals were nulliparous and non-pregnant.
Age at the beginning of the study: 8 — 12 weeks
Number of animals: 5 mice per test group
The animals were derived from a controlled full barrier maintained breeding system (SPF). According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals are bred for experimental purposes.
Vehicle:
other: acetone
Concentration:
Based on the results of the preliminary test the test item was assayed at three concentrations: 6.25%, 12,5% and 25%.
The preparations were made immediately prior to each dosing.
No. of animals per dose:
5 mice per test group
Details on study design:
A preliminary test was performed first to detect the highest possible application solution.
For this purpose, two animals were treated by topical application with a test item concentration of 25% (suspended in AOO) to the entire dorsal surface of each ear.
One further animal was treated with 100% AQO and served as negative control.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoe, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in the animal.

The animals were randomly selected.
Identification was ensured by cage number and individual marking (tail).

Prior to the application and once a day thereafter all animals were observed in order to detect specific clinical signs or reactions to. treatment.

The animals were weighed prior to the application and at the end of the test period.

3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime
Topical Application
Each mouse was treated by topical application of 25 uL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days.

Administration of 3H-methyl thymidine
Five days after the first topical application all mice were dosed with 20 pCi tI-methyl thymidine by intravenous injection (tail vein) of 250 pL of 3H- methyl thymidine, diluted to a working concentration of 80 pCi/mL.

Preparation of cell suspension
Approximately 5 hours after tI-methyl thymidine-injection all mice were sacrificed. The draining “auricular lymph nodes” were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in 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 approx. 1 mL 5% TCA at approx. 4 °C overnight for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 5 mE scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of incorporated 3H-methyl thymidine
The 3H-methyl thymidine — incorporation was measured in a 13-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.

Reliability Check
The recent reliability check was performed in February 2009. The raw data of this study are kept in the I3SL archives (BSL Project ID 090012E). The reliability checks are audited by the QA-unit periodically.
Positive control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity> 98%, lot 057K0052) 1% in AOO
The stimulation index after the application of the positive control substance was 13.0, confirming the reliability of the test system.

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3=c+[(3-d)/(b-d)]x(a-c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a ‘sensitize? 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 lymph nodes of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
Positive control substance(s):
other: P-Phenylenediamine (CAS 106-50-3)
Statistics:
The recent reliability check was performed in February 2009. The raw data of this study are kept in the BSL archives (BSL Project ID 090012E). The reliability checks are audited by the QA-unit periodically.
Positive control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity> 98%, lot 057K0052) 1% in AOO
The stimulation index after the application of the positive control substance was 13.0, confirming the reliability of the test system.
Positive control results:
Reliability Check
The recent reliability check was performed in February 2009. The raw data of this study are kept in the BSL archives (BSL Project ID 090012E). The reliability checks are audited by the QA-unit periodically.
Positive control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity> 98%, lot 057K0052) 1% in AOO
The stimulation index after the application of the positive control substance was 13.0, confirming the reliability of the test system.
Parameter:
SI
Remarks on result:
other: The stimulation index at a concentration of 6.25% was 1.1 The stimulation index at a concentration of 12.5% was 1.8 The stimulation index at a concentration of 25% was 0.9
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.
Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The EC3 value (derived by linear interpolation) could not be stated, as all measure points were below the stimulation index of three.
Results of radioactivity determination are supported by the means of the lymph node weights per group, which showed no significant difference compared to the negative control.
Consequently, according to OECD 429 and the OECD-GHS (Globally Harrnonised System of Classification and Labelling of Chemicals), the test item is not expected to have sensitising properties and therefore is no dermal sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The study followed OECD testing guideline 429 and GLP (BSL 2009d). Due to the overall insolubility, the test item was suspended in AOO, and 25% was the highest achievable concentration. Lymph node proliferation was measured via 3H-thymidine incorporation. None of the three doses induced a simulation index of above 3. There was no dose-response. Overall, the substance is did not cause sensitization in the LLNA.


Migrated from Short description of key information:
The subtance did not cause sensitization in the local lymph node assay in mice at concentrations of up to 25% (BSL 2009d).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available study is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin sensitization under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.